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Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes [1st ed.]
 9789811563324, 9789811563331

Table of contents :
Front Matter ....Pages i-ix
Introduction (Sai Kishore Ravi, Swee Ching Tan)....Pages 1-25
Augmenting Photocurrent Using Photoproteins of Complementary Optical Characteristics (Sai Kishore Ravi, Swee Ching Tan)....Pages 27-40
Interfacing Photoproteins with Mechanoresponsive Electrolytes for Enhancing Photocurrent and Stability (Sai Kishore Ravi, Swee Ching Tan)....Pages 41-64
Integrating the Light Reactions of a Photoprotein and a Semiconductor for Enhanced Photovoltage (Sai Kishore Ravi, Swee Ching Tan)....Pages 65-77
Role of Band-Structure Approach in Biohybrid Photovoltaics—A Path Beyond Bioelectrochemistry (Sai Kishore Ravi, Swee Ching Tan)....Pages 79-110
Prolonged Charge Trapping in Photoproteins and Its Implications for Bio-Photocapacitors (Sai Kishore Ravi, Swee Ching Tan)....Pages 111-125
Photoproteins Tapping Solar Energy to Power Sensors (Sai Kishore Ravi, Swee Ching Tan)....Pages 127-140
Bio-Schottky Semi-Artificial Photosynthetic Devices (Sai Kishore Ravi, Swee Ching Tan)....Pages 141-156
Future Directions (Sai Kishore Ravi, Swee Ching Tan)....Pages 157-166
Back Matter ....Pages 167-172

Citation preview

Green Energy and Technology

Sai Kishore Ravi Swee Ching Tan

Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes

Green Energy and Technology

Climate change, environmental impact and the limited natural resources urge scientific research and novel technical solutions. The monograph series Green Energy and Technology serves as a publishing platform for scientific and technological approaches to “green”—i.e. environmentally friendly and sustainable—technologies. While a focus lies on energy and power supply, it also covers “green” solutions in industrial engineering and engineering design. Green Energy and Technology addresses researchers, advanced students, technical consultants as well as decision makers in industries and politics. Hence, the level of presentation spans from instructional to highly technical. **Indexed in Scopus**.

More information about this series at http://www.springer.com/series/8059

Sai Kishore Ravi Swee Ching Tan •

Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes

123

Sai Kishore Ravi Materials Science and Engineering National University of Singapore Singapore, Singapore

Swee Ching Tan Materials Science and Engineering National University of Singapore Singapore, Singapore

ISSN 1865-3529 ISSN 1865-3537 (electronic) Green Energy and Technology ISBN 978-981-15-6332-4 ISBN 978-981-15-6333-1 (eBook) https://doi.org/10.1007/978-981-15-6333-1 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Photosynthesis, a process by which plants and algae convert solar energy, water, and carbon dioxide into biomass, is a key player that sustains nearly every form of life on earth. Central to this process is the photosynthetic pigment-protein complexes that transduce sunlight into biologically useful forms of energy through a photochemical charge separation that has a quantum efficiency close to 100%. Integrating these natural pigment–protein complexes in bio-hybrid architectures for solar energy harvesting is attractive due to their global abundance and environmental compatibility. Despite the ability of the protein complexes to achieve near-unity quantum efficiency in their native environments, achieving a similar performance by integrating them in device environments, without losing their integrity, has been challenging. In this book, a few different approaches to constructing bio-hybrid devices are presented that aid in achieving enhanced photocurrent and photovoltage generation using the photosynthetic protein complexes. The book targets the readership of students, academics and industrial practitioners who are interested in alternative solar technologies and is primarily a compilation of the first author’s doctoral work at the National University of Singapore. A brief introduction on the key processes in plant/bacterial photosynthesis is presented in Chap. 1 with a specific focus on the structure and function of a purple bacterial photosynthetic system. With this background, the Chapter chronicles the different possible ways of integrating the photosynthetic proteins in a device set-up and discusses the various existing ways of photocurrent enhancement under two broad classes, which are, respectively, charge-transport enhancement and light-harvesting enhancement. Based on a comprehensive review of the progress made in this area, the chapter also discusses some of the existing challenges and possible research opportunities in the field of bio-hybrid photovoltaics. A few different approaches of enhancing the photocurrent, stability and photovoltage in these bio-hybrid are discussed in the subsequent chapters (Chaps. 2–4). Firstly, the possibility of enhancing the photocurrent by improving the light-harvesting ability (or more specifically, the spectral coverage) of the device is probed. With this background, Chap. 2 demonstrates the photocurrent enhancement v

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possible by combining photoproteins of complementary absorption characteristics in a tandem device structure. Followed by this, the possibility of improving the device stability along with photocurrent enhancement is explored. Based on this, Chap. 3 discusses the role of a mechanoresponsive electrolyte in preserving the protein stability in the device. The chapter highlights the mechanism of charge conduction in different photoprotein systems and demonstrates the possibility of generating high photocurrents in protein multilayers. While a vast majority of works in the literature focusses only on photocurrent enhancement methods, approaches for enhancing the photovoltage in photoprotein-based bio-electrochemical devices is often scarce. Throwing light on this research gap, Chap. 4 discusses the possibility of photovoltage enhancement in a new hybrid system that cascades the photo-generation process in the photoprotein with that of a semiconductor. With the importance of the choice of material bandgap highlighted in Chap. 4, a comprehensive review of how the band-structure approach could be used in bio-photovoltaics is presented in Chap. 5. As protein multilayers were found to be favourable both for the photocurrent and photovoltage generation, Chaps. 3 and 4 employed dense films of protein multilayers. To cast more light on the fundamental light-harvesting properties of the protein multilayers, a systematic study on thick protein films without electron transfer mediators has been carried out. This forms the basis for Chap. 6, which discusses the new phenomenon of prolonged charge trapping in photoprotein multilayers with direct microscopic evidence. This chapter outlines the effect of concurrent charge photo-generation and storage in protein multilayers and demonstrates the effect in a device setting. Chapter 7 throws light on the possibility of using photoproteins as a power source in hybrid tactile sensors and photosensors and Chap. 8 talks about synergetic effects in a semi-artificial photosynthetic device. Chapter 9 presents a short account of the possible future directions which is bio-photoelectrochemical cells, and self-powered sensors and human–machine interfaces. Singapore

Sai Kishore Ravi Swee Ching Tan

Acknowledgements We would like to thank our collaborator, Dr. Mike Jones (University of Bristol) whose support and insight to the research projects have been pivotal to the formation of this book. We would also like to extend our sincere appreciation to all the students and researchers of Swee Lab, National University of Singapore, for their contributions, and Ministry of Education, Singapore, for the financial support to our research projects.

Contents

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1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Prospects of Photosynthetic Proteins in Solar Energy Harnessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Major Phases of Photosynthesis . . . . . . . . . . . . . . . . . . 1.1.2 Key Photosynthetic Processes/Subunits for Biohybrid Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3 Structure and Function of Purple Bacterial Photosynthetic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Ways of Integrating Proteins in Devices . . . . . . . . . . . . . . . . . 1.3 Manipulation of Electron Transfer Processes . . . . . . . . . . . . . . 1.3.1 Effect of Protein Orientation . . . . . . . . . . . . . . . . . . . . . 1.3.2 Effect of Electron Transport Mediators . . . . . . . . . . . . . 1.3.3 Pigment Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Enhancement of Light Harvesting Ability . . . . . . . . . . . . . . . . 1.4.1 Increased Protein Loading . . . . . . . . . . . . . . . . . . . . . . 1.5 Scope for Further Research . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Research Trend and Gaps . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7.1 Stability Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7.2 Discrepancies in Excitation Light Intensity . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2 Augmenting Photocurrent Using Photoproteins of Complementary Optical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Construction and Working of Bio-Tandem Cells . . . . . . . . . . . 2.3.1 Photoproteins with Red or Green Carotenoid Pigments . 2.3.2 Device Fabrication and Characterization . . . . . . . . . . . . 2.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3 Interfacing Photoproteins with Mechanoresponsive Electrolytes for Enhancing Photocurrent and Stability . . . . . . . . . . . . . . . . . . 3.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Construction Protocol for Bio-Hybrid Cells with Phase-Changing Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Isolation of Photoproteins . . . . . . . . . . . . . . . . . . . . . . . 3.3.2 Protein Deposition and Characterisation . . . . . . . . . . . . 3.3.3 Synthesis and Characterisation of the Electrolyte . . . . . . 3.3.4 Device Fabrication and Photochronoamperometry . . . . . 3.3.5 Cell Fabrication Using Mechano-Responsive Electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 The Mechanism of Photocurrent Generation . . . . . . . . . 3.4.2 Structure and Mechanism of RC-LH1 Complexes . . . . . 3.4.3 Factors Limiting Photocurrent Density in RC-LH1 Cells . 3.4.4 Mechanism of Electron Donation to the Working Electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.5 Charge Conduction Through RC Multilayers . . . . . . . . . 3.4.6 Stability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 Integrating the Light Reactions of a Photoprotein and a Semiconductor for Enhanced Photovoltage . . . . . . . . . . . . 4.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Construction and Working of Protein-Semiconductor Hybrids . 4.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5 Role of Band-Structure Approach in Biohybrid Photovoltaics— A Path Beyond Bioelectrochemistry . . . . . . . . . . . . . . . . . . . . . . 5.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Review of Types of Photosynthetic Proteins . . . . . . . . . . . . . . 5.3 Photo-Bioelectrochemical Cells . . . . . . . . . . . . . . . . . . . . . . . 5.4 Band-Structure Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Interfacial Electronic Structure in Biophotovoltaics . . . 5.4.2 Bioinspired OPVs and OPV-Inspired BPVs . . . . . . . . . 5.5 New Design Principles in Biophotovoltaics . . . . . . . . . . . . . . 5.5.1 Photoprotein Bulk Heterojunction Cells . . . . . . . . . . . 5.5.2 Photoprotein-Semiconductor Hybrid Cells . . . . . . . . . . 5.5.3 Photoprotein Plasmonic Cells . . . . . . . . . . . . . . . . . . . 5.5.4 Photoprotein Tandem Cells . . . . . . . . . . . . . . . . . . . .

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79 79 79 81 82 83 87 91 91 95 98 99

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5.6 Directions for Future Bio-Photovoltaics . . . . . . . . . . . . . . . . . . . . 102 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 6 Prolonged Charge Trapping in Photoproteins and Its Implications for Bio-Photocapacitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Biophotonic Power Cell: Construction and Photovoltage Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Origin of Voltage Build-up . . . . . . . . . . . . . . . . . . . . . 6.3.2 Kelvin Probe Microscopy: Surface Potential Build-up and Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3 Microscale and Macroscale Capacitance Measurements . 6.4 Proof-of-Concept Demonstration . . . . . . . . . . . . . . . . . . . . . . . 6.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7 Photoproteins Tapping Solar Energy to Power Sensors . 7.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Self-powered Tactile Sensors . . . . . . . . . . . . . . . . . . . 7.4 Bio-hybrid Tactile Sensors Powered by Photoproteins References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8 Bio-Schottky Semi-Artificial Photosynthetic Devices . . . . . 8.1 Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Establishment of an In-Plane Potential Gradient on a Bio-Schottky Electrode . . . . . . . . . . . . . . . . . . . . 8.4 Enhanced Charge Transfer by Thixotropic Electrolytes . 8.5 Synergistic Photo-Electric Effect in Bio-Schottky Cells . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Enhancement of In Vitro Protein Stability . . . . . . . . . . . 9.2 Understanding the Energy Level Alignment and Electron Transfer Pathways in Biohybrid Devices . . . . . . . . . . . . 9.3 Future Sensors and Human-Machine Interfaces . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Appendices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Chapter 1

Introduction

1.1 Prospects of Photosynthetic Proteins in Solar Energy Harnessing The diminishing reserves of fossil fuels and the environmental concerns in extracting the carbonaceous fuels from the earth’s crust necessitate our independence from the non-renewable energy resources [1–3]. A promising alternative to these fuels is to make use of carbon-free and profusely available solar energy [1–3]. Solar energy is harvested by different approaches namely direct solar energy—electricity conversion, solar energy—chemical energy conversion and solar energy—thermal energy conversion [4]. The first approach includes different types of solar cells while the second includes photoelectrochemical water splitting [5] for hydrogen production [6] and photoelectrochemical/photocatalytic reduction [7] of CO2 to liquid fuels. These two approaches have witnessed considerable improvements due to bioinspiration [8, 9]. One of the major developments in the domain of solar cells was Michael Gratzel’s work on Dye-Sensitized Solar Cells (DSSCs) [10–13]. As opposed to the conventional Si solar cells, where the semiconductor performs both the tasks of light absorption and charge carrier transport, the functions in the Gratzel cell are decentralized and the light absorption is performed by an organic sensitizer (dye) held in a mesoporous and nanocrystalline scaffold [10–13]. This design of a DSSC is an analog of natural photosynthesis, where the function of chlorophyll is adopted by the synthetic dye and a cyclic electron flow as in photosynthesis has been facilitated by a redox mediator [8, 9]. Nevertheless, DSSCs with organic ionic conductors that function both as a light-harvester and redox mediator have also been reported [14]. The domain of solar-fuel generation also has similar bioinspiration, where the water-oxidation catalysts used in fuel cells are analogs of the oxygen evolving complex present in photosystem II of higher plants [8]. Bioinspiration is emerging as a promising approach in engineering sustainable devices, as the biological systems and mechanisms, in the process of continuing © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_1

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1 Introduction

evolution, have been engineered by nature to be highly efficient, making them worthy models for design and engineering [15]. Some microbes and plants found in nature have a superior system and mechanism for light harvesting and energy conversion. Their quantum efficiencies [16] are higher than that of the man-made solar cells. [17– 19] Photosynthesis is an exemplary model for solar cell research as it is the prime mover powering the biological world, the mechanism behind the energy storage in fossil fuels and the sustainer of the earth’s oxygenated atmosphere [20]. The architecture and the internal circuitry of the photosynthetic systems are very sophisticated that the initial light-driven steps have ≈ 100% quantum efficiency [17, 21, 22]. Bioinspiration of photosynthesis in solar cells has instigated novel research perspectives which are, in close pace, moving towards devising a high-efficiency solar cell. Artificial photosynthesis is one novel approach that tries to emulate the natural photosynthetic systems by employing intricate biomolecular complexes to execute the light harvesting and charge separation [23]. Extensive research is being done to make synthetic complexes that match the sophistication and functions of the natural photosynthetic complexes. Though there is good progress in the supramolecular research emulating natural complexes, the mimics are still a far cry from the molecular circuitry of the photosynthetic protein complexes [24, 25]. This limitation thus gives rise to a new idea of making hybrid or semi-artificial [26] devices involving the biomolecular complexes effectively interfaced with manmade materials [27]. This research perspective has gathered much interest in the recent years and there is good progress in the number of research studies (Fig. 1.1) utilizing the photovoltaic abilities of natural photosynthetic systems for various device applications like solar cells, photodetectors, biosensors and solar fuel cells [27].

1.1.1 Major Phases of Photosynthesis In general, at the first phase of photosynthesis, photon/light absorption takes place aided by photosynthetic pigments namely chlorophyll and a few other accessory pigments like carotenoids [30]. It is the architecture or the arrangement of these pigments that greatly supports the feasibility of photosynthesis rather than just the chemistry of the pigments [30]. Considering the intensity of solar light and the dimensions of the pigment molecules, it has been estimated that a single chlorophyll molecule can only absorb ten photons per second, thus signifying the need for a welldesigned arrangement of pigment molecules apposite for an efficient photon absorption process [30]. It is an interesting fact that the photosynthetic pigments perform different roles at different sites of the photosynthetic apparatus. Some pigments are involved in a photophysical action (light absorption) while some carry out photochemical reactions. It was found that only less than 1% of the pigments in the photosynthetic apparatus are photochemically active and the rest of the pigments

1.1 Prospects of Photosynthetic Proteins in Solar Energy Harnessing

3

Fig. 1.1 Research trend in the field. Number of publications on photosynthetic biocomplexes utilized for solar energy harvesting. The plot includes the studies on photosynthetic apparatus from photosynthetic bacteria (RCs/RC-LH1/other subsystems/whole cell), higher plants (photosystems) and photosynthetic algae (subsystems/whole cell) for photovoltaic, solar fuel and sensor applications [28, 29]

are involved only in the light absorption [30, 31]. The photon absorption creates an electronic excited state in an antenna pigment molecule, which is migrated from one molecule to the other and finally trapped within a site called the RC by various mechanisms [30]. The arrangement of pigments can often be referred to as an antenna system, as the arrangement enables collection and concentration of light suitable to facilitate an energy conversion [30]. The photophysical properties of the pigments are finetuned by nature by an elegantly engineered biopolymer material called protein that offers the biological systems the required degree of specificity, efficiency and control to accomplish a biological function [28]. Thus, the antenna systems exist as an assemblage of pigment and protein where the photosynthetic pigments are bound to the proteins in highly specific associations to ensue in an effective light absorption [30, 31]. The antenna complex concentrates the collected light energy to the RC where the photochemical reaction takes place [30, 31]. The structure of the antenna complex is not unique, because different photosynthetic organisms, as a part of evolution, adapt their light gathering systems in different ways to suit their environments [32]. The mechanism of energy transfer to RCs varies with the type of antenna system (the type depends on the relative arrangement of pigments with the lipid membrane) present in the photosynthetic apparatus of an organism [27]. In principle, the function of an antenna system, regardless of the species, is to efficiently absorb the light energy and efficiently deliver it to the RC. The RC is a complex organization of pigment

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molecules and redox active cofactors held in a precise three-dimensional configuration by a protein scaffold [33]. The RC is embedded in a photosynthetic membrane as a multi-subunit protein complex containing chlorophyll and other cofactors, with the extremely hydrophobic peptides threading the membrane back and forth [30]. The general process that occurs in an RC is presented in Fig. 1.2. The pigments in the RC are chemically identical to those in the antenna complex but the environment in the RC renders these pigments fit for photochemical functions [30]. In general, the photochemistry is affected by a special dimer of pigments (P) in the RC that acts as the primary electron donor for the subsequent electron transport process and it is into this dimer the antenna complex concentrates the light energy, making it electronically excited [30]. As the excited electronic state is a highly reducing agent, it rapidly loses an electron to an acceptor molecule (A) and generates an ion pair state P+ A− [30]. In this primary process of photosynthesis, the electronic excitation energy is transformed into a chemical redox energy which is highly prone to be lost as heat, as the physical proximity of the highly oxidizing species P+ with the highly reducing species A− may easily deem a backflow of electrons to P+ from A− [30]. But such a recombination is tactfully avoided by nature through a series of extremely expeditious secondary reactions that spatially separate the positive and negative charges [30, 32]. In anoxygenic phototrophs, RCs exist as structurally separated operational units whereas, in oxygenic phototrophs, RCs exist as an integral part of larger complexes called photosystems which are capable of oxidizing water [31, 32]. There are two different photosystems namely PS I and PS II; both of which are found only in

Fig. 1.2 General electron transfer scheme in photosynthetic reaction centers. Light excitation promotes a pigment (P) to an excited state (P*), where it loses an electron to an acceptor molecule (A) to form an ion-pair state P+ A− ; secondary reactions separate the charges, by transfer of an electron from an electron donor (D) and from the initial acceptor A to a secondary acceptor (A ) [30]. This spatial separation prevents the recombination reaction. [30] (Adapted with permission from Ref. [29, 30])

1.1 Prospects of Photosynthetic Proteins in Solar Energy Harnessing

5

oxygenic phototrophs like cyanobacteria, algae, and plants, where they carry out the oxygenic photosynthesis by a coordinated sequence of actions [34].

1.1.2 Key Photosynthetic Processes/Subunits for Biohybrid Devices Photosynthesis in organisms involve a series of processes led by different components described above, but only the initial steps involving the light absorption and charge separation are essentially the most useful phases for solar cell applications. The primary stages of photosynthesis involve a sequence of actions starting with photon absorption which is an extremely swift process taking place in a few femtoseconds followed by fast photochemical reactions (few ns to ps) and electron transport processes (few μs) [30, 35]. For biohybrid solar cells, the biocomplexes (RCs or photosystems) are intended to perform only these initial phases of photosynthesis in a manmade material environment. The initial steps are then followed by much slower biochemical (few ms) and physiological and ecological reactions involving synthesis and transport of stable products spanning a few seconds, which are not of direct use for solar cells though of some use in biofuels [30, 35]. The types of photosynthetic biocomplexes that can be used for solar energy harnessing have increased in the recent years thanks to the advancements in biochemistry and genetic engineering which have made it possible to extract different functional units of photosynthetic apparatus from different species and to improve their functions by genetic modifications. The RCs are some of the most widely studied photosynthetic components for employment in solar cells. While some studies use only the core RCs for the purpose, there are also a few studies on using the RCs with the surrounding light harvesting complexes, in view of achieving improved performance [21, 27, 36–42]. Photosystems I and II are also studied for photoelectrochemical applications. Some biochemical separations from photosystems are also being used, an example of which is the membrane fragments of PS II called PSII particles (BBY- or KM-) [43]. Biocomplexes of a higher structural level like chloroplasts [44] and chromatophores [45, 46] are also used in biohybrid devices. Apart from utilizing the function of a protein biocomplex in a device, it has also been a well-known approach to use the photosynthetic pigments like chlorophyll and its derivatives in devices like DSSCs [47–50].

1.1.3 Structure and Function of Purple Bacterial Photosynthetic Proteins The RCs in anoxygenic phototrophs like purple bacteria have a simple and wellunderstood structure (Fig. 1.3) compared to the photosystems of cyanobacteria and

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1 Introduction

Fig. 1.3 RC and RC-LH1—structures and cofactors. a Structure of RC—The ten cofactors (spheres) are held in place by a scaffold consisting of PufL and PufM (ribbons) [33]. b The BChl, BPhe and ubiquinone cofactors (sticks) form two membrane-spanning branches. Mg atoms of BChl and non-heme Fe are shown as spheres; the arrows show the route of electron transfer [33]. c Exploded view of component parts [55]. d and e Structures of RC-LH1—The central RC is surrounded by an LH1 antenna pigment-protein comprising an inner ring of 16 α-polypeptides (cyan ribbons) and an outer ring of 16 β-polypeptides (magenta ribbons), each of which has a single membrane-spanning α-helix; sandwiched between these concentric LH1 protein cylinders is a ring of 32 BChls (shown as spheres alternating red/orange) [41] (a, b and d—Reproduced with permission from Ref. [33]; e—Reproduced with permission from Ref. [55]; e—Reproduced with permission from Ref. [41]) [29]

higher plants [51]. Though the structures of photosystems are more complex than RCs, there are some structural similarities between the RCs and the photosystems that facilitate a better understanding of the highly intricate photosystems [51]. It has been found that the PS II shares some similarities with the RC in purple bacteria (i.e. RC Type II) while the PS I resembles some structural aspects of the RC in green sulfur bacteria (i.e. RC Type I) [52–54]. In a strict sense, an RC has to be defined as a minimal unit capable of photochemical charge separation between the primary electron donor and the primary electron acceptor which is then followed by stabilization of the separated charges [43]. The photosystems are sometimes referred to as reaction centers, but the minimal unit responsible for the photochemistry is often not easily isolatable [32, 43, 52]. Photosystems are in a way different from RCs as they are not the minimal units performing the photochemical reactions, being a complex assemblage of several constituents like pigments, antenna, and proteins that are present in addition to the core system primarily carrying out the photochemistry [43].

1.1 Prospects of Photosynthetic Proteins in Solar Energy Harnessing

7

Several attempts have been made to biochemically separate the smallest structural units from the photosystems to make them as simple as RCs but they pose a few functional limitations [43]. Since the RCs of purple bacteria have been extensively studied and are devoid of some of the structural complexities found in the photosystems, understanding their in vitro behavior becomes more promising for various device applications. These RCs are also known to be more robust than those found in the photosystems of algae and higher plants [33]. Though in a true sense, the photosystems are not to be classified as RCs, they have nevertheless been researched for use in bio-hybrid devices over years and are discussed in a few recent reviews [56, 57]. A comprehensive elucidation of the structural model and characterization of bacterial RCs deciphering their functions and mechanisms is available in several research articles [16, 33, 51, 52, 55, 58, 59]. One of the widely used purple bacterial RCs for photovoltaic applications is that of Rhodobacter sphaeroides. The RC complex of this bacteria contains three polypeptides namely H, L and M that encase ten cofactors which are namely four Bacteriochlorophyll (BChl), two Bacteriopheophytin (BPhe), two ubiquinone molecules, a photoprotective carotenoid and a non-heme iron atom [30]. The structure of RC and the arrangement of its subunits and cofactors are often described with respect to the photosynthetic membrane of which the RC was inherently a part of and from which it has been isolated. The structure of the membrane is available in the literature [30] and is not discussed here. The two polypeptides L and M also referred to as PufL and PufM, are arranged around an axis of 2-fold rotational pseudo-symmetry that runs perpendicular to the plane of the photosynthetic bilayer membrane [30, 55] and form a scaffold that holds the cofactors in a precise configuration [33, 55]. The BChl, BPhe, and the ubiquinone molecules are arranged at the interface of the L and M polypeptides in two membrane spanning branches named A and B that are related by a twofold pseudo-symmetry [33, 55]. The cofactors that are located in the A and B branches are denoted by a subscript A and B respectively. Near the periplasmic side of the membrane, two of the BChl molecules form a dimer called ‘special pair’, the two closely spaced and excitonically coupled molecules of which are called PA and PB , as they are located in the A and B branch respectively, which are shown by the yellow carbons in the Fig. 1.3 [33, 55, 60]. Near the special pair there exist two monomeric bacteriochlorophyll molecules named BA and BB which are often called as accessory bacteriochlorophylls, depicted by the green carbons in the Fig. 1.3 [33, 55]. These are then followed by the two BPhes (HA and HB ) and the two quinones (QA and QB ). The single carotenoid is embedded in M-polypeptide, adjacent to BB [33, 55]. The non-heme iron is located right on the symmetry axis between the two quinones [33, 55]. The structure of RC and the location of its cofactors is shown in Fig. 1.3a, b respectively. Though it is possible to isolate the RC as a discrete fully-functional photochemical unit, the so-called “RC-LH1 core complex” having an LH1 antenna pigment protein encircling the RC (Fig. 1.3d, e), found to naturally exist in all characterized photosynthetic purple bacteria, is promising to enhance the light absorption and hence the photoelectric performance in devices [33, 41]. In Rhodobacter

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1 Introduction

sphaeroides the LH1 antenna consists of a large number of polypeptides that are basically of two types named α and β, each of which has a single transmembrane helix together with BChl and carotenoid pigments, which performs the function of light absorption [33]. In nature, the LH1 protein forms a hollow cylinder that only partially surrounds the RC due to the presence of a PufX polypeptide [41, 61], which can be removed by genetic engineering to obtain a more thermally stable RC-LH1 that has an enlarged LH1 antenna completely surrounding the central RC core [41]. When light is incident on the RC complex, the photochemical process begins by the formation of a singlet excited state of the BChl special pair [55, 60]. In the case of RC-LH1, the light is first absorbed by the BChls and the carotenoids of the LH1 protein and the excitation energy is passed to the BChl special pair (P) that acts as a trap for the electronic energy in the RC core, thereby forming an excited singlet state P* . In RCs without LH1 the P* is directly formed by the light absorption of BChl molecules present in the special pair [55, 60]. The P* formed is a strong reducing agent and acts as the primary electron donor that transfers an electron to the adjacent BA molecule forming the P+ B− A radical within 3 ps. The in about 1 ps and further transferred electron is then passed to HA forming P+ H− A in approximately 200 ps [33, 60]. This is followed by a rather to QA forming P+ Q− A slow charge separation process in which the electron is transferred to the secondary quinone QB in a span of several microseconds aided by a few other electron or proton carriers [60]. The formation of such a charge separated state P+ Q− B is the key principle utilized by the photosynthetic biohybrid solar cells for the photocurrent generation. A way of achieving this is by reduction of the photo oxidized special pair P+ by an electrode with the electrons being delivered to the counter electrode by an electrolyte containing an electron mediator, thereby generating a photocurrent. Different approaches have been attempted in the past few decades in devising an efficient method by which the separated charges are utilized for photocurrent generation in these photoelectrochemical cells.

1.2 Ways of Integrating Proteins in Devices One of the earliest attempts of incorporating reaction centers in solar cells was by Janzen and Seibert in 1980 when they constructed a photoelectrochemical cell with RCs coated on SnO2 coated glass electrode [62, 63]. By modification of the electrode and electrolyte, the electron transfer processes were improved to achieve a photocurrent of ≈0.5 μA/cm2 [62, 63]. In these works, RCs were immobilized by a rather simple method of dipping the electrode in a concentrated suspension of RCs thereby the RCs are physically adsorbed to the electrode when dried [62, 63]. Although no attempt was made to orient the RCs in these pioneering works, it had been predicted that a better photoresponse could be possible with controlled orientation [62, 63].

1.2 Ways of Integrating Proteins in Devices

9

A number of perspectives then engendered to control RC orientation, one being the widely used method of making SAMs. Oriented immobilization of RC monolayers on platinum electrode [64] and pyrolytic graphite electrode [65] modified by organic functional groups has been studied. With the use of bifunctional agents with condensed aromatic groups and cysteine thiol groups, the photocurrent was found to increase greatly as the RCs were oriented by these linkers [65]. Different bifunctional agents have been used to establish site specific binding of RCs with electrodes [66, 67]. The importance of selecting the chemical linker has also been realized in effectively adsorbing RCs on electrode [36]. The terminating group in the chemical linker used for the SAM had been found to affect adsorption of RC complexes on electrode [36]. The complexes exhibited a higher stability and were well adsorbed to a gold electrode with amino group terminated SAM while they were partially stable with carboxyl ended chemical linkers and were found to be greatly denatured when SAM with terminal methyl groups was used [36]. Langmuir Blodgett (LB) method was widely being used to coat electrodes with RC layers and attempts were made to control the orientation of the RCs on the electrodes [68–72]. The LB method involves consecutive crossing of an air-water interface by which a compact monomolecular layer of amphiphilic molecules is coated on the substrate with a well-defined molecular arrangement and orientation [73]. Artificial lipid bilayers called liposomes were also used to immobilize the proteins on the electrode as the liposome film on the electrode can provide nearly native environment for the proteins [73]. The drawbacks of physical and chemical binding were overcome by precoating the electrode with a Self Assembled Monolayer (SAM), where an ultrathin ordered film is prepared based on spontaneous molecular assembly using bifunctional reagents thereby providing an easy way to control the orientation and conformation of protein on the electrode surface [73, 74]. Attaching a genetically engineered poly-histidine (His) tag to RC has been found to be of great use to control the orientation of RCs on the electrode surface [75]. This is generally achieved by coating the electrode with a commercially available Ni2+ resin called Ni-NTA (Ni-Nitrilotriacetic acid) which has a very high affinity to His-tagged proteins [25, 36, 76, 77].

1.3 Manipulation of Electron Transfer Processes 1.3.1 Effect of Protein Orientation The need for oriented RC layers on electrode for improved performance has been felt even in the earliest attempts of RC immobilization in solar cell but the photoelectric performances of different orientations were not extensively studied until recently. Two opposite RC orientations, one with primary donor (P-side) facing the electrode

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1 Introduction

Fig. 1.4 Effect of protein orientation. a Two possible ways of RC binding and ET pathways between RC and electrode. P-primary electron donor (special pair), B-monomeric bacteriochlorophyll, Hbacteriopheophytin, QA and QB -primary and secondary electron acceptors (quinones) (Adapted with permission from Ref. [78]). b Photoinduced- and dark-electron transfers in RC-Cyt-SAMGold electrode (Reproduced with permission from Ref. [79]). c Different orientations of RC with cytochrome c on NTA SAM modified electrode [29]

and the other with the acceptor (H subunit side) facing the electrode have been elaborately studied [78]. The two orientations of the proteins on a carbon coated gold grid electrode are shown in Fig. 1.4a. In the first orientation, the RC is bound to the electrode by means of a bifunctional linker with one end having an NTA group charged with Ni2+ suitable to bind with the His tag of RC and the other end having a pyrene group to attach to the carbon electrode and in the second type, N-(1-pyrene)iodoacetamide was used as a linker that binds to the RC H-subunit through a single cysteine group [78]. Ubiquione-10 and cytochrome-c (reduced by Na2 S2 O4 ) were used as diffusible electron transfer mediators. Two profound conclusions were drawn from the photoelectric studies of these two orientations: (1) The photosynthetic RCs act as photo rectifiers making the photocurrent always flow in one direction i.e. from the primary donor to the primary acceptor. This applies well to both the orientations in Fig. 1.4a, where photocurrent is anodic if the H subunit side faces the electrode and cathodic if the P side faces the electrode. (2) The orientation of RCs with the P side bound to electrode exhibits a higher photocurrent and reaches the photochemical steady state approximately an order of magnitude faster than that orientation with the H-subunit bound to electrode [78]. Reasonable explanations were put forth for the decreased photocurrent observed in the case of H subunit facing the electrode. The difference in surface coverage of

1.3 Manipulation of Electron Transfer Processes

11

RCs could not be a reason as equally high surface coverage of RCs was ensured for both the orientations [78]. Though the length of the bifunctional linker used to bind H subunit to the electrode is shorter in length (4 Å) than that used to bind P-side to the electrode (12 Å), the electron transfer efficiency in the former orientation is lesser [78]. However, X-ray crystallographic studies revealed that the actual distance between the final electron acceptor and the electrode bound to H subunit is 28 Å considering both the thickness of H subunit (24 Å) and the linker length (4 Å) [78]. It has been estimated that a variation of 20 Å in the distance between the electron donor and the electron acceptor in a protein would change the electron transfer rate by 1012 fold [80]. Thus, the mediocre performance of the orientation with H subunit bound to the electrode is attributed to the higher electron tunneling distance between the electron acceptors and the electrode, owing to the presence of the relatively thick H subunit [78]. A similar account on the importance of the protein orientation to minimize the distance of electron transfer pathway is elucidated by Kondo et al. [36] with the study of photoelectrodes modified by RC-LH1 isolated from Rhodopseudomonas palustris, where the orientation of RC complex with the H-chain facing the electrode was found to be more favourable than the opposite orientation [36]. The effect of the dependence of electron transfer kinetics on the distance between the electron acceptor and the electrode has been systematically studied using a series of MHisRCs modified Ni-NTA SAM-coated gold electrodes having SAMs of different thicknesses with the number (n) of methylene units in the liker molecule (n = 3, 6, 10 and 15) being the measure of SAM thickness (Fig. 1.4b) [79]. In the photoelectric measurements, two electron transfer mediators were added namely Ubiquinone (Q2 ) that acts an electron acceptor and cytochrome c that acts as an electron donor and also serves as a conductive wire in coupling the working electrode with the RC’s special pair [79]. The photoelectric studies proved a significant dependence on linker lengths and thus the SAM thicknesses. The photocurrent was found to be independent of the distance (linker length) when the RCs are at shorter distances from the electrode and it decreases to a great extent with distance from the electrode. A maximum steady state photocurrent of 167 A/cm2 was observed when 7-carboxyheptyl disulphide acid having 6 methylene units was used as the linker. The linkers of lengths 3 and 10 methylene units yielded photocurrents of 161 and 158 nA/cm2 which are still close to the maximum photocurrent obtained, but there was a drastic decrease in the photocurrent to about 25 nA/cm2 when the linker length was 15 methylene units. The study highlights the importance of protein’s proximity to the electrode to achieve a sound RC-electrode junction for high photoelectric efficiency [79]. These observations are also in congruence with the photoelectrochemical cell reported by Kondo et al. [36] where the photoelectric performance of RC-LH1 immobilized on a gold electrode coated with SAMs of different linker lengths of alkanethiols NH2 (CH2 )n SH (n = 2, 6, 8, 11) was studied [36]. The photocurrent was found to be maximum for the linker length n = 6 and decreasing with increasing linker length [36]. The photocurrent decrease was due to the increase in the distance between the electrode and the RC-LH1 with the maximum current at a separation distance of

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1 Introduction

1 nm corresponding to the linker length n = 6 and lower currents for n = 8 and 11 that resulted in higher separation distances of 1.4 and 2.1 nm respectively [36]. It is interesting to note that the maximum photocurrent was not observed at the lowest linker length (n = 2) as the adsorption of the RC complexes to the electrode was poor. It was found that the adsorption of the complexes increased with increasing linker length which being a conflicting condition for improving the electron transfer efficiency, a trade off was essential and was achieved at a linker length of 6 methylene units. The effectiveness of cytochrome c in improving the electrical coupling of RC and electrode has been studied. An Ni-NTA SAM-coated gold electrode was used to immobilize the RCs with His-tagged M subunits [17]. A time-dependent improvement in photocurrent was observed on addition of cytochrome c to the electrolyte and the photocurrent increased 20–40 times higher than the initial value after a few minutes of incubation which was observed to occur with both the oxidized and the reduced forms of cytochrome c [17]. Cytochrome c was assumed as sitting on SAM surface and the RC was assumed to have a single point of contact with the SAM. In the absence of cytochrome, three possible RC orientations were explained (Fig. 1.4c). In the first case, RC was assumed to lie on the SAM surface. In the second case, RC was assumed to be oriented to SAM in the same way if cytochrome were present whereas in the third case, RC was assumed to stand on the surface of SAM with its primary donor facing the SAM surface [17]. With the assumption that the open area around RC was surrounded by water in all the three cases, it was found that the electron transfer could be possible in the absence of cytochrome only when the RC is close to the standing position [17]. The addition of cytochrome c was found to offer a shorter electron tunneling path and a more effective electron transfer, thus improving the photoelectric performance acting as a conductive wire connecting the RC’s special Pair P and the electrode [17]. Further to this study, Mahmoudzadeh et al. [81] reported the study of photoelectric performances of different configurations of RC modified photoelectrodes with varying RC-electrode distances, in light of their electron tunneling probabilities and mechanisms [81, 82].

1.3.2 Effect of Electron Transport Mediators The selection of electrolyte and its constituents are also vital for the photoelectric performance but by and large, the addition of appropriate electron transfer mediators have been found to significantly enhance the electron transfer efficiency and hence the photocurrent. The use of ubiqinone-10 as a diffusionally mobile electron transfer mediator in the solution was also found to improve the electron transfer between the RC and the electrode thereby increasing the photocurrent [64, 65]. Cytochrome c, ferrocene [83] and methyl viologen [36] have also been used for the purpose. It is a general approach to improve the electron transfer by adding two electron transfer mediators to the electrolyte, one acting as an electron donor and the other acting as

1.3 Manipulation of Electron Transfer Processes

13

an acceptor. Ubiquinone and cytochrome c are often added to the electrolyte to serve as an electron acceptor and donor respectively [19, 78, 79, 81]. Takshi et al. [83] studied the photoelectric performance of a photoelectrochemical cell with the RCs dissolved in the electrolyte. Since it was found that a significant fraction of electron transfer occurs through electron transfer mediators even when the RCs are directly attached to the electrode, this study attempted to improve the performance by making the charge transfer fully diffusion controlled and without the use of any direct electron transfer as the RCs are not bound to the electrode [83, 84]. Two electron transfer mediators namely ferrocene and methyl viologen that were added to the electrolyte prevent the charge recombination by effecting faster redox reactions and transfer the charges to the electrodes [83]. The reactions involve oxidation of Cp2 Fe to Cp2 Fe+ by an electron transfer from ferrocene to P and reduction of MV2+ to MV+ by the electron transfer from QB to methyl viologen [37, 83]. Unlike the observation in the above-discussed work, the RCs added to the electrolyte were still found to adhere to the electrode without the use of any chemical linkers or tags. Employing a single redox mediator can achieve this direct electrical contact between the RCs and the electrode which may be understood as an electrolyte that by itself also a good electron transfer mediator [41]. The photoelectrochemical cell employed a mixture of the protein and the mediator N,N,N ,N -tetramethylp-phenylenediamine (TMPD). The FTO electrode acted most preferentially as the working electrode as the RCs being hydrophilic in nature tend to adhere more to the hydrophilic FTO electrode than to the relatively hydrophobic Pt electrode [41]. Thus, a majority of RC-LH1 complexes were believed to bind to the FTO electrode with any of the two possible terminals of the complex and the electrons were shuttled from the other terminal of the complex to the Pt counter electrode [41]. The choice of electrolyte and electron transfer mediators also play a major role in the photocurrent generation and it was demonstrated that a ~30-fold increase in the open circuit voltage is possible by a simple manipulation of the electrolyte connecting the protein to the counter electrode, with an approximately linear relationship being observed between the vacuum potential of the electrolyte and the open-circuit voltage [42]. It was for the first time that the RCs were used in an electrolyte-less energy device when Rupa Das et al. reported the photoelectric performance of an RC immobilized solid state set up [25]. Surfactant-like peptides [85–88] have been used to stabilize the RC complexes and a subnanometer thick layer of the amorphous organic semiconductor has been deposited in between the RC and the metal electrode to serve as a solid state antenna enhancing the light absorption [25]. Rupa Das et al. constructed an RC based solid state electronic device with a conductive ITO-coated glass electrode, coated further by a nanolayer of the gold anode (with a Cr adhesion layer in between Au and ITO) to which MHisRCs are oriented by an Ni-NTA SAM on the gold surface [25]. The RC layer is further coated by a preferentially electron transporting fullerene C60 followed by a layer of bathocuproine (BCP) and finally by a layer of silver that acts as a cathode. This solid state device has exhibited the highest photocurrent of 0.12 mA/cm2 under an excitation intensity of 10 W/cm2 [25].

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1 Introduction

1.3.3 Pigment Substitution Highly enhanced photocurrent was possible using pigment exchanged RC adsorbed onto the nanostructured WO3 –TiO2 matrix [73, 89]. A pigment replaced RC mutant containing spinach pheophytin in place of bacteriopheophytin was used to alter the energetics and kinetics of the electron transfer process. Such RCs (RC-Phe) entrapped in the nanoporous electrode exhibited a significant delay in the excitation transfer and the relatively slower charge separation was attributed to the higher energy level of P+ Phe− than that of P+ Bchl− [73, 89]. Native RCs and pheophytin replaced RCs (RCPhe ) were also studied for their photoelectric performance in SAM-coated gold electrodes [90]. The cells with pheophytin replaced RCs yielded a photocurrent 15% higher than that of native RCs owing to increased electron injection. The electron transfer process for the mutant RC is found to be different from native RCs and it is shown in Fig. 1.5. The enhanced photoelectric performances are principally effected by the higher population and longer lifetime of P* or P+ BChl− in the mutant RC as opposed + to wild-type RCs [90]. The wider energy gap between P+ Q− A and P/P was also found to contribute to the superior photoelectric performance of mutant RCs [90].

Fig. 1.5 Effect of pigment substitution. a Electron transfer in native RC (Adapted with permission from Ref. [33]). b Electron transfer in the mutant RCphe . c Molecular structure of bacteriopheophytin sp in native RC. d Molecular structure of spinach pheophytin (HA ) in the mutant RCphe (Adapted with permission from Ref. [89]) [29]

1.4 Enhancement of Light Harvesting Ability

15

1.4 Enhancement of Light Harvesting Ability While enormous efforts have been directed at improving the photocurrent generation by altering the electron transfer processes, very limited work has so far been done on improving the light harvesting ability of the devices. Among them, the most common and simplistic approach adopted is to increase the protein loading in the device.

1.4.1 Increased Protein Loading Immobilization of RCs on nanocrystalline electrodes has been found to improve the photoelectric performance of the devices. RCs have been immobilized in a nanoporous-nanocrystalline TiO2 film coated on ITO-glass [91, 92]. The larger surface area in the porous matrix facilitates higher adsorption of RCs leading to an increased photochemical activity even without the use of any chemical linkers. A photocurrent of 8 μA/cm2 was achieved with a biophotovoltaic cell with the RC modified nanoporous TiO2 working electrode [91, 92]. RCs were also immobilized on a tailored three-dimensional (3D) wormlike mesoporous WO3 -TiO2 electrode that had a number of features favorable for an enhanced photoelectric performance notably the well-matched energy levels of WO3 -TiO2 with RCs [91, 92]. The immobilized RC had retained the natural function and activity due to the mesoporous structure that had open pores of size ≈7 nm matching the dimension of RC, ideal hydrophilic surface and suitable surface charge [73]. The performance stability of the RC immobilized mesoporous electrode was reasonably good that the photocurrent decreased only 15% after a continuous illumination of 1 h which is promising for constructing bioelectronic devices though inadequate for solar cells [73]. Novel materials with hexagonal honeycomb structured pores are attractive for loading protein complexes of any size as it is possible to control the diameter of the tubular pores by chosen process routes [93, 94]. RCs [93] and light harvesting complexes [95] from a thermophilic purple photosynthetic bacterium, Thermochromatium tepidum, were successfully adsorbed to a folded-sheet silica mesoporous material (FSM) and the binding of RCs to FSM of different pore sizes was studied. Interestingly, it was found that the protein complex was capable of retaining the photosynthetic function inside the material only when the pore size matched the size of the complex [93, 95]. The interior surface of the pores being hydrophobic, the pores are expected to provide the protein complex an environment similar to the hydrophobic membrane [93, 95]. FSMs of different pore diameters namely 2.7, 7.9 and 9 nm were used to study the photochemical capability of the proteins in the porous matrix. The RC may be understood as a cylindrical unit with 5 × 7 nm cross section along the membrane surface and a 13 nm height normal to the membrane [93]. A maximum absorption of RC was evident in FSM with 7.9 pore diameter that fits the RC well as the pore diameter closely matches the cross-sectional dimensions of the reaction center

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1 Introduction

complex [93]. While it was possible to adsorb 0.29 g of RCs per gram of silica in FSM with 7.9 nm diameter, the maximum protein adsorption possible in the other two FSMs were much lower [93]. An intact structure and photochemical activity were found to be possible only with the FSM of fitting pore size which was also witnessed when light harvesting complexes LH2 were loaded to various FSMs [95]. The light harvesting complex adsorbed well with the FSM with 7.9 nm pores as the dimensions of the LH2 complex fit to the pore size [95]. The adsorption was higher than that obtained for the RC as the size of the LH2 is more close to the pore diameter than that of RC, which is suggestive of an enhanced photoelectric performance possible if RCs with LH2 can be immobilized to these porous materials owing to a higher adsorption capacity.

1.5 Scope for Further Research One apparent downside of most cells above described in scaling up to a biohybrid technology for device applications is the use of rare and extremely expensive metal electrodes like platinum/gold. Not many studies have attempted the use of a different counter electrode other than Pt with very few exceptions. This demands a better alternative material for the counter electrode that can make the comparable performance as that with Pt. From recent studies, it is also evident that an expensive material like Pt is not the only choice for the counter electrode, but much efficient photobioelectrochemical cells can be designed with lower cost by making a pragmatic choice of the back electrode and the electrolyte by optimizing their work function and the redox potential respectively. The use of alternative materials for constructing photo-bioelectrochemical cells is a potential research avenue which is also promising in developing cost-effective bio-hybrid applications with improved performance. From a comprehensive review of previous reports on the photo-bioelectrochemical cells, potential research opportunities and the challenges to be addressed have been identified.

1.6 Research Trend and Gaps While enormous efforts have been directed to control the orientation of RCs by chemically modifying the electrodes, in recent years, improved photoelectric performance has been proved possible even without any linkers and by more novel immobilization techniques with no effort to orient the RCs on electrode [41, 96]. Adding the RCs to the electrolyte is one such approach that just uses bare electrode where the photocurrent generation is aided by electron transfer mediators. Hollander et al. [39] showed that a high photocurrent density of 3980 nA/cm2 [39] was possible when the bare electrode was dipped in the solution containing RCs and electron transfer mediators, an approach similar to that used in the pioneering works [62, 63] where

1.6 Research Trend and Gaps

17

simple dipping of electrode in RC suspension yielded around 300 nA/cm2 [62] the only difference in these recent works being the use of the electron transfer mediators that increase the efficiency of electron transfer and hence the photocurrent. RCs with light harvesting complex are also being increasingly studied for their photovoltaic applications. The maximum photocurrent density reported so far for biohybrid solar cells with RC-LH1 is 45 μA/cm2 obtained for RC-LH1 from Rhodopseudomonas acidophila [21]. However, the highest photocurrent reported for RC so far is 120 μA/cm2 which is obtained for an RC based solid state device [25]. The progress in the photoelectric performance of the RC based biohybrid solar cells achieved over decades is worthwhile to be understood for adopting more effective research strategies. The progress of steady state photocurrent densities achieved in the field can be grouped under four main approaches as presented in the roadmap (Fig. 1.6). The various improvement strategies adopted so far by researchers are also presented alongside the photocurrents as this may be handy to compare and devise future techniques to improve the performance of these solar cells (Fig. 1.6). One classic approach is the adsorption of proteins to an unfunctionalized electrode which after a great number of years have gathered interest as they have been proved to be one of the effective methods when used with a higher protein loading (i.e. the amount of protein) and a minimal protein-electrode distance. The pioneering works of Janzen and Seibert [62, 63] directed the research more towards protein orientation on electrodes which was then utilized by a majority of researchers by attaching the proteins to electrodes functionalized by chemical linker molecules and by engineering special tags in the protein to ensure uniform orientation. The progress achieved by this approach of ‘preferential linking to electrode’ has been less appreciable though it spanned more than a decade. Over this period, there were also a few attempts to improve photocurrent by increasing the protein content

Fig. 1.6 Progress in photocurrent generation in photosynthetic protein based photovoltaic devices [29]

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1 Introduction

in the solar cell which was promising and yielded more attractive results with the idea of using nanoporous materials tailor-made to match the size and energy levels of the proteins. A recently developed approach is the employment of RCs in electrolyte which has shown a great progress in a short time span especially due to the use of alternative electrolytes. It is also evident from the road map that using RC-LH1 has also been a very useful strategy to obtain better photoelectric performance. Unlike other strategies, using very high light intensity may not be very useful as solar cells ultimately are to use the sunlight, which is discussed in the next section. As also notable in the roadmap, some aspects like the counter electrode material and the protein stability in the solar cell have rarely been explored and are worthy to be our future research avenues in this field. Recent studies on biohybrid photovoltaic or photo-powered applications throw light on steps forward in taking device performance beyond this roadmap [26, 97–106].

1.7 Challenges 1.7.1 Stability Problem Though enormous efforts have been taken to improve the photoelectric performance of these protein-based solar cells, the useful life period of these cells are way lesser than the conventional solar cells. This is evident with most of the reported works on photosynthetic protein based solar cells. Though high photocurrents are achieved with these cells, they are short-lived and they can hardly produce any photocurrent after a week which is indicative of some kind of degradation with time [83]. As the major photovoltaic components in these solar cells are biomolecular complexes, it is vital to understand their vulnerabilities in a foreign environment. The biomolecular complexes often lack a protective environment in the solar cells, which deteriorates their functionalities, ensuing in a short-lived solar cell. The in vitro stability of the reaction centers need to be improved to devise a more useful and realistic solar cell. As RCs are isolated from their native environment they are prone to conformational changes as the stabilizing effect offered by the membrane lipids is lost. Lipids play a vital role in affecting the biophysical and electron transfer properties and promote structural stability and flexibility, binding the light harvesting cofactors and filling the intra-protein cavities [33]. The stability of these biomolecules is mostly affected by two main stress factors—light and temperature that cause their denaturation [107]. Denaturation of proteins involves a loss of structural integrity that occurs due to the separation of secondary structural subunits and unfolding of domains outside the membrane [108]. The structural integrity of a reaction center can be predicted from the absorption spectrum. As RCs denture, the characteristic absorbance bands of the RC cofactors are seen displaced and shifted, that is suggestive of unfolding of RC, where some of

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the cofactors are no more bound to it [108, 109]. The RCs in their native heterogeneous environment of lipids, have a greater resistance to thermal denaturation and are stable up to 70 °C [108]. When isolated, the possible structural changes associated with the thermal effects can be understood by studying the energetics and kinetics of the denaturation process [108, 110]. Hughes et al. [108] proposed a kinetic model for the thermal denaturation of the Rba. Sphaeroides RCs that demonstrates the likelihood of an intermediate state with respect to heating times [108]. When RCs were held at a high temperature for a very short period and cooled down to room temperature, the spectral properties lost at the high-temperature state were regained, while this did not happen so with longer holding periods at the high temperature [108]. Since it is known that a complete reversibility from the denatured state is impossible, there must be some intermediate state from which complete reversibility to native state was possible [108]. As shown in Fig. 1.7, at low temperatures, RCs are in the native state (N) which on heating follow a kinetic pathway to the denatured state (D) involving an off-pathway intermediate state (I) [108]. The intermediate state is interpreted as a misfolded RC with a distorted structure but with a significant fraction of cofactors still bound to RCs in such a way that the transition from I to N is reversible and this reversible transition is coupled to an irreversible transition to denatured state where the cofactors are unbound from the RCs and the polypeptides are separated leading to the unfolding of the protein [108]. On continuous illumination with intense light, a light-induced denaturation occurs by either or all of the three mechanisms—(1) Singlet oxygen sensitization, (2) Reduction of quinone (QA ) to quinol (QA H2 ) and (3) Localized heating [107]. The temperature induced denaturation has also been found to be linked with light stress as an intense illumination often creates thermal effects [107]. It has been found that, on continuous illumination, the light-induced changes in the redox states of the RC

Fig. 1.7 Protein denaturation model. A schematic of thermal denaturation of RCs as described in Ref. [108]. The spheres of various colors represent the RC cofactors and the ribbons represent the polypeptides (The scheme is illustrative and does not represent the true structure of RC) [29]

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1 Introduction

have a greater effect on its thermal stability and in turn its photochemical activity compared to the effect of the light intensity and the duration of illumination [107]. Detailed mechanisms of the light-induced and temperature induced damage in RC has been discussed in the literature [108, 109].

1.7.2 Discrepancies in Excitation Light Intensity There is a major disparity in the light intensity used for illuminating the solar cell, which greatly influences the value of photocurrent generated. The light intensity used in literature ranges from 0.1 mW/cm2 to as high as 105 times greater intensities (Fig. 1.8). As these research works are oriented towards devising an efficient biohybrid solar cell, it would be realistic to use intensities comparable to the intensity of natural sunlight. A relatively very high value of 120 μA/cm2 was obtained when the RC implanted photovoltaic device was illuminated with a laser light of intensity 10 W/cm2 [25] which is far greater than that of sunlight, a value comparable to light intensity from 100 suns as mentioned by Kamran et al. [21]. As suggested by Henry Snaith [111], the light source used in the characterization of any solar cell must closely match the terrestrial solar spectrum, which is possible by using a xenon lamp with appropriate light filters [111].

Fig. 1.8 Intensity and wavelength range adopted in literature for solar cell illumination [29]

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75. Nakamura C et al (2000) Self-assembling photosynthetic reaction centers on electrodes for current generation. In: Finkelstein M, Davison BH (eds) Twenty-first symposium on biotechnology for fuels and chemicals, applied biochemistry and biotechnology. Humana Press, Totowa, NJ, pp 401–408 76. Goldsmith JO, Boxer SG (1996) Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag. Biochim Biophys Acta Bioenerg 1276(3):171–175 77. Trammell SA et al (2004) Orientated binding of photosynthetic reaction centers on gold using Ni-NTA self-assembled monolayers. Biosens Bioelectron 19(12):1649–1655 78. Trammell SA et al (2006) Effect of protein orientation on electron transfer between photosynthetic reaction centers and carbon electrodes. Biosens Bioelectron 21(7):1023–1028 79. Trammell SA et al (2007) Effects of distance and driving force on photoinduced electron transfer between photosynthetic reaction centers and gold electrodes. J Phys Chem C 111(45):17122–17130 80. Moser CC et al (1992) Nature of biological electron transfer. Nature 355(6363):796–802 81. Mahmoudzadeh A et al (2011) Photocurrent generation by direct electron transfer using photosynthetic reaction centres. Smart Mater Struct 20(9) 82. Page CC et al (1999) Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature 402(6757):47–52 83. Takshi A et al (2010) A photovoltaic device using an electrolyte containing photosynthetic reaction centers. Energies 3(11):1721–1727 84. Takshi A, Madden JD, Beatty JT (2009) Diffusion model for charge transfer from a photosynthetic reaction center to an electrode in a photovoltaic device. Electrochim Acta 54(14):3806–3811 85. Valery C, Artzner F, Paternostre M (2011) Peptide nanotubes: molecular organisations, selfassembly mechanisms and applications. Soft Matter 7(20):9583–9594 86. Hamley IW (2014) Peptide nanotubes. Angew Chem Int Ed 53(27):6866–6881 87. Colherinhas G, Fileti E (2014) Molecular description of surfactant-like peptide based membranes. J Phys Chem C 118(18):9598–9603 88. Tsutsumi H, Mihara H (2013) Self-assembly of designed peptides and their nanomaterials applications. Amino Acids Pept Proteins 38:122 89. Lu YD et al (2006) Manipulated photocurrent generation from pigment-exchanged photosynthetic proteins adsorbed to nanostructured WO3 -TiO2 electrodes. Chem Commun (7):785– 787 90. Xu J et al (2007) Sensitively probing the cofactor redox species and photo-induced electron transfer of wild-type and pheophytin-replaced photosynthetic proteins reconstituted in selfassembled monolayers. J Solid State Electrochem 11(12):1689–1695 91. Lu YD et al (2005) Photoelectric performance of bacteria photosynthetic proteins entrapped on tailored mesoporous WO3 -TiO2 films. Langmuir 21(9):4071–4076 92. Lu Y et al (2005) Bio-nanocomposite photoelectrode composed of the BacteriaPhotosynthetic reaction center entrapped on a nanocrystalline TiO2 matrix. Sensors 5(4):258–265 93. Oda I et al (2010) Photosynthetic electron transfer from reaction center pigment-protein complex in silica nanopores. Langmuir 26(16):13399–13406 94. Inagaki S et al (1999) Novel mesoporous materials with a uniform distribution of organic groups and inorganic oxide in their frameworks. J Am Chem Soc 121(41):9611–9614 95. Oda I et al (2006) Function of membrane protein in silica nanopores: incorporation of photosynthetic light-harvesting protein LH2 into FSM. J Phys Chem B 110(3):1114–1120 96. Mirvakili SM et al (2014) Photoactive electrodes incorporating electrosprayed bacterial reaction centers. Adv Funct Mater 24(30):4789–4794 97. Ravi S et al (2018) Photosynthetic bioelectronic sensors for touch perception, UV-detection, and nanopower generation: toward self-powered E-skins. Adv Mater 30(39):1802290 98. Ravi SK et al (2020) Bio-photocapacitive tactile sensors as a touch-to-audio braille reader and solar capacitor. Mater Horizons 7(3):866–876 99. Ravi SK et al (2019) Photosynthetic apparatus of Rhodobacter sphaeroides exhibits prolonged charge storage. Nat Commun 10(1):1–10

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Chapter 2

Augmenting Photocurrent Using Photoproteins of Complementary Optical Characteristics

2.1 Brief Overview Research on solar energy conversion by photosynthetic proteins in a device setting has been primarily directed toward optimising charge separation and mediation, with much less attention paid to manipulating the absorption cross-section of the light harvesting proteins to boost current output. This chapter [1, 2] explores the benefits of incorporating optically-complementary variants of a photosynthetic protein in a stacked, tandem solar cell architecture that utilises a conductive polymer, poly(3,4ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS), as a transparent electrode material. Two naturally-occurring red and green versions of a bacterial reaction centre/light harvesting 1 protein were used that vary only in the type of light harvesting carotenoid that feeds the central reaction centre module with excited state energy. In green/red tandem cells assembled with optically-complementary layers, a photocurrent was obtained that was consistently up to ≈20% stronger than could be obtained from tandem cells assembled with two optically-identical layers or cells in which the two types of protein were simply mixed. In addition, the use of PEDOT:PSS as an electrode material resulted in a 12-fold enhancement in photocurrent density compared to that achievable with platinum. The findings provide insights into the design of cost-effective photo-bioelectrochemical cells with superior photoelectric performance that exploit the diversity of naturally occurring photosynthetic pigments in an optimised manner.

2.2 Introduction RCs from purple photosynthetic bacteria such as Rhodobacter (Rba.) sphaeroides [3], and the larger RC-LH1 complexes they form with the LH1 light harvesting protein [4], are a popular choice for the construction of photo-bioelectrochemical © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_2

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cells and other solar-powered biohybrid applications [2, 5–17]. Previous studies utilising these proteins have been aimed at enhancing photocurrent generation by improving the effectiveness of protein-electrode electron transfer processes in threeelectrode cells [18–23] or, to a lesser extent, in two-electrode cells [24–26]. Manipulating protein orientation and the nature of chemical or biochemical linkers for immobilizing proteins on electrodes has been the primary approach to improving electron-transfer, along with the use of alternative electrode/electrolyte combinations based on energy-level considerations to achieve current enhancement (see [5, 6] for reviews). In contrast, there have been almost no attempts to enhance photocurrents by augmenting the natural light-harvesting abilities of photosynthetic bacteria and therefore the spectral range covered in biohybrid devices. A characteristic of natural photosynthetic pigments is that they have strong absorbance bands in some regions of the UV-visible-nearIR spectrum but little or no absorbance in other regions. Plasmonic enhancement of photocurrent generation by purple bacterial RC-LH1 complexes on nanostructured metal electrodes has been achieved [27], but this does not change the wavelengths of light absorbed. A few attempts have been made to increase the optical absorption cross-section of photosynthetic RCs or LH complexes by attaching tailored molecular fluorophores and photoluminescent quantum dots, but these have not been scaled up for photocurrent generation at a device-level [28–32]. Fabrication of such partially-synthetic photovoltaic proteins complicates device construction, adds to cost, and can involve the use of materials that are not environmentally friendly or constitute a limited resource. An alternative approach to enhancing spectral coverage is to employ a stacked tandem device architecture in which photovoltaic proteins with natural pigments that have complementary absorption characteristics incorporated into different layers of the device. This work employs two variants of RC-LH1 complexes which incorporate either the native red carotenoid spheroidenone (RC-LH1red ) or the green carotenoids neurosporene, hydroxyneurosporene and methoxyneurosporene (RC-LH1green ) (Fig. 2.1a–d). This is achieved using a “green strain” of Rba. sphaeroides containing a spontaneous mutation in the crtD gene encoding methoxyneurosporene dehydrogenase, which halts carotenoid synthesis prematurely [33]. The two, otherwise identical, pigment-proteins have different absorption characteristics in the blue to yellow region of the visible spectrum (Fig. 2.1e), 17 molecules of spheroidene per complex giving rise to a single broad band between 400 and 600 nm, and 17 molecules of neurosporene and its derivatives to narrower, more intense absorbance between 400 and 500 nm with distinctive maxima at 429 nm, 454 nm and 485 nm [34]. As the tandem cell architecture requires a transparent rear electrode for the front cell, the option of using poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was explored. This transparent polymer has attracted considerable interest in photovoltaics owing to its good electronic conductivity achievable on chemical treatment and also due to its low cost [35, 36]. In this work, optically-complementary RC-LH1red and RC-LH1green proteins were encapsulated in sub-cells formed from a sandwich of FTO-glass and PEDOT:PSS electrodes, and assembled either individually or in a tandem, parallel architecture

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Fig. 2.1 Proteins of complementary optical characteristics. a RC-LH1red complex viewed parallel to the photosynthetic membrane, with the 16 LH1 carotenoids carotenoid shown as red spheres, LH1 BChls in yellow and all other components in white. The RC is shown as a solid object and LH1 proteins as ribbons. b The LH1 carotenoids (green) and BChls (yellow) power charge separation in the central RC by passing excited state energy to the P BChls (orange carbons). The nearest four BChls and two carotenoids have been removed. Views of c RC-LH1red and d RCLH1green complexes perpendicular to the photosynthetic membrane; only the carotenoid pigments differ between complexes. e Visible region absorbance spectra of RC-LH1red and RC-LH1green complexes in solution, normalized to BChl absorbance at 875 nm

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(Fig. 2.2). By measuring photocurrents from a variety of cell configurations, current enhancements brought about by the tandem architecture and the advantages of using transparent polymeric electrode materials are demonstrated.

Fig. 2.2 Schematic showing the device terminals. The two terminals in a a RC-LH1green sub-cell, b a RC-LH1red sub-cell, c a parallel tandem cell, d a series tandem cell e a 3D schematic of the tandem device architecture

2.3 Construction and Working of Bio-Tandem Cells

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2.3 Construction and Working of Bio-Tandem Cells 2.3.1 Photoproteins with Red or Green Carotenoid Pigments DNA encoding pufLM modified with a poly-histidine tag at the C-terminus of pufM [37] was cloned into plasmid pvBALM, which is a derivative of broad-host-range vector pRK415 containing a 6.0 kb section of Rba. sphaeroides DNA encoding pufQBALM. The resulting plasmid, termed pvBALMt, was introduced into Rba. sphaeroides strains DD13 and DD13/G1 [33] by conjugative transfer. This produced transconjugant strains expressing His-tagged PufX-deficient RC-LH1 complexes with either red or green carotenoid pigments. Protein complexes were purified as described in detail previously [27] and were stored as concentrated solutions in 20 mM Tris (pH 8.0)/0.04% (w/v) DDM at −80 °C. The schematic models of PufXdeficient RC-LH1 complexes in Fig. 2.1 were based on the X-ray crystal structure of the similar complex from Thermochromatium tepidum [4]. In some schematics all components other than the carotenoid ring was shown in white. In others the colour code was as follows: LH1 α-polypeptide—cyan ribbon; LH1 β-polypeptide— magenta ribbon; LH1 BChls—yellow sticks; RC H-polypeptide—pink surface; RC L-polypeptide—beige surface; RC M-polypeptide—green surface.

2.3.2 Device Fabrication and Characterization The electrodes were prepared by spin coating 2 layers of PEDOT:PSS (Clevios PH1000) atop of pre-cleaned substrates. The post-treatment of PEDOT:PSS was done with 8M methanesulfonic acid at 160 °C following by rinsing with DI-water for three times as reported elsewhere [38]. FTO glasses (2 cm × 2 cm) were cleaned by sequentially sonicating in acetone, isopropyl alcohol and deionised water before the cell fabrication. The intermediate electrode in the tandem cell was prepared by depositing the same PEDOT:PSS on the non-conductive face of FTO glass. Two rectangular cavities were formed on assembling the three electrodes using a thermoplastic spacer (Surlyn 50 μm) between each pair, followed by heating at 70 °C. After cooling, 5 μL aliquots of protein solution were injected into the top and bottom cavities which were then sealed with epoxy resin. The same procedure was followed for sub-cells formed from two electrodes. The protein solutions comprised RC-LH1 complexes at a final concentration 50 μM, in 20 mM Tris (pH 8.0)/0.04% DDM buffer supplemented with 10 mM Q0 and 1 mM TMPD. Photocurrents from an active area of 2 mm × 2 mm were measured under white light illumination (100 mW cm−2 ) using K2400 source meter (Keithley). The absorbance of the protein solutions and the transparency of the cells were measured using a Shimadzu UV-Vis spectrophotometer.

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Sub-cell and Tandem-Cell Performance

Sub-cells were constructed by connecting front FTO-glass and rear PEDOT:PSS electrodes with a 50 μm thick U-shaped thermoplastic spacer. Tandem cells were constructed with a middle electrode comprising an FTO glass substrate with PEDOT:PSS coated on the non-conductive side (Fig. 2.2e). The conductive PEDOT:PSS films prepared by spin-coating had a smooth and homogeneous topography (Fig. 2.3) with a root-mean-square roughness of ≈1 nm„ a mean thickness of 34 ± 4 nm and a transmittance of 93% at 550 nm. Consistent smoothness of these surfaces is known to be favorable for the application as electrode materials in several electronic devices [39]. Solutions of 50 μM protein injected into the cavity of each sub-cell included 10 mM ubiquinone-0 (Q0 ) and 1 mM TMPD as electrolytes [24]. Assembled sub-cells and tandem cells were visibly transparent (Fig. 2.4), with transmittances at 550 nm of 82% for the RCLH1red sub-cell (Fig. 2.4b) and 87% for the RC-LH1green sub-cell (Fig. 2.4a). The lower transmittance of the former was consistent with the higher absorbance of spheroidene at this wavelength compared to neurosporene (Fig. 2.1e). A green/red

Fig. 2.3 AFM topograms of the PEDOT:PSS film over 25 μm2 (left) and 4 μm2 (right) areas

Fig. 2.4 Active areas of sub-cells and a tandem cell on a coloured background. a Sub-cell with RC-LH1green , b sub-cell with RC-LH1red , c green/red tandem cell

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(top/bottom) tandem cell (Fig. 2.4c) had a transmittance at 550 nm of 59%, intermediate between that of a green/green tandem cell (61%) and a red/red tandem cell (56%), and again consistent with the higher absorbance of spheroidenone at 550 nm. Both the electrode materials (FTO and PEDOT:PSS) had a very minimal absorbance in the range of 400–600 nm where the two proteins exhibit complementary absorption characteristics (Appendix A: Fig. A.1). All cells were two terminal devices (Fig. 2.2) and in the case of the tandem cell both front electrodes were connected together as were both back electrodes, producing a parallel tandem architecture. In an earlier work on photocurrent generation by RC-LH1red complexes Pt was employed as the rear electrode material [24]. Here, both red and green sub-cells with PEDOT:PSS as the rear electrode showed significantly higher average steady-state photocurrents (≈5 μA/cm2 —Fig. 2.5a, triangles and squares, respectively) compared with identical cells made with a 25 nm thick Pt rear electrode (0.3 to 0.4 μA/cm2 — Fig. 2.5a, circles and diamonds, respectively). In addition to being more cost-effective therefore, PEDOT:PSS was also a more functionally effective alternative to Pt for collection of electrons following charge separation in the photoactive proteins. Measurements of red/red and green/green tandem cells fabricated with PEDOT:PSS demonstrated the drawbacks of adding more layers of an opticallyidentical material in an attempt to obtain larger photocurrents from a given footprint. Over multiple measurements (Fig. 2.5d) the photocurrents obtained from these tandem cells were only 44% (red/red) and 59% (green/green) larger than currents obtained from the equivalent single sub-cells. This demonstration of the law of diminishing returns caused us to explore a tandem architecture in which higher-energy absorbing RC-LH1green complexes were stacked on top of lower-energy absorbing RC-LH1red complexes. One such green/red tandem cell exhibited a maximum peak current density of ≈58 μA/cm2 and a steady-state photocurrent density of ≈8.6 μA/cm2 (Fig. 2.5a inset, blue stars). Over multiple measurements this steady state output was ≈74% greater than the steady-state photocurrent density produced by either the red and green sub-cells (see Fig. 2.5a, inset), and this current increase was significantly higher than could be achieved from green/green or red/red tandem cells (Fig. 2.5c, inset). To further examine possible benefits of the controlled heterogeneity offered by the tandem structure, a cell was prepared with an equimolar mixture of red and green RCLH1 proteins, the individual concentrations being the same as used for the component proteins in the green/red tandem cell. This “mixed cell” yielded a ≈41% lower photocurrent compared to the green/red tandem cell (Fig. 2.5a, inset, purple/triangles compared to blue/stars). This lower photocurrent is attributed to shading of green complexes by red complexes in the mixed cell configuration. The green/red tandem cell and the two corresponding sub-cells produced average steady-state open-circuit photovoltages of ≈3 mV, with only a negligible difference of 0.1 mV between the two sub-cells (Fig. 2.5b). The factors that give rise to this voltage, including the potentials of the electron transfer components with the RC-LH1 complex, are not expected to be affected by the type of carotenoid present.

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Fig. 2.5 Output of sub-cells and tandem cells. a Photocurrent density from green and red sub cells with either PEDOT:PSS (solid) or Pt (dotted) back electrodes, a green/red mixture cell and a green/red tandem cell. b Open circuit voltage from green and red sub-cells and a green/red tandem cell. c Comparison of photocurrent density from tandem cells with green/green, red/red and green/red configurations. d Comparison of steady-state photocurrent densities produced by the sub-cells, tandem cells and the mixed cell with mean photocurrents (average ± standard error; n = 3 cells; All the measurements were performed using a solar simulator with a standard light intensity of 100 mW/cm2 under AM 1.5 conditions.)

In exploring ways to supplement the tandem effect, a tandem cell with a higher protein loading was tested. To avoid any absorption losses caused by shading by the top cell the protein loading was doubled only in the bottom RC-LH1red cell. Experiments with sub-cells containing RC-LH1green complexes at 50 μM and RC-LH1red complexes at 100 μM showed that doubling the RC-LH1red concentration boosted the current output by ≈50% (Appendix A, Figure A.2, red compared with green). However in the tandem architecture, doubling the RC-LH1red concentration in the lower cell to 100 μM produced a photocurrent that was increased by only ≈20% compared to previous green/red tandem cells with a 50 μM concentration for both proteins (Appendix A, Figure A.2, blue compared with Fig. 2.5a, blue). Doubling the RC-LHred concentration in the lower cell resulted in a lower overall transmittance of

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38% (Appendix A, Figure A.3), indicating the constraint of maintaining the transparency of the cells for practical applications and the need for a trade-off between the transmittance and photocurrent in such devices. External quantum efficiency (EQE) action spectra were measured to confirm the source of photocurrent and to further demonstrate the photocurrent enhancement resulting from the improved light absorption cross-section in the tandem cell (Fig. 2.6a). These showed bands at 375 nm, 600 nm and above 700 nm attributable to the RC-LH1 bacteriochlorophyll (BChl) pigments. Bands at 805 and 875 nm are attributable to the BChls of the RC and the surrounding LH1 ring, respectively. In

Fig. 2.6 Spectral response and energy diagram. a EQE action spectra for the red and green sub-cells and the green/red tandem cell; inset shows the carotenoid region of the tandem cell EQE spectrum compared to the solution absorbance spectrum of an equimolar red + green mixture. b Energy level diagram and proposed mechanism of photocurrent generation

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the 430–580 nm region the EQE spectrum of the green/red tandem cell showed spectral features attributable to the carotenoids in the green, upper cell between 430 and 510 nm and the red, lower cell in the region around 560 nm. A maximum EQE at 870 nm of 1.3% was obtained for the green/red tandem cell, while the individual green and red sub-cell had an EQE of 1.1% and 0.85%, respectively, at this wavelength (Fig. 2.6a). The suggested mechanism for photocurrent generation is shown in Fig. 2.6b. Photo-oxidation of the primary electron donor BChls in the RC (P → P*), principally following excitation transfer from the BChls and carotenoids of the LH1 antenna, results in charge separation to reduce the QB ubiquinone. In line with previous studies [24], maximal photocurrents were obtained using a mixture of Q0 and TMPD. It is likely that Q0 enhanced electron transfer from the QB quinone binding site to the rear electrode whilst TMPD mediated between the front electrode and the photooxidised RC. Another salient point is that PEDOT:PSS presents less of a potential drop for electron transfer from Q0 (or TMPD) than Pt (Fig. 2.6b), which accounts for the much higher steady-state currents obtained in sub-cells with PEDOT:PSS as opposed to Pt. The spin-coated films of PEDOT:PSS were found to have a smooth and homogeneous surface (Fig. 2.3) which should favour the conductive properties of the film and hence charge transfer. In the case of this work, it was decided to connect the two sub-cells of the tandem cell in parallel rather than in series. Although both tandem architectures are theoretically proficient in achieving a high light-absorption cross section for a given footprint, output is limited by stringent current-matching criteria. To avoid current losses in parallel-connected tandem cells the component sub-cells should ideally produce equal photovoltages, whilst to avoid voltage losses in series-connected tandem cells the component sub-cells should ideally produce equal photocurrents. As the Voc of these biohybrid photoelectrochemical cells is dependent on the energy-levels of the mediators or components of the RC, which are unaltered by a change from red to green light harvesting carotenoids, achieving an approximately equal Voc in the two sub-cells was easier than achieving an equal Jsc , as the latter is affected by the efficiencies of light capture and energy transfer. Hence, the parallel-connected tandem architecture provided the best approach to enhancing the photocurrent generation without altering the footprint of the cell. The modular nature of the LH/RC system in which solar energy harvesting and charge separation are undertaken by different cofactors, enables this approach where these two processes can be manipulated independently of one another. The photocurrent of a parallel-connected tandem cell can be equal to the sum of the two sub-cell currents if light harvesting by the two is fully complementary. However in the present case the photocurrent of the tandem cell was 12% lower than this sum, which can be attributed to the reduced incident light intensity for the bottom (red) sub-cell between 400 and 500 nm and also to a minor difference in Voc between the two sub-cells that results in a circulating current, reducing the overall photocurrent output. To further examine the relative benefits of a parallel versus serial configuration, a serial green/red tandem cell was fabricated (Fig. 2.2d) and the JSC and VOC compared to those from green and red sub-cells. As expected an additive effect on VOC was

2.3 Construction and Working of Bio-Tandem Cells

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Fig. 2.7 Open circuit voltages from green and red sub-cells and a series tandem cell

Fig. 2.8 Photocurrent densities from green and red sub-cells and a series tandem cell

observed (Fig. 2.7), but the JSC obtained was lower than for either sub-cell (Fig. 2.8), in contrast with the enhancement seen for the tandem cell with the parallel architecture (Fig. 2.5a). The average power density (calculated as the product of JSC and VOC ) was 39 nW/cm2 in case of the parallel cell and 25 nW/cm2 in case of the serial cell.

2.4 Summary In conclusion, this work introduced a new approach to photocurrent generation in a biohybrid device by integrating complementary photosynthetic proteins in different layers of a stacked tandem architecture, with the proteins tuned to absorb more photons of a higher energy positioned in the top layer of the device. To realize this, PEDOT:PSS was employed as a cost-effective transparent rear electrode for the cells,

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which produced the additional benefit of a 12–16 fold enhancement in photocurrent over the Pt electrodes. The proof-of-principle tandem biohybrid photoelectrochemical cell constructed using optically-complementary photovoltaic proteins produced a photocurrent that was 88% of the theoretical output of the two component subcells, an output that was higher than could be achieved by simply doubling the amount of either individual protein in the same cell footprint. The complementary absorption characteristics being exhibited only in a narrow wavelength range of 100 nm, a considerable photocurrent addition has been obtained, which indicates the possibility of further enhancement in photocurrent by designing and employing pigment-proteins of wider complementary absorption range. In future work, it is promising to achieve higher output by increasing the number of layers and the variety of complementary light-harvesters in the device.

References 1. Ravi SK (2018) Solar energy harvesting with photosynthetic pigment-protein complexes. National University of Singapore 2. Ravi SK et al (2017) Enhanced output from biohybrid photoelectrochemical transparent tandem cells integrating photosynthetic proteins genetically modified for expanded solar energy harvesting. Adv Energy Mater 7(7):1601821 3. Feher G et al (1989) Structure and function of bacterial photosynthetic reaction centres. Nature 339:111–116 4. Niwa S et al (2014) Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å. Nature 508(7495):228–232 5. Ravi SK, Tan SC (2015) Progress and perspectives in exploiting photosynthetic biomolecules for solar energy harnessing. Energy Environ Sci 8(9):2551–2573 6. Kim Y et al (2014) Hybrid system of semiconductor and photosynthetic protein. Nanotechnology 25(34):342001 7. Yehezkeli O et al (2014) Photosynthetic reaction center-functionalized electrodes for photobioelectrochemical cells. Photosynth Res 120(1–2):71–85 8. Ravi S et al (2018) Photosynthetic bioelectronic sensors for touch perception, UV-detection, and nanopower generation: toward self-powered E-skins. Adv Mater 30(39):1802290 9. Ravi SK et al (2020) Bio-photocapacitive tactile sensors as a touch-to-audio braille reader and solar capacitor. Mater Horiz 7(3):866–876 10. Ravi SK et al (2019) Optical shading induces an in-plane potential gradient in a semiartificial photosynthetic system bringing photoelectric synergy. Adv Energy Mater 9(35):1901449 11. Ravi SK et al (2019) Photosynthetic apparatus of Rhodobacter sphaeroides exhibits prolonged charge storage. Nat Commun 10(1):1–10 12. Ravi SK al (2018) Bio-photoelectrochemical cells: protein immobilization routes and electron transfer modes. In Photosynthetic protein-based photovoltaics. CRC Press, pp 141–159 13. Ravi SK et al (2018) Emerging role of the band-structure approach in biohybrid photovoltaics: a path beyond bioelectrochemistry. Adv Func Mater 28(24):1705305 14. Singh VK et al (2018) Biohybrid photoprotein-semiconductor cells with deep-lying redox shuttles achieve a 0.7 V photovoltage. Adv Funct Mater 28(24):1703689 15. Ravi SK et al (2018) A mechanoresponsive phase-changing electrolyte enables fabrication of high-output solid-state photobioelectrochemical devices from pigment-protein multilayers. Adv Mater 30(5):1704073 16. Ravi SK, Tan SC (2018) Electronics, photonics, and device physics in protein biophotovoltaics. In: Photosynthetic protein-based photovoltaics. CRC Press, pp 161–224

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17. Tan SC (2018) Photosynthetic protein-based photovoltaics. CRC Press 18. den Hollander M-J et al (2011) Enhanced photocurrent generation by photosynthetic bacterial reaction centers through molecular relays, light-harvesting complexes, and direct protein-gold interactions. Langmuir 27(16):10282–10294 19. Caterino R et al (2015) Photocurrent generation in diamond electrodes modified with reaction centers. ACS Appl Mater Interfaces 7(15):8099–8107 20. Gebert J et al (2015) Electron transfer to light-activated photosynthetic reaction centers from Rhodobacter sphaeroides reconstituted in a biomimetic membrane system. J Phys Chem C 119(2):890–895 21. Kondo M et al (2012) Photocurrent and electronic activities of oriented-his-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode. Biomacromol 13(2):432–438 22. Yaghoubi H et al (2012) The role of gold-adsorbed photosynthetic reaction centers and redox mediators in the charge transfer and photocurrent generation in a bio-photoelectrochemical cell. J Phys Chem C 116(47):24868–24877 23. Takshi A, Madden JD, Beatty JT (2009) Diffusion model for charge transfer from a photosynthetic reaction center to an electrode in a photovoltaic device. Electrochim Acta 54(14):3806–3811 24. Tan SC et al (2012) Generation of alternating current in response to discontinuous illumination by photoelectrochemical cells based on photosynthetic proteins. Angew Chem Int Ed 51(27):6667–6671 25. Tan SC et al (2012) Increasing the open-circuit voltage of photoprotein-based photoelectrochemical cells by manipulation of the vacuum potential of the electrolytes. ACS Nano 6(10):9103–9109 26. Tan SC et al (2013) Superhydrophobic carbon nanotube electrode produces a near-symmetrical alternating current from photosynthetic protein-based photoelectrochemical cells. Adv Func Mater 23(44):5556–5563 27. Friebe VM et al (2016) Plasmon-enhanced photocurrent of photosynthetic pigment proteins on nanoporous silver. Adv Func Mater 26(2):285–292 28. Dutta PK et al (2014) Reengineering the optical absorption cross-section of photosynthetic reaction centers. J Am Chem Soc 136(12):4599–4604 29. Nabiev I et al (2010) Fluorescent quantum dots as artificial antennas for enhanced light harvesting and energy transfer to photosynthetic reaction centers. Angew Chem Int Ed 49(40):7217–7221 30. Milano F et al (2012) Enhancing the light harvesting capability of a photosynthetic reaction center by a tailored molecular fluorophore. Angew Chem Int Ed 51(44):11019–11023 31. Dutta PK et al (2014) A DNA-directed light-harvesting/reaction center system. J Am Chem Soc 136(47):16618–16625 32. Maksimov E et al (2013) Photophysical properties of hybrid complexes of quantum dots and reaction centers of purple photosynthetic bacteria Rhodobacter sphaeroides adsorbed on crystalline mesoporous TiO2 films. Nanotechnol Russ 8(7–8):423–431 33. Jones MR et al (1992) Mutants of Rhodobacter sphaeroides lacking one or more pigmentprotein complexes and complementation with reaction-centre, LH1, and LH2 genes. Mol Microbiol 6(9):1173–1184 34. Chi SC et al (2015) Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway. Biochim Biophys Acta (BBA) Bioenergetics 1847(2):189–201 35. Alemu D et al (2012) Highly conductive PEDOT: PSS electrode by simple film treatment with methanol for ITO-free polymer solar cells. Energy Environ Sci 5(11):9662–9671 36. Yoon DH et al (2016) PEDOT: PSS as multi-functional composite material for enhanced Li-air-battery air electrodes. Sci Rep 6 37. Swainsbury DJ et al (2014) Evaluation of a biohybrid photoelectrochemical cell employing the purple bacterial reaction centre as a biosensor for herbicides. Biosens Bioelectron 58:172–178

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38. Ouyang J (2013) Solution-Processed PEDOT:PSS films with conductivities as indium tin oxide through a treatment with mild and weak organic acids. ACS Appl Mater Interfaces 5(24):13082– 13088 39. Xia Y, Ouyang J (2011) PEDOT: PSS films with significantly enhanced conductivities induced by preferential solvation with cosolvents and their application in polymer photovoltaic cells. J Mater Chem 21(13):4927–4936

Chapter 3

Interfacing Photoproteins with Mechanoresponsive Electrolytes for Enhancing Photocurrent and Stability

3.1 Brief Overview Exploitation of natural photovoltaic reaction centre pigment-proteins in biohybrid architectures for solar energy harvesting is attractive due to their global abundance, environmental compatibility and near-unity quantum efficiencies. However, it has been challenging to achieve high photocurrents in a device setup due to limitations imposed by low light absorbance by protein monolayers and/or slow long-range diffusion of liquid-phase charge carriers. In an attempt to enhance the photocurrent density achievable by pigment-proteins, this chapter [1, 2] shows an alternative solid-state device architecture enabled by a mechanoresponsive gel electrolyte that can be applied under non-denaturing conditions. The phase-changing electrolyte gel provides a pervading biocompatible interface for charge conduction through highly-absorbing protein multilayers that are fabricated in a simple fashion. Assembled devices exhibited enhanced current stability and a maximal photo-response of ≈860 μA cm−2 , a five-fold improvement over the best of previous comparable devices under standard illumination conditions. Photocurrent generation was enhanced by directional energy transfer through extended layers of light harvesting complexes, mimicking the modular antenna/transducer architecture of natural photosystems, and by metastable radical pair formation when photovoltaic reaction centres were embedded throughout light harvesting regions of the device.

3.2 Introduction As part of the development of more varied, sustainable and eco-friendly lightharvesting technologies there is growing interest in either mimicking or directly exploiting natural photosynthetic complexes [1, 3–17]. In addition to inspiring the design of molecular systems for artificial photosynthesis [18], photosynthetic © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_3

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pigment-proteins have been directly interfaced with electrodes for potential applications in photo-bioelectrochemical cells, biosensing, photodetection, solar fuel synthesis and biocomputing [5, 19–21]. Much of this effort has focused on the reaction centre (RC) complex from purple photosynthetic bacteria [22], and the larger RC-LH1 complex that is assembled between this RC and the so-called LH1 light harvesting protein [23] (Fig. 3.1). This RC facilitates a multi-step charge separation between a pair of bacteriochlorophylls (P) and a quinone electron acceptor (QB ) with a very high quantum efficiency [22, 24, 25] (Figs. 3.1 and 3.2a). Photocurrents from these proteins have typically been studied in open, three-electrode cells incorporating a protein-coated working electrode immersed in a buffer containing a mobile, small molecule electrolyte [26– 36]. Two-electrode sealed liquid cells with one or more mobile mediators have also been constructed [37–39]. Proteins are typically adhered to the working electrode by drop casting or adsorption from solution, although electrospraying [34] and laser induced forward transfer [40] have also been explored. Photocurrents from such devices frequently show an initial spike of “peak current” which decays over seconds or minutes to a significantly lower, steady-state output [27, 30, 31, 33–35, 37–39]. Proposals that this decline is due to limitations imposed by slow mediator diffusion [31, 41, 42] have been corroborated by measurements with a rotating disk working electrode to provide mixing [33]. Effective electrical connection of these proteins to both the working and counter electrodes often requires the assembly of mediato r-accessible protein monolayers at the surface of the working electrode, resulting in relatively low absorption of actinic light. Photocurrent densities achieved with RC and RC-LH1 complexes have typically occupied the range from a few μA cm−2 to several tens of μA cm−2 with, as discussed below, a couple of reports of higher currents [33, 43]. Progression from liquid to solid-state device architectures [44] could create new opportunities for enhancement of photocurrent output and elimination of photocurrent decline due to diffusional limitations. However, interfacing proteins with suitable conductive materials presents considerable challenges as this often requires one or more coating steps involving elevated temperatures or high-velocity deposition. As an example, three thermal evaporation steps were used to deposit electron acceptor materials on the protein layer in the only solid-state RC photo-bioelectrochemical device described to date [43]. Given the sensitivity of pigment-proteins to thermal or mechanical stress, it is important to develop fabrication processes for interfacing conductive materials with proteins under non-denaturing conditions. Similar concerns affect a wider range of synthetic light harvesting materials that exhibit sensitivity to stress conditions such as elevated temperature. Furthermore, the use of protein multilayers to achieve high absorbance raises the question of how proteins in different layers can be electrically connected to both electrodes. One approach to increasing absorbance is to use nanostructured electrode materials in combination with a diffusible mediator [33, 45] or entrapment in a redox hydrogel [46], but such nanostructuring to achieve an increased electrode surface area adds complexity to cell fabrication procedures.

3.2 Introduction

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Fig. 3.1 Structure and mechanism of the RC-LH1 and RC complexes. a Representation of the RCLH1 complex from Tch. tepidum [23] viewed parallel to the plane of the photosynthetic membrane (represented by a grey box). The central RC is shown as a solid object with the protruding Hpolypeptide shown in pink. The RC is surrounded by helical, membrane-spanning LH1 α- and β-polypeptides (cyan and magenta ribbons, respectively) that each bind one bacteriochlorophyll (BChl) pigment, shown as alternating red and orange spheres, and one carotenoid (not shown). b View of the complex from the periplasmic side of the membrane (bottom in a)) showing how concentric cylinders of 16 LH1 α- and β-polypeptides hold in place a ring of 32 BChls. The Land M-polypeptides of the central RC are shown in beige and green. c View of the bacteriochlorins and ubiquinones of the RC-LH1 complex with the four nearest LH1 BChls removed. FRET (red arrow) from the LH1 BChls to the P BChls of the RC (yellow carbons) triggers electron transfer (green arrows) to the HA bacteriopheophytin (pink carbons) and the QA and QB ubiquinones (cyan carbons). The brown sphere is a structural iron, the magenta spheres are the Mg atoms of the RC BChls and the “accessory” BChls are shown with green carbons. Hydrocarbon side chains of all cofactors have been removed for clarity

In this work a range of new solid-state photo-bioelectrochemical cells were constructed based on multilayers of an engineered variant of the Rhodobacter (Rba.) sphaeroides RC-LH1 complex or its component LH1 and RC pigment-proteins (see Sect. 3.4.2). Employing a similar RC-LH1 complex, a photocurrent of ~160 μA cm−2 was recently obtained when the protein was drop-casted onto a nanostructured roughsilver electrode in a three-electrode liquid cell employing a rotating disk electrode [33]. This was comparable to a photocurrent density of ≈120 μA cm−2 achieved by a RC solid state device exposed to extremely strong (10 W cm−2 ) monochromatic excitation [43]. The photo-bioelectrochemical cells described here were designed with a view to improve the steady-state photocurrent output achievable under standard

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illumination conditions by eliminating limitations imposed by slow mediator diffusion, whilst also enhancing light absorption through a multilayer architecture that can be fabricated under entirely benign conditions using planar electrode materials and simple deposition procedures. The new design for a solid-state, two electrode RC-LH1 cells combined, for the first time, a gel-state interface between electrodes, a permeating disulfide-thiolate (T2 /T− ) electron carrier and spin-coated multilayers of protein. A variety of cell architectures were explored to address the question of whether, in a man-made threedimensional photosystem, light harvesting and charge separating functions work best when combined in all layers in the device or when confined to separate layers. Investigation of the latter included placing separately purified LH1 and RC proteins in different layers to mimic natural two-dimensional photosystems in which specialized light harvesting proteins (the antenna) pass harvested excited state energy to a smaller number of photovoltaic RC proteins (the transducers) through directional energy transfer (Fig. 3.2b). The so-called “fast recombining” RC and RC-LH1 complexes that, due to a single amino acid change (AM260W), assemble without a ubiquinone at the QA electron acceptor site in the RC, were also used [47]. In AM260W RCs the recombination lifetime of the final product of charge separation (P+ H− A , ~17 ns [48]) is eight orders of magnitude shorter than that in the native RC (P+ Q− B , ~1–2 s), but the absorbance spectrum and gross structure of the RC or RC-LH1 complex are not affected. Proteins in the multilayers were connected to the electrodes using a conductive interface based on succinonitrile (N≡C–CH2 –CH2 –C≡N), a highly polar organic plastic crystalline material which, due to lattice defects and rotational vacancies, can provide a non-conducting matrix for a conducting salt [49]. This matrix was suffused by an equimolar mixture of the N-tetramethylammonium (+ NMe4 ) salt of 5-mercapto-1-methyltetrazole (T− ) and di-5-(1-methyltetrazole) disulfide (T2 ) [50, 51], producing a plastic gel-phase material with a high ionic and molecular diffusivity [52]. While gels are typically liquefied by heating, crucially for work with labile pigment-proteins it was discovered that this material undergoes a reversible gel-toliquid transition under mechanical vibration. As sonication did not produce significant heating of the succinonitrile/electrolyte mix, this provided a means of permeating layers of photoactive protein with the electrolyte matrix at room temperature (Fig. 3.2c).

3.3 Construction Protocol for Bio-Hybrid Cells with Phase-Changing Gels 3.3.1 Isolation of Photoproteins All proteins were isolated from strains of Rba. sphaeroides lacking the peripheral LH2 light harvesting protein that were grown under dark/semiaerobic conditions.

3.3 Construction Protocol for Bio-Hybrid Cells with Phase-Changing Gels

45

PufX-deficient RC-LH1 complexes were isolated from photosynthetic membranes using the detergent n-dodecyl β-D-maltopyranoside (DDM), purified as described previously [33] and stored at −80 °C as a concentrated solution (A873 ≈ 600 absorbance units) in 20 mM Tris (pH 8.0)/0.04% (w/v) DDM. RCs were isolated from membranes from a strain also lacking LH1 using the detergent n-dodecyl-N,Ndimethylamine-N-oxide (LDAO), purified as described previously [29] and stored at −80 °C as a concentrated solution (A803 ≈ 350 absorbance units) in 20 mM Tris (pH 8.0)/0.1% (w/v) LDAO. LH1 complexes were isolated from membranes from a strain also lacking RCs, purified as described elsewhere [53] using the detergent octyl glucoside, detergent exchanged into 0.04% (w/v) DDM, and stored at −80 °C as a concentrated solution (A873 ≈ 600 absorbance units) in 20 mM Tris (pH 8.0)/0.04% (w/v) DDM.

3.3.2 Protein Deposition and Characterisation An area of 1 cm2 of conductive FTO-glass (TEC 15, MTI Corporation, USA) was coated with RC-LH1 protein by placing a 5 μL drop of concentrated protein solution in the centre of the glass and spinning at 300 rpm for 20 s followed by 800 rpm for one minute. Multiple cycles of coating were carried out with a waiting time of four minutes between cycles to allow partial drying. AFM topography and phase profiles were obtained in tapping mode using a Bruker Dimension ICON—Nanoscope V Controller. Absorbance spectra of deposited protein were recorded using a Shimadzu UV-Vis spectrophotometer. The thickness of the protein films was determined with an Alpha Step D-500 Profiler (KLA Tencor).

3.3.3 Synthesis and Characterisation of the Electrolyte NMe4 T− and T2 were synthesized as described elsewhere [49] and dissolved in molten succinonitrile at 80 °C to equal final concentrations. The dynamic viscosity of the electrolytes was measured using an MCR 702 TwinDrive Rheometer (Anton Paar) with a rotational test employing a parallel plate of 2.5 cm diameter. Cyclic voltammetry was carried out on equimolar mixtures of + NMe4 T− and T2 using AUTOLAB potentiostat (Metrohm). +

3.3.4 Device Fabrication and Photochronoamperometry Ti counter or working electrodes were prepared by sputtering titanium on a clean silicon substrate to a thickness of 350 Å. FTO glasses were cleaned by sequentially sonicating in acetone, isopropyl alcohol and deionised water before spin coating

46

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

as above. The gel electrolyte was sonicated for 1–3 min using an Elmasonic S30H ultrasonic water bath (Elma) to change its viscosity to that of a liquid and a volume of 10 μL was drop-casted onto the protein film. After a waiting period of 2 min to enable permeation of the electrolyte into the protein multilayer the coated working electrode was sandwiched with the cleaned counter electrode and the sides sealed with epoxy resin. Photocurrents were measured under white light illumination (100 mW cm−2 ) using K2400 source meter (Keithley). Average photocurrent densities are compiled in Table S1 (Supporting Information). All currents had similar profiles with a sharp

3.3 Construction Protocol for Bio-Hybrid Cells with Phase-Changing Gels

47

Fig. 3.2 Properties and fabrication of photo-bioelectrochemical cells. a The Rba. sphaeroides RC-LH1 complex (inset) comprises a central RC (solid object) surrounded by a cylindrical LH1 protein (ribbons). Energy absorbed by the LH1 bacteriochlorophylls (ring of alternating red and orange spheres) and carotenoids (not shown) flows by resonance transfer (red arrows) to the P bacteriochlorophyll pair of the RC (sticks, yellow carbons), initiating charge separation (green arrows) to HA (sticks, pink carbons), QA and QB (sticks, cyan carbons) (also see Fig. 3.1 and Sect. 3.4.2). b In natural photosystems, light energy harvested by an extended antenna pigmentprotein system is concentrated by directional energy transfer onto a smaller number of photovoltaic reaction centre pigment-proteins, where energy is trapped through charge separation. In the present study, this is mimicked in three dimensions by coating a base layer of RC-LH1 proteins with multilayers of LH1 antenna proteins (not drawn to scale). This demonstrates the possibility of expansive collection of photons by extended layers of light harvesting antennae that concentrate the excitonic energy to a smaller number of energy traps, which is analogous to the mechanism of a satellite dish that collects weak signals from a wide space and concentrates them on a sensor at a focal point. c To achieve device fabrication the gel-phase succinonitrile/T2 /T− electrolyte was liquefied by sonication and applied to one or more protein multilayers that had been spin-coated on the working electrode (Refer movie B1—see Appendix B for movie link and description). During the subsequent resting period the electrolyte soaked into the protein coating after which the counter electrode was placed on the gel surface and the cell sealed with epoxy resin. Individual complexes are expected to be oriented randomly throughout the protein film. Spin coating can be used to lay down complex architectures, such as that illustrated where a base coating of RC-LH1 complexes (bottom layer) is overlaid with upper coatings of LH1 complexes (three upper layers)

onset and decline on light on/off and a steady level under illumination. EQE action spectra were recorded using a QE/IPCE measurement system (Zolix).

3.3.5 Cell Fabrication Using Mechano-Responsive Electrolytes Prepared salts of the T2 /T− redox couple (Fig. 3.3a) were used as an equimolar mixture and had a vacuum potential of −4.8 eV (Fig. 3.4). Both + NMe4 T− and T2 were dissolved in molten succinonitrile at a concentration of 0.2, 0.4, 0.6 or 0.8 M (Fig. 3.3b). The gels formed on cooling exhibited a mechano-induced transition to liquid phase that reversed on resting (Refer movie B1—see Appendix B for movie link and description). Concentrations of + NMe4 T− and T2 of 0.8 M represented an upper limit beyond which the succinonitrile mix did not form a gel. The viscosities of the gels were measured by rheometry at a low shear rate of 0.005 s−1 for 30 s, producing a plateau with a slight slope (Fig. 3.3c). The 0.2 M electrolyte produced a gel state with the highest viscosity, higher T2 /T− concentrations producing progressively lower viscosities. After 30 s gels were sheared at an increasing rate which confirmed non-Newtonian shear thinning behaviour with final liquid viscosities in the mPa s range (Fig. 3.3c, right). Charge flux through the electrolytes was measured by electrochemical impedance spectroscopy, the decreasing diameter of the semicircles in the resulting Nyquist plots (Fig. 3.3d) indicating an enhancement of ion flux and charge transfer to the counter electrode as the concentrations of T2 and T− were

48

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

a

b

c

d

1000 Shear rate 0.2 M 0.4 M 0.6 M 0.8 M

10

100 1

10 0.1

1

gel

0.1 0.01 0

30

60

90

120 150 Time (s)

180

210

0.6 M

150

200 250 Z' (Ohm)

0.8 M

0.8 M 0.6 M 0.4 M 0.2 M

40

Z'' (Ohm)

Viscosity (Pa.s)

1000

0.4 M

60 50

100

Shear rate (s-1)

10000

0.2 M

30 20

0.01

10

1E-3

0 0

50

100

300

350

400

Fig. 3.3 Characterization of the gel electrolyte. Characterization of the gel electrolyte. a The electrolyte was an equimolar mixture of + NMe4 T− and T2 in succinonitrile. b Images of gel phase electrolytes formed from increasing equimolar mixtures of + NMe4 T− and T2 . Concentrations above 0.8 M were not used as this resulted in a liquid electrolyte rather than the desired gel. c Change in viscosity on increased shear rate for different concentrations of electrolyte. The 0.2 M, 0.4 M, 0.6 M and 0.8 M electrolytes had viscosities of ~75 kPa s, ~8 kPa s, ~2 kPa s and ~0.3 kPa s, respectively, in the gel form. d Nyquist plots for dummy cells comprising different concentrations of electrolyte between two Ti electrodes. Inset: Equivalent circuit model for the Nyquist plot. Rs , Cdl and Rct denote series resistance, double layer capacitance and charge transfer resistance, respectively

increased. An electrolyte concentration of 0.8 M was selected for the fabrication of most devices to maximise charge transfer whilst retaining a gel phase when dissolved in succinonitrile. The first set of working electrodes were fabricated by depositing multilayers of RC-LH1 protein onto a conductive FTO glass substrate through up to twenty cycles of spin coating and partial drying (Fig. 3.5a). Achieving an optimal coverage of protein on the 1 cm2 substrate required use of a protein stock solution at a concentration of around 150 μM (600 absorbance units at 875 nm); at this concentration the protein solution (in a 20 mM Tris (pH 8.0)/0.04% n-dodecyl β-D-maltopyranoside buffer) was very viscous but could still be pipetted. The most consistent coverage was achieved with a two-stage spin cycle and a four minute period of partial drying between, and it was possible to deposit up to twenty coats before adding further layers became impracticable. The use of spin coating freed-up selection of the material for the working electrode, as no functionalization or direct binding interactions were needed to attach proteins. In addition, as described below, it enabled the controlled deposition of successive multilayers made up from different proteins. Atomic force microscopy (AFM) of a RC-LH1 multilayer deposited by a single cycle of spin

3.3 Construction Protocol for Bio-Hybrid Cells with Phase-Changing Gels

49

Fig. 3.4 Cyclic voltammogram (CV) of the electrolyte. CV of an equimolar mixture of T2 /T− in dimethyl sulphoxide obtained with glassy carbon as the working electrode and a Pt wire as the counter electrode. The reference electrode was Ag/AgCl in 3 M KCl, with 0.5 M tetrabutylammonium perchlorate as the supporting electrolyte (0.21 V relative to normal hydrogen electrode (NHE)). Inset: Determination of vacuum potential from the redox potential obtained with a Ag/AgCl reference electrode, based on the peak anodic potential (EPA ) and peak cathodic potential (EPC ). Calculation of vacuum potential was based on the convention that 0 V (vs NHE) is equivalent to − 4.5 eV on an absolute scale [54]

coating revealed an upper surface comprising particles with dimensions and spacings consistent with closely-packed RC-LH1 complexes (Fig. 3.5b). Absorbance spectra showed that the amount of protein adhering after the first coating was greater than after subsequent coatings (Fig. 3.5c). Beyond ten cycles the measured absorbance spectrum became distorted due to the thickness of the protein multilayer and contributions from light scattering (Fig. 3.5c). Profilometry showed that the thickness of the deposited protein multilayer increased from ≈4 μm for a single coating to ≈45 μm for twenty coatings (Fig. 3.6). Assuming that an individual RC-LH1 complex in its detergent micelle has a mean diameter of around 15 nm, consistent with the particle separation in the image in Fig. 3.5b and atomic-level structural information on RC-LH1 complexes, these thicknesses would be consistent with multilayers comprising 267 and 3000 protein molecules, respectively. The thickness of protein film deposited by spin-coating showed very good reproducibility, enabling comparisons across multiple cells and investigation of the relative merits of equivalent loadings of RC-LH1 and LH1 proteins as light harvesting materials (Fig. 3.7). Importantly, the absorbance spectra of deposited proteins did not vary significantly in line shape from that in solution, demonstrating that the pigment-proteins had not been damaged by spin-coating and subsequent treatments.

50

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

Fig. 3.5 Spin-coated protein layer—structural and optical characteristics. a Images of RC-LH1 films on FTO-glass prepared by one or more cycles of spin-coating, taken against a coloured background. b AFM phase image of the surface of an RC-LH1 multilayer film formed by a single cycle of spin coating. c Absorbance spectra of RC-LH1 films. The detector became saturated for the thickest films producing spectral distortion that was also contributed to by light scatter Fig. 3.6 Thickness of deposited RC-LH1 multilayers. Error bars represent standard error (n = 3)

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors

51

Fig. 3.7 Absorbance spectra of five-coating cells. Comparison of near-infrared absorbance spectra of cells fabricated from five cycles of spin coating using either RC-LH1, RC or LH1 proteins

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors Cells for photochronoamperometry were constructed by drop-casting 10 μL of sonicated, liquid-phase electrolyte onto a protein-coated FTO-glass electrode (Fig. 3.2c). After allowing the liquid electrolyte to spread across the entire protein area and soak into the protein coating, the Ti-coated back electrode was brought into contact with the electrolyte layer and the cell was sealed with epoxy resin. Photochronoamperometry revealed that the photocurrent density in response to one sun illumination increased as the concentrations of T2 /T− increased in cells fabricated with a single coating of RC-LH1 complexes (Fig. 3.8a). All subsequent experiments were therefore performed with 0.8 M electrolyte, which had the lowest viscosity and highest charge flux (Fig. 3.3d). The photocurrent density increased as the amount of deposited RC-LH1 protein increased (Fig. 3.8b), up to an average of 780 μA cm−2 for an electrode fabricated by twenty cycles of spin coating; the highest output achieved with an individual cell of this type was 860 μA cm−2 (Fig. 3.8b). Cells yielded only a negligible photocurrent of 0.5 μA cm−2 without the protein coating (Fig. 3.8b), demonstrating that the RCLH1 complexes were the source of the substantial photocurrent. With greater than one cycle of spin coating the photocurrent density was linearly proportional to the measured thickness of the RC-LH1 multilayer (Fig. 3.8c). An action spectrum of external quantum efficiency (EQE) versus excitation wavelength for an RC-LH1 cell formed from a twenty cycles of spin coating was compared to the solution RC-LH1 spectrum (Fig. 3.8d). A peak EQE of ≈13% was observed attributable to the characteristic LH1 absorbance. In the near-infrared the absorbance spectrum principally comprises a prominent band centred at 875 nm attributable to 16 LH1 bacteriochlorophylls and two RC bacteriochlorophylls and a lower band

250 200 150 100 50 0 10

c

30

40

d

3.0 Thickness Absorbance

20 cycles*

1.5

20

1.0 5 cycles

0.5 0 200

0.0

100 0

Protein Base and Upper coatings

1 RC + 4 RC

1 RC-LH1 + 4 RC-LH1

1 RC + 4 LH1

1 RC-LH1

1 RC-LH1 + 4 AM260W RC-LH1

200

1 LH1

300

0

0

10

20 Time (s)

30

40 14

Solution Absorption spectrum EQE - 20 cycles

12

0.10

10

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8

0.06

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400

300 400 500 600 700 800 Photocurrent Density ( A/cm2)

1 RC

Photocurrent Density ( A/cm2)

400

200

0.00

1 cycle

600 500

Absorbance (a.u.)

10 cycles

400

0.12

2.5 2.0

600

0.14

f Percentage photocurrent density (%)

Thickness ( m)

40

e

20 Time (s)

Absorbance (a.u.)

0

20 cycles 10 cycles 5 cycles 1 cycle Without RC-LH1

800

EQE (%)

300

1000

0 500

600 700 800 Wavelength (nm)

900

100

100

80

80

60

60 40

40 20

Transparent dummy cell Spin coated film under ambient conditions

20

Percentage 875 nm absorbance (%)

0.8 M 0.6 M 0.4 M 0.2 M

Photocurrent Density Jsc ( A/cm2)

b 350

1 RC-LH1 + 4 LH1

a

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes … Photocurrent Density Jsc ( A/cm2)

52

0

0 day 0

day 9 day 18 Time

day 28

Fig. 3.8 Photocurrents and stability of photo-bioelectrochemical cells. a Photocurrent density over 20 s illumination as a function of electrolyte concentration for a RC-LH1 multilayer formed by a single cycle of spin-coating. b Photocurrent density over 20 s illumination as a function of multiple cycles of spin-coating for RC-LH1 complexes with 0.8 M electrolyte. c Variation of photocurrent density with the thickness and absorbance at 875 nm of the RC-LH1 multilayer; *the absorbance value corresponding to 20 cycles of spin coating is a slight underestimate as the spectrophotometer detector reached saturation. d EQE action spectrum for a 20 cycle RC-LH1 cell compared to the absorbance spectrum of RC-LH1 complexes in solution; both exhibit bands specific to (mainly) LH1 at 875 nm and the RC at 805 nm. e Photocurrent densities for different protein base and upper coatings, with 0.8 M electrolyte. f Protein and photocurrent stability over 28 days at ambient illumination and temperature

at 805 nm attributable to two RC bacteriochlorophylls. In the action spectrum the relative contribution of the (mainly) LH1-specific band was strongly reduced relative to the shorter wavelength RC-specific band. This under-representation of LH1 to an action spectrum of photocurrent generation has been seen previously [27] and interpreted as showing that the photocurrent output is limited by electron transfer in the cell rather than light harvesting by LH1 (see Sect. 3.4.3 for a discussion). Action

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors

53

Fig. 3.9 EQE action spectrum for a single cycle RC-LH1 cell. The EQE spectrum is compared to the absorbance spectrum of RC-LH1 complexes in solution; both exhibit bands specific to (mainly) LH1 at 875 nm and the RC at 805 nm

spectra recorded for RC-LH1 cell fabricated by a single cycle of spin coating also showed this phenomenon (Fig. 3.9), confirming that it was not due to self-shading by LH1 pigments in the thicker protein multilayers.

3.4.1 The Mechanism of Photocurrent Generation The observed anodic photocurrent implied that photo-excitation of the pigmentprotein coating produced electron donation to the FTO-glass working electrode from a sufficiently reducing excited or anion state (Fig. 3.10, cyan and blue arrows), with re-reduction by the Ti counter electrode of the resulting oxidized pigment(s), most probably P+ (Fig. 3.10, gold). As the vacuum potential of T2 /T− (Fig. 3.10, yellow) was much deeper than that of FTO-glass or Ti, it was concluded that the T2 /T− facilitated electron transfer from the counter electrode to the photo-oxidised species in the protein coating (Fig. 3.10, magenta arrows). One expectation is that the ease of penetration of the 0.8 M electrolyte should be greater than that of lower concentrations as its liquid state viscosity is the closest to that of water at room temperature (Fig. 3.3c), and this may have contributed to the higher current seen with 0.8 M T2 /T− compared to other concentrations. Given the similarity in vacuum potential of FTO-glass and Ti (Fig. 3.10f), a prediction is that cells of a reverse electrode configuration should be functional. This was the case, a photocurrent of −270 μA cm−2 being obtained when RC-LH1 complexes were spin-coated onto a Ti back electrode (single round) and FTO-glass was used as the counter electrode (Appendix B, Figure B1). This was almost identical, but with reverse polarity, to the average of 287 μA cm−2 obtained for an equivalent cell with a single coating of RC-LH1 complexes on FTO-glass. The modular nature of the Rba. sphaeroides photosystem was exploited to investigate the mechanism of charge injection into the working electrode, by comparing the average current from a single coating of purified RCs, or a single coating of purified LH1 light harvesting pigment-proteins, with the 287 μA cm−2 obtained from a single

54

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

direct RC excita on

Vacuum poten al (eV)

-3.5

FRET 50 ps

P*

LH1 excita on LH1*

3 ps HA30 ns

-4.0

200 ps QA-

Ti

100 μs

-4.5

100 ms

Q B-

FTO

1-2 s

T2/T -5.0

P+

Fig. 3.10 Mechanism of photocurrent generation. Mechanism corresponding to the cells with an FTO-glass working electrode: vacuum potentials of reducing and oxidizing components of the RC are shown in purple and brown, respectively. Direct excitation of the RC, or excitation of LH1 pigments followed by FRET to the RC (magenta arrow), triggers charge separation by promoting a pair of bacteriochlorophyll cofactors in the RC (P) into their first singlet excited state (P*— + − rainbow arrow). Charge separation (green arrows) proceeds through the radical pairs P+ H− A , P QA − + and P QB (see Fig. 3.2a for the spatial arrangement of these cofactors and Sect. 3.4.2 for a more detailed description) at each stage rapid forward electron transfer (green arrows) outcompetes slow radical pair recombination (grey dashed arrows). The mechanism of multistep charge separation and associated lifetimes for separation and recombination events have been reviewed [22, 24, 25]. Reduction of P+ mediated by the electrolyte (red arrows) would isolate a long-lived anion state, − most probably Q− B in wild-type RCs and HA in fast-recombining AM260W RCs. Cyan and blue arrows indicate possible processes for reduction of the working electrode by RCs or LH1 complexes, respectively, some or all of which can operate depending on which protein complex makes up the base coating of the device

coating of the combined RC-LH1 complex. An average current of 74 μA cm−2 was obtained with LH1 (Fig. 3.8e), demonstrating that some charge injection can happen directly through an LH1 excited state (LH1*) acting as a sensitizer (Fig. 3.10, blue arrow), as this protein does not itself carry out charge separation. The mechanism would be equivalent to that proposed to operate in TiO2 -based solar cells employing light harvesting proteins as the photosensitizer [55–57]. A strongly-oxidising LH1 cation will result that can be re-reduced, in the present case, by the permeating T2 /T− electrolyte. Cells with a single coating of purified RCs gave an average current of 175 μA cm−2 (Fig. 3.8e). This demonstrated that electron injection must also be able to occur from a RC excited or anion state (Fig. 3.10, cyan arrows). As the lowest-energy RC excited state (P*) has a lifetime of only 3–5 ps it seems more likely that this state was one or more of the multiple anions produced, sequentially, during charge separation within

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors

55

the RC (Fig. 3.10, green arrows). The identity of this anion was explored further using fast-recombining RCs, as discussed in Sect. 3.4.4. Although either RCs or LH1 proteins could individually drive a photocurrent, the strongest output was obtained using the combined RC-LH1 complex. Data from more strongly pigmented, multi-coating cells showed that, although LH1 and RCLH1 complexes have similar absorbance spectra, the latter was still much more effective at generating a photocurrent. Cells fabricated with either one or ten coatings of LH1 produced currents of 74 and 114 μA cm−2 , respectively, whilst cells fabricated with either one or ten coatings of RC-LH1 produced currents of 287 and 603 μA cm−2 , respectively (Table 3.1). This was despite similar light harvesting capacities, as judged by the intensity of the LH1 absorbance band at 875 nm. The mechanism of photocurrent generation by multilayers of photosynthetic protein is a little explored and poorly understood area, but is likely to involve energy transfer between protein layers. To clarify how different layers supported photocurrent generation in multilayer cells, four coatings of LH1 complexes were deposited over a single base coating of either RC-LH1 or RC complexes. Average photocurrents were 453 and 428 μA cm−2 respectively (Fig. 3.8e and Table 3.1), much higher than the 287/175 μA cm−2 that could be obtained with just a single base coating of RC-LH1 or RC complexes. This strongly suggested that the additional LH1 coatings play a light harvesting function, passing excited state energy to the base coating to power electrode reduction. Table 3.1 Average photocurrent densities for cells fabricated from one or more coatings of photosynthetic protein Base coating

Additional coatings

Photocurrenta (μA cm−2 ) Standard errora (μA cm−2 )

RC-LH1



287

29

RC-LH1

4 RC-LH1

468

27

RC-LH1

4 LH1

453

38

RC-LH1

4 AM260W RC-LH1 317

38

LH1



LH1

9 LH1

RC



175

21

RC

4 LH1

428

33

138

11

AM260W RC –

74

10

114

21

RC

4 RC

304

42

RC

4 AM260W RC

221

30

AM260W RC 4 AM260W RC

173

32

AM260W RC 4 LH1

433

34

1 RC-LH1

9 RC-LH1

603

48

1 RC-LH1

19 RC-LH1

780

47

a Average

of at least 3 measurements

56

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

These current enhancements supported the proposal that the photochemical process that produced the current was electron injection into the working FTO-glass electrode from the adjacent base coating of protein, followed by reduction of the photo-oxidised protein by the permeating T2 /T− electrolyte. The alternative mechanism, reduction of photo-excited bacteriochlorophylls at the protein/electrolyte interface, would be expected to produce strongly-reducing LH1 bacteriochlorophyll anions. It is unlikely that subsequent migration of electrons through multilayers of LH1 protein to the working electrode would be efficient in the absence of suitable mediators. An interesting finding was that the average current of 468 μA cm−2 obtained by overlaying a single base coating of RC-LH1 complexes with four further RC-LH1 coatings was almost identical to the 453 μA cm−2 achieved using four additional coatings of LH1 protein (Fig. 3.8e). The implication is that having additional charge separating RCs in the upper coatings did not waste harvested energy. This was intriguing, as a feature of natural photosystems that supports their high quantum efficiencies is that unproductive charge separation is avoided in the extensive light harvesting regions of the photosystem, being limited to a small number of specialist RC proteins where charge separation is electrically connected to an external protonmotive electron transfer chain to achieve energy conservation. Two possible explanations could be put forward for this apparent lack of energy wastage. The first, which seems unlikely, is that the rate of LH1 → LH1 energy transfer between adjacent RC-LH1 complexes in the upper coatings of the cell is much faster than the rate of LH1 → RC energy transfer (trapping) within individual RCLH1 complexes (lifetime of around 50 ps), and so wasteful charge separation in the upper coatings is not competitive with productive energy migration through hundreds or thousands of LH1 antenna proteins. The second is that charge separation takes place in the upper layers but is not wasteful, instead making a positive contribution to the photocurrent through the creation of metastable radical pairs throughout the bulk of the device, such that any decrease in light harvesting efficiency due to trapping is compensated for. To investigate this point further, cells were fabricated in which a base coating of RC-LH1 complexes was overlaid with four coatings of fast-recombining AM260W RC-LH1 complexes. The resulting average photocurrent was 317 μA cm−2 , substantially lower than the 468 μA cm−2 obtained with four additional coatings of normal RC-LH1 or the 453 μA cm−2 obtained with four additional coatings of LH1 protein (Fig. 3.8e). This can be attributed to the ~17 ns charge separated state formed in the AM260W RCs being less effective in supporting charge conduction through the bulk of the protein multilayer than the 108 longer-lived charge separated state formed in the native RC. The study also examined the impact of charge separation taking place throughout the bulk of a protein multilayer by fabricating cells with either one or five coatings of RCs. Average currents were 175 and 304 μA cm−2 , respectively (Fig. 3.8e). As RCs have evolved to trap excitation energy through highly-efficient ultrafast charge separation it is likely that the current enhancement on adding more RCs was due to charge separation in the upper coatings and RC to RC electron transfer, rather than a

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors

57

light harvesting effect involving inter-RC energy transfer. As discussed in Sect. 3.4.5, experiments with multilayer cells fabricated from fast-recombining AM260W RCs supported this interpretation. Reducing the lifetime of the longest-lived RC radical pair by eight orders of magnitude substantially reduced the photocurrents that were produced from RC multilayers.

3.4.2 Structure and Mechanism of RC-LH1 Complexes The RC-LH1 complex from Rba. sphaeroides is embedded in the photosynthetic bilayer membrane (Fig. 3.1) and comprises a central RC surrounded by a cylindrical LH1 light-harvesting protein [58]. The native complex is dimeric due to the presence of the PufX protein [59] which prevents complete encirclement of the LH1 around the RC, facilitating dimer formation. Removal of PufX by gene deletion results in a monomeric complex in which the LH1 pigment-protein completely encircles the RC [60]. This simplifies purification and maximises the number of light harvesting pigments that surround each RC at 32 bacteriochlorophylls (BChls) and 32 carotenoids (as opposed to 28 and 28, respectively, in the presence of PufX) [58]. This version of the RC-LH1 complex engineered for maximal light harvesting was used in the present study. The structure of this PufX-deficient RC-LH1 from Rba. sphaeroides is similar to that of the RC-LH1 complex from Thermochromatium (Tch.) tepidum, for which a high resolution X-ray crystal structure has been determined [23], and this latter structure is shown in Figs. 3.1 and 3.2. In Rba. sphaeroides both the RC and the LH1 ring can assemble independently of one another, and the present study exploited bacterial strains in which the RC-LH1, RC or LH1 complex is the sole pigment-protein that assembles in the photosynthetic membrane, unwanted components having been removed by gene deletions [61]. The LH1 pigment-protein comprises concentric cylinders of 16 α- and βpolypeptides (Fig. 3.1b) that encase 32 carotenoids (not shown) and a ring of 32 BChls (Fig. 3.1, BChl macrocycles shown alternating red and orange spheres) which give rise to the major absorbance band at 875 nm (Figs. 3.5c and 3.8d). Light energy absorbed by this pigment system across the visible spectrum creates excited states which convert, on an ultrafast time scale, to the lowest energy transition in the system, the first singlet excited state of the LH1 BChls [62]. This LH1* state decays with a lifetime of ~30–60 ps by Förster resonance energy transfer (FRET) to the P BChls of the RC (Fig. 3.1c, red arrow), forming their first singlet excited state (P*). In the absence of a RC the excited state lifetime of the BChls of the LH1 antenna is a few nanoseconds. The carotenoids and bacteriochlorins of the RC can also absorb light energy, the initial excited state formed relaxing to P* on a sub-picosecond time scale [62]. Irrespective of whether energy is transferred within the RC, or from a surrounding LH1, the formation of P* triggers a membrane-spanning charge separation through the RC (Fig. 3.1c, green arrows). P* decays with a lifetime of 3–5 ps through electron transfer to the HA bacteriopheophytin, H− A passes its electron to the QA ubiquinone

58

3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

with a lifetime of ~200 ps, and Q− A reduces the QB ubiquinone with a lifetime of ~100 μs. The system therefore evolves through the excited and radical pair states P* + − + − → P+ H− A → P QA → P QB . If forward electron transfer is blocked at any stage a slower charge recombination occurs (grey dotted arrows in Fig. 3.10) with loss of energy as heat. Reduction of P+ by an external donor on a time scale faster than this recombination will produce a long lived anion state, usually Q− B.

3.4.3 Factors Limiting Photocurrent Density in RC-LH1 Cells As illustrated in Fig. 3.8d, in the action spectrum of photocurrent generation by RC-LH1 complexes the contribution of energy absorbed by the LH1 component relative to that absorbed directly by the RC was significantly lower that theoretically possible based on their absorbance spectra. This suggests that only a fraction of the energy absorbed by the LH1 pigments is used to drive the photocurrent. The likely explanation for this is that the photocurrent density in this device configuration was limited by the rate of electron transfer into, or out of, the RC charge separation chain. In order for energy transferred from LH1 to the RC to drive a metastable charge separation that can oxidise or reduce an external donor/acceptor (usually at least a microsecond time scale reaction) the RC has to be “open” with the P BChls in the reduced state and the QA quinone in the oxidised state (PQA ). If, instead, the RC − is in a “closed” P+ QA , P+ Q− A or PQA state, energy transfer from LH1 to the RC results in energy dissipation as heat through a variety of mechanisms. As a result, the contribution of LH1 to the photocurrent is likely to be limited by the relative rates of energy trapping by each RC versus the rate at which that RC is returned to an open state by electron flux into the P+ “terminal” (via the permeating T2 /T− electrolyte) and electron efflux from the quinone “terminal” (which is likely to be the limiting process in the system). An implication of this is that it may be possible to further improve device performance by making electron transfer from RC quinone acceptors to the working electrode more efficient, such that the limiting factor becomes energy harvesting rather than charge conduction.

3.4.4 Mechanism of Electron Donation to the Working Electrode As described earlier, cells fabricated with a single coating of purified RCs supported an average 175 μA cm−2 photocurrent, implying that either the lowest energy excited − singlet state of the RC pigments (P*) or a subsequently formed anion state (H− A , QA − + or QB ) donates an electron to the working electrode, with reduction of P by the electrolyte (Fig. 3.10). As the P* state has a lifetime of only 3–5 ps, one or more of

3.4 Photoactivity in the Biohybrid Cells and the Controlling Factors

59

− − the longer-lived H− A , QA or QB states would seem the more likely donor. It is also possible that the doubly reduced quinone state QB H2 could be formed after reduction of P+ and a second charge separation, but for simplicity this is not included in the scheme in Fig. 3.10. Some insight into the possible electron donor came from use of fast-recombining AM260W RCs [61]. Cells fabricated with a single coating of AM260W RCs supported a photocurrent of 138 μA cm−2 , somewhat lower than the 175 μA cm−2 achieved with wild-type RCs (at comparable protein loadings). This result clarified that a substantial current could be supported without formation of the relatively − + − long-lived radical pairs P+ Q− A or P QB . One interpretation of this could that HA is capable of donating electrons to the working electrode on a timescale faster than P+ H− A recombination (lifetime ~17 ns). However an additional factor could be that the permeating T2 /T− electrolyte is able to reduce P+ on a time scale faster than the lifetime of P+ H− A recombination, and so the RC anion is stabilised irrespective of + − + − whether charge separation reaches the P+ H− A , P QA or P QB stage. Interestingly, when four coatings of LH1 complexes were overlaid on a base coating of AM260W RCs the current obtained, 433 μA cm−2 , was not significantly different to that obtained with an equivalent cell employing wild-type RCs in the base coating (428 μA cm−2 ). This reinforces the finding that the RCs making up the base coating did not need to carry out a full charge separation and stabilization process in order to support a photocurrent.

3.4.5 Charge Conduction Through RC Multilayers It was intriguing to observe that a RC multilayer could support a substantial photocurrent, and making the multilayer thicker further increased the current output (from 175 μA cm−2 for one coating to 304 μA cm−2 for five coatings). The mechanism through which this current enhancement is likely to have occurred has interesting possible implications. The six bacteriochlorin and one carotenoid pigments of each RC can act as light harvesting pigments, and indeed strains of Rba. sphaeroides devoid of light harvesting complexes can grow photosynthetically provided they are supplied with a sufficiently high light intensity [63]. However, extensive spectroscopic studies have shown that photoexcitation of any of the seven RC pigments initiates femtosecond energy transfer to form P* and subsequent picosecond charge separation. Given that the RC pigments are buried within the RC protein, to provide insulation for controlled electron transfer, it seems inconceivable that long-range energy transfer between RCs in a multilayer could compete with short-range energy transfer and charge separation within a single RC. How then do RCs not in immediate contact with the working electrode contribute to the photocurrent if energy transfer between RCs is not a significant process? The answer may be that indicated by the finding that photocurrents were lower when upper coatings of protein were formed from “fast recombining” AM260W RCs. Cells formed from a base coating of wild-type RCs and four upper coatings of

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3 Interfacing Photoproteins with Mechanoresponsive Electrolytes …

AM260W RCs gave a lower average current (221 μA cm−2 ) than cells formed from five coatings of wild type RCs (304 μA cm−2 ), and cells formed from five coatings of AM260W RCs gave an even lower current (173 μA cm−2 ). This indicates a role for charge separation in supporting current flow throughout the RC multilayers, this being much less effective when the lifetime of the radical pair formed as the end product of charge separation is reduced by eight orders of magnitude by mutation. This role is likely to involve electron transfer between adjacent RCs in multilayers. Such electron transfer could be feasible; the P and QB cofactors are buried within the RC protein at depths of about 6 and 20 Å, respectively, compared with an internal P/quinone separation of around 21 Å, and so the distances for intra- and inter-protein charge recombination are of a similar order. Inter-RC electron transfer is not a natural phenomenon as RCs are housed in a two-dimensional membrane system with each RC surrounded by an LH1 pigment-protein. However, it may be an important feature of a three-dimensional RC film where the question of whether Q− B → P+ electron transfer occurs within a single protein or between adjacent proteins in a disordered multilayer is expected to be determined by relative distances for electron tunnelling and the balance between the driving force for the transfer and the relative reorganisation energies for intra- and inter-protein electron transfer.

3.4.6 Stability Studies Finally, in addition to enabling very stable photocurrent densities during short periods of continuous illumination (Fig. 3.8a, b), studies with sealed and dummy cells suggested that the long-term stability of the RC-LH1 complex was enhanced by embedding it in the succinonitrile/T2 /T− gel. A dummy transparent RC-LH1/T2 /Tcell employing two FTO-glass electrodes showed 39% drop in absorbance at 875 nm over 28 days of storage at room temperature and a continuous ambient illumination of ~2 W m−2 , indicating slow degradation of the LH1 pigment-protein (Figs. 3.8f and 3.11). In good agreement with this the photocurrent obtained from an equivalent cell with a Ti counter electrode showed a 27% decrease in current density (Fig. 3.8f). For a spin coated RC-LH1 film on FTO-glass not incorporated into a cell with electrolyte the 875 nm absorbance dropped by 73% over the same period (Figs. 3.8f and 3.11), suggesting a protective effect of the gel electrolyte.

3.5 Summary In conclusion, the protein-based photo-bioelectrochemical cells described above combined a number of innovations in cell design, fabrication and materials that produced a maximum five-fold improvement in photocurrent amplitude compared to the highest previously-reported current from a comparable device [33]. Our previous

3.5 Summary

61

Fig. 3.11 Stability of RC-LH1 complexes under continuous illumination. Decay of the native absorbance spectrum of RC-LH1 complexes: a in a transparent dummy RC-LH1/T2 /T− cell constructed using two FTO-glass electrodes; b in a spin coated RC-LH1 film on FTO-glass not incorporated into a cell. Pigment-protein degradation gives rise to absorbance decreases at 420– 570 nm for carotenoid and at 590 and 875 nm for BChl. The appearance of BChl breakdown products produces broad absorbance increases in the region between 650 and 800 nm. The drop in LH1 absorbance at 875 nm is accompanied by a blue shift of the absorbance band due to increasing overlap with underlying RC absorbance at 800 nm (visible) and 865 nm (not visible)

research with this particular RC-LH1 complex in liquid state photoelectrochemical cells with diffusible electrolytes produced steady-state photocurrents ranging between 0.15 and 8 μA cm−2 [15, 37, 38] and the maximally two orders of magnitude increase in current density seen in this work can be attributed to a combination of the dense protein multilayers achievable with spin coating and the charge conduction enabled by a high concentration of permeating gel phase electrolyte. Spin coating also facilitated controlled deposition on unfunctionalised electrodes of multiple multi-layers of different types of photosynthetic protein, making possible exploration of photocurrent mechanism and evaluation of strategies for maximising output. The architecture of some devices followed natural principles in which a limited number of photovoltaic centres are fed with excited state energy by a larger light harvesting system, but the work also revealed that metastable charge separation

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throughout all layers can bring about current enhancements. The succinonitrile/T2 /T− electrolyte material removes commonly-observed diffusive restrictions on the steadystate photocurrent density and, due to its vibration-induced phase transition, could be combined with labile biological materials in a simple way without inducing protein damage. In broader terms this methodology could provide a means of interfacing an ionic electrolyte with any photoactive species that cannot tolerate exposure to the harsh conditions often required for device fabrication.

References 1. Ravi SK et al (2018) A Mechanoresponsive phase-changing electrolyte enables fabrication of high-output solid-state photobioelectrochemical devices from pigment-protein multilayers. Adv Mater 30(5):1704073 2. Ravi SK (2018) Solar energy harvesting with photosynthetic pigment-protein complexes. National University of Singapore, Singapore 3. Croce R, van Amerongen H (2014) Natural strategies for photosynthetic light harvesting. Nat Chem Biol 10(7):492–501 4. Scholes GD et al (2011) Lessons from nature about solar light harvesting. NatChem 3(10):763– 774 5. Ravi SK, Tan SC (2015) Progress and perspectives in exploiting photosynthetic biomolecules for solar energy harnessing. Energy Environ Sci 8(9):2551–2573 6. Kim Y et al (2014) Hybrid system of semiconductor and photosynthetic protein. Nanotechnology 25(34):342001 7. Yehezkeli O et al (2014) Photosynthetic reaction center-functionalized electrodes for photobioelectrochemical cells. Photosynth Res 120(1–2):71–85 8. Ravi S et al (2018) Photosynthetic bioelectronic sensors for touch perception, UV-detection, and nanopower generation: toward self-powered E-skins. Adv Mater 30(39):1802290 9. Ravi SK et al (2020) Bio-photocapacitive tactile sensors as a touch-to-audio braille reader and solar capacitor. Mater Horizons 7(3):866–876 10. Ravi SK et al (2019) Optical shading induces an in-plane potential gradient in a semiartificial photosynthetic system bringing photoelectric synergy. Adv Energy Mater 9(35):1901449 11. Ravi SK et al (2019) Photosynthetic apparatus of rhodobacter sphaeroides exhibits prolonged charge storage. Nat Commun 10(1):1–10 12. Ravi SK et al (2018) Bio-photoelectrochemical cells: protein immobilization routes and electron transfer modes, in photosynthetic protein-based photovoltaics. CRC Press, Boca Raton, pp 141–159 13. Ravi SK et al (2018) Emerging role of the band-structure approach in biohybrid photovoltaics: a path beyond bioelectrochemistry. Adv Func Mater 28(24):1705305 14. Singh VK et al (2018) Biohybrid photoprotein-semiconductor cells with deep-lying redox shuttles achieve a 0.7 V photovoltage. Adv Func Mater 28(24):1703689 15. Ravi SK et al (2017) Enhanced output from biohybrid photoelectrochemical transparent tandem cells integrating photosynthetic proteins genetically modified for expanded solar energy harvesting. Adv Energy Mater 7(7):1601821 16. Ravi SK, Tan SC (2018) Electronics, photonics, and device physics in protein biophotovoltaics, in photosynthetic protein-based photovoltaics. CRC Press, Boca Raton, pp 161–224 17. Tan SC (2018) Photosynthetic protein-based photovoltaics. CRC Press, Boca Raton 18. Xie X et al (2014) Photocurrent generation based on a light-driven proton pump in an artificial liquid membrane. Nat Chem 6(3):202–207 19. Yehezkeli O et al (2012) Integrated photosystem II-based photo-bioelectrochemical cells. Nat Commun 3:742

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20. Efrati A et al (2016) Assembly of photo-bioelectrochemical cells using photosystem Ifunctionalized electrodes. Nat Energy 1:15021 21. Schrantz K et al (2017) Hematite photoanode co-functionalized with self-assembling melanin and C-phycocyanin for solar water splitting at neutral pH. Catal Today 284:44–51 22. Feher G et al (1989) Structure and function of bacterial photosynthetic reaction centres. Nature 339:111–116 23. Niwa S et al (2014) Structure of the LH1-RC complex from thermochromatium tepidum at 3.0 Å. Nature 508(7495):228–232 24. Zinth W, Wachtveitl J (2005) The first picoseconds in bacterial photosynthesis —ultrafast electron transfer for the efficient conversion of light energy. ChemPhysChem 6(5):871–880 25. Jones MR (2009) The petite purple photosynthetic powerpack. Biochem Soc Trans 37:400–407 26. Ham M-H et al (2010) Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate. Nat Chem 2(11):929–936 27. den Hollander M-J et al (2011) Enhanced photocurrent generation by photosynthetic bacterial reaction centers through molecular relays, light-harvesting complexes, and direct protein-gold interactions. Langmuir 27(16):10282–10294 28. Sumino A et al (2013) Electron conduction and photocurrent generation of a lightharvesting/reaction center core complex in lipid membrane environments. J Phys Chem Lett 4(7):1087–1092 29. Swainsbury DJ et al (2014) Evaluation of a biohybrid photoelectrochemical cell employing the purple bacterial reaction centre as a biosensor for herbicides. Biosens Bioelectron 58:172–178 30. Yaghoubi H et al (2014) Hybrid wiring of the rhodobacter sphaeroides reaction center for applications in bio-photoelectrochemical solar cells. J Phys Chem C 118(41):23509–23518 31. Caterino R et al (2015) Photocurrent generation in diamond electrodes modified with reaction centers. ACS Appl Mater Interfaces 7(15):8099–8107 32. Gebert J et al (2015) Electron transfer to light-activated photosynthetic reaction centers from rhodobacter sphaeroides reconstituted in a biomimetic membrane system. J Phys Chem C 119(2):890–895 33. Friebe VM et al (2016) Plasmon-enhanced photocurrent of photosynthetic pigment proteins on nanoporous silver. Adv Func Mater 26(2):285–292 34. Mirvakili SM et al (2014) Photoactive electrodes incorporating electrosprayed bacterial reaction centers. Adv Func Mater 24(30):4789–4794 35. Yaghoubi H et al (2015) Large photocurrent response and external quantum efficiency in biophotoelectrochemical cells incorporating reaction center plus light harvesting complexes. Biomacromol 16(4):1112–1118 36. Kondo M et al (2012) Photocurrent and electronic activities of oriented-his-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode. Biomacromol 13(2):432–438 37. Tan SC et al (2012) Generation of alternating current in response to discontinuous illumination by photoelectrochemical cells based on photosynthetic proteins. Angew Chem Int Ed 51(27):6667–6671 38. Tan SC et al (2012) Increasing the open-circuit voltage of photoprotein-based photoelectrochemical cells by manipulation of the vacuum potential of the electrolytes. ACS Nano 6(10):9103–9109 39. Tan SC et al (2013) Superhydrophobic carbon nanotube electrode produces a near-symmetrical alternating current from photosynthetic protein-based photoelectrochemical cells. Adv Func Mater 23(44):5556–5563 40. Chatzipetrou M et al (2016) Functionalization of gold screen printed electrodes with bacterial photosynthetic reaction centres by laser printing technology for mediatorless herbicide biosensing. Electrochem Commun 64:46–50 41. Katz E (1994) Application of bifunctional reagents for immobilization of proteins on a carbon electrode surface: oriented immobilization of photosynthetic reaction centers. J Electroanal Chem 365(1–2):157–164

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42. Takshi A, Madden JD, Beatty JT (2009) Diffusion model for charge transfer from a photosynthetic reaction center to an electrode in a photovoltaic device. Electrochim Acta 54(14):3806–3811 43. Das R et al (2004) Integration of photosynthetic protein molecular complexes in solid-state electronic devices. Nano Lett 4(6):1079–1083 44. Vaghasiya JV et al (2018) Dual functional hetero-anthracene based single component organic ionic conductors as redox mediator cum light harvester for solid state photoelectrochemical cells. J Mater Chem A 6(11):4868–4877 45. Yaghoubi H et al (2017) A ZnO nanowire bio-hybrid solar cell. Nanotechnology 28(5):054006 46. Sokol KP et al (2016) Rational wiring of photosystem II to hierarchical indium tin oxide electrodes using redox polymers. Energy Environ Sci 9(12):3698–3709 47. Ridge JP et al (1999) Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides. Photosynth Res 59(1):9–26 48. Gibasiewicz K et al (2011) Mechanism of recombination of the P+ H− A radical pair in mutant rhodobacter sphaeroides reaction centers with modified free energy gaps between P+ B− A and P+ H− . J Phys Chem B 115(44):13037–13050 A 49. Alarco P-J et al (2004) The plastic-crystalline phase of succinonitrile as a universal matrix for solid-state ionic conductors. Nat Mater 3(7):476–481 50. Wang M et al (2010) An organic redox electrolyte to rival triiodide/iodide in dye-sensitized solar cells. Nat Chem 2(5):385–389 51. Xu X et al (2012) Disulfide/thiolate based redox shuttle for dye-sensitized solar cells: an impedance spectroscopy study. J Phys Chem C 116(48):25233–25241 52. Hwang D et al (2013) Highly efficient plastic crystal ionic conductors for solid-state dyesensitized solar cells. Scientific Reports 3:3520 53. Fiedor L et al (2001) Excitation trap approach to analyze size and pigment–pigment coupling: reconstitution of LH1 antenna of rhodobacter sphaeroides with ni-substituted bacteriochlorophyll. Biochemistry 40(12):3737–3747 54. Zoski, CG (ed) (2006) Handbook of electrochemistry. Elsevier, Amsterdam 55. Fu Q et al (2014) The photoelectric performance of dye-sensitized solar cells fabricated by assembling pigment–protein complexes of purple bacteria on nanocrystalline photoelectrode. Mater Lett 129:195–197 56. Yu D et al (2015) Enhanced photocurrent production by bio-dyes of photosynthetic macromolecules on designed TiO2 film. Sci Rep 5:9375 57. Nagata M et al (2012) Immobilization and photocurrent activity of a light-harvesting antenna complex II, LHCII, isolated from a plant on electrodes. ACS Macro Lett 1(2):296–299 58. Qian P et al (2013) Three-dimensional structure of the rhodobacter sphaeroides RC-LH1-PufX complex: dimerization and quinone channels promoted by pufX. Biochemistry 52(43):7575– 7585 59. Holden-Dye K, Crouch LI, Jones MR (2008) Structure, function and interactions of the PufX protein. Biochim Biophys Acta Bioenerg 1777(7–8):613–630 60. Walz T et al (1998) Projection structures of three photosynthetic complexes from rhodobacter sphaeroides: LH2 at 6 Å, LH1 and RC-LH1 at 25 Å. J Mol Biol 282(4):833–845 61. Jones MR et al (1992) Mutants of Rhodobacter sphaeroides lacking one or more pigmentprotein complexes and complementation with reaction-centre, LH1, and LH2 genes. Mol Microbiol 6(9):1173–1184 62. Blankenship RE (ed) (2013) Molecular mechanisms of photosynthesis. Wiley, Hoboken 63. Jones MR et al (1992) Construction and characterization of a mutant of Rhodobacter sphaeroides with the reaction center as the sole pigment-protein complex. Biochemistry 31(18):4458–4465

Chapter 4

Integrating the Light Reactions of a Photoprotein and a Semiconductor for Enhanced Photovoltage

4.1 Brief Overview Photosynthetic proteins transduce sunlight into biologically-useful forms of energy through a photochemical charge separation that has a close to 100% quantum efficiency, and there is increasing interest in their use as sustainable materials in biohybrid devices for solar energy harvesting. This chapter [1, 2] presents a new strategy for boosting the open-circuit voltage of photoelectrochemical cells based on a bacterial photosynthetic pigment-protein by employing highly oxidising redox electrolytes in conjunction with an n-type silicon anode. Illumination generates electron-hole pairs in both the protein and the silicon electrode, the two being connected by the electrolyte which transfers electrons from the reducing terminal of the protein to photogenerated holes in the silicon valence band. A high open circuit voltage of 0.6 V was achieved with the most oxidizing electrolyte TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy), and this was further improved to 0.7 V on surface modification of the silicon electrode to increase its surface area and reduce reflection of incident light. The photovoltages produced by these biohybrid protein/silicon cells are comparable to those typical of dye-sensitized solar cells and Si solar cells.

4.2 Introduction The Rba. sphaeroides RC, and the RC-LH1 macromolecular complex it forms with the LH1 light harvesting protein, are tractable and well characterised systems that have been used extensively to explore the molecular basis of solar energy conversion during natural photosynthesis [3–7]. The light harvesting bacteriochlorophyll (BChl) and carotenoid pigments of the LH1 protein absorb sunlight across the visible and near-infrared spectral regions and focus excited state energy on an electron transfer chain in the central RC domain (Fig. 4.1a). Arrival of excitation energy at a pair of © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_4

65

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Fig. 4.1 General mechanism of charge separation and photovoltage generation by RC-LH1 complexes in a sandwich-configuration biohybrid cell. a View of the cofactors of the RC-LH1 complex with the six nearest LH1 cofactors removed. The LH1 light harvesting pigments comprise 32 BChls (alternating blue/purple) and 16 carotenoids (red). Energy transfer to the P BChls of the RC (yellow sticks) triggers charge separation (dotted arrow) through the RC cofactors (sticks) to the terminal QB ubiquinone acceptor (cyan sticks). After charge separation to form P+ Q− B , electrons enter the complex through reduction of P+ (magenta arrow) and leave the complex through oxidation of Q− B (cyan arrow). It should be noted that in some device configurations current flow may be dependent on double reduction and protonation of QB to form QB H2 ; for simplicity this is not shown or discussed in the text. b Mechanism of photocurrent generation in a photoelectrochemical cell in which RC-LH1 (or RC) proteins interface directly with a FTO-glass anode and a diffusing electrolyte carries charge to the anode. The VOC is determined by the potential gap between the P/P+ redox couple in the protein and the electrolyte. With electrolytes that have potentials intermediate + between those of P/P+ and QB /Q− B there is also the possibility that they will carry charge to P from − either the FTO electrode or from QB , affecting the observed VOC

4.2 Introduction

67

BChls in the RC protein (denoted P) triggers a meta-stable separation of electrical charge through a four-step transfer of an electron across the RC to a dissociable ubiquinone (QB ), forming the radical pair P+ Q− B [3–5, 8]. This charge separation takes place in a few hundred microseconds and has a lifetime of over a second, with a difference in potential of around 0.5 V between the P/P+ and QB /Q− B redox couples (Fig. 4.1b). As the lowest energy singlet excited state of the P BChls (denoted P*) has an energy of 1.45 eV relative to the ground state, this means that around one third of the energy required to trigger charge separation is conserved in the final radical pair. The remainder is sacrificed to ensure that each step of charge separation is effectively irreversible and that losses through charge recombination are minimised. In the main, published studies of solar energy conversion by RCs and RC-LH1 complexes in biohybrid photoelectrochemical cells have focussed on the mechanism of photocurrent generation and the control of protein/electrode interactions, with relatively little attention given to the open circuit voltage (V OC ) or its optimisation. A handful of studies have reported values of V OC that range between 7 and 140 mV [9–14]. In typical device architectures the V OC of a cell based on a RC or RC-LH1 protein is expected to be limited by the potential gap between the molecular species receiving electrons from the cathode and the species donating electrons to the anode. These may be components of the protein, usually the P+ and Q–B redox centres at either end of the internal charge separation chain, or one or more electrolytes that provide the electrical connection to one or both electrodes. In the schematic in Fig. 4.1b, which is based on previous works [15–18], where the P+ species in RC-LH1 complexes adjacent to an FTO-glass anode receives electrons directly, and a mediator moves charge from the “negative terminal” of the RC-LH1 complex to the counter electrode. The V OC of such a cell is expected to be dependent on the potential gap between the P/P+ couple that interfaces with the cathode and the mediator that interfaces with the anode, and in support of this it has been shown that the V OC in such cells can be manipulated in a systematic fashion using electrolytes of different potential, producing values of up to 200 mV in cells with RC-LH1 complexes [17]. A drawback of this device architecture, however, is the possibility that the mediator can enable futile recombination of electrons between the reducing and oxidising terminals of the RC (dotted arrow in Fig. 4.1b), negatively affecting both the photocurrent and photovoltage. In addition, it is possible that such a mediator could also facilitate electron flow from the FTO cathode to P+ . In such a scenario, with the same mediator interfacing the photoprotein with both electrodes, the V OC would be expected to be very low. In this work, a new strategy is explored in which light-induced charge separation in RC-LH1 complexes adhered to a FTO-glass cathode was connected to a second photo-transition in an n-doped silicon (n-Si) anode in a sandwich-style, two electrode device architecture. Illumination of n-Si produces holes in its valence band at a potential much more oxidising than the P/P+ couple in the RC. The protein/FTO cathode and n-Si anode were connected by an electrolyte solution containing either the nitroxide-containing organic radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), the metallocene couple ferrocene/ferrocenium, or the iodide/triiodide

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(I− /I3 − ) couple commonly used in dye-sensitized solar cells. Each of these electrolytes has a potential considerably more oxidising than P/P+, and so cannot mediate unwanted electron transfer between the reducing and oxidising terminals of the RC protein or between the protein and the cathode. In addition, the study evaluated both planar silicon and silicon that had been textured with micrometre scale pyramidal surface structure to increase the electrode area and reduce photon reflection. It was found that such cells exhibit open circuit voltages of up to 700 mV under illumination. The basis of the high voltages generated by these novel biohybrid cells is discussed.

4.3 Construction and Working of Protein-Semiconductor Hybrids Before construction of photoelectrochemical cells the redox energy levels of the three electrolytes were determined by cyclic voltammetry (Fig. 4.2a). In good agreement with reported values [19], ferrocene and TEMPO each had one redox pair at 0.63

Fig. 4.2 Cell components and performance. a Cyclic voltammograms of the three electrolytes tested. Each was a 10 mM solution in γ-butyrolactone with 0.1 M lithium perchlorate, recorded at a scan rate of 100 mV/s. b Voc (Inset: steady-state photovoltage in TEMPO cell over 400 s). c Jsc produced by RC-LH1 cells with different electrolytes under illumination. d Absorbance spectrum of RC-LH1 proteins in solution compared with an EQE action spectrum for an RC-LH1 cell with TEMPO

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69

Table 4.1 Photovoltaic performance of the RC-LH1 cells with different electrolytes and a planar n-Si counter electrode Electrolyte

Electrolyte vacuum potential (eV)

J sc (μA/cm2 )

V aoc (mV)

ηb (%) × 10−3

TEMPO/TEMPO+

−5.35

1.1

620

0.68

I− /I3 −

−5.21

0.1

460

0.05

Fc/Fc+

−5.13

0.3

320

0.10

a Values

refer to the photo-induced changes in Voc (i.e. light voltage minus dark voltage). Refer Table C1 in Appendix C

b The

power conversion efficiencies (η) of the cells were estimated from η =

JSC ×VOC Pin

× 100,

without considering fill factors due to the transient nature of photocurrent. Pin = 100 mW/cm2

and 0.87 V (vs. NHE), respectively. The iodide/triiodide system had two redox pairs at 0.71 and 0.79 V, the first of which corresponded to the I− /I3 − couple. TEMPO possessed the highest redox potential relative to that for oxidation of the RC primary electron donor, followed by I− /I3 − and ferrocene. Equivalent vacuum potentials are compiled in Table 4.1 RC-LH1 cells were fabricated by drop casting 4 μL of protein solution onto a cleaned FTO surface followed by vacuum drying to produce a protein film. The electrolyte solution was then dropped onto the protein film, and the back electrode brought in contact. The current and voltage outputs of the three types of cell were measured without any applied bias. With 0.2 M ferrocene, a steady-state photoinduced V OC of ≈320 mV was obtained (Fig. 4.2b), over and above a dark voltage of −50 mV (see Table C1 in Appendix C). This was accompanied by a peak transient J sc of −8.8 μA/cm2 that decayed in a couple of seconds to a lower steady-state value of ≈−0.3 μA/cm2 (Fig. 4.2c). Such decays have been previously attributed to limitations imposed by electrolyte diffusion [9, 15, 20, 21] and this has been confirmed experimentally [22]. Cells fabricated with 0.2 M I− /I3 − as electrolyte produced a steady-state photoinduced V OC of ≈460 mV (Fig. 4.2b and Table C1 in Appendix C) and a peak J SC of −3.0 μA/cm2 that decayed to a low steady state level of ~−0.1 μA/cm2 (Fig. 4.2c). With 0.2 M TEMPO, cells produced a steady state photo-induced V OC of around 620 mV (Fig. 4.2b and Table C1 in Appendix C) and a peak J SC of ≈−7.7 μA/cm2 that decayed to a steady-state photocurrent of ≈−1.1 μA/cm2 (Fig. 4.2c). As is evident from the data compiled in Table 4.1 the J SC (both the peak and steady-state) did not show any dependence on the vacuum potential of the electrolyte, as might be expected, but the photo-induced V OC increased as the electrolyte became more oxidising, in an approximately linear fashion (Table 4.1). The time taken for the V OC to stabilise also increased as the electrolyte became more oxidising; the likely reason for this is discussed below. In this cell architecture the photoprotein effectively creates an electric potential to drive electrons out of the n-Si wafer used as the anode. To confirm that the RC-LH1 complexes were the source of the observed photoresponses an external quantum

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efficiency (EQE) action spectrum was recorded for a TEMPO cell and compared to the absorbance spectrum of the RC-LH1 complex in solution (Fig. 4.2d). The absorbance spectrum showed a dominant absorbance band at 874 nm, attributable to the 32 BChl light harvesting pigments of the LH1 antenna protein and the pair of P BChls of the RC, and a band at 805 nm attributable to the two monomeric BChls of the RC. The EQE spectrum had a maximum of 1.0% at 860 nm and the positions of its bands corresponded reasonably well with absorption spectrum of the RC-LH1 complex in solution. In addition to confirming that the RC-LH1 complex was responsible for driving the photocurrent, the line shape of the EQE spectrum provided insight into factors limiting that photocurrent. The low amplitude of the longest wavelength band in the EQE spectrum, attributable (mainly) to the LH1 BChls (i.e. 875 nm absorption peak), relative to the adjacent band at 805 nm attributable to the RC indicated the likelihood that the photocurrent is not limited by light harvesting by the antenna pigments. This response characteristic has been observed previously in photoelectrochemical cells employing RC-LH1 complexes and its origin discussed [23]. A power conversion efficiency (η) for each of the cells was estimated by multiplying the J SC by the V OC and dividing by the incident light intensity of 100 mW/cm2 (Table 4.1). This modified method was used as it was not possible to determine a fill factor from a full I-V curve due to the transient nature of the photocurrents (see Ref. [24] for a previous discussion). The highest value of η was obtained with TEMPO, which gave the highest V OC and J SC , and the lowest η was with I− /I3 − which gave the lowest J SC (Table 4.1). The function of the oxidising electrolytes was to shuttle charge from the protein adhered to the FTO-glass electrode to the n-Si counter electrode. To better understand the electrochemical characteristics of the n-Si/electrolyte interaction electrochemical impedance spectroscopy (EIS) was performed on symmetrical cells. The Nyquist plots (Fig. 4.3a) were obtained from the symmetrical cells fabricated by sandwiching an aliquot of electrolyte solution between two identical n-Si wafers (Fig. 4.3b). The modelled equivalent circuit used for data fitting is shown in Fig. 4.3c. Typically, the high frequency range of the impedance curve is attributed to the series resistance (Rs ) which includes the bulk resistance of the counter electrode and the contact resistance. The middle frequency range can be assigned to the resistance capacitance networks of the electrode/electrolyte interface. This includes the charge transfer resistance (Rct ) which refers to the barrier through which the electron must pass between the electrode surface and the adsorbed species, and is a measure of the rate of electron exchange between the redox electrolyte and the counter electrode. The low frequency range can be assigned to the diffusion impedance of the redox electrolyte. The diameters of the semicircles in the Nyquist plots provide a good way to compare the charge transfer resistance as the smaller the semicircle, the lower the charge transfer resistance and the faster charge transfer kinetics. As seen from Fig. 4.3a, symmetrical cells with ferrocene had a much larger charge transfer resistance than cells with I− /I3 − or TEMPO. Despite the similar, low charge transfer resistances obtained for I− /I3 − and TEMPO, the steady-state photocurrent obtained with I− /I3 − was 11-fold lower than that obtained with TEMPO. This is attributed to the

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Fig. 4.3 Interactions of the electrolytes with the planar n-Si electrode. a Nyquist plots of EIS data for symmetrical cells using different electrolytes. The inset shows the complete EIS spectrum obtained. b Schematic of the symmetrical cells. c Equivalent circuit model used to fit the impedance spectra of symmetrical cells

irreversibility of the redox process associated with the I− /I3 − electrolyte (Fig. 4.2a). Although the first redox curve in the I− /I3 − CV trace can be described by the process 3I− ↔ 3I− + 2e− , there was no reduction of I3 − back to I− in γ-butyrolactone (Fig. 4.2a). Were the same to be the case in the protein photoelectrochemical cells then the oxidation of the QB ubiquinone would be inhibited, breaking the electronic circuit and stopping charge flow, resulting in a poor photocurrent. The observation that the photocurrent obtained with ferrocene was also lower than that obtained with TEMPO can be attributed to the high charge transfer resistance associated with ferrocene. As mentioned above, the time required for stabilisation of the photo-induced V OC depended on the electrolyte used (Fig. 4.2b). For ferrocene, which gave the lowest final V OC , attainment of a steady value was very rapid. In contrast for TEMPO, which gave the highest final V OC , stabilisation took around 300 s (see inset to Fig. 4.2b), with an intermediate result for iodide. The most likely explanation is that this was a consequence of the size of the energy-level difference between the quinone QB site of the protein and the electrolyte. For TEMPO, the deepest lying redox electrolyte, electron-transfer might be expected to be slowest because this energy gap was the largest, resulting in a gradual build-up of photovoltage over 200–300 s. For iodide and ferrocene the build-up was more rapid because the energy gap between the QB and electrolyte was smaller, being most rapid for ferrocene where the energy gap was smallest and the steady state was reached within ≈1 s. To explore the possibility of further improving cell performance the n-Si counter electrode was textured to produce micrometer scale pyramidal structures at its surface (Fig. 4.4a). Protein photoelectrochemical cells were constructed with TEMPO as the electrolyte and with either flat or textured n-Si as the back electrode. Allowing for a dark voltage of 210 mV, a photo-induced V OC of ≈700 mV was obtained from RC-LH1 cells with a textured back electrode (Fig. 4.4b and Table C1 in Appendix

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Fig. 4.4 Voltage and current enhancements from texturing the silicon counter electrode. a Scanning electron microscopy image of the textured n-Si electrode showing pyramidal structures. b VOC . c JSC from RC-LH1 cells with 0.2 M TEMPO as electrolyte and a planar or textured Si counter electrode. d Nyquist plots of EIS data for these cells over the frequency range 10 Hz to 0.3 MHz. The inset shows the complete EIS spectrum for the cell with a plain n-Si electrode with the frequency range extended to 1 MHz. e Schematic of the RC-LH1 cells. f Equivalent circuit model used for fitting the impedance spectra of the cells. g In cells formed from planar Si a significant portion of the incoming light is reflected back into electrolyte. h Enhanced light absorption by a textured n-Si electrode occurs because the pyramidal structures scatter light back to the surface enabling multiple instances of absorption and producing minimal reflection

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Table 4.2 Photovoltaic performance and charge conduction for RC-LH1/TEMPO cells with a planar or textured n-Si electrodes Anode

V oc (mV)

J sc (μA/cm2 )

η (%) × 10−3

Planar Si

620

1.1

Textured Si

700

8.2

Rs (k)

Rct (k)

0.68

7.5

23.7

5.75

2.9

3.2

C), a ≈13% improvement over that obtained with planar n-Si. The amplitude of the steady-state photocurrent density from the textured n-Si cells was eight-fold larger than that obtained with planar Si (Fig. 4.4c and Table 4.2). The dark current exhibited by the cells with a textured n-Si anode was not seen for cells with a planar electrode (Fig. 4.4b) and likely arose from trapped charges at the textured silicon surface. Replacing planar n-Si with textured n-Si gave a ≈eight fold increase in power conversion efficiency (Table 4.2). This is principally attributed to enhanced charge transfer and enhanced light absorption by the n-Si. To corroborate the first of these, EIS was performed on the protein cells used for photocurrent and photovoltage measurements. The EIS spectra (Fig. 4.4d) obtained from the protein cells (Fig. 4.4e) were fitted using an equivalent circuit model (Fig. 4.4f, Fig. C1 in Appendix C). The equivalent circuit model is chosen based on the impedance characteristics of the individual components in the cell (Fig. C1 in Appendix C). However, as it is seen that the circuit model fitted still shows minor deviations from the EIS Nyquist spectra, a more profound physical understanding of the solid-state protein layers and their interactions with electrolytes is needed to arrive at a circuit model with a better fit. The fitted Nyquist plots revealed a seven-fold drop in charge transfer resistance Rct when the plain Si was replaced with textured Si (Table 4.2). It is likely that this was due to the increase in the interfacial area between the electrode and the electrolyte. Regarding the second effect, it is well known that texturing the Si surface with random pyramidal structures minimizes reflection losses (Fig. 4.4g) and so enhances light absorption by the Si. The proposed mechanism of photo-induced charge transfer in the biohybrid cells is illustrated in the energy diagram in Fig. 4.5. Illumination of the RC-LH1 protein oxidises the special pair bacteriochlorophylls (P+ ) and reduces the terminal ubiquinone (Q–B ), with P+ being re-reduced by the FTO-electrode in a process that would not be expected to involve any of the three evaluated deep-lying electrolytes. In parallel, light not absorbed by the protein layer triggers a photoexcitation in n-Si generating an electron-hole pair. The hole in the valence band is subsequently filled by the electrolyte, completing the circuit. In this novel architecture the maximum possible V OC is determined by the potential gap between the P/P+ couple in the photoprotein, at −5.0 eV, and the conduction band of the n-Si at ~−4.3 eV, a span of around 0.7 eV which is good agreement with the photovoltage observed in the cell with a textured n-Si counter electrode and the deepest lying TEMPO electrolyte. In addition to providing a means of shuttling electrons between the protein and the n-Si, the use of electrolytes with deep-lying vacuum potentials avoided the possibility of the electrolyte facilitating short-circuit

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Fig. 4.5 Schematic of charge transport through the biohybrid cell. Incident light triggers a primary photochemical reaction in the RC-LH1 complex involving photoexcitation of the special pair (P → P*) and charge separation to form P+ and Q− B . Unabsorbed light triggers auxiliary photoexcitation in n-Si resulting in excitation of an electron into the conduction band (EC ) and leaving a hole in the valence band (EV ). The two photo-reactions are coupled by the electrolytes with deep-lying vacuum potentials which fill holes in the n-Si valence band using electrons collected from the Q− B cofactors in the RC-LH1 complex

electron flow between the Q–B and P+ terminals of the protein, or being involved in electron flow from the FTO-glass cathode to P+ . Regarding overall cell performance, a point to note is that the simple approach taken to fabrication of the working electrode in these cells, vacuum drying a protein layer directly onto a cleaned but unfunctionalised FTO-glass surface, did not provide a mechanism to control protein orientation at the electrode surface. The orientation depicted in Figs. 4.1 and 4.5, with the side of the protein normally exposed to the bacterial periplasm closest to the FTO-surface, places the P/P+ redox center closest to the FTO-glass to support a cathodic photocurrent. This part of the protein surface is very flat, and the P bacteriochlorophylls are located close to the surface. However, it is likely that proteins in the vacuum dried layer are oriented more randomly than this, and that this is a factor that limits the magnitude of the photocurrents produced by these devices. For example, proteins oriented with their cytoplasmic side closest to the FTO could support an anodic current through electron donation from the RC quinones, such that the observed photocurrent is the sum of cathodic and anodic processes. It

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should be noted that the protein surface on the cytoplasmic side of the membrane is more strongly contoured than on the periplasmic side due to the presence of the H domain of the RC (coloured as a pink solid surface in Figs. 4.1 and 4.5). Also, the quinones are more deeply buried in the protein interior than is the case for the P bacteriochlorophylls, as the function of the H domain is to insulate the quinone binding site from the extramembrane aqueous phase. Another factor that cannot be ruled out is some detachment of individual proteins from the vacuum dried layer during cell assembly and performance, although these vacuum dried protein films were reasonably resistant to rinsing using deionised water. Although free protein in solution would not be expected to support a photocurrent, it could affect cell performance by adhering to the counter electrode and so interfering with electrode/electrolyte interactions. In future work, therefore, it may be possible to boost the photocurrent from this type of device through better control of protein orientation and adherence at the working electrode, although this would have the trade-off of making cell fabrication more complex. In addition, the mechanisms through which protein multilayers support photocurrents are poorly understood, particularly with regard to those proteins not in immediate contact with either electrode or electrolyte, and a better understanding of this architecture should also enable improvements in cell design to boost output.

4.4 Summary In conclusion, high photovoltages have been achieved through a device design incorporating two parallel photoexcitation reactions at a FTO-glass/protein cathode and an n-doped silicon anode. The highest V OC of ~0.7 V was obtained with the deepest lying electrolyte, TEMPO, in conjunction with a textured n-Si anode, producing a power conversion efficiency of 5.7 × 10−3 . This raises the photovoltage achievable in a device employing purple bacterial photoproteins to a level comparable to that typical of silicon heterojunction and dye-sensitized solar cells [25]. The challenge now is to seek mechanisms to enhance the photocurrent achievable in such cells to similarly raise the power conversion efficiency of these biohybrid devices. New device architectures are promising in achieving this and widening the application scope of photoproteins [18, 26–34].

References 1. Ravi SK (2018) Solar energy harvesting with photosynthetic pigment-protein complexes. National University of Singapore, Singapore 2. Singh VK et al (2018) Biohybrid photoprotein-semiconductor cells with deep-lying redox shuttles achieve a 0.7 V photovoltage. Adv Funct Mater 28(24):1703689 3. Feher G et al (1989) Structure and function of bacterial photosynthetic reaction centres. Nature 339:111–116

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4. Zinth W, Wachtveitl J (2005) The first picoseconds in bacterial photosynthesis—ultrafast electron transfer for the efficient conversion of light energy. ChemPhysChem 6(5):871–880 5. Jones MR (2009) The petite purple photosynthetic powerpack. Biochem Soc Trans 37:400–407 6. Niwa S et al (2014) Structure of the LH1-RC complex from thermochromatium tepidum at 3.0 Å. Nature 508(7495):228–232 7. Qian P et al (2013) Three-dimensional structure of the Rhodobacter sphaeroides RC-LH1-PufX complex: dimerization and quinone channels promoted by PufX. Biochemistry 52(43):7575– 7585 8. Blankenship RE (ed) (2013) Molecular mechanisms of photosynthesis. Wiley, Hoboken 9. Takshi A, Madden JD, Beatty JT (2009) Diffusion model for charge transfer from a photosynthetic reaction center to an electrode in a photovoltaic device. Electrochim Acta 54(14):3806–3811 10. Yaghoubi H et al (2015) Large photocurrent response and external quantum efficiency in biophotoelectrochemical cells incorporating reaction center plus light harvesting complexes. Biomacromolecules 16(4):1112–1118 11. Yaghoubi H et al (2017) A ZnO nanowire bio-hybrid solar cell. Nanotechnology 28(5):054006 12. Das R et al (2004) Integration of photosynthetic protein molecular complexes in solid-state electronic devices. Nano Lett 4(6):1079–1083 13. Lu YD et al (2005) Photoelectric performance of bacteria photosynthetic proteins entrapped on tailored mesoporous WO3 -TiO2 films. Langmuir 21(9):4071–4076 14. Xu J et al (2007) Sensitively probing the cofactor redox species and photo-induced electron transfer of wild-type and pheophytin-replaced photosynthetic proteins reconstituted in selfassembled monolayers. J Solid State Electrochem 11(12):1689–1695 15. Tan SC et al (2012) Generation of alternating current in response to discontinuous illumination by photoelectrochemical cells based on photosynthetic proteins. Angew Chem Int Ed 51(27):6667–6671 16. Tan SC et al (2013) Superhydrophobic carbon nanotube electrode produces a near-symmetrical alternating current from photosynthetic protein-based photoelectrochemical cells. Adv Funct Mater 23(44):5556–5563 17. Tan SC et al (2012) Increasing the open-circuit voltage of photoprotein-based photoelectrochemical cells by manipulation of the vacuum potential of the electrolytes. ACS Nano 6(10):9103–9109 18. Ravi SK et al (2017) Enhanced output from biohybrid photoelectrochemical transparent tandem cells integrating photosynthetic proteins genetically modified for expanded solar energy harvesting. Adv Energy Mater 7(7):1601821 19. Jeena V, Robinson RS (2012) Convenient photooxidation of alcohols using dye sensitised zinc oxide in combination with silver nitrate and TEMPO. Chem Commun 48(2):299–301 20. Caterino R et al (2015) Photocurrent generation in diamond electrodes modified with reaction centers. ACS Appl Mater Interfaces 7(15):8099–8107 21. Katz E (1994) Application of bifunctional reagents for immobilization of proteins on a carbon electrode surface: oriented immobilization of photosynthetic reaction centers. J Electroanal Chem 365(1–2):157–164 22. Friebe VM et al (2016) Plasmon-enhanced photocurrent of photosynthetic pigment proteins on nanoporous silver. Adv Funct Mater 26(2):285–292 23. den Hollander M-J et al (2011) Enhanced photocurrent generation by photosynthetic bacterial reaction centers through molecular relays, light-harvesting complexes, and direct protein-gold interactions. Langmuir 27(16):10282–10294 24. Ciesielski PN et al (2010) Photosystem I—based biohybrid photoelectrochemical cells. Biores Technol 101(9):3047–3053 25. Vaghasiya JV et al (2018) Dual functional hetero-anthracene based single component organic ionic conductors as redox mediator cum light harvester for solid state photoelectrochemical cells. J Mater Chem A 6(11):4868–4877 26. Ravi S et al (2018) Photosynthetic bioelectronic sensors for touch perception, UV-detection, and nanopower generation: toward self-powered E-skins. Adv Mater 30(39):1802290

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27. Ravi SK et al (2020) Bio-photocapacitive tactile sensors as a touch-to-audio braille reader and solar capacitor. Mater Horiz 7(3):866–876 28. Ravi SK et al (2019) Optical shading induces an in-plane potential gradient in a semiartificial photosynthetic system bringing photoelectric synergy. Adv Energy Mater 9(35):1901449 29. Ravi SK et al (2019) Photosynthetic apparatus of Rhodobacter sphaeroides exhibits prolonged charge storage. Nat Commun 10(1):1–10 30. Ravi SK et al (2018) Bio-photoelectrochemical cells: protein immobilization routes and electron transfer modes. In: Photosynthetic protein-based photovoltaics. CRC Press, Boca Raton, pp 141–159 31. Ravi SK et al (2018) Emerging role of the band-structure approach in biohybrid photovoltaics: a path beyond bioelectrochemistry. Adv Funct Mater 28(24):1705305 32. Ravi SK et al (2018) A mechanoresponsive phase-changing electrolyte enables fabrication of high-output solid-state photobioelectrochemical devices from pigment-protein multilayers. Adv Mater 30(5):1704073 33. Ravi SK, Tan SC (2018) Electronics, photonics, and device physics in protein biophotovoltaics, in photosynthetic protein-based photovoltaics. CRC Press, Boca Raton, pp 161–224 34. Tan SC (2018) Photosynthetic protein-based photovoltaics. CRC Press, Boca Raton

Chapter 5

Role of Band-Structure Approach in Biohybrid Photovoltaics—A Path Beyond Bioelectrochemistry

5.1 Brief Overview Emulation of natural photosynthesis is central to modern photovoltaic research eying sustainable and economic ways of solar energy harvesting. Natural photosynthetic systems have succeeded in efficiently harvesting solar energy which is key to the sustenance of life on earth. With numerous advances in understanding the structure and function of the natural photosystems, the last decade has witnessed new perspectives in developing Bio-inspired Photovoltaics. Interestingly, Organic Photovoltaics (OPVs) adopting photosynthetic design principles and Biohybrid Photovoltaics (BPVs) adopting solid-state device architectures have now converged at a juncture. Several reports in the past few years point to a new scope of improvement in OPVs and BPVs stemming from mutual inspiration. At this juncture, there are new perspectives by which a BPV can be designed that were previously limited only to the conventional optoelectronics. Treating natural photosynthetic pigment-proteins as optically- and electronically-functional materials in any photovoltaic design, from the band-theory viewpoint, is a promising direction for advancing BPVs beyond the boundaries of Bioelectrochemistry. This chapter presents an overview of selected reports on BPVs in the recent years utilizing new design concepts based on bandtheory and its associated principles. In light of this, the scope of band-structure approach in BPVs is discussed eliciting prospective research directions.

5.2 Review of Types of Photosynthetic Proteins Photosynthesis, the process by which plants and algae turn solar energy, water, and carbon dioxide into biomass, plays a decisive role in sustaining nearly every form of life on earth, underpinning the entire heterotrophic food web [1–3]. Evolutionary pressure over billions of years has enabled photosynthesis to operate even in extreme © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_5

79

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5 Role of Band-Structure Approach in Biohybrid …

physiological temperatures and under minimal light flux backed by the structural adaptations in the photosynthetic light harvesting systems [4, 5]. The human race now eyes on photosynthesis for bio-inspiration, driven by the strong need to develop a more varied, sustainable, economic, and eco-friendly photovoltaic technology [4– 7]. Not surprisingly, photosynthesis has been of irrefutable research significance in recent times as the design principles by which nature has mastered the solar energy capture is intriguing while enticing with the otherwise unattainable heights of technological advancements in the field of photovoltaics. Against a background of concerns over catering the growing energy needs by cost-effective and sustainable solutions, there is increasing interest in developing artificial photosynthetic systems [8, 9] and in direct exploitation of natural photosynthetic pigment-proteins for photovoltaics [10, 11]. Natural photosynthetic pigment-proteins facilitate photoinduced energy transfer and charge separation processes with near-unity high quantum efficiencies (event per photon absorbed) and diverse systems have been subjected to in-depth structural and functional characterization [12]. As well as inspiring the design of man-made materials and molecular systems [13] to achieve artificial photosynthesis, a wide variety of photosynthetic complexes and biomolecules have been interfaced with man-made materials for potential applications in photovoltaics, biosensing, photodetection, solar fuel synthesis and biocomputing [11, 12, 14–25]. Different photosynthetic proteins and antenna complexes from various species of plants, algae, and bacteria have been integrated into optoelectronic and photoelectrochemical devices. The photosynthetic reaction center proteins from purple bacteria are some of the most widely studied and relatively well-understood among all [3, 6, 26]. Numerous reports on reaction center based photoelectrochemical cells are found in literature [6]. While core reaction center proteins were widely used in a number of photoelectrochemical cells, in the recent years the use of isolated light harvesting complexes (LH1) and the use of reaction centers along with the surrounding antenna complexes (RC-LH1) have also become common. The progress in the use of purple bacterial photosynthetic proteins for photocurrent generation has been discussed in a few recent reviews [3, 6, 27]. In contrast to the simple and well-studied structure of proteins in purple bacteria, the protein complexes from oxygenic phototrophs like plants, algae, and cyanobacteria are far more complex and sophisticated [26, 28–30]. Oxygenic photosynthesis often involves two main protein complexes namely Photosystem 1 (PS1) and Photosystem 2 (PS2) which though share several similarities with the purple bacterial reaction centers are not the minimal functional units responsible for the photochemical reaction [26, 28–31]. The minimal core units are not easy to isolate from these photosystems, unlike the reaction centers which are per se the minimal functional units performing the photochemical charge separation [31]. A number of comprehensive reviews are also available in the literature on the use of photosystems 1 and 2 in biophotovoltaic devices and other related applications [32–34]. In addition to PS1 and PS2, LHC2 which is a light harvesting complex extracted from oxygenic phototrophs has also been used in bio-photovoltaic devices in recent times. Other than this, there are also reports on biophotovoltaics using other light harvesting pigment-protein complexes like Phycocyanin (PC) [35, 36] and proton-pump based photosynthetic proteins like Bacteriorhodopsin (bR) [37–39].

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81

Fig. 5.1 Types of photosynthetic pigment-proteins and antenna complexes commonly used in biohybrid devices: PS1-Photosystem 1, RC: reaction centre (refers to purple bacterial reaction centre unless otherwise specified), PS2: Photosystem RC-LH1: Purple bacterial reaction centre with its antenna complex LH1. LHC2: Light harvesting antenna complex that forms a part of PS2 supercomplex/megacomplex in algae and higher plants. PC: Phycocyanin, bR- Bacteriorhodopsin. Structures of RC-LH1, PS1, RC and PS2 are adapted with permission [40] Copyright 2007, Elsevier. Structure of LHC2 is adapted with permission [41] Copyright 2011, Elsevier. Structure of Phycocyanin is adapted with permission [42] Copyright 2007, Elsevier. Structure of Bacteriorhodopsin is adapted with permission [38] Copyright 2010, Nature Education

The approach in the majority of these studies has been to construct a biophotoelectrode comprising a man-made material coated with a layer of photosynthetic protein, in an electrochemical set-up. The photosynthetic complexes generally used in the biohybrid devices are RC, RC-LH1, PS1, PS2, bR, LHC2 and PC (Fig. 5.1) which depending on the application have been used for chemical sensing, water splitting and for electricity generation.

5.3 Photo-Bioelectrochemical Cells Electricity generation is often the direct application of the photoinduced charge separation found in photosynthetic proteins. Most bioelectrochemical cells constructed for photocurrent generation are three electrode cells where the proteins are either

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immobilized on the working electrode or suffused in a solution while the flow of electrons between the working and counter electrode is achieved through the use of a small molecule mediators and redox couples [43–48]. In some cases two electrode sealed cells (without any reference electrode) have been constructed with, again, a mobile mediator carrying charge between the two electrodes [12, 49, 50]. In addition to the common chemical immobilization techniques using linker molecules, proteins are also adhered to the working electrode by methods like drop casting and electrospraying [51–53]. Photocurrents obtained with such systems often show an initial spike of “transient photocurrent” which decays over time scales of seconds or minutes to a lower, steady-state value [12, 43–53]. It has been proposed that this period of transient current represents a decay to a steady state where the current density is limited by mediator diffusion [45, 46, 54, 55]. Several findings point to a drawback of bio-photoelectrochemical cells where the current output can be limited by slow diffusive processes rather than the capacity of the photovoltaic proteins to absorb light energy and separate charge [45, 46, 54, 55]. This chapter discusses the prospects and perspectives in applying the bandstructure approach in photosynthetic light-harvesting devices. As part of this, our review briefly presents the electronic processes in natural photosynthetic systems contrasting with that in the emerging photovoltaic systems thereby throwing light on how band-structure approach can aid in bridging the photosynthetic research with the emerging photovoltaic technologies taking inspirations from each other.

5.4 Band-Structure Approach The conventional biophotoelectrochemical cells being predominantly diffusioncontrolled, the device structure is often decided mainly by the choice of the electrolyte redox potential and by the mode of immobilization of the photoactive species. However, employing a band-structure approach in constructing devices holds a huge potential in improving the device performance, with more precise control over the charge transport in the device by means of energy level alignment at various interfaces in the device [56–58]. Unlike that in electrochemical cells, the band-structure approach in biohybrid devices essentially treats every component of the device as an electronically functional material [58, 59]. Akin to most optoelectronic devices, the function stems from the material-interfaces in the device [58, 60, 61]. For instance, in an electroluminescent device electrons are injected from an electrode to the electron transport layer (ETL) and holes are injected from the other electrode into the hole transport layer (HTL) after which the carriers recombine to emit light [62–64]. In an organic solar cell, there forms a Schottky barrier at the metal-organic material interface resulting in band bending at the interface where the photogenerated electron-hole pairs get separated [58, 65–68]. The interfacial electronic structure hence forms a crucial part of the functioning of any optoelectronic device. While the band-structure approach has been extensively used in the design and construction of

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83

inorganic optoelectronic devices, the approach is still emerging in modern optoelectronics where the inorganic building blocks in the devices are increasingly getting replaced by their organic counterparts in view of the numerous advantages offered by the organic solids ranging from the low material cost to ease of property-tunability [61, 69, 70]. In recent years, organic/organic interfaces and organic/metal interfaces are largely studied with respect to energy level alignment and interfacial electronic structure so as to understand and improve the charge transport processes in organic optoelectronic devices [57, 71–73]. The approach is becoming especially popular in a wide range of emerging optoelectronic devices like organic light-emitting diodes, organic photovoltaics, organic field-effect transistors and perovskite solar cells. A brief review of the conceptual aspects associated with interfacial electronic structure in optoelectronic devices and the role of energy-level selection of the different functional layers would be worthwhile for propelling the biophotovoltaics in a whole new direction.

5.4.1 Interfacial Electronic Structure in Biophotovoltaics Energy level alignment is an important concept of Band-structure approach that has been given a great attention in the conventional optoelectronics and in emerging organic optoelectronics, however, it is still relatively a new concept in biophotovoltaics. As in most biophotovoltaic devices, the biomolecules involved (which might be proteins, pigments, light-harvesting complexes or thylakoids) are often interfaced with a metal electrode. All the prevailing conditions applicable for a metal/organic interface in deciding the interfacial electronic structure holds good for a metal/biomolecule interface. However, the role of interfacial electronic structure in the biophotovoltaic device performance is not studied. To understand the similarities and contrasts of a natural photosynthetic machinery with an organic photovoltaic device (OPV) is paramount for applying the bandstructure approach in biophotovoltaics. OPVs consist of active light-absorbing layers with domains of conjugated polymers or molecules behaving as electron donors and acceptors. On illumination, the excitons at the donor/acceptor interface dissociate into electrons and holes which then get separated by moving into the donor and acceptor domains [5]. In a subtle similarity with this, in natural photosynthesis, sunlight is captured by light harvesting complexes equipped with pigments like chlorophylls (Chl) and carotenoids (Car), after which the excitation energy is funneled into a reaction center which essentially is an interface between an acceptor and a donor [5, 6, 67]. While the photosynthetic architecture in nature is highly sophisticated with the presence of highly ordered arrays of core reaction center super-complexes (Fig. 5.2a–c) in the photosynthetic membrane rather than a random distribution, it hard to achieve such an ordered arrangement in organic photovoltaics [5, 7, 74]. In organic photovoltaics, the acceptor and donor domains exist as a disordered mixture (Fig. 5.2d) in contrast to the intricate molecular circuitry of light-harvesters and charge carrying cofactors seen in photosynthetic proteins [5, 6, 61, 67, 75].

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Fig. 5.2 Contrasts in structural sophistication between the natural photosynthetic systems and the plastic photovoltaic systems: a The architecture of a PS2-LHC2 supercomplex found in Spinach. b A cartoon diagram showing the ordered arrangement of the various subunits of the spinach PS2LHC2 supercomplex (a, b—figures are reproduced with permission [76] Copyright 2016, Nature Publishing Group). Description of the subunits is beyond the scope of this review and can be found from literature [76]. c A model showing a tight organization of light harvesting antenna complexes around the photosystem in PS2-LHC2 supercomplex found in an algae Chlamydomonas reinhardtii (reproduced with permission [77] Copyright 2014, Elsevier). d Schematic of a polymer solar cell showing only the active layer—where there are randomly mixed domains of electron acceptors (shown as spheres) and donors (shown as rectangular flakes); the two rectangular substrates on the either side of the active layer are just a representation of neighbouring layers which can either be electrodes or can be cathodic/anodic interlayers used for charge extraction and transport (Adapted with permission [75] Copyright 2013, Nature Publishing Group)

While there are dedicated networks in a photosynthetic complex for exciton transport and charge transport, the two functions are not separated in an OPV [5, 6, 61, 67] (Fig. 5.3a, b). The efficacy of the charge photogeneration and separation is another feature in a photosynthetic protein that is in marked contrast with that in OPVs. The charge photogeneration in such proteins is a result of a redox relay or cascade [67] as shown in Fig. 5.3a. Light absorption leads to the formation of a short-lived molecular singlet excited state (typically lasting for a few nanoseconds) in the primary donor species (P) after which the charge separation ensues from a chain of energetically downhill electron transfer from the donor excited state to the adjacent acceptor molecules [6, 78, 79] (Fig. 5.3c, d). Following the initial charge separation, the secondary electron transfer reactions result in a long-lived charge separated state that lasts even up to a few seconds [6, 67, 79] (Fig. 5.3d). Though there have been several studies on the electron transfer dynamics and recombination pathways in molecular mimics of photosynthetic proteins involving molecular donor/acceptor redox relays, the studies were predominantly limited to dilute solutions [67]. However, in OPVs, the donor and acceptor molecules are typically solid films that essentially give rise to percolation pathways which aid in providing an electrical ‘wiring’ of the photogenerated charges at the donor/acceptor interface to the device electrodes [67, 80, 81]. In a striking contrast to the architecture of the photosynthetic protein which allows

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Fig. 5.3 Excitation energy transfer and charge separation in a photosynthetic system and a plastic photovoltaic system: a Cartoon showing the migration of excitation energy from the antenna complexes to the reaction center in a PS2-LHC2 supercomplex (The position and arrangement of the antenna complexes and the cofactors are not drawn to precision; the cartoon is created based on the structural information of PS2 supercomplexes reported in literature [86–88]). b Cartoon showing exciton migration and charge dissociation at the donor/acceptor interface in a plastic solar cell. c On light absorption, the exciton generated diffuses to the interface, where the electron transfer to the acceptor occurs resulting in a charge transfer (CT) state. For clarity, the CT state in the cartoon is shifted down along the vertical axis of the page. The electron and hole are initially separated by a distance called thermalization length ‘a’. Theoretical predictions indicate the dependence of the probability of full charge dissociation on the ratio a:rc where rc is the Coulomb capture radius which is represented by a circle (spherical in 3D) for simplicity neglecting the anisotropy [67]. c Schematic representation of the structure of PS2 reaction center. d Schematic showing the energetics and kinetics of charge separation in the reaction center (Adapted with permission [67] Copyright 2010, American Chemical Society). e Energy level diagram of the donor-acceptor interface in a plastic solar cell showing interfacial electron-hole pairs in charge-transfer (CT) state. The energy of the CT state depends on the Coulombic attraction between the two charge carriers and hence on their spatial separation, as shown by the dotted curve. EBexc and EBCT are the binding energies associated with the exciton and CT states [67]. Cartoons b, c, and e are created based on the descriptions and depictions available in reference article [67]

a cascade of electron flow through multiple donor/acceptor sites, in an OPV device, the interfaces are formed only by one type of donor and acceptor molecules ruling out the possibility of such redox relays (Fig. 5.3b). Unlike in the natural photosynthesis where the coulombic attraction between the photogenerated electron-hole pair is efficiently avoided by the redox relay, in an OPV device this is achievable only when the energy offset between the donor and acceptor LUMO (Lowest unoccupied molecular orbital) levels is greater than the coulombic binding energy of the photogenerated exciton into order to enable the primary electron transfer reaction to be

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energetically downhill [67, 82]. However, in most practical cases, the physical proximity of the donor and acceptor molecules at the charge separation interface often limits the electron-hole spatial separation to be in the same order of magnitude as the size of the molecules concerned (0.5–1 nm) that proves in ineffective in completely overcoming the coulombic attraction [67, 83] (Fig. 5.3e). Several recent studies focus on overcoming this limitation in OPVs by understanding the interfacial electronic structure and by engineering the energy-level alignment in the device interfaces [56, 84, 85]. With the recent developments in biophotovoltaics focussing more on solid-state devices, understanding the interfacial energetics becomes crucial. With differences only in the level of sophistication, the interfaces formed by a simple organic molecule or by a photosynthetic complex with an electrode can fairly be understood by the same of set of governing principles established by the band theory of solids. For an interface formed by an organic layer in an OPV, the possibility of an energy barrier at the interface is often investigated [89–91]. When a thick organic layer is interfaced with an electrode, there is a chance of interfacial band-bending which stems from the non-equilibrium state created when materials of two different work functions are brought into contact [58, 92, 93]. In order to reach an electrical equilibrium where the Fermi levels of the two materials are to be at the same level, charge redistribution occurs around the metal/organic interface [58, 94–96]. The flow/distribution of charges into either side of the interface continues until the Fermi energies in the bulk of the two materials are aligned, resulting in a diffusion layer with band bending [58, 92, 93]. This results in a built-in potential in the device. While the same effect is possible in photoprotein/electrode interface, the interfacial electronic structure can no more be treated as the junction of two solids of different work functions. As a photoprotein complex is an assemblage of polypeptides and pigments and other cofactors each having a distinct Fermi level, it isn’t possible to assign it a single value of work-function. Though when a photoprotein is interfaced with an electrode ideally it is only one part of the protein with a specific organic molecule that is intended to be in contact with the electrode, in most practical cases it is hard to ensure this. An exception to this would be those devices employing ordered monolayers of proteins with genetic tags and functionalized electrodes, however, these architectures are far from practical high-output biophotovoltaics where high loading/concentration of light absorbing units is necessary [6]. Hence simplification of the protein/electrode interface to an organic/electrode interface is unsound in any biophotovoltaic device with high photoprotein loading. The interfacial electronic properties (especially the energy barrier at the interface) are often studied by different kinds of in-device photoelectron spectroscopies under ultra-high vacuum (UHV) [58, 91]. These studies haven’t been so far attempted in biophotovoltaic devices due to the challenges in preserving the structural integrity of the photoproteins under the test conditions.

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5.4.2 Bioinspired OPVs and OPV-Inspired BPVs Though the in-device interfacial electronic structures are not explored in the field of biophotovoltaics, in the recent few years there are a number of reports adopting design principles based on ‘band gap approach’ [11, 97–103]. Stepping beyond the conventional bio-electrochemical cells, in these reports, attempts are made either to construct solid-state multi-layered devices with exciton/charge transfer modes decided purely based on band-structure approach or to introduce certain new design principles based on the band-gap approach in a biohybrid device retaining a basic electrochemical cell design. The first category of devices is solid-state photovoltaic cells integrated with photosynthetic proteins which include design principles adopted both from the natural photosynthesis and from the conventional photovoltaics. The second category includes liquid photo-bioelectrochemical cells with certain specific design principles based on the band-gap approach incorporated in them to result in performance enhancement. The first category includes Biophotovoltaic cells (BPVs) inspired from OPV device architectures. To envision new perspectives by which the BPVs can be inspired from OPVs, it is important to understand the research trends in the BPVs and OPVs independently. Several recent reviews elaborate the progress and the recent research trends in these fields [3, 6, 27, 61, 104–107]. While BPVs adopting device architectures from OPVs is certainly a new research trend, the motivation to incorporate natural photosynthetic design principles in the conventional photovoltaics isn’t new. The entire field of dye-sensitized solar cells (DSSCs) was given rise to by a simple design inspiration from the natural photosynthesis. The design of a DSSC is in a few ways analogous to the natural photosynthesis, where the functions of light harvesting and charge separation by the antenna complexes and the reaction center are mimicked by the dye and the semiconductor [6, 108]. A nanoporous/nanocrystalline semiconductor is employed to increase the number of dye molecules in contact with the semiconductor in a unit volume, which loosely imitates the photosynthetic protein complexes where a well-structured array of pigments lie in a close proximity to the reaction centre, harvesting light energy and funnelling the excitation energy into the reaction centre as opposed to the excited dye molecules in DSSCs injecting the electrons into the conduction band of the semiconductor. While OPV is still an emerging research field, many efforts have already been directed to emulate the light harvesting mechanisms from natural photosynthesis foreseeing huge improvements. The light harvesting antennae complexes in the photosynthetic proteins capture the photonic energy from sunlight and funnel the excitation energy into reaction centers on a timescale of 10-100 picoseconds [7, 109]. The light harvesting is driven by the transfer of electronic excitation energy stored (within nanoseconds) in the excited state chromophores in the network of light absorbing molecules to the target chromophores or energy traps which are nothing but the reaction centre [6, 7, 79, 110]. To mimic this arrangement in an artificial light-harvesting device is challenging as the time scale largely constrains the size of the chromophore arrays attached to the trap/target reactive site [7]. In other words, there is a limitation on how far the excitation energy can travel in organic films

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which is defined by the exciton diffusion length in organic solar cells [7, 111, 112]. The solar energy harvesting in the natural light harvesting complexes is realized by the more sophisticated structural arrangement of chromophores which works more efficiently than a typical organic solar cell containing a disordered mixture of donor and acceptor molecules. The design principles that are apparent in natural photosynthesis and the dynamics of excitation energy transfer provide a great inspiration in designing new OPVs. In an attempt to mimic the energy transfer kinetics of the natural photosynthetic system in a non-biohybrid photovoltaic cell, phycobilisome-photosystem (Fig. 5.4a) has recently been studied and compared with an artificial system emulating certain design features in the photosystem [113]. The inspiration in designing an artificial

Fig. 5.4 Bioinspired organic solar cell: a Cartoon showing the structural architecture of the Phycobilisome (PBS) antenna complex found in the cyanobacteria ‘Acaryochloris marina’. b Schematic structure of a P3HT-va-CNT OPV device (va-CNT: vertically aligned carbon nanotube). c Cartoon showing the exciton generation and migration through the PC and APC trimers towards the reaction center. d Energy level diagram of the P3HT-va-CNT OPV. e Exciton migration and charge separation in the OPV (1) Photoexcitation of an electron into the donor LUMO creates a hole in HOMO. (2) Formation of an exciton as the photogenerated electron and hole are Coulombically bound. (3) Hopping of electron onto the LUMO of the acceptor as it has a higher electron affinity while the hole still remains at the donor HOMO. There is a formation of the geminate pair together with the electron existing in the acceptor molecule. (4) Complete dissociation of exciton into charge carriers followed by polaron hopping towards the respective electrodes (electrons → anode and holes → cathode). Adapted with permission [113] Copyright 2017, American Chemical Society

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photosynthetic system was the ability of certain cyanobacterial bacteria surviving in deep aquatic zones to perform photosynthesis even in low light intensity. As opposed to the high efficiency of the natural photosystem in solar energy to chemical energy conversion, such quantum efficiencies are often unachievable. One possible reason for this could be the poor charge carrier generation upon exciton dissociation. In order to address this a P3HT-CNT based Organic Photovoltaic cell was constructed using poly(3-hexyl)thiophene (P3HT) as the photoactive donor and vertically aligned single-walled carbon nanotubes as the acceptor. The phycobilisome (PBS) antenna complex from the cyanobacteria ‘Acaryochloris marina’ performs the primary light harvesting followed by funnelling of the excitation energy into the reaction centre of the photosystem 2 (PS2) protein, where the light harvesting efficiency is as high as 95% which is attributed to the architecture of the antenna complex with a well-structured and ordered array of pigments. The PBS antenna system has a rod-like architecture with pigment proteins stacked as hexameric disks connected by non-pigment linker proteins (Fig. 5.4a). The hexamer proximal to the thylakoid membrane has two trimers namely phycocyanin (PC) pigment and allophycocyanin (APC) pigment while the rest are composed only of phycocyanin trimers. Though not in specific, the design of the P3HT-CNT system in a way mimics the PBS photosystem forming organized arrays of photoactive donors (i.e. P3HT) the biological analogs of which are the PC and the APC pigments in the PBS photosystem (Fig. 5.4b). Despite the rough similarity in the device architecture with an organized array of pigments, the P3HT-CNT system could achieve efficiency only in the range of 10–15% which is still not significantly higher than the conventional organic solar cells. The formation of excitons and the excitation energy transfer from the PBS antenna complex to the reaction center in PS2 in the cyanobacterial system is shown in Fig. 5.4c. The PC pigments initially absorb the incident light that is followed by a downhill transfer of excitation energy where the energy transfer follows the scheme of PC → APC → PBS-TE → RC. PBS-TE is the lowest energy state in the PBS antenna system that funnels the excitation energy into the reaction center of PS2 hence called the terminal emitter. While in the bio-inspired OPV system, P3HT absorbs the incident light, get excited (i.e. gains energy to push an electron from the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) resulting in the formation of an exciton). Within the exciton, the electron and the hole are bound by a coulombic attraction the binding energy associated with which has to be overcome to enable charge separation. In the OPV, the exciton dissociation occurs at the P3HT/CNT interface as the CNT has a higher ionization potential than that of the exciton (Fig. 5.4d). The π-election in the CNT exerts a coulombic force on the exciton which is greater than the coulombic attraction force binding the electron and the hole within the exciton. An electrostatic potential gradient is formed at the P3HT/CNT interface that attracts the electron into the CNT LUMO forming a geminate pair with the electron existing in the CNT LUMO, while the hole remains in the P3HT HOMO. After dissociation, these charge carriers reach the respective electrode by polaron hopping (Fig. 5.4e). Organic solids do not favor high charge carrier mobility as the mean free path of the charge carriers is in the

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same order of magnitude as that of the lattice constant, hence resulting in hopping transport of carriers. To emulate photosynthesis the hopping distance has to be minimized which has been attempted by the use of the vertically aligned CNTs as the CNT-electron acceptors are expected to reduce the mean free path in the OPV due to the ballistic charge transport properties along the CNT side wall. On comparing the natural PBS system with the bio-inspired OPV system through time-resolved spectroscopic methods, several interesting conclusions have been drawn on the exciton generation and migration kinetics. Though the time taken for exciton formation in both the natural and OPV systems can roughly be equaled, the time span of the subsequent downhill energy transfer into the RC is three orders of magnitude shorter than the transport time from P3HT to the electrode. This recent study mainly points to the inefficiency of OPVs caused by the exciton trapping at the donor/acceptor interface and the need for a better emulation of the photosynthetic system in photovoltaics. Other than the use of fully artificial photosynthetic systems in OPV cells, borrowing certain components from natural photosynthetic complexes for integration into the photovoltaic device has also been attempted. These are basically biohybrid devices (BPVs) that adopt the device architectures from OPVs. Recently a photosynthesis-inspired OPV has been reported with use of photosynthetic pigments as active layers in the device [102]. A linear Carotenoid (Car) pigment lycopene have been used as the electron donor and two different derivatives of Chlorophyll (Chl1: methyl 32 ,32 -dicyanopyropheophorbide-a, Chl2: methyl 131 -deoxo131 -(dicyanomethylene)pyropheophorbide-a) as acceptors (Fig. 5.5). Unlike the P3HT-CNT system discussed earlier, there is no ordered arrangement of donors and acceptors in this device architecture. Instead, the donor and acceptor materials are blended to form an active layer. This is a method used typically in a special type of OPVs called Bulk-heterojunction (BHJ) cells which are discussed in the next section.

Fig. 5.5 A biohybrid solar cell (BPV) with architecture inspired from OPVs: a Schematic showing the device structure of the BPV with lycopene as donor and chlorophyll derivatives (Chl1 or Chl2) as acceptor, Ca and MoO3 as cathodic and anodic interlayers. b Energy level diagram of the BPV. Adapted with permission [102] Copyright 2015, Royal Society of Chemistry

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Moreover, another design feature inspired from typical OPVs is the use of holeextracting and electron-extracting interlayers like MoO3 and Ca (Fig. 5.5a) [114]. The use of low work function materials like Ca, Ba and Mg as cathode interlayers is common in OPVs as this helps in reducing the electrode work function thereby enabling efficient electron extraction [114]. The interlayer also prevents the cathode materials like Al and Ag from penetrating into the active layer during deposition (typically by thermal evaporation) acting as a buffer layer [114]. Similarly, transition metal oxides like MoO3 , V2 O5 , and WO3 are used as transparent anode interlayers as these n-type semiconductors help in reducing the energy barrier for hole transport at the interface owing to the energy level alignment with the organic photoactive layer [114, 115]. Two different devices have been tried using the natural-pigment based BHJ approach, (a) Lycopene: Chl1 and (b) Lycopene: Chl2 [102]. In both the device architectures studied, there is one basic design principle that is followed as per the band-structure approach. In both cases, the energy offset between the donor and acceptor LUMO levels was maintained greater than the exciton binding energy which is typically in the range of 0.3–0.5 eV (Fig. 5.5b) [113]. Though the exciton dissociation could be efficient in both the cases owing to this energy offset, the device structure with Chl2 as the acceptor has been found to have better photovoltaic performance due to the higher electron mobility in Chl2 than that in Chl1 [102]. The two main factors pointed here are the carrier mobility and the exciton binding energy, the interplay between which has to be studied in any device for a deeper understanding and for designing cells with better photovoltaic performance. The crucial role played by these two factors in OPVs has led to the creation of a separate class of OPVs with Bulk hetero junctions which of late has also been used with photoproteins integrated into the device.

5.5 New Design Principles in Biophotovoltaics 5.5.1 Photoprotein Bulk Heterojunction Cells Mobility is the speed at which the charge carrier traverses through a medium under an electric field reviews [116–118]. Exciton binding energy is the strength of interaction between the electron and hole Coulombically bound within the exciton [116, 119– 121]. Both these factors play an important role in deciding the OPV efficiency by influencing carrier recombination. Three major types of recombination (Radiative, non-radiative and Auger) have been discussed in detail in several reviews [116, 117, 122]. Reducing the losses stemming from each of these recombination types is crucial for the OPV performance. However, this can be avoided only by maintaining the active layer defect-free, which is challenging. The defects in the active layer bring rise to new energy states within the band gap called electronic traps [116, 123]. These traps are potential sites for recombination to occur and hence the defect density in the material has to be minimized [124, 125].

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Since reducing the density of traps beyond an extent in extremely hard, an alternative approach called ‘Heterostructuring’has been developed, where such losses are minimized by separating the electrons and holes into two different phases formed by intermixing of the donor and acceptor materials [116]. Bulk heterojunctions resulting from the intermixing enable rapid dissociation/charge-separation of the photogenerated exci-tons at interfaces, reducing radiative recombination losses [116, 126, 127]. The change in strategy from conventional bilayer heterojunctions to bulkheterojunctions has taken the performance of OPVs to a new height and has also been inspiring other branches optoelectronics. Several attempts have been in recent years to introduce the BHJ strategy in Biophotovoltaic devices. Bulk Heterojunction (BHJ) solar cells have an architecture similar to the bilayer OPVs (Fig. 5.6a) in terms of materials used (i.e. both use an electron donor and an acceptor) but the only unique feature in a BHJ cell is that the electron acceptors and donors are blended together (Fig. 5.6b), and hence it still follows the same four stages involved in the working of any typical OPV (Fig. 5.4d). In addition to the use of natural pigment molecules in BHJ devices, attempts have also been made to integrate antenna complexes reaction centers and photosystems into BHJ-OPVs. Different device architectures have adopted photosynthetic pigments or proteins to serve different purposes in the photovoltaic cell. (1) A basic approach has been to use

Fig. 5.6 BHJ device architectures: a A simple bilayer OPV with donors and acceptors as separate layers. b A BHJ OPV with the donors and acceptors intermixed. c A BHJ OPV with additional interfacial materials on the either of the active BHJ layer. Different approaches in utilizing photosynthetic pigments/proteins in a BHJ OPV: d Photoproteins used as a donor in a bilayer OPV with C60 as an acceptor and ETL. e Photosynthetic pigments used as both acceptors and donors in a BHJ architecture. f Use of photoproteins as an interlayer in a BHJ that has a separate donor/acceptor concoction. The use as ‘anodic interlayer’ is specified considering a conventional OPV structure, the same protein layer would act as a ‘cathodic interlayer’ in an inverted OPV structure

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photosynthetic proteins/pigments as a photoactive layer/electron donor in a bilayerOPV device with a conventional electron acceptor (or an electron transporting layer) coated over it [128]. (2) Another approach is to use photosynthetic pigments/proteins both as donors and acceptors blended together to result in a bulk-heterojunction structure [102]. (3) Another recent approach is to use the proteins, not as the primary photoactive layer but to employ as an interlayer in a typical BHJ device [99, 100]. The use of anodic and cathodic interlayers are common in BHJ OPVs to enhance the charge extraction and transport (Fig. 5.6c). The use of additional interfacial materials has several vital purposes in OPVs; they can be used to control the interfacial energy level alignment, adjust the built-in electric field and to control the surface energy and surface recombination [114] The use of photoproteins in an OPV device structure has been attempted a decade back but with a typical bilayer-OPV structure [128]. This is an example of the first approach where photosynthetic reaction centers (RCs) from purple bacteria have been used the photoactive layer in the OPV with C60 as a separate layer coated on it that accepts the photogenerated electrons from the RC layer and transports them to a silver cathode (Fig. 5.6d) [128]. Using the second approach BHJ devices have been constructed with photosynthetic pigments like lycopene and chlorophyll as electron donors and acceptors (Fig. 5.6e) [102]. Photosystems with peptide linkers have been used in the third approach in BHJ-OPVs to act as an anodic interlayer (Fig. 5.6f) [100]. The use of photosystem as the only photoactive layer in an OPV structure has been reported in the two different reports. Oriented PS1 proteins on Au-coated ITO electrode have been used for this purpose, where the PS1 proteins were genetically modified with special tags that tend to attach in a particular orientation to the functionalized anode [128]. An organic semiconductor tris(8-hydroxyquinoline) aluminum (Alq3 ) was used as the electron transporting layer interfacing the PS1 layer to an Al/Ag cathode [128]. Though significant advancement from wet electrochemical cells was put forward, the effect of PS1 in determining the device open-circuit voltage has been unknown. Recently, a high open-circuit voltage of 0.76 V was achieved with a similar PS1 based OPV [99]. In this case, a dense layer of PS1 proteins was used with no special immobilization technique to orient the proteins on the electrode (Fig. 5.7a). A transparent semiconducting poly triarylamine polymer (PTAA) was used a hole-conducting medium, which makes the device structure different from the conventional OPV where the electron is typically driven to the Al/Ag electrode. The working mechanism of this inverted OPV device involves (1) light absorption by PS1, (2) Photogeneration of electrons and holes after charge separation in PS1 reaction center, (3) Migration of the electrons to the ITO electrode through the titanium oxide layer. (4) Simultaneous migration of the photogenerated holes from PS1 → PTAA → MoO3 → Al. PTAA serves as the hole transporting layer while the additional MoO3 layer aids in better hole extraction as it known to reduce the energy barrier for hole transport [99]. The electron transfer from the PS1 → TiOx → ITO might have been facilitated by the corresponding reduction in the energy level of the

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Fig. 5.7 Photoprotein-integrated BHJ devices: a Schematic representation of a PS1-integrated BHJ-OPV where PS1 is the only photoactive layer. b Energy level diagram of the PS1-BHJ-OPV. Effect of using PS1 layer in BHJ-OPV where PS1 is not the dominant photoactive layer, but serves as an anodic interfacial layer: Variation in open circuit voltage in MEH-PPV PCBM BHJ devices with PS1 (without orientated immobilization) used as an interfacial layer between the BHJ and ITO, with LiF (c) and MoO3 (d) as cathodic interfacial layers. e Use of oriented PS1 in a MEH-PPV PCBM BHJ device to serve as an anodic interfacial layer. f Effect of the PS1 interlayer on the cathode work function. Figure 5.7a–d are recreated based on the descriptions and depictions presented in article [99] and Fig. 5.7e, f are recreated based on the descriptions and depictions presented in article [100]

LUMO of the materials at each step from PS1 to ITO (Fig. 5.7b) [99]. However, the exact mode of electron and hole transport in any bio-organic photovoltaic device is still unclear. Another approach in integrating PS1 in a BHJ device has been to modify the electrode work function that ultimately affects the device open circuit voltage (Voc ). This has been proven possible by orienting the proteins in a desirable way on to the electrode. In an earlier attempt, even without special immobilization techniques, the PS1 proteins (though not 100% of them) were found to have the tendency to orient themselves with P700 site facing up and FB site adhering to the ITO in a BHJ device of structure ITO/PS1/MEH-PPV:PCBM/(LiF or MoO3 )/Al, where the BHJ is formed by the blend of the conjugated polymer MEH-PPV (poly[2-methoxy-5-(2’ethylhexyloxy)-p-phenylene vinylene]) and the fullerene derivative PCBM ([6,6]phenyl-C61 -butyric acid methyl ester) [99, 100] (Fig. 5.7c, d). Based on the type of interlayer (Electron-extracting LiF or Hole-extracting MoO3 ) coated on the BHJ, conventional- and inverted- OPV structures were constructed with differences only in the direction of charge transport. However, the presence of PS1 in the device caused a significant performance difference between the two cases. This can be better understood by considering the protein layer under illumination as a layer of electric dipoles on the ITO electrode as light absorption results in a charge separated

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state in PS1 with electrons staying at the FB site and the holes staying at the P700 site with a spatial separation in between the opposite charges [99]. The presence of electric dipoles on a conductive material is known to affect its work function. Since the work function difference between the two electrodes in an OPV determines its open circuit voltage, the Voc in the two cases (A, ITO/PS1/MEH-PPV:PCBM/LiF/Al B, ITO/PS1/MEH-PPV:PCBM/MoO3 /Al) was differently affected by the change in electrode work function caused by the PS1 dipole layer [99]. While the PS1 dipole orientation with electrons accumulating at ITO (i.e. at the FB site) had a positive effect on the case B (the inverted architecture) where photogenerated electrons from the BHJ tend to migrate towards ITO (due to the presence of the high work function MoO3 at the other electrode), the orientation had a negative effect on case A as there is a deleterious combination of photogenerated holes (from BHJ) migrating towards ITO and the photogenerated electrons (from PS1) staying on ITO [99]. The presence of PS1 dipoles reduces the work-function of ITO that results in an increase in ‘work function difference (ϕ)’ between the ITO and MoO3 /Al and a decrease in ϕ between ITO & LiF/Al, hence affecting the case B negatively [99]. In another recent work, this effect has been confirmed by using a fully oriented PS1 layer in the same BHJ structure. Employing peptide anchors, almost 100% of the PS1 proteins were oriented with the FB site facing ITO and P700 site facing the BHJ (Fig. 5.7e) achieved by phage display technique which popularly used in biochemistry and bioinformatics [100]. The use of peptides resulted in a greater work function shift (Fig. 5.7f) which had a positive effect on the device Voc when an inverted OPV structure was employed (i.e. with MoO3 /Al as the second electrode) [100]. While band structure approach is a crucial in any optoelectronic device, adapting device architectures from such devices (like OPVs), where controlling interfacial electronic structures, energy level alignment and electrode work-functions are popular tools, it has become inevitable for biophotovoltaics to equip with similar approaches. The above sections concentrated primarily on the band-structure approach in the perspective of interfacial materials and energy level alignment for controlling exciton/charge transport. The following three sections would cast light on the other ‘band-structure’ approaches or more specifically ‘band-gap’ approaches that are promising in biophotovoltaics. In these sections, the discussion is based on the performance improvement caused not just by adopting solid-state multilayer device structures like in OPVs but by the use other band-gap approaches in the emerging biohybrid solar cells.

5.5.2 Photoprotein-Semiconductor Hybrid Cells This section casts light on a new strategy for enhancing the open-circuit voltage of a photo-electrochemical cell integrated with photosynthetic pigment-protein by cascading the light-harvesting reaction of the protein with that of a semiconductor. In a very recent report, this has been achieved by the use highly oxidizing redox

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electrolytes as juncture medium enabling the cascade effect. This has been demonstrated using purple bacterial RC-LH1 complexes [129]. On photoexcitation, the special pair of BChls in the RC protein (denoted as P) initiates a meta-stable charge separation by a four-step electron transfer reaction across the RC to a cofactor called ubiquinone (QB ) resulting in a radical pair P+ Q− B (Fig. 5.8a, b). In any typical biophotoelectrochemical cell, the V OC would be limited by the potential gap between the molecular species receiving charge carriers from one electrode and the species receiving the opposite charge carrier from (or in other words, donating the same charge carriers to) the electrode [129]. Based on this, the V OC of the RC-based bioelectrochemical cell has been found to depend on the potential gap between the P/P+ couple that interfaces with the cathode and the electron transport mediator (or electrolyte) that interfaces with the anode. Hence, manipulating the electrolyte redox potential has been proven successful in controlling the V OC [50]. However, there is still a drawback with many bioelectrochemical cells because of the choice of redox potential that facilitates futile recombination reactions (as shown by the dotted arrow in Fig. 5.8b), which has a negative effect on the device performance. In the recent report, the photoinduced charge separation in the protein complexes has been cascaded with a second photo-transition in an n-doped silicon (n-Si) anode [129]. On illumination, the n-Si has photogenerated holes in its valence band and the photogenerated electrons in the conduction band. At the same time, there are photogenerated electrons at the QB site of the protein complexes. It is to be noted that the holes in the silicon’s valence band are at a more oxidizing potential than the P/P+ couple in the RC. The protein-coated FTO photoelectrode (cathode) and the n-Si anode were connected by an electrolyte that was either A. TEMPO (2,2,6,6tetramethyl-1-piperidinyloxy), B. ferrocene/ferrocenium, or C. the iodide/triiodide (I− /I3 − ) couple [129]. The redox potential of all these electrolytes are such that they are more oxidizing than P/P+ , and hence preventing undesirable electron transfer reactions between the oxidizing and reducing cofactors of the RC protein or between the RC and the FTO. The cascade mechanism in the cell is depicted in Fig. 5.8c. These electrolytes aid in migrating the photogenerated electrons from the protein to fill the photogenerated holes in the Si valence band. The electron excited to the conductor band reaches the electrode-contact, flows through the external circuit and reaches the FTO photoelectrode where the P+ in the RC gets re-reduced, completing the circuit. Moreover, as planar Si has reflection losses, the surface has been textured with micro pyramids that increased the Si light absorption and minimized the losses due to reflection [129]. The increased surface area ensuing from texturing also has resulted in a better charge transfer at the electrode/electrolyte interface (Fig. 5.8d, e). In this architecture, VOC has been determined by the energy level difference between the P/P+ couple in the RC and the conduction band of the n-Si which amounts to 0.7 eV. The observed photovoltage of 0.6 V in case of using the planar Si anode had a good match with the theoretical prediction, moreover, a further increment of 0.1 V has also been found to result from the use of textured Si [129]. These hybrid devices are not to be mistaken for bio-sensitized solar cells (BSSCs) which still employ the combination of semiconductors, proteins, and redox electrolytes. However, the BSSCs are in principle biohybrid analogs of dye-sensitized

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Fig. 5.8 Photoprotein semiconductor biohybrid cells: a View of purple bacterial reaction center with its surrounding light harvesting complex. On photoinduced charge separation (i.e. P+QB−), electrons enter the complex by means of reduction of P+ (magenta arrow) and exit the complex by oxidation of QB− (cyan arrow). b The working mechanism of an RC or RC-LH1 integrated biophotoelectrochemical cell with proteins interfaced directly onto FTO and with a diffusing electrolyte transporting charge to the anode. The potential gap between the P/P+ redox couple in the protein and the electrolyte determines the VOC in the device. In conventional bioelectrochemical cells, the redox potentials of the electrolytes typically lying in between those of P/P+ and QB/QB−, the possibility of recombination of electrons from the electrolyte to the P+ cannot be ruled out. c Schematic of the working mechanism of biohybrid cell: Incident photon initiates a primary photochemical reaction in the RC-LH1 complex which involves photoexcitation of the special pair (P → P*) and charge-separation to form P+ and QB−. Remnant light unabsorbed by the protein triggers an auxiliary photoexcitation in n-Si resulting in excitation of an electron into the conduction band (EC) while leaving a hole in the valence band (EV). The two photo-reactions are cascaded by the highly oxidizing electrolytes which replenish the photogenerated holes in the n-Si valence band with those electrons collected from the RC-LH1 complex. d Cells with planar Si in reflection losses (a considerable fraction of the incident light gets reflected back into the electrolyte). e Reduced reflection loss and higher light absorption by replacing a plain Si with a textured one (the pyramidal structures scatter the incoming light back to the surface facilitating multiple instances of light absorption). Bio-sensitized Solar Cells: f Schematic of the bio-sensitized solar cell with genetically modified Green fluorescent protein GFPdopa sensitizing the TiO2 . g working mechanism of the BSSC. Figure 5.8a–e reproduced from article [129], Fig. 5.8f, g reproduced with permission [130] Copyright 2017, American Chemical Society

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solar cells and do not involve any cascade effect discussed above. Nevertheless, the use of photosynthetic pigments and proteins for sensitization of semiconductors in like TiO2 [131, 132] ZnO [133, 134], Fe2 O3 [135] etc. DSSC-type devices has also been a popular approach. Recently, using this approach, Green Fluorescent Protein (GFP) and its genetically engineered designer version (GFPdopa) bio-sensitized solar cells (Fig. 5.8f) have been developed [130]. The designer protein exhibited superior adsorption on the TiO2 surface by means of oriented immobilization and the device had an open circuit voltage of 0.6 V which is comparable to that in conventional DSSCs. As opposed to the cascade effect in the protein-semiconductor hybrid cells, the working mechanism is the same as that of any typical DSSC, i.e. an electron gets injected into the semiconductor conduction band as the sensitizer gets photoexcited and the photogenerated hole in the protein/sensitizer gets filled by the redox electrolyte (Fig. 5.8g).

5.5.3 Photoprotein Plasmonic Cells Another recent approach in biophotovoltaics has been to favor broadband optical absorption in the device by using plasmonic nanostructures. This approach though closely related to the band theory is a separate field per se. Plasmonics, a branch of Nano-photonics deals with confinement of electromagnetic field over a dimension of the order of the wavelength of light and its interaction with the free electrons in a metal or metallic nano structures [136]. An enhanced light-matter interaction is achieved due to localized incident radiation at the interface or in nano structures [137]. Plasmonic effects are evident in both bulk and nano materials. Properties which require interfaces like surface plasmon resonance, hot spot generation are predominantly espied in nano particles [138, 139]. Equivalently, bulk plasmon effects are observed due to propagating longitudinal surface waves through the bulk of material [140]. The brilliant colors of noble nano materials are attributed to the surface plasmon oscillation, oscillating in the frequencies usually in visible region [141]. A lucid modification in size and introduction of anisotropy in shape modifies the optical properties of the nanoparticles drastically [142]. The same phenomenon is not observed when the size of particles is larger. When the size of particles falls below the mean free path of free electrons, there is scattering from surface alone. When incident light resonates with surface plasmon oscillations, the free electrons in the metal are subjected to oscillations [141, 143–145]. Polarisation and oscillation in resonance with the frequency of light causes fixed oscillation and the same is referred as surface plasmon effect [146]. A change in size, shape and dielectric constant of the either nanoparticle and or the surrounding base material influences the oscillating frequency [147]. When the wavelength of incident radiation is in the resonance range of the nanoparticle, its gets strongly scattered or absorbed causing constructive interference between the waves at resonating wavelengths [139]. In resonance frequency range, polarisation enabled interaction of light with matter occurs at a larger area of cross-section. Losses in the effect are attributed to surface plasmon

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resonance absorption whereas plasmon assisted scattering enhances the efficiency of the solar cells [148]. Encouraged by such optoelectronic surprises given by metal nanostructures, SPR effects have been extended to a wide range of photovoltaics and of late to biophotovoltaics [46, 149, 150]. Several recent reports have attempted to enhance the output of the biohybrid solar cells through the use of surface plasmons [46, 98, 101]. A nano-bio hybrid cell has been recently reported with Silver (Ag) nanoparticles (NP) which aid in photocurrent enhancement assisted by surface plasmon effect [98]. The device architecture involved a typical structure similar to that in Fig. 5.6f where the photosynthetic complexes are used as interlayers and not as primary photoactive layers. The plasmon enhancement has been demonstrated using LHC2 antenna complexes in an inverted BHJ structure ITO/ZnO/Interlayer/PIDTT-DFBT:PC71 BM/MoO3 /Ag where LHC2Ag NP nano biohybrids served as the interlayer. PIDTT-DFBT:PC71 BM blend formed the BHJ where PIDTT-DFBT (poly(indacenodithieno[3,2-b]thio-phenedifluorobenzothiadiazole) served as the donor polymer and PC71 BM ([6,6]-phenylC71 -butyric acid methyl ester) as the acceptor (Fig. 5.9a). As the nanoparticles were incorporated into the light harvesting complexes the photoactivity of the LHC2 complex was enhanced significantly due to the localized surface plasmon resonance of the Ag NPs. These nanoparticles enhanced the performance of the device mainly by means of two factors: 1. broadband optical absorption enhancement caused by the complementary absorption of the Ag NPs, 2. increased light harvesting by the LHC2 complexes due to the LSPR effect [98] (Fig. 5.9b). Additionally, the nanoparticles also are known to act as nano optical antennas which will act as a center for electromagnetic coupling for the proteins and nano particles resulting in enhanced light absorption over the entire spectrum. Based on this approach, a self-assembly of protein (RC-LH1) on silver nano structured substrate yielded a 2.5 fold improvement in current density in a recent report [46]. The device structure involved Ag nanostructures loaded with RC-LH1 complexes, with cytochrome c as an electron transfer mediator. The charge transfer mechanism involves the transfer of electrons from the silver substrate to the cytochrome c and further to the reaction center and then getting captured in the quinol oozing out of the protein. Plasmon effect increased the overall number of photogenerated electrons hence yielding high photocurrets [46].

5.5.4 Photoprotein Tandem Cells While most of the reported bio-photoelectrochemical cells devices focus on photocurrent enhancement by improving the electron transfer in three-electrode or twoelectrode cells interfacing the proteins with an electrolyte or electron transporter [6, 128], there has been very little efforts so far on enhancing photocurrent by increasing the light-harvesting ability and the spectral range of the protein-integrated devices. Use of surface plasmon effect in biophotovoltaics in one such attempt to broaden the light absorption range. There were also a few attempts in the past to increase

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Fig. 5.9 Photoprotein-plasmonic cells: a An Inverted BHJ device with Ag-NP-LHC2 nano biohybrids as an interlayer. b The synergetic effect of the two factors contributing to the enhanced photocurrents. Reproduced with permission [98] Copyright 2016, Royal Society of Chemistry

the optical absorption cross-section of the photosynthetic reaction centers by means of tagging tailored molecular fluorophores and photoluminescent quantum dots to the protein complexes though not scaled up at a device-level for photocurrent generation [151–153]. While the enhancement in light-absorption has been realized by Förster resonance energy transfer (FRET) from the attached fluorophore to the core reaction centre complex [152], it is still challenging to realize the same with an RC-LH1 complex, which if made possible, could lead to a significant enhancement in photocurrent generation as a result of the spectral response broadened by the fluorophore complementing the role of the LH1 protein.

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An alternative approach to overcome this is to have a stacked device architecture where pigments of complementary absorption characteristics can be incorporated into the proteins at different layers of the device, rather than all to a single protein [11]. This architecture would need a transparent rear electrode so as to let the incident light reach the complementary absorption layer beneath. Recently, a proofof-principle bio-tandem cell has been reported employing two kinds of RC-LH1 complexes with one having the native red carotenoids in the LH1 protein (RC-LH1red ) and the other engineered to have green carotenoids in the LH1 (RC-LH1green ) for the photo-bioelectrochemical tandem cell to demonstrate the enhancement in light absorption and hence the photocurrent (Fig. 5.10a, b) [11]. The two pigment-proteins having optically complementary absorption characteristics in the visible spectrum (Fig. 5.10c) were contained in two sub-cells connected in parallel so as to realize addition of photocurrents in the tandem-structured cell (Fig. 5.10d) where the front and rear electrodes for each sub-cell were FTO and PEDOT:PSS respectively [11]. Two possible tandem architectures—series and parallel, though theoretically are proficient in achieving high light-absorption cross section for a given footprint, series combination of sub-cells are limited by stringent current-matching criteria

Fig. 5.10 Photoprotein tandem cells: Views of a RC-LH1green and b RC-LH1red complexes perpendicular to the photosynthetic membrane; only the type of carotenoid pigments differ between the two complexes. c Absorption spectra of RC-LH1red and RC-LH1green complexes in solution, in the visible spectrum. d Cartoon of a biohybrid tandem device architecture for extended lightharvesting with the complementary absorption in blue to yellow region. e Energy level diagram and mechanism of photocurrent generation. Reproduced from article [11]

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[11, 154]. To avoid current and voltage losses in parallel-connected and seriesconnected tandem cells respectively, their component sub-cells should ideally have equal photovoltage (in case of parallel connection) or equal photocurrent (in case of series connection). While Voc of the biohybrid photoelectrochemical cells is often energy-level dependent, achieving approximately equal Voc in the two subcells was easier than achieving equal Jsc , since Voc is not as sensitive as Jsc to the light-harvesting nature of the proteins [11]. Hence, the parallel-connected tandem architecture has been suggested as a better alternative in enhancing the photocurrent generation without altering the footprint of the cell. Though ideally the photocurrent of the parallel-connected tandem cell can be 100% of the sum of the two sub-cell currents, there was ≈10% loss in the overall photocurrent of the tandem cell, which has been attributed to the reduced incident light intensity for the bottom sub-cell and also to any minor difference in Voc between the two subcells that would result in an undesirable circulating current, reducing the overall photocurrent output [11]. The device employed two electron transport mediators TMPD (N, N, N , N -tetramethylp-phenylenediamine) and Q0 (ubiquinone-0) and the working mechanism involved the photoinduced charge separation in the RC followed migration of electrons from the QB site to the PEDOT:PSS counter electrode through the Q0 electrolyte, while the electron entering the device from the external circuit is used by the TMPD mediator to reduce the P+ (Fig. 5.10e) [11] The use of a highly transparent PEDOT:PSS film has been found to be effective as a rear electrode in biohybrid photoelectrochemical cells in addition to facilitating the tandem construction. The work function of PEDOT:PSS is closer to the vacuum potential of Q0 as compared to Pt and it offered less of a potential drop for the electron transfer from Q0. The use of PEDOT:PSS resulted in a 12-fold enhancement in photocurrents over the conventional Pt electrode a maximum of 88% photocurrent addition has been demonstrated in the tandem cell with a parallel configuration [11]. The study has highlighted the photocurrent enhancement possible by integrating different pigment-proteins at different layers of a photovoltaic device with the one with the biggest bandgap (shortest wavelength) on top and lower bandgap materials below.

5.6 Directions for Future Bio-Photovoltaics As solar energy capture has been mastered by natural photosynthetic systems since times immemorial, emulating their photosynthetic machinery and their design principles in man-made photovoltaic devices has always been of a huge research interest. While numerous attempts are made in designing materials/structures imitating the photosynthetic proteins, the sophistication and ordered arrangement of antenna complexes and reaction centers are still unparalleled. Direct integration of photosynthetic proteins in devices which are typically known as Biophotovoltaics became popular in the last decade with a number of successful reports on photocurrent enhancement. Though there was a good understanding of the structure of proteins

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and the energy/charge transfer pathways within, appreciation of device-level structure and mechanism has rather been inadequate which has been a general drawback with the field. This restricted most photoprotein-BPVs to employ device structure of a simple liquid electrochemical cell as it offered a better control over protein stability and a relatively easier understanding of the charge transport in the device. However, advancements in photovoltaic performance from these wet electrochemical cells have not been satisfactory even after a decade of research. An interesting research trend that brought has brought a new perspective to BPVs is the juncture of bio-inspiration in OPVs with solid-state BPVs. The overview of works presented in this review essentially sheds light on this juncture and the benefits it can bring to BPVs. The use of solid-state protein films in the BPV has been proven more effective than the wet electrochemical cells, though the number of reports on fully solid-state BPVs is extremely low. Of late, since OPVs have also been eying on bio-inspiration and on adapting photosynthetic proteins for better performance, the efforts to develop solid-state biohybrid solar cells are now extended from both the fields. While the use of band-gap approach/band-structure approach in device design and construction is common in OPVs, the approach is almost alien to BPVs. The study of interfacial electronic structure and energy level alignment between various layers (which are based on the band theory of solid) were crucial in OPVs, such studies were not attempted in BPVs as they remained predominantly as liquid Bio-electrochemical cells. From the recent trend in BPVs and in the Bio-inspired OPVs, the use of bandstructure approach would be crucial for any breakthrough advancement in BPVs. To highlight this prospect, this review presented an overview of selected works in the last few years where the use band-structure approach either directly or indirectly boosted the device performance. This approach would be promising for pushing the photoelectric efficiency of BPVs way higher than the status quo. This can be realized by a few major initiatives: (1) To develop thin solid-state BPVs with protein layers safeguarded from harsh fabrication conditions like high temperatures, denaturing chemical ambience etc. by developing new encapsulation materials that can prevent the proteins from denaturation while still allowing light transmission and charge conduction. (2) To understand the interfacial electronic structure at the protein/electrode interface. While this has been studied in organic/metal interfaces, no clear account of the effect of the protein complexes on the interfacial electronic structure is available in the literature. (3) To study the energy level alignment between the device layers in a BPV and the effect of the proteins on it. This is especially promising as it would open up new research directions on the use of photosynthetic proteins in photovoltaics other than its use as a photoactive layer. This would enable to precisely tune the energy levels and hence the charge transport pathway in the device by introducing new interfacial materials and biomolecular surface modifiers. Such studies would also aid in a better understanding of the mode of charge transport (e.g. tunneling, hopping etc.) in the device facilitating a well-informed concoction the device architectures. (4) To broaden the light harvesting range of BPVs by employing other bandgap-related approaches. This includes developing multilayer-tandem devices and incorporating

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auxiliary light-harvesters in the device by combining protein complexes with semiconductors, quantum dots and with plasmonic nanoparticles. In light of the promising research avenues put forth by the band-structure approach, the scope for advancing the biophotovoltaics by developing new device architectures has become much wider and the future progress in BPVs in a path beyond bioelectrochemistry is certainly bright.

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Chapter 6

Prolonged Charge Trapping in Photoproteins and Its Implications for Bio-Photocapacitors

6.1 Brief Overview Biophotonic processes in nature have been excellent working models for light harvesting and energy-conversion which has led to integration of natural/artificial photosynthetic systems in photoelectrochemical and photovoltaic cells. Though engineered photosynthetic proteins have been immensely researched for enhanced light harvesting and photocurrent generation, their potential as charge storage systems have not been investigated before. This chapter [1, 2] outlines a recently reported phenomenon of prolonged charge storage in purple bacterial photoprotein multilayers. Based on this, the concept of Biophotonic power cell is presented which not only generates charges on photoabsorption but also essentially traps the photogenerated charges. Several previous attempts to achieve concurrent charge generation and storage have been made by combining photovoltaic layers with capacitors/batteries in a hybrid device. However, both these functions are possible through a single material system in this biophotonic power cell which generates charges upon photoabsorption amounting to a voltage as high as 0.45 V, and traps the charges effectively for almost 1560s before decay. Charge build-up on protein multilyers upon photoabsorption and external injection are studied by Kelvin Probe Force Microscopy and Scanning Capacitance Microscopy. The capacitance in the biophotonic power cells ranged from 0.1 to 0.2 F/m2 , utilizing which powering of an external load is demonstrated.

6.2 Introduction The processes of energy harvesting, conversion and storage underlie many existing energy technologies, [3–5] These three processes are usually realized separately through a modular device architecture [6], with each requiring optimization and effective interfacing to achieve a high overall efficiency [7]. Typically, solar, thermal, © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_6

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chemical or mechanical energy harvested through a variety of mechanisms is converted into electrical energy and, if not used directly, stored using a capacitor or a battery [8, 9]. As there are losses involved at each stage, considerable efforts have gone into the development of hybrid systems where two or more of the three processes are combined. In some cases, energy harvesting and conversion systems might be one and the same, but devices typically have a distinct system performing the energy storage function. An emerging example of a hybrid system is the ‘selfcharging’ power cell or supercapacitor, the basic function of which is to harvest and convert ambient energy into electricity and continuously charge a battery or capacitor to ensure a sustainable supply of power [10, 11]. The majority of these power cells are based on piezoelectric [12, 13] or triboelectric [14–16] nanogenerators that harvest ambient (bio) mechanical energy and store converted energy in supercapacitors or batteries. Fewer attempts have been made to develop self-charging power cells that harvest light energy, these mainly involving the hybridization of dye-sensitized solar cells with supercapacitors [17, 18]. Hybrid devices have also been described in which Li-ion batteries are photocharged by perovskite solar cells [19], and recently a hybrid self-charging power cell was reported that combined a photoelectrochemical cell with a redox-flow battery [20]. These hybrid power cells typically contain a light-harvesting/conversion system and an electrical energy storage system combined into a single device architecture, sometimes with a common electrode. This study makes use of the biophotonic processes in the natural photosynthetic systems to realize a self-charging power cell (Fig. 6.1a). In a general sense, Biophotonic processes refer to those phenomena related to the interaction of biological matter with photons [21]. However, the most primary and widespread example of biophotonic processes are those photophysical and photochemical processes taking place during the initial steps of photosynthesis [21, 22]. These steps involve the interaction of light-harvesting antenna complexes with incoming photons, generation of excitation energy, ultrafast energy transfer (on a picosecond timescale) to efficiently move this energy to the site of charge separation called reaction center which acts as an energy trap [23, 24]. Photogenerated charges being the product of these biophotonic processes, the possibility to use the same complexes to act a charge trap is studied in this work. Based on this, photoprotein-based Biophotonic Power Cells (BPC) are constructed in which the light harvesting, energy conversion, and charge storage processes are integrated within a single, multi-functional material. This has been realized through the use of a pigment-protein complex from a natural bacterial photosystem that absorbs energy across the solar spectrum and transiently stores harvested energy through charge separation [25–28]. The particular photoprotein used, the so-called PufX-deficient reaction centre-light harvesting 1 (RC-LH1) complex from the photosynthetic bacterium Rhodobacter (Rba.) sphaeroides (Fig. 6.1b), is an integral membrane pigment-protein made up from a central reaction centre (RC) domain (Fig. 6.1b, c, cyan) surrounded by a cylindrical LH1 antenna complex (Fig. 6.1b, d) [25, 26, 29, 30].

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Fig. 6.1 Photosynthetic proteins, charge separation and cell architecture. a Cartoon illustrating the concept of biophotonic power cell. b The RC-LH1 complex comprises a central reaction centre charge separation domain (cyan) surrounded by a cylindrical light harvesting (LH1) domain. c The RC can be isolated as a separate complex and is shown as a solid object with a transparent surface in the plane of the membrane to reveal the electron transfer cofactors. Photoexcitation of the P BChl pair initiates a four step charge separation via a BChl, bacteriopheophytin (BPhe) and ubiquinone (UQ) to reduce the QB UQ on the opposite side of the photosynthetic membrane. d The LH1 cylinder can be isolated as a separate complex and is formed from 32 BChls (alternating red/orange) and 32 carotenoids (yellow) held in place by a protein scaffold (green). e Assembled cells comprised a multilayer of purified RC-LH1 proteins (green LH1 with cyan RC) sandwiched between n-Si and FTO-glass electrodes. f Under illumination a photovoltage is produced due to light-activated protein/electrode redox interactions, but current does not flow through the protein multilayer in the absence of mobile mediators

Following the absorption of solar energy by the bacteriochlorophylls (BChls) and carotenoids of the LH1 domain (Fig. 6.1d), the key energy conversion event is a four-step electron transfer between a pair of BChl molecules (P) at one end of the RC protein and a ubiquinone molecule (QB ) at the opposite end (Fig. 6.1c) [27, 28]. This forms a charge-separated state (P+ Q− B ) around 1 µs after photoexcitation that is stable for a few seconds (Fig. 6.1c). To fabricate a simple device that would generate and trap the photogenerated charges, films of concentrated photoprotein of varying thickness were sandwiched between a transparent fluorine-doped tin oxide (FTO) glass front-electrode and an n-doped silicon (n-Si) back electrode (Fig. 6.1e, f). The work demonstrates that the resulting BPC carries out solar energy harvesting, energy conversion and energy storage in a single, integrated architecture.

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6.3 Biophotonic Power Cell: Construction and Photovoltage Generation BPCs were assembled by drop casting a 20–1000 µL aliquot of concentrated RCLH1 protein solution into a well formed from one or more layers of plastic paraffin film (Parafilm M) adhered to an FTO-glass slide. This enabled formation a protein multilayer of regular area, and thicknesses between 0.1 and 2 mm. After partially drying the protein under a vacuum a precleaned n-type silicon counter electrode was sandwiched with the protein-coated FTO electrode and the cell was sealed. The result was a densely-packed protein multilayer formed in the absence of any additional electrolyte to act as a charge carrier. For reference, a single RC-LH1 complex has a maximum diameter of ~13 nm [25], which means that a closely-packed, 0.1 mmthick multilayer should be the equivalent of a minimum of ~7700 stacked protein monolayers, and ~154,000 monolayers for a 2 mm film. Five RC-LH1 BPCs of varying thickness were charged by exposure to one sun illumination for 200 s under open-circuit conditions. The photovoltage achieved increased with the thickness of the protein multilayer up to 500 µm, beyond which the maximum photovoltage dropped (Fig. 6.2a). In addition to the highest photovoltage under this standard illumination period (≈0.37 V; Fig. 6.2b), the 500 µm

Fig. 6.2 Charging and discharging of RC-LH1 BPCs. a, b Effect of protein film thickness on photocharging and discharging. c, d Effect of photocharging time on the photovoltage and discharge time. In a and c, grey = light-off and white = light-on

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film exhibited the second longest dark discharge time (Fig. 6.2b). For these 500 µm cells, the photovoltage increased as the photo-charging time was increased (Fig. 6.2c, d), as did the dark discharge time (Fig. 6.2d).

6.3.1 Origin of Voltage Build-up Taking into account vacuum potentials, and a well-established understanding of the photochemistry of RC-LH1 complexes [27, 28], the observed photovoltage is attributed to net oxidation of RC-LH1 proteins at the FTO electrode (Fig. 6.3, left) and net reduction of RC-LH1 proteins at the n-Si electrode (Fig. 6.3, right), producing trapped charges on opposite sides of the protein multilayer. The initiating event, in either case, is the photogeneration of the radical pair P+ Q− B (Fig. 6.1c), which has a lifetime of 3–5 s in purified RC-LH1 complexes [31–33], and which relaxes by recombination at the P BChls. Net oxidation of protein at the FTO-glass electrode would be achieved if Q− B in a suitably oriented RC-LH1 protein is able to reduce the + FTO more rapidly than either P+ Q− B recombination or reduction of P by the FTO (Fig. 6.3, left). At the photoactive n-Si electrode (Fig. 6.3, right), a net reduction of

Fig. 6.3 Photoexcitation and electrochemical activity at the two electrodes in an RC-LH1 BPC. In photo-excited protein layers near the FTO electrode (left), trapped positive charges (initially P+ ) accumulate as Q− B formed by intra-RC charge separation (green arrow) donates electrons to the FTO (red arrow). In photo-excited protein layers near the n-Si electrode (right), trapped negative charges + (initially Q− B ) accumulate as P formed by intra-RC charge separation (green arrow) is reduced by the photoactive n-Si electrode (red arrow). In the absence of an electrolyte, these protein/electrode interactions cause the build-up of a potential difference over time. At either electrode, the process responsible for the generation of trapped positive or negative charges would be expected to be in competition with wasteful competing reactions (dashed black arrows)

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the adjacent protein would be possible if electrons from the conduction band of the n-Si were able to reduce P+ more rapidly than either P+ Q− B recombination or donation of an electron to the conduction band of the n-Si by Q− B. The observed gradual build-up of the photovoltage (Fig. 6.2c) indicated that the density of trapped charges increased over several hundred seconds. This can be attributed to multiple oxidation or reduction events within individual complexes and/or propagation of trapped electrons or holes deeper into the multilayer through slow inter-protein electron transfer. Regarding the former, it has been estimated from electrochemical titrations that an LH1 ring can store up to eight BChl cations without undergoing irreversible photo-oxidative damage [34]. In addition, RCs contain a single dissociable QB ubiquinone that can undergo double reduction, the second reduction being accompanied by double protonation, and preparations of RC-LH1 complexes typically contain 10–15 molecules of ubiquinone in the space between the RC and the surrounding LH1 ring that can exchange with the quinone at the QB site [31]. Gradual penetration of long-lived trapped charges into the photoactive protein multilayer through redox interactions between adjacent proteins is also possible, and could be a factor in the slow discharge of the photovoltage on terminating the illumination period (Fig. 6.2d). A factor in the use of RC-LH1 complexes as a material for charge storage is that it is known from experiments employing conductive atomic force microscopy that individual complexes conduct electrical current under an applied bias [35–37]. Such experiments typically interrogate a monolayer of protein oriented on a conductive surface, and it has been suggested that electron tunneling across an LH complex is facilitated by the carotenoid cofactors [35–37]. Given this, a possible reason that the photovoltage supported by an RC-LH1 multilayer increased as its thickness increased up to 500 µm (Fig. 6.2b) is that the effective resistance of the multilayer also increased, enabling the build-up of a larger potential difference. Such an explanation seems more plausible than positing that thicker layers produced higher voltages because they were more absorbing, as photochemical activity leading to trapped charges would be expected to be confined to a minority of the structure comprising (probably several) protein layers close to either electrode, rather than the entire thickness of the multilayer. The decrease in photovoltage achieved by films thicker than 500 µm (Fig. 6.2b) is attributed to poor light penetration to the n-Si back electrode and its adjacent RC-LH1 proteins that lessens the accumulation of negative charge at this interface. The Rba. sphaeroides RC-LH1 photosynthetic complex is modular (Fig. 6.1b– d), and the RC and LH1 proteins can be purified as separate, functional entities. BPCs formed from isolated RCs or LH1 complexes also generated a photovoltage (Fig. 6.4a). On comparing charge-discharge characteristics for 500 µm protein films, it was found the photovoltage reached at the end of a standard 200 s illumination was the highest for the combined RC-LH1 complex and lowest for the LH1 protein (Fig. 6.4a). The light harvesting capacity of isolated RCs is limited to its six bacteriochlorins and one carotenoid (Fig. 6.1c), but each RC-LH1 complex has an additional 32 BChl and 32 carotenoid light-harvesting pigments (Fig. 6.1b and d).

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Fig. 6.4 Photocharging and dark discharging of RC-LH1, LH1 and RC films. a Bulk chargedischarge characteristics of 500 µm films exposed to 100 s illumination. b–d KPFM surface potential maps of b RC, c LH1 and d RC-LH1 films before, during and after a 2 min period of illumination. e–g Changes in surface potential before, during and after a 2 min period of illumination of e RC, f LH1 and g RC-LH1 films

The lower photovoltage achieved by a film of RCs is therefore likely attributable to a strongly diminished light harvesting capacity even though, as they are smaller than an RC-LH1 complex, the concentration of RCs in a packed multilayer would be expected to be greater. Although the pigment content of the isolated LH1 antenna protein is closer to that of RC-LH1 complexes, the absence of the RC means that light harvesting is not translated into a meta-stable charge separation, but rather energy is lost as emission and heat. Nevertheless, a photovoltage is still obtained because LH1 is capable of acting in a manner akin to an organic semiconductor, injecting excited electrons into the FTO electrode and becoming positively charged, or receiving electrons from the n-Si electrode and becoming negatively charged (Fig. 6.5). However, the number of photo-accumulated charges is likely lower than in RC or RC-LH1 complexes because the lifetime of the LH1 excited state (estimated as 680 ps at room temperature [38]) is very short relative to the 1–5 s lifetime of the charge-separated state formed in RC and RC-LH1 complexes.

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Fig. 6.5 Photoexcitation and electrochemical activity at the two electrodes in an LH1 BPC. In photo-excited LH1 layers near the FTO electrode (left), trapped positive charges accumulate as excited state electrons are donated to the FTO (red arrow). In photo-excited LH1 layers near the n-Si electrode (right), trapped negative charges accumulate as photo-excited LH1 is reduced by the photoactive n-Si electrode (red arrow). In the absence of an electrolyte, this causes build-up of a potential difference between electrodes. At either electrode, the process responsible for generation of trapped positive or negative charges would be expected to be in competition with competing reactions (dashed black arrows)

6.3.2 Kelvin Probe Microscopy: Surface Potential Build-up and Decay Charging and discharging of the different types of BPC were further investigated by recording the surface potentials of the three types of protein film by Kelvin Probe Force Microscopy (KPFM). Films coated on an FTO substrate were scanned using a conductive Pt/Ir probe before, during and after illumination. For all three films, the surface potential measured along a 1000 nm trajectory increased after light-on and decayed gradually after light-off (Fig. 6.4b–d), confirming the generation of a photovoltage. In good accord with the trend in overall device photovoltage (Fig. 6.4a), the averaged surface potential shift (Fig. 6.4e–g) was highest for the RC-LH1 films (~30 mV) and lower for LH1 or RC films (~15 mV). The time for relaxation of the surface potential shift was also longest for RC-LH1 films (~15 min) and shortest for RC films (~3 min) (Fig. 6.4e–g). These relaxation times of minutes confirmed that the potential shifts were due to the photo-generation of trapped charges rather than photochemistry within individual proteins, where relaxation occurs in a few seconds. The KPFM and macroscopic photovoltage measurements indicated that all three proteins can generate and store charge. However, the extent of charge accumulation by each protein under these conditions was determined by its light-harvesting capacity and photochemical activity rather than its innate charge storage capacity.

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As a result, the discharge times in Figs. 6.2 and 6.4 did not necessarily convey the relative charge storage capacities of the three proteins as each was charged to different extent under illumination. To investigate the capacitance of each of the proteins at a macroscopic level, galvanostatic charge-discharge measurements and cyclic voltammetry (CV) were performed on the two electrode devices (Fig. 6.6a–f). Near-symmetrical charge-discharge responses were obtained over a 0–1 V voltage window at an applied current of 2 µA/cm2 , indicating a good capacitive behavior (Fig. 6.6a). The LH1 photoprotein exhibited the largest area under the curve, indicating the highest capacitance, followed by RC-LH1 and then RC. Galvanostatic charge–discharge responses of the LH1 cell over a range of applied currents from 2 to 12 µA/cm2 were also near-symmetrical, confirming the capacitive behavior (Fig. 6.6b).

6.3.3 Microscale and Macroscale Capacitance Measurements Cyclic voltammetry confirmed the higher capacitance of the LH1 cells compared to the RC-LH1 and RC cells, the area of the near-rectangular CV plot being the highest for LH1 (Figs. 6.6c and 6.7).  It is known that the capacitance is proportional to the area under the CV curve ( iv—where i and v are the current and applied potential, respectively). Using the galvanostatic charge-discharge responses, the specific capacitance can be determined from the applied current, the active area and the slope of the discharge curve. From the charge–discharge profiles the specific capacitance were found to be the highest for LH1 films (≈0.19 F/m2 ) followed by RC-LH1 films (≈0.14 F/m2 ) and RC films (≈0.11 F/m2 ) (Fig. 6.6d). Over a 2–12 µA/cm2 range of applied current, the LH1 cells exhibited a maximal capacitance at 2 µA/cm2 and the capacitance did not drop beyond 0.12 F/m2 over the entire range (Fig. 6.6e). These differences in capacitance were confirmed on the microscopic level using Scanning Capacitance Microscopy (SCM) measurements in which the same amount of injected charge was made available to each type of protein film by applying a range of drive voltages from an external source. The determined gradient of capacitance between the AFM tip and the sample, V(dC/dZ) , is an indirect measure of the dielectric constant of the sample. SCM maps were recorded over a defined area of RC-LH1, LH1 or RC film at six different drive voltages and converted into average values of V(dC/dZ) to establish uniformity (Fig. 6.8). A plot of the capacitance gradient averaged across the scanned film area as a function of drive voltage (Fig. 6.6f) showed that V(dC/dZ) was highest for LH1 films and lowest for RC films at each drive voltage, in agreement with the trend in specific capacitances derived from charge–discharge and CV measurements (see above). Despite these measured capacitances exhibiting the order LH1 > RC-LH1 > RC, the observed photovoltage displayed the trend RC-LH1 > RC > LH1, consistent with the RC-LH1 complex being superior to RCs in light harvesting and superior to LH1 complexes in the translation of short-lived excited states into long-lived charge-separated states.

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Fig. 6.6 Capacitances of the protein BPCs. a Galvanostatic charge–discharge over a 0–1 V range for RC, LH1 and RC-LH1 BPCs at an applied current density of 12 µA/cm2 . b Galvanostatic charge– discharge curves for an LH1 BPC at different current densities. c Cyclic voltammetry curves for the three BPCs. d Specific capacitance of the three BPCs; capacitance presented is the average of at least three measurements and the error bar denotes standard deviation. e Specific capacitance of an LH1 BPC at different applied current densities. f Capacitance gradient V(dC/dZ) as a function of drive voltage for the three types of cell

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Fig. 6.7 Cyclic voltammetry curves at scan rates of 50, 100, 200 and 500 mV/s for an LH1 BPC

Fig. 6.8 Scanning capacitance microscopy (SCM) maps. Data are shown for films formed from. a RC. b RC-LH1. c LH1 complexes. Each step in the map corresponds to an applied drive potential of (left to right) 0, 100, 250, 500, 750 and 1000 mV. d Plot of capacitance gradient as a function of horizontal position for the three films

6.4 Proof-of-Concept Demonstration Finally, the utility of the 500 µm RC-LH1 BPCs was demonstrated through their ability to power a low-consumption LED display. In the demonstration depicted in Fig. 6.9, a single RC-LH1 cell was charged by a constant current of 10 µA for 50 s (Fig. 6.9a–c). This provided sufficient charge to power an LED display for approximately 1 s (Fig. 6.9d). In a second experiment of this type (Fig. 6.10) three

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Fig. 6.9 Storage of externally injected charges by a single RC-LH1 cell. a A single RC-LH1 cell was connected to source meter. b Charge was injected into the cell by applying a constant current of 10 µA. c The cell was charged for 50 s. d The charged cell powered an LED display for approximately 1 s

Fig. 6.10 Storage of externally injected charges by a bank of three RC-LH1 cells. Three RC-LH1 cells were connected in series and then were connected to a source meter and the two end-terminals of the cells were connected to the LED display (top left). Charges were injected into the cell by applying a constant current of 1 mA for 2 s during which the LED display was powered, but still under the control of the power source (top centre). When the current input was then turned off (top right), the cells were allowed to discharge by powering the LED display connected in the circuit. The LED display was powered for up to 5 s while gradually fading off (remaining panels)

RC-LH1 cells connected in series were charged by a 1 mA current for 2 s, and were able to power an LED display for up to 5 s. Photo-charging of a bank of 4 RC-LH1 cells at 1 sun illumination for 5 min powered an LED display for about a second (Fig. 6.11).

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Fig. 6.11 Storage of photo-generated charges by a bank of 4 RC-LH1 cells. a 4 RC-LH1 cells connected in series were exposed to one sun illumination for 5 min. The cells were then connected to an LED display. The cell electrodes had static accumulated charges that surged into the display terminal as a lead from the cells comes in contact, which resulted in a short-lived current that powered the LED display for about a second, discharging the cells. b LED display before connecting the cells. c LED display after connecting the cells

6.5 Summary Looking to future developments, a key feature of the BPCs described in this chapter was the use of protein multilayers to generate the photovoltage and trap charges. As these films were fabricated by simply drop-casting a concentrated solution of detergent-solubilized protein into a pre-formed well it is to be expected that individual proteins had a random orientation within the multilayer. One way to possibly boost the photovoltages obtained may be to control the orientation of individual RC-LH1 proteins throughout the multilayer such that there is alignment of the dipoles created between the P and QB termini of the RC on photoexcitation. Large photovoltages obtained from micron thick crystals of the Photosystem I (PSI) reaction centre from Pisum sivatum (pea) in response to strong (1.1 W cm−2 ) 660 nm laser excitation have been attributed to a superposition of photoinduced dipoles across uniformly oriented PSI proteins within the crystal lattice [39, 40]. In the present case, imposing a uniform alignment on the RC-LH1 complexes within a film could both maximise desired redox interactions at each electrode surface (i.e. QB oxidation at the FTOglass anode or P+ reduction at the n-Si cathode) and align the dipoles created across individual RC-LH1 complexes, both of which could potentially lead to a higher photovoltage. However, it is worth noting that achieving such control over protein orientation in a thick, multilayer film is challenging, and an attractive feature of the unoriented protein films used in the present work is their simplicity of fabrication.

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References 1. Ravi SK (2018) Solar energy harvesting with photosynthetic pigment-protein complexes. National University of Singapore, Singapore 2. Ravi SK et al (2019) Photosynthetic apparatus of Rhodobacter sphaeroides exhibits prolonged charge storage. Nat Commun 10(1):902 3. Xue X et al (2014) Flexible self-charging power cell for one-step energy conversion and storage. Adv Energy Mater 4(5):1301329 4. Chen T et al (2012) An integrated “energy wire” for both photoelectric conversion and energy storage. Angew Chem Int Ed 51(48):11977–11980 5. Wang ZL, Wu W (2012) Nanotechnology-enabled energy harvesting for self-powered micro/nanosystems. Angew Chem Int Ed 51(47):11700–11721 6. Ramadoss A et al (2015) Piezoelectric-driven self-charging supercapacitor power cell. ACS Nano 9(4):4337–4345 7. Lu K (ed) (2014) Materials in energy conversion, harvesting, and storage. Wiley 8. Xue X et al (2012) Hybridizing energy conversion and storage in a mechanical-toelectrochemical process for self-charging power cell. Nano Lett 12(9):5048–5054 9. Kim Y-S et al (2015) Highly porous piezoelectric PVDF membrane as effective lithium ion transfer channels for enhanced self-charging power cell. Nano Energy 14:77–86 10. Niu S et al (2015) A universal self-charging system driven by random biomechanical energy for sustainable operation of mobile electronics. Nat Commun 6:8975 11. Qi Y, McAlpine MC (2010) Nanotechnology-enabled flexible and biocompatible energy harvesting. Energy Environ Sci 3(9):1275–1285 12. Xu S, Hansen BJ, Wang ZL (2010) Piezoelectric-nanowire-enabled power source for driving wireless microelectronics. Nat Commun 1:93 13. Wu F et al (2016) Energy scavenging based on a single-crystal PMN-PT nanobelt. Sci Rep 6:22513 14. Zhu G et al (2014) Radial-arrayed rotary electrification for high performance triboelectric generator. Nat Commun 5:3426 15. Chun J et al (2016) Boosted output performance of triboelectric nanogenerator via electric double layer effect. Nat Commun 7:12985 16. Zi Y et al (2016) Effective energy storage from a triboelectric nanogenerator. Nat Commun 7:10987 17. Skunik-Nuckowska M et al (2013) Integration of solid-state dye-sensitized solar cell with metal oxide charge storage material into photoelectrochemical capacitor. J Power Sources 234:91–99 18. Chen H-W et al (2010) Plastic dye-sensitized photo-supercapacitor using electrophoretic deposition and compression methods. J Power Sources 195(18):6225–6231 19. Xu J, Chen Y, Dai L (2015) Efficiently photo-charging lithium-ion battery by perovskite solar cell. Nat Commun 6:8103 20. Liao S et al (2016) Integrating a dual-silicon photoelectrochemical cell into a redox flow battery for unassisted photocharging. Nat Commun 7:11474 21. Zazubovich V, Jankowiak R. (2015) Biophotonics of photosynthesis. Photonics, Volume 4: Biomedical Photonics, Spectroscopy, and Microscopy, pp. 129 22. Coles D et al (2017) A nanophotonic structure containing living photosynthetic bacteria. Small 13(38):1701777 23. Dahlberg PD et al (2017) Mapping the ultrafast flow of harvested solar energy in living photosynthetic cells. Nat Commun 8:988 24. Ravi SK et al (2018) A mechanoresponsive phase-changing electrolyte enables fabrication of high-output solid-state photobioelectrochemical devices from pigment-protein multilayers. Adv Mater 30(5):1704073 25. Niwa S et al (2014) Structure of the LH1-RC complex from thermochromatium tepidum at 3.0 Å. Nat 508(7495):228–232 26. Qian P et al (2013) Three-dimensional structure of the rhodobacter sphaeroides RC-LH1-PufX complex: dimerization and quinone channels promoted by PufX. Biochem 52(43):7575–7585

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27. Jones MR (2009) The petite purple photosynthetic powerpack. Biochem Soc Trans 37:400–407 28. Zinth W, Wachtveitl J (2005) The first picoseconds in bacterial photosynthesis—Ultrafast electron transfer for the efficient conversion of light energy. ChemPhysChem 6(5):871–880 29. Siebert CA et al (2004) Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX. EMBO J 23(4):690–700 30. Walz T et al (1998) Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 Å, LH1 and RC-LH1 at 25 Å. J Mol Biol 282(4):833–845 31. Dezi M et al (2007) Stabilization of charge separation and cardiolipin confinement in antenna– reaction center complexes purified from Rhodobacter sphaeroides. Biochim Biophys Acta (BBA)-Bioenerg 1767(8):1041–1056 32. Comayras F, Jungas C, Lavergne J (2005) Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides, I. Quinone domains and excitation transfer in chromatophores and reaction center antenna complexes. J Biol Chem 280(12):11203–11213 33. Francia F et al (2004) Light-harvesting complex 1 stabilizes P+ Q-B charge separation in reaction centers of Rhodobacter sphaeroides. Biochem 43(44):14199–14210 34. Kropacheva TN, Hoff AJ (2001) Electrochemical oxidation of bacteriochlorophyll a in reaction centers and antenna complexes of photosynthetic bacteria. J Phys Chem B 105(23):5536–5545 35. Sumino A et al (2013) Electron conduction and photocurrent generation of a lightharvesting/reaction center core complex in lipid membrane environments. J Phys Chem Lett 4(7):1087–1092 36. Kondo M et al (2012) Photocurrent and electronic activities of oriented-his-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode. Biomacromol 13(2):432–438 37. Stamouli A et al (2004) The electron conduction of photosynthetic protein complexes embedded in a membrane. FEBS Lett 560(1–3):109–114 38. Monshouwer R et al (1997) Superradiance and exciton delocalization in bacterial photosynthetic light-harvesting systems. J Phys Chem B 101(37):7241–7248 39. Volotsenko I et al (2015) Evidence for deep acceptor centers in plant photosystem I crystals. J Phys Chem B 119(4):1374–1379 40. Toporik H et al (2012) Large photovoltages generated by plant photosystem I crystals. Adv Mater 24(22):2988–2991

Chapter 7

Photoproteins Tapping Solar Energy to Power Sensors

7.1 Brief Overview Energy self-sufficiency is an inspirational design feature of biological systems that fulfill sensory functions. Plants such as ‘touch-me-not’ (Mimosa pudica) and ‘Venus flytrap’ (Dionaea muscipula) not only sustain life by photosynthesis but also execute specialized sensory responses to touch. Photosynthesis enables these organisms to sustainably harvest and expend energy, powering their sensory abilities. Photosynthesis therefore provides a promising model for self-powered sensory devices such as electronic skins (e-skins). While the natural sensory abilities of human skin have been emulated in man-made materials for advanced prosthetics and soft-robotics, most reported e-skins do not incorporate phototransduction and photosensory functions that could extend the sensory abilities of human skin. This chapter [1] throws light on a proof-of-concept bioelectronic device integrated with natural photosynthetic pigment-proteins that not only shows an ability to sense touch stimuli but also to sense low-intensity UV radiation and generate an electrical power. The scalability of this photoprotein-based sensing device is demonstrated through the fabrication of flexible, multi-pixel bioelectronic sensors capable of touch registration and tracking. The polysensory abilities, energy self-sufficiency and additional nanopower generation exhibited by this bioelectronic material make it particularly promising for applications such as smart e-skins and wearable sensors, where the photo-generated power can enable remote data transmission.

7.2 Introduction Central to our ability to detect and evaluate the myriad objects we come into direct contact with daily is the sense of touch enabled by human skin [2–6]. Tactile sensing is a common attribute among animals, but responses to mechanical stimuli can be © Springer Nature Singapore Pte Ltd. 2020 S. K. Ravi and S. C. Tan, Solar Energy Harvesting with Photosynthetic Pigment-Protein Complexes, Green Energy and Technology, https://doi.org/10.1007/978-981-15-6333-1_7

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varied. In humans, for example, the response to a touch can be physiological or psychological in addition to physical. It is known that tactile sensing by skin is facilitated by receptors and sensory neurons [2, 5, 6], and the mechanisms through which stimuli are detected by the receptors and the information distributed by neurons are active areas of research [7]. These receptors enable sensitivity to varied pressure, vibration, strain, temperature and humidity stimuli that includes recognition of irritation and pain [2, 8]. The skins of some animals exhibit attributes not displayed by humans, including the well-known capabilities for camouflage and communication through environmentally-triggered colour changes most commonly associated with chameleons [9] and cephalopods [10]. An appealing attribute of the sensory abilities of biological systems is that they are powered by energy autonomously harvested from ambient sources without the need of any persistent external power supply such as is necessary for man-made electronic sensors. Sensory functions in certain plants such as “touch-me-not” (Mimosa pudica) are particularly inspirational as models for electronic sensors as they exhibit energy self-sufficiency, their sensory abilities being sustained by just by a fraction of the energy harvested by photosynthesis. While attempts have been made to mimic the complex biological sensory systems of the human skin in synthetic electronic skins, the rationale of the work reported here is to look at how photosynthesis can be used to enhance the sensory abilities of an electronic skin and make it self-contained in terms of the energy required for its sensory functions.

7.3 Self-powered Tactile Sensors Synthetic electronic skins are being developed for a variety of potential applications including biomimetic prosthetics, soft robotics, health monitoring, energy harvesting and enhanced sensing [2, 6, 11–17]. A fundamental goal is emulation of the tactile sensing function of natural skins in electronic devices [18–26]. Tactile sensing has been implemented through a variety of working principles in touch screen technologies and artificially intelligent systems including electronic skins [18–23, 27], with resistive and capacitive sensors being the most commonly employed components in commercial touch screens. Resistive sensors comprise two parallel conductors separated by spacer pads, touch being sensed by a change in resistance at a local point where the two conductors are brought into contact [27–29]. In a resistive-touch mobile phones, as a stylus is used to apply a pressure at a point, the two electrodes come in contact essentially completing the circuit at that point which aids in locating which part of the touch panel is triggered. Capacitive touch panels typically consist of a glass sheet coated with a transparent conductive film which, when touched by a conductor (e.g. a human finger), experiences an electrostatic field distortion that is detected as a change in capacitance [27]. Alternative sensing mechanisms such as piezoresistivity [30], piezoelectricity [31, 32], piezotronics [33–35], triboelectricity [14, 36, 37], tribotronics [27] and field-effect transistors [20] have also been employed in materials fabricated to mimic biological skin. Many of these approaches

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require the sensing material to be connected to a continuous or rechargeable power source to provide a practical sensing function, and so there is also interest in development of solar cells, energy harvesters and batteries that can be incorporated into electronic skins [11, 12, 38, 39]. The engineering of self-powered electronic systems is also of great interest [40], and approaches to the development of self-powered electronic systems for sensor applications have been described in comprehensive reviews [41–43]. Mimicry of the multiple and diverse sensory abilities of animal skins presents considerable challenges. Many recent reports have either focused on improving the sensitivity of an electronic skin to a mechanical or thermal stimulus, or have been aimed at developing a more versatile electronic skin by increasing the number of sensory abilities it displays. As an example of the former an ultra-sensitive piezoresistive electronic skin has been reported that is able to detect pressures lower than 1 Pa [19]. A noteworthy example of the latter is a description of a stretchable prosthetic skin based on silicon nanoribbon electronics that can sense pressure, temperature, strain and humidity [44]. Electronic skins that mimic the optical properties of chameleon and cephalopod skins have also been attempted [9, 10, 12, 45]. In addition to its pressure and temperature sensing attributes, human skin displays a complex set of responses to natural sunlight and artificial light sources [29]. It is known that the exposure of skin to UV radiation can cause DNA damage and skin cancer [46–48], and UV exposure also contributes to compositional and morphological changes associated with skin ageing [49]. The development of electronic skins with light sensing and/or photoprotective functions is of particular relevance to skin prostheses and wearable materials for health monitoring, most obviously where exposure to UV or strong visible light needs to be avoided. In addition to the need to widen the sensory abilities of electronic skins for prosthetics and wearable electronic systems, to realize practical systems for daily use, the development of energy self-sufficiency in electronic skins has become highly desirable. Autonomous functioning of electronic skins without any continuous power supply has hence become an important challenge. Taking inspiration from the energy management and self-sufficiency achieved in sensory plants, where solar energy harvesting not only powers tactile sensing and light sensing functions but also a plethora of other biological functions, the biohybrid sensor demonstrates the rationale of engineering future electronic skins that are not only energetically self-contained but also generate surplus power that could be expended on allied electronic functions. The work explores the development of a self-powering bioelectronic system that is sensitive to touch and has additional light-responsive functions that can warn of excessive levels of harmful solar UV radiation. The flexible sensor described here is based on a photosynthetic protein that has multiple absorbance bands that span the biologically-relevant wavelengths of the solar spectrum. The properties of this natural, environmentally benign and biodegradable photovoltaic pigment-protein enabled three functionalities, power generation, touch sensing and UV detection, in a flexible bioelectronic device for prospective e-skin applications.

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7.4 Bio-hybrid Tactile Sensors Powered by Photoproteins The bioelectronic device was assembled from a flexible top electrode comprising an indium tin oxide coated polyethylene terephthalate film (ITO-PET) which is a commonly used electrode in flexible electronics [18, 24], a blend of photosynthetic protein and electron transfer mediator, and a flexible base electrode comprising a gold-coated PET film (Fig. 7.1a). The photosynthetic protein was the Rhodobacter sphaeroides RC-LH1 reaction centre/light harvesting complex, which has multiple absorbance bands between 200 and 950 nm (Fig. 7.1b). The mediator was 100 mM ubiquinone-0 (Q0) dispersed in a gel matrix formed from succinonitrile (SCN) and water. Solutions of concentrated protein (100 μM) and Q0-SCN were mixed in a 5:1 ratio (v/v) to form a two-phase system (Fig. 7.1a). The resulting composite material was transparent across the visible spectrum with a maximum transmittance of ≈50% between 520 and 550 nm (Fig. 7.1c). The decline in transmittance below 500 nm in Fig. 7.1c is attributable to the carotenoids of the RC-LH1 complex that absorb between 400 and 500 nm (Fig. 7.1b). The dip in transmittance at 590 nm in Fig. 7.1c is due to a minor absorbance band of the bacteriochlorophylls, the main absorbance of which

Fig. 7.1 Device architecture, optical properties and mechanism. a Device architecture (ITOPET/RC-LH1/Q0-SCN/Au-PET). b Absorption spectrum of the RC-LH1 pigment-protein in solution. c Transmittance of the device across the visible spectrum, inset: image of a section of device (under red dotted-line) covering a mobile phone screen to illustrate transparency; Scale bar: 2 cm. d Energy diagram showing how photoexcitation of the RC-LH1 complex (rainbow arrow) elicits an intra-protein charge separation (red arrows) and direct or Q0-mediated charge transport to the electrodes (cyan arrows)

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lies outside the visible with major bands peaking at 376 and 874 nm (Fig. 7.1b). The absorbance of the protein around 275 nm (Fig. 7.1b) is attributable to aromatic amino acids, principally tryptophan. The two-phase RC-LH1/Q0-SCN formulation enabled the material to not only function as a bio-photoelectrochemical cell [50, 51] but also as an electrochemical capacitor. Cells assembled in ambient light displayed a built-in potential difference of −530 to −600 mV between the two electrodes. This is attributed to the electrochemical interactions between the electrodes and the cell contents, with the possibility of contributions from photoactivity of the RC-LH1 protein. Photoexcitation is expected to initiate trans-protein charge separation within the RC-LH1 complex, forming a cation on a pair of bacteriochlorophyll molecules at one terminal of the protein (P+ ) and an anion on a quinone at the opposite terminal (Q–B ) (Fig. 7.1d). Consistent with their vacuum potentials, the ITO electrode was capable of direct electron transfer to adjacent P+ sites, whilst the Au base electrode was able to accept electrons either directly from Q–B or, after electron transfer across the heterojunction, from the Q0 entrapped in the SCN plastic crystalline phase (Fig. 7.1d). In the protein/Q0-SCN blend the high capacitance of the protein-only phase was offset by the moderate conductance of the Q0-SCN phase. The use of SCN, a plastic crystalline material with a high dielectric constant (κ = 55 at room temperature), aided in the build-up of the potential difference of the cell. SCN has been used previously in electrochemical double layer capacitors [52] but to the best of our knowledge has not been used for an electronic skin. Touch sensing was based on modulation of this base voltage difference (V OC ). The application of pressure brought the electrodes into contact at the point of touch, resulting in a localized low-resistance path for electron flow in the device that reduced the V OC to zero (Fig. 7.2a–c). The tactile sensing capability of the material was tested by applying either instantaneous or continuous loads to an area of 0.6 cm2 . For both types of load no response was observed below 0.2 N of applied force, but an average voltage response of 0.65 mV was seen between 0.2 and 0.4 N (Fig. 7.2d). This voltage shift was constant during an applied load. A 0.5 N load produced a larger average voltage shift of 24 mV that relaxed to the pre-load level over ~15 s (Fig. 7.2e), and progressively higher instantaneous loads in the range 0.6–1.0 N produced progressively greater initial voltage changes that relaxed over ~100 s. As the instantaneous load was increased to 1 N, the measured voltage shifted to 0 V (Fig. 7.2e). To confirm the formation of a low-resistance path under an applied load, I-V curves were recorded by cyclic voltammetry under continuous-load and no-load conditions. In contrast to the no-load condition (Fig. 7.2f, blue), under a 1 N load a high current was obtained at all non-zero applied voltages and the I-V curve became a straight line indicating ohmic behavior (Fig. 7.2f and inset). A plot of the initial voltage shift as a function of instantaneously applied pressure revealed three response regimes (Fig. 7.2g). The material could sense pressures as low as 3000 Pa, which lies within the low-pressure regime typical of a sensation such as a gentle finger touch (1000–10,000 Pa), with a graded response between ~7000 and 10,000 Pa. There was also a high voltage response of up to 600 mV in the medium-pressure regime above 10,000 Pa.

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Fig. 7.2 Touch sensing: a Touch sensing is based on deformation of the top electrode and the protein/Q0-SCN blend. b The blend supports a VOC between the PET-ITO and PET-Au electrodes. c The VOC drops to zero in response to a touch that brings the electrodes into contact. d Voltage changes in response to the application and removal of a 0.2–0.4 N continuous load. e Voltage changes in response to instantaneous loads of 0.5–1 N. f IV characteristics showing a drop in resistance on application of a 1 N load. g Force dependence of the voltage response. Beyond 1 N load, there was no further increase in the voltage response. The area to which the load was applied was 0.6 cm2 , from which the corresponding pressures were calculated

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The voltage response to applied pressure was relatively rapid, highly reproducible and was only seen when the photosynthetic protein was included in the material (Fig. 7.3a). In addition to supporting a much higher base voltage the protein/Q0SCN blend was largely liquid and deformable in contrast to a pure Q0-SCN phase which was gel-like and non-deformable. Under a ~1.0 s load of 1 N the shift to 0 V was complete within ~0.5 s and, if reapplied in a cyclical fashion, this zero V OC was restored in a few tenths of a second (Fig. 7.3a). Applying instantaneous force at a fixed frequency allowed the V OC to be oscillated between selected values in a highly reproducible fashion over different time scales; e.g. between 0 and −0.15 V utilizing partial relaxation of the touch response (Fig. 7.3b) or between 0 and − 0.57 V utilizing near full relaxation over a longer period (Fig. 7.3c). The graded responses could be used for different purposes if integrated into an electronic device;

Fig. 7.3 Voltage responses to repetitive touches. a Utilization of a 0.1 V touch response to achieve a reaction time of