Immunosensing for Detection of Protein Biomarkers [1st Edition] 9780081020005, 9780081019993

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Immunosensing for Detection of Protein Biomarkers [1st Edition]
 9780081020005, 9780081019993

Table of contents :
Content:
Front Matter,Copyright,About the Authors,PrefaceEntitled to full text1 - Introduction, Pages 1-30
2 - Signal amplification for immunosensing, Pages 31-75
3 - Electrochemical immunosensing, Pages 77-110
4 - Functional nanoprobes for immunosensing, Pages 111-142
5 - Chemiluminescent immunoassay, Pages 143-169
6 - Electrochemiluminescent immunosensing, Pages 171-206
7 - Multianalyte immunoassay, Pages 207-237
8 - Fast immunoassay, Pages 239-267
9 - Proximity hybridization regulated immunoassay, Pages 269-286
Index, Pages 287-293

Citation preview

Immunosensing for Detection of Protein Biomarkers

Immunosensing for Detection of Protein Biomarkers Huangxian Ju Guosong Lai Feng Yan

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2017 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-08-101999-3 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Glyn Jones Acquisition Editor: Glyn Jones Editorial Project Manager: Thomas Van Der Ploeg Senior Production Project Manager: Priya Kumaraguruparan Cover Designer: Miles Hitchen Typeset by SPi Global, India

About the Authors

Huangxian Ju Changjiang Scholar, Professor of Chemistry, and Director of the State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, P. R. China [email protected] Guosong Lai Chutian Young Scholar in Hubei Province Professor of Chemistry, Hubei Normal University Huangshi, P. R. China [email protected] Feng Yan Leading Talent in Jiangsu medicine. Deputy Director, Department of Clinical Laboratory and the Province Key Laboratory of Cancer Molecular Biology and Translational Medicine, Jiangsu Cancer Hospital; Senior Researcher, Jiangsu Institute of Cancer Prevention and Cure Professor, Nanjing Medical University Nanjing, P. R. China [email protected]

Preface

Immunosensing involves the coupling of immunoreactions to appropriate ­transducers for producing analytical signals, which leads to excellent specificity and high ­sensitivity for selective detection of protein biomarkers in real samples. In recent ­decades, rapid development has seen made in immunosensing and immunoassay methods with various detection formats and wide applications in different fields, such as clinical, industrial, environmental, food, and agricultural analyses. In the medical field, considerable multidisciplinary efforts have been devoted to the development of precise, rapid, sensitive, and selective immunosensing of disease biomarkers and/or biomarkers panel for meeting emerging needs of early screening and diagnosis of diseases, monitoring of curative effect, and reliable point-of-care diagnostics in precision medicine, although there are still problems concerning the assay of analytes in clinical application due to the stability of immunosensing devices and nonspecific adsorption from complex sample matrix. A large number of research works in immunosensing methodology have been reported (more than 78,000 since 2000). These methods are generally simple to utilize and easy to realize with automation, digitization, and miniaturization. Therefore, immunosensing is now becoming one of the most widely used analytical technologies in protein biomarker detection. Although a large number of academic papers in immunosensing and immunoassay have been published in different journals recently, it is still difficult or time-­consuming for researchers, especially the beginners, to have a good understanding of the principles, methods, and research progress of immunosensing in a wide scope. The goal of this book is to not only offer a survey of the principles and methods of immunoassay and immunosensing, but also to present the latest achievements and detection strategies in different aspects such as electrochemical immunosensors, n­ anoprobe-based immunoassay, chemiluminescence immunoassay, electrochemiluminescent immunoassay, multianalyte immunoassay, optical imaging immunoassay, signal amplification for immunoassay, and so on. More importantly, based on the experience of these authors, the aim is also to bridge the common gap between research literature and new research ideas in order to develop immunosensing methodology. This material is presented in nine chapters, covering all the authors’ study topics of immunosensing methodology. Some experts who received the PhD degree from Ju’s group participated in the writing of some chapters, including Dr. Wei Cheng (Chapter 2), Dr. Zhanjun Yang (Chapter 5), Dr. Dajie Lin (Chapter 6), Dr. Zhifeng Fu (Chapter 7), Dr. Jie Wu (Chapter 8), and Dr. Kewei Ren (Chapter 9). This book also contains the previously published works of Dr. Zong Dai, Dr. Jiehua Lin, Dr. Dan Du, Dr. Lina Wu, Dr. Xuan Liu, Dr. Wenwen Tu, Dr. Shengyuan Deng, Dr. Chen Zong, Dr. Hong Liu, Dr. Chuan Leng, Miss Hua Yu, Miss Fang Tan, Miss Qiunan Xu, Miss

xPreface

Lisong Wang, Miss Jie Xu, and Mr. Jie Li during the time they studied in this group. We are very grateful to all these members for their contribution. This book is one of the monograph series published by the first author (Huangxian Ju) or with the coauthors, including Electroanalytical Chemistry and Biosensing Technologies (Science Press, 2006, Chinese), Bioanalytical Chemistry (Science Press, 2007, Chinese), Electrochemical Sensors, Biosensors and Their Biomedical Applications (Academic Press, Elsevier, 2007, English, and Chemical Industry Press, 2009, Chinese), NanoBiosensing—Principles, Development and Application (Springer, 2011, English, and Science Press, 2012, Chinese), and Nucleic Acid Detection: Methods for Analysis of DNA and microRNA (Intellectual Property Press, 2015, Chinese). This book offers a good reference for a broad audience, including peer researchers and graduate students who have similar research interests. It can provide readers with new research ideas to develop immunosensing methodology. The book can also be used as a graduate-level textbook for those studying for the master degree in analytical chemistry and clinical laboratory. We are fortunate to have the opportunity to undertake this project. We warmly acknowledge the gracious support of our families. Finally, we also thank Elsevier’s editors for doing a remarkable job to publish this book. Huangxian Ju Nanjing, PR China Guosong Lai Huangshi, PR China Feng Yan Nanjing, PR China

Introduction

1

Millions of people throughout the world face the risk of malignancies, which have been one of the leading causes of mortality. In cancer, as tumors develop, the cells or the organs can release specific proteins into the circulation system. The levels of these proteins as tumor-related antigens in serum are associated with the stages of tumors and can therefore be used as tumor biomarkers for screening and clinical diagnosis of cancer [1]. Hence, reliable, sensitive testing for these tumor biomarkers is crucial in early clinical diagnosis and in the evaluation of the recovery of patients with certain tumor-associated diseases. Compared with the conventional biochemistry-, immunology-, and molecular biology–based methods, immunosensors, which combine the unique advantages of immunoassay and biosensor, have been recognized as significant and received rapid development in the last decades [2,3]. This chapter briefly introduces the main principles of immunoassay and immunosensor as well as signal labels and the immobilization method of immunoreagents during the immunoassay of protein analytes. Future perspectives on immunosensors in the field of protein analysis are also evaluated.

1.1 Immunoassay Immunoassay is a highly selective bioanalytical method that measures the presence or concentration of analytes ranging from small molecules to macromolecules in a solution through the use of an antibody or an antigen as a biorecognition agent. The theoretical basis of immunoassay is the antibody-antigen immunoreaction as well as its coupling to appropriate transducers for producing an analytical signal. Thus high specificity is the unique advantage of immunoassay methods, which results from the use of purified ­antibodies and antigens as analytical reagents. An antibody is a protein (immunoglobulin) produced by b-lymphocytes (immune cells) in response to stimulation by an anti­ gen. Immunoassays measure the formation of antibody-antigen complexes by labeling or ­labeling-free format [4]. Due to the signal transduction and amplification by using a labeling system (e.g., enzyme label), high sensitivity can be also achieved for immunoassays.

1.1.1 Antigen and antibody The science of immunology is based on an organism’s ability to generate the biological effect known as the immune response. In higher forms of life, particularly in mammals, the immune system is a complex mechanism in which identification and communication take place in the blood and lymph. When a foreign substance (antigen) enters the body of an advanced animal, certain proteins (antibodies) are synthesized to identify the invader and to prohibit its harmful effects. Antibodies show very high specificity and binding constants toward their corresponding antigens. Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00001-3 Copyright © 2017 Elsevier Ltd. All rights reserved.

2

Immunosensing for Detection of Protein Biomarkers

Antibodies are divided into five classes (immunoglobulin IgG, IgA, IgE, IgM, and IgD) based on their structures and biological functions. Of the five classes of antibodies, IgG is the class used the most frequently for immunoassays because it exists at the highest level and is readily available. Generally, the structure of IgG is represented by a Y-shaped figure consisting of four polypeptide units (Fig. 1.1). Two of them are identified and known as the heavy chains with a molecular weight of 55,000–60,000 Da. The other two sequences are light chains with a molecular weight of 20,000–24,000 Da. The two double-ended segments of the Y are denoted as Fab fragments and are the sites at which antibody binds with antigen. The variable and hypervariable regions of Fab create an active portion that recognizes a specific area of the antigen. The singular segment at the other end of the Y shape is known as the Fc fragment, which cannot bind with antigen but has the ability to affix to the cell surface and to pass through the placenta [5]. The antigen molecule detected by immunoassay is often referred to as an “analyte.” It may be the natural antigens including such macromolecule substances as proteins and nucleic acids, or the haptens as some substances with low molecular weight, typically 4 ng/mL). Yu and Knoll [42] proposed an immunosensor for hCG based on the diffraction of surface plasmon. The inherent self-referencing mechanism of surface diffraction was found to be very effective for compensating fluctuations of the bulk. Although optical biosensors are highly sensitive, they are bulky, expensive, and require dedicated personnel to perform the tests. Additionally, colorimetric, fluorescence, and luminometric type of sensors require difficult labeling procedures that depend on indirect indicator-based signal schemes.

1.2.2 Electrochemical immunosensors In this case, an electrical signal in terms of change in currents and/or voltages is measured, which shows significant differences in magnitude if antigen-antibody complexes are formed. Based on their operating principle, electrochemical immunosensors can employ potentiometric (measuring of electrode potential or voltage differences), amperometric (measuring of current), capacitative (measuring of reciprocal capacitance),

8

Immunosensing for Detection of Protein Biomarkers

and conductimetric (measuring of conductivity or electrochemical resistance) transducers converting the chemical information into a measurable electrical signal. The single analyte enzyme-linked electrochemical immunosensor, developed in the 1990s, stands as a perfect example [43,44]. Most approaches feature antibodies attached to the sensor surface, so that antigen capture, enzyme-labeled secondary antibody binding, and detection are all done on the same surface. The most selective and sensitive electrochemical immunoassays employ the sandwich format. Thus, electrochemical immunosensors exclude biosensors, which require light, mechanical motion, or use of magnetic particles. Due to their low cost, low power, and ease of miniaturization, electrochemical immunosensors provide great promise for applications such as point-of-care diagnosis and biological warfare agent detection [45]. In Chapter 3, detailed samples will be presented to introduce the applications of electrochemical immunosensors.

1.2.3 Mass-sensitive immunosensors The study of mass-based immunosensors mainly focuses on microbalances of piezoelectric crystals [46]. The piezoelectric quartz-crystal device comprises a quartz-­ crystal wafer with different thicknesses sandwiched between two metal electrodes, which connect the device to an external oscillator circuit. The resonant frequency of quartz-crystal microbalance (QCM) depends on the mass of the crystal surface as well as the mass of any layer confined to the electrode area of the crystal. The change of crystal frequency can reflect a tiny change in mass on the electrode surface. Currently the piezoelectric immunosensors are used more and more for the determination of tumor markers in clinical diagnosis due to the advantages of label-free and real-time detection. Chou et al. [47] proposed a piezoelectric immunosensor for human ferritin by immobilizing antihuman ferritin antibody on a gold disk of a QCM. Human ferritin could be determined in a linear range from 0.1 to 100 ng/mL. Zhang’s group [48] presented a multichannel 2 × 5 model of piezoelectric quartz microarray immunosensor for quantitative detection of hCG in serum or urine samples. Compared with a one-channel immunosensor, this 2 × 5 model of microarray immunosensor could provide eight times higher detection speed for hCG assay. In recent years, microcantilever sensors, another kind of mass-based sensor, have attracted much attention for improved performance of QCM measurements [49]. Microcantilever sensors can sensitively monitor the molecular adsorption, which bends the microcantilever and changes its resonant frequency. If two surfaces of the cantilever are chemically different, different molecular adsorption will produce different surface stress between the top and bottom surfaces of the cantilever. When specific binding of biomolecules occurs on the surface of microcantilever, intermolecular nanomechanical force induces the cantilever to bend, and this can be observed as changes in cantilever deflection. Microcantilever sensors do not require any label or reporter molecule to signal the presence of a molecule on a biosensor surface. In particular, nanomechanical sensors have the advantage of high sensitivity with small area compared with other label-free biosensors (e.g., the quartz-crystal device). Wee et al. [50] prepared a label-free PSA immunosensor using self-sensing piezoresistive

Introduction9

microcantilevers. Electrical detection of antigen-antibody specific binding was accomplished through a direct nanomechanical response of microfabricated self-sensing microcantilevers. This cantilever immunosensor was used for the detection of PSA and C-reactive proteins and shown to be effective for clinic application. Wu and coworkers [51] developed a label-free immunosensor for PSA with microcantilevers using a polyclonal antiprostate antibody as a covalent linker. In this system, the PSA could be detected in the concentration range from 0.2 ng/mL to 60 μg/mL.

1.3 Immobilization method of immunoreagents As most immunoassays are performed in heterogeneous format, a key feature of immunosensors is the use of a solid substrate to couple either the antibody or antigen. To fabricate a sensitive and reliable immunosensor, a large variety of rigid materials such as chemical electrodes, polymer membranes or beads, microtiter plates, and magnetic bead can be applied as a proper matrix for binding the recognition biomolecules. The immobilization method of immunoreagents will decide their amount, distribution, orientation, and even bioactivity on substrates, thus directly affecting the performance of the immunosensor such as repeatability, stability, specificity, and detection limit. Commonly, the methods of physical adsorption, polymer entrapment, covalent binding, and several oriented immobilization approaches can be used for the immobilization of immunoreagents by combination with the characters of the solid substrate.

1.3.1 Physical adsorption The physical adsorption method adsorbs proteins on the solid substrate via noncovalent interactions, mainly electrostatic force, ionic bond, hydrogen bond, and hydrophobic interaction. The method is simple and generates little effect on bioactivity. However, physical immobilization often results in random orientation and weak attachment due to the weak attachment between biomolecules and the substrates. Generally, optical ELISA involves the nonspecific adsorption of the recognition elements on the bottom of polystyrene wells [52]. All the immunoassay steps (the series of reaction, washing, enzymatic reaction, and colorimetric measurement) are performed on this solution/ well interface. For electrochemical immunosensor preparation, capture antibodies can be directly adsorbed onto rough carbon electrode surfaces by passive adsorption [53,54]. The coating of a layer of polyethylene glycol on the electrode surface assists in the antibody immobilization by adsorption [55], which, obviously, can improve the performance of the electrochemical immunosensor. To enhance the attachment of proteins with electrode surface, Wilson and Rauh designed an electrochemically grown iridium oxide (IrOx) thin film matrices for antibodies adsorption [56]. The three-­ dimensional porous and hydrous matrix offered better antibody stability and higher antibody loading, thus improving the recognition efficiency of the immunosensor. Recently, nanoparticles have attracted growing attention regarding immobilization of immunoreagents due to their large specific surface area, strong adsorption capacity, and excellent biocompatibility [57]. Compared to the naked surfaces of bulk materials,

10

Immunosensing for Detection of Protein Biomarkers

the adsorption of protein biomolecules onto the surfaces of nanoparticles can generally retain their bioactivity. Since most of the nanoparticles carry charges, they can electrostatically adsorb biomolecules with different charges. Besides the common electrostatic interaction, some nanoparticles can also immobilize biomolecules by other interactions. For example, Au NPs can immobilize proteins through the inherent interaction between the gold atoms and the amine groups and cysteine residues of proteins [58,59]. So Au NPs are popularly applied to the electrode surface for immobilizing capture antibody during the immunosensor preparation [60–62].

1.3.2 Polymer entrapment To ensure high stability and bioactivity of immunosensors, various polymers with a three-dimensional structural network are often used for the entrapment of immunoreagents. One typical polymer for the encapsulation of biomolecules is gel matrices with porous surface and high capacity. Ju’s group [63,64] prepared a novel, titania sol-gel, thin film by a vapor deposition method. This porous matrix with excellent biocompatibility provided an excellent environment to immobilize the antigen for the competitive electrochemical immunoassay of carbohydrate antigen 19-9 (CA 19-9) [63] and carcinoma antigen-125 (CA 125) [64], respectively. They also used an organically modified silicate sol-gel [65] to immobilize the HRP-labeled antibody to achieve the direct electrochemistry of HRP. Based on the direct immunoreaction at the immunosensor to inhibit the direct electrochemical signal, novel reagentless immunosensors were constructed for electrochemical measurement of CEA [66] and hCG [67]. Hydrogels are another widely used polymer for the protein encapsulation during immunosensor preparation. Commonly used hydrogels are chitosan, dextran, and commercial carboxymethyl cellulose. Ju’s group prepared an Au NPs-doped cellulose acetate membrane [68] and an Au NPs-doped chitosan membrane [69] for immobilizing antigens, thus developing two electrochemical immunosensors for the measurement of CA 125 and CEA, respectively. The conducting polymers with high electroconductivity and aqueous compatibility also served as excellent gel materials for the encapsulation of antigens or antibodies for immunosensor preparation. Sadik et al. [70] reported a simple method for immunosensor construction by means of electrochemical polymerization of pyrrole with antibodies at a platinum-disk working. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the interaction between human serum albumin and the antibody-immobilized polypyrrole immunosensor successfully.

1.3.3 Covalent binding Covalent bonds are mostly formed between side-chain-exposed functional groups of proteins and suitably modified transducer surface, resulting in an irreversible binding and producing a high surface coverage [71,72]. One of the most commonly used methods for covalent immobilization is to couple the antibody randomly via their free amino groups to an activated sensor surface (Fig. 1.5). Chemical coupling agents, such as carbodiimides and succinimidyl esters, may be used to activate carboxylic acids on sensor surfaces.

Introduction11

COOH

NH2

a

b

CONH

OH

+

–NH2 Antibody with free amino group

c

NHCO

Fig. 1.5  Schematic illustration of covalent immobilization of antibodies to sensor surfaces via their free amino groups. Reaction (a) involves activation of carboxylic acids (COOH), achieved with carbodiimides and succinimyl esters. Reaction (b) shows amine surfaces (NH2), which can be activated using isothiocyanates, epoxides, or aldehydes. Reaction (c) shows alcohol surfaces that can be activated using periodate oxidation, isothiocyanates, epoxides, aldehydes, and cyanogen bromide.

Amines or alcohols can be activated by isothiocyanates, epoxides, glutaraldehyde (GA), or other aldehydes. Oxidation of alcohols is achieved with periodate to yield aldehydes, which react readily with amines. In addition, conversion of alcohols to a highly reactive ester by cyanogen bromide allows for further reaction with amines of antibody. For example, the ultrasonication treatment of carbon nanotubes (CNTs) in concentrated acid condition can produce abundant carboxyl groups on their surface. When CNTs are modified on the electrode surface, the carbodiimide/N-hydroxysuccinmide system is commonly applied to link antibodies with the activated carboxyl groups [60]. Another approach is to use bifunctional cross-linking reagents [73]. The cross-linking reagents contain two different reactive groups, thereby providing a means of covalently linking two dissimilar target groups on the sensor surface and protein biomolecules. A wide variety of these linkers such as (3-aminopropyl)triethoxysilane (APTES) [74], (3-glycidoxypropyl)-trimethoxysilane (GPTMS) [75], 3-mercaptopropyltrimethoxysilane (MPTMS) [76], diazonium cation [77], and various thiol derivatives [78–80] are commercially available to cover a broad range of functional groups necessary. For example, the silanization reaction of APTES at the hydroxyl group-containing substrate (e.g., glass, electrode, and microwell plate) can provide amino groups at the surface. Then antibodies can be coupled to the substrate via the cross-linking of GA (Fig. 1.6). OH OH APTES OH OH OH

O O Si O O Si O

NH2

GA

NH2

O O Si O O Si O

O N=C H

CH

N=C H

CH O

–NH2

O O Si O O Si O

N=C H

C=N H

N=C H

C=N H

Fig. 1.6  Schematic illustration of immobilization of antibody by the bifunctional cross-linking reagents of APTES and GA.

12

Immunosensing for Detection of Protein Biomarkers

1.3.4 Oriented immobilization In some cases, immobilization may lead to partial or complete loss of protein activity due to random orientation and structural deformation. Therefore, oriented immobilization is very important for the improvement of the immunosensor performance. Oriented immobilization means that the captured antibodies or antigens are immobilized in such a way that their recognition sites are uniformly arranged and exposed to the sample solution. We know that antibodies consist of two polypeptide heavy chains and two polypeptide chains, which together form a Y-shaped conformation. The arms of the Y-shaped molecule are antigen binding fragment (Fab), and the vertical portion is called the fragment-crystallizable (Fc). If antibodies are attached on the surface randomly, there are four possible orientations (Fig. 1.7A): end-on (Fc attached to the support), head-on (Fabs attached to the support), sideways-on (one Fc and one Fab attached to the support), and flat-on (all three fragments attached to the support) [73,81]. As the active sites of antibodies are on the Fab segment, the ideal orientation of the immobilized antibody is end-on. In this case, the Fc adsorbs onto the substrate surface, and the Fabs orient to the analyte solution. So far several approaches have been developed to control the orientation of antibodies to improve the analytical performances of immunosensors. One technique is site-oriented immobilization, in which Fabs of antibody are prepared and self-assembled onto gold surfaces via native thiol groups located at the hinge region [82]. While the thiols are attached to the surface of sensor, the antigen binding site is away from the transducer surface, resulting in a higher accessibility and activity of the immobilized receptor molecules. As shown in Fig. 1.7B, by using half-antibody or Fab (most often Fab fragments are used), the antigen binding site is available to the analyte solution, resulting in high antigen affinity efficiency. Fab fragments can be prepared in several ways. The simplest and most widely used way is to digest intact antibodies with pepsin, bromelain, or ficin [83], yielding F(ab)2 fragments and several Fc peptides. Another way is by employing gene engineering [84]. It allows the production of recombinant Fab fragments having different characteristics; for example, terminal cysteine residues can be included. Immobilization of Fab fragments was first proposed by Jimbo and Saito in 1988 [85]. Lu et al. described the comparison of random and oriented immobilization of antibody fragments. It showed the increased antigen binding activity of Fab fragments in comparison to randomly oriented Fab fragments by 2.7 times [86]. g a

(A)

h

i

b c

e

d

(B)

f

(C)

Fig. 1.7  Illustration of (A) different orientation of randomly immobilized antibodies: end-on (a), head-on (b), sideways-on (c), flat-on (d); (B) oriented immobilization of antibody fragments: half-antibody (e), and Fab (f); (C) immobilization of antibodies through different bioaffinity systems: avidin-biotin (g), protein A/G (h), and DNA-directed (i) immobilization.

Introduction13

Another approach to achieve oriented antibody immobilization was developed on different bioaffinity interactions [71,73]. A number of affinity systems including ­avidin-biotin, protein A/G, lectin-sugar, and DNA-directed immobilization have been employed for the oriented and homogeneous attachment of antibodies onto different substrates (Fig. 1.7C). Avidin is a tetrameric glycoprotein soluble in aqueous solutions and stable over wide pH and temperature ranges. It can bind up to four molecules of biotin. The bond formation is very rapid and unaffected by pH, temperature, organic solvents, enzymatic proteolysis, and other denaturing agents. Streptavidin is a closely related tetrameric protein with similar affinity to biotin. However, streptavidin has a lower degree of nonspecific binding, especially lectin binding due to its lack of glycosylation and its acidic isoelectric point, whereas it exhibits more highly specific binding to biotin than avidin. Streptavidin exhibits pH-dependent biotin binding with a strong interaction in acidic conditions (pH 4–5), and it dissociates at a higher pH. Biotin, which is also known as vitamin H, vitamin B7, or coenzyme R, comprises a ureido unit that binds avidins and a thiophene unit with a carboxyl group at the tip of a valeric acid side-chain. The carboxyl group can be derivatized to conjugate antibodies, enzymes, and other molecules without significantly affecting its biological activity. Since biotin is a small molecule, its conjugation to macromolecules does not affect conformation, size, or functionality. Biotin is often conjugated to antibodies during immunoassays. After immobilization of an avidin layer onto the substrate through proper approaches, the avidin-biotin reaction can be exploited to attach the biotinylated antibodies. Ju et al. immobilized streptavidin on a three-dimensional ordered nanoporous silica film by using GPTMS as a linker. Based on the high-selectivity recognition of streptavidin to biotin-labeled antibody, a novel immunosensor is constructed for highly efficient chemiluminescent immunoassay of CA 125 in the concentration range from 0.5 to 400 U/mL [87]. Antibody binding proteins of protein A and protein G can be also used to control the oriented antibody immobilization. These proteins bind specifically with the Fc regions of different types of IgG antibodies with high affinity [88,89]. This approach leaves the antigen binding sites of the immobilized antibodies to be exposed to the analyte solution, thus significantly improving the antigen binding capacity, sensitivity, and stability of immunosensors. The first use of Fc binding proteins for immunosensing applications was reported by Muramatsu and coworkers in 1987 [90]. They designed a QCM-based piezoelectric biosensor for human IgG detection by a protein A layer immobilized via APTES. It was demonstrated that the system could be used for determining concentrations of IgG of different subclasses. Nowadays, this method has been commonly used for immunosensor preparation based on different forms of signal transduction strategies [91–93].

1.4 Signal labels used in immunoassay Since label-free detection of the interaction between antigens and antibodies has become available with optical (e.g., SPR), electrochemical (e.g., electrochemical impedance, amperometry), and mass-sensitive (e.g., QCM) transducers, the immunological

14

Immunosensing for Detection of Protein Biomarkers

binding events are accompanied by only small physicochemical changes. The lack of sufficient sensitivity for detecting analytes at low concentrations is a major impediment to development of label-free immunosensors [94]. Thus various detectable labels or tags are commonly coupled to one of the immunoreagents (i.e., analyte or antibody) to amplify the immunological interactions. The use of these labels in immunoassay systems results in assay methods with extremely high sensitivity and low limits of detection. So far, a large variety of signal labels, especially enzymes, noble-metal nanoparticles, various electrochemically or optically active agents, and QDs have been popularly used for sensitive signal transduction in immunosensors.

1.4.1 Enzymes Enzymes are protein biocatalysts that can catalyze specific biological reactions at high efficiency. For example, HRP may cause the conversion of 10−7 molecules of substrate per minute. It is a common strategy for construction of biosensors by the use of enzymatic amplification. During the preparation of immunosensors, enzyme biomolecules are often labeled with an antibody or antigen to visualize the binding event [95]. Due to enzymatic reaction amplification, trace amount of the analyte would bring about enlargement of the signal response for sensitive quantitative measurement. Different enzymes such as HRP, ALP, and glucose oxidase (GOx) are commonly chosen for enzyme labels in immunoassay systems. HRP is an important metalloenzyme that can conveniently serve as a suitable label for immunoassay. It can be activated by hydrogen peroxide, which oxidizes the iron heme HRP enzyme to the FeIVO form [96]. This enzymatic reaction can catalyze the oxidation of a wide variety of organic and inorganic substrates such as 3,3′,5,5′-tetramethyl benzidine (TMB), 2,2-azino-di(ethylbenzothiazoline-6-sulfonic acid) (ABTS), uric acid, hydroquinone, ascorbic acid, p-phenylene diamine (PPD), o-phenylene diamine (OPD), p-aminophenol (PAP), o-aminophenol (OAP), 1,5-­ dihydroxynaphthalene (DHN), thionine, methylene blue, methylene green, and other dyes [94,97,98]. Based on the quantitative capture of the HRP labels on the immunosensor and the HRPcatalytic reactions, colorimetry (ELISA) [99–101] or voltammetry [102–105] can be applied to detect the enzymatic products for signal transduction. As HRP can catalyze the oxidation of luminol to 3-aminophthalate via several intermediates for enhancing the chemiluminescence of luminol, HRP also serves as a powerful label in ­chemiluminescence-based immunoassays [106–109]. Additionally, the enzymatic reactions of HRP labels can consume or generate the coreactants for the ECL emission of different materials [31,33–35,110], thus enabling the successful development of ECL immunosensors for protein measurement. ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules with an optimum activity around pH 8–10 [111]. Substrates, such as phenyl phosphate, p-aminophenyl phosphate (PAPP), p-nitrophenyl phosphate, 1-naphthyl phosphate, 2-phospho-l-ascorbic acid (AAP), and 3-indoxyl phosphate, have been widely employed in ALP-based immunoassay systems. After enzymatic hydrolysis reactions, electroactive organic radical could be produced on the immunosensor surface for voltammetric measurement [112,113]. Chailapakul et al. [114]

Introduction15 AAP

ALP

ALP

AA

AARe O

NH C

O C

NH O

NH C

AAP

O

NH C

BDD

O C

NH O

NH C

AAOx

2e–

BDD

ALP

ALP conjugated GaMIgG

GaMIgG

MIgG

Fig. 1.8  Schematic illustration of the amperometric enzyme immunosensor based on the poly-o-ABA-modified electrode. Reprinted with permission from A. Preechaworapun, T.A. Ivandini, A. Suzuki, A. Fujishima, O. Chailapakul, Y. Einaga, Development of amperometric immunosensor using boron-doped diamond with poly (o-aminobenzoic acid), Anal. Chem. 80 (2008) 2077–2083.

reported a protein immunosensor developed at an electropolymerized o-aminobenzoic acid (o-ABA) modified boron-doped diamond (BDD) electrode. AAP was selected as the substrate of the ALP label to detect mouse IgG, as it can produce l-ascorbic acid (AA), which is sensitive to the electrochemical detector, does not foul the electrode, and has excellent stability (Fig. 1.8). Čadková et al. [115] employed ALP-labeled antibody for electrochemical measurement of human epididymis protein 4, a promising biomarker for diagnosis of ovarian cancer. The enzymatic reaction of ALP resulted in the hydrolysis of its PAPP substrate to electroactive PAP, which provided excellent electrochemical signal tracing at the immunosensor by square-wave voltammograms. On the other hand, the inherent reduction ability of the ALP-hydrolyzed products can also serve as reduction agents to induce the deposition of silver nanoparticles on the electrode surface for electrochemical stripping analysis. This led to the development of some new ALP label–based electrochemical immunosensing strategies for sensitive measurement of protein biomarker [116,117]. Similar to HRP label, the enzymatic reaction of ALP label can also be employed for ELISA and chemiluminescence-based immunoassays [118–120]. GOx can catalyze the oxidation of β-d-glucose to d-glucono-1,5-lactone and H2O2, using molecular oxygen as the electron acceptor. As the enzymatically generated H2O2 can act as coreactants of ECL materials or electron donors of photoelectrochemical materials, several ECL and photoelectrochemical immunosensing methods have been developed based on the signal tracing of the GOx label [121–123]. By combination with the electron mediators such as ferrocene [124], Prussian blue [125], and p-benzoquinone [126] for facilitating the electron transfer, the GOx label is also popularly applied for the sensitive signal tracing of the electrochemical immunosensor. Additionally, the electron mediator-participated enzymatic reaction of GOx label can also induce to the deposition of Au NPs for electrochemical stripping analysis [127],

16

Immunosensing for Detection of Protein Biomarkers

which enables the development of another sensitive electrochemical signal tracing strategy for GOx-based immunoassay. Other enzymes such as urease [128], catalase [129], acetyl cholinesterase [130], and galactosidase [131] can also be employed as useful signal labels of immunosensors with proper signal transduction approaches.

1.4.2 Noble-metal nanoparticles The noble-metal nanoparticles, especially colloidal Au NPs, Ag NPs, Pt NPs, and Pd NPs, have been widely used as important nonenzymatic labels in immunosensors due to their excellent biocompatibility and stability, tunable surface for convenient functionalization, and outstanding optical/electrochemical properties for signal tracing. One simple strategy for the measurement of the noble-metal NP labels is their direct electrochemical stripping analysis. The conventional method requires extremely severe conditions (e.g., highly concentrated HNO3-HCl or HBr-Br2) to dissolve the Au NP labels into gold (III) ions for electrochemical reduction measurement [132,133]. So the pretreatment step is complicated and time-consuming. This step can be replaced by the electrochemical preoxidization of Au NPs in HCl to produce electroactive gold (III) ions, which has been successfully applied to tracing the electrochemical signal of immunosensors [134,135]. For example, Ju’s group [134] reported a simple, sensitive, and low cost multiplexed immunoassay by combining a disposable chip with Au NP as an electrochemical label (Fig. 1.9). The immunosensors array as the

W1

W2 Adsorption of capture antibodies

W1

W2

BSA blocking

W1

W2

Preoxidation Incubation of IgG mixture

W1

W2

Incubation of AuNP labeled antibodies

W1

Mouse antihuman IgG

Human IgG

Rabbit antigoat IgG

Goat IgG

BSA

W2

DPV detection

W1

W2

AuNP labeled rabbit antihuman IgG

AuNP labeled rabbit antigoat IgG

Fig. 1.9  Schematic representation of preparation of immunosensors array and analytical procedure for simultaneous detection of human IgG and goat IgG. Reprinted with permission from C. Leng, G.S. Lai, F. Yan, H.X. Ju, Gold nanoparticle as an electrochemical label for inherently crosstalk–free multiplexed immunoassay on a disposable chip, Anal. Chim. Acta. 666 (2010) 97–101.

Introduction17

disposable chip was first prepared by immobilizing capture antibodies on different screen-printed carbon working electrodes by passive adsorption. With a sandwich mode, the analytes were then bound to the corresponding capture antibodies for further capture of the gold nanoparticle-labeled antibodies. Au NP labels were finally electrooxidized in 0.1 M HCl to produce AuCl 4 - for differential pulse voltammetric detection. Using human IgG and goat IgG as model targets, under optimal conditions this method achieved linear ranges from 5.0 to 500 and 5.0 to 400 ng/mL with limits of detection of 1.1 and 1.6 ng/mL, respectively. Compared with Au NPs, Ag NPs can be directly detected using an electrochemical stripping detection due to their lower oxidation potential and facile stripping conditions. Particularly, solid-state voltammetry of the electrochemical stripping analysis of Ag NPs can bring on a larger peak current at a lower potential, which greatly benefits the Ag NP as an useful electrochemical label for sensitive signal tracing of immunosensors [136,137]. Additionally, the noble-metal NPs (e.g., Au NPs, Pt NPs, and Pd NPs) with different sizes, shapes, and structures exhibit distinctive capability in catalyzing oxidation, reduction, hydrogenation, or dehydrogenation of a variety of molecules, which were employed as excellent electrochemical labels for designing another kind of nonenzymatic electrochemical signal tracing strategy for immunosensors [138–140]. For example, Ying and coworkers [139] described a sensitive electrochemical immunosensing strategy based on a platinum-catalyzed hydrogen evolution reaction for sensitive monitoring of PSA (Fig.  1.10). To enhance the sensitivity of the immunoassay, the Pt NP labels quantitatively captured onto the immunosensor surface were further applied for seed-mediated Pt deposition, which greatly enhanced the loading of platinum catalysts. Moreover, the platinum catalysts were closer to the gold electrode surface after the Pt enhancement step, which quickens the electron transfer from the solution up to the electrode surface. The practical applicability of the platinum enhancement strategy using hydrogen evolution was successfully employed for the sensitive electrochemical measurement of PSA. Besides electrochemical methods, the noble-metal nanoparticles such as Au NP and Ag NP labels can also be used for optical immunoassays. For example, Yang’s group [141] used specifically modified silver nanoparticles (Ag NPs) as the signal elements, setting up a label-free, rapid, and sensitive colorimetric immunoassay for the synthetic peptide fragment of neurogenin. The detection procedure is based on an antiaggregation mechanism, by which neurogenin 1 inhibited the aggregation of the probe in the presence of NaClO4 salt. Jiang et al. [142] reported a colorimetric immunosensing method for the convenient detection of human immunodeficiency virus in blood serum based on the aggregation of Au NPs for color change. The Cu (I) ion was from CuO nanoparticles subjected to immunoreaction, which could act as a catalyst to induce the aggregation of Au NPs functionalized with azide and alkyne groups. Additionally, these noble-metal NPs can also be used for optical immunosensing by employing the signal transduction approaches such as surface plasmon resonance [143], fluorescence [144], surface-enhanced Raman scattering [145], and chemiluminescent [146].

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Immunosensing for Detection of Protein Biomarkers

Gold electrode Platinum enhancement solution

1 M KCI+ 10 mM HCI Pt-catalyzed process Key:

11-MUOH

Antibody

16-MHA

Secondary antibody with Pt nanoparticle

PSA analyte

Fig. 1.10  Schematic of the electrochemical PSA biosensor based on Pt-catalyzed hydrogen evolution for signal tracing. Reprinted with permission from J. Zhang, B. Ting, M. Khan, M. Pearce, Y. Yang, Z. Gao, et al. Platinum nanoparticles label-mediated deposition of Pt catalyst for ultrasensitive electrochemical immunosensors, Biosens. Bioelectron. 26 (2010) 418–423.

1.4.3 Electrochemically or optically active agents Some agents with special electrochemical or optical activity are also popularly employed for signal tracing of immunoassay. For example, ferrocene and its derivatives are attractive components as electroactive labels, which have been confirmed to be extremely useful for the construction of sensitive electrochemical biosensors [147,148]. As early as 1941, Coons et al. [149] discovered the possibility of coupling antibodies to fluorescent dyes (e.g., rhodamines) without changing their specificity. Currently, the use of dye excited in the red or the near infrared (NIR) part (600–1000 nm) of the electromagnetic spectrum (e.g., FITC [150]) to conjugate the immunoreagents for quantitative fluorescent signal tracing has become popular and commercially available. Additionally, a large variety of agents such as TMB [151], methylene blue [152], fluorescein diacetate [153], luminol [154], and tris(2,2′-bipyridyl) ruthenium [155] with excellent electrochemical, fluorescent, chemiluminescent, or ECL properties can also be used for preparing various nanolabels for immunoassay. This approach not only simplifies the complicated immunolabeling process but also greatly improves the signal transduction.

Introduction19

1.4.4 Quantum dots QDs are defined as semiconductor nanocrystals composed of IIB–VIA groups (e.g., CdSe, CdTe, CdS, and ZnSe) or IIIA–VA elements (e.g., InP and InAs). As their radius is less than or equal to the excitation Bohr radius, QDs exhibit unique optical properties, electricity, magnetism, and catalysis, which make them attract a great deal of attention in the bioassay field. Because the surface of QDs can be easily modified for immobilizing binding molecules, QDs can function as useful signal labels in immunoassay. For example, when QDs are used as electrochemical labels, the quantitatively captured nanocrystals can be dissolved by acid solution to release a large amount of metallic ions. Then anodic stripping voltammetry will be used to measure the ions for achieving sensitive electrochemical signal transduction. Wang’s group [156] used three kinds of QDs (ZnS, CdS, and PbS) as signal labels to develop a multiplexed electrochemical immunoassay protocol (Fig. 1.11). The different stripping peaks with corresponding peak currents provide well-resolved electrochemical responses for simultaneous measurements of different proteins successfully. Due to their size-controlled fluorescence, high fluorescence quantum yields, and stability against photobleaching, QDs can be also used as fluorescent labels for protein

(B)

Ab1

ZnS-Ab1 CdS-Ab2 PbS-Ab3

Ag3 Ag2 Ag1

Ab2

(A)

(C)

Ab3

Cd

Current(µA)

Zn

Pb

Acid dissolution

(D)

M° M2+

M2+

E (V)

Fig. 1.11  Multiprotein electrical detection protocol based on different inorganic colloid nanocrystal tracers. (A) Introduction of antibody-modified magnetic beads; (B) binding of the antigens to the antibodies on the magnetic beads; (C) capture of the nanocrystal-labeled secondary antibodies; (D) dissolution of nanocrystals and electrochemical stripping detection. Reprinted with permission from G. Liu, J. Wang, J. Kim, M.R. Jan, Electrochemical coding for multiplexed immunoassays of proteins, Anal. Chem. 76 (2004) 7126–7130.

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measurement. Tu and his workers developed a CdSe/ZnS QDs-based optical immunosensor for human serum albumin detection [157]. The detection limit of the fluorescence immunosensor developed in this study was ~3.2 × 10−5 mg/mL. Kerman’s group developed a QDs-based immunosensor for the detection of PSA using fluorescence microscopy [158]. Fluorescence imaging of the substrate surface illuminated the QDs and provided a very sensitive tool for the detection of PSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. As semiconductors, QDs can also be excited by chemical and electrochemical reactions. The light emission of QDs controlled by the applied electrode potential enables them to be applied as excellent ECL labels for bioassays [159]. The use of CdTe QDs as the luminophor and K2S2O8 as the coreactant is the most popular system in sandwich-enhanced ECL immunoassays. Li et  al. [160] developed an ECL immunoassay by using CdTe QDs as labels (Fig. 1.12). The electrode modification

-S-CH2-COOH HS-CH2-COOH

-S-CH2-COOH

TGA

-S-CH2-COOH

NH2Ab1

BSA

-S-CH2-COOH NPGL electrode -S-CH2-CO-NH-

-S-CH2-CO-NH Ag

-S-CH2-CO-

Ab2

HOOC-

CdTe QDs

HO

O

-S-CH2-CO-NH-S-CH2-CO-NH-S-CH2-CO-S-CH2-CO-NH-

C-

-NH-OC- C

O

HO

HO

-COOH

HO

-COOH

O

C-

H OO -C -COOH -C OO H

COOH

C-

-COOH

O

-S-CH2-CO-S-CH2-CO-NH-

-S-CH2-CO-NH

HO

-S-CH2-CO-NH-

O

C-

-NH-OC-

-COOH

C-

O

HO

H O O -C -COOH -C O O H

COOH

-S-CH2-CO-NH

H O O -C -COOH -C O O H

Fig. 1.12  Schematic representation of steps for immunoassembly and QDs labeling on NPGL electrode. Reprinted with permission from X.Y. Li, R.Y. Wang, X.L. Zhang, Electrochemiluminescence immunoassay at a nanoporous gold leaf electrode and using CdTe quantun dots as labels, Microchim. Acta. 172 (2011) 285–290.

Introduction21

by the nanoporous gold leaf (NPGL) greatly improved the sensitivity of the method for CEA detection. The relation between ECL intensity and CEA concentration was linear in the range from 0.05 to 200 ng/mL, and a detection limit of 0.01 ng/mL was obtained. Wang’s group reported a near infrared ECL immunosensor by using CdTe/ CdS QDs tagged silica nanospheres as signal amplification [161]. The protocol successfully fulfilled the ultrasensitive detection of human IgG with a detection limit down to 87 fg/mL.

1.5 Perspective Immunosensors have been used for the detection of various analytes other than protein biomarkers. Their wide application indicates a great future in the clinical diagnosis field for the accurate and convenient measurement of protein biomarkers owing to their outstanding advantages (e.g., no need for separation or pretreatment) over the other analytical methods. However, since the application of biosensor depends basically on the analytical performance, a crucial aspect in future immunosensors will focus on the improvement of their specificity and sensitivity. Besides the design of new approaches to improve the oriented immobilization of immunoreagents, the development and application of improved molecular recognition elements are also a valuable way to enhance the specificity of immunosensors. In this respect, biotechnology and genetic engineering offer the possibility of tailor binding molecules with predefined properties. Through combinatorial screening or design of the amino acid sequence of the binding region of the antibody, some recombinant antibodies or polypeptide are produced to replace the conventional antibodies. Meanwhile, a large kind of synthetic nucleic acids (aptamers) can be screened by the technique of systematic evolution of ligands by exponential enrichment (SELEX) and used as novel recognition elements to bind in a manner conceptually similar to antibodies to a wide array of target molecules with high affinity and specificity. Additionally, the future of immunosensors will be continually influenced by the inclusion of novel nanomaterials, which provide powerful tools to improve the performance of immunosensors. Taking advantage of their excellent compatibility, controllable morphology, and outstanding conductive properties, they can provide an ideal immunosensing platform for accelerating the signal transduction. Meanwhile, the unique optical/electrochemical properties and high specific area of nanomaterials will also enable the development of a large variety of signal tracing strategies with ultrahigh sensitivity. Furthermore, the relatively low cost, small sample consumption, and convenient operation of immunosensors make them possess great potential for practical applications. For example, an immunosensing device can be designed for point-of-care testing of protein biomarkers aimed at the convenient diagnosis of various diseases. It is expected that the huge commercial value in this field will drive their continued development in the coming years, and more point-of-care testing platforms will find wide applications that will benefit health care. In addition, new achievements in biotechnology will be widely adopted into immunoassay. For example, the array-based

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Immunosensing for Detection of Protein Biomarkers

multiple analytes detection, microfluidics, and lab-on-chip techniques will be combined with future immunosensors. These combinations will greatly push forward the development of immunosensing devices for fulfilling the needs of high throughput, automatization, and miniaturization in the health care assessment and disease diagnosis fields.

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Introduction23

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Introduction25

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[145] X.F.  Gu, Y.R.  Yan, G.Q.  Jiang, J.  Adkins, J.  Shi, G.M.  Jiang, et  al., Using a silver-­ enhanced microarray sandwich structure to improve SERS sensitivity for protein detection, Anal. Bioanal. Chem. 406 (2014) 1885–1894. [146] Z.P. Li, C.H. Liu, Y.S. Fan, Y.C. Wang, X.R. Duan, A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels, Anal. Biochem. 359 (2006) 247–252. [147] S. Prabhulkar, S. Alwarappan, G. Liu, C.Z. Li, Amperometric micro- immunosensor for the detection of tumor biomarker, Biosens. Bioelectron. 24 (2009) 3524–3530. [148] D. Liu, Z. Gao, Q. Luo, Z.S. Wu, Conductive three-dimensional ordered nano-gold film: ultrasensitive electrochemical sensing platform for clinical immunoassay, Talanta 82 (2010) 1175–1180. [149] A.H. Coons, H.J. Creech, R.N. Jones, Immunological properties of an antibody containing a fluorescent group, Proc. Soc. Exp. Biol. Med. 47 (1941) 200–202. [150] A.E. Boyer, M. Lipowska, J.M. Zen, G. Patonay, V.C.W. Tsang, Evaluation of near infrared dyes as labels for immunoassays utilizing laser diode detection: development of near infrared dye immunoassay (NIRDIA), Anal. Lett. 25 (1992) 415–428. [151] R.  Akter, C.K.  Rhee, M.A.  Rahman, Sensitivity enhancement of an electrochemical immunosensor through the electrocatalysis of magnetic bead-supported non-enzymatic labels, Biosens. Bioelectron. 54 (2014) 351–357. [152] F.Y.  Kong, X.  Zhu, M.T.  Xu, J.J.  Xu, H.Y.  Chen, Gold nanoparticle/DNA/methylene blue nanocomposites for the ultrasensitive electrochemical detection of carcinoembryonic antigen, Electrochim. Acta 56 (2011) 9386–9390. [153] W.Y. Cai, I.R. Gentle, G.Q. Lu, J.J. Zhu, A.M. Yu, Mesoporous silica templated biolabels with releasable fluorophores for immunoassays, Anal. Chem. 80 (2008) 5401–5406. [154] X.Y.  Yang, Y.S.  Guo, A.G.  Wang, Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen, Anal. Chim. Acta 666 (2010) 91–96. [155] X.  Yang, R.  Yuan, Y.Q.  Chai, Y.  Zhuo, L.  Mao, S.R.  Yuan, Ru(bpy)32+-doped silica nanoparticles labeling for a sandwich-type electrochemiluminescence immunosensor, Biosens. Bioelectron. 25 (2010) 1851–1855. [156] G. Liu, J. Wang, J. Kim, M.R. Jan, Electrochemical coding for multiplexed immunoassays of proteins, Anal. Chem. 76 (2004) 7126–7130. [157] M.C. Tu, Y.T. Chang, Y.T. Kang, H.Y. Chang, P. Chang, T.R. Yew, A quantum dot-based optical immunosensor for human serum albumin detection, Biosens. Bioelectron. 34 (2012) 286–290. [158] K. Kerman, T. Endo, M. Tsukamoto, M. Chikae, Y. Takamura, E. Tamiya, Quantum dotbased immunosensor for the detection of prostate-specific antigen using fluorescence microscopy, Talanta 71 (2007) 1494–1499. [159] D. Bera, L. Qian, T. Tseng, P. Holloway, Quantum dots and their multimodal applications: a review, Materials 3 (2010) 2260–2345. [160] X.Y.  Li, R.Y.  Wang, X.L.  Zhang, Electrochemiluminescence immunoassay at a nanoporous gold leaf electrode and using CdTe quantun dots as labels, Microchim. Acta 172 (2011) 285–290. [161] J. Wang, H. Han, X. Jiang, L. Huang, L. Chen, N. Li, Quantum dot-based near-­infrared electrochemiluminescent immunosensor with gold nanoparticle-graphene nanosheet hybrids and silica nanospheres double-assisted signal amplification, Anal. Chem. 84 (2012) 4893–4899.

Signal amplification for immunosensing

2

Achieving high sensitivity is one of the major goals in developing novel immunosensing methods for the detection of protein biomarkers because a few molecules of important protein are sufficient to regulate the biological functions of cells and trigger disease processes. Moreover, numerous pathological protein biomarkers are present at very low levels during the early stages of disease development, such as in acute myocardial infarction, cancer, and infectious diseases. Therefore, the ultrasensitive techniques for protein detection play essential roles not only in the elucidation of molecular mechanisms of life processes and many diseases, but also in promoting the early diagnosis of diseases and facilitating health care. Traditional immunological techniques, such as the enzyme-linked immunosorbent assays (ELISA), radio-­ immunoassays (RIA), and fluorescence immunoassays are able to provide relatively high sensitivity and low limits of detection. But most of them are only capable of detecting abundant proteins. The ultrasensitive detection of proteins is particularly challenging due to the need of affinity ligands for specific target recognition and the absence of polymerase chain reaction (PCR)-like protocols for exponential amplification of target molecules. Recently, tremendous advances have been achieved in the exploration of signal amplification strategies for the development of ultrasensitive protein biosensors. Two groups of strategies have been applied to couple with protein biosensing protocols, when protein targets are recognized by affinity ligands (antibodies or aptamers). One group of strategies attempts to transfer protein target-binding events into a large number of reporter molecules or detection probes for signal readout. These strategies commonly employ nano/micromaterials as vehicles for multilabeling or DNA/RNA polymerase amplification for increasing the abundance of the detection probes. The other group of strategies uses nano/micromaterials or enzyme mimics as catalytic tools to obtain enhanced detection signal. This chapter focuses on several important strategies of signal amplification for highly sensitive protein biosensing.

2.1 Multilabeling for signal amplification Immunological techniques, such as ELISA [1], RIA [2] and fluorescence immunoassays [3], and chemoluminescence immunoassays [4] have been conventionally applied in a wide range of areas, including disease diagnosis, clinical therapy, environmental monitoring, and food analysis. In these traditional immunological methods, the labeled antibodies that usually are linked with various labels, such as Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00002-5 Copyright © 2017 Elsevier Ltd. All rights reserved.

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Immunosensing for Detection of Protein Biomarkers

enzymes, illuminant dyes, and radioactive elements at a ratio of 1:1, are employed to specifically recognize the target proteins and indicate detection signals with competitive or sandwich mode [5]. There is no doubt that the low ratio of signal label to antibody results in a limited detectable signal and relatively low sensitivity of these traditional immunoassays. The sensitivity of any analytical strategy is determined by the correlation between the intensity of the output signal and the analyte concentration. Therefore, in recent years, great efforts have been made to amplify the signal output and to obtain high sensitivity of immunosensing by using multilabeling strategies. These multilabeling strategies have been explored to increase the ratio of signal tags to specific recognition ligands with the application of novel nanomaterials [6,7], polymers/polymerization [8], and DNA nano-assembly [9] as carriers loading numerous reporter molecules. Nano/micromaterials, such as quantum dots (QDs), nanogold particles, nanotubes, nanowires, microbeads (microspheres), and microcrystals, have earned increasing attention in recent years in biosensing applications due to their special optical, electrochemical, or magnetic characteristics and high surface-to-volume ratios. One of the important reasons for the usage of nano/micromaterials as functionalized nanoprobes to improve the sensitivity is the amplification of target recognition events [10]. Nano/ micromaterials with high surface-to-volume ratios have been used as carriers labeling numerous reporter molecules by immobilizing enzymes, nucleic acids, or noble-metal nanoparticles on nano/microprobes. The large number of reporter molecules on the surface of nano/microparticles produces an enhanced signal for the detection of the target molecules. This will be described in Section 2.2 of this chapter and in Chapter 4 in detail. Similarly, polymeric materials can also be used to design multilabeling strategies for amplifying the signal and enhancing the detection sensitivity, which hold abundant sites or groups for functionalization with probe molecules in their interiors or on their surfaces. For example, the polymeric nano/microspheres can be excellent candidates for vehicles to enhance the probe loading due to their good conductivity, biocompatibility, and functionality. Dong et  al. [11] has developed an electrochemical signal amplification platform by applying CdTe QDs-tagged polybeads. The polybead templates were prepared simply by the emulsifier-free copolymerization of styrene with acrylic acid to form poly(styrene-co-acrylic acid) cores and the layer-by-layer deposition of a poly(sodium 4-styrenesulfonate) layer and two layers of poly(allyaminehydrochloride) on the cores, which facilitated the chemisorption of negatively charged mercaptoacetic acid capped CdTe QDs on the templates (Fig.  2.1). The enormous coverage (9.54 × 103 QDs per polybead) and the unique properties of the QDs make the polybeads a candidate for an effective signal amplification platform for biosensing by labeling of DNA or protein. Yuan et  al. [12] proposed a multilabeling strategy for sensitive detection of tumor necrosis factor-alpha (TNF-α) using QDs-polymer-functionalized silica nanosphere as the label. The poly(glycidyl methacrylate) functionalized silica nanospheres led to a further increase of CdTe QD loading per immunoassay event, resulting in 10.0- and 5.5-fold increased signals of the electrochemiluminescence

Signal amplification for immunosensing33

PAH

PSS

PAH

PSA

CdTe

Streptavidin

Fig. 2.1  Schematic diagram of the assembly process for the preparation of CdTe QDs-tagged polybeads for signal amplification. Reprinted with permission from H.F. Dong, F. Yan, H.X. Ji, D.K.Y. Wong, H.X. Ju, Quantum-dot-functionalized poly(styrene-co-acrylic acid) microbeads: step wise self-assembly characterization, and applications for sub-femtomolar electrochemical detection of DNA hybridization, Adv. Funct. Mater. 20 (2010) 1173–1179.

(ECL) and square-wave voltammetry (SWV) measurements, respectively, in comparison with the unamplified method. Wei et al. [13] also developed a robust immune-label based on encapsulation of Fe3O4 nanoparticles by poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymeric vesicles. This displayed a high catalytic activity of the PEG-PLA-Fe3O4 toward H2O2. With the conjugation of a large amount of antibodies onto the hybrid vesicle’s surface, the immune-labels were successfully used for the fabrication of ultrasensitive sandwich immunosensing of prostate-specific antigen (PSA). Polymerization-based amplification (PBA) has also been demonstrated to be a highly effective multilabeling strategy for signal amplification, which amplifies molecular recognition events by the application of radical polymerization with macromolecule-linked dynamic growth [14]. In the PBA strategy, ligand-target binding reactions induce the immobilization of small initiator molecules on the measuring substrate surface. These initiating molecules, as polymerization reaction centers, can trigger in situ polymerization after the addition of the monomer solution and other reaction necessities. The in situ polymerization provides numerous active sites for the binding of different tags for a wider measurement technique [15,16]. Cheng’s group [17] designed a surface plasmon resonance (SPR) biosensing method for detection of bacterial cholera toxin (CT) with high degree signal amplification by the use of functional gold nanoparticles (AuNP) in combination with in situ atom transfer radical polymerization (ATRP) reaction (Fig. 2.2). The biotin-AuNP surface was functionalized with ATRP initiator that triggers localized growth of poly(hydroxyl-ethyl methacrylate) brush, contributing to marked SPR signal enhancement and quantitative measurement of CT with a detection limit of 160 aM.

34

Immunosensing for Detection of Protein Biomarkers

Au

EDC/NHS S HN NH

H2N

O

N H

O

O O

OH O

TEA, DMF NH2

O Br

Br

O 16-MHDA AuNP

AuNP-hydroxyl-biotin

AuNP-initiator-biotin

(A) CT

Biotin anti-CT

Avidin

Calcinated Au

Calcinated Au AuNP-initiator-biotion

HEMA

(B)

Catalyst Calcinated Au

Calcinated Au

Fig. 2.2  Schematic representation of (A) AuNP surface modification with initiator and biotin and (B) in situ AuNP coupling by biotin/avidin interaction and surface ATRP reaction for SPR signal amplification in CT detection. Reprinted with permission from Y. Liu and Q. Cheng, Detection of membrane-binding proteins by surface plasmon resonance with an all aqueous amplification scheme, Anal. Chem. 84 (2012) 3179−3186.

Liu’s group also reported a series of polymerization-assisted signal amplification strategy for ultrasensitive detection of proteins [18–20]. For example, a multilabeling strategy was proposed based on the combination of tyramide signal amplification (TSA) and PBA. The surface-initiated ATRP of glycidyl methacrylate was triggered by the initiator-coupled antibody immobilized on the surface of biosensor via traditional sandwich immunoreactions (Fig. 2.3). Growth of long chain polymeric materials provided numerous epoxy groups for subsequent coupling of horseradish peroxidase (HRP), which in turn significantly increased the loading of QDs labeled tyramide in the presence of hydrogen peroxide due to the TSA mechanism. As a result, ECL and SWV measurements showed 9.4- and 10.5fold increases in detection signal in comparison with the unamplified method, respectively.

Signal amplification for immunosensing35

QD T

P HR

QD

QD T

T

QD

T

T

QD T

QD QD

Ag

QD T

QD-tyramide

QD

T

QD

P

QD

QD

P

T

HRP

T

HR

ECL QD T

HR

P Ag

HRP

QD

T

D

T

QD

Ag

Q

HR

O2 H2

QD T

QD

CdTe QDs

ATRP

Route 3. Dual amplification HRP

Ag

QD QD

Ab1

T

QD

PGMA

GMA

Ag

Ag T

GCE

Ag

InitiatorAb2

QD-tyramide

D

T

Q

QD

Route 2.Polymerization-assisted amplification PAB

HRP

QD

Ag

HRP-Ab2

Ab1

QD

T

T

Ag HRP

GCE

T

QD

PAB

H

HRP

QD

T

Route 1. Common ELISA with TSA

QD T

O2 2

Signal amplification

SWV

Fig. 2.3  Schematic representation of typical TSA based on common ELISA using QD-tyramide conjugates as labels (Route 1), the polymerization-assisted amplification in sandwich immunoassay via surface-initiated ATRP, and subsequent direct binding of CdTe QDs (Route 2), and the sandwich immunoassay using QD-tyramide conjugates as labels via surface-initiated ATRP and TSA (Route 3). Reprinted with permission from L. Yuan, L.L. Xu, S.Q. Liu, Integrated tyramide and polymerization-assisted signal amplification for highly-sensitive immunoassay, Anal. Chem. 84 (2012) 10737–10744.

2.2 Nanoparticles as signal labels for signal amplification High affinity of antigens-antibodies generally ensures the specificity. Similarly, appropriate labels are usually employed for signal amplification to guarantee sensitivity in immunoassay. In fact, enzyme-labeled immunoassays possess remarkable status than others. However, the limitation owing to the 1:1 ratio between the enzyme label and antibody leads to restricted amplification. Nanotechnology has been considered as an innovative approach to provide excitingly new possibilities for advanced development of new analytical tools and instrumentation. Nanoparticles such as carbon nanomaterials, semiconductor nanoparticles, and metal oxide nanostructures, having the advantages of high surface-to-volume ratio, favorable conductivity, good biocompatibility, and unique optical properties, are considered as potential signal labels to stimulate the development of protein biomarkers signal amplification detection for immunosensors [21,22]. Moreover, nanoparticles label research could realize ideal signal amplification to overcome the sterical limitation of traditional enzymes [23]. The enormous signal enhancement associated with the use of nanomaterials amplifying labels and with the formation of nanoparticle-antibody-antigen assemblies provides the basis for ultrasensitive immunosensor and immunoassays [24].

36

Immunosensing for Detection of Protein Biomarkers

Electrochemical immunosensors offer high sensitivity, uncomplicated instrumentation, and capability in protein determination. Nanotechnology has opened the possibility of the use of nanomaterial labels for further enhancement of the sensitivity of electrochemical immunosensors based on the characteristics of electroactivity [25] and catalytic activity [26] of the nanoparticles, involving various electrochemical approaches such as amperometric [27], potentiometric [28], impedimetric [29], and capacitometric analysis [30]. Due to its good biocompatibility with biomolecules and easy functionalization, AuNPs have been frequently utilized in immunoassays. AuNP is an attractive electroactive label for developing an enzyme-free immunosensing strategy since it can be directly measured by electrochemical stripping analysis or by inducing silver deposition for amplifying the electrochemical stripping signals. Ju’s group [31] utilized double strand DNA@Au nanoparticle as signal label and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator to develop an ultrasensitive enzyme-free electrochemical immunosensor based on hybridization chain reaction (HCR) for ­ ­detection of carcinoembryonic antigen (CEA) (Fig. 2.4). They used a chitosan/AuNP

n

n

n

n cycles HCR

I-DNA

H1

B-DNA1

S-DNA

H2

B-DNA2

Ab1

CEA

BSA

GA

SA-Ab2 n

CS/AuNP n

n

n

GCE

n

(A)

n

n

AuNP

n

n

n

Current

n

n

n

n

n

dsDNA@AuNP

(B)

RuHeX

SWV Potential

Fig. 2.4  Schematic diagram of (A) the preparation of dsDNA@AuNP tag and (B) the immunosensor fabrication and electrochemical immunoassay procedure. Reprinted with permission from Y. Ge, J. Wu, H. Ju, S. Wu, Ultrasensitive enzyme-free electrochemical immunosensor based on hybridization chain reaction triggered double strand DNA@Au nanoparticle tag, Talanta 120 (2014) 218–223.

Signal amplification for immunosensing37

(CS/AuNP) nanocomposite film modified glass carbon electrode to immobilize the capture antibody, and a dsDNA@AuNP complex formed with HCR as a signal tag to label the signal antibody. After the sandwiched immunoreaction, hexaammi-­ neruthenium (III) chloride (RuHex) was used a DNA intercalator to be assembled in the dsDNA for obtaining the electrochemical signal. Consequently, both the dsDNA@ AuNP tag and AuNPs-promoted electron transfer enhanced the sensitivity of the immunosensor. Under optimal conditions, the proposed sensing platform showed a wide linear detection range from 10 fg mL−1 to 10 ng mL−1 along with a detection limit of 3.2 fg mL−1 for CEA. Another principle is to take advantage of nanoparticles acting as the catalyst reacts with a large quantity of substrate, which strengthens the electrochemical signal. Moreover, nanoparticles label on electrode surface can mediate the deposition of metallic nanoparticles to further increase the loading of either this class of electrochemically detectable species or electrocatalysts [32,33]. Zhao et al. [34] developed an ultrasensitive multiplexed immunoassay method via using streptavidin/nanogold/ carbon nanohorn (SA/Au/CNH) as a novel signal tag to induce silver enhancement for signal amplification (Fig. 2.5). Carbon nanohorn has been used as an excellent nanocarrier to load a large amount of AuNPs based on the unique features of large surface area, plentiful inner nanospaces, highly defective horns, good electrical conductivity. To realize multiplexed detection of a-fetoprotein and CEA, nanocomposite formed via numerous AuNPs modified with streptavidin loading on the carbon nanohorn as signal tag to recognize biotinylated signal antibody, and then the SA/Au/CNH tag was captured on the immunoconjugates immobilized in disposable screen-printed electrodes to induce silver deposition and amplify the electrochemical stripping signals. Through

SPCE

Antigens W1

W2

Biotinylated antibodies

W1

W2

Ag

LSV detection

Silver deposition SA/Au/CNH

W1

W2

W1

W2

Fig. 2.5  Schematic representation of sandwich-type immunoassay procedure. Reprinted with permission from C. Zhao, J. Wu, H. Ju, F. Yan, Multiplexed electrochemical immunoassay using streptavidin/nanogold/carbon nanohorn as a signal tag to induce silver deposition, Anal. Chim. Acta 847 (2014) 37–43.

38

Immunosensing for Detection of Protein Biomarkers

sandwich-type immunoreaction and biotin-streptavidin affinity reaction amplifying sensing signals, the proposed immunosensor showed wide linear ranges with the detection limits down to 0.024 and 0.032 pg mL−1, respectively. Prior to this, they also designed an electrochemical trace tag based on nanogoldenriched carbon nanohorn structure (nanoAu/CNH) for highly efficient detection of tumor markers [35]. With a sandwich format, the nanoAu/CNH labeled antibody was conjugated on the immunosensor for electrochemical measurement of tumor marker by electrooxidizing the nanoAu at +1.3 V and then cathodic potential scan in 0.1 M HCl. The nanoAu/CNH label as well as the disposable immunosensor proved to possess potential application in point-of-care testing for protein detection with high sensitivity. Lin et al. [36] further utilized nanogold functionalized mesoporous carbon foam (Au/MCF) as signal label for sensitive electrochemical immunosensing of biomarker (Fig. 2.6). The high loading of nanogold on MCF caused a C-Au synergistic silver enhancement and hence greatly amplified the detection signal and improved the detection sensitivity. Through a sandwich-type immunoreaction, Au/MCF tags were immobilized on an electrochemically reduced grapheneoxide/chitosan film modified glassy carbon electrode, and induced a silver deposition process monitored by the electrochemical stripping signal. The Au/MCF-mediated silver enhancement along with the graphene-promoted electron transfer led to high detection sensitivity of CEA with a detection limit down to 0.024 pg mL−1.

HAuCl4 NaBH4

MCF

AuNPs/MCF

(A)

Graphene oxide

Ab2/AuNPs/MCF

Chitosan

Current

Electroreduction

LSV

Ag+

in KCl

HQ

BSA

Potential

(B)

Ab1

Ab2

BSA

CEA

AgNPs

Fig. 2.6  Schematic representation of (A) the preparation of Ab2/Au/MCF and (B) the immunosensor fabrication and sandwich-type immunoassay procedure. Reprinted with permission from D. Lin, J. Wu, H. Ju, Y. Feng, Nanogold/mesoporous carbon foam-mediated silver enhancement for graphene-enhanced electrochemical immunosensing of carcinoembryonic antigen, Biosens. Bioelectron. 52 (2014) 153–158.

Signal amplification for immunosensing39

In comparison to AuNP, the silver nanoparticle (AgNP) can be directly detected by anodic stripping analysis in KCl at a more negative potential and produces a relatively sharper peak and can be used as a nanolabel for immunosensing [37]. Lin et al. [38] combined surface-initiated enzymatic polymerization (SIEP) and the subsequent deposition of streptavidin functionalized silver nanoparticles (AgNPs) to propose a cascade signal amplification strategy for electrochemical immunosensing of protein (Fig. 2.7). It was accomplished by integrating an advanced amplification technique, SIEP, with biobarcode nanoparticle probe, and streptavidin functionalized AgNPs. In the first step of constructing, the immunosensor capture antibody was covalently immobilized on a chitosan modified glass carbon electrode. With a sandwich immunoassay, the biobarcode nanoparticles with numerous oligonucleotides were captured on the immunosensor surface, which catalyzed at the 3′-OH group of DNA addition of deoxynucleotides (dNTP) containing biotinylated deoxyadenosine 5′-triphosphate (biotin-dATP) by terminal deoxynucleotidyl transferase (TdT). This process results in the formation of many long single-stranded DNAs labeled with numerous biotins. Through the binding between biotin and streptavidin, a large number of streptavidin functionalized AgNPs were assembled. Anodics tripping voltammetric detection of Ag was performed in KCl solution for the cascade signal amplification, in which the method showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. A low detection limit down to sub pg mL−1 level has been achieved.

AuNP

(A)

(B)

Au

AgNP

dNTP (bio-dATP)

Ab2

Au

Au Ab1 AFP

Silver deposition

DNA Au

LSV

Streptavidin

in KCI TdT

(C) Fig. 2.7  Schematic representation of (A) the preparation of Ab-biobarcode AuNP probe, (B) the preparation of streptavidin functionalized AgNPs, and (C) the SIEP signal amplification strategy. Reprinted with permission from D. Lin, C. Mei, A. Liu, H. Jin, S. Wang, J. Wang, Cascade signal amplification for electrochemical immunosensing by integrating biobarcode probes, surface-initiated enzymatic polymerization and silver nanoparticle deposition, Biosens. Bioelectron. 66 (2015) 177–183.

40

Immunosensing for Detection of Protein Biomarkers

Optical-based technologies, such as chemiluminescent (CL), ECL, fluorescence, SPR, and Raman scattering technologies, are the most popular and convenient tools for immunoanalysis due to the advantages of applying visible radiation, nondestructive operation mode, and rapid signal generation and reading [39,40]. The development of nanotechnology supplies a large amount of excellent materials for signal generation and transmission, bioactive species loading, biorecognition process, and catalytic activity. AuNPs and AgNPs, with unique properties, such as highly resonant particle plasmons, direct visualization of single nanoclusters by scattering of light, catalytic size enhancement by silver deposition, and excellent biological tags by functioned modify, exhibit distinguished application in image and visualization of the biological process. QDs, considered as excellent semiconductor material, possess the unique characteristic of high-fluorescence quantum yields, photostability and tunable fluorescence bands, playing a significant role as fluorescence labels for biorecognition processes and biochemical assays. Shourian et al. [41] designed a multifunctionalized AuNPs as signal label for highly sensitive immunoassay of hepatitis B surface antigen (HBsAg). The AuNPs are functionalized by bearing biotin and luminol molecules, and then the multifunctionalized AuNPs coupling with streptavidin modified secondary antibody, which immobilized in polystyrene well via the primary monoclonal antibodies interaction with HBsAg (Fig. 2.8). The CL signal is produced by oxidation of luminol molecules in the presence of HAuCl4 as catalyst and H2O2 as oxidant. The immunosensor showed about 40 times lower detection limit toward HBsAg relative to previous work [42]. In such case, the nanoparticles act as both a solid carrier to load a large number of reporters/markers and as labels to amplify the CL signal for the protein detection. QDs, as the most representative semiconductor material, possess intelligent optical properties and favorable biocompatibility, exhibiting valuable potential in bioapplications. Based on mesoporous SiO2 coated carbon nanotubes (mCNTs)-QDs nanocomposites and electrochemiluminescence-energy transfer (ECL-ET) technology, Zhu’s

Ab2

HP-biotin

GNP

HBs-Ag MUA-luminol

hn

Ab1

STR

H2O2 HAuCl4

Fig. 2.8  The immune sandwich formation for detection of HbsAg. Reprinted with permission from M. Shourian, H. Ghourchian, M. Boutorabi, Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles, Anal. Chim. Acta 895 (2015) 1–8.

Signal amplification for immunosensing41

CTAB

(1) PEI

(1) SiO2 growth

(2) NIR QDs

(2) CTAB removal

mCNTs

mCNTs-QDs

(A)

Ab

1

ECL-ET

SA

(B)

)B

Au N

(1

Rs

-Ab

2

EA

)C

(2

Fig. 2.9  Construction of the ECL immunosensor. (A) Schematic representation of preparation procedure for mCNTs-QDs composites. (B) Schematic illustration of the stepwise immunosensor fabrication process. Reprinted with permission from L. Li, Y. Chen, Q. Lu, J. Ji, Y. Shen, M. Xu, R. Fei, G. Yang, K. Zhang, J. Zhang, J. Zhu, Electrochemiluminescence energy transfer-promoted ultrasensitive immunoassay using near-infrared-emitting CdSeTe/CdS/ZnS quantum dots and gold nanorods, Sci. Rep. 3 (2013) 776–776.

group [43] fabricated a versatile biosensor by employing the energy tunable CdSeTe/ CdS/ZnS double shell QDs as donor and gold nanorods (GNRs) as acceptor (Fig. 2.9). Surprisingly, they proposed a microwave-assisted production of high-quality NIRemitting CdSeTe/CdS/ZnS QDs with successive epitaxial CdS and ZnS double shells. The double CdSeTe/CdS/ZnS QDs manifested perfect energy match with GNRs as the donor-quencher pair. Due to the mCNTs-QDs and ECL-ET technology twofold signal amplification, this approach provided a sensitive response to CEA in a wide range from 0.001 to 200 pg mL−1 with a detection limit of 0.5 fg mL−1. Recently, surface-enhanced Raman scattering (SERS) imaging has been reported as a novel tool for glycan detection due to its advantages of high sensitivity, nondestructive and noninvasive features, and fingerprinting capability on chemical structures [44]. Song et al. [40] designed a novel gold nanoflower (AuNF) as a bridge to recognize target cell surface sialic acids (SAs), which are widely expressed on higher eukaryote cell surfaces and take part in diverse biological processes [45–47], and then assemble poly(N-acetylneuraminic acid) (PNA) and 5,50-dithiobis(2-nitrobenzoic acid) modified poly(amidoamine) (PAMAM)-encapsulated AuNPs (DAuNPs) to form a single-core multisatellite nanostructure for the global imaging of SAs on living cells (Fig. 2.10). The nanostructure possesses enormous “hot spots” because of the coarse surface and dense tips of the AuNFs, which produce a strong plasmonic coupling effect for the enhancement of the SERS signal. The proposed strategy has been employed to monitor the dynamic change in SA levels on cell surfaces and is suitable for other cell lines.

42

Immunosensing for Detection of Protein Biomarkers

DTNB 3-MPBA PSA

(A) AuNFs

AuNF probe

SA

HAuCl4 NaBH4

(B) PAMAM

DAuNPs

DAuNP probe Laser

SERS imaging

(C) Fig. 2.10  Schematic illustration of (A) the preparation of the AuNF probe, (B) the preparation of the DAuNP probe, and (C) SERS imaging of the cell surface SAs based on plasmonic coupling of the core AuNF with the satellite AuNPs. 3-MPBA represents 3-mercaptophenylboronic acid. Reprinted with permission from W. Song, L. Ding, Y. Chen, H. Ju, Plasmonic coupling of dual gold nanoprobes for sers imaging of sialic acids on living cells, Chem. Commun. 52 (2016) 10640–10643.

2.3 Enzyme mimics for signal amplification Enzyme-label possessed remarkable status in conventional immunoassays. High loading of enzymes on the nanoparticles is also the popular strategy of multilabeling for signal amplification in the biosensing field. However, the stability of protein enzyme is limited due to easy denaturation during their storage and immobilization procedure. Furthermore, the preparation and purification of biomolecules are usually timeconsuming and expensive [48,49]. Artificial mimicking enzymes that possess high catalytic activity and distinct substrate selectivity are highly desired for developing new catalytic reactions and bioanalysis systems due to their simpler syntheses and preparation, better stability, and easier modification than natural enzymes [50]. Therefore, lots of researches have focused on the development of enzyme mimetics-based signal amplification for immunosensing, which employs coordination compounds with peroxidase activity, such as hemin and porphyrin, and catalytic/electroactive nanoparticles, especially metal and semiconductor nanoparticles as labels [51–55]. As the catalytic centers of many enzymes, porphyrins, especially metalloporphyrins, have been widely used for the development of artificial enzymes. Hemin, an

Signal amplification for immunosensing43

Fe (III)-protoporphyrin IX, as the catalytic center of peroxidase, myoglobin and hemoglobin could conjugate to G-quadruplex, a single-stranded guanine-rich nucleic acid, to form DNAzyme with peroxidase-mimicking activity [56,57]. Compared with the native enzyme of HRP, the catalytic activity of DNAzyme is slightly lower [58]. However, due to its easy synthesis, convenient storage, and easy assembly as a molecule for signal readout, G-quadruplex/hemin has attracted substantial recent research interest as an amplifying mimic bioenzyme label for biosensing applications [59– 62]. Designing of specific biosensors by direct combining the G-quadruplex/hemin DNAzyme with an antiprotein aptamer is a simple and effective method for protein detection [63,64]. However, their sensitivity is limited. So, the application of nanomaterials as carriers to load multiplex G-quadruplex/hemin and ligands (antibody or aptamer) is an effective strategy for signal amplification. Typically, Ju’s group [65] designed a multilayer G-quadruplex/hemin DNAzyme wrapped gold nanoparticle (M-DNAzyme/AuNP) tag for ultrasensitive CL image analysis of proteins (Fig.  2.11). The M-DNAzyme/AuNP tag was prepared by assembling a high ratio of alkylthiol-capped signal DNA containing multiple G-quadruplex sequences to biotinylated DNA on AuNPs and then reacting with hemin to form multilayer hemin/G-quadruplex DNAzyme units. It could be bound to the biotinylated secondary antibody of sandwich immunocomplex by biotinstreptavidin conjugation to catalyze a CL reaction on a protein array, which produced strong CL emission. By combining with a disposable protein array, an ultrasensitive and high-throughput multiplex CL immunoassay method was proposed for simultaneous detection of four cancer biomarkers, including α-fetoprotein, human chorionic gonadotrophin-β, carcinoma antigen 125, and CEA, with the limits of detection of 2.7 × 10−5 ng mL−1, 1.1 × 10−5 IU mL−1, 1.7 × 10−5 U mL−1, 2.0 × 10−5 ng mL−1, respectively. Similarly, a G-quadruplex/hemin-Pt nanoparticle (NP) was used as enzyme mimetics probe for fabricating ultrasensitive photoelectrochemical (PEC) immunoassay [66]. The G-quadruplex/hemin-Pt NP probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant due to its peroxidase-mimicking activity. The oxidation product of HQ, a polymer with abundant electron-accepting groups, could efficiently inhibit the photocurrent of CdS QDs through photoinduced electron transfer and steric hindrances effect, resulting in a highly sensitive PEC immunosensor. Ju’s group [67] also prepared a hemin functionalized graphene sheet via the noncovalent assembly of hemin on nitrogen-doped grapheme (NG). The hemin@NG could act as an oxygen reduction catalyst to produce sensitive ECL quenching of QDs due to the annihilation of dissolved oxygen, the ECL coreactant, by its electrocatalytic reduction. The hemin@NG was proved to produce higher catalytic efficiency than hemin@ GO to quench the cathodic ECL emission of QDs. With a sandwich immunoassay format using an immunosensor constructed by immobilization of bidentate-chelated CdTe QDs and capture antibody, the hemin@NG as a tracing tag showed higher sensitivity (Fig. 2.12). With the use of CEA as a model, the proposed ultrasensitive immunoassay method could detect the protein biomarker down to the subpicomolar level with a detection range over five orders of magnitude. Moreover, this method showed acceptable reliability and stability (Fig. 2.13). Eight measurements of ECL emission

Immunosensing for Detection of Protein Biomarkers

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Streptavidin Biotinylated antibody

Hemin

Fig. 2.11  Schematic diagrams of (A) preparation of M-DNAzyme/AuNP, (B) sensor preparation and CL immunosensing procedure, and (C) multiplex CL imaging immunoassay of four tumor markers using an immunosensor array. Reprinted with permission from C. Zong, J. Wu, J. Xu, H. Ju, F. Yan, Multilayer hemin/G-quadruplex wrapped gold nanoparticles as tag for ultrasensitive multiplex immunoassay by chemiluminescence imaging, Biosens. Bioelectron. 43 (2013) 372–378.

from the immunosensor at 0.1 ng mL–1 CEA upon continuous cyclic scans showed coincident signal with relative standard deviation of 1.7%. Manganese porphyrin, manganese(III) meso-tetrakis(4-N-methylpyridyl)-porphyrin (MnTMPyP), which contains manganese as the central metal atom, has also showed peroxidase-like activity and much better catalytic performance than iron porphyrin [68,69]. Xu et al. [70] designed a new avenue for preparation of mimicking

Signal amplification for immunosensing45

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8000

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Fig. 2.12  Schematic illustration of hemin@Ng as signal tag for QD-based ECL immunoassay. Reprinted with permission from S. Deng, J. Lei, Y. Huang, Y. Cheng, H. Ju, Electrochemiluminescent quenching of quantum dots for ultrasensitive immunoassay through oxygen reduction catalyzed by nitrogen-doped graphene-supported hemin, Anal. Chem. 85 (2013) 5390–5396.

1.0

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Fig. 2.13  (A) ECL responses of the QDs-based immunosensor to CEA at 0.1, 1.0, 10, 100 pg mL−1, 1.0, and 10 ng mL−1 (from top to bottom) in air-saturated 0.1 M pH 8.0 PBS. Inset: calibration curve. (B) Continuous cyclic scans of the immunosensor in air-saturated 0.1 M pH 8.0 PBS after incubation with 10 ng mL–1 of CEA and then hemin@NG-labeled Ab2. Reprinted with permission from S. Deng, J. Lei, Y. Huang, Y. Cheng, H. Ju, Electrochemiluminescent quenching of quantum dots for ultrasensitive immunoassay through oxygen reduction catalyzed by nitrogen–doped graphene-supported hemin, Anal. Chem. 85 (2013) 5390–5396.

enzyme with peroxidase-like activity by loading MnTMPyP in the dsDNA scaffold. It was demonstrated that MnTMPyP-dsDNA complex possessed high catalytic activity and much quicker catalytic kinetics and better stability with exposure to light irradiation and high temperature than both HRP and hemin/G-quadruplex DNAzyme. The groove binding of MnTMPyP to the dsDNA scaffold efficiently maintained the

46

Immunosensing for Detection of Protein Biomarkers

+

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+ HCR

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Antigen O

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CL MnTMPyP NH2 O

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OH OH

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O

Fig. 2.14  Schematic illustration of amplifying synthesis of MnTMPyP-dsDNA enzyme mimic as label for CL biosensing. Reprinted with permission from J. Xu, J. Wu, C. Zong, H. Ju, F. Yan, Manganese porphyrindsDNA complex: a mimicking enzyme for highly efficient bioanalysis, Anal. Chem. 85 (2013) 3374−3379.

catalytic activity of the MnTMPyP center and improved its stability. By combining with an isothermal HCR and in situ formation of MnTMPyP-dsDNA, a highly efficient CL immunosensing method was proposed (Fig.  2.14). After a sandwich immunoreaction, a biotinylated DNA strand, which was bound to biotinylated signal antibody by streptavidin, triggered the HCR and growth of MnTMPyP-dsDNA on the immunocomplex. The in situ, HCR-assisted enzyme formation brought numerous enzymatic catalytic centers, MnTMPyP, on the immunocomplex, resulting in significant CL signal amplification and highly sensitive CL detection. Using CEA as the model target, the proposed CL immunoassay method showed a wide linear range from 10 pg mL−1 to 100 ng mL−1 with a detection limit of 6.8 pg mL−1. Due to the outstanding advantages of MnTMPyP-dsDNA, this novel enzyme mimic can be extended to combine with other amplification strategies and nanomaterial carriers for application in different fields. Nanoparticles with enzyme-like activities, especially metal and semiconductor nanoparticles, can be used directly as electroactive labels for the electrochemical amplification detection of proteins. Fe3O4 NPs and Fe3O4 NPs-based hybrid materials can exhibit enhanced peroxidase-like activity [71–73]. Wang et  al. [74] synthesized Au NPs doped Fe3O4 (Au@Fe3O4) NPs by a facile one-step solvothermal method. The peroxidase-like activity of Au@Fe3O4NPs was effectively enhanced due to the synergistic effect between the Fe3O4NPs and Au NPs. On this basis, an efficient colorimetric aptasensor has been developed for the detection of ochratoxin A using the intrinsic dual functionality of the Au@ Fe3O4 NPs as signal indicator. Wei et  al. [13] developed an enzyme mimic immuno-label by loading Fe3O4NPs into a polymeric vesicle, poly(ethylene glycol) epoly (lactic acid) (PEG-PLA), followed by

Signal amplification for immunosensing47

conjugating secondary antibody (Ab2) onto the vesicle’s surface. The resulting Ab2@ PEG@PLA@Fe3O4 demonstrated high catalytic activity toward H2O2, and the sensitivity of the sandwich-type immunosensor using this label for prostate PSA detection increased greatly. Metallic alloy nanomaterials with enzyme-like activities have also attracted great attention due to their better catalytic properties than their monometallic counterparts, such as Pt-Au nanoparticles [75], Au-Pd nanostructures [76], and AuPd bimetallic nanoprobes [77]. These metallic alloy nanomaterials with excellent electrocatalytic performance, large surface area, and favorable biocompatibility provide ideal nanoprobes for electrochemical immunosensor. Hou et  al. [76] designed a DNAzymefunctionalized gold-palladium hybrid nanotag (AuPd-DNA) for highly sensitive and selective impedimetric immunosensor of prostate-specific antigen (PSA) with triple signal amplification. The signal was amplified based on the AuPd-DNA toward the catalytic precipitation of 4-choloro-1-naphthol (4-CN). DNAzyme could catalyze the oxidation of 4-CN, while the AuPd hybrid nanostructures could not only provide a large surface coverage for immobilization of biomolecules but also promoted 4-CN oxidation (Fig. 2.15). The produced insoluble benzo-4-chlorohexadienone via 4-CN was coated on the electrode surface, and hindered the electron transfer between the solution and the electrode, thereby increasing the faradaic impedance of the base electrode and improving the sensitivity of impedimetric immunosensor of PSA. Ju’s group [77] also presented a modular labeling strategy for e­ lectrochemical immunoassay via supramolecular host-guest interaction between β-cyclodextrin (β-CD) and adamantine (ADA) (Fig. 2.16). The ADA labeled antibody (ADA-Ab) was synthesized via amide reaction, and the number of ADA moieties loaded on single antibody was calculated to be ~7. The β-CD functionalized AuPd bimetallic nanoparticles (AuPd-CD) were synthesized in aqueous solution via metal-S chemistry. After the ADA-Ab was bound to antigen electrode surface with a competitive immunoreaction, AuPd-CD as signal tag was immobilized onto the immunosensor by

O

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Fig. 2.15  Schematic illustration of DNAzyme-functionalized gold-palladium hybrid nanostructures for triple signal amplification of impedimetric immunosensor. Reprinted with permission from L. Hou, Z. Gao, M. Xu, X. Cao, X. Wu, G. Chen, D. Tang, DNAzyme-functionalized gold–palladium hybrid nanostructures for triple signal amplification of impedimetric immunosensor, Biosens. Bioelectron. 54 (2014) 365–371.

48

Immunosensing for Detection of Protein Biomarkers COOH

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Fig. 2.16  Schematic representation of the preparation of (A) ADA-Ab conjugate, (B) β-CD functionalized AuPd bimetallic nanoparticles, and (C) electrochemical immunoassay procedure of detection of a small molecule. Reprinted with permission from L.S. Wang, J.P. Lei, R.N. Ma, H.X. Ju, Host-guest interaction of adamantine with a β-cyclodextrin-functionalized AuPd bimetallic nanoprobe for ultrasensitive electrochemical immunoassay of small molecules, Anal. Chem. 85 (2013), 6505–6510.

the host-guest interaction, leading to large loading of AuPd nanoparticles. The highly efficient electrocatalysis of AuPd nanoparticles toward NaBH4 oxidation produced an ultrasensitive response to chloramphenicol as a model of small molecule antigen. The immunoassay method showed a wide linear range from 50 pg mL−1 to 50 μg mL−1 and a detection limit of 4.6 pg mL−1 (Fig. 2.17). The specific recognition of antigen to antibody resulted in good selectivity of the proposed method. The host-guest interaction strategy provided a universal labeling approach for ultrasensitive detection of small molecule targets. Chang et al. [78] used Pt NPs and DNAzyme functionalized polymer nanospheres as enzyme mimics nanolabel for triple signal amplification strategy for highly sensitive electrochemical immunosensing. First, electroactive polymer nanospheres were synthesized by infinite coordination polymerization of ferrocene dicarboxylic acid, which could generate strong electrochemical signals due to plentiful ferrocene molecules. Further, the polymer nanospheres were functionalized by Pt NPs and G-quadruplex/ hemin DNAzyme with the ability of catalyzing H2O2, which contributes to enhance the electrochemical signals. Yuan’s group [79] also developed an ultrasensitive ECL immunosensor based on the synergetic amplification of ECL of luminol by heminreduced graphene oxide and AgNPs decorated reduced graphene oxide. Yuan’s group [80] further fabricated a synergetic signal amplification strategy based on the amplification of hemin/G-quadruplex functionalized Pt@Pd nanowires

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Signal amplification for immunosensing49

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Fig. 2.17  (A) LSV responses and (B) calibration curve of the CAP immunosensor in 0.1 M borate buffer (pH 11) containing 5 mM NaBH4 after incubation with CAP ranging from 5 × 10−3 to 5 × 105 ng mL−1 (from top to bottom). Scan rate: 50 mV s−1. Reprinted with permission from L.S. Wang, J.P. Lei, R.N. Ma, H.X. Ju, Host-guest interaction of adamantine with a β-cyclodextrin-functionalized AuPd bimetallic nanoprobe for ultrasensitive electrochemical immunoassay of small molecules, Anal. Chem. 85 (2013), 6505–6510.

(Pt@PdNWs) (Fig. 2.18). The synthesized Pt@PdNWs possessed a large surface area, which could effectively improve the immobilization amount of hemin/G-quadruplex DNAzyme produced via HCR. In the electrolyte of PBS (pH 7.0) containing NADH, the hemin/G-quadruplex acted as an NADH oxidase, where NADH was oxidized to NAD+ with concomitant formation of high concentration of H2O2, and the generated H2O2 was next electrocatalytically reduced by hemin/G-quadruplex and Pt@PdNWs to obtain a conspicuously enhanced electrochemical signal. With these synergetic amplification factors, the electrochemical immunosensor exhibited a wide linear range from 0.001 ng mL−1 to 100 ng mL−1 with a detection limit of 0.24 pg mL−1.

2.4 PCR for signal-amplified immunosensing PCR, invented by Kary B. Mullis, is a historical breakthrough in molecular biology, which exponentially generates up to a billion copies of the target within just a few hours by sequential iteration of process, including heat denaturation, specific hybridization or annealing of short oligonucleotide primers to single-stranded DNA, and synthesis by DNA polymerase [81]. In 1992, PCR was first introduced into immunoassays by Sano et  al. [82]. This new technology, named immuno-PCR or IPCR, combined the versatility and flexibility of ELISA with the exponential signal amplification power of PCR. In the time since the original report, many researchers have worked to improve the assay workflow of IPCR by focusing on the key aspects of DNA-antibody conjugates, target binding, and assay readout [83].

50

Immunosensing for Detection of Protein Biomarkers

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Fig. 2.18  (A) Scanning electron microscope (SEM) image of Pt@PdNWs; (B) preparation procedure of Pt@PdNWs/Ab2/S0 bioconjugation; (C) schematic illustrations of the immunosensor fabrication process and the detection principle. Reprinted with permission from Q. Wang, Y. Song, Y. Chai, G. Pan, T. Li, Y. Yuan, R. Yuan, Electrochemical immunosensor for detecting the spore wall protein of Nosema bombycis based on the amplification of hemin/G-quadruplex DNAzyme concatamers functionalized Pt@Pd nanowires, Biosens. Bioelectron. 60 (2014) 118–123.

In an initial work, a streptavidin-protein A fusion protein was constructed as a bridge of biotin-DNA complex and antibody based on the specific bind of protein A to the Fc fragment of IgG and streptavidin to the biotin-DNA complex (Fig. 2.19I). Although this strategy has been used successfully, the application of this technique was limited due to the limited availability of the fusion proteins. To overcome this limitation and extend the application of immuno-PCR, streptavidin or avidin was used to join both the biotinylated DNA reporter and the biotinylated antibody (Fig. 2.19II), since the streptavidin (avidin) and biotinylated antibodies are readily available [84,85]. Another strategy for constructing antibody-DNA conjugates was direct covalent linkage of the DNA to the antibody (Fig. 2.19III), which offered a number of advantages in IPCR as they do not require lengthy preincubation steps before the actual analysis can take

Signal amplification for immunosensing51

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Fig. 2.19  Different strategies for coupling antibodies and DNA for use in IPCR: (I) A streptavidin-protein A fusion protein, (II) streptavidin or avidin used to join both the biotinylated DNA reporter and the biotinylated antibody, (III) antibody-DNA direct conjugates using chemical methods, (IV) phage display mediated immuno-polymerase chain reaction (PD-IPCR) or liposome mediated PCR assay, and (V) barcodes biofunctionalized nanoparticles for IPCR. Reprinted with permission from K.P.F. Janssen, K. Knez, D. Spasic, J. Lammertyn, Nucleic acids for ultra–sensitive protein detection, Sensors 13 (2013) 1353–1384.

place [86]. Furthermore, unique DNA nucleotides were covalently coupled to each of the analyte-specific antibodies, enabling the analysis of multiple analytes. More recently, in order to achieve higher sensitivity, some modified methods of the immuno-PCR were developed to present multiple reporter DNAs at each binding site. Phage display mediated immuno-polymerase chain reaction (PDIPCR) technology was designed to combine the advantages of immuno-PCR and phage display [87]. In this technology, the antibody fragments, such as single chain fragment variable [87], variable domain of heavy chain antibodies (VHH) [88], were expressed on the surface of a phage particle as a recognition element (Fig. 2.19IV). While, the genome of phage M13 served as the reporter DNA molecule for PCR amplification [89]. While the defect of PD-IPCR that the limited molecular weights of peptides displayed on phages causes the lower binding affinity to target protein. Thus, Zhang et al. [90] coupled a thiolated antibody with the free amino groups on surface of T7 phase by using a crosslinker, sulfosuccinimidyl-4(N-­maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC), which proposed a novel and universal version of immuno-PCR based on the natural phage nanoparticles. Furthermore, liposomes encapsulated with reporter DNA and functionalized with recognition elements were applied in IPCR, leading to impressive limits of detection as low as 0.1 aM for Botulinum toxin [91]. Mirkin’s group [92] demonstrated for the first time that short DNA oligonucleotides biofunctionalized nanoparticles can be used as “barcodes” and coupled with PCR assay for the identification of protein targets in a homogeneous sandwich (Fig. 2.20). AuNP were immobilized with a polyclonal antibody and a large number of single-stranded DNA molecules. A complementary DNA strand was hybridized to serve as a barcode for the subsequent detection. After a sandwich and magnetic separation, the barcode DNA probes from the nanoparticles that were bound to the proteins were released by dehybridization, and were subsequently identified and quantified by the incorporation of PCR. The assay was able to measure 3 aM of PSA, showing a sensitivity of six orders of magnitude greater than conventional ELISA for PSA. This biobarcode strategy

52

Immunosensing for Detection of Protein Biomarkers

Step 1. Target protein capture with MMP probes

Step 2. Sandwich captured target proteins with NP probes

Target protein (PSA) 13 nm NPs for bio-bar-code PCR 30 nm NPs for PCR-less method Step 5. Chip-based detection of bar-code DNA for protein identification Ag Au

Step 4. Polymerase chain reaction Bar-code DNA

Step 4. PCR-less detection of bar-code DNA from 30 nm NP probes

Step 3. MMP probe separation and bar-code DNA dehybridization

M Magnetic field

Fig. 2.20  Overview of the biobarcode immunoassay. Reprinted with permission from J. Nam, C.S. Thaxton, C.A. Mirkin, Nanoparticle-based biobarcodes for the ultrasensitive detection of proteins, Science 301 (2003) 1884–1886.

was further used to enhance the sensitivity of IPCR for the detection of Hantaan virus nucleocapsid protein [93]. Aptamers are versatile oligonucleotide biorecognition elements whose target selectivity and affinity can rival antibodies. Recently, the aptamer specificity has been combined with PCR sensitivity in various approaches for the ultrasensitive detection of proteins, including a proximity ligation assay (PLA) [94,95], nuclease protection assay [96], capillary electrophoresis (CE) [97], the use of target-modified magnetic microparticles [98], and quantitative reverse transcription (RT)-PCR [99]. For example, Zhang et al. [97] described an ultrasensitive, aptamer-based, affinity-PCR technique for the determination of trace amounts of proteins (Fig. 2.21). In this affinity aptamer amplification assay, the sample was first incubated with an aptamer that was able to bind to the target protein. The protein-aptamer complex was then separated from the unbound aptamer by CE. CE fractions were collected at 30 s intervals, and

CE

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Fig. 2.21  Schematic of a protein assay using PCR amplification of an affinity aptamer. Reprinted with permission from H. Zhang, Z. Wang, X.F. Li, X.C. Le, Ultrasensitive detection of proteins by amplification of affinity aptamers, Angew. Chem. Int. Ed. 45 (2006) 1576–1580.

Signal amplification for immunosensing53

each ­fraction was subjected to PCR analysis. Amplification of the protein-bound aptamer by PCR dramatically enhanced the sensitivity of the analysis of the corresponding target protein. This method has been applied to the determination of trace amounts of HIV-1 reverse transcriptase (RT). It was able to detect 30 fM of HIV-1 RT in a 10 nL sample, representing ~180 molecules of the protein.

2.5 Rolling circle amplification for amplified immunoassay Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic process, which synthesizes a long, repetitive ssDNA via special polymerase. A typical RCA reaction requires two reaction steps of ligation and polymerization. T4 DNA ligase helps circularization of DNA template, and Phi29 DNA polymerase initiates an RCA polymerization from the prime-template hybrids, which generate long ssDNA with many tandem copies of the complement to the circularized molecule in a few minutes [100]. In contrast to the PCR, which requires a thermal cycler and thermostable DNA polymerases, RCA is an isothermal amplification, which can be conducted in solution, on a solid support or in a complex biological environment [101]. We can monitor and detect the RCA process and products with a variety of signal readout techniques. Gel electrophoresis is the most common way to analyze RCA products [102,103]. Especially, easy visualization can be achieved by labeling detection reagents with fluorophores, or colored products can be catalytically deposited by enzymes like HRP attached to the RCA products. Furthermore, an important feature of linear RCA is that the product of amplification remains immobilized on the target molecule, which permits easy detection of localization signals [104]. Since its discovery in the mid 1990s [105,106], due to these attractive features, RCA has been developed extensively to establish sensitive detection methods for DNA [107,108], RNA [109,110], DNA methylation [111], single nucleotide polymorphism [112], small molecules [113], proteins [114], and cells [115]. In 2000, Ward et  al. first described an adaptation of the RCA for the detection of protein, termed “immuno-RCA” [116]. The present report has demonstrated that immuno-RCA could process reproducible measurements of lower-abundance proteins and intranuclear proteins that are difficult to measure or to visualize by using conventional signal detection methods. More recently, RCA has been explored as an important strategy for signal enhancement in immunoassay due to the sensitivity, simplicity, and versatility of the RCA technique [117]. For example, Cheng et  al. [118] introduced the biotin-streptavidin system to bind primers to antibody and combined the RCA technique with oligonucleotide functionalized QDs, and anodic stripping voltammetric test, realizing detection of protein target at ultralow concentration (Fig. 2.22). The QDs-tagged RCA product with micrometer length could be observed with transmission electron microscopic and fluorescence microscopic images (Fig.  2.23). Cheng’s group used human vascular endothelial growth factor (VEGF) as a model protein to attain an outstanding detection limit of 0.27 aM with a

54

Immunosensing for Detection of Protein Biomarkers

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Fig. 2.22  Schematic representation of the cascade signal amplification strategy for protein detection. Reprinted with permission from W. Cheng, F. Yan, L. Ding, H. Ju, Y. Yin, Cascade signal amplification strategy for subattomolar protein detection by rolling circle amplification and quantum dots tagging, Anal. Chem. 82 (2010) 3337−3342.

Fig. 2.23  (A) TEM and (B) fluorescence microscopic images of QD-tagged RCA product. Reprinted with permission from W. Cheng, F. Yan, L. Ding, H. Ju, Y. Yin, Cascade signal amplification strategy for subattomolar protein detection by rolling circle amplification and quantum dots tagging, Anal. Chem. 82 (2010) 3337−3342.

wide linear range from 1 aM to 1 pM (Fig. 2.24). The low detection limit meant that this method could quantitatively detect protein down to 16 molecules in a 100 μL sample. In this work, the RCA and the multiplex binding system presented numerous superior features, including remarkable amplification efficiency, very little nonspecific adsorption, and low background signal.

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Signal amplification for immunosensing55

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10–12

Fig. 2.24  (A) Anodic stripping voltammograms of cadmic cation responding to 10−12, 10−13, 10−14, 10−15, 10−16, 10−17, 10−18, and 0 M VEGF (left to right). (B) The quantitative dynamic range of the designed method. Reprinted with permission from W. Cheng, F. Yan, L. Ding, H. Ju, Y. Yin, Cascade signal amplification strategy for subattomolar protein detection by rolling circle amplification and quantum dots tagging, Anal. Chem. 82 (2010) 3337−3342.

PLA is another way to attach primer to protein, which allows for detection of individual proteins or protein complexes, under the circumstances of antibodies attached to DNA strands that participate in ligation and RCA initiation. Landegren et al. described the method of in situ PLA, applying to visualization of protein interactions in both tissue sections and in vitro cells [104]. In situ PLA (Fig. 2.25), identification of target molecules based on two recognition events, realizing localized detection reactions, by using DNA ligation products served as template for highly sensitive protein detection. In Chapter 9, the proximity hybridization regulated immunoassay will be described in detail. Immuno-RCA converted antibody-antigen recognition events to nucleic acid signal amplification. A long DNA production containing hundreds of copies of the circular DNA sequence that remained attached to the Ab in the immuno-RCA assay, which can be detected via a variety of signal-reporting labels in suit, including direct incorporation fluorophores or by hybridization of fluor-labeled complementary oligonucleotide probes and so on. For example, using a highly fluorescent SYBR Green I intercalated the RCA product, Xue et al. [119] proposed a novel cascade fluorescence signal amplification strategy based on RCA-aided assembly of fluorescent DNA nanotags as fluorescent labels for detection of protein target at ultralow concentration (Fig. 2.26). Moreover, Huang et al. [120] adopted a novel protein detection technique by using SPR and RCA. When RCA amplification products, which were characterized by oligonucleotide tags bound to the probes modified on the sensor chip surface, the change of mass on the surface produced the SPR signal. From measuring the change in SPR signal, target protein from the solution can be detected. In addition, immuno-RCA was coupled with nanoparticle-based amplification format for pursuing higher sensitivity and selectivity [121,122]. Nanomaterials such as nanoparticles, carbon nanotubes, and nanohybrid materials have demonstrated that their presence can significantly enhance the protein detection signal and overcome the problem of lower-abundance protein analyses [114,121,123,124].

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Proximity probe binding

Rolling circle amplification

Circularization and ligation of connector oligonucleotides

Detection of rolling circle product

Fig. 2.25  Schematic presentation of in situ PLA. Reprinted with permission from O. Söderberg, K.J. Leuchowius, M. Gullberg, M. Jarvius, I. Weibrecht, L.G. Larsson, U. Landegren, Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay, Methods 45 (2008) 227–232.

For example, AuNPs modified primers have been proven to effectively initiate the RCA reactions. When the target protein different epitopes binding to the detection antibody and capture antibody by a specific sandwich immunoreaction, RCA reaction was triggered by AuNPs, which were attached to both the detection of antibody and DNA primer [121]. Using biotinylated 2′-deoxyuridine 5′triphosphate (biotin-dUTP) and avidin-horseradish peroxidase (Av-HRP), RCA products generated enzymatic catalysis-based colorimetric signals, which quantitatively reflected the immunological target recognition event (Fig.  2.27). This on-nanoparticle, rolling circle amplification (nanoRCA) strategy combined the advantages of immuno-RCA and nanoparticle-based assays, and allowed the visual detection of 30 molecules of CEA, with at least 1010-fold selectivity over a range of

Signal amplification for immunosensing57

1 Target

Primer

1 BT-Ab 2 Strepavidin

2 BSA blocking

Circular template

RCA Fluorescent DNA nanotags

Probe SYBR Green I

Fig. 2.26  Illustration of the immunoassay using assembled cascade fluorescent DNA nanotags based on RCA. Reprinted with permission from Q. Xue, Z. Wang, L. Wang, W. Jiang, Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification, Bioconjugate. Chem. 23 (2012) 734–739.

S

S

S S S

S

SH-DNA tag

Antigen

Detection antibody

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Capture antibody

Biotin-dNTPs

Gold nanoparticle

Phi29 polymerase

Magnetic nanoparticle

Avidin-HRP

RCA Color

S

S

S

TMB

TMB

S S

S

S

S

S

S

Color

Ag

Fig. 2.27  Schematic illustration of the nanoRCA strategy. Reprinted with permission from J. Yan, S. Song, B. Li, Q. Zhang, Q. Huang, H. Zhang, C. Fan, An on-nanoparticle rolling-circle amplification platform for ultrasensitive protein detection in biological fluids, Small 6 (2010) 2520–2525.

noncognate proteins. Moreover, a multiwalled carbon nanotube (MWCNT)-based rolling circle amplification system (CRCAS) was established via colorimetric and CL assays for the highly sensitive and specific detection of protein markers [123]. CRCAS allowed in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single-strand DNA (ssDNA) where a large number of

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nanoparticles or proteins could be loaded, forming a nanobiohybridized 3D structure with a powerful signal amplification ability. Aptamers, a synthetic DNA/RNA nucleic acid single strand commonly used as another type of affinity ligand, have been selected in  vitro from random sequence libraries against a variety of analytes, including small molecules [125,126], proteins [127,128], even whole viruses [129], with high affinity and specificity based on special changes of secondary and tertiary structure without depending on base-pairing to the target. Compared to Abs, aptamers possess several merits, such as simple synthesis, easy labeling, and good stability, thus more likely substituting antibody for protein recognition in immuno-RCA. King et al. first introduced a class of circular DNA aptamers (“captamers”) to combine with the proximity extension reaction of RCA for highly sensitive protein analysis [130]. However, the need for two binding sites of aptamers for proximity extension reaction of RCA limits its application to other proteins with only one aptamer. Therefore, Ellington et al. [131] developed a novel type of conformation-switching aptamer strategy for RCA, in which once protein target interacted with aptamer that can be circularized. The proposed method of combining real-time aptamer-based RCA would not only be fast and quantitative but could also potentially be carried out in heterogeneous solutions without having to wash away unbound affinity reagents or DNA templates. This innovative strategy can be universally adapted to almost aptamer and does not require multiple affinity reagents, unlike sandwich or proximity assays (Fig. 2.28).

Phi 29

PDGF, T4 DNA ligase PDGF

(A)

C G

G

A C C

Inactive

A C C T C T

A

G T

T C T CA C A G C A G

CT CT C A CC

CT

T A AG C AT C A C C A T G AT C C G

G

3′ T A G T G G T A C T A G G C T C T A G C C 5′

T G G GT

G

T AG G TA G

T G T T G T

C A A T T G T C G TG G T Probe T A T A PDGF C A T TG sequence TG G CCTGTGG G GAT CG G A T C AT A G G C GGACA CTC C T A G C C 5′ 3′ T A G T A G A T T T A C G CA C Primer T C G C C A C T C binding Ligation C G T C T CAC C

Active

(B)

Fig. 2.28  Design of the conformation-switching aptamer for RCA. (A) General design principles. (B) Sequence and secondary structure of the designed conformation-switching aptamer for PDGF. Reprinted with permission from L. Yang, C.W. Fung, E.J. Cho, A.D. Ellington, Real-time rolling circle amplification for protein detection, Anal. Chem. 79 (2007) 3320–3329.

Signal amplification for immunosensing59

Some aptamer-based RCA systems have also been designed based on aptamer with dual functions of protein recognition and RCA priming. Savran et al. [132] presented a robust, label-free, self-assembled optical diffraction biosensor that combined RCA and magnetic microbeads as a signal enhancement method to detect platelet-derived growth factor B-chain (PDGF-BB). As shown in Fig. 2.29, a secondary aptamer bound to the immobilized protein in a classic sandwich assay style. The secondary aptamer also acted as a primer resulting in a circular template for RCA. By conjugating biotinylated probes to streptavidin-coated microbeads on the RCA-amplified concatemers, it proved possible to obtain visual confirmation of the binding reaction and produced diffraction modes upon illumination with a laser. At the same time, Cheng et al. [133] designed an aptamer-initiated RCA to facilitate the development of RCA for protein targets, which used only one aptamer to serve

Aptamer-primer

Padlock probe

PDGF-BB

Ligase

RCA

Probe

Phi29

SA-bead

(A)

SA-bead

(B) Fig. 2.29  (A) Schematic of RCA-based microbead detection assay in combination with aptamers. (B) Self-assembled streptavidin (SA)-coated beads on the RCA-based micropattern form diffraction gratings that yield diffraction modes upon illumination with a laser. Reprinted with permission from J. Lee, K. Icoz, A. Roberts, A.D. Ellington, C.A. Savran, Diffractometric detection of proteins using microbead-based rolling circle amplification, Anal. Chem. 82 (2009) 197–202.

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Aptamer

Circular DNA

Target protein

Epoxy-coated slide

(2) Scan

Phi29 RCA

AuNP probe

(1) Silver enhancement

Fig. 2.30  Schematic representation of the designed aptamer-initiated RCA strategy for protein detection. Reprinted with permission from W. Cheng, L. Ding, Y. Chen, F. Yan, H. Ju, Y. Yin, A facile scanometric strategy for ultrasensitive detection of protein using aptamer–initiated rolling circle amplification, Chem. Commun. 46 (2010) 6720–6722.

as both the primer to form aptamer-circular DNA duplex for RCA and the recognition element to recognized target protein. The principle of the scheme was shown as follows (Fig. 2.30): In the presence of target protein, the bound circular DNA could be substituted by a target-induced strand release process, because of the higher affinity of selected aptamer to its protein. The unchanged aptamer-circular DNA duplex, whose amount depended on the quantity of target protein, could initiate RCA. Subsequently, complementary AuNP probes hybridized with the long DNA strand that contained hundreds of tandem-repeat complementary sequences of the circular DNA. Finally, the simple scanometric readout was finally performed with a scanner for detection signals after silver enhancement. This work significantly improved the sensitivity of colorimetric detection by combining AuNP probe with simple scanometric readout with the 10 fM of the limit of detection (LOD) of human VEGF 165, as shown in Fig. 2.31. Up to now, several techniques including fluorescence [134–136], electrochemistry [137,138], colorimetry [139], SPR [140], and ECL [141] have been applied to detection of immuno-RCA products. These immuno-RCA sensors based on different detection approaches are listed in Table 2.1.

2.6 HCR for amplified immunoassay DNA-based signal amplification techniques have been developed for improving sensitivity of immunoassay, such as PCR, RCA, etc. However, PCR needs complicated thermal cycles and strict laboratory conditions, which largely limit the application of PCR in point-of-care testing. In addition, RCA, an isothermal amplification reaction without thermal cycles, often depends on multiple enzymes, and the steps require ligation and polymerization, which are time consuming and costly. Therefore, with great attention focused on enzyme-free isothermal amplification strategies, HCR amplification shows great potential in signal amplification [142–144]. In HCR, two sets of DNA monomer hairpin structure (H1 and H2) are designed to be partially complementary,

Signal amplification for immunosensing61

(A)

A

B

C

D

E

F

G

40

Relative intensity

60

80

100

120

(B)

0

10–14

10–13 10–12 10–11 Concentration (M)

10–10

10–9

Fig. 2.31  (A) Scanometric image of the spots responding to 0, 10−14, 10−13, 10−12, 10−11, 10−10, and 10−9 M VEGF (A–G), and (B) quantitative dynamic range of the designed strategy. The error bars represent the standard deviations calculated from three different spots. Reprinted with permission from W. Cheng, L. Ding, Y. Chen, F. Yan, H. Ju, Y. Yin, A facile scanometric strategy for ultrasensitive detection of protein using aptamer–initiated rolling circle amplification, Chem. Commun. 46 (2010) 6720–6722. Table 2.1  Immuno-RCA sensors based on different detection approach Approach

Target protein

Detection limit

Ref.

Fluorescence Fluorescence Fluorescence Electrochemistry Electrochemistry Colorimetry Surface plasmon resonance Electrochemiluminescence

Thrombin PDGF-BB Thrombin PDGF-BB PDGF-BB CEA Thrombin Thrombin

30 pM 6.8 pM 10 pM 6.3 pM 1.6 fmol 2 pM 0.78 aM 1.2 aM

[134] [135] [136] [137] [138] [139] [140] [141]

which provides building blocks via amplifying short sequences of oligonucleotides, and achieves an enzyme-free alternative for selective and specific extension at room temperature. The single-stranded DNA that can be programmed to self-assemble into complex structures triggers a chain of alternating hairpin molecules’ hybridization reaction in which H1 and H2 hairpins sequentially open to assemble into a long, nicked,

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double-stranded amplification polymer driven by the free energy of base pair formation [142]. HCR can also play an important role in biosensing applications acting as an amplifying signal transducer. These distinct mechanisms have made HCR a favorite strategy for detection of diverse classes of targets, including nucleic acids [145,146], proteins [147,148], and small molecules [149], by designing a variety of HCR initiator molecular probes, such as nucleic acid probes [150], aptamers [151], antibodies [152], functionalized nanoparticles [153], and DNAzymes [154]. Performing HCR in immunoassay has become an important tool for the detection of proteins because HCR can achieve molecular signal amplification in a multiplexed, isothermal, enzyme-free way. Combining electrochemical devices with immuno-HCR offers an attractive route for electrochemical transduction of protein recognition events. For example, based on an enzyme-free dual amplification strategy for protein assay, which coupled HCR with toehold-mediated click chemical ligation induced DNA strand displacement reaction, Yang et al. [155] obtained a wide linear dynamic range from 10 fM to 10 nM and a very low detection limit of 30 fM due to the dual amplification. This assay was relatively simple and inexpensive for proceeding in isothermal conditions with enzyme-free, providing a better performance in protein detection. In order to further achieve ultrasensitive detection, the coupling of nanoscale materials with HCR developed a unique strategy for sensitive measurements of protein. Ju’s group has developed a series of HCR-based signal amplification methods for biosensing [31,70,145,146]. For example [156], they designed a DNA nanopolylinker with high loading of signal molecules as a three-dimensional nanoprobe, which was prepared by rationally engineering dsDNA polymerization on initiator DNA modified AuNP via an HCR with two kinds of fluorescein isothiocyanate (FITC)-labeled DNA hairpins (Fig. 2.32). The sequences of used oligonucleotides included: Initiator DNA: 5′-AGTCTAGGATTCGGCGTGGGTTAA T15-SH-3′ Spacer DNA: 5′-SH-T15-3′ FITC-H1: 5′-FITC-TTAACC CACGCCGAATCCTAGACT CAAAGT AGTCTAGGATT CGGCGTG-FITC-3′ FITC-H2: 5′-FITC-AGTCTAGGATTCGGCGTG GGTTAA CACGCCGAATCCTAGAC T ACTTTG-FITC-3′ Biotin-DNA1: 5′-TTAACCCACGCCGAATCCTAGACT T5-biotin-3′ Biotin-DNA2: 5′-biotin-T5 CACGCCGAATCCTAGACTACTT TG-3′

The biotinylated core-shell nanoprobe was immobilized on the immunosensor surface, and the FITC molecules then bound enzyme-labeled anti-FITC antibody to catalyze a silver deposition process via a classic sandwich type and a biotin-streptavidin affinity system. Therefore, following this cascade signal amplification, this method showed satisfactory results for linear detection range over five orders of magnitude for CEA with a detection limit of 1.2 fg mL−1 (Fig. 2.33). MWCNTs were also used as carrier conjugated with DNA for HCR to develop an ultrasensitive and selective electrochemical immunosensor [157]. Zhang et al. [158] fabricated a novel signal amplification electrochemical aptasensor based on HCR and a hemin/G-quadruplex DNAzyme electrocatalytic system (Fig.  2.34). AuNPs electrode surface were modified with the complementary thrombin binding aptamers, then thrombin aptamers containing the sequence of initiator that

n

n

Signal amplification for immunosensing63

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AuNP

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Biotin-DNA1

Biotin-DNA2

(A)

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3-IP + Ag+

(5)

(4)

Potential CS WE

(B)

BSA

Ab1

Target

Streptavidin

CS WE Biotin-Ab2

Anti-FITC-ALP

Fig. 2.32  (A) Synthesis of the DNA nanopolylinker. (B) Schematic diagram of the amplified electrochemical immunoassay using the DNA nanopolylinker probe: (1) structure of immunosensor, (2) sandwich-type immunoreaction of target protein and biotin-labeled antibody, (3) binding of the nanopolylinker probe to immunocomplex through streptavidinbiotin reaction and ALP-labeled anti-FITC to the probe, (4) enzymatic induced silver deposition, and (5) linear sweep voltammetry for stripping analysis of the deposited AgNPs. Reprinted with permission from L. Tong, J. Wu, J. Li, H. Ju, F. Yan, Hybridization chain reaction engineered DNA nanopolylinker for amplified electrochemical sensing of biomarkers, Analyst 138 (2013) 4870–4876.

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40

30

–10

Current (µA)

Current (µA)

0

a –20 i

20

–30 10 –40

(A)

–0.1

0.0 0.1 0.2 Potential (V)

0.3

(B)

10–3 10–1 101 10–5 Concentration (ng mL–1)

Fig. 2.33  (A) LSV responses and (B) calibration curve of the proposed method for CEA detection. Curves a–i are for 0, 10−6, 10−5, 10−4, 10−3, 10−2, 0.1, 1, 10 ng mL−1 CEA (n = 3 for error bars). Reprinted with permission from L. Tong, J. Wu, J. Li, H. Ju, F. Yan, Hybridization chain reaction engineered DNA nanopolylinker for amplified electrochemical sensing of biomarkers, Analyst 138 (2013) 4870–4876.

CTBA

HT

Dp Au

Thrombin aptamer

GCE NAD+

NADH

H2O2

O2

MB(red)

MB(ox) e



+ I + II

Hemin

MB

Thrombin

Fig. 2.34  Schematic illustration of the electrochemical aptasensor based on HCR with hemin/G-quadruplex DNAzyme-amplification. Reprinted with permission from J. Zhang, Y. Chai, R. Yuan, Y. Yuan, L. Bai, S. Xie, L. Jiang, A novel electrochemical aptasensor for thrombin detection based on the hybridization chain reaction with hemin/G-quadruplex DNAzyme-signal amplification, Analyst 138 (2013) 4558–4564.

triggered HCR, forming hemin/G-quadruplex structure by intercalating hemin into two induced single-stranded DNA (ssDNA). This work exhibited good specificity, acceptable reproducibility and sensitivity, with a linear calibration range of 0.01– 50 nM and a detection limit of 2 pM. HCR has been coupled with the optical-based immunosensors, including fluorescent [159,160], electrochemiluminescence [161], and bioluminescence [147] for signal

Signal amplification for immunosensing65

PDGF-BB

H1 5 3

5 3

H2 5

HP

SG 5 3

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+ 5

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H1

+

5

3

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H2

PDGF-BB 5 5 3 5

H1

3

ON

5 5

H2 5

GO

5

SG

OFF

Fig. 2.35  Schematic illustration of the fluorescence assay for PDGF-BB based on the target-triggered HCR amplification. Reprinted with permission from X. Wang, A. Jiang, T. Hou, H. Li, F. Li, Enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification, Biosens. Bioelectron. 70 (2015) 324–329.

amplification by loading large amounts of reporting molecules. For example, Song et al. [159] introduced streptavidin quantum dots (SA-QD) as reporting molecules for the detection of PDGF-BB via HCR based on an aptameric system. In addition, Wang et al. [162] designed a label-free fluorescence aptasensing strategy based on targettriggered HCR amplification and used the superior fluorescence quenching ability of GO to the fluorescence indicator, SYBR Green I (SG), absorbed to ssDNA (Fig. 2.35). In the presence of PDGF-BB, a helper DNA probe consisting of an aptamer sequence reconfigured to the active Y structure that binds PDGF-BB and subsequently triggered HCR. The SG then bound with the double strands of the HCR product and gave a strong fluorescence signal, because HCR product cannot be adsorbed onto a graphene oxide surface. So this strategy provided a high sensitive fluorescence detection for PDGF-BB with a LOD down to 1.25 pM.

2.7 Perspective The analysis of specific proteins at ultra-low levels usually has to be carried out in the presence of a huge excess of other high abundant molecules since the abundance of a variety of proteins present in cells can vary by as much as 106-fold. Though tremendous advances have been achieved in the exploring of signal amplification strategies for the development of ultrasensitive protein biosensors, ultrasensitive detection of specific proteins at ultra-low levels in real sample is still particularly challenging. Bioconjugated nanomaterials have demonstrated broad potential for the amplified transduction of protein recognition events. The variability of the preparation of nanomaterials and their biofunctionalization often affect the reproducibility and quantification of these sensors, especially for the real samples. Thus extensive effort is still urgently needed to improve the practicability of the nanomaterial-based signal amplification strategies.

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DNA polymerase-based signal amplification techniques have significantly improved the sensitivity of immunoassay by using DNA antibody labeling technologies or aptamer chemistries. A drawback of polymerase-based methods is the increased reagent cost and a strict requirement to enzymatic oligonucleotide replication. Enzymefree isothermal amplification strategies such as HCR, catalyzed hairpin assembly, and DNA-fueled molecular machine may be promising candidates for developing simple and practical signal amplification strategies for protein biosensing in medical diagnosis and treatment. Additionally, most of the present signal amplification methods for protein analysis are compatible with the detection of an individual or several proteins. The whole genome sequencing could easily be accomplished with the development of high throughput sequencing technologies, while there is no comparable high throughput technique to the demands of proteomics. Thus the exploration of new signal amplification techniques with high throughput and high sensitivity is still in urgent demand for the validation and routine application of protein biomarkers.

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Electrochemical immunosensing

3

3.1 Introduction Among various immunoassay techniques, electrochemical immunosensors have gained considerable interest as bioanalytical devices and have played an important role in the detection of protein biomarkers [1]. They are attractive tools because they are robust, economical to mass produce, and can achieve high sensitivity with small sample consumption. Unlike spectroscopic-based techniques, electrochemical immunoassay methods are not affected by sample turbidity, color, quenching, or interference from absorbing and fluorescing compounds commonly found in biological samples. Furthermore, the required instrumentation is relatively simple and can be miniaturized easily to circuit board levels with very low power requirements, facilitating the development of disposable devices and methodologies for point-of-care diagnostic applications [2,3]. Electrochemical immunosensors combine antibody-antigen interaction with different electrochemical measurement technologies, including amperometric, potentiometric, impedance, capacitive, and conductometric measurements [4]. For potentiometric immunosensors, ion-selective electrodes (ISEs) are often used for monitoring the surface charge or potential change upon immunoreaction on the interface of the detection device. Ding et  al. [5] reported the construction of a potentiometric immunosensor based on the immobilization of primary antibodies with a carboxylated poly(vinyl chloride) matrix at the outer layer of an ISE membrane. After sandwich immunoreaction followed by enzymatic reaction of the captured alkaline phosphatase (ALP) labels on the sensing surface, the open circuit potential of the ISE was measured for potentiometric determination of human IgG. Electrochemical impedance immunosensors, based on impedance measurements of the electrical equivalent circuit of the oscillator, can characterize the electrical properties of immunoassay systems nondestructively without the need for reagents and a separation step. Zhu et al. [6] fabricated a biosensor for detecting carcinoembryonic antigen (CEA) using Faradic impedance spectroscopy. CEA antibody was immobilized on an Au NPs-modified glassy carbon electrode (GCE) by physical absorption. The tests revealed significant changes in the electron-transfer resistance of the 3 - /4 Fe ( CN )6 probe as the concentration of CEA changed. Capacitive immunosensors are based on altering electrical conductivity at a constant voltage, caused by immunoreaction that specifically generates or consumes ions. Fernandez-Sanchez et al. [7] developed a disposable, noncompetitive capacitive immunosensor for protate-specific antigen (PSA) measurement by integrating a single-step, lateral-flow immunoassay and impedance detection of the specific affinity event with a pH-sensitive, polymer-based electrochemical transducer. This approach was particularly useful for designing a rapid, single-use immunosensor for sensitive detection of PSA. Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00003-7 Copyright © 2017 Elsevier Ltd. All rights reserved.

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The group also prepared a quick, quantitative, disposable, impedance immunosensor for f-PSA and t-PSA [8]. Although the measurements of the immunosensor were inferred from impedance, it used capacitance measurement ultimately. Because they can offer direct and label-free measurements, capacitive immunosensors are attractive for practical applications. Amperometric immunosensors are the most promising and most interesting immunosensors because of their high sensitivity and wide linear range associated with the reduction or oxidation of an electroactive species involved in the recognition process. They usually apply a constant potential and monitor the current generated by electrochemical reaction. Since most analytes (proteins) cannot intrinsically act as redox partners in an electrochemical reaction, electroactive indicators such as various enzyme labels are often used in these systems [9]. Through the enzymatically catalytic reaction, sensitive amperometric current can be produced for tracing the immunoreaction of the analyte at the immunosensing electrode. Based on the difference of immunoreaction format, different electrochemical immunoassay or immunosensing methods have been developed. This chapter focuses on the introduction of electrochemical immunosensors based on direct immunoreaction, competitive immunoreaction, sandwich immunoreaction, and direct electrochemistry of redox enzymes for the sensitive immunosensing of protein biomarkers.

3.2 Direct immunoreaction-based immunosensors Upon immunoreaction on the electrochemical immunosensor surface, antibody-­ antigen immunocomplex is formed, which results in changes in physical properties such as potential [10], capacitance [11], conductance [12], impedance [13], and mass [14]. Thus various label-free electrochemical immunosensing methods can be simply constructed. Yuan et al. [15] prepared a 1,1′-bis-(2-mercapto)-4,4′-bipyridinium dibromide, a kind of sulfhydryl viologen functionalized Au NP for electrode modification. After antibody modification, an electrochemical immunosensor was constructed for direct immunoassay of the protein biomarker of alpha-fetoprotein (AFP). Based on the electron transfer inhibition of the immunocomplex to hexacyanoferrate ([Fe(CN)6]4−/[Fe(CN)6]3−) as electrochemical probe, a label-free electrochemical immunosensing method was successfully developed. Elshafey et al. [16] also reported a label-free electrochemical immunosensor for accurate quantification of the level of p53 antibodies in serum. As shown in Fig. 3.1, the immunosensor was prepared based on self-assembly of Au NPs on an electrochemically reduced graphene oxide (GO)-modified screen-printed carbon electrode (SPCE). This ­hybrid interface provided a large surface area for the effective immobilization of p53 antigens with excellent bioactivity and stability, which led to high sensitivity of the immunosensor. Electrochemical impedance spectroscopy (EIS) represents a powerful method for probing the interfacial reaction mechanisms of modified electrodes, providing a rapid approach for monitoring the dynamics of biomolecule interaction [17]. Thus,  EIS

Electrochemical immunosensing79

NH2

O

NH2

OH

OH

O

O

O O OH O

O

OH

O

NH

O

O

NH

O

OH

(2) NH2

Electrochemical reduction

(1) EDC/NHS

S

S

S

S

O NH

SH

O

OH O

SH

SH

OH

O

OH

NH2

p53

O NH

O

O

O NH (1)

O

O NH

O SWV

(2)

AntiP53

(3)

OH O

SPCE

O

O OH

O

O OH O

SH

OH

O OH

O

OH

Cysteamine

GO

AuNPs

p53 protein

BSA

Anti-p53 Ab

NH2

Fig. 3.1  Schematic illustration for fabrication of the p53-Ab immunosensor. Reprinted with permission from R. Elshafey, M. Siaj, A.C. Tavares, Au nanoparticle decorated graphene nanosheets for electrochemical immunosensing of p53 antibodies for cancer prognosis, Analyst 141 (2016) 2733–2740.

is often used for investigating chemical transformations and processes associated with conductivity changes in an electrochemical circuit. The blocking effect caused by the formation of the antibody-antigen immunocomplex results in an increased charge-transfer resistance of the electrode for a redox probe [18]. Wang et  al. [19] employed staphylococcal protein A assembled on an Au NP-modified electrode for immobilizing antibody to fabricate an immunosensor. Based on the well-defined oriented antibody surface for protein analyte recognition and its hindrance to the redox probe of hexacyanoferrate, an electrochemical impedance immunosensing method was developed for CEA measurement. On an Au NP/Protein G modified electrode, well-oriented antibodies were also immobilized to obtain an immunosensor [20]. This method was successfully used for electrochemical impedimetric immunosensing of a cancer biomarker of epidermal growth factor receptor. A simple strategy to develop amperometric immunoassay comes from the early enzyme-linked immunosorbent assay (ELISA) method that is based on the colorimetric reaction catalyzed by horseradish peroxidase (HRP) and measures the signal with spectrophotometry. Ju et al. [21] proposed an amperometric method for the determination of a-1-fetoprotein (AFP) in human serum by using a HRP label in an ELISA. The method was based on the electrochemical determination of enzymatic reaction product with differential pulse voltammetry at a gold disk electrode. The assay consisted of two successive steps. The first step is a conventional HRP-mediated ELISA for the formation of electroactive 2,2′-diaminoazobenzene by means of the o-phenylenediamine-H2O2-HRP system. At the second step,

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2,2′-diaminoazobenzene exhibited a sensitive voltammetric response at −0.19 V in pH 2.0 phosphate-buffered saline (PBS). The peak current is proportional to the concentration of AFP in the range of 0.5 ± 400 ng mL−1 (R = 0.9993) under optimum conditions. The sensitivity of this method was higher than that of the traditional spectrophotometric ELISA procedure. Besides, some electroactive agents such as thionine [22–24], methylene blue [25], Azure I [26], toluidine blue [27], ferrocene [28], and Prussian blue (PB) [29] can be immobilized on the electrode surface for indicating the sensitive amperometric current signal change as the results of the direct immunoreaction. For example, Yuan et al. [22] prepared an Au NP-thionine doped carbon paste electrode for immunosensor preparation. After assembly of antibodies on the electrode surface, the obtained immunosensor was used for direct immunoreaction with the CA 125 antigen. Based on the inhibition of the amperometric current of the thionine indicator, a new electrochemical immunoassay method was developed successfully. Qiu et al. [28] covalently bounded ferrocene to the chitosan long chain and used this electroactive biopolymer to construct a label-free amperometric immunosensor. As shown in Fig. 3.2, the chitosan-ferrocene (CS-Fc) composite modified on the surface of a GCE provided a biocompatible surface to assemble Au NPs for antibody immobilization, leading to obtaining an electrochemical immunosensor. The covalent grafting of ferrocene also avoided its leakage, thus ensuring its excellent stability. Based on direct immunoreaction at this immunosensor, it showed excellent analytical performance for sensitive detection of hepatitis B surface antigen (HBsAg). It should be noted that some electroactive agents can also serve as useful electron mediators of redox enzymes to promote the electrode transfer rate between the enzyme active site and electrode interface [30–32]. Based on the mediator-participated enzyme reaction, more sensitive electrochemical signals can be provided for electrochemical immunoassay. For example, Ju et al. [33] co-immobilized thionine and HRP-labeled CEA onto a GCE surface through covalently binding of glutaraldehyde to obtain an electrochemical immunosensor (Fig. 3.3). The immobilized thionine displayed a surface-controlled electrochemical process with a fast electron transfer rate, and could be used as an electron transfer mediator for enzymatic activity detection of the HRP-labeled antibody to CEA. Based on the inhibition of the electron transfer

BSA

GCE

Antibody

GCE

Au NPs

GCE

GCE

GCE

CS-Fc

Fig. 3.2  The stepwise fabrication process of the CS-Fc based electrochemical immunosensor. Reprinted with permission from J.D. Qiu, R.P. Liang, R. Wang, L.X. Fan, Y.W. Chen, X.H. Xia, A label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix, Biosens. Bioelectron. 25 (2009) 852–857.

Electrochemical immunosensing81

OH COOH

Electrochemical

O

H

H

O

OH

pretreatment

OH COOH

HO

HO O

CH

CHO

O

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CH

COOH

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COOH

COOH O

CH

CHO

O

H2N S H2N

N H

O NH2

CH

S CH2 HN

N H

NH2

COOH

Fig. 3.3  The procedure for preparation of the co-immobilization of thionine and HRP-labeled antibody based immunosensor. Reprinted with permission from Z. Dai, F. Yan, H. Yu, X.Y. Hu, H.X. Ju, Novel amperometric immunosensor for rapid separation−free immunoassay of carcinoembryonic antigen, J. Immunol. Methods 287 (2004) 13−20.

between the activity center of HRP and immobilized thionine after direct incubation with CEA solution, the immunosensor could be simply used to detect CEA from 0.5 to 167 ng mL−1. The immunosensor showed good accuracy and acceptable precision, reproducibility, and storage stability. Ju et  al. [34] also prepared a disposable amperometric immunosensor by entrapping HRP-labeled AFP antibody in chitosan membrane to modify an SPCE. The HRP-AFP antibody incorporated chitosan membrane showed a uniform porous structure with little change of pore size compared with chitosan membrane and aggregates of trapped biomolecules with a regular distribution (Fig.  3.4). The electrochemical experiments demonstrated that the entrapped HRP remained excellent electrocatalytic performance toward H2O2 reduction under the electron mediating of thionine added in substrate solution. Based on the similar inhibition of the enzymatic activity by the formation of immunocomplex through direct immunoreaction after incubation with the sample at 30°C for 35 min, a low detection limit of 0.74 ng mL−1 was achieved for AFP measurement. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6% and 4.2% at 10 and 100 ng mL−1, respectively.

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Fig. 3.4  Scanning electron microscopic (SEM) images of (A) bare SPCE, (B) chitosan film, and (C) HRP-AFP antibody/chitosan modified SPCE. Reprinted with permission from H. Yu, F. Yan, Z. Dai, H.X. Ju, A disposable amperometric immunosensor for α-1-fetoprotein based on enzyme-labeled antibody/chitosan-membranemodified screen-printed carbon electrode, Anal. Biochem. 2004, 331 (2004) 98−105.

3.3 Competitive immunoreaction-based immunosensors The competitive immunoassay format can also be used for the electrochemical immunosensing of protein biomarkers. After one-step competitive immunoreaction at the immunosensor, the electrochemical labels could be quantitatively captured on the electrode surface to produce corresponding signal transduction. Costa-Garcı́́a [35] reported a competitive voltammetric immunosensing method for the determination of mouse IgG. The carbon paste electrode provided a solid support for physical adsorption of goat anti-mouse IgG antibody to obtain the immunosensor, which was then used for competitive immunoassay with IgG and ALP-labeled IgG. As ALP can catalyze the hydrolysis of its 3-indoxyl phosphate substrate to produce sensitive electrochemical oxidation signal at the electrode [36], this method provided an effective approach for the protein analyte measurement. Ju et al. [37] reported an electrochemical immunosensor for CA 19-9 based on the immobilization of CA 19-9 with a titania sol-gel. The titania sol-gel film was fabricated by a vapor deposition method [38], which provided a homogeneous porous matrix with excellent biocompatibility for immobilizing the antigen biomolecules. After incubation with the mixed solution of CA 19-9 sample and its HRP-labeled antibody, the HRP labels were quantitatively captured on the immunosensor to catalyze the reduction of its H2O2 substrate with the participation of the electron mediator of catechol (Fig. 3.5). Under optimal conditions, the current decrease of the immunosensor showed a good linearity to the CA 19-9 concentration in the range of 3–20 U mL−1 for quantitative measurement (Fig. 3.6). Ju et al. [39] also prepared an electrochemical immunosensor by immobilizing CA 125 with an Au NPs-doped cellulose acetate membrane (Fig. 3.7). A competitive immunoassay format was employed to detect CA 125 antigen with HRP-labeled CA 125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates.

Electrochemical immunosensing83

and in 0.1 M PBS pH 6.6 30ºC 120 min

CA19-9

Immunosensor for CA19-9

HRP labeled CA19-9 antibody

H2O2 Catechol(red)

Catechol(ox)

Immunoassay of CA19-9

Fig. 3.5  Principle of the CA 19-9 immunosensor based on immobilized antigen, competitive immunoreaction, and electrochemical detection. Reprinted with permission from D. Du, F. Yan, S.L. Liu, H.X. Ju, Immunological assay for carbohydrate antigen 19-9 using an electrochemical immunosensor and antigen immobilization intitania sol-gel matrix, J. Immunol. Methods 283 (2003) 67−75. 120

1 2

Current (µA)

100

3 4

80 5 6 7 8

60 40 0.8

0.6

0.4

0.2

0.0

−0.2

E (V)

Fig. 3.6  Differential pulse voltammograms of the immunosensor in 0.1 M PBS pH 6.6 containing 0.5 mM catechol and 0.5 mM H2O2 after incubation in 1.0 mL pH 6.6 incubation solutions containing 50-μL HRP-labeled CA 19-9 antibody and (1) 0, (2) 3.0, (3) 6.0, (4) 10.0, (5) 16.0, (6) 20.0, (7) 25.0, and (8) 30.0 U mL−1 CA 19-9 at 30°C for 120 min. Reprinted with permission from D. Du, F. Yan, S.L. Liu, H.X. Ju, Immunological assay for carbohydrate antigen 19-9 using an electrochemical immunosensor and antigen immobilization intitania sol-gel matrix, J. Immunol. Methods 283 (2003) 67−75.

After the immunosensor was incubated with a mixture of HRP-labeled CA 125 antibody and CA 125 sample at 35°C for 50 min, the amperometric response decreased with an increasing CA 125 concentration in the sample solution (Fig. 3.8). The decreased percentage of the electrocatalytic current was proportional to CA 125 concentration ranging from 0 to 30 U mL−1 with a detection limit of 1.73 U mL−1 (S/N = 3). Similarly, they also reported the preparation of two electrochemical immunosensors for the protein biomarker of hCG based on the immobilization of hCG antigen on

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Immunosensing for Detection of Protein Biomarkers

Au-CA125

+

CA membrane

+

Incubation at 35°C for 50 min

In incubation solution

In 0.1 M pH 5.5 PBS OPD(OX) CA125

HRP labeled CA125 antibody H 2 O2 OPD(RED)

Colloidal gold nanoparticles

Fig. 3.7  Principle of CA 125 immunosensor based on immobilized antigen, competitive immunoreaction, and electrochemical detection. Reprinted with permission from L.N. Wu, J. Chen, D. Du, H.X. Ju, Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane, Electrochim. Acta 51 (2006) 1208−1214.

a chitosan/Au NPs composite [40] and an Au NP-doped three-dimensional silica solgel [41] modified electrodes, respectively. Through competitive immunoreaction, the HRP-labeled antibodies were captured onto the immunosensor surface, and showed good enzymatic activity for the oxidation of o-phenylenediamine by H2O2, thus providing sensitive amperometric current response through enzymatic reaction (Fig. 3.9). With a competitive mechanism, an amperometric method for immunoassay of hCG up to 30 mIU mL−1 with a relatively low detection limit of 0.26 mIU mL−1 at 3σ was

Electrochemical immunosensing85 1

Current (µA)

25

2 3

20

4

15

5 6

10 5

7,8 9 –0.4

–0.6

–0.8

E (mV)

Fig. 3.8  Differential pulse voltammograms of the immunosensors in 0.1 M pH 5.5 PBS containing 1.5 mM OPD and 1.0 mM H2O2 after incubated in 50 μL solutions containing HRP labeled CA 125 antibody and (1) 0, (2) 5.0, (3) 10.0, (4) 15.0, (5) 20.0, (6) 25.0, (7) 30.0, (8) 35.0, and (9) 40.0 U mL−1 CA 125 at 35oC for 50 min. Reprinted with permission from L.N. Wu, J. Chen, D. Du, H.X. Ju, Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane, Electrochim. Acta 51 (2006) 1208−1214.

Cross-linked chitosan film

Colloidal gold nanoparticals

hCG

NH2 2

HRP conjugated hCG antibody

hCG

NH2

+ 2H2O2

NH2 + 4 H2O

N N

2e-

2H+

H2N NH2 NH NH

Glassy carbon electrode

H2N

Fig. 3.9  Schematic diagram of the hCG immunosensor based on electrochemical detection using o-phenylenediamine as an electron transfer mediator. Reprinted with permission from J. Chen, F. Yan, D. Du, J. Wu, H.X. Ju, Electrochemical immunoassay of human chorionic gonadotrophin based on its immobilization in gold nanoparticles−chitosan membrane, Electroanalysis 18 (2006) 670−676.

developed [40]. The Au NPs introduced onto the immunosensor resulted in promotion of the enzyme-catalytic activity, leading to the sensitive measurement of the protein biomarker with satisfactory results.

86

Immunosensing for Detection of Protein Biomarkers CEA/Au colloid/chitosan membrane

+ Incubation Substrates

+

Washing

Sample CEA

Au colloid

Detection

HRP-labeled anti-CEA

Fig. 3.10  Schematic diagram of the flow immunoassay procedure at a CEA/Au NPs/chitosan nanocomposite based immunosensor. Reprinted with permission from J. Wu, J.H. Tang, Z. Dai, F. Yan, H.X. Ju, N.E. Murr, A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen, Biosens. Bioelectron. 22 (2006) 102−108.

c

d a

e

Computer Potentiostat

Carrier

f

Substrate

P

V

EFC

Waste

P

b

(A)

(B)

Fig. 3.11  Schematic diagrams of (A) three-electrode SPCE system and (B) the flow system. (a) Nylon sheet, (b) silver ink, (c) graphite auxiliary electrode, (d) graphite working electrode, (e) Ag/AgCl reference electrode, (f) insulating dielectric. P, peristaltic pump; V, eight-port rotary injection valve; EFC, electrochemical flow-through cell. Reprinted with permission from J. Wu, J.H. Tang, Z. Dai, F. Yan, H.X. Ju, N.E. Murr, A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen, Biosens. Bioelectron. 22 (2006) 102−108.

To improve the operation of electrochemical immunosensors, Ju et al. [42] designed a disposable immunosensor and used it for competitive immunoassay by combination with a flow injection system. The immunosensor was prepared based on the modification of an SPCE by a CEA/Au NPs/chitosan nanocomposite (Fig.  3.10) [43]. As shown in Fig. 3.11, the immunosensor was inserted in a flow system with an injection of sample and HRP-labeled CEA antibody. The CEA immobilized on the immunosensor trapped the labeled antibody to produce detectable current signal upon injection of substrates. The current response decreased proportionally to the CEA concentration in the range of 0.50−25 ng mL−1 with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng mL−1 (S/N = 3) (Fig. 3.12). Since the system was capable of continuously carrying out all steps, including incubation, washing, enzymatic reaction

160 120

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Electrochemical immunosensing87

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800

Fig. 3.12  Differential pulse voltammograms of immunosensor in stop flow system for (1) 5, (2) 10, (3) 15, (4) 20, (5) 30, and (6) 40 ng mL−1 CEA concentrations (A) and FIA diagram at −0.4 V at different CEA concentrations (B) from competitive immunoassay of CEA under optimal conditions. Inset: linear relation between relative current response and CEA concentration. Reprinted with permission from J. Wu, J.H. Tang, Z. Dai, F. Yan, H.X. Ju, N.E. Murr, A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen, Biosens. Bioelectron. 22 (2006) 102−108.

and determination, this method had the advantages of miniaturization, portability, and programmable operation without the need of a skilled operator for potential commercial applications. Carbon nanofiber (CNF) is a novel carbon nanomaterial possessing much larger functional surface area and higher ratio of surface active groups to volume over carbon nanotubes (CNTs) [44]. Ju et al. [45] prepared a novel amperometric immunosensor based on the covalent linking of CA 125 antigen and thionine at a carboxylated CNF modified GCE (Fig. 3.13). After competitive immunoassay, the HRP labels captured onto the immunosensor surface catalyzed its substrate reaction with the electron mediating of thionine immobilized on the electrode surface. The electrode modification of CNF provided an excellent platform with large active sites and fast electron transfer rate for sensitive electrochemical immunosensing. The differential pulse voltammetric peak current decreased linearly with an increasing CA 125 concentration from 2 to 75 U mL−1 in the incubation solution (Fig. 3.14). The CA 125 immunosensor showed good precision, high sensitivity, acceptable stability, and reproducibility with a detection limit of 1.8 U mL−1. This strategy avoided the trouble of adding a mediator to the sample solution; thus it showed great potential in clinical assays. To achieve higher sensitivity, some nanoprobes have also been applied for competitive electrochemical immunosensing in recent years. Neuron-specific enolase (NSE) is

88

O COOH OH

CNF

C NH EDC+NHS

COOH OH

H2N

N H

C NH2

C NH

O C OH

NH

S

+

N H

NH2

Detection solution with H2O2

N H

NH2

2e

2e

OH O C OH

H2O2

CA125 antibody

S NH 2e

N H

NH2

CA125 antigen NH2

Fig. 3.13  Principle of CA 125 immunosensor based on co-immobilization of antigen and thionine, competitive immunoreaction, and electrochemical detection. Reprinted with permission from L.N. Wu, F. Yan, H.X. Ju, An amperometric immunosensor for separation-free immunoassay of CA125 based on its covalent immobilization coupled with thionine on carbon nanofiber, J. Immunol. Methods 322 (2007) 12−19.

Immunosensing for Detection of Protein Biomarkers

O

C NH Washing

NH

OH

O

OH

NH2

S

O S

H2N

+

OH

Electrochemical immunosensing89

60 Peak current (µA)

Current (µA)

60 40 20 0

0.3

(A)

0.0

40

20

0

–0.3 –0.6 –0.9

(B)

E (V)

20

40

60

80

CA125 concentration (U mL–1)

Fig. 3.14  (A) DPV of the immunosensor in 0.2 M pH 7.0 PBS containing 4.0 mM H2O2 after incubated with 0, 2.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75 U mL−1 CA 125 (from highest to lowest peak current) and (B) calibration curve for CA 125 immunoassay. Reprinted with permission from L.N. Wu, F. Yan, H.X. Ju, An amperometric immunosensor for separation-free immunoassay of CA125 based on its covalent immobilization coupled with thionine on carbon nanofiber, J. Immunol. Methods 322 (2007) 12−19.

a widely used biomarker that often expresses in serum of serious cancer patients [46]. Ju et al. [47] designed an electrochemical immunosensor for detection of NSE by immobilizing NSE covalently functionalized single-walled CNTs (SWCNTs) on a GCE. As shown in Fig. 3.15, the immunosensor was used for competitive immunorecognition of anti-NSE primary antibody and then Au NP labeled with ALP-conjugated secondary antibody. The CNT modification on the immunosensor surface not only provided abundant antigen domains for electrochemical immunoassay, but also greatly enhanced the electrochemical signal along with the multienzyme catalytic signal amplification of the gold nanoprobe. Thus this immunosensor was amenable to direct quantification of target protein with a wide range of concentration in complex clinical serum specimens.

EDC+NHS

GCE

Current (µA)

+

α-NP

E (V)

Detection

SWNT

Incubation step II NSE

BSA

Incubation step I AuNP

Anti-NSE

AP-anti-IgG

Fig. 3.15  Schematic representation of the designed electrochemical immunosensor for NSE detection. Reprinted with permission from T.X. Yu, W. Cheng, Q. Li, C.H. Luo, L. Yan, D.C. Zhang, Y.B. Yin, S.J. Ding, H.X. Ju, Electrochemical immunosensor for competitive detection of neuron specific enolase using functional carbon nanotubes and gold nanoprobe, Talanta 93 (2012) 433−438.

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Zhu et  al. [48] fabricated a nanocomposite of electrochemically reduced GO and gold-palladium bimetallic nanoparticles on a carbon electrode surface with a simple electrodeposition method. This platform was used for immobilizing the antibody of human interleukin-6 (IL-6), an important cancer biomarker cytokine associated with the development of multiple myeloma and leukemia [49], to obtain an electrochemical immunosensor. An Ag NP-functionalized polystyrene was used for labeling IL-6 antigen and used for competitive immunoassay. Based on the electrochemical stripping analysis of the Ag NP labels, this method immunosensor was used to detect IL-6 in serum samples with satisfactory results. Similarly, Akter et al. [50] also prepared an electrochemical immunosensor for competitive immunosensing CEA based on the immobilization of a monoclonal CEA antibody on a protein attached-Au NPs modified electrode. An electroactive compound of 3,3′,5,5′-tetramethylbenzidine (TMB) [51] was bound to the carboxylated magnetic bead for labeling CEA and used for competitive immunoreaction. Based on the electrocatalytic oxidation of ascorbic acid by the multiple TMB labels quantitatively captured onto the immunosensor, this method showed excellent performance for nonenzymatic measurement of CEA.

3.4 Sandwich-type immunosensors Sandwich immunoassay is another format that is popularly used for the sensitive electrochemical immunosensing of protein biomarkers. Among sandwich immunoassays, the ELISA is the most common technique [52,53]. ELISA tests utilize a capture antibody for solid phase immobilization (on the microtiter wells) and a signal antibody conjugated with an enzyme label as a signal generator. The test sample is allowed to react sequentially with the two antibodies, resulting in the antigen molecules being sandwiched between the solid phase and enzyme-linked antibodies. Of the most commonly used ELISA enzymes, HRP is the most desired antibody label due to it being the smallest and most stable. Based on the spectroscopic detection of a colored reagent of the enzymatic reaction products, a calibration curve can be constructed for the protein measurement. In fact, this spectroscopic detection strategy has been conveniently replaced by electrochemical method by Ju et al. [21]. To avoid the addition of electron mediator in the substrate solution, Ju et al. [54] also fabricated a thionine monolayer modified gold electrode (Fig. 3.16) and used it as a working electrode to detect the electrocatalytic signal of the HRP labels that were quantitatively captured to the microwell through sandwich immunoreaction. A calibration curve with two linear ranges from 0.6 to 17 and 17 to 200 ng mL−1 and a detection limit of 0.2 ng mL−1 for CEA determination were obtained in pH 4.2 PBS containing 2.0 mmol L−1 H2O2 and 0.5 mol L−1 NaCl. The precision and reproducibility are acceptable with the intraassay CV of 4.9% and 5.9% at 10 and 100 ng mL−1 CEA concentrations, respectively, and the interassays CV of 7.8% at 100 ng mL−1 CEA. The response of thionine-modified electrode shows only 1.6% decrease after 100 replicate measurements, and the storage stability is acceptable in a pH 7.0 PBS at 4oC for 1 week. This strategy is much simpler and more convenient for sensitive CEA levels over traditional assays.

Electrochemical immunosensing91

S HS

CH2

CH2

NH2

S Au

Au

S S

O

S S Au

S S

CH2 CH2 CH2 CH2

NH

C

CH2 CH2

NH2

CH2

NH2

CH2 CH2

O

Cl C

C

Cl

O

NH2

Cl

H N

O NH2 NH

H2N

O C

S

NH2

Cl C

CH2 CH2

CH2

NH2

CH2

C

CH2 CH2

CH2

O NH2 H N O

S S Au

S S

CH2 CH2 CH2 CH2

NH

C

CH2 CH2

O NH2 NH

CH2 CH2

C

N H

S H N

NH2

N H

S

NH2

O C

C O

NH2

Fig. 3.16  The procedure for preparation of thionine-modified gold electrode. Reprinted with permission from Z. Dai, J. Chen, F. Yan, H.X. Ju, Electrochemical sensor for immunoassay of carcinoembryonic antigen based on thionine monolayer modified gold electrode, Cancer Detect. Prev. 29 (2005) 233−240.

Most sandwich electrochemical immunoassays were performed at modified e­lectrode-based electrochemical immunosensors. For example, Preechaworapun et al. [55] developed a boron-doped, diamond electrode-based electrochemical immunosensor for sandwich immunoassay of mouse IgG. In order to construct the base of the immunosensor, o-aminobenzoic acid was electropolymerized on the electrode surface by cyclic voltammetry for covalently immobilizing capture antibody. After sandwich immunoreaction, signal antibodies labeled with ALP could be quantitatively captured onto the immunosensor surface. Through the enzymatic reaction of ALP to catalyze hydrolysis of its substrate of 2-phospho-l-ascorbic acid [56,57], an electroactive species of ascorbic acid was obtained, which was determined by amperometric detection. This method showed a low detection limit and a wide linear range for mouse IgG measurement. Based on this ALP signal tracing mechanism, the enzymatic reaction product of ascorbic acid can also employed to catalyzed deposition of Ag NPs, thus proposing a novel electrochemical signal transduction strategy for sandwich immunosensing of protein analyte by electrochemical silver stripping analysis [58]. The enzymatic reaction of ALP labeled on the signal (secondary) antibody has been used for the direct detection of protein or P-glycoprotein on cell surface by immobilizing

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the cells on gold nanoparticle modified electrode. Ju's group [59] proposed a strategy to detect P-glycoprotein on cell membrane and quantify the cell number using electrochemical immunoassay by effective surface immunoreactions and immobilization of cells on a highly hydrophilic interface, which was constructed by adsorption of colloidal gold nanoparticles on a methoxysilyl-terminated (Mos) butyrylchitosan modified GCE (Au-CS/GCE) (Fig. 3.17). Atomic force microscopy studies proved that the nanoparticles adsorbed on Mos-butyrylchitosan were efficient in preventing the cell

CH2OCOC3H7 O O OMe n Hydrolysis H7C3OCO N (CH2CH CO(CH2)3 Si OMe)y O H(2−y) CH3

OMe

CH2OCOC3H7 O O OH n H7C3OCO N (CH2CH CO(CH2)3 Si OH)y O H(2−y) CH3

Mos-butyrylchitosan

OH

R

R OH OH OH OH Cross-linking on electrode surface HO Si R-Si-OH + O Oxidated glassy OH Dehydration carbon electrode

R O

Si O

R O

Si OH O

Mos-butyrylchitosan

O R-O-P- O− + H2O O−

1-NP

R- OH + HPO42− e-

H+

R= O

Glassy carbon electrode

Mos-butyrylchitosan

Colloidal gold nanoparticals

P-glycoprotein on cell surface

K562/ADM cell

P-glycoprotein monoclonal antibody

Secondary AP conjugated antibody

Fig. 3.17  Preparation of Au-CS/GCE and mechanism for determination of P-glycoprotein on K562/ADM cell surface by electrochemical enzyme-linked immunoassay. Reprinted with permission from D. Du, H.X. Ju, X.J. Zhang, J. Chen, J. Cai, H.Y. Chen, Electrochemical immunoassay of membrane P-glycoprotein by immobilization of cells on gold nanoparticles modified on a methoxysilyl-terminated butyrylchitosan matrix, Biochemistry 44 (2005) 11539–11545.

Electrochemical immunosensing93

leakage and retaining the activity of immobilized living K562/ADM leukemic cells. The incubation with P-glycoprotein monoclonal antibody and then the secondary ALP conjugated antibody introduced ALP onto the K562/ADM cell immobilized on Au-CS/ GCE. The bound ALP led to an amperometric response of 1-naphthyl phosphate. Under optimal conditions the response was proportional to the logarithm of cell concentration in the range from 5.0 × 104 to 1.0 × 107 cells mL−1 with a detection limit of 1.0 × 104 cells mL−1 (Fig. 3.18). The results were comparable to flow cytometric analysis of P-glycoprotein expression. This proposed method was practical, convenient, and significant in the clinic and cytobiology. To enhance the sensitivity of the sandwich immunosensors, various nanomaterials are commonly used for electrode modification and immunosensor preparation [60–65]. Mitra et al. [64] used Au NPs for covalently attaching antibodies to develop an electrochemical immunosensor, and employed it for achieving the sensitive immunoassay of human serum albumin successfully. The electrochemical detection was done by using HRP as enzyme label and TMB as electroactive mediator. Through a layer-by-layer assembly method as shown in Fig. 3.19, Li et al. [65] fabricated an uniform and stable multiwalled CNT and chemically reduced graphene nanocomposite electrode interface and used it to construct an electrochemical immunosensor for sensitive sandwich immunoassay. HRP-conjugated antibody was used for probing the electrochemical signal with the participation of hydroquinone as the electron mediator. The nanomaterials' modification exhibited significant improvement on the electron transfer rate, mass a b c

−4 −6 −8

−10

d e f

8

g

ip (µA)

Current (µA)

−2

4

h

0 3

0.8

4 5 6 7 log c (cells mL–1)

0.6

0.4 E (V)

i

0.2

0.0

Fig. 3.18  DPV curves of 0.25 mM 1-NP in pH 7.4 PBS at ALP-P-glycoprotein-K562/ ADM-Au-CS/GCEs prepared with different cell concentrations of (a) 0, (b) 50,000, (c) 100,000, (d) 200,000, (e) 500,000, (f) 1,000,000, (g) 2,000,000, (h) 5,000,000, and (i) 10,000,000 cells mL−1. Inset: plot of DPV peak current versus logarithm of cell concentration. Reprinted with permission from D. Du, H.X. Ju, X.J. Zhang, J. Chen, J. Cai, H.Y. Chen, Electrochemical immunoassay of membrane P-glycoprotein by immobilization of cells on gold nanoparticles modified on a methoxysilyl-terminated butyrylchitosan matrix, Biochemistry 44 (2005) 11539–11545.

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1

3

2

Repeat step 1 to 4 Three times

4

PDDA

Graphene

Carbon nanotubes

Ab1

Ag

Ab2-HRP

Fig. 3.19  Schematic illustration of the fabrication of graphene-CNT electrode interface and the construction of the sandwich electrochemical immunosensor. Reprinted with permission from Y. Liu, Y. Liu, H.B. Feng, Y.M. Wu, L. Joshic, X.Q. Zeng, J.H. Li, Layer-by-layer assembly of chemical reduced graphene and carbon nanotubes for sensitive electrochemical immunoassay, Biosens. Bioelectron. 35 (2012) 63–68.

transfer, and specific surface area on the electrode interface, which greatly improved the analytical performance of the electrochemical immunosensors. Besides enzyme labels, the noble-metal nanoparticles such as Au NPs [66,67], Ag NPs [68], Pt NPs [69], and quantum dots [70,71] can also be used for sandwich signal tracing of electrochemical immunosensor through electrochemical stripping analysis or electrocatalysis. Among these sandwich electrochemical immunoassays, various electroactive labels are popularly used to produce sensitive amperometric signal corresponding to the sandwich immunorecognition events. In fact, the electron transfer resistance caused by the formation of antibody-antigen immunocomplex on the immunosensor surface can weaken this kind of signal enhancement in some degree, which may limit any improvement in analytical performance of these methods. In this regard, the electrochemical signal-inhibition format has also been employed as an alternative approach for sensitive sandwich immunosensing. Lai et al. [72] synthesized an Ag NPs-chitosan (Ag NPs-CS) composite and used it to construct an electrochemical immunosensor (Fig.  3.20). An antibody labeled with silica nanosphere was prepared for sandwich immunoassay at the immunosensor. As silica possesses low semiconducting conductivity, its capture onto the immunosensor surface through sandwich immunoreaction increased the electron transfer obstruction, thus leading to the current decrease of silver stripping analysis [73,74]. Based on this mechanism, a novel electrochemical immunosensing method was developed for protein analyte measurement. Additionally, they also prepared a graphene-thionine nanocomposite and used it to construct an immunosensing platform [75]. The good reducing property of thionine ensures the one-step reduction of GO. The planar aromatic structure of

Electrochemical immunosensing95

GCE S1

LSV in KCI

Ag NPs-CS

Au NP

S0

Silica

Anti-HlgG

HlgG

BSA

Fig. 3.20  Schematic representation of the preparation process of the Ag NPs-CS based immunosensor and sandwich electrochemical immunoassay strategy. Reprinted with permission from C.Y. Yin, G.S. Lai, L. Fu, H.L. Zhang, A.M. Yu, Ultrasensitive immunoassay based on amplified inhibition of the electrochemical stripping signal of silver nanocomposite by silica nanoprobe, Electroanalysis 26 (2014) 409–415.

thionine ensures its noncovalent π-π stacking interaction with graphene to obtain the nanocomposite simply [76,77]. By using the antibody-functionalized silica captured onto the immunosensor surface via sandwich immunoreaction to inhibit the current of the thionine indicator, a new electrochemical immunosensing method was also developed for sensitive measurement of protein biomarker. Moreover, to enhance the electrochemical signal response of each immunorecognition event for achieving ultrasensitive measurement of low-abundant protein biomarkers in early stage of diseases, the nanoprobes that can load high-content signal labels have been widely designed to replace the conventional single-label probes for electrochemical signal tracing of sandwich immunosensors recently [78–80]. So far various nanomaterials have been successfully used as versatile nanocarriers for preparing a large variety of enzyme [81–85] and nonenzyme [86–90] labels functionalized nanoprobes for electrochemical immunoassays. Due to the multiple-label signal amplification of the nanoprobes, the sensitivity of these methods has been greatly improved. Detailed applications of the nanoprobes for sandwich electrochemical immunosensing of protein biomarkers will be introduced based on references in Chapter 4. In recent years, some redox cycling has also been designed to combine with ALPand β-galactosidase (Gal)-based immunoassay for improving the sensitivity. Among these redox cycling strategies, a nonenzymatic signal amplification strategy such as electrochemical-chemical (EC) and electrochemical-chemical-chemical (ECC) cycling can generate high signal-to-background ratio with the expense of minimum incubation time and minimum concentration of enzyme labels, thus greatly improving the practicality of immunoassay. Similar to the typical electrocatalytic mechanism,

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the appropriate redox cycling can greatly enhance the concentration of signal species during electrochemical process. This process needs a reducing agent to convert the oxidized signal species into its reduced state. Although excellent immunosensor preparation can assure quick electron transfer and low background, the features of signal generating species and the reducing agent at electrode are important preconditions. Inspired by the dual enzymatic activity of tyrosinase (Tyr), oxygenation activity and oxidase activity, and its excellent performance for electrochemical biosensing of catechol and phenol, Ju's group [91] designed a Tyr-responsive nonenzymatic redox cycling for significantly amplifying the electrochemical signal produced from Tyr-labeled immunoassay. In the designed detection system, phenol and NADH were used as enzyme substrate and reducing agent, respectively. Both the low electroactivity of phenol and high oxidation overpotential of NADH reducing agent at immunosensor surface led to negligible background. The oxygenation activity of tyrosinase could convert lowly electroactive phenol to highly electroactive catechol to trigger a NADH-related nonenzymatic EC catalysis (Fig. 3.21), which greatly increased the voltammetric response and thus produced high signalto-noise ratio. Unlike the dual enzyme redox cycling of Gal-tyrosinase, the oxygenation activity of tyrosinase and following nonenzymatic redox cycling could be performed without the need of additional enzyme and catalyst support. By coupled with a conveniently prepared immunosensor for CEA, the proposed method showed excellent performance with a detectable range from 1.0 pg mL−1 to 0.1 μg mL−1 and a sub pg mL−1-level detection limit, as shown in Fig. 3.22. The acceptable accuracy and good reproducibility of the proposed immunoassay method indicated its superior suitability in clinical diagnosis.

Capture antibody

Antigen

Chitosan GCE

Current (mA)

NAD+

NADH

0

GCE

GCE

BSA

OH

Tyrosinase conjugated antibody

O2

-10 O

-20 -0.2 0 0.2 0.4 E vs Ag/AgCI (V)

O

e-

OH

OH

GCE

H2O2

GCE

Fig. 3.21  Schematic illustration of preparation of electrochemical immunosensor and immunoassay procedure with redox cycling. Reprinted with permission from Md. R. Akanda, H.X. Ju, A tyrosinase responsive-redox cycling for amplified electrochemical immunosensing of protein biomarker, Anal. Chem. 88 (2016) 9856–9861.

Electrochemical immunosensing97

Current (µA)

−5 −10 −15 −20 −25 −30

(A)

25 10 µg mL–1

20

1 µg mL–1 100 ng mL–1 10 ng mL–1 1 ng mL–1 100 pg mL–1 10 pg mL–1 1 pg mL–1 100 fg mL–1

Current (µA)

0

0.0

10 5

zero

−0.2

15

0.2

E vs Ag/AgCl (V)

0.4

Zero + 3SD Zero

0

10−13 10−12 10−11 10−10 10−9 10−8 10−7 10−6 10−5

0.6

(B)

Concentration of CEA (g mL–1)

Fig. 3.22  (A) Cyclic voltammograms of immunosensors in 10 mM PBS (pH 7.4) containing 5.0 mM phenol + 5.0 mM NADH after incubation with target CEA at marked concentrations and then Tyr-conjugated anti-CEA for 30 min at 4°C. (B) Calibration plot of the response at 0.4 V versus CEA concentration. Reprinted with permission from Md. R. Akanda, H.X. Ju, A tyrosinase responsive-redox cycling for amplified electrochemical immunosensing of protein biomarker, Anal. Chem. 88 (2016) 9856–9861.

3.5 Direct electrochemistry of enzyme for immunosensing Enzyme labels (e.g., HRP) are commonly used for the electrochemical signal tracing of immunosensors through their biocatalytic reaction. Although the enzyme conjugate amplifies the amperometric responses of the immunosensors, one mediator is always required to add in the detection solution or immobilized onto the immunosensor surface to accelerate the electron transfer on the electrode surface [92–95]. For HRP-label based amperometric immunosensors, one substrate such as hydroquinone [94] or o-­ aminophenol [95] is often added to the detection solution as a mediator to transfer the electron between HRP and H2O2. The addition of non-immunoreagents causes the immunoassay system to be more complex, increases the analytical time and expense, and thus limits the practical application of immunosensors, particularly in the clinical online diagnosis. To eliminate this problem, one effective approach is to use the direct electrochemistry of the enzyme labels to develop reagentless electrochemical immunosensors. To achieve the direct electrochemistry of the redox protein (enzyme), the important challenge is to expose the active sites of proteins for facilitating the electron transfer between their redox centers and electrode surface [96,97]. Based on the direct electrochemical signal of bound HRP with regard to the Fe(III) to Fe(II) conversion, Ju et al. [98] introduced a new mechanism for reagentless amperometric immunoassay of the tumor marker of CA 125. Similar to Fig. 3.5, the immunosensor was prepared by embedding antigen with the titania sol-gel film. After incubating the immunosensor in PBS containing HRP-labeled CA 125 antibody and analyte, the HRP-labeled CA 125 antibody was competitively conjugated with the immobilized CA 125, leading to the formation of an HRP-modified surface (Fig. 3.23). The active center of the bound HRP could exchange

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Immunosensing for Detection of Protein Biomarkers

and in 0.1 mol L–1 pH 7.0 PBS

Immunosensor for CA125

CA125 HRP labeled CA125 antibody

Immunosensor for CA125 assay

Fig. 3.23  Principle of reagentless amperometric immunosensor based on immobilized antigen, competitive immunological reaction and direct electrochemistry of HRP label. Reprinted with permission from Z. Dai, F. Yan, J. Chen, H.X. Ju, Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125, Anal. Chem. 75 (2003) 5429−5434.

electrons with the electrode directly with a rate constant of 3.04 ± 1.21 s−1. Based on this competitive recognition mechanism and the direct exchange of electrons between the immobilized HRP and electrode to produce detectable current signal, CA 125 could be determined in the linear range of 2–14 U mL−1 with a limit of detection (LOD) of 1.29 U mL−1 at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7% and 5.5% at 8 and 14 units mL−1 CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL−1. The storage stability was acceptable in a pH 7.0 PBS at 4°C for 15 days. The proposed method provided a new promising platform for clinical immunoassay. For example, when using this strategy for the measurement of the glycoprotein hormone of HCG, the rate constant for direct electron transfer of HRP labeled to anti-HCG-HCG-titania sol-gel membrane modified electrode was 1.35 ± 0.4 s−1, and this method showed a detection limit of 1.4 mIU mL−1, a good accuracy and acceptable precision and reproducibility with an intraassay CV of 4.7% at 5.0 mIU mL−1 and an interassay precision of 8.1% obtained at 10 mIU mL−1 without the addition of any electron mediator or H2O2 substrate in the detection solution [99]. Most immunosensors based on the direct electrochemistry of enzymes work with the format of direct immunoassay. In these methods, the enzyme tracers are often immobilized with proper matrix or techniques to achieve their direct electrochemistry. Ju et al. [100] proposed another strategy to construct a reagentless immunosensor by encapsulating HRP-labeled antibody-adsorbed Au NPs in the titania sol-gel matrix. Due to the biocompatible immobilization environment, the HRP-labeled antibody was uniformly distributed in the sol-gel matrix (Fig.  3.24), and the HRP tracer showed stable and well-defined redox peaks. Based on the electron transfer inhibition by the immunocomplex formed by direct immunoreaction, this reagentless immunosensor was successfully used for HCG measurement in the linear range of 0.5–30 mIU mL−1 with an LOD of 0.3 mIU mL−1 (Fig. 3.25). This group then used an organically modified silicate sol-gel to immobilize the HRP-labeled antibody to fabricate a reagentless immunosensor (Fig. 3.26) [101]. Use of the ormosil sol-gel provided a hydrophilic interface for retaining the activity of the bound HRP-anti-CEA. The immobilized HRP showed direct electron transfer at about −35 mV with a rate constant of 5.94 ± 0.4 s−1.

Electrochemical immunosensing99

Fig. 3.24  Scanning electron micrographs of sol-gel (A), nano-Au-SG (B), HRP-anti-hCG/ SG (C, D) and HRP-anti-hCG/nano-Au-SG (E, F) membranes at ×3.0 K (A–C, E) and ×10 K (D, F). Reprinted with permission from J. Chen, J.H. Tang, F. Yan, H.X. Ju, A gold nanoparticles/ sol-gel composite architecture for encapsulation of immunoconjugate for reagentless electrochemical immunoassay, Biomaterials 27 (2006) 2313−2321.

(b) (c)

8

(d)

6

(e) (f) (g) (h) (i)

4 2

(A)

−200

−400

80

40

Decrease percentage (%)

Current (µA)

10

Decrease percentage (%)

(a)

0

50

80

40 60 30

0

2

40

4

10

20

30

HCG concentration (mIU mL−1)

−600

20

0

40

60

HCG concentration (mIU mL−1)

(B)

E (mV) vs SCE

Fig. 3.25  DPV of hCG/HRP-anti-hCG/nano-Au-SG/GCE obtained by incubating immunosensor in mixture of 5 μL (a) 0, (b) 0.5, (c) 5.0, (d) 10.0, (e) 20.0, (f) 30.0, (g) 40.0, or (h) 50.0 mIU mL−1 hCG with 40 μL dilute solution and 40 μL hCG zero buffer at 30°C for 40 min (A) and plot of decrease percentage of peak current vs. hCG concentration in standard solution for preparation of incubation solution (B). Insets in B show linear calibration. Reprinted with permission from J. Chen, J.H. Tang, F. Yan, H.X. Ju, A gold nanoparticles/ sol-gel composite architecture for encapsulation of immunoconjugate for reagentless electrochemical immunoassay, Biomaterials 27 (2006) 2313−2321. OH

H2N O

NH2(CH2)3Si(OC2H5)3 +Si(CH3CH2O)4

(1)

H2N

Si HO

Si

NaOH

HO

Si

O

OH

Hydrolysis

H2N

OH

Si

Si

O

H2N O

NH2

Si

Si OH HO Si

Si(OR) + H2O « Si(OH) + ROH

OH HO

OH

NH2

Si(OR)+(HO)Si « Si-O-Si+ ROH Si(OH)+(HO)Si « Si-O-Si+ ROH

(2)

(3)

Fig. 3.26  Fabrication procedure of ormosil sol-gel film: (1) hydrolysis of two precursors under weak basic condition, (2) formation of ormosil sol-gel, and (3) preparation of bioactive surface. Reprinted with permission from F. Tan, F. Yan, H.X. Ju, A designer ormosil gel for preparation of sensitive immunosensor for carcinoembryonic antigen based on simple direct electron transfer, Electrochem. Commun. 8 (2006) 1835−1839.

Electrochemical immunosensing101

By a simple one-step immunoreaction between CEA in serum sample solution and the immobilized HRP-anti-CEA, the differential pulse voltammetric peak current of HRP decreased linearly with an increasing CEA concentration in two ranges of 0.5–3.0 and 3.0–120 ng mL−1 with an LOD of 0.4 ng mL−1 at 3σ (Fig. 3.27), which was sufficient to measure clinically relevant CEA levels (>3.0 ng mL−1). When this strategy was used for hCG measurement [102], the HRP tracer immobilized in the ormosil material showed a large rate constant of 15.8 ± 3.8 s−1 of direct electron transfer on the electrode. By a simple one-step immunoreaction between hCG in sample solution and the immobilized HRP-anti-hCG, the DPV peak current of HRP decreased linearly with an increasing hCG concentration from 0.5 to 50 mIU mL−1, and a relatively low LOD of 0.3 mIU mL−1 was obtained for clinical applications (Fig. 3.28). Similarly, Du et al. [103] entrapped the HRP-labeled antibody adsorbed Au NPs in a carbon paste electrode to develop an amperometric immunosensor. The Au NPs modified in carbon paste effectively preserved the activity of the immobilized biomolecules. Based on the block of the direct electron transfer of HRP, this immunosensor showed excellent performance for the measurement of CA 19-9 in serum samples. GOx is another important redox protein (enzyme) that is commonly used in the biosensing field. Based on the exposure of its FAD cofactor for facilitating the electron transfer, the direct electrochemistry of GOx has been extensively studied [104–107]. By combination with proper immobilization of GOx and antibody on the modified electrodes for direct immunoreaction, the direct electrochemical behavior of GOx (A)

8 4 0

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Fig. 3.27  DPV of HRP-anti-CEA/sol-gel/GE after incubation in 50 μL solutions containing 10 μL 0.5, 1.0, 2.0, 3.0, 12, 30, 45, 60, 90, and 120 ng mL−1 CEA (from highest to lowest peak current) at 30°C for 35 min. Inset: (A) plot of DPV peak current versus CEA concentration; (B) linear calibration for 0.5–3.0 ng mL−1 CEA. Reprinted with permission from F. Tan, F. Yan, H.X. Ju, A designer ormosil gel for preparation of sensitive immunosensor for carcinoembryonic antigen based on simple direct electron transfer, Electrochem. Commun. 8 (2006) 1835−1839.

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Fig. 3.28  DPV of hCG/HRP-anti-hCG/sol-gel/GCE in 0.1 M pH 7.0 PBS after incubated in 50 μL solutions containing 10 μL 0.5, 1.0, 2.5, 5.0, 20.0, 27.5, 35.0, 42.5, 50.0, 75.0, 100 and 150 mIU/mL hCG at 30oC for 40 min (from highest to lowest peak current). Insets: plot of DPV peak current versus hCG concentration. Reprinted with permission from F. Tan, F. Yan, H.X. Ju, Sensitive reagentless electrochemical immunosensor based on anormosil sol-gel membrane for human chorionic gonadotrophin, Biosens. Bioelectron. 22 (2007) 2945−2951.

can also be used for electrochemical immunosensing of protein biomarkers. Yuan et al. [108] reported a novel strategy for the construction of reagentless immunosensors based on the direct electrochemistry of GOx. As shown in Fig. 3.29, a GCE was first modified with a layer of a nanocomposite containing CNTs and core-shell ­organosilica@chitosan nanospheres (OrgSi@CS) [109], which was followed by the adsorption of Pt nanoclusters and GOx successively. After the repeated adsorption of

GCE

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(c) GOx

(b) Pt NCs

(d) Pt NCs

(e) Anti-CA15-3 FAD

(f) GOx

FADH2

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Fig. 3.29  Illustration of the preparation process of GOx-based immunosensor. Reprinted with permission from W.J. Li, R. Yuan, Y.Q. Chai, S.H. Chen, Reagentless amperometric cancer antigen 15-3 immunosensor based on enzyme-mediated direct electrochemistry, Biosens. Bioelectron. 25 (2010) 2548–2552.

FAD

FADH2

Electrochemical immunosensing103

Pt nanoclusters for immobilizing the capture antibody, GOx was further applied to ­blocking the possible remaining active sites, resulting in the final preparation of the electrochemical immunosensor. The GOx modified on the immunosensor exhibited good direct electron transfer with a rate constant of 4.89 s−1. The peak current of the direct electrochemistry of GOx decreased with the increase of the antigen concentration, which was successfully used for the direct immunosensing of CA 15-3 with satisfactory results. Similarly, they also combined a CNT nanocomposite and a magnetic core-shell gold nanosphere for immobilizing GOx and capture antibody and thus constructed another GOx-based electrochemical immunosensor [110]. The direct electron transfer of GOx was employed for the reagentless immunosensing CA 19-9 in two linear ranges of 0.01–1.11 U mL−1 and 11.11–476.11 U mL−1 with a relatively low detection limit of 0.004 U mL−1.

3.6 Perspective Electrochemical immunosensor is an excellent biosensing platform for accurate measurement of protein biomarkers that combines the unique advantages of electroanalytical methods, specific immunorecognition reaction, and biosensor devices. The properties of electrochemical immunosensors such as high sensitivity, small sample consumption, and low detection cost give it great potential for point-of-care testing (POCT). Similar to the handheld blood sensor, considerable researches in the field of electrochemical immunosensing will drive fast development in the design of useful POCT devices for convenient measurement of protein biomarkers. Meanwhile, new achievements developed in the areas of microelectronics or even nanoelectronics can also be used in biosensors. For example, by using the microfabrication techniques, some electrode arrays can be designed for multianalyte immunoassay. By combination with the microfluidic chips, the miniaturization and automatization of devices are expected to have a marked impact on immunosensor technology. Furthermore, to meet the need for accurate measurement of ultra-low levels of protein biomarkers, a crucial aspect in future immunosensors is to improve their analytical performance, especially the sensitivity of the methods. Then, more and more signal amplification technique, for example, molecular biological techniques and nanosignal amplification strategies, will be extensively adopted into the immunosensing field. In conclusion, the rapid development in immunosensors will make them play more and more important roles for accurate detection of protein biomarkers in the clinical diagnosis field.

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Functional nanoprobes for immunosensing

4

As tumor biomarkers in body fluid or tissues usually show low expression levels in the early stages of disease, the development of immunosensing methods with ultra-high sensitivity is urgently required in this field. In recent years, the unique optical, electronic, and mechanical properties of nanomaterials have offered excellent prospects in designing various ultrasensitive signal tracing strategies for immunosensors. Compared with the conventional approaches (e.g., the use of special electrical, optical, catalytic, and magnetic properties of nanomaterials for sensitive signal transduction of immunosensors), the use of nanoprobes that are artificially designed from various nanomaterials, as versatile signal labels, have shown great success in improving the sensitivity of immunosensors drastically. This chapter first introduces the signal tracing and amplification mechanism of nanoprobes as signal labels of immunosensors. Then the use of different kinds of nanoprobes including enzyme-functionalized nanoprobes, noble-metal nanoparticlefunctionalized nanoprobes, and quantum dot (QD) functionalized nanoprobes for ultrasensitive immunosensing of protein biomarkers is introduced based on detailed examples.

4.1 Nanoprobe as signal labels As the direct antigen-antibody immunoreaction can only cause limited change of detectable signal for quantitative immunoassay, the labeling immunosensing in two modes such as sandwich-type and competitive-type immunoassays has been commonly used for detection of protein biomarkers. In a competitive format, unlabeled analyte (usually the antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay. Typically, the detectable signal decreases with the increase of analyte concentration, which requests a high background signal toward zero analyte [1]. In contrast, sandwich-type immunoassay gives the higher level of sensitivity and specificity because of the use of a couple of match antibodies [2]. The measurement of the labeled reagent (usually the antibody) is directly proportional to the amount of antigen present in the sample, with the result that the detectable signal increases with the increasing target analyte. Therefore, the sandwich-type immunoassay is commonly the most popular choice for developing sensitive immunosensing methods. Typically, an indicator system (signal label or tag) is required to involve in sandwich immunoassays for producing the detectable signal corresponding to sandwich immunorecognition events. In the conventional methods, different labels (e.g., enzyme, dye) are usually linked with signal antibodies at a ratio of 1:1 to obtain the labeled antibodies [3]. This label process often needs a complicated chemical Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00004-9 Copyright © 2017 Elsevier Ltd. All rights reserved.

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reaction and a separation step resulting in relatively high analytical cost. Meanwhile, the detectable signal is also limited by the low content of signal label conjugated with labeled antibodies. The rapid development of nanotechnology and the versatile applications of nanomaterials in the bioassay field provide exciting new possibilities for achieving immunoassay with better performance. One major advantage in using nanomaterials lies in the possibility of controlling and tailoring their unique properties to meet the needs of nanoprobe preparation for signal tracing in sandwich immunoassay [4–6]. Due to the excellent biocompatibility of nanomaterials (e.g., the noble-metal nanoparticles, different carbon nanomaterials), antibodies labeled with these nanomaterials can retain excellent bioactivity for highly efficient biorecognition reactions. Furthermore, the high specific surface area of these nanomaterials makes them useful nanocarriers for loading high-content signal labels, resulting in the successful preparation of a kind of functionalized nanoprobe at a high ratio of label to antibody. When using these nanoprobes for signal tracing of sandwich immunoassay, the amount or concentration of analytes can be determined through the detection of those signal labels loaded on the nanoprobes that were quantitatively captured on the immunosensing surface via sandwich immunoreaction [7]. The enormous signal enhancement associated with the use of multilabel nanoamplification strategy provides the basis for the ultrasensitive sandwich immunosensing.

4.2 Enzyme-functionalized nanoprobes Due to the highly efficient bioactivity of enzymatic reactions, enzyme labels are frequently functionalized with various nanomaterials for the preparation of nanoprobes in immunoassays. The unique properties of the enzyme-functionalized nanoprobes enable the development of a large variety of signal transduction strategies for ultrasensitive immunosensing of protein biomarkers. Carbon nanotube (CNT) is a kind of sp2 hybridized carbon nanomaterial with one-dimensional nanosized fibrous and tubular structure. Due to their remarkable structural, electronic, mechanical, chemical, and physical properties, CNTs, including single-wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes (MWCNTs), have attracted enormous interest since their discovery. Their unique electroconductivity and small size also make them an ideal nanocarrier of enzymes for nanoprobe preparation and biosensing applications [8,9]. For example, Rusling's group reported the combination of electrochemical immunosensors using SWCNT forest platforms with a multi-HRP functionalized MWCNT based nanoprobe for the highly sensitive detection of a cancer biomarker [10]. The 20–30 nm long terminally carboxylated SWCNT forests self-assembled on Nafion-iron oxide [11] provided an ideal electrochemical immunosensing platform for efficient and direct electrical communication. The nanoprobe was prepared through covalently linking high ratio of horseradish peroxidase (HRP) labels and secondary antibodies onto the surface of the carboxylated MWCNT nanocarrier. When used for the signal tracing of sandwich immunoreaction at the constructed immunosensor, greatly enhanced sensitivity was

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achieved based on the multilabel signal amplification (about 90 catalytic labels per binding event on the immunosensor surface) and the enzyme-catalytic amperometric response from the HRP labels (Fig. 4.1). This approach provided a low detection limit of 4 pg mL−1 (100 amol mL−1) for prostate-specific antigen (PSA) measurement in serum and tissue lysates. Rusling's group also constructed an SWCNT forest based electrochemical immunosensor for measurement of IL-6 biomarker and compared the signal amplification between the biotinylated Ab2 bound to streptavidin-HRP and the multi-HRP functionalized MWCNT nanoprobe [12]. The results showed that the former could provide 14–16 labels per antigen binding event for signal amplification. However, Ab2 attached to carboxylated MWCNT nanocarrier with 106 HRP labels per 100 nm gave a high sensitivity with a detection limit of 0.5 pg mL−1 (25 fM) for IL-6 in 10 μL of calf serum. As a new carbon nanomaterial with two-dimensional sheet structure, graphene has received considerable attention in the biosensing field in the last few years [13]. Because of its unique structure and distinct physical properties, graphene owns extraordinary electron transfer capabilities, high surface area, good conductivity, and fine biocompatibility [14]. Besides its common use as conductive nanomaterials for constructing sensitive biosensing platforms, the large specific surface area of graphene also enables it to act as an ideal nanocarrier for nanoprobe preparation. Du et al. [15] reported the preparation of an HRP functionalized graphene oxide (GO) based nanoprobe for ultrasensitive immunosensing of p53392 protein, a potential biomarker of ovarian neoplasms (Fig. 4.2). GO with good biocompatibility and large surface area possesses abundant oxygen-containing functional groups for the surface carboxylation and covalent linking of detection antibody and high-content HRP labels. Based on the

Ab1

HRP (label)

SWNT forest Ab2 - HRP nanotube

Voltage + H2O2

Signal

Fig. 4.1  Illustration of detection principles of the immunosensor after treating with HRP-SWCNT nanoprobes to obtain amplification by providing numerous enzyme labels per binding event. Reprinted with permission from X. Yu, B. Munge, V. Pate, G. Jensen, A. Bhirde, J.D. Gong, S.N. Kim, J. Gillespie, J.S. Gutkind, F. Papadimitrakopoulos, J.F. Rusling, Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers, J. Am. Chem. Soc. 128 (2006) 11199–11205.

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H2O2

2e–

2e–

HRP-p53392 Ab2-GO conjugate

Thionine

phospho-p53392 antigen phospho-p53 Ab1

2e–

H2O2 + HRP(Red)

HS - (CH2)11 - (OCH2CH2)6 - OCH2-CO-NHS AuNPs-SPCE HRP(Ox) + H2O

HRP(Ox) + Thionine(Red)

HRP(Red) + Thionine(Ox)

Thionine(Ox) + 2H+ + 2e–

Thionine(Red)

Fig. 4.2  Schematic illustration of the multienzyme labeling amplification strategy using HRP-p53392Ab2-GO conjugate. Reprinted with permission from D. Du, L.M. Wang, Y.Y. Shao, J. Wang, M.H. Engelhard, Y. Lin, Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy for ultrasensitive electrochemical immunoassay of phosphorylated p53 (S392), Anal. Chem. 83 (2011) 746–752.

signal tracing of the designed HRP-GO nanoprobe and the amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide, this method was successfully used for electrochemical measurement of phospho-p53392 concentration in the range of 0.02–2 nM with the detection limit of 0.01 nM. As HRP can catalyze oxidation of its 4-chloro-1-naphthol substrate to induce the production of an insoluble precipitate, the HRP functionalized GO nanoprobe can also be used to develop an impedimetric immunosensors [16]. Upon sandwich immunoreaction followed by enzymatic reaction induced by the quantitatively captured nanoprobe, a layer of insoluble benzo-4-chlorohexadienone product was deposited on the electrode surface, thus hindering the electron transfer process of the redox probes between the base electrode and the solution and enhancing the sensitivity of the impedimetric immunosensors successfully. Lai et al. also employed this enzymatic reaction to develop an ultrasensitive electrochemical immunosensor based on the amplified inhibition of the electrochemical signal of ferrocene indicator immobilized on the immunosensor [17]. As shown in Fig.  4.3, the immunosensor was constructed on a chitosan-ferrocene (CS-Fc) composite and Au NPs modified electrode. Due to the production of the benzo-4-chlorohexadienone precipitate caused by the sandwich capture of HRP-GO nanoprobe on the immunosensor, the electrochemical signal of ferrocene decreased with the increase of analyte concentration. Compared with the direct immunoreaction-based label-free method reported previously by Qiu et al. [18], the multi-HRP signal amplification greatly enhanced the sensitivity for the electrochemical measurement of carcinoembryonic antigen (CEA). The gold nanostructures at various sizes and shapes (mostly Au NPs) have high specific surface area, high surface energy, high conductivity, and offer numerous

Functional nanoprobes for immunosensing115 CS-Fc

SPCE

Au N

Ps

OH

H2O HRP

CI O

DPV

S1 S0

H2O2 H CI

(Insoluble)

Ab1

HR

P-G

O-

Ab

2

CEA

Fig. 4.3  Schematic representation of the preparation process of the CS-Fc based immunosensor and the electrochemical detection strategy of the method. Reprinted with permission from G.S. Lai, H. Cheng, D.H. Xin, H.L. Zhang, A.M. Yu, Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay, Anal. Chim. Acta 902 (2016) 189–195.

adsorption sites to the biomolecules such as antibodies, enzymes, and proteins, which make them an ideal choice for the biosensor construction. Gold nanomaterials are commonly used to modify electrode surface to improve the conductivity and hence the sensitivity. Numerous researches have been focused on the use of Au NPs coating on electrode to improve the interaction of biomolecule with the electrode surface [19–21]. Because of the narrow size distribution, good biocompatibility, and ease of modification with thiol groups, Au NPs could also serve as excellent nanocarriers for the immobilization of a large number of enzyme labels for nanoprobe preparation, which provide more amplification of the analytical signal in a single recognition reaction. Recently, various enzyme-functionalized gold nanoprobes have been designed for the development of a large variety of ultrasensitive electrochemical immunosensing methods [22–27]. For example, Zhu et al. [22] reported the combination of an electrochemical immunosensor using Au NPs/CNTs platform with an HRP-functionalized Au NP probe for the sensitive detection of human IgG. The hybrids of Au NPs/CNTs covered on the electrode surface provided an effective antibody immobilization matrix and made the immunosensor hold high stability, bioactivity, and sensitivity. Based on the multi-HRP catalytic amplification of the nanoprobe, this immunosensor provided a wide linear response range from 0.125 to 80 ng mL−1 with a detection limit of 40 pg mL−1. Lai et  al. [24] designed a GOx and ferrocene dually functionalized nanoporous gold nanosphere (pAu NS) probe and used it for ultrasensitive CEA at a gold electrode based aptasensor successfully (Fig. 4.4). The pAu NS was synthesized through a sacrificial template method [25]. The large specific surface area of this nanocarrier

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MCH coo−

er

Fc

Fc

A

Fc-SH

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Gold electrode pAu NS

Fc

A CE

m pta

Fc

Fc coo− Fc

Ab

x

GO

Fc Fc

DP

V

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uc

Fc

Fc

Fc

Fc

os

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Fig. 4.4  Schematic representation of the preparation process of the aptasensor and the pAu NS based nanoprobe as well as the electrochemical detection strategy of the method. Reprinted with permission from H. Cheng, L.L. Xu, H.L. Zhang, A.M. Yu, G.S. Lai, Enzymatically catalytic signal tracing by a glucose oxidase and ferrocene dually functionalized nanoporous gold nanoprobe for ultrasensitive electrochemical measurement of a tumor biomarker, Analyst 141 (2016) 4381–4387.

enabled the simultaneous loading of high-content GOx labels and electron mediator of ferrocene to obtain a useful functionalized nanoprobe. The enzymatically catalytic reaction of the nanoprobe provides sensitive electrochemical signal tracing for the CEA aptasensor. Both the immobilization of the ferrocene mediator on the nanoprobe and the highly specific aptamer recognition decrease the background current signal, resulting in the ultrasensitive measurement of CEA in a wide linear relationship of five orders of magnitude and a low detection limit down to 0.45 pg mL−1. Ju's group [26] reported an ultrasensitive multiplexed immunosensing method developed by combination with an alkaline phosphatase (ALP)-labeled antibody functionalized Au NP probe and the enzyme-Au NP catalyzed deposition of Ag NPs at a disposable immunosensor array (Fig. 4.5). After sandwich immunoreaction at the immunosensor, the Au NP/ALP nanoprobes were quantitatively captured onto the immunosensing surface, and the enzymatic reaction of the nanoprobes induced the catalytic deposition of Ag NPs. The silver deposition process was catalyzed by both ALP and Au NPs, which amplified the detection signal. Based on the anodic stripping analysis of Ag NPs in KCl solution, this multiplexed immunosensor showed excellent analytical

Functional nanoprobes for immunosensing117

W1

W1

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W2

3-

IP

/A

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O

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Au NP

Ag NP

Au NPs

3

Ag NPs

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(2)

3-IP + Ag

NH2 NH2 NH2 NH2 NH2

(1) GA

W1

+

GA

Glutaraldehyde

CS

Chitosan

3-IP 3-indoxyl phosphate W1

W2

Fig. 4.5  Schematic representation of preparation of immunosensor array and detection strategy by sandwich immunoassay and linear sweep voltammetric stripping analysis of enzymatically deposited Ag NPs. Reprinted with permission from G.S. Lai, F. Yan, J. Wu, C. Leng, H.X. Ju, Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction, Anal. Chem. 83 (2011) 2726–2732.

performance for simultaneous ultrasensitive measurement of the model analytes of human IgG and mouse IgG with wide linear ranges over four orders of magnitude with the detection limits down to 4.8 and 6.1 pg mL−1, respectively (Fig. 4.6). Similarly, Lai et al. [27] also employed the enzymatically catalytic deposition of Au NPs to develop an ultrasensitive electrochemical immunosensing method by combination with gold stripping analysis at a CNT-based immunosensor. The gold nanoprobe was prepared by loading signal antibody and high-content GOx on the nanocarrier of gold nanorod. After sandwich immunoreaction, the GOx-Au NR nanoprobe quantitatively captured onto the immunosensor surface induced the deposition of Au NPs via the enzymatically catalytic reaction. Based on the electrochemical stripping analysis of the Au NR nanocarriers and the enzymatically produced Au NPs, a sensitive electrochemical signal was obtained for the immunoassay. Both the GOx-induced deposition of Au NPs by the nanoprobe and the sensitive electrochemical stripping analysis on the CNTs based sensing surface greatly amplified the signal response, leading to the ultrasensitive measurement of CEA in a wide linear range from 0.01 to 100 ng mL−1 with a detection limit down to 4.2 pg mL−1. As HRP can catalyze the oxidation of aniline in a weak acid condition to produce the electroactive polymer of polyaniline, Lai et al. [28] reported an ultrasensitive immunosensing method based on the electrochemical measurement of polyaniline (PAn), which was catalytically produced by the Au NP/HRP nanoprobe at an immunosensor

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0

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Fig. 4.6  Linear sweep stripping voltammetric curves of Ag NPs deposited at immunosensors (A, C) and calibration curves (B, D) for simultaneous multiplexed detection of HIgG (A, B) and MIgG (C, D) using the proposed strategy. Curves a–g and h–n are for 5 pg mL−1 to 250 ng mL−1 HIgG and MIgG at W1 and W2, respectively. Reprinted with permission from G.S. Lai, F. Yan, J. Wu, C. Leng, H.X. Ju, Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction, Anal. Chem. 83 (2011) 2726–2732.

(Fig. 4.7). The immunosensor was constructed on a reduced GO/Au NPs nanocomposite modified electrode. After catalytically produced electroactive PAn on the immunosensor surface by the quantitatively captured Au NP/HRP nanoprobe via sandwich immunoreaction, the electrochemical measurement of PAn provided convenient signal tracing for the ultrasensitive measurement of the protein analyte of human IgG. In addition, the enzymatically catalytic reaction of the gold nanoprobes can also be used for ultrasensitive signal tracing by the colorimetric [29,30], chemiluminescent [31], electrochemiluminescent (ECL) [32], or photoelectrochemical (PEC) [33] method. For example, with the aid of the enzymatic catalysis of 3,3′,5,5′-tetramethylbenzidine and o-phenylenediamine by HRP, the signal tracing of Au NP/HRP probe was also combined with a magnetic bead-based sandwich immunoreaction to develop a multiplexed colorimetric immunoassay method [30]. Two antigens of CEA and alpha-fetoprotein (AFP) could be detected even with the naked eye. The detection limit obtained from the spectrophotometric measurements was as low as 0.02 ng mL−1. Zhang's group [31]

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Anti-HlgG H

G

rG

O

/A u

N Ps

lg

SPCE

V

DP

be

ro

p no

Na

PAn

Aniline H2O2

Fig. 4.7  Schematic representation of the preparation of immunosensor and sandwich immunoassay based on the electrochemical measurement of PAn catalytically deposited by an HRP-Au NP nanoprobe. Reprinted with permission from G.S. Lai, H.L. Zhang, T. Tamanna, A.M. Yu, Ultrasensitive immunoassay based on electrochemical measurement of enzymatically produced polyaniline, Anal. Chem. 86 (2014) 1789−1793.

employed the Au NP/HRP nanoprobe for enhancing the sensitivity of chemiluminescence (CL) immunoassay. After magnetic bead-based sandwich immunoreaction, the Au NP/HRP probes were quantitatively captured on the immunocomplex. The enzymatic reaction of the high-content HRP labels greatly amplified the CL of luminol to achieve the sensitive detection of AFP with a detection limit of 0.01 ng mL−1. Li's group [32] applied the multilabel signal amplification of the GOx functionalized gold nanorod probe to develop a cathodic ECL immunosensor. The immunosensor was constructed on a graphene-chitosan nanocomposite modified electrode. The functionalized graphene with large surface area and fast electron transfer ability provided an excellent sensing platform. The enzymatic reaction of the gold nanoprobe greatly amplified the ECL signal of luminol, which led to the ultrasensitive measurement of PSA biomarker at a low operation potential. PEC immunosensing is a new immunosensing technology. Ju's group [33] used CL as the exciting light source to construct a universal PEC immunosensor by combination with a reduced GO-CdS nanocomposite platform and the signal triggering of a luminol and HRP-Ab dually functionalized Au NP probe (Fig. 4.8). After sandwich immunoreaction, the gold nanoprobe could be quantitatively captured onto the immunosensing platform to produce corresponding CL through the multi-HRP catalyzed luminol-H2O2 system. Based on the recording of the PEC current of the CdS nanocomposite excited by the CL, the typical time-based photocurrent response of the light source-free PEC immunosensor to analyte was demonstrated at different

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H2O2 + PIP

e–

FTO

Photocurrent (nA)

e–

CdS

e– h+ H2O2

Time (s)

Oxidation product

RGO

CdS QDs Ab1

CEA

Luminol functionalized AuNPs HRP labeled Ab2

CL emission

Fig. 4.8  Schematic illustration of the photoelectrochemical platform excited by chemiluminescence for immunoassay. Reprinted with permission from W.W. Tu, W.J. Wang, J.P. Lei, S.Y. Deng, H.X. Ju, Chemiluminescence excited photoelectrochemistry using graphene-quantum dots nanocomposite for biosensing, Chem. Commun. 48 (2012) 6535–6537.

concentrations of CEA (Fig. 4.9), which showed a good linear relationship between the photocurrent and the logarithm value of CEA concentration ranging from 0.05 to 20 ng mL−1. The coefficients of variation for five times measurements of 0.1 and 5.0 ng mL−1 CEA using five CL-excited PEC immunosensors were 8.2% and 7.6%, respectively, indicating acceptable precision and fabrication reproducibility. Since the normal level of CEA in human serum is in the range of 3–5 ng mL−1, this PEC platform was suitable for practical application. The assay results of clinical serum samples, compared with the reference values obtained by a commercial ECL test, showed an acceptable agreement, with relative errors less than 14.4%. The unique properties of Au NP also make it easy to be hybridized with other nanomaterials such as CNT [34], graphene [35,36], carbon nanosphere [37,38], TiO2 nanosphere [39] for preparing more versatile enzyme-functionalized nanoprobes. For example, Ju's group [34] designed a GOx functionalized CNT/Au NPs nanoprobe through a layer-by-layer assembly method and used it to develop an ultrasensitive multiplexed electrochemical immunosensing method by combination with a disposable immunosensor array (Fig.  4.10). The immunosensor array was constructed through the stepwise assembly of colloidal Prussian blue (PB), Au NPs, and captured antibodies on screen-printed carbon electrodes. The enzymatic reaction of the nanoprobe provided sensitive electrochemical signal tracing of the immunosensors. The colloidal PB acted as a mediator to catalyze the reduction of H2O2 produced in the GOx enzymatic reaction. Both the high-content GOx and CNT/Au NPs in the nanocomposite tracer amplified the detectable signal of the immunosensor. Using CEA and AFP as model analytes, the simultaneous multiplexed immunoassay method

Functional nanoprobes for immunosensing121

Photocurrent (nA)

400 300

300

200

200

100

100

0

0 0

(A)

150

300

-1

450

(B)

Time (s)

0

1

2

Log (CCEA / ng mL-1)

Fig. 4.9  (A) Photoelectrochemical responses of the immunosensor to 0, 0.05, 0.1, 0.5, 5, 20, and 50 ng mL−1 CEA at the applied potential of +0.2 V, and (B) linear calibration. Reprinted with permission from W.W. Tu, W.J. Wang, J.P. Lei, S.Y. Deng, H.X. Ju, Chemiluminescence excited photoelectrochemistry using graphene-quantum dots nanocomposite for biosensing, Chem. Commun. 48 (2012) 6535–6537.

H2SO4/HNO3

PDDA

(A)

(B) PB-PDDA-CS

Au NP

GOx

Antibody

Antigen

Fig. 4.10  Schematic representation of (A) preparation procedure of CNT/Au NPs/GOx nanoprobe and (B) preparation of immunosensors and sandwich electrochemical immunoassay. Reprinted with permission from G.S. Lai, F. Yan, H.X. Ju, Dual signal amplification of glucose oxidase-functionalized nanocomposites as trace label for ultrasensitive simultaneous multiplexed electrochemical detection of tumor markers, Anal. Chem. 81 (2009) 9730–9736.

using the immunosensor array and the designed tracer showed linear ranges of three orders of magnitude with the detection limits down to 1.4 and 2.2 pg mL−1, respectively. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The dual signal amplification of GOx-functionalized nanocomposites provided a promising ultrasensitive simultaneous multiplexed immunoassay approach for clinical applications.

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To avoid the effect of the large background current from the PB immobilized on the electrode surface, they [38] further designed a GOx and PB dually functionalized nanoprobe for the electrochemical immunosensing of the protein biomarker (Fig. 4.11). First, a mesoporous carbon nanosphere (MCN) was synthesized by using mesoporous silica nanosphere as a hard template in combination with a hydrothermal carbonization method [40]. After in situ deposition of high-content PB on the MCN nanocarrier, Au NPs were statically assembled on its surface further to immobilize signal antibody and high-content GOx resulting in the successful preparation of the nanoprobe. Based on the sandwich signal tracing with this nanoprobe composite, the PB-mediated GOx catalytic reaction provided sensitive electrochemical response for immunoassay of human IgG in a wide concentration range from 0.01 to 100 ng mL−1 with a low detection limit of 7.8 pg mL−1. In addition, other nanomaterials such as bionanosphere [41,42], magnetic bead [43], and some nanohybrids [44–47] can also act as useful nanocarriers for preparation of various enzyme-functionalized nanoprobes in immunoassay. By immobilizing capture antibodies on Au NP-CNT-chitosan modified screen-printed carbon electrodes, and loading signal antibodies and high-content GOx on amino-functionalized silica nanosphere, Ju et  al. [42] developed an ultrasensitive multiplexed electrochemical method with a sandwich-type immunoassay by the enzymatic signal amplification with ferrocenecarboxylic acid as electron transfer mediator and the accelerated electron transfer by CNTs (Fig. 4.12). This method showed wide linear ranges with the detection limits down to 3.2 and 4.0 pg mL−1 for CEA and AFP, respectively.

K3Fe(CN)6

(1) PDDA

FeCl3

(2) Au NPs

MCN DPV

Anti-HlgG

HlgG

SPCE

SPCE

Glucose

PW

H2O O2

PB

H2O2

Gluconolactone GOx

GOx

Glucose

PB

Au NPs

Fig. 4.11  Schematic representation of the preparation of MCN-PB based nanoprobe and the electrochemical detection strategy of the immunoassay method. Reprinted with permission from G.S. Lai, H.L. Zhang, A.M. Yu, H.X. Ju, In situ deposition of Prussian blue on mesoporous carbon nanosphere for ultrasensitive electrochemical immunoassay, Biosens. Bioelectron. 74 (2015) 660–665.

Functional nanoprobes for immunosensing123 d f c b

Anti-CEA

a

H2N

g e W1

APTES

Anti-AFP CEA AFP

W2

GOx

H2N

W2

W1

W2

or

W1

Au NPs

+

W2

(2)

W1

SiO2 nanosphere CNTs

(1) GA (4 h)

W2

W2

FCA, glucose

NH2

CS W1

W1

NH2

W1

W2

Fig. 4.12  Schematic representation of the sandwich-type electrochemical multiplexed immunoassay using GOx-functionalized silica nanosphere as trace tags. (a) Nylon sheet, (b) silver ink, (c) graphite auxiliary electrode, (d) Ag/AgCl reference electrode, (e) W1, (f) W2, and (g) insulating dielectric. Reprinted with permission from G.S. Lai, J. Wu, C. Leng, H.X. Ju, F. Yan, Disposable immunosensor array for ultrasensitive detection of tumor markers using glucose oxidasefunctionalized silica nanosphere tags, Biosens. Bioelectron. 26 (2011) 3782–3787.

4.3 Noble-metal nanoparticle-functionalized nanoprobes Due to the low oxidation potential for facile electrochemical stripping analysis, Ag NP is often functionalized with nanocarriers such as CNT [48], graphene [49,50], and carbon nanosphere [51] for designing different nanoprobes in electrochemical immunoassays. For example, Ju's group [48] designed a streptavidin-functionalized Ag NPs-enriched CNT/Ag NP as trace tag for the ultrasensitive multiplexed measurements of tumor markers using a disposable immunosensor array (Fig. 4.13). Due to the inherent reduction property of carboxylated CNT [52], Ag NPs were in situ deposited on this support from silver ions in solution without the addition of any other reduction agent, which was further used for linking antibody through the streptavidin assembly. Through a sandwich-type immunoreaction at the immunosensor, the high-content Ag NPs were captured onto the immunosensor surface to further induce the silver deposition for greatly amplifying the detection signal. Based on the electrochemical stripping detection of the Ag NPs at the immunosensor, this method showed ultra-high sensitivity with wide detection ranges and the detection limits down to 0.093 and 0.061 pg mL−1 for simultaneous electrochemical measurement of CEA and AFP (Fig. 4.14).

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Immunosensing for Detection of Protein Biomarkers

H2SO4

AgNO3

HNO3 W1

AA

LSV

Ag+

W2

in KCI

or

W1

W2

then blocking with BSA

W1

Ag+ AA

W2

AA + AgNO3 W1

W2

W1

W2

Anti-CEA

Biotin-anti-CEA

CEA

Anti-AFP

Biotin-anti-AFP

AFP

W1

Ag NPs

W2

Streptavidin CNTs

Fig. 4.13  Schematic representation of preparation of immunosensor array and trace tag, and detection strategy by linear-sweep stripping voltammetric analysis of Ag NPs on the immunosensor surface. Reprinted with permission from G.S. Lai, J. Wu, H.X. Ju, F. Yan, Streptavidin-functionalized silver-nanoparticle-enriched carbon nanotube tag for ultrasensitive multiplexed detection of tumor markers, Adv. Funct. Mater. 21 (2011) 2938–2943.

As the Au NP label modified on electrode surfaces can be electrochemically preoxidized in an HCl solution to produce electroactive gold (III) ions for electrochemical reduction measurement, it can also be loaded on different nanomaterials for the nonenzymatic electrochemical signal tracing in immunosensing [53–56]. Ju's group [53] employed the carbon sphere (CNS) synthesized with a hydrothermal method to electrostatically load Au NPs and thus prepared a gold nanoprobe (Fig. 4.15). With a sandwichtype immunoassay format, the analyte and then CNS/AuNPs labeled antibody were bound to the immunosensor. The bound AuNPs were finally electrooxidized in 0.1 M HCl to produce AuCl 4 - for differential pulse voltammetric detection. The high loading capability of AuNPs on CNS for the sandwich-type immunorecognition led to obvious signal amplification. Using human IgG as model target, the DPV signal of AuNPs after being electrooxidized at optimal potential of +1.40 V for 40 s showed a wide linear dependence on the logarithm of target concentration ranging from 10 pg mL−1 to 10 ng mL−1 (Fig. 4.16). The detection limit was around 9 pg mL−1. The immunosensor showed excellent analytical performance with cost-effectiveness, good fabrication reproducibility, and acceptable precision and accuracy. Lai et  al. [55] also reported the preparation of a gold nanoprobe for the electrochemical immunoassay based on in situ deposition of Au NPs on a polydopamine (PDA) functionalized silica nanosphere followed by the labeling of signal antibodies. The PDA film, self-polymerized on the

Functional nanoprobes for immunosensing125

0

24 a

Current (µA)

–5

20 g

–10

16

–15

12

–20

8

–25 0.2

(A)

0.1 0.0 Potential (V)

–0.1

0

(B)

1E–5 1E–4 1E–3 0.01 0.1 1 10 CEA concentration (ng mL–1)

20 h

–5 Current (µA)

4

n

–10

16 12

–15 8

–20

(C)

0.2

0.0 0.1 Potential (V)

–0.1

1E–5 1E–4 1E–3 0.01 0.1 1 10 AFP concentration (ng mL–1)

(D)

Fig. 4.14  Linear sweep stripping voltammetric curves of Ag NPs (A, C) and calibration curves (B, D) for simultaneous multiplexed detection of CEA (A, B) and AFP (C, D) using the proposed strategy. Curves a-g and h-n are for CEA and AFP at concentrations from 0.02 pg mL−1 to 5.0 ng mL−1 at W1 and W2, respectively. Reprinted with permission from G.S. Lai, J. Wu, H.X. Ju, F. Yan, Streptavidin-functionalized silver-nanoparticle-enriched carbon nanotube tag for ultrasensitive multiplexed detection of tumor markers, Adv. Funct. Mater. 21 (2011) 2938–2943.

silica surface at weakly alkaline condition [57], possesses abundant catechol groups. So AuCl 4 - can be adsorbed onto its surface and in situ reduced to Au NPs without the addition of any other reducing agent. When used in sandwich immunoassay at a CNTs based immunosensor, the high-content Au NPs on the nanoprobe and the electron transfer acceleration of CNTs greatly amplified the detection signal. The stripping voltammetric detection of Au NPs for immunosensing could also be achieved with gold nanorods (AuNRs) superstructure [58]. The amplification pathway firstly used a thio-β-cyclodextrin (SH-β-CD) functionalized gold nanoparticle to label signal antibody, and then in situ assembled multilayer SH-β-CD end-functionalized AuNRs to sandwich immunocomplex on immunosensor surface by using 4,4,4,4(21H, 23H-porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) as a bridge to achieve simple and convenient host-guest reaction (Fig. 4.17). The built end-to-end AuNRs superstructure showed excellent performance for the signal amplification in ­connection

126

Immunosensing for Detection of Protein Biomarkers SPCE

1. 2.

CH2OH O OH H OH H H OH H OH

PDDA

Incubation

180 °C, 6 h H

H2SO4 / HNO3

a ub

n

tio

c

In 1. Preoxidation 2. DPV PEG

Rabbit anti-HlgG

BSA

HIgG

Au NP

CNS

Fig. 4.15  Signal-amplification strategy for sensitive electrochemical immunoassay by using CNS/Au NPs as a tracing tag. Reprinted with permission from Q.N. Xu, F. Yan, J.P. Lei, C. Leng, H.X. Ju, Disposable electrochemical immunosensor using carbon sphere/gold nanoparticles composite as label for signal amplification, Chem. Eur. J. 18 (2012) 4994–4998. 1.5

Current (µA)

2.0

1.5

1.0

1.0

0.5

0.5 0.0 0.1

(A)

0.2

0.3

0.4

Potential (V)

0.5

(B)

0.01

1

100

CHlgG (ng mL–1)

Fig. 4.16  (A) DPV responses to 0, 0.001, 0.01, 0.1, 0.5, 1, 5, 10, and 100 ng mL−1 HIgG from low to high using the proposed label and immunosensor. (B) Calibration curve. Reprinted with permission from Q.N. Xu, F. Yan, J.P. Lei, C. Leng, H.X. Ju, Disposable electrochemical immunosensor using carbon sphere/gold nanoparticles composite as label for signal amplification, Chem. Eur. J. 18 (2012) 4994–4998.

Functional nanoprobes for immunosensing127

(B)

(A)

GCE

GCE

BSA

GCE

GCE

GCE

Chitosan

GCE

GCE

n cycles

i GCE

Preoxidation n

DPV E

Ab1

Ab2

AFP

PDDA/CNTs

SH-β-CD

AuNP

TCPP

CTAB-AuNR

(C) Fig. 4.17  Schematic representation of the preparation of (A) CD-Ab2/AuNPs and (B) CD/AuNRs tags; (C) immunosensor fabrication and sandwich-type immunoassay with AuNRs superstructure based signal amplification. Reprinted with permission from D.J. Lin, J. Wu, H.X. Ju, F. Yan, Signal amplification for electrochemical immunosensing by in situ assembly of host-guest linked gold nanorod superstructure on immunocomplex, Biosens. Bioelectron. 45 (2013) 195–200.

with the electrochemical biosensing by preoxidation and then voltammetric analysis of gold element. Using AFP as an analyte, the immunosensor was constructed by covalently binding capture antibody to chitosan-carbon nanotubes-poly(diallyldimethylammonium chloride) modified electrode. The superstructure rich in AuNRs brought an enhanced detection sensitivity of protein, which could detect AFP in a linear range of 0.5 pg mL−1 to 0.5 ng mL−1 with a detection limit down to 0.032 pg mL−1 (Fig. 4.18). The immunoassay exhibited good stability and acceptable reproducibility and accuracy. At 50 pg mL−1 AFP, the relative standard deviations examined with five immunosensors prepared with the same and different glassy carbon electrodes were 5.7% and 7.2%, respectively. The in situ superstructure assembly could be extended to other labeled recognition systems, providing a promising novel avenue for signal amplification and potential applications in bioanalysis and clinical diagnostics. As Au NPs possessing good biocompatibility for functionalization with biomolecules can catalyze the deposition of Ag NPs on electrode surface for the convenient and sensitive silver stripping analysis [59], the gold nanoprobe can be combined with silver enhancement reaction for the ultrasensitive nonenzymatic electrochemical immunoassays [60]. Ju's group [60] reported the combination of a poly(styrene-co-acrylic acid) microbead (PSA)-Au NPs based nanoprobe and a graphene nanocomposite

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Current (µA)

30 0

15 0 0.7

(A)

0.5 ng mL-1

0.6

0.5 Potential (V)

0.4

Current (µA)

50

45

40 30 20

10 1E-5 1E-4 1E-3 0.01 0.1 CAFP (ng mL-1) (B)

1

10

Fig. 4.18  DPV responses (A) and calibration curve (B) of immunosensor for the detection of AFP at concentrations of 0, 0.5, 5 and 50 pg mL−1, and 0.5 ng mL−1, respectively (error bars were obtained by thrice repetitive measurements). Reprinted with permission from D.J. Lin, J. Wu, H.X. Ju, F. Yan, Signal amplification for electrochemical immunosensing by in situ assembly of host-guest linked gold nanorod superstructure on immunocomplex, Biosens. Bioelectron. 45 (2013) 195–200.

based immunosensing platform for developing a triple signal amplification strategy in ultrasensitive immunosensing of cancer biomarker (Fig. 4.19). The in situ synthesis of numerous Au NPs on the PSA nanocarrier ensured the catalyzed deposition of high-content Ag NP at the immunosensor for anodic stripping analysis. The introduction of graphene on immunosensor surface efficiently accelerated the electron transfer signal. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect CEA with a linear range of 0.5 pg mL−1 to 0.5 ng mL−1 and a detection limit down to 0.12 pg mL−1. When noble-metal nanoparticles are hybridized with the other nanomaterials (mostly carbon nanomaterials), the synergistic effect often improves their catalytic ability greatly. Therefore, this unique catalytic property makes them serve as powerful nanolabels for ultrasensitively probing the immunorecognition events by combination with the nanoprobe signal amplification. For example, Ju's group [61] reported the preparation of a platinum nanodendrite (PtNDs) functionalized graphene nanosheets (PtNDs@GS) based nanoprobe and employed it in an excellent electrocatalytic reduction of dissolved oxygen to develop an ultrasensitive immunosensing method for human IgG measurement. As shown in Fig. 4.20, the PtNDs@GS hybrid was prepared in situ by reducing K2PtCl4 with ascorbic acid in an aqueous solution of reduced GO. After labeling with antibody, the obtained nanoprobe was used for sandwich immunoreaction at a PEG-modified electrode-based immunosensor. Upon the electrocatalytic reduction of oxygen by the nonenzymatic nanoprobe, this method showed excellent analytical performance for the ultrasensitive detection of human IgG biomarkers. Due to the excellent electrocatalytic reduction ability of Pd NP toward dissolved oxygen, it was statically conjugated with CNT to prepare a CNT/Pd NPs based nanoprobe for immunosensing [62]. The immunosensor was constructed by assembling the capture antibody on gold nanoparticles decorated graphene nanosheets modified screen-printed carbon working electrode. With a sandwich immunoassay mode, the

PSA

PDDA

PSS

Y

HAuCl4

PDDA

NaBH4

(A) Graphene oxide

Chitosan

Current

Electroreduction

LSV

Ag+

in KCl

HQ

BSA

Potential

Y Anti-CEA

Y

Anti-CEA

BSA

CEA

AuNPs

AgNPs

(B) Fig. 4.19  Schematic representation of (A) the preparation of PSA-Au NPs tracing nanoprobe, and (B) immunosensor fabrication and sandwich immunoassay procedure. Reprinted with permission from D.J. Lin, J. Wu, M. Wang, F. Yan, H.X. Ju, Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing, Anal. Chem. 84 (2010) 3662–3668.

(A)

GO

PVP+hydrazine

K2PtCl4

Ammonia+heating

Ascorbic acid

SPCE

G

PE

H2O

O2 e–

DP

V

PVP

PEG

RaHIgG

PtNDs

HIgG

BSA

(B) Fig. 4.20  Schematic representation of (A) the preparation of PtNDs/graphene based nanoprobe and (B) the signal amplification strategy for sensitive electrochemical immunoassay. Reprinted with permission from Q.N. Xu, L.S. Wang, J.P. Lei, S.Y. Deng, H.X. Ju, Platinum nanodendrite functionalized graphene nanosheets as a non-enzymatic label for electrochemical immunosensing, J. Mater. Chem. B 1 (2013) 5347–5352.

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palladium nanoparticle decorated carbon nanotubes were captured to the immunocomplex and showed strong electrocatalytic activity toward oxygen reduction (Fig. 4.21). The use of CNT carrier offered a high amount of Pd NPs on each immunoconjugate, hence amplifying the detectable signal from the electroreaction of dissolved oxygen. The graphene nanosheets and Au NPs improved the electronic conductivity and the hydrophilicity of electrode surface for immobilization of the capture antibody, respectively. Under optimal conditions, a linear detection range from 50 pg mL−1 to 10 ng mL−1 and a limit of detection of 44 pg mL−1 (0.3 pM) were achieved for human IgG. Using dissolved oxygen as a signal reporter, the detection process avoided deoxygenation. The specificity of the immunosensor was examined by testing the amperometric response toward the interfering substances such as BSA, CEA, AFP, and glucose prepared in blank phosphate buffer, which did not show any change, indicating good specificity of the proposed immunosensing method. The inter-assay precision of the immunosensor evaluated using five chips showed the coefficients of variation of 8.3% and 2.6% for 1 and 5 ng mL−1 HIgG. By combination with a graphene/Au NPs modified electrode based immunosensor, this nanoprobe enabled the development of another ultrasensitive nonenzymatic electrochemical immunosensing method. As dissolved oxygen is the coreactant of the ECL of CdTe QDs, Ju's group [63] designed a Pd nanoprobe and used it to electrocatalytically consume the dissolved oxygen to develop an ECL immunosensing method. As shown in Fig.  4.22, highcontent Pd NPs were produced and encapsulated in the interior structure of polyami-

H2SO4/HNO3

PDDA

PDDA PSS

SPCE O2 H2O DPV Rabbit antihuman IgG BSA

Human IgG

Pd NP

Graphene-Au NPs

Fig. 4.21  Schematic representation of the sensitive electrochemical immunoassay using CNT/ Pd NPs as label. Reprinted with permission from C. Leng, J. Wu, Q.N. Xu, G.S. Lai, H.X. Ju, F. Yan, A highly sensitive disposable immunosensor through direct electro-reduction of oxygen catalyzed by palladium nanoparticle decorated carbon nanotube label, Biosens. Bioelectron. 27 (2011) 71–76.

Functional nanoprobes for immunosensing131

O HO

HO O

O OH

O OH

OH O OH O

1) EDC 2) NaBH4 PdCl4–

Ab1

HN O

HN O O N H

HN O

EDC+NHS

H N O

O NH

Ab2

BSA

HN O

O N H

CEA

H N O

O NH

QD PdNP

O NH

O NH

HN O O N H

hv

PAMAM-G5

H N O

O NH O NH

Incubation

HN

HN O

hv

O N H O

O

HN

O H N O

NH O NH

Incubation

GCE

GCE 2e O2 +

2QD

2QD•–

H2O2+2QD•–

2e O2 H2O2 H2O2+ 2QD* 2QD*+2OH–

GCE

2e 2QD O2 H2O2 O2+2QD•– 2QD*+ H2O2 4e PdNP 2H O 2 2e

Fig. 4.22  Schematic representation of preparations of tracing nanoprobe, and ECL annihilation strategy by electrocatalytic reduction toward dissolved oxygen at the PdNPs@ PMM5/SWNH nanohybrid. Reprinted with permission from S.Y. Deng, J.P. Lei, H.X. Ju, Electrocatalytic reduction of coreactant by highly loaded dendrimer-encapsulated palladium nanoparticles for sensitive electrochemiluminescent immunoassay, Chem. Commun. 48 (2012) 9159–9161.

doamine dendrimer (PMM5), which was further covalently anchored on the SWCNH with highly defective cone-shaped horns and large surface area to obtain the PdNPs@ PMM5/SWNH nanohybrids for nanoprobe preparation. Upon the quantitative capture of the nanoprobes on a QD-based immunosensor, the dissolved oxygen was consumed by the electrocatalytic reduction reaction, leading to the decrease of the ECL intensity for the ultrasensitive immunosensing of CEA in a wide linear range over six orders of magnitude ranging from 100 ng mL–1 to 1 pg mL–1 with detection limit down to 0.47 pg mL−1. Based on the ECL of QDs and the electrocatalytic consumption of coreactant dissolved oxygen, more immunosensing strategies have been developed by this group, which will be introduced in Chapter 6. The graphene/silver nanocomposite also showed excellent electrocatalytic reduction toward H2O2, which made it act as a nonenzymatic signal amplification label for the nonenzymatic electrochemical measurement of CEA [64]. Meanwhile, due to the strong ability in enhancing the PEC activity of the CdS:Mn/TiO2, an antibody functionalized graphene-silver nanocomposite was prepared to serve as signal amplification probe for developing a signal-on PEC immunosensor [65]. The electron transport and exciton recycle reduced the possibility of electron-hole recombination and greatly improved the phototocurrent conversion efficiency of the sensing matrix. The as-prepared immunosensor showed excellent performance for CEA measurement with a wide linear range from 1.0 pg mL−1 to 100 ng mL−1 and a low detection limit of 1.0 pg mL−1.

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4.4 QD functionalized nanoprobes Because QDs possess excellent feasibility for surface modification, they can conveniently link with various nanocarriers for preparing a large variety of functionalized nanoprobes for immunoassay. First, the electrochemical stripping analysis of metallic ions released from QDs enables their wide applications in ultrasensitive electrochemical immunoassays [66–70]. For example, Liu's group [66] reported the preparation of a CdTe QDs functionalized silica nanosphere probe for the magnetic electrochemical immunoassay of AFP. The nanoprobe was prepared based on the carbodiimide coupling of the as-synthesized 3-mercaptopropionic acid coated QDs on the aminofunctionalized silica nanocarriers at an average diameter of 200 nm. To achieve the simultaneous detection of two analytes, they employed the CdSe and PbS QDfunctionalized silica to label the signal antibodies of rabbit immunoglobulin IgG and CEA, respectively [67]. The well-defined electrochemical stripping peaks from the QD labels enabled the simultaneous measurements of the two protein analytes at a gold substrate based immunosensor. Shiddiky et al. [68] reported the preparation of a CdSe QD-functionalized GO probe for the electrochemical immunoassay of epithelial cell adhesion molecule (EpCAM) antigen at an indium tin oxide (ITO) electrode based immunosensor. The nanoprobe was prepared based on the carbodiimide coupling of amino-functionalized QDs and carboxylated GO nanocarrier. The high loading of the QD labels per sandwich immunoreaction on the release of metallic ions for anodic stripping analysis greatly enhanced the sensitivity of these methods for the measurement of protein biomarkers. As a kind of ECL luminophore with numerous advantageous features including high quantum yield, low photobeaching, and high photochemical stability, QDs have been widely used for ECL immunoassays. Most of the ECL immunoassays of QDs are based on the quenching, or enhancement, of ECL intensities via the well-researched coreactant ECL systems that took S2 O8 2- , H2O2, SO32- as the coreactants [71]. CdTe QDs as the luminophore and K2S2O8 as the coreactant is the most widely used system in sandwich-enhanced ECL immunoassays. For example, Liu's group [72] reported a versatile immunosensor using a CdTe QDs coated silica nanosphere as a label for ultrasensitive detection of rabbit IgG. The signal amplification from the high loading of CdTe QDs on silica provided 6.6fold enhancements in ECL signals for the protein analyte detection compared to the unamplified method. Zhang's group [73] reported a novel ECL immunosensor for the sensitive detection of HCG using CdTe QDs functionalized nanoporous PtRu alloys as labels for signal amplification. Due to signal amplification from the high loading of CdTe QDs, 4.67-fold enhancements in ECL signal for HCG detection were achieved compared to the unamplified methods. Han's group [74] reported a near-infrared ECL immunosensor for the ultrasensitive detection of a biomarker by combination with the use of CdTe/CdS QDs tagged silica nanospheres for signal amplification and a graphene-Au NPs hybrid for immunosensor construction. The ECL response from CdTe/CdS QDs enhanced 16.8-fold compared to the unamplified protocol and successfully fulfilled the ultrasensitive detection of human IgG with a detection limit

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CdCl2

Ab2

NaHTe

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(A)

(B)

Cd(NO3)2

Ab1

Na2S

BSA

MWNTs

CdTe

PSA

Ab

PSA

BSA

CdTe/MWNTs/Ab2

TiO2 NTs

CdS

Fig. 4.23  (A) The preparation of CdTe-MWNTs composites. (B) The principle of the CdS-TiO2 nanotube array electrode for PSA detection using CdTe-MWNTs composites as ECL quenchers. Reprinted with permission from C.Y. Tian, W.W. Zhao, J. Wang, J.J. Xu, H.Y. Chen, Amplified quenching of electrochemiluminescence from CdS sensitized TiO2 nanotubes by CdTe-carbon nanotube composite for detection of prostate protein antigen in serum, Analyst 137 (2012) 3070–3075.

of 87 fg mL−1. Yu et al. [75] reported a novel ECL immunosensor based on a prepared 3D paper device using CdTe QDs coated carbon microspheres as signal probes. Both the multiple labels signal amplification of the nanoprobe and the gold-silver nanocompositefunctionalized graphene modified on the sensing platform greatly improved the sensitivity of the method. The ECL quenching principles of QDs have also been employed in sandwich ECL immunoassays for protein analysis. Xu's group [76] reported an ECL immunoassay method for ultrasensitive detection of PSA, by remarkably efficient energy-transfer induced ECL quenching from the CdS QDs sensitized TiO2 nanotube array to the activated CdTe QDs functionalized MWCNTs nanocomposite (Fig. 4.23). The coupling of TiO2 and CdS resulted in a cathodic ECL intensity 14.7 times stronger than that of the pure TiO2 electrode, which could be efficiently quenched by the CdTe-MWCNTs. The ECL intensity decrement was logarithmically related to the concentration of the PSA in the range of 1.0 fg mL−1 to 10 pg mL−1 with a detection limit of 1 fg mL−1.

4.5 Other signal label functionalized nanoprobes Recently, other labels such as biobarcode oligonucleotides [77–80], metal oxide nanoparticles [81–84], and some electroactive/optical agents [85–89] have conjugated with nanocarriers for nanoprobe preparation, leading to the construction of a large variety of signal transduction strategies for the ultrasensitive immunosensing of protein biomarkers.

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Nam et al. [77] designed a biobarcode DNA functionalized Au NP probe for PSA detection by combination with magnetic beads based immunoassay system. As shown in Fig. 4.24, this system relied on magnetic nanoparticles coated with antibodies that specifically bind the target protein of PSA, Au NP probes encoded with biobar DNA, and antibodies that can sandwich the target captured by magnetic separation system. Upon sandwich immunoreaction followed by dehybridization of the oligonucleotides captured onto the magnetic nanoparticle surface, the determination of the target protein was achieved by identifying the oligonucleotide sequence released from the magnetic immunoassay system. Because the nanoparticle probe carries with a large number of oligonucleotides per protein binding event, there is a substantial amplification and PSA can be detected at a 30 amol concentration, which is six orders of magnitude more sensitive than clinically accepted conventional assays. Wang et al. [78] also designed a biobarcode DNA functionalized polystyrene nanoprobe for the electrochemical immunoassay of IgG analyte at a magnetic platform, and the signal transduction was conducted through the electrochemical detection of the acid-released purine bases at a pyrolytic graphite electrode by adsorptive chronopotentiometry. In addition, Zhang's group [79] reported the use of a biobarcode DNA functionalized Au NP probe for electrochemical immunoassay of thrombin by combination of the DNA hybridization of QD-labeled barcode for electrochemical stripping analysis. Chen's group [80] reported an ultrasensitive nonenzyme immunosensing strategy for CEA measurement by combination with the signal tracing of a biobarcode DNA functionalized Au NP probe and an in situ DNA-based hybridization chain reaction. Due to the unique mimic enzymatic property of Fe3O4 nanoparticles, Wei et al. [81] prepared poly(ethylene glycol)-poly(lactic acid) polymeric vesicles for simultaneous encapsulation of Fe3O4 NPs and signal antibody to obtain a novel nanoprobe. The high sensitivity of the nanoprobe toward electrocatalytic reduction of H2O2 provided excellent signal transduction of the electrochemical immunosensor for sandwich immunoassay of PSA. Based on the copper-mediated reaction, antibodies functionalized CuO nanoparticle probe could also be used for ultrasensitive immunoassay of protein biomarker. Based on the release of copper ion from CuO labels for click reaction, Qu et al. [82] reported a novel immunoassay method for protein measurement allowing readout by the naked eye. The Cu acts as a catalyst that induces aggregation of AuNPs functionalized with azide and alkyne groups, which can be seen as a color change. Based on the copper ion-induced quenching of the photocurrent of QDs, Ju's group [83] developed a novel PEC immunosensor for the simple and sensitive sandwich immunoassay of AFP at a CdTe QDs modified F-doped tin oxide (FTO) based immunosensor. As shown in Fig. 4.25, the detection signal is produced by dissolving the CuO NPs to release copper ions, which can induce to the formation of exciton trapping of QDs for blocking the escape of photoelectrons and thus leading to a “signal off” PEC method for sensitive immunoassay. Layer-by-layer assembly by polyelectrolytes is a simple technique for the encapsulation of different molecules with high content. By applying this method, Mak [85] prepared a high-content ferrocene microcrystals encapsulated probe, and Yu [86]

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SH

1.

2. Bovine serum albumin 3. Nanoparticle (NP) probe

1. 2. Bovine serum albumin

Magnetic microparticle (MMP) probe Gold nanoparticle

SH

Monoclonal anti-PSA

capture DNA

Polyclonal anti-PSA

(A) Step 1. Target protein capture with MMP probes

Bar-code DNA Amine-functionalized magnetic particle

Step 2. Sandwich captured target proteins with NP probes

Target protein (PSA) 13 nm NPs for Bio-bar-code PCR 30 nm NPs for PCR-less method Step 5. Chip-based detection of bar-code DNA for protein identification Ag Au

(B)

Step 4. PCR-less detection of bar-code DNA from 30 nm NP probes

Step 4. Polymerase chain reaction Bar-code DNA

Step 3. MMP probe separation and bar-code DNA dehybridization

M Magnetic field

Fig. 4.24  The biobarcode assay method: (A) Probe design and preparation; (B) PSA detection and barcode DNA amplification and identification. Reprinted with permission from J.M. Nam, C.S. Thaxtion, C.A. Mirkin, Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins, Science, 301 (2003) 1884–1886.

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hv

CB QD VB

eexciton

-

O2 e-

h+

FTO Dissolved with acid Cu2+

CB QD

hv

VB

FTO

CuO

Trapping site

Fig. 4.25  Schematic illustration of the photoelectrochemical immunoassay using the CuO NPslabeled antibody and copper ion-induced formation of exciton trapping on a CdTe/FTO electrode. Reprinted with permission from G.M. Wen, H.X. Ju, Ultrasensitive photoelectrochemical immunoassay through tag induced exciton trapping, Talanta 134 (2015) 496–500.

prepared an organic dye fluorescein diacetate (FDA) encapsulated probe. Based on the signal tracing of the probes by electrochemical and fluorescent methods respectively, they both achieved the ultrasensitive immunoassay of protein biomarkers successfully. The synthesis of silica nanoparticles in water-in-oil (W/O) can also trap high-content signal labels for nanoprobe preparation. Rusling's group [87] employed this method to prepare a [Ru(bpy)3]2+-doped silica nanoparticle probe, which was used to achieve the ultrasensitive ECL signal tracing at a SWCNT based immunosensor. The detection limit of the method was 40 pg mL−1 for PSA measurement.

4.6 Perspective The multilabel signal amplification of functionalized nanoprobes greatly improves the sensitivity of the immunosensing methods for achieving the accurate measurement of

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ultra-low levels of protein biomarkers. However, successful realization of this kind of signal amplification strategy requires proper attention to the nonspecific adsorption issue that commonly affects the analytical performance of immunosensors. Besides proper washing and surface blocking steps that should be employed to decrease nonspecific adsorption, more researches will focus on improving or controlling the surface functionalization of nanomaterials by biomolecules for enhancing the efficiency and selectivity of the biorecognition of nanoprobes. For nanoprobes to play a bigger role, it is also desirable to seek more novel nanomaterials with sufficient binding sites for functionalization, especially low-toxicity, eco-friendly alternatives such as silicon and carbon nanomaterials. For example, the mesoporous nanomaterials often possess a higher specific surface area, which can load higher content labels to improve the amplification ability of nanoprobes. Due to the diverse properties of different nanomaterials, utilizing two or more types of nanomaterials can enhance the good qualities as well as offset the insufficiency of each individual nanomaterial, which can produce better results than that using only one type of nanomaterial. Additionally, greater understanding of the properties and action mechanisms of nanomaterials and nanoprobes will not only enhance the biosensing capabilities compared to conventional methods but will also bring out new signal transduction or operation approaches for immunosensing in future. Mimicking biological signal transduction, nanomaterials-based autocatalytic systems, in which each step produces a product that acts not just as a template or as a stoichiometric trigger, but rather is a catalyst (or activates a catalyst) to produce more products, provide another opportunity for signal transduction and amplification with proper approaches. In conclusion, with the demand in life sciences and clinical diagnosis, the ultimate goal of this field is the utilization of nanomaterials to not only enhance analytical performance of immunosensing methods but also bring out new approaches such as miniaturization, reagentless biosensing, and single-molecule detection for practical applications.

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Further Reading [1] D. Bera, L. Qian, T. Tseng, P. Holloway, Quantum dots and their multimodal applications: a review, Materials 3 (2010) 2260–2345.

Chemiluminescent immunoassay

5

5.1 Introduction Chemiluminescent immunoassay (CLIA), an efficient combination of chemiluminescent system and the immunoreaction, has gained increasing attention [1–4] in different fields since it was presented at the end of the 1970s by Halmann and Velan, including life science, clinical diagnosis, environmental monitoring, food safety, and pharmaceutical analysis [5], due to its high sensitivity, good specificity, wide range of applications, simple equipment, and wide linear range. It is mainly a labeling immunoassay technology due to the extremely high detection sensitivity [6] and generally uses a chemiluminescent reagent or enzyme to label antigen or antibody. Upon the immunoreaction of the capture immunological reagent with the target, the chemiluminescent reaction produces chemiluminescent emission, and the intensity is directly proportional to the concentration of analytes in a sample. The most commonly used chemiluminescent reagents include luminol, 4-methoxy-4-(3-­phosphatephenyl)-spiro(1,2-dioxetane-3,2-adamantane) (AMPPD) and their derivatives, acridinium ester, and enzyme molecules such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP). Recently, some fluorescence dyes such as Cy5, and nanoparticles such as Au nanoparticles (AuNPs) and quantum dots (QDs) have also been used as the labels to the chemiluminescent system. Luminol and its derivatives can be oxidized by H2O2 in the presence of HRP to produce the excited species and then to emit the chemiluminescence (CL) signal, while AMPPD is decomposed to unstable intermediate in the presence of ALP to emit the CL signal. Among CLIA methods, heterogeneous CLIA has become very popular in various fields because of its much higher sensitivity in comparison with homogeneous mode [7]. Heterogeneous CLIA usually requires solid-phase materials to immobilize capture antibody probes [8]. Compared with conventional solid-phase supports, magnetic beads (MBs) have demonstrated many advantages in CLIA [9–13]: free suspension in the immunoreagents or CL substrate for rapid immunoreaction or CL reaction, large surface area to accelerate reaction and allow binding more capture antibodies for enhancing the sensitivity, easy and rapid isolation in a magnetic field, and effective concentration of trace amount of analyte when the MBs are collected. In recent years, MBs based CLIA have been widely used in clinical diagnosis, environmental monitoring, and food safety [14]. CLIA can conveniently combine with flow injection analysis, which has been proved to be a potential approach for the development of automated CLIA. This assay mode combines all the advantages of high sensitivity, high precision, high speed, and high selectivity [15]. Flow injection CLIA can be performed in both homogeneous and heterogeneous systems, especially more suitable for heterogeneous systems because the separation step can be easily performed on line in the flow injection immunoassay system [16]. Recently, it has been widely applied in many fields including Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00005-0 Copyright © 2017 Elsevier Ltd. All rights reserved.

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e­ nvironmental monitoring, food safety, pharmaceutical analysis, and clinical diagnosis [17–19] due to its small sample-cost, reduced sample handling, acceptable reusability, good reproducibility, shorter time, and easy automation for high sample throughput. Capillary electrophoresis as a power separation tool has been exploited for use in the CLIA field. Capillary electrophoresis CLIA, which combines the high separation efficiency of capillary electrophoresis with the specificity of immunoassay, has been proved to be a powerful assay technique for the separation and analysis of biological samples [20]. Compared to the conventional CLIA, this assay technique shows obvious advantages such as high efficiency, less samples, short analysis time, and easy automation. It has been successfully applied to determine tumor markers, hormones, and abuse drugs [21,22]. With the development and application of new types of solid-phase materials, automation analysis, and efficient separation technique, CLIA methods can be developed for the sensitive detection of protein biomarkers. This chapter focuses on the introduction of MBs CLIA, flow injection CLIA, and capillary electrophoresis for CLIA.

5.2 Magnetic beads CLIA MBs CLIA combines the advantages of the CLIA and MBs, and has attracted great attention in different fields over the past decades [14]. Based on the immobilization of antibodies on the surface of MBs with functional group, the MBs CLIA method can be easily developed for rapid and sensitive detection of protein markers. Employing MBs as the second antibody separation agent and the first-antibody ­solid-surface, Lin et al. [23–26] developed high sensitive CL enzyme immunoassay methods for the α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen 50 (CA 50) in human serum with high sensitivity, specificity, rapidity, and reproducibility. The double-sandwiched immunocomplex was first formed through the reaction among antifluorescein isothiocyanate (FITC) antibody coated magnetic particles (MPs), FITC-labeled anti-AFP antibody, AFP antigen, and ALPlabeled anti-AFP antibody. The subsequent CL reaction of ALP with AMPPD gave light intensity that was directly proportional to the amount of analyte present in the samples. After several physicochemical parameters, including the concentration of FITC-labeled anti-AFP antibody, the dilution ratio of ALP-labeled anti-AFP antibody, the volume of MPs and substrate, the immunoreaction time and other variables relevant to the immunoassay, were optimized, the proposed methods showed a sensitivity of 3.0 ng mL−1, low cross reactivities, and an assay time of 1 h. The linear range for AFP was 0–1200 ng mL−1 through using MPs, which was useful for samples with extremely high AFP concentrations without dilution while avoiding the hook effect. The intra- and interassay precision was