Aggregation-induced emission : materials and applications v2 9780841231559, 0841231559, 9780841231573, 0841231575

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Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.fw001

Aggregation-Induced Emission: Materials and Applications Volume 2

Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.fw001

Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

ACS SYMPOSIUM SERIES 1227

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.fw001

Aggregation-Induced Emission: Materials and Applications Volume 2 Michiya Fujiki, Editor Nara Institute of Science and Technology Nara, Japan

Bin Liu, Editor National University of Singapore Singapore

Ben Zhong Tang, Editor Hong Kong University of Science and Technology Kowloon, Hong Kong

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.fw001

Library of Congress Cataloging-in-Publication Data Names: Fujiki, Michiya, editor. | Liu, Bin, 1974- editor. | Tang, Ben Zhong, editor. Title: Aggregation-induced emission : materials and applications Volume 2 / Michiya Fujiki, editor, Nara Institute of Science and Technology, Nara, Japan, Bin Liu, editor, National University of Singapore, Singapore, Ben Zhong Tang, editor, Hong Kong University of Science and Technology, Kowloon, Hong Kong. Description: Washington, DC : American Chemical Society, [2016]- | Series: ACS symposium series ; 1226 | Includes bibliographical references and index. Identifiers: LCCN 2016039915 (print) | LCCN 2016040743 (ebook) | ISBN 9780841231580 (v. 2) | ISBN 9780841231573 (ebook) Subjects: LCSH: Luminescence. | Aggregation (Chemistry) | Photoemission. Classification: LCC QC476.5 .A35 2016 (print) | LCC QC476.5 (ebook) | DDC 541/.35--dc23 LC record available at https://lccn.loc.gov/2016039915

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2016 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.pr001

Preface In December 2015, the AIE community gathered in Honolulu, Hawaii, USA and enjoyed an engaging symposium entitled, “Aggregation-Induced Emission: Materials and Applications,” co-organized by Bin Liu, Ben Zhong Tang, and myself. This symposium offered a unique opportunity to bring together distinguished experts from different areas to share their exciting results and the latest developments in the field, which formed the foundation of this two-volume e-book published by the American Chemical Society (ACS). Aggregation-induced emission (AIE) stands for an intriguing phenomenon in which a series of non-emissive molecules in solutions are induced to emit strongly in the aggregate or solid state. The concept of AIE was first coined by Ben in 2001, when he and his co-workers serendipitously discovered that 1-methyl-1,2,3,4,5-pentaphenylsilole was almost non-emissive in ethanol solution but became extremely bright in water-ethanol mixtures. This seminal paper appeared in Chemical Communications (2001, 1740), which was immediately highlighted by Chemical and Engineering News (2001, 79, 29) of the ACS as an unusual phenomenon that is opposite to the behavior of traditional luminophores. Over the past 15 years, AIE has grown into a research field with high visibility and broad impact across both science and technology. The e-books of Aggregation-Induced Emission: Materials and Applications summarize the recent advances in AIE research, ranging from fundamentals, such as design, synthesis, and optical properties of AIE-active molecules, to mechanism studies supported by modeling and experimental investigations, and further to promising applications in the fields of energy, environment, and biology. Because of the large amount of excellent research, the contents have to be divided into two volumes, with Volume 1 focusing on materials and Volume 2 placing greater emphasis on applications. The topics covered in Volume 1 include: New mechanisms and theoretical understanding of AIE phenomena; Vibration-induced emission; The art of restriction of molecular rotation; Domino synthesis of AIE molecules; Small molecule AIE systems; and Mechanochromic AIE materials. The topics covered in Volume 2 include: AIE polymers; AIE-induced chirogenesis; Room-temperature phosphorescent AIE molecules; Liquid crystalline AIE molecules; AIE materials for energy devices; New chemo- and biosensors with AIE molecules; Cell structure and function imaging with AIE molecules; and AIE materials in drug delivery and therapy. These e-books offer readers an excellent perspective of the significant progress recently made in the field of AIE research. They are essential for chemists, physicists, materials scientists, and biologists who work on optic and ix Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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photonic materials and their applications. The book also provides an easy entry to researchers who are new to the field. Each volume should satisfy your curiosity and may stimulate new ideas in your own future work. I cannot end this preface without expressing my gratitude to all the people who have made contributions to these e-books. We thank all the authors and reviewers for their dedicated work and also the editorial team from ACS, especially Bob Hauserman, Jack Nestor, Elizabeth Hernandez, and Arlene Furman, whose efforts and support made these e-books possible.

Michiya Fujiki, Ph.D. Nara Institute of Science and Technology Graduate School of Materials Science 8916-5 Takayama, Ikoma Nara 6300192 Japan

Bin Liu, Ph.D. Department of Chemical and Biomolecular Engineering 4 Engineering Drive 4 National University of Singapore Singapore, 117576

Ben Zhong Tang, Ph.D. Hong Kong University of Science and Technology Chemistry Clear Water Bay, Kowloon Hong Kong

x Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Chapter 1

Pure Organic Luminogens with Room Temperature Phosphorescence Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch001

Shuqin Wang,1,2 Wang Zhang Yuan,*,1 and Yongming Zhang*,1 1School

of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China 2Office of Research Management, Shanghai Jiao Tong University, Shanghai 200240, China *E-mails: [email protected] (W.Z.Y.); [email protected] (Y.Z.)

Recent progress in the fields of pure organic luminogens with room temperature phosphorescence (RTP) is reviewed. Besides basic molecular design considerations, varying strategies adopted to rigidify molecular conformations of the luminogens and to isolate oxygen, such as chelation, (co)crystallization, doping/trapping in rigid matrix, crosslinking, and creation of host-guest interactions, are summarized. Some new phenomena, concepts, and strategies like crystallizationinduced phosphorescence (CIP), directed heavy atom effect (DHAE), and cocrystallization utilizing halogen bonding are emphasized. Moreover, exciting advancements in persistent RTP and efficient RTP from solutions have also been highlighted. Meanwhile, their promising applications are also briefly mentioned.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Introduction Luminogens with room temperature phosphorescence (RTP) enjoy much broader applications compared to their fluorescent counterparts due to the involvement of longlived triplet manifold. Besides the well known applications in high efficiency electroluminescent devices (1, 2), RTP luminogens offer new opportunities in bioimaging, which allows better monitoring of cellular phenomena through excluding the interference from shortlived cellular auto-fluorescence background (3, 4). Additionally, emerging new applications, such as cellular hypoxia imaging, photodynamic therapy, temperature monitoring, oxygen/ozone-sensing, solvent detection, and security inks, have also been demonstrated (5–10). RTP phosphors, however, are essentially confined to inorganics or organometallic complexes (4, 11, 12), pure organic luminogens are difficult to receive efficient RTP (13–15), despite they take the advantages of low cost, versatile molecular design, facile functionalization and good processability. This is because the rate of phosphorescence is slow owing to the occurrence of spin flip, which is quantum mechanically forbidden. During such durable time, triplet excitons could easily lose their energies through thermally vibrational and collisional processes and exposure to such quenchers as oxygen and moisture. Therefore, phosphorescence from pure organic luminogens has typically been limited to cryogenic (e.g. 77 K) and inert conditions for a long time (15). To suppress vibrational relaxations of the triplet manifold, several approaches have been developed, including the use of special solvents, adsorption onto solid substrate or embedded into silica glass, and inclusion into surfactants or cyclodextrins, however, normally special and complex fabrication techniques are required and only instrument detectable signals are obtained (16–19). In 2007, Zhang and Fraser reported the interesting persistent RTP from the pure organic materials of difluoroboron dibenzoylmethane polylactide (20). In 2010, Tang and Yuan discovered that some pure organic luminogens such as benzophenone and its derivatives, methyl 4-bromobenzoate, and 4,4′-dibromobiphenyl exhibit no emission in solution, in polymeric films, or on thin-layer chromatography (TLC) plates, but become highly phosphorescent in the crystalline state at room temperature and ambient conditions, exhibiting crystallization-induced phosphorescence (CIP) characteristics (13). Restriction of intramolecular motions (RIR) by effective intermolecular interactions in the crystals and isolation from oxygen and moisture are ascribed for the boosted phosphorescence emission. Discovery of the CIP phenomenon paves the way for the fabrication of new efficient pure organic RTP luminogens through crystal engineering. In 2011, Bolton and Kim reported similar phenomenon of enhanced RTP from pure organic luminogens utilizing mixed crystals and halogen bonding (14). Since then, more and more reports concerning on the RTP from pure organic chromophores are demonstrated, by the utilization of (co)crystallization, doping or trapping into rigid matrix, intermolecular interaction, and singlet fission (15, 21–42). In this chapter, we summarized the recent progress of these exciting works, aiming to give a brief but clear picture on this renewed area.

2 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Fundamental Considerations To achieve efficient RTP from pure organic luminogens, several internal and external requirements should be fulfilled. Firstly, intersystem crossing (ISC) from the lowest excited singlet (S1) state to the triplet manifold (Tn) should be highly efficient. According to El-Sayed’s rule, ISC can be greatly promoted through efficient spin–orbit coupling (SOC) by the effective mixing of the singlet and triplet states of different molecular orbital (MO) configurations (43–45). Normally, carbonyls, nitrogen heteroatoms, heavy halogens, and/or small singlet–triplet splitting energy (ΔEST) are favorable to enhance the ISC process (14, 43–48). Meanwhile, radiationless relaxation from the lowest excited triplet (T1) state to ground (S0) state must be substantially impeded. Adequate suppression of vibrational and collisional dissipations is perhaps the most important and challenging aspect. What’s more, as triplet excitons are highly susceptible to triplet oxygen (5, 20, 49), which can facilitate the triplet-triplet quenching process, isolation of the luminogens from oxygen is a must. In short, considerable ISC, restricted molecular motions (rigidified conformations), and free of quenchers (particularly oxygen) are essential to receive efficient RTP from pure organic luminogens. Below, we summarized the recent endeavors in obtaining efficient pure organic RTP luminogens based on such fundamental aspects.

Difluoroboron Chelates Chelation of the dyes is an effective strategy to enforce conformation rigidity and charge redistribution in molecular systems (9, 20, 50–52). Difluoroboron β-diketonate (BF2bdk) luminogens are of great interest due to their intriguing properties in both solution and solid states (20, 50–54). Such skeleton provides the luminogens with rigid conformation as well as large Stokes shift, making them highly emissive in both solution and solid states (20, 50). Moreover, BF2 chelates are capable to impede intramolecular twisting of the aromatic-carbonyl moiety, thus offering phosphorescence in rigid environments (5, 9, 20, 50–54). Fraser and Zhang fabricated a series of BF2bdk dyes combined with biocompatible and biodegradable poly(lactic acid) (PLA) (Figure 1), which exhibit intense fluorescence, thermally activated delayed fluorescence (TADF) and oxygen-sensitive RTP (5, 9, 20, 51–54). The photophysical properties of BF2bdkPLA molecules can be well modulated by molecular structure, polymer molecular weight, halide substituents and their placement, as well as external temperature and oxygen. As the polymer molecular weight increases, dye−dye interactions and medium polarity are decreased, resulting in blue-shifted fluorescence with much shorter lifetime, longer phosphorescence lifetime, and larger singlet to triplet energy gap. While heavy atom normally promotes phosphorescent emission, systems without heavy atoms enjoy much longer phosphorescence lifetimes, which is favorable for highly sensitive, concentration independent time-resolved oxygen sensing. Specifically, with BF2dbmPLA (dbm = dibenzoylmethane) and its halogenated, heavy atom congener, BF2dbm(I)PLA, they demonstrated that emission wavelength, relative F/P intensity, and oxygen 3 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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sensitivity are tunable with heavy atom substitution and polymer molecular weight. They also showed that these materials can be exploited for cellular, tissue, and in vivo imaging studies (e.g., tumor hypoxia). Compared to dbm systems, the naphthyl materials with extended conjugation display redshifted absorbance, fluorescence, and phosphorescence. Through systematic variation of the chain length of PLA combined with heavy atom substitution, a new method for quantifying tumour hypoxia is demonstrated. For the iodine-substituted derivative, BF2dnm(I)PLA, clearly distinguishable fluorescence (green) and phosphorescence (orange) peaks are present, making it ideal for ratiometric oxygen-sensing and imaging. These dual emissive polymers are also ready to be fabricated as nanoparticles, which hold intense fluorescence and long-lived RTP, making them suitable for bioimaging and sensing (51).

Figure 1. Chemical structures of dual-emissive boron diketonates and β-hydroxyvinylimineboron compounds. (A) Foluorescence (F) and phosphorescence (P) emissions of the BF2dbmPLA film. Reprinted with permission from ref (20). Copyright 2007 American Chemical Society. (B) TEM and emission images of BF2dbmPLA nanoparticles (NPs). Reprinted with permission from ref (51). Copyright 2008 American Chemical Society. (C) Emission images of BF2dnm(I)PLA NPs. Reprinted with permission from ref (10). Copyright 2015 American Chemical Society. Besides BF2bdk chelates, Koch and coworkers reported a novel family of β-hydroxyvinylimineboron compounds (Figure 1, B-β-HOVI-1−7) that show high efficiency and color tunable RTP emission arisen from singlet fission (29). Variation of the molecular scaffold and fine-tuning of the electronic nature of the substituents were adopted to tailor the photophysical properties in solutions, neat solids, and doped PMMA films. Impressively, an absolute quantum yield over 100% is obtained for B-β-HOVI-3 and B-β-HOVI-4 in both solution and PMMA, 4 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and B-β-HOVI-7 in PMMA. These remarkably high efficiencies are believed to the singlet fission, which is favored by such collective factors as strong π–π stackings, permanent molecular dipole moment, and the presence of a functional group that aids to promote radical character in the excited state.

Figure 2. Chemical structures of BP and its derivatives, MBP, and DBBP′. Photographs of (A) DFBP in solutions (from left to right: n-hexane, THF, DCM, acetonitrile, ethanol), in PMMA films, and on TLC plates at room temperature and 77 K, (B) crystals of different compounds taken under 365 nm UV light. (C) Fragmental molecular packing and intermolecular interactions in DFBP crystals. Reprinted with permission from ref (13). Copyright 2010 American Chemical Society.

(Co)Crystallization-Induced Phosphorescence Conventional luminogens normally suffer from ACQ problems, which is even serious for triplet emitters. In sharp contrast to ACQ, in 2010, Tang and Yuan discovered the CIP phenomenon, which offers a new strategy to efficient pure organic RTP luminogens through crystal engineering (13). A series of organic luminogens, including BP and its derivatives, MBB, and DBBP′ are induced to emit RTP upon crystallization (Figure 2) in high efficiencies (Φp up to ~40%), with lifetimes ranging from 19.2 μs to 4.8 ms (13). These luminogens are practically nonemissive when they are dissolved in solvents, doped into polymer films, and dotted on TLC plates, because active intramolecular motions under these conditions effectively annihilate their triplet excitons via nonradiative rotational and vibrational relaxation channels. In the crystalline state, intramolecular motions are suppressed by the crystal lattices and effective intermolecular interactions. For example, in DFBP crystals, numbers of ·C−H···O (2.724 Å) and C−H···F (2.777, 2.796 Å) short contacts form a firmly 3D interaction network (Figure 2C), which strikingly restricts the molecular motions. The physical constraints and multiple intermolecular interactions synergistically rigidify the molecular conformations of the luminogens, thus making them highly phosphorescent in the crystalline state even at room temperature. Such 5 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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rigidification effect induced by crystallization is comparable and somewhat even better than that of the conventional cryogenic cooling (Figure 2A). Generally, carbonyl group and nonplanar conformation are favorable for effective spin-orbit coupling. BZL with more carbonyl groups and even more twisted conformation compared to BP is thus expected to be CIP-active. Exactly, similar to BP, BZL and its derivatives demonstrate CIP characteristics with high RTP efficiencies at crystalline states (Figure 3A). For example, while BZL is nonluminescent in solution and when dotted on TLC plates, its crystals emit distinct green light at 521 nm with a lifetime of 142.02 μs. Furthermore, we found a unique phenomenon of crystallization-induced dual emission (CIDE), namely, simultaneously boosted fluorescence and phosphorescence upon crystallization, in a group of pure organic aromatic acids and esters even without any metal- or heavy atoms (25). Notably, two triplet-involved relaxations of delayed fluorescence and RTP are activated. Specifically, long afterglow from TPA and IPA after the stop of UV irradiation is observed (Figure 3B), which is rarely found for pure organic luminogens. To further explore luminogens with persistent RTP, we designed CZBP combined typical CIP compound BP with carbazole, and heavy bromine atom was also introduced for comparison (Figure 3C). It is found that persistent RTP can be rationally achieved based on CIP and severe crystallization, and heavy atom effect is not the predominant factor for the relatively short lifetimes of BCZBP and DBCZBP (26). Perfect crystal with dense molecular packing and effective intermolecular interactions isolates the triplet excitons from quenching sites, and moreover significantly blocks the high energy vibrational dissipations, thus yielding persistent RTP.

Figure 3. Chemical structures of BZL and its derivatives, some aromatic acids and esters, as well as CZBP and its derivatives. (A-C) Photographs of the crystals taken under UV irradiation. (A) Reprinted with permission from ref (24). Copyright 2013 Science China Press and Springer-Verlag Berlin Heidelberg. (B) Reprinted with permission from ref (25). 2015, published by the Royal Society of Chemistry. (C) Reprinted with permission from ref (26). Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. 6 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Hetero sulfur (S) and tellurium (Te) containing pure organic luminogens with CIP or aggregation-induced phosphorescence characteristics were also developed (9, 28, 55, 56). As shown in Figure 4, a series of persulfurated benzene molecules are nonemissive in both air-equilibrated and oxygen-free solutions at room temperature, but in a striking contrast, strong green RTP can be obtained in the solid state with efficiencies up to unity (28). This is a consequence of decreased intramolecular motions, however, conformational and rotamer factors along with substituents might also effect. He et al. demonstrated the first examples of tellurophenes capped with pinacolboronates (BPin) exhibiting RTP in the solid state under ambient conditions (Figure 4) (55). Furthermore, these luminogens readily form emissive host-free films that can be directly cast from THF solution. They revealed that both TeII and proximal BPin units are required for the RTP emission. Subsequently, new phosphorescent BPin-appended benzo[b]tellurophenes two phenyl/BPin substituted tellurophene isomers, with tunable emission colors have been achieved (56).

Figure 4. Chemical structures of persulfurated benzene molecules and tellurophenes and emission characteristics of B-Te-6-B in solution and in the solid state. The photographs are reprinted with permission from ref (55). Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Besides above systems, very recently, there are increasing reports on pure organic CIP compounds. People try to evaluate the effects of substituents (22), heavy atom (46, 47), and mechanical stimuli on the photophysical properties of the phosphors, and endeavored to explore their potential applications. Some examples are shown in Figure 5. Shimizu and coworkers reported a new class 7 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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of pure organic CIP luminogens of 1,4-bis(aroyl)-2,5-dibromobenzenes. The bis(aroyl)-benzene derivatives are nonemissive either in solution or in doped polymeric films. On the contrary, aided by effective intermolecular interactions as C=O···H, Br···Br, C=O···Br, F···F, S···H, and MeO···H, their crystals exhibit intense RTP under ambient conditions, with tunable emission color from blue to green and luminescent quantum yields of 5~18% (22). For another example, Shi and Zhao reported a concise approach to obtain pure organic CIP luminogens with efficiency up to 21.9% by manipulating heavy-atom interaction based on a class of dibromobenzene derivatives. It is found that PhBr2C6Br2 and PhBr2C8Br2with two more bromine atoms show much higher luminescence efficiency than their PhBr2C6 and PhBr2C8 counterparts, owing to the increased intermolecular heavy-atom interaction in crystals (46). Maity and coworkers reported the bright green RTP from BaA crystals, despite its planar structure (27).

Figure 5. Chemical structures of some CIP pure organic luminones developed by Shimizu (22), Shi (46), and Maity (27) and the corresponding photographs of the crystals taken under UV irradiation. Upper graph: Reprinted with permission from ref (22). Copyright 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Lower first 4 graphs: Reprinted with permission from ref (46). Copyright 2016 American Chemical Society. Lower last graph: Reprinted with permission from ref (27). Copyright 2016 Elsevier B.V.

Apart from conventional conjugated compounds, efficient RTP was also observed by us in aggregated natural compounds and polymers without classic chromophores, such as rice, starch, cellulose, bovine serum albumin (BSA), and some other carbohydrates (23). We ascribed such unusual emission to the clustering of electron rich groups, whose lone pair electrons can be overlapped in clusters. Electron overlapping and sharing extend the effective conjugation and rigidify the molecular conformations, thus making the luminogens easier to be excited with higher efficiency compared to their dilute solutions. Soon after our report on the CIP phenomenon of pure organic luminogens, Kim and coworkers reported efficient purely organic phosphors generated through a ‘directed heavy atom effect’ (DHAE) and crystal engineering in 2011 (14). Planar chromophores with triplet-producing aromatic aldehydes and triplet-promoting bromine (Figure 6) were utilized. These phosphors demonstrate CIP characteristics. However, their planar conformations (e.g. Br6A) render the homocrystals with low (2.9% for Br6A) RTP efficiency due to the excimer-induced self-quenching. Brighter RTP is achieved by a mixed crystal design strategy. Analogous compounds in which the aldehyde group is replaced 8 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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by bromine are adopted as the hosts, which isolate the aldehyde molecules and prevent their self-quenching in the resulting intermixed crystals, thus affording higher RTP efficiencies. For example, with 1 wt% Br6A, Br6A–Br6 mixed crystals give a remarkably enhanced RTP efficiency of 55%. Importantly, variation of the aromatic building block and/or bridging units finely tunes the RTP emission color from blue to orange for the mixed crystals, with lifetimes of 0.1~6.4 ms and efficiencies of 0.5~28% (Figure 6).

Figure 6. Structures of various brominated aromatic aldehydes and corresponding dibromo compounds and the photographs of their cocrystals taken under UV irradiation. Reprinted with permission from ref (14). Copyright 2011 Rights Managed by Nature Publishing Group.

Jin et al. (31–34) and d’Agostino et al. (57) successfully fabricated cocrystals between 1,4-diiodotetrafluorobenzene (DITFB) and polycyclic aromatic hydrocarbons [PAHs, like naphthalene (Nap), phenanthrene (phe), pyrene (Pyr), carbazole (Cz), fluorine (Flu), dibenzofuran (Dbf), and dibenzothiophene (Dbt)], diphenylacetyl (DPA), and trans-stilbene (tStb) with RTP emissions (Figure 7). Halogen bonding between DITFB and other compounds is crucial to the formation of the cocrystals. Herein, the conformer DITFB plays vital roles as follows: (1) serves as halogen bonding donor to link the chromophores; (2) heavy iodine atoms promote spin–orbit coupling of the compounds, thus inducing their phosphorescence emission; (3) behaves as a ‘solid diluent’, reducing self-quenching of the luminogens. Therefore, halogen bonding combined with multiple intermolecular interactions provides a new strategy to obtain RTP cocrystals with modulated emissions. As depicted in Figure 7B, efficient green and orange RTP emissions are observed in the cocrystal of Nap-DITFB and Phe-DITFB, respectively (34). Specifically, d’Agostino et al. prepared the cocrystals of DPA-DITFB, DPA-2DITFB, tStb-DITFB, and tStb-2DITFB by mechanochemical methods. As a result of the external heavy atom effect and effective intermolecular interactions, and depending on the stoichiometry, DPA-DITFB and tStb-DITFB cocrystals exhibit both fluorescence and RTP, whereas the other two emit exclusive RTP (57). Such facile cocrystal strategy utilizing halogen bonding can well expand to other systems. 9 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 7. (A) Chemical structures of the conformer DITFB and other luminogens. (B) Phosphorescent excitation (blue lines) and emission spectra of Nap-DITFB (green) and Phe-DITFB (orange) cocrystals. Inset panels are the photographs of the cocrystals taken under UV irradiation. Reprinted with permission from ref (34). Copyright 2012 Royal Society of Chemistry.

Dopping/Trapping in Rigid Matrix For pure organic luminogens, to suppress vibrational dissipations and to isolate oxygen and/or moisture, doping or trapping in rigid environment is another choice. Hirata and coworkers explored organic host-guest materials with efficient persistent RTP (35, 58, 59). As depicted in Figure 8, through doping the highly deuterated aromatic compounds in amorphous steroidal matrix (such as β-estradiol) with rigidity and oxygen barrier properties, the nonradiative relaxation of triplet excitons is minimized. Firstly, deuteration greatly reduces the nonradiative decay rate (knr) of the guest molecules. Moreover, substitution with a secondary amino-group does not increase knr of the guest, but promotes the ISC process. Consequently, the host-guest systems afford both a nonradiative relaxation from the T1 state at less than 10-1 s-1 at room temperature and effective ISC process. Therefore efficient RGB RTP emissions with efficiencies > 10% are obtained and moreover with the lifetimes > 1 s in air. This approach allows for the fabrication of both amorphous and persistent pure organic RTP luminogens at the same time, which will surely find diverse applications in OLEDs, thermal sensing, recording, and security inks. 10 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 8. Material design for efficient persistent RTP from pure organic luminogens in air. Reprinted with permission from ref (35). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Suitable polymeric matrix may also be used to create rigid and oxygen barrier environment. Lee and Kim reported bright RTP by embedding a pure organic phosphor, namely Br6A (see Figure 6) into an amorphous glassy isotactic PMMA (iPMMA) matrix (60). Compared to atactic and syndiotactic PMMA (aPMMA and sPMMA), the reduced beta (β)-relaxation of iPMMA most efficiently suppresses vibrational triplet decay of the embedded luminogens and allows them to achieve bright RTP with efficiency of 7.5%. They also fabricated a microfluidic device integrated with a novel temperature sensor based on this composite system (60). Following their success of physical blending, they further utilized chemical bonding to construct bright RTP systems. Figure 9 illustrates the involved pure organic phosphors and the design concept. Covalent linking between phosphors and a polymer matrix not only increases the matrix rigidity, and moreover effectively reduces nonradiative relaxations of embedded phosphors. Consequently, collision frequency and the Dexter-type triplet energy transfer processes and vibronic mixing between zero-order electronic states of T1 and S0 are decreased, thus yielding efficient RTP from pure organic luminogens in a variety of amorphous polymer matrices (36). As demonstrated in their work, Diels–Alder click chemistry results in cross-linked luminogen doped polymer system with efficient RTP (up to ~28% quantum yield), which is around 2~5 times higher than that of physically blended Br6A/polymer system without covalent linkages. 11 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 9. Chemical structures of the designed phosphor and polymer and description of covalent crosslinking strategy. Reprinted with permission from ref (36). Copyright 2015 Rights Managed by Nature Publishing Group.

Similar to Kim’s work, Reineke and Baldo reported efficient, simultaneous fluorescence and RTP (74% yield) from a single molecule ensemble of N,N′-bis(4-benzoyl-phenyl)-N,N′-diphenyl-benzidine [(BzP)PB], which was diluted into PMMA. Such RTP from (BzP)PB is efficient (50% yield) and long lasting (208 ms lifetime) with extremely low knr for the triplet state (2.4 × 10 s−1). Interstingly, in contrast to the general trend–an increase of phosphorescence for (BzP)PB/PMMA going from 77 to 293 K is observed (40). Later, they proposed a new more general method to observe RTP from pure organics (15). Through controlling nonradiative rates by engineering a polymeric and energetically inert host matrix embedded with the target molecule, RTP is readily observable for a wide variety of molecules with functionalities spanning multi-exciton generation (singlet exciton fission), OLED host materials, and TADF emitters. And very recently, Zhou and Zhang reported rather an impressive work on the fabrication of fluorescent and phosphorescent single-component dual emissive materials (SDMs) (61). On the basis of a general design principle, N,N-hydroxyethylamino benzophenone (K1) is covalently incorporated into waterborne polyurethanes (WPU) and results in SDMs. The luminescent properties of SDMs are dependent on the chromophores concentration: increased concentrations generate progressively narrowed singlet-triplet energy gaps which can be well explained by the polymerization-enhanced intersystem crossing (PEX) model (62). Namely, polymerization of luminophores results in exciton coupling and subsequently enhanced forward and reverse ISC processes. Theoretical calculation for the WPU system suggests that the presence of K1 aggregates indeed enhances the crossover from excited singlets to triplets. This work sheds lights on the fabrication of diverse pure organic polymeric RTP luminogens. 12 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Unlike above rigid steroidal and polymeric matrices, Dong reported the utilization of inorganic host to receive persistent RTP from carbon dots (CDs) (37). Upon dispersing the CDs into a potash alum KAl(SO4)2·x(H2O) host, the resulting composite powders exhibit green RTP (500 nm) with a lifetime of 707 ms and an average lifetime of 655 ms (37). Such RTP emission is originated from triplet excited states of aromatic carbonyls (C=O) on the surface of CDs (7, 37), however, without KAl(SO4)2·x(H2O), no RTP is observed. Therefore, the matrix must have effectively rigidified the surface structure of CDs, thus suppressing the energy loss of C=O bonds from rotational or vibrational dissipations. It is also noticed that both crystal water and KAl(SO4)2 molecules play crucial roles in rigidifying the surface C=O bonds of CDs. Meanwhile, the KAl(SO4)2·x(H2O) matrix also shows good oxygen barrier performance, diminishing the oxygen contact and quenching of the triplet excitons.

Creating of Intermolecular Interactions between Luminogens and Hosts Doping in rigid matrices is effective to yield RTP. Specifically, if proper intermolecular interactions between the luminogens and hosts were constructed, it would be even better to create rigid surroundings. Our story may start from Al-Attar and Monkman’s work on the RTP of water soluble conjugated polymers (WSCP). In 2012, they discovered and described a simple but useful method to fabricate near perfect isolation of dense luminescent WSCP chains (see Figure 10 for examples) using caging within such polymeric surfactants as poly(vinyl alcohol) (PVA) and poly(vinylpyrrolidone) (PVP) (63). The PVA or PVP surfactant is ready to breaks up WSCP chain aggregates in solution, moreover, the in-situ formation of hydrogen bonds between PVA or PVP molecules upon drying firmly locks in the isolation of the WSCP, thus preventing aggregation and yielding long term stability to the resultant systems. Such perfect isolation and rigid locking by hydrogen bonds of the matrix render the WSCP luminogens highly emissive, and moreover, for the first time, unprecedented RTP from conjugated polymers is observed, thus offering an ideal system to study the triplet dynamic and energy transfer in conjugated polymers. Despite there seems no typical intermolecular interactions such as hydrogen bonding between the guest and host, this work inspires the researchers to further develop novel RTP systems through building specific interactions between luminogens and matrices.

Figure 10. Chemical structure of some water soluble conjugated polymers and naphatic acids. 13 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Gahlaut studied the photophysical properties of 1-naphthoic acid (1-NpA) and 2-naphthoic acid (2-NpA) (Figure 10) in three polymeric hosts of PVA, PMMA, and cellulose acetate (CA) (42). In case of PVA, besides fluorescence, strong RTP of 1-NpA is detected, which can be remarkably quenched by oxygen. In PMMA, weak phosphorescence is observable only under inert atmosphere and no RTP is noticed in CA. For 2NA, except weak RTP in PVA, no phosphorescence signal is recorded in the other two matrices. The presence of strong and weak hydrogen bonded conformers and polymer heterogeneity are invoked to explain above emission behaviors. While both species are present in PVA, possibly only weakly bonded one exists in PMMA and CA (42). The effective hydrogen bonding between the surface aromatic C=O groups and PVA chains also generates dual emissive CDs, which was first reported by Deng and coworkers (7). Dispersing CDs into a PVA matrix, besides well documented and studied fluorescence, persistent green RTP (~500 nm) with a lifetime of around 380 ms is observed. As illustrated in Figure 11, For CDs in solution, triplet emission from C=O groups is not optimal owing to remarkable nonradiative quenching processes, only fluorescence is hence observed. Upon dispersing into PVA, besides the general rigid polymeric matrix and oxygen isolation effects, abundant hydrogen bonds between PVA molecules and C=O units play an essential role, which further effectively rigidify C=O bonds, impeding their intramolecular motions and thus promoting the RTP emission. Subsequently, in 2014, Kwon et al. reported a design strategy for tailoring intermolecular interactions to enhance RTP from pure organic luminogens in PVA at ambient conditions (41). Figure 12 shows the involved compounds and schematic illustration of the designed principle. Halogen bonding between adjacent G1 luminogens and hydrogen bonding between the phosphor G1 and the PVA matrix are present in the resulting composite film. While the former facilitates ISC processes as well as suppresses the vibration of the phosphor, the latter effectively restricts the vibrational dissipations, thus enabling intense RTP with efficiency up to 24% (41). Furthermore, water can be used to modulate the strength of halogen and hydrogen bonding in the G1–PVA system, yielding unique reversible switch of phosphorescence to fluorescence. This property renders the system utilizable as a ratiometric water sensor.

Figure 11. Proposed RTP mechanism of CDs dispersed in PVA. Reprinted with permission from ref (7). Copyright 2013 Royal Society of Chemistry. 14 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 12. (A) Chemical structures of Br6A, G1, and PVA. (B) Phosphorescence image of G1 in PVA100 under UV illumination and schematic illustration of phosphorescence processes in the G1–PVA composite film. Reprinted with permission from ref (41). Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 13. The steady-state photoluminescence (left) and ultralong phosphorescence (right) spectra of a series of reported molecules. The insets show the corresponding photographs taken under UV irradiation (left) and after the stop of irradiation (right). Reprinted with permission from ref (6). Copyright 2015, Nature Publishing Group. 15 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Persistent RTP Persistent RTP has significant fundamental importance and promising applications in high density data recording, high contrast background independent imaging, and anti-counterfeiting. Unfortunately, it is typically observed in inorganic materials, especially in the rare-earth element containing crystals. For pure organic luminogens, to achieve persistent RTP, it is the prerequisite that kr must be at a suitable level. Based on proper kr, nonradiative relaxations including vibrational dissipation and oxygen/moisture quenching should be strictly impeded. Recently, researches endeavored to explore general strategies to persistent RTP luminogens (64–70). So far, however, no universal mechanism is derived, possibly because it is still difficult to predict the kr values of designed luminogens. In this part, we summarized the currently reported long lasting RTP systems. As we have mentioned above, BF2bdkPLA molecules (5, 9, 20, 51–54), deuterated aromatic compounds (35, 58, 59), and CDs (7, 37) are promising to produce persistent RTP under proper conditions. Embedding suitable luminogens in rigid polymeric systems is also hopeful to generate persistent RTP (15, 40). Meanwhile, serve crystallization with dense molecular packing based on CIP luminogens is also helpful to achieve long lasting RTP (25, 26). What is more, in 2015, An et al. realized ultralong RTP (lifetime: 0.23~1.35 s, efficiency: 0.08~2.1%) through effective stabilization of triplet excitons by strong coupling in H-aggregates of a group of pure organics (Figure 13) (6). The longest lifetime of 1.35 s is observed in DECzT crystals under ambient conditions. Meanwhile, through tailoring the molecular structure of the luminogens, the emission color of persistent RTP can be readily tuned from green (515 nm) to red (644 nm) (Figure 13) (6). There are also other scattered examples of pure organic persistent RTP systems (Figures 14 and 15) (64–70). Zhang and coworkers reported the interesting dual-emissive property of BF2EMO molecules in the crystalline state, which exhibit green RTP after ceasing the irradiation (Figure 14A) (64). Single-crystal structure analysis reveals the formation of multiple hydrogen bonds between neighboring molecules in crystals, which may significantly enhance the conformation rigidity of the luminogens. Li et al. reported a carbazole-based benzophenone derivative DCZBP (Figure 14B), whose cocrystal with chloroform exhibits weak RTP lasting for > 1.7 s (65). Removal of chloroform will eliminate the RTP emission, which can be switched on upon fuming with chloroform vapors. Dual emission were also observed by Kuno (IPA, TPA, PMA, and TPM) (66), Yang (CZBBP, CZDPS, and CZBDPS) (68), and Xue (CBA) (64), in different systems (Figure 15). Despite their similar phenomena or even resembling structures, no general mechanism for persistent RTP is reached.

16 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 14. (A) Chemical structure of BF2EMO and its crystal emission photographs. (B) Steady-state and delayed emission spectra of BF2EMO at room temperature in air. A and B are reprinted with permission from ref (64). Copyright 2014 American Chemical Society. (C) RTP spectrum of DCZBP crystal obtained through recrystallization from chloroform solution. (D) RTP decay curve of DCZBP crystal at 553 and 600 nm and its crystal emission photographs. C and D are reprinted with permission from ref (65). Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Normally, above persistent RTP emissions are in low yields, because the ultralow radiative process cannot compete with the other fast transitions. Until very recently, Xue et al. proposed a new strategy to explore efficient persistent RTP from carbazole derivatives through an intermolecular moderate heavy atom effect (47). Isolation with flexible alkyl chains rather than direct connection between the heavy atom (Br) and carbazole moieties is adopted to avoid strong internal heavy atom affect (Figure 16). Seven among eight designed compounds exhibit persistent RTP in their crystals (lifetime: 20~340 ms, efficiency: 1.6~39.5%). Notably, CC6PhBr demonstrates persistent RTP (lifetime: 200 ms) with efficiency as high as 39.5%. Moreover, white light emission with efficiency of 72.6% is achieved from it owing to the presence of strong blue fluorescence and yellow-orange phosphorescence.

17 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 15. Chemical structures of a series of pure organic luminogens with persistent RTP emission and photographs of their crystals. Left panel is reprinted with permission from ref (66). 2015, published by the PCCP Owner Societies. Right panel is reprinted with permission from ref (68). Copyright 2016 John Wiley and Sons.

Figure 16. Design principle, molecular stacking of carbazole crystals, molecular structures of reported CCnBr and CCnPhBr compounds and photographs of their crystals taken under 365 nm UV light with fluorescence and phosphorescence quantum yields and average phosphorescence lifetimes indicated. Reprinted with permission from ref (47). 2016, published by the Royal Society of Chemistry. 18 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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RTP from Solutions Due to strong vibrations and collisions in solvents, efficient RTP emission from solutions is difficult to achieve even in oxygen free solutions. However, there is also some exciting progress. The realization of efficient RTP in solution will further facilitate their applications in sensing and imaging. Herein, we list two impressive examples. The first example is a fluorene derivative, 7-bromo-9,9didodecylfluorene-2-carbaldehyde (Br–FL–CHO) reported by Xu and coworkers, which exhibits RTP in conventional oxygen free organic solvents (49). Its absolute RTP efficiency reaches 5.9% in chloroform. The directly-linked bromo and formyl substituents may promote the ISC from S1 state to T1 state, and the rigid fluorene framework may suppress the nonradiative process in solution. They also prepared other fluorene derivatives to compare the substituent effect on emission properties (Figure 17). None of these luminogens, however, can generate RTP in solution. Meanwhile, due to the rigidity of Br-FL-CHO, its doped PMMA film also shows bright RTP emission, which lasts for several days even upon exposure to oxygen.

Figure 17. Chemical structure of fluorenen derivatives and luminescent photographs of Br–FL–CHO taken under Ar (left) and air (right) in CHCl3 at 298 K. Reprinted with permission from ref (49). Copyright 2013 Royal Society of Chemistry.

Figure 18. Schematic illustration of the reversible inclusion of the binary IQC[5]/CB[7] system and corresponding photographs of the aqueous solutions. Reprinted with permission from ref (71). Copyright 2016 John Wiley and Sons. 19 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The second example is from Gong and coworkers. They reported a cururbit[7]uril (CB[7]) based pH-controlling molecular shuttle encoded by a visible RTP signal without any deoxidant, which is generated by complexation of CB[7] and bromo-substituted isoquinolines, for example IQC[5] (Figure 18), in aqueous solution (71). In the acidic medium, the CB[7] host shuttles along the axial guest, and only weak RTP emission is observed, whereas deprotonation of IQC[5] makes the CB[7] wheel locate on the phosphor group, leading to increased RTP by more than six times. 1H NMR data indicate that the internal void space of CB[7] is mainly occupied by a nitrogen heterocycle and a heavy atom at high pH values. Moreover, the switching process along with the visible RTP signal is recyclable by adjusting the pH between ~4.0 and 7.0.

Conclusions and Perspectives There is growing interest in high-efficiency pure organic RTP luminogens owing to the fundament importance and promising applications, and significant advances have been achieved in recent years. Based on the concepts of spin-orbit coupling, new kinds of possible phosphorescent luminogens were designed by taking advantages of carbonyl groups, hetero atoms, and heavy atoms. To receive efficient RTP, varying strategies were adopted to rigidify the molecular conformations and to isolate oxygen. Novel phenomena and new strategies such as CIP, DHAE, chelation, and cocrystallization utilizing halogen bonding have been observed or proposed. Moreover, persistent RTP and efficient RTP from solutions have also been demonstrated. Due to the sensitivity of triplet excitons to oxygen and moisture, these pure organic luminogens have found their applications in biological hypoxia imaging, temperature, water and ion sensing, document security, and smart optical recording. Moreover, OLEDs utilizing these pure organic RTP luminogens were fabricated (72–74). Despite the relatively low performance, it paves the way for future optoelectronic applications. Further exploration in these renewed area will not only reveals more underlying fundamentals on the dynamics of excitons, but also offers new intriguing applications.

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60. Lee, D.; Bolton, O.; Kim, B. C.; Youk, J. H.; Takayama, S.; Kim, J. Room Temperature Phosphorescence of Metal-Free Organic Materials in Amorphous Polymer Matrices. J. Am. Chem. Soc. 2013, 135, 6325–6329. 61. Zhou, C.; Xie, T.; Zhou, R.; Trindle, C. O.; Tikman, Y.; Zhang, X.; Zhang, G. Waterborne Polyurethanes with Tunable Fluorescence and Room-Temperature Phosphorescence. ACS Appl. Mater. Interfaces 2015, 7, 17209–17216. 62. Sun, X.; Wang, X.; Li, X.; Ge, J.; Zhang, Q.; Jiang, J.; Zhang, G. Polymerization-Enhanced Intersystem Crossing: New Strategy to Achieve Long-Lived Excitons. Macromol. Rapid Commun. 2015, 36, 298–303. 63. Al-Attar, H. A.; Monkman, A. P. Room-Temperature Phosphorescence From Films of Isolated Water-Soluble Conjugated Polymers in Hydrogen-Bonded Matrices. Adv. Funct. Mater. 2012, 22, 3824–3832. 64. Zhang, X.; Xie, T.; Cui, M.; Yang, L.; Sun, X.; Jiang, J.; Zhang, G. General Design Strategy for Aromatic Ketone-Based Single-Component Dual-Emissive Materials. ACS Appl. Mater. Interfaces 2014, 6, 2279–2284. 65. Li, C.; Tang, X.; Zhang, L.; Li, C.; Liu, Z.; Bo, Z.; Dong, Y. Q.; Tian, Y.-H.; Dong, Y.; Tang, B. Z. Reversible Luminescence Switching of an Organic Solid: Controllable On–Off Persistent Room Temperature Phosphorescence and Stimulated Multiple Fluorescence Conversion. Adv. Opt. Mater. 2015, 3, 1184–1190. 66. Kuno, S.; Akeno, H.; Ohtani, H.; Yuasa, H. Visible Room-Temperature Phosphorescence of Pure Organic Crystals via a Radical-IonPairMechanism. Phys. Chem. Chem. Phys. 2015, 17, 15989–15995. 67. Clapp, D. B. The Phosphorescence of Tetraphenylmethane and Certain Related Substances. J. Am. Chem. Soc. 1939, 61, 523–524. 68. Yang, Z.; Mao, Z.; Zhang, X.; Ou, D.; Mu, Y.; Zhang, Y.; Zhao, C.; Liu, S.; Chi, Z.; Xu, J.; Wu, Y.-C.; Lu, P.-Y.; Lien, A.; Bryce, M. R. Intermolecular Electronic Coupling of Organic Units for EfficientPersistent Room-Temperature Phosphorescence. Angew. Chem., Int. Ed. 2016, 55, 2181–2185. 69. Xue, P.; Sun, J.; Chen, P.; Wang, P.; Yao, B.; Gong, P.; Zhang, Z.; Lu, R. Luminescence Switching of a Persistentroom-Temperature Phosphorescent Pure Organicmolecule in Response to External Stimuli. Chem. Commun. 2015, 51, 10381–10384. 70. Yuan, J.; Tang, Y.; Xu, S.; Chen, R.; Huang, W. Purely Organic Optoelectronic Materials with Ultralong-Lived Excited States under Ambient Conditions. Sci. Bull. 2015, 60, 1631–1637. 71. Gong, Y.; Chen, H.; Ma, X.; Tian, H. A Cucurbit[7]uril Based Molecular Shuttle Encoded by Visible Room-Temperature Phosphorescence. ChemPhysChem 2016, 17DOI:10.1002/cphc.201500901. 72. Bergamini, G.; Fermi, A.; Botta, C.; Giovanella, U.; Di Motta, S. D.; Negri, F.; Peresutti, R.; Gingras, M.; Ceroni, P. A Persulfurated Benzene Molecule Exhibits Outstanding Phosphorescence in Rigid Environments: from Computational Study to Organic Nanocrystals and OLED Applications. J. Mater. Chem. C 2013, 1, 2717–2724. 25 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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73. Chaudhuri, D.; Sigmund, E.; Meyer, A.; Röck, L.; Klemm, P.; Lautenschlager, S.; Schmid, A.; Yost, S. R.; Voorhis, T. V.; Bange, S.; Höger, S.; Lupton, J. M. Metal-Free OLED Triplet Emitters by Side-Stepping Kasha’s Rule. Angew. Chem. 2013, 125, 13691–13694. 74. Kabe, R.; Notsuka, N.; Yoshida, K.; Adachi, C. Afterglow Organic LightEmitting Diode. Adv. Mater. 2016, 28, 655–660.

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Chapter 2

Luminogenic Polymers with AIE Characteristics Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch002

Anjun Qin,*,1 Ming Chen,2 and Ben Zhong Tang*,1,2 1State

Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China 2Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China *E-mails: [email protected] (A.Q.); [email protected] (B.Z.T.)

Since conceptually proposed by Tang et al. in 2001, the aggregation-induced emission (AIE) has caused tremendous interests among scientists worldwide. Currently, hundreds of AIE-active luminogens have been designed and prepared, but most of them are small molecular ones. However, the AIE-active polymers possess charming advantages, such as mutable and tunable molecular structure, excellent film-foming ability and facile processability, thus, are potentially applied in diverse high-tech areas, like fluorescent chemo- and bio-sensors, optoelectronic devices and so on. In this chapter, we first summarize the types of the presented AIE-active polymers by incorporating AIE-active units, such as tetraphenylethene (TPE), multi-phenyl substituted siloles and distyrylanthracene (DSA). In addition, a new kind of luminogenic polymers containing unconventional chromophore, such as poly(amido amine) (PAMAM), poly(amino ester) and poly[(maleic anhydride)-alt-(vinyl acetate)], are also discussed. Our aim is to establish a structure-property relationship of AIE-active polymers, and further demonstrate how to control the AIE-activity of the polymers via intelligent and rational molecular design.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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1. Introduction Over the past decades, luminogenic polymers have received considerable attention owing to their potential applications in fluorescent chemo- and bio-sensor, organic light-emitting diodes (OLEDs) and organic lasers, etc. (1–4) Up to now, most developed polymeric luminogens emit intensely in their diluted solution, while the emission is weakened or even quenched upon aggregation. In other words, they suffer from notorious aggregation-caused quenching (ACQ) effect (5). For example, polyfluorene (PFO), which was regarded as one of the most acceptable candidates for blue emitter in OLED, tends to form excimers during device fabrication and operation, therefore leading to red-shift its emission peak and lower its luminescent efficiency (6, 7). Plenty of efforts have been devoted to alleviate the negative ACQ effect caused by aggregation. However, the reported chemical approaches, such as design macromolecule in branched and spiro structure or covalently graft bulky cube onto polymer chain always encounter fussy synthetic steps and painful separation process, whereas, physical doping of luminogens as guest in host inclines to produce phase separation with prolonged operation time (8–11). Actually, such measures only meet with limited success. We all know aggregation is an intrinsic process of molecules in condensed phase. It is envisaged but required urgently whether the aggregation could play a positive instead of negative role in enhancing luminescence. In 2001, Tang et al. found that multi-phenyl substituted silole derivatives are non-emissive in dilute solution but emit intensely upon aggregation. Since the emission of siloles was induce by the aggregation, Tang thus coined this unique photo-physical phenomenon as aggregation-induced emission (AIE) (12, 13). Afterwards, restriction of intramolecular rotation (RIR) has been rationalized as the cause of AIE effect, which has been proved not only by a large amount of experiments but also by theoretical analysis (14–17). Besides silole derivatives, other AIE-active luminogens, such as tetraphenylethene (TPE), tetraphenylpyrazine (TPP), distyrylanthracene (DSA), and tetraphenylpyrrole have been developed subsequently (18–20). Based on these archetypal AIE-active luminogens (AIEgens), lots of work has been conducted to fit various research interests (21–23). In contrast, less work has been focused on AIE-active polymer in spite of its unique advantages of high molecular weight. In device operation, conjugated polymer always possesses a higher glass-transition temperature (Tg), which plays a decisive role in improving device stability. The solid film fabricated by spin-coating, static casting and ink-jet printing instead of vapor deposition could greatly reduce the cost in practical application. And the viscoelastic polymer film is very suitable for utilizing as flexible large-area flat panel display (24, 25). It is also worth noting that polymer is usually partially crystalline or even hard to crystallize due to its piled molecular chain in disorder upon aggregation. To some degree, it favors the formation of porosity in polymer, especially with the branched, hyperbranched or network structures, which provides the void to bind with analyte and induce the signal switch when function as fluorescent sensor (26). For example, hyperbranched AIE-active polymer always shows an unique superamplification quenching 28 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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effect as turn-off sensor to detect explosives, which is much sensitive than small molecule probes. Last but not least, the structure of polymer is multivariate and easy to tailor as it is need. For instance, one or more of the functional groups or moieties with hydrophily or lipophilicity, characteristics of luminescence, and response to photon, temperature or pH, and so on, could be incorporated and coordinated into the polymer, which is of great significance to construct stimuli-responsive materials (27, 28). All these demonstrate the attractive virtues of luminogenic polymer, which is always difficult to achieve in single small molecules. As enlightened by the previous researches, one of the most feasible strategies to design AIE-active polymers is to link, insert and graft typical AIE-active units into or onto the polymer chains. Indeed, by utilization of free radical polymerization, polycondensation, Suzuki polycoupling, ring-opening polymerization, metathesis polymerization, click polymerization, and post-functionalization reaction, etc., a wide variety of AIE-active polymers including polyolefins, polyimides, polyacetylenes, polytrizoles and post-modified polymers with linear, dendritic and hyperbranched structures have been prepared (29). The species diversity, corresponding synthetic methods and diversified applications have been summarized in detail in our previous reviews (29, 30), which thus will be less described here. Instead, in this chapter, we mainly discuss TPE, silole and other typical AIE cores based AIE-active polymers with varying molecular architectures, which include: (a) linear, dendritic, hyperbranched and network structures, (b) partially- and fully-conjugated structures, and (c) AIE-active units linked, grafted and inserted structures. Note that some cluster luminogens without typical luminogenic units, such as poly(amido amine) (PAMAM), poly(amino ester) and poly[(maleic anhydride)-alt-(vinyl acetate)], are introduced here (31–33). However, polymer systems, such as supramolecular polymers and metal-organic frameworks (MOFs) generated by non-covalent interaction are not within the topic. Our aim is to establish a structure-property relationship of AIE-active polymers. We hope, with the aid of the discussion in this chapter, more and more AIE-active polymers with exciting properties could be designed and prepared, which will in turn promote the development of luminogenic polymer in high-tech applications in diverse areas.

2. TPE-Based AIE-Active Polymers Currently, TPE is regarded as the most famous star molecule in AIE research due to its high emission efficiency in solid state, easy preparation and convenient post-functionalization. Recently, more interests have been inclined to the study of AIE-active polymers by incorporating TPE as luminescent units. Thanks to a burgeoning polymerization technique of click polymerization, AIE-active polymers could be prepared with high molecular weights in excellent yields (34, 35). TPE-containing 1,4-regioregular polytriazoles 4 and 5 could be obtained by the organo-soluble Cu(PPh3)3Br-catalyzed click polymerization of diyne 1 and diazides 2 or 3 (Scheme 1). Photo-physical property investigation indicated that both 4 and 5 are AIE-active. In solution state, they are non-luminescent 29 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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because the peripheral phenyl rings of TPE units could freely rotate to annihilate the excitation state energy, whereas, upon aggregation, such rotation is partially restricted accompanying with the emission renovated evidently, with their PL properties resembling to that of TPE. The reason for this emission behavior is that the TPE units incorporated in the polytriazoles are linked with aliphatic alkyl chains, which results in no electron communication between TPE and formed triazole rings (36).

Scheme 1. Synthetic routes to TPE-containing polytriazoles 4 and 5.

It is believed that the residue of copper species in polytriazoles, which probably could coordinate with the form triazole rings and be hard to be completely removed, may cause fluorescence quenching in optoelectronic field and even toxicity in biological application. To overcome this difficulty, one of the approaches of using supported Cu(I) catalyst (CuI@A-21) to catalyze the click polymerization was reported, which not only greatly reduces the metallic residues in the resulting polytriazoles but also makes the catalyst recyclable. More importantly, polytriazole 6 prepared by CuI@A-21 catalyzed click polymerization exhibits increased emissive efficiency in comparison with 5, where both of them possess the same molecular structures (Figure 1) (37). This result demonstrates that the quenching of the fluorescence could be alleviated by reducing the copper residues in the polymers, which further guides to design AIE-active polymer with metal-free or catalyst-free polymerizations. Along this line, the AIE-active polytriazoles 9-11 were prepared by metal-free click polymerizations of activated diynes and TPE-containing diazides (Scheme 2) (38). The electron-withdrawing property of carbonyl group adjacent to alkyne makes it more reactive than normal ones, thus prone to polymerize with diazide in the absence of metal-catalyst systems, which may prompt the application of functional polytriazoles in optoelectronic and biological fields. 30 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 1. (a) PL spectra of 6 in THF/water mixtures with various water fractions. Polymer concentration: 10 μM; λex: 326 nm. (b) Changes of quantum yield (ΦF) of 5 and 6 in THF/water mixtures with various water fractions. Inset: the structure of CuI@A-21.

Scheme 2. Synthetic routes to TPE-containing polytriazoles 9-11. Besides the partially conjugated polytriazoles, the conjugated polytriazoles of 15, 16, 20 and 21 could also be synthesized via Cu(PPh3)3Br-catalyzed click polymerization of aromatic diynes and diazides (Scheme 3). Owing to the presence of TPE unit, 16 and 21 display aggregation-enhanced emission (AEE) characteristics. In solution state, a weak emission of both polytriazoles could be discerned due to the enhanced molecular stiffness, making the phenyls less easy to rotate to annihilate the excitons. It is worth noting that the emission peaks of the aggregates of two polymers formed in THF/water mixture with 90% water fraction located at around 485 nm, which is red-shifted compared with aforementioned aliphatic alkyl chains containing polytriazoles, manifesting that 31 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the triazole ring could enhance the conjugation length of emitters. Moreover, the similar emission behaviors of 16 and 21 indicate that negligible influence on emission could be exerted by exchanging the substituents on the triazole rings. However, when two phenyl rings of TPE units in 16 and 21 are replaced by hydrogen atoms, both 15 and 20 suffer from ACQ effects. The less congested phenyl rings in stilbene units induce the molecule to adopt a planar conformation, which might favor the formation of π-π interaction in the aggregate states, and thus leads to the quenching of the emission. All these findings suggest that TPE is an ideal unit to construct AIE-active conjugated polymer, and a subtle change in substituents, especially the number of phenyl rotors, may have a great impact on the photo-physical properties of polymers (39).

Scheme 3. Synthetic routes to TPE-containing polytriazoles 15, 16, 20 and 21. In some occasions, polymers with long wavelength emission are much desired because of their huge application potential in biological field. One of the most used strategies to design red emissive AIEgens is to incorporate both strong electron-donating and electron-withdrawing groups into a molecule to induce intramolecular charge-transfer process. As a result, the highest occupied molecular orbital (HOMO) energy level is elevated, while the lowest unoccupied molecular orbital (LUMO) energy level is reduced, which coherently cause a narrow energy gap and resulted red emission. As shown in Scheme 4, alternative copolymer 24 was synthesized via classical Suzuki polycoupling of 22 and 23. In 24, diethylamine and diazosulfide moieties serve as donor and acceptor, respectively, which is consistent with the design principle of red emissive polymers. 24 gave a deep-red emission at 665 nm in thin solid film with ΦF of 6.9%. Compared to its low ΦF (< 1%) in solution, 24 thus features AEE-activity. The contained TPE units in 24 are responsible for overcoming the ACQ factors brought from both diazosulfide and fluorine units (40).

Scheme 4. Synthetic route to red emissive TPE-containing polytriazole 24. 32 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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TPE units could also function as pendant group and be grafted on both flexible and rigid polymer main-chains. For example, 25 and 26 could be prepared from radical copolymerization of TPE-containing vinyl monomer and post-functionalization of TPE unit onto a preformed polymer, respectively (Chart 1). Moreover, the amount of TPE units in polymers could be fine-tuned to fulfill the functionalities as sensors of temperature, pH and bovine serum albumin (BSA). Because only one phenyl ring in TPE unit is dragged onto the main chain, less rotation could be restricted in solution, whereas, the flexible main-chain promotes the full restriction of motion of TPEs in aggregate state, both of which makes resulting polymers behave like archetypal TPE core (41, 42).

Chart 1. Molecular structures of TPE-based polymers 25 and 26.

The TPE unit could also be attached to a rigid polymer main-chain but different emission behaviors were observed (Chart 2). It is interesting to note that the AIE-activity of TPE could remain when it was attached to the rigid polyacetylene main-chain (27) spaced via an alkyl chain, demonstrating that the involved flexible chain plays a crucial role in determining AIE behavior. While, when the spacer is removed, the photo-physical property of 28 is much different. Firstly, the TPE may conjugate with polyacetylene backbone, as evidenced by the emission of polymer in solution was peaked at the longer wavelength of 613 nm, which is more than 60 nm red-shifted than that of 27. Secondly, the AIE effect is discounted seriously, with only a maximum PL increment about 2.8-fold probably because the TPE units are fastened to backbone rigid strand, which makes the whole molecule like a “brush”. Upon aggregation, the polymer chains are hard to pack compactly, where plenty of free volume is produced to induce the rotation of phenyl rings in TPE unit (43). Such negative effect could be alleviated in certain degree when the rigid strand of 28 is replaced by less regular conjugated backbone, such as polydiphenylamine. As expected, the AIE effect of 29 and 30 is improved, and the fluorescence enhancement of 22-fold and 8-fold are recorded, respectively. Additionally, when one more phenyl ring was inserted between polydiphenylamine main-chain and TPE unit, the AIE-activity of 30 is lessened obviously. It is probably due to that the extended conjugated branch-chain may cause more free volume under aggregation (44). 33 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 2. Molecular structure of TPE-based polymers 27-30. The TPE units could also be attached to polymers as terminal groups (45, 46). Are they also AIE-active? Here, we take 31 as an example (Chart 3). Its structure includes a hydrophilic poly(ethylene glycol) (PEG) and a lipophilic end group embracing TPE as luminescent unit. The structure of 31 facilitates it to form spherical micelles in aqueous solution with the hydrophobic TPE units as the core and hydrophilic PEG as the shell. Thus, the TPE terminals behave like self-aggregation, enabling the polymer to be AIE-active.

Chart 3. Molecular structure of TPE end-capped polymer 31. The influence of end-capped TPE units of polymers/oligomers on the emission behavior was also investigated (Chart 4). When the ACQ fluorene unit was covalently bound with two TPE units, the new compound is AIE-active because the peripheral TPE rotors could dissipate the excitation energy transferred from the fluorene units. When the number of fluorene units was increased to 5, the ΦF of 32 in solution was enhanced accordingly by 6-fold, suggesting that the free rotation of TPE units is not enough to quench the emission from five fluorene units (47). This study could guide for further molecule design of AIE-active polymers/oligomers.

Chart 4. Molecular structure of TPE end-capped oligomer 32. 34 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The TPE units has also been incorporated into hyperbranched polymers. For example, hyperbranched polytriazoles 35 and 36 were prepared by Cu(I)-catalyzed click polymerization of triazides 32 or 33 and diyne 34 (Scheme 5), which display dramatic AIE effect with fluorescence enhancement about 347 and 229-fold, respectively. The ΦF of the aggregate of 35 formed in THF/water mixture with 90% water fraction is up to 38.31%, which is much higher than that of linear analogs of 4-6 and 9-11. The flexible alkyl spacers weaved into the hyperbranched polymer increase their solubility and make them non-emissive in their good solvents. While, in the aggregate state, the alkyl chains may facilitate the TPE units to accumulate more tightly and enable them to emit intensely. Furthermore, more flexible alkyl chains (judged by length), more AIE effect could be achieved (48).

Scheme 5. Synthetic route to TPE-containing hyperbranched polytriazoles 35 and 36. The influence of molecular rigidity of a hyperbranched polymer could be proved by luminescent property of polytriazole 39, which is also prepared by Cu(I)-catalyzed click polymerization (Scheme 6). When inspected the molecular structure of 39, we could find that tetra- and di-substituted TPE units are knitted by triazole rings. The whole molecule is conjugated and therefore its conformation is much rigid than that of 35 and 36, where the phenyl rings in TPE are much harder to rotate in solution. Its branched structures also hamper a tight aggregation of polymer chains. It is also in accordance with the increased ΦF of 4.31% in solution and decreased ΦF of 23.83% in its aggregates compared to 35 (49). More complicated emission behaviors have been recorded from 40-42 (Chart 5). Unlike 39, the enlarged molecular rigidity after polymerization could only provide soluble 40-42 with molecular weights around 4000. The fluorescence enhancement of 40, 41 and 42 was measured to be 2309-, 15- and 4.4-fold, respectively, which is mainly determined by their different ΦF in solution (0.01%, 1.1% and 5.2%). These results manifest that the conjugated joints between branching points have a profound influence on AIE effect of a hyperbranched conjugated polymer. It is speculated that the phenyl rings of TPE units are more restricted by near carbazole and phenyl units due to their strong steric effect, which further reduce their motion in solution. Also, the low molecular weights 35 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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make the hyperbranched polymer possess less generations, which leads to less congestion between branching chains. Therefore, upon aggregation, all the polymer chains are easy to pile up compactly, even though inserting a partial chain of one polymer into the interspace of another one, which agrees with their similar experimental values of ΦF in aggregate state (50).

Scheme 6. Synthetic route to TPE-containing hyperbranched polytriazole 39.

Chart 5. Molecular structures of hyperbranched polymer 40-42. Another topological structure of crosslinked network 43 was prepared via free radical polymerization of TPE containing diacrylates (Chart 6). By fine-tuning the molecular weight, 43 is surprisingly soluble in commonly used organic solvents. 43 is weakly luminescent in solution, which is probably because the RIR process is partially activated when TPE units are knitted into the network and easy to be affected by adjacent chains, which somehow differs from that of TPE incorporated linear and hyperbranched polymers with flexible spacers. Moreover, such flexible network could further restrict the aggregation of luminogens upon aggregation, making the network AEE-active (51). 36 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 6. Molecular structure of TPE-containing crosslinked polymer 43. TPE could also be utilized as a unit to construct conjugated microporous polymers (CMPs) with extended π-conjugation, which have receive much interest for their unique applications in gas storage, light harvesting/emission, catalysis and sensing, etc. (52) To improve the microporosity, morphologies and dispersibility of CMP, 45 has been prepared by mini-emulsion polymerization of dibromo substituted TPE and 1,3,5-triethynyl benzene via Pd-catalyzed Sonogashira polycoupling. Solvothermal treatment of generated hyperbranched prepolymer 44 readily produced CMP 45 (Scheme 7). Although the intraconnection is inevitably present in 45, it could be dispersed in THF, making it feasible to investigate its photo-physical properties both in solution and aggregate states. From 44 to 45, the maximum emission wavelength is red-shifted from 500 to 520 nm, and the ΦF increases from 1.03 to 3.25%, respectively. These results demonstrate that the effective π-conjugation has enlarged but more restriction of rotatable phenyl rings has occurred after solvothermal treatment. Surprisingly, the ΦF of 45 in solid state is determined to be as high as 58.0% (53). Similar high value of 40% was also recorded in CMP film prepared form electrochemical polymerization of four N-carbazole decorated TPE derivatives (54). It seems hard to understand why such effective luminescence could be obtained even when TPE rotors located into hollow three-dimensional rigid scaffolds. Nevertheless, improved floating ability of π-electron in three-dimensional electronic cloud channels may cause a larger transition behavior and result in higher emission efficiency.

Scheme 7. Synthetic route to TPE-containing conjugated microporous polymer 45 from hyperbranched polymer 44. 37 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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3. Silole-Based AIE-Active Polymer Compared to TPE-containing AIE-active polymer, multi-phenyl substituted silole-based polymers are much rare, probably because silole derivatives are troublesome in preparation and purification, and unstable under basic condition. In early studies, multi-phenyl substituted siloles were synthesized by lithiation of diphenylacetylene, followed by the reaction between the intermediate of dilithiotetraphenylbutadiene and chloro-silicane (55). Thus, the substituents at 1,1-positions of silole derivatives were easy to be decorated to generate monomers suitable for polymerization. For example, polysilole 47 can be prepared by reduction of 1,1-dichloro-2,3,4,5-tetraphenylsilole 46 and lithium in THF (Scheme 8). Due to the large steric effect of main chain caused by direct linking of silicon atoms, 47 can only be obtained as an oligomer with a degree of polymerization of around 15. The highest PL intensity of 47 in THF/water mixture with 99% water fraction was recorded to be 18-fold stronger than that in pure THF, indicative of an AEE-activity. Furthermore, compared to the first reported AIEgen of 1-methyl-2,3,4,5-pentaphenylsilole (MPPS), a bathochromic shift of maximum emission about 20 nm was observed in 47, which is ascribed to a slight increase in σ-conjugation along the silicon-silicon main-chain (56, 57). However, another alternative copolymer of 48 (Chart 7) with spirobifluorene units linked to 1,1-position of silole units through carbon-carbon double bond centered its emission at near 500 nm in film, which is much similar to that of MPPS. These results indicate that extended conjugation of silole derivatives through 1,1-position may pose a subtle influence on the emission wavelength, while their AEE-activity could be kept (58).

Scheme 8. Synthetic route to silole-based polymer 47. Actually, it has been proved theoretically and experimentally that the photo-physical property of silole derivatives is mainly dominated by central silole ring and 2,5-substituted groups, but less affected by 1,1-substituted ones, due to the sp3 hybrid of silicon atom (59, 60). Thanks to the synthetic method of intramolecular reduction of diethynylsilane, the substituents at 2,5-position of silole derivatives can be readily tuned or decorated (61). Conjugated polymer 49 could be readily prepared by Sonogashira polycoupling of 2,5-diethynylsilole and 1,4-diiodobenzene derivative (Chart 7). Due to the conjugated effect, the adjacent alkynyl groups may cause partial restraints of rotation of phenyl rings and enhance the molecular luminescence in solution. Indeed, the ΦF values of 49 in solution and aggregate states were measured to be 8.0 and 12.3%, respectively, suggesting 49 is AEE-active (62). 38 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 7. Molecular structures of silole-based polymers 48 and 49. Silole-based units could function as pendants to endow polymers with AIE effect. The silole-containing polymer 50 was readily synthesized by atom transfer radical polymerization (ATRP) of styrene and silole-containing vinyl monomers, in which α-(2-bromo-2-methylpropoyloxy)-PEO functionalizes as an initiator, and CuBr and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) serve as catalyst (Chart 8). 50 exhibited a concentration dependent emission behavior. Thanks to its contained PEG unit, no emission could be detected when 50 was at low concentration in aqueous solution, whereas the emission intensity was increased prominently at higher concentration by the formation of micelles. This result suggests that regulating the manner of luminescent polymer aggregation by rational molecular design could also make it AIE-active (63).

Chart 8. Molecular structure of silole-polymer 50. Silole derivative could also be attached to the rigid strand of polyacetylene main-chain through alkoxyl spacers (51, Chart 9). According to the experimental results of polyacetylene bearing TPE units as side groups, it is believed the AIE effect of silole-containing polymers will not be influenced by the rigid main-chain, since the existence of long flexible alkoxyl chains. However, the ΦF of aggregates of 51 formed in chloroform/methanol mixture with 90% methanol fraction was measured to be only 3.0%, though an increase of ΦF about 20-fold could still be obtained when compared to its solution state. It is probably due to the interaction between silole units and polyacetylene main-chains formed upon aggregation, which induced the quenching of emission from silole-based luminogens. 52 is 39 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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an analogue of 51, only with its alkoxy spacer removed. 52 gave a faint red emission at 652 nm, which was attributed to the inefficient radiative decay from polyacetylene backbone. However, with an addition of poor solvent of methanol into the system, only slight emission enhancement was observed, manifesting 52 is AIE-inactive. The direct attachment of silole moieties to the rigid polymer backbone can not pack well under aggregation, where the phenyl rotors can still rotate vigorously to decay excitons (64).

Chart 9. Molecular structures of silole-based polymers 51 and 52. 53 is a quasi-linear AIE-active polyethyleneimine (PEI) end-capped with silole units (Chart 10). The average grafting ratio of silole units is about 4.5, which is much less than conventional AIE-active polymers. Similar to 50, the amphipathy 53 could form nanoparticles in water with hydrophobic silole units as core and hydrophilic PEI chain as crown. The self-assemble structures induced a remarkable emission with ΦF of around 12% due to RIR effect. Further reaction of 53 with 2,3-dimethylmaleic anhydride (DMA) readily produced surface charge-switchable light-up functional nanoparticle, which could be used for targeted biological imaging and selective restraint of cancer cell (65).

Chart 10. Molecular structure of silole end-capped polymer 53. The silole units were also incorporated into hyperbranched polymers. TaCl5-Ph4Sn catalyzed polycyclotrimeriztion of 1,1-diethynyl silole and 1-hexyne readily produced hyperbranched poly(phenylenesilolene) of 54 (Chart 11). Contrary to the expectation, 54 is AIE-inactive although the AIE-active silole units were presented. Its emission intensity in chloroform/methanol mixture 40 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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was weakened gradually with progressive addition of methanol. This is because the 3D rigid scaffold of 54 can not induce a compact intermolecular and intramolecular aggregation, where plenty of free volume was left to provide enough space for free rotation of phenyl rings and for nonradiatively deactivating excitons. Interestingly, cooling-enhanced light emission is THF solution of 54 was observed, suggesting that the phenyl rings inside hyperbranched polymer in solution is rotatable at room temperature, and such motion is prone to freeze at lower temperature, therefore populating excitons decayed via radiative transition channel (66). As discussed above, the conjugated phenyl linkers in 54 have a negligible effect on the electronic property of silole unit, the abnormal luminescent behavior of 54 may be basically determined by the hindering effect of 2,5-position substituted phenyl rings against rotation.

Chart 11. Molecular structure of hyperbranched poly(phenylenesilolene) 54.

By altering ethynyl groups from the 1,1- to 2,5-positions of silole, hyperbranched poly(phenylenesilolene) 56 was prepared under similar polymerization conditions (Scheme 9). Unlike 54, each silole unit of 56 was knitted at its 2,5-positions,which make the whole polymer chain much less congested. Accordingly, the interior phenyl rings can partially rotate, and the polymer chains are easy to be compressed upon aggregation to activate RIR process. As a result, the ΦF of aggregates of 56 formed in THF/water mixture with 90% water fraction is 2.5-fold higher that that in THF, further confirming its AEE-activity, though such effect is weak as displayed in 49 (67). Therefore, both substitution position and polymer chain rigid play important roles in determining the AEE effect of silole-containing hyperbranched polymers.

41 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 9. Synthetic route to hyperbranched poly(phenylenesilolene) 56.

4. Other Typical AIEgen-Based Polymers Except for the most commonly used AIE cores of TPE and silole, other cores, such as distyrylanthracene (DSA), tetraphenylpyrrole, tetraphenylthiophene (TPT), and tetraphenylpyrazine (TPP), have been generated according to RIR mechanism. In principle, all of them could be incorporated into polymers to enables them AIE-active. The development of new types of AIE-active polymers will not only enrich AIE family, but also boost their high-tech applications. Therefore, such research is full of significance, but, still in its infant stage.

a. DSA-Based AIE-Active Polymers DSA is an AIE-active archetypal molecule with its emission centered at near 520 nm in aggregate state (68). The red-shifted maximum emission wavelength of DSA, in comparison to TPE and silole, makes it more favorably applicable in biological imaging. Thus, the construction of DSA-based AIE-active polymers is always associated with biological application. For example, polymer 57 was prepared by AIBN-initiated radical copolymerization of three vinyl monomers with one of them having DSA pendant groups (Chart 12). A very weak emission with ΦF of 0.08% was detected when 57 was dissolved in dimethyl sulphoxide (DMSO). After adding poor solvent of water into the DMSO solution, the emission sustained to enhance. The emission intensity of the aggregates of 57 formed in DMSO/water mixture with 98% water fraction was recorded to be 7.9%, which is 98-fold stronger than that in DMSO. Such remarkable AIE effect of 57 is much like its small molecule derivative, exhibiting a feasible strategy to design AIE-active polymer with DSA as pendants. 57 could be self-assembled into green fluorescent micelles and be used for cell imaging (69).

42 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 12. Molecular structure of DSA-based polymer 57. DSA unit could also serve as a joint to knot two polymer chains together (58, Chart 13). Different from 57, 58 is AEE-active. It is emissive in THF with a ΦF value of 9.3%, which was increased to 17.0% for the aggregates formed in THF/water mixture with 90% water fraction. The AEE effect of 58 is probably ascribed to the end-capped folic acid moieties, which are insoluble in THF, thus induce the polymer chains to form coil conformation, and restrict the rotation of the phenyl rings in DSA. Nevertheless, the introduction of folic acid units onto the polymer may improve its functionalities, especially in targeting cancer cells (70). It is apparent that the properties of attached functional groups as well as the structure of polymer can greatly influence the photo-physical behaviors of DSAcontaining polymers.

Chart 13. Molecular structure of DSA-based polymer 58.

b. 2,4,6-Triphenylpyridine-Based AIE-Active Polymers Besides siloles, other heterocycle-based AIEgens are much desirable owing to their unique electronic properties. A new heterocycle-based AIEgen, named 2,4,6-triphenylpyridine, has been developed recently. Much attention has been inclined to the study of its polymers. 2,4,6-Triphenylpyridine was first used to self-polymerize or copolymerize with fluorene units, and conjugated polymers of 59 and 60 were produced, respectively (Chart 14). The AIE property investigation showed that very weak fluorescent enhancement was observed both in 59 and 60, implying that 2,4,6-triphenylpyridine is not a powerful unit for the design of AIE-active conjugated polymers. Meanwhile, the extended conjugation could red-shift the emissions of polymers into blue-purple light region, which is much preferable to display application, in comparison to 2,4,6-triphenylpyridine archetypal molecule, whose maximum emission is located around 360 nm (71). 43 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 14. Molecular structures of 2,4,6-triphenylpyridine-based polymers 59 and 60. Following question is how to endow 2,4,6-triphenylpyridine-containing polymers with much notable AIE effect? It seems the enhanced rigidity of polymer will deteriorate its AIE effect. Thus, 2,4,6-triphenylpyridine unit was grafted onto the flexible polystyrene as side chains to produce 61 (Chart 15). As 2,4,6-triphenylpyridine units were directly connected by methylene, the rotation of the phenyl rings is free in solution, which has efficiently dissipated the energy of excited state and make the polymer non-emissive, whereas, the flexible main-chain may favor them to pack tightly to induce much enhanced emission in the aggregate state. The ΦF values of 61 in solution and aggregate states were deduced to be 25 and 78%, respectively, proving that such strategy is accessible (72). Moreover, by utilizing diamino-substituted 2,4,6-triphenylpyridine as initiator, 2,4,6-triphenylpyridine unit knotted polymer polytyrosine 62 could be prepared through living ring-opening polymerization of L-tyrosine-N-carboxyanhydride (Chart 15). Similar to 61, the flexible “wing” of 62 could give rise to almost 1-fold of fluorescent enhancement from solution to aggregate states. It is noteworthy that maximum emission wavelength of 62 has red-shifted to around 500 nm compared to 2,4,6-triphenylpyridine, which is mainly derived from a intramolecular charge transfer from peripheral secondary amine to central pyridine ring (73).

Chart 15. Molecular structures of polymers 59 and 60 with 2,4,6-triphenylpyridine unit as side group or knot. 44 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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c. Tetraphenylthiophene (TPT)-Based AIE-Active Polymer Tetraphenylthiophene (TPT) is also a heterocycle-based AIEgen, whose structure resembles that of multi-phenyl substituted siloles, with the silicon atom replaced by sulfur atom. The subtle change of molecular structure, as a result, leads to a huge transition of emission wavelength. The emissions of TPT and silole derivatives are peaked around 400 and 490 nm, respectively (74). The strategy of utilizing TPT to design AIE-active polymers is similar to that of 2,4,6-triphenylpyridine. After grafting TPT unit onto polyethylene as side chains, the resultant polymer 63 exhibits a typical AEE characteristic with more than 8-fold of emission enhancement in aggregate against solution states (Chart 16) (75). Also, by using the method similar in the synthesis of 62, TPT end-capped poly(γ-benzyl-L-glutamate) 64 was obtained. However, only 1-fold fluorescent enhancement of 64 was observed, which is nearly as same as that of 62 (76).

Chart 16. Molecular structures of TPT-based AIE-active polymers 63 and 64.

d. Nitrilevinylphenothiazine-Based AIE-Active Polymers Nitrilevinylphenothiazine-based AIEgen usually emits an orange-yellow light peaked at near 580 nm due to the involvement of strong donor-acceptor groups. Lots of works have been done in designing nitrilevinylphenothiazine-based AIE-active polymers, and exploring their applications in biological imaging. A linear polymer of 65 was prepared by radical copolymerization of nitrilevinylphenothiazine-containing vinyl monomer and glycidyl methacrylate (GM), followed by ring-opening reaction of GM and glucosamine (GLU) (Chart 17). The produced polymer is amphiphilic with hanging nitrilevinylphenothiazine units and glycosyl groups as hydrophobic and hydrophilic moieties, respectively. Meanwhile, the soft alkyl main-chains may help tune the molecular conformation to self-assemble into nanoparticles with core-shell structure, where the lipophilic luminogen aggregates were inside. The ΦF of assembled nanoparticles is as high as 41%, which is rare among the present red-emissive self-assemble materials. As the fluorescent intensity of 65 in diluted solution is very low, its AIE effect is much remarkable, indicative of the practicability to cultivate AIE-active polymers based on nitrilevinylphenothiazine core (77). 45 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 17. Molecular structures of nitrilevinylphenothiazine derivative-based polymers 65-67. Another linear polymer 66 was synthesized by ring-opening polymerization of amino group substituted nitrilevinylphenothiazine and 4,4′-oxydiphthalic anhydride as hydrophobic chain segment, which was then decorated with two PEO chains at terminals as hydrophilic segments. Different from 65, all the luminogenic units in 66 are located in the central chain segments instead of hanging as side groups. Such amphiphilic structure is very prevailing in construction of self-assemble materials. Thanks to RIR process, the formed nanoparticles showed strong red emission at 600 nm (78). In many cases, the nanoparticles self-assembled by aforementioned polymers are subject to disperse at new working conditions below the critical micelle concentration, which hampers their practical biomedical application. To avoid this negative effect, cross-linked amphiphilic polymer like 67, was proposed to make the self-assemblies much more stable. Using nitrilevinylphenothiazine units and PEI as hydrophobic and hydrophilic segments, respectively, 67 is prone to self-assemble into nanoparticles in aqueous solution. The formed nanoparticles give strong emission at 580 nm, with a ΦF of 40%, which is ascribed to the AIE effect of nitrilevinylphenothiazine units in the hydrophobic core. Such emission efficiency is similar with that of 65, demonstrating that the incorporation of nitrilevinylphenothiazine units into the polymer does not influence their aggregation behavior (79). e. Phenyl-Substituted Quinolone-Based AIE-Active Polymer An interesting strategy for designing AIE-active polymer is to postfunctionalize a non-emissive polymer with another non- or weakly luminescent molecules. For instance, both poly(4-acety styrene) 68 and 2-aminobenzophenone 69 are not typical fluorophores. However, after condensation reaction, 70 became AEE-active due to the new generated luminescent phenyl-substituted quinolone 46 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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derivative (Scheme 10). The ΦF of 70 in THF and THF/water (1:9 v/v) mixture were recorded to be 2.1 and 3.7%, respectively. Moreover, by complexing bulky camphorsulfonic acid (CSA) with the quinoline unit of 70, the AEE effect of 71 has almost no change, but the emission efficiencies both in solution and aggregate states increase remarkably, and their ΦF values reached 11.6 and 38.1%, respectively. The hampered intramolecular rotation caused by CSA moiety is the reason for the emission enhancement. Moreover, 71 emits a blue-green light peaked at 500 nm, which has a bathochromic shift about 100 nm compared to that of 70. This phenomenon could be ascribed to the narrowed energy gap manipulated by the lowed LUMO energy level after protonation of quinoline ring (80).

Scheme 10. Synthetic route to phenyl-substituted quinolone-based AIE-active polymer 71.

f. Boron Ketoiminate or Boron Diiminate-Based AIE-Active Polymers Recently, much attention has been focused on organoboron-based AIEgens due to their high emission efficiency in aggregate state and unique electronic property (81). Besides small molecules, AIE-active organoboron-based polymers have also been investigated. For examples, 72-75 are alternative copolymers comprising of boron ketoiminate units and fluorine units. The difference is the R groups connected on the phenyl rings (Chart 18), which make the maximum emission wavelengths of their nanoparticles change from 521 nm to 661 nm. All the polymers here emit faintly with their ΦF < 1% in solutions. However, the values increase notably to be 7-39% in their aggregate states (82). It is worth noting that the enhancement of electron donating ability of substituents of the polymers could not only red-shift their emission, but also lessen their emission efficiency in aggregate states, which is derived from the less electron cloud overlap between ground and excited states of the repeating units (83). Other organoboron-based conjugated polymers 76-78 are different in their backbones (Chart 18). The photoluminescence (PL) measurement showed that the ΦF values of 76-78 in solutions are below 1%, and increase remarkably to 6-14% in their aggregate states, which is similar to that of 74. It is thus concluded that changing the substituents on the phenyl rings connected to the nitrogen atoms, instead of the aromatic repeating units, is a much effective approach to fine-tune the emission behavior of boron ketoiminate unit-based AIE-active polymers (84).

47 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 18. Molecular structures of boron ketoiminate-based conjugated polymers 72-78. By replacing the oxygen with nitrogen atom in phenyl substituted boron ketoiminate moiety, new boron diiminate-based polymers 79-86 were obtained, which featured AIE characteristics (Chart 19) (85). Similar with 72-75, by changing the electronic properties of functional groups in phenyl rings attached to nitrogen atoms, the maximum emission wavelengths of 79-86 varied from 509 nm to 628 nm. The ΦF values of 79-83 in films are in the range of 2-11%, which are much higher than that of their solution states (≤ 1%) (86, 87).

Chart 19. Molecular structures of boron diiminate-based conjugated polymers 79-86.

5. Luminescent Polymer Containing Unconventional Fluorophore It is a general understanding that the emission of polymers is stemmed from their containing fluorophores, otherwise, the polymers are non-emissive. This understanding is, however, not a general dogma. The new luminescent polymers containing no conventional fluorophores are also emissive as reported in recent years, though their mechanism is still under debate. 48 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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An example of such polymer is glacodynamer 87, which was prepared from polycondensation of bishydrazide and oligosaccharide-containing dialdehyde (Chart 20). Both monomers show no fluorescence, however, the yielded polymer displayed remarkable emission at 457 nm in water. The unexpected fluorescent property of 87 is probably due to the tightly packed structure of polymer chains while the inner aromatic chromophores is isolated and inflexibly held in the hydrophobic core. The reason why a deep blue light could be observed by a less conjugated system was not given, but suppression of motion of polymer chains plays a crucial role in enhancing the emission. When the polymer solution heated from 23 to 85 °C, the emission was quenched notably, suggesting that RIR may be one of the factors that induce the polymer to emit. In addition, the dynamic character of these glycodynamers was revealed through covalent exchange reactions of a monomeric component by another, which gave rise to the regulation of the luminescent properties of glycodynamers (88, 89).

Chart 20. Molecular structure of glycodynamer 87.

It is occasionally found that poly(N-isopropyl acrylamide) (PNIPAM, 90) prepared from addition-fragmentation chain transfer (RAFT) polymerization of N-isopropyl acrylamide mediated by the initiator of 89 (Scheme 11), gave a strong fluorescence at near 410 nm in solution. Since no conventional fluorescent species are involved in the polymer, this newfangled emission phenomenon is thus much interesting. After plenty of experimental investigations and theoretical calculation, it is reported that an electron transition between ground state located in phenyl ring and excited state located in benzene ring and adjacent carbonyl group with π-π interaction contributes to the emission. The fluorescence is intensified with the increase of polymerization time, as the longer polymer may form more compact coiled nanostructures, in which the π-π interaction could be 49 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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much stabilized and effectively isolated, and more motions of such emissive π-π interaction- distortion, collision or rotation could be restrained. More importantly, it has been a general strategy to design fluorescent polymers with phenyl unit containing RAFT agent, since the fluorescent 91 and 92 could also be obtained readily by polymerization of methyl acrylate and N,N-dimethylacrylamide, respectively (90).

Scheme 11. Synthetic routes to polymers 90-92. In above two examples, no extended conjugated system is involved and responsible for the visible luminescence, but the phenyl rings undoubtedly participated for the emission. However, some systems even contain no aromatic moieties, but still emit intensely. That is an important and interesting issue that should be clarified. Poly(amido amine) (PAMAM), one of the earliest developed emissive polymers containing no aromatic groups, is extremely worthy being discussed here. It is first reported that Au8 nanoclusters encapsulated on PAMAM dendrimers can induce an obvious emission with peaks at 450 nm. It is believed that the emission is stemmed from the Au8 nanoparticles (91), but this conclusion was ruled out when similar PL behavior was observed without addition of any Au species. Furthermore, simple treatment of OH-terminated PAMAM dendrimers with oxidant of (NH4)2S2O8 (PS) in methanol/water mixture can generate emission, which probably results from the produced blue-emissive chemical species after oxidation reaction of peripheral hydroxyl groups. Similarly, Au3+ is also apt to oxidize OH-terminated dendrimer in the preparation of encapsulated nanoparticles to make it emissive. It was concluded from above experiments that the hydroxyl end groups instead of backbone of PAMAM dendrimer play a vital role in forming the luminescent centers although the underneath mechanism is to be explored (92). Subsequently, besides OH-terminated PAMAM dendrimer, it was reported that NH2 and carboxylate-terminated PAMAM showed similar PL behaviors at pH = 6. For OH-terminated PAMAM dendrimer, increase of the generations from G2 to G4 remarkably enhanced the emission due to the crowed peripheral functional groups, which induces the dendrimers to form a densely packed globular structure. The PL intensity of PAMAM dendrimer (e.g., NH2-G4) could further be intensified when the pH value of solution decreased, and reach maximum at pH ≈ 2.5. According to the reports, several factors could be supposed to explain the behavior: (a) the incorporated tertiary amine groups will be protonated at low pH, while dendritic chains with charge repulse each other, therefore making the structure of PAMAM dendrimer much more rigid; (b) much effective hydrogen bonds could be formed under acid conditions in the dendrimer; 50 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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(c) some emissive species were generated by the reaction of functional groups in polymer chains mediated by acid. It was thus believed that backbone of the dendrimer was crucial for enhancing the emission when hyperbranched PAMAM is subject to acidification (93). To gain deeper insight into the emissive source of PAMAM, triethylamine (TEA) is selected as control for PL studies because it is a typical amino-branching unit inside PAMAM dendrimers. After treating TEA and G4-PAMAM dendrimer with oxidant of ammonium persulfate (APS) in aqueous solution for 3 days, both resulting small molecule and polymer exhibit similar emission with peaks at near 450 nm, manifesting that the tertiary amino-branching points inside the dendrimer are indispensable for emission. Nevertheless, the ΦF of APS-treated TEA and PAMAM are recorded to be 0.25 and 52%, respectively, while each tertiary amino unit of latter was deduced to contribute the luminescent efficiency of 0.84%. This result means 2.4-fold of emission enhancement was occurred when tertiary aminobranching points were incorporated into a dendrimer with more generations. In other words, with the increase of dendrimer generations, tertiary amines located much more compactly into the polymer interior, while the emissive centers are concentrated and restrained in a confined dendrimer “pocket”, thus inducing an enhanced emission due to the lack of rational and translational freedom. It was also proposed a physical interaction (or called as “exciplex”) generated between a tertiary amino unit and a doped oxygen molecule or the formation of a peroxyl radical from two partners may cause intrinsic emission of tertiary amino moieties in blue light region (94). Very recently, the emission behavior of PAMAM has been directly related to the AIE. Linear (l) and hyperbranched (hb) PAMAMs (95 and 96) were prepared by Michael-type polycondensation of acrylamide 93 and amino-substituted piperazine 94 in DMF and water, respectively (Scheme 12). The dilute aqueous solutions of 95 and 96 were nearly non-emissive probably because no oxidation process was involved according to the previous explanation. However, after adding a large amount of poor solvent of acetone into its solution, the emission boosts greatly with peak at around 450 nm, indicative of a typical AIE behavior. The following question is why a significant emission could be observed in such system since no conventional fluorophores was involved and no oxidation or protonation processes was occurred. Theoretical calculation revealed that the lone-pair electron and delocalized π electron dedicated by nitrogen atom and carbonyl group, respectively, could generate a variety of intra- and interchain clusters, where conjugated systems are enlarged though n-π and π-π interactions, which responded to the emission in the aggregate state. Obviously, the emission behavior of PAMAMs here is different from previous ones: (a) no oxidizing agent and acid participated in enhancing the emission; (b) both l- and hb-PAMAM are highly emissive; (c) the emissive wavelength of PAMAMs could be fine-tuned in the range of 463-570 nm by altering the excitation wavelength from 380 to 530 nm, probably due to the various emissive centers determined by different conjugation extent of clusters (95). Another interesting finding is that the intra- and inter-molecular aggregation of carbonyl groups in silicon-containing PAMAMs is an exclusive cause for their luminescence. Si-PAMAMs 97 with generations from G0 to G2 were prepared by 51 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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aza-Micheal and amidation reactions (Scheme 13). Similar to above descriptions, all Si-PAMAM dendrimers gave strong blue emission at around 435 nm in methanol without treatment of any oxidant. The 29Si NMR spectra measurements demonstrated that N→Si coordination bonds existed in Si-PAMAM dendrimers, which facilitate the carbonyl groups to pack together. The emission intensities of polymers enhance promptly as the generations are increased due to more carbonyl groups are involved and crowed conformations. It is worth noting that these polymers possess the unique property of AEE feature. When addition of water into their THF solution, the carbonyl groups tend to aggregate more tightly, and the extended conjugation of cluster induced a red-shifted emission. At the same time, the aggregation well suppressed the rotational or vibrational relaxation of clusters, thereby caused an enhanced emission (31).

Scheme 12. Synthetic routes and cartoon structures of PAMAMs 95 and 96.

The emission mechanism of PAMAMs seems to be complicated. Similar situation is encountering in poly(amino ester)s (PAEs, 100-103) with various terminals, which were synthesized by Michael addition reactions (Scheme 14). The emission of PAEs in aqueous solution may be ascribed to the combination of acidification and protonation of polymer chain. It was explained as the compacted spatial morphologies of polymers become very open at acid atmosphere, and oxygen molecules are easy to approach to the interior tertiary amine branching points. However, such rule does not hold if the PAE, such as 101, is prone to hydrolyze at low pH. The photo-physical properties of 100-103 can not be affected significantly by varying the end groups at neutral pH, and a linear PAE with similar compositions does not show emission at all. These results indicate that a coexistence of tertiary amines/carbonyl moieties in the compacted dendritic architecture could be a key factor for displaying blue emission, and oxidation dose not play an indispensable role in this process. While, reasonable oxidation of hyperbranched PAE may act positively in enhancing emission (32). In another work of sulfur-containing hyperbranched PAE, it was reported that the emission was strengthened with increase of polymerization time owing to the decreased interior mobility of tertiary amine units. Surprisingly, a switching of emission wavelength from 440 to 560 nm was observed after oxidation. Moreover, the emission at 560 nm was enhanced remarkably with prolonging the oxidation time, which was confirmed to stem from the formed ≡N→O groups as proved both by NMR and FT-IR analyses (96). 52 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 13. Synthetic routes to silicon-containing PAMAMs 97. The unique emission behaviors of hyperbranched PAMAM and PAE were also reported in poly(propyl ether imine) (PETIM) in despite of their different structures. When excited at 330 nm, the polymer is emissive with a peak at 390 nm. Furthermore, increasing the generation, oxidation and protonation of PETIM could also remarkably enhance its emission (97). 53 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 14. Synthetic routes and cartoon structures of PAE 100-103.

Interestingly, sole nitrogen-containing polymer of poly(ethyleneimine) (PEI) was also reported to be strongly emissive. The photo-physical properties of 107G0 to 107-G3 (Scheme 15) are also similar to that of PAMAM and PAE. It is undisputed that the amine groups in PEI are the source for generating fluorescence. Increasing the generations of PEI is favorable for emission because the crowed conformation will decrease the mobility of amine moieties. In addition, a notable emission enhancement of PEI after oxidation is directly attributed to a complex formed between amino groups and oxygen as proved by comparative elemental analysis of untreated and treated products (98). Aforementioned emissive polymers containing no conventional chromophores must be designed with much congested dendritic/hyperbranched structure. This strategy is, however, not a general rule. For example, poly(N-vinylpyrrolidone) (PVP, 108) is highly emissive with maximum wavelength at around 380 nm in aqueous solution after polymerization, while its monomer of N-vinyl-2-pyrrolidone (NVP) only shows a very faint emission. Through structural and spectra investigation, it was concluded that the pyrrolidone ring in PVP could be hydrolyzed to generate a product with a secondary amine and a carboxylate spaced by propylidene, in which the stable N→O compound is readily formed between the secondary amine and oxygen molecule (Scheme 16). The resulted secondary amine oxide moiety was regarded as the new luminogens responsible for emission. The PL of PVP and its oxidized hydrolyzate showed stimuli response to metal ions and acid/base, and very possibly some other chemicals (99). 54 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 15. Synthetic routes to PEI 107.

Scheme 16. The oxidized hydrolyzate processes of PVP 108.

As discussed above, in PAMAMs (such as Si-PAMAMs 97), the aggregation of interior carbonyl groups is an intrinsic cause for the emission and AIE process. Such concept has been accepted in the studies of a series of linear polymers containing succinic anhydride (SA) groups. For instance, SA end-capped polyisobutene (PIBSA, 112) (Chart 21) is non-emissive in dilute solution, but exhibits a strong blue-green emission in concentrated heptane solution or viscous fluid, indicating that it is AIE-active. In heptane, the aggregation of carbonyl groups is formed as discerned by UV-vis spectra, where their vibro-rotational motions are restricted and thus increases the radiative recombination of excitons. Moreover, increase the number of SA functional groups in polymer chains may induce more effective aggregation in PIBSA, and thus pose a much significant AIE effect (100). 55 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 21. Molecular structure of SA end-capped PIB 112 and its proposed aggregation mode.

Similarly, it has reported that the colloidal nanoparticles of poly[(maleic anhydride)-alt-(vinyl acetate)] (PMV, 113) emits intensely at 419 nm, whereas, only weak emission was observed in solution (101). Very recently, such phenomenon of PMV was expatiated in detail. To address this issue, poly(maleric anhydride) (PMAh, 114) and poly(vinyl acetate) (PVAc, 115) were prepared for comparison (Chart 22). The experiments showed that PMAh dissolved in THF gave remarkable blue emission centered at 390 nm, whereas, no fluorescence was observed for both PVAc and maleric anhydride (MAh). It is because the bulky anhydride groups of PMAh make the molecular chain much rigid, which in turn favors the formation of heterodox clusters via collection of plenty of carbonyl groups, thereby inducing intense emission. Hence, it is believed the emission behavior of PMAh is closely related to the carbonyl clusters. PMV also possesses intriguing solvatochromism behavior. It appears colorless and gives a blue emission in aromatic and oxygenic solvents of THF, acetone, toluene and dioxane, whereas, it is magenta and displays a redder emission in polar solvents of N-methyl-2-pyrrolidone (NMP), DMSO and dimethylformamide (DMF) etc., indicating that certain interactions between polymer and solvent molecules have taken place. For comparison, polymers 115 and 116 were selected. It was concluded that the vinyl acetate (VAc) moieties in PMV is crucial for its solvatochromic process, and MAh cluster is the origin of emission of PMV (33). 56 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chart 22. Molecular structures of PMV 113 and other polymers 114-117 for control.

6. Conclusion AIE-active polymers could be designed readily by incorporation of typical AIE-active units, especially of TPE, silole, DSA and boron ketoiminate moieties, into the polymer chains. Generally, introduction of flexible alkyl into polymer main-chains or branching chains may help to keep the AIE effect of resultant products. In conjugated polymers, it is feasible to realize longer emissive wavelength through enlarging effective conjugation or reasonable construction of donor-acceptor structures. Nevertheless, as the conjugation extended, the polymer rigidity increased, thereby making them AEE instead of AIE active. We also summarized the unique AIE-activity of some polymers without typical fluorophores. In most systems, the incorporated nitrogen atom or carbonyl group with lone-pair electrons play a vital role in determining the emission. In the former, tertiary amine groups existed in PAMAM, PAE, PETIM and PEI dendrimers are responsible for the emission, while protonation or oxidation of nitrogen atom may notably strengthen the fluorescence. Increase the generations of dendrimers also acts positively. The emission of these polymers is mainly related to increased rigidity of branching chains or congested environment of tertiary amine, which reduce the rotational and vibrational relaxation of luminescent species. Whereas, in the latter, the formed clusters of carbonyl groups in the aggregate state, mostly in PMV, dominate the emission because of restriction of molecular motion of groups. Though the photo-physical phenomenon of these polymers is quite interesting and some mechanisms were proposed, the underneath mechanism is to be further explored, which will promote the development and application of these systems. It is worth noting that the AIE-active polymers possess quite a few advantages over AIE-active low molecular weight luminogens. For instance, there are numerous possibilities to fine-tune the molecular structure, topology, and morphology as well as functionalities of the polymers. Moreover, the excellent film-forming ability of the AIE-active polymers could facilitate the fabrication large-area films via convenient and simple processes like spin-coating 57 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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or static-casting. In addition, the covalently bonding of the repeating units in polymers often endows the resulting materials with decent mechanical strengths. However, the emission efficiency of AIE-active polymers is generally not as high as AIE-active low molecular weight luminogens mostly because the metal catalyst residues could not be completely removed, which might act as quenchers. What’s more, the property and application of AIE-active polymers should be further explored. Thus, to design AIE-active polymer with very effective emission and to explore their property and applications are still challengeable and will receive keen interest in future research. We hope that with pilot of this chapter, more AIE-active polymers with desired structures and properties could be designed rationally and intelligently, to enrich or embellish the applications in the areas of polymer light-emitting diodes (PLED), chemical and biological probes, biological imaging, porous material, stimuli-responsive material, and so on.

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67. Liu, J.; Zhong, Y.; Lam, J. W. Y.; Lu, P.; Hong, Y.; Yu, Y.; Yue, Y.; Faisal, M.; Sung, H. H. Y.; Williams, I. D.; Wong, K. S.; Tang, B. Z. Macromolecules 2010, 43, 4921–4936. 68. He, J.; Xu, B.; Chen, F.; Xia, H.; Li, K.; Ye, L.; Tian, W. J. Phys. Chem. C 2009, 113, 9892–9899. 69. Lu, H.; Su, F.; Mei, Q.; Zhou, X.; Tian, Y.; Tian, W.; Johnson, R. H.; Meldrum, D. R. J. Polym. Sci. Pol. Chem. 2012, 50, 890–899. 70. Zhang, Y.; Chen, Y.; Li, X.; Zhang, J.; Chen, J.; Xu, B.; Fu, X.; Tian, W. Polym. Chem. 2014, 5, 3824–3830. 71. Yang, C. M.; Lee, I. W.; Chen, T. L.; Chien, W. L.; Hong, J. L. J. Mater. Chem. C 2013, 1, 2842–2850. 72. Lai, C. T.; Hong, J. L. J. Mater. Chem. 2012, 22, 9546–9555. 73. Mohamed, M. G.; Lu, F. H.; Hong, J. L.; Kuo, S. W. Poly. Chem. 2015, 6, 6340–6350. 74. Lai, C. T.; Hong, J. L. J. Phys. Chem. B 2010, 114, 10302–10310. 75. Chien, R. H.; Lai, C. T.; Hong, J. L. J. Phys. Chem. C 2011, 115, 5958–5965. 76. Li, S. T.; Lin, Y. C.; Kuo, S. W.; Chuang, W. T.; Hong, J. L. Poly. Chem. 2012, 3, 2393–2402. 77. Wang, K.; Zhang, X.; Zhang, X.; Ma, C.; Li, Z.; Huang, Z.; Zhang, Q.; Wei, Y. Poly. Chem. 2015, 6, 4455–4461. 78. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Poly. Chem. 2014, 5, 689–693. 79. Wang, K.; Zhang, X.; Zhang, X.; Yang, B.; Li, Z.; Zhang, Q.; Huang, Z.; Wei, Y. Colloids Surf., B 2015, 126, 273–279. 80. Chou, C. A.; Chien, R. H.; Lai, C. T.; Hong, J. L. Chem. Phys. Lett. 2010, 501, 80–86. 81. Hu, R.; Lager, E.; Aguilar-Aguilar, A.; Liu, J.; Lam, J. W. Y.; Sung, H. H. Y.; Williams, I. D.; Zhong, Y.; Wong, K. S.; Peña-Cabrera, E.; Tang, B. Z. J. Phys. Chem. C 2009, 113, 15845–15853. 82. Yoshii, R.; Nagai, A.; Tanaka, K.; Chujo, Y. Chem. Eur. J. 2013, 19, 4506–4512. 83. Dai, C.; Yang, D.; Fu, X.; Chen, Q.; Zhu, C.; Cheng, Y.; Wang, L. Polym. Chem. 2015, 6, 5070–5076. 84. Dai, C.; Yang, D.; Zhang, W.; Fu, X.; Chen, Q.; Zhu, C.; Cheng, Y.; Wang, L. J. Mater. Chem. B 2015, 3, 7030–7036. 85. Yoshii, R.; Hirose, A.; Tanaka, K.; Chujo, Y. Chem. Eur. J. 2014, 20, 8320–8324. 86. Yoshii, R.; Hirose, A.; Tanaka, K.; Chujo, Y. J. Am. Chem. Soc. 2014, 136, 18131–18139. 87. Hirose, A.; Tanaka, K.; Yoshii, R.; Chujo, Y. Polym. Chem. 2015, 6, 5590–5595. 88. Ruff, Y.; Lehn, J. M. Angew. Chem., Int. Ed. 2008, 47, 3556–3559. 89. Ruff, Y.; Buhler, E.; Candau, S. J.; Kesselman, E.; Talmon, Y.; Lehn, J. M. J. Am. Chem. Soc. 2010, 132, 2573–2584. 90. Yan, J. J.; Wang, Z. K.; Lin, X. S.; Hong, C. Y.; Liang, H. J.; Pan, C. Y.; You, Y. Z. Adv. Mater. 2012, 24, 5617–5624. 61 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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91. Zheng, J.; Petty, J. T.; Dickson, R. M. J. Am. Chem. Soc. 2003, 125, 7780–7781. 92. Lee, W. I.; Bae, Y.; Bard, A. J. J. Am. Chem. Soc. 2004, 126, 8358–8359. 93. Wang, D. J.; Imae, T. J. Am. Chem. Soc. 2004, 126, 13204–13205. 94. Chu, C. C.; Imae, T. Macromol. Rapid Commun. 2009, 30, 89. 95. Wang, R. B.; Yuan, W. Z.; Zhu, X. Y. Chinese J. Polym. Sci. 2015, 5, 680–687. 96. Sun, M.; Hong, C. Y.; Pan, C. Y. J. Am. Chem. Soc. 2012, 134, 20581–20584. 97. Jayamurugan, G.; Umesh, C. P.; Jayaraman, N. Org. Lett. 2008, 10, 9–12. 98. Yemul, O; Imae, T. Colloid. Polym. Sci. 2008, 286, 747–752. 99. Song, G.; Lin, Y.; Zhu, Z.; Zheng, H.; Qiao, J.; He, C.; Wang, H. Macromol. Rapid Commun. 2015, 36, 278–285. 100. Pucci, A.; Rausa, R.; Ciardelli, F. Macroml. Chem. Phys. 2008, 209, 900–906. 101. Xing, C. M.; Lam, J. W. Y.; Qin, A.; Dong, Y.; Häuβler, M.; Yang, W. T.; Tang, B. Z. Polym. Mater. Sci. Eng. 2007, 96, 418–419.

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Chapter 3

Aggregation-Induced Chirogenesis of Luminescent Polymers Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch003

Michiya Fujiki* Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan *E-mail: [email protected]

Herein we demonstrate aggregation-induced enhancement (AIEnh) in chiroptical signals, including circular dichroism (CD), optical rotation dispersion (ORD), and circularly polarized luminescence (CPL), of inherently emissive σand π-conjugated polymers in a homogeneous solution. This is in sharp contrast to the idea of aggregation-induced emission (AIE)-CPL materials utilizing non-emissive latent luminophores in a homogeneous solution. A restricted intramolecular rotation along C–C and Si–Si single bonds is a common idea of AIEnh- and AIE-CPL phenomena. To efficiently enhance the CD, ORD, and CPL signals, the choice of a surrounding fluidic medium with a tuned refractive index (RI) is critical because the chiral optofluidic effects play a key role in the AIEnh chiroptical effects. We showcase several AIEnh-CPL, AIEnh-CD, and AIEnh-ORD systems from optically active polymer aggregates obtained by: (i) optically active σ-conjugated polysilanes with chiral substituents; (ii) optically inactive polysilanes induced by limonene solvent chirality; (iii) optically inactive π-conjugated polymers induced by limonene solvent chirality; (iv) optically inactive photochromic π-conjugated polymer upon excitation of left- and right-handed circularly polarized light sources; and (v) optically inactive non-photochromic π-conjugated polymer upon excitation of left- and right-handed circularly polarized light sources. Our experiments should provide artificial models of an open-flow coacervates suspension in an optically tuned optofluidic medium in the photoexcited and ground states with © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the aid of chirally shining AIEnh materials. The slowed leftand right-handed circularly polarized light of the structure suspension in a medium was assumed to be responsible for the enhanced CPL signals. Moreover, our photoexcited-induced aggregation-induced enhancement and inversion in CPL experiments shed light on open energy flow chiral systems by tuning the RI of the fluidic medium, regardless of the non-aggregated molecules, oligomers, and polymers in the ground state. AIE-CPL materials of several luminogens with large rotational freedom are further enhanceable with the help of achiral and chiral fluidic media with tuned RIs.

Introduction Historical Background Since the mid-19th century, the origin of homochirality on Earth has been one of the greatest mysteries in the modern scientific community (1–10). Living organisms are tempo-spatial and in a metastable state as a consequence of far-from-equilibrium systems (11) because life is a low-entropy open system (12). If life existed in the past on Earth, a curious question is whether stereogenic centers and/or stereogenic bonds were identical to those of our current life on Earth (1). However, answering this question remains difficult because there is a lack of fossil records and chiral molecular evidence. Moreover, the snowball earth hypothesis with the analysis of paleomagnetism asserts that Prokaryotes only inhabited Precambrian eras for approximately 2,000 million years (13, 14). Recent studies claimed that marked depletion of the 13C in 13C-/12C-isotopic ratio is direct evidence of the existence of methanogenic microbes in the Archaean era 3,500–3,800 million years ago (15–17), based on the fact that lighter 12C-containing substances in living organisms are enriched during their entire lifetime. So far, many scientists have invoked several plausible hypotheses from the primordial era to address the question. Scientists have long argued about the possibility that the circularly polarized radiation sources existing in our universe, such as γ-ray, X-ray, and vacuum UV, may become a trigger for the left-right selection of biomolecular substances (2–8). A subtle imbalance in L-/D-amino acids can catalyze the asymmetric generation of carbohydrates with a high ee, resulting in the homochiral biological world (18). A nearly racemic substance of 10–5 % ee can significantly amplify the ee value to reach nearly 100 % ee with the help of the Soai reaction (19). Coacervate Hypothesis – The Origin of Life From the 1920s–1930s, Oparlin (20) and Haldane (21) independently hypothesized that the cell-wall free coacervate might be a prototype of living cells during the chemical evolution of life. The coacervate refers to spherical-like aggregates surrounded by fluidic water. The diameter of the coacervate typically 64 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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ranges from 1 to 100 μm, almost identical to that of living cells. The aggregates made of stable organic substances were postulated to propagate with time, followed by spontaneous growth and metabolism. The hypothesis relies on spontaneous self-organization resulting from non-covalent interactions, including electrostatic, hydrogen bonding, and van der Waals forces. The hypothesis was, however, mostly abandoned because the coacervate did not evolve into living organisms. The reason why is that most coacervates are made of rigid hard particles surrounded by water in the dark. The confinement of external photon source energy and low-entropy chiral chemical substances in the coacervate are crucial for the chemical evolution of life, followed by propagation of life. If the coacervates are assumed to adopt a dynamic structure, this soft matter system might be adaptable to any changes by external chemical and physical biases. This idea led us to design a cell-wall free, semi-artificial coacervate model consisting of chain-like synthetic polymers surrounded by a mixture of chiral and achiral organic solvents. This open-flow system prompted us to investigate the tempo-spatial molecular chirality transcription of the soft aggregates. In particular, optically active polymer aggregates in the ground and photoexcited states are detectable by CD and CPL spectroscopies because photoluminescent aggregation, known as aggregation-induced emission (AIE), has become popular in recent years (22–25). Aggregation-Induced Emission (AIE) vs Aggregation-Induced Enhancement (AIEnh) in Chiroptical Signals In 2001, Tang et al. reported the first AIE effect of 1-methyl- 1,2,3,4,5pentaphenylsilole. The silole revealed an abrupt enhancement in the quantum yield (Φ) of photoluminescence (PL) from an ultraweak emissive state (≈ 0.06 %) in homogeneous ethanol solution to a highly emissive state (≈ 90 %) when the silole formed aggregate suspension in water–ethanol cosolvent (22). In 2003, this finding led to an important concept that AIE is caused by significantly restricted intramolecular rotations of multiple C–C bonds between the five peripheral phenyl rings and the silole core (23). The restricted motion is made feasible by an increase of solvent viscosity and a decrease of solution temperature. This concept was viable for polymer aggregates made of polyacetylenes substituted with 1,2,3,4,5-pentaphenylsilole in acetone-water cosolvents (24). In 2011, Tang et al. demonstrated the first AIE-circular dichroism (AIE-CD) and AIE-circularly polarized luminescence (AIE-CPL) of a silole derivative bearing two D-sugar moieties using n-hexane and dichloromethane as cosolvent (25). The optically active silole revealed a high Kuhn’s anisotropy in the CPL and CD signals in the UV-visible region. The magnitude of gCPL in a microfluidic channel reaches ≈ –0.32 at 500 nm (25). More recently, Tang et al. reported AIE-CPL of tetraphenylethylene bearing two L-valines in dichloroethane-methanol cosolvent (26). SEM/TEM images of the tetraphenylethylene suggested that the one-dimensional helical fibers are responsible for the AIE-CPL, which is of the order of gCPL ≈ –5×10–3. At approximately the same time, we reported the first aggregation-induced enhancement (AIEnh) in chiroptical signals, as proven by the gigantic enhanced 65 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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CD amplitudes of several σ-conjugated polysilanes (27–30), π-conjugated polymers, including chiral polythiophenes (31–33) and supramolecules of polyfluorene with polysaccharide (34), and cofacial π-π stacks of phthalocyanines (35–37). When used as building blocks, these polymers and molecules are inherently highly luminophoric and/or inherently chromophoric probes in the UV-visible region in homogeneous solutions. These results stimulated us to explore several AIEnh phenomena in CPL, CD, and optical rotation dispersion (ORD) since 2010. However, we did not fully understand the plausible reasoning for these AIEnh-CD, AIEnh-CPL, and AIEnh-ORD phenomena until recently. We assumed that the restriction of intramolecular and/or intermolecular rotations, known as ro-vibrational and translational modes, are commonly responsible for the AIE-CPL signal and AIEnh-CD/CPL/ORD signal amplitudes in the framework of the adiabatic relaxation process from Sn- (n = 1,2...) to S0-states. Chiral Optofluidics In 2006, optofluidics was coined as a new fusion concept of integrated optics and microfluidics (38–41). Optofluidics offers unique μm-scale liquid-based devices with great flexibility. Optofluidics is analogous to the corresponding solid-state devices with respect to the ability to (i) tailor several optical properties (particularly the refractive index (RI)) of the fluidic medium, (ii) obtain optically smooth interfaces between the media with immiscible droplets/aggregates, and (iii) easily confine photon energy into an optical cavity. Whiteside et al. demonstrated ultralow threshold multi-colored lasing action using rhodamine 560/rhodamine 640-doped microdroplets of benzyl alcohol (nD = 1.54, 20–40 μm in diameter) dispersed in fluorinated solvent (C7F15OC2H5, nD = 1.29) (42). This heterogeneous liquid device acts as an efficient optical cavity with whispering gallery mode (WGM) (43, 44). These noticeable advantages offer a great opportunity to more freely design the low-reflection-loss photoluminescent polymer aggregates with a high RI surrounded by a lower RI fluidic medium to fulfill a specific resonance condition. For example, the Fabry–Pérot cavity formed between Bragg grating reflectors in a planar microfluidic geometry revealed a resonantly enhanced transmission peak at a very specific RI value of the fluidic medium (45). It is expected that the photoluminescent aggregates with a high RI act as an optical cavity when surrounded by a fluidic medium with a lower RI that is properly tuned. Note that linearly polarized light is a superposition of pseudoscalar left (l-) and right (r-) circularly polarized light (CP-light) carrying an angular momentum of integer ±ℏ (46–48). Previously, Ghosh et al. (49) and Silverman et al. (50) independently indicated that CP-light signals can magnify the optical rotation of an isotropic chiral medium by several orders of magnitude when a coupled geometry of multiple prismatic cuvettes is filled with limonene, carvone, and camphorquinone-containing methanol. As an application of optofluidics, Mortensen et al. theoretically showed that the slow light of colloidal particles filled with a liquid medium allows for significant enhancement of the CD signal amplitude at the edges of the optical band gap (51, 52). This phenomenon is spectroscopically detectable as ORD signals in an RI-tuned fluidic medium. The 66 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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ORD spectrum detects differences in light speed between the l-CP and r-CP light of optically active aggregates as a function of the incident wavelength of the land r-CP light. Their surprising outcomes combined with the optofluidics and AIE stimulated us to investigate the chiral optofluidics of μm-sized photoluminescent polymer aggregates utilizing inherently highly photoluminescent σ- and π-conjugated polymers as the source of materials surrounded by optically inactive fluidic medium and/or by optically active fluidic media containing limonene (52–58). Herein, we employed a series of AIEnh in CPL, CD, and ORD experiments regarded as artificial models of open-flow coacervates dispersed in an optofluidic medium in the photoexcited and ground states with the aid of chirally shining AIE-related materials.

AIEnh-CD and AIEnh-CPL in the Ground and Photoexcited States Steady-State CD and CPL Spectroscopies Steady-state CPL and PL spectroscopic data provide information about the photoexcited but short-lived chiral species, whereas steady-state CD and UV-vis spectra dictate long-lived chiral species at ambient and/or lower temperatures. Based on a modified Jablonski diagram and Kasha’s rule of chiral luminophores (Figure 1), the short-lived chiral species (S1, S2 …) upon incoherent unpolarized photoexcitation are first generated by the Franck-Condon scenario of the order of 10–15 sec, followed by non-radiative relaxation process associated with ro-vibrational modes to the lowest vibronic state (S1-state with ν´=0) of the order of 10–11–10–12 sec, finally relaxing from the low-entropy chiral S1-state (ν´=0) to the high-entropy chiral ground S0-state (ν=0,1,2,3 ...) along with CPL radiation of the order of 10–9–10–6 sec. For example, an absolute temperature of 300 K equals 0.0259 eV (208 cm–1, 0.60 kcal mol–1, 0.00095 Hartrees). When luminescent molecules and polymers are excited at 400 nm (in a vacuum and in an air) by a light source, the 400-nm energy corresponds to 3.10 eV (25,000 cm–1, 71.5 kcal mol–1, 35,970 K, 0.114 Hartrees). CPL and CD spectroscopic methods can therefore detect different chiral information. CPL signals can dictate chiral species at 35,970 K, although CD signals provide chiral information about species at 300 K. When one can ensure the identity between the chiral photoexcited and ground states, an enantiopair of rigid luminophoric chromophores possessing very restricted intramolecular and/or intermolecular rotations at 35,970 K is needed. Chiral aggregate made of luminophores is one candidate to realize WGM-based chiroptical resonators with a pair of (+)- and (–)-sign AIEnh-CPL signals. This idea predicts that, although a non-rigid chiral luminophore possessing substantial ro-vibrational freedom in the ground state reveals weak CD signal and/or non-detectable CD signal (so-called cryptochirality), the luminophores may not emit CPL signals and solely emit unpolarized PL signals due to an equal probability of left- and right-handed photoexcited chiral structures. But, if a non-rigid chiral luminophore can produce a certain chiral aggregate in a 67 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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restricted translational freedom, one can detect this spatio-temporal, dynamic chiral aggregate as AIEnh-CPL signals. The lifetime of spatio-temporal species is on the order of ≈ 1 –10 nsec, which enables us to detect steady-state AIEnh-CPL signals.

Figure 1. Schematic Jablonski diagram, Kasha’s rule, and Franck-Condon rule of chiral luminophore without optical cavity effects. A representative example is CPL-active pyrene excimer systems. This phenomenon may be photoexcited induced aggregation-induced enhancement in CPL (PI-AIEnh-CPL). Recently, Imai, Fujiki, and coworkers have proved that several CD-silent and/or ultraweak CD-active pyrene-containing molecules and oligomers reveal intense pyrene excimer origin CPL signals on the order of |gCPL| ≈ 10–2, as discussed in a later section. However, reliable computer calculation to predict plausible photoexcited states of the chiral aggregates remains a major challenge. The underlying problem of the huge computational cost should be solved. An open question is whether optofluidics is actually valid in AIEnh-CD and AIEnh-CPL beyond an extension of AIE and the associated restricted intramolecular rotations. To address this question, whether the amplitudes in CD and CPL signals of the polymer aggregates are resonantly enhanced at very specific RI values of the surrounded medium had to be elucidated. Optically oriented film and/or flowing vortex conditions of the anisotropic chromophores and luminophores often cause unfavorable chiroptical signals, leading to an apparent sign inversion and alteration in the absolute magnitudes of the CD and CPL signals. To avoid these spectroscopic difficulties due to optically anisotropic film and/or vortex flowing conditions, the optically anisotropic aggregates suspended in optically isotropic fluidic medium due to the random orientation in the medium must be measured. 68 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Additionally, CPL/CD chiroptical signals (λ values, amplitudes, and signs), as well as PL/PL excitation (PLE) signals (λ values and amplitudes), are not significantly affected by Rayleigh scattering, which causes an unfavorable increment in the background UV-vis signals in proportion to λ–4 (λ: wavelength of light in a vacuum). Instrumental knowledge is one of the crucial factors to characterize an inherently chiral aggregate in the ground and photoexcited states. The RI under unpolarized incident light should be noted because incident light largely depends on the wavelength of the incident light. ORD spectroscopy can measure the difference in RI between left- and right-CP light. Unpolarized light is a superposition of left-CP (r-CP) and right-CP (l-CP) light. Thus, a fine tuning of the RI is crucial when chiral optofluidics plays a key role in AIEnh chiroptical signals. We showcased several typical examples in the following.

AIEnh-CD, AIEnh-ORD, and AIEnh-CPL Aggregates From Optically Active Polysilane with Chiral Substituents Herein, we show evidence that fine control of the RI value of the surrounding solvents is crucial to enhance AIEnh-CD, AIEnh-ORD, and AIEnh- CPL signals of helical polysilane aggregates bearing chiral substituents, including p-(S)-2-methylbutoxypheneyl-n-propyl-polysilane (1S) (27), poly(n- decyl-(S)3-methylpentylsilane) (2S) (29), poly(n-decyl-(S)-2-methylbutylsilane) (3S) (53), poly(n-dodecyl-(S)-2-methylbutylsilane) (4S) (53), and poly(n-dodecyl-(R)2-methylbutylsilane) (4R) (53) (Chart 1). In this section, we focused on mainly 3S aggregates as a function of the surrounding cosolvents to continuously tailor the RI value, whereas the RI value of the 3S aggregate was assumed to be ≈1.7 (53).

Chart 1. Chemical structures of semiflexible and rodlike polysilanes bearing chiral substituents. 69 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The AIEnh-CD, AIEnh-ORD, and AIEnh-CPL spectra of the 3S aggregates (weight-average molecular weight (Mw) = 8.5×104, weight-averaged degree of polymerization (DPw) = 311) are given in Figure 2a–2c. From Figure 2a, the 3S aggregates clearly showed negative bisignate exciton couplet Cotton CD bands arising from the lowest Siσ–Siσ* transition at 323 nm. The magnitudes of the gCD values reach –0.31 at 325 nm and +0.33 at 313 nm. These gCD values correspond to 15.5 % left-circular polarization and 16.5 % right-circular polarization because the ideal left- and right-circular polarizations in the absorption of gCD are ±2.0, respectively.

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Figure 2. (a) AIEnh-CD and UV spectra, (b) AIEnh-ORD and UV spectra, and (c) AIEnh-CPL and PL spectra of the 3S aggregates. (d) The gCD values of the 3S, 4S, and 4R aggregates vs the RI value of methanol-tetrahydrofuran (THF) cosolvent. Here, the AIEnh-CD and AIEnh-CPL spectra were normalized by dimensionless Kuhn’s anisotropy in the ground and photoexcited states (59, 60) (e) Schematic Jablonski diagram of chiral 3S aggregates. (f) Possible explanation for the AlEnh-CPL scenario by confining left- and right-circularly polarized light in chiral the 3S aggregates in an optofluidic medium (53). Reproduced with permission from ref. (53). Copyright 2011 American Chemical Society.

From Figure 2b, the 3S aggregates clearly showed negative bisignate Cotton ORD bands at the 323-nm transition. The speed of r-CP light at 330 nm is greatly slowed relative to that of l-CP light at 320 nm. Conversely, the speed of l-CP light at 320 nm is greatly slowed relative to that of r-CP light at 320 nm. Therefore, the RI values between r-CP light and l-CP light are strongly dependent on the wavelength. Higher RI values result in slower CP-light than other CP-light associated with wavelength shortening, while the wavenumber is unchanged regardless of the RI value. 71 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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In Figure 2c, the 3S aggregates clearly showed only a negative CPL band at the 330-nm PL band associated with a very small Stokes’ shift (5 nm, 466 cm–1). The small Stokes’ shift means that the structural alteration due to ro-vibrational modes of 3S aggregates in the photoexcited state is minimal. The magnitude of the gCPL value was –0.65 at 330 nm. This gCPL value corresponds to 32.5 % right-elliptical circular polarization, whereas the ideal left- and right-circular polarization in emission occurs at gCPL values of ±2.0, respectively. The AIEnh-CD and AIEnh-CPL characteristics of 3S are almost identical to those of 4S and 4R. However, these AIEnh-CD and AIEnh-CPL characteristics of 3S, 4S, and 4R strongly depend on the nD value of the methanol-THF cosolvent, as shown in Figure 2d. The gCD value of the 3S aggregates is resonantly enhanced when nD = 1.374. Similarly, the gCD values of the 4S and 4R aggregates are resonantly enhanced when RI = 1.359 for 4S and nD = 1.365 for 4R. These RIdependent resonance effects are a typical feature of chiral optofluidics, where a marked difference in speed between l- and r-CP light travelling in μm-scale colloidal particles dispersed in a tuned RI liquid medium causes enhanced CD signals in the absorption mode, followed by enhanced CPL signals in the emission mode (51, 52). Based on Figure 2a–2c, a modified Jablonski diagram of the 3S aggregates combined with the Kasha rule and exciton coupling theory is schematically given in Figure 2e (55). An optical cavity of l- or r-CP light due to a large difference in their RI values is crucial. This idea might be applicable to other polysilane aggregates carrying chiral substituents (27, 29). Figure 2f displays a possible explanation for the AlEnh-CPL signals in an optical cavity in WGM mode due to an efficient confinement of left- and rightcircularly polarized light in the chiral 3S aggregates with a high RI surrounded by optofluidic medium with a lower RI (53). The optically active aggregate acts as an optical cavity for l- and r-CP light separation. Firstly, l- and r-CP light (so-called natural light) at 317 nm (3.90 eV) simultaneously excite 3S aggregates with a high RI (n2) surrounded by a liquid with a lower RI. In this case, the RI value for l-CP light (n1(l)) at 317 nm is higher than that for r-CP light (n1(r)) at 317 nm. We hypothesized that n1(l) and n1(r) are 1.8 and 1.6, respectively, and that n2 is 1.4. In this case, the critical angles of refraction (θc) for l- and r-CP light by Snell’s law are estimated as 51° and 61°, respectively. The difference in the θc angles of l- and r-CP light acts as a chiroptical filter to sort l- and r-CP light sources at the polymer-liquid interface. Therefore, fine tuning the RI at the specific wavelength of the surrounding medium is a critical factor. This idea is valid for heterogeneous suspension systems but not for homogeneous solution systems. In other words, when the RI values of the aggregates and surrounding medium are identical, no refraction and no scattering at the polymer-liquid interface occurs. The heterogeneous system is apparently transparent. One of the examples is optically transparent TPX® (Mitsui Chemicals, poly(4-methyl1-penetene). The reason for the transparency is that the RI values of the crystalline and non-crystalline TPX® are almost identical. The l- and r-CP light in the polymer at 317 nm slow to 1.67 × 108 m sec1 and 1.88 × 108 m sec-1, respectively. However, the wavelength of 317 nm of 72 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the incident l- and r-CP light in a vacuum becomes 176 nm and 198 nm in the polymer, respectively. As a result, the incident l-CP light at 317 nm becomes greatly slowed l-CP light of 176 nm. Similarly, the incident r-CP light at 317 nm become a slightly slower light of 198 nm in the polymer. If 176 nm l-CP light was employed, multiple total internal reflections will occur efficiently (12 times, for example) in the aggregates rather than the 198 nm r-CP light. Increasing the number of total internal reflections of CP light at the polymer-liquid interface increases the opportunity for light-matter interactions. The slowed l-CP light shifts to longer-wavelength, lower-energy r-CP light at 330 nm with a change in CD sign due to energy migration in the aggregates. The faster r-CP light at 317 nm migrates to the lower energy r-CP light at 330 nm without a change in CD sign. Due to the great suppression of the photoexcited aggregates at 330 nm, r-CP emission at 325 nm occurs from the lowest photoexcited S1 state with a minimal Stokes’ shift. This phenomenon is spectroscopically detectable as ORD signals in a RI-tuned fluidic medium. The ORD spectrum detects differences in light speed between the l-CP and r-CP light of optically active aggregates as a function of the incident wavelength of l- and r-CP light in a vacuum. From Optically Inactive Polysilanes Induced by Limonene Chirality Optically active polysilane aggregates are generated by adding poor solvent to a homogeneous solution of the corresponding polysilane carrying chiral substituents. However, this methodology requires expensive chiral source materials and multiple, time-consuming synthetic steps when chiral substituents are introduced to polysilanes. Herein, we demonstrate more facile, inexpensive, and environmentally friendly approaches to yield AIEnh-CPL and AIEnh-CD polymer aggregates (Chart 2). Previously, achiral 5 showed couplet-like AIEnh-CD with the help of (S)and (R)-1-phenylethyl alcohol and several other alkyl alcohols when the alcohol was used as solvent (29). It was noted that 5 in homogeneous solution adopts a CD-silent state (mirror symmetry) resulting from a racemic mixture of dynamic twisting between the left- and right-hand helices. During aggregation by adding a poor solvent (methanol), CD-silent 5 provided AIEnh-CD 5 as a consequence of mirror symmetry breaking, in which the CD sign is determined by the alcohol chirality. The chiral CH/O interaction is assumed to be responsible for the AIEnhCD (Chart 2) (29). This report prompted further testing of whether three CD-silent, rod-like dialkylpolysilanes (7, 8, 9) can provide the corresponding AIEnh-CD (7, 8, 9) and AIEnh-CPL (8, 9) in the presence of inexpensive terpenes, (S)- and (R)limonene (54). We assumed that non-covalent intermolecular chiral CH/π and van der Waals attractive interactions induce AIEnh-CD and AIEnh-CPL signals. In these cases, precise control of the RI of the surrounding solvents including limonene is critical as well. The AIEnh-CD and UV spectra of aggregates 7 and 8 are given in Figure 3a and 3b, respectively. For aggregate 7 in 10R-containing solvent, the magnitudes of the bisigned gCD values at 327 nm and 309 nm were +0.022 and –0.031, respectively. For aggregate 7 in 10S-containing solvent, the gCD values at 327 nm 73 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and 309 nm were –0.021 and –0.033, respectively. Compared to the AIEnh-CD values of aggregate 7, the AIEnh-CD values of aggregate 9 were comparable, but aggregate 8 was decreased by one third. For aggregate 8 in 10R-containing solvent, the magnitudes of the bisigned gCD values at 331 nm and 317 nm were –0.007 and +0.010, respectively. In 10S-containing solvent, the gCD values at 331 nm and 317 nm were +0.005 and –0.007, respectively. The degree of circular polarization of 7, 8, and 9 was rather weak, on the order of 1.0–1.5 % circular polarization.

Chart 2. Chemical structures of CD-silent polysilanes and chiral solvents (1-phenylethyl alcohol and limonene).

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Figure 3. AIEnh-CD and UV spectra of (a) aggregate 7 and (b) aggregate 8. (c) The gCD value of aggregate 7 vs Mw of 7. (d) AIEnh-CPL and PL spectra of aggregate 8. The gCD values of (e) aggregate 7 and (f) aggregate 8 as a function of the limonene-containing solvent. Reproduced with permission from ref. (54). Copyright 2012 Royal Society of Chemistry.

The absolute magnitudes of the AIEnh-CD values of 7, 8, and 9 had intense Mw dependence. A representative example of 7 is given in Figure 3c. When the Mw of 7 was 2.7×104, the gCD value was maximized; 8 and 9 had similar Mw-dependent AIEnh-CD effects (54). Only 8 and 9, with weaker gCD values, had AIEnh-CPL signals of ≈ 0.005 as the absolute gCPL values (Figure 3d). However, aggregate 7 did not have PL and CPL due to unresolved reasons. From Figure 3a–3b, it is evident that the CD sign of aggregate 7 is opposite of that of aggregate 8 when the same limonene chirality was employed as the solvent. However, the CD sign of 7 inverted at the specific RI value (nD = 1.36) of the limonene-containing solvent (Figure 3e). The gCD value resonantly enhanced twice at nD = 1.35 and 1.39. The CD sign of 8 was unchanged by the RI value of limonene-containing solvent but had an abrupt transition at nD = 1.41 (Figure 3f). Thus, subtle structural changes in the side chains of dialkylpolysilanes significantly affect the PL and CPL characteristics as well as the chiroptical sign.

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From Optically Inactive π-Conjugated Polymers Induced by Limonene Chirality

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Three CD-silent aggregates, 7, 8, and 9, in homogeneous solution provided the corresponding AIEnh-CPL (8, 9) and AIEnh-CD (7, 8, 9) in the presence of (S)and (R)-limonene (54). These results stimulated us to further test the possibility that achiral (CD-silent) π-conjugated photoluminescent polymers reveal AIEnhCD and AIEnh-CPL effects in the presence of (S)- and (R)-limonene (55, 56, 61–65).

Chart 3. Chemical structures of CD-silent π-conjugated polymers. Herein, we demonstrated the first successful limonene chirality transfer experiment of AIEnh-CD and AIEnh-CPL poly[(9,9-dioctylfluorenyl-2,7-diyl)alt-bithiophene] (PF8T2, 14) among several π-conjugated polymers (Chart 3). The AIEnh-CD and AIEnh-CPL characteristics of π-conjugated polymers (15, 17, 18, 20, 21, 22) are similar to those of 14. The AIEnh-CD and AIEnh-CPL characteristics (gCD and gCPL) of these aggregates were resonantly enhanced at the RI value of the limonene-containing solvent. The UV-vis and PL spectra of 14 in homogeneous chloroform are shown in Figure 4a. The PL spectrum has at least three well-resolved vibronic bands located at 500, 534, and 578 nm with ≈1350 cm–1 spacing. The corresponding UV-vis absorption band has a structureless broad band peaking at 457 nm associated with a weak shoulder at 478 nm. This indicates that 14 adopts a highly ordered πconjugated structure (a low entropy state) in the photoexcited state. Note that 14 is in a considerably disordered π-conjugated state (a high entropy state) in the ground state. These UV-vis and PL spectral features in homogeneous solution are typical characteristics of random coiled polysilanes (66, 67). The rotational freedom with an equal probability between left-and-right twisting modes of C–C, Si–Si, and Si–C single bonds in the ground state is responsible for the broadened 76 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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UV-vis absorption bands, resulting in no detectable Cotton CD band. Typically, the barrier heights of these single bonds are on the order of 1–2 kcal mol–1 based on our calculations (54, 55, 57, 63). During the aggregation process in the presence of limonene, 14 revealed AIEnh-CD and AIEnh-CPL spectra due to the loss of rotational freedom, as evident from Figure 4b and 4d. The magnitude of the bisigned gCD reached –0.085 at 510 nm and +0.042 at 394 nm (10R) and +0.114 at 510 nm and –0.041 at 394 nm (10S). Concurrently, the magnitude of the bisigned gCPL reached +0.012 at 489 nm and –0.058 at 511 nm (10R) and –0.010 at 489 nm and +0.056 at 511 nm (10S). In the presence of 10R, the (–)-sign 511 nm-CPL band originates from the (–)-sign 510 nm-CD band, while the (+)-sign 489 nm-CPL band originates from the (+)-sign 394 nm-CD band. The presence of 10S as the solvent provides the opposite chiroptical signs of 10R.

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Figure 4. (a) UV-vis and PL spectra of 14 in homogeneous chloroform solution. (b) AIEnh-CD and UV spectra of unfiltered aggregate 14 in limonene-chloroform-methanol tersolvent. (c) AIEnh-CPL and PL spectra of unfiltered 14 in limonene-chloroform-methanol tersolvent. (d) The gCD value at 510 nm (the first Cotton band) as a function of the RI of the mixed solvent. (e) The gCD value of unfiltered 14 as a function of the limonene ee value. (f) The gCD value of 14 as a function of the aggregate size. Reproduced with permission from ref. (61). Copyright 2012 Royal Society of Chemistry. Similarly, the gCD value of 14 aggregates is resonantly enhanced at the nD = 1.44 of the limonene-containing solvent, as shown in Figure 4c. However, the gCD value varied extremely as a function of the ee value of limonene, exhibiting the socalled negative cooperative effect, as plotted in Figure 4e. This result indicates that enantiopure homochiral limonene needs the highest gCD value. We were aware that 78 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

the gCD value tends to decrease when the aggregate size decreases, as seen in Figure 4f. A larger size of aggregate may offer an advantage to facilitate a morphologydependent resonance condition in WGM-based chiroptical resonators, as expected by theory (68) and experiment (42).

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From Optically Inactive Photochromic π-Conjugated Polymer Induced by Circularly Polarized Photon Chirality We demonstrated AIEnh-CD and AIEnh-CPL effects utilizing CD-silent σand π-conjugated polymers in the presence of (S)- and (R)-limonene. The gCD and gCPL signals are resonantly enhanced at the specific RI values of the optofluidic solvents. These results led us to further test the possibility of fully controlled absolute asymmetric synthesis (AAS) of two π-conjugated polymers as aggregate forms 14 (57) and 16 (58) by tuning the RI of the solvents (Chart 4).

Chart 4. Chemical structures of CD-silent π-conjugated polymers and alcohols used as a lower RI solvent with the aid of fully controlled AAS experiment using an r- and l-CP light source. Fully controlling refers to all chiroptical modes of chiroptical polarization, depolarization, inversion, retention, and switching. Historically, the possibility of AAS using an r- and l-CP light source was proposed independently by LeBel in 1874 and van’t Hoff in 1894 (3, 4). This conjecture was experimentally proven by Kuhn and Broun in 1929 (69). Their pioneering works led to many AAS studies over 150 years because specific, expensive chemicals may be not needed (70). However, researchers have long believed that an l-CP light source produces left-handed molecules preferentially or vice versa because the product chirality is determined solely by the handedness of CP light. In this section, we demonstrate the capability of r- and l-CP light-controlled chiroptical polarization, depolarization, inversion, retention, and switching of μm-sized aggregates made of 14 and 16 in achiral optofluidic media, as proven by the AIEnh-CD and AIEnh-CPL spectra. The AIEnh-CD and UV spectra of aggregate 16 upon excitation with an rand l-CP light source at 436 nm are given in Figure 5a. The gCD values at the first and second Cotton bands are –0.025 at 500 nm and +0.021 at 367 nm (r-CP light), whereas they are +0.025 at 509 nm and –0.027 at 364 nm (l-CP light). However, the AIEnh-CPL and AIEnh-PL signals were not feasible because 16 is not emissive due to an aggregation-caused quenching (ACQ) effect (22–26). Note that nonaggregate 16 and unsubstituted azobenzene in chloroform are weakly emissive. 79 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The negative-couplet AIEnh-CD 16 generated by r-CP light at 436 nm turned into nearly zero AIEnh-CD upon l-CP light excitation at 436 nm for 5–10 min (Figure 5b). Further l-CP light excitation for 51 min led to an ideal, mirror-image, positivecouplet AIEnh-CD signal (Figure 5b). An alternative excitation between the r- and l-CP light source enables chiroptical inversion between the positive- and negativecouplet AIEnh-CD signals (57). The Arrhenius plots of aggregate 16 and unsubstituted azobenzene during thermal cis-to-trans isomerization indicated that the activation energy (Ea) from cis-16 to trans-16 in chloroform-methanol cosolvent is ≈22 kcal mol–1 (57, 71), which is slightly higher than that of azobenzene of ≈18 kcal mol–1 (Figure 5c). The Ea value of aggregate 16 may be responsible for the long-term thermal chiroptical stability at ambient temperature. From the activation enthalpy–activation entropy (ΔH‡_ΔS‡) relationship of the Eyring plot, the thermally excited (cis-to-trans) isomerization, possibly, the photoexcited (trans-to-cis) isomerization of 16 main chains in the aggregates, may obey the rotation mechanism of azobenzene moieties rather than the inversion mechanism (57). Regardless of 16, with considerably restricted rotational freedom, the photon chirality of the r- and l-CP light source should induce the generation, inversion, and retention of AIEnh-CD signals in the aggregate.

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Figure 5. (a) AIEnh-CD and UV spectra of aggregate 16 upon excitation with an r- and l-CP light source at 436 nm. (b) AIEnh-CD of 16 initially generated by l-CP light at 436 nm, followed by l-CP light excitation at 436 nm. (c) The Arrhenius plots of 16 and unsubstituted azobenzene during thermal cis-to-trans isomerization. (d) Alcohol-dependent thermal stability of the CP-light source induced cis-16 aggregate at 25 °C. The gCD values as a function of the RI values of (e) non-branched alcohol and chloroform cosolvents and (f) isoalcohol and chloroform cosolvents. Reproduced with permission from ref. (57). Copyright 2013 Royal Society of Chemistry.

However, the degree of thermal chiroptical stability is greatly dependent on the nature of the alcoholic solvents. The gCD value of 16 in non-branched alcohols and isopropanol tends to diminish in two days from the half-life of the original gCD value (Figure 5d). In particular, methanol had a short lifetime of 4–5 hrs. The gCD value of 16 in isobutanol was notably unchanged for at least two days. Thus, the proper choice of alcohol is another crucial factor to generate and retain the chiroptical properties of the aggregates. More importantly, fine control of the RI value of the alcoholic chloroform solvents is important regardless of the nonbranched and branched alcohols (Figure 5e–5f). Regardless of the r- and l-CP light source excitation, the gCD values of aggregate 16 are resonantly enhanced at nF = 1.382 (methanol), 1.404 (ethanol), 1.410–1.412 (n-propanol), 1.418 (n-butanol), 1.426 (n-pentanol), 1.405–1.411 81 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

(isopropanol), and 1.415 (isobutanol). The resonance points of the gCD values are subtly altered by the nature of the alcohol, a longer alkyl chain tends to shift to a larger nF value.

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From Optically Inactive Non-Photochromic π-Conjugated Polymer Induced by Circularly Polarized Photon Chirality The knowledge and understanding of photochromic, but non-emissive aggregate 16 led us to design a fully CP-light controlled AIEnh-CPL and AIEnh-CD polymer aggregates. To achieve this goal, highly photoluminescent but non-photochromic 14 was chosen for the AAS experiments using a solely wavelength-dependent r- and l-CP light source.

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Figure 6. AIEnh-CD and UV spectra of aggregate 14 excited with an (a) r-CP light source at 546 nm and an l-CP light source at 365 nm and (b) an l-CP light source at 546 nm and an l-CP light source at 365 nm. (c) AIEnh-CPL and PL spectra of aggregates 14 excited with an r- and l-CP light source at 546 nm. (d) The gCD values as a function of the nD values of the cosolvents. (e) AIEnh-CD inversion of aggregate 14 excited with an r- and l-CP light source at 546 nm. (f) Thermal stability of aggregate 14. The gCD value as a function of solvent temperature. Reproduced with permission from ref. (58). Copyright 2015 Royal Society of Chemistry. The AIEnh-CD and UV spectra of aggregate 14 after excitation with an l-CP light source at 546 nm and 365 nm and an r-CP light source at 546 nm and 365 nm are given in Figure 6a–6b, respectively. The positive sign couplet CD spectrum induced by l-CP light source at 546 nm is completely inverted compared to that of the l-CP light source at 365 nm. Similarly, the negative sign couplet CD spectrum induced by the r-CP light source at 546 nm is completely inverted compared to that of the r-CP light source at 365 nm. CP light excitation at 313 nm and 405 nm gave similar trends as the 365-nm excitation, while CP light excitation at 436 nm and 577 nm had similar trends as 546-nm excitation. For the same l- (and r-) CP light, the choice of shorter (UV) and longer (visible) wavelengths of CP light switched the sign in the chiroptical polarization of aggregate 14. The handedness of CP-light, whether left or right, was not a deterministic factor for the AIEnh-CD sign of aggregate 14. 83 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The AIEnh-CPL and AIEnh-PL spectra of aggregate 14 by excitation with an l- and r-CP light source at 546 nm are given in Figure 6c. A positive sign couplet CPL spectrum induced by r-CP light source at 546 nm and negative sign couplet CPL spectrum induced by l-CP light source at 546 nm can be observed. The magnitude of gCPL is weak and on the order of 10–3. Upon r-CP light excitation, aggregate 14 had weak positive-sign CPL at 570 nm arising from the positive-sign CD at 540 nm, while the aggregates showed weak negative-sign CPL at 518 nm originating from the negative-sign broad CD at 380 nm. The handedness of the CP light and its irradiating wavelength successfully allowed the generation of CPLactive 14 aggregates (quantum yield ≈ 8 %) on the order of |gCPL| = (2−4)×10–3 at 540 nm. The AIEnh-CD of aggregate 14 generated by r-CP light at 546 nm for 30 nm irradiation completely inverted from a positive-sign couplet to a negative-sign couplet by solely l-CP light at 546 nm for prolonged 120 nm irradiation, as shown in Figure 6d. Massless photon chirality carrying angular momentum is an efficient chiral physical force that enables chirality to be induced and inverted from the aggregates made of achiral substances at ambient temperature. By optofluidically tuning the RI of the cosolvents, the CP light-induced AIEnh-CD signals were resonantly enhanced at a specific nD =1.412, regardless of the r- and l-CP light source excitation (Figure 6e). The AIEnh-CD signals of aggregate 14 were thermally stable and unchanged at 25 °C for at least seven days (Figure 6f). For comparison, detectable CD signals of non-aggregate 14 in homogeneous CHCl3 solution before and after prolonged irradiation with r-CP light at 546 nm were confirmed. The restricted C–C rotational freedom along with efficient confinement of the CP light source in the aggregates as the optical cavity are critical factors in designing CP light-driven AAS experiments. Recent theoretical study (72) shows that a chiroptical enhancement is possible when an optically active aggregate with an ideal chiral sphere efficiently interacts with the surrounding chiral molecules. This means that the AIEnh-CPL and CD signals of chirally assorted, soft-matter aggregates may be further enhanced with the help of an optically tuned chiral fluidic medium. CP light plays a key role in the migration and delocalization of photoexcited energy in optically active macro-aggregates containing ~108 molecules of chlorophyll under excitation of incoherent unpolarized sunlight (73–76). This chiral aggregate requires the existence of three stereogenic centers at the peripheral positions of the chlorophylls and two chiral stereogenic centers in the long alkyl tail of the chlorophylls. Notably, the CP light-related photophysical and biological properties of the chiral aggregates are adaptable to any changes in osmotic pressure, Mg2+/K+ ions, sunlight intensity, and temperature (73–76), because the chiral macro-aggregates are surrounded by fluidic aqueous medium named stroma within the chloroplasts. The adaptability to external chiral chemical substances and/or chiral physical forces appears essential in the chemical evolution and propagation of life (76) by acquiring several sophisticated biological functions during the entire lifetime. Our experiments provide artificial models of an open-flow coacervates suspension in an optically tuned optofluidic medium in the photoexcited and ground states with the aid of chirally shining AIEnh materials (77). 84 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Photoexcited-Induced Enhancement and Inversion of CPL Signals Recently, we demonstrated several PI-AIEnh-CPL molecular systems (25–29) by using fluidic and non-fluidic media (Chart 5) (78–82). These rather simple molecules (25–29), however, can adopt 7,000–24,000 conformations due to free rotations along the C–C, C–O, and C–N single bonds with low rotational barriers in the ground state (80, 81). The system is an extension of the spatio-temporal, open-flow energy transition from low- to high-entropy states that should obey the arrow of time (second law of thermodynamics) (80). These conformationally labile molecular systems in the ground state did not have detectable CD signals and/or had ultraweak CD signals (80, 81). In particular, 29 revealed abrupt CPL signals during photoexcitation (80), and, more surprisingly, 25 and 26 had chiroptical sign inversion characteristics between the CPL and CD signals (81). These phenomena are applicable to designing elaborate photoexcited-induced AIE systems with restricted motion that are non-aggregated luminogens in the tuned RI of the fluidic medium without the addition of a precipitation solvent.

Chart 5. Chemical structures of CPL-active but CD-silent, ultraweak CD-active bi-pyrene-containing molecules in a low-viscosity solvent. 85 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Conclusion In this chapter, we demonstrated the aggregation-induced enhancement of the AIEnh-CD, AIEnh-ORD, and AIEnh-CPL signals of σ- and π-conjugated polymer aggregates. The starting polymers studied here are inherently highly emissive in homogeneous solution. The restricted intramolecular and/or intermolecular rotations are critical to AIEnh and aggregation-induced emission (AIE) phenomena in the ground and photoexcited states. Herein, we emphasized that, to efficiently enhance the CD, ORD, and CPL signals, the choice of surrounding fluidic medium with a tuned RI is a critical factor because the chiral optofluidic effects play a key role in these AIEnh chiroptical signals. We showcased several examples of optically active polymer aggregates obtained from (i) optically active alkylarylpolysilanes and dialkylpolysilanes with chiral substituents, (ii) optically inactive alkylarylpolysilanes and dialkylpolysilanes by solvent chirality, including limonene, (iii) optically inactive, polyfluorene analogs induced by limonene chirality, (iv) optically inactive, photochromic poly(fluorene-alt-azobenzene) induced by circularly polarized photon chirality, and (v) optically inactive, non-photochromic poly(fluorene-alt-bithiophene) induced by circularly polarized photon chirality. Our results suggest that the finely tuned RI of a fluidic medium in the ground and photoexcited states causes resonantly enhanced AIE-CPL signals by utilizing the ideally spherical chiral aggregates made of ultraweak emissive luminogens. Moreover, our recent PI-AIEnh-CPL experiments shed light on open energy flow chiral systems by tuning the RI of the fluidic medium, regardless of the non-aggregated molecules, oligomers, and polymers in the ground state.

Acknowledgments The author is grateful for the financial support from a Grant-in-Aid for Scientific Research (16655046, 21655041, 22350052, 23651092, 26620155, and 16H04155). The author expresses special thanks to his coworkers, including Dr. Yoko Nakano, Kana Yoshida, Yoshifumi Kawagoe, Dr. Yang Liu, Dr. Ayako Nakao, Dr. Nozomu Suzuki, Dr. Makoto Taguchi, Prof. Kotohiro Nomura, Dr. Mohamed Mehawed Abdellatif, Dr. Nor Azura Abdul Rahim, Abd Jalil Jalilah, Fumiko Ichiyanagi, Prof. Seiji Shinkai, Dr. Takao Noguchi, Dr. Masanobu Naito, Dr. Hiroshi Nakashima, Dr. Seiji Toyoda, Prof. Wei Zhang, Prof. Yonggang Yang, Prof. Julian R. Koe, and Prof. Yoshitane Imai.

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Chapter 4

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New Chemo-/Biosensors Based on the Aggregation-Induced Emission Mechanism Xue You, Guanxin Zhang,* Chi Zhan, Yuancheng Wang, and Deqing Zhang* Beijing National Laboratory for Molecular Sciences, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China *E-mails: [email protected] (D.Z.); [email protected] (G.Z.)

A series of fluorescent sensors have been constructed by utilizing the aggregation-induced emission feature of silole and tetraphenylethylene molecules. In this chapter, we discuss representative chemo-/biosensors for biomacromolecules, small molecules and ions. The new enzymatic assays based on aggregation-induced emission mechanism are also described. In addition, new detection methods for gamma radiation are introduced.

1. Introduction Chemo-/biosensors that can realize sensitive and selective detection of relevant chemical and biological species are rather important for various areas including biological sciences, biotechnology, food industry and environmental monitoring (1, 2). Among the sensing modules fluorescent sensing is now becoming a versatile analytical approach with various advantages including high sensitivity, convenient pretreatment, low costs, visualization and usually it can be employed in real time and in situ (3, 4). For the past several decades fluorescent sensors have been extensively investigated to meet the increasing demanding needs of various areas. Normally, the fluorescence signals of these sensing systems are modulated upon interactions with analytes through either photo-induced electron transfer (PET) or intramolecular charge transfer (ICT) or fluorescence resonance energy transfer (FRET) mechanisms (5). It is noted that

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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most of fluorophores employed for sensing exhibit aggregation-caused emission quenching (ACQ) effect (6), which sometimes limits their sensing performances. As referred in previous chapters, Tang and coworkers reported a few fluorophores such as 1-methyl-1, 2, 3, 4, 5-pentaphenylsilole (silole) and tetraphenylethylene (TPE) show abnormal emission behavior (7, 8); they are almost non-emissive in good solvent, but become strongly fluorescent after aggregation. Such emission behavior was referred as to aggregation-induced emission (AIE) (9). We and others have successfully utilized these AIE fluorophores to construct new chemo-/biosensors by manipulating their aggregation and deaggregation. In this chapter, we will introduce representative examples of chemo-/biosensors based on the aggregation-induced emission mechanism. These include i) sensors for biomacromolecules, ii) enzymatic assay, iii) sensors for metal cations and anions, iv) detection of small molecules, and v) sensing gamma-ray radiation.

2. Sensors for Biomacromolecules Biomacromolecules such as nucleic acids, proteins and glycoaminoglycan play very important roles in biological processes. Hence, selective and sensitive sensors for biomacromolecules are highly demanding for bioscience research, biotechnology, clinical diagnose and medical and pharmaceutical industries as well. These biomacromolecules often contain charged moieties and it is expected that silole or TPE derivatives entailing oppositely charged moieties will aggregate upon mixing with these biomacromolecules, leading to fluorescence enhancement. In this way, sensors for biomacromoleules can be designed by appropriately manipulating the aggregation and deaggregation of AIE fluorophores such as silole and TPE molecules (Scheme 1).

Scheme 1. Molecular structures of compounds for biomacromolecules sensing and enzymatic assay based on aggregation-induced emission. 94 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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For instance, some of us successfully utilized silole 1 with an ammonium group for sensing heparain (10), a highly sulfated linear glycoaminoglycan (GAG) consisting of repeating units of 1→4-linked pyranosyluronic acid and 2-amino-2-deoxyglucopyranose residues (see Scheme 2). As a negatively charged biomacromolecule heparin is used for the anticoagulant therapy and thus it is crucial to monitor and control its level and activity during and after surgery (11, 12). Silole 1 was found to be soluble in aqueous solution and it was almost non-emissive. After mixing with heparin the fluorescence of silole 1 increased gradually and heparin with concentration as low as 23 nM was detected with silole 1 (Figure 1A). The interferences from other biomolecules ( e.g. chondroitin sulfate and hyaluronic acid as well as dextran) can be largely eliminated by adjusting the pH value of the solution (Figure 1B). Furthermore, silole 1 can be utilized to probe the interaction of protamine and heparin.

Scheme 2. Illustration of the fluorescence turn-on sensor for heparin based on the AIE feature of silole 1. Reproduced with permission from reference (10). Copyright 2008 Royal Society of Chemistry.

Figure 1. A) Fluorescence spectra (λex. = 370 nm) of silole 1 [5 ×10-5 M in HEPES buffer solution (5.0 mM), pH = 7.4] in the presence of different amounts of heparin (from 0 to 16 μM); B) Variation of the fluorescence intensity of silole 1 [5 ×10-5 M in HEPES buffer solution (5 mM), pH = 7.4] at 480 nm vs. concentrations of chondroitin sulfate(ChS), hyaluronic acid (HA), dextran (DeX) and heparin. Reprinted with permission from ref (10). Copyright 2008 Royal Society of Chemistry. The fluorescence of silole 1 was switched on upon addition of either single stranded DNA (ssDNA) or double-stranded DNA (dsDNA) (13). Tang and his coworkers reported that TPE 1 with four ammonium moieties was successfully utilized as an external fluorescent reporter to monitor folding processes of G-rich DNA (14). The transformation of random coil conformation into G-quadruplex led to the fluorescent spectral redshift for TPE 1, thus a visual distinguishment of G-quadruplexes from other DNA conformations was achieved with TPE 1. This sensing scheme may become applicable for biomedicical studies, especially 95 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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for high-throughput quadruplex targeting anticancer drug screening. Some of us and coworkers used silole 1 to detect G-quadruplex structure formation by an Exonuclease I hydrolysis assay (15). As illustrated in Scheme 3, the fluorescence of silole 1 was lighted up after binding with either ssDNA or the folded G-quadruplex. However, after further introducing Exonuclease I to the ensemble the emission of silole 1 became weak in the presence of ssDNA, whereas it remained strong in the presence of the folded G-quadruplex. This is because ssDNA can be hydrolyzed by Exonuclease I while the folded G-quadruplex cannot be fragmented by Exonuclease I. Alternatively, the ensemble of silole 1 and Exonuclease I can be utilized to investigate the G-quadruplex stabilizers like inorganic alkali metal cations (e.g. Na+, K+) or some small organic molecules. Accordingly, this ensemble may be applicable for the real-time monitoring of G-rich DNA folding process and identifying G-quadruplex binding ligands as anticancer drugs. Tang and coworkers synthesized TPE 2 with thymine, which was successfully used to differentiate ssDNA from dsDNA and ssDNA without the presence of adenine (16). They claimed the hydrogen bonding between adenine and the thymine bases induced the aggregation of TPE 2 and fluorescence enhancement.

Scheme 3. Illustration of the fluorescence variation of silole 1 after mixing with ssDNA and G-quadruplex DNA in the presence of Exo. I. Reprinted with permission from reference (15). Copyright 2009 Elsevier. AIE fluorophores were also investigated for sensing proteins. Tang and coworkers synthesized TPE 3 with two negatively charged groups and studied its emission spectral variation upon interactions with proteins such as human serum albumin (HSA) (17) and bovine serum albumin (BSA) (18). The results reveal that TPE 3 not only can selectively detect these proteins, but also can be used to monitor their conformation changes. For instance, TPE 3 was almost non-emissive in the buffer solution, but it became emissive and its fluorescence intensity increased gradually upon addition of BSA. After the further addition of sodium dodecyl sulfate (SDS) to the solution, which can induce the unfolding process of BSA, the emission of TPE 3 was dramatically reduced. Similarly, HSA with concentration down to 1.0 nM can be detected with TPE 3, and the 96 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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three-step conformation transition induced by GndHCl (guanidine hydrochloride) can be traced with TPE 3. Milk as one kind of vital protein sources is extremely necessary for human heath especially for children (19). Casein is one of the most essential proteins in milk composed of twenty necessary amino acids (20). Some of us and coworkers established a rapid assay for casein quantification in milk powder with TPE 3 (21). The fluorescence enhancement was observed in the presence of casein with concentration from 10 μg/mL to 5000 μg/mL, and in range from 20 μg/mL to 1250 μg/mL the emission intensity of TPE 3 increased linearly. In addition, this assay for casein quantification was explored in real sample with a pretreatment to get rid of the fat in full milk powder. As a type of integrin, αvβ3 has been the focus of intensive research due to its major role in several distinct processes, including osteoclast mediated bone resorption, angiogenesis, pathological neovascularisation and tumour metastasis (22). Especially, its expressions level is well associated with the state of solid tumors (23). Thus, specific detection of integrin αvβ3 plays an important role in early detection and treatment of rapidly growing solid tumors. Liu, Tang and coworkers designed silole 2 (Scheme 1) as a specific light-up probe for integrin αvβ3, in which the two cyclic RGD (cRGD) tripeptides were appended (24). Owing to the high affinity and specificity of cRGD to integrin αvβ3, the fluorescence of solution of silole 2 was switched on upon addition of integrin αvβ3. However, when other proteins having no specific interaction with silole 2 were added, the soluition still remained in the dark state. These results indicate that the specific binding between silole 2 and integrin αvβ3 can dramatically restrict the intramolecular rotations of the aromatic rotors, leading to fluorescence light-up of the probe. Moreover, it has been further demonstated that it might be applied in discriminating integrin αvβ3-positive cancel cells from integrin αvβ3-negative cancer cells.

3. Enzymatic Assays Enzymes can catalyze biochemical reactions involved in almost all metabolic processes in organisms to sustain life. However, a malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can often lead to diseases (25). Thus, development of efficient enzymatic assays is vital not only for medical diagnostics and drug discovery, but also for studies of functions of enzymes. By taking advantage of the AIE feature of silole and TPE molecules, a series of new enzymatic assays have been constructed. As stated above silole and TPE molecules with charged moieties can form fluorescent aggregates with biomacromolecules with oppositely charged groups such as DNA and proteins. Since these biomolecules can be hydrolyzed into small fragments by the respective enzymes, the fluorescent aggregates can be disassembled after incubation with the respective enzymes and accordingly the fluorescence of the ensemble becomes weak again. In this way, label-free fluorometric assays for enzymes can be established with these AIE molecules. With such strategy, 97 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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some of us described a fluorometric assay for nuclease with silole 1 (13). The design rationale is illustrated in Scheme 4. As expected, the fluorescence of silole 1 increased after the addition of ssDNA (see Figure 2A). Interestingly, such fluorescence enhancement was found to be dependent on the length of ssDNA; the longer was ssDNA, the more significantly increased the fluorescence of silole 1 under the same condition. After incubation with nuclease the fluorescence of the ensemble of silole 1 and ssDNA decreased gradually (see Figure 2B). Furthermore, the fluorescence of silole 1 was more significantly reduced by increasing the concentration of nuclease. In this way, the activity of nuclease can be assayed with silole 1. Additionally, this fluorometric assay was successfully utilized for screening inhibitors of nuclease.

Scheme 4. Illustration of fluorescence turn-on detection of DNA and label-free nuclease assay based on aggregation-induced enhanced emission (AIE) feature of silole 1. Reprinted with permission from reference (13). Copyright 2008 American Chemical Society.

Figure 2. A) The fluorescence spectra of silole 1 (2.0×10-5 M) in the presence of different amounts of ssDNA. B) Fluorescence spectrum of silole 1 (2.0×10-5 M) containing ssDNA (20 μL, 5 μM) and those after cleavage by nuclease S1 ([nuclease S1] = 50 U/mL) at 37°C for different periods. Reprinted with permission from reference (13). Copyright (2008) American Chemical Society. With the same strategy, label-free continuous fluorometric assays for trypsin were developed by some of us, Tang and their coworkers (26, 27). Trypsin, as one class of protease, is the most important digestive enzyme produced by pancreas, and it is involved in the digestive enzyme activation cascade that induces the transformation of other pancreatic proenzymes into their active forms within the intestine and then initiates autodigestion (28). Therefore, facile and convenient assays for trypsin activity allow for rapid and effective prescreening of drug candidates. An ensemble of TPE 4 and a positively charged peptide, Arg6, was selected to construct trypsin activity assay by some of us (26). The design rationale is illustrated in Scheme 5. Fluorescent aggregates are 98 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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expected to be formed after mixing TPE 4 and Arg6. But, the aggregates will be disassembled after incubation with trypsin and thus the fluorescence is weakened. The fluorescence of the ensemble of TPE 4 and Arg6 decreased gradually after incubation with trypsin (see Figure 3); the fluorescence was more significantly reduced by increasing the concentration of trypsin. Thus, a new fluorometric assay for trypsin was constructed with the ensemble of TPE 4 and Arg6. In addition, the ensemble was also demonstrated the utility for screening inhibitors of trypsin. In a similar way, Tang and coworkers demonstrated the application of the ensemble of BSA and TPE 5 for label-free detection of protease (27).

Scheme 5. Design rationale for trypsin activity assay and inhibitor screening based on AIE feature of TPE 4. Reprinted with permission from reference (26). Copyright 2010 American Chemical Society.

Figure 3. A) Fluorescence spectra of TPE 4 (60.0 μM) in PBS buffer solution (2.0 mM, pH = 8.5) in the presence of different amounts of Arg6 peptide; B) Fluorescence spectra of the ensemble of TPE 4 (60.0 μM) in PBS buffer solution (2.0 mM, pH = 8.5, containing CaCl2 (10.0 μM)) and Arg6 peptide (10.0 μM) in the presence of trypsin incubated at room temperature for different times. Reprinted with permission from reference (26). Copyright 2010 American Chemical Society. Meanwhile, enzyme-catalyzed amplification of small bio-fragments into biomacromolecules has provided another platform for enzyme activity assay. For instance, a telomerase-substrate-oligonucleotide can be extended by telomerase to DNA with longer sequence, which is able to switch on the fluorescence of AIE molecules bearing positively charged groups as stated above (13). Accordingly, a new assay for telomerase can be established by making use of AIE feature of TPE as demonstrated by Xia, Lou and coworkers (29). It is noted that telomerase is one of the most common biomarkers for cancers and its detection is closely related to early diagnosis of cancers and cancer therapeutics (30). The solution of TPE 5 telomerase-substrate-oligonucleotide (TS primer) was weakly fluorescent due to less negatively charged sites within TS primer. However, after addition of telomerase the fluorescence intensity increased. This is attributed to the fact 99 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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that the TS primer can be elongated to longer DNA strand which offer more negatively charged sites to bind TPE 5 and hence form aggregates. This assay was not only successfully employed to detect telomerase activity from different cell lines with high sensitivity and specificity, but also employed to analyze the untreated real urine samples of bladder cancer patients. They further improved this fluorometric assay by using quencher group (Dabcyl (4-(4-(dimethylamino) phenylazo) benzaoic acid) labeled TS primer to enhance the signal-to-background ratio (31). Apart from biomacromolecules amphiphilic molecules with oppositely charged head groups can also induce the aggregation of AIE molecules with charged groups. Thus, by properly choosing the amphiphilic molecules which can be good substrates of certain enzymes or can be generated in situ in the presence of the enzymes, label-free fluorometric enzymatic assays can be established with AIE molecules and the amphiphilic molecules (32). For instance, a new fluorometric assay for AChE was established with TPE 3 with two negatively charged groups and myristoylcholine (33). It is noted that AChE plays an important role in the regulation of the neutral response system, and it is highly relevant to Alzheimer’s disease (AD) (34). As expected TPE 3 was weakly emissive in aqueous solution, but the fluorescence intensity increased after mixing with myristoylcholine. This is likely owing to the aggregation of TPE 3 induced by myristoylcholine, an amphiphile with a positively charged head group as illustrated in Scheme 6. Myristoylcholine can be hydrolyzed into myristic acid and choline in the presence of AChE. Hence, the fluorescent aggregates formed between TPE 3 and myristoylcholine are expected to be disassembled because of the electrostatic repulsion as illustrated in Scheme 6. As depicted in Figure 4, the fluorescence of the ensemble of TPE 3 and myristoylcholine was gradually reduced after incubation with AChE. In this manner, a new assay for AChE was constructed with the ensemble of TPE 3 and myristoylcholine, which was also useful for screening the inhibitors of AChE.

Scheme 6. Design rationale for AChE activity assay and inhibitor screening based on AIE. Reprinted with permission from reference (33). Copyright 2009 American Chemical Society. Alternatively, some of us and coworkers devised a fluorescence “turn-on” assay for AChE with the ensemble of TPE 3, acetylthiocholine (ATC) and the maleimide with a long alkyl chain (35). The design rationale is illustrated in Scheme 7 and explained as follows: ATC is expected to be hydrolyzed into thiocholine which will quickly react with the maleimide to generate the 100 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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amphiphile with one positively-charged unit; as a result, the aggregation of TPE 3 takes place, leading to the enhancement of the fluorescence of TPE 3. As shown in Figure 5, the fluorescence of TPE 3 increased after incubation with AChE and it was incremented more significantly by enhancing the concentration of AChE in the ensemble. Similarly, the ensemble can be utilized for screening the inhibitors of AChE.

Figure 4. A) Fluorescence spectra of TPE 3 [20 μM in PBS (10 mM) buffer solution, pH = 8.0] in the presence of different amounts of myristoylcholine; B) Fluorescence spectra of the ensemble of TPE 3 [20 μM in PBS (10 mM) buffer solution, pH = 8.0] and myristoylcholine (25 μM) in the presence of AChE (0.5 U/mL) incubated at 25 °C for different periods. Reprinted with permission from reference (33). Copyright 2009 American Chemical Society.

Scheme 7. Design rationale for fluorescence turn-on assay for AChE activity and inhibitor screening. Reprinted with permission from reference (35). Copyright 2009 American Chemical Society. Monoamine oxidase B (MAO-B) catalyzes the oxidative deamination of biogenic and xenobiotic amines and plays an important role in the catabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues. Recent studies indicate that Alzheimer’s disease and Parkinson’s disease are both associated with elevated levels of MAO-B in the brain (36). Hence, MAO-B and its analogues became key therapeutic targets for a variety of brain disorders. Moreover, inhibitors of MAO-B are currently being explored as potential drugs for clinical treatment of Parkinson’s and Alzheimer’s diseass. Some of us and coworkers described a new direct continuous fluorometric turn-on assay for MAO-B by taking the advantage of the AIE behavior of silole 1 (37). The design rationale is illustrated in Scheme 8. Heptylamine can be oxidized 101 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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by MAO-B into heptylaldehyde which can react with NaHSO3 to generate the amphiphile with a negatively-charged moiety that is able to induce the aggregation of silole 1, leading to fluorescence enhancement. Figure 6 shows the fluorescence of silole 1 increased after incubation with MAO-B and the emission became stronger after incubation with higher concentration of MAO-B. Also, this ensemble was successfully utilized to screen inhibitors of MAO-B.

Figure 5. A) Fluorescence spectra of the ensemble of TPE 3 [20 μM in HEPES (10 mM) buffer solution, pH=7.35], TPE 3 (30 μM) and ATC (30 μM) in the presence of AChE (0.1 U/mL) incubated at room temperature for different periods; B) Variation of the fluorescence intensity at 490 nm vs. the reaction time for the ensemble of TPE 3 [20 μM in HEPES (10 mM) buffer solution, pH=7.35], TPE 3 (30 μM) and ATC (30 μM) in the presence of different concentrations AChE. Reprinted with permission from reference (35). Copyright 2009 American Chemical Society.

Scheme 8. Design rationale for monoamine oxidase activity assay and inhibitor screening based on AIE. Reprinted with permission from reference (37). Copyright 2010 Royal Society of Chemistry. Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of biomolecules, including nucleotides, proteins, and alkaloids. It has received much attention because of its wide applications in clinical practices (38). For instance, serum ALP activity is routinely used as a diagnostic indicator of several diseases including bone disease (osteoblastic bone cancer, Paget’s disease and osteomalacia), liver dysfunction, breast and prostatic cancers, and diabetes. Therefore, the development of convenient assay methods for monitoring ALP activity is extremely important and valuable, not only for clinical diagnoses but also for biomedical research. A series of enzymatic assay for ALP were constructed by utilization of AIE fluorophores (39–43). For instance, some of us and coworkers reported a 102 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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label-free fluorometric assay to monitor the activity of ALP (39). Addition of ATP to the solution of silole 1 led to fluorescence enhancement. In comparison, the fluorescence of silole 1 kept weak after addition of either ADP or AMP or pyrophosphate by adjusting the pH and ionic strength of the solution (see Figure 7A). Thus, silole 1 can discriminate ATP from ADP, AMP and pyrophosphate. By considering this feature, silole 1 can be used to build a fluorometric assay for ALP which catalyses the hydrolysis of ATP. The fluorescence of silole 1 in the presence of ATP was reduced gradually after incubation with ALP and the fluorescence intensity decreased more quickly upon incubation with ALP of higher concentration (see Figure 7B).

Figure 6. (left) Fluorescence spectra of the ensemble of silole 1 (7.5×10-5 M), heptylamine (2.0 × 10-4 M) and NaHSO3 (1.0 × 10-4 M) in the mixture of HEPES buffer (10 mM, pH = 7.4) and THF (200:1, v/v) in the presence of MAO-B (2.2 μg/mL) after incubation for different times at room temperature. (right) Variation of the fluorescence intensity at 467 nm vs. the reaction time for the ensemble of silole 1 (7.5×10-5 M), heptylamine (2.0×10-4 M) and NaHSO3 (1.0×10-4 M) in the mixture of HEPES buffer (10 mM, pH = 7.4) and THF (200:1, v/v) in the presence of different concentrations of MAO-B. Reprinted with permission from reference (37). Copyright 2010 Royal Society of Chemistry.

Figure 7. A) Variation of the relative fluorescence intensity of silole 1 (8×10-5 M in 10 mM, pH = 9.0 Tris buffer solution) vs. the respective concentrations of ATP, ADP, AMP and pyrophosphate. B) The plot of the fluorescence intensity at 470 nm of silole 1 [8×10-5 M in tris buffer solution (10 mM, pH = 9.0)] containing ATP (5.0 μM) vs the hydrolysis reaction time for the selected concentrations of ALP. Reprinted with permission from reference (39). Copyright 2008 American Chemical Society. 103 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Han and coworkers reported an assay for ALP with the ensemble of TPE 6 and monododecylphosphate as an amphiphile with a negatively charged head group (40). The fluorescence of TPE 6 increased after addition of monododecylphosphate. But, the fluorescence intensity of the solution decreased after incubation with ALP. This is ascribed to the fact that monododecylphosphate was hydrolyzed to form dodecanol, and accordingly the aggregates formed between TPE 6 and monododecylphosphate were dissociated. In this way, the ensemble of TPE 6 and monododecylphosphate was used to monitor the activity of ALP. The enzymatic assays discussed above are mostly based on two-component ensemble or even three-component ensemble in a few cases. Although they display good sensitivity and selectivity, they may not suitable for relevant cellular studies. Recently, more AIE molecules with enzyme responsive units have been devised and investigated for sensing enzymes. For instance, some of us and coworkers reported a new fluorometric turn-on assay for ALP activity and inhibitor-screening by manipulating the aggregation and deaggregation of TPE 7 in aqueous solution (41). The sensing mechanism is illustrated in Scheme 9. TPE 7 entails a –PO3H2 group, which endows its good water solubility, and accordingly it is expected to be weakly emissive in aqueous solution. But, the –PO3H2 group can be removed in the presence of ALP to generate a more hydrophobic compound (TPE 7′) and hence aggregation will occur, leading to fluorescence enhancement. In this way, TPE 7 can be employed for the fluorescence turn-on assay for ALP activity. As shown in Figure 8, the buffer solution of TPE 7 emitted very weakly. However, the green fluorescence emission was switched on quickly after addition of ALP. ALP at concentrations as low as 18 mU /mL can be assayed with TPE 7. Further results clearly indicate that TPE 7 can be utilized not only for ALP activity assay but also for the corresponding inhibitor screening. More importantly, this probe is biocompatible and can be applied for detection of ALP in living cells. Liu and coworkers reported TPE 8 with two –PO3H2 groups for ALP assay. The results reveal that the detection limit can reach 0.2 U/L and the assay is also successfully carried out in diluted serum with a linear range up to 175 U/L, demonstrating its potential application in clinical analysis of ALP levels in real samples (42). In fact, besides TPE and silole derivatives, Liu and Tang et al have also discovered that a phosphorylated chalcone derivative can be used for the fluorescence turn-on assay for ALP activity based on excited-state intramolecular proton transfer (ESIPT) and AIE processes (43).

Scheme 9. Illustration of the design rationale for the fluorometric assay with TPE 7 for ALP. Reprinted with permission from reference (41). Copyright 2013 Royal Society of Chemistry. 104 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 8. The fluorescence spectra of TPE 7 [10 μM in Tris-HCl (10 mM) buffer solution, pH = 7.4] in present of ALP (0.1 U/mL) after incubation at 25 °C for different times. Reprinted with permission from reference (41). Copyright 2013 Royal Society of Chemistry. It is known that carboxylesterases are a group of isoenzymes commonly distributed in mammalian organs, and they catalyze the hydrolysis of carboxyl ester. As a result, they play an important role in detoxification of narcotics or chemical toxin clearance (44). Moreover, they serve as important drug candidates for protein-based therapeutics or drug targets for chemotherapeutic prodrug activation. Li, Chen and coworkers developed a new fluorometric assay for carboxylesterase with TPE 9 with four carboxylic acid groups (45). TPE 9 was found to be weakly emissive in aqueous solution. After incubation with carboxylesterase TPE was transformed into a more hydrophobic TPE fluorophore which aggregated and the fluorescence from TPE was intensified accordingly. In this way, a new assay for carboxylesterase was constructed by taking advantage of the AIE feature of TPE. The results indicate that TPE 9 display a high sensitivity towards carboxylesterase with a detection limit as low as 29 pM. As ‘executioner’ proteins in the cell, caspases are essential for apoptosis, or programmed cell death. Failure of apotosis is one of the main contributions to tumour development and antoimmune diseases such as ischemia or Alzheimer’s disease (46). Thus, caspases have caught much attention as potential therapeutic targets. Tang, Liu and coworkers designed a new live-cell-permeable fluorescent turn-on probe TPE 10 for caspase, which contains a TPE skeleton and a caspase-specific Asp-Glu-Val-Asp (DEVD) peptide (47). In aqueous solution, the molecule is almost nonfluorescent owing to its highly water solubility coming from the hydrophilic peptide sequence. However, after specific cleavage of DEVD by caspase-3/-7, aggregation occurs due to the poor water solubility of TPE residues, leading to fluorescence enhancement. The results reveal that this probe is capable of detecting caspase-3/-7 activities both in solution and in living cells. Moreover, this probe can be used for real-time apoptosis imaging and in situ apotosis-related drug screening with high fluorescence contrast.

4. Sensors for Metal Cations and Anions Heavy and transition metal (HTM) ions play an important role in many biological and environmental processes. For instance, Zn2+ and Cu2+ are essential for biochemical reactions like catalysis, transport or biosynthesis at trace level (< 1.0 μM). Some heavy metal ions, like Cd2+, Pb2+ or Hg2+, are potentially 105 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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carcinogenic or mutagenic and many HTM ions affect the toxicity of organic xenobiotics through interaction with metabolizing enzymes or protein synthesis. Similarly, anions also possess some fundamental roles in a wide range of biological and chemical processes, such as the transport of hormones, proteins biosynthesis, DNA regulation, and the activity of enzymes (48). For instance, PPi (pyrophosphoric acid) is the product of ATP hydrolysis under cellular condition (49). However, some anions are fatal. Cyanide is one of the most rapidly acting and powerful poisons (50). Thus, the detection and quantification of ions are very important in many applications, including environmental monitoring, waste management, developmental biology and clinical toxicology. Up to now, various sensing modules for ions have been reported by using AIE molecules. In general, the sensing mechanism is based on the modulation of aggregation and deaggregation of AIE molecules through either metal ions coordination or chemical reaction or others. In this part, sensors for cations and anions with silole and TPE molecules will be discussed.

Scheme 10. Chemical structures of silole and TPE-based probes for ions sensing. Firstly, we will introduce representative sensing systems for metal ions with AIE molecules. It is known that Zn2+ is the second most abundant transition metal ion in human body after iron, it is involved in a number of biological processes, such as brain function and pathology, gene transcription, immune function and mammalian reproduction. Zinc deficiency is associated with many diseases. Meanwhile, consumption of excess zinc ion can also cause ataxia, lethargy and copper deficiency (51). Therefore, sensitive and selective chemosensors for Zn2+ are highly desirable. Some of us and coworkers reported TPE 11 (see Scheme 10) containing four -N(CH2COO-)2 groups for fluorescence detection of Zn2+ in aqueous solution (52). TPE 11 was rather weakly fluorescent in aqueous solution, but the addition of Zn2+ induced fluorescence enhancement as shown in Figure 9. The detection limit was evaluated to be as low as 9.66 × 10-8 M. Such 106 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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fluorescence enhancement is attributed to the intermolecular coordination of Zn2+ with four N(CH2COO-)2 groups which on one hand leads to aggregation, and on the other hand inhibits the photoinduced electron transfer. Further experiments indicate that this probe displays high selectivity toward Zn2+ over most competing metal ions including Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Pb2+, Mn2+ and Hg2+( Figure 9). In a similar way, Tang and coworkers designed and investigated terpyridine functionalized TPE 12 (see Scheme 10) for sensing Zn2+ (53). As their results indicated, the emission of TPE 12 in aqueous solution was quenched upon addition of Zn2+ accompanied with a red-shift (from greenish blue to yellow) of emission color. However, no color change could be observed in the presence of other cations. Thus, this probe could discriminate of Zn2+ ions from other metal ions.

Figure 9. (left) Fluorescence spectra of TPE 11 (10 μM) upon addition of Zn2+ in HEPES buffer (50 mM, pH = 7.4, 0.1 M NaCl); (right) Cation selectivity profiles of TPE 11 in the presence of various metal cations in HEPES buffer. Reprinted with permission from Reference (52). Copyright 2011 American Chemical Society. It is known that copper is also an essential element for living systems. However, excessive amount of copper ion will have adverse effect. An elevated content of copper ion in body is often associated with severe neurodegenerative diseases (54). In addition, Cu2+ could also be regarded as an environmentally pollutant. Thus detection of Cu2+ could be of significant importance both in biology and environmental protection. So far, probes for Cu2+ base on the TPE motif have been developed by different research groups through diverse detecting mechanisms. For example, TPE 13 containing Schiff base macrocycle (see Scheme 10) was synthesized for sensing Cu2+ (55). TPE 13 shows AIE and ESIPT (excited-state intramolecular proton transfer) effects. TPE 13 emitted red fluorescence with a maximum at 595 nm after aggregaion into nanofibers in an aqueous solution. But, the fluorescence became gradually weak upon addition of Cu2+. In addition, a color transition from colorless to yellow brown at the 10 μM level makes it possible to judge the Cu2+ level by naked eye. A fluorescence turn-on sensor for Cu2+ was established on the basis of click reaction between TPE 14 with four azide groups (see Scheme 10) and diethylene glycol dipropionlate in the presence of sodium ascorbate. Such cross-linking reaction will result in formation of aggregates and fluorescence enhancement. This sensor exhibits good sensitivity and selectivity toward Cu2+ with a detection limit of 1.0 μM (56). Many proteins use ferric ions for oxygen transport, electron transport and as a catalyst in oxido-reductase reactions. Conversely, excess amounts of ferric ions in a living cell can catalyze the production of reactive oxygen species (ROS) via 107 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the Fenton reaction, which can damage lipids, nucleic acids and proteins. The cellular toxicity of ferric ions has been connected with serious diseases, including Alzheimer’s, hepatic cirrhosis, Huntington’s and Parkinson’s disease (57). Thus, the detection of Fe3+ has attracted a lot of interests. For instance, TPE 15 (see Scheme 10) bearing coordiantion ligands as binding sites was designed and synthesized for sensing ferric ion (58). TPE 15 was strongly fluorescent in the mixture of THF/H2O, but the fluorescence decreased dramatically upon addition of ferric ion. The detection limit was estimated to be as low as 0.7 μM. Moreover, the interferences from other metal ions can be neglected, thus TPE 15 is selective toward Fe3+ over other metal ions. Similarly, TPE 16 with imidazole groups can also be utilized as fluorescence turn-off sensor for Fe3+ (59). Detection of Al3+ with AIE molecules was also explored. Some of us and coworkers reported TPE 17 (see Scheme 10) with an acetic acid group for the detection of Al3+ in vitro and in vivo (60). TPE 17 was weakly emissive in aqueous solution while it became strongly emissive after mixing with Al3+. As the results manifest, the binding of carboxyl group within TPE 17 with Al3+ induced the aggregation and therefore switched on the fluorescence. This probe enjoys high sensitivity for Al3+ with the detection limit of as low as 21.6 nM. Furthermore, it can distinguish Al3+ from other 15 metal ions (Ba2+, Fe2+, Ca2+, Mg2+, Cr3+, Li+, Zn2+, Hg2+, Pb2+, Na+, Ag+, Fe3+, K+, Ni2+, and Cu2+). Moreover, TPE 17 was also successfully applied to imaging and real-time monitoring Al3+ in living HeLa cells. In fact, besides detection of Al3+ fluorescence sensors for other trivalent metal cations have also caught much attention. Because these trivalent metal cations have their own biological significance and environmental importance. For example, Cr3+ is very important trace element in human nutrition. However, several in vitro studies indicate that high concentration Cr3+ in the cell can lead to DNA damage (61). Thus, the detection of other trivalent metal cations is also important. The pyridinyl-functionalized tetraphenylethene (TPE-PY, see Scheme 10) demonstrated colorimetric (blue to red) and ratiometric fluorescent responses to trivalent metal cations (Cr3+, Fe3+, Al3+) over a variety of mono and divalent metal cations (62). Mercury ions act as severe environmental pollutants, and several diseases are known to be associated with mercury contamination. Silver has numerous applications beyond currency, such as in solar panels, water filtration, jewelry and ornaments, etc. However, Hg2+ and Ag+ have some adverse biological effects, bioaccumulation and toxicity (63). Thus, development of sensitive and selective chemosensors for Ag+ and Hg2+ in various media is of considerable importance for environmental protection and human health. Based on the consideration that adenine and thymine show specific binding ability towards Ag+ and Hg2+, respectively, TPE 18 (see Scheme 10) with two adenine groups and TPE 19 with two thymine groups were appended, were investigated for sensing Ag+ and Hg2+, respectively (64). As illustrated in Scheme 11, the coordination of adenine and thymine motifs with Ag+ and Hg2+, respectively, will yield the respective coordination polymers which tend to aggregate due to the poor solubility; thus, the fluorescence from TPE framework is switched on. As shown in Figure 10, the TPE fluorescence increased gradually after addition Ag+ and Hg2+ to the solutions 108 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

of TPE 18 and TPE 19, respectively. Ag+ and Hg2+ with concentrations down to 0.34 μM and 0.37 μM, respectively, can be analyzed with TPE 18 and TPE 19.

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Scheme 11. Illustration of the design rationale for the fluorometric assay with TPE 18 and TPE 19 for Ag+ and Hg2+ based on the AIE feature. Reprinted with permission from Reference (64). Copyright 2008 The American Chemical Society.

Figure 10. (left) The fluorescence spectra of TPE 18 (5.60×10-5 M) in H2O/THF (5:1, v/v) in the presence of increasing amounts of AgClO4; (right) the fluorescence spectra of TPE 19 (1.34×10-4 M) in H2O/CH3CN (2:1, v/v) in the presence of increasing amounts of Hg(ClO4)2. Reprinted with permission from Reference (64). Copyright 2008 The American Chemical Society. Tang and coworkers employed TPE 20 (see Scheme 10) entailing benzothiazolium with iodide as counter anion for sensing Hg2+ (65). Due to intramolecular charge transfer (ICT) from electron-donating TPE group to electron-accepting benzothiazolium unit, a red emission was observed for TPE 20 either in the solution state or in the aggregation state, though their emission efficiencies were very low owing to the heavy atom effect of iodide. However, the red fluorescence was switched on upon addition Hg2+. This is ascribed to the formation of HgI2 which prevents the quenching effect of the iodide. Moreover, a solid film of TPE 20 was able to monitor the level of Hg2+ in aqueous solution with a detection limit of 1.0 μM. Lead is recognized as one of the most hazardous and poisonous metals to humans. However, it has been widely used in various industrial products and fields such as storage batteries, lead wires, paints, alloys, high quality glasses, soldering of electronic devices and foundries; thus the risk of Pb2+ entering into human bodies increases. Accumulation of Pb2+ in human bodies will induce a series of health problems such as hemotoxic effects, reproductive dysfunction, and nephropathies, etc. More dangerous is that mental retardation may happen when Pb2+ accumulates in children (66). Considering its adverse biological effect, development of sensors with high sensitivity and selectivity is crucial important. Chatterjee and coworkers reported TPE 21 for selective detection of Pb2+ ions (67). The -PO3H2 not only promotes the solubility of TPE 21 in the mixture of 109 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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water and THF, but also can bind Pb2+ strongly. The resulting coordination of Pb2+-complex owns poor solubility in the solvent, hence triggers the aggregation and the emission from the TPE framework is switched on. The limit of detection of TPE 21 for Pb2+ was estimated to be 10 ppb and no remarkable fluorescent response towards other metal ions (Ca2+, Fe2+, Mg2+, Ag+, Mn2+, Fe3+ etc.) was observed. Cyanide (CN-) is one of the most toxic anions and does great harm to human health as well as the environment. Cyanide tends to bind with iron species to form cytochrome-c oxidase which inhibits the electron-transport chain resulting in hypoxia (50). Consequently, sensitive and simple sensor for cyanide is highly desired. Some of us and coworkers developed a fluorescence turn-on sensor for cyanide assay in aqueous solution with the ensemble of silole 1 and a hydrophobic compound (2,2,2-trifluoro-N-(4-heptylphenyl)acetamide) that can reacts with cyanide to form an amphiphile with a negatively charged head group (68). As illustrated in Scheme 12, the amphiphile generated in situ will cause the aggregation of silole 1 via electrostatic and hydrophobic interactions, and as a result the fluorescence of silole 1 is turned on. The fluorescence of silole 1 increased after the addition of cyanide, and the limit of detection of silole 1 toward cyanide was estimated to be 7.74 μM. Meanwhile, no appreciable fluorescence enhancement was observed for silole 1 in the presence of other anions including OAc-, Br-, Cl-, F-, H2PO4-, HSO4-, N3- and NO3-.

Scheme 12. Illustration of the design rationale for the fluorometric assay with silole 1 for cyanide. Reprinted with permission from Reference (68). Copyright 2009 The American Chemical Society. Some of us further reported another fluorescence probe for cyanide with TPE 22 (see Scheme 10) containing an indolium moiety which renders its water solubility (69). As expected, TPE 22 exhibited rather weak fluorescence in aqueous solution. After addition of cyanide the fluorescence intensity of the solution increased gradually. Other anions could not induce such fluorescence enhancement. The selectivity of TPE 22 toward cyanide is owing to the reaction between cyanide and the indolium unit to generate a neutral compound which will aggregate in aqueous solution. Cyanide with concentration down to 91 nM 110 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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cyanide can be detected with TPE 22. Moreover, a paper test strip was established for sensing cyanide with TPE 22. Trogler and coworkers reported colloidal suspensions of silole 3 for selectively sensing carcinogenic chromium(VI) analyte (70). The fluorescence of these emissive particles was efficiently quenched by CrO42-. The authors suggest the electron transfer from the excited state of silole 3 to CrO42- is responsible for the fluorescence quenching. CrO42- with concentration as low as 0.1 ppm can be detected and common anions such as NO3-, NO2-, SO42- and ClO4- posed no interferences. As discovered by Atilgan and coworkers, pyridinium functionalized tetraphenylethene with iodide ion as a counter anion (TPE 23, see Scheme 10) can be used as highly selective senor for fluoride ion (71). TPE 23 shows negligible fluorescence in the red region owning to the heavy atom effect of iodide anion and good water solubility. However, addition of fluoride ion led to fluorescence enhancement with a blue shift at 470 nm. This can be understood as follows: i) the small size and electronegative character of fluoride ion leads to ion exchange between fluoride and iodide ion to generate a salt with poor solubility. As a result, aggregation occurs and the fluorescence emerges quickly; ii) the small size of the fluoride anion may give rise to more twisted configuration for the TPE framework in the aggregation state, leading to fluorescence blue-shift as reported previously (72). The results demonstrate that TPE 23 can be utilized for selective detection of fluoride ion with concentration down to 6.0 × 10−4 M.

5. Detection of Small Molecules Sensing H2S Hydrogen sulfide (H2S) is a crucial biological molecule with ill smell as the third recognized gasotransmitter besides nitric oxide (NO) and carbon monoxide (CO) (73). Endogenous sulfide shows critical functions in the cardiovascular, immune and nervous systems (74). Moreover, it is known that the dysfunction of H2S is related to various diseases such as Alzheimer’s disease and diabetes (75). Hence, detection of H2S is crucial for biological research and diagnose of diseases. Tang and coworkers developed a new sensing approach for H2S with TPE 24 (76), which was found to be non-emissive both in the solution and aggregate states. But, the azide group in TPE 24 (see Scheme 13) can be transformed into amine to form the corresponding amino-substituted TPE which can emit upon aggregation. Thus, TPE 24 is potentially useful for sensing H2S. After the solution of TPE 24 in DMSO-HEPES buffer was incubated with NaHS for short period, a commonly used hydrogen sulfide source, the fluorescence increased significantly. Interestingly, the detection threshold concentration of H2S was found to dependent on the concentration of TPE 24. The sensing of H2S with TPE 24 was not interfered by NO, biothiols and relevant anions.

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Scheme 13. Chemical structures of silole and TPE probes for sensing small molecules.

Sensing H2O2 It is well known that the reactive oxygen species (ROS), such as singlet oxygen (1O2), hydroxy radicals (.OH), superoxide anions (O2.-) and hydrogen peroxide, play rather important roles in biological processes. Irregular metabolism of ROS appears to a potential cause of cell damage (77). Among ROS hydrogen peroxide (H2O2) mostly exists in living organisms and its amount becomes a common indicator signaling oxidative stress (78). For these reasons various electrochemical and optical sensors for H2O2 have been developed. Some of us and coworkers described a H2O2 sensor with TPE 25 (see Scheme 13) (79). The sensing mechanism is illustrated in Scheme 14. The presence of pyridinium in TPE 25 renders it water soluble and thus TPE 25 is expected to be weakly fluorescent. The oxidation of phenylboronic pinacolester with H2O2, followed by hydrolysis and 1,6-elimination of p-quinone-methide, will induce the transformation of TPE 25 into the pyridine-substituted TPE. In comparison with TPE 25, the pyridine-substituted TPE is expected to show low solubility in aqueous solutions, thus aggregation will occur and turn on the fluorescence of TPE 25. Figure 11 shows the fluorescence spectra of TPE 25 after incubation with different concentration of H2O2. Clearly, the fluorescence intensity was enhanced by increasing the concentration of H2O2 and such fluorescent enhancement was easily observed by naked eyes under UV light irradiation (see the inset of Figure 11). H2O2 with concentration down to 180 nM can be detected with TPE 25. 112 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 14. The design mechanism for the fluorescence turn-on detection of H2O2. Reproduced by permission of Elsevier. Reprinted with permission from Reference (79). Copyright 2014 Elsevier.

Figure 11. Fluorescence spectra of TPE 25 (5.0 μM) after incubation with different amount of H2O2. Reprinted with permission from Reference (79). Copyright 2014 Elsevier. Alternatively, Liu and coworkers reported TPE 26 (see Scheme 13) with four L-tyrosine moieties for sensing H2O2 (80). Because of the presence of four Ltyrosine moieties, TPE 26 is water soluble and thus it is rather weakly fluorescent in aqueous solution. The L-tyrosine moieties in TPE 26 can be cross-linked in the presence of horseradish peroxidase (HRP) after addition of H2O2. Accordingly, aggregation of TPE 26 will take place and thus its fluorescence is lighted up. The fluorescence intensity of the ensemble of TPE 26 and HRP is initially weak, but it increased after addition of H2O2. Such fluorescence enhancement was not observed for TPE 26 in the presence of other ROS species. The results indicate the ensemble of TPE 26 and HRP can be utilized to detect H2O2 selectively and sensitively. Recently, Tang and coworkers have reported TPE 27 with two boronic pinacol ester groups for fast and selective detection of H2O2 (81). Sensing D-Glucose D-Glucose (Glu) plays a crucial role in biological process and it acts as the energetic spring of living cells. Abnormal levels of Glu indicates biological dysfunctions. Thus, molecular sensors for Glu have received continuous attentions and in fact various sensing systems have been invented for past several decades. Tang and coworkers developed a new sensor for Glu with TPE 28 (see Scheme 13) with two boronic acid groups (82). In the alkaline condition TPE 28 was soluble and thus it emitted hardly. After addition of Glu the fluorescence of TPE 28 increased gradually. Interestingly, TPE 28 kept weakly emissive 113 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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after incubation with either D-fructose (Fru) or D-galactose (Gal) or D-mannose (Man) under the same condition as for Glu. The selectivity of TPE 28 toward Glu is owing to the presence of two cis-diol units in Glu which can react with the two boronic acid groups in TPE 28 to generate rigid oligomers, as a result the fluorescence of TPE 28 is lighted up. In comparison, Fru, Gal and Man just contain one cis-diol unit and thus oligomerization reaction cannot occur. Some of us, Liu and coworkers successfully utilized sensors for H2O2 to detect Glu in the presence of glucose oxidase which can specifically oxidize Glu to generate H2O2 (79, 80, 83). For instance, some of us prepared an amphiphilic copolymer combining hydrophilic and hydrophobic blocks including TPE luminogen (83), which self-assembled into emissive polymeric micelles in aqueous solutions. The ensemble of such polymeric micelles, iodide and glucose oxidase can be used to sense Glu based on the following mechanism: Glu is oxidized by glucose oxidase to generate H2O2 which can further oxidize iodide to form I2 that can diffuse into the hydrophobic parts of micelles to quench the fluorescence.

Sensing L-Lactic acid L-Lactic acid (LA) is one of the most significant metabolites in clinical analysis and the food industry, and it is also an indicator for many diseases. It is known that LA is over-expressed under hyoxia, poor perfusion of tissue, acute circulatory shock, as well as liver damage (84). Thus, selective and sensitive detection of LA is highly desirable. Some of us constructed a new sensor for LA with silole 1 (85). The design rationale is illustrated in Scheme 15. Aggregation of silole 1 can be induced by amphiphiles with oppositely charged units. Such an amphiphile can be generated in situ by the cascade reactions among LA, lactate and dodecanoic hydrazine (DH). The fluorescence of the ensemble of silole 1, lactate and dodecanoic hydrazine (DH) increased after incubation with different amounts of LA as shown in Figure 12. LA with concentration down to 9.2 μM can be detected, and interferences from saccharides, amino acids, and ascorbic acid are negligible.

Scheme 15. Illustration of the design rationale for the fluorescence turn-on detection of LA by employing the AIE feature of silole. Reprinted with permission from Reference (85). Copyright 2012 The American Chemical Society. 114 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 12. Fluorescence spectra of the ensemble of silole 1 (50 µM) and DH (0.3 mM) containing LOD (0.25 U/mL) in the presence of different amounts of LA. Reprinted with permission from Reference (85). Copyright 2012 The American Chemical Society. Sensing CO2 CO2 nowadays draws wide attentions for its effect in various areas such as climate. The emission of CO2 contributes a considerable part to the global warming. Moreover, CO2 exists in many places like mines, rivers, and even in human body. Thus, to monitor the change of CO2 level becomes attractive. In 2010, Tang and his coworkers came up with a CO2 assay method with silole 4 in the presence of dipropylamine (DPA) (86). The sensing mechanism is based on the reaction between DPA and CO2, which yields a carbamate ionic liquid with high polarity and viscosity. The transformation of DPA into a polar and viscose medium will induce the aggregation of silole 4, resulting in the fluorescence enhancement. The results reveal that silole 4 and DPA can be used to detect CO2 and the sensing process is specific, quantitativeand interferent tolerant. Similarly, the ensemble of tetraphenylethylene, 1,8-diazabi-cyclo-[5,4,0]-undec-7-ene (DBU) and 5-amino-1-petanol was successfully utilized to detect CO2 (87). Sensing Biothiols Cellular thiols are essential biomolecules associated with various biological processes including antioxidant defense, cell signaling and cell proliferation (88). Cysteine (Cys), Homocysteine (Hcy) and glutathione (GSH) are the most common biothiols. Amounts of chemical or biological probes have been developed to detect these biothiols (89). However, new sensing methods for sensitive and selective detection of Cys, Hcy and GSH are still demanding. In particular, it is still challenging to discriminate between Cys and Hcy efficiently because of their structural similarity. Series of sensors for biothiols were constructed by taking advantage of the aggregation-induced emission feature of silole and TPE. For instance, Tang and coworkers reported silole 5 (see Scheme 13) with two aldehyde groups which were able to discriminate Cys, Hcy and GSH (90). silole 5 was weakly emissive in the H2O/DMSO medium, and it showed different fluorescence response after addition of Cys and Hcy. The addition of Cys led to fluorescence increment immediately and eventually reached a 13 fold enhancement after reaction for 100 minutes. However, the fluorescence of silole 5 remained almost unchangeable within hours after addition of Hcy, and only started to gradually 115 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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increase after three days. This may be explained by the different reaction kinetics for the aldehyde groups in silole 5 with Cys and Hcy to form the thiazinane and thiazolidine, respectively. The reaction product of silole 5 with Cys exhibited low solubility in H2O/DMSO medium and as a result its fluorescence was switched on. In comparison, GSH showed a significant quenching effect on the fluorescence of silole 5 owing to the good solubility of the resultant product in the medium. Therefore, silole 5 can be used to discriminate Cys, Hcy and GSH. Tang and his coworkers further developed TPE 29 (see Scheme 13) with two aldehyde groups. In comparison with silole 5, TPE 29 excelled as an AIE probe for Cys, Hcy and GSH in terms of faster response, higher fluorescence enhancement and sensitivity, and better specificity and selectivity (91). Tang and coworkers designed and synthesized TPE 30 (see Scheme 13) for selective sensing biothiols by considering the reaction of thiols with α, βunsaturated ketone (92). The nucphilic additions of Cys, Hcy and GSH with TPE 30 led to reaction products with different solubilities in acetonitrile/phosphate buffer, thus induced different fluorescence responses. The fluorescence of TPE 30 was shifted from yellow to blue and the intensity at 455 nm enhanced after addition of Hcy, whereas the fluorescence was very faint in the detection medium after reactions with either Hcy and GSH. The fluorescence intensity increased almost linearly with the concentration of Hcy in the range between 1.5 and 18.0 μM, and Hcy with concentration down to 0.3 μM could be detected with TPE 30. The blue-shift of the emission spectrum is understandable as the 1,4-addition reaction between Cys and TPE 30 results in the disruption of molecular conjugation. Such selective fluorescence response of TPE 30 toward Cys, Hcy and GSH can be attributed to the fact that Hcy is more hydrophobic than Cys and GSH, and the adduct product of TPE 30 and Hcy thus possesses poor solubility and forms aggregates in detection media. By employing similar strategy TPE 31 was examined for sensing biothiols (93). The emission color of TPE 31 was altered from red to intense blue after introducing Hcy to the buffer solution, whereas the buffer solution became weak blue after addition of Cys. Under the same condition, the red emission of TPE 31 was kept, but its intensity was slightly weakened after addition of GSH. All these changes in emission color and intensity can be easily distinguished by naked eyes. It is clear that TPE 31 shows high selectivity to Hcy over Cys, GSH and other amino acids in weakly basic buffer solution. Tang and coworkers also reported TPE 32 for distinguishing GSH from Cys and Hcy (94). The thiol-ene reaction of TPE 32 with Cys, Hcy and GSH can take place quickly in the ethanol/water medium. After introducing GSH to the solution of TPE 32 the emission was blue-shifted and the intensity was gradually enhanced. In comparison, no emission enhancement was observed after reaction of TPE 32 with either Cys or Hcy under the same condition. Such selective fluorescence response of TPE 32 toward GSH can be attributed to the hydrophilicity of GSH which causes the relevant adduct to be less hydrophobic and then easily aggregate in the ethanol/water solution. They further utilized TPE 32 for real time monitoring of glutathione reductase (GR) activity based on the ensemble of TPE 32 and GSSG (oxidative product of GSH). Moreover, TPE 32 was successfully applied to intracellular thiols recognition in the live HeLa cells. 116 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Liu and coworkers designed TPE 33 as a light-up probe for cell specific intracellular thiol imaging (95). TPE 33 (see Scheme 13) entails a targeted cRGD peptide, which endows TPE 33 with good water solubility. The selection of cRGD is based on the fact that cRGD exhibits high binding affinity towards αvβ3 integrin, as mentioned above. The TPE fluorogen and cRGD is linked with a thiol-specific cleavable disulfide linker. TPE 33 was found to be almost non-fluorescent in aqueous media. After reaction with GSH the disulfide bond was cleaved, the released TPE fluorogen aggregated, leading to fluorescence enhancement. Furthermore, TPE 33 was successfully utilized in intracellular thiol imaging with U87-MG human glioblastoma cell and MCF-7 breast cancer cell. They also reported TPE 34 for fluorescence turn-on detection of biothiols (96). TPE 34 entails a short peptides with five aspartic acid residues and as a result it becomes soluble in aqueous solution. The disulfide linker enables TPE 34 to be responsive to biothiols. The results reveal that TPE 34 is almost non-emissive in aqueous solution, but its emission is switched on after reaction with GSH.

6. Sensing Gamma-Ray Radiation Gamma-radiation is one kind of ionizing radiation which exists in many areas such as nuclear science, medicine, metallurgy, the food industry, and the environment (97). Gamma-radiation is known to be very hazardous to human health. Furthermore, the growing violence in terrorist attacks leads to greater awareness of the threat of nuclear and radiological terrorism (98). Therefore, it is highly desired and urgent to develop facile and easily-operated methods for sensitive and quick detection of gamma-radiation. Some of us and coworkers demonstrated the application of silole 1 for sensing gamma-radiation (99). The sensing was achieved with the polymer containing sulfone groups (–SO2–) in the main chain and –COONa groups in the side chain as shown in Scheme 16. The fluorescence of silole 1 was lighted up in the presence of this polymer owing to the electrostatic and hydrophobic interactions. But, the fluorescence intensity of the ensemble of silole 1 and the polymer decreased gradually after the polymer was exposed to gamma-radiation. Such fluorescence decrease was induced by the defragmentation of the polymer by release of SO2 upon exposure to gamma-ray radiation due to relative weakness of C–S bonds in the main chain; accordingly, the aggregates formed between silole 1 and the polymer were disassembled, leading to fluorescence decrease. Obviously, as shown in Figure 13, the fluorescence intensity of the ensemble solution decreased gradually after the solution of the polymer was exposed to increasing doses of gamma-ray radiation. Gamma-ray radiation as low as 0.13 kGy could be detected with the ensemble of silole 1 and the polymer.

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Scheme 16. Illustration of the design rationale for the fluorescence detection of gamma-ray radiation. Reprinted with permission from Reference (99). Copyright 2011 Royal Society of Chemistry.

Figure 13. Fluorescence spectrum (λex. = 370 nm) of the aqueous solution of Silole 1 (1.0 × 10 -5 M) and polymer (15 mgL-1) and those after exposure to different doses of gamma-ray radiation at room temperature. Reprinted with permission from Reference (99). Copyright 2011 Royal Society of Chemistry.

TPE 35 (see Scheme 17) was designed and synthesized for sensing gammaradiation (100). The design rationale is illustrated in Scheme 17 and explained as follows: i) TPE 35 with an indole moiety is expected to weakly emissive in organic solvents such as CHCl3 and CH2Cl2; ii) however, when the indole moiety is transformed into the indolium by protonation, the fluorescence of TPE 35 will be lighted up as protonated product (TPE 35-HCl) is anticipated to show low solubility in CHCl3 and CH2Cl2 and hence aggregation will occur. Furthermore, the emission will be red-shifted since the indolium within the protonated product is electron-withdrawing; iii) it is known that the halogenated solvents such as CHCl3 and CH2Cl2 are able to decompose into free radicals and recombine to yield HCl upon gamma-ray irradiation. As shown in Figure 14 the CHCl3 solution of TPE 35 became red-emissive and its intensity increased gradually after exposure to different dosages of gamma-radiation. Interestingly, the fluorescence intensity at 640 nm increased almost linearly with the dosage of gamma-rays in the range of 0.0 – 8.0 Gy. The limit of detection of TPE 35 for gamma-radiation was estimated to be 0.023 Gy. Thus, this facile and convenient fluorescence turn on detection of gamma-radiation based on compound TPE 35 is much more sensitive. 118 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 17. Chemical structures of TPE 35 and TPE 35-HCl and design rationale for the fluorescence turn-on detection of gamma-radiation. Reprinted with permission from Reference (100). Copyright 2015 Royal Society of Chemistry.

Figure 14. Fluorescence spectra of the CHCl3 solution of TPE 35 (3.0 mL, 10.0 μM) recorded at increasing dosages of gamma-rays. Reprinted with permission from Reference (100) . Copyright 2015 Royal Society of Chemistry.

7. Summary and Perspective In this chapter, new fluorometric chemo-/biosensors on the basis of aggregation-induced emission mechanism have been introduced and discussed. These sensors are constructed by manipulating the aggregation and deaggregation of silole and tetraphenylethylene molecules which exhibit aggregation-induced emission. The sensing modules include: i) silole and tetraphenylethylene molecules with charged groups interact with either biomacromolecules or amphiphilic molecules to form emissive aggregates which can be disassembled by enzyme-catalyzed reactions; ii) silole and tetraphenylethylene molecules with reactive groups which can react selectively with analytes to change the hydrophobic properties of the fluorophores; iii) silole and tetraphenylethylene molecules with functional groups which can lead to form oligomers or polymers through coordination with metal ions or chemical reactions. These new fluorometric sensors are unique in comparison with conventional ones, which are relied on photoinduced electron transfer, intramolecular charge transfer and fluorescence resonance energy transfer mechanisms, in terms of the following aspects: i) they are based on the aggregation and deaggregation mechanism; ii) most of these sensors can be carried out aqueous solutions which are highly desirable for biosensing; iii) the fluorescent probes based on silole and tetraphenylethylene are easily synthesized. 119 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Further improvements on sensing performances of these sensors are certainly necessary in order to apply these sensors for detection of interesting anlytes in real samples. This is particular true for detection of biomolecules in real biological samples. For this purpose, construction of fluorescent sensor arrays with AIE fluorophore deserves more investigations. This is because fluorescent sensor arrays with the assistance of statistical methods have shown strong discriminatory power in identifying chemically or structurally similar analytes from complex samples. Bioimaging has been reported with these AIE molecules, but the combination of sensing and imaging by using AIE molecules deserve further investigations. In addition to sensing, further exploration of functions of AIE molecules such as phototherapy and drug delivery are also appealing.

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Chapter 5

AIEgens-Functionalized Porous Materials for Explosives Detection Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch005

Dongdong Li and Jihong Yu* State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, P. R. China *E-mail: [email protected]

Explosives detection has become one of the current pressing concerns in global security. In the past decades, many chromophore-functionalized materials have been developed for the detection of explosives because they are more simple, sensitive, and cost-effective compared to other real time analytical methods. Aggregation-induced emission luminogens (AIEgens), a novel class of luminophores, show amplified sensing performance for the detection of explosives. In recent years, many AIEgens have been introduced into porous materials, including metal organic frameworks (MOFs), porous organic polymers (POPs) and mesoporous materials to constitute a new type of chromophore-functionalized porous materials. These materials with excellent photoluminescence emission properties and porous structure, exhibit a high sensitive detection performance to explosives. This chapter summarizes a wide range of AIEgens-functionalized porous materials and their sensing performance to explosives.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Introduction The reliable and accurate detection of explosives in groundwater or seawater has become increasingly important and urgent issue in modern society. So far, many real time analytical methods have been used for the detection and quantification of explosives, such as trained canine teams, gas chromatography, ion mobility spectrometry (IMS), surface-enhanced Raman spectroscopy, and so on (1). However, none of these methods is ideal for the detection of explosives due to certain features such as more complicated, lack of selectivity, high cost, and time-consuming. Fluorescence-based detection of explosives by harnessing organic dyes has drawn much more attention because they offer many benefits over other common detection techniques, such as good portability, high sensitivity, and selectivity (2). To date, various fluorescent chemosensors, including conjugated polymers, nanomaterials, and metal organic frameworks have been developed for the detection of nitroderivatives, especially, trinitrotoluene (TNT) and 2,4-dinitrotoluene (DNT), etc (3, 4). While traditional fluorescent dyes often suffer from the aggregation-caused quenching (ACQ) effect when dispersed in poor solvent or incorporated into solid matrix materials, resulting in drastically negative effects on the efficiency and sensitivity of the sensors. Aggregation-induced emission luminogens (AIEgens), which are nonemissive in their dilute solution, but luminesce intensively upon molecular aggregation, have drawn increasing research interest because of their striking turn-on fluorescence phenomenon (5). Restriction of intramolecular motion (RIM), including restricted intramolecular rotation (RIR) and restricted intramolecular vibration (RIV), is proposed as the main cause for their turn-on fluorescence phenomenon (6). Since AIE-active materials were found by Tang and coworkers, they have been widely used as efficient electroluminescent materials, sensitive chemosenors, and bioprobes, etc (7–10). Particularly, many AIEgens have been employed in the sensitive detection of nitroaromatics (11, 12). This is possibly because AIEgens are electron-rich molecules, which have Lewis acid-base interactions with electron acceptors nitroaromatic compounds. Furthermore, the AIE aggregate based sensors contain many cavities, which are suitable for the explosive molecules to enter and interact with the chromophores, making them quickly response to the explosives. Tang and coworkers synthesized a series of AIE-active 3D hyperbranched conjugated polymers, significantly increased the sensitivity and selectivity to picric acid (PA) detection (13–15). These results demonstrate that the pore structure plays an important role in explosives detection. Porous materials can be classified into inorganic open frameworks, such as zeolites and mesoporous materials, metal organic frameworks (MOFs), and porous organic polymers (POPs) according to the skeleton component (16). Their well-defined pore size, large surface area, high pore volume, and easily modifiable structures have drawn great interests of scientists. So far, many functional groups have been introduced into the porous materials and used in various fields such as catalysis, separation, gas storage, adsorption, and so on (17–19). In previous work, our group introduced AIEgens tetraphenylethene (TPE) into mesoporous materials and POPs via covalent bonds. The synthesized materials combine the 130 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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unique properties of the AIEgens and porous materials, demonstrating excellent performance in biomedicine and explosives detection. Many of these applications are built on the unique character that porous materials can rapidly associate analytes inside the pores via physical diffusion and/or chemical interaction. Other groups also designed and synthesized many AIEgens-functionalized porous materials, showing excellent performance in optical device and chemical sensing. In this chapter, we summarize the synthesis of AIEgens-functionalized porous materials (including MOFs, POPs, and mesoporous materials) to make the readers understand their structure easily, and further put forward their efficient sensing performance to explosives on the basis of structure. Figure 1 presents some representative explosive molecules that are detected by AIEgens-functionalized porous materials.

Figure 1. Chemical structures of the explosives and explosive-like substances. NB (nitrobenzene), NT (4-nitrotoluene), NP (4-nitrophenol), NBD (4-nitrobenzaldehyde), TNT (2,4,6-trinitrotoluene), PA (picric acid), DNCB (2,4-dinitrochlorobenzene), DNT (2,4-dinitrotolunene), 5-ATZ (5H-tetrazol-5-amine), NTO (5-nitro-2,4-dihydro-3H-1,2,4-triazole-3-one), HAT (3-hydrazinyl-4H-1,2,4-triazol-4-amine dihydrochloride), AT (4H-1,2,4-triazol-4-amine), DAT (4H-1,2,4-triazole-3,4-diamine hydrochloride).

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AIEgens-Functionalized MOFs for Explosives Detection Metal organic frameworks (MOFs), highly crystalline hybrid materials that combine metal ions with rigid organic ligands, have emerged as an important class of porous materials. Since they were discovered, MOFs have attracted considerable attention due to their facile preparation, tunable pore size, and easy functionalization of surfaces. So far, many MOFs materials have been widely used in gas storage and separation, heterogeneous catalysis, chemical sensing, optoelectronics, biomedicine, and so on (17, 20). Luminescent MOFs combine the merits of large internal surface areas and readable fluorescence signals, providing for sensing certain species, such as volatile organic compounds (VOCs), explosive compounds, and toxic metal ions (21, 22). However, the low fluorescence efficiency of many luminescent MOF-based sensors undermines the sensitivity greatly, which make them incapable for sensing applications. That’s because the commonly used organic luminescent ligands in MOFs often subject to emission quenching when assembled into coordination complexes.

Figure 2. Molecular structure of the organic luminescent ligands with AIE properties used in MOFs. AIEgens are exactly opposite to the commonly used organic luminescent ligands, showing high fluorescence quantum yields in the solid state. As a star molecule in AIE systems, tetraphenylethene, an intriguing chromophore featuring AIE characteristics, has emerged as a popular building block to construct luminescent MOFs. That’s because TPE can be easily decorated with carboxylate 132 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and pyridine coordination groups, which are thermally stable and water-stable. Up to now, many organic ligands based on TPE have been synthesized, as shown in Figure 2. These organic ligands can couple with metal ions to form a variety of luminescent MOFs, including layer-structured compounds and three-dimensional compounds. Dincã and coworkers synthesized the first MOFs materials based on TPE by the coordination of AIEgen 1 to Zn2+ and Cd2+ ions (Figure 3) (23). After anchoring TPE to metal ions within a rigid matrix, the rotation of the phenyl rings can be restricted, making the materials emit blue light centered at 480 and 455 nm. The Brunauer Emmett Teller (BET) surface areas of the materials are 317 and 244 m2/g, respectively, confirming that the porous structure can accommodate small guest molecules. Although the fluorescence quantum yields are only 1.0 and 1.8% for both compounds, they demonstrate the possibility by using AIE type chromophores to construct coordination assemblies with sustainable porosity. Further works will focus on improving the fluorescence quantum yields and extending their applications.

Figure 3. Portions of the X-ray crystal structures of Zn based MOF depicting (a) side and (b) top views of the two-dimensional sheets, and of Cd based MOF depicting (c) the Cd4 secondary building unit and (d) the truncated three dimensional structure. Reproduced with permission from Reference (23). Copyright 2011 American Chemical Society.

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On the basis of previous work, Zhou and coworkers employed new extended TPE-based AIEgen 2 and crystallized it with Zr(IV) to form a robust tetravalent zirconium MOF (Figure 4) (24). The twisted AIEgen 2 conformation of the crystals induced bright blue fluorescence emission at 470 nm. More importantly, the quantum yield of crystals is as high as unity (99.9 ± 0.5%) in the solid state, which is primarily attributed to the immobilization of the AIEgen 2 as it is strongly coordinated to Zr(IV), preventing the torsional relaxation. These rigidifying methodology and high fluorescence quantum yield of crystals making them suitable for potential applications in molecular electronics and/or sensor technologies.

Figure 4. The synthesis of MOF from TPE-based AIEgen 2 and Zr(IV). Insert show the photos of AIEgen 2 and MOF under UV light. Reproduced with permission from Reference (24). Copyright 2011 American Chemical Society. Besides these two MOFs materials, other compounds with similar organic ligands were also synthesized (25). Du and coworkers chose AIEgen 1 and crystallized it with Co(II) to form a compound with a (4,8)-connected scu framework (26). Magnetic studies reveal that this compound is paramagnetic with weaker anti-ferromagnetic coupling compared with normal syn-anti carboxylate magnetic pathway, which is due to the non-planarity of the Co–O–C–O–Co unit. Meanwhile, the same AIEgen 1 can also be incorporated with Zn(II) to form a zinc-based MOF, which maintains its fluorescence up to 350 °C, close to the decomposition temperature of AIEgen 1 (400 °C) (27). Notably, the high temperature fluorescence of this compound enables the selective detection of gaseous ammonia at 100 °C by causing fluorescence shifts in its emission maximum without the interference of ethylenediamine, N,N-diethylformamide, and water vapors. Many other AIEgens 3-6 based on TPE were also synthesized and incorporated with metal ions to form AIE-active MOFs (28, 29). To construct highly porous MOFs for applications, especially for gas storage, Zhou and coworkers designed dendritic AIEgen 3 (30). Then they synthesized robust and porous MOF containing AIEgen 3 and a Cu2-paddlewheel structural motif by using a Zn2-paddlewheel based MOF as a template to prearrange the linkers for the Cu2-based MOF target. The obtained materials show a type I isotherm for N2 134 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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sorption at 77 K and 1 bar, revealing the microporous nature of the framework. The Langmuir surface area and total pore volume of the compound are 2615 m2 g−1 and 0.94 cm3 g−1, respectively, showing excellent gas (H2, CO2) adsorption capacity. Tang and coworkers also reported 2D layered MOFs composed of Zn4O-like secondary building units and AIEgen 6 (31). Compared with the previously reported ligands, AIEgen 6 has two dangling phenyl rings without carboxylate groups that will remain unrestricted even after the formation of MOFs. So the synthesized compound exhibits responsive turn-on fluorescence to various VOCs, such as benzene, toluene, o-xylene, m-xylene, p-xylene, and mesitylene. That’s because the motion of phenyl rings can be restricted through molecular interactions with analytes, leading to responsive turn on emission. Although so many TPE-based luminescent MOFs have been reported, and some of them exhibit sensing abilities toward ammonia or VOCs, their applications in explosives detection are still rarely explored. As is known, many luminescent MOFs are excellent candidates for explosives detection. The pores in luminescent MOFs can promote efficient mass transport, thus enhancing the interactions of explosive molecules with chromophore. In addition, the combination of effective electron and energy transfers can significantly improve the sensitive and selective detection of explosives. As AIEgens are electron-rich molecules, the AIE-based luminescent MOFs can also show excellent performance in explosives detection. Zhao and coworkers prepared TPE-based porous MOF 1 comprised of the AIEgen 2 and Zr6 metal cluster SBUs (Figure 5) (32). Each AIEgen 2 links four parallel arranged zinc carboxylate bridge chain SBUs through shared carboxylate groups, forming a 3D framework with 1D rhombus pores. After undergoing a solvent exchange process with dichloromethane, followed by drying under vacuum at 50 °C, they obtained the activated 1 sample with micro-porosity. It is worth noting that the emission wavelength of the compound varies apparently when exposed to different VOCs, such as benzene, m-xylene, and mesitylene. More importantly, the photoluminescence (PL) of the compound can be quenched greatly when exposed to the vapors of NB and DNT for 1 week. Although most of the AIE-MOFs materials were not used for explosives detection and the SBUs performed not so efficiently in sensing nitroaromatic explosives, they demonstrate their great potential of sensing nitroaromatic explosives, which may inspire more researchers to do these research areas. We introduce the preparation of AIE-MOFs is to make the readers further understand the sensing performance from the structure aspect. Unlike the detection of some well known carbon-based explosives, the demand for a facile and sensitive detection method for five-membered-ring energetic heterocyclic compounds is also urgent. Because five-membered-ring heterocyclic compounds such as NTO is one of the new explosive compounds used in insensitive munitions (IM) developed to replace traditional explosives, TNT. IMs are designed to detonate when fired but not in response to unplanned stimuli, such as mechanical shock and high temperatures (33, 34). Wang and coworkers synthesized AIEgen 7 and made it coordinate with Mg2+, Ni2+, and Co2+ to produce three MOFs compounds (TABD-MOF-1, -2, and -3) with the fluorescent quantum yields of 38.5%, 1.12%, 0.15%, respectively (35). The obvious hypsochromic shifts can be attributed to the stronger ligand-to-metal charge 135 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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transfer (LMCT) effect by rational alteration of the metal ions. Remarkably, the TABD-MOF-3 can selectively sense the powerful explosive NTO with turn-on fluorescence and the minimum amount of NTO detectable by the naked eye is as low as 10 μL of a 1 × 10−6 M solution, corresponding to a visible detection limit of ca. 6.5 ng/cm2 (Figure 6). Furthermore, AT, DAT, HAT, and 5-ATZ, which are source materials for the synthesis of high energy-density materials, can cause remarkable emission turn on. The present new AIE-MOF sensing method shows advantages of universality, high sensitivity, and eases of visualization and may shed light on the development of new probes for turn-on chemo/biosensing applications.

Figure 5. (a) X-ray crystal structure of the MOF 1 with 1D rhombus channels; right: the 1D zinc carboxylate bridge chain. (b) PL spectra of 1, activated 1 and activated 1 with selected guest molecules, excited at 365 nm. (c) PL spectra of activated 1 before and after exposure to the vapors of NB and DNT. Reproduced with permission from Reference (32). Copyright 2015 Royal Society of Chemistry.

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Figure 6. (a) Photographs of TABD-MOF-3 deposited paper strips upon addition of THF solution of NTO at different concentrations under UV light. (b) Fluorescence spectra of TABD-MOF-3 in THF upon addition of NTO solution at different concentrations followed by addition of hexane. (c) Fluorescence enhancement efficiencies ((I−I0)/I0) obtained from different analytes by TABD-MOF-3. Excitation wavelength: 360 nm. Reproduced with permission from Reference (35). Copyright 2014 American Chemical Society.

AIEgens-Functionalized POPs for Explosives Detection Porous organic polymers (POPs) are a new generation of porous materials constructed from light elements, such as H, B, C, N, and O, which are linked by strong covalent bonds. So far, many POPs including covalent organic frameworks (COFs), polymers of intrinsic microporosity (PIMs), hyper-cross-linked polymers (HCPs), conjugated microporous polymers (CMPs), and porous aromatic frameworks (PAFs) have been developed (36). The excellent physical and chemical stability, low framework density, and various structural features have made them good candidates for gas storage and separation, catalysis, sensors, and so on (37). It is worth noting that the luminescent POPs have attracted much attention recently due to their promising applications in light harvesting, photocatalysis, sensing, and photovoltaic devices (38, 39). However, manipulation of strong light emission into POPs remains difficult due to the ACQ effect. So far, several POPs based on AIE building blocks have attracted much attention due to their potential applications in chemical sensing and bio-probes, owing to their enhanced emission in the aggregate form or solid state.

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Figure 7. Molecular structure of the building blocks with AIE properties used in POPs. Thanks to the efficient preparation, the propeller-like structure of TPE molecule is expected to be a novel building block for the design of porous materials with special properties (Figure 7). In 2011, Han and coworkers synthesized TPE-based POPs through a Suzuki coupling polycondensation and oxidative coupling polymerization (Figure 8) (40). All the POPs exhibit strong photoluminescence properties with the maximum peaks ranged from 530 to 610 nm. Their BET surface areas vary between 472 and 810 m2 g–1 and the pore widths are mainly centered at 0.58 or 0.67 nm, proving the existence of porous structure. This work demonstrates that AIEgens can be promising building blocks for designing porous polymers with special properties.

Figure 8. Preparation of POPs by Suzuki coupling polymerization and oxidative coupling polymerization. Reproduced with permission from Reference (40). Copyright 2011 Royal Society of Chemistry. 138 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 9. Schematic representation of the synthesis of conjugated polymers CMPs with core–shell architecture. Reproduced with permission from Reference (42). Copyright 2011 Royal Society of Chemistry. Since then, Jiang and coworkers developed highly luminescent CMPs with the same AIEgen 8. Due to the crosslinking nature of CMPs, it can suppress the rotation of TPE units, thus allowing for high luminescence in both solution and solid states (41). The absolute fluorescence quantum yield of the CMPs was as high as 40% when the Yamamoto reactions were carried out at 2 h. The microporous structure can be confirmed by high resolution tunneling electron microscopy (HR-TEM) and N2 sorption isotherm measurements. This work suggests that the CMP architecture provides a new platform for the design of highly luminescent materials. In addition, they also developed a core-shell strategy for achieving color-tunable and -controllable light emission while retaining high luminescence efficiency (Figure 9) (42). By fixing the core size and changing the shell thickness, they successfully tuned the light emission from deep blue to sky blue, near white, and green, and the fluorescence quantum yield reaches up to 32%. Many other AIEgens 10-12 based on TPE were also used to synthesize AIE-active POPs (43). Zheng and coworkers developed a straightforward, cost-effective, and environmentally-friendly method to prepare stable organic molecular cages based on AIEgen 10 (44). The compound exhibits a good CO2 uptake capacity of 12.5 wt% and a high selectivity for CO2 over N2 adsorption of 80 with the BET surface area of 432 m2/g. This study opens new opportunities for the development of efficient cage-based porous materials in gas storage, catalysis, and chemosensors. Jiang and coworkers synthesized a hyper-cross-linked CMP via Friedel–Crafts alkylation of AIEgen 11 and/or 1,1,2,2-tetraphenylethane-1,2-diol (TPD) using a formaldehyde dimethyl acetal crosslinker promoted by anhydrous FeCl3 (45). The materials also show high CO2/N2 selectivity with the increased TPD content, suggesting an efficient 139 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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strategy for the design of organic microporous polymers for post-combustion carbon capture. Besides the capability of gas storage, the TPE-based luminescent POPs also show excellent performance for the detection of explosive molecules. In 2010, Tang and coworkers developed a polymer with 3D globular structure, which offered more diffusion channels for the excitons to migrate, allowing them to be quickly annihilated by the PA quenchers (46). When the PA concentration is increased to 0.12 mM, virtually no light is emitted from the polymer nanoaggregates in the 90% aqueous mixture and the static quenching constants (K) can be up to 1.45×105 M–1. The superamplification effect in the explosive detection process can be attributed to the pore structure and the electron donor ability of the fluorescent polymers. As the POPs materials based on the AIEgen possess a 3D topological structure with molecular cavities or voids, allowing the efficient diffusion of explosives and very large band-gap energy with a deep-blue emission. The POPs are thus expected to serve as an excellent fluorescent sensor for the detection of electron-deficient quenchers such as nitroaromatic explosives.

Figure 10. Representative ideal molecular structures for luminescent POPs synthesized by reacted AIEgen 13 with potassium vinyltrifluoroborate. Reproduced with permission from Reference (47). Copyright 2014 Royal Society of Chemistry. Recently, our group reported a novel one-pot synthetic strategy for the straightforward preparation of luminescent POPs based on the palladium catalyzed tandem Suzuki–Heck C–C coupling reactions of AIEgen 13 with potassium vinyltrifluoroborate (Figure 10) (47). Their luminescence can be adjusted from blue to green by selecting the aromatic halides and alternating the ratio of monomers and their BET surface areas change from 318 to 693 cm2 140 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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g–1. It is found that the luminescences of POPs are quenched upon the addition of different analytes such as DNCB, DNT, NT, and PA. Particularly, the POPs exhibit relatively sensitive sensing ability towards PA than other compounds, and the emissions could be quenched quickly upon the addition of PA. The faster rate of fluorescence quenching can be explained by the photo-induced energy transfer quenching mechanism. Other luminescent microporous organic polymers were also developed by our group via the palladium catalyzed Suzuki-Heck cascade coupling reactions of 4-vinylphenylboronic acid with AIEgen 8 (48). The synthesized polymer exhibits strong yellow emission, which shows selective quenching toward PA compared with other nitroaromatic analytes. These excellent performances suggest that the AIEgens functionalized POPs can be used as potential chemical sensor for explosives. Apart from these porous polymers containing only C, H elements, many other kinds of POPs have also been synthesized and are used for the detection of explosives. Octavinylsilsesquioxane was also used as building unit to react with AIEgen 8 to construct a class of luminescent porous inorganic-organic hybrid polymers though the Heck coupling reaction (Figure 11) (49). The BET surface areas can be up to 685 m2 g−1. The polymer shows poor abilities in sensing DNCB, DNT, NT, and NP, but has sensitive quenching behavior toward PA. The overlap of the PA absorption spectrum with the emission spectra of polymer indicates that there exists energy transfer process between them, which can be used to explain the reason of the rapid fluorescence quenching.

Figure 11. Syntheses of luminescent porous inorganic-organic hybrid polymers by the Heck coupling reaction of AIEgen 8 with octavinylsilsesquioxane, and their luminescence in ethanol (0.05 mg mL−1) in the presence of various amounts of PA. Reproduced with permission from Reference (49). Copyright 2015 Royal Society of Chemistry. 141 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Fluorescent cross-linked polymer with strong blue emission was prepared efficiently through a one-step polycondensation AIEgen 14 with cyclophosphazene by Han and coworkers (Figure 12) (50). Its fluorescence can be quenched by both the nitroaromatics TNT and PA significantly. The fluorescence quenching detection to PA is more sensitive than to TNT in the suspension or solid-state. That is because a spectral overlap occurs between the emission of polymer and PA absorption in wavelength ranging from 350 to 480 nm which prompts the energy transfer from the excited state of the polymer to the ground state of PA, resulting in the efficient fluorescence quenching.

Figure 12. Representative conjugated microporous organic polymer constructed from AIEgen 14, and their photos in the absence (a), in the presence of TNT (100 ppm) (b) and PA (50 ppm) (c) under UV light (365 nm) illumination. Reproduced with permission from Reference (50). Copyright 2011 Royal Society of Chemistry.

Hydrogen-bondings have great importance in life, such as in the double helix of DNA and stabilization of secondary structure of protein. Chen and coworkers designed and fabricated a fluorescent hydrogen-bonded organic framework HOF-1111 by using AIEgen 15 as building block (Figure 13) (51). The obtained materials showed high thermal stability and 3D structure, which can be used for sensing of aromatic compounds via a fluorescence quenching and enhancement mechanism. NB showed the highest quenching efficiency of 73%, while the quenching efficiency values of other analytes such as NP, NBO and DNCB were less than 60%. The highest quenching efficiency of NB can be attributed to not only the electron with drawing ability but also the high vapor pressure. Furthermore, fluorescence enhancement was observed when using benzene (BE), toluene (TO), para-xylene (PX), and trimethylbenzene (TM) as the detected objects due to their good electron-donating ability and relatively high vapors pressure. 142 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 13. (a) Local hydrogen bonding environments of AIEgen 15. (b) Quenching efficiency of HOF-1111 exposed to explosives. (c) Fluorescence enhancement of HOF-1111 exposed to aromatic compounds containing electron-donating groups. Reproduced with permission from Reference (51). Copyright 2015 American Chemical Society.

AIEgens-Functionalized Mesoporous Materials for Explosives Detection Mesoporous materials are important porous materials with pore diameters in the range of 2-50 nm. Since they were discovered in 1990s, synthesis and applications of mesoporous materials have received intensive attention because of their highly ordered structures, larger pore size, and high surface areas (52–54). So far, many mesoporous materials have been widely used in various fields such as separation, catalysis, sensors, and devices (18). Chromophore-functionalized mesoporous materials can potentially serve as a new generation of fluorescent chemo-/bio-sensor with remarkable sensitivity (55). That is because the pore size of mesoporous materials is large enough, which can rapidly associate analytes and drugs inside the pores via physical diffusion and/or chemical interaction. Thus the adsorbed molecules in the pores can interact efficiently with fluorophores in/on the pore wall, leading to dramatically enhanced sensing performance. So far, many AIEgens-functionalized mesoporous materials combining the unique properties of mesoporous materials and AIEgens have been developed. Our group developed the first AIEgen-functionalized mesoporous silica by post grafting AIEgen 16 on SBA-15 (56). The synthesized materials combine the unique properties of the AIEgen and porous materials, proving to be an excellent fluorescence probe for potential applications in drug delivery. In addition, we also introduced AIEgen 17 into bioactive hydroxyapatite (HAp) via co-condensation approach to form AIEgen-functionalized HAp (MHAp-FL) (57) (Figure 14). The PL intensity of the materials varies greatly with the loading and release of drugs ibuprofen (IBU), suggesting that the drug release process can be tracked 143 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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in terms of the change of PL intensity (Figure 15). These results suggest that this multifunctional material may be used as an excellent drug carrier in future bioapplications.

Figure 14. Molecular structure of the building blocks with AIE properties used in mesoporous materials.

Figure 15. (a) Fluorescence spectra of MHAp-FL with IBU released at different percentages. (b) The plot of PL intensity as a function of cumulative release amount of IBU. Reproduced with permission from Reference (57). Copyright 2013 Royal Society of Chemistry. AIEgens-functionalized mesoporous materials with other emission colors have also been synthesized for biomedicine. Shi and coworkers synthesized a novel type of green fluorescent mesoporous silica nanoparticles by hybridizing mesoporous silica nanospheres with AIEgen 18 (58); Tian and coworkers fabricated a novel type of folate-functionalized yellow fluorescent mesoporous silica nanoparticles with AIEgen 19 as core (59); Wei and coworkers used the AIEgen similar to 19 and cationic surfactant cetyltrimethyl ammonium as structure-directed template to fabricate uniform yellow luminescent mesoporous silica nanoparticles (60, 61). All these mesoporous materials show excellent performance in cell imaging or drug delivery, suggesting their potential applications in biomedicine. In addition, our group also demonstrated a strategy to integrate AIE and ACQ chromophores in periodic mesoporous organosilicas (PMOs) for high-efficiency multicolor emission (62). The high-quality white light can be obtained by fine tuning of ACQ dyes and AIE-PMOs and the quantum 144 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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yield is up to 49.6%. These high-performance multicolor luminescent materials can be applied in solid-state lighting, biomedicine, and other areas. It is expected that the explosive molecules can transfer into the mesopores more easily and interact with the chromophores, which may lead to dramatically enhanced sensing performance. Our group attempted to apply AIEgen-functionalized mesoporous SBA-15 for the detection of explosives (Figure 16) (63). The PL intensity of materials decreases significantly with the increasing loading amount of PA. The PL quenching can be clearly discerned at a level as low as 1.7 µM or 0.4 ppm and the quenching constants can be up to 2.5 × 105 M–1, much higher than that of TPE itself in the THF/water mixture (1: 9 v v–1) of 3.4 × 104 M–1, as well as those of other linear conjugated polysiloles reported in the literature (2 × 104 M–1) (64). The rapid fluorescence quenching response can be explained by the photo-induced electron transfer and/or energy transfer quenching mechanism. Importantly, this probe is recyclable after washing with proper solvents, thus proving to be a promising candidate for practical explosive detection in an environmentally friendly manner.

Figure 16. Reversible fluorescence quenching mechanism of TPE-functionalized mesoporous materials with PA based on photo-induced electron transfer and/or energy transfer. Reproduced with permission from Reference (63). Copyright 2012 Royal Society of Chemistry. However, the bulk solid materials such as SBA-15 cannot guarantee better dispersibility in water as a result of large size, which may limit their further applications in chemical detection. Mesoporous silica nanoparticles show excellent dispersibility in water solution. So we further investigated the detection capacity of AIEgens-functionalized mesoporous silica nanoparticles with pore diameters of 2.4 nm for explosives in water (Figure 17) (65). The PL intensity decreases significantly with the increasing loading amount of NB, NT and PA, and the detection limits are 0.43, 0.77, and 1.11 ppm, respectively. It is noting that the sensitivity of materials to the explosives show the sequence of NB > NT > PA. That’s because the addition of an electron-donating –CH3 group will decrease the electron-withdrawing ability of the nitro group and the larger molecular size of PA is 7.8 Å, which may limit its quick diffusion into the pores and adsorption around the TPE fluorophores. Whereas the smaller molecules NB (4.6 Å) and NT (4.6 Å) can access to the pores more easily, leading to higher quenching constants. These results suggest that the large pores of the materials play an important role in the explosives detection. We further prepared mesoporous silica nanoparticles with the pore size of 5.7 nm and used them for the sensing of explosive vapors. 145 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 17. Molecular modeling of PA, NT and NB using Materials Studio program. Reproduced with permission from Reference (65). Copyright 2014 Elsevier Ltd. We modified AIEgen 20 to mesoporous silica nanoparticles via carbonnitrogen double bond and then fabricated them into a film by dip-coating method (66). The fluorescence of the film is quenched significantly by nearly 40% in 2 s and 62% in 10 s, respectively, after exposing to DNT vapors (ca. 100 ppb), as shown in Figure 18. The fluorescence quenching almost reach a balance after 30 s with the fluorescence quenching efficiency up to 74%. The large pore size of the nanoparticles is favored for the DNT molecules to be transported into the pores quickly and effectively adsorbed around fluorophores 20 by the formation of a DNT-amine complex via acid-base interaction. The close vicinity between 20 and DNT greatly facilitates the electron transfer process and increases the chemosensory efficiency, thus enhancing the fluorescence quenching efficiency.

Figure 18. (a) Time-dependent fluorescence spectra of AIEgen-functionalized mesoporous silica nanoparticles film in DNT saturated vapor. (b) Time-course of fluorescence quenching efficiency of AIEgen-functionalized mesoporous silica nanoparticles film exposed to DNT vapor; the intensity is monitored at 485 nm. Reproduced with permission from Reference (66). Copyright 2015 Royal Society of Chemistry. 146 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Summary As a class of nonconventional fluorescent materials, AIEgens have aroused great attention because of their emission turn-on nature, instead of quenching. A number of AIE molecules have been used for the fabrication of AIEgens-based porous materials including MOFs, POPs, and mesoporous materials. The obtained materials combined the strong luminescence of the AIEgens and high pore volume and large surface areas of porous materials, providing potential applications in solid-state lighting, biomedicine, gas storage, and chemosensing. Particularly, these AIEgens-functionalized porous materials show sensitive and selective detection of explosives compared to other probing methods. The reasons can be explained that the fluorescent materials have proven to be excellent candidate for the rapid detection of explosives in virtue of the high sensibility, simplicity, short response time, and the ability to be applied in both solution and solid phase. Particularly, the AIEgens are electron-rich molecules, which have Lewis acid-base interactions with electron acceptors of nitroaromatic compounds, improving the capacity of photo-induced electron and/or energy transfer. AIEgens-functionalized porous materials show highly efficient fluorescence in the solid state, avoiding the interference of noise. Furthermore, the large pores and ordered structure of porous materials allow the explosive molecules to enter and interact with the chromophores, leading to high rapid fluorescence quenching response to the explosives. Thus, AIEgens-functionalized porous materials usually have superamplification effect in the detection process, showing a promising application in sensoring of explosives. However, so far most of the AIEgens-functionalized porous materials have been focused on the detection of PA in solution. The highly efficient detection of TNT or other explosives vapors from packed bombs or landmines under real circumstances is of great significance in national defence security. In addition, the fluorescence quenching is still the main method in fluorescence-based explosives detection. Other phenomena, such as the fluorescence enhancement or change of luminescence color in the presence of analytes, would be a more desirable method to improve the sensitivity of detection. It is noting that the fluorescence of the materials may be quenched by other electron-deficient compounds except nitrated explosives. Developing a fluorescent sensor that can selectively detect and differentiate different explosives is also highly desirable. As a kind of novel material, AIEgens-functionalized porous materials provide a good choice to solve these challenges, showing a promising and bright future.

Acknowledgments This work is supported by the State Basic Research Project of China (Grant No: 2014CB931802) and the National Natural Science Foundation of China (Grant Nos.: 21320102001 and 21501063).

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Chapter 6

Liquid Crystalline AIE Luminogens: Properties and Applications Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch006

Dongyu Zhao* Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of the Ministry of Education, School of Chemistry and Environment, Beihang University, Beijing 100191, P. R. China *E-mail: [email protected]

Aggregation-Induced Emission (AIE)-active liquid crystals (LCs) is a charming research area. Melding the advantages of AIE and LC, AIE-active LCs not only solve the annoying fluorescence quench naturally accompanying the formation of a mesophase but also exhibit a lot of distinguishing features. Despite the promising prospects of AIE-active LCs, the relative research is still rarely reported since the requirements for the LC and AIE characteristic are hard to fulfill simultaneously in one molecule. In this chapter, we summarized the state-of-the-art development of AIE LCs based on different chromphore cores, including silole, tetraphenylethylene and other AIEgen. The relationship between phase behavior and luminescence properties, as well as the aggregate state of the mesophase and luminescence properties are emphatically discussed. The versatile applications of this composite luminescent liquid crystal material are also introduced.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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1. Introduction Luminescent liquid crystals (LE-LCs) has drawn growing attention because of the basic phenomenological interest and attractive technological applications such as anisotropic light-emitting diodes (1), polarized organic lasers (2), light-emitting liquid-crystal displays (LCDs) (3, 4), information storage (5), sensors (6) and one-dimensional semiconductors (7). The combination of intrinsic light-emitting properties with its unique self-organizing features endows the LE-LCs plenty of novel advantages. For example, LE-LCs can be used to manufacture stimuli-responsive luminescent materials owing to the sensitive response of LCs to external force stimuli, such as grinding, smearing and pressing. Among the variety of applications of LE-LCs, the linearly or circularly polarized luminescence is of especial importance for construction of highly efficient emissive LCDs. As an emissive display, the luminescent LCD fabricated from LE-LCs may enjoy quite a few advantageous features. For instance, the power-consuming extra backlight, polarizers and color filters would be obviated and thus enhance the power efficiency remarkably. Moreover, the LE-LCs allows simpler device design and hence device brightness, efficiency, contrast ratio and viewing angle of LCDs would be substantially increased (8). Despite these thrilling prospects of LE-LCs, achieving LCs with light emission characteristic remains a big challenge due to the aggregation caused emission quenching (ACQ) for traditional organic luminophores. Similarly, chromophoric mesogens are usually composed of rigid conjugated segments to self-assemble into LC phases. However, the dense packing of these rigid units probably suffer the ACQ, leading to the quench of fluorescence. Therefore, avoidance of the quench of the LC materials in aggregate state becomes a primary issue. Moreover, the efficiency of luminescence in the liquid crystalline phase remains in question, since aggregation or self-organization is a natural process in forming a mesophase which will also suffer the fluorescence quench. Aggregation-induced emission (AIE) is a fantastic photophysical phenomenon (9, 10) proposed by Tang et al. in 2001. In sharp contrast to annoying ACQ effect (11), AIE molecules usually exhibit brightly emission in the aggregate or solid state, which can be explained by the restriction of intramolecular motion (RIM) (12, 13) mechanism. Therefore, the AIEgens are expected to be promising candidates for developing LE-LCs. Up to now, a number of efforts have been made to develop AIE-based LE-LCs. Scientists incorporated different AIE cores with mesogens, leading to new AIE molecules with preservation of their mesomorphic properties (14–26). Conventional AIE luminogens (Scheme 1) such as silole, tetraphenylethylene (TPE), and cyanostilbene are used to construct the AIE-LCs. Some unusual AIE core are also explored to develop the AIE-LCs. The peripheral flexible side chains including alkyl and alkoxy chain are often introduced into the AIE-LC molecules as pendants. In this chapter, the state-of-the-art development of AIE-LCs is introduced, including their phase types, structure-property relationship, and their technological applications.

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Scheme 1. The molecule structure of representative AIE molecules.

2. Materials and Methods 2.1. Silole-Based AIE-LCs Since the rigid group and flexible group are two indispensable parts of a LC molecule, we try to summarize the LC from the point of rigid group, which is often also the luminescent unit in the molecule. Usually, the molecules delivering pronounced AIE effect possess propeller-shaped structures. As a well-known AIE core, silole with the non-coplanar geometry was used to construct the AIE-LC molecules. Wan et al. (14) described the preparation of two new AIE-LCs based on silole containing amide units and alkoxy chains. As homologues, these two silole-based compounds could be prepared in the same way with three procedures through a key intermediate compound, 2,5-bis(4-amidophenyl)-3,4,-diphenylsilole, accompanied with a coupling reaction (Figure 1A). Although containing a non-planar structure, the twisted tetraphenylsiole cores self-assembled into liquid-crystalline phases over a wide temperature range with the aid of the amide units and the alkoxy chains. In the DSC curves, both 1a and 1b showed two phase transitions during the heating and cooling processes, indicating undoubtedly these are two enantiotropic LCs. Moreover, increasing the aliphatic side-chain length of the tetraphenylsilole derivatives from dodecyloxy to hexadecyloxy lowered the melting point and clearing point, and the temperature range of the LC phase also decreased (146 °C for 1a, 122 °C for 1b). POM of 1a and 1b also showed anisotropic textures. As shown in Figure 1B, the textures of compound 1a obtained at 112.4 °C and 60.6 °C belong to smectic mesophase; however, the texture of 1b at 127.2 C and 67.6°C showed that compound 1b is an atypical LC molecule, indicating that the mesomorphic packing arrangements of LCs are sensitive to the length of the substituted alkoxy chains. In addition to the mesospheric properties, the compound 1a can thermo-reversibly form stable organogel in cyclohexane. And the emission of gel was much stronger than that of the solution, almost 100 times as much as which (Figure 1C, up). The drastic enhancement of fluorescence intensity after gelation could be observed by the naked eye (Figure 1C, down) and be explained by the 153 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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RIM mechanism. After aggregation, the intramolecular rotation of the phenyl substituents was hindered which blocked the non-radiative decay and resulted in the stronger PL intensity in the gel state. This gelation will induce diverse ordered aggregates which may have potential applications in explosive and ion detections.

Figure 1. (A) The molecule structure of LE-LC compounds 1a and 1b. (B) Optical micrographs of compounds 1a and 1b between crossed polarizers: liquid crystalline texture for 1a at 112.4 °C and 60.6 °C and 1b at 127.2 °C and 67.6 °C. (C, top) Fluorescence spectra of 1a in gel state and CH2Cl2 solution with the same concentration (14 mg/mL); photographic images of corresponding gel and solution observed upon UV irradiation at 365 nm (C, bottom). (Reprinted with permission from ref. (14). Copyright 2010 Royal Society of Chemistry.) 2.2. TPE-Based AIE-LCs TPE is another typical AIE luminogen. The advantages of TPE such as facile synthesis, high solid-sate efficiency, and versatile functionalization approaches make it an ideal candidate for constructing novel functional AIEgens. To date, several research groups have reported a few LC examples carrying TPE units. In general, four mesogenic pendants are usually needed to link with TPE to form the LC molecule. Yuan W Z et al. (15) reported the synthesis and characteristic of TPE4Me. As shown in Scheme 2, TPE4Me was prepared through a nucleophilic substitution and followed by a McMurry coupling, affording AIEgen-mesogens with both luminogenic and mesogenic properties (Scheme 2). In the THF/water mixture, TPE4Me demonstrated typical AIE-active features. At water fractions (fw) ~20% level, weak PL signals were recorded while in 90% aqueous mixture, the PL intensities at 380 and 450 nm increased by about 16 and 42 times (Figure 2A, 2B), respectively, attributed to the aggregation of luminogenic TPE4Me molecules. In the solid state, the absolute ΦF was as high as 67.4 ±5.0%. Upon cooling form the isotropic state, two anisotropic 154 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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mesomorphic textures of fan-shaped texture and focal conic texture emerged at 190°C and 100°C, respectively, as recorded by POM (Figure 2C, 2D). Combined with the DSC trace, it can be concluded that TPE4Me exhibited two mesomphoric phases and the phase transitions at 194.2°C and 140.8°C were associated to the isotropic–LC and LC–LC transitions, respectively. As suggested by WXRD analysis, although the disc-like TPE cores are twisted in conformation, the central TPE cores are still capable of packing together to generate face-on aligned columns. In the meantime, the peripheral mesogens self-assemble into a tetragonal smectic building blocks which were orthogonal to the TPE columns, resulting in a unique biaxially oriented mesomorphic structure, as shown in Figure 2E-2G.

Scheme 2. Synthetic route of TPE4Me. (Reprinted with permission from ref. (15). Copyright 2012 Royal Society of Chemistry.) Another kind of propeller-shaped TPE derivatives were prepared (16), which contain a TPE core and multiple long alkoxyl chains, linked by four 1,2,3-triazolyl groups between the TPE unit and substituted phenyl rings through click reaction (Figure 3A). As molecular design, the triazolyl group with polar character was expected to reinforce the microphase separation and stable LC phases would hence be produced. Since the space-filling of alkyl chains could affect the packing of propeller-shaped mesogens, the number of peripheral dodecyl chains was altered from eight in TPE-LC1 to 12 in TPE-LC2 to manipulate the packing structure of the propeller-like mesogens. The thermal properties of TPE-LC1 and TPE-LC2 were studied with the conventional methods of DSC analysis and POM observation. In the DSC curve of TPE-LC1, two reversible first-order transitions were observed during both the heating and the cooling scan, suggesting it was an entropic liquid crystal. Such a thermal behavior of TPE-LC1 was also verified by POM photographs obtained in the cooling process. Upon cooling from the isotropic state to 181 °C, the LC phase displayed a typical fan-like texture associated with the columnar phase of discotic LCs at 181 °C (Figure 3B). When it was continuously cooled, the second LC phase was attained at 159 °C and the fanlike texture remained unchanged till to room temperature except the texture color due to the variation of the sample thickness by temperature change (Figure 3C) , implying that there was significant structural change with the LC-to-LC transition. In comparison with TPE-LC1 possessing eight dodecyl chains, TPE-LC2 with twelve dodecyl chains showed only one LC phase with a fanlike texture that is an indicator of the formation of a columnar phase (Figure 3D). 155 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 2. (A) PL spectra of TPE4Mes in THF and THF/water mixtures; (B) Plots of the emission intensities at 450 and 380 nm versus water fractions (fw) for TPE4Mes in THF and THF/water mixtures. Concentration =4 mM. Excitation wavelength=310 nm. The inset graph is TPE4Mes in THF and 10/90 THF/water mixture taken under UV illumination. (C) and (D): POM images recorded on cooling TPE4Me to (C) 190 and (D) 100 °C from its isotropic state. (E)-(G): Schematic illustration of the biaxially oriented packing model of TPE4Me in the low temperature phase. (E) side view perpendicular to the side-chain mesogens, (F) top view, and (G) side view along the side- chain mesogens of the model. (Reprinted with permission from ref. (15). Copyright 2012 Royal Society of Chemistry.)

Owing to intrinsic AIE features, the propeller-like aromatic mesogens TPE-LC1 and TPE-LC2 exhibited their respective thermochromic behaviors upon heating and the emission color changes were associated with the conformational variations. Under 365 nm UV light irradiation, the emission color of TPE-LC1 was sky blue in the rectangular columnar (Colrec) phase, at a temperature of 170 °C, the color change to emerald which should be correlated with the hexagonal columnar (Colhex) phase (Figure 4A). Further rising the temperature, the emission turned to be dimmer. For TPE-LC2, with the temperature increasing, the fluorescence color exhibited a continuous change from sky blue to green in the Colhex phase, accompanied with a fluorescence decrease. The temperature-variable small and wide angle X-ray scattering methods were employed to study the microstructures of the propeller-like mesogens. The results revealed that unusual intercolumnar transformation and the packing dependency of the fluorescence property occurred in TPE-LC1 (Figure 4B-4D). The transition from Colrec to Colhex phase was conformed to first-order transition rather than the “second-order” transitions that usually occurs in discotic or polycatenar LCs based on a common tilt mechanism (Figure 4B). At the same time, TPE-LC1 adopted an unprecedented zigzag stacking in the Colrec phase. The fluorescence color change from sky blue to green was thus reversibly observed during the LC transition to Colhex in TPE-LC1, due to the planarization of the propeller mesogen. Taking full advantage of this reversible color change, some stimuli-responsive materials could be rationally prepared. 156 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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From AIE behaviors and mesophoric properties of luminescent compounds TPE-LC1 and TPE-LC2, we can make a conclusion that the peripheral flexible side chains play an important role on the phase transition and the type of mesophase, which will in turn lead to the different emission wavelength due to different stacking fashions of mesophases. Tang group also developed another TPE-functionalized molecule, named TPE-PPE that behaved as an AIE-LC (17). Conjugating four mesogenic units to the TPE core (Figure 5A), TPE-PPE was simply prepared through the Sonogashira coupling reaction between a brominated TPE and 4-ethynylpropylbenzene. From POM observation, an atypical anisotropic texture emerged during the heating scan (Figure 5B) and the mesophase appeared within the range from 218 to 228 °C was identified as sematic LC by XRD analysis. Attributed to the TPE unit, TPE-PPE exhibited evident AIE activity in the THF/water mixture: it is almost nonluminescent in pure THF while addition of water into its THF solution intensively enhanced its light emission, exhibiting a bright emission with its maximum at 530 nm.

Figure 3. (A) Molecular structures of TPE-LC1 and TPE-LC2 with the propeller-like TPE unit. The double-ended arrow indicates the torsional variation by the rotation of the phenyl rotor. Temperatures are given in °C. The values in parentheses are the enthalpy change (kJmol-1) of each transition. Optical textures of (B) TPE-LC1 at 181 °C, (C) TPE-LC1 at 159 °C, and (D) TPE-LC2 at 131 °C. Colrec, rectangular columnar; Colhex, hexagonal columnar; I, isotropic liquid phase. (Reprinted with permission from ref. (16). Copyright 2014 John Wiley and Sons.)

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By dissolving 0.1 wt% of TPE-PPE into the nematic LC host, the linearly polarized emission was obtained on the unidirectional orientated LC cell. Firstly, for the nonpolarized emission, it was found that photoluminescence of the LC cell with 1.0 wt% concentration TPE-PPE was stronger than those of 0.5 wt% and 0.1 wt%, showing the evident AIE behavior (18). Second, the polarized fluorescence did not depend on the concentration of the luminescent dye. The photoluminescence polarization ratio of the LC cell reached to 4.16 in the directions perpendicular and parallel to the azimuthal direction. Utilizing the emissive anisotropy of TPE-PPE, two kinds of photoluminescent liquid crystal displays (PL-LCD) were fabricated with patterned ITO glass substrates and only one polarizer. For the first device, at the electric field-off state, the device emitted no light under UV illumination. However, when an electric field was applied on the device, the letters of ‘CDR HKUST’ with yellowish-green color were clearly seen. The off/on performance of this LE-LC devices could be easily switched by an electric field (Figure 6A). Moreover, the second PL-LCD based on the photoalignment technology has potential application in anti-counterfeiting (Figure 6B). This approach simplified device design, lowered the energy consumption and increased brightness of the LCD.

Figure 4. (A) Emission color change of TPE-LC1 as a function of temperature under 365 nm UV light at a heating rate of 10 °C min−1. (B) Models of “tilt” and “zigzag” stackings. (C) The molecular organization in the zigzag stacking of the Colrec phase of TPE-LC1 and (D) schematics for the variation in the degree of interdigitation between propeller-like mesogens at the intercolumnar transition. (Reprinted with permission from ref. (16). Copyright 2014 John Wiley and Sons.) 158 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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In addition to the linearly polarized emission, circularly polarized luminescence based on AIE-active TPE-PPE was attained (19). By dissolving TPE-PPE into chiral nematic LCs (N*LC), luminescent N*LC containing selective light reflection and circularly polarized luminescence could be attained. Like the TPE-PPE/N-LC system, the concentration of TPE-PPE had no influence on the CPL degree of the LC composites but was essential for the emission intensity, owing to the AIE effect in the system. Based on the selective reflection and CPL behaviors of the TPE-PPE/N*LC composite, a reflective-luminescent N*LC display device was constructed which could work under both sunlight and total darkness conditions (Figure 7). Such a work has paved the way for high efficiency LE-LC devices with low power consumption.

Figure 5. (A) Chemical structure of TPE-PPE. (B) POM photograph recorded on heating TPE-PPE to 222 °C. (Reprinted with permission from ref. (17). Copyright 2015 John Wiley and Sons.)

Besides the symmetric TPE derivatives, the dissymmetric TPE-based molecules also could form mesomphoric phase (20). Derived from tetraphenylethylene and gallic acid, Miao Luo et al. reported synthesis of two single substituted compounds of TPE, luminescent LCs P4 and P5, and the synthetic route is shown in Scheme 3. POM and DSC measurements indicated that compounds P4 and P5 exhibited enantiotropic LC behaviors under heating and cooling processes. The temperatures of melting point and clearing point of P5 were higher than that of P4, owing to the additional phenyl unit. Both the POM (Figures 8A, 8B) and WXRD revealed that P4 and P5 probably belongs to smectic LC phase. Attributed to their twisted structure, P4 and P5 exhibited strong AIE activity. In Comparison with P4, P5 had bigger molecular size and steric hindrance (Figure 8C), and thus showed a stronger AIE activity with an I/I0 ratio of 101 than the 44 of P4 (Figure 8D). Moreover, P4 and P5 possessed different gelation behavior in organic solvents. Also, the gelation would induce fluorescence enhancement for both P4 and P5. In the case of the same concentration, P5 was almost non-luminescent in n-hexane but became much more emissive (the fluorescence intensity increased by almost 120 times) in the gel state, due to the formation of self-assembly aggregates (Figure 8E). The fluorescence of P5 can reversibly modulated between on and off states by the gel-solution transition via alternate 159 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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cooling and heating cycles. This thermo-responsive AIEgen-based organogels have potential applications in information storage, optical devices and biological applications.

Figure 6. (A) Photograph of the LE-LCD device in the electric field-off and field-on states using the light-emitting LC mixture. The LC mixture = Nematic LC PA0182 + 0.1 wt% TPE-PPE. (B) The structure and the photographs of the LE-LCD with photo-patterned alignment using light-emitting LC mixture. There are two regions in the device that are aligned orthogonally. In the electric field-off state, the regions with and without figures in the device will be alternately bright and dark, viewing through the rotatable polarizer under UV irradiation. In the electric field-on state, both the two regions will be light-emitting and the figures disappeared under UV irradiation. (Reprinted with permission from ref. (17). Copyright 2015 John Wiley and Sons.) 160 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 7. Photographs of the reflective-luminescent display device with an applied DC electric field (60 V) under sunlight and under UV-light irradiation using the light-emitting N*LC mixture. The LC mixture = N*LC-3+0.5 wt% TPE-PPE. The wavelength of the excitation light was 365 nm. (Reprinted with permission from ref. (19). Copyright 2016 John Wiley and Sons.)

Scheme 3. Synthetic routs for LE-LCs P4 and P5. (Reprinted with permission from ref. (20). Copyright 2014 Elsevier.) In addition to the AIE behaviors and gelation ability, the piezofluorochromic properties were also recorded. For compound P4, there was no obvious piezofluorochromic property; However, compound P5 exhibited the clear emission color change from blue to blue-green upon pressing, and the fluorescence could be recovered through heating or solvent fuming. In consideration of molecular structure and molecular aggregation, since P5 was more flexible than P4 due to the one more phenyl ring in the linkage, the crystalline-amorphous phase transformation turned to be easily happened and thus the piezofluorochromic properties were easy to be induced. 161 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 8. Optical micrographs of compounds P4 and P5 between crossed polarizers: (A) liquid crystalline texture for P4 at 120 °C; (B) liquid crystalline texture for P5 at 128 °C. (C) PL spectra of P5 in THF/water mixtures with different water fractions (inset: the images of P5 were taken at room temperature under 365 nm UV light in THF and 90% water); (D) Change in PL peak intensities of P4 and P5 with different water fractions in THF/water mixtures (concentration 10 mM, excitation wavelength 365 nm). (E) Fluorescence spectra of P5 in gel state and solution state in n-hexane with the same concentration (20 mg/mL), inset: Fluorescence images of P5 for various temperatures. (Reprinted with permission from ref. (20). Copyright 2014 Elsevier.) 2.3. Cyanostilbene-Based AIE-LCs Besides the usual AIE luminescent cores including sisole and TPE, there are still unusual light-emissive cores that were utilized in constructing AIE LCs. For example, cyanostilbene is a special AIEgen, it could form both smectic and columnar luminescent liquid crystals, depending on the substituent groups on the luminescent core. Lu H et al. (21) has reported a cyanostilbene-based AIEgen, (Z)-CN-APHP, with a dodecyloxy tail directly linking with the cyanostilbene core (Figure 9A). The introduction of electron-donating -NH2 group was aimed to enhance compatibility with LC molecules and made CN-APHP form a gel in the liquid crystalline phase. The result of POM and DSC suggested that (Z)-CN-APHP was an enantiotropic LC (Figure 9B). Under heating, (Z)-CN-APHP entered the liquid crystalline phase with the melting point at ~80 °C and clearing point at ~112 °C, respectively. The focal conic fan-shaped texture obtained at 75°C during the cooling process illustrated that (Z)-CN-APHP has a smectic mesophase. As a specific AIEgen, cyanostilbene unit in (Z)-CN-APHP underwent the E−Z isomerization process after irradiation at 365 nm in the LC phase (90 °C), accompanied by the transition from intensive green luminescence with a ΦF of 19.5% to the light bluet fluorescence with a radical low ΦF of 9.2% (Figure 9C). As revealed by the XRD analysis, before irradiation of the thin film of (Z)-CN-APHP, rod-like shaped Z-isomer showed a well-ordered liquid crystalline phase and turned to an obvious amorphous structure after 2 hours UV 162 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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irradiation, leading to the E-isomer. The strongly twisted two rings of bent-shaped E-isomer prevented the ordered packing of the molecules in the aggregation state. Moreover, the transition from Z-isomer to E-isomer reduced its effective π-conjugation length, resulting in the hypsochromic shift of ~24 nm, the decrease of photoluminescence intensity and the loss of LC phase (Figure 9D). As predicted, this E−Z isomerization process of AIE-LC molecule between LC state and amorphous state will induce many optical applications. The fluorescent molecule-dispersed liquid crystals was prepared by mixing 16.7% (Z)-CN-APHP with 83.3% positive liquid crystals E7. After filling into the unidirectional rubbed LC cell, the Z- E-isomerization process occurred upon UV irradiation and led to phase separation as a result of the poor compatibility of E-isomers with liquid crystals, forming the so-called fluorescent-molecule dispersed liquid crystals (FMDLC) (Figure 9E, 9F). Between the applied electric field-on (30v) and electric field-off states, the photoluminescence intensity of FMDLC could be switched repeatedly, owing to the different internal scattering of the excitation light (Figure 9G). Take advantages of the internal scattering-based mechanism, this modulation could also be utilized to develop other light-emissive liquid crystal systems, such as fluorescent polymer dispersed liquid crystals and fluorescent polymer stabilized liquid crystals.

Figure 9. (A) Schematic illustration of Z−E isomerization of (Z)-CN-APHP upon UV irradiation in the liquid crystal phase. (B) DSC curves of (Z)-CN-APHP at a heating/cooling rate of 10°C per minute. (C) Photo images of (left) unirradiated and (right) irradiated films of 521a on the quartz cells. (D) POM images of the (left) unirradiated film and (right) irradiated film of (Z)-CN-APHP. (E) Fluorescence emission spectra of FMDLC at field-off (0 V) and field-on (30 V) states. (F) Repeated switching of photoluminescence between the field-off (0 V) and field-on (30 V) states. (G) Schematic illustration of the mechanism of electrically switched photoluminescence of FMDLC. (Reprinted with permission from ref. (21). Copyright 2014 Royal Society of Chemistry.) 163 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Another cyanostilbene-based AIE-LC compound, namely, (Z)-2,3bis(3,4,5-tris (dodecyloxy)phenyl) acrylonitrile (GCS, Scheme 4), was also developed to explore the potential use of cyanostilbene compounds by virtue of the photoisomerization property (22). GCS was synthesized via the Knoevenagel reaction of 3,4,5-tris(dodecyloxy)benzaldehyde and 2-(3,4,5-tris(dodecyloxy)phenyl)acetonitrile. The molecular structure of GCS consists of a cyanostilbene moiety and the tris-(dodecyloxy) groups as peripheral flexible side chains which were directly attached to the mesogenic core to facilitate mesomorphic organization.

Scheme 4. Molecule structure of LE-LC GCS. (Reprinted with permission from ref. (22). Copyright 2014 John Wiley and Sons.) In studying the liquid crystalline properties of GCS, a typical focal-conic fan-shaped texture which is an indicator of a columnar hexagonal (Colh) phase was observed in the cooling process with POM (Figure 10A, 10C). The formation of the Colh phase was further confirmed by XRD patterns of the GCS in LC phase temperature, demonstrating a two-dimensional hexagonal lattice with p 6 mm symmetry. Near room temperature (RT), the isothermal crystallization of GCS was observed, which transformed the columnar hexagonal LC phase to the crystalline phase with a tetragonal arrangement under prolonged natural cooling (Figure 10B, 10D), implying the limited mesophase stability of GCS induced by intermolecular forces. Usually, azo materials were utilized to fabricate SRG (surface relief grating) through photo-isomerization of azobenzene resulting from the photoinduced mass migration; however, fluorescent SRG is scarce because the azobenzenes are generally non-fluorescent while fluorescent dyes that doped into azobenzene systems are prone to suffer from quenching by the non-fluorescent azobenzene components. Taking advantages of the E-Z photo-isomerization of GCS, fluorescence patterning was obtained based on photoinduced mass transfer. It was found that, under UV light irradiated and at 39 (±1) °C the crystalline phase totally disappeared, resulting in the dramatic decrease in the fluorescence intensity (Figure 10E, 10F). Thin film of GCS was thus prepared by spin-coating and then irradiated with a non-polarized Hg lamp at 365 nm through a micropatterned-photomask to fabricate the SRG. The maximum diffraction efficiency reached to almost 30%. Figure 10G shows the AFM image of the SRG. Regular sinusoidal undulation was observed clearly, consistent with the spacing of the photomask (Figure 10H). Because of the intrinsic AIE feature of GCS, strongly fluorescent micropattern with high contrast was generated (Figure 10I), thus avoided the concentration quenching. 164 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 10. (A) POM image of the Colh phase of GCS obtained at 30 °C during cooling. (B) POM image of the crystal phase of GCS obtained at room temperature during cooling. (C) XRD patterns of the GCS in LC phase at 28 °C and (D) in the crystal phase. The insets in (C) show the magnified diffraction peaks and the 2D pattern image. (E) POM image and (F) Fluorescence optical microscopic image of the crystalline film. (G) Topographical AFM image and (H) height profile along the white line. (I) Fluorescence optical microscopy image. (Reprinted with permission from ref. (22). Copyright 2014 John Wiley and Sons.) Besides GCS containing single cyanogroup, Park et al. also synthesized another AIEgen (GDCS) which is a phasmidic molecule comprising dicyanostilbene moieties and the terminal trisdodecyloxy fragments (23). Different from GCS, GDCS contains two cyanostilbene in its molecular structure (Figure 11A). Similarly, GDCS also exhibited a luminescent columnar hexagonal phase at room temperature. As revealed by the POM observation, a pseudo focal-conic fan-shaped texture of a hexagonal columnar (Colh) phase emerged in both heating and cooling cycles (Figure 11B), illustrating it is an enantiotropic LC. DSC trace also confirmed the enantiotropic characteristic with the phase-transitions of the heating and cooling processes (Figure 11C). From the calculated optimized molecular structure of GDCS based on the XRD data, a pair of GDCS molecules self-assembled into a molecular disk in a side-by-side disposition, driven by the secondary bonding interactions of the lateral polar 165 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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cyano group. And thus, the hexagonal structure of columnar LC phase was organized from the supramolecular disks. Owing to the AIE feature of GDCS, the fluorescence efficiency (ΦF) in THF solution, LC phase and crystalline (ΦF = 0.45) state was 1.1 × 10−2, 0.25 and 0.45, respectively, demonstrating an obvious increasing tendency. During the cooling process from 200 °C to RT, the PL spectra of GDCS in condensed state under 365 nm UV light underwent a bathochromic shift, followed by a gradually increased PL intensity, from liquid state to liquid crystal state and to the crystalline state (Figure 12A-12C). Above the clearing point, the molecule entered the isotropic state and complete absence of light emission was observed. In addition, the LC and crystalline state emitted intense green and yellow lights, respectively. The thermochromic fluorescence of GDCS were attributed to the peculiar intra- and intermolecular interactions of its dipolar cyanostilbene units. The hierarchical mesomorphic organization of the GDCS was comprehensively presented in Figure 12D, explaining the PL behaviors of the GDCS material in different phases.

Figure 11. (A) Molecular structures of GDCS. (B) Pseudo focal-conic fan-shaped texture of GDCS observed by POM in the Colh phase at 40 °C on the cooling process. (C) DSC trace of GDCS on heating/cooling rate of 10 °C per minute. (Reprinted with permission from ref. (23). Copyright 2012 John Wiley and Sons.) The specific molecular stacking processes in the LC and crystalline phases lead to the restriction of the molecular disks’ rotational motions, making it present an AIE effect. This deep insight of the structure-property relationship would provide guidance for the design of the LE-LC with efficient luminescence in the LC state. Moreover, since the different stacking fashion will result in the variety of excited-state molecular coupling and thus the different emission colors. This 166 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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provides the possibility to develop a probe that can distinguish the phases of thermotropic liquid crystals.

Figure 12. (A) Photos of GDCS in condensed state during the cooling process from 200 °C to RT under 365 nm UV light. (B) PL wavelength maximum shift behavior and (C) PL intensity change behavior depending on the temperature at the cooling process. (D) Schematic representation of the hierarchical mesomorphic organization of GDCS molecule concurrent with the intra- and intermolecular actions related to the emission characteristics. (Reprinted with permission from ref. (23). Copyright 2012 John Wiley and Sons.) 2.4. AIE-Active Liquid Crystalline Polymers (AIE-LCPs) In addition to AIE-LC small molecule, AIE active liquid crystalline polymers (AIE-LCPs) can also be achieved by incorporating AIE mesogens into the polymer backbones. Yuan et al. reported liquid crystalline polytriazoles with high solidstate emission efficiencies by click polymerization (24), affording polymers LCP1 and LCP2 et al. characterized by different spacer lengths.

Scheme 5. Molecular structures of aggregation-Induced emission active liquid crystalline polymers. (Reprinted with permission from ref. (24). Copyright 2011 American Chemical Society.) 167 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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As shown in scheme 5, the long alkyl chains and the TPE group endowed these polymers with good solubility, high regioregularity and obvious AIE activity. The photophysical properties of these polytriazoles showed obvious structural dependence, and those having longer spacer length showing lower solid-state ΦF. Besides, the mesomorphic properties was closely relative to the spacer length. LCP1 that possesses rigid main chains belong to nematic LC phase, while those polytriazoles with longer flexible spacers formed smectic phases through better mesogenic packing (LCP2a and LCP2b; Figure 13B, 13E). It seems the polymers with longer spacer lengths prefer to exhibit nematicity due to its better mesogenic packing.

Figure 13. Mesomorphic textures observed on cooling (A) LCP1a to 159.9 °C, (B) LCP2a to 89.9 °C, (C) LCP2c to 94.9 °C, (D) LCP2b to 250.6 °C, (E) LCP2b to 69.8 °C, and (F) LCP2d to 114.9 °C from their melting states at a cooling rate of 1 °C/min. Photos in parts A, B, D, and E are taken after application of a shearing force. (Reprinted with permission from ref. (24). Copyright 2011 American Chemical Society.) 2.5. Other AIE-LCs In above AIE-LCs, their AIE feature is well preserved no matter whether the core is silole, TPE, or cyanostilbene. In addition to these common AIE cores, some unusual AIEgens were also explored to construct AIE-LCs. Fujisawa K. et al. have synthesized several liquid-crystalline gold complexes that comprised a rigid body and a flexible alkoxy tail, showing smectic phase and AIE characteristics (25). During the phase transitions between LC and isotropic phases as well as crystalline and LC phases, the fluorescence color could be reversibly switched. The stronger emission of LC complexes in solid states demonstrated their potential application in light-emitting devices. Kana Tanabe et al. reported the AIE-active luminescent ionic liquid crystals that have hexagonal or rectangular columnar structures in their LC phase (26). The fluorescent color covering the visible region could be easily tuned by changing electron-donating and electron-accepting moieties of the 168 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

compounds, providing the luminescent materials potential in organic fluorescent sensors and optoelectronic applications.

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3. Conclusion In general, the research on AIE-active LCs is an attractive area with thrilling prospects. By integrating the advantages of light emission and liquid crystal, many photofunctional liquid crystal materials were achieved such as stimuli-responsive luminescent LCs and organogelators. More importantly, the polarized fluorescence of the LE-LCs with AIE properties are very useful for fabrication of novel luminescent optoelectronic devices. Despite the achievements, research in this area is still not enough. The mesophase of these luminescent LC materials is very limited, preventing many important applications such as stimuli-responsable luminesence; the fluorescence anisotropy of these LCs is urgently needed to be enhanced. Therefore, the next step of research in this area should be enrichment in the variety of AIE-LCs. In addition, the real-world technological applications of these superior LC materials should be explored further.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant 51203005) and the New Teacher Fund for Doctor Station and the Ministry of Education of China (Grant 20121102120045).

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21. Lu, H. B.; Qiu, L. Z.; Zhang, G. Y.; Ding, A. X.; Xu, W. B.; Zhang, G. B.; Wang, X. H.; Kong, L.; Tian, Y. P.; Yang, J. X. E. Electrically Switchable Photoluminescence of Fluorescent-Molecule-Dispersed Liquid Crystals Prepared via Photoisomerization-Induced Phase Separation. J. Mater. Chem. C 2014, 2, 1386–1389. 22. Park, J. W.; Nagano, S.; Yoon, S. J.; Dohi, T.; Seo, J. W.; Seki, T.; Park, S. Y. High Contrast Fluorescence Patterning in Cyanostilbene-Based Crystalline Thin Films: Crystallization-Induced Mass Flow Via a Photo-Triggered Phase Transition. Adv. Mater. 2014, 26, 1354–1359. 23. Yoon, S. J.; Kim, J. H.; Kim, K. S.; Chung, J. W.; Heinrich, B.; Mathevet, F.; Kim, P.; Donnio, B.; Attias, A. J.; Kim, D. H.; Park, S. Y. Mesomorphic Organization and Thermochromic Luminescence of Dicyanodistyrylbenzene-Based Phasmidic Molecular Disks: Uniaxially Aligned Hexagonal Columnar Liquid Crystals at Room Temperature with Enhanced Fluorescence Emission and Semiconductivity. Adv. Funct. Mater. 2012, 22, 61–69. 24. Yuan, W. Z.; Yu, Z. Q.; Tang, Y. H.; Lam, J. W. Y.; Xie, N.; Lu, P.; Chen, E. Q.; Tang, B. Z. High Solid-State Efficiency Fluorescent Main Chain Liquid Crystalline Polytriazoles with Aggregation-Induced Emission Characteristics. Macromolecules 2011, 44, 9618–9628. 25. Fujisawa, K.; Okuda, Y.; Izumi, Y.; Nagamatsu, A.; Rokusha, Y.; Sadaike, Y.; Tsutsumi, O. Reversible Thermal-Mode Control of Luminescence from Liquid-Crystalline Gold(I) Complexes. J. Mater. Chem. C 2014, 2, 3549–3555. 26. Tanabe, K.; Suzui, Y.; Hasegawa, M.; Kato, T. Full-Color Tunable Photoluminescent Ionic Liquid Crystals Based on Tripodal Pyridinium, Pyrimidinium, and Quinolinium Salts. J. Am. Chem. Soc. 2012, 134, 5652–5661.

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Chapter 7

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Aggregation-Induced Emission Luminogens (AIEgens) for Non-Doped Organic Light-Emitting Diodes Han Nie, Jian Huang, Zujin Zhao,*,1 and Ben Zhong Tang*,1,2,3 1State

Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China 2Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China 3Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Hong Kong, China *E-mails: [email protected] (Z.Z.); [email protected] (B.Z.T.)

Organic light-emitting diodes (OLEDs) have shown great potential in full-color flat panel displays and solid-state white lighting. For the fabrication of high-performance OLEDs, the light-emitting materials are of high importance. However, most conventional luminescent materials generally suffer from aggregation-caused quenching (ACQ) problem, which weakens the fluorescence of their solid films, and thus, undermines OLED performance. Recently, a new kind of luminogens with aggregation-induced emission characteristics (AIEgens) is found to be free of ACQ problem. They can fluoresce strongly in solid films and perform outstandingly in non-doped OLEDs. Herein, representative AIEgens with fluorescence color covering from blue to red and varied carrier transport ability for the application in efficient non-doped OLEDs are described.

1. Introduction Organic light-emitting diodes (OLEDs) are being highly expected as the next generation technology for flat panel displays and solid-state lighting because they have excellent self-emitting properties, simple manufacture procedures © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and flexible characteristics (1–3). Owing to the enormous effort devoted by a great number of scientists, more and more efficient OLEDs with various emission color have been obtained (4). In order to realize a further breakthrough, development of efficient and stable organic light-emitting materials with excellent optoelectronic properties is of significant importance. Many conventional luminescent materials showing robust emissions in the molecularly dispersive state usually experience serious quenching problem, and thus present weak or nearly no emissions in condensed phase or solid state, which is referred to as notorious aggregation-caused quenching (ACQ) effect (5). When these traditional luminescent molecules aggregate, the strong intermolecular π−π stacking interactions will promote the formation of delocalized excitons, which leads to low luminescence quantum efficiencies, and thus ACQ phenomenon. In OLEDs, the light emitters are practically fabricated into solid thin films, so the ACQ problem has impeded the progress of OLEDs field to some extent. To alleviate ACQ effect, various chemical, physical and/or engineering approaches have been proposed. Commonly, the luminogens are introduced with bulky groups or are doped into host materials at low concentration to suppress the aggregate formation. These methods, however, often end up with limited success and even are accompanied by some side effects in many cases (5). For example, the bulky groups can twist the conformations and shorten the effective conjugation lengths of the luminogens, which will barricade the charge transport in OLED devices. Because aggregate formation is a natural process when the luminogens are located in close proximity, the host-guest blending system may intrinsically suffer from phase separation, resulting in the limitation of efficiency and stability for OLEDs. Tang et al. reported a unique photophysical phenomenon from 1-methyl1,2,3,4,5-pentaphenylsilole (MPPS, Figure 1, 1) in 2001, whose weak emission in dilute solution was turned on by the formation of aggregates (6). This phenomenon was termed as aggregation-induced emission (AIE) that is essentially opposite to ACQ effect. During the past decade, lots of studies have been done to draw a clear picture on this novel AIE process by systemically experimental measurements and theoretical calculations (7). These studies had proposed that the restriction of intramolecular motions (RIM), including rotations (RIR) and vibrations (RIV), is the main cause for AIE effect. In the isolated state, the active intramolecular motions can enhance the nonradiative decay rate of the excited state. In the aggregated state, however, these nonradiative decay channels are blocked due to restricted intramolecular motions by the spatial constraints and interactions from the surrounding molecules. Moreover, the AIE molecules usually have twisted conformations which can also prevent the π−π stacking interactions. Consequently, they exhibit stronger emissions in the aggregated state. Since the first report of AIE phenomenon, many investigators with different backgrounds have focused their attentions on AIE research because of its potential significance in fundamental understanding and technological application. So far, a wide range of luminogens with AIE characteristics (AIEgens) have been developed and utilized in many frontier fields based on the understanding of AIE mechanism (8). By merging AIE elements and conventional ACQ chromophores (ACQphores) through molecular engineering, many of the resulting luminogens 174 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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presented AIE or aggregation-enhanced emission (AEE) characteristics with high emission efficiencies in the aggregated state. Therefore, AIE effect provides researchers with an effective strategy to address ACQ problem and develop efficient solid-state emitters for the construction of excellent non-doped OLEDs. As the most common AIE-active building blocks, silole (9) and teraphenylethene (TPE) (10) have been widely used to construct new fluorescent AIEgens with high fluorescent quantum yield (ΦF) values in solid films, which have been extensively employed to fabricate stable, efficient and simplified non-doped fluorescent OLEDs. The emission color of the fabricated OLEDs covered the entire visible region, and some of the devices performed excellently with high efficiencies approaching or reaching the theoretical limit of fluorescent OLEDs. In order to provide guidance for the further development of efficient solid state luminescent materials, in this chapter, silole-based and TPE-based AIEgens employed mainly as emitting materials for OLEDs with various EL emissions will be discussed. Then we will summarize the recent advances of multifunctional AIEgens, which can function efficiently as light emitter and carrier transporter simultaneously in OLEDs.

2. Silole-Based AIEgens Since the AIE behavior of MPPS (1) was first reported, silole has become an effective unit for the building of fluorescent AIEgens with high ΦF values in the aggregated state. In silole derivatives, the unique σ*-π* conjugation between the σ* orbital of two exocyclic C-Si σ-bonds and the π* orbital of the butadiene portion lowers the LUMO (lowest unoccupied molecular orbital) energy levels, which enables siloles to have good electron affinity and fast electron mobility. Therefore, silole derivatives can be adopted to transport electrons in optoelectronic devices. According to reported data, silole derivatives also exhibit excellent thermal and morphological stabilities as well as good solubility in common solvents (9). Therefore, siloles would certainly be the promising candidates of light-emitting materials in high-performance OLEDs. For example, MPPS had showed outstanding electroluminescence (EL) properties in early report (11). By utilizing dibenzosilole and TPE as the building blocks, a series of new fluorescent AIEgens (2-4) were achieved, whose chemical structures are presented in Figure 1 (12). These AIEgens possessed high thermal stabilities and good EL properties (Table 1). It is noteworthy that the EL spectra of OLEDs based on compounds 2 and 3 were located on deep-blue (CIE 0.16, 0.12) and sky blue (CIE 0.20, 0.33) region, respectively. Among them, compound 3 had the best EL efficiencies, and its OLED device with a configuration of ITO/MoO3 (10 nm)/NPB (60 nm)/3 (15 nm)/TPBi (30 nm)/LiF (1 nm)/Al had a low turn-on voltage (Von) at 3.0 V, a maximum luminance (Lmax) of 27161 cd m-2, a high maximum current efficiency (ηC,max) of 8.04 cd A-1, a maximum power efficiency (ηP,max) of 6.17 lm W-1, and a good maximum external quantum efficiency (ηext,max) of 3.38%. The device constructed from 4 radiated bright green EL at 512 nm with a Lmax of 28718 cd m-2 and displayed relatively high efficiencies shown in Table 1. From this work, it can be found that the EL properties of AIEgens can be easily tuned 175 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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by fine structural modulations, such as altering linkage mode and sharing benzene ring between the main building blocks.

Figure 1. Chemical structures of representative silole-based AIEgens with good EL performances. Their EL maxima are given in parentheses. As mentioned in Introduction section, ACQ effect is a knotty problem for the application of common luminescent materials in OLEDs, which is difficult to thoroughly solve without any negative effects by traditional approaches. It would be nice and highly desirable if the ACQ problem is eliminated, while the valuable properties of these conventional dyes are maintained at the same time. During the past few years, this desirability had been realized through transforming ACQphores to AIEgens on the basis of numerous AIE studies, which could not only address ACQ problem but also expand the AIE systems. One of the approaches on ACQ-to-AIE transformation is the replacement of the moieties of AIEgens with ACQ units. In addition, the electronic structures 176 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and photophysical properties of silole derivatives can be easily modulated by the substituents at the 2,5-positions. With these considerations, a series of silole derivatives 5-14 (Figure 1) were designed and successfully synthesized. When the ACQ-active triphenylamine (TPA) units were introduced into the 2,5-positions of silole ring, the resulting luminogen 5 exhibited typical AEE characteristics with a high film-state ΦF value of 74% (13). The multilayered OLED fabricated from 5 [ITO/NPB (60 nm)/5 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)] was turned on at a low bias of 3.1 V, and radiated a yellow emission of 544 nm with a Lmax of 13405 cd m-2. The device displayed good EL performance with ηC,max, ηP,max and ηext,max of 8.28 cd A-1, 7.88 lm W-1 and 2.42%, respectively. It is worth mentioning that the simplified OLED [ITO/5 (80 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)] built by using 5 simultaneously as light-emitting layer (EML) and hole-transporting layer (HTL) showed comparable EL efficiencies (7.60 cd A-1, 6.94 lm W-1, and 2.26%), revealing the good hole-transporting capability of 5 due to the presence of TPA groups. Another hole-transporting group, carbazole, is always used to construct efficient organic semiconductors. To investigate the influence of bonding pattern between substituents and silole ring on the EL property, two isomers 6 and 7 comprising of same building blocks (silole ring and carbazole units) through different bonding patterns were obtained (14). The powders of these two materials presented strong fluorescence with much higher ΦF values of 56-65% than those in THF solutions (2.3-6.0%), indicating AEE characteristics. And their non-doped OLEDs emitted yellow EL spectra peaking at 548-552 nm and displayed moderate EL performance (Table 1). Fluorene-based substituents are generally beneficial to the generation of efficient light emitters for OLEDs because they are intensely emissive and thermally stable. We developed several fluorene-substituted siloles (8-10) shown in Figure 1, which gave good thermal stabilities and high ΦF values up to 88% in solid films (15, 16). Consequently, the non-doped OLEDs based on them showed excellent EL performance. As shown in Table 1, compound 10 presented the best EL properties and its OLED with a simple and non-optimized configuration already performed very well. The OLED built from 10 with a further optimized configuration [ITO/MoO3 (5 nm)/NPB (60 nm)/10 (20 nm)/TPBi (60 nm)/LiF (1 nm)/Al (100 nm)] displayed a low Von of 3.3 V and radiated a strong yellow emission of 540 nm (CIE 0.36, 0.57) with a high Lmax of 37800 cd m-2. Notably, the ηC,max, ηP,max and ηext,max of this device were measured to be extraordinarily high with the values of 18.3 cd A-1, 15.7 lm W-1 and 5.5%, respectively, which reached the theoretical limit of fluorescent OLEDs. This breakthrough demonstrated the great potential of AIE emitters in OLED applications. Planar fluorescent chromophores (PFCs) are conducive to transport carriers in optoelectronic devices but can lead to undesirable ACQ effect. The introduction of representative PFC groups, anthracene and pyrene, into the 2,5-positions of silole ring produced some new silole derivatives (11-14) (15). These siloles were AEE-active and the OLEDs fabricated from them had good performance with EL emissions ranging from yellow to orange (Table 1). For instance, when compound 13 was employed as light-emitting materials to construct EL device, the Lmax, ηC,max, ηP,max and ηext,max attained by the resulting OLED were 49000 cd m-2, 9.1 cd A-1, 7.1 lm W-1 and 3.0%, respectively. 177 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 1. Electroluminescent performances for some representative silole-based AIEgens AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

2

ITO/MoO3 (10 nm)/NPB (60 nm)/2 (15 nm)/TPBi (30 nm)/LiF (1 nm)/Al (100 nm)

432

0.16, 0.12

3.7

4411

1.39

1.18

1.21

(12)

3

ITO/MoO3 (10 nm)/NPB (60 nm)/3 (15 nm)/TPBi (30 nm)/LiF (1 nm)/Al (100 nm)

488

0.20, 0.33

3.0

27161

8.04

6.17

3.38

(12)

4

ITO/MoO3 (10 nm)/NPB (60 nm)/4 (15 nm)/TPBi (30 nm)/LiF (1 nm)/Al (100 nm)

512

0.21, 0.37

3.5

28718

7.40

5.57

2.92

(12)

5

ITO/NPB (60 nm)/5 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

544

0.39, 0.53

3.1

13405

8.28

7.88

2.42

(13)

5

ITO/5 (80 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

548

0.40, 0.57

3.1

14038

7.60

6.94

2.26

(13)

6

ITO/NPB (60 nm)/6 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

552

0.41,0.56

4.6

28240

4.50

1.91

1.44

(14)

7

ITO/NPB (60 nm)/7 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

548

0.39,0.57

4.6

17280

4.26

2.23

1.35

(14)

8

ITO/NPB (60 nm)/8 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

552

0.40,0.53

4.8

2790

6.9

4.4

2.2

(15)

9

ITO/NPB (60 nm)/9 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

556

0.42,0.51

5.6

1900

8.1

4.6

2.9

(15)

10

ITO/NPB (60 nm)/10 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

544

0.37, 0.57

3.2

31900

16.0

13.5

4.8

(16)

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AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

10

ITO/MoO3 (5 nm)/NPB (60 nm)/10 (20 nm)/TPBi (60 nm) /LiF (1 nm)/Al (100 nm)

544

0.36, 0.57

3.3

37800

18.3

15.7

5.5

(16)

11

ITO/NPB (60 nm)/11 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

568

0.45,0.52

3.3

21100

5.6

4.6

2.0

(15)

12

ITO/NPB (60 nm)/12 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

582

0.48,0.50

4.4

7660

3.9

2.8

1.5

(15)

13

ITO/NPB (60 nm)/13 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

546

0.36,0.53

3.5

49000

9.1

7.1

3.0

(15)

14

ITO/NPB (60 nm)/14 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

544

0.35,0.51

4.6

5170

2.9

1.5

0.9

(15)

Abbreviations: λEL = electroluminescence maximum; Von = turn-on voltage at 1 cd m−2 ; Lmax = maximum luminance; ηC,max = maximum current efficiency; ηP,max = maximum power efficiency; ηext,max = maximum external quantum efficiency; CIE = Commission Internationale de I’Eclairage coordinates; NPB = N,N′-di(1-naphthyl)-N,N′-diphenylbenzidine; TPBi = 1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene; Alq3 = Tris-(8-hydroxyquinoline)aluminum. NPB functions as hole-transporting layer (HTL), TPBi serves as electron-transporting layer (ETL) and hole-blocking layer (HBL), Alq3 functions as ETL and MoO3 serves as hole-injection layer (HIL).

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3. TPE-Based AIEgens Amongst various AIEgens, TPE (15) is another star molecule, and possesses a very simple molecular structure but exhibits outstanding AIE characteristics with a ΦF value of 49% in solid state. When the naked TPE was used as light emitter to fabricate OLED, the generated device radiated a deep blue EL emission of 445 nm but showed inferior performance with the Lmax, ηC,max and ηext,max of only 1800 cd m-2, 0.45 cd A-1 and 0.4%, respectively (17). In the past decade, through the facile combination of TPE units and other functional groups, plenty of TPE derivatives have been created and their PL and EL properties have been improved significantly compared to the TPE parent. The OLEDs made from these TPE-based AIEgens emitted efficiently and displayed colorful emissions covering the whole range of visible lights. Herein, we will categorize some representative TPE-based AIEgens by EL emissions and give their EL performance data. The development of stable and efficient luminescent materials emitting three primary colors (red, green and blue) is crucial for OLEDs to be applied in full-color displays and solid-state lighting sources. The performance of blue OLEDs is often inferior to that of red and green ones due to the inherent wide band gap of blue emitters. Therefore, it is very challenging to hunt stable and efficient blue or deep blue emitting materials and devices for the realization of commercial applications. Recently, a great deal of effort has been invested to search highly efficient pure organic blue fluorescent materials and OLEDs owing to the inferior stability and short longevity of the electrophosphorescent devices (18). Since the conventional fluorophores would undergo ACQ effect in solid films, the blue fluorescent AIEgens could be good candidates for the construction of efficient blue OLEDs. As previously discussed, TPE showed a deep blue EL emission but a bad EL property. Frequently, spirofluorene and carbazole groups are used to develop efficient blue emitters. Thus Li and co-workers directly attached spirofluorene or carbazole moieties to TPE unit through sharing a benzene ring or Carbon–Nitrogen bonds, and the generated AIEgens (16 and 17) exhibited a good balance between blue light emission and improved EL properties (19). The performances of OLEDs based on 16 and 17 are listed in Table 2. Besides spirofluorene and carbazole groups, phenanthro[9,10-d]imidazole (PI) is another valuable building block for the fabrication of efficient blue fluorophores. In addition, triphenylethene is also a useful AIE unit, which has a shorter conjugation length and radiates a bluer solid-state emission relatively to TPE. The merging of triphenylethene units and a phenanthro[9,10-d]imidazole (PI) group by molecular engineering produced an efficient deep blue AIEgen 18 with a high ΦF value of 94% in solid film state (20). Compound 18 showed the best EL property among the deep blue AIEgens (Table 2). Its non-doped OLED device with a configuration of ITO/NPB (40 nm)/18 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm) emitted a bright deep blue light with a peak at 450 nm and CIE coordinates of (0.15, 0.12) as well as a Lmax of 16400 cd m-2. The ηC,max, ηP,max, and ηext,max of this device were as high as 4.9 cd A-1, 4.4 lm W-1 and 4.0%, respectively. In addition, this OLED presented good stability, as demonstrated by the high efficiencies of 3.7 cd A-1, 3.1 lm W-1 and 3.0% at the luminance of 1000 cd m-2. As mentioned above, ACQ-to-AIE transformation is a feasible proposal to obtain efficient emitters for the application in OLEDs. 180 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Another effective approach of ACQ-to-AIE transformation is the decoration of ACQphores with AIE archetypes. The fluorophores 19-22 shown in Figure 2 are nice examples for this approach. Attaching two TPE units to anthracene, a blue ACQphore, yielded a new blue AIEgen 19 (21). The optimized nondoped OLED based on 19 [ITO/HATCN (150 nm)/NPB (20 nm)/19 (10 nm)/Bepp2 (40 nm)/LiF (1 nm)/Al (200 nm)] was turned on at a low voltage of 2.8 V and displayed an intense deep blue emission with CIE coordinates of (0.17, 0.14) and a high Lmax of 17721 cd m-2. The device exhibited good EL performance with an appreciable ηP,max of 4.3 lm W-1 and a low efficiency roll-off (22). By decorating the anthracene ring of 19 with a tert-butyl group, a new luminogen 20 was developed by Shu et al. (23) For compound 20, the ΦF value of solid film (89%) was much higher than that of its dilute solution (6%), which indicated the typical AEE characteristics. The non-doped OLED employing 20 as light-emitting layer emitted a deep blue EL of 456 nm (CIE 0.14, 0.12) and performed excellently with high ηC,max and ηext,max of 5.3 cd A-1 and 5.3%, respectively, reaching the theoretical limitation of fluorescent OLEDs. Pyrene is one other traditional blue fluorophore, whose emission in solid film is always quenched severely due to ACQ effect. Attaching TPE unit(s) to the periphery of pyrene afforded two AIE/AEE-active fluorophores (21 (24) and 22 (25)) which showed strong emissions in the solid state with extremely high ΦF values up to unity. The OLEDs constructed from 21 or 22 both radiated sky blue emissions and exhibited pretty good performance. The EL efficiencies of the device based on 21 were recorded to be 7.3 cd A-1, 5.6 lm W-1 and 3.0%. Compound 22 presented better EL property than 21. And its optimized OLED with a configuration of ITO/NPB (60 nm)/22 (26 nm)/TPBi (20 nm)/LiF (1 nm)/Al (100 nm) displayed fairly high Lmax, ηC,max, ηP,max, and ηext,max of 36300 cd m-2, 12.3 cd A-1, 7.0 lm W-1, and 4.95%, respectively. Connecting two TPE units by simple linkage modes generated AIEgens 23 (26) and 24 (27) (Figure 2), which showed efficient PLs with ΦF values as high as 100% in the solid state. As shown in Table 2, when 23 or 24 were utilized as emitting layers to fabricate multilayer OLEDs, the resulting EL devices displayed sky-blue ELs at 488 nm and comparable EL performance. Efficient green emitters can be easily achieved through rational fusing of TPE unit and pyrene groups at the molecular level. Luminogens 25 (28) and 26 (24) shown in Figure 3 were the good examples for this strategy. 26 was an AIE-active molecule possessing strong emission with a high ΦF of 100% in solid phase and thermally very stable. Its multilayer OLED radiated bright green light at 516 nm with a Lmax of 25500 cd m-2, showing good EL performance with a ηext,maxof 2.1%. The ΦF value of 25 in dilution solution (9.8%) was much higher than that of TPE (0.24%), which derived from the relatively stiffer molecular structure due to higher steric congestion. Its ΦF value was enhanced to unity in the amorphous film, indicating that 25 was AEE-active. The EL device based on 25 with a configuration of ITO/NPB (60 nm)/25 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm) showed the EL peak at 516 nm and superior EL performance with Lmax, ηC,max and ηext,max of 49830 cd m-2, 10.2 cd A-1 and 3.3%, respectively. Linking two TPEs to the benzo-2,1,3-thiadiazole core created a yellowish green emitter 27 with excellent thermal and morphological stability as well as high solid-state ΦF value (89%) (29). The non-doped OLED fabricated by adopting 27 as emitter exhibited 181 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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a yellowish green emission peaking at 540 nm, a Lmax of 13540 cd m-2 and a ηC,max of 5.2 cd A-1.

Figure 2. Chemical structures of representative TPE-based blue AIEgens with good EL performances. Their EL maxima are given in parentheses.

Figure 3. Chemical structures of representative TPE-based green and yellow AIEgens with good EL performances. Their EL maxima are given in parentheses. In the field of OLEDs, it is equally important to develop efficient red emitters and devices for the full-color displays and lighting sources. However, many conventional red fluorophores containing planar polycyclic aromatic hydrocarbon (PAHs) units generally undergo strong ACQ problem in the solid state, whose film-state emission efficiencies are always unsatisfactory (30). Taking advantage of AIE effect is emerging as a promising strategy to address this issue. On the basis of the abundant experience on exploitation of efficient solid emitters with blue, green and yellow colors, Tang’s group had created a series of efficient solid red emitting materials, whose molecular structures are shown in Figure 4. The benzo-2,1,3-thiadiazole moiety, a heterocyclic ring and a famous strong electron-withdrawing group, is widely used to tune effectively the emission colors of the materials and thus develop red emitters. In addition, the introduction of thiophene ring can prompt the intramolecular charge transfer as well as elongate molecular conjugation, which are helpful for red-shifting the emissions of luminogens in many cases (29). Making use of AIE-active TPE 182 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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units, benzo-2,1,3-thiadiazole and thiophene rings as building blocks, several efficient solid red fluorophores (28-29) were prepared (29). 28 and 29 emitted orange-red and red emissions and presented relatively high ΦF values up to 55% in the solid state. In Table 2, the performance data of OLEDs based on them are listed in detail. The device of 28 radiated strong orange-red EL at 592 nm with a Lmax of 8330 cd m-2, a ηC,max of 6.4 cd A-1 and a high ηext,max of 3.1%. The device of 29 emitted at red region (668 nm) but the device performance was poor. When one more TPE unit was added into the conjugated backbone of 29 molecule to suppress the intermolecular interactions, a new luminogen 30 was generated (31). 30 presented better PL, EL properties and thermal stability than 29. The non-doped OLED of 30 [ITO/NPB (60 nm)/30 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)] displayed a red light at 650 nm (CIE 0.67, 0.32) and higher efficiencies (2.4 cd A-1 and 3.7%) than those measured from 29-based device. Because of good hole-transporting property and electron-donating capability, arylamines units are usually utilized to fabricate excellent solid red emitting materials (30). Thus another set of red AIEgens 31 and 32 were recently created (30). The film-state ΦF values of 31 and 32 were 48.8% and 63.0%, respectively. When they were adopted to build non-doped OLEDs, the resulting devices both had a red EL at 604 nm and showed outstanding EL performance. The device based on 32 displayed superior performance with excellent Lmax, ηC,max, ηP,max and ηext,max of 16396 cd m-2, 7.5 cd A-1, 7.3 lm W-1 and 3.9%, respectively, compared to those of the device based on 31 (15584 cd m-2, 6.4 cd A-1, 6.3 lm W-1 and 3.5%). At 1000 cd m-2, high efficiency values of up to 4.6 cd A-1, 2.6 lm W-1 and 2.4% were also achieved based on these two OLEDs. What’s more, their potential hole-transporting properties enabled the simplified OLEDs utilizing them as both EMLs and HTLs to perform well.

Figure 4. Chemical structures of representative TPE-based red AIEgens with good EL performances. Their EL maxima are given in parentheses. 183 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 2. Electroluminescent performances for some representative TPE-based AIEgens AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

16

ITO/NPB (60 nm)/16 (30 nm)/TPBi (20 nm)/LiF (1 nm)/Al (100 nm)

466

0.18, 0.24

2.6

8196

3.33

2.10

-

(19)

17

ITO/NPB (40 nm)/17 (10 nm)/ TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

462

0.17, 0.21

3.3

6179

2.80

2.51

-

(19)

18

ITO/NPB (60 nm)/18 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

463

0.15, 0.15

3.2

20300

5.9

5.3

4.4

(20)

18

ITO/NPB (40 nm)/18 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

450

0.15, 0.12

3.2

16400

4.9

4.4

4.0

(20)

19

ITO/HATCN (150 nm)/NPB (20 nm)/19 (10 nm)/Bepp2 (40 nm)/LiF (1 nm)/Al (200 nm)

449

0.17, 0.14

2.8

17721

-

4.3

-

(22)

20

ITO/PEDOT/TFTPA (30 nm)/20 (40 nm)/TPBi (40 nm)/Mg:Ag (100 nm)/Ag (100 nm)

456

0.14, 0.12

4.9

4165

5.3

2.8

5.3

(23)

21

ITO/NPB (60 nm)/21 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

484

-

3.6

13400

7.3

5.6

3.0

(24)

22

ITO/NPB (60 nm)/22(40 nm)/TPBI (20 nm)/LiF (1 nm)/Al (100 nm)

492

-

4.7

18000

10.6

5.0

4.04

(25)

22

ITO/NPB (60 nm)/22 (26 nm)/TPBi (20 nm)/LiF (1 nm)/Al (100 nm)

488

-

3.6

36300

12.3

7.0

4.95

(25)

23

ITO/NPB (60 nm)/23 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

488

-

4.0

11180

7.26

-

3.17

(26)

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185

AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

24

ITO/NPB (60 nm)/24 (20 nm)/TPBi (40 nm) /LiF (1 nm)/Al (100 nm)

488

-

4.2

10800

5.8

3.5

2.7

(27)

25

ITO/NPB (60 nm)/25(20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

516

-

3.2

49830

10.2

9.2

3.3

(28)

26

ITO/NPB (60 nm)/26 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

516

-

4.6

25500

6.0

2.7

2.1

(24)

27

ITO/NPB (60 nm)/27 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

540

-

3.9

13540

5.2

3.0

1.5

(29)

28

ITO/NPB (60 nm)/28 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

592

-

5.4

8330

6.4

2.9

3.1

(29)

29

ITO/NPB (60 nm)/29 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

668

-

4.4

1640

0.4

0.5

1.0

(29)

30

ITO/NPB (60 nm)/30 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

650

0.67, 0.32

4.2

3750

2.4

-

3.7

(31)

Continued on next page.

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Table 2. (Continued). Electroluminescent performances for some representative TPE-based AIEgens AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

31

ITO/NPB (80 nm)/31 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

604

-

3.2

15584

6.4

6.3

3.5

(30)

32

ITO/NPB (80 nm)/32 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

604

-

3.2

16396

7.5

7.3

3.9

(30)

Abbreviations: λEL = electroluminescence maximum; Von = turn-on voltage at 1 cd m−2 ; Lmax = maximum luminance; ηC,max = maximum current efficiency; ηP,max = maximum power efficiency; ηext,max = maximum external quantum efficiency; CIE = Commission Internationale de I’Eclairage coordinates; NPB = N,N′-di(1-naphthyl)-N,N′-diphenylbenzidine; TPBi = 1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene; Alq3 = Tris-(8-hydroxyquinoline)aluminum; TFTPA = tris[4-(9-phenylfluoren-9-yl)phenyl]amine; PEDOT = poly(styrenesulfonate)-doped poly(3,4-ethylenedioxythiophene); HATCN = 1,4,5,8,9,11-hexaazatriphenylene hexacarbonitrile; Bepp2 = bis[2-(2-hydroxyphenyl)-pyridine] beryllium. NPB and TFTPA functions as hole-transporting layer (HTL), TPBi and Bepp2 serve as electron-transporting layer (ETL) and hole-blocking layer (HBL), Alq3 functions as ETL, HATCN, PEDOT and MoO3 serve as hole-injection layer (HIL).

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4. Multifunctional AIEgens Besides high solid-state emission efficiencies, AIEgens can also be endowed with good carrier-transporting abilities by introducing hole- and/or electron-dominated units into their structures. The materials with multiple functionalities (p-type or n-type AIE emitters) can simultaneously act as EMLs and HTLs or electron-transporting layers (ETLs) in OLED devices, which are very appealing for the development of OLEDs as they can simplify the device configurations, shorten the fabrication process and cut down the manufacture cost (8). These attractive merits encourage material scientists to design multifunctional materials in a new way. Many AIEgens with excellent hole- or electron-transporting property have been developed and illustrated in Figure 5 as examples. The performance data of the OLEDs based on them are summarized in Table 3. Triphenylamine (TPA) segment is famous for its good hole-transporting capability and has been widely utilized to construct efficient p-type light emitters. As presented in Figure 5, fusing TPA group(s) and TPE unit(s) by different linkage modes generated a series of TPE-TPA adducts (33-40) that had not only high ΦF values but also excellent hole mobility in the film state. By directly attaching TPA unit(s) to TPE core, two new AIEgens (33 and 34) were obtained (32). They showed strong emissions in film phase with outstanding ΦF values up to unity. In addition, the amorphous film of 34 exhibited a reasonably high hole mobility of 5.2×10-4 cm2 V-1 s-1 attained by time-of-flight technique, manifesting that 34 was a good p-type emitter (10). Therefore, a simplified OLED fabricated using 34 simultaneously as EML and HTL [ITO/34 (60 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF/Al (200 nm)] emitted strong green EL with a Lmax of 33770 cd m-2 and presented appreciable efficiencies with the ηC,max, ηP,max and ηext,max of 13.0 cd A-1, 11.0 lm W-1 and 4.4%, respectively. These performance data were better than those measured from the device of 34 with NPB as the additional HTL. The simplified OLED based on 33 also displayed comparable performance than the one with “standard” configuration. Since NPB is a commercialized and useful hole-transporting material in the field of optoelectronic device, facilely inserting a TPE unit into the conjugated backbone of NPB produced a stable and efficient AIEgen (35) with a high film-state ΦF of 98% and good hole-transporting property (33). The non-doped multilayer OLED fabricated utilizing 35 as emitting material [ITO/NPB (40 nm)/35 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)] radiated green EL at 512 nm, and showed a high ηC,max of 11.9 cd A-1 and a pretty good ηext,max of 4.0%. Even better EL efficiencies (ηC,max = 13.1 cd A-1 and ηext,max = 4.2%) were obtained in its bilayer device without NPB [ITO/35 (60 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)]. Decorating TPA and DTPA cores with three and four TPE units generated two compounds 36 and 37, respectively, whose optical and electrical properties were systematically investigated (34). 36 and 37 emitted nearly no light in their dilute THF solutions with the ΦF values of merely 0.42% and 0.55%, while in condensed phase the ΦF values were improved significantly to 91.6% and 100%, respectively, indicative of excellent AIE characteristics. As shown in Table 3, the OLED device using 36 as both EML and HTL presented higher EL efficiencies than its “standard” 187 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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device. 37 also showed good multifunctional properties as both efficient emitter and hole-transporter in the OLED. By modifying the TPA core of 36 with three methyl groups, a blue-shifted AIEgen (38) with a high ΦF value of 64% in the aggregate state and a hole-transporting potential was developed (35). The simplified OLED of 38 without NPB [ITO/MoO3 (10 nm)/38 (75 nm)/TPBi (35 nm)/LiF (1 nm)/Al (100 nm)] emitted sky blue EL at 469 nm (CIE 0.18, 0.25) and showed good performance with the Lmax, ηC,max, ηP,max and ηext,max up to 15089 cd m-2, 6.51 cd A-1, 6.88 lm W-1 and 3.39%, respectively, which were comparable to those (13639 cd m-2, 8.03 cd A-1, 7.04 lm W-1 and 3.99%) received from its multilayer device using NPB as HTL. By incorporating a hole-dominate N,N,N′,N′-tetraphenyl-p-phenylenediamine (PDA) or TPA core and TPE moieties, two starburst luminogens 39 and 40 were created by Adachi and co-workers (36). Thanks to the AIE effect, the ΦF values of solid films for 39 and 40 were as high as 56% and 73%, respectively. In addition, by means of the space-charge-limited current (SCLC) technique, the hole mobilities of their amorphous thin films were measured to be more than 10-2 cm2 V-1 s-1, being much higher than that of NPB, due to the presence of PDA or TPA segments and the spontaneous molecular orientations of the molecules. Hence, the bilayer EL devices of 39 and 40 [ITO/ 39 or 40 (65 nm)/BPhen (35 nm)/LiF (0.8 nm)/Al (70 nm)] performed extremely well as indicated by the data given in Table 3. When NPB was used as additional HTL to balance carriers, the resulting triple-layer devices exhibited remarkably high ηext,max values of up to 5.9%, which was contributed by the efficient solid-state luminescence and the enhancement of the hole mobility and the light out-coupling efficiency due to the horizontal orientation characteristics. Among the fabricated EL devices, the triple-layer device based on 40 showed the best performance with a current efficiency of 15.9 cd A-1 and a power efficiency of 16.2 lm W-1 at the luminance of 100 cd m-2. Surprisingly, its external quantum efficiency remained nearly 5% when the luminance was increased to ca. 10000 cd m-2. Compared with efficient p-type light emitters, the n-type organic semiconductors with fast electron mobility and excellent solid-state emission are rare and in urgent need. As mentioned earlier, silole derivatives have electron-transporting potential because of the unique electronic structures. Numerous good works have demonstrated that siloles are excellent solid emitters owing to their AIE attributes. So, decorating silole with functional groups by molecular engineering would create many efficient n-type solid emitters with AIE characteristics. On the basis of these considerations, we designed and prepared two silole-based AIEgens 41 and 42 that were comprised of a silole ring and dimesitylboryl groups (37). Thanks to the presence of the vacant pπ orbital on the boron center, the dimesitylboryl group is inherently electron-deficient and beneficial for reducing the LUMO energy level and thus elevating the electron-transporting capacity of the molecule. 41 and 42 possessed low-lying LUMO energy levels with the values of -3.06 and -3.10 eV, respectively, indicative of their good electron-transporting potentials. Moreover, the solid films of 41 and 42 presented high ΦF values about 60%. Based on these excellent integrated properties, their double-layer OLEDs employing 41 or 42 as both emitter and electron-transporter [ITO/NPB (60 nm)/41 or 42 (60 nm)/LiF (1 nm)/Al (100 188 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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nm)] were constructed, which presented outstanding performances (Table 3) with high ηC,max, ηP,max and ηext,max values up to 13.9 cd A-1, 11.6 lm W-1 and 4.35%, respectively. These EL performance data were much higher than those measured from their multilayered devices with an additional TPBi as ETL. Significantly, the simplified device based on 42 showed a small efficiency roll-off when luminance increased, and a high current efficiency of 7.0 cd A-1 was maintained at a luminance of 1000 cd m-2. These good results demonstrated that 41 and 42 were excellent n-type solid-state emitters. When the electron-withdrawing dimesitylboryl or diphenylphosphoryl functional groups were introduced into silole 10, two new silole derivatives 43 and 44 were prepared (38, 39). They emitted intense fluorescence in solid state with high ΦF values up to 88% and exhibited good thermal stabilities. They were also potential n-type solid emitters as indicated by their EL performance data listed in Table 3. Attaching a dimesitylboryl group to TPE unit could produce a new AIEgen molecule 45 (40). The presence of dimesitylboryl portion enabled 45 to act as both emitter and electron-transporter in OLED devices. As shown in Table 3, the triplelayer OLED of 45 radiated green EL at 496 nm and displayed good EL efficiencies (ηC,max = 5.78 cd A-1 and ηext,max = 2.3%). Superior EL performance was achieved from its simplified double-layer OLED [ITO/NPB (60 nm)/45 (60 nm)/LiF (1 nm)/Al (100 nm)] with the higher ηC,max and ηext,max of 7.13 cd A-1 and 2.7%, respectively. 2,5-Diaryl-1,3,4-oxadiazole (Oxa) is an electron-deficient aromatic group and another widely used moiety to construct electron-transporting materials. 46 containing Oxa group and TPE unit showed strong solid-state emission and high thermal stability (41). The 46-based OLED with a configuration of ITO/NPB (60 nm)/46 (60 nm)/LiF (1 nm)/Al (200 nm) presented better EL performance than that of the standard OLED device, which revealed the fine electron-transporting ability of 46 (Table 3). Apart from above p-type and n-type AIE emitters, bipolar AIE systems including both electron donors (D), acceptors (A) and AIE elements have been designed wisely and achieved. These bipolar AIE luminescent materials could not only function as excellent emitters but also balance carriers in OLEDs, which may improve the EL efficiencies and help to simplify the device configurations. Hence, by inserting TPE or two more phenyls attached TPE (TPEBPh) into a D-A framework containing diphenylamino as the electron donor and dimesitylboron as the electron acceptor, two nice bipolar AIE molecules (47 and 48) were developed (42). These two bipolar AIEgens showed highly satisfactory PL and EL properties (Table 3). For instance, 48 possessed a weak D-A interaction and fluoresced intensely in film state with an extremely high ΦF of 94%. The trilayer OLED fabricated from 48 [ITO/NPB (60 nm)/48 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)] was turned on at a low basis of 3.2 V and emitted bright green EL with a high Lmax of 49993 cd m-2. Its appreciable ηC,max, ηP,max, and ηext,max were measured to be 15.7 cd A-1, 12.9 lm W-1 and 5.12%, respectively. Its bilayer EL device without the NPB layer showed better performance (ηext,max up to 5.35%), demonstrating that 48 was an excellent p-type emitter. It is noteworthy that the OLEDs of 48 emitted efficiently and exhibited the small roll-off in EL efficiencies. When the luminance increased to 1000 cd m-2, the ηexts of its trilayer and bilayer devices were as high as 4.75% and 4.45% respectively. On the basis 189 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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of these good results, the authors thought that the hole mobility of 48 film should be a crucial factor for its outstanding EL property. So, the hole-transporting abilities of the thin films of 48 and NPB were evaluated by SCLC technique. As a result, the hole mobility values of 48 and NPB thin films were almost at the same level. Compounds 49-52 were new bipolar AIEgens based on TPE building block attached with N-ethyl-carbazole groups and dimesitylboron or (dimesitylboranyl)phenyl units by different linkage modes (43). The devices with the configuration of ITO/HATCN (20 nm)/NPB (40 nm)/49-52 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (80 nm) radiated strong blue or green emissions with the Lmax up to 65150 cd m-2 and displayed good EL performances as shown Table 3. Another series of donor-AIE-accepter system (53-55) was reported in 2015, utilizing TPE as AIE-active core, TPA groups as electron donor and (dimesitylboryl)phenyl blocks as electron accepter (44). As illustrated in Figure 5, these novel bipolar TPE deviratives showed star-shaped conformation, which is good for the materials to be fabricate into good films by spin-coating technique. Moreover, they had very high film-state ΦF values up to 95%. So the solution-processed non-doped OLEDs employing these star-shaped AIEgens as emitters were constructed. The OLED based on 53 performed best and showed a Lmax of 11665 cd m-2 and a high ηC,max up to 8.3 cd A-1. Driven at 1000 cd m-2, this device presented slight efficiency roll-off and its current efficiency could also maintain at the value of 6.2 cd A-1.

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Figure 5. Examples of representative multifunctional AIEgens. Their EL maxima are given in parentheses. 191 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

192

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Table 3. Electroluminescent performances for some representative multifunctional AIEgens AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

33

ITO/NPB (40 nm)/33 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (200 nm)

492

-

3.6

15480

8.6

5.3

3.4

(32)

33

ITO/33 (60 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (200 nm)

492

-

4.2

26090

8.3

4.9

3.3

(32)

34

ITO/NPB (40 nm)/34 (20 nm)/ TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (200 nm)

514

-

3.4

32230

12.3

10.1

4.0

(32)

34

ITO/34 (60 nm)/TPBi (10 nm)/Alq3 (30 nm) /LiF (1 nm)/Al (200 nm)

512

-

3.2

33770

13.0

11.0

4.4

(32)

35

ITO/NPB (40 nm)/35 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

512

-

3.7

11981

11.9

8.9

4.0

(33)

35

ITO/35 (60 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

516

-

3.9

12607

13.1

7.8

4.2

(33)

36

ITO/NPB (60 nm)36 (20 nm)/TPBi (10 nm) /Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

493

-

5.4

1662

3.1

1.1

1.2

(34)

36

ITO/36 (80 nm)/TPBi (10 nm) /Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

499

-

4.5

6935

4.0

1.9

1.5

(34)

37

ITO/37 (30 nm)/TPBi (10 nm) /Alq3 (30 nm)/LiF (1 nm)/Al (100 nm)

488

-

4.1

10723

8.0

5.2

3.7

(34)

38

ITO/MoO3 (10 nm)/NPB (60 nm)/38 (15 nm)/TPBi (35 nm) /LiF (1 nm)/Al (100 nm)

480

0.17, 0.28

3.1

13639

8.03

7.04

3.99

(35)

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193

AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

38

ITO/MoO3 (10 nm)/38 (75 nm)/TPBi(35 nm)/LiF (1 nm)/Al (100 nm)

469

0.18, 0.25

2.9

15089

6.51

6.88

3.39

(35)

39

ITO/NPB (40 nm)/39 (25 nm)/ Bphen (35 nm)/LiF (0.8 nm)/Al (70 nm)

515

-

2.6

58300

-

-

4.5

(36)

39

ITO/39 (65 nm)/Bphen (35 nm) /LiF (0.8 nm)/Al (70 nm)

510

-

2.6

48300

-

-

3.6

(36)

40

ITO/NPB (40 nm)/40 (25 nm)/ Bphen (35 nm)/LiF (0.8 nm)/Al (70 nm)

523

-

2.4

53600

-

-

5.9

(36)

40

ITO/40 (65 nm)/Bphen (35 nm)/LiF (0.8 nm)/Al (70 nm)

523

-

2.4

54200

-

-

4.5

(36)

41

ITO/NPB (60 nm)/41 (20 nm)/ TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

548

0.39, 0.55

5.4

15200

8.4

4.1

2.62

(37)

41

ITO/NPB (60 nm)/41 (60 nm)/LiF (1 nm)/Al (100 nm)

524

0.33, 0.56

4.3

12200

13.9

11.6

4.35

(37)

42

ITO/NPB (60 nm)/42 (20 nm)/ TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

552

0.40, 0.54

7.5

9610

6.6

2.4

2.13

(37)

42

ITO/NPB (60 nm)/42 (60 nm)/LiF (1 nm)/Al (100 nm)

520

0.30, 0.56

3.9

13900

13.0

10.5

4.12

(37)

43

ITO/NPB (60 nm)/43 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

554

0.41, 0.56

3.8

48348

12.3

8.8

4.1

(38)

43

ITO/NPB (60 nm)/43 (40 nm)/TPBi (20 nm)/LiF (1 nm)/Al (100 nm)

554

0.41, 0.56

4.6

34080

10.1

5.9

3.3

(38)

Continued on next page.

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Table 3. (Continued). Electroluminescent performances for some representative multifunctional AIEgens AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

44

ITO/NPB (60 nm)/44 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

542

0.38, 0.56

3.1

19019

9.2

9.0

3.1

(39)

44

ITO/NPB (60 nm)/44 (60 nm)/LiF (1 nm)/Al (100 nm)

544

0.38, 0.56

3.1

16656

8.7

8.6

2.9

(39)

45

ITO/NPB (60 nm)/45 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

496

-

6.3

5581

5.78

3.4

2.3

(40)

45

ITO/NPB (60 nm)/45 (60 nm)/LiF (1 nm)/Al (100 nm)

496

-

6.3

5170

7.13

3.2

2.7

(40)

46

ITO/NPB (60 nm)/46 (20 nm)/TPBi (10 nm)/Alq3 (30 nm)/LiF (1 nm)/Al (200 nm)

466

-

4.4

2800

1.5

1.1

0.7

(41)

46

ITO/NPB (60 nm)/46 (60 nm)/LiF (1 nm)/Al (200 nm)

476

-

3.2

7000

2.4

2.2

1.0

(41)

47

ITO/NPB (60 nm)/47 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

544

0.35, 0.55

3.3

42924

10.5

9.40

3.24

(42)

47

ITO/47 (80 nm)/TPBi (40 nm) /LiF (1 nm)/Al (100 nm)

544

0.37, 0.54

3.3

7942

11.9

9.90

3.73

(42)

48

ITO/NPB (60 nm)/48 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (100 nm)

516

0.27, 0.51

3.2

49993

15.7

12.9

5.12

(42)

48

ITO/48 (80 nm)/TPBi (40 nm) /LiF (1 nm)/Al (100 nm)

516

0.25, 0.50

3.2

13678

16.2

14.4

5.35

(42)

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195

AIEgen

Device configuration

λEL (nm)

CIE

Von (V)

Lmax (cd m-2)

ηC,max (cd A-1)

ηP,max (lm W-1)

ηext,max (%)

Ref.

49

ITO/HATCN(20 nm)/NPB (40 nm)/49 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (80 nm)

513

0.23, 0.46

4.2

65150

8.60

5.07

3.28

(43)

50

ITO/HATCN(20 nm)/NPB (40 nm)/50 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (80 nm)

500

0.20, 0.34

5.2

16410

4.49

2.57

2.16

(43)

51

ITO/HATCN(20 nm)/NPB (40 nm)/51 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (80 nm)

489

0.19, 0.30

4.9

14980

2.53

0.99

1.26

(43)

52

ITO/HATCN(20 nm)/NPB (40 nm)/52 (20 nm)/TPBi (40 nm)/LiF (1 nm)/Al (80 nm)

524

0.25, 0.52

4.8

30210

9.96

5.43

2.73

(43)

53

ITO/PEDOT:PSS(40 nm)/53 (70 nm)/TPBi (30 nm)/Ba (4 nm)/Al (120 nm)

543

0.37, 0.54

3.4

11665

8.3

7.5

2.6

(44)

54

ITO/PEDOT:PSS(40 nm)/54 (70 nm)/TPBi (30 nm)/Ba (4 nm)/Al (120 nm)

532

0.35, 0.53

3.4

7290

6.3

5.9

2.1

(44)

55

ITO/PEDOT:PSS(40 nm)/55 (70 nm)/TPBi (30 nm)/Ba (4 nm)/Al (120 nm)

521

0.34, 0.50

8.1

838

1.8

0.6

0.6

(44)

Abbreviations: λEL = electroluminescence maximum; Von = turn-on voltage at 1 cd m−2 ; Lmax = maximum luminance; ηC,max = maximum current efficiency; ηP,max = maximum power efficiency; ηext,max = maximum external quantum efficiency; CIE = Commission Internationale de I’Eclairage coordinates; NPB = N,N′-di(1-naphthyl)-N,N′-diphenylbenzidine; TPBi = 1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene; Bphen = 4,7-diphenyl-1,10-phenanthroline; Alq3 = Tris-(8-hydroxyquinoline)aluminum; PEDOT:PSS = poly(3,4-ethylenedioxythiophene)–poly-(styrenesulfonic acid); HATCN = 1,4,5,8,9,11-hexaazatriphenylene hexacarbonitrile. NPB functions as hole-transporting layer (HTL), TPBi and Bphen serve as electron-transporting layer (ETL) and hole-blocking layer (HBL), Alq3 functions as ETL, HATCN, PEDOT:PSS and MoO3 serves as hole-injection layer (HIL).

Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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5. Conclusion and Outlook Since the first report of AIE phenomenon, numerous fluorescent AIEgens with high ΦF values in the solid state have been created for the construction of stable and efficient non-doped OLEDs. The emission colors of these devices have covered the whole range of visible lights. The efficiencies of some OLEDs have approached or reached the theoretical limit range of fluorescent OLEDs. This chapter has summarized the applications of siloe-based and TPE-based AIEgens for non-doped fluorescent OLEDs with various emission colors and the multifunctional AIEgens that played multiple roles in OLEDs. The successes of AIE effect in improving the performance of common fluorophors (the first-generation luminescent materials) for OLEDs demonstrate the great academic and practical significance of AIE research. However, there is still much room for further improvement of efficiencies of fluorescent OLEDs because 75% of the generated excitons (triplet excitons) remain unemployed. Hence, many current efforts have been devoting to fabricating efficient OLEDs based on pure organic thermally activated delayed fluorescence (TADF) materials (the third-generation luminescent materials) which can make full use of singlet and triplet excitons. The TADF materials, however, also have some disadvantages, such as severe efficiency roll-off in OLEDs, and doping technique is usually required for OLED fabrication based on TADF materials. Integrating of both AIE and TADF effects within a molecule could be a promising strategy to construct more robust luminescent materials for high-performance non-doped OLEDs.

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32. Liu, Y.; Chen, S.; Lam, J. W. Y.; Lu, P.; Kwok, R. T. K.; Mahtab, F.; Kwok, H. S.; Tang, B. Z. Chem. Mater. 2011, 23, 2536–2544. 33. Qin, W.; Liu, J.; Chen, S.; Lam, J. W. Y.; Arseneault, M.; Yang, Z.; Zhao, Q.; Kwok, H. S.; Tang, B. Z. J. Mater. Chem. C 2014, 2, 3756–3761. 34. Yuan, W. Z.; Lu, P.; Chen, S.; Lam, J. W. Y.; Wang, Z.; Liu, Y.; Kwok, H. S.; Ma, Y.; Tang, B. Z. Adv. Mater. 2010, 22, 2159–2163. 35. Huang, J.; Sun, N.; Yang, J.; Tang, R.; Li, Q.; Ma, D.; Li, Z. Adv. Funct. Mater. 2014, 24, 7645–7654. 36. Kim, J. Y.; Yasuda, T.; Yang, Y. S.; Adachi, C. Adv. Mater. 2013, 25, 2666–2671. 37. Chen, L.; Jiang, Y.; Nie, H.; Lu, P.; Sung, H. H. Y.; Williams, I. D.; Kwok, H. S.; Huang, F.; Qin, A.; Zhao, Z.; Tang, B. Z. Adv. Funct. Mater. 2014, 24, 3621–3630. 38. Quan, C.; Nie, H.; Hu, R.; Qin, A.; Zhao, Z.; Tang, B. Z. Chin. J. Chem. 2015, 33, 842–846. 39. Quan, C.; Nie, H.; Zhao, Z.; Tang, B. Z. Organic Light Emitting Materials and Devices XIX, Proc. SPIE 9566, 95660C, September 22, 2015. 40. Yuan, W. Z.; Chen, S.; Lam, J. W. Y.; Deng, C.; Lu, P.; Sung, H. H. Y.; Williams, I. D.; Kwok, H. S.; Zhang, Y.; Tang, B. Z. Chem. Commun. 2011, 47, 11216–11218. 41. Liu, Y.; Chen, S.; Lam, J. W. Y.; Mahtab, F.; Kwok, H. S.; Tang, B. Z. J. Mater. Chem. 2012, 22, 5184–5189. 42. Chen, L.; Jiang, Y.; Nie, H.; Hu, R.; Kwok, H. S.; Huang, F.; Qin, A.; Zhao, Z.; Tang, B. Z. ACS Appl. Mater. Interfaces 2014, 6, 17215–17225. 43. Shi, H.; Xin, D.; Gu, D.; Zhang, P.; Peng, H.; Chen, S.; Lin, G.; Zhao, Z.; Tang, B. Z. J. Mater. Chem. C 2016, 4, 1228–1237. 44. Chen, L.; Zhang, C.; Lin, G.; Nie, H.; Luo, W.; Zhuang, Z.; Ding, S.; Hu, R.; Su, S.-J.; Huang, F.; Qin, A.; Zhao, Z.; Tang, B. Z. J. Mater. Chem. C 2016, 4, 2775–2783.

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Chapter 8

AIE Luminogens for Visualizing Cell Structures and Functions Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch008

Sean Lim,1 Ben Zhong Tang,2 and Yuning Hong*,1 1School

of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia 2Department of Chemistry, Hong Kong University of Science and Technnology, Clear Water Bay, Kowloon, Hong Kong *E-mail: [email protected]

Organic fluorogens with aggregation-induced emission (AIE) characteristics have demonstrated their potential to be ideal candidates for live cell imaging. Opposite to conventional organic dyes, the AIE luminogens are nonluminescent when molecularly dissolved but highly emissive upon aggregation. As small molecules, the AIE luminogens normally enter cells through diffusion, accumulate in the target location, and generate light emission. Inherently, they possess large Stokes shift with appreciable brightness and they are resistant to photobleaching, owing to the formation of aggregates inside the cells. The utilization of AIE dyes for visualizing the structures and dynamics of subcellular organelles such as mitochondria, lysosomes, and lipid droplets and for monitoring cell functions such as intracellular pH and viscosity will be discussed in this chapter.

Introduction Cells are the smallest units of life yet highly organized. To decode the sophisticated cellular processes and their association with diseases, it is essential to analyze changes of the microenvironment and dynamics of the intracellular compartments on site and in time (1). Among many analytical tools, fluorescence imaging is a non-invasive method that allows direct visualization with superior sensitivity and unraveling spatiotemporal resolution, which is well suited for © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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studying events at the cellular and molecular levels (2–4). However, even with the recent advancement in instrumentation, many cellular processes still remain invisible, and this calls for further exploration of versatile fluorescent imaging agents (5). In general, fluorescent protein, synthetic dyes, and fluorescent nanoparticles are the three prominent types of fluorescent probes for imaging (6–8). Genetically encoded fluorescent proteins enjoy the high specificity to the target of interest. Despite of the unwanted oligomerization problem, most of the fluorescent proteins are photolabile as compared to synthetic dyes and nanoparticles (9). Fluorescent nanoparticles such as quantum dots demonstrate excellent photostability and high luminescence quantum efficiency (10). However, they usually enter the cells via endocytosis processes, which limit their distributions and sensing regions to the endocytic compartments instead of the entire cytoplasm, not to mention that some of them are even cytotoxic. Organic fluorophores are the most widely used agents for cell imaging because of their simple operation and rich variety (7). They are small in size and less likely to perturb the functions of target molecules. Through robust organic synthesis, the chemical and photophysical properties of the organic fluorophores could be tailored for a specific application (11). Although numerous structures with different functionalities have been made, most of them are built on a limited selection of the conventional chromophores, such as fluorescein, coumarin, cyanine and boron-dipyrromethene (BODIPY) (12). These derivatives inherit the shortcomings of the parental fluorophores and suffer from small Stokes shift, concentration-quenching and poor photostability problems. Therefore, it is desirable if we can expand the pool of the core chromophores and develop new fluorescent probes that can inherently overcome these problems. In our search of the alternatives, luminogens with aggregation-induced emission (AIE) characteristics have attracted our attention. Opposite to conventional dyes, the AIE luminogens are non-emissive when molecularly dissolved, but become highly fluorescent in the aggregate state owing to the restriction of their intramolecular motions (13–16). The AIE dyes collect the merits of both small organic dyes and fluorescent nanoparticles. They can enter the cells through diffusion as small molecules and no complicated and time-consuming transfection process is required. Inside the cells, the dye molecules may accumulate in the target location to form aggregates and generate light emission. As aggregates, they are more resistant to photo-bleaching than conventional dyes which have to be used in very dilute solutions (15). These compelling attributes of the AIE luminogens may provide a new platform for the development of a new generation of fluorescent probes for cell imaging. In this chapter, a series of AIE luminogens and their applications in specific organelle imaging and intracellular environment sensing will be discussed.

Fluorescence Imaging of Cell Structures Mitochondria Mitochondria are the membrane-bound organelle widely existing in most eukaryotic cells, with various pivotal functions under both physiological 200 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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status and pathological conditions (17). Prominently, mitochondria, known as “powerplants” for the cell, supply the most of cellular energy currency - adenosine triphosphate (ATP). The production of ATP involves a series of electron-transport systems to the oxidative phosphorylation reaction. Accompanying with this process, reactive oxygen species (ROS) are generated; the accumulation of ROS in mitochondria will raise the risk of cell damage and cell death (18). The programmed cell death, as called apoptosis, is initiated by the permeabilization of the mitochondrial outer membrane and the release of the pro-apoptotic proteins to the cytosol (19). Owing to the central role in the life and death of the cell, the characteristics of mitochondria have become a important indicator for cell functions.

Morphology Tracking Mitochondria are dynamic organelles that often change their shapes and distribution (20). Under normal physiological conditions, mitochondria form physically interconnected networks in order to deliver nutrition, transfer metabolites and redistribute calcium. However, in pathological (e.g. hyperglycemia, apoptosis, etc.) or diseased conditions (Alzheimer’s ischemic and hemorrhagic stroke, etc.), the mitochondria can undergo fragmentation to form short, round mitochondria (19). Tracking the dynamics of mitochondria morphology can thus provide direct evidence for studying the physiological changes and pathological mechanism of mitochondria disordered diseases (21). To follow the change of mitochondria morphology over time, fluorescent probes with high specificity and photostability are highly desirable. However, most of the conventional dyes suffer from poor photostability and hence imaging over an extended period of time is not possible. The AIE fluorogens, which often consist of multiple phenyl rings, are hydrophobic and tend to form nanoaggregates when dispersed in aqueous buffer solution or cell culture media (22). These nanoaggregates possess better photostability than single fluorescent molecules because even the outermost layer of the nanoaggregates are damaged by excitation light, it forms a protective layer to avoid the contact of the inner part to oxygen species and hamper further photobleaching. The photostability of the AIE active dyes is improved, which allow the tracking of mitochondria dynamics for a long period of time. Triphenylphosphonium (TPP) moiety was found to be a mitochondriatargeting group. Linking TPP with tetraphenylethene (TPE), a typical AIE luminogen, generated a mitochondria-targeting AIE fluorogen (1, Figure 1) (23). Cytotoicity of 1 was evaluated using an MTT assay, which showed only little effect of 1 on cell viability. The selectivity of 1 to mitochondria was examined by the co-staining experiment with the commercial MitoTracker Red FM (MT) (Figure 1A-C). The results suggested that the perfect colocalization of the signals from 1 and MT with the Pearson’s correlation coefficient (Rr) of 0.96. The photostability was investigated using confocal fluorescent imaging to produce continuous scans of HeLa cells stained with 1 or MT. The loss of emission intensity (%) from the cells was measured over the time frame. The signal loss 201 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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from 1 was less than 20% during 50 scans, while for MT, about 75% of the signals lost during only 6 scans (Figure 1D). The results demonstrated the superior photostability of the AIE fluorogen.

Figure 1. Fluorescent images of HeLa cells stained with (A) 1 and (B) MitoTracker Red FM (MT). (C) Merged image. (D) Signal loss (%) of fluorescent emission of 1 (solid circle) and MT (open circle)with increase number of scans. Inset: fluorescent imagings of 1-stained cells with increase number of scans. Reproduced with permission from ref. (23). Copyright (2013) American Chemical Society. The approach demonstrated above allows the use of high fluorophore concentration to compensate the photobleaching effect encountered by most conventional probes (23). In order to enrich the palette of the AIE mitochondria dyes, a yellow-emissive AIE dye, 2 was developed (24). With the positive pyridinium moiety, 2 exhibits excellent specificity to mitochondria, comparable to that of MT. Similar as 1, 2 exhibited excellent photostability and biocompatibility. The morphological dynamics of mitochondria was monitored by using 2 under 202 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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physiological and pathological conditions. To model the changes in mitochondrial morphology in cells of diabetes patients, 2 was used to staine HeLa cells, which then were exposed to high concentration of D-glucose. Under such hyperglycemia condition, short, vesicular structure of mitochondria were found, which were completely different from the elongated, tubular structure in the untreated cells (Figure 2). This mitochondrial fragmentation was found to be reversible, as after treatment of the cells the mitochondria gradually returned to its elongated state.

Figure 2. Hyperglycemic-induced mitochondria fragmentation of HeLa cells. The cells was stained with 2 and then incubated with 40 mM d-glucose solution for (A,B) 0, (C,D) 15, (E,F) 60 and (G,H) 120 min, respectively.

Membrane Potential Measurement The potential across the mitochondrial membrane is measured by the parameter ΔΨm, reflecting the proton gradient that is maintained to drive respiration and ATP synthesis in mitochondria (25). Changes in this potential is therefore indicative of mitochondrial health and is closely related to cellular function. Existing dyes that are selective to mitochondria exhibit aggregation-caused quenching (ACQ) properties, hence only a dilute solution of the dye can be used without causing quenching. This in turn leads to photobleaching by the excitatory light source and reduced emission. Furthermore, using such dyes to measure ΔΨm means that their concentrations in the mitochondria is constantly changing based 203 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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on the proton gradient – if there is an increased concentration of dye within the cell, emission readings could go either way, leading to inaccurate measurements of ΔΨm (26).

Figure 3. (A) Changes in fluorescence intensity of 3-stained HeLa cells upon treated with oligomycin and then CCCP. Inset: snapshots of the cell images taken upon the addition of the stimulants. (B) Fluorescent intensity of the unstained HeLa cells (blank), oligomycin treated and CCCP treated 3-stained HeLa cells analysed by flow cytometry. (C) Confocal images of mouse sperm cells stained with 3 showing the mitochondria and (D) Hoechst 33342 indicating nuclei. (E) The merged picture of panel C and D and the bright field image. Scale bar: 20 μm. Reproduced with permission from ref. (27). Copyright (2015) Royal Society of Chemistry. The abovementioned AIE-active mitochondria dye, 1 and 2, are basically insensitive to the change in ΔΨm. In our recent finding, the indolium salt of TPE derivative, 3, was reported to be mitochondria specific and sensitive to ΔΨm (27). 3 has long excitation (534 nm) and emission wavelength (585 nm) owing to the 204 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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intramolecular charge transfer between the donor and acceptor groups. Thanks to its AIE property, 3 also possesses superior photostability and when used in cell imaging. The viability of 3 for real-time ΔΨm tracking in HeLa cells was investigated, manipulating ΔΨm prior to the addition of the dye by adding oligomycin or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to increase and decrease ΔΨm, respectively. As shown in Figure 3A, 3 was able to accumulate in the mitochondria, showing an increase in its fluorescence intensity when oligomycin was added, then a decrease when CCCP was added. The advantage of using the AIE-active 3 is that the fluorescence values can be used to establish a direct correlation between intensity and ΔΨm. The method of measuring ΔΨm change is also applicable by using flow cytometry, showing similar results to those generated by confocal microscopy – highlighting the potential of 3 for high throughput analysis of mitochondrial cells (Figure 3B). Furthermore, ΔΨm is a critical parameter determining the viability and fertilization potential of a sperm, the germ cell of males (28). To demonstrate the utility of 3 in this aspect, 3 was used to stain mouse sperm cells, which contained a large concentration of mitochondria (Figure 3C-E). By utilizing its specificity and sensitivity to ΔΨm, identification of energetic and non-energetic sperm cells was possible by quantifying their fluorescence intensities. Further uses of 3 as a probe will include drug screening for tumors with increased ΔΨm, and apoptotic cells with decreased ΔΨm.

Dual Functional Mitochondrial Probe The use of fluorophores to target and illuminate cellular structures has garnered the interest of researchers in recent times. Related to this is the growing field of photodynamic therapy (PDT) - the use of photosensitive species that produce ROS and induces apoptosis in cells (29). Existing photosensitizers are mostly porphyrin and phenylthiazinium derivatives that suffer from long irradiation time and limited specificity, owing to the permeability of the nucleus being tightly controlled (30). In light of this, targeting the mitochondria as a way to induce apoptosis is a favorable solution owing to the essential role of mitochondria in apoptosis. Isoquinolinium functionalized TPE derivative (4), with the positive charge, can target and visualize mitochondria in live and fixed cells with high selectivity comparable to that of MT (31). Intriguingly, this compound can also serve as a photosensitizer to promote the ROS generation in the mitochondrial region upon photoirradition, which induces cell apoptosis, as exemplified by the fragmention of mitochondria in cells (Figure 4). The photosensitizing property of 4 was quantified by using H2DCF-DA, a commercially available ROS probe which fluoresces at 530 nm in the presence of ROS. When irradiated by 365 nm UV light, emission from H2DCF-DA in a solution containing 4 intensified over time, suggesting the ROS generation by 4.

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Figure 4. Fluorescence images of HeLa cells stained with 4 for 10 min and exposed to UV irradiation for 10 s. Reproduced with permission from ref. (31). Copyright (2014) Royal Society of Chemistry.

Cytotoxicity of 4 was evaluated using a lactate dehydrogenase (LDH) assay in HeLa cells, which measures the amount of LDH released from the damaged membrane of dead cells. Results showed that 4 in cells did not alter their viability under the concentration up to 1 μM. However, upon exposure to UV radiation, even 500 nM of 4 can cause significant cytotoxicity with only about 50% of cell viability. The results were further confirmed by using a cell impermeable dye, propidium iodide, as it only stains cells with a damaged membrane. Red emission recorded from cells exposed to UV irradiation indicated that ROS generation had indeed killed these cells, while cells not exposed to UV irradiation could not be stained in this manner. The dual functions of 4, as a mitochondria imaging agent and photosensitizer for PDT, offer the possibility of image-guided therapy by generating ROS to induce cell apoptosis and tracking the mitochondrial dynamics at the same time to monitor the efficiency of the PDT.

Lysosomes Studying lysosomal activities provides important information relating to autophagy – the disassembly of unwanted and unnecessary components that accompany cell destruction – which in turn shows a relationship to ageing, longevity, as well as cancers and neurodegenerative diseases (32). The autophagosomes responsible for the delivery of such components fuses with lysosomes, which are then degraded by hydrolases within them. Hence a lysosome probe will be useful for the study of autophagy activities under pathological conditions.

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Visualising Autophagy Processes

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A lysosome-targeting AIE dye (5) has been developed based on the fluorophore with excited-state intramolecular proton transfer (ESIPT) property and a morpholino moiety for lysosome targeting (Figure 5) (33). Because of its ESIPT characteristics, 5 exhibits two different wavelengths of yellow emission: keto emission produces a longer wavelength while enol emission produces a shorter one. This is caused by the intramolecular hydrogen bond formed in 5 in its keto form, which is absent in its enol form.

Figure 5. (A−E) Fluorescence images of 5-stained HeLa cells before and after rapamycin treatment (50 µg/mL) for different periods of time. (F) Enlarged region of interest of panel E. Scale bar: (A−E) 30 μm, (F) 10 μm. λex = 400−440 nm. Reproduced with permission from ref. (33). Copyright (2015) Wiley. MTT assay was used to assess the cytotoxicity of 5, which revealed that after 24 hour incubation with 5 the HeLa cells still showed 80% viability that implies low interference of 5 on cell growth. Then the lysosome targeting ability was investigated by staining HeLa cells with both 5 and the commercial LysoTracker Red DND-99 (LTR). The results showed that both signals overlap very well (Rr = 0.90) meaning 5 was indeed lysosome selective. It was found that 5 exhibited higher contrast and brighter emission in comparison with LTR; by introducing both dyes to cells it was found that they compete for the same site but 5 possesses higher contrast, providing evidence for its superior affinity to lysosomes. Furthermore, similar as the other AIE dyes, 5 possesses better photostability than LTR. 207 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

5 was then used to visualize autophagy under physiological conditions by employing the use of mammalian target of rapamycin (mTOR), an autophagy suppressant. Rapamycin is a lipophilic antibiotic that binds to mTOR and is used to induce autophagy in prion diseases, by activating autophagy and prion protein degradation (34). 5 was used to visualize HeLa cells treated with rapamycin; during the autophagy process, lysosome count increased and fused with the autophagosome to form an autolysosome which can be clearly visualized by the emission of 5 (Figure 5).

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Lipid Droplets Lipids are stored in cells in the form of lipid droplets (LD), which also regulate lipid metabolism and transfer, protein degradation and signal transduction (35). Failure to regulate these processes are linked to a number of diseases, and abnormal LD amount is used as a biomarker in diseases such as infection by the hepatitis C virus. Hence monitoring LD volume and concentration is important in biomedical research to pinpoint diseases – mainly liver related diseases at the early stage (36).

Figure 6. HeLa cells stained with (A-C) 6 and (D-F) Nile red. Images taken under bright field (A,D) and photoexcitation (B,E) by fluorescence microscopy. Panels C,F show merged images. Reproduced with permission from ref. (38). Copyright (2014) Royal Society of Chemistry. 208 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Current tomography scanning techniques that detect LD levels and liver diseases are relatively insensitive and inaccurate, only able to pick up on diseases in their later stages (37). Fluorescent dyes that can selectively illuminate LDs could improve the sensitivity in the diagnosis process. TPE derivative decorated with the electron donating and accepting groups (6) showing polarity dependent flurorescence were synthesized and used for the staining of LDs (38). The emission of 6 is found to be blue-shifted with the increase of hydrophobicity of the solvents. Due to the lipophilicity of the dye, 6 can quickly target LDs in cells (10-15 minutes after incubation), which can be seen as spherical objects in the cytosol under fluorescence microscopy (Figure 6A-C). A parallel experiment was conducted using Nile red dye, which is commonly used to stain LDs. However, compared to 6, the Nile red dye was not selective to LDs; it also stained other organelles such as mitochondria, as seen in Figure 6D-E. As lipid content is an important parameter to determine the value of algaes as a biofuel, 6 can also be used for the screening of algaes (38). 6 was introduced into the green algae Nano by diffusion, after which the diameter of the emission region can be used to quantify the size of LDs within the cytoplasm. In this way, the viability of algae as an environmentally friendly fuel can be assessed.

Fluorescence Imaging of Cellular Environment Intracellular pH The pH of a cell is an important indicator of its overall health, as it regulates cellular functions such as apoptosis, proliferation and protein signaling (39). Monitoring the intracellular pH is possible through the use of a number of methods such as microelectrodes, NMR spectroscopy and optical microscopy. Fluorescent dyes are powerful pH probes due to their high sensitivity and resolution, yet currently available ones have narrow spans that do not cover the entire pH range in physiological environment (40). Alternative approach by doping multiple pH-sensitive dyes into nanoparticle matrixes has been reported to achieve the full-range pH sensing (41). However, the uneven distribution of nanoparticles in endocytic compartments restricts their sensing area over the entire cytoplasm. To circumvent these problems, an AIE active, pH responsive fluorogen based on TPE-cyanine adduct (7) was synthesized, which is cell permeable and has a broad pH-sensing range (Figure 7) (42). 7 can react with either OH- or H+ to produce red or blue emissions of varying intensity (Figure 7A). The aqueous solution of 7 under neutral condition was weakly fluorescent in red region owing to its amphiphilicity. In acidic conditions, the sulfonate group of 7 was protonated (7A), resulting in poor solubility in water and thus higher fluorescence intensity because of the AIE property. Under alkaline conditions, the OH- can act as a nucleophile to break down the conjugation of 7 (7B), thus changing the emission color from red to blue. The presence of 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), the most abundant phospholipid in cellular membrane, facilitates the nucleophilic reaction and promotes the transition of red-to-blue emission at around pH 6.5, much lower than the one in the absence of DOPC (~pH 10) (Figure 7B). The ratio of the blue-to-red emission 209 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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can thus be correlated to the pH change in the physiological conditions from pH 4.5 to 8. In cell imaging, fluorescence signals of 7 were collected in two channels – the blue channel by excited at 405 nm and the red channel by excited at 488 nm. The intracellular pH can thus be mapped by using the ratio of the fluorescence intensity from the two channels, which revealed the most acidic compartment-lysosomein pseudo red color and the most alkaline compartment-mitochondria in pseudo blue color (Figure 7C). This ratio to pH relationship was further investigated using cells incubated in HEPES buffer, which kept the cell at a constant pH 7; analysis showed less than 15% of the cell was acidic. Introducing acetic acid to the living cells increased this percentage to 20%, while Dulbecco’s Modified Eagle Medium (DMEM, pH 8.5) caused the acidic regions to shrink in size. Flow cytometry can also be used to track intracellular pH by using 7 for a high-throughput pH sensing, which could be of use in a variety of assays.

Figure 7. (A) Fluorescent response of 7 to pH change. Structures of 7 and corresponding 7A and 7B after reacting with H+ and OH- respectively. (B) Plot of the ratio of the blue-to-red emission (I489/I615) of 7 in buffer solutions with different pH in the presence of DOPC. Inset: the corresponding fluorescence photographs. (C) Ratiometric fluorescence image of HeLa cells stained with 7, shown in pseudo colors from red to blue indicating low to high blue-to-red emission ratios. Scale bar 20 µm. Reproduced with permission from ref. (42). Copyright (2013) American Chemical Society. 210 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Intracellular Viscosity Intracellular viscosity is an important indicator of cell function; it affects processes such as transportation and signal transduction, and abnormalities in viscosity is associated with a range of diseases (43). Current methods in place to measure intracellular viscosity include fluorescence recovery after photobleaching (FRAP), which employs the use of a high powered laser to bleach an area of a cell, and by measuring the recovery rate the viscosity can then be deduced (44). However, FRAP can only focus on a small area of interest in the cell and it is not an efficient method to study the intracellular viscosity in the entire cytoplasmic region. Using an AIE active fluorescent probe, it is possible to correlate its fluorescence intensity and lifetime to cell viscosity, due to its mechanism of fluorescence by restriction of intramolecular motions being affected by its surroundings (45). 7 was chosen as a potential probe, which when exposed to basic conditions is transformed into 7B and exhibiting blue emission (Figure 7). The blue emission from 7B was found to be weak in pure ethylene glycol solvent, but increasing the viscosity by increasing the glycerol fractions caused enhanced emission, due to reducing the intramolecular motions of the dye and hence inhibiting the non-radiative pathways of decay. Consistent with the change in fluorescence intensity, the fluorescence lifetime was also prolonged with the increase of viscosity. To mimic the membrane environment in cells, artificial lipid vesicles were fabricated and 7B was used to quantify the fluidity within them. DOPC, 1,2diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) with cholesterol – membrane lipid components with different degrees of saturation – were used to represent membranes with different fluidity (Figure 8A). As DOPC has unsaturated bonds, it packs less tightly than DOPC with cholesterol, and the saturated tails of DHPC imply it can pack closer to each other. DSPC with the long saturated fatty acid chains pack the closest and leads to the highest viscosity among these models. Similar as the results in solution, 7B exhibited longer lifetime with the increase of membrane rigidity, or in other word, the decrease of membrane fluidity (Figure 8B). Following this, 7 was introduced into the cytoplasm of live HeLa cells from comparing fluorescent images with Nile red – a lipid droplet (LD) selective stain – was found to accumulate around these LDs as well as other organelles due to the lipophilicity of 7. In order to map the fluorescence lifetime of the stained regions in the cell, two photon excitation was used so as to minimize interference from intrinsic autofluorescence (Figure 8C). From the results gathered, a variety of viscosity was shown to exist within the cytoplasm, with the fluorescence lifetime much shorter in LDs than around structures such as tubular mitochondria. Different from most of the membrane-bound organelles with lipid bilayer membranes, LDs are surrounded by a lipid monolayer, which is less rigid. The hydrophobic AIE dyes can dissolve in the lipid pool stored inside LDs where the dye molecules can enjoy free intramolecular motions, leading to shorter fluorescence lifetime. 211 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 8. (A) The proposed packing modes of membranes with different lipid compositions. (B) Fluorescece lifetime of 7 stained lipid vesicles. (C) FLIM images of HeLa cells stained with 7 by using two-photon excitation. Reproduced with permission from ref. (45). Copyright (2015) Wiley.

Conclusion and Perspective In this chapter, we have introduced a family of imaging agents with aggregation-induced emission (AIE) properties and their applications in visualizing the cell structures and monitoring the change in cellular environment. Conventional fluorescent dyes suffer from concentration-quenching effect, and thus only very dilute concentrations of these imaging agents can be used. The small number of these dye molecules can be easily photobleached under the harsh excitation light source, resulting in poor photostability which is not suitable for long-term tracking of the cell events. As alternatives, the AIE dyes exhibit superior photostability, which allows the observation of the dynamic processes in live cells over a long period of time. These dyes show excellent biocompatibility, posing little effect on the cell viability. Through structural modification, specific targeting for organelle imaging and morphological tracking has been realized. Most of our early work on sensing and imaging were based on the change of single-wavelength fluorescence intensity, which might not be suitable for quantitative analysis. The uneven dye distribution and other technical artifacts may also affect the intensity. To overcome these problems, we later on developed AIE luminogens for ratiometric imaging of intracellular pH and lifetime imaging of intracellular viscosity, from which quantative analysis became possible. Dual functional AIE dyes that can monitor and manipulate cell activities have also been developed. A couple of AIE dyes that can induce cell apoptosis through 212 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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photoexcitation have been found, which could be alternative to the current photodynamic therapy systems. In addition to the few examples shown in this chapter, the small molecular AIE luminogens have also been found for nucleic acids in cells (46), long-term cell tracing (22, 47), evaluation of bacteria viability assay (48), enzyme activity detection in cells (49), etc. On the other hand, AIE dyes functionalized with short peptides for specific targeting of proteins of interest and AIE nanoparticles for in vivo imaging have been reported by Liu et al (50, 51) and will be covered in the other chapters of this book. Because of the multiple advantages of the AIE dyes, new imaging agents based on AIE materials should be developed. For zooming into the cells, with the advancement in imaging techniques such as super-resolution microscopy, these new dyes would be useful to visualize biological structures and dynamic intracellular events beyond the diffraction barriers. For zooming out of the cells, the AIE luminogens could be further modified for tissue and in vivo imaging through multi-photon excitation. In perspective, multifunctional AIE materials synthesized by combining fluorescence with other modalities (e.g., magnetic resonance imaging) or functionalities (e.g., photodynamic therapy) will be explored as new generation of theranostic reagents.

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Chapter 9

AIE Nanoparticles for in Vitro and in Vivo Imaging Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch009

Duo Mao and Dan Ding* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, P. R. China *E-mail: [email protected]

In recent years, fluorogens with aggregation-induced emission (AIE) characteristics have attracted considerable research interest in design and preparation of fluorescent organic nanoparticles (NPs) for bioimaging. So far, plenty of AIE NPs have been successfully utilized for in vitro and in vivo fluorescence imaging applications, which show outstanding performances by virtue of their unique advantages in terms of high brightness, excellent photostability, free of random blinking, facile cell internalization and superb cellular retention as well as negligible cytotoxicity and in vivo toxicity. In this chapter, the recent status of the development of AIE NPs for in vitro fluorescence imaging was summarized according to their utilizations, including non-specific cell imaging, targeted cancer cell imaging, specific organelle imaging, in vitro long-term cell tracking, and bacterial imaging. Furthermore, we also discuss the in vivo applications of AIE NPs in in vivo fluorescence imaging of tumors, intravital two-photon fluorescence imaging, in vivo long-term cell tracking as well as in vivo dual-modality imaging. The perspectives for the future investigation of advanced AIE NPs for bioimaging are discussed in this chapter as well.

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Introduction In recent years, considerable research interest has focused on the biocompatible nanostructured materials for in vitro and in vivo imaging (1–3). Versatile imaging modalities, including fluorescence imaging, magnetic resonance imaging (MRI) and nuclear imaging (such as positron emission tomography (PET) and single photon emission computed tomography (SPECT)), et al., along with the corresponding imaging contrast agents have been becoming increasingly prosperous (4–6). Among them, fluorescence imaging has been extensively investigated and attracted great attentions as this technique holds the advantages of high sensitivity, excellent temporal resolution, good safety, large in vitro and in vivo throughputs and manoeuvrable imaging instruments (7). To date, various fluorescent materials, which include organic dyes (8), fluorescent proteins (9) and inorganic semiconducting quantum dots (QDs) (10), have been widely employed for the purpose of in vitro and in vivo bioimaging. However, each material suffers its own limitations, such as low molar absorptivity, small Stokes shift, limited photobleaching thresholds or high cytotoxicity (11, 12). Exploration of alternative fluorescent materials with improved properties is highly desirable. Fluorescent organic nanoparticles (NPs) have recently emerged as a new generation of nanoprobes for bioimaging, which show such merits as flexible synthetic approaches, good biocompatibility and facile surface functionalization (13, 14). Besides, the NP probes are able to passively accumulate into many disease regions (i.e., tumor or inflammatory sites) by the enhanced permeability and retention (EPR) effect (15). As highly emissive probes are always desirable for high contrast imaging, it would be ideal that the brightness of the fluorescent organic NP is proportional to the amount of its doped fluorophores. Unfortunately, due to the well-known aggregation-caused quenching (ACQ) effect, conventional fluorophores that often possess planar aromatic structures suffer from severe emission quenching at high dye loading contents in NPs. This is because the doped dye molecules are prone to aggregation in NPs when a high fluorophore loading is achieved, which leads to π-π stacking as well as other non-radiative pathways and thus quenches the light emission (16). Tang’s group has recently developed a novel class of organic fluorogens with aggregation-induced emission (AIE) signature, which is exactly opposite to the ACQ effect (17, 18). The AIE fluorogens (AIEgens) are brightly emissive in the aggregate state by virtue of the restriction of intramolecular motion (RIM) mechanism (19). Consequently, the emergence of AIEgens has well addressed the concerns of emission quenching resulting from the high fluorogen loading in NPs, which has also opened up new opportunities for fabricating super-bright fluorescent organic NPs applied for bioimaging. In this chapter, we sum up the recent status and advances in the development of AIE NPs for in vitro and in vivo imaging. Considering that there are vast amounts of successful examples of AIE NPs for bioimaging, we divide them into two sections: 1) in vitro and 2) in vivo imaging applications. We then summarize each of the two sections in detail according to the utilizations of AIE NPs. Finally, the perspectives for the future exploration of advanced AIE NPs for bioimaging are discussed. 218 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

AIE NPs for in Vitro Imaging So far, AIE NPs have been extensively investigated for in vitro fluorescence imaging, as AIE-involved imaging systems exhibit such features as high fluorescence, excellent photostability, superb biocompatibility, and free of random blinking (20). In this section, the in vitro imaging applications of AIE NPs will be summarized in terms of non-specific cell imaging, targeted cancer cell imaging, specific organelle imaging, in vitro long-term cell tracking, and bacterial imaging.

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Non-Specific Cell Imaging Cell imaging with fluorescent probes is of great importance for providing the scientists and clinicians with sights and insights into the function and mechanism of biological species and processes in cells (21). The development of AIE NPs offers a new avenue for cell imaging with high brightness and excellent photostability. To date, a variety of AIE NPs including pure AIE fluorogen nanoaggregates (NPs formed by only small AIE molecules without using any encapsulation matrix) (22), AIEgen-based silica NPs (23–25), AIEgen-based block co-polymer NPs (26) and in situ AIEgen-polymerized NPs (27–36) et al., were utilized for non-specific cell imaging. For example, several research groups focused on the design and synthesis of AIEgen-based silica NPs, as silica NPs are known to be hydrophilic, biocompatible and size-tunable (23–25). AIEgen-based silica NPs that are colloidally stable could be prepared via the reactions between tetraphenylethene (TPE)- (compound 1) or silole-functionalized siloxane (compound 2) and tetraethoxysilane (TEOS) (23). The size of the obtained NPs is uniform and can be tunable by altering the reaction conditions (45-295 nm). The fluorescent AIE-based silica NPs have been demonstrated to be non-toxic against the cancer cells and internalized into the cell cytoplasm for imaging (Figure 1). Moreover, in situ AIEgen-polymerized NPs refer to covalently bonded AIE-polymer NPs, which were prepared by in situ polymerizing AIEgen into a polymer chain. Wei’s group has made a great effort on such NPs. Through synthesizing different polymerizable AIEgens, they employed a variety of polymerization methods such as emulsion polymerization (27, 28), reversible addition-fragmentation chain transfer (RAFT) polymerization (29–31), anhydride ring-opening polymerization (32–35), and cross-linked polymerization (36) to generate a library of amphiphilic AIE-polymers, which were able to self-assemble into fluorescent NPs in aqueous solution. These in situ AIEgen-polymerized NPs show the advantages of compact structure without dye leaking and surface coating detachment in harsh biological environments as well as good cytocompatibility and high cell uptake efficiency in cellular imaging application.

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Figure 1. (A) Chemical structures of Compounds 1 and 2, as well as the synthetic route to 1- or 2-based silica NPs. Fluorescence images of HeLa cancer cells stained with (B) 1- and (C) 2-based silica NPs. [1] = 8 μM; [2] = 6 μM. Adapted with permission from ref (23). Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Targeted Imaging of Cancer Cells Targeted imaging of cancer cells is highly valuable for recognition of the class and subclass of cancers, diagnosis and treatment of cancers at early stage, and evaluation of the anticancer efficacy of drugs (2). One of the most efficient strategies to promote the selectivity and targeting effect of the fluorescent NPs toward cancer cells is to modify the NP surfaces with specific targeting ligands. Generally, the targeting ligands possess selective interaction with receptors that are overexpressed in the cell membrane (11, 37). In the field of AIE NPs, many a ligand, including folic acid (38–40), RGD peptide (41) and biotin (42) et al. have been widely utilized for targeted cancer cell imaging. For instance, several groups selected folic acid as the targeting ligand to functionalize the surface of AIE NPs, as there is high binding affinity between folic acid and folate receptors. The folate receptors have been well established to be overexpressed in many kinds of cancer cells, whereas at a low expression level in normal cells (43). Liu and co-workers reported the design and preparation of compound 3-based AIE NPs with folic acid-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethyleneglycol) (DSPE-PEG5000-folic acid) and DSPE-PEG2000 as the encapsulation matrix (Figure 2A) (38). The 3-encapsulated AIE NPs have a spherical morphology with 220 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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mean hydrodynamic size of around 85 nm, high quantum yield of 24%, and low cytotoxicity against MCF-7 breast cancer cells. Besides, such AIE NPs exhibit much superior selectivity toward MCF-7 cancer cells to NIH/3T3 normal cells. The fluorescence intensity of 3-encapsulated AIE NP-stained MCF-7 cancer cells can be further enhanced with the increase of folic acid density on the NP surface, which saturates when the feed ratio of DSPE-PEG5000-folic acid in the matrix is 40% (Figure 2B).

Figure 2. (A) Chemical structures of Compound 3, DSPE-PEG2000 and DSPE-PEG5000-folic acid, as well as the schematic illustration of the formation of 3-encapsulated AIE NPs. (B) Confocal images of MCF-7 cancer cells after incubation with 3-encapsulated AIE NPs with various folic acid densities on the NP surfaces for 2 h at 37 °C. The feed ratio of DSPE-PEG5000-folic acid in the matrix is 0%, 20%, 40% and 60%, respectively. Adapted with permission from ref (38). Copyright 2011 Royal Society of Chemistry. Specific Organelle Imaging Fluorescent probes that are capable of visualizing specific organelles have been attracting considerable research interest, as the organelles play key roles on cell function, growth and death (44, 45). In particular, mitochondrion is an important organelle that is found in nearly all eukaryotes and generates the energy currency of the cells (46). As mitochondria are involved in plenty of human 221 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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diseases, which include mitochondrial disorders, cardiac dysfunction, and heart failure (47), specific mitochondrion imaging is particularly valuable. Recently, a series of AIEgens with different emission colors have been explored for specific mitochondrial imaging (48–52). In general, these AIEgens can form NPs in aqueous solution without any encapsulation matrix. Taking compound 4 as an example, 4 is composed of one TPE and two triphenyphosphonium (TPP) moieties, and TPP has been extensively reported as a mitochondria-specific targeting ligand (48). Additionally, 4 appears as AIE NPs in aqueous solution with an average diameter of about 144 nm. These blue color emissive NPs of 4 have been demonstrated to be able to clearly stain the mitochondria (reticulum structures) of live HeLa cells, which was further confirmed by the good co-localization of NPs with commercial mitochondria probe MitoTracker red FM (Figure 3A).

Figure 3. Chemical structures of 4-6. (A) Fluorescent images of 4 (5 μM) and MitoTracker red FM (50 nM) co-stained HeLa cells. Reprinted with permission from ref (48). Copyright 2013 American Chemical Society. (B) Fluorescent images of 5 (5 μM) and MitoTracker red FM (100 nM) co-stained HeLa cells. Reprinted with permission from ref (49). Copyright 2013 Royal Society of Chemistry. (C) Confocal images of 6 (5 μM) and Mito-GFP (CellLight® Mitochondria-green fluorescent protein) co-stained HeLa cells. Reprinted with permission from ref (50). Copyright 2015 Royal Society of Chemistry. 222 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Subsequently, Tang’s group developed AIEgens of 5 and 6 with yellow and red fluorescence, respectively (49, 50). Each of the two AIEgens consists of a large hydrophobic moiety donated by TPE motif and other π–conjugated groups as well as a positively charged head. Thanks to such intelligent molecular design, both the NPs of 5 and 6 can vividly visualize the mitochondria with extremely high resolution, using MitoTracker red FM and CellLight® Mitochondria-green fluorescent protein (Mito-GFP) as the co-staining agent, respectively (Figures 3B and 3C). It is important to note that all of the aforementioned mitochondria-specific AIE NPs show excellent photostability with high photobleaching resistence, making them ideal probes for long-term monitoring. Noteworthy is that in addition to mitochondria-staining AIE NP probes, the ones that can selectively visualize other organelles such as lysosome (53) and lipid droplets (54) have also been developed with bright emission, high resolution and large photobleaching threshold. Given these facts, AIEgens can serve as promising fluorescent materials in creating photostable fluorescent NP probes for specific organelle imaging.

In Vitro Long-Term Cell Tracking Fluorescent probes that have the ability in continuous tracking the cells over a long period of time have received great attention (7). GFP and its variants have been extensively reported as genetic cell tagging for cell tracing (55); however, the GFP labeling method suffers from high cost and safety issues owing to the introduction of random insertional mutation at integration sites (56). In addition to GFP, inorganic semiconducting QDs have been widely utilized as cell trackers, as they hold the advantages of high brightness, good photostability and nanoscale sizes that enable superb retention in living cells without leaking out from cytoplasm (57, 58). So far, several QD-based cell tracing reagents have been commercialized. Nevertheless, the oxidative degradation of the heavy metal components followed by heavy metal ions releasing makes QDs rather toxic against cells (12). As a result, only relative low concentrations of QDs can be safely used to label cells, which undoubtedly influence the long-term tracking effect. Moreover, as compared to organic NPs, inorganic QDs are more inclined to aggregate in biological systems, leading to fluorescence quenching (59). These concerns significantly limit the practical application of QDs in long-term cell tracking. Recently, a series of AIE NPs have been explored for in vitro cell tracking application, which exhibit such merits as bright emission, high cell labeling efficiency, excellent photostability, low cytotoxicity, superb retention in live cells without leaking during proliferation, as well as long cell tracking period (60–64). For instance, Tang’s group reported an AIE-active chitosan-TPE conjugate (compound 7) by covalent conjugation of isothiocyanate-bearing TPE molecules onto chitosan chains (60). The chitosan-TPE conjugates can be readily internalized by HeLa cancer cells, which have been testified to be capable of rooting within the cells and efficiently tracing the living HeLa cells for 15 passages (Figure 4). The remarkable long-term cell tracking capability 223 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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of chitosan-TPE conjugates is much superior to that of commercial CellTracker Green CMFDA, which was reported to track the cells for no more than 3 passages. For another example, Liu, Tang and co-workers synthesized an AIEgen (8) with high far-red/near-infrared (FR/NIR) fluorescence in the aggregate state (61). Then 8 was encapsulated using DSPE-PEG2000 and its amine end-capped DSPEPEG2000-NH2 as the matrix, affording 8-doped AIE NPs. The surfaces of NPs were further functionalized by cell penetrating peptide HIV-1 Tat via carbodiimidemediated coupling. The resulted 8-doped AIE-Tat NPs have spherical morphology with a mean size of around 30 nm, high quantum yield of 24% as well as strong photobleaching resistance. Both the flow cytometry and confocal imaging studies indicate that the 8-doped AIE-Tat NPs can efficiently track MCF-7 cancer cells up to 12 generations, whereas the commercial Qtracker® 655 is only able to trace 5-6 generations of the cells, revealing the far superior in vitro cell tracking capacity of 8-doped AIE-Tat NPs over Qtracker® 655 that is well-known as a good long-term cell tracker (Figures 5A and 5B). Furthermore, the retention of 8-doped AIE-Tat NPs in the labeled cells was also investigated. The NP-stained MCF-7 cells were mixed with untreated cells at 1:1 ratio, which were subsequently co-cultured for 24 h. Flow cytometry analysis indicates that the ratio of MCF-7 cells with and without fluorescence is still nearly 1:1, revealing that 8-doped AIE-Tat NPs would not easily leak out from the labeled cells (Figure 5C). Given these facts, AIE NPs hold great promise as a new generation of fluorescent cell trackers for long-term reporting the fate of cells.

Figure 4. Chemical structure of 7. Fluorescent images of HeLa cells stained by 7 at various passages. Adapted with permission from ref (60). Copyright 2013 American Chemical Society. 224 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 5. Chemical structure of 8. Flow cytometry histograms and confocal images of (A) 8-doped AIE-Tat NPs-stained and (B) Qtracker® 655-stained MCF-7 cells, followed by subculture for designated generations. Control: untreated MCF-7 cells. (C) Flow cytometry histograms and confocal images of a mixture of 8-doped AIE-Tat NPs-stained MCF-7 cells and unlabeled cells, which were co-cultured for 24 h. Adapted with permission from ref (61). Copyright 2013 Rights Managed by Nature Publishing Group.

In Vitro Bacterial Imaging In addition to cellular imaging, AIE NPs also show good performance in bacterial imaging and tracking (65–67). Tang’s group developed a highly emissive and photostable AIEgen (9) that can differentiate dead and live bacteria (65). As shown in Figures 6A-L, upon incubation of the live as well as dead bacteria with 9 (formed NPs in aqueous solution), it is found that only dead bacteria are visualized, while there is almost no detectable fluorescence in the live bacteria. This is because the AIE NPs of 9 is live bacterial cell-impermeable, 225 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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which is not capable of entering and lighting up the live bacteria. On the other hand, as the membranes of dead bacteria are broken, the AIE NPs of 9 would readily penetrate into the bacterial protoplasm and interact with the intracellular DNA strands, which significantly activate the fluorescence of 9 by virtue of the restriction of intramolecular rotation mechanism. Moreover, thanks to the unique capability in discrimination of live and dead bacteria, 9 can serve as an efficient probe on evaluating the effectiveness of different bactericides. To date, besides 9 that emits blue fluorescence, the AIEgens with yellow, orange and red emission colors have also been explored for bacterial imaging (66, 67).

Figure 6. Chemical structure of 9. Images of 9-stained live and dead (A-D) E. coli, (E-H) S. epidermidis and (I-L) B. subtilis. (A, C, E, G, I, K): bright-field images; (B, D, F, H, J, L): fluroescent images. Adapted with permission from ref (65). Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

AIE NPs for in Vivo Imaging Besides in vitro fluorescence imaging, great efforts have also been focused on the in vivo imaging of AIE NPs (68). Actually, many AIE NPs give excellent performances in a variety of in vivo imaging applications, evolving as an alternative fluorescence probe for in vivo use. In this section, the in vivo imaging of AIE NPs will be summarized in terms of in vivo fluorescence imaging of tumors, intravital two-photon fluorescence imaging, in vivo long-term cell tracking as well as in vivo dual-modality imaging. In Vivo Fluorescence Imaging of Tumors In vivo fluorescence imaging of tumors is of undoubtedly vital importance for cancer diagnosis and therapeutics (10). As for such use, fluorescent probes would possess several necessary qualities to meet the requirements: 1) intense emission in the FR/NIR region (>650 nm) that enables bioimaging with low biological background fluorescence and high tissue penetration (69); 2) optimal nanoscale size, as it has been well established that the nanomaterials are capable 226 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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of preferentially accumulating into tumors via the enhanced permeability and retention (EPR) effect, known as "passive" targeting (15); 3) low cytotoxicity and in vivo toxicity; and 4) "active" tumor-targeting ability through functionalizing the surfaces of the probes with tumor-targeting ligands (5). The emergence of AIEgens sweeps away our concerns of emission quenching caused by a high fluorogen loading in NPs, which endows AIEgens with great potential in fabricating NP probes with extremely high fluorescence and photostability. Consequently, AIE NPs become ideal probes applied for in vivo fluorescence imaging.

Figure 7. Chemical structure of 10. (A) Schematic illustration of the preparation of 10-loaded BSA NPs. (B) Time-dependent in vivo non-invasive fluorescence images of H22 tumor-bearing mice post intravenous injection of 10-loaded BSA NPs. To set up the tumor-bearing mouse model, H22 cancer cells were subcutaneously inoculated into the left axillae of the ICR mice. The circle indicate the location of tumor. Adapted with permission from ref (70). Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA. 227 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Taking 10 for example, Tang and co-workers reported a 10-loaded bovine serum albumin (BSA) NP with intense FR/NIR fluorescence (emission maximum at 668 nm) (70). To prepare the NPs, tetrahydrofuran (THF) solution containing 10 was added into the aqueous solution of BSA. During NP formation, the molecules of 10 aggregate and entangle with the hydrophobic sections of BSA biopolymers, becoming the inner part of NPs. On the other hand, the ionized carboxylic groups of BSA serve as the outer layers and stabilize the NPs in aqueous solution, which was confirmed by the negative zeta potential of -29 mV. Finally, the aqueous suspension of 10-loaded BSA NPs were obtained by cross-linking of BSA matrix with glutaraldehyde and removal of THF (Figure 7A). Through tuning the encapsulation amounts of 10 in NPs, the resulted NPs have particle sizes ranging from about 100-150 nm and high quantum yields up to 12% (utilizing Rhodamine 6G in ethanol as the standard). Noteworthy, 10-loaded BSA NPs can selectively visualize tumor tissues at about 28 h after intravenous injection of the NPs into murine hepatoma-22 (H22) tumor-bearing mice (Figure 7B), by virtue of the suitable NP size that enables prolonged blood circulation and prominent EPR effect in vivo. It is also important to note that 10-loaded BSA NPs are able to be easily excreted from the body through biliary pathway, that is, from liver, bile duct, intestine, to feces, indicating that the NPs would not long-term reside in the mice, causing possible in vivo side toxicity. This was testified by the obvious fluorescent signals in the collected feces from tumor-bearing mice post intravenous administration of 10-loaded BSA NPs. For another instance, to further enhance the brightness of 10-loaded BSA NPs in FR/NIR region, a fluorescence resonance energy transfer (FRET) strategy was employed (41). A conjugated polymer, poly[9,9bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorenyldivinylene] (PFV), was selected as the FRET donor owing to its π-conjugated backbones and large absorption coefficients. PFV and 10 (FRET acceptor) were co-encapsulated utilizing BSA as the polymer matrix, affording PFV/10 co-loaded BSA NPs (Figure 8A). Subsequently, the surfaces of the NPs were functionalized by arginine-glycine-aspartic acid (RGD) peptide (Figure 8A), endowing the NPs with active targeting ability, as RGD peptide is a well-acceptable targeting ligand to selectively bind to integrin receptors overexpressed in many a cancer cell (5). The resulted PFV/10 co-loaded BSA-RGD NPs have a large Stokes shift of about 223 nm as well as low cytotoxicity against HT-29 colon cancer cells. More importantly, as compared to 10-loaded BSA NPs, the FR/NIR fluorescence of PFV/10 co-loaded BSA-RGD NPs is amplified for 5.3 times (Figure 8B), thanks to the good spectral overlap and close proximity between PFV and 10 within BSA matrix, which achieve efficient FRET. Using H22 tumor-bearing mice as model animals, PFV/10 co-loaded BSA-RGD NPs and PFV/10 co-loaded BSA NPs (used as a control) were intravenously injected into the mice, respectively, followed by non-invasive in vivo fluorescence imaging. By virtue of the amplified FR/NIR signal and good EPR effect, both NPs with and without RGD functionalization can passively target and clearly visualize the tumors in vivo (Figures 8C and 8D). Noteworthy, the fluorescence signals of the tumors from PFV/10 co-loaded BSA-RGD NP-injected mice are significantly higher as compared to that of tumors from PFV/10 co-loaded BSA NP-injected 228 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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mice at all tested time points after administration (Figures 8C and 8D). As H22 tumors have been reported to overexpress integrin receptors, the in vivo imaging data reasonably illustrate that the PFV/10 co-loaded BSA-RGD NPs can image tumors in vivo in a high-contrast and specific manner due to both active and passive targeting effects.

Figure 8. (A) Chemical structure of PFV and schematic illustration of the fabrication of PFV/10 co-loaded BSA-RGD NPs. (B) Photoluminescence (PL) spectra of various NPs in water. Time-dependent in vivo non-invasive fluorescence images of H22 tumor-bearing mice after intravenous injection of (C) PFV/10 co-loaded BSA NPs and (D) PFV/10 co-loaded BSA-RGD NPs, respectively. The circles in (C) and (D) indicate the locations of tumors. Adapted with permission from ref (41). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Intravital Two-Photon Fluorescence Imaging Two-photon fluorescence imaging is a powerful technique that generates high energy visible fluorescence from low energy irradiation in the NIR region, which allows much deeper imaging of living tissues (up to around 1 millimeter in depth) as compared to conventional one-photon fluorescence imaging (71). For effective two-photon fluorescence imaging, high two-photon absorption (TPA) cross-section (δ) and large two-photon action cross-section (ηδ, η is the fluorescence quantum yield) are needed. So far, many AIE NPs have been 229 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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demonstrated as TPA materials (72–75). Compared with commercially available two-photon imaging agents such as QD-based probes and Evans Blue, several AIE NPs have been proved to possess higher δ and ηδ as well as show better performance in intravital two-photon fluorescence imaging.

Figure 9. Chemical structure of 11. (A) Two-photon absorption spectra of different agents in aqueous solution. (B) Intravital two-photon fluorescence imaging of blood vessels in various tissues from mice after administration of 11-encapsulated DSPE-PEG2000 NPs. Scale bar is 50 μm. Excitation: 800 nm; signal collected at 542±27 nm. Adapted with permission from ref (72). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Taking 11 for example, 11-encapsulated DSPE-PEG2000 NPs were prepared with spherical morphology and a mean hydrodynamic diameter of about 33 nm (72). Thanks to the AIE signature of 11, such AIE NPs show a high η of 62% (using Rhodamine 6G in ethanol as the standard), large δ and ηδ values (higher 230 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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than QD655 and Evans Blue (Figure 9A)), free of random blinking and strong photobleaching resistance. The utility of 11-encapsulated DSPE-PEG2000 NPs in real-time two-photon fluorescence imaging of blood vasculature in the brain, bone marrow and ear skin tissues of living mice was then investigated. From the results depicted in Figure 9B, it is reasonable to conclude that the 11-encapsulated DSPE-PEG2000 NPs can serve as an outstanding two-photon fluorescence imaging agent for in vivo deep-tissue blood vessels imaging application. Finally, the 11-encapsulated DSPE-PEG2000 NPs have been testified to show negligible in vivo toxicity to the mice after intravenous injection, indicating that they are safe probes for in vivo use. Considering these unique advantages, AIE NPs are very promising materials for next generation of intravital two-photon fluorescence imaging probes. In Vivo Long-Term Cell Tracking In addition to good performance in in vitro cancer cell tracing, 8-doped AIE-Tat NPs also show a unique merit in in vivo long-term cancer cell tracking (61). The 8-doped AIE-Tat NP-stained C6 glioma cells were subcutaneously injected into the flank of live mice. As a reference, another group of mice were subcutaneously inoculated with Qtracker® 655-stained C6 cells (C6 cells were pre-incubated with 2 nM 8-doped AIE-Tat NPs or Qtracker® 655 for 4 h). As displayed in Figure 10, 8-doped AIE-Tat NPs can continuously monitor the labeled C6 glioma cells for 21 days, whereas there is almost no detectable fluorescence signal in the Qtracker® 655-stained C6 cells at day 7 post-injection, revealing that 8-doped AIE-Tat NPs possess much superior in vivo cell tracking ability to the most popular commercial cell tracker, Qtracker® 655.

Figure 10. Representative in vivo fluorescence images of the mice after subcutaneously injected with (A) 8-doped AIE-Tat NP-stained and (B) Qtracker® 655-stained C6 glioma cells for designated time intervals. Adapted with permission from ref (61). Copyright 2013 Rights Managed by Nature Publishing Group. 231 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Besides, Liu and co-workers employed 8-doped AIE-Tat NPs to track the fate of stem cells in vivo as well (76). Stem cells are undifferentiated biological cells that can differentiate into specialized cells and possess paracrine effects. In the field of stem cells, one of the most urgent issues is to develop efficient stem cell trackers, which are capable of precisely reporting the fate of transplanted stem cells over a long period of time. Generally, reporter gene labeling and exogenous imaging agent labeling are the main strategies for stem cell tracking (77). The reporter gene labeling method needs to transfect genetic material and modify DNA, which holds the advantage of being able to precisely and quantitatively report the distribution and proliferation of the labeled stem cells. However, this strategy is hard to implement clinically because of several factors in terms of complex cell manipulation, concern of insertional mutagenesis and safety issues, etc.. On the other hand, exogenous imaging agents as stem cell trackers may be more promising for clinical translation. To date, iron oxide nanoparticles have been extensively studied for stem cell tracking through MRI technique. However, they have been reported to suffer from low sensitivity and decreased MRI signal owing to cell proliferation and cell exocytosis (78). As compared to MRI, fluorescence imaging technique shows the advantages of high sensitivity, excellent temporal resolution as well as maneuverable instruments. Considering the unique virtues of AIE NPs in cell tracing application, Liu and co-workers investigated the feasibility of 8-doped AIE-Tat NPs in tracking of the transplanted adipose-derived stem cells (ADSCs) (76). Encouragingly, the 8-doped AIE-Tat NPs exhibit very low cytotoxicity and in vivo side toxicity, excellent retention in living ADSCs as well as negligible interference on ADSC plurpotency (Figure 11A), paracrine (Figure 11B) and in vivo treatment efficacy (Figure 11C). Using ischemic hind limb bearing mice as model animals, the 8-doped AIE-Tat NPs show an excellent performance in precisely and quantitatively reporting the fate of the transplanted ADSCs for 6 weeks (Figure 11D), which represents the longest in vivo cell tracking duration among the currently available exogenous fluorescent cell trackers. Furthermore, through the labeling of 8-doped AIE-Tat NPs, it is found that in the ischemic tissues, the transplanted ADSCs can secrete angiogenic factors and differentiate into necessary cells to participate in neovascularisation, which thus provides important information on how ADSCs contribute to ischemia therapy. Based on these exciting results, it is reasonable to say that AIE NP-based cell trackers hold great promise for potential clinical translation.

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Figure 11. (A) Chondrogenic, adipogenic, and osteogenic differentiation capacities and (B) paracrine analyses of ADSCs labeled with and without 8-doped AIE-Tat NPs. (C) Representative photographs of ischemic hind limb-bearing mice after 30 days post treatments with saline, ADSCs labeled with and without 8-doped AIE-Tat NPs, respectively. (D) Representative time-dependent in vivo fluorescence images of the ischemic hind limb-bearing mouse that was intramuscularly injected with 8-doped AIE-Tat NP-labeled ADSCs. Adapted with permission from ref (76). Copyright 2014 American Chemical Society.

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Dual-Modality Imaging In recent years, great research interest has been focused on dual-modality imaging, which can overcome the limitations of either imaging modality when used alone (79). The NPs with the capabilities for both fluorescence imaging and MRI are particularly valuable. The main advantages of fluorescence imaging is the high sensitivity, temporal resolution as well as large in vitro and in vivo throughputs even at cellular level; however, the poor anatomical resolution makes fluorescence imaging hard to display the exact spatial location of a three-dimensional (3D) target (80). On the other hand, MRI holds the advantages of excellent spatial resolution that clearly offers 3D information, but MRI suffers from low sensitivity toward contrast agents (81). Therefore, as mentioned above, these two imaging techniques are complementary in principle. Recently, AIE systems have also been extended to enjoy dual functions of fluorescence imaging and MRI. Tang and co-workers developed an AIEgen 12 that consists of a hydrophobic TPE and two hydrophilic gadolinium (Gd) diethylenetriaminepentaacetic acid moieties (Figure 12) (82). It has been well acceptable that Gd-based agents provide positive contrast information under MRI. The amphiphilic 12 self-assembles into highly fluorescent AIE NPs in aqueous solution with a mean size of about 165 nm. Besides fluorescent cell imaging, such dual-functional AIE NPs shows similar longitudinal relaxivity in water as commercial Magnevist®. The in vivo MRI imaging study reveals that the dual-functional AIE NPs can act as a liver specific MRI contrast agent to continuously image liver tissue for at least 150 min (Figure 12).

Figure 12. Chemical structure of 12 and Axial T1-weighted MR images through the liver of rat after intravenous injection of the NPs of 12 (0.1 mmol/kg Gd3+) for designated time intervals. Adapted with permission from ref (82). Copyright 2014 American Chemical Society. 234 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 13. Chemical structure of 13. (A) Schematic illustration of the formation of 13/gold NPs co-loaded DSPE-PEG2000 NPs. (B) In vivo fluorescence images ex vivo fluorescence images of CT26 tumor-bearing mice after intravenous injection of 13/gold NPs co-loaded DSPE-PEG2000 NPs for 6, 12, and 24 h. The arrows and circles indicate tumor regions. 1: Liver; 2: Spleen; 3: Kidney; 4: Heart; 5: Lung; 6: Tumor; 7: Brain; 8: Intestine. (C) CT images (top row: stereo images; bottom row: sectional images) of CT26 tumor-bearing mice before and after intravenous injection of 13/gold NPs co-loaded DSPE-PEG2000 NPs for 6, 12, and 24 h. The circles indicate tumor regions. Adapted with permission from ref (84). Copyright 2014 Elsevier Ltd. 235 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Similarly, X-ray computed tomography (CT) is an imaging modality with high spatial resolution and unlimited penetration depth, but possesses an inherent limitation of low sensitivity (83). Liang and co-workers thus designed and synthesized a complementary dual-modal fluorescence/CT imaging NP probe via co-encapsulating a red emissive AIEgen 13 and gold NPs employing DSPE-PEG2000 as the matrix (Figure 13A) (84). It is known that conventional fluorescent dyes would be significantly quenched by gold NPs; however, thanks to the AIE feature, 13 can effectively overcome the strong fluorescence quenching of shielding-free gold NPs. In vivo imaging studies reveal that the 13/gold NPs co-loaded DSPE-PEG2000 NPs display good tumor-targeting ability and are able to act as a safe and efficient probe for dual-modal fluorescence/CT imaging (Figures 13B and 13C). On the basis of these successful examples, AIEgens can serve as ideal fluorescent materials to be the fluorescent component of dual-modal imaging nanoprobes.

Conclusions and Perspectives In this chapter, recent progress of AIE NPs for in vitro and in vivo imaging has been summarized and discussed. A vast amount of successful in vitro and in vivo examples verify that AIE NPs are highly promising materials for next generation fluorescent probes. As compared to the nanoprobes based on conventional fluorescent materials, AIE NPs show such advantages as remarkably high brightness due to the opposite ACQ effect, strong resistance to photobleaching, nonblinking signature, excellent cellular retention ability, large two-photon cross-section as well as negligible cytotoxicity and in vivo toxicity. In addition, AIE NPs also exhibit merits of facile synthesis, flexible surface functionalization, effective cell uptake and permeability, long blood circulation time as well as passive and active tumor-targeting ability, making them accessible to various bioimaging applications. Future work of AIE NPs for bioimaging will mainly focus on the development of smart and multifunctional AIE NPs. For example, many AIEgens can efficiently generate reactive oxygen species (ROS) under light irradiation (85–87). These AIEgen-based NPs can serve as both emitters and photosensitizers, which would be used for tumor tissue detection and subsequent imaging-guided photodynamic therapy. Moreover, exploration of smart stimuli-responsive (i.e., enzyme, pH, or ROS) AIE NPs that can selectively activate the fluorescence in the disease sites is also highly desirable. Although some efforts have been devoted to develop AIE-active dual-modal imaging NPs, there is still large room for creating advanced nanoprobes. We believe AIE NPs have been, are, and will provide the scientists and clinicians with new sights and insights in the areas of life science and biomedical engineering.

Acknowledgments The authors gratefully acknowledge financial support provided by the National Natural Science Foundation of China (31571011). 236 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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60. Wang, Z.; Chen, S.; Lam, J. W. Y.; Qin, W.; Kwok, R. T. K.; Xie, N.; Hu, Q.; Tang, B. Z. Long-term fluorescent cellular tracing by the aggregates of AIE bioconjugates. J. Am. Chem. Soc. 2013, 135, 8238–8245. 61. Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing. Sci. Rep. 2013, 3, 1150. 62. Feng, G.; Tay, C. Y.; Chui, Q. X.; Liu, R.; Tomczak, N.; Liu, J.; Tang, B. Z.; Leong, D. T.; Liu, B. Ultrabright organic dots with aggregation-induced emission characteristics for cell tracking. Biomaterials 2014, 35, 8669–8677. 63. Li, K.; Zhu, Z.; Cai, P.; Liu, R.; Tomczak, N.; Ding, D.; Liu, J.; Qin, W.; Zhao, Z.; Hu, Y.; Chen, X.; Tang, B. Z.; Liu, B. Organic dots with aggregation-induced emission (AIE dots) characteristics for dual-color cell tracing. Chem. Mater. 2013, 25, 4181–4187. 64. Qin, W.; Li, K.; Feng, G.; Li, M.; Yang, Z.; Liu, B.; Tang, B. Z. Bright and photostable organic fluorescent dots with aggregation-induced emission characteristics for noninvasive long-term cell imaging. Adv. Funct. Mater. 2014, 24, 635–643. 65. Zhao, E.; Hong, Y.; Chen, S.; Leung, C. W. T.; Chan, C. Y. K.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Highly fluorescent and photostable probe for long-term bacterial viability assay based on aggregation-induced emission. Adv. Healthcare Mater. 2014, 3, 88–96. 66. Zhao, E.; Chen, Y.; Chen, S.; Deng, H.; Gui, C.; Leung, C. W. T.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z. A luminogen with aggregation-induced emission characteristics for wash-free bacterial imaging, high-throughput antibiotics screening and bacterial susceptibility evaluation. Adv. Mater. 2015, 27, 4931–4937. 67. Zhao, E.; Chen, Y.; Wang, H.; Chen, S.; Lam, J. W. Y.; Leung, C. W. T.; Hong, Y.; Tang, B. Z. Light-enhanced bacterial killing and wash-free imaging based on AIE fluorogen. ACS Appl. Mater. Interfaces 2015, 7, 7180–7188. 68. Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 2013, 46, 2441–2453. 69. Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 2003, 7, 626–634. 70. Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications. Adv. Funct. Mater. 2012, 22, 771–779. 71. Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 2003, 300, 1434–1436. 72. Ding, D.; Goh, C. C.; Feng, G.; Zhao, Z.; Liu, J.; Liu, R.; Tomczak, N.; Geng, J.; Tang, B. Z.; Ng, L. G.; Liu, B. Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging. Adv. Mater. 2013, 25, 6083–6088. 73. Gao, Y.; Feng, G.; Jiang, T.; Goh, C. C.; Ng, L. G.; Liu, B.; Li, B.; Yang, L.; Hua, J.; Tian, H. Biocompatible nanoparticles based on 241 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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85. Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B. Targeted and image-guided photodynamic cancer therapy based on organic nanoparticles with aggregation-induced emission characteristics. Chem. Commun. 2014, 50, 8757–8760. 86. Yuan, Y.; Zhang, C. J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific lightup bioprobe with aggregation-induced emission and activatable photoactivity for the targeted and image-guided photodynamic ablation of cancer cells. Angew. Chem., Int. Ed. 2015, 54, 1780–1786. 87. Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Targeted bioimaging and photodynamic therapy of cancer cells with an activatable red fluorescent bioprobe. Anal. Chem. 2014, 86, 7987–7995.

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Chapter 10

AIE Nanoprobes for Multi-Photon in Vivo Bioimaging Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch010

Yalun Wang, Hequn Zhang, Nuernisha Alifu, and Jun Qian* State Key Laboratory of Modern Optical Instrumentation, Centre for Optical and Electromagnetic Research, Zhejiang University, 310058 Hangzhou, China *E-mail: [email protected]

Deep-tissue bioimaging is highly important in medical research and clinical applications. Multi-photon luminescence (MPL) imaging with excitation wavelength in the near-infrared (NIR) range is found to be an effective way to obtain large imaging depth of tissues. AIE nanoparticles with high fluorescence brightness, good biocompatibility and photobleaching resistance are ideal nanoprobes for MPL imaging. In this chapter, we introduced the concept of tissue penetration, optical tissue windows, and MPL imaging, and summarized some of the progresses in multi-photon in vivo bioimaging based on AIE nanoprobes.

1. Background 1.1. Optical Tissue Windows Bioimaging is of great significance to medical research and clinical practices (1). Since the beginning of the 20th century, various imaging techniques have been developed, and X-rays, ultrasonic imaging, magnetic resonance imaging (MRI), positron emission tomography (PET) are some of the most promising ones (2). However, there’s exposure to radiation in X-rays and PET (3, 4), and the details in ultrasonic imaging and MRI are not well recognized (5, 6). Optical imaging, with high spatial resolution, abundant spectral information and radiation free feature, is very ideal for biological applications (7). However, the depth of optical bioimaging is usually a problem, due to the limited penetration depth of visible light in biological tissues (8). © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The attenuation of light in bioimaging is mainly from the optical scattering and absorption in tissue. Photon scattering can be scaled as λ-α, where λ is the wavelength of light and α = 0.2-4 for different types of tissues (9). Thus light with longer wavelengths would suffer less loss from scattering, and it is better for deep-tissue imaging. For light absorption in tissue, as water is the main component for nearly all the tissues, its light absorption is in dominant (10). The optimal wavelength for deep-tissue imaging is the trade-off between the tissue scattering and absorption, and these wavelengths can be classified into some optical tissue windows. Spectral rang in 750-900 nm, which is named as the first near-infrared (NIR-I) window, is very useful for deep-tissue imaging (11). The tissue scattering in this range is smaller than those in the commonly used ultraviolet (UV) and visible ranges, and the water absorption in this range is also very small. Thus, large light penetration depth can be realized in this window, and many relevant work has been performed (12–15).

Figure 1. The calculated attenuation length in mouse cortex. Dashed line, absorption length of water. Dashed-dotted line, scattering length of mouse brain cortex. Solid line, combined attenuation length. Reproduced with permission from reference (10). Copyright (2013) Nature Publishing Group. Spectral range in 1000-1700 nm, which is defined as the second near-infrared (NIR-II) window, is even better for deep-tissue imaging (16). According to the difference of water absorption, NIR-II window can be devided into NIR-IIa (1000-1400 nm) and NIR-IIb (1450-1700 nm) windows (17). In NIR-IIa window, the tissue scattering is smaller than that in visible and NIR-I ranges, and the water absorption is medium, slightly larger than those in visible and NIR-I ranges. In NIR-IIb window, the tissue scattering is the smallest. Although the water absorption is strong, the greatly reduced light scattering could compensate it. Thus, light in NIR-II window could have better light penetration capability than light in visible and NIR-I ranges. Horton et al. had calculated the attenuation of light in mouse cortex from 700-2000 nm, and found that the light near 1700 nm had the largest penetration depth, as shown in Figure1 (10). Bioimaging based on NIR-II window is very promising, and some pioneer works have been devoted on it (17–19). 246 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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1.2. Multi-Photon Imaging In a bioimaging process, light usually travels twice in the tissue. First, the excitation light penetrates into the tissue to excite the fluorophores. Second, the emitted signals come out from the tissue, and are collected by the detector. In a multi-photon luminescence (MPL) imaging process, the excitation wavelength usually locates in the aforementioned optical tissue windows, and the signal is usually collected in the visible wavelength range. Compared with NIR bioimaging, which directly collects singals in NIR range, MPL imaging has some advantages. First, much more fluorophores with visible emission can be chosen than those with NIR emission (20, 21). Second, optical detectors usually have better response in the visible range than in NIR range. Third, a better imaging resolution can be obtained from visible emitted signals, according to the Abbe’s formula ε = 0.61λ/NA (where ε is the resolution limit, λ is the wavelength, and NA is the numerical aperture of the objective lens). Besides, MPL is a typical nonlinear optical effect, and the out-of-focus signals would be greatly reduced in MPL imaging process. Two-photon luminescence (2PL) is a process in which a fluorophore molecule absorbs two photons with the same longer wavelength simultaneously, and is stimulated to the excited state. After fast relaxation, the fluorophore molecule returns to its ground state and emits a photon with shorter wavelength, as shown in Figure 2(a). The probability of two-photon absorption occurrence is characterized by two-photon absorption (2PA) cross-section (22). For convience, its unit is usually in GM, where 1 GM = 10-50cm4s. The product of 2PA cross-section and fluorescence quantum yield is defined two-photon action cross-section, which describes the two-photon brightness of a fluorophore (23).

Figure 2. A schematic illustration of multi-photon luminescence process. (a) Two-photon luminescence, (b) three-photon luminescence. In three-photon luminescence (3PL), three photons with the same longer wavelength are absorbed simultaneously by a fluorophore, and a photon with shorter wavelength is emitted, as shown in Figure 2(b). The probabitility of three-photon absorption occurrence is characterized by three-photon absorption (3PA) cross-section, and it’s usually in unit of cm6GW2 (22). As the occurrence probability of higher-order nonlinear optical process is much smaller, 2PL and 3PL are usually more feasible in bioimaging. 247 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Compared with conventional one-photon luminescence (1PL) imaging with short wavelength excitation, MPL imaging with excitation in NIR spectral window has many advantages (24–26). (1) Longer-wavelength excitation light has less tissue scattering, and much larger penetration depth could be obtained. (2) The photodamage of tissues under longer-wavelength excitation in MPL imaging, would be greatly reduced, compared with that under UV/blue wavelength exictation in 1PL imaging. (3) It’s much easier to separate the excitation light from the shor-wavelength signals in MPL imaging, and the signal-to-noise ratio would be improved accordingly. (4) The fluorescence intensity has a square or higher-order power dependence on the intensity of exicitation light, and the focusing point would be rather small. Thus, the out-of-focus fluorescence could be greatly reduced, which is very helpful to improve the imaging quality. (5) With the rather small focusing point, MPL imaging has inherent ability of sectioning, and the complicated pin-hole structure in 1PL confocal microscope could be eliminated. As the nonlinear optical coefficient of most materials is low, high instaneous power density is needed to excite enough MPL signals. To avoid photodamage to tissues, pulsed laser sources are commonly utilized in MPL imaging. In addition, due to the limitation of high excitation power density, MPL imaging is usually applicable in microscopic imaging with focusing illumination, and not so proper for macro imaging requiring wide field illumination. MPL signals are usually colleted by highly sensitive photo-multiplier tubes (PMTs) via non-descanned detection (NDD) mode. Since the first development of 2PL scanning microscope in 1990s, MPL imaging has been widely applied in cancer cell detection, neuron cell imaging, vascular imaging and so on (27–32). 1.3. AIE Nanoprobes for in Vivo MPL Imaging As the endogenous fluorescence of bio-tissues is usually very weak, exogenous fluorophore is needed to acquire good contrast in bioimaging. To date, various types of nanoprobes have been developed for 2PL imaging, including small organic dyes (33), fluorescent proteins (34), inorganic semiconductor quantum dots (35), and metal nanoparticles (36). However, the fluorescence stability of fluorescent proteins is limited (37), and there is potential toxicity in quantum dots (38). Metal nanoparticles are susceptible to photo-thermal damage (39), and common organic dyes suffer from aggregation-caused quenching (ACQ) when their concentration is high. In 2001, a new type of organic dyes with aggregation-induced emission (AIE) property was developed, and it is very promising for MPL in vivo imaging (40). First, the multi-photon absorption cross-section of AIE nanoprobes can be designed very high, which is very beneficial to MPL imaging. Second, the biocompatibility of these organic dyes is very good. Third, the fluorescence stability is greatly improved as there are many molecules inside each AIE nanoprobe. AIEgens are usually encapsulated into nanoparticles for bioimaging. One commonly used routine is the modified nanoprecipitation method, in which AIEgens are incorporated into the polyethylene glycol (PEG) matrix 248 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

(41). A micelle and silica coprotection strategy was proposed to improve the quantum yield, by providing a more hydrophobic environment for AIEgens (42). AIEgens in dimethyl sulfoxide (DMSO) can also be directly injected to form nanoaggregates spontaneously for in vivo imaging (43). In this chapter, we will summarize some progresses in MPL in vivo imaging based on AIE nanoprobes.

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2. AIE Nanoprobes for in Vivo 2PL Imaging with Excitation Wavelength in NIR-I Window Compared to 1PL imaging with excitation in the ultraviolet (UV) and visible wavelength ranges, 2PL imaging with its excitation wavelength in NIR-I window (750-900 nm) has many advantages. First, it has very small water absorption, and the tissue scattering was not so distinct. Thus the penetration depth of NIR-I light in biological tissues could be greatly improved. In addition, the photodamage towards tissues would be greatly reduced, as NIR-I photons have less energy than the UV and blue photons. Furthmore, as the focusing point of excitation light is very small in 2PL imaging, the out-of-focus fluorescence can be greatly reduced, and the signal-to-noise ratio can be obviously improved. 2PL imaging with excitation wavelength in NIR-I window has been widely applied in cell, tissue, and in vivo imaging (44, 45). A mode-locked Ti:Sapphire femtosecond (fs) laser is widely used in 2PL imaging (46). It has an average output power of as high as several watts and a pulse width of ~100 fs. In addition, its output wavelength can be tuned from 700 to 1040 nm, which perfectly covered the NIR-I window (47). When fs excitation with its wavelength in NIR-I window is adopted, 2PL is the main nonlinear optical process, which has been widely applied in bioimaging. 2PL in vivo imaging based on NIR-I window and AIE nanoprobes is very proming due to the good penetration capability of the excitation light, as well as the excellent optical property of AIE nanoparticles, and there have been many relevant work on this field (43, 46, 48).

Figure 3. (a) Chemical structure and molecular geometry of BTPEBT. (b) Fluorescence spectra of BTPEBT in THF/water mixtures with water fractions from 50 to 90 vol%, excitation wavelength λex = 418 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons. 249 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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In 2013, Ding et al. contributed the first report on real-time two-photon intravital vasculature imaging with AIE nanodots (46). A type of AIEgen BTPEBT was synthesized with donor-(π-conjugated-bridge)-acceptor (D-π-A) structure, which was assumed to be benefit for large two-photon cross-section, as shown in Figure 3(a). The AIE characteristics of BTPEBT was investigated by dissolving it in different tetrahydrofuran (THF)/water mixtures. With increasing volume fraction of water from 50% to 90%, the aggregation of BTPEBT increased and the fluorescence upon excitation of 418 nm was intensified according, illustrating its AIE property, as shown in Figure 3(b). The hydrophobic BTPEBT molecules were incorporated into hydrophilic nanoparticles together with 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) by a modified nanoprecipitaion method, as indicated in Figure 4(a). These nanodots were found to have spherical morphology with a mean size of 29 nm on transmission electron microscopy (TEM), as shown in Figure 4(b).

Figure 4. (a) The schematic illustration of BTPEBT nanodots fabrication. (b) TEM image of BTPEBT nanodots. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons.

Figure 5. (a) The normalized absorption and fluorescence spectra of BTPEBT nanodots, λex = 425 nm. (b) The measured 2PA cross-section of BTPEBT nanodots, QD655, and Evans Blue, λex = 800-960 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons. 250 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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These BTPEBT nanodots beared the absorption peak at 425 nm and emission peak at 547 nm, with a large Stokes shift of 122 nm, as shown in Figure 5(a). The 2PA cross-section of BTPEBT nanodots was measured via a two-photon induced fluorescence method, and it was found to be 10.2×104 GM at 810 nm, even larger than those bright quantum dots (QDs), as shown in Figure 5(b). With a high quantum yield of 62±1%, the two-photon action cross-section of BTPEBT was very high, and it was very helpful to 2PL imaging. The BTPEBT nanodots were then utilized for mouse brain imaging under an excitation of 800 nm from a Ti:Sapphire fs laser. As shown in Figure 6, the blood vessels could be visualized as deep as 400 μm, which was beyond the pia matter. From the 3D reconstruction, the mouse brain vascular system could be recognized.

Figure 6. The 2PL mouse brain imaging. (A-C) At different times. (D-I) At different depths. (J) 3D reconstruction. λex = 800 nm. Reproduced with permission from reference (46). Copyright (2013) John Wiley and Sons.

Parallelly, Wang and Qian et al. (our group) realized in vivo two-photon functional bioimaging with AIE nanodots (48). A type of AIEgen TPETPAFN (TTF) with D-π-A-π-D structure was synthesized, as shown in Figure 7(a). The TTF molecules were encapsulated by the molecules of 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-methoxy-(polyethylene glycol)-5000 (DSPE-mPEG5000) to form nanoparticles, as indicated in Figure 7(b). By varying the amount of TTF added, the diameter of TTF nanodots could be tuned. As shown in Figure 8, with 30 wt% of TTF in reactants, the mean size of TTF nanodots was 30 nm, while for TTF nanodots with 50 wt% of TTF in reactants, their mean size was 100 nm. The percentage of TTF in TTF nanodots could be defined as loading ratio. For different loading ratios, the aggregation states of TTF in nanodots would be different. The AIE property of TTF was studied by measuring the fluorescence of TTF nanodots with different loading ratios. As shown in Figure 9, when increasing the loading ratio of TTF nanodots, the fluorescence centered at 624 nm was intensified, indicating the AIE characteristics of TTF. 251 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 7. (a) The chemical structure of TTF. (b) A schematic illustration for the preparation of TTF nanodots. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Figure 8. TEM images of TTF nanodots with different TTF in reactants. (a) 30 wt% of TTF, (b) 50 wt% of TTF. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

TTF nanodots were used for functional imaging of ear blood vessels under a two-photon scanning microscope. As shown in Figure 10(a)-(d), the blood vessels in mouse ear could be clearly viewed under the excitation of an 800 nm fs laser. By tracking red blood cells (RBCs) with time (49), its instantaneous velocity could be obtained, as shown in Figure 10(e). By studying the fluorescence from the blood post injection, a blood circulation half-life of about 4h was observed for TTF nanodots. 252 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 9. Absorption and fluorescence spectra of TTF nanodots with different loading ratios, λex = 365 nm. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Figure 10. (a) Bright field, (b) 1PL, (c) 2PL, (d) merged images of TTF nanodots stained mouse ear. λex = 543 nm for 1PL, λex = 800 nm for 2PL. (e) A line scan along capillaries was used to determine RBC’s instantaneous velocity (dx/dt). (f) Blood circulating kinetics of TTF nanodots in mice. Reproduced with permission from reference (48). Copyright (2014) Nature Publishing Group.

Recently, Qian et al. (our group) implemented deep-tissue in vivo neuron imaging with the 2PL of AIE nanoaggregates (43). A type of AIEgen called TPETPP was synthesized (50), and its molecular structure was shown in Figure 11(a). As shown in Figure 11(b), there was almost no fluorescence in its benign organic solvent, e.g., DMF, DMSO, while there was strong fluorescence in its solid state, indicating the AIE property of TPE-TPP. 253 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 11. (a) Chemical structure of TPE-TPP. (b) The fluorescence of TPE-TPP in DMSO, DMF, and solid state under UV irradiation. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

The TPE-TPP nanoaggregates were formed by mixing DMSO solution of TPE-TPP with water, and their typical TEM images were shown in Figure 12(c). The absorption and fluorescence spectra of TPE-TPP were centered at 320 nm and 480 nm, with a large Stokes shift of 160 nm, as shown in Figure 12(a)-(b). Under the fs laser excitation of 740 nm, 2PL spectra of TPE-TPP was very similar to its 1PL spectra.

Figure 12. (a) Absorption and (b) Fluorescence spectra of TPE-TPP nanoaggregates, λex = 320 nm. (c) A typical TEM image of TPE-TPP nanoaggregates. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

Neuron imaging is of great significance in neuron/brain research. TPE-TPP nanoaggregates were applied in primary neurons imaging with a two-photon fluorescence microscope. As shown in Figure 13, there was strong 2PL signals from the TPE-TPP treated primary neurons (Figure 13(a)), and it coincided very well with the bright field channel (Figure 13(b)-(c)), indicating the effective staining of TPE-TPP nanoaggregates on neurons.

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Figure 13. 2PL images of TPE-TPP treated primary neurons.(a) fluorescence channel, (b) bright field channel, (c) merged. λex = 740 nm. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America.

Figure 14. 2PL images of TPE-TPP stained microglia in mouse brain at different depths. (a) Top section, (b) middle section, (c) bottom section, (d) 3D reconstruction. λex = 740 nm. Reproduced with permission from reference (43). Copyright (2015) Optical Society of America. 2PL in vivo brain-microglia imaging was further conducted by microinjecting TPE-TPP into the mouse brain at a depth of 300 μm. As shown in Figure 14(a)-(c), the morphologies of the microglia at different sections of the in vivo mouse brain could be clearly discriminated. From the 3D reconstruction in Figure 14(d), the whole microglia was vividly demonstrated. Due to the resistance to photobleaching of TPE-TPP nanoaggregates, those microglia could be utilized for long-time dynamic observation. To improve the fluorescence quantum yield, Geng and Liu et al. proposed a micelle and silica coprotection strategy to synthesize PFBT-F127-SiO2 nanoparticles (42), as shown in Figure 15(a)-(b). These PFBT-F127-SiO2 255 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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nanoparticles had a high quantum yield of 75%, larger than that of PFBT-DSPE nanoparticles (40%). Also, the 2PA cross-section of PFBT-F127-SiO2 nanoparticles was large, with 1085 GM at 810 nm. These stable and biocompatible PFBT-F127-SiO2 nanoparticles were applied for 2PL imaging of mouse brain, and an imaging depth of 500 μm was obtained, as shown in Figure 15(c).

Figure 15. (a) Chemical structure of F127 and PFBT. (b) The schematic illustration of PFBT-F127-SiO2 nanoparticles fabrication. (c) 3D reconstruction of mouse brain imaging with PFBT-F127-SiO2 nanoparticles. λex = 800 nm. Reproduced with permission from reference (42). Copyright (2014) American Chemical Society. Geng and Liu et al. further applied micelle and silica coprotection method to synthesize TTF-F127-SiO2 nanoparticles (51), as shown in Figure 16(a). The TTFF127-SiO2 nanoparticles had a high quantum yield of 50%, much larger than that of TTF-F127 nanoparticles (24%). In addition, the TTF-F127-SiO2 nanoparticles had a high 2PA cross-section of 900 GM at 840 nm. TTF-F127-SiO2 nanoparticles were applied in 2PL imaging of mouse tibial muscle, and the blood vessels at a depth of 80 μm could still be visualized clearly, as shown in Figure 16(b).

Figure 16. (a) A schematic illustration of TTF-F127-SiO2 nanoparticles. (b) The z-projected image of mouse tibial mussel blood vessels stained with TTF-F127-SiO2 nanoparticles. λex = 810 nm. Reproduced with permission from reference (51). Copyright (2015) Royal Society of Chemistry. 256 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 17. (a) Chemical structure of DPP-based compounds. (b) Fluorescence spectra of DPP-2 in THF/water mixtures with different water fractions, λex = 455 nm. (c) 3D reconstruction of DDP-2 nanoparticles stained mouse ear blood vessels. λex = 800 nm. Reproduced with permission from reference (52). Copyright (2015) John Wiley and Sons. To improve the 2PA cross-section, Gao and Hua et al. synthesized DPP-2 with AIE properties (52), as shown in Figure 17(a)-(b). DPP-2 molecules were found to have a very large 2PA cross-section of 8100 GM at 800 nm, which was much larger than that of most commercial dyes (hundreds of GM). The quantum yield of DPP-2 in solid state was 11%. Furthermore, hydrophobic DPP-2 molecules were encapsulated into hydrophilic nanoparticles via a modified nanoprecipitation method, by using DSPE-PEG-Mal as the matrix, and the 2PA cross-section of DPP-2 nanoparticles reached 5.34×105 GM at 810 nm. The red emissive DPP-2 nanoparticles were applied in mouse ear imaging, and the blood vessels deep into 76 μm could be clearly viewed, as shown in Figure 17(c). Besides, Zhao et al. synthesized TPE-decorated BODIPY luminogens, PIPBT-TPE, and PITBT-TPE with large 2PA cross-sections (53, 54). They were encapsulated into nanoparticles together with DSPE-PEG and were utilized for mouse brain and ear vascular imaging. Blood vessels with good contrast could be visualized.

3. AIE Nanoprobes for in Vivo 2PL Imaging with Excitation Wavelength in NIR-IIa Window Excitation light in NIR-IIa window (1000-1400 nm) has less tissue scattering than that in conventional NIR-I window, and a deeper penetration depth can be anticipated. Wang and Cai et al. had studied the focal spots of 1040 nm and 800 nm laser beams at some representative depths of biological tissue via the Monte Carlo simulation (55). As shown in Figure 18, the 1040 nm laser beam has a better focusing than 800 nm laser beam in biological tissue, and excitation at 1040 nm would be more appropriate for deep-tissue imaging. So far, the 2PL imaging depth record (as deep as 1.6 mm in live mouse cortex) was achieved by utilizing 257 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Alexa680-Dextran (non-AIE) as the fluorophore, and the fs excitation wavelength was in NIR-IIa window (56). Besides, lower autofluorescence and better signal to background ratio of 2PL imaging could also be obtained under longer-wavelength excitation.

Figure 18. Simulation of the light intensity distribution of 1040 nm and 800 nm laser beams in biological tissue at various vertical depths. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America.

There are some types of pulse laser sources in NIR-IIa window. An optical parameter oscillator (OPO, 1000-1600 nm, 76 MHz, ~200 fs), pumped by a modelocked Ti:sapphire fs laser (800 nm, 76 MHz), is an alternative (56–58). The tunable output wavelengths from 1000-1600 nm makes it very useful in NIR-II applications. The average output power of the fs OPO is not very high, about hundreds of milliwatts. A large-mode-area ytterbium-doped photonic crystal fiber (PCF) oscillator (1040 nm, 50 MHz, 150 fs) (55), with high output power of several watts, is very appropriate for the applications at 1040 nm. In addition, it has a relatively low price and is easy to operate. A Cr:forsterite fs laser (1230 nm, 110 MHz, 130 fs) is outstanding for applications at 1230 nm (59). When fs excitation with its wavelength in NIR-IIa window is adopted, 2PL will be excited from some red emitted fluorophores (55), while 3PL will be excited from some blue emitted fluorophores (60). There are not so many reports on AIE nanoprobes assisted MPL imaging in NIR-IIa window, and a lot of opportunaties still exists. Wang and Qian et al. (our group) implemented deep-tissue in vivo imaging with AIE nanodots, under the 1040 nm fs excitation (55). A red emissive AIEgen BODIPY-TPE (BT) was synthesized (61), and its chemical structure was shown in Figure 19(a), with the propeller-shaped TPE as the donor. As shown in Figure 19(b), when increasing the volume fraction of water from 65% to 95%, the fluorescence of BT in THF/water mixture near 600 nm was intensified, indicating the AIE characteristics of BT. The hydrophobic BT molecules were incorporated into hydrophilic nanodots by a modified nanoprecipitation method with DSPE-mPEG5000 as the matrix. BT nanodots had an absorption peak at 522 nm and an emission peak at 620 nm, with 258 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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a large Stokes shift of 98 nm, as shown in Figure 20(a). The 2PA cross-section of BT nanodots at 1040 nm was found to be 2.9×106 GM, and it was much larger than those at 770-860 nm, as shown in Figure 20(b).

Figure 19. (a) Chemical structure of BT. (b) Fluorescence spectra of BT in THF/water mixtures with water fractions from 65 to 95 vol%, λex = 420 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America.

Figure 20. (a) The absorption and fluorescence spectra of BT nanodots, λex = 420 nm. (b) The 2PA cross-section of BT nanodots at various wavelengths. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America

The biodistribution and clearance of BT nanodots in mice were studied by collecting the characteristic fluorescence from the major organs at various times after the injection of nanodots. As shown in Figure 21, BT nanodots mainly accumulated in the liver, reached maxima about 12 hours post injection, and were then cleared out gradually. 259 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 21. (a) Fluorescence images of liver at various time points. (b) Fluorescence intensities of different organs at various time points. λex = 523 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America. The biocompatible BT nanodots were utilized for in vivo mouse brain vasculature imaging under the excitation of 1040 nm fs laser (from a large-mode-area ytterbium-doped PCF oscillator). As shown in Figure 22, the major blood vessels and small capillaries could be visualized clearly as deep as 700 μm, which was deeper than the 2PL imaging depth in most reported work performed in NIR-I window.

Figure 22. 3D reconstructed 2PL in vivo images of BT nanodots stained mouse brain blood vessels with different visual angles. λex = 1040 nm. Reproduced with permission from reference (55). Copyright (2015) Optical Society of America. There is plenty of room to achieve deeper 2PL imaging NIR-IIa window. The improvements in imaging systems and nanoprobes could result in larger imaging depth. Recently, our group found TTF nanodots had a larger 2PA cross-section than BT nanodots at 1040 nm, and it was then used for in vivo mouse brain imaging under the fs excitation at this wavelength. As shown in Figure 23, the blood vessels could be clearly viewed to a depth of 810 μm.

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Figure 23. 3D reconstructed 2PL in vivo images of TTF nanodots stained mouse brain blood vessels, with different visual angles. λex = 1040 nm.

4. AIE Nanoprobes for in Vivo 3PL Imaging with Excitation Wavelength in NIR-IIb Window Excitation light in NIR-IIb window (1450-1700 nm) has the lowest tissue scattering compared with it in NIR-I and NIR-IIa windows. Although the water absorption is strong in NIR-IIb, the reduced tissue scattering can compensate it effectively. Cai et al. had studied the focal spots of 800-1680 nm laser beams at a 1.5 mm depth of biological tissue via the Monte Carlo simulation (62). As shown in Figure 24, the 1680 nm laser beam has the best focusing intensity than others, exhibiting the superiority of light excitation in the NIR-IIb window. An imaging depth of 1.3 mm (in living mouse brain) was achieved in 3PL microscopy based on Texas Red (non-AIE), where the fs excitation in NIR-IIb window was adopted (10). Besides, low autofluorescence and good contrast can be also obtained when NIR-IIb fs excitation is utilized. There are not so many types of pulsed laser sources, whose wavelengths are in NIR-IIb window. The aforementioned optical parameter oscillator (OPO, 10001600 nm, 76 MHz, ~200 fs) covers partial NIR-IIb window (58). However, the average power of the fs output is very low ( about hundreds of millwatts). A fs laser (FLCPA-01C, Calmar Laser, 1560 nm, 1 MHz, 400 fs), which takes advantage of rare-earth ion doped fiber as the active medium, can be adopted as the fs excitation source in NIR-IIb window (63). It is cost-effective, and easy to operate. Moreover, its average output power can reach one watt. Soliton self-frequency shift (SSFS) in a photonic crystal rod, which was pumped by a turnkey energetic fibre laser, could reach the optimal spectral window near 1700 nm (1675 nm, 1 MHz, 65 fs) (10). It was very helpful to deep-tissue imaging, although required lots of optical experience.

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Figure 24. Simulation of the focal spots of various laser beams at a depth of 1.5 mm of biological tissue. (a) 800 nm, (b) 1040 nm, (c) 1280 nm, (d) 1440 nm, (e) 1560 nm, (f) 1680 nm. Reproduced with permission from reference (62). Copyright (2013) Electromagnetics Academy. When the fs excitation wavelength was in NIR-IIb window, 3PL is the main nonlinear optical process, and fluorophores with large 3PA coefficients are favored for bioimaging applications. There are very few reports on AIE nanoprobes assisted MPL imaging in NIR-IIb window, and the longest attenuation length of light in tissues would give those who carry out their work in NIR-IIb window a surprise. Zhu and Qian et al. (our group) proposed a new protocol to encapsulate AIE molecules into nanoparticles and implemented 3PL in vivo imaging of mouse ear with these AIE nanoparticles (64). A type of AIEgen TTF as referred was synthesized, and it was encapsulated by nano graphene oxide (NGO) to form nanoparticles, as shown in Figure 25. The stability and emission efficiency of TTFNGO NPs was improved and the size could be tuned by controlling the amount of NGO added.

Figure 25. The protocol for the synthesis of TTF-NGO nanoparticles. Reproduced with permission from reference (64). Copyright (2016) American Chemical Society. 262 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The biocompatible TTF-NGO nanoparticles were utilized for 3PL in vivo imaging, under the excitation of a 1560 nm fs laser. As shown in Figure 26, the structure of blood vessels in the mouse ear at various vertical depths could be clearly recognized.

Figure 26. 3PL microscopic imaging of the ear blood vessels of a mouse. (a) At different depths, (b) 3D reconstruction, λex = 1560 nm. Reproduced with permission from reference (64). Copyright (2016) American Chemical Society.

Figure 27. 3PL microscopic imaging of brain blood vessels of a mouse at various depths, λex = 1560 nm. Reproduced with permission from reference (63). Copyright (2015) John Wiley and Sons. 263 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Qian et al. (our group) performed 3PL in vivo imaging of mouse brain with AIE nanodots (63). The same type of AIEgen TTF was synthesized, as shown in Figure 7(a). It was encapsulated into nanodots following the same routine, as shown in Figure 7(b). The high-order nonlinear optical effect of TTF nanodots were systematically studied and they were found to have strong 3PL under the excitation of 1560 nm. TTF nanodots were further used for 3PL imaging of mouse brain, by utilizing a 1560 nm fs laser as the excitation. As shown in Figure 27, the brain blood vessels at as deep as 550 μm can be clearly visualized. Recently, by optimizing the optical devices, e.g. using objective lens of high transmittance in NIR-IIb, we have improved the imaging depth of TTF stained mouse brain to 1000 μm. As shown in Figure 28, plenty of blood vessels and small capillaries could be vividly visualized with high contrast at various depths.

Figure 28. 3PL microscopic imaging of brain blood vessels of a mouse at various depths with improved optical devices, λex = 1560 nm.

5. Summary In this chapter, the motivation, mechanism, and some application examples of AIE nanoprobes assisted MPL in vivo imaging were introduced. Various types of AIEgens were synthesized, encapsulated into nanoparticles, and applied in in vivo imaging. The blood vessels in mouse ear and brain could be visualized clearly with a vertical depth up to hundreds of micrometers. The excitation wavelengths were classified into NIR-I, NIR-IIa, and NIR-IIb windows, according to the tissue scattering and absorption. The water absorption in NIR-I window is very small, and most MPL microscopy was carried out in this window. The tissue scattering is smaller in NIR-IIa window, and a better light penetration capability could be obtained. With the lowest tissue scattering, imaging in NIR-IIb window is very promising, although the optical devices should be specially designed. To get larger imaging depth and higher image contrast, AIEgens with large multi-photon absorption cross-section and quantum yield are highly required. With fluorophore of high brightness, fluorescence is much easier to be excited 264 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and collected, and the deep tissues are more likely to be visulized. In addition, red and far-red emissions are beneficial to deep-tissue imaging, as fluorescence with longer wavelengths is easier to come out from the tissue to be detected. Furthermore, the compatibility and photo-stability of AIE nanoprobes are both helpful to long-term in vivo imaging. MPL imaging system still needs optimization to obtain larger imaging depth. As the scattering coefficients are not the same in different tissues, the best fs excitation wavelengths for them is also different. In addition, the factors of the multi-photon absorption cross-sections of the nanoprobes and the pulsed laser sources should also be considered when choosing the excitation. In NIR-II, the lenses and mirrors need special coatings to obtain good transmittance or reflection. The properties of the pulsed laser source, such as the output power, the repetition frequency, and the duty cycle are also very important for deep-tissue imaging (56). Nanoprobe-assisted bioimaging is a multidisciplinary research area, and it needs the effort from those who do their work on materials, optics, biology, etc. MPL in vivo imaging with AIE nanoprobes is a small and promising field of bioimaging, and it will give us much deeper images if we do deep on it.

Acknowledgments This work was supported by National Basic Research Program of China (973 Program; 2013CB834704), the National Natural Science Foundation of China (61275190), the Fundamental Research Funds for the Central Universities, and the Open Fund of the State Key Laboratory of Luminescent Materials and Devices (South China University of Technology).

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62. Cai, F.; Yu, J.; He, S. Vectorial electric field Monte Caro simulations for focused laser beams (800 nm-2220 nm) in a biological sample. Prog. Electromagn. Res. 2013, 142, 667–681. 63. Qian, J.; Zhu, Z.; Qin, A.; Qin, W.; Chu, L.; Cai, F.; Zhang, H.; Wu, Q.; Hu, R.; Tang, B. Z.; He, S. High-order non-linear optical effects in organic luminogens with aggregation-induced emission. Adv. Mater. 2015, 27, 2332–2339. 64. Zhu, Z.; Qian, J.; Zhao, X.; Qin, W.; Hu, R.; Zhang, H.; Li, D.; Xu, Z.; Tang, B. Z.; He, S. Stable and size-tunable aggregation-induced emission nanoparticles encapsulated with nanographene oxide and applications in three-photon fluorescence bioimaging. ACS Nano 2016, 10, 588–597.

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Chapter 11

AIEgens for Drug Delivery Applications

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Jing Liang,1 Youyong Yuan,1 and Bin Liu*,1,2 1Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117585 2Institute of Materials Research and Engineering, 3 Research Link, 117602 Singapore *E-mail: [email protected]

Lack of therapeutic efficiency and prevalence of side effects are the major problems in therapy development, which are usually caused by low drug concentration, poor biodistribution and limited drug targeting ability. Drug delivery systems (DDS) that can selectively deliver therapeutic agents to target site of action is highly demanded. This necessitates a controlled drug release mechanism which can respond to characteristic stimuli in target cells or tissues and a signal reporter that can keep track of drug trafficking, release and activation behaviors. AIEgens have demonstrated excellent versatility, biocompatibility and sensitivity as biosensing and bioimaging probes, which are highly compatible with DDS for tracking of drug location, signaling of drug activation and monitoring of therapeutic effects. These DDSs can easily incorporate other functional elements to achieve multi-modal therapy and imaging.

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Introduction As one of the most important applications in the biomedical realm, drug delivery is receiving increasing research interest, not only driven by the thriving nanotechnology, but also the need for personalized medicine to achieve higher therapeutic efficiency. Efficient drug delivery can significantly improve clinical effectiveness and reduce the treatment cycle. Drug delivery system (DDS) that enables controlled drug release at targeted sites is crucial for achieving good therapeutic effect with minimal systemic toxicity. A typical DDS consists of a carrying vehicle loaded with therapeutical agents via encapsulation, covalent and noncovalent binding. The carriers are usually biocompatible hydrogels, dendrimers, liposomes, micelles and inorganic nanoparticles (carbon nanotubes, gold, silver and silica) (1), which can be further functionalized with elements to improve their performance in longevity, cell uptake, stimuli-responsiveness and visual tracking ability. Drug delivery can be achieved via simple desorption of the loaded drug. However, to achieve on-demand drug delivery with improved drug release efficiency, DDSs are usually designed to respond to specific stimuli which causes physiochemical changes of the carriers to release cargo (2). Flurescence tagging is one of the most powerful tools for drug tracking due to its high sensitivity, versatility and good compatibility with biosamples. Fluorescence reporters are useful in revealing the information of biodistribution and drug delivery kinetics of the administered drug, which is crucial for therapy development and evaluation. Conventional DDSs based on quantum dots and small molecule dyes have limited application due to their high cytotoxicity or aggregation-caused quenching (ACQ) nature (3, 4). The fluorescence tag may also be hydrolyzed during intracellular tracking or affect the uptake behaviors of the naked drug. AIEgens with unique aggregation-induced emission (AIE) characteristics have emerged as promising materials for drug delivery applications. A series of light-up probes and nanoparticle probes have been developed based on AIEgens for biosensing and bioimaging (5–8), which are promising to be transformed into theranostic platforms by introducing drug or prodrug elements. Both specificity and controlled drug release functions are essential for increasing local drug concentration in pathological sites with enhanced therapeutic efficiency and reduced side toxicity. The cell specificity can be achieved by functionalization of targeting ligands which may have specific interaction with cell surface markers or preferential accumulation in target organelles. For nanoparticle based DDS, the enhanced permeation and retention (EPR) effect may also contribute to the targeting effect to tumor sites (9). On the other hand, the controlled drug release or on-demand drug delivery can be achieved by employing a stimuli-responsive mechanism. In light of the fact that many disease conditions such as cancer are associated with abnormalities in the microenvironment including change in acidity, temperature, redox potential and enzyme activity (10), chemical moieties that are responsive to these stimuli can be integrated into the system to allow for stimuli-regulated drug release. Furthermore, DDS can be designed to be responsive to external stimuli such as light, ultrasound 272 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

and magnetic field to introduce a different modality therapy and imaging such as photothermal and photodynamic therapy and magnetic resonance imaging (MRI). This chapter reviews the examples of AIEgens in theranostic applications, with a focus in delivering of chemotherapeutic agents. The examples are organized according to working mechanisms, each having different approaches in achieving therapeutic effects.

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DDS Based on Light-Up Probes AIEgen based light-up probes here refer to molecular probes that are typically are nonemissive in aqueous media and become highly emissive upon interaction with target analytes (11). The fluorescence turn on based on solubility change is especially useful for sensing of bioanalytes such as enzymes and small molecule reductant that may lead to cleavage of the AIEgen conjugates. Inspired by these designs, a few theranostic probes have been developed based on a Pt prodrug. Cisplatin is one of the most effective drugs used by clinic to treat a wide spectrum of malignancies as they can bind to DNA and cause cell apoptosis (12). However, the use of cisplatin is limited due to its severe side effects (13). As an alternative, the non-toxic Pt(IV) has been found to be an effective prodrug which can be activated by reducing agents such as ascorbic acid to resume the latent cytotoxicity (14). Taking advantage of this mechanism and AIE effect, light-up theranostic probes have been constructed. The first example of such probes was demonstrated for in situ monitoring of therapeutic response in 2014 (15). As shown in Figure 1A, the theranostic system is comprised of four major elements: (i) the chemotherapeutic prodrug platinum(IV), which can be reduced to active toxic Pt(II) form intracellularly; (ii) a tetraphenylsilole (TPS) unit with AIE property; (iii) an Asp-Glu-Val-Asp (DEVD) peptide sequence which can be cleaved by caspase specifically upon apoptosis activation; and (iv) a cRGD tripeptide as a targeting ligand that can bind αvβ3 integrin surface biomarker. This probe is essentially nonemissive due to good water solubility and it can be specifically taken up by cancer cells that overexpress αvβ3 integrin (Figure 1B). Upon entering the cells, the prodrug is reduced to its Pt(II) counterpart which triggers the cell apoptosis and activates caspase-3 to cleave the DEVD peptide and turn on fluorescence. The light-up response is thus able to real-time monitor the therapeutic effect of anticancer drugs in the early stage. Knowing how and when drugs are activated in cells are also important in therapy development and pharmacokinetic studies. Liu’s group has developed two theranostic AIE probes for in situ monitoring of drug activation. One example is based on a tetraphenylethene pyridinium (PyTPE) AIEgen conjugated to both Pt prodrug and cRGD targeting ligand, which is able to light-up upon drug activation and release of the highly fluorescent residue (16). The other example demonstrated a TPE-based probe that can simultaneously track two drugs, namely Pt prodrug and doxorubicin (DOX), which can produce synergistic anticancer effect (17). It takes advantage of the efficient energy transfer between TPE and DOX to report the drug location and activation through fluorescence changes. 273 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 1. Chemical structure of targeted theranostic platinum (IV) prodrug with built in apoptosis sensor (A) and schematic illustration of the probe for in situ evaluation of therapeutic response (B). Adapted with permission from ref. (15). Copyright 2014 American Chemical Society. Combinational therapy using multiple therapeutic modality can not only enhance therapeutic efficiency, but also offers additional advantages in overcoming drug resistance and inducing anticancer immunity (18). Recently, Liu’s group reported the combinatorial photodynamic and chemotherapy based on a light-up probe incorporating both a Pt prodrug and an AIE active photosensitizer (PS) (19). Upon entering the targeted cell, the probe is reduced by intracellular glutathione to generate cytotoxic cisplatin for chemotherapy, yielding a highly fluorescent PS residue that can not only report drug activation, but also generation reactive oxygen species (ROS) for photodynamic therapy (PDT) to kill cisplatin resistant cells. Another strategy to achieve targeted drug delivery and reduced systemic toxicity is by targeting altered redox status associated with certain organelles, in particular, mitochondria in cancer cells. Mitochondria play vital functions in eukaryotic cells and they are featured by negative membrane potential (20). As mitochondria in cancer cells usually have more negative membrane potential as compared to those in normal cells, targeting mitochondria offers a promising approach for cancer therapy with improved efficiency. As an example, Liu’s group demonstrated the selective cancer killing with a mitochondria targeting probe with both AIE and excited-state intramolecular proton transfer (ESIPT) characteristics (21). As shown in Figure 2, the probe AIE-mito-TPP consists of an AIE+ESIPT active fluorogen and two units of triphenylphosphonium (TPP) with lipophilicity and delocalized positive charge which can preferentially accumulate in mitochondria. It was found that HeLa cells incubated with the probe showed higher cytotoxicity as compared to fibroblast NIH-3T3 cells and cell viability further reduces with increasing probe concentration. Results further revealed that 274 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the probe accumulated in mitochondria can decrease their membrane potential, causing generation of ROS and inhibition of ATP to achieve cancer cell killing. A similar strategy was used for combination chemotherapy and PDT using an AIE active PS (22). Among the various probes prepared, the probe with one TPP group (TPECM-1TPP) demonstrated only significant cytotoxicity under light irradiation while the one with two TPP groups (TPECM-2TPP) demonstrated high cytotoxicity both in dark and under light irradiation which are due to depolarization of mitochondria membrane potential and ROS generation. The findings elucidate the importance of molecular design in realizing mitochondria targeted therapy.

Figure 2. Molecular structures of mitochondria targeting AIE probes. A number of AIE probes have been reported to have specificity for mitochondria (23–27). While most of them are applied for mitochondria imaging and tracking due to their good biocompatibility, those who have high cytotoxicity have the potential to be used for the other purpose—killing the cells for chemotherapy applications. For example, Tang’s group has designed a mitochondria targeting probe TPE-In (23), which leads to significant reduction of cell viability upon staining HeLa cells with the probe. The anti-cancer effect was allegedly attributed to interaction of the probe with DNA due to the planarity of the probe. Organic Nanoparticle Based DDS Nanoparticle based DDSs typically consist of a drug molecule that is either covalently linked to an AIEgen or encapsulated within an AIEgen or AIEgen-polymer complex shell. Such DDSs usually are designed to have pH-responsiveness which enable drug release upon change in pH in the targeted organelles. 275 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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For example, Liang’s group has reported a self-indicating nanoparticle DDS based on an AIEgen for visualization of spatiotemporal drug release (28). A negatively charged TPE and the positively charged DOX were self-assembled into TPE-DOX nanoparticles (TD NPs) in aqueous medium via electrostatic interaction (Figure 3). These TD NPs were found to be pH responsive: at neutral pH, the TD NPs display weak fluorescence due to FRET from TPE to DOX; at lower pH, the charge reversion of TPE leads to dissociation of the NPs and subsequent recovery of blue fluorescence from TPE and red fluorescence from DOX. Confocal fluorescence images of the cells stained by TP NPs show that blue emission from TPE is observed in cytoplasm region and red emission from DOX is mainly localized in nucleus region, with some signal overlap with TPE in the lysosome. After 2 h incubation, nearly all DOX are translocated into nucleus. It was thus concluded that when taken up by cancer cells, TD NPs are transported into lysosomes where drug release occurs due to the low pH value of 5.5. The dissociated DOX will then enter nucleus indicated by red emission while the TPE remains in cytoplasm with blue emission. Therefore, by tracking the spatiotemporal transition the colors, the subcellular location of TD NPs, sites of drug release and action can be clearly visualized. The TD NPs were found to be more effective in inhibiting cancer cell growth as compared to free DOX at the same concentration. The DDS designed here is advantageous over conventional dyes and quantum dots based systems as it is free of ACQ effect, cytocompatible and does not affect drug functions. Based on a similar principle, another nanoparticle-based DDS was also reported by the same group using drug loaded self-assembly micelles (29). The nanocarrier is an amphiphilic polymer comprised of TPE and polyethylene glycol (PEG) conjugate. The nanomicelles were formed through hydrophobic interaction between TPE and DOX to leave the hydrophilic PEG arms as water soluble shell. Drug release is triggered when the micelles are transported into lysosomes, in which the protonation of DOX leads to electrostatic repulsion between drug molecules and reduced hydrophobic interaction between DOX and TPE that facilitates drug dissociation. It was found that the DOX-loaded micelles show higher anticancer efficiency than that of free DOX. The above two examples are nanoparticles with drug and AIEgen associated based on electrostatic or hydrophobic interactions and the drug release is triggered by pH-induced disruption of these interactions. Another approach to fabricate nanoparticles based DDSs is through covalent conjugation between the drug and AIEgen, and the pH responsiveness can be achieved using a pH sensitive linker. As an example, Liang’s group designed a probe with a TPE and DOX linked by a pH responsive hydrazone linker for dual-color fluorogenic drug tracking (30). The hydrazine bond is known to be cleavable under low pH environment and it does not produce pendent residues that may affect the drug properties (31, 32). The TPE-DOX conjugates can self-assemble into nanoparticles and they display weak fluorescence due to FRET between the two. Once they are in lysosomes, the hydrazine bond will be disrupted, releasing both free DOX and TPE to evoke a dual-color fluorescence response. By observing the dual color recovery, the kinetic drug release in live cells can be captured. 276 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 3. Schematic illustration of TD NP formation and mechanism of drug delivery. Adapted with permission from ref. (28). Copyright 2015 John Wiley and Sons. Based on similar principles, Liu and coworkers also reported the AIE DDSs using the pH responsive hydrazine linker (33, 34). Instead of linking the AIEgen and drug, the linker is designed to form the bridge between AIEgens and a hydrophilic polymer. As shown in Figure 4, the TPE can be either conjugated with a PEG chain or multiple TPE molecules can be conjugated with a dextran polysaccharide to form a copolymer. These amphiphilic conjugates are then used to encapsulate DOX to serve as a drug delivery carrier. The encapsulating polymer of the DDSs are biocompatible and they help to prevent premature drug release during blood circulation and minimize side effects. Upon cell uptake through endocytosis, they are both transported via endo/lysosomes in which pH-regulated drug delivery take place. The DDSs show dose-dependent cytotoxicity in HeLa cells and they are useful for both drug tracking and controlled drug delivery. The pH responsive DDSs illustrated above all target the lysosomal low pH environment within the cells. Liu and Tang recently has reported a nanoparticle probe Net-TPS-PEI-DMA for targeting tumor extracellular acidic microenvironment and its therapeutic applications (35). The tumor extracellular region (pH 6.5–7.2) is known to be more acidic than the blood and normal tissues (pH ~7.4), thus providing a hallmark for targeting tumors for drug release. In this work, the nanoparticle probe consists of a TPS AIEgen and a pH responsive charge-reversible polymer polyethyleneimine (PEI) modified with 2,3-dimethylmaleic anhydride as shown in Figure 5. The probe is negatively charged and almost nonemissive at physiological pH (7.4). Once it is in the acidic tumor environment, its charge is reversed which enables its electrostatic interaction with the positively charged cell membranes and components to turn on fluorescence. Unusually, this is the first example of nanoparticle based AIE probe that shows light-up response. The nanoparticle probe was also found to cause 277 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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cytotoxicity to cancer cell through suppression of Akt pathway and activation of the apoptotic pathway. The selective inhibition of cancer cells over normal cells renders them an effective tool for image-guided tumor therapy.

Figure 4. Schematic illustration of drug-loaded micelles based on TPE-polymer conjugates for cell imaging and pH-controlled drug delivery. Reproduced with permission from ref. (33, 34). Copyright 2015 the Royal Society of Chemistry.

Figure 5. Chemical structure of the probe Net-TPS-PEI-DMA and schematic illustration of the light-up nanoparticle probe for cancer cell imaging. Reproduced with permission from ref. (35). Copyright 2015 the Royal Society of Chemistry. 278 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Inorganic Nanoparticle Based DDS In addition to the stimuli-responsive DDSs, there are also delivery systems based on simple physical desorption of drugs. In such systems, an AIEgen and drug molecules are usually coloaded in a mesoporous structure and the drug molecules are slowly released in the desired location via physical desorption while the AIEgen can be used for imaging and drug tracking. For example, in Yu’s work in 2011, a mesoporous silica structure SBA-15 grafted with BTPE showed strong blue emission and was further loaded with a model drug ibuprofen (Figure 6) (36). The blue emission is enhanced after drug loading due to additional inhibition of intramolecular rotations. The drug delivery of the complex nanostructure was tested in simulated body fluid and decreased fluorescence was observed with increasing drug release due to lessened AIE effect. Thus the change of fluorescence intensity can be used to monitor drug release process. A similar strategy was reported based on an AIEgen bridged hollow hydroxyapatite nanocapsules for delivery of ibuprofen drugs in cancer cells (37). The nanostructure showed good biocompatibility and drug dosage dependent fluorescence changes.

Figure 6. Synthetic route for preparing mesoporous SBA-15 loaded with AIEgen and drug molecules. Reproduced with permission from ref. (36). Copyright 2011 the Royal Society of Chemistry. Similar work was reported by Tao and Wei using drug and AIEgen co-loaded mesoporous silica nanoparticles for cancer imaging and therapy (38). The AIEgen 9,10-distyrylanthracene derivative and cancer killer agent cetyltrimethyl ammonium bromide (CTAB) form amphiphilic complex first and they serve as structure-directed template for forming the mesoporous nanocomposite. Results show that A549 cells incubated with the nanocomposites yielded bright fluorescence and the cell viability dropped as a result of drug release and the cytotoxicity increases with increasing nanocomposite concentration.

Conclusions The unique physiological properties of AIEgens allow them to play different roles in DDS. The switching on and off of AIE light-up probes are dynamically linked to its solubility state regulated by drug release or enzyme activation. Capitalizing on fluorescence energy transfer between AIEgens and fluorescent drugs, multicolor response can be used for dynamic monitoring of fluorogen-drug separation. A number of Pt prodrug based theranostic probes have been designed and demonstrated for tracking of single and multiple drug activation, image-guided therapy in targeted cells and evaluation of therapeutic response. 279 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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AIEgens can not only serve as image contrast agent, but also as therapeutic agent if they exhibit cytotoxicity or photoactivity. Examples have been demonstrated for AIE photosensitizers for combination of chemotherapy and photodynamic therapy through targeting the mitochondria of cancer cells. When used in solid form, AIEgens display exceptionally high brightness with good photostability, allowing them to be used for self-indicating DDS. A simple strategy to track drug delivery was introduced, which involves encapsulation of both AIEgens and drug molecules with silica or other inorganic structures. The drug release can be monitored by measuring the change of fluorescence associated with desorption of drugs. A group of work have been demonstrated for spatiotemporal drug release using AIE-drug or AIE/polymer-drug nanoparticles through pH responsive mechanism. The nanoparticle based DDS are known to be better uptaken by cells via endocytosis and show homing effect to tumor tissues due to EPR effect. The examples covered in this review are bound to inspire the rational design of more advanced DDS based on AIEgens such as combitorial therapeutics. Through variation of therapeutic agents, recruitment of targeting elements and adoption of different internal and external stimuli, the applications of AIE DDS will be greatly expanded.

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Editors’ Biographies

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Michiya Fujiki Michiya Fujiki has been a Professor at the Graduate School of Materials Science, Nara Institute of Science and Technology in Japan since 2002. Previously, he worked for the research and development division of Nippon Telegraph and Telephone (NTT) Corporation from 1978 to 2002, and also served as a principal investigator of the JST-CREST program, directed by Emeritus Prof. Hideki Sakurai (Tohoku University), from 1998 to 2003. Prof. Fujiki is interested in chiroptics of σ-/π-conjugated polymers, oligomers, molecules, and atoms in the ground and photoexcited states. He has over 300 publications and an h-index of 48.

Bin Liu Bin Liu is the Dean’s Chair Professor in the Department of Chemical and Biomolecular Engineering, National University of Singapore. Her research focuses on the development of organic nanomaterials with explorations on their sensing, imaging, and device applications. She has over 260 publications and an h-index of 58. Bin Liu has received many prestigious awards and was named among The World’s Most Influential Minds by Thomson Reuters. Dr. Liu is a Fellow of the Royal Society of Chemistry and serves as an Associate Editor of Polymer Chemistry.

Ben Zhong Tang Ben Zhong Tang is Chair Professor in the Department of Chemistry and the Division of Biomedical Engineering at the Hong Kong University of Science & Technology. He is interested in polymer chemistry, materials science, and biomedical engineering. He has published more than 800 papers with an h-index of 96. He has been listed as one of the most cited researchers in the areas of both chemistry and materials science. Dr. Tang was elected to the Chinese Academy of Sciences and the Royal Society of Chemistry. He is now serving as Editor-in-Chief of Materials Chemistry Frontiers.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Subject Index

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A Aggregation-induced chirogenesis, 63 AIEnh-CD, AIEnh-ORD, and AIEnh-CPL aggregates, 69 aggregate 14, AIEnh-CD and UV spectra, 82f aggregate 16, AIEnh-CD and UV spectra, 80f aggregates, AIEnh-CD and UV spectra, 74f AIEnh-CD and UV spectra, 70f CD-silent π-conjugated polymers, chemical structures, 76c CD-silent π-conjugated polymers and alcohols, chemical structures, 79c CD-silent polysilanes and chiral solvents, chemical structures, 74c CPL-active but CD-silent, chemical structures, 85c homogeneous chloroform solution, UV-vis and PL spectra, 77f limonene chirality, 73 semiflexible and rodlike polysilanes, chemical structures, 69c AIEnh-CD and AIEnh-CPL, 67 schematic Jablonski diagram, 68f Aggregation-induced emission luminogens (AIEgens) multifunctional AIEgens, 187 representative multifunctional AIEgens, electroluminescent performances, 192t representative multifunctional AIEgens, examples, 191f silole-based AIEgens, 175 representative silole-based AIEgens, chemical structures, 176f silole derivatives, photophysical properties, 176 some representative silole-based AIEgens, electroluminescent performances, 178t TPE-based AIEgens, 180 representative TPE-based blue AIEgens, chemical structures, 182f representative TPE-based green and yellow AIEgens, chemical structures, 182f representative TPE-based red AIEgens, chemical structures, 183f

some representative TPE-based AIEgens, electroluminescent performances, 184t AIE nanoparticles, 217 AIE NPs for in vivo imaging 10, chemical structure, 227f 11, chemical structure, 230f 12, chemical structure, 234f 13, chemical structure, 235f ADSCs, paracrine analyses, 233f dual-modality imaging, 234 fluorescence imaging technique, 232 intravital two-photon fluorescence imaging, 229 10-loaded BSA NPs, brightness, 228 mice, in vivo fluorescence images, 231f PFV, chemical structure, 229f tumors, in vivo fluorescence imaging, 226 in vivo long-term cell tracking, 231 introduction, 218 in vitro imaging, AIE NPs 4-6, chemical structures, 222f 7, chemical structure, 224f 8, chemical structure, 225f 9, chemical structure, 226f cancer cells, targeted imaging, 220 compound 3, chemical structures, 221f compounds 1 and 2, chemical structures, 220f non-specific cell imaging, 219 specific organelle imaging, 221 in vitro long-term cell tracking, 223 AIE nanoprobes background mouse cortex, calculated attenuation length, 246f multi-photon imaging, 247 multi-photon luminescence process, schematic illustration, 247f optical tissue windows, 245 in vivo MPL imaging, AIE nanoprobes, 248 BT, chemical structure, 259f BT nanodots, absorption and fluorescence spectra, 259f BT nanodots, 3D reconstructed 2PL in vivo images, 260f light intensity distribution of 1040 nm and 800 nm laser beams, simulation, 258f

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liver at various time points, fluorescence images, 260f NIR-IIb window, AIE nanoprobes for in vivo 3PL imaging, 261 brain blood vessels, 3PL microscopic imaging, 264f brain blood vessels of a mouse, 3PL microscopic imaging, 263f ear blood vessels of a mouse, 3PL microscopic imaging, 263f synthesis of TTF-NGO nanoparticles, protocol, 262f various laser beams at a depth of 1.5 mm of biological tissue, simulation of the focal spots, 262f TTF nanodots, 3D reconstructed 2PL in vivo images, 261f in vivo 2PL imaging, AIE nanoprobes, 249 BTPEBT, chemical structure and molecular geometry, 249f BTPEBT nanodots, normalized absorption and fluorescence spectra, 250f BTPEBT nanodots fabrication, schematic illustration, 250f DPP-based compounds, chemical structure, 257f F127 and PFBT, chemical structure, 256f nanodots with different TTF in reactants, TEM images, 252f 1PL and 2PL, bright field, 253f 2PL mouse brain imaging, 251f TPE-TPP, absorption and fluorescence spectra, 254f TPE-TPP, chemical structure, 254f TPE-TPP stained microglia in mouse brain, 2PL images, 255f TPE-TPP treated primary neurons, 2PL images, 255f TTF, chemical structure, 252f TTF-F127-SiO2 nanoparticles, schematic illustration, 256f TTF nanodots, absorption and fluorescence spectra, 253f in vivo 2PL imaging with excitation wavelength, AIE nanoprobes, 257

C Chemo-/biosensors enzymatic assays, 97

AChE activity assay and inhibitor screening, design rationale, 100s design rationale for the fluorometric assay with TPE 7, illustration, 104s ensemble of silole 1, fluorescence spectra, 103f ensemble of TPE 3, fluorescence spectra, 102f fluorescence turn-on assay for AChE activity, design rationale, 101s fluorescence turn-on detection of DNA, illustration, 98s monoamine oxidase activity assay, design rationale, 102s relative fluorescence intensity of silole 1, variation, 103f silole 1 in the presence of different amounts of ssDNA, fluorescence spectra, 98f TPE 3, fluorescence spectra, 101f TPE 4, fluorescence spectra, 99f TPE 7, fluorescence spectra, 105f trypsin activity assay and inhibitor screening, design rationale, 99s introduction, 93 metal cations and anions, sensors, 105 Al3+ fluorescence sensors, 108 design rationale for the fluorometric assay, illustration, 109s design rationale for the fluorometric assay with silole 1 for cyanide, illustration, 110s silole and TPE-based probes for ions sensing, chemical structures, 106s TPE 11, fluorescence spectra, 107f TPE 18, fluorescence spectra, 109f sensing gamma-ray radiation, 117 aqueous solution of silole 1, fluorescence spectrum, 118f CHCl3 solution of TPE 35, fluorescence spectra, 119f design rationale for the fluorescence detection of gamma-ray radiatio, illustration, 118s TPE 35 and TPE 35-HCl, chemical structures, 119s sensors for biomacromolecules, 94 compounds for biomacromolecules, molecular structures, 94s fluorescence turn-on sensor for heparin, illustration, 95s silole 1, fluorescence spectra, 95f silole 1, fluorescence variation, 96s small molecules, detection, 111 aldehyde groups, different reaction kinetics, 115

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TPE-functionalized mesoporous materials, reversible fluorescence quenching mechanism, 145f AIEgens-functionalized MOFs, 132 carbon-based explosives, detection, 135 MOF from TPE-based AIEgen, synthesis, 134f MOF 1 with 1D rhombus channels, x-ray crystal structure, 136f organic luminescent ligands, molecular structure, 132f TABD-MOF-3 deposited paper strips, photographs, 137f X-ray crystal structures of Zn based MOF, portions, 133f AIEgens-functionalized POPs, 137 AIEgen 14, representative conjugated microporous organic polymer constructed, 142f AIEgen 15, local hydrogen bonding environments, 143f building blocks with AIE properties used in POPs, molecular structure, 138f luminescent POPs, representative ideal molecular structures, 140f luminescent porous inorganic-organic hybrid polymers, syntheses, 141f Suzuki coupling polymerization, preparation of POPs, 138f synthesis of conjugated polymers CMPs, schematic representation, 139f introduction, 130 explosives and explosive-like substances, chemical structures, 131f

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design rationale for the fluorescence turn-on detection of LA by employing the AIE feature of silole, illustration, 114s ensemble of silole 1, fluorescence spectra, 115f fluorescence turn-on detection of H2O2, design mechanism, 113s silole and TPE probes for sensing small molecules, chemical structures, 112s TPE 25, fluorescence spectra, 113f

D Drug delivery applications, AIEgens, 271 introduction, 272 drug-loaded micelles, schematic illustration, 278f electrostatic or hydrophobic interactions, 276 inorganic nanoparticle based DDS, 279 light-up probes, DDS based, 273 mitochondria targeting AIE probes, molecular structures, 275f organic nanoparticle based DDS, 275 preparing mesoporous SBA-15, synthetic route, 279f probe Net-TPS-PEI-DMA, chemical structure, 278f targeted theranostic platinum, chemical structure, 274f TD NP formation and mechanism of drug delivery, schematic illustration, 277f

E

L

Explosives detection, 129 AIEgens-functionalized mesoporous materials, 143 AIEgen-functionalized mesoporous silica nanoparticles film in DNT saturated vapor, time-dependent fluorescence spectra, 146f building blocks with AIE properties, molecular structure, 144f Materials Studio program, molecular modeling of PA, NT and NB, 146f MHAp-FL with IBU, fluorescence spectra, 144f

Liquid crystalline AIE luminogens, 151 introduction, 152 representative AIE molecules, molecule structure, 153s materials and methods, 153 aggregation-Induced emission active liquid crystalline polymers, molecular structures, 167s Colh phase of GCS, POM image, 165f compounds P4 and P5 between crossed polarizers, optical micrographs, 162f

293 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ix002

cooling, mesomorphic textures observed, 168f GDCS., molecular structures, 166f GDCS in condensed state, photos, 167f LE-LC compounds, molecule structure, 154f LE-LCD device in the electric field-off, photograph, 160f LE-LC GCS, molecule structure, 164s LE-LCs P4 and P5, synthetic routs, 161s reflective-luminescent display device, photographs, 161f TPE-LC1 and TPE-LC2, molecular structures, 157f TPE-LC1 as a function, emission color change, 158f TPE4Me, synthetic route, 155s TPE4Mes in THF and THF/water mixtures, PL spectra, 156f TPE-PPE, chemical structure, 159f Z-E isomerization, schematic illustration, 163f Luminogenic polymers, 27 luminescent polymer containing unconventional fluorophore, 48 glycodynamer 87, molecular structure, 49c PAE 100-103, synthetic routes and cartoon structures, 54s PAMAM, emission behavior, 51 PAMAMs 95 and 96, synthetic routes and cartoon structures, 52s PEI 107, synthetic routes, 55s PMV 113 and other polymers 114-117 for control, molecular structures, 57c polymers 90-92, synthetic routes, 50s PVP 108, oxidized hydrolyzate processes, 55s SA end-capped PIB 112 and its proposed aggregation mode, molecular structure, 56c silicon-containing PAMAMs 97, synthetic routes, 53s other typical AIEgen-based polymers, 42 boron diiminate-based conjugated polymers 79-86, molecular structures, 48c boron ketoiminate -based conjugated polymers 72-78, molecular structures, 48c DSA-based polymer 57, molecular structure, 43c

DSA-based polymer 58, molecular structure, 43c nitrilevinylphenothiazine derivative-based polymers 65-67, molecular structures, 46c phenyl-substituted quinolone-based AIE-active polymer 71, synthetic route, 47s polymers 59 and 60 with 2,4,6-triphenylpyridine unit as side group or knot, molecular structures, 44c TPT-based AIE-active polymers 63 and 64, molecular structures, 45c 2,4,6-triphenylpyridine-based polymers 59 and 60, molecular structures, 44c silole-based aie-active polymer, 38 hyperbranched poly(phenylenesilolene), molecular structure, 41c hyperbranched poly(phenylenesilolene) 56, synthetic route, 42s silole-based polymer 47, synthetic route, 38s silole-based polymer 51 and 52, molecular structure, 40c silole-based polymers 48 and 49, molecular structures, 39c silole end-capped polymer 53, molecular structure, 40c silole-polymer 50, molecular structure, 39c TPE-based AIE-active polymers, 29 hyperbranched polymer 40-42, molecular structures, 36c THF/water mixtures with various water fractions, PL spectra, 31f TPE-containing conjugated microporous polymer 45 from hyperbranched polymer 44, synthetic route, 37s TPE-containing crosslinked polymer 43, molecular structure, 37c TPE-containing hyperbranched polytriazole 39, synthetic route, 36s TPE-containing hyperbranched polytriazoles 35 and 36, synthetic route, 35s TPE-containing polytriazoles 9-11, synthetic routes, 31s TPE-containing polytriazoles 15, 16, 20 and 21, synthetic routes, 32s TPE-containing polytriazoles 24, synthetic routes, 32s TPE-containing polytriazoles 27-30, synthetic routes, 34c

294 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

series of pure organic luminogens, chemical structures, 18f rigid matrix, dopping/trapping, 10 designed phosphor and polymer, chemical structures, 12f efficient persistent RTP from pure organic luminogens, material design, 11f RTP from solutions, 19 fluorenen derivatives, chemical structure, 19f reversible inclusion of the binary IQC[5]/CB[7] system, schematic illustration, 19f

TPE-containing polytriazoles 31, synthetic routes, 34c TPE-containing polytriazoles 4 and 5, synthetic routes, 30s TPE-containing polytriazoles 25 and 26, synthetic routes, 33c

Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ix002

R Room temperature phosphorescence, 1 (Co)crystallization-induced phosphorescence, 5 BZL and its derivatives, chemical structures, 6f conformer DITFB and other luminogens, chemical structures, 10f persulfurated benzene molecules and tellurophenes, chemical structures, 7f some CIP pure organic luminones, chemical structures, 8f various brominated aromatic aldehydes, structures, 9f difluoroboron chelates, 3 BP and its derivatives, chemical structures, 5f dual-emissive boron diketonates and β-hydroxyvinylimineboron compounds, chemical structures, 4f fundamental considerations, 3 luminogens and hosts, creating of intermolecular interactions, 13 Br6A, G1, and PVA, chemical structures, 15f CDs dispersed in PVA, proposed RTP mechanism, 14f series of reported molecules, steady-state photoluminescenceand ultralong phosphorescence, 15f some water soluble conjugated polymers, chemical structure, 13f persistent RTP, 16 BF2EMO and its crystal emission photographs, chemical structure, 17f carbazole crystals, design principle, molecular stacking, 18f

V Visualizing cell structures cell structures, fluorescence imaging, 200 6 and Nile red, HeLa cells, 208f dual functional mitochondrial probe, 205 HeLa cells, fluorescent images, 202f HeLa cells, hyperglycemic-induced mitochondria fragmentation, 203f HeLa cells stained with 4, fluorescence images, 206f lipid droplets, 208 lysosomes, 206 mitochondria, 200 morphology tracking, 201 3-stained HeLa cells, changes in fluorescence intensity, 204f 5-stained HeLa cells, fluorescence images, 207f cellular environment, fluorescence imaging intracellular pH, 209 intracellular viscosity, 211 membranes with different lipid compositions, proposed packing modes, 212f 7 to pH change, fluorescent response, 210f

295 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.