Aggregation-Induced Emission: Applications [2 ed.] 9781118701584, 9781118701614

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RED BOX RULES ARE FOR PROOF STAGE ONLY. DELETE BEFORE FINAL PRINTING.

AGGREGATION-INDUCED EMISSION EDITORS ANJUN QIN

Department of Polymer Science and Engineering, Zhejiang University, China

BEN ZHONG TANG

Department of Chemistry, The Hong Kong University of Science and Technology, China Aggregation-Induced Emission (AIE) is a novel photophysical phenomenon which offers a new platform for researchers to look into the light-emitting processes from luminogen aggregates, from which useful information on structure–property relationships may be collected and mechanistic insights may be gained. The discovery of the AIE effect opens a new avenue for the development of new luminogen materials in the aggregate or solid state. By enabling light emission in the practically useful solid state, AIE has the potential to significantly expand the technological applications of luminescent materials. Aggregation-Induced Emission: Applications is the first book to explore the high-tech applications of AIE luminogens, including technological utilizations of AIE materials in the areas of electroluminescence, mechanochromism, chiral recognition, ionic sensing, biomolecule detection, and cell imaging. Potential applications in room temperature phosphorescence, liquid crystals, circularly polarized luminescence, and organic lasing are also introduced in this volume.

This book is essential reading for scientists and engineers who are designing optoelectronic materials and biomedical sensors, and will also be of interest to academic researchers in materials science, physical and synthetic organic chemistry as well as physicists and biological chemists.

APPLICATIONS

Topics covered include: • AIE materials for electroluminescence applications • Liquid crystals with AIE characteristics • Mechanochromic AIE materials • Chiral recognition and enantiomeric differentiation based on AIE • AIE and applications of aryl-substituted pyrrole derivatives • New chemo-/biosensors with AIE-active molecules • AIE luminogens for in vivo functional bioimaging • Applications of AIE materials in biotechnology

AGGREGATION-INDUCED EMISSION

APPLICATIONS

EDITORS QIN TANG

EDITORS ANJUN QIN AND BEN ZHONG TANG

AGGREGATION-INDUCED EMISSION APPLICATIONS

Aggregation-Induced Emission: Applications

Aggregation-Induced Emission: Applications

Edited by

ANJUN QIN Department of Polymer Science and Engineering, Zhejiang University, China

AND BEN ZHONG TANG Department of Chemistry, The Hong Kong University of Science and Technology, China

This edition first published 2013 # 2013 John Wiley & Sons, Ltd Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom.

Library of Congress Cataloguing-in-Publication Data applied for. Print ISBN: 9781118701768 A catalogue record for this book is available from the British Library. Set in 10/12pt Times by Thomson Digital, Noida, India.

Contents

List of Contributors Preface 1

2

3

AIE or AIEE Materials for Electroluminescence Applications Chiao-Wen Lin and Chin-Ti Chen 1.1 Introduction 1.2 EL Background, EL Efficiency, Color Chromaticity, and Fabrication Issues of OLEDs 1.3 AIE or AIEE Silole Derivatives for OLEDs 1.4 AIE or AIEE Maleimide and Pyrrole Derivatives for OLEDs 1.5 AIE or AIEE Cyano-Substituted Stilbenoid and Distyrylbenzene Derivatives for OLEDs 1.6 AIE or AIEE Triarylamine Derivatives for OLEDs 1.7 AIE or AIEE Triphenylethene and Tetraphenylethene Derivatives for OLEDs 1.8 White OLEDs Containing AIE or AIEE Materials 1.9 Perspectives References Crystallization-Induced Phosphorescence for Purely Organic Phosphors at Room Temperature and Liquid Crystals with Aggregation-Induced Emission Characteristics Wang Zhang Yuan, Yongming Zhang and Ben Zhong Tang 2.1 Crystallization-Induced Phosphorescence for Purely Organic Phosphors at Room Temperature 2.1.1 Introduction 2.1.2 Molecular luminogens with crystallization-induced phosphorescence at room temperature 2.2 Liquid Crystals with Aggregation-Induced Emission Characteristics 2.2.1 Luminescent liquid crystals 2.2.2 Aggregation-induced emission strategy towards high-efficiency luminescent liquid crystals 2.3 Conclusions and Perspectives References Mechanochromic Aggregation-Induced Emission Materials Zhenguo Chi and Jiarui Xu 3.1 Introduction 3.2 Mechanochromic Non-AIE Compounds 3.3 Mechanochromic AIE Compounds

xi xiii 1 1 2 7 10 14 17 17 31 36 37

43

43 43 44 52 52 53 57 58 61 61 62 64

vi Contents 3.4

4

5

6

Conclusion References

Chiral Recognition and Enantiomeric Excess Determination Based on Aggregation-Induced Emission Yan-Song Zheng 4.1 Introduction to Chiral Recognition 4.2 Chiral Recognition and Enantiomeric Excess Determination of Chiral Amines 4.3 Chiral Recognition and Enantiomeric Excess Determination of Chiral Acids 4.3.1 Enantiomeric excess determination of chiral acids using chiral AIE amines 4.3.2 Enantiomeric excess determination of chiral acids using a chiral receptor in the presence of an AIE compound 4.4 Mechanism of Chiral Recognition Based on AIE 4.4.1 Mechanism of chiral recognition by a chiral AIE monoamine 4.4.2 Mechanism of chiral recognition by a chiral AIE diamine 4.5 Prospects for Chiral Recognition Based on AIE References AIE Materials Towards Efficient Circularly Polarized Luminescence, Organic Lasing, and Superamplified Detection of Explosives Jianzhao Liu, Jacky W.Y. Lam and Ben Zhong Tang 5.1 Introduction 5.2 AIE Materials with Efficient Circularly Polarized Luminescence and Large Dissymmetry Factor 5.2.1 Aggregation-induced circular dichroism 5.2.2 AIE, fluorescence decay dynamics and theoretical understanding 5.2.3 Aggregation-induced circularly polarized luminescence 5.2.4 Supramolecular assembly and structural modeling 5.3 AIE Materials for Organic Lasing 5.3.1 Fabrication of nano-structures 5.3.2 Lasing performances 5.4 AIE Materials for Superamplified Detection of Explosives 5.4.1 Hyperbranched polymer-based sensor 5.4.2 Mesoporous material-based sensor 5.5 Conclusion References Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives Bin Tong and Yuping Dong 6.1 Introduction 6.2 Luminescence Properties of Triphenylpyrrole Derivatives in the Aggregated State 6.3 Applications 6.4 Aggregation-Induced Emission of Pentaphenylpyrrole 6.5 AIEE Mechanism of Pentaphenylpyrrole 6.6 Conclusion References

82 83

87 87 88 91 91 98 101 102 102 104 105

107 107 107 108 110 113 115 118 118 119 121 122 127 127 128

131 131 132 136 147 150 152 152

Contents vii 7

8

9

10

Biogenic Amine Sensing with Aggregation-Induced Emission-Active Tetraphenylethenes Takanobu Sanji and Masato Tanaka 7.1 Introduction 7.1.1 Biogenic amines 7.1.2 Sensing methods for biogenic amines 7.2 Fluorimetric Sensing of Biogenic Amines with AIE-Active TPEs 7.2.1 Design for fluorimetric sensing of biogenic amines 7.2.2 Sensing studies and statistical analysis 7.2.3 Determination of histamine concentration 7.2.4 Fluorimetric sensing of melamine with AIE-active TPEs 7.3 Summary and Outlook References New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules Based on the Aggregation and Deaggregation Mechanism Ming Wang, Guanxin Zhang and Deqing Zhang 8.1 Introduction 8.2 Cation and Anion Sensors 8.3 Fluorimetric Biosensors for Biomacromolecules 8.4 Fluorimetric Assays for Enzymes 8.5 Fluorimetric Detection of Physiologically Important Small Molecules 8.6 Miscellaneous Sensors 8.7 Conclusion and Outlook References Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing Qi Chen and Bao-Hang Han 9.1 Introduction 9.2 Carbohydrate-Bearing AIE-Active Molecules 9.2.1 Carbohydrate-bearing siloles 9.2.2 Carbohydrate-bearing phosphole oxides 9.2.3 Carbohydrate-bearing tetraphenylethenes 9.3 Luminescent Probes for Lectins 9.4 Luminescent Probes for Enzymes 9.5 Luminescent Probes for Viruses and Toxins 9.6 Conclusion Acknowledgments References Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging Jun Qian, Dan Wang and Sailing He 10.1 Introduction 10.2 AIE Dyes for Macro In Vivo Functional Bioimaging 10.2.1 AIE dye-encapsulated phospholipid–PEG nanomicelles 10.2.2 AIE dye-encapsulated nanomicelles for SLN mapping of mice 10.2.3 AIE dye-encapsulated nanomicelles for tumor targeting of mice

157 157 157 157 158 158 158 162 163 163 164

165 165 166 169 173 180 183 185 185

189 189 190 191 192 193 195 199 203 205 205 205 209 209 210 210 210 216

viii Contents

10.3

10.4

11

12

10.2.4 Other types of AIE-nanoparticles for in vivo functional bioimaging Multiphoton-Induced Fluorescence from AIE Dyes and Applications in In Vivo Functional Microscopic Imaging 10.3.1 Two- and three-photon-induced fluorescence of AIE dyes 10.3.2 AIE dye-encapsulated nanomicelles for two-photon blood vessel imaging of live mice 10.3.3 AIE dye-encapsulated nanomicelles for two-photon brain imaging of live mice Summary and Perspectives Acknowledgments References

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics for Protein Sensing Jing Liang, Haibin Shi, Ben Zhong Tang and Bin Liu 11.1 Introduction 11.2 In Vitro Detection of Integrin avb3 Using a TPS-Based Probe 11.2.1 Detection mechanisms 11.2.2 Synthesis and characterization of the TPS-2cRGD probe 11.2.3 Detection of integrin in solutions 11.2.4 In vitro sensing of integrin in cancer cells 11.3 Real-Time Monitoring of Cell Apoptosis and Drug Screening with a TPE-Based Probe 11.3.1 Design principles 11.3.2 Synthesis and characterization of Ac-DEVEK-TPE probe 11.3.3 Detection of caspase and kinetic study of caspase activities in solutions 11.3.4 Imaging of cell apoptosis and screening of apoptosis-inducing agents 11.4 In Vivo Monitoring of Cell Apoptosis and Drug Screening with PyTPE-Based Probe 11.4.1 Working principles 11.4.2 Synthesis and characterization of DEVD-PyTPE probe 11.4.3 Monitoring of caspase activities in solutions 11.4.4 In vitro and in vivo imaging of cell apoptosis 11.5 Conclusion Acknowledgments References Applications of Aggregation-Induced Emission Materials in Biotechnology Yuning Hong, Jacky W.Y. Lam and Ben Zhong Tang 12.1 Introduction 12.2 AIE Materials for Nucleic Acid Studies 12.2.1 Quantitation and gel visualization of DNA and RNA 12.2.2 Specific probing of G-quadruplex DNA formation 12.3 AIE Materials for Protein Studies 12.3.1 Quantitation and PAGE staining of proteins 12.3.2 Fluorescence immunoassay by AIE materials 12.3.3 Monitoring of the unfolding/refolding process of human serum albumin 12.3.4 Monitoring and inhibition of amyloid fibrillation of insulin

221 223 223 227 230 232 234 234

239 239 240 241 241 243 244 245 245 246 247 248 251 251 252 253 253 255 255 256 259 259 260 260 262 263 263 266 266 267

Contents ix 12.4

12.5

Index

AIE Materials for Live Cell Imaging 12.4.1 AIE bioprobes for long-term cell tracking 12.4.2 AIE nanoparticles for cell staining Conclusion References

269 269 269 271 272 275

List of Contributors

Chin-Ti Chen Institute of Chemistry, Academia Sinica, Taiwan Qi Chen National Center for Nanoscience and Technology, China Zhenguo Chi PCFM Laboratory and DSAPM Laboratory, State Key Laboratory of Optoelectronic Materials and Technologies, School of Chemistry and Chemical Engineering, Sun Yat-sen University, China Yuping Dong College of Materials Science and Engineering, Beijing Institute of Technology, China Bao-Hang Han National Center for Nanoscience and Technology, China Sailing He Center for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, China Yuning Hong Department of Chemistry, The Hong Kong University of Science and Technology, China Jacky W.Y. Lam China

Department of Chemistry, The Hong Kong University of Science and Technology,

Jing Liang Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Chiao-Wen Lin Institute of Chemistry, Academia Sinica, Taiwan Bin Liu Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Jianzhao Liu Department of Chemistry, The Hong Kong University of Science and Technology, China Jun Qian Center for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, China Takanobu Sanji Chemical Resources Laboratory, Tokyo Institute of Technology, Japan Haibin Shi Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Masato Tanaka Chemical Resources Laboratory, Tokyo Institute of Technology, Japan

xii List of Contributors Ben Zhong Tang Department of Chemistry, Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology, Hong Kong, and Institute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, China Bin Tong College of Materials Science and Engineering, Beijing Institute of Technology, China Dan Wang Center for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, China Ming Wang Beijing National Laboratory, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, China Jiarui Xu PCFM Laboratory and DSAPM Laboratory, State Key Laboratory of Optoelectronic Materials and Technologies, School of Chemistry and Chemical Engineering, Sun Yat-sen University, China Wang Zhang Yuan China

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,

Deqing Zhang Beijing National Laboratory, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, China Guanxin Zhang Beijing National Laboratory, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, China Yongming Zhang School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, China Yan-Song Zheng School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, China

Preface

The discovery of new natural phenomena, the unveiling of new physical laws, the development of new methodologies, and the generation of new knowledge are at the core of scientific research. From this viewpoint, the study of light-emitting behaviors of luminogens in an aggregate state is a challenging yet important topic because it may lead to the creation of new photophysical knowledge. Since the 1950s, studies have shown that the fluorescence of a number of luminophores became weaker or even completely quenched in concentrated solutions or in the solid state. This common photophysical phenomenon is widely known as ‘concentration quenching’ or ‘aggregation-caused quenching’ (ACQ) of light emission. The ACQ process has been studied in great detail, and mature theories have been established. The ACQ effect, however, is harmful in practice, because luminophores are usually used as solid films or aggregates in real-world applications, which hinders them from realizing their full potential. Numerous processes have been employed and many approaches have been developed to prevent the luminophores from aggregating, but these efforts have met with only limited success. The difficulty lies in the fact that chromophore aggregation is an intrinsic natural process when luminophore molecules are located in close vicinity in the condensed phase. Exactly opposite to the ACQ effect, in 2001 we observed a unique luminogen system in which aggregation played a constructive, instead of destructive, role in the luminescence process: a molecule named 1-methyl-1,2,3,4,5-pentaphenylsilole was found to be almost nonemissive in dilute acetonitrile solution but became highly fluorescent when a large amount of water was admixed with acetonitrile. Because water is a poor solvent of the hydrophobic silole luminogen, addition of water to acetonitrile causes the silole molecules to aggregate in aqueous media. As the light emission is induced by aggregate formation, we coined the term aggregation-induced emission (AIE) for the phenomenon. In the past decade, a large variety of molecules with propeller shapes have been found to show the AIE effect, indicating that AIE is a general, rather than special, photophysical phenomenon. On the basis of our experimental results, we have rationalized that the restriction of intramolecular rotation (RIR) is the main cause of the AIE phenomenon. In the solution state, intramolecular rotation of the aromatic rotors of the AIE luminogens is active, which serves as a relaxation channel for the excited states to decay nonradiatively. In the aggregate state, however, the intramolecular rotation is restricted owing to the physical constraint involved, which blocks the nonradiative pathway and opens the radiative channel. The novel AIE phenomenon offers a new platform for researchers to look into the light-emitting processes from luminogen aggregates, from which useful information on structure–property relationships may be collected and mechanistic insights may be gained. Such information and insights will be instructive to the structural design for the development of new efficient AIE luminogens. Furthermore, the discovery of the AIE effect overturns the general belief of ‘concentration quenching’ or ACQ of luminescence processes,

xiv Preface opens a new avenue for the development of new luminogen materials in the aggregate or solid state and may spawn new models or theorems for photophysical processes in solution and aggregate states. As AIE is a photophysical effect concerning light emission in the practically useful solid state, AIE studies may also lead to hitherto impossible technological innovations. In AIE systems, one can take great advantage of aggregate formation, instead of fighting against it. The AIE effect permits the use of highly concentrated solutions of luminogens and their aggregates in aqueous media for sensing and imaging applications, which may lead to the development of fluorescence turn-on or light-up nanoprobes. A probe based on AIE luminogen nanoaggregates is in some sense the organic version of inorganic semiconductor quantum dots, but are superior to the latter in terms of wider molecular diversity, readier structural tunability, and better biological compatibility. Attracted by this intriguing phenomenon and its promising applications, a number of research groups throughout the world have enthusiastically engaged in AIE studies, and exciting progress has already been made. In response to an invitation from the Wiley editors, we embarked on the preparation of two volumes dedicated to the study of AIE – this volume, Aggregation-Induced Emission: Applications and the related volume, Aggregation-Induced Emission: Fundamentals. In this volume, we invited a group of active researchers in the area to contribute on the exploration of high-tech applications of AIE luminogens. The technological utilization of AIE materials in the areas of electroluminescence, mechanochromism, chiral recognition, ionic sensing, biomolecule detection, and cell imaging is covered. Their potential applications in room-temperature phosphorescence, liquid crystals, circularly polarized luminescence, organic lasing, and so on are also introduced in this volume. This book is expected to be a valuable reference to readers who are now working or planning to be involved in the areas of research on organic optoelectronic materials and biomedical sensors. Although we have tried our best to make this book comprehensive, some important work may have inadvertently been omitted, owing to the limitations on the size of the book and the rapid developments in this area of research. The book may contain some overlapping contents in different chapters and possibly even some errors. We hope the readers will provide us with constructive comments, so that we may modify and improve the book in its next edition. We would like to thank all the authors who have contributed to this book. Without their enthusiastic support, the foundation of this book could not have been be laid. We also thank the Wiley in-house editors, Sarah Hall, Sarah Tilley, and Rebecca Ralf, for their enthusiastic encouragement and technical support. We hope that this book will serve as a ‘catalyst’ to stimulate new efforts, to trigger new ideas, and to accelerate the pace in the research endeavors on the design of new AIE luminogen systems, the establishment of new theoretical models, and the exploration of innovative applications. Anjun Qin Department of Polymer Science and Engineering Zhejiang University, China Ben Zhong Tang Department of Chemistry, Division of Biomedical Engineering The Hong Kong University of Science and Technology China

1 AIE or AIEE Materials for Electroluminescence Applications Chiao-Wen Lin and Chin-Ti Chen Institute of Chemistry, Academia Sinica, Taiwan

1.1 Introduction The science and technology of organic light-emitting diodes (OLEDs) have been developing and progressing for more than 30 years since a small team led by Tang at Kodak invented the first thin-film-based highefficiency OLED [1]. Nowadays, OLEDs have reached a stage where they are ready to be one of the main types of display in the marketplace, as is evident from the market demand for smartphones and tablets along with Samsung’s Galaxy production line of AMOLED mobile devices. Several breakthroughs and discoveries, either intentionally or simply by serendipity, have brought OLEDs beyond being just a research niche in the laboratory. In this chapter, we illustrate one such serendipity, namely the aggregation-induced emission (AIE) or aggregation-induced enhanced emission (AIEE) found for a certain kind of fluorescent materials that leads to an electroluminescence (EL) efficiency of nondopant devices comparable to that of dopantbased OLEDs, the fabrication of which process is more complicated and less easy to control. AIE or AIEE is an inverse effect (see NPAFN and NPAMLI shown in Figure 1.1a) with respect to the more common aggregation-caused quenching (ACQ) or concentration-quenching effect (see Nile Red, DCM1, and TPP shown in Figure 1.1b) that takes place for most fluorophores in the solid state [2]. The difference between AIE and AIEE effects is the relative intensity of the fluorescence [or more generally photoluminescence (PL)] in solution, which is very much solvent dependent. If the chosen solvent enables no or nearly no PL of the material, any PL observed in the solid state is called AIE effect. If solution PL is observed but it is less intense than that in the solid state, the material is sad to show an AIEE effect. Since OLED devices are fabricated in a thin solid film structure, the common ACQ effect impairs the solid-state material PL or EL of OLEDs, the PL quantum yield or the brightness (electroluminance, L) of OLEDs, and hence the EL efficiency of OLEDs. Accordingly, materials that display PL having AIE or AIEE characteristics, instead of

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

2 Aggregation-Induced Emission: Applications

Figure 1.1 From left to right: fluorescence image of (a) NPAFN and NPAMLI and (b) Nile Red, DCM1, and TPP in solution (CH2Cl2) and in the solid state. Reproduced with permission from [2], # 2004 American Chemical Society.

ACQ, are very desirable and valuable for high-performance OLEDs fabricated by a simpler fabrication process. In a survey of the literature, we found many PL materials showing an AIE or AIEE effect but only a few of them have been reported with EL data, that is, their OLEDs were not fabricated and tested. For those have been applied in OLEDs, according to their chemical structure, we classify AIE or AIEE materials into five main categories and an extra category. The first two are five-membered heterocyclic compounds, namely silicon-containing silole derivatives (Section 1.3), imide-containing maleimide derivatives and nitrogen-containing pyrrole derivatives (Section 1.4), the third type is cyano-substituted stilbenoid and distyrylbenzene derivatives (Section 1.5), the fourth type is triarylamine-based derivatives (Section 1.6), and the fifth type is tri- or tetraphenylethene derivatives (Section 1.7). Finally, we group the white OLEDs containing AIE or AIEE materials into an extra category (Section 1.8). Fluorescent materials showing the AIE or AIEE effect are advantageous with respect to the solid-state PL quantum yield, which is one of four key factors that are decisive for achieving high EL efficiency, the external quantum efficiency (EQE or hEXT), of OLEDs. Therefore, after this introductory section, the chapter begin with the background to EL, EL efficiency, color chromaticity, and fabrication issues of OLEDs. The rest of the chapter considers the six categories of AIE or AIEE molecular materials outlined above.

1.2 EL Background, EL Efficiency, Color Chromaticity, and Fabrication Issues of OLEDs A typical architecture of an OLED device is illustrated in Figure 1.2. The diode device is composed of two electrodes, anode and cathode, sandwiching a hole-transporting layer (HTL), light-emitting layer (EML), and electron-transporting layer (ETL) at the center.

AIE or AIEE Materials for Electroluminescence Applications 3

Figure 1.2 Typical architecture of an OLED device. ITO, indium tin oxide.

The anode is usually transparent, enabling EL from the device, and it is usually an indium tin oxide (ITO)-coated glass substrate. For the cathode, low work function metals, such as Al and Ca, or a metal alloy, such as Mg–Ag, are common choices. To facilitate charge injection and reduction of the driving voltage, injection layers are sometimes inserted adjacent to the electrodes. For electron injection, inorganic ionic substance such as LiF, CsF, or Cs2CO3 and low work function metal such as Ba or Cs are commonly adopted as the electron injection layer (EIL) in OLED fabrication. Owing to the usually 6.5 eV as for BCP and BPhen, it is useful for the hole-blocking layer (HBL) between the light-emitting layer (EML) and ETL to confine or enhance the charge recombination on EML. Using the charge balance factor (g), the partition ratio of emissive states (rST), that is, exciton in singlet or triplet state (25 or 75%), PL quantum yield (hPL), and the light out-coupling efficiency (j), the basic equation for EQE or hEXT of the OLED can be written as [4] hEXT ¼ grST hPL j Figure 1.3 depicts schematically each factor or component in Equation 1.1.

Scheme 1.3 ETL materials mentioned in the chapter.

(1.1)

AIE or AIEE Materials for Electroluminescence Applications 5

Figure 1.3 Schematic depiction of each term (g, rST, hPL, j) in Equation 1.1. A photograph of a turn-on red lightemitting OLED is included for illustration purposes.

Whereas the charge balance (g) depends on the charge carrier mobility and energy level alignment of each material in an OLED device, the solid-state PL quantum yield (hPL) is directly related to the AIE or AIEE effect of the material. The theoretical maximum hEXT that one OLED can achieve depends on the light out-coupling (j) of the device and the nature of the emitting light (rST), either fluorescence or phosphorescence or both, of the materials used in OLEDs. For the first approximation, j is proportional to 1/(2n2), where n is the refractive index of the light-emitting layer and is commonly in the range 1.5–1.7 for most PL and EL materials. Accordingly, j  0.17–0.22 and hEXT 20% is the theoretical prediction of the maximum hEXT. In fact, by utilizing both the phosphorescence and fluorescence energy in OLEDs, hEXT values beyond theoretical limit and approaching 30% have been realized [5–7.] Even for OLEDs showing only fluorescence-based EL, hEXT  5%, the top limit predicted by theory, has also been exceeded. One of the highest hEXT values of 8% for a fluorescence-based OLED was reported with EML using silole compounds [8], the fluorescent materials showing AIE. Incidentally, two other commonly used units for EL efficiency are cd m2 for the current efficiency (hC) and lm W1 for power efficiency (hP). As mentioned earlier, particularly in the solid state, light-emitting materials showing the AIE or AIEE effect can directly contribute to the high hPL, which is beneficial for promoting the brightness (L, electroluminance) and hEXT of the OLED. Whereas it is irrelevant to rST or j in Equation 1.1, light-emitting materials showing the AIE or AIEE effect do not necessarily have a high g. Therefore, there are numerous OLEDs that show decent to exceptionally good hEXT values, but there are even more OLEDs that show poor hEXT, even though the device contains AIE or AIEE materials as the EML. Before moving on to the next section, the PL or EL color specification is worth noting here. The RGB color specification is one of the quality checks for full-color OLED displays. Figure 1.4 shows a typical standardized 1931 CIE (Commission Internationale de l’Eclairage) color chromaticity diagram [9].

6 Aggregation-Induced Emission: Applications

Figure 1.4 A typical 1931 CIE color chromaticity diagram [9].

Considering the wide color gamut range of a display, it is highly desirable that the materials used in an OLED display should exhibit a color purity of red, green, or blue that is as high as possible. This issue is a challenge to be overcome particularly for blue and red. Moreover, the problems associated with blue and red light-emitting material differ. It is relatively difficult to acquire pure or deep blue-emitting material because of the red-shifting emission, either PL or EL, caused by material aggregation in the solid state, which applies also to AIE or AIEE materials. For red light-emitting materials, red-shifted PL or EL is satisfactory in terms of red color purity. It is the emission intensity that is usually impaired due to the material aggregation in the solid state, which is most serious for red light-emitting materials [2]. However, the AIE or AIEE effect of red light-emitting materials can alleviate the problem of reduced emission intensity. Moreover, in

AIE or AIEE Materials for Electroluminescence Applications 7 order to reduce the adverse ACQ of light-emitting materials, OLED fabrication often utilizes a doping process. Unfortunately, this fabrication process is relatively problematic in terms of uniformity and reproducibility, thus hindering a high production yield in volume fabrication [2]. AIE or AIEE materials can take advantage of a ‘nondoping process’ in OLED fabrication and hence are more feasible for volume production of OLED devices. Finally, for white OLEDs (WOLEDs) in lighting applications, the EL efficiency under lighting conditions (L  1000 cd m2) should be examined, because most of the OLED devices exhibit efficiency ‘rolloff’ at high brightness, and most exhibit peak or maximum EL efficiency (hEXT, hC, or hP) at low current density or low brightness. Such low brightness may be acceptable for small-sized displays (such as those on smartphones or the display panel of laptop computers), but is insufficient for lighting applications. In addition, for lighting applications, the color rendering index (CRI) is an important specification of WOLEDs. The CRI is a quantitative measure of the ability of a light source to reproduce the colors of various objects faithfully in comparison with an ideal or natural light source, that is, sunlight. A CRI of 100 represents the maximum value, which is defined for sunlight. An incandescent lamp is a poor light source because of its low efficiency, but it has an excellent CRI of >95, almost as high as for sunlight. A light source with CRI >80 is usually required for general lighting applications. Normally, a two-color-white light source rarely has CRI >80 and a three-color white light system is necessary to achieve CRI >80 for practical lighting applications.

1.3 AIE or AIEE Silole Derivatives for OLEDs The first literature report on OLEDs based on a series of silole-based small-molecule compounds, DMTPS, MPClTPS, MPTPS, and HPS (Table 1.1) as EML, by Tang et al., appeared in 2001 [10]. The performance of the OLEDs was rather poor (maximum brightness Lmax 100 nm] and such an EL band has substantial intensity (more than one-third of the peak intensity at lEL max ¼ 464 nm) extending far beyond 550 nm. Even though EL peaked at a relatively short wavelength of 464 nm, the color of the HPS2,4 OLED is

10 Aggregation-Induced Emission: Applications unlikely to be authentic blue and more possibly green–blue or blue–green as for most silole-based AIE or AIEE materials. The silole HPS2,4 has built-in steric hindrance due to the isopropyl substituent at the ortho-position of two phenyl rings forcing a twist on the p-conjugation and shortening the EL wavelength. Based on fundamental intuition, the twisted conformation should be helpful in reducing the exciplex formation with the HTL (such as TPB and NPB), which usually results in a red-shifted emission wavelength. Therefore, the higher than normal EL intensity around 550 nm may be partly due to Alq3, the material used as the ETL in an HPS2,4 OLED.

1.4 AIE or AIEE Maleimide and Pyrrole Derivatives for OLEDs In 2002, Chen and co-workers reported a red OLED based on NPAMLI, a maleimide fluorophore, as the EML in a nondopant device [20], namely a red OLED fabricated without the application of a doping process. This is one of the first long-wavelength (>600 nm) AIE or AIEE materials to be reported for OLED application and yet the OLED exhibited reasonably good performance. The NPAMLI OLED exhibited Lmax  8000 cd m2 and hEXT ¼ 2.4% (Table 1.2), and such a performance is comparable to that of red OLEDs fabricated with a doping process. It is worth mentioning that the device was fabricated without an HTL because NPAMLI has the capability of transporting holes in an OLED. NPAMLI shares a common structural feature, namely 2-naphthylphenylamine, with NPB (Scheme 1.2), one of the most widely used materials for HTLs. In fact, when the paper was first published it was not realized that the maleimide NPAMLI is indeed one of the materials that show an AIE or AIEE effect. The image shown in Figure 1.1a demonstrating the AIEE effect (dichloromethane solution versus solid state) was taken two years later in 2004. To provide the missing evidence thus far, Figure 1.5 displays the AIE effect of NPAMLI in acetonitrile solution with increasing amount of water addition (from left to right).

Figure 1.5 Fluorescence images of NPAMLI in acetonitrile–water mixture with water fractions of 0, 20, 50, 60, 70, and 80% from left to right.

AIE or AIEE Materials for Electroluminescence Applications 13

Figure 1.6 Fluorescence images of AsNPAMLI in acetonitrile–water mixture with water fractions of 0, 20, 50, 60, 70, and 80% from left to right.

Recently, our laboratory synthesized an asymmetric NPAMLI, the red–orange AsNPAMLI (see Table 1.2 for its structure). Although its OLED application awaits exploration, we have clearly demonstrated its AIE effect in solution (Figure 1.6). More maleimide compounds bearing indole substituents (Table 1.2), either symmetrical (INMLI series) or asymmetric (AsINMLI series) [22, 24], were subsequently reported for OLED application. However, all of these indole-substituted maleimide OLEDs show shorter EL wavelengths in the orange to red–orange region and their performances are all inferior to that of the first reported NPAMLI OLED. One non-maleimide-based material listed in Table 1.2 is a five-membered heterocylic pyrrole derivative, NPANPy [23]. Pyrroles are nitrogen-containing five-membered cyclic dienes similar to siloles except that the silicon is the heteroatom of the five-membered ring structure. Structure-wise, tetraarylsubstituted pyrrole derivatives have a propeller-like molecular conformation very similar to that of tetraaryl-substituted silole derivatives. Recent studies have revealed that a propeller-like molecular structure is vital for the AIE or AIEE effect caused by the restriction of intramolecular bond rotation in the solid state. It is not surprising that such pyrrole derivatives exhibit stronger fluorescence intensities than those in dichloromethane solution [23], a typical AIEE effect found for structurally similar silole derivatives. Unlike the electron-deficient nature of the silole derivatives, pyrrole derivatives are electron rich and seldom produce red-shifted exciplex emission with the HTL. Therefore, it is relatively easy for pyrrole derivatives to achieve blue EL when fabricated as the EML in OLEDs. Provided that low-lying HOMO TPBI is used as the ETL, authentic blue EL with 1931 CIEx,y (0.16, 0.14–0.17) can be readily obtained (Table 1.2). However, the EL efficiency is not very good (none of the hEXT values of the blue emissions is more than 1.5%).

14 Aggregation-Induced Emission: Applications

1.5 AIE or AIEE Cyano-Substituted Stilbenoid and Distyrylbenzene Derivatives for OLEDs The observation of the enhanced fluorescence on cyano-substituted stilbenoid and distyrylbenzene derivatives (CN-DSB) in the solid state can be traced back as early as that of silole derivatives. In fact, one of the first reported CN-DSB OLEDs was observed in 2002 by Luo et al. [25]. However, the device was fabricated with CN-DSBx (Table 1.3) by a doping process and the reported OLED performance was far from satisfactory. Shortly after, in 2003, Chen and co-workers reported high performance (maximum hEXT  2.4%) nondopant red–orange OLEDs containing a dicyano-substituted, 2-naphthylphenylamine-appended stilbenoid, NPAFN (Table 1.3) [26]. Once again, similarly to the case of the NPAMLI OLED, NPAFN OLEDs performed better without including NPB as the HTL. As shown in Figure 1.1a, NPAFN exhibits a pronounced AIE or AIEE effect. The AIE or AIEE effect of NPAFN has recently been confirmed in solution, as shown in Figure 1.7. Several years later, in 2008, Chen and co-workers developed the second generation of NPAFN, PhSPFN and FPhSPFN (Table 1.3) [27]. With longer p-conjugation between donor and acceptor moieties, PhSPFN and FPhSPFN OLEDs both display EL at longer wavelength, corresponding to authentic red color chromaticity, 1931 CIEx,y (0.67, 0.30) and (0.66, 0.34), respectively. The maximum hEXT of the FPhSPFN OLED reaches 3.1%, one of the highest among AIE or AIEE nondopant red OLEDs, only exceeded by TTPEBTTD [maximum hEXT ¼ 3.7%, CIEx,y (0.67, 0.32)] [28]. In this case, AIE and AIEE take place for PhSPFN and FPhSPFN, respectively (Figure 1.8). The fluorine ortho-substituent of FPhSPFN enhances the restriction of intramolecular bond rotation and hence increases the fluorescence intensity not only in the solid state but also in solution.

Figure 1.7 Fluorescence images of NPAFN in acetonitrile–water mixture with water fractions of 0, 20, 50, 60, 70, and 80% from left to right.

AIE or AIEE Materials for Electroluminescence Applications 17

Figure 1.8 Fluorescence images of PhSPFN (a) and FPhSPFN (b) in solution (dichloromethane) and in the solid state.

Although EFPAFN in Table 1.3 has a longer EL wavelength and hence better chromaticity of red color, in terms of EL efficiency [29], FPhSPFN is by far one of the best red AIE or AIEE materials for OLED application. In Table 1.3, CN-DPASDB exhibits a fairly high hC of 4.0 cd A1 but it is a yellow OLED [31], mostly not a desirable color for display applications.

1.6 AIE or AIEE Triarylamine Derivatives for OLEDs Triarylamines represent the smallest family of AIE or AIEE materials, although they are often present in the molecular structure of long wavelength-emitting AIE or AIEE materials (see examples in Section 1.4 and Section 1.5). A triarylamine has an electron-rich character and is therefore suitable as a strong electron donor in structural design, readily raising the HOMO energy level and narrowing the emission bandgap energy of the molecular compound. Basically, a triarylamine molecule possesses a nonplanar molecular structure, similar to the propeller-like structural feature of most AIE or AIEE materials. Two types of triarylamine-based AIE or AIEE materials have been demonstrated for OLED application (Table 1.4). Constructed with an appropriate electron acceptor segment, two of them (SBCN and M1) exhibit near-IR emission [32, 33], although their OLED performance is rather poor, not very different from those of other near-IR OLEDs reported in the literature.

1.7 AIE or AIEE Triphenylethene and Tetraphenylethene Derivatives for OLEDs As summarized in this section, a wide variety of chemical structures have been reported for tri- and tetraphenylethene derivatives. This group of organic fluorophores is one of the latest discovered AIE or AIEE

18 Aggregation-Induced Emission: Applications Table 1.4 Summary of reported OLEDs containing triarylamine-based AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

EML CZCHO SBCHO CZCN SBCN EML CZCN SBCN M1

ITO/PEDOT:PSS/EML/TPBI/Ba/Al 505 (–, –) green–blue 7.2 542 (–, –) green 9.3 644 (–, –) red–orange 11.6 724 (–, –) near-IR 8.6 ITO/EML/TPBI/Ba/Al 646 (–, –) red–orange 10.9 724 (–, –) near-IR 8.6 ITO/MoO3/NPB/M1/BCP/Alq3/LiF/Al 1050 (–, –) near-IR 5 ITO/MoO3/NPB/M1/TPBI/LiF/Al 1050 (–, –) near-IR –

a

1

In units of W Sr

Von (V)

Lmax (cd m2) hEXT (%)

hC (cd A1)

hP (lm W1) Ref.

804 330 111 55

2.09 1.33 0.61 0.05

3.03 1.13 0.30 0.03

– – – –

140 69

0.93 0.05

0.47 0.02

–

60a

0.05

–

–

–

0

–

–

32

33

2

m .

materials, although this family is the largest in number. However, their OLED application was documented long before the invention of AIE or AIEE terminology. The first compound considered in this section is DPVBi (Table 1.5), which was first commercialized by Idemitsu Kosan Co. as one of the most widely used blue fluorophores in the mid-1990s [34]. Apart from being a derivative of triphenylethene, DPVBi can also be looked as upon as a diphenylsubstituted stilbenoid dimer. In fact, the tetraphenylethene moiety can also be considered as a crossconjugated stilbenoid. The triphenylethene moiety exhibits a similar propeller shape of to the tetraphenylethene moiety. As illustrated by other examples shown in Section 1.5, stilbenoid compounds are well known for the free rotor effect on the radiationless transitions that cause serious fluorescence quenching in solution state [35, 36]. The AIE or AIEE effect of DPVBi has never been shown until now (see Figure 1.9). Accordingly, in terms of the AIE or AIEE effect and structural similarity, triphenylethene derivatives can be considered as one member of the large tetraphenylethene family. According to our survey, the best nondopant DPVBi OLED was probably reported by Park and co-workers in 2007 (Table 1.5) [41]. The device was fabricated as a control OLED and it was an authentic blue device with an EL wavelength at 465 nm corresponding to 1931 CIEx,y (0.15, 0.16). The device has a maximum hC of 3.92 cd A1 and a maximum hP of 1.61 lm W1, which are good compared with other OLEDs in Table 1.6 having a similar blue color purity. In addition to the early work on DPVBi, triphenylethene and tetraphenylethene have been attached to anthracene (Table 1.5), one of the first organic fluorophores studied for EL by Pope et al. in the 1960s [45].

20 Aggregation-Induced Emission: Applications

Figure 1.9 Fluorescence images of DPVBi in THF–water mixture with water fraction 0, 20, 40, 60, and 80% from left to right. Its solid-state fluorescence image is shown in front of the solution samples.

Table 1.6 Summary of reported OLEDs containing DPFv-NH2, DPFV-OMe, TPE, TPEPh, BTPE, or TDPVBi AIE or AIEE materials. AIE or AIEE lmaxEL, CIEx,y, color code fluorophore (nm), (x, y), color EML DPFv-NH2 DPFv-OMe EML TPE TPEPh BTPE TDPVBi

Von (V)

ITO/NPB/EML/BCP/Alq3/LiF/Al 485 (–, –) green–blue – 520 (–, –) green – ITO/NPB/EML/BCP/Alq3/LiF/Al 445 (–, –) deep blue 2.9 476 (–, –) green–blue 5 ITO/NPB/BTPE/TPBI/Alq3/LiF/Al 488 (–, –) green–blue 4 ITO/NPB/TDPVBi/TPBI/LiF/Al 468 (0.16, 0.21) blue 3.8

Lmax (cd m2) hEXT (%)

hC (cd A1)

hP (lm W1) Ref.

5000 449

– –

1.90 –

0.7 –

1800 10680

0.4 2.56

0.45 5.15

0.35 –

11180

3.17

7.26

3.81

31170

3.8

6.2

3.94

47

48

49 42

AIE or AIEE Materials for Electroluminescence Applications 21 DPVPA is bistriphenylethene-attached anthracene [43] and TPVAn, BTPPA, and BTBPPA are bistetraphenylethene-attached anthracene derivatives [41, 44]. Among all the compounds listed in Table 1.6, the nondopant TPVAn OLED shows the highest fluorescence-based EL efficiency (hEXT ¼ 5.3%). In addition, its blue color purity is excellent, 1931 CIEx,y (0.14, 0.12), corresponding to an authentic blue color. This is not surprising because the solution PL quantum efficiency of 9,10-diphenylanthracene is very high and close to unity [46]. The bulky propeller-like tetraphenylethene substituent prevents the anthracene moiety from close contact and red shifting the emission wavelength. On the other hand, bistetraphenylethene substituents contribute to the strong AIE or AIEE effect that simply preserves the high PL quantum efficiency of the material in the solid state. DPFvNH2 and DPFvNH2 are two-ring-fused versions of tetraphenylethene, although the propeller molecular shape remains and the AIE or AIEE effect is observed [47]. DPFvNH2 and DPFvNH2 OLEDs have been fabricated and tested, and the results were not very good (Table 1.6). Both devices have a significantly red-shifted EL at 485 and 520 nm, respectively, and weak EL has been observed for DPFvNH2 OLEDs, Considering the red-shifted and weak EL, it can be suspected that DPFvNH2 forms an excimer in the thin-film device. Interestingly, PhBAPN is a three-ring-fused version of tetrephenylethene and the propeller-like structure of DPFvNH2 and DPFvNH2 no longer exists in this compound (Table 1.6), so nor is the AIE or AIEE effect. A PhBAPN OLED was reported with an even weaker excimer EL (Lmax ¼ 219 cd m2) at 556 and 580 nm [47]. Whereas TPE is the parent structure of tetraphenylethene, TPEPh is probably the simplest tetraphenylethene derivative (Table 1.6) [48], being a single phenyl-substituted TPE. In the literature, the solid-state PL wavelength of TPEPh was demonstrated to be morphology dependent. It is 454 and 503 nm for crystalline and amorphous TPEPh, respectively, although its EL wavelength is at 476 nm and it is green–blue. The EL efficiency of green–blue TPEPh is just moderate (hEXT ¼ 2.56%, hC ¼ 5.15 cd A1). The parent compound TPE has a very short EL wavelength at 445 nm (indicative of a deep blue color) and nearly overlaps with the PL spectrum of its crystal. However, the device’s Lmax is only 1800 cd m2 and none of the EL efficiency criteria is over 0.5 (%, cd A1, or lm W1), although a very low turn-on voltage (L of the device equal to 1 cd m1) of 2.9 V was observed for a TPE OLED. BTPE is a single covalent bond-connected dimer form of the parent TPE structure (Table 1.6) [49]. Like TPE or TPEPh mentioned above, BTPE is one of the tetraphenylethene derivatives having a relatively simple chemical structure. Its solid-state PL also behaves similarly to TPEPh, with the PL wavelength being morphology dependent, 445 and 499 nm for crystalline fibers and amorphous film, respectively. Similarly to a TPEPh OLED, the BTPE EL wavelength at 488 nm (green–blue) is between the PL wavelengths of crystalline and amorphous samples, although its OLED performance is better than that of TPEPh OLEDs. On the other hand, TDPVBi can be considered as a triphenylethene version of BTPE bearing two extra propeller-like moieties (Table 1.6) [42]. It also can be considered as an ‘upgraded’ version of DPVBi because of the two extra propeller-like moieties. Concerning the OLED performance, TDPVBi has a much shorter EL wavelength at 468 nm corresponding to a green–blue color, 1931 CIEx,y (0.16, 0.21), which is bluer than that of the TPEPh OLED. The EL efficiency of the TDPVBi OLEDs is also in general better than that of TPEPh OLEDs. TPESiPh3, (TPE)3SiPh2, and (TPE)2SiPh are hybrid structures of TPE and tetraphenylsilane (Table 1.7) [50], which is known for the amorphous feature of the OLED material that can extend the morphological stability of the thin-film structure in OLEDs [51, 52]. However, from the reported OLED data for these materials, as more TPE units are attached to the tetraphenylsilane, the performance of the OLEDs becomes worse, not only the EL efficiency and maximum brightness but also the EL wavelength (becoming longer with less blue color purity). Among the series, the least TPE-containing TPESiPh3 has the best-performing OLED. It is an authentic blue OLED with a short EL wavelength at 452 nm and Lmax ¼ 5672 cd cm2, but its EL efficiency is not poor (hEXT ¼ 1.6%, hC ¼ 2.1 cd A1, hP ¼ 1.1 lm W1).

22 Aggregation-Induced Emission: Applications Table 1.7 Summary of reported OLEDs containing silane-based TPESiPh3, (TPE)2SiPh2, (TPE)3SiPh AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

EML TPESiPh3 (TPE)2SiPh2 (TPE)3SiPh

ITO/NPB/EML/TPBI/LiF/Al 452 (–, –) blue 472 (–, –) green–blue 484 (–, –) green–blue

Von (V)

Lmax (cd m2) hEXT (%) hC (cd A1) hP (lm W1)

5 8 7

5672 3635 1081

Ref. 50

1.6 0.7 0.8

2.1 1.4 1.6

1.1 0.4 0.47

In contrast to an earlier report on triarylamine-based AIE or AIEE materials by He and co-workers [42], 0 0 0 Tang et al. clearly showed that triphenylamine (TPA) and N4,N4,N4 ,N4 -tetraphenylbiphenyl-4,4 -diamine, a TPA dimer (DTPA), are ACQ materials instead of AIE or AIEE materials (Table 1.8) [53]. After attaching three TPE units to TPA or four TPE units to DTPA, 3TPETPA and 4TPEDTPA become typical AIE or AIEE materials. However, regarding their OLEDs, 3TPETPA peculiarly exhibits multiple EL bands at 493 and 511 nm (in a device containing NPB as HTL) or 499 and 513 nm (in a device without NPB HTL), which are both red shifted from PL wavelength in solution (THF–water mixture) of 484 nm. The OLED of 4TPEDTPA behaves normally with a single EL band at 488 nm, which is green–blue not much different from the PL wavelength in solution (THF–water mixture) of 486 nm. Without the red-shifted EL and anomalous emission band, 4TPEDTPA OLEDs outperform 3TPETPA OLEDs. Pyrene is another fluorophore known for notorious aggregation even in the solution state. Pyrene is a typical ACQ material because of its rigid and flat molecular structure. There have been two approaches to chemical modification of pyrene with a TPE moiety (Table 1.9). The first involves surrounding the pyrene structure with four propeller-like TPE moieties like that of TTPEpy [54]. The second approach is to incorporate pyrene moieties, either one or two, into TPE as part of the structure like those of TPPyE and DPDPyE [55]. Both approaches are successful in changing pyrene from an ACQ material to an AIE or AIEE material. However, considering the performance of the OLEDs, the former approach, namely TTPEpy OLEDs, can reach a high EL efficiency (hEXT ¼ 4.98%, hC ¼ 12.3 cd A1, hP ¼ 7.0 lm W1) and substantially outperforms the latter (TPPyE and DPDPyE). TPPyE and DPDPyE OLEDs are also worse in blue color purity and their EL wavelength is in the range 504–516 nm, which is longer than the 488–492 nm of green-blue TTPEpy OLEDs. The next group in the triphenylethene family is DPVP2Mst, DPVPP2Mst, and DPVP4Mst (Table 1.10) [56], which can be considered as an improved version of DPVBi in terms of blue color purity. With two central methyl substituents twisting the biphenyl structure, the effective p-conjugation length of the molecule is reduced, the bandgap energy is increased, and the emission wavelength is shortened. An authentic blue EL, 1931 CIEx,y (0.15, 0.17), was acquired for a DPVPP2Mst OLED. As the number of triphenylethene

AIE or AIEE Materials for Electroluminescence Applications 23 Table 1.8 Summary of reported OLEDs containing triphenylamine-based 3TPETPA or 4TPEDTPA AIE or AIEE materials. Von (V) Lmax (cd m2) hEXT (%) hC (cd A1) hP (lm W1) Ref.

AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

3TPETPA

ITO/NPB/EML/TPBI/Alq3/LiF/Al 493, 511 (–, –)green–blue 5.4 ITO/EML/TPBI/Alq3/LiF/Al 499, 513 (–, –)green–blue 4.5 ITO/EML/TPBI/Alq3/LiF/Al 488 (–, –) green–blue 4.1

4TPEDTPA

53 1662

1.2

3.1

1.1

6935

1.5

4.0

1.9

10723

3.7

8.0

5.2

moieties attached increases, the AIE or AIEE effect intensifies the solid-state blue emission of these bimesitylene fluorophores. With Bim-DPAB (Scheme 1.2) as the HTL in the device, the EL efficiency of a DPVPP2Mst OLED was increased (hEXT ¼ 4.7%, hC ¼ 5.4 cd A1, hP ¼ 2.02 lm W1). The molecular hybrid structure of TPE and silole, two well known AIE or AIEE moieties, seems to be interesting and worth OLED testing. Tang and co-workers prepared 2,5-BTPEMTPS and 3,4-BTPEMTPS after the first failed attempt at the preparation of TPE hybrid hexaphenyl-substituted siloles (Table 1.11) [49]. The silole and TPE hybrids 2,5-BTPEMTPS and 3,4-BTPEMTPS [57] are in fact a pair of structural isomers. Both of their OLEDs exhibit green–yellow EL, that of the 2,5-isomer having a longer wavelength at 552 nm than that of the 3,4-isomer at 520 nm. The not very good performance of nondopant OLEDs can be improved by making a dopant device with BTPE as the host material for 2,5-BTPEMTPS after optimizing the dopant concentration. A similar molecular hybrid approach was applied to TPE and triphenylethene by Chi and co-workers (Table 1.11). [58] One of such hybrid molecules is (VP)3-(TPE)3 and its OLED has been tested. However, (VP)3–(TPE)3 OLEDs perform poorly without EL efficiency data. The device requires a relatively high 6.0 V to be turned on and Lmax is only 1908 cd m2 Its EL color is not blue but green–blue at 474 nm, corresponding to 1931 CIEx,y (0.18, 0.31). In order to increase the hole-transporting ability of TPE or triphenylethene AIE or AIEE materials, arylamine moieties are a potent structural feature to be incorporated in the structure [59]. We have seen that 3TPETPA and 4TPEDTPA mentioned earlier (Table 1.8) are two examples following the principle of

24 Aggregation-Induced Emission: Applications Table 1.9 Summary of reported OLEDs containing pyrene-based 3TPETPA or 4TPEDTPA AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), Von (V) Lmax (cd m2) hEXT (%) hC (cd A1) hP (lm W1) Ref. (x, y), color

TTPEPy 40 nm 26 nm EML TPPyE DPDPyE EML TPPyE DPDPyE

ITO/NPB/TTPEPy (x nm)/TPBI/LiF/Al 488 (–, –) green–blue 3.6 492 (–, –) green–blue 4.7 ITO/NPB/EML/TPBI/LiF/Al 508 (–, –) blue–green 4.6 520 (–, –) green 5.3 ITO/NPB/EML/TPBI/Alq3/LiF/Al 504 (–, –) blue–green 4.6 516 (–, –) blue–green 3.2

54 36300 18000

4.95 4.04

12.3 10.6

7.0 5.0

15450 45550

1.4 2.9

3.7 9.1

2.1 4.1

25470 49830

2.0 3.3

4.0 10.2

2.7 9.2

55

Table 1.10 Summary of reported OLEDs containing bimesitylene-based DPVP2Mst, DPVPP2Mst, or DPVP4Mst AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

EML DPVP2Mst DPVPP2Mst DPVP4Mst EML DPVP2Mst DPVPP2Mst DPVP4Mst EML DPVP2Mst DPVPP2Mst DPVPP2Mst

ITO/NPB/EML/BCP/Alq3/LiF/Al 448 (0.15, 0.09) deep blue 4.5 462 (0.10, 0.16) blue 4.5 452 (0.16, 0.13) blue 5.0 ITO/NPB/EML/TPBI/LiF/Al 452 (0.14, 0.08) deep blue 4.5 452 (0.14, 0.11) blue 4.5 452 (0.15, 0.12) blue 5.5 ITO/NPB/CBP/EML/TPBI/LiF/Al 450 (0.15, 0.10) deep blue 5.0 456 (0.15, 0.13) blue 5.0 ITO/Bim-DPAB/DPVPP2Mst/TPBI/LiF/Al 460 (0.15, 0.17) blue 4.5

Von (V)

Lmax (cd m2)

hEXT (%)

hC (cd A1)

hP (lm W1)

5415 6129 1466

1.52 1.07 1.06

1.22 1.38 1.15

0.55 0.49 0.39

4575 9031 1727

1.51 1.95 1.08

1.29 1.80 1.07

0.66 0.95 0.43

7599 9500

2.12 2.11

1.90 2.24

0.93 1.10

15361

4.70

5.40

2.02

Ref. 56

AIE or AIEE Materials for Electroluminescence Applications 25 Table 1.11 Summary of reported OLEDs containing TPE–silole hybrid 3,4-BTPEMTPS or 2,5-BTPEMTPS AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

EML 3,4-BTPEMTPS 2,5-BTPEMTPS 2,5-BTPEMTPS x ¼ 10 x ¼ 20 (VP)3-(TPE)3

ITO/NPB/EML/TPBI/LiF/Al 520 (–, –) green 6.2 3980 552 (–, –) green–yellow 5.2 12560 ITO/NPB/BTPE: 2,5-BTPEMTPS (x wt%)/TPBI/LiF/Al 540 (–, –) green–yellow 4.8 8894 540 (–, –) green–yellow 4.6 10480 ITO/NPB/(VP)3-(TPE)3/Alq3/LiF/Al 474 (0.18, 0.31) green–blue 6.0 1908

Von (V)

Lmax (cd m2)

hEXT (%)

hC (cd A1)

hP Ref. (lm W1)

1.66 1.98

4.96 6.40

2.05 2.45

2.13 2.18

6.64 7.02

3.36 3.24

–

–

–

57

58

structural design herein. More TPE or triphenylethene AIE or AIEE materials containing an arylamine moiety are known in the literature. Arylamine-containing C4 [60], TPATPE [61], 2TPATPE [61], TPE-2Cz [62], TPECa [63], and TPECaP [63] are all such materials. Their OLED data are all summarized in Table 1.12. Except for TPE-2Cz, most of the OLEDs show green–blue to blue–green EL with wavelengths in the range 484–514 nm. The TPE-2Cz OLED has an EL wavelength at 462 nm corresponding to 1931 CIEx,y (0.17, 0.21), approximately blue, but its EL efficiency is only moderate (hC ¼ 2.80 cd A1, hP ¼ 2.51 lm W1). Fluorene (or the more rigid and bulky 9,90 -spirobifluorene) is the repeating unit of high-performance blue light-emitting polymers, polyfluorenes (PFs). The hybrid or fused structure of TPE and fluorene (or 9,90 spirobifluorene) can take good advantage of the AIE or AIEE effect and high PL efficiency of blue emission. Three sets of such materials, BTPEBCF [64], SFTPE [62], and Fn-TPE (n ¼ 1–5) [65], are known in the literature (Table 1.13). Whereas SFTPE, F1-TPE, and F2-TPE OLEDs exhibit EL with wavelengths shorter than 470 nm, an empirical benchmark of blue color, BTPEBCF, F3-TPE, F4-TPE, and F5-TPE OLEDs all exhibit EL at wavelengths 476–508 nm, indicative of green–blue color. Regardless of the unsatisfactory blue color purity, the EL efficiency of BTPEBCF is the best; hC and hP can reach 7.9 cd A1 and 3.7 lm W1, respectively. Organoboron compounds are particularly attractive and promising owing to their unique properties stemming from the pp–p conjugation between the vacant p orbital on the boron atom and the p orbital of the p-conjugated framework. Organoboron compounds also have electron-transporting properties [66, 67]. A very high blue EL efficiency (hEXT >6%) has been reported for D–p–A-type blue fluorophores containing dimesitylboron as electron acceptor [68]. In the literature, two TPE-containing organoboron compounds

26 Aggregation-Induced Emission: Applications Table 1.12 Summary of reported OLEDs containing arylamine-based TPE derivative AIE or AIEE materials. AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

C4

ITO/NPB/C4/Alq3/LiF/Al 474 (–, –) green–blue ITO/NPB/EML/TPBI/Alq3/LiF/Al 492 (–, –) green–blue 514 (–, –) blue–green ITO/EML/TPBI/Alq3/LiF/Al 492 (–, –) green–blue 513 (–, –) blue–green ITO/NPB/TPE-2Cz/TPBI/LiF/Al 462 (0.17, 0.21) blue ITO/NPB/EML/TPBi/Alq3/LiF/Al 484 (–, –) green–blue 488 (–, –) green–blue 488 (–-, –-) green–blue ITO/EML/TPBI/LiF/Al 496 (–, –) green–blue 490 (–, –) green–blue

EML TPATPE 2TPATPE EML TPATPE 2TPATPE TPE-2Cz EML TPECa TPECaP TTPECaP EML TPECaP TTPECaP

Von (V)

Lmax hEXT (cd m2) (%)

hC (cd A1)

hP (lm W1)

6.0

548

–

2

–

3.6 3.4

15480 32230

3.4 4.0

8.6 12.3

5.3 10.1

4.2 3.2

26090 33770

3.3 4.4

8.3 13.0

4.9 11.0

3.3

6179

–

2.80

2.51

4.0 3.8 4.0

7508 11060 13650

1.8 1.7 1.8

3.8 3.5 3.8

2.7 2.9 2.1

4.4 3.6

12930 9048

2.2 2.3

5.5 6.3

3.8 4.1

Ref. 60 61

62 63

have been reported for OLED applications, TPEMesB and BTPEPBN (Table 1.14) [69, 70]. For TPEMesB OLEDs, it has been shown that the EL efficiency is in fact higher without using an ETL (TPBI) in the device, demonstrating the electron-transporting property in addition to the green–blue light-emitting function of TPEMesB AIE or AIEE materials. For BTPEPBN OLED, no EL wavelength was reported, but ‘sky blue’ color was mentioned for its PL, probably in the solid state. A BTPEPBN OLED was reported with low EL efficiency (hEXT ¼ 1.52%, hC ¼ 4.43 cd A1, hP ¼ 1.64 lm W1), although it is better than the OLED

AIE or AIEE Materials for Electroluminescence Applications 27 Table 1.13 Summary of reported OLEDs containing fluorene- or 9,90 -spirobifluorene-based TPE derivative AIE or AIEE materials. Von (V) Lmax (cd m2) hEXT (%) hC (cd A1) hP (lm W1) Ref.

AIE or AIEE fluorophore

lmaxEL, CIEx,y, color code (nm), (x, y), color

BTPEBCF

ITO/NPB/BTPEBCF/TPBI/LiF/Al 508 (0.24, 0.42) blue–green 5.5 ITO/NPB/BTPEBCF/TPBI/Alq3/LiF/Al 502 (0.21, 0.37) blue–green 4.5 ITO/NPB/SFTPE/TPBI/LiF/Al 466 (0.18, 0.24) blue 2.6 ITO/PEDOT/EML/TPBI/LiF/Al 468 (–, –) blue 5.8 468 (–, –) blue 5.5 488 (–, –) green–blue 6.4 476 (–, –) green–blue 6.2 480 (–, –) green–blue 4.8

SFTPE EML F1-TPE F2-TPE F3-TPE F4-TPE F5-TPE

64 13760

2.6

7.2

2.8

6400

2.9

7.9

3.7

8196

–

3.33

2.10

1300 1100 1050 1050 120

– – – – –

2.6 1.5 1.6 1.8 0.2

1.0 0.55 0.5 0.6 0.06

62 65

with the same organoboron material lacking two TPE substituents [70]. This is yet further experimental evidence supporting the AIE or AIEE effect of a TPE moiety that is beneficial for EL efficiency. Regarding electron transport of OLED materials, the other well-known electron transporting material is 2,5-diphenyl1,3,4-oxadiazole structural moiety, and it has been attached with two TPE units to form OXa-pTPE and OXa-mTPE (Table 1.14) [71]. Although the AIE or AIEE effect has been demonstrated for both OXa-pTPE and OXa-mTPE compounds, high EL efficiency (hEXT ¼ 5.0%, hC ¼ 9.79 cd A1, hP ¼ 9.92 lm W1) has been achieved with the dopant OLEDs, of which OXa-pTPE or OXa-mTPE are used as the host material for BUBD-1 blue dopant. Compared with the EL spectra of BUBD-1 reported elsewhere [72], the EL spectra shown in the paper on OXa-pTPE and OXa-mTPE OLEDs [71] indicate that it is a co-emission EL from both host and dopant and it is green–blue in color, 1931 CIEx,y (0.15, 0.34). One large family of TPE derivatives is derived from attaching to or being attached with various polycyclic aromatic hydrocarbons (PAHs). TPTPE (or thiophene-based TPTDPE) and BTPTPE are the fused TPE dimer and trimer (Table 1.15), respectively, via one common benzene ring, the fundamental repeating unit of PAHs [73]. Whereas the trimer BTPTPE OLED exhibits very short wavelength EL at 448 nm, indicative of an authentic blue color, the TPTPE (or thiophene-based TPTDPE) OLED has a green–blue EL at

28 Aggregation-Induced Emission: Applications Table 1.14 Summary of reported OLEDs containing organoboron- or 1,3,4-oxadiazole-based TPE derivative AIE or AIEE materials. AIE or AIEE lmaxEL, CIEx,y, color code (nm), fluorophore (x, y), color TEPDMesB

BTPEPBN Oxa-pTPE Oxa-mTPE

Von (V) Lmax (cd m2) hEXT (%) hC (cd A1) hP (lm W1) Ref.

ITO/NPB/TEPDMesB/LiF/Al 496, 512 (–, –) green–blue 6.3 5581 ITO/NPB/TEPDMesB/TPBI/LiF/Al 496, 512 (–, –) green–blue 6.3 5170 – –, – (–, –) green–blue – – ITO/NPB/Oxa-pTPE:BUBD-1/TPBI/Alq3/Al 500 (0.15, 0.34) green–blue 5.1 10070 ITO/NPB/Oxa-mTPE:BUBD-1/TPBI/Alq3/Al 500 (0.15, 0.33) green–blue 6.25 7734

69 2.3

5.78

3.4

2.7

7.13

3.2

1.52

4.43

1.64

5.0

9.79

9.92

–

9.82

7.96

70 71

488 nm (512 nm for thiophene-based TPTDPE). The TPTPE OLED shows better EL efficiency than the other two. However, the green–blue EL efficiency of TPTPE is only fair (hEXT ¼ 2.7%, hC ¼ 5.8 cd A1, hP ¼ 3.5 lm W1). PAHs such as pyrene, anthracene, phenanthrene, and naphthacene have been singly attached to TPE forming TPEPy, TPEAn, TPEPa, TPENp, and TPE-2-Np (Table 1.15) [74]. They are also doubly attached to TPE in forming TPEBPy, TPEBAn, TPEBPa, TPEBNp, and TPEB-2-Np (Table 1.15) [74]. Although the solid-state PL of TPENp has the shortest wavelength at 469 nm, TPEPa has the shortest EL wavelength at 464 nm, a possible blue color. The EL of TPENp is red shifted to 480 nm and becomes green–blue. A similar red shift, from the smallest þ2 nm of TPEB-2-Np to the largest þ30 nm of TEPBAn, are common for these two types of TPE derivatives, except one TPEPa, which has a blue-shifted rather than a red-shifted EL. All of the non-dopant OLEDs containing the materials mentioned here have EL efficiencies ranging from poor (hEXT ¼ 1.3%, hC ¼ 2.4 cd A1, hP ¼ 1.1 lm W1 for TPENp OLED) to above average (hEXT ¼ 3.0%, hC ¼ 7.3 cd A1, hP ¼ 5.6 lm W1 for TPEPy OLED). However, the performance of TPEPy OLED is not as good as that of another pyrene-containing TPE derivative OLED (TTPEPy) reported earlier, and both OLEDs have comparable green–blue color. The latest member joining the family may be the mono-, di-, and triphenylethene n-hexyloxybenzene derivatives PhTPE, Ph2TPE, and Ph3TPE reported by Li and co-workers [75] (Table 1.15). It must be because of the steric hindrance twisting the coplanarity between the TPE unit and ortho-substituted nhexyloxybenzene that the Ph2TPE OLED shows the shortest EL wavelength at 457 nm, an authentic blue

AIE or AIEE Materials for Electroluminescence Applications 31 corresponding to 1931 CIEx,y (0.16, 0.15). Similarly to several of the TPE derivatives shown earlier, Ph2TPE OLED has an unusually blue-shifted EL compared with its solid-state PL at 494–498 nm. Nevertheless, the EL efficiency of the Ph2TPE OLED is not good (hC ¼ 2.3 cd A1, hP ¼ 1.7 lm W1) and that of Ph3TPE OLED [also with a blue EL at 467 nm and 1931 CIEx,y (0.17, 0.20)] is better (hC ¼ 3.7 cd A1, hP ¼ 2.5 lm W1). As we have seen with the many triphenylethene or TPE derivatives so far, they mostly exhibit EL with green–blue, blue–green, green, and even yellow–green color, and these are not the desirable colors in display applications. From this survey, it is evident that authentic blue AIE or AIEE materials containing a triphenylethene or TPE moiety are difficult to come by. The last group in the family of triphenylethene or TPE derivatives are the AIE or AIEE effects shown in the other extreme of visible spectra, namely the long wavelengths in the orange, red, and even near-IR region. In order to acquire a long emission wavelength, either PL or EL, the triphenylethene or TPE moiety has to be p-conjugated to a strong electron acceptor. From a survey of the literature, it was found that the benzo[c][1,2,5]lthiadiazole (BTZ) moiety seems to be the most potent electron acceptor for long-wavelength PL or EL (Table 1.16). For EL wavelengths longer than 650 nm, a benchmark for red color, thiophene-bridged triphenylethene and BTZ is necessary and it requires two thiophene bridges (like BTPEBTTD) instead of one thiophene (like BTPETTD) or two benzene bridges (BTPETD) (Table 1.16) [78]. This is because the thiophene ring has a lower bandgap energy than the benzene ring. Similarly, for V2BV2 and T2BT2 (Table 1.16) [79], a C C double bond as the connecting p-conjugation bridge is not good enough to extend the EL wavelength beyond 650 nm, even though there is a strong electron donor triphenylamine between the triphenylethene and BTZ. Another approach is to increase the electron-deficient power of BTZ by changing to [1,2,5]thiadiazolo[3,4-g]quinoxaline (QTD) of TPEQTD or MTPEQTD, or benzo[1,2-c;4,5-c0 ]bis[1,2,5]thiadiazole (BBTD) of TPEBBTD or MTPEBBTD (Table 1.16). [80] These four compounds (TPEQTD, MTPEQTD, TPEBBTD, and MTPEBBTD) have EL wavelengths in the range 706–864 nm, within the near-IR region. However, their EL Lmax are all low and none of their hEXT values is over 1%. The most successful design for red EL is the branched version of BTPEBTTD, that is, TBTPEBTTD (Table 1.16) [28]. TBTPEBTTD has an EL wavelength at 650 nm, 1931 CIEx,y (0.67, 0.32), at the edge of red color on the chromaticity diagram. It was reported with hEXT as high as 3.7%, superior to the 3.1% of the FPhSPFN OLED [27], one of the most efficient nondopant red OLEDs based on AIE or AIEE materials.

1.8 White OLEDs Containing AIE or AIEE Materials Although its AIE or AIEE effect was demonstrated above, the blue fluorophore DPVBi was utilized in RGB multi-color OLED displays as early as 1997 [81]. For white EL used for lighting, there are a number of materials and device configurations for generating white EL from a DPVBi-containing OLED (Table 1.17) [38, 82–86]. In terms of EL efficiency and Lmax, a DPVBi blue-emitting layer doped with yellow–orange rubrene is probably the best approach, as reported by Li and Shinar [83] Such a WOLED exhibits EL with 1931 CIEx,y (0.27, 0.31) near-white chromaticity, Lmax as high as 50100 cd m2, hEXT 4.0% and hP 3.9 lm W1. However, since it is a two-color-white WOLED, it is not possible for the CRI to be high (the original paper did not report the CRI value of the device). The second kind of AIE or AIEE material being used in WOLEDs is the orange–red fluorophore NPAFN (Table 1.3 and Table 1.17) [87]. Taking advantage of its strong AIE or AIEE effect reported by Chen and coworkers, NPAFN enabled the first all-nondopant three-color WOLED to be obtained. Because of its nondopant nature, the NPAFN WOLED showed almost constant white chromaticity with 1931 CIEx,y (0.34, 0.38) and a good CRI of 80, although its EL efficiency was only fair (hEXT ¼ 3.4%, hP ¼ 9.1 cd A1 at 1000 cd m2).

36 Aggregation-Induced Emission: Applications The next WOLED containing AIE or AIEE materials to be reported was the efficient blue fluorophore TPVAn (Table 1.5 and 1.17) [44]. It adopted a two-color-white approach including the commercially available red dopant DCJTB. Although it has good white chromaticity [1931 CIEx,y (0.33, 0.39)], Lmax (30 000 cd m2), and EL efficiency (hEXT ¼ 3.4%, hC ¼ 9.1 lm W1 at 100 mA cm2 and >2000 cd m1), its CRI cannot be high, which was not mentioned in the reported paper. Two other WOLEDs are based on all-AIE or -AIEE materials as the nondopant light-emitting layers appear. As shown in Table 1.17, Ma and co-workers reported all-AIE or -AIEE material-containing WOLEDs with blue TDPVBi (Table 1.6) and yellow CN-DPASDB (Table 1.3) [30]. At almost at the same time, Tang and co-workers reported WOLEDs with green–blue TTPEPy (Table 1.9) and orange BTPETTD (Table 1.16) [76]. Under lighting condition 1000 cd m2, the best hC of both sets of the WOLEDs can reach 7 cd A1, which is the highest among all fluorescence-based WOLEDs in Table 1.17. The white color chromaticity of TTPEPy/BTPETTD WOLED is 1931 CIEx,y (0.42, 0.39) or (0.40, 0.42), which is somewhat off from standard white 1931 CIEx,y (0.33, 0.33). Surprisingly, these TTPEPy/BTPETTD WOLEDs exhibit very high CRI, 85 or 90, which is extremely rare for two-color-white WOLEDs. According to the paper on the TTPEPy/BTPETTD WOLED [76], the multiple-emission peaks centered at 524, 492, and 472 nm were attributed to TTPEPy (492 nm) and impurities (524 and 472 nm), resulting in a broad EL band and hence unusually high CRI. In terms of EL efficiency, TDPVBi/CN-DPASDB WOLEDs are comparable to TTPEPy/BTPETTD WOLEDs, although the former are superior in white color chromaticity. The orange BTPETTD AIE or AIEE material has been used in two other sets of WOLEDs. First, it was assembled with a non-AIE or -AIEE blue emitter, 4,40 -bis(1-phenyl-1H-phenanthro[9,10-d]imidazol-2-yl) biphenyl (DPPi) into a bilayer WOLED (Table 1.17) [77]. Owing to the uncommon energy level alignment between BTPETTD and DPPi, the blue emission is due to direct recombination of excitons in DPPi, whereas the red emission originates not only from the direct recombination of excitons in BTPETTD but also from a color down-conversion process by absorbing blue emission and re-emitting red photons. The combination of blue emission and red emission yields an efficient and extremely stable white color, regardless of the driving voltages. Once again, multiple-emission bands were observed for such WOLEDs and an unusually high CRI of 92 was achieved with excellent white color chromaticity, 1931 CIEx,y (0.31, 0.31). Second, BTPETTD has been used as a color down-conversion (CCL) capping layer of a top-emitting blue phosphorescence OLED (having FIrpic as the green–blue phosphorescence dopant) in generating goodquality white EL, 1931 CIEx,y (0.34, 0.35) (Table 1.17) [88]. At 914 cd m2, a common lighting condition, the phosphorescence–fluorescence hybrid white EL efficiency (hC ¼ 17–18 cd A1, hP ¼ 6–7 lm W1) is better than that of most fluorescence-based WOLEDs. The last pair of compounds for WOLEDs is OTB (blue emission at 433 nm) and T4AC (yellow emission at 551 nm), reported by Gao and co-workers [89] (Table 1.17). Compared with those commonly seen with AIE or AIEE effects, the chemical structures of OTB and T4AC are unconventional and previously unknown. Yellow T4AC was reported with a large Stokes shift of 182 nm without self-absorption in the solid state, which is attributed to the excited-state intramolecular proton transfer property, providing the potential for solid-state emitters. Nevertheless, although good quality and stability of the white EL were reported (Table 1.17), the EL efficiency of the WOLED was rather poor under lighting conditions (hC  1 cd A1).

1.9 Perspectives As shown in this chapter, it has been demonstrated repeatedly that the AIE or AIEE effect is effective in promoting the EL efficiency of nondopant OLEDs. Without AIE or AIEE materials, nondopant OLEDs, which are simple and easy to fabricate, show insufficient EL efficiency for practical application. In terms of

AIE or AIEE Materials for Electroluminescence Applications 37 EL color purity, it is more successful for red than blue color; although there are many more AIE or AIEE materials available for green–blue or blue–green EL color, authentic blue EL of AIE or AIEE materials is still rare and needs further research and development. Although EL polymers derived from AIE or AIEE small-molecule materials have been reported in the literature, the AIE or AIEE effect of a small molecule does not necessarily remain in the polymeric form [90, 91]. They are not included in this chapter mainly because their OLED performances are far from satisfactory. Polymeric AIE or AIEE materials have potential for OLED applications but their device performances need improvement or a better design is needed for polymeric AIE or AIEE materials. Similarly, all or partial AIE or AIEE material-based WOLEDs are difficult to come by and not many of them have been reported. For practical lighting conditions, none of them is good enough in terms of EL efficiency. For lighting application of OLEDs, phosphorescence-based materials have a better chance of success, and this is one potential material that may be suitable for demonstrating the AIE or AIEE effect, although so far there are no literature reports.

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2 Crystallization-Induced Phosphorescence for Purely Organic Phosphors at Room Temperature and Liquid Crystals with Aggregation-Induced Emission Characteristics Wang Zhang Yuan1, Yongming Zhang1 and Ben Zhong Tang2 1 2

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, China Department of Chemistry, The Hong Kong University of Science and Technology, China

2.1 Crystallization-Induced Phosphorescence for Purely Organic Phosphors at Room Temperature 2.1.1 Introduction Phosphorescence is distinguished from fluorescence by an electronic transition from the excited triplet state rather than the excited single state to the singlet ground state, resulting in much longer lifetimes (usually in the microseconds to milliseconds range) compared with those of fluorescence (typically on the order of nanoseconds) owing to the spin-forbidden nature of such a transition [1]. Phosphorescent materials have attracted increasing attention not only because of the fundamental importance of the triplet state involved electronic transition processes, but also owing to their promising applications in bioimaging [2], chemoand bioassays [3], protein structure and dynamics indicators [4], organic light-emitting diodes (OLEDs) [5], photovoltaic devices, and so on. However, for organic phosphors, although organometallic chelates such as iridium (Ir), platinum (Pt), gold (Au), and osmium (Os) heavy atom complexes can be highly phosphorescent, attributable to the large spin–orbit coupling effect [6–8], few purely organic materials are capable of emitting efficient room-temperature phosphorescence, for the following reasons: first, the highly bonded

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

44 Aggregation-Induced Emission: Applications nature of the electrons in metal-free organic materials leaves them little freedom and less impetus to emit from triplet states [9]; second, the singlet-triplet mutual transitions are spin-forbidden; thirdly, molecular motions can effectively annihilate the triplet state excitons during their long residence time through detrimental processes such as thermal perturbations, intramolecular motions, solute–solute and solute–solvent collisions, and intermolecular interactions with quenching moieties such as oxygen and humidity [10]. Therefore, phosphorescence from purely organic luminogens has usually been observed under conditions free from the above interfering effects, typically at cryogenic temperatures (e.g. 77 K) in rigid ‘glasses’ of frozen (mixture) solvents free of oxygen. Although proteins and some other organic compounds in micelles, cyclodextrin complexes [11], and polymeric matrixes [12], and on filter paper or silica show room temperature phosphorescence (RTP), normally with only instrument-detectable intensity, and efficient RTP from purely organic phosphors is rarely observed. To achieve efficient RTP from purely organic phosphors, in which intersystem crossing (ISC) should be highly efficient, translational, vibrational, and rotational dissipations must be effectively depressed. Taking these factors into consideration, groups capable of inducing or enhancing ISC are highly desirable. Moreover, motion-rich solution states of the phosphors must be avoided in order to decrease unexpected collisional and vibrational deactivations. We have endeavored to find and create such systems with considerable ISC and restricted molecular motions. It is envisioned that solid states may help to prevent active molecular motions. However, most fluorescent dyes can be highly emissive in dilute solutions but become weakly luminescent or even nonemissive in condensed solutions or as solid aggregates owing to the formation of detrimental species such as excimers and exciplexes, exhibiting the deleterious concentration quenching effect and aggregation-caused quenching (ACQ) effect. Although various chemical, physical, and engineering approaches have been utilized, attempts to tackle the notorious ACQ effect have met with only limited success. In most cases, aggregation is impeded only partially or temporarily, because aggregate formation is a natural process when luminogenic molecules are located nearby in the condensed phase. Fortunately, in 2001, we observed the intriguing phenomenon of aggregation-induced emission (AIE), which is opposite to the ACQ effect: a series of silole molecules, such as 1-methyl-1,2,3,4,5-pentaphenylsilole, that were nonemissive in dilute solutions were induced to become strong emitters by aggregate formation [13]. Systematic studies of the influence of internal and external changes on the emission behaviors of the AIE luminogens clearly suggested that restriction of intramolecular rotation (RIR) is the main cause of the AIE effect [14, 15]. Based on the discovery and successful exploration of the AIE phenomenon [16, 17], particularly inspired by the special case of crystallization-induced emission (CIE) [18], we subsequently discovered that some purely organic phosphors such as benzophenones exhibited no emission in solution, in polymeric films, or on thin-layer chromatography (TLC) plates, but turned out to be highly phosphorescent in the crystalline state at room temperature and ambient conditions [19]. Aromatic ketone groups are the internal source of the phosphorescence; meanwhile, RIR and molecular motions caused by various intermolecular interactions are the essential factors to boost the phosphorescence emission [19]. Similarly to AIE and CIE, we termed this phenomenon ‘crystallization-induced phosphorescence’ (CIP) [19]. The new discovery of the CIP phenomenon breaks the traditional concept that efficient RTP is almost unavailable for purely organic phosphors under ambient conditions, and paves the way for the fabrication of new, efficiently emissive, purely organic phosphors. In this chapter, we first summarize the recent progress in CIP studied. 2.1.2 Molecular luminogens with crystallization-induced phosphorescence at room temperature Aromatic ketones constitute a wide variety of purely organic compounds that have been well known for decades to be phosphorescent owing to the spin–orbit coupling of the carbonyl oxygen [20]. For example, benzophenone (BP) crosses from the first singlet excited state (S1) to the first triplet excited state (T1) with

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 45

Figure 2.1 Photographs of various benzophenone solutions (a) and crystals (b) under 365 nm UV irradiation. Reprinted with permission from [19], # 2010 American Chemical Society.

almost unity efficiency [21]. Therefore, we first checked the benzophenone emissions in solution and the solid state. It is practically nonluminescent in various solvents [n-hexane, tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile, ethanol], poly(methyl methacrylate) (PMMA) films, and as an amorphous solid solute on a TLC plate at room temperature. However, unprecedentedly, its single crystals emit bright blue light upon 365 nm UV irradiation under ambient conditions (Figure 2.1). The time-resolved photoluminescence (PL) spectrum of the crystals revealed their long-lived excited state with a mean lifetime (kti) of 312.9 ms, indicating the phosphorescent nature of the emissions. The results clearly suggest that crystallization induced the efficient phosphorescence of BP. Such a unique CIP phenomenon is understandable because in solutions and polymeric films, and even on a TLC plate, BP molecules can rotate freely, which favors nonradiative decay of its excitons as heat, and no emission is therefore observed. In the crystal state, various rotations are restricted or locked by the ordered molecular packing lattices, van der Waals forces, and specific intermolecular interactions such as C–H  O hydrogen bonds [H  O distances of 2.578 and    2.673 A, less than the sum of the Bondi radii of H (1.20 A) and C (1.55 A)], rigidifying the molecular conformations. Such restricted RIR process greatly suppressed the nonradiative pathways of the excitons, subsequently making them decay mainly radiatively, and thus turning on the phosphorescence emission of BP at room temperature [19]. Detailed measurements of the PL emission spectra and quantum yields (Ff and Fp for fluorescence and phosphorescence quantum yields, respectively) of BP in THF at different temperatures and its crystals at room temperature showed that BP only gives detectable fluorescent signals (310, 380 nm, Ff ¼ 0.001%)

46 Aggregation-Induced Emission: Applications

Scheme 2.1 Structures of some purely organic phosphors with ‘crystallization-induced phosphorescence’ (CIP) characteristics at room temperature [19].

and high-efficiency phosphorescence (420, 449, 483 nm, Fp ¼ 15.9%) in THF and the crystalline state at room temperature. Such results confirm the CIP nature of BP. It is also noted that whereas only phosphorescence is observed in the crystals, dual fluorescence (310 nm) and phosphorescence (410, 440, 472 nm) in THF at 77 K are detected, implying that crystals are more effective than cryogenic ‘solid glass’ in generating efficient phosphorescence. Encouraged by the discovery of the CIP behavior of BP, we continued to check whether it was a general phenomenon. We studied the halogen- and amino-substituted derivatives of BP (Scheme 2.1, 2–6) [19]. Similarly to BP, these luminogens are all nonemissive in dilute solutions and as amorphous films but are induced to emit phosphoresce (kti 19.2–4800 ms) efficiently at room temperature by crystal formation, thus testifying to their CIP characteristics. Whereas 4,40 -difluorobenzophenone (DFBP, 2) emits blue phosphorescence with maxima at 409, 436 and 467 nm, its analogs 4,40 -dichlorobenzophenone (DCBP, 3) and 4,40 dibromobenzophenone (DBBP, 4) with heavier substituents show longer wavelength yellow emissions with maxima at 421, 448, 483, and 517 nm and 428, 457, 486, 495, and 535 nm, respectively [19]. Further, 4-bromobenzophenone (BBP, 5) and 2-aminobenzophenone (ABP, 6) give bright blue and bluish green light, respectively, in the crystalline state. Figure 2.2 shows vivid photographs of the crystals of 3–6 under normal laboratory lighting and 365 nm UV irradiation at room temperature. Clearly, all these crystalline samples emit bright phosphorescence at room temperature. Further quantum yield measurements using an integrating sphere gave phosphorescence Fp values for 2–6 of 39.7, 8.30, 12.0, 6.70, and 8.60%, respectively. Table 2.1 summarizes the absorption and emission parameters of these CIP-active phosphors. The CIP effects of BP and its halogenated derivatives indicate that the carbonyl group and heavy atoms are essential structural parameters that make them phosphorescent. The nonplanar molecular conformation of the compounds is another factor that favors spin–orbit coupling and promotes the phosphorescence process [22, 23]. Keeping these structural features (carbonyl group, heavy atom, and nonplanar conformation) in mind, we searched for new potential CIP luminogens other than BP derivatives. It was found that methyl 4-bromobenzoate (MBB, 7), and 4,40 -dibromobiphenyl (DBBP0, 8) (Scheme 2.1) are also strongly luminescent in the crystalline state under ambient conditions, although their dilute solutions and the spots on TLC plates are practically nonemissive at room temperature. PL decay spectra of the crystals revealed the longlived excited states of both compounds, confirming the phosphorescence attribute of the emissions. Thereby, MBB and DBBP0 are also CIP-active phosphors at room temperature. The above results suggest that crystal engineering is an effective and powerful approach to generate highefficiency phosphorescence from suitable purely organic phosphors at room temperature. Therefore, to explore the mechanistic features, the crystal structures and molecular packing of the CIP compounds were

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 47

Figure 2.2 Photographs of crystals of phosphors DCBP (3), DBBP (4), BBP (5), and ABP (6) taken under (a–d) normal laboratory lighting and (e–h) 365 nm UV illumination at room temperature. Reprinted with permission from [19], # 2010 American Chemical Society.

investigated. Figure 2.3 shows examples of the molecular packing arrangements in the crystals of 3–6. Similarly to 1 and 2, all phosphors adopt nonplanar molecular conformations, which are fixed by various intermolecular interactions. In addition to typical C–H  O hydrogen bonds (H  O distances 2.711, 2.614, and  2.754 A for 3, 4, and 5, respectively), specific C–H  X (X ¼ Cl, Br) hydrogen bonds and C–Br  BrC halogen bonding are present in the crystals of the halogen-substituted BPs. C–H  Cl and C–H  Br hydro gen bonds with distances of 2.895, 2.939, and 3.236 A are observed in the crystals of 3 and 5, whereas C–  Br  Br–C halogen bonding with a distance of 3.788 A and an angle of 153.81 is seen in the crystal of 4.  Intermolecular N–H  O hydrogen bonds with a distance of 2.118 A are formed in the crystal of ABP, thanks to its amino group. For these phosphors, the C ¼ O groups and heavy halogen atoms, together with twisted conformations, are favorable for efficient spin–orbit coupling and thus phosphorescence-emitting

48 Aggregation-Induced Emission: Applications Table 2.1 Photophysical properties of CIP luminogens.a Compound

lab (nm)

lem (nm)

F (%)b Solution Crystal

BP DFBP DCBP DBBP BBP ABP MBB DBBP0

269 259 266 268 263 236 243 269

420, 449, 483 409, 436, 467 421, 448, 483, 517 428, 457, 486, 535 421, 450, 482 479 338, 476, 504 480

0.001 0.003 0.091 0.565 0.010 0.001 0.044 0.001

15.9 39.7 8.30 12.0 6.70 8.60 5.90 13.9

Phosphorescence decayc A1 (%)/t 1 (ms)

A2 (%)/t 2 (ms)

A3 (%)/t 3 (ms)

kti (ms)

27/55.6 56/660.4 99.5/16.8 79/2127.8 18/25.1 39/24.5 76/585.2 57/240.4

47/226.7 44/2107.1 0.5/488.5 21/14855.2 44/66.2 61/220 24/11991.7 43/1555.9

26/735.9

312.9 1296.9 19.2 4800.6 93.0 143.8 3322.8 806.1

38/156.3

a Abbreviations: lab ¼ absorption maximum; lem ¼ emission maximum; F ¼ quantum yield; A ¼ molecular fraction; t ¼ phosphorescence lifetime; kti ¼ mean lifetime. b Determined in acetonitrile solution (1 mM) using 9,10-diphenylanthracene as standard (F ¼ 90% in cyclohexane) or in the crystalline state using a calibrated integrating sphere. c Measured in the crystalline state. Reprinted with permission from [19], # 2010 American Chemical Society.

capability. Moreover, multiple effective intermolecular interactions rigidify the molecular conformations and block the rotational and vibrational deactivation of the phosphorescence, thus giving remarkably enhanced phosphorescence of the crystals of such compounds at room temperature. Based on our discovery and mechanistic understanding, several instructive conclusions can be drawn with regard to high-efficiency purely organic phosphors at room temperature. (1) Internal structural characteristics such as aromatic carbonyl groups, heavy atoms (e.g. halogens), and twisted molecular conformations are beneficial to spin–orbit coupling, thus increasing the chance of ISC and promoting the phosphorescence-emitting probability. (2) High-efficiency RTP from purely organic phosphors can be realized through rational crystal engineering. Depressing the rotational and vibrational deactivation channels by various intermolecular interactions, such as C–H  O, N–H  O, C–H  p, and C–H  X (X¼F, Cl, Br) hydrogen bonds and halogen bonding (C–X  X–C, X ¼ F, Cl, Br, I) is essential to turn on the RTP. (3) The twisted conformations of the luminogens prevent the formation of detrimental excimers and exciplexes, and are thus helpful in generating high-efficiency RTP phosphors in the crystalline state. After our work, Kim and co-workers also reported efficient purely organic phosphors generated through a ‘directed heavy atom effect’ and crystal engineering [24]. Unlike our twisted molecules, planar chromophores containing triplet-producing aromatic aldehydes and triplet-promoting bromine were utilized for the crystal design. Scheme 2.2 shows the structures of such luminogens. Similarly to our observations, these compounds are nonphosphorescent in solution, but emit phosphorescence with modest to high quantum yields in the crystalline state under ambient conditions, exhibiting typical CIP characteristics. The mechanism for such CIP emitters is also ascribed to the decreased thermal deactivation process in the crystals. However, owing to the planarity of the phosphors (e.g. Br6A, Scheme 2.2), the RIR efficiency for the crystals with a single component is low (only 2.9% for Br6A) because of the excimer-induced rampant selfquenching. To achieve brighter phosphorescence emission, Kim and co-workers utilized a mixed crystal design strategy to overcome the self-quenching problem. To maintain the ordered lattice and phosphorescence emission of the aldehyde crystals, a compound that features the same halogen bonding motif without

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 49

Figure 2.3 Perspective view of packing arrangement in crystals of (a) DCBP (3), (b) DBBP (4), (c) BBP (5), and (d) ABP (6), with C–H  O, C–H  Cl, C–Br  Br–C, C–H  Br, and N–H  O intermolecular interactions marked by dotted lines. Reprinted with permission from [19], # 2010 American Chemical Society.

optical interference with the phosphorescence is required. For these reasons, analogous compounds in which the aldehyde group was replaced with a bromine atom were chosen as the hosts. Such hosts act as optically inert matrixes that prevent self-quenching by isolating each chromophore molecule in the cocrystals, thus enhancing the phosphorescence efficiencies. For example, with a 1 wt% content of Br6A, Br6A– Br6 mixed crystals give a greatly improved quantum yield of 55%.

50 Aggregation-Induced Emission: Applications

Scheme 2.2 Structures of various brominated aromatic aldehydes and corresponding dibromo compounds [24].

Based on the above results, a series of mixed crystals (Scheme 2.2 and Figure 2.4) of halogenated aromatic aldehyde dopants (BrC6A, BrS6A, Np6A) and analogous bis-halogenated aromatic hosts (BrC6, BrS6, Np6) were designed and prepared to produce RTP (kti 0.1–6.4 ms, Fp 0.5–28%) with tunable colors from blue to orange, testifying to the generality of the ‘directed heavy atom effect’ and cocrystal design strategy. In an effort to explore novel purely organic phosphorescent materials, Yong et al. found that some internal salts and sodium salts based on an aromatic carbonyl-containing organic radical, namely 2-(imidazo [1,2-a]pyridin-2-yl)-2-oxoacetic acid, exhibit excitation wavelength-independent white and blue RTP in the solid state, with efficiencies of 5.2 and 7.5%, respectively (Scheme 2.3) [25]. Although no solution quantum yields were reported, the crystal structure indicates that multiple intermolecular interactions help to rigidify the molecular configurations. Such rigidification may greatly enhance the solid-state phosphorescence. In a subsequent study, Yong et al. synthesized a group of organic conjugated compounds based on (E)-3-benzylideneimidazo[1,2-a]pyridin-2(3H)-one (Scheme 2.4) [26]. They examined the emission characteristics of these compounds in both the solution and solid states, and long kti values of 16.8–18.5 and 22.9–29.0 ms, respectively, were obtained, indicating their phosphorescence nature. Crystal structure analysis indicates that the ordered columnar stacking arrangements, intermolecular close contacts, and lateral intermolecular hydrogen bonding interactions between neighboring columns are present in the crystalline states. Such interactions, on the one hand, decrease the rotational and vibrational deactivations, leading to higher phosphorescence efficiencies, and on the other, yield emission color changes compared with those in DMF. The Fp values of 14–20 in DMF are as low as 0.016, 0.016, 0.017, 0.0021, 0.0018, 0.017, and 0.018%, respectively, whereas their solid-state phosphorescent emissions are remarkably enhanced, with Fp values of 1.3, 1.2, 1.7, 1.0, 1.1, 1.5, and 1.2%, respectively. For these molecules, the phosphorescence originates from the aromatic carbonyl group-enhanced spin–orbit coupling and subsequent promoted ISC. The boosted solid-state phosphorescence is also ascribed to the RIR of the compounds. Recently, Jin and co-workers successfully utilized cocrystallization between 1,4-diiodotetrafluorobenzene (1,4-DITFB, 21) and polycyclic aromatic hydrocarbons (PAHs, e.g. 22–29) (Scheme 2.5) [27–30] to fabricate room temperature phosphorescent cocrystals, in which various C–H  p and C–H  F contacts and moreover C–I  p halogen bonding are involved. For example, 1,4-DITFB (21) and fluorene (22) can easily form monoclinic cocrystal 21/22 with 1:1 stoichiometry, multiple intermolecular interactions, includ ing C–I  p halogen bonds (I  C distances 3.409 and 3.534 A, within the sum of the van der Waals radii of  I and C) and C–H  p interactions (H  C distances 2.707 and 2.837 A, within the sum of the van der Waals radii of H and C) are involved (Figure 2.5). There are no p–p stacks (unfavorable for emission) between

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 51

Figure 2.4 Photographs of (mixed) crystals of Br6A, BrC6A–BrC6, Br6A–Br6, BrS6A–BrS6, and Np6A–Np6 under 365 nm UV irradiation. In the mixed crystals, the aldehyde content is 1 wt%. The lifetime and quantum efficiency of the mixed crystals at room temperature are indicated as the white texts. Reprinted with permission from [24], # 2011 Nature Publishing.

Scheme 2.3 Structures of compounds 13a and 13b [25].

52 Aggregation-Induced Emission: Applications

Scheme 2.4 Structures of compounds 14–20 [26].

1,4-DITFB or fluorene units. Here, 1,4-DITFB plays vital roles as follows: (1) iodine atoms act as heavy atom perturbers to increase the spin–orbit coupling of the PAHs, thus inducing their phosphorescence emission; (2) serve as halogen bonding donor or cement to link the luminogens together; and (3) behave as ‘solid-dilution agents’ to isolate the PAH building blocks, making them free of self-quenching. Such characteristics help to induce efficient phosphorescent emission at room temperature. Therefore, it is believed that the halogen bonding combined with other multiple weak interactions provides a new strategy to obtain phosphorescent cocrystal materials at room temperature with modulated emissions, which is particularly suitable for the rigid and planar PAHs.

2.2 Liquid Crystals with Aggregation-Induced Emission Characteristics 2.2.1 Luminescent liquid crystals Luminescent liquid crystals (LCs) have attracted widespread interest because of their fundamental importance and technological implications [31–33].The combination of the intrinsic light-emitting capability and the spontaneous self-organization attribute within a liquid crystalline phase is of crucial importance for optoelectronic applications, such as in stimuli-responsive smart materials [34], anisotropic light-emitting diodes, and emissive LC displays [35]. The fluorescent LCs may emit linear or circularly polarized light when aligned [36], which may be utilized for the construction of lighting and orientating layers in LC optical display devices, thus obviating the use of polarizing sheets and absorbing color filters. The color and brightness of the light emitted by the liquid crystalline luminogens could be manipulated by external fields, which may lead to the development of readily tunable electrochromic and optical switching systems. Therefore, the utilization of fluorescent LCs can simplify the device and moreover substantially increase the device brightness, contrast, efficiency, and viewing angle [37, 38].

Scheme 2.5 Structures of 1,4-DITFB (21) and compounds 22–29 [27–30].

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 53

Figure 2.5 (a) Crystal cell unit and (b) detailed C–I  p and C–H  p bonds for cocrystal 20/21. Reprinted with permission from [27], # 2012 American Chemical Society.

Despite the promising prospects for fluorescent LCs, the fabrication of high emission efficiency LCs is intractable. In the mesophases, particularly those formed by disk-like building blocks, the chromophoric mesogens are regularly packed and undergo intense intermolecular interactions, which often quench their light emissions due to the formation of detrimental excimers and/or exciplexes [39]. The light emission is often enhanced at the sacrifice of ordered molecular packing, which is the basic requirement in the formation of liquid crystalline phases, thus making the synthesis of efficient fluorescent LCs a daunting task. 2.2.2 Aggregation-induced emission strategy towards high-efficiency luminescent liquid crystals We are interested in the design and synthesis of both molecular and polymeric functional LCs [40–42], particularly light-emitting LCs. Through rational molecular design, a wide variety of side-chain liquid crystalline polyacetylenes carrying chromophoric and mesogenic pendants have been successfully synthesized and systematically investigated in our laboratory. Recently, based on our discovery of the AIE phenomenon, we tried to fabricate such novel luminescent LCs whose emission is enhanced rather than quenched upon self-assembly or aggregation. It is envisioned that the introduction of AIE-active dyes into LCs may achieve such goals, thus solving the contradictory requirements for mesophase formation (self-assembly nature, aggregation) and high emission efficiency (avoiding aggregation). With careful design, we successfully obtained a group of AIE-active polymers and molecules [43, 44]. Tetraphenylethene (TPE) was selected as the basic luminophoric unit for such compounds owing to its

54 Aggregation-Induced Emission: Applications

Scheme 2.6 Structures of some AIE-active liquid crystalline polymers (AIE-LCPs) [43].

facile synthesis and functionalization, high solid-state emission efficiency, and AIE-active characteristics. Scheme 2.6 shows a group of TPE- and biphenyl-containing AIE-active liquid crystalline polymers (AIELCPs) [43]. These polytriazoles [P30(x)–P32(x), x ¼ 5, 10] were synthesized by efficient click polymerization between biphenyl-containing diazides and diynes carrying TPE units. Figure 2.6 illustrates the textures of such polymers observed by polarized optical microscopy (POM) upon cooling from their melting states with or without shearing. The birefringence clearly suggests the liquid crystalline nature of the polymers.

Figure 2.6 POM images observed on cooling (a) P30(5) to 159.9  C, (b) P31(5) to 89.9  C, (c) P32(5) to 94.9  C, (d) P30(10) to 250.6  C, (e) P31(10) to 69.8  C, and (f) P32(10) to 114.9  C from their melting states at a cooling rate of 1  C min1. The photographs in (a), (b), (d), and (e) were taken under shearing. Reprinted with permission from [43], # 2011 American Chemical Society.

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 55 It is noted that no birefringence is observed when P30(x) and P31(x) are slowly cooled from their melted states without external forces. Anisotropic domains, however, emerged under shearing (Figure 2.6a, b, d, and e), indicating that the liquid crystalline domains of these polymers are too small to be observed without external stimuli. Such a phenomenon can be ascribed to the high rigidity and strong inter- and intramolecular interactions of the polymer chains, which greatly restrict the movement and packing arrangements of the mesogenic units. Nevertheless, when a shear force is applied, such small domains can merge together and align along the shear direction, thus making them observable under POM. The solid-state emission quantum yields of the polymers can be as high as 63.7%, which is remarkable compared with those of reported luminescent LCs. The photophysical properties of the AIE-LCPs are sensitive to their molecular structures and the solid-state efficiency decreases with increasing spacer length. For example, the solid-state efficiencies of P30(5), P31(5), and P32(5) gradually decrease from 63.7% to 35.2% and then to 28.2% with increasing flexible spacers in the repeating units. The spacer length also impacts on the mesomorphic properties. Whereas polymers P30(x) possess stiff main chains and therefore exhibit nematicity, their counterparts P32(x) with longer flexible spacer groups in the repeat units show better mesogenic packing and form more ordered smectic phases. For these main-chain polymeric LCs, owing to the high rigidity of the polymer chains, P30(x) and P31(x) exhibit nematic mesomorphic phases, whereas P32(x) with much longer flexible chains show smectic phases. Figure 2.7 illustrates temperature-variable wide-angle X-ray diffraction (WAXD) diagrams for P32(10), from which mesophase formation and the developing processes can be traced. Apparently, at 30  C, three peaks and one diffuse halo at 2u ¼ 2.63, 5.47, 7.77, and 20.76 are observed. When gradually heated to 130  C, the former three reflections disappear, revealing that the isotropic state is reached and the mesogens are now randomly dispersed. However, these peaks reappear upon cooling. As evidenced in Figure 2.7b, during the cooling process, at temperatures 100  C, three former reflections are distinctly

Figure 2.7 X-ray diffractograms of P32(10) at different temperatures obtained during the heating and cooling cycles. Reprinted with permission from [43], # 2011 American Chemical Society.

56 Aggregation-Induced Emission: Applications

Scheme 2.7 Structures of two molecular AIE-active liquid crystals (AIE-LCs) [44].

observed and even a sharp peak at 2u ¼ 20.21 . Further, the liquid crystalline feature is kept even at 40  C, at which temperature the glass state is formed. From the XRD profile, it is reasonable to assign its mesogenic phase as smectic. The successful fabrication of AIE-LCPs testifies to the feasibility of our strategy. However, the highly rigid and strong intermolecular interactions of the polymer chains mean that they can only form small domains. To improve the mobility of the molecules and obtain better and more regular mesophases, several molecular AIE-active liquid crystals (AIE-LCs) were designed and prepared. In our molecular design, TPE and 1-cyclohexyl-4-(phenylethynyl)benzene were employed as luminogenic and mesogenic units, respectively. Two examples of such molecules are shown in Scheme 2.7, where single and quadruple calamitic mesogens are attached to a TPE unit to yield 33 and 34, respectively. Thermal analysis and POM observations testify to their liquid crystalline attributes. Considering 34, for example, when cooled from its isotropic state to 190  C, an anisotropic fan-shaped mesomorphic texture emerges from the homotropic dark background (Figure 2.8a), suggesting its liquid crystalline nature and highly ordered molecular packing. On further cooling from such a mesomorphic phase to 100  C, it does not crystallize but transforms into a more ordered mesophase (Figure 2.8b). DSC analysis revealed two first-order phase transitions upon cooling and heating (Figure 2.8c and d). Two exothermic peaks at 194.2 and 140.8  C are observed for 34 at a cooling rate of 10  C min1, whereas two endothermic peaks are recorded at 143.1 and 201.0  C during the subsequent heating process. Clearly, the higher and lower temperature peaks are associated with the isotropic–LC and LC–LC mutual transitions, respectively. It is notable that the enthalpy change in the higher temperature transition (190  C) is as high as 26 kJ mol1 [44], implying that a highly ordered mesophase is formed upon cooling, which is consistent with the POM observations. Such high enthalpy is caused by the effective packing and intermolecular interactions between TPE cores. Further, even at room temperature, a liquid crystalline glass but not crystal is formed for 34. Further 1D and 2D WAXD patterns revealed a unique biaxially oriented mesomorphic structure of its mesophase. The AIE characteristics are well preserved in these liquid crystalline systems. The solution/solid-state efficiencies for 33 and 34 are 0.68/55.4% and 0.99/67.4%, respectively, indicating their AIE feature. The mesogenic and luminogenic properties of the molecules may enable them to find novel applications in advanced optoelectronic devices. For example, judicious utilization of highly fluorescent LCs may help to fabricate new optical display systems with a simplified device structure and wide viewing angle, while circumventing the use of power-hungry backlighting. Similarly, Lai and co-workers synthesized two 2,3,4,5-tetraphenylsilole-based molecules with long-chain alkoxydiacylamido groups (Scheme 2.8) [45]. Such compounds induce gelation of only hydrocarbon solvents and show AIE characteristics in the gel state, in contrast to the weak emission in solution. DSC and POM studies indicated that both compounds 35a and 35b exhibit stable liquid crystalline phases over a wide temperature range, thus proving that they are AIE-LCs. Interestingly, these new silole derivatives can

Crystallization-Induced Phosphorescence for Purely Organic Phosphors 57

Figure 2.8 POM images recorded on cooling 34 to (a) 190 and (b) 100  C from its isotropic state and DSC thermograms of 34 during (c) cooling and (d) subsequent heating cycles at different scan rates. Reprinted with permission from [44], # 2012 The Royal Society of Chemistry.

self-assemble into one-dimensional fibers and even two-dimensional molecular monolayered aggregates. Hydrogen bonding and p-stacking interactions are the main driving forces for the formation of such aggregates. These unique properties make them promising candidates in advanced applications.

2.3 Conclusions and Perspectives There is growing interest in high-efficiency room temperature phosphors and AIE-LCs owing to their potential applications in optoelectronic devices. The discovery of the CIP phenomenon for purely organic phosphors opens up a new approach for the fabrication of bright phosphors at room temperature through

Scheme 2.8 Structures of silole-based liquid crystals [45].

58 Aggregation-Induced Emission: Applications versatile crystal engineering. In crystals, the rotational, vibrational, and torsional deactivation paths are blocked via effective intermolecular interactions such as hydrogen bonds, halogen bonds, and C–H  p contacts. For twisted and planar phosphors, crystallization and cocrystallization strategies can be utilized to obtain efficient RTP. However, more general and detailed design strategies for the population of phosphors need to be further developed in the future. For AIE-LCs, the results clearly suggest the feasibility of combining the high efficiency of AIE-active luminogens and the mesomorphic properties of mesogens through rational molecular design. Future research may generate room temperature AIE-LCs that are more suitable for optical display applications. Alternatively, endowing a stimuli-responsive nature to the AIE-LCs may also generate numerous high-efficiency smart liquid crystalline materials for other applications.

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Crystallization-Induced Phosphorescence for Purely Organic Phosphors 59 18. Dong, Y., Lam, J.W.Y., Qin, A., Li, Z., Sun, J., Sung, H.H.-Y., Williams, I.D., and Tang, B.Z. (2007) Switching the light emission of (4-biphenylyl)phenyldibenzofulvene by morphological modulation: crystallization-induced emission enhancement. Chem. Commun., 40–42. 19. Yuan, W.Z., Shen, X.Y., Zhao, H., Lam, J.W.Y., Tang, L., Lu, P., Wang, C., Liu, Y., Wang, Z., Zheng, Q., Sun, J.Z., Ma, Y., and Tang, B.Z. (2010) Crystallization-induced phosphorescence of pure organic luminogens at room temperature. J. Phys. Chem. C, 114, 6090–6099. 20. Kearns, D.R., and Case, W.A. (1966) Investigation of singlet ! triplet transitions by the phosphorescence excitation method. III. Aromatic ketones and aldehydes. J. Am. Chem. Soc., 88, 5087–5097. 21. Lamola, A.A., and Hammond, G.S. (1965) Mechanisms of photochemical reactions in solution. XXXIII. Intersystem crossing efficiencies. J. Chem. Phys., 43, 2129–2135. 22. Adams, J.E., Mantulin, W.W., and Huber, J.R. (1973) Effect of molecular geometry on spin–orbit coupling of aromatic amines in solution. Diphenylamine, iminobibenzyl, acridan, and carbazole. J. Am. Chem. Soc., 95, 5477– 5481. 23. Schmidt, K., Brovelli, S., Coropceanu, V., Beljonne, D., Cornil, J., Bazzini, C., Caronna, T., Tubino, R., Meinardi, F., Shuai, Z., and Bredas, J.-L. (2007) Intersystem crossing processes in nonplanar aromatic heterocyclic molecules. J. Phys. Chem. A, 111, 10490–10499. 24. Bolton, O., Lee, K., Kim, H.-J., Lin, K.Y., and Kim, J. (2011) Activating efficient phosphorescence from purely organic materials by crystal design. Nat. Chem., 3, 205–210. 25. Yong, G.-P., Zhang, Y.-M., She, W.-L., and Li, Y.-Z. (2011) Stacking-induced white-light and blue-light phosphorescence from purely organic radical materials. J. Mater. Chem., 21, 18520–18522. 26. Yong, G., She, W., and Zhang, Y. (2012) Room-temperature phosphorescence in solution and in solid state from purely organic dyes. Dyes Pigments, 95, 161–167. 27. Gao, H.Y., Zhao, X.R., Wang, H., Pang, X., and Jin, W.J. (2012) Phosphorescent cocrystals assembled by 1,4-diiodotetrafluorobenzene and fluorene and its heterocyclic analogues based on CI  p halogen bonding. Cryst. Growth Des., 12, 4377–4387. 28. Shen, Q.J., Wei, H.Q., Zou, W.S., Sun, H.L., and Jin, W.J. (2012) Cocrystals assembled by pyrene and 1,2- or 1,4-diiodotetrafluorobenzenes and their phosphorescent behaviors modulated by local molecular environment. CrystEngComm, 14, 1010–1015. 29. Gao, H.Y., Shen, Q.J., Zhao, X.R., Yan, X.Q., Pang, X., and Jin, W.J. (2012) Phosphorescent co-crystal assembled by 1,4-diiodotetrafluorobenzene with carbazole based on C–I  p halogen bonding. J. Mater. Chem., 22, 5336–5343. 30. Shen, Q.J., Pang, X., Zhao, X.R., Gao, H.Y., Sun, H.-L., and Jin, W.J. (2012) Phosphorescent cocrystals constructed by 1,4-diiodotetrafluorobenzene and polyaromatic hydrocarbons based on C–I  p halogen bonding and other assisting weak interactions. CrystEngComm, 14, 5027–5034. 31. L€ussem, G., and Wendorff, J.H. (1998) Liquid crystalline materials for light-emitting diodes. Polym. Adv. Technol., 9, 443–460. 32. Camerel, F., Bonardi, L., Schmutz, M., and Ziessel, R. (2006) Highly luminescent gels and mesogens based on elaborated borondipyrromethenes. J. Am. Chem. Soc., 128, 4548–4549. 33. Perez, A., Serrano, J.L., Sierra, T., Ballesteros, A., de Saa, D., and Barluenga, J. (2011) Control of self-assembly of a 3-hexen-1,5-diyne derivative: toward soft materials with an aggregation-induced enhancement in emission. J. Am. Chem. Soc., 133, 8110–8113. 34. Sagara, Y., and Kato, T. (2008) Stimuli-responsive luminescent liquid crystals: change of photoluminescent colors triggered by a shear-induced phase transition. Angew. Chem., 120, 5253–5256. 35. Grell, M., and Bradley, D.D.C. (1999) Polarized luminescence from oriented molecular materials. Adv. Mater., 11, 895–905. 36. Hayasaka, H., Tamura, K., and Akagi, K. (2008) Dynamic switching of linearly polarized emission in liquidcrystallinity-embedded photoresponsive conjugated polymers. Macromolecules, 41, 2341–2346. 37. Weder, C., Sarwa, C., Montali, A., Bastiaansen, C., and Smith, P. (1998) Incorporation of photoluminescent polarizers into liquid crystal displays. Science, 279, 835–837. 38. Chen, S.H., Shi, H., Conger, B.M., Mastrangelo, J.C., and Tsutsui, T. (1996) Novel vitrifiable liquid crystals as optical materials. Adv. Mater., 8, 998–1001.

60 Aggregation-Induced Emission: Applications 39. Jenekhe, S.A., and Osaheni, J.A. (1994) Excimers and exciplexes of conjugated polymers. Science, 265, 765–768. 40. Lam, J.W.Y., and Tang, B.Z. (2003) Liquid-crystalline and light-emitting polyacetylenes. J. Polym. Sci. Part A: Polym. Chem., 41, 2607–2629. 41. Lam, J.W.Y., and Tang, B.Z. (2005) Functional polyacetylenes. Acc. Chem. Res., 38, 745–754. 42. Liu, J., Lam, J.W.Y., and Tang, B.Z. (2009) Acetylenic polymers: syntheses, structures, and functions. Chem. Rev., 109, 5799–5867. 43. Yuan, W.Z., Yu, Z.-Q., Tang, Y., Lam, J.W.Y., Xie, N., Lu, P., Chen, E.-Q., and Tang, B.Z. (2011) High solid-state efficiency fluorescent main chain liquid crystalline polytriazoles with aggregation-induced emission characteristics. Macromolecules, 44, 9618–9628. 44. Yuan, W.Z., Yu, Z.-Q., Lu, P., Deng, C., Lam, J.W.Y., Wang, Z., Chen, E.-Q., Ma, Y., and Tang, B.Z. (2012) High efficiency luminescent liquid crystal: aggregation-induced emission strategy and biaxially oriented mesomorphic structure. J. Mater. Chem., 22, 3323–3326. 45. Wan, J.-H., Mao, L.-Y., Li, Y.-B., Li, Z.-F., Qiu, H.-Y., Wang, C., and Lai, G.-Q. (2010) Self-assembly of novel fluorescent silole derivatives into different supramolecular aggregates: fibre, liquid crystal and monolayer. Soft Matter, 6, 3195–3201.

3 Mechanochromic Aggregation-Induced Emission Materials Zhenguo Chi and Jiarui Xu PCFM Laboratory and DSAPM Laboratory, State Key Laboratory of Optoelectronic Materials and Technologies, School of Chemistry and Chemical Engineering, Sun Yat-sen University, China

3.1 Introduction Mechanochromic fluorescent (mechanofluorochromic) materials change their emission colors (spectra) when an appropriate pressure or other mechanical force is applied. As a class of ‘smart’ materials, they possess mechanical responsiveness that provides a fundamental basis for fluorescence switches, mechanosensors, indicators of mechanohistory, security papers, optoelectronic devices, and data storage in various fields [1–4]. Mechanofluorochromic behavior can generally be achieved by either chemical or physical structural change. Although the modification of molecular structures containing open/closed cyclic forms [5] and double-bond E/Z configurations [6] is the most common approach for tuning the emissions of fluorescent compounds, limited success has been achieved in switching the fluorescence of solid-state materials with high efficiency and reproducibility [5, 7]. Chemical structural change is implemented using chemical reactions, such as bond breaking or formation at the molecular level. In these cases, a relatively high pressure is necessary to promote the chemical reactions [8]. Moreover, insufficient conversion, irreversible reactions, or loss of the fluorescence capability of the compound may frequently occur during solid-state chemical reactions, which have been considered a drawback in such systems. By contrast, the fluorescent properties of molecules in the solid state depend on molecular arrangement, conformational flexibility, and intermolecular interactions. Any modification of the molecular packing and conformation of the fluorophore would affect the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels and alter the fluorescent properties. Therefore, controlling the mode of molecular packing (aggregation states) to achieve dynamic control of highly efficient and reversible solidstate fluorescence is more attractive for both fundamental research and practical applications because of the low pressure demand and good reversibility. However, organic mechanofluorochromic materials that are Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

62 Aggregation-Induced Emission: Applications dependent on changes in physical molecular packing modes are extremely rare. This rarity may be attributed to two major issues [2]. First, predicting and designing materials that exhibit polymorphism are difficult. Each identified compound is an isolated event, which makes the identification of a general characteristic difficult. Second, the fluorescence efficiency of organic fluorescent materials often becomes very weak when they are in the solid state because of the aggregation-caused quenching (ACQ) effect. Consequently, observing the mechanofluorochromic phenomenon becomes difficult. In 2001, Tang and co-workers [9] reported on aggregation-induced emission (AIE) materials, which are an important class of anti-ACQ materials that emit more efficiently when they are in the aggregated state than in the dissolved form. Since then, AIE materials have attracted considerable research attention owing to their potential application in various fields, such as organic light-emitting devices (OLEDs) and chemosensors [10–19]. A number of AIE compounds with different AIE moieties have been found to possess mechanochromic emission properties. Hence the use of AIE is important in the synthesis of various mechanofluorochromic materials. The chapter summarizes recent advances in organic mechanochromic fluorescent materials, which include mechanochromic non-AIE and mechanochromic AIE materials.

3.2 Mechanochromic Non-AIE Compounds Dye-doped polymers are a representative family of mechanochromic fluorescent materials. Depending on the dye self-assembly characteristics and miscibility with polymers, fluorophores are generally believed to start to aggregate into supramolecular assemblies above a particular concentration through weak noncovalent interactions among the planar aromatic backbones, such as p–p interactions. Upon tensile deformation, the macromolecular chain slippage and reorganization promote the breakdown of noncovalent interactions among fluorophore molecules (Scheme 3.1, dyes 1 and 2) and their molecular mixing within the polymer matrix, which results in the transformation from an excimer to a monomer and significant blue shift in emission (Figure 3.1). However, not all dye-doped polymer systems have mechanochromic fluorescent properties because of the complexity of molecular interactions not only between dye molecules but also between dye molecules and matrix polymer molecules [3]. Based on the strict criteria for dye and polymer matrices, mechanochromic fluorescent polymer blends are rather limited [20–28]. As mentioned above, although dye-doped polymers are a representative family of mechanofluorochromic materials, reports on the successful preparation of these polymer systems are very limited. Therefore, a great deal of attention has been paid to the mechanofluorochromic properties of single-component small organic dye molecules. Pyrene-based dye 3 [29], liquid crystalline dye 4 [30, 31], heteropolycyclic dye 5 [32, 33], difluoroboron b-diketonate derivative 6 [34–37], polypeptide-based dendron 7 [38, 39], triphenylamine aldehyde dye 8 [40], dibenzofuran derivative 9 [41], anthrylpyrazole derivative 10 [42], diphenylacrylonitrile derivative 11 [43], cholesterol-appended quinacridone derivative 12 [44], perylene bisimide dye 13 [45], naphthoisoindole derivative 14 [46], and dicyanodistyrylbenzene derivative 15 [47] (Scheme 3.2) were studied in depth CN

OMe

CN

MeO

MeO OMe MeO

1

NC

OMe

2 Scheme 3.1

NC

Mechanochromic Aggregation-Induced Emission Materials 63

Figure 3.1 Emission images of the blends (a) linear low-density polyethylene (LLDPE)–dye 1 and (b) LLDPE–dye 2 stretched at room temperature under UV irradiation (365 nm). Reproduced with permission from [2], # 2012 The Royal Society of Chemistry.

O

C12H25Ο

O

N H

ΟC12H25

N H

O

C12H25Ο

ΟC12H25

O

O

O

O

C12H25Ο O

3

O

HN

Ar

C12H25Ο F

O

F B

O

5

R1

O O

Ar= pyrenyl, anthryl

ΟC12H25

O

O C12H25Ο

R2

6

O

ΟC12H25

O O

O

4

O

N

NH

O

O

O N

ΟC12H25 O

R1

R2

O O

H N

H N

C12H25O

ΟC12H25

R1, R2 = H, alkyl, alkoxy, aryl, etc. H3C

NBu2

R1 = H, CN, COOMe, COOH, t-butyl N

O

R2 = H, n-butyl, benzyl, 5-nonyl

N N

CHO

N Ο

Ο HN

Ο

H N

Ο N H

Ο Ο

7

Si

Ο

N

Ο

CF3 N

H

NC

Ο F3C

Ο Ο

Si Si O O Si O Si

O

O

N

N

O

O

13

Si Si O O O Si Si

CF3 N

NC O

O O O

N C4H9

Si O Si OO Si Si

N

R

CN

15

N

O

O

14

R= donor group

Scheme 3.2

F3C

CF3

H

N

nN

N n

11

F3C

Si O O O Si

O

CF3

Ο

O

O

H H

CN N H

S

10

F3C

HN Ο

Si

Ο

9

8

H3C

H N

N N

O

N

O

O

12

n = 4-10

O

H

H

64 Aggregation-Induced Emission: Applications and it was found that they showed mechanofluorochromism. However, no correlation with molecular structure was observed. Each identified compound is an isolated event, which makes the identification of a general characteristic difficult. This is why few organic small molecules with mechanofluorochromic properties were reported. However, in recent years, the new finding that AIE compounds have mechanochromic properties has changed everything.

3.3 Mechanochromic AIE Compounds As a class of stimuli-responsive materials, AIE compounds exhibit an off–on switching property of emission intensity by external stimuli, such as organic solvent vapor, pressure, heat, and so on [48–51]. Tang and co-workers reported that several AIE compounds possess bright–dark switching properties between crystalline and amorphous states [52, 53], which is a basis for the theoretical explanation of the mechanochromic phenomena of many AIE compounds. In 2008, to investigate the AIE mechanism further, pressure stimuli were first applied to hexaphenylsilole film and its device [54]. It was found that the photoluminescence (PL) and electroluminescence (EL) of hexaphenylsilole were enhanced by pressurization in the solid state, which, to the best of our knowledge, was the first attempt to carry out a mechanochromic investigation of an AIE compound. The cyano-substituted distyrylbenzene fluorescence core has often been used to prepare AIE compounds. Dye 16 (Scheme 3.3) [55] has a highly enhanced fluorescence emission in the solid state compared with that in solution that exhibits AIE properties. In the solid state, the dye formed highly fluorescent ‘molecular sheets,’ which were assisted by multiple C

H  N and C

H  O hydrogen bonds with stacking and shear-sliding capabilities via external stimuli. The ‘molecular sheets’ exhibited two-color fluorescence that switched in response to pressure, temperature, and solvent vapor. Based on structural, optical, photophysical, and computational studies, two different phases were identified, the metastable green-emitting G-phase and the thermodynamically stable blue-emitting Bphase. In the G-phase, the antiparallel coupling of the local dipoles kinetically stabilized a structure with moderate excitonic coupling but with efficient excimer formation. Upon annealing, a smooth slip of the molecular sheets with a low activation barrier formed the B-phase with a head-to-tail arrangement of the local dipoles (Figure 3.2). The dye was successfully fabricated as a rewritable fluorescent optical recording medium, that is, a poly(methyl methacrylate) (PMMA)–dye 16 blend film, which exhibited fast-responding and reversible multi-stimuli fluorescence switching (Figure 3.3). Yoon and Park [56] designed and synthesized dyes 17 and 18 (Scheme 3.3) to investigate the change in the mechanochromic phase via direct crystalline structure analysis. The two dyes possess simpler molecular CN

O

O

CN

16 CN

CN

NC

NC

18

17 Scheme 3.3

Mechanochromic Aggregation-Induced Emission Materials 65

Figure 3.2 Illustration of two different modes of slip-stacking in dye 16 molecular sheets, dictated by different ways of antiparallel/head-to-tail coupling of local dipoles. Reproduced with permission from [55], # 2010 American Chemical Society.

Figure 3.3 Photographs of the fluorescence writing/erasing cycle of dye 16–PMMA film. Reproduced with permission from [55], # 2010 American Chemical Society.

structures than dye 16 and were expected to possess highly crystalline properties, which was advantageous in preparing single crystals. Both dyes exhibited very high solid-state fluorescence quantum yields because of the characteristics of the AIE properties and also their capacity for polymorphic two-color emission. The polymorphic fluorescent phases were obtained using various sample preparation methods, such as suspension preparation, drop casting, solution recrystallization, melt solidification, thermal annealing, and mechanical grinding. Dye 17 undergoes a phase transition from phase I with a short emission wavelength to phase II with a long emission wavelength. Based on the analysis of single-crystal structures, the head-to-tail coupling of the local dipoles and the multiple C

H  p and C

H  N interactions in phase I were responsible for the specific molecular stacking architecture with weak excited-state dimeric coupling. In phase II, the crystals exhibited efficient excited-state dimeric coupling that was attributed to the substantial p–p overlap, which includes the antiparallel coupling of local dipoles. The different molecular packing structures produced differently colored

66 Aggregation-Induced Emission: Applications fluorescence emission. The reciprocal transformation between phases I and II with the application of thermal and mechanical stimuli resulted in reversible changes in the emission colors. Although the aforementioned mechanochromic AIE systems were reported, reading between the lines in the references, the existence of a structural relationship between the AIE compound and the mechanochromic nature was not completely recognized. Almost at the same time, we synthesized and reported a number of new mechanochromic compounds with AIE properties. These compounds were called piezofluorochromic AIE (PAIE) compounds at that time. A possible mechanism was also proposed to explain the PAIE phenomenon. Numerous reported AIE compounds such as typical triphenylethylene, tetraphenylethylene, silole, and cyanodistyrylbenzene derivatives [14, 57–61] possess a common structural feature that is characterized by multiple phenyl peripheries that are linked to an olefinic core via rotatable carbon–carbon single bonds to form an AIE moiety. The steric effect between the phenyl rings forces the AIE moieties or the molecules to have a twisted conformation. This twisted conformation and the weak p–p interactions cause the molecular packing to be relatively loose and generate a number of defects (cavities) that result in a low lattice energy. The cavities are the weakest parts of the crystalline structures. Two structural features, the low lattice energy and cavity formation, caused the crystal to be easily destroyed by the planarization of the molecular conformation or slip deformation when an external pressure is applied. The planarization of molecular conformation resulted in increased molecular conjugation, which caused a red shift in the PL spectrum [62]. Such a mechanism is entirely different from that proposed by Park and co-workers [55]. Based on the aforementioned hypothesis, a number of AIE compounds synthesized in our laboratory exhibited mechanofluorochromism. The established common structure–property relationships will be helpful in identifying and synthesizing more novel mechanofluorochromic materials. The butterfly-shaped AIE dyes 19 and 20 derived from tetraphenylethylene and carbazole (Scheme 3.4) were synthesized by Chi and co-workers [63]. Two different aggregates were obtained from the solutions of dye 19 in different solvent systems via rotary evaporation. The sample obtained from dichloromethane–nhexane mixed solvent (1:3 v/v) solution was a white crystalline aggregate with a strong blue emission (451 nm). The sample obtained from the dichloromethane solution was a light-green amorphous aggregate with a strong green emission (479 nm). The results indicated that dye 19 has a better polymorph-forming

N

19

20

N

Scheme 3.4

Mechanochromic Aggregation-Induced Emission Materials 67 ability. However, dye 20 only produced a blue-emitting crystal under the same concentration conditions. When crystalline samples of dye 19 were briefly pressed in an infrared pellet at 1500 psi for 1 min, ground using a mortar and pestle, or when their melt was quenched by liquid nitrogen, all samples were converted to their amorphous form. The emission of the pressed (ground) sample was at 479 nm, whereas that of the quenched sample was at 493 nm. hence the quenched sample had a longer PL wavelength than the pressed sample. The results indicated that dye 19 possessed solid morphology-alterable emission and mechanofluorochromic properties. However, dye 20 had no such properties because its molecules had excellent crystallization capability. Although dye 20 evaporated from either dichloromethane or dichloromethane–n-hexane (1:3 v/v) solutions, it was always formed in the crystalline state and not in the amorphous state. In other words, if an AIE compound has a strong crystallizability to form stable crystals, no such change from the crystalline to amorphous phase will occur. Therefore, the compound would not be mechanofluorochromic. The PL peak intensity of the ground sample decreased rapidly from 30 to 120 s when fumed with dichloromethane. The intensity gradually increased with prolonged fuming time after 120 s (Figure 3.4). The permeation of a good solvent during fuming could weaken the interaction of the packing molecules because of solvation, which results in increased intramolecular rotational and vibrational motions, increased nonemissive decay of the excited-state energy, and decreased PL intensity. Simultaneously, the molecules underwent a solvent-induced crystallization process. As the degree of crystallization increased, the intramolecular vibrations and rotations were gradually restricted. Moreover, the nonemissive decay of the excited-state energy gradually weakened, which resulted in a substantial increase in PL intensity. Thus, the two opposite effects resulted in a V-shaped curve depending on which effect played the dominant role in the overall PL behavior [63]. This finding indicated that (1) the crystallization of the ground amorphous sample could be induced by solvent vapor to achieve a reversible change from the amorphous to crystalline state and (2) the amorphous phase was a metastable phase and was immediately converted to a more stable crystalline phase via solvent-induced crystallization. Single-crystal analysis results indicated that the molecules were packed via the synergetic effect of weak p–p and C

H  p interactions that form lamellar layer structures. The layers were connected via the antenna parts of the butterfly-shaped molecules with weak p–p interactions (partially p-overlapping). The

Figure 3.4 PL peak intensity and wavelength of dye 19 versus fuming time with dichloromethane. Reproduced with permission from [63], # 2012 The Royal Society of Chemistry.

68 Aggregation-Induced Emission: Applications

Figure 3.5 Molecular packing of dye 19 in single crystals: (a) capped sticks style and (b) spacefill style showing inclusion of dichloromethane–n-hexane between the layers (the hydrogen atoms have been omitted for clarity). Reproduced with permission from [63], # 2012 The Royal Society of Chemistry.

interfaces between the layers were relatively loose, and a number of defects (cavities) were formed that were filled with solvent molecules (Figure 3.5). As mentioned earlier, the structural features caused the crystal to be easily destroyed by the planarization of the molecular conformation or slip deformation when an external pressure is applied, which results in mechanofluorochromism. As mentioned previously, the structure of dye 19 crystals has a relatively loose molecular packing and contains a number of defects. This finding was supported by a study of dye 21 (Scheme 3.5), which was

NC

CN NC

Si

21 Scheme 3.5

CN

Mechanochromic Aggregation-Induced Emission Materials 69

Figure 3.6 X-ray crystal structures of dye 21 for (a) crystal R at 173 K, perspective view of the framework and the one-dimensional channel in the c-axis; the guest acetone molecules inhabit the one-dimensional channels highlighted in the inset; (b) crystal O at 133 K, a perspective view in the azimuth angle of
45 to the a-axis to show the guest hexane molecules in the one-dimensional channels. Adapted with permission from [64], # 2012 The Royal Society of Chemistry.

designed and synthesized by Tang and co-workers in 2012 [64]. Dye 21 is a multi-substituted silole that contains 1-phenyl-2,2-dicyanoethene moieties. Such a strategy is helpful in designing and constructing organic soft porous crystals with other conjugated building blocks and in developing novel smart and stimuli-responsive photo/electronic materials. Two single crystals (O, orange and R, red) were successfully obtained in appropriate conditions. The crystallographic data indicated that crystals O and R had reasonable hollow structures inside, in which different solvent molecules were selectively encapsulated (Figure 3.6). Multi-stable crystalline states with different fluorescent colors increased the potential of dye 21 to respond to external stimuli. As expected, the orange powder was dried in a vacuum oven and subsequently changed into a yellow–orange solid, which had a yellow–orange emission color (YO-form, lem ¼ 576.5 nm). After grinding, the YO-form was converted to a vivid cherry-colored solid that showed strong red fluorescence upon UV irradiation (R-form, lem ¼ 600.5 nm). After thermal annealing at 150  C (below its melting point, 228  C), the red solid immediately turned yellow–orange. The YO-form solid recovered from the R-form continued to emit an intense yellow–orange fluorescence (lem ¼ 566 nm), which is approximately equal to that of the original O-form. The mechanochromic luminescence was readily reproducible during the cycle of the grinding–annealing operation. The typical X-ray diffraction (XRD) pattern of the pristine YO-form solid exhibited a number of recognizable diffraction peaks that can be ascribed to a partially crystallized solid. After grinding, most diffraction peaks disappeared or became vague, while a number of weak new peaks at different diffraction angles emerged. This phenomenon indicated that the initial crystal lattice was significantly disrupted by mechanical force, and the crystalline size of the new polymorph is fairly small. When the ground R-form crystal was heated, the solid again turned into the YO-form with a recovered XRD pattern. Hence the mechanofluorochromism of dye 21 should also be ascribed to a reversible change of molecular packing or the transformation of crystal forms that was induced by grinding and heating. As discussed previously, the crystals formed from dye 20 are very stable because of its strong crystallizability. Hence the change from the crystalline to the amorphous phase is difficult, which results in nonmechanofluorochromism. The initial state is important for mechanofluorochromism. If a dye is noncrystalline, the mechanofluorochromic phenomenon is often difficult to determine because no solid phase exists with a degree of order worse than the amorphous structure. This hypothesis was confirmed by experimental results on the series of dyes 22–25 (Scheme 3.6), which were designed and synthesized by Chi and co-workers in 2011 [65]. All compounds were AIE active because they contain a triphenylethylene or distyrylanthracene AIE moiety. The distyrylanthracene derivatives 22 and 24 exhibited obvious mechanofluorochromic phenomena (Figure 3.7). Before and after grinding, the emission wavelength increased from 534 to

70 Aggregation-Induced Emission: Applications

N N N N N N

23

N

22

N

N N N N

N

N N

N N N

N

25

N N

N N

N N

24 N N N

Scheme 3.6

572 nm and from 566 to 580 nm for dyes 22 and 24, respectively. However, the diphenylanthracene derivatives 23 and 25 were non-mechanofluorochromic. The initial states of dyes 22 and 24 were crystalline to some extent, whereas those of dyes 23 and 25 were amorphous, as confirmed by XRD. No room for further development upon pressure application was observed for the packing structures of the initially amorphous dyes 23 and 25 because mechanofluorochromic behavior depends on the packing change from the crystalline to the amorphous state. Consequently, the two compounds were not mechanofluorochromic. In this study, the existence of a relatively stable crystalline state is a prerequisite for mechanofluorochromism. In other words, AIE compounds with strong crystallizability or noncrystallizability are unsuitable for use as mechanofluorochromic materials. Therefore, designing mechanofluorochromic compounds remains a crucial issue. Dyes 26, 27, and 28 (Scheme 3.7), which contain triphenylamine–anthrylenevinylene and tetraphenylethene moieties with AIE natures, were also synthesized by Chi and co-workers [66]. When the as-synthesized sample of dye 27 was briefly ground using a mortar and pestle, the luminescence of the sample changed from yellow (561 nm) to orange–red (583 nm). This result indicated that the compound was mechanofluorochromism active. Although the molecular structures of the three compounds were very similar, only dye 27 exhibited an obvious mechanofluorochromic behavior. The emission spectra of dyes 26 and 28 showed minimal changes before and after grinding. In the former case there was some degree of crystallizability, whereas in the latter the structure was almost amorphous, as confirmed by the XRD results. The results indicated that the dependence of the initial state on molecular structure is significant for mechanofluorochromism.

Mechanochromic Aggregation-Induced Emission Materials 71

Figure 3.7 Images of (a) dye 22 and (b) dye 24 taken at room temperature under 365 nm UV irradiation: (left) assynthesized samples or annealed samples (at 300  C, for 5 min); (right) pressed (1500 psi for 5 min) or ground sample. Dye 22 (c) and dye 24 (d) were cast on filter-paper and ‘SU’ and ‘AIE’ were written with a metal spatula at room temperature under ambient light (left) and UV light (right). Reproduced with permission from [65], # 2011 American Chemical Society.

For AIE dye 16, the two-color fluorescence switching behavior was explained by the interchange between the metastable green-emitting G-phase and the thermodynamically stable blue-emitting B-phase with different modes of local dipole coupling (antiparallel and head-to-tail arrangements, respectively). The interchange was facilitated by the two-directional shear-sliding capability of molecular sheets formed via intermolecular multiple C

H  N and C

H  O hydrogen bonds. The structure of dye 29 had no such hydrogen bonds. If the dye is mechanofluorochromism active, the mechanism proposed by Park and coworkers [55] is not suitable for explaining this mechanofluorochromic phenomenon. The mechanofluorochromic behavior of this class of AIE compounds could be explained by the proposed mechanism based on the planarization of the AIE molecular conformation. Dye 29 (Scheme 3.8), which was derived from tetraphenylethylene and divinylanthracene, was designed and synthesized by Chi and co-workers [67]. The dye is AIE active because it contains tetraphenylethylene and distyrylanthracene AIE units. The dye exhibited significant mechanofluorochromic activity. After grinding, the emission wavelength increased from 506 to 574 nm (red shifted by 68 nm). The XRD results indicated that the mechanofluorochromism resulted from the reversible morphological change between the crystalline and amorphous structures. The differential scanning calorimetry (DSC) results showed that the pressed sample had a significant cold-crystallization peak at 336  C. This peak indicated the existence of a

72 Aggregation-Induced Emission: Applications

N N

26

N N

27

N

N

28

Scheme 3.7

metastable-state aggregate in the pressed sample, which could be converted into a more stable state through annealing. Cold-crystallization transition of the pressed sample seems to be a common feature of numerous mechanofluorochromic compounds. Subsequently, Chi and co-workers [68] successfully synthesized two distyrylanthracene derivatives capped with triphenylethylene groups (dyes 30 and 31), and another derivative capped with tetraphenylethylene groups (dye 32) (Scheme 3.8). Similarly to dye 29, all compounds exhibited strong AIE activities. However, only the derivatives capped with tetraphenylethylene groups (29 and 32) showed significant mechanofluorochromic properties and the ability to revert to their original forms upon annealing because of the crystalline–amorphous phase transformation (Figure 3.8). AIE compounds that contain sterically hindering groups can potentially exhibit distinct mechanofluorochromic activities. This was further confirmed

Mechanochromic Aggregation-Induced Emission Materials 73

30

29

31 32

Scheme 3.8

by the different mechanofluorochromic properties of the AIE dyes 33–35 (Scheme 3.9) [69]. Dye 33 had no mechanofluorochromic property. However, significant red shifts of up to 24 and 32 nm were observed in dyes 34 and 35, respectively, after grinding. The AIE dye 36 (Scheme 3.10) studied by Chi and co-workers [70] had neither a heteroatom nor C

H  N and C

H  O hydrogen bond interactions in its molecular structure, similarly to dye 29.

Figure 3.8 Images of 30 (a), 31 (b), 29 (c) and 32 (d) taken at room temperature under (left column) natural light and (right column) UV light after pressing and annealing. Reproduced with permission from [68], # 2012 The Royal Society of Chemistry.

74 Aggregation-Induced Emission: Applications

N N

33

34

N N N N N N

35

Scheme 3.9

36

37

38 Scheme 3.10

Nevertheless, the dye possessed a mechanofluorochromic nature. However, the analogs of AIE dye 36, namely AIE dyes 37 and 38, do not exhibit mechanofluorochromic phenomena. The XRD results demonstrated the discrepancy that resulted from the different morphologies of the dyes, namely that the analogs were amorphous and dye 36 was crystalline (Figure 3.9). The molecular conformation and packing in the

Mechanochromic Aggregation-Induced Emission Materials 75

Figure 3.9 XRD curves of the as-synthesized sample of dyes (a) 36, (b) 37, and (c) 38. Reproduced with permission from [70], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

crystal were obtained through single-crystal analysis. The dihedral angle values between the aryl rings in the single crystal indicated that the molecule possessed a very twisted conformation in both the free and crystalline states. The values of the dihedral angles in the single crystal were greater than those in the isolated free molecule, and the difference suggested the existence of a strong twist stress for the molecule in the crystalline state. The typical cofacial p–p stacking of the molecules was practically impossible because of the highly twisted conformation. The molecules were packed via weak C

H  p interactions in the crystal cell, which led to the relative looseness of the molecular packings. Such looseness resulted in the formation of several cavities. This feature of the crystal structure enabled the compound to exhibit pronounced mechanofluorochromism. The PL spectrum of the as-synthesized dye 36 sample red shifted from 454 m to 482 nm after grinding. The spectroscopic properties and morphological structures of dye 39 (Scheme 3.11) reported by Chi and co-workers [71] were reversibly and repeatedly exhibited upon pressing, grinding, annealing, or fuming. The mechanofluorochromic nature was also generated through phase transformation, which was confirmed by XRD and DSC. The crystalline structure showed that the dye molecules were packed in a head-to-head manner. The backbone of the molecule deviated considerably from the plane. Typical cofacial p–p stacking became impossible because of the highly twisted conformation and the steric hindrance of the bulky

S N

F3C CN

N

N F3C

S

40 39 Scheme 3.11

76 Aggregation-Induced Emission: Applications phenothiazinyl groups in the molecule. The molecules were packed in clusters via weak C

H  S, S  p, and C

H  p interactions. The former was also used to bind the clusters to form lamellar layers. These layers were connected via weak and sparse p–p interactions and partial p-overlaps from the phenyl rings in the phenothiazinyl groups. Hence the layer–layer and cluster–cluster interfaces were readily destroyed via slip deformation by an external force or stimulus, which facilitated mechanofluorochromism. The AIE dye 40 (Scheme 3.11) with a D–A electron structure was synthesized by Wang and co-workers [72]. The fluorescent colors of this dye can be conveniently switched using various environmental stimuli, which include mechanical force, organic vapor, heat, acid, and base. Grinding and heating treatments effectively induced fluorescence changes from red–orange to yellow by tuning the molecular packing in the solid state. XRD measurements confirmed that the unground sample was a well-ordered microcrystalline-like structure, whereas the ground sample was amorphous. Upon exposure to trifluoroacetic acid (TFA) vapor, the sublimed or heated sample (orange) was converted into a blue emissive state, which could be recovered by triethylamine (NEt3) vapor. A chloroform solution of dye 40 emitted nearly unobservable fluorescence at room temperature and intense yellowish green fluorescence in frozen conditions (77 K). After addition of TFA, the solution changed into a weak green emissive state. This phenomenon is considered to be the effect of protonation. Two crystallization-induced emission enhancements (also a typical AIE phenomenon), shown by dyes 41 and 42 (Scheme 3.12), were reported by Dong and co-workers [73]. Dye 41 forms two types of crystals that emit green and yellow light with quantum yields of 82.1 and 56.2%, respectively. By contrast, the amorphous state of the crystal emitted rather weak orange light with an efficiency lower than 1%. The fluorescence of the crystals of both 41 and 42 could be turned into ‘dark’ and ‘bright’ upon grinding and annealing. The yellow-emitting crystal could be converted into a green-emitting crystal by heating. The emission of dye 41 could be repeatedly switched between ‘dark’ and ‘bright’ by controlling the transition between the amorphous state and the green-emitting crystal via heating and cooling. The emission of the ground sample of dye 41 could spontaneously return to green emission at room temperature, whereas ground dye 42 remained dark for over 24 h under the same conditions. This phenomenon resulted from the different molecular flexibilities that governed the capability for molecular motion in the solid state. Sun and co-workers [74] reported on E and Z stereoisomers of dyes 43 and 44 (Scheme 3.13). The molecules were expected to be mechanofluorochromically active because of their propeller-shaped tetraphenylethylene moiety. Moreover, the molecules exhibited multiple chromic effects, which included mechano-, thermo-, vapo-, and chronochromisms. The as-synthesized dye 43 was an offwhite solid with a blue emission (447 nm). This dye changed to a pale-yellow powder with a bluish green emission (477 nm) after grinding, which showed a mechanofluorochromic effect. The ground sample was transformed back to the off-white solid with a blue emission after heating at 120  C for

O

O

42

41 Scheme 3.12

Mechanochromic Aggregation-Induced Emission Materials 77 Ν Ν Ν (CH2)6O O(H2C)6

Ν Ν Ν

Ν Ν Ν

(CH2)6O Ν

43

Ν Ν

(CH2)6O

44

Scheme 3.13

1 min. The as-synthesized dye 44 was a pale-yellow solid with a bluish green emission (460 nm). Mechanical grinding of the solid caused little change in the physical appearance or emission color. The grinding shifted its emission peak from 460 to 470 nm, which indicated that grinding a mainly amorphous solid might not result in significant changes in the morphologic structure and in the fluorescence spectrum. The XRD patterns of the as-prepared solid of the dyes demonstrated that the crystallization capability of the Z-isomer was lower than that of the E-isomer, which accounted for their obviously different chromic behaviors. The mechano- and thermochromisms were associated with the morphology transformations between the crystalline and amorphous phases. In addition to grinding, pressurization could also cause a red shift in the PL spectrum of the E-isomer, although it was small (8 nm). The significant difference in the extents of the mechanochromic effects indicated that shearing (grinding) was highly efficient in causing a larger change in the morphological structure and emission spectrum compared with compression (pressurization). The isomer showed a novel chronochromic phenomenon, in which its emission spectrum changed with time. The chronochromism indicated that the ground sample was in a metastable state, which slowly transformed back to the thermodynamically stable crystalline state at room temperature. Dye 43 also exhibited a solventdependent vapochromic effect. The ground sample of this dye was sensitive to volatile polar solvents, such as chloroform, dichloromethane, and tetrahydrofuran. After exposure to chloroform vapor for 1 min, the bluish green emission of the ground sample quickly reverted to the blue emission of its crystals caused by the solvent-induced crystallization. Zhang and co-workers [75] reported a series of cyanostilbene derivatives and found that the recrystallization of dye 45 (Scheme 3.14) from ethanol resulted in a light-blue powder with a faint luminescence (FF 10 (Table 4.1, entries 2–6).

Figure 4.1 Change of fluorescence spectra of L-6 (2.0  10
3 M) with increasing content of water in ethanol. Inset: fluorescence intensity versus water content. Reproduced with permission from [18], # 2009 American Chemical Society.

90 Aggregation-Induced Emission: Applications Table 4.1 Enantioselectivity and state of mixtures of amine enantiomers with L-6 in aqueous solution Entry 1

Amine NH2

HO Ph

2 3

I(1R,2S)-7/I(1S,2R)-7

262

Sus/Sol

I(2S)-8/I(2R)-8

10

Sus/Sol

I(1R,2R)-9/I(1S,2S)-9

18

Sus/Sol

I(2R)-10/I(2S)-10

17

Sus/Sol

I(2R)-11/I(2S)-11

455

Sus/Sol

I(R,R)-12/I(S,S)-12

18

Sus/Sol

Ph

7

NH2

HO H

Statea

Enantioselectivity

Ph 8

NH2

H2N Ph

9

4

Ph

NH2 H3C

Ph

10

5

O N H

NH2

11

6

NH2 NH2

12 a

Enantiomer 1/enantiomer 2; Sus ¼ suspension; Sol ¼ solution.

Under the same conditions as for L-6, interaction of D-6 with the chiral amines led to opposite results. For instance, mixture of D-6 with (1S,2R)-7 formed a suspension whereas that of D-6 with (1R,2S)-7 gave rise to a clear solution in aqueous ethanol. Therefore, the enantioselective aggregation was the result of inherent chiral recognition by the chiral receptor. The fluorescence intensity of mixture of L-6 and the two enantiomers of 7 increased with increase in the molar percentage of one enantiomer (1R,2S)-7 when the total concentration of mixture of the two enantiomers of 7 was kept constant. Similarly, the fluorescence intensity of a mixture of D-6 and the two enantiomers of 7 increased with increase in the molar percentage of one enantiomer (1S,2R)-7 (Figure 4.3). As a result, two calibration curves were drawn from which the enantiomeric L-6+(1R,2S )-7

Intensity (a. u.)

400

300 L-6+(1S,2R )-7

200

100

L-6+(1R,2S )-7

L-6+(1S,2R )-7

X10

0 400

500 Wavelength (nm)

600

Figure 4.2 Fluorescence spectra of a mixture of L-6 and enantiomers of chiral amine 7 in aqueous ethanol. Inset: photographs of suspension and solution illuminated with a portable UV lamp. Reproduced with permission from [18], # 2009 American Chemical Society.

Chiral Recognition and Enantiomeric Excess Determination 91

Figure 4.3 Change of fluorescence intensity of a mixture of L-6 and 7 (molar ratio 1:1) with enantiomer content. [L-6] ¼ 5  10
4 M; the total concentration of the two enantiomers of 7 was 5  10
4 M). Reproduced with permission from [18], # 2009 American Chemical Society.

composition of chiral amine 7 could be obtained [18]. Using the same method, the enantiomeric composition of other amines such as 8, 9, 10, 11, and 12 could also be determined. This result indicated potential application in high-throughput analysis of the enantiomer purity of chiral amines.

4.3 Chiral Recognition and Enantiomeric Excess Determination of Chiral Acids 4.3.1 Enantiomeric excess determination of chiral acids using chiral AIE amines In order to recognize the enantiomers of chiral carboxylic acids, the chiral AIE amines 13 and 14 containing an optically pure diphenylaminoethanol group were synthesized by a method similar to that used for 6 (Scheme 4.2). The chiral AIE amine 13 could efficiently differentiate the enantiomers of mandelic acid (15) and 2,3-dibenzoyltartaric acid (22). Whereas a mixture of (1S,2R)-13 and (S)-15 in 1,2-dichloroethane led to a suspension, a mixture of (1S,2R)-13 and (R)-15 remained a clear solution. Under the same conditions, mixing (1S,2R)-13 and D-22 gave a suspension, but mixing (1S,2R)-13 and L-22 led to a clear solution. The fluorescence of the suspension was stronger than that of the solution, which resulted in high enantiomer selectivity of up to 598 [I(S)-8/I(R)-8] and 196 [ID-22/IL-22], respectively. The enantiomeric composition could be determined from a calibration curve of fluorescence intensity change with one enantiomer content. The chiral receptor 13 failed to discriminate between enantiomers of other convenient chiral carboxylic acids [20].

Scheme 4.2 Structure of chiral AIE amines containing a-aromatic cinnamonitrile fluorophore.

92 Aggregation-Induced Emission: Applications When an a-aromatic cinnamonitrile fluorophore was attached with a chiral cyclohexyldiamine group, the resultant chiral AIE amine 14 showed not only exceptionally high enantioselectivity but also broad applicability for a large variety of chiral carboxylic acids. For three standard chiral a-hydroxycarboxylic acids, mandelic acid (15), 2-chloromandelic acid (16) and 3-phenyllactic acid (17), (R,R)-14 could differentiate their enantiomers with very high enantioselectivity. A mixture of (S)-15 or (R)-16 with (R,R)-14 led to precipitates, but a mixture of (R)-15 or (S)-16 with (R,R)-14 in 1,2-dichloroethane remained in solution. The fluorescence intensity ratios (IS/IR) for the two enantiomers of 15 and 16 were 16 865 and 261, respectively. For 3-phenyllactic acid (17), a mixture of (S)-17 and (R,R)-14 gave a suspension and a mixture of (R)-17 with (R,R)-14 led to a clear solution, which resulted in a fluorescence intensity ratio [I(S)-17/I(R)-17] of 1000 (Table 4.2, entries 1-3) [21]. For chiral carboxylic acids bearing methyl, alkoxy, amide, or acyloxy instead of a hydroxyl group at the a-position, (R,R)-14 could also differentiate their enantiomers (Table 4.2, entries 4–7). Upon mixing with (R,R)-14, one enantiomer of naproxen (18), 2-tetrahydrofuroic acid (20), or pyroglutamic acid (21) gave a Table 4.2 Enantioselectivity and state of mixtures of the two carboxylic acid enantiomers with (R,R)-14 Entry 1

Statea

Acid

Enantioselectivity

OH

I(S)-15/I(R)-15

16865

Pre/Sol

OH

I(R)-16/I(S)-16

261

Pre/Sol

I(S)-17/I(R)-17

1000

Sus/Sol

I(S)-18/I(R)-18

26

Sus/Sol

I(S)-19/I(R)-19

30

Sticky/Sol

I(S)-20/I(R)-20

410

Sus/Sol

I(S)-21/I(R)-21

249

Sus/Sol

I(R,R)-22/I(S,S)-22

528

Sus/Sol

OH

O 15

2

Cl

OH

O 16

3

OH OH 17

O

4

OH O

O 18

5

OH O

19

6

OH O

O

20

7

OH O

N H

O 21

8

O

HO O O O O

22

OH

O

Chiral Recognition and Enantiomeric Excess Determination 93 Table 4.2 (continued ) Entry

Acid

9

Statea

Enantioselectivity

HO

O

O O

I(R,R)-23/I(S,S)-23

2240

Sus/Sol

I(S)-24/I(R)-24

769

Sus/Sol

I(S)-25/I(R)-25

117

Sus/Sol

I(S)-26/I(R)-26

55

Sus/Sol

I(S)-27/I(R)-27

10

Sus/Sol

I(S)-28/I(R)-28

59

Sus/Sol

I(R)-29/I(S)-29

18

Pre/Sol

I(S)-30/I(R)-30

1717

Pre/Sol

I(S)-31/I(R)-31

287

Sus/Sol

I(S)-32/I(R)-32

59

Gel/Sol

O O OH O

23

10

O

OH OH

HO O

24

11

O O

NH OH

HO O

O

25

12

O O

NH OH O

26

13

O O Ph

NH OH O

27

14

O O

NH OH

HO O

28

15

O O

NH OH

S O

29

16

O NH H

S

OH 30

O

17

COOH S S

31

18

COOH

32

Enantiomer 1/enantiomer 2; Pre ¼ precipitates; Sus ¼ suspension; Sol ¼ solution.

a

94 Aggregation-Induced Emission: Applications suspension and the other enantiomer led to solution, which resulted in fluorescence intensity ratios (IS/IR) of 26, 410, and 249 for 18, 20, and 21, respectively. For ibuprofen (19), a sticky solution rather than a suspension or precipitate formed for a mixture of (S)-19 and (R,R)-14 whereas a mixture of (R)-19 and (R,R)-14 remained a clear solution. Under excitation, the emitting intensity of the sticky solution was 30-fold larger than that of the clear solution. In addition to monoacids, diacid enantiomers were also recognized by the chiral AIE compound (R,R)-14 (Table 4.2, entries 8–11). For (R,R)-2,3-dibenzoyltartaric acid (22), (R,R)-2,3-di-p-toluoyltartaric acid (23), (S)-malic acid (24) and (S)-N-Boc-glutamic acid (25), a mixture of one enantiomer and (R,R)-14 produced a suspension but a mixture of the other enantiomer and (R,R)-14 remained a solution. The suspension and solution had fluorescence intensity ratios of 528, 2240, 769, and 117 for 22, 23, 24, and 25, respectively. For the two enantiomers of all the selected N-protecting amino acids, (R,R)-14 also exhibited a strong chiral recognition ability (Table 4.2, entries 12-16). The fluorescence intensity ratios of a suspension of (S)amino acid and (R,R)-14 versus a solution of (R)-amino acid and (R,R)-14 were 55, 10, and 59 for N-Bocalanine (26), N-Boc-phenylalanine (27), and N-Boc-serine (28), respectively. For N-Boc-methionine (29), and N-acetylcysteine (30), the fluorescence intensity ratios of the precipitate formed by one enantiomer versus solution resulting from the other enantiomer were 18 and 1717, respectively. Most impressively, the reagent (R,R)-14 can even discriminate the enantiomers of a carboxylic acid with a chiral center remote from the carboxylic group (Table 4.2, entries 17 and 18). Thioctic acid (31) has the chiral center at the e-position, but one enantiomer gave a suspension and the other enantiomer led to a solution with a fluorescence intensity ratio (IS/IR) of up to 287 when they were mixed with (R,R)-14 in a mixed solvent of 1,2-dichloroethane and n-hexane. Interestingly, a mixture of (R,R)-14 and (S)-perillic acid [(S)-32] formed a gel in mixed 1,2-dichloroethane and n-hexane whereas that of (R,R)-14 and (R)-32 remained a clear solution. The fluorescence intensity of the gel was stronger than that of the solution with an intensity ratio of up to 59. A different enantioselective aggregation for the chiral acids was obtained if (S,S)-14 instead of (R,R)-14 was used as a chiral receptor. Thus, by using both (R,R)-3 and (S,S)-3, the enantiomeric composition of the chiral carboxylic acids could be quantitatively measured. As a representative example, the enantiomeric composition of mandelic acid (15) could be determined by the chiral AIE amine 14 as shown in Figure 4.4 [21].

Figure 4.4 Change of fluorescence intensity of a mixture of (R,R)-14 or (S,S)-14 and 15 with enantiomer content of 15 in 1,2-dichloroethane. [(R,R)-14] ¼ [(S,S)-14] ¼ [(R)-15] þ [(S)-15] ¼ 2.0  10
3 M. Reproduced with permission from [18], # 2009 American Chemical Society.

Chiral Recognition and Enantiomeric Excess Determination 95

Scheme 4.3 Synthesis of chiral AIE diamines 34 containing a tetraphenylethylene fluorophore.

In order to develop chiral AIE receptors with better properties, especially to improve the sensitivity of the receptor for chiral recognition of chiral acids, the chiral AIE diamines (1S,2R)-34 and (1R,2S)-34 containing the convenient fluorophore tetraphenylethylene (TPE) having an AIE effect were synthesized by the method shown in Scheme 4.3. Due to the AIE effect, TPE and its derivatives have been the most commonly used materials as chemical/biological sensors. Therefore, it was hoped the chiral diamines bearing TPE would demonstrate exceptional properties in chiral recognition based on the AIE effect. Probably bearing two amine groups, (1S,2R)-34 was found to be an especially excellent chiral sensor for chiral diacids [22]. Mixed with (1S,2R)-34, one enantiomer of 2,3-dibenzoyltartaric acid (22), 2,3-di-ptoluoyltartaric acid (23), malic acid (24), N-Boc-glutamic acid (25), and N-Cbz-aspartic acid (35) produced precipitates or suspensions but the other enantiomer gave a solution. The suspension or the suspended precipitates fluoresced strongly whereas the solution did not emit, which resulted in fluorescence intensity ratios of 20, 25, 14, 20, and 12 for 22, 23, 24, 25, and 35, respectively (Table 4.3, entries 1–5). (1S,2R)-34 also exhibited an excellent enantioselectivity for monoacids, namely 46, 16, 13, and 5.0 for mandelic acid (15), 2-chloromandelic acid (16), pyroglutamic acid (21), and N-Boc-serine (28), respectively (Table 4.3, entries 6–9). In addition, even for a strong acid, camphorsulfonic acid (36), (1S,2R)-34 could also discriminate its two enantiomers with a good enantioselectivity of 5.6 (Table 4.3, entry 10). Owing to the effect of solvent polarity on the aggregation of solutes, it is not difficult to envision that the concentration of the solutes at which the solutes start to aggregate will decrease if a good solvent is mixed with a nonsolvent. Therefore, there is a possibility to decrease the concentration (to increase the sensitivity) at which the enantiomers can be efficiently discriminated. As expected, in chloroform, which is a good solvent for 34, the concentration at which the enantiomers of 23 could be discriminated was >3.0  10
4 M. As the concentration of the mixture of 34 and 23 increased, they were also easy to aggregate and the enantioselectivity gradually increased (Figure 4.5a) . When hexane, a bad solvent for 34 and 23, was added to chloroform (volume ratio 2:1), the concentration at which the enantiomers of 23 could be discriminated was lowered to 2.0  10
5 M with a high enantioselectivity up to 22 (Figure 4.5b, ~). With a greater volume ratio of hexane to chloroform (4:1), the concentration of the enantiomer could be lowered even to 3.0  10
6 M with an enantioselectivity of up to 9.0 (Figure 4.5b, ). At low concentrations in the region of 10
6 M, all mixtures of (1S,2R)-34 and enantiomers of 23 seemed to be almost a clear solution. However, even under a portable 365 nm UV lamp, the mixture of (1S,2R)-34 and D-23 emitted strong blue light but that of (1S,2R)-34 and L-23 showed no fluorescence at this low concentration.

96 Aggregation-Induced Emission: Applications Table 4.3 Enantioselectivity of (1S,2R)-34 resulting from two enantiomers of chiral acids Entry

Acid

1

Enantioselectivity

Statea

HO

O

O O O

20 (D/L)

Pre/Solb

25 (D/L)

Pre/Solb

14 (D/L)

Sus/Solc

20 (D/L)

Sus/Solc

12 (L/D)

Sus/Solc

46 (R/S)

Sus/Sole

16 (S/R)

Sus/Solc

13 (D/L)

Sus/Solc

5.0 (D/L)

Sus/Sold

5.6 (D/L)

Sus/Sold

O OH O

22

2

HO

O

O O O O OH O

23

3

O

OH OH

HO O

24

4

O O

NH OH

HO O

O

25

5

O NH

C6H5 CH2O O

OH O

HO

35

6

OH OH O

15

7

Cl

OH OH O

16

8

OH O

N H

O 21

9

O O

NH OH

HO

28

O

10 O 36

SO3H

Enantiomer 1/enantiomer 2; Pre ¼ precipitates; Su ¼ suspension; Sol ¼ solution. In CHCl3. In H2O–THF. d In CH2Cl2. e In CH2Cl2–hexane. a b c

Chiral Recognition and Enantiomeric Excess Determination 97

Figure 4.5 Change of enantioselectivity for two enantiomers of 23 with concentration of (1S,2R)-34 in different solvent(s): (a) CHCl3; (b) ~, concentration axis n ¼ 5, CHCl3–hexane (1:2 v/v); , concentration axis n ¼ 6, CHCl3–hexane (1:4 v/v). [D-23]:[(1S,2R)-34] ¼ [L-23]:[(1S,2R)-34] ¼ 1:1. Reproduced with permission from [21], # 2011 The Royal Society of Chemistry.

When 3.3  10
5 M solutions of 23 with varying enantiomeric ratios were tested with (1S,2R)-34 at the same concentration, the fluorescence intensity increased with increasing molar percentage of D-23 with respect to the two enantiomers of 23 (Figure 4.6). Moreover, the fluorescence intensity change was sensitive to variations of the enantiomeric composition, especially at low molar percentages of D-23 of less than 10%. Similarly, owing to inherent chiral recognition, the fluorescence intensity of the same experiment with (1R,2S)-34 increased with increasing molar percentage of L-23. It also showed a sharp increase provided that a small amount of L-23 was added to D-23, indicating the high sensitivity of the chiral sensor. Therefore, the enantiomeric purity of chiral acid 23 could be obtained

Figure 4.6 Change of fluorescence intensity of (1S,2R)-34 () and (1R,2S)-34 (~) with enantiomeric content of
5 D-23 in the two enantiomers of 23. [(1S,2R)-34] ¼ [(1R,2S)-34] ¼ [D-23] þ [L-23] ¼ 3.3  10 M in CHCl3– hexane (1:2 v/v). Reproduced with permission from [21], # 2011 The Royal Society of Chemistry.

98 Aggregation-Induced Emission: Applications from either of two calibration curves as shown in Figure 4.6 [22]. The high sensitivity is especially suitable for chiral drug screening in which the amount of the chiral drugs or intermediates that can be obtained is often very small. 4.3.2 Enantiomeric excess determination of chiral acids using a chiral receptor in the presence of an AIE compound Molecular organogels produced by organic small molecules denoted organogelators are attracting intense interest owing to their great scientific and technological relevance in chemistry, materials, biology, medicine, and so on. Just like an AIE compound in a sticky organic liquid which has stronger fluorescence emission than that in a convenient solvent, the fluorescence of an AIE compound present in an organic gel was stronger than that of the AIE compound in solution. Moreover, in different phase states, including suspensions, precipitates, gels, and solution, AIE compounds could display different fluorescence intensities. Therefore, simple AIE compounds can be also applied to differentiate the enantiomers of chiral acids if a chiral amine can enantioselectively form a gel with one enantiomer of the chiral acid but give other phase state with the other enantiomer of the acid. It was found that a mixture of chiral amine 7 and a wide variety of carboxylic acids was excellent organogelator for a large number of organic liquids. Outstandingly, (1S,2R)-7 or (1R,2S)-7 formed a gel with only one enantiomer of many chiral carboxylic acids. For example, a 1:1 mixture of (1S,2R)-7 and (S)-mandelic acid (15) in 1,2-dichloroethane gave rise to a transparent gel (TGel) but a mixture of (1S,2R)-7 and (R)-15 only led to a suspension. When (1R,2S)-7 was used as base, the opposite result was obtained. For other chiral carboxylic acids in Table 4.4, only one enantiomer resulted in a transparent or opaque gel (OGel) whereas the other enantiomer led to suspensions or precipitates [23]. Table 4.4 Fluorescence intensity ratios of 37a in mixtures of (1S,2R)-7 and two enantiomers of chiral acids (molar ratio 1:1, 10 mM) in 1,2-dichloroethane Entry

Statea

Tg ( C)b

Enantioselectivity

Sus

/

I(R)-15/I(S)-15

32

TGel

35

TGel

31

I(S)-17/I(R)-17

14

Sus

/

OH

Sus

/

I(R)-26/I(S)-26

23

OH

TGel

48

Chiral acid OH

1

OH

(R)-15

O

OH

2

OH

(S)-15

O

OH OH

(R)-17

3

O OH

(S)-17

OH

4

O Boc NH

5

(R)-26 O

Boc NH

6

(S)-26

O

Chiral Recognition and Enantiomeric Excess Determination 99 Table 4.4 (continued ) Entry

Statea

Tg ( C)b

Enantioselectivity

Sus

/

I(R)-28/I(S)-28

78

TGel

30

OH

TGel

50

I(S)-29/I(R)-29

15

OH

Sus

/

TGel

34

I(S)-30/I(R)-30

6.6

P

/

P

/

I(R)-18/I(S)-18

46

TGel

28

OGel

24

I(R)-19/I(S)-19

1.2

P

/

OGel

65

I(R)-27/I(S)-27

4.2

Sus

/

Chiral acid O

7

O (R)-28

NH OH

HO O O

8

O

(S)-28

NH OH

HO O

Boc NH

(R)-29

9

S O Boc NH

(S)-29

10

S O

O

11

NH

(R)-30

H

S

OH O O

12

NH

(S)-30 H

S

OH O

OH

(R)-18

13

O

O

OH

14

(S)-18

O

O

OH

(R)-19

15

O

OH

(S)-19

16

O

Boc NH

17

(R)-27

Ph

OH O

Boc NH

18

OH

(S)-27 Ph O

TGel ¼ transparent gel; OGel ¼ opaque gel; Sus ¼ suspension; P ¼ precipitates. Tg, temperature at which the gel disassembled on heating.

a b

100 Aggregation-Induced Emission: Applications

Scheme 4.4 Structures of AIE compounds 37 and 38.

On addition of 37a, the suspension from a mixture of (1S,2R)-7 and (R)-15 emitted fluorescence much stronger than the TGel resulting from (1S,2R)-7 and (S)-15. The fluorescence intensity ratio [I(R)-15/I(S)-15] of 37a in a suspension and in TGel arising from the two enantiomers of mandelic acid was up to 32 (Table 4.4, entry 1). In the solution obtained by heating the gel, 37a showed no fluorescence (Figure 4.7). For other acids, one enantiomer gave TGel or OGel and the other enantiomer led to suspensions or precipitates. The fluorescence intensity of 37a in precipitates was much larger than that in TGel, and the fluorescence intensity in OGel was larger than that in suspensions or precipitates, which gave enantioselectivities of 14, 23, 78, 15, 6.6, 46, 1.2, and 4.2 for phenyllactic acid (17), N-Boc-alanine (26), N-Boc-serine (28), N-Boc-methionine (29), N-acetylcysteine (30), naproxen (18), ibuprofen (19), and N-Boc-phenylalanine (27), respectively (Table 4.4, entries 2–18). On addition of a nonsolvent for the acid–base complex, the concentration at which the enantiomers could be discriminated decreased. In 1,2-dichloroethane, 0.5 mM of enantiomers of 26 could be differentiated with an enantioselectivity of 4.7. If a mixed solvent of hexane and 1,2-dichloroethane (0.2:3.8 v/v) for the 7–26 complex was used, 0.25 mM of enantiomers of 26 could be differentiated with a high enantioselectivity of up to 8. Therefore, the enantiomeric composition of the chiral carboxylic acids could be quantitatively measured in a mixture of a good solvent and a bad solvent. As a

Figure 4.7 Fluorescence spectra of 37a in mixture of enantiomers of 15 and (1S,2R)-7. Solid line, in a suspension from (1S,2R)-7 and (R)-15; dashed line, in TGel from (1S,2R)-7 and (S)-15; dotted line, in solution obtained by heating TGel. [(1S,2R)-7] ¼ [15] ¼ 5[37a] ¼ 10 mM. Reproduced with permission from [22], # 2012 The Royal Society of Chemistry.

Chiral Recognition and Enantiomeric Excess Determination 101

Figure 4.8 Change of fluorescence intensity of a mixture of (1R,2S)-7 or (1S,2R)-7, 37a, and 26 with enantiomer content of 26 in 1,2-dichloroethane and hexane (0.2/3.8 ml). ([(1R,2S)-7] ¼ [(1S,2R)-7] ¼ [(R)-26] þ [(S)-26] ¼ 5 [37a] ¼ 2.5  10
4 M). Reproduced with permission from [22], # 2012 The Royal Society of Chemistry.

representative example, in the presence of 20 mol% of 37a, the enantiomeric composition of 26 could be determined using two calibration curves of the change in the fluorescence intensity of 37a with enantiomer content of 26 (Figure 4.8). Other AIE compounds, such as 37b, 38a, and 38b, also emit different fluorescence intensities in different phase states. Therefore, by addition of a simple AIE compound and a commercially available chiral amine, the enantiomeric composition of chiral carboxylic acids can be determined. This avoids the extensive effort and cost of synthesizing complex chiral receptors. Pu and co-workers found that a chiral 1,10 -bi-2-naphthol–amine receptor enantioselectively precipitated together with one enantiomer of a-hydroxycarboxylic acids in benzene containing 0.4 vol.% of dimethoxyethane. The precipitates could emit stronger fluorescence than the solution resulting from the receptor and the other enantiomer, which could be used to determine the enantiomeric composition of the a-hydroxycarboxylic acids, although both the receptor and the acids were not AIE compounds [24].

4.4 Mechanism of Chiral Recognition Based on AIE Why do some organic compounds exhibit the AIE effect? A process of restriction of intramolecular rotation (RIR) is an extensively accepted and applicable mechanism. According to this mechanism, compounds that show an AIE effect usually have a twisted conjugation structure. In the twisted conjugation structure, multiple bulky substituents are attached to a double bond and their repulsive forces drive the substituents away from the conjugation plane of the double bond. Hence the bond connecting the substituents and the double bond does not have character of a conjugation bond, which makes it easy to rotate. If fewer substituents (only two or even one) are attached to the double bond, the substituents can conjugate well with the double bond, and the bond connecting the substituents and the double bond will have the character of conjugation bond, which makes it difficult to rotate. Therefore, in solution, compounds with a twisted conjugation structure show no emission and those with a well-conjugated structure fluoresce strongly because the

102 Aggregation-Induced Emission: Applications nonradiative relaxation arising from intramolecular rotation in the twisted conjugation structure renders it non-fluorescent. When these compounds with a twisted conjugation structure aggregate, intramolecular rotation is restricted and nonradiative relaxation arising from intramolecular rotation is annihilated, which will open up the radiative channels and render the compounds fluorescent. Meanwhile, the twisted conjugation structure prevents the compound from forming an excimer, which usually results in quenching in the solid state, so they have AIE properties, whereas the conjugation structure can easily form an excimer in the solid state and shows an aggregation-caused quenching (ACQ) effect [10–12]. The AIE effect that is manifested in chiral recognition also has an RIR mechanism. Especially the AIE effect displayed by 37 and 38 in sticky solutions, gels, suspensions, and precipitates formed by other compounds, as well as by 14 in sticky solution and gel formed by itself, confirmed the RIR process. Now the question is why the chiral AIE compounds can selectively aggregate together with only one enantiomer of chiral analytes. Using the enantioselective aggregation by receptor 14 and 34 as an example demonstrates the mechanism as follows. 4.4.1 Mechanism of chiral recognition by a chiral AIE monoamine The selective interaction of (R,R)-14 with the enantiomers of chiral carboxylic acid 15 was demonstrated by 1 H NMR titration and 2D NOESY experiments. The 1 H NMR titration in CDCl3 showed that (R,R)-14 with 15 formed a 1:1 complex in which the proton of the carboxylic was transferred to the amino group of the amine. The association constants are (1.20  0.33)  104 and (1.81  0.08)  103 M
1 for the (R,R)-14–(S)-15 and (R,R)-14–(R)-15 complexes, respectively. The 2D NOESY spectra (Figure 4.9c and d) of the complexes of (R,R)-14 with enantiomers of 15 showed that the a-hydroxyl group of (S)-15 points towards the exterior of the (R,R)-14–(S)-15 complex, but the a-hydroxyl group of (R)-15 points towards the interior of the (R,R)-14–(R)-15 complex (Figure 4.9a and b). The a-hydroxyl group would have more steric repulsion than the a-proton when pointing towards the interior; therefore, the association constant of the (R,R)-14–(S)-15 complex is larger. Moreover, the a-hydroxyl group pointing towards exterior of the (R,R)-14–(S)-15 complex could increase the interaction between complexes through hydrogen bonding between the hydroxyl group of 15 in one complex and the phenoxy or carbonyl group of 14 in the neighboring complex, which would result in further aggregation of the complexes and give more intensive fluorescence [21]. 4.4.2 Mechanism of chiral recognition by a chiral AIE diamine The mechanism of chiral recognition by the chiral AIE diamine 34 was demonstrated by the interaction of (1S,2R)-34 with D-23 or L-23. 1 H NMR titration indicated that both D-23 and L-23 formed a 1:1 complex with (1S,2R)-34. The association constants of the (1S,2R)-34–D-23 complex and the (1S,2R)-34–L-23 complex were 6.3  104 and 1.3  105 M
1, respectively, indicating different bonding forces of these two enantiomers to (1S,2R)-34. ESIþ mass spectra revealed that the interaction of 34 and 23 easily formed a 22–32 tetramer (m/z 2459.1, M þ 3) in addition to the 34–23 complex. In the 2D NOESY spectra of 34–23 complexes, there were obvious intermolecular NOEs between the toluoyl protons of the acid and the protons of the substituted phenyl ring of the TPE part of the amine (between Hb and Hc) (Scheme 4.5), demonstrating that the toluoyl group of the acid and the TPE part of the amine were close to each other. However, no intermolecular NOEs between methyl protons of the toluoyl group and phenyl protons of the TPE part were found. Therefore, in the 34–23 complex the acid 23 approached the diamine 34 from outside the two amino groups rather than between these two amino groups of 34 (Scheme 4.5). The intermolecular NOEs also excluded the possibility of forming a bridge between 34 and 23. By dipole–dipole attraction of two acid– base ion pairs and hydrogen bonds, the 34–23 complexes can be converted into a tetramer complex A. By

Chiral Recognition and Enantiomeric Excess Determination 103 Hd H d

O

Ha

N HbHc NH3

Hg

O He

CN

Hf

O Hk O C Hj

Hd

N

O

Hd

Hh

Hi

Hb Hc Ha NH3 O Hk O C OH

Hl

HO

O NO2

Hg

He

Hf

Hi

Hl

Hj

Hm

CN Hh

NO2

Hm

(b)

(a) Hk Hc HI

He

Hk

ppm

He

HI Hc

ppm 4.7

4.8 Hj

4.9

4.8 Hj 4.9

5.0

7.4

7.2

7.0

5.0 7.4

ppm

7.0

ppm

(d)

(c)

O

O (R, R)-14-(S)-15 complex

O

7.2

O

(R, R)-14-(R)-15 complex

H

H O O

H

O

OH O O O O

H

H

aggregation

(e)

non-aggregation

(f)

Figure 4.9 Scheme showing the interaction and selective aggregation of (a) (R,R)-14 with (S)-15 and (b) of (R,R)14 with (R)-15. The arrows represent observed intermolecular nuclear Overhauser effects (NOEs). Portions of the 2D NOESY spectra of 1:1 mixtures of (c) (R,R)-14 and (S)-15 and (d) (R,R)-14 with (R)-15. (e) Schematic illustration of aggregation and photograph of suspension and (f) schematic illustration of nonaggregation and photograph of solution. Reproduced with permission from [22], # 2012 The Royal Society of Chemistry.

104 Aggregation-Induced Emission: Applications

Scheme 4.5 The main intermolecular NOEs between 34 and 23 in the 34–23 complex and probable mechanism of aggregate formation.

further acid–base interaction in the x-direction, tetramer complexes A form a 1D network B, which can stack side-by-side in the y- and z-directions to give a 3D nano-rod (Scheme 4.5). In the case of the tetramer complexes that do not have a sufficient interaction force between them, or are easily soluble, the 1D network B cannot form and no further aggregation occurs. With different binding forces of 34 to 23 and different solubilities of the 342–232 tetramer, one enantiomer results in aggregates and the other leads to no or less aggregates. In aggregates, the RIR process will lead to light emission from 34, and the emission will be stronger if one enantiomer makes 34 aggregate more [22].

4.5 Prospects for Chiral Recognition Based on AIE Compared with other methods, chiral recognition based on the AIE effect has exhibited exceptionally high enantioselectivity and wide applicability. By adjusting the ratio of good solvent to nonsolvent, very high sensitivity in the quantitative determination of enantiomer purity can also be obtained. The chiral AIE receptor is easy to be prepared, and even many commercially available chiral compounds or chiral resolution agents are also potential chiral receptors for the determination of enantiomeric composition. Therefore, chiral recognition based on the AIE effect will have practical applications in enantiomer analysis, especially

Chiral Recognition and Enantiomeric Excess Determination 105 the high-throughput analysis of enantiomeric composition in asymmetric synthesis catalyst screening and chiral drug development. Although successful results have been achieved in chiral recognition and enantiomeric excess determination of chiral acidic and chiral basic compounds, the development of chiral receptors based on the AIE effect for neutral chiral analytes is still a challenge owing to the weaker interactions between the receptor and the neutral analytes. To widen the applicable area, especially applicability to neutral chiral analytes, the following strategies could be considered according to the RIR mechanism of the AIE effect. 1. Using a chiral AIE metal complex. The enantiomers of neutral chiral analytes, such as alcohols, aldehydes, ketones, esters, and so on, have the possibility to coordinate with chiral AIE metal complexes. The difference in coordinating ability will result in different solubility or different aggregation, which will lead to different fluorescence intensity for a pair of enantiomers [25]. 2. Using a chiral AIE macrocyclic compound with an AIE fluorophore as part of the cycle. Macrocyclic compounds, such as crown ethers, calixarenes, and cucurbiturils, have a cavity which can easily include a neutral molecule due to multiple intermolecular interactions. After a chiral analyte has been included in the cavity of a chiral AIE macrocyclic compound, the rotation of the cycle of the latter will be restricted due to the multiple interactions, which will lead to fluorescence emission or fluorescence enhancement. Because of the chirality of the macrocycle, the two enantiomers of a chiral analyte will have the possibility of being included in the cavity to different extents, which will lead to different RIR processes and make the AIE macrocycle emit different intensities of fluorescence. 3. Using a chiral macrocyclic compound. If a simple AIE compound could be included in the cavity of a macrocycle, it will show fluorescence emission or fluorescence enhancement due to being confined in the cavity. When a chiral analyte is added, it has the possibility of replacing the included AIE compound and releasing the AIE compound into solution, which will make the fluorescence of the AIE compound decrease. Owing to the chirality of the cavity of the macrocyclic compound, the two enantiomers of a chiral analyte will show different abilities to replace the included AIE compound, which will lead to different fluorescence intensities of the AIE compound. Cyclodextrin or some artificial chiral macrocycles [2, 26] can be used as chiral macrocyclic compounds. According to this idea, the chiral cavity in proteins, DNA, and even enzymes can also be exploited to recognize enantiomers. In conclusion, there are many approaches to chiral recognition based on the AIE effect, and it is expected that more and more excellent chiral receptors based on the AIE effect will be developed as the result of continuing research studies.

References 1. Blaschke, G., Kraft, H.P., and Markgraf, H. (1980) Chromatographische Racemattrennung des Thalidomids und anderer Glutarimid-Derivate. Chem. Ber., 113, 2318–2322. 2. Ema, T., Tanida, D., and Sakai, T. (2007) Versatile and practical macrocyclic reagent with multiple hydrogenbonding sites for chiral discrimination in NMR. J. Am. Chem. Soc., 129, 10591–10596. 3. Zheng, Y.S., and Zhang, C. (2004) Exceptional chiral recognition of racemic carboxylic acids by chiral calix[4] arenes bearing optical pure a,b-amino alcohol groups at lower rim. Org. Lett., 6, 1189–1192. 4. Bobbitt, D.R., and Linder, S.W. (2001) Recent advances in chiral detection for high performance liquid chromatography. Trends Anal. Chem., 20, 111–123. 5. Welch, C.J., Hyun, M.H., Kubota, T., Schafer, W., Bernardoni, F., Choi, H.J., Wu, N.J., Gong, X.Y., and Lipshutz, B. (2008) Microscale HPLC enables a new paradigm for commercialization of complex chiral stationary phases. Chirality, 20, 815–819.

106 Aggregation-Induced Emission: Applications 6. Traverse, J.F., and Snapper, M.L. (2002) High-throughput methods for the development of new catalytic asymmetric reactions. Drug Discov. Today, 7, 1002–1012. 7. Hembury, G.A., Borovkov, V.V., and Inoue, Y. (2008) Chirality-sensing supramolecular systems. Chem. Rev., 108, 1–73. 8. Pu, L. (2004) Fluorescence of organic molecules in chiral recognition. Chem. Rev., 104, 1687–1716. 9. Pu, L. (2012) Enantioselective fluorescent sensors: a tale of BINOL. Acc. Chem. Res., 45(2), 150–163. 10. Hong, Y., Lam, J.W.Y., and Tang, B.Z. (2011) Aggregation-induced emission. Chem. Soc. Rev., 40, 5361–5388. 11. Wu, J., Liu, W., Ge, J., Zhang, H., and Wang, P. (2011) New sensing mechanisms for design of fluorescent chemosensors emerging in recent years. Chem. Soc. Rev., 40, 3483–3495. 12. Wang, M., Zhang, G., Zhang, D., Zhu, D., and Tang, B.Z. (2010) Fluorescent bio/chemosensors based on silole and tetraphenylethene luminogens with aggregation- induced emission feature. J. Mater. Chem., 20, 1858–1867. 13. Liu, Y., Tang, Y., Barashkov, N.N., Irgibaeva, I.S., Lam, J.W.Y., Hu, R., Birimzhanova, D., Yu, Y., and Tang, B.Z. (2010) Fluorescent chemosensor for detection and quantitation of carbon dioxide gas. J. Am. Chem. Soc., 132, 13951–13953. 14. Liu, Y., Deng, C., Tang, L., Qin, A., Hu, R., Sun, J.Z., and Tang, B.Z. (2011) Specific detection of D-glucose by a tetraphenylethene-based fluorescent sensor. J. Am. Chem. Soc., 133, 660–663. 15. Zhang, Y., Gu, H., Yang, Z., and Xu, B. (2003) Supramolecular hydrogels respond to ligand receptor interaction. J. Am. Chem. Soc., 125, 13680–13681. 16. Zheng, Y.-S., Ji, A., Chen, X.-J., and Zhou, J.-L. (2007) Enantioselective nanofiber-spinning of chiral calixarene receptor with guest. Chem. Commun. 3398–3400. 17. Zheng, Y.-S., Ran, S.-Y., Hu, Y.-J., and Liu, X.-X. (2009) Enantioselective self-assembly of chiral calix[4]arene acid with amines. Chem. Commun. 1121–1123. 18. Zheng, Y.-S., and Hu, Y.-J. (2009) Chiral recognition based on enantioselectively aggregation-induced emission. J. Org. Chem., 74, 5660–5663. 19. Li, D.-M., and Zheng, Y.-S. (2011) Single-hole hollow nanospheres from enantioselective self-assembly of chiral AIE carboxylic acid and amine. J. Org. Chem., 76, 1100–1108. 20. Zheng, Y.-S., Hu, Y.-J., Li, D.-M., and Chen, Y.-C. (2010) Enantiomer analysis of chiral carboxylic acids by AIE molecules bearing optically pure aminol groups. Talanta, 80, 1470–1474. 21. Li, D.-M., and Zheng, Y.-S. (2011) Highly enantioselective recognition of a wide scope of carboxylic acids based on enantioselectively aggregation-induced emission. Chem. Commun., 47, 10139–10141. 22. Liu, L.-L., Song, S., Li, D.-M., and Zheng, Y.-S. (2012) Highly sensitive determination of enantiomeric composition of chiral acids based on aggregation-induced emission. Chem. Commun., 48, 4908–4910. 23. Li, D.-M., Wang, H., and Zheng, Y.-S. (2012) Light-emitting property of simple AIE compounds in gel, suspension and precipitates, and application to quantitative determination of enantiomer composition. Chem. Commun., 48, 3176–3178. 24. Liu, H.-L., Hou, X.-L., and Pu, L. (2009) Enantioselective precipitation and solid-state fluorescence enhancement in the recognition of a-hydroxycarboxylic acids. Angew. Chem. Int. Ed., 48, 382–385. 25. You, L., Pescitelli, G., Anslyn, E.V., and Bari, L.D. (2012) An exciton-coupled circular dichroism protocol for the determination of identity, chirality, and enantiomeric excess of chiral secondary alcohols. J. Am. Chem. Soc., 134, 7117–7125. 26. Carrillo, R., Lopez-Rodrίguez, M., Martın, V.S., and Martın, T. (2009) Quantification of a CH–p interaction responsible for chiral discrimination and evaluation of its contribution to enantioselectivity. Angew. Chem. Int. Ed., 48, 1–7.

5 AIE Materials Towards Efficient Circularly Polarized Luminescence, Organic Lasing, and Superamplified Detection of Explosives Jianzhao Liu, Jacky W. Y. Lam and Ben Zhong Tang Department of Chemistry, The Hong Kong University of Science and Technology, China

5.1 Introduction Materials with aggregation-induced emission (AIE) features have opened up enormous opportunities for their optoelectronic and sensory applications in the aggregated state. In this chapter, we report our recent efforts in developing AIE-active materials for applications in generating efficient circularly polarized luminescence with large dissymmetry factors, organic lasing, and superamplified detection of explosives. The design rationales, typical materials synthesized, and their working performances are introduced and discussed.

5.2 AIE Materials with Efficient Circularly Polarized Luminescence and Large Dissymmetry Factor Circularly polarized luminescence (CPL) reflects the chirality of materials in the excited electronic state. Stereochemical, conformational, and three-dimensional structure information on chiral materials can be identified from their spectroscopic CPL signatures [1
3]. CPL is characterized by differential spontaneous emission of left- and right-handed circularly polarized (LCP and RCP) light upon photo- or electro-excitation [DI(l) ¼ IL(l) – IR(l), where IL(l) and IR(l) denote the emission intensities of the LCP and RCP

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

108 Aggregation-Induced Emission: Applications components, respectively] [1, 2]. Chiral materials with efficient CPL are very useful for biosensing and optoelectronic applications, such as probing biomacromolecular targeting/binding events by reading the change of CPL signal, and devices for stereoscopic optical information processing, display, and storage [4
7]. All these technological applications require materials with high CPL performance. Two essential parameters are utilized to evaluate the performance of CPL-active materials: (1) the emission dissymmetry factor gem, which is defined as 2(IL
IR)/(IL þ IR) and whose value is in the range
2 to 2, and (2) the emission efficiency, especially in the condensed phase for practical use. So far, various chiral systems, such as lanthanide ion complexes [8
11], transition metal complexes [12
14], organic molecules [15
18], synthetic polymers [19
27], biomacromolecules [4], and crystals [16], have been reported with CPL observations. However, most of them were studied in the solution phase and found with small gem values (10
5–10
2), except for some rare earth compounds [8
11] and liquid crystalline conjugated polymers [5, 23]. From solution to condensed phase, the performance becomes even worse because aggregation of chiral luminophores normally populates the nonradiative pathways and thus quenches the light emission to a great extent. In this context, the development of new chiral luminescent systems with both high emission efficiency and large dissymmetry factor in the condensed phase has been a challenging task. Organic chiral p-conjugated molecules are one of the promising candidate materials for use in advanced electronic CPL devices owing to their tailored synthetic feasibility, low cost, and high flexibility and processability. In principle, helically organized luminophores can produce a CPL signal upon photo- or electroexcitation [1, 2]. A common strategy is to synthesize molecules with chiral moieties at the peripheries and a planar p-conjugated luminophore in the core, as shown in Figure 5.1a, and a helical assembly can be formed under the driving force of p–p stacking interactions. Owing to the formation of detrimental excimers or exciplexes induced by p–p stacking interactions in the aggregate state, the CPL of the assembly suffers from an intrinsic aggregation-caused quenching (ACQ) effect [28], leading to poor emission efficiency and spectral instability. From the practical application point of view, high emission efficiency and spectral stability are essential. To overcome the serious limitation of ACQ, efficient solid emitters obtained by a judicious construction strategy are needed. Recently, an exactly opposite phenomenon to the ACQ effect was observed by Tang’s group [29]. A family of propeller-shaped p-conjugated molecules, for example, silole and tetraphenylethene, which are weakly or nonemissive in dilute solutions, are induced to emit intensively by aggregate formation, with the fluorescence quantum efficiency (FF) enhanced by more than two orders of magnitude [29, 30]. With this inspiration, we conceived the idea that an appropriate combination of AIE-active luminophores and chiral moieties may open up a new avenue to creating high-performance CPL-active materials that ideally meet the requirements of high emission efficiency and large dissymmetry factor in the aggregated state (Figure 5.1b). As a proof of concept, we designed a molecule 1 containing an AIE-active silole core and chiral sugar pendants as shown in Scheme 5.1 [31]. This molecule has no circular dichroism (CD) and is nonemissive in solution. Upon aggregation, 1 can easily assemble into hierarchical helical structures and display simultaneous aggregation-induced CD (AICD) and AIE characteristics, giving absolute FF and gem values of up to 81.3% and 0.32 in the solid state, respectively. To the best of our knowledge, this system represents the optimum result among organic chiral p-conjugated molecule systems in terms of emission efficiency, dissymmetry factor, and spectral stability. 5.2.1 Aggregation-induced circular dichroism The molecule 1 in dichloromethane (DCM) solution shows two absorption bands located at 360 and 279 nm, corresponding to the absorptions of the silole core and the peripheral triazolylphenyl rings, respec
1  in DCM). CD tively (Figure 5.2a). It has specific optical rotation ð½a23 D Þ of þ24.6 (c ¼ 0.26 g dl

Circularly Polarized Luminescence, Organic Lasing, and Superamplified Detection of Explosives 109

Figure 5.1 Comparison of molecular design strategies for making CPL-active materials. CPL (output lem) is generated from helical assemblies of luminescent chiral molecules bearing (a) conventional planar and (b) AIE luminophores under excitation with polarized/nonpolarized light (input lex).

spectroscopy, which measures the differential absorption of LCP and RCP light [De(l) ¼ eL(l) – eR(l), where eL(l) and eR(l) are the molar extinction coefficients of LCP and RCP light, respectively], mirrors the structural information of the ground electronic state of a system [32]. Figure 5.2b shows the CD spectra of 1 in DCM solutions with different concentrations and all of them are CD silent in the absorption region. Addition of hexane, a nonsolvent for 1, to the DCM solution can cluster the molecules into aggregates and form a suspension. At a concentration of 10
5 M, aggregation of 1 leads to the emergence of Cotton effects at 278 and 249 nm attributed to the absorptions of triazolylphenyl group (Figure 5.2c), showing an AICD effect. As the concentration increases from 1  10
5 to 2  10
4 M, a new peak at 340 nm appears and grows stronger with respect to the peak at 278 nm. This new peak is considered to be associated with the absorption transition of the silole core, suggesting the occurrence of chirality transfer such that the chiral sugar pendant together with the triazolylphenyl group induces the silole core to be helically arranged with a preferred screw sense. From 2  10
5 to 2  10
4 M, the absorption dissymmetry factor at 360 nm, gabs ¼ 2[eL(l) – eR(l)]/[eL(l) þ eR(l)] [32], is increased from 1.59 to 2.23  10
3. Figure 5.2d illustrates the CD spectra of cast films of 1 dispersed in a poly(methyl methacrylate) (PMMA) matrix with different weight fractions (wt%). Below 2.5 wt%, no CD signal is detected. When more than 5 wt% of 1 is loaded, there is a trend that the intensity of the CD signal increases with the amount loaded. It is understandable that at a small loading ratio, 1 exists in an isolated state in the polymer matrix, whereas with a larger amount loaded, the molecules tend to aggregate due to the intrinsic phase separation effect, and the population of chiral aggregates with helical structures accordingly becomes larger. In addition, the two AICD peaks originally

110 Aggregation-Induced Emission: Applications

Scheme 5.1 Synthetic route to chiral AIE-active molecule 1 with CPL activity according to the strategy shown in Figure 1b. An ORTEP drawing of the single-crystal structure of intermediate 6 (CCDC 845739) at 40% probability is shown.

at 340 and 278 nm in DCM–hexane suspensions are red shifted to 380 and 300 nm, respectively, and the former peak becomes more intense, indicative of the formation of a more conjugated structure in the helical assemblies and thus the enhanced chirality transfer effect. Natural evaporation of neat 1 in 1,2-dichloroethane (DCE) solution on a quartz substrate leads to the formation of a non-smooth film, which shows a similar spectral profile to those mentioned above. 5.2.2 AIE, fluorescence decay dynamics and theoretical understanding As expected, the designed molecule 1 is AIE active: it shows faint photoluminescence (PL) when molecularly dissolved in its good solvents but are induced to fluoresce intensively when aggregated in poor solvents (Figure 5.3). The fluorescence intensity and FF value of 1 remain almost unchanged until 80 vol.% of poor solvent had been added. Subsequently, the fluorescence efficiency rises abruptly. In pure solutions, the FF value of 1 is merely 0.6%, whereas in the DCM–hexane mixture with 90 vol.% hexane, its FF value rises to 31.5%, as can be clearly discerned from the fluorescence images shown in Figure 5.3b. It is amazing that

Circularly Polarized Luminescence, Organic Lasing, and Superamplified Detection of Explosives 111

Figure 5.2 Properties of electronic transition and circular dichroism. (a) Absorption spectrum of 1 in DCM. (b, c) CD spectra of 1 with different concentrations in (b) DCM solution and (c) DCM–hexane (1:9 v/v) mixture. (d) CD spectra of 1 with different weight fractions dispersed in a PMMA matrix prepared by drop casting of their mixed DCE solutions. Concentration of PMMA ¼ 10 mg ml
1. The CD spectrum of neat 1 prepared by natural evaporation of its DCE solution of 2 mg ml
1 on a quartz substrate is also shown for comparison.

the FF value in the solid thin-film state can reach 81.3%. Meanwhile, the profiles of the PL spectra remain unchanged from the solution to aggregated state. The AIE effect can be quantified by the extent of emission enhancement (aAIE), as defined by the following equation: aAIE ¼

FFðaggrÞ FFðsolnÞ

(5.1)

112 Aggregation-Induced Emission: Applications

Figure 5.3 Photoluminescent property and theoretical calculation. (a) PL spectra of 1 in DCM–hexane mixtures with different volume fractions (fH) of hexane. Concentration ¼ 10–5 M; excitation wavelength ¼ 356 nm. (b) Fluorescence quantum yield (FF) of 1 versus solvent composition of the DCM–hexane mixture. The FF values were estimated using 9,10-diphenylanthracene as standard (FF ¼ 90% in cyclohexane). The FF value of a thin cast film (FF,f) of 1 determined by integrating sphere is given in (b) for comparison. Inset in (b): fluorescent photographs of 1 in DCM–hexane mixtures with fH of 0 and 90 and its powder under a hand-held UV lamp with an excitation wavelength of 365 nm. (c) Time-resolved fluorescence decay curves of 1 in DCM solution with a concentration of 10–5 M and its cast thin film from a DCE solution of 2 mg ml
1. (d) Plot of calculated reorganization energy versus the normal-mode wavenumber of TPS.

Circularly Polarized Luminescence, Organic Lasing, and Superamplified Detection of Explosives 113 where FF(aggr) and FF(soln) are the quantum efficiencies in the aggregated (e.g. the FF value in the thin-film state) and solution states, respectively. According to this equation, the corresponding aAIE factor of 1 is about 136. To deepen our understanding of the AIE phenomenon of 1, we studied its emission dynamics by the timeresolved technique. Lifetime is a key kinetic parameter for PL decay. In dilute solution, the excited state of 1 decays in a single-exponential fashion, suggesting that all of the excited molecules relax through the same pathway (Figure 5.3c). The lifetime is as short as 30 ps. In the solid thin-film state, the PL decay is much slower and the decay dynamics are better fitted by a double-exponential function. This indicates that two relaxation pathways are involved in the decay process. For example, 39% (A1) and 61% (A2) of the excitons of 1 decay via the fast and slow channels with lifetimes of 0.46 ns (t 1) and 2.49 ns (t2), respectively. The weighted mean lifetime (t) is 1.70 ns. These results show good consistency with the steady-state PL studies. In addition, molecule 1 suffers from less intermolecular excitonic coupling effect induced by the p–p stacking interaction due to their twisted structure. Therefore, both the emission lifetime and efficiency are enhanced. A mechanistic picture for understanding the cause of the AIE effect is essential especially from the viewpoint of quantum mechanics. AIE-active silole has a twisted structure and is conformationally unstable (strained) owing to the repulsion of sterically crowded phenyl rings. The intramolecular vibrations and rotations (torsions) are very active in the free state. It was conjectured that these low-frequency motions constitute the major nonradiative energy dissipation pathways in the solution state. These motions are suppressed in the aggregated state, leading to recovery of the radiative electronic transitions. We employed Shuai and co-workers’ method to calculate how these low-frequency motions affect the fluorescence efficiency of 1 [33
35]. To reduce the difficulty of the calculation, we extracted the central luminogenic part 1,1dimethyl-2,3,4,5-tetraphenylsilole (TPS) as a model (Figure 5.3d). The ground-state geometry (S0) was optimized at the level of the density functional theory (DFT) in the Gaussian 03 program [36] and then the time-dependent DFT method was applied to optimize the first single excited-state geometry (S1). The B3LYP functional and 6–31G(d) basis set were used. At the equilibrium geometries, the vibrational frequencies and the normal vibrational modes of S0 electronic states were calculated by analytic energy gradients, and those for S1 were obtained by numerical energy gradients. Under the harmonic approximation by considering the Duschinsky rotation effect [33], the reorganization energy, which is the energy required for structural adjustments from initial to final coordinate, was calculated. The map of specific normal modes that contributed to the total reorganization energy from the excited to the ground state is shown in Figure 5.3d. The high-frequency modes (1200–1700 cm
1) are related to the stretching vibrations of the
1 C

C or C

C bonds, whereas the low-frequency modes (10-fold was triggered with the addition of 1.6 equiv. of Al3þ. Figure 6.13 shows the dependence of the changes in emission intensity on the Al3þ concentration. It exhibited a three-phase increment with increase in Al3þ concentration, a slow growth below 50 mM, a rapid growth in the range 50–130 mM, and a suspended manner subsequently. Notably, there is a good linear relationship between the changes in PL intensity and the square of Al3þ concentration (R2 ¼ 0.9979, 0–130 mM). Its quantitative detection range is much more broader than that of TPP-COO [31], indicating that this approach is more applicable for the quantitative detection of Al3þ. In addition, the detection limit of TPP-3COO towards Al3þ was estimated as 5.3 mM, which met the limit for drinking water according to the WHO standard (7.41 mM). To evaluate further the specificity of TPP-3COONa towards Al3þ, we carried out fluorescence titration with other cations, including Al3þ, N(CH3)þ, Au3þ, Cr2þ, Ni2þ, Cd2þ, Hg2þ, Fe3þ, Pb2þ, Agþ, Ce2þ, Cu2þ, Zn2þ, Ba2þ, Mg2þ, Ca2þ, and Kþ, with a concentration of 500 mM to make sure of its maximum fluorescent response. Although TPP-3COO shows a positive response towards Au3þ, Cr2þ, Pb2þ, and Cu2þ and a negative response towards Hg2þ, Fe3þ, and Agþ, it is clearly seen that the most striking effects are observed for Al3þ, confirming that the probe has a selective response to Al3þ(Figure 6.14). Compared with the detection result in pure water, the ratio of H2O to THF (96 : 4 v/v) used in the sensory experiments was chosen because of its relatively higher selectivity and stronger amplification ability owing to the formation of TPP3COO AIE aggregates induced by THF (Figure 6.14). Compared with TPP-COO, TPP-3COO exhibits improved selectivity due to its relative higher solubility and COO density. To confirm further the single selectivity of TPP-3COO for Al3þ in practical applications, we chose Kþ, Mg2þ, Ca2þ, and Ba2þ as

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 141

Figure 6.12 Emission spectra of TPP-3COO (100 mM) in H2O–THF (96 : 4 v/ v) upon addition of Al3þ. The Al3þ concentrations are 0, 10, 20, 30


160 mM, from bottom to top.

Figure 6.13 Emission intensities of TPP-3COO (100 mM) in H2O–THF (96 : 4 v/ v) as a function of [Al3þ] and [Al3þ]2 (0–170 mM). Excitation wavelength: 340 nm. Inset: linear relationship between emission growth ratio and Al3þ concentration from 0 to 130 mM. Curve-fit equation: (I – I0)/ I0 ¼ 0.27809 þ 5.67207  104[Al3þ]2, R2 ¼ 0.99786.

142 Aggregation-Induced Emission: Applications

Figure 6.14 Maximum fluorescence response of TPP-3COO (100 mM) upon addition of different metal ions (500 mM) in H2O–THF (96:4 v/ v) (black column) or pure water (red column): (A) Al3þ; (B) N(CH3)4þ; (C) Au3þ; (D) Cr2þ; (E) Ni2þ; (F) Cd2þ; (G) Hg2þ; (H) Fe3þ; (I) Pb2þ; (J) Agþ; (K) Ce2þ; (L) Cu2þ; (M) Zn2þ; (N) Ba2þ; (O) Mg2þ; (P) Ca2þ; (Q) Kþ; (R) Al3þ þ Ba2þ þ Mg2þ þ Ca2þ þ Kþ.

interfering ions, some of which have a relatively high concentration in biological tissues and drinking water. As shown in Figure 6.14, the experimental results indicated that the fluorescence intensities of TPP3COO at 460 nm enhanced by Al3þ are not much affected by the presence of the interfering ions, thus providing a potential application for biological detection and water quality monitoring. The ion recognition properties of TPP-3COO were also studied by using the absorption technique. The UV–vis spectra of TPP-3COO in H2O–THF (96:4 v/v) show only one p–p transition band centered at 329 nm, which can be assigned to an aryl-substituted pyrrole moiety (Figure 6.15) . Upon addition of Al3þ (0–60 mM), the absorbance at 329 nm decreased gradually with a slight red shift whereas the absorbance at 346 nm remained constant with increase in Al3þ concentration. Further, a low-energy (LE) shoulder developed at 391 nm although a new absorption band was not obviously observed (Figure 6.15). The presence of a well-defined isosbestic point at 346 nm indicated that interconversion between the uncomplexed and complexed species occurs. In addition, the transmittance of the solution decreased upon titration with Al3þ and reached its minimum value when the Al3þ concentration was >200 mM (Figure 6.16), which implies that the amount of aggregates has increased and/or the size of the aggregates has become larger with increase in Al3þ content. These conclusions were further confirmed by DLS measurements. The average particle size of TPP-3COO in H2O–THF (96:4 v/v) solution increased from 50 to 130 nm upon addition of Al3þ (500 mM) (Figure 6.17). Hence an AIE mechanism contributed to the ‘turn-on’ detection of Al3þ with TPP-3COO. In order to elucidate the effect of Al3þ on aggregate formation, the morphology of TPP-3COO aggregates in H2O–THF (96:4 v/v) without and with Al3þ was examined by transmission electron microscopy (TEM). TEM image of the sample without Al3þ showed amorphous and incompact aggregates because of the electrostatic repulsion between carboxylate ions (Figure 6.18a). The diameters of the approximate spheres range from 15 to 100 nm with an average of 45.3 nm, which are consistent with that from DLS analysis (Figure 6.17, line a). Part of the nanospheres further clumped together to form nanoclusters with

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 143

Figure 6.15 Absorption response of TPP-3COO (100 mM) to titration of Al3þ in H2O–THF (96 : 4 v/ v). The total [Al3þ] increases from 0 to 60 mM along the direction of the arrow. Inset: linear relationship between absorbance and Al3þ concentration at 329 and 391 nm.

Figure 6.16 Transmittance changes of TPP-3COO (100 mM) in H2O–THF (96 : 4 v/ v) upon titration of Al3þ. The total [Al3þ] increased from 0 to 500 mM along the direction of the arrow.

random morphologies. In the Al3þ-containing sample, the TEM image, however, revealed that the introduction of Al3þ leads to a morphological transition of the aggregates in H2O–THF (96:4 v/v). Aggregates composed of spheres, capsules, and ribbons were observed (Figure 6.18). Especially the morphology of spheres and capsules is more regular and more compact than that of TPP-3COO aggregates in H2O–THF (96:4 v/v)

144 Aggregation-Induced Emission: Applications

Figure 6.17 Particle size distribution of TPP-3COO [100 mM, H2O–THF (96:4 v/ v) solution] (a) without and (b) with the addition of Al3þ (500 mM) determined by DLS.

without Al3þ, which indicates that the introduction of Al3þ induces TPP-3COO to form regular aggregates. The diameters of the spheres and capsules range from 23 to 71 nm with an average of 47 nm. For the ribbons, their average width and length are about 30 and 120 nm, respectively. The results also explain the corresponding attributions of the two peaks shown in Figure 6.17, line b. According to previous studies, reducing the anionic repulsion between Al3þ and amphiphilic TPP-3COO headgroups permits closer packing to fabricate spheres and can provide an additional impetus for capsule formation [78, 79]. Because the concentration of Al3þ was much higher than that of TPP-3COO here, the decrease in charge of partial unilamellar vesicle

Figure 6.18 TEM images of TPP-3COO (100 mM) in H2O–THF (96:4 v/ v) (a) without and (b) with 500 mM Al3þ.

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 145

Figure 6.19 Fluorescence spectra of TPP-3COO (100 mM) in H2O–THF (96 : 4 v/ v) in the presence of 500 mM Al3þ at different times.

bilayers could, in turn, induce the stacks of lamellae to form ribbons by decreasing the repulsive force between anionic bilayers [80–82]. The time-dependent fluorescence intensity of an H2O–THF (96:4 v/v) solution of TPP-3COO with respect to Al3þ was evaluated. Because the minimum response time is limited by the manipulation time, all the measurements with different Al3þ contents were started at 10 s. As shown in Figure 6.19 and Figure 6.20, the emission intensity increased up to the maximum after the addition of Al3þ in just a few

Figure 6.20 Fluorescence response of Al3þ with different concentrations (5–500 mM) versus time.

146 Aggregation-Induced Emission: Applications seconds and then remained constant over an extended period of time. This performance is much faster than that of TPP-COO [31]. We attribute this phenomenon to the fact that compared with TPP-COO aggregates, there are more carboxylate groups that can bind with Al3þ through electrostatic attraction existing on the surface of TPP-3COO aggregates. This provides a potential ‘zero-wait’ detection method for Al3þ. We consider that the fluorescence enhancement on addition of Al3þ can be ascribed to a synergetic effect based on the AIE mechanism. On the one hand, according to the literature, both experimental results and theoretical calculations revealed that the bidentate complex formed between carboxylate groups and Al3þ is more stable than the monodentate complex [83, 84]. When at least one carboxylate group in TPP-3COO is used to form a bidentate complex with Al3þ ion, the pyrrole ring easily constructs a coplanar conformation with the benzyl ring, which further favors aggregation and suppresses intramolecular rotation-caused nonradiative decay, that is, an AIE mechanism (Scheme 6.3). Thus, the PL intensity of TPP-3COO is enhanced with increase in Al3þ concentration. On the other hand, little and loose aggregates are considered to be formed in the initial TPP-3COO solution [100 mM, H2O–THF (96:4 v/v)], which can be proved by the DLS data and TEM images (Figure 6.17 and Figure 6.18). Owing to its small size, high charge density,

Scheme 6.3 Sensing process based on the AIE mechanism.

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 147 and oxophilicity, Al3þ will immediately approach close to the carboxylate groups present on the surface of these aggregates and then form a chelation complex with lower solubility through electrostatic attraction. This can not only increase the amount and the size of the aggregates but also compact them. All these effects can suppress intramolecular rotation-caused nonradiative decay and boost the fluorescence emission based on the AIE mechanism (Scheme 24.3) [85].

6.4 Aggregation-Induced Emission of Pentaphenylpyrrole The photophysical properties of pentaphenylpyrrole (PPP) during aggregation were also studied owing to the similarity of its structure to that of silole [86]. The UV–vise absorption spectra and PL spectra of PPP were measured in H2O–THF mixtures with different volume fractions of water and the final concentrations were kept constant at 1  105 mol l1 according to the previous methods [87]. As can be seen from Figure 6.21, when the water content in the H2O–THF mixture is 60 vol.%, the absorbance of PPP hardly changes. At 60% water content, the absorption peak of PPP is bathochromically shifted with a large decrease in intensity. At high water contents (90%), the absorption peaks of PPP are shifted back to the position of its THF solution with moderate decreases in intensity. The absorption spectra of PPP in the H2O–THF mixtures with 70% water contents contain light-scattering tails in the longwavelength region, suggesting that the molecules of PPP have clustered into nano-aggregates in the poor solvents [87, 88]. The morphologies of PPP aggregates obtained in H2O–THF solution with different volume fractions of water were observed by field-emission scanning electron microscopy (FE-SEM) measurements (Figure 6.22). The experimental results show that the sizes of most PPP aggregates obtained in H2O– THF mixtures with 70 and 80% volume fractions of water are about 140–300 nm  80–100 nm and 200– 400 nm  50–100 nm with irregular rectangles, respectively. Hence the absorption spectra of PPP in the H2O–THF mixtures with 70 and 80% water contents revealed the light-scattering tails in the long-wavelength region. However, the size of PPP aggregates (20–30 nm diameter) obtained in H2O–THF with a 90% volume fraction of water is much smaller than that obtained with 70 and 80% water contents. This

Figure 6.21 UV spectra of PPP in H2O–THF mixtures with different volume fractions of water. PPP concentration: 5  105 mol l1.

148 Aggregation-Induced Emission: Applications

Figure 6.22 FE-SEM of PPP aggregates obtained in H2O–THF mixtures with (a) 70%, (b) 80% and (c) 90% volume fractions of water. PPP concentration: 5  105 mol l1.

caused a decrease in light-scattering tails in the long-wavelength region observed in the absorption spectrum for the solvent with a 90% water content (Figure 6.21). Upon photoexcitation, the dilute THF solution of PPP shows a PL spectrum with an emission peak at 386 nm (Figure 6.23). When water is continually added to the THF solution of PPP while keeping the luminogen concentration unchanged at 1  105 mol l1, the PL intensity of PPP increases slowly when the water content in the H2O–THF mixtures is ‘low’ (60%) but increases rapidly when the water content is ‘high’ (>60%) (Figure 6.23). Since water is a poor solvent for PPP, the molecules of PPP must have aggregated in the H2O–THF mixtures with high water contents, in agreement with the observation of the light-scattering tails in the absorption spectra discussed above (Figure 6.21). Evidently, the emission of PPP is spectacularly boosted by aggregation; in other words, PPP is AIEE active. Compared with the PL spectrum of silole, a moderate blue shift ( 15 nm) in the emission peak is observed when the water fraction is increased from 60 to 80%. However, the PL intensity decreases and a red shift of the emission peak from 372 to 378 nm occurs when the water volume fraction is increased from 80 to 90%. Careful inspection of the PL spectra of PPP in the H2O–THF mixtures reveals an increase/decrease in the intensity ratio and a blue/red shift in the emission peak with increase in the water volume fraction from 60 to 90% that agree well with the absorbance spectra shown in Figure 6.21. This is probably due to the change in the packing mode of the PPP molecules in the aggregates. In the mixtures with ‘low’ water content, the dye molecules may steadily assemble in an ordered fashion to form more emissive, bluer crystalline aggregates. In the mixtures with ‘high’ water contents, the dye molecules may, however, quickly agglomerate in a random way to form less emissive, redder, and smaller particles (Figure 6.22).

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 149 Water fraction 0% 60% 200 70% 80% 90% 150

100

PL Intensity (I/Io )

PL Intensity (a.u.)

250

0 10 20 30 40 50 60 70 80 90

Water fraction / %

50

0 320

370

420 Wavelength / nm

470

520

Figure 6.23 PL spectra of PPP in H2O–THF mixtures. The inset illustrates the relationship between PL intensity of PPP at the maximum intensity and the water volume fraction in H2O–THF mixtures. PPP concentration: 1  105 mol l1. Excitation wavelength: 310 nm.

To investigate further the process involved in the AIEE phenomenon, we studied the relationship between the PL intensity of PPP and the aggregation time in H2O–THF mixture with an 80% water volume fraction. Water was injected into the THF solution of PPP with vigorous stirring at the room temperature, and the PL was measured after stirring for 2 min. The PL spectra show that the rate of fluorescence enhancement is first order with respect to time, and at the same time the maximum peak gradually blue shifts from 381 to 374 nm (Figure 6.24). It is assumed that, initially, the mechanical shear stress leads to a greater chance of valid collisions, hence a portion of the dye molecules cluster together to form tiny particles. The portion of

Figure 6.24 Dependence of the PL intensity of PPP on aggregation time in H2O–THF (80 : 20 v/ v). PPP concentration: 1  105 mol l1. Excitation wavelength: 310 nm.

150 Aggregation-Induced Emission: Applications the dye molecules remaining in the solvent mixture then gradually deposits on to the initially formed particles in a way similar to recrystallization. The size of the PPP aggregates and the degree of arrangement between PPP molecules in the aggregates can be increased with increase in the aggregation time, which are likely to enhance the RIR effect. This is the reason why the peak was blue shifted and enhanced.

6.5 AIEE Mechanism of Pentaphenylpyrrole The crystal structures of the fluorophores in the aggregated state are the most direct evidence to help us to explain the mechanism of AIEE. Single crystals of PPP were grown by slow crystallization in a H2O–THF mixture. Crystals of high quality were used for X-ray diffraction (XRD) analysis. The crystal structure of PPP belongs to the monoclinic system with space group P2(1)/c (Table 6.1). In the crystal, PPP adopts a highly twisted conformation with torsion angles between the pyrrole group and the neighboring phenyl groups of 40.09, 53.23, 56.18, 48.04, and 60.07 (Figure 6.25a) . As shown in Figure 6.25b, the molecules of PPP are packed into molecular columns that are perpendicular to the plane of the central pyrrole rings.  The distance between two molecules within one column for PPP is 5.14 A, which is too large to undergo p–p interactions. As shown in Figure 6.25c, no face-to-face p–p stacking exists but rather edge-to-face interactions such as aromatic CH


p hydrogen bonding in the crystal structure [31, 34, 89]. The delocalized system of sp2-hybridized covalent bonds can act as an acceptor group, and hydrogen atoms serve as proton donors for the formation of aromatic CH


p hydrogen bonds. The difference in the torsion angles is caused by the CH


p interaction, which in turn stabilizes the twisted conformation of the dye molecule, Table 6.1 Crystal data and structure refinement parameters for PPP. Empirical formula Formula weight Temperature Wavelength Crystal system, space group Unit cell dimensions

Volume Z, calculated density Absorption coefficient F(000) Crystal size Theta range for data collection Limiting indices Reflections collected/unique Completeness to theta ¼ 27.59 Absorption correction Max. and min. transmission Refinement method Data/restraints/parameters Goodness-of-fit on F2 Final R indices [I > 2s(I)] R indices (all data) Largest diff. peak and hole

C34H25N 447.55 296(2) K  0.71073 A Monoclinic, P2(1)/n  a ¼ 10.76300(10) A, a ¼ 90  b ¼ 9.89870(10) A, b ¼ 95.4160(10)  c ¼ 23.7757(3) A, g ¼ 90  2521.75(5) A3 4, 1.179 Mg m3 0.068 mm1 944 0.53  0.41  0.26 mm 1.72–27.59 13  h  14, 12  k  12, 30  l  30 23331/5805 [R(int) ¼ 0.0290] 99.4% Semiempirical from equivalents 0.9826 and 0.9651 Full-matrix least-squares on F2 5805/0/316 1.022 R1 ¼ 0.0536, wR2 ¼ 0.1615 R1 ¼ 0.0662, wR2 ¼ 0.1750  0.357 and 0.578 e A3

Aggregation-Induced Emission and Applications of Aryl-Substituted Pyrrole Derivatives 151

Figure 6.25 (a) Molecular structure of PPP. (b) Stacking image of PPP. The hydrogen atoms have been omitted for clarity. (c) Schematic intermolecular interactions in the crystal of PPP. The interaction distance of the C–H


p  center is 2.86 A . Atoms: carbon, gray; hydrogen, white; nitrogen, yellow.

helping to hinder the rotation of the s-bond between the phenyl rings and the pyrrole group. This structural rigidification may have made the crystals stronger emitters [16–31]. The fluorescence quenching of PPP in solution might be understood as dominant nonradiative decay. In dilute solution, twisting of the s-bond between the phenyl and the pyrrole group might facilitate the approach between the excited and ground states of PPP and, thus, the occurrence of efficient rapid radiationless decay. Once the water content is above 70%, however, the degree of internal conversion is insignificant because the rigid environment (C–H


p bond formation) restricts intramolecular rotations of PPP, resulting in the observed enhanced emission. Furthermore, crystallographic analysis indicated that the  long molecular distance ( 5.14 A) reduces the distance-dependent intermolecular quenching effects to produce intense fluorescence in the aggregated state. Therefore, the enhanced emission of PPP is attributed to the synergistic action of the RIR effect with the twisted geometry configuration. Based on B3LYP/6–31G calculations, Liu and co-workers found that the filled p-orbitals (or HOMOs) and the unfilled orbitals (or LUMOs) are mainly dominated by orbitals originating from the silole ring and the two phenyl groups at the 2,5-positions in all cases, while the LUMOs have significant orbital density at the two exocyclic s-bonds on the ring silicon, implying that s –p conjugation plays an important role [24]. The HOMO and LUMO of PPP were calculated using the B3LYP/6– 31þG basis set (Figure 6.26). The results revealed that the HOMO of PPP is similar to that of 1,1,2,3,4,5-hexaphenylsilole (HPS) [24, 30], which is located on the core ring (pyrrole or silole) and

152 Aggregation-Induced Emission: Applications

Figure 6.26 Molecular orbital amplitude plots of HOMO and LUMO energy levels of PPP calculated using the B3LYP/ 6–31þG basis set.

also on the phenyl rings. The LUMO wavefunction of PPP, however, is found to be localized on the both the pyrrole ring and three phenyl rings at the 1,2,5-positions with a staggered distribution, which is much more complicated than that in HPS. This may cause a decrease in fluorescence in the solid state, leading to a weaker AIEE response compared with HPS.

6.6 Conclusion The relationship between the structures and light-emitting properties of a series of aryl-substituted pyrroles was studied during aggregation. Comparing the optical properties and aggregated structures of these pyrroles, it is suggested that the more twisted configuration that prevented a parallel orientation of conjugated chromophores, combined with the RIR effect, was the main cause of the AIE or AIEE phenomena. With the introduction of carboxylic groups, triphenylpyrrole salts with AIE properties can be used as chemosensors for the high-sensitivity, high-selectivity, turn-on detection of Al3þ ions in aqueous solution.

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7 Biogenic Amine Sensing with AggregationInduced Emission-Active Tetraphenylethenes Takanobu Sanji and Masato Tanaka Chemical Resources Laboratory, Tokyo Institute of Technology, Japan

7.1 Introduction 7.1.1 Biogenic amines Food freshness is important both for food appearance and for food safety, especially concerning meat and fish. People usually check the freshness of food in the home or shop by sight and/or smell. Although these senses are important, it is difficult to quantify and externalize the freshness using these alone. Hence food freshness sensors are highly desirable. Biogenic amines, which are produced by decarboxylation of amino acids, are found in virtually all living organisms and play a significant role in regulating cell growth and differentiation [1]. Increased production of biogenic amines, for example, is caused by abnormal rapid cell proliferation, where the biogenic amine levels can serve as markers of health complications, including cancer and bacterial infection [2]. In addition, several biogenic amines, such as histamine, putrescine, and cadaverine, are associated with spoilage of raw and processed foods, causing food poisoning [3]. Accordingly, biogenic amine levels can serve as probes of food freshness. In this context, the detection and identification of biogenic amines are important for health and food safety. 7.1.2 Sensing methods for biogenic amines Several methods exist for detecting biogenic amines, including chromatographic techniques such as gas chromatography and liquid chromatography [4], which are highly sensitive but often require sample pretreatment and relatively long analysis times, making them unsuitable for routine and on-site use. Other methods involve the use of enzymes [5], antibodies [6], and fluorescent molecules [7]. Lavigne and co-workers demonstrated a colorimetric method for the detection and identification of amines using carboxylic acid-functionalized polythiophene [8]. Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

158 Aggregation-Induced Emission: Applications In this chapter, as an alternative, fluorimetric sensing for the detection and identification of biogenic amines using aggregation-induced emission (AIE)-active tetraphenylethenes (TPEs) is presented [9].

7.2 Fluorimetric Sensing of Biogenic Amines with AIE-Active TPEs 7.2.1 Design for fluorimetric sensing of biogenic amines The design for sensing biogenic amines is depicted in Figure 7.1. TPE was used as the sensory element. TPE is AIE active, that is, it shows no emission in solution but intense emission in a state of aggregation [10]. Because a carboxylic acid interacts with amines through hydrogen bonding and/or electrostatic interactions, carboxylic acid-modified TPEs display emission, that is, ‘turn on’ fluorescence when they recognize amines and then form aggregates. TPEs 1–3 (Scheme 7.1) were easily synthesized; they have different linking units with the carboxylic acid groups. 7.2.2 Sensing studies and statistical analysis A solution of 1 (10 mM) was practically nonluminescent. However, after addition of 1,4-butanediamine (BDA, putrescine) to the solution, the emission intensity at 480 nm gradually increased (Figure 7.2). The fluorescence intensity at 480 nm showed an
80-fold increase ([BDA] ¼ 50 mM) relative to the original value. The fluorescence signal increased almost linearly with increase in amine concentration and reached a constant value. As shown in the inset, the intense emission after addition of BDA was clear to the naked eye. However, after filtration with a 0.45 mm pore-size membrane, the solution returned to being nonluminescent.

Figure 7.1 Schematic diagram of fluorescence ‘turn-on’ sensing of amines with TPEs. Reproduced with permission from [9], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

Biogenic Amine Sensing with Aggregation-Induced Emission-Active Tetraphenylethenes 159

Scheme 7.1 Structures of amines used as analytes and carboxylic acid-modified TPEs 1–3.

The dramatic enhancement of the fluorescence intensity upon addition of BDA can be attributed to aggregation triggered by divalent interactions of BDA with 1 bearing carboxylic acid groups, as illustrated in Figure 7.1. The detection limit of BDA was estimated to be at the parts per billion level, evaluated from a signal-tonoise ratio >3, which is notably more sensitive than previously reported colorimetric sensors [8]. In total, 10 amines were examined as sensing targets: five a,v-diamines, 1,2-ethylenediamine (EDA), 1,3-propanediamine (PrDA), BDA, 1,5-pentanediamine (PeDA, cadaverine), and 1,6-hexanediamine (HDA); two polyamines, spermidine (SpermD) and spermine (SperM); and three aromatic amines, histamine (HistA), tryptamine (TryptA), and phenethylamine (PhenA) (Scheme 7.1). Addition of each amine to a dichloromethane solution of 1 turned on the fluorescence of the solution, but the fluorescence intensity was dependent on the amine (Figure 7.3). For example, with addition of the a,v-diamines, which differ only by a methylene unit, the fluorescence increased with increasing chain length of the amines. However, HDA did not follow this trend. With addition of polyamines, 1 showed a high fluorescence; however,

160 Aggregation-Induced Emission: Applications

Figure 7.2 Fluorescence spectra of 1 upon addition of BDA in dichloromethane ([1] ¼ 10 mM, lex ¼ 350 nm). The inset shows photographs of 1 and the mixture with BDA under irradiation with UV light ([1] ¼ 10 mM, [BDA] ¼ 10 mM). Reproduced with permission from [9], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

addition of aromatic amines resulted in only weak fluorescence. The fluorescence responses of 1 in the presence of the amines were evident to the naked eye, as shown in the inset in Figure 7.3. The fluorescence responses of 1 to the amines result from multiple inter- and intramolecular interactions between the carboxylic acid side chains on the TPE and the amine via electrostatic and/or hydrogen-bonding interactions, as shown in Figure 7.1. Indeed, it seemed to be associated with the pKa value of each amine. Furthermore, the three TPEs 1–3 exhibited different fluorescence response patterns in the presence of the amines. This is because their binding ability to the amines is dependent on the length and degree of flexibility of the tether. The sensitivity of the TPEs to the amines also depended on the medium used. For example, the assay of 1 was examined in dichloromethane and those of 2 and 3 in a mixture of dichloromethane and hexane. However, the assays did not work in water because of their solubility. Because the fluorescence response pattern is unique to each of the amines and TPEs, a statistical analysis was performed to discriminate the amines. A sensor array based on analyte-specific patterns arising from the differential binding affinity of a set of nonspecific receptors is attractive for this purpose, because this sensing approach simplifies the sensor design [11]. Linear discriminant analysis (LDA) is one of the most powerful statistical methods for finding the linear combination of features that best separates two or more classes of objects [12]. For the statistical analysis, the fluorescence intensities of the three TPEs 1–3 were monitored after addition of the 10 different amines at two different concentrations (10 and 50 mM). Eight replicates were analyzed for each amine at each concentration, for a total of 48 measurements for each amine. Thus, 480 measurements overall (3 TPEs  10 analytes  2 concentrations  8 replicates) were considered for LDA. The LDA results were projected in two dimensions. Figure 7.3b shows the canonical scores plot for the first two factors of the fluorescence response patterns of 1–3. The first two canonical factors contained 80.1%

Biogenic Amine Sensing with Aggregation-Induced Emission-Active Tetraphenylethenes 161

Figure 7.3 (a) Fluorescence spectra of 1 upon addition of EDA, PrDA, BDA, PeDA, HDA, SpermD, SperM, HistA, TryptA, and PhenA ([1] ¼ 10 mM, [amine] ¼ 10 mM, lex ¼ 350 nm). The inset shows photographs of 1 and the mixture with BDA, SpermD, and HistA under irradiation with UV light. (b) LDA canonical scores plot for the first two factors of fluorescence response patterns of TPEs 1–3 against the amine analytes. Reproduced with permission from [9], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

162 Aggregation-Induced Emission: Applications and 16.9% of the variation, respectively, representing 97.0% in total, which is a very good value. The canonical patterns were clustered into 10 different groups, although the groupings were subtle for some amines. In this plot, the a,v-diamines (except EDA) and the polyamines are at the middle right and top right, respectively, and the aromatic amines are at the left. The leave-one-out classification option was used to estimate the predictive ability of the LDA model; it showed the classification accuracy to be 98%, correctly identifying 78 out of the 80 samples. The two errors occurred for the classification of HistA and TryptA, which showed weak fluorescence against the TPEs. 7.2.3 Determination of histamine concentration Next, quantification of the amount of a biogenic amine was examined. HistA is the most prevalent biogenic amine in tuna fish and potentially hazardous to health. The US Food and Drug Administration (FDA) set the safe maximum concentration of histamine in tuna at 50 ppm [13]. When tested in a ‘tuna fish matrix,’ which was extracted from canned tuna with dichloromethane–hexane and spiked with HistA, addition of the analyte to a solution of 2 increased the fluorescence at 480 nm (Figure 7.4). The ‘turn-on’ fluorescence was clear to the naked eye. The fluorescence intensity at 480 nm increased linearly with increase in HistA concentration in the range 0–100 ppm. The line obtained could be used as a calibration curve for quantification of the amount of HistA in the analyte. It is important to note that the detection sensitivity of this assay is better than the safe concentration level (50 ppm) of HistA in tuna set by the FDA.

Figure 7.4 Changes in the fluorescence intensity of 2 at 480 nm upon addition of HistA in a ‘tuna fish matrix’ ([2] ¼ 10 mM, lex ¼ 350 nm). The inset shows photographs of 2 and the mixture with HistA (0, 20, 50, and 100 mM) under irradiation with UV light. Reproduced with permission from [9], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

Biogenic Amine Sensing with Aggregation-Induced Emission-Active Tetraphenylethenes 163

Figure 7.5 (a) Structure of cyanuric acid-modified TPE, 4, for melamine sensing. (b) Schematic representation of ‘turn-on’ fluorescence sensing of melamine using 4 based on AIE. (c) Photographs of 4 (10 mM) with extracts from melamine-contaminated infant powdered milk (blank, 1, and 10 ppm, from left to right) in acetonitrile solution. Reproduced with permission from [14], # 2012 Wiley-VCH Verlag GmbH.

7.2.4 Fluorimetric sensing of melamine with AIE-active TPEs In conjunction with the detection and identification of biogenic amines, a sensor for melamine has recently been developed. A few years ago, melamine was found as a contaminant in milk, milk products, and infant formula in China. Owing to increasing public concern, selective detection methods for melamine are highly desirable. An AIE-active TPE with cyanuric acid moieties, 4, was designed (Figure 7.5) [14]. This approach was adopted because melamine and cyanuric acid combine to form a stable adduct through multivalent hydrogen-bonding interactions. TPE integrated with cyanuric acid moieties 4 displayed intense emission, that is, ‘turned on’ fluorescence, when it recognized melamine in real powdered milk. The limit of detection was below the safe concentration level of melamine (1 ppm) in real powdered milk set by the FDA.

7.3 Summary and Outlook A fluorimetric sensing array for the detection and identification of biogenic amines using carboxylic acidmodified TPEs 1–3 based on the AIE mechanism has been demonstrated. In the sensing array, TPEs 1–3

164 Aggregation-Induced Emission: Applications showed unique fluorescence response patterns against 10 amines, which were classified with 98% accuracy using LDA. The HistA concentration was estimated by a further analysis of the fluorescence intensity in a ‘tuna fish matrix.’ This sensing array is a highly sensitive and convenient detection method for biogenic amines at hazardous levels and thus could serve as a food freshness sensor for on-site use. However, further experiments, for example, testing in a more complex environment and aqueous solutions, are required to demonstrate the practical use of this assay.

References 1. (a) Tabor, H., and Tabor, C.W. (1983) Polyamines, Academic Press, New York; (b) Zappia, V., and Pegg, A.E. (1988) Progress in Polyamine Research: Novel Biochemical, Pharmacological, and Clinical Aspects, Plenum Press, New York. 2. (a) Russell, D.H. (1971) Nat. New Biol., 233, 144–145; (b) Fujita, K., Nagatsu, T., Shinpo, K., Maruta, K., Teradaira, R., and Nakamura, M. (1980) Clin. Chem., 26, 1577–1582. 3. (a) ten Brink, B., Damink, C., Joosten, H.M.L.J., and Huis in’t Veld, J.H.J. (1990) Int. J. Food Microbiol., 11, 73–84; (b) Santos, M.H.S. (1996) Int. J. Food Microbiol., 29, 213–231. 4. (a) Ruiz-Capillas, C., and Moral, A. (2001) J. Food Sci., 66, 1030–1032; (b) Rossi, S., Lee, C., Ellis, P.C., and Pivarnik, L.F. (2002) J. Food Sci., 67, 2056–2060. 5. Cheng, S.G.G., and Merchant, Z.M. (1995) Biosensors in food analysis, in Characterization of Foods: Emerging Methods (ed. A.G. Gaonkar), Elsevier Science, New York, Chapter 14. 6. Merchant, Z.M., and Cheng, S.G.G. (1995) Developments in characterization of foods using antibodies, in Characterization of Foods: Emerging Methods (ed. A.G. Gaonkar), Elsevier Science, New York, Chapter 15. 7. (a) Greene, N.T., and Shimizu, K.D. (2005) J. Am. Chem. Soc., 127, 5695–5700; (b) Montes-Navajas, P., Baumes, L.A., Corma, A., and Garcia, H. (2009) Tetrahedron Lett., 50, 2301–2304; (c) Satrijo, A., and Swager, T.M. (2007) J. Am. Chem. Soc., 129, 16020–16028; (d) McGrier, P.L., Solntsev, K.M., Miao, S., Tolbert, L.M., Miranda, O.R., Rotello, V.M., and Bunz, U.H.F. (2008) Chem. Eur. J., 14, 4503–4510; (e) Ikeda, M., Yoshii, T., Matsui, T., Tanida, T., Komatsu, H., and Hamachi, I. (2011) J. Am. Chem. Soc., 133, 1670–1673. 8. (a) Nelson, T.L., O’Sullivan, C., Greene, N.T., Maynor, M.S., and Lavigne, J.J. (2006) J. Am. Chem. Soc., 128, 5640–5641; (b) Maynor, M.S., Nelson, T.L., O’Sullivan, C., and Lavigne, J.J. (2007) Org. Lett., 9, 3217–3220; (c) Nelson, T.L., Tran, I., Ingallinera, T.G., Maynor, M.S., and Lavigne, J.J. (2007) Analyst, 132, 1024–1030. 9. Nakamura, M., Sanji, T., and Tanaka, M. (2011) Chem. Eur. J., 17, 5344–5349. 10. For a review of AIE, see Hong, Y., Lam, J.W.Y., and Tang, B.Z. (2009) Chem. Commun., 4332–4353. 11. (a) Albert, K.J., Lewis, N.S., Schauer, C.L., Sotzing, G.A., Stitzel, S.E., Vaid, T.P., and Walt, D.R. (2000) Chem. Rev., 100, 2595–2626; (b) Collins, B.E., and Anslyn, E.V. (2007) Chem. Eur. J., 13, 4701–4708. 12. Jurs, P.C., Bakken, G.A., and McClelland, H.E. (2000) Chem. Rev., 100, 2649–2678. 13. FDA (1996) Compliance Policy Guides 7108.240, Section 540.525, US Food and Drug Administration, Washington, DC. 14. Sanji, T., Nakamura, M., Kawamata, S., Tanaka, M., Itagaki, S., and Gunji, T. (2012) Chem. Eur. J., 18, 15254–15257.

8 New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules Based on the Aggregation and Deaggregation Mechanism Ming Wang, Guanxin Zhang and Deqing Zhang Beijing National Laboratory, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, China

8.1 Introduction The detection of chemical and biological species plays a significant role in biomedical diagnostics, environmental monitoring, and national security areas [1–4]. In general, fluorescent sensors are able to report the presence of a single or a mixture of species by modulating the physicochemical properties (e.g. emission) upon the recognition of analytes via either ICT (intramolecular charge transfer) or PET (photoinduced electron transfer) mechanisms [5, 6]. Traditional organic or polymeric fluorophores for sensing usually suffer from the aggregation-caused emission quenching (ACQ) effect, and as a result they are only emissive in dilute solutions, whereas the aggregation quenches their emission severely due to the formation of species such as excimers and exciplexes [7, 8]. The ACQ effect compromises the sensitivity and selectivity of fluorescent sensors by weakening the reporting emission signals and limiting the use of concentrated solutions. Therefore, luminogens that remain strongly emissive and possess high quantum yields in aggregated or solid states hold enormous potential for fluorescent sensing, particularly with regard to developing solid sensors and aqueous sensors for trace amounts of biological samples, where the fluorescent dyes commonly used are prone to aggregate and thus result in emission quenching. As discussed in previous chapters, a few fluorophores such as 1-methyl-1,2,3,4,5-pentaphenylsilole (silole) and tetraphenylethene (TPE) show abnormal emissive behaviors; they are almost nonfluorescent in solutions, but they become strongly emissive after aggregation. Such an unusual fluorescent feature was termed aggregation-induced emission (AIE) by Tang and co-workers. AIE molecules have been intensively

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

166 Aggregation-Induced Emission: Applications explored for optoelectronic materials in the past decades. Simultaneously, new chemo-/biosensors have been devised by taking advantage of the AIE feature of certain fluorophores. The sensing mechanism is based on the manipulation of the respective aggregation and deaggregation of AIE molecules. In this chapter, we focus on chemo-/biosensors with silole and TPE molecules mainly including (1) cation and anion sensors, (2) fluorimetric detection of biomolecules, (3) fluorimetric assays for enzymes, and (4) fluorimetric detection of physiologically important small molecules.

8.2 Cation and Anion Sensors TPE molecules with appropriate ligands have been successfully employed for sensing cations. The molecular design rationale is illustrated in Scheme 8.1 and explained as follows: coordination with metallic cations leads to the formation of coordination polymers, and as a result aggregation of TPE fluorophores occurs, thus switching on the emission. We previously reported the selective sensing Agþ and Hg2þ with compounds 1 and 2 bearing adenine and thymine moieties (see Scheme 8.2), respectively [9]. As expected, both 1 and 2 were weakly emissive in THF–H2O or H2O–CH3CN, but their fluorescence intensities gradually increased on addition of Agþ and Hg2þ to the solutions of 1 and 2, respectively (see Figure 8.1). Hence 1 and 2 can be utilized for fluorescence turn-on detection of Agþ and Hg2þ, respectively. For instance, Hg2þ with a concentration as low as 0.37 mM can be detected with 2. Moreover, the interferences from other metallic cations can be neglected. Similarly, fluorescence enhancement was detected for terpyridine-functionalized TPE (3, Scheme 8.2) after mixing with Zn2þ [10]. Compound 4, bearing two imidazole moieties, was potentially useful for sensing Fe3þ as its fluorescence increased in the presence of Fe3þ [11]. Compound 5 was reported for fluorescence turn-on detection of Zn2þ in aqueous solution [12]. The binding of 5 with Zn2þ will on the one hand inhibit the intramolecular PET, and on the other lead to cross-linking of TPE cores, hence molecules of 5 aggregate, leading to fluorescence enhancement (see Figure 8.2). Only slight fluorescence enhancement was detected for 5 in the presence of other cations. Furthermore, the corresponding ethyl ester compound was utilized for intracellular Zn2þ imaging since it was able to penetrate into NIH 3T3 cells easily and hydrolyzed to 5 by the intracellular esterase. Apart from coordination of metal cations, the formation of covalent bonds facilitated by metal cations can also induce the aggregation of TPE fluorophores. For instance, Cu2þ-catalyzed click reaction of the

Scheme 8.1 Molecular design rationale for cation sensors with TPE molecules.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 167

Scheme 8.2 Structures of compounds for cation and anion sensors based on AIE.

TPE derivative 6 and the diethylene glycol dipropiolate 7 in the presence of sodium ascorbate induced the cross-linking of 6 and 7, leading to aggregation of TPE fluorophores and a dramatic fluorescence enhancement [13]. Therefore, the ensemble of 6 and 7 can be used for the selective detection of Cu2þ because of the high specific catalytic ability of Cu2þ towards click reactions. A different sensing approach for Hg2þ was based on the ensemble of 8 and thymine-rich single-stranded DNA (ssDNA) [14]. Owing to the electrostatic and hydrophobic interactions, fluorescent aggregates were

Figure 8.1 (a) Fluorescence spectra of 1 (5.60  105 M) in H2O–THF (5:1 v/v) in the presence of increasing amounts of AgClO4 (from 0 to 108 mM). (b) Fluorescence spectra of 2 (1.34  104 M) in H2O–CH3CN (2:1 v/v) in the presence of increasing amounts of Hg(ClO4)2 (from 0 to 214 mM). Reproduced with permission from [9], # 2008 The American Chemical Society.

168 Aggregation-Induced Emission: Applications

Figure 8.2 (a) Fluorescence spectra (lex ¼ 340 nm) of 5 (10 mM) on addition of Zn2þ in HEPES buffer (50 mM, pH ¼ 7.4, 0.1 M NaCl). (b) Cation selectivity profiles of 1 (10 mM) in the presence of various metal cations in HEPES buffer (50 mM, pH ¼ 7.4, 0.1 M NaCl). Reproduced with permission from [12], # 2011 The American Chemical Society.

formed for 8 in the presence of ssDNA. In the presence of Hg2þ, the ssDNA was folded into a hairpin-like structure that exhibited a strong interaction with 8; accordingly, more fluorescence enhancement was observed for the ensemble of 8 and ssDNA after addition of Hg2þ. Chemosensors for anions were also constructed by taking advantage of the AIE behavior of silole molecules. We developed a fluorescence turn-on sensor for cyanide assay in aqueous solution with the ensemble of 9 and 10 [15]. As illustrated in Scheme 8.3, 10 can be transformed into the amphiphile after reaction with

Scheme 8.3 Illustration of the design rationale for the fluorescence turn-on detection of cyanide by making use of the AIE feature of silole.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 169 CN in aqueous solution; assembly of the amphiphile into the micelles will cause the aggregation of 9 via electrostatic and hydrophobic interactions and turn on the fluorescence of 9. The detection limit for cyanide was as low as 7.74 mM, which is lower than the concentration of cyanide in the blood of fire victims. No appreciable fluorescence enhancement was observed for 9 in the presence of other anions including OAc, Br, Cl, F, H2PO4, HSO4, N3, and NO3. Trogler and co-workers reported the formation of well-dispersed emissive nanoparticles by injection of the THF solution of 11 (see Scheme 8.2) into water [16]. The fluorescence of these emissive particles was efficiently quenched by CrO42. It was suggested that the electron transfer from the excited state of 11 to CrO42 accounts for the fluorescence quenching. CrO42 at concentrations as low as 0.1 ppm can be detected and common anions such as NO3, NO2, SO42, and ClO4 posed no interferences.

8.3 Fluorimetric Biosensors for Biomacromolecules Tang and co-workers first reported the fluorescence ‘light-up’ of 12 (see Scheme 8.4) after mixing with DNA [17]. Fluorescence enhancement of up to 16-fold was observed for 12 in the presence of 300 mg ml1calf thymus DNA (ctDNA). They attributed this to the electrostatic binding of negatively charged DNA with 12 with a positively charged moiety, and as a result molecules of 12 were docked on the surface of DNA and aggregation of TPE fluorophores occurred. They further reported compound 13 with four positively charged moieties, which was able to discriminate the folded DNA G-quadruplex from its random-coil structure [18]. This is based on the observation that the fluorescence of 13 is weaker after mixing with the G-quadruplex than that in the presence of the respective ssDNA; moreover, the fluorescence spectrum of 13 is more red shifted after the addition of the G-quadruplex. The results also revealed that the alkyl chain length and molecular structure of the

Scheme 8.4 Structures of compounds for biosensors based on AIE.

170 Aggregation-Induced Emission: Applications

Scheme 8.5 Illustration of the variation of the fluorescence of 9 after mixing with ssDNA and G-quadruplex DNA in the presence of exonuclease. I.

ammonium unit play critical roles in discriminating the G-quadruplex from its random-coil structure [19]. A short hydrophobic alkyl chain and also a more bulky ammonium chelating unit are important in Gquadruplex sensing. Steady-state and time-resolved emission spectral studies suggested that the specific binding of 13 with the G-quadruplex can be ascribed to their structural matching. Theoretical modeling further indicated that molecules of 13 may be docked on the grooves of the G-quadruplex surface with the aid of electrostatic interactions. Note that the G-quadruplex is recognized as a potential target to identify anticancer drugs, hence a simple and convenient G-quadruplex sensing strategy is highly desirable [20]. Zhou and co-workers reported the sensing of G-quadruplex DNA with 9 [21]. As illustrated in Scheme 8.5, the fluorescence of 9 was significantly enhanced after addition of either ssDNA or the folded G-quadruplex. However, the fluorescence of the ensemble of 9 and ssDNA became weak again after introducing exonuclease I to the solution because the hydrolysis of ssDNA and accordingly the fluorescent aggregates were disassembled. The enzymatic hydrolysis of ssDNA was inhibited by forming the Gquadruplex with stabilization of a specific ligand; accordingly, the silole–ssDNA–exonuclease I complex emitted differently in the absence and presence of G-quadruplex stabilization ligands. The label-free fluorescence probing of the G-quadruplex with 9 allowed real-time monitoring of the G-rich DNA folding process and also the identification of the G-quadruplex binding ligand as an anticancer drug. Tang and co-workers demonstrated that the fluorescence of the TPE derivative 12 with two cationic moieties could be also turned on after binding with proteins, but the sensitivity for sensing proteins with 12 was low [17]. They further designed the TPE derivative 14 with two anionic moieties for protein detection and quantitation [22]. Bovine serum albumin (BSA) at a concentration as low as 500 ng ml1 was successfully detected with 14. Furthermore, the protein detection with 14 was not subject to interferences from the miscellaneous bioelectrolytes in artificial urine, hence it was possible to determine the human serum albumin

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 171 (HSA) level in body fluids with 14 [23]. Based on the finding that the fluorescence of 14 cannot be switched on by denatured proteins that lack hydrophobic cavities, the fluorescence enhancement of 14 in the presence of albumin is attributed to the hydrophobic interactions involving hydrophobic pockets within BSA. Therefore, 14 and relevant AIE molecules provide an intriguing possibility of ‘seeing’ the hydrophobic pockets of proteins and active sites of enzymes. In addition, the light-up feature of 14 by albumin further allowed its application as a protein staining reagent in polyacrylamide gel electrophoresis (PAGE) assay. Alternatively, the incorporation of TPE moieties into the side chains of conjugated cationic polyelectrolytes 15 further improved the detection sensitivity for proteins [24]; for instance, BSA at ultralow concentrations (0–0.6 ppm) can be analyzed with polymer 15. AIE molecule 14 with two anionic moieties was also demonstrated to be a versatile tool in monitoring protein conformational transitions and fibrillation [23]. The nonemissive probe 14 was aggregated and its emission was enhanced upon addition of HSA; however, the denatured HSA in the presence of guanidine did not trigger the aggregation of 14. By utilizing the AIE feature of 14 and the F€ orster resonance energy transfer (FRET) from HSA to 14, the unfolding process of HSA was monitored and revealed to be a multistep transition process involving molten globule intermediates, as illustrated in Scheme 8.6. The protein amyloid fibrillation is associated with a variety of diseases such as type II diabetes and Alzheimer’s disease, hence monitoring of protein amyloidosis is of great value in early disease diagnosis [25]. Tang and co-workers found that 14 interacted differently with native and fibril insulin, thus leading to different degrees of fluorescence enhancement [26]. The probe 14 cannot bind with native insulin efficiently and remains nonemissive after mixing with native insulin in a buffered solution; however, 14 becomes strongly fluorescent after incubation with insulin fibril. Such distinct emission behaviors of 14 in the presence of native and fibrillar forms of insulin permit the ex situ monitoring of amyloidogenesis kinetics and high-contrast fluorescence imaging of protein fibrils. Further, the binding of 14 with insulin can inhibit the nucleation process and impede the formation of protofibril. This results from the hydrophobic interactions

Scheme 8.6 The proposed mechanism for fluorescence probing of the guanidine hydrochloride-induced HSA unfolding process with 14.

172 Aggregation-Induced Emission: Applications of 14 and hydrophobic residues of insulin that partially stabilize the unfolded insulin and obstruct the formation of critical oligomeric species in the protein fibrillogenesis process. Apart from the application of AIE molecules with charged moieties in sensing proteins based on the respective electrostatic and hydrophobic interactions, TPE and silole molecules with specific recognition moieties have been investigated for the selective detection of proteins. For instance, TPE derivatives modified with saccharides were reported to be strongly emissive in the presence of lectin or cholera toxin; this was attributed to the saccharide–protein interactions which induce the aggregation of AIE molecules [27–29]. Moreover, these AIE molecules were extended to probe protein– protein interactions [30]. Liu and co-workers recently reported the selective sensing of specific biomarker protein with silole derivative 16 functionalized with cyclic arginine–glycine–aspartic acid (cRGD) [31]. The fluorescence of 16 was enhanced dramatically after mixing with integrin avb3. This is due to the specific binding of cRGD in 16 towards integrin avb3 and as a result the intramolecular torsions and rotations within the silole moiety are restricted. In comparison, proteins with low affinity to cRGD did not boost the emission of 16. Cancer cell lines HT-29 and MCF-7 were selected to examine the in vitro detection of integrin avb3 with 16. The overexpression of integrin avb3 on the HT-29 cell surface induced probe 16 to emit after extracellular binding; therefore, the high binding affinity of 16 towards HT-29 suggested the potential to utilize 16 as a specific probe to discriminate integrin avb3-positive cancer cells from avb3-negative cancer cells and also applications in cell biology and in vivo tumor diagnosis. Heparin is a highly sulfated linear glycoaminoglycan (GAG) consisting of repeating units of 1 ! 4linked pyranosyluronic acid and 2-amino-2-deoxyglucopyranose residues. It is crucial to monitor and control the level and activity of heparin during and after surgery and to manipulate the amount of heparin for the anticoagulant therapy in order to avoid complications such as hemorrhage or thrombocytopenia induced by heparin overdose [32]. Zhang and co-workers studied the fluorescence spectral variation of AIE molecule 9 in the presence of heparin (see Scheme 8.7) [33]. The fluorescence of 9 was significantly enhanced after addition of heparin, and heparin with concentrations as low as 23 nM can be detected with 9. The interferences from other biomolecules can be largely eliminated by adjusting the pH of the solution. Furthermore, 9 can be utilized to probe the interaction of protamine and heparin.

Scheme 8.7 (a) Illustration of the fluorescence turn-on sensor for heparin based on the AIE feature of 9.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 173

Figure 8.3 Fluorescence spectra of the ensemble of 17 (12.0 mM) and 18 (13.2 mM) in HEPES buffer (5.0 mM, pH ¼ 7.4) solution after the addition of different amounts of heparin (from 0.0 to 18.0 mM). Each solution was mixed and left for 1.5 min before recording the fluorescence spectra; the excitation wavelength was 340 nm. Reproduced with permission from [34], # 2012 The Royal Society of Chemistry.

By combining the respective AIE and ACQ behaviors of TPE and anthracene, a ratiometric fluorescence detection of heparin was realized with compounds 17 and 18 [34]. As depicted in Figure 8.3, the fluorescence intensity at 421 nm due to anthracene emission decreased and that at 497 nm due to TPE emission gradually increased after increasing the concentration of heparin in the solution. In a similar way, Wang and co-workers reported the detection of heparin with the cationic conjugated polyfluorene probe 19 featuring TPE moieties [35]. The electrostatic interaction of 19 and heparin induced the aggregation of polymeric chains, and accordingly the energy transfer between TPE and polyfluorene moieties turned on the emission of TPE.

8.4 Fluorimetric Assays for Enzymes Silole and TPE molecules can form fluorescent aggregates with certain biomolecules such as DNA and proteins. It is well known that these biomolecules can be cleaved by the respective enzymes. Thus, the fluorescent aggregates can be disassembled after incubation with the respective enzymes and accordingly the fluorescence of the ensemble becomes weak again. In this way, label-free fluorimetric assays for enzymes can be established with these AIE molecules. Zhang and co-workers described a fluorimetric assay for nucleases with AIE molecule 9 (Scheme 8.8) [36]. As expected, the fluorescence of 9 increased after the addition of ssDNA (Figure 8.4). Interestingly, the fluorescence enhancement is dependent on the length of ssDNA, as depicted in Figure 8.4; the longer the ssDNA, the more significant is the increase in the fluorescence of 9 under the same conditions. After incubation with nuclease, the fluorescence of the ensemble of 9 and ssDNA gradually decreased (Figure 8.4). Furthermore, the fluorescence of 9 was more significantly reduced by increasing the concentration of nuclease. In this way, the activity of nuclease can be assayed with 9. Additionally, this fluorimetric assay was successfully utilized for screening inhibitors of nuclease. Adenosine triphosphate (ATP) was also able to induce the aggregation of 9 and turn on the fluorescence of 9 [37]. Moreover, 9 was able to discriminate ATP from ADP, AMP, and pyrophosphate by adjusting the pH and ionic strength of the solution so that the fluorescence was only enhanced in the presence of ATP. Accordingly, a label-free fluorimetric assay for the phosphatase (CIAP) was constructed with 9. Proteases are involved in a number of physiological processes, and their inhibitors are widely used for disease treatment [38]. Hence facile and convenient protease activity assay methods allow for rapid and

174 Aggregation-Induced Emission: Applications

Scheme 8.8 (a) Illustration of fluorescence turn-on detection of DNA and label-free nuclease assay based on the AIE feature of 9. (b) Structure of silole with a quaternary ammonium moiety (9) used for DNA detection and nuclease assay. (c) DNA sequence used in this study.

Figure 8.4 (a) Fluorescence spectra of 9 (2.0  105 M) in the presence of different amounts of ssDNA2 (from 0 to 0.3 mM). (b) Variation of the relative fluorescence intensity (I/I0) of 9 (2.0  105 M) versus the concentration of DNA with different lengths (5-mer to 30-mer). (c) Fluorescence spectrum of 9 (2.0  105 M) containing ssDNA2 (20 ml, 5 M) and those after cleavage by nuclease S1 ([nuclease S1] ¼ 50 U ml1) at 37  C for different periods. (d) Inhibition effect of pyrophosphate on ssDNA2 cleavage by nuclease S1: [ssDNA2] ¼ 5.0 mM, [nuclease S1] ¼ 50 U ml1, and [pyrophosphate] ¼ 1.2 mM. Reproduced with permission from [36], # 2008 American Chemical Society.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 175 Arg6 Trypsin TPE

De-aggregaion Weakly Fluorescent

TPE-Arg6 Complex Strongly Fluorescent SO3– NA +

O

20 O O H 2N

O HN

NH H 2N

NH2

O HN

NH H2N

NH2

O HN

NH H2N

NH2 H2N

O HN

NH NH2

O HN

NH H2N

NH2 H2N

OH

NH NH2

Scheme 8.9 Design rationale for trypsin activity assay and inhibitor screening based on AIE.

effective prescreening of drug candidates. As a proof of concept for the protease assay with AIE molecule 20, a positively charged peptide, Arg6, was selected and served as a substrate of trypsin and a template to induce the aggregation of 20 (Scheme 8.9). As depicted in Figure 8.5, the fluorescence of the ensemble of 20 and Arg6 gradually decreased after incubation with trypsin; the fluorescence was more significantly reduced by increasing the concentration of trypsin. The authors further demonstrated the capability of this trypsin assay with 20 in screening protease inhibitors [39]. An alternative fluorimetric trypsin assay was developed using BSA as the protease substrate and 8 as the fluorescent probe [40].

Figure 8.5 (a) Fluorescence spectra of 20 (60.0 mM) in phosphate-buffered saline (PBS) buffer solution (2.0 mM, pH ¼ 8.5) in the presence of different amounts of Arg6 peptide (from 0.0 to 10.0 mM). (b) Fluorescence spectra of the ensemble of 20 (60.0 mM) in PBS buffer solution [2.0 mM, pH ¼ 8.5, containing CaCl2 (10.0 mM)] and Arg6 peptide (10.0 mM) in the presence of trypsin (8.0 mg ml1) incubated at room temperature for different times. Reproduced with permission from [39], # 2010 American Chemical Society.

176 Aggregation-Induced Emission: Applications

Figure 8.6 (a) Fluorescence spectra of 14 [20 mM in PBS (10 mM) buffer solution, pH ¼ 8.0] in the presence of different amounts of myristoylcholine (from 0 to 32 mM). (b) Fluorescence spectra of the ensemble of 14 [20 mM in PBS (10 mM) buffer solution, pH ¼ 8.0] and myristoylcholine (25 mM) in the presence of AChE (0.5 U ml1) incubated at 25  C for different periods. Reproduced with permission from [41], # 2009 American Chemical Society.

Amphiphilic molecules can form micelles, which can also induce the aggregation of AIE molecules with oppositely charged units. By properly choosing the amphiphilic molecules that either can be good substrates of certain enzymes or can be generated in situ in the presence of the enzymes, label-free fluorimetric enzymatic assays can be established with AIE molecules and the amphiphilic molecules. For instance, myristoylcholine can induce the aggregation of 14 and thus turn on the fluorescence of 14. As depicted in Figure 8.6, the fluorescence of 14 gradually increased after the addition of myristoylcholine. However, the fluorescence of the ensemble of myristoylcholine and 14 gradually decreased after the introduction of acetylcholinesterase (AChE) into the ensemble, because myristoylcholine was hydrolyzed into myristic acid and choline in the presence of AChE, and as a result the fluorescent aggregates were disassembled (Scheme 8.10). Therefore, a label-free fluorimetric assay for AChE was constructed with the ensemble of myristoylcholine and 14. Moreover, this ensemble was also successfully applied for screening the inhibitors of AChE [41]. Notably, AChE inhibitors are currently widely used for the treatment of Alzheimer’s disease, hence reliable and simple AChE approaches for screening inhibitors of AChE are of great value in identifying potential therapeutics for this disease [42].

Scheme 8.10 Design rationale for AChE activity assay and inhibitor screening based on AIE.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 177

Scheme 8.11 Design rationale for AChE activity assay and inhibitor screening based on the AIE properties of 14.

Zhang and co-workers further developed a fluorescence ‘turn-on’ AChE assay with the ensemble of 14, acetylthiocholine (ATC), and the maleimide 20. As illustrated in Scheme 8.11, ATC is expected to be hydrolyzed into thiocholine, which will quickly react with the maleimide to generate the amphiphile with one positively charged unit (21); as a result, the aggregation of 14 occurs and the fluorescence of 14 will be switched on. Figure 8.7 also shows the fluorescence enhancement for the ensemble after incubation with

Figure 8.7 (a) Fluorescence spectra of the ensemble of 14 [20 mM in HEPES (10 mM) buffer solution, pH ¼ 7.35], 20 (30 mM), and ATC (30 mM) in the presence of AChE (0.1 U ml1) incubated at room temperature for different periods. (b) Variation of the fluorescence intensity at 490 nm versus the reaction time for the ensemble of 14 [20 mM in HEPES (10 mM) buffer solution, pH ¼ 7.35], 20 (30 mM), and ATC (30 mM) in the presence of different concentrations of AChE (0, 0.005, 0.01, 0.02, 0.05, 0.1 U ml1). Reproduced from [43], # 2009 American Chemical Society.

178 Aggregation-Induced Emission: Applications

Scheme 8.12 Preparation of emissive core–shell particles and design rationale for AChE activity assay and inhibitor screening based on TPE silica nanoparticles.

AChE. Obviously, as shown, the fluorescence increased more significantly by enhancing the concentration of AChE in the ensemble. Again, the ensemble can be utilized for screening the inhibitors of AChE [43]. More recently, emissive core–shell silica nanoparticles incorporating TPE were created for a fluorimetric AChE assay [44]. As illustrated in Scheme 8.12, the co-hydrolysis of TPE-silane 22 and tetraethyl orthosilicate (TEOS) formed emissive core–shell silica nanoparticles due to the restricted internal rotation of TPE. The fluorescence of silica nanoparticles was quenched in the presence of positively charged acetylcholine derivative 23, which incorporated the dabcyl [4-(dimethylamino)-40 -carboxyazobenzene] chromophore as the fluorescence quencher. As depicted in Figure 8.8, the emission of the silica nanoparticles was quenched after the addition of 23. However, further addition of AChE to the solution resulted in gradual fluorescence

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 179

Figure 8.8 (a) Fluorescence spectra of the silica particles (27.6 mg ml1 in 50 mM PBS buffer solution, pH ¼ 8.5) in the presence of different amounts of 23 (from 0 to 5.0 mM). Inset: photographs of the corresponding buffer solutions of silica particles (27.6 mg ml1) in the absence (A) and presence (B) of 23 (4.0 mM). (b) Fluorescence spectra (lexc. ¼ 334 nm) of the silica particles (27.6 mg ml1) and 23 (4.0 mM) in phosphate buffer solutions (50 mM, pH ¼ 8.5) containing AChE (2.0 U ml1) after incubation at 37  C for different times. Inset: photographs of the corresponding solutions of silica particles and 23 containing AChE (2.0 U ml1) before (A) and after (B) incubation at 37  C for 30 min under UV illumination (365 nm). Reproduced with permission from [44], # 2012 The Royal Society of Chemistry.

enhancement because the fluorescence quenching unit was cleaved from 23 via the AChE-catalyzed hydrolysis of 23. Fluorimetric assay with AIE molecule 9 was also established for monoamine oxidase (MAO-B). It is known that MAO-B can catalyze the aerobic oxidative deamination of amines to produce aldehydes and hydrogen peroxide, hence MAO plays significant roles in maintaining the balance of neurotransmitters, and also dietary and biogenic amines [45]. Zhang and co-workers reported a direct continuous fluorimetric MAO-B assay by taking advantage of the two reactions: (1) MAO-B-catalyzed oxidation of heptylamine to heptanal and (2) nucleophilic addition of NaHSO3 to heptanal to generate the amphiphile with a negativelycharged moiety (see Scheme 8.13). As shown in Figure 8.9, the ensemble of 9, heptylamine, and NaHSO3

Scheme 8.13 Design rationale for monoamine oxidase activity assay and inhibitor screening based on aggregation-induced-emission.

180 Aggregation-Induced Emission: Applications

Figure 8.9 (a) Fluorescence spectra of the ensemble of 9 (7.5  105 M), heptylamine (2.0  104 M), and NaHSO3 (1.0  104 M) in a mixture of HEPES buffer (10 mM, pH ¼ 7.4) and THF (200:1 v/v) in the presence of MAO-B (2.2 mg ml1) after incubation for different times at room temperature. (b) Variation of the fluorescence intensity at 467 nm versus the reaction time for the ensemble of 9 (7.5  105 M), heptylamine (2.0  104 M), and NaHSO3 (1.0  104 M) in a mixture of HEPES buffer (10 mM, pH ¼ 7.4) and THF (200:1 v/v) in the presence of different concentrations of MAO-B (0.25, 0.5, 1, 2.0 mg ml1); the excitation wavelength was 370 nm. Reproduced with permission from [46], # 2010 The Royal Society of Chemistry.

became emissive after incubation with MAO-B. Also, this ensemble was successfully utilized to screen the inhibitors of MAO-B [46].

8.5 Fluorimetric Detection of Physiologically Important Small Molecules Small molecules such as D-glucose, L-lactic acid, and thiols are involved in various physiological processes, and abnormal levels of these molecules are associated with various diseases. Accordingly, the development of sensitive and selective detection methods for these small molecules is highly desirable. By manipulating the aggregation and deaggregation of AIE molecules, new fluorimetric detection methods for physiologically important small molecules have been described in the past few years. Tang and co-workers investigated TPE derivative 24 (Scheme 8.14) with two boronic acid groups for the selective sensing of D-glucose [47]. As shown in Figure 8.10, the fluorescence of 24 gradually increased after incubation with D-glucose. Their studies revealed that such fluorescence enhancement occurs because D-glucose can bind two boronic acid groups and as a result oligomerization of 24 occurs, which leads to aggregation of TPE fluorophores and fluorescence enhancement. Moreover, almost no fluorescence enhancement was detected for 24 after incubation with D-fructose, D-galactose, or D-mannose. Hence selective detection of D-glucose was achieved with 24. AIE molecule 9 was also applied to the fluorescence turn-on detection of L-lactic acid [48]. This is based on tandem enzymatic and chemical reactions leading to the generation of the amphiphile with –COOH: (1) L-lactic acid was oxidized to pyruvic acid by lactate oxidase; (2) pyruvic acid underwent condensation with dodecanoic hydrazine (Scheme 8.15). The fluorescence of 9 can be switched on after exposure to the in situ-generated amphiphile as indicated in Figure 8.11, where the fluorescence spectra of the ensemble of 9, lactate oxidase, and dodecanoic hydrazine are displayed after addition

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 181

Scheme 8.14 Structures of compounds for sensing physiologically-important small molecules.

of different amounts of L-lactic acid. This fluorimetric method with 9 can be used to detect L-lactic acid at concentrations as low as 9.2 mM, and interferences from saccharides, amino acids, and ascorbic acid are negligible. Tang and co-workers described the TPE derivative 25 with a maleimide group for sensing thiol-containing molecules [49]. The fluorescence of 25 was quenched due to the maleimide group. However, the AIE fluorescent behavior of 25 was restored after reaction with thiol-containing molecules such as L-cysteine, glutathione, and proteins. However, 25 still remained nonemissive after exposure to other amino acids without thiol groups. The cell imaging of thiol levels in living cells with 25 was examined by incubating 25 with

Figure 8.10 (A) Variation in the FL intensity (I) of 24 (50 mM) at 485 nm as a function of the concentration of saccharide in a carbonate buffer containing 2 vol.% dimethyl sulfoxide (pH 10.5). I0 is the intensity in the absence of a saccharide. Inset: photographs of solutions of 24 in carbonate buffers containing 5 mM saccharide taken under UV illumination. (b) FL responses of 24 (50 mM) to saccharides (4 mM) (red bars) or to Glu in the presence of another saccharide interferent (0.1 mM) (green bars). Reproduced with permission from [47], # 2011 American Chemical Society.

182 Aggregation-Induced Emission: Applications

Scheme 8.15 Illustration of the design rationale for the fluorescence turn-on detection of L-lactic acid by taking advantage of the AIE feature of silole.

HeLa cells; strong emission from the cytoplasm was visualized rather than that from the nuclei, probably owing to the high glutathione (GSH) level in the cytoplasm. TPE derivatives 26 bearing two carboxylic acid groups were reported to aggregate and emit differently in the presence of a variety of biologically relevant amines (biogenic amines) [50]. The multivalent interactions including hydrogen-bonding and/or electrostatic interactions induced aggregation of 26 differently and biogenic amines could therefore be discriminated by the distinct fluorescence behavior of 26.

Figure 8.11 Fluorescence spectra of the ensemble of compound 9 (50 mM) and dodecanoic hydrazine (0.3 mM) containing lactate oxidase (0.25 U ml1) in the presence of different amounts of L-lactic acid (0–0.22 mM). Reproduced with permission from [48], # 2012 American Chemical Society.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 183

8.6 Miscellaneous Sensors The rapid and reliable detection of explosives such as 2,4,6-trinitrobenzene (TNT) and 2,4-dinitrotoluene (DNT) has attracted intense interest owing to public security concerns. TPE and silole-containing molecules and polymers were utilized to construct fluorescent sensors for explosives. The sensing mechanism is based on the fact that explosive nitroaromatics such as DNT, TNT, and picric acid (PA) can quench the emission of TPE and silole efficiently. Polymers 27 and 28 [51–53] incorporating TPE moieties and also 29 [54] containing silole moieties (Scheme 8.16) were examined for sensing these explosive chemicals. Yu and coworkers prepared TPE-modified mesoporous materials that permitted the supersensitive detection of explosives [55]. Furthermore, such mesoporous materials containing TPE moieties were recyclable by washing the explosives with suitable solvents. Therefore, these emissive mesoporous materials are promising for practical explosive detection. We developed a facile and convenient fluorescent detection method for gamma-radiation based on the AIE feature of silole [56]. The detection was based on the AIE molecule 9 and the negatively charged polymer 30 containing sulfone and carboxylate groups (Scheme 8.17). The fluorescence of 9 increased after mixing with polymer 30, as shown in Figure 8.12. This is likely due to the electrostatic and hydrophobic interactions between the polymer and 9. Interestingly, polymer 30 was easily degraded upon exposure to gamma-radiation due to the relative weakness of CS bonds as illustrated in Scheme 8.17. As a result, the fluorescence of 9 became weak after mixing with the polymer that was exposed to gamma-radiation for a certain time. The fluorescence intensity of 9 was dependent on the radiation dose exposure of polymer 8.

R

N

N N O

O N N

N

R .

Ar

Ar

Ar

Ar

n

R = –CH 2 –, –O(CH 2 )6– 27

Si

Ar

Si

Si n

OC6H13 Ar =

N C 6H13

C4H9

C6H13

C6H13O

S

Ar =

28 C6H13

C 6H13

29

Scheme 8.16 Structures of compounds for detecting explosives based on AIE.

184 Aggregation-Induced Emission: Applications

Scheme 8.17 Illustration of the design rationale for the fluorescence detection of gamma-radiation based on 9 and polymer 8.

Figure 8.12 (a) Fluorescence spectra (lex. ¼ 370 nm) of the aqueous solution of 9 (1.0  105 M) in the presence of different amounts of polymer 30 (from 0 to 15 mg l1) at room temperature. (b) Fluorescence spectrum (lex. ¼ 370 nm) of an aqueous solution of 9 (1.0  105 M) and polymer 30 (15 mg l1) and those after exposure to different doses of gamma-radiation (0.13–40 kGy) at room temperature. Reproduced with permission from [56], # 2011 The Royal Society of Chemistry.

New Chemo-/Biosensors with Silole and Tetraphenylethene Molecules 185

8.7 Conclusion and Outlook The past decade has witnessed great achievements in discovering AIE molecules and exploring their functionalities, such as designing light-emitting diodes, chemical and biological probes, and stimuli-responsive soft materials [57–59]. Various chemo-/biosensors have been constructed with these AIE molecules. In this chapter, we summarized chemo-/biosensors with silole and TPE molecules based on the aggregation and deaggregation mechanism. The sensing targets include cations/anions, biomacromolecules, enzymes, physiologically important small molecules, and explosive chemicals. These chemo-/biosensors possess the following features: (1) these sensors are based on the manipulation of aggregation and deaggregation, hence the sensing mechanism is different from those of traditional sensors; (2) the covalent connection of either silole or TPE to biomolecules is not necessary, and the detection can be carried out in a label-free manner; (3) the detection procedure is usually very convenient: just mix and detect; (4) detection is mostly carried out in aqueous solutions. It is known that the aggregation of gold nanoparticles leads to red shifts of plasmon resonance absorption. By taking advantage of this spectral change upon aggregation of gold nanoparticles, a number of chemo-/biosensors have been established [4, 60, 61]. In contrast, chemo-/biosensing with AIE molecules is still in its infancy and deserves further investigations in the following directions: 1. AIE molecules functionalized with recognition moieties for highly selective and sensitive sensors. Most of the reported sensors with AIE molecules are based on electrostatic and hydrophobic interactions, which lead to low sensitivity and selectivity for these sensors. The incorporation of recognition moieties into AIE molecules allows the aggregation and deaggregation to be controlled by specific target–ligand interactions. Accordingly, both selectivity and sensitivity are expected to be improved. The decoration of AIE molecules with specific ligands may allow the identification of tumor cells and early-stage cancer diagnosis. 2. AIE molecules with emission at longer wavelengths. Most of the reported AIE molecules show either blue or green emission. Because of the background emission, fluorescent probes for biosensors are preferred to have red and even near-infrared emission. Therefore, the design and studies of AIE molecules that emit in the red and even longer wavelength region will receive more and more attention. 3. Application of AIE molecules in fluorescence imaging. Fluorescence imaging is becoming a major tool in biotechnology and the life sciences. By taking advantage of the abnormal fluorescent behavior, the application of AIE molecules in fluorescence imaging has been demonstrated in several cases. The extension of chemo-/biosensors to fluorescence imaging will become the major application area for AIE molecules.

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9 Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing Qi Chen and Bao-Hang Han National Center for Nanoscience and Technology, China

9.1 Introduction Carbohydrate-mediated biological interactions play a crucial role in numerous biological processes such as cell growth, recognition and differentiation, cancer metastasis, inflammation, and bacterial and viral infection [1, 2]. As a model system of natural carbohydrates, synthetic glycoconjugates have been demonstrated to be an important, well-defined tool for investigating carbohydrate-based biological events and related biosensing [3, 4]. Despite the wide range and importance of carbohydrates in biology, the difficulties in studying carbohydrate
protein interactions have hindered any effort to develop a mechanistic understanding of carbohydrate structure and function. Among these difficulties, the weak binding affinities of carbohydrate
protein interactions with dissociation constants in the millimolar range and the availability of fluorescent complex glycoconjugates are two major challenges. The affinity and specificity of these interactions strongly depend on multivalency. There is therefore intense interest in the design and application of multivalency of carbohydrates known as the ‘glycoside-cluster effect’ [5, 6]. Several multivalent models have already been proposed for sialyl LewisX and globotriaosyl antigens, in which polymers, dendrimers, and starfish models have been widely examined. Some of carbohydrate clusters exhibited improved bioactivities. For example, a dimeric Tn antigen glycolipid has been shown to be highly immunogenic [7] and a divalent galabioside was 100 times more efficient than the monomer in inhibiting hemagglutination by Gram-positive bacteria [8]. Fluorescence-based assays have also attracted much attention, because they are highly sensitive, convenient, cost-effective, and easily scaled up to high-throughput screening format [9]. Especially fluorescent

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

190 Aggregation-Induced Emission: Applications glycoconjugates that possess both fluorescent scaffolding and reporting carbohydrate ligands are advantageously employed in carbohydrate
protein interaction studies and biosensing [10, 11]. However, aggregation-caused fluorescence quenching of traditional dyes often takes place when they are dispersed in aqueous media or interact with biomacromolecules, resulting in drastic adverse effects on the efficiencies and sensitivities of biosensors or bioprobes [12]. To overcome this problem, carbohydrate-functionalized molecules with aggregation-induced emission (AIE) characteristics, possessing both fluorescent dye and reporting carbohydrate ligands, are usefully applied in carbohydrate-mediated interaction studies and biosensor applications owing to their intrinsic optical properties, high sensitivities to minor stimuli, and good biocompatibilities [13, 14]. AIE-active materials provide a unique platform for exploiting novel optical materials and sensors, owing to their enhanced emission in aggregate form or the solid state [15, 16]. Integration of sugar moieties with AIE-active dyes can not only improve the water solubility and biocompatibility of the dyes, but also provide specific ligands to bind biomacromolecules. Carbohydratebearing AIE-active molecules show almost no luminescence in dilute aqueous solution when they exist in the molecular state. However, aggregation derived from specific carbohydrate
protein binding or selective enzyme-induced hydrolysis can ‘turn on’ the photoluminescence (PL) owing to the AIE feature, and this effect can be used to study carbohydrate-mediated interactions and related biosensing.

9.2 Carbohydrate-Bearing AIE-Active Molecules Carbohydrate-bearing AIE-active molecules generally contain three parts: AIE-active core structure, linker, and carbohydrate ligands (Figure 9.1). Silole is the first AIE-active core structure used in designing AIEactive glycoconjugates [17]. Phosphole oxide [18] and tetraphenylethene (TPE) [13, 14] are also suitable core molecules used in the preparation of AIE-active glycoconjugates. Most AIE-active glycoconjugates are based on the TPE core structure, owing to the simple preparation and facile functionalization [19, 20]. Depending on the target biomacromolecule, various carbohydrate ligands such as monosugars (mannose, galactose, and glucosamine) [14, 18
20], disaccharides (lactose and cellobiose) [17, 21], and oligosaccharides (globotriaoside and 60 -sialyllactoside) [13, 17] were introduced into AIE-active core molecules. Further, the water solubility and biocompatibility of AIE-active core molecules are improved, which are beneficial to biosensing in aqueous media. Oligo(ethylene glycol) [20], triazolyl [13, 14], and alkyl groups [17] have been used as the linkers to connect these carbohydrate ligands with AIE-active core molecules. Among these approaches, the formation of a triazolyl ring by Cu(I)-catalyzed azide/alkyne ‘click’ ligation

(OH)n AIE Core

AIE Core linker n(HO)

linker

O

m

Phosphole oxide, silole, TPE oligo(ethylene glycol), triazolyl, alkyl

O

monosugar, disaccharide, oligosaccharide

Figure 9.1 General schematic structures of carbohydrate-bearing AIE-active molecules.

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 191 shows major advantages such as nearly quantitative yields, high tolerance to other functional groups and insensitivity to the reaction solvent [22]. 9.2.1 Carbohydrate-bearing siloles Siloles (silacyclopentadienes) have recently drawn much attention owing to their unique optical and electronic properties, which can be attributed to the low-lying LUMO level associated with the s –p conjugation arising from the interaction between the s orbital of the silicon atom and p orbital of the butadiene moiety [17, 23]. Silole derivatives are weakly fluorescent in solution, however, they exhibit strong fluorescence upon aggregation and this intriguing phenomenon is referred to as AIE, which was first reported by Tang and co-workers in 2001 [24]. Since then, new organic materials, devices, and sensors based on silole derivatives and the AIE phenomenon have been described [15]. In 2007, Hatano et al. reported the first synthesis of a luminescent glycocluster containing a silole moiety as a luminophore and its unique optical properties in aqueous solution [17]. As shown in Scheme 9.1, the silole core 1,1-diallyl-2,3,4,5-tetraphenylsilole (2) was synthesized from a known intermediate (1) in 50%

Ph Ph

Ph

Ph

1) HSiCl3/H2PtCl6

Allyl Grignard Ph Cl

Ph

Si 1

Ph

Ph

Si

Si

2

Si

Ph

4 R = OH

MsCl/Py

5 R = OMs

1) c-Hex2BH Ph

2) NaOH/H2O2 R

NaBr

Ph

Si

Si

3 OR

RO

O

RO

RO O

SAc

7 or 8

Basic condition

7 Sug = AcGb3

RO

RO Ph Ph Sug

RO

Ph

O

OR O RO

O

OR

O

O

O RO RO

Si

S

10 Sug = Lac

Scheme 9.1 Preparation of carbohydrate-bearing siloles.

AcGb3: R = Ac Gb3: R = H

RO

OR

9 or 10

9 Sug = Gb3

O

O RO

Ph

Si

Si 3

OR

RO

8 Sug = AcLac

S

6 R = Br

R

Si 3

Sug

3

3

3

Ph

Ph

2) Allyl Grignard

Ph

Si

Cl

Ph

Ph

Sug

3

AcLac: R = Ac Lac: R = H

192 Aggregation-Induced Emission: Applications overall yield. Hydrosilylation of 2 with trichlorosilane using H2PtCl6 as a catalyst and the succeeding Grignard reaction provided the first generation of silole-core dendrimers (3). The resulting dendrimer 3 was treated with dicyclohexylborane followed by hydrolysis with hydrogen peroxide in alkaline solution to afford a hexahydroxy derivative 4, which further underwent successively O-mesylation and replacement with bromide anions, giving 6 in 67% yield. A coupling reaction between the silole-core dendrimer and a peracetylated globotriaose (Gb3) derivative was achieved by nucleophilic substitution of the terminal bromide on the dendrimer 6 with a thiolate anion generated from 7 by treatment with sodium methoxide. The silole-core glycodendrimer 9 was afforded after deprotection by a combination of Zempl
en conditions and saponification. Its structure was confirmed by NMR, UV–vis, and PL spectra, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The corresponding silole core glycodendrimer containing lactosyl ligands 10 was also prepared from 8 according to similar methods. 9.2.2 Carbohydrate-bearing phosphole oxides Phosphacyclopentadiene (phosphole) is an interesting building block for the construction of conjugated systems, where an interaction between the butadiene p orbital and the low-lying s (P
R) orbital takes place in the ring [18]. Phosphole also possesses a central phosphorus atom, which retains versatile reactivity. This offers the possibility of tuning the electronic properties of the materials by simple chemical modifications, such as oxidation. Sanji et al. found that phosphole oxide-cored dendrimers do not show emission in solution but produce intense emissions in the aggregated or crystal states [25]. When phosphole oxide aggregates from a well-dispersed state, it will change the conformation from a twisted state to a coplanar state, and the coplanarization of phosphole oxide could lead to better conjugation between its peripheries and core and thus show enhanced fluorescence emission. Synthetic routes to phosphole-cored glycoconjugates are shown in Scheme 9.2 [18, 25]. A Ti(II)mediated cyclization of 1,7-diyne 11 leading to an intermediate titanacyclopentadiene followed by subsequent addition of dichlorophenylphosphine gave phosphole 12 in 83% yield. The oxidation of phosphole 12 MeO

OMe CH2 4

MeO

MeO

1) Ti(O-i-Pr)4 i-PrMgCl

OMe H2O2

P Ph

2)PhPC l 2

OMe

11

MeO

OMe 12

OMe

MeO

OH

HO BBr3

MeO

P Ph O

OMe

HO

13

OH

14

SugO

OSug

HO 17 Sug

SugO

P Ph O

P Ph O

OSug

HO HO HO

17 and 18 18 Sug

HO

AcO AcO O AcO NH AcO 15 BF3-EtO2 OCCl3

AcO OAc O AcO 16 AcO OAc SnCl4

HO O

OH O HO

Scheme 9.2 Preparation of carbohydrate-bearing phosphole oxide.

NaOMe/MeOH

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 193 with hydrogen peroxide afforded phosphole oxide 13 in good yield. Demethylation of 13 with boron tribromide afforded 2,5-bis(3,5-dihydroxyphenyl)-substituted phosphole oxide 14, The mannopyranoside derivative 17 was synthesized by glycosylation of 14 with acetyl-protected mannopyranoside 15 followed by deprotection. Galactopyranoside congener 18 was also synthesized similarly from peracetylated galactose 16. Importantly, 17 and 18 are water soluble, which is usually required for biosensing in aqueous media. 9.2.3 Carbohydrate-bearing tetraphenylethenes Since Tang’s group reported the AIE features of tetraphenylethene-based luminophores, TPE-based AIEactive materials have already shown practical applications in chemosensors and bioprobes, owing to its efficient preparation and facile functionalization. Tetraphenylethene derivatives show strong fluorescence in the aggregated state. Kato’s [13] and Han’s [21] groups have designed and synthesized tetraphenylethene derivatives bearing alkyne groups that were used to synthesize fluorescence oligosaccharide probes. The merit of this preparation approach is being able to introduce carbohydrate ligands to probe compounds by ‘click chemistry,’ a Cu(I)-catalyzed azide–alkyne cycloaddition method, and can facilitate the manufacture of various biosensors. This reaction is very suitable for the preparation of various kinds of probes because it has high efficiency and proceeds in a variety of solvents, including aqueous alcohol or organic co-solvent and water. The TPE derivatives were prepared as shown in Scheme 9.3. Compound 19 was first synthesized by a McMurry coupling reaction between two molecules of 4,40 -dihydroxybenzophenone. However, the yield of this reaction was poor (only 34%) [13]. Further studies showed that McMurry coupling of 4,40 -dimethoxybenzophenone to produce 20 and then demethylation can give 19 smoothly. Compared with the straightforward coupling of 4,40 -dihydroxybenzophenone, the yield of the two-step preparation is much higher (up to 84%) [21] The poor yield of the one-step preparation may stem from some side reactions induced by the active hydroxyl groups during the McMurry reaction. Compound 19 was converted to tetrapropargyl compound 21 by reaction with 3-bromo-1-propyne under basic conditions. Various sugar moieties such as lactoside (22 and 24), 60 -sialyllactoside (SL, 23), and cellobioside (25) were introduced into TPE by click chemistry between the propargyl group of TPE derivative 21 and the azide group of the aglycone of oligosaccharide compounds as shown in Figure 9.5.

HO

O

OH

HO

OH

O

K 2CO3

reflux

HO

19

OH

O

BBr3 /CH2Cl2 H3CO

OCH3

O TiCl3 /Zn/THF

H3CO

OCH3

O

Br

TiCl3 /Zn/THF

reflux

H3CO

20

OCH3

Scheme 9.3 Preparation of TPE derivative bearing alkyne groups.

21

O

194 Aggregation-Induced Emission: Applications N N

N N O n(HO) 21

Sug N

Cu(I)

O

N Sug

O

N3

or O n(HO)

Cu(I) N3

OH OH O O HO HO OH

O

Sug N

and deprotection

O

N N OH OH

HO

OH O OH

O

COOH

AcHN 10

OH

HO

21

HO

Sug OH OH O O HO HO OH 24

OH O

HO HO

OH O OH

N Sug N N

22~25

O O

O OH HO 23

OH O OH

O 10

OH O HO

O OH

OH 25

Scheme 9.4 Preparation of carbohydrate-bearing TPE derivatives 22–25.

Grafting of glucosamine hydrochloride moieties to a TPE motif furnished a novel cationic water-soluble tetraphenylethene derivative, GH-TPE (31) [14]. With AIE properties, GH-TPE can be used for fluorimetric detection of alkaline phosphatase through enzyme-triggered deaggregation of the ensemble of GH-TPE and substrate. The synthetic route to GH-TPE is shown in Figure 9.6. Propargylation of the known TPE derivative 26 afforded the propargyl-attached TPE 27 under basic conditions. Cu(I)-catalyzed ‘click’ ligation between 27 and azido-functionalized glucosamine derivative 28 furnished the sugar-bearing TPE smoothly. Formation of the triazole ring is confirmed by the chemical shift at 7.93 ppm (single peak) in the 1 H NMR spectrum and two peaks at 122.2 and 144.3 ppm in the 13 C NMR spectrum. After removing all the acetyl groups and deprotecting N-Boc groups, the desired water-soluble GH-TPE was obtained in excellent yield by a one-pot procedure. For control studies, glucopyranosyl-bearing TPE (Glu-TPE, 32), and mannopyranosyl-bearing TPE (CTPE-2, 33), were also prepared by similar methods from 29 and 30, respectively. It is well known that the affinity and specificity of carbohydrate-mediated biological interactions depend strongly on multivalency due to the so-called glycoside cluster effect. Therefore, the TPE-based multivalent glycoconjugate CTPE-1 [19] was synthesized according to the synthetic route shown in Scheme 9.6. Treatment of the known TPE derivative 34 with excess NaN3 gave azido-functionalized TPE 35 quantitatively. Cu(I)-catalyzed ‘click’ ligation between azido-attached mannoside 30 and the known tetrakis(2-propynyloxymethyl)methane (36) furnished the propargyl-bearing mannopyranoside cluster 37, which was reacted with 35 through a cycloaddition reaction again to afford the peracetylated TPE-based multivalent glycoconjugate 38. After global deacetylation under Zempl
en conditions, the desired glycocluster CTPE-1 was obtained in excellent yield. Glycosyl-bearing luminophores are often used to assess glycosidase capability. Therefore, cellobiosyl- or lactosyl-attached TPEs (CTPE-3 and CTPE-4) were synthesized from propargyl

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 195

Br

OH

O

deprotection

Cu(I)

O

HO

28~30

K 2 CO 3 AcO 26

27

AcO AcO

R1 O

R3

R2

Sug O N N Sug N

N N N

28 R1 = H, R 2=NHBoc, R3=N3

O

29 R1 = H, R2=OAc, R3=N3 30 R1 = OAc, R2=H, R3=

S

N3

31~33

Sug

HO HO HO

O NH3CI 31

HO HO HO

O OH 32

HO HO O HO HO S 33

Scheme 9.5 Preparation of carbohydrate-bearing TPE derivatives 31–33.

glycosides (39 and 40) through ‘click’ ligation and Zempl
en deacetylation to detect specific glycosidases based on the AIE effect. All the synthetic TPE-based glycoconjugates are soluble in aqueous solution and were well characterized by 1 H NMR, 13 C NMR, IR, and mass spectrometry. To facilitate fluorescence turn-on sensing with AIE-active materials as a detection tool, a modification is required to improve the sensitivity to analytes. Two possible principles to enhance the sensitivity towards concanavalin A (Con A) can be examined: (1) elongation of the linker between the sugar and the AIE molecules to endow the interaction with flexibility and to decrease the entropic cost of the interaction, and (2) an increase in the number of sugar units installed to bind more efficiently multivalent Con A [20]. Scheme 9.7 shows the modification for the mannose-modified TPEs [20]. A mannopyranoside derivative 44 was synthesized by the reaction of 4-hydroxy-substituted TPE 19 and acetyl-protected mannopyranoside 15 followed by deprotection. NMR analysis showed that the incorporated mannopyranosides involve the a-isomer. A TPE with mannopyranoside linked by a diethylene glycol unit (45) was prepared by the reaction of 15 and diethylene glycol-tethered TPE 43 followed by deprotection. A TPE with eight mannopyranoside units linked by diethylene glycol chains (49) was also synthesized from tetrakis(3,5dihydroxyphenyl)ethene (47) as a starting material that was easily obtained by the McMurry coupling of 3,30 ,5,50 -tetramethoxybenzophenone (46) followed by demethylation. The products were fully characterized by spectroscopic methods and dissolved in water completely, which is usually required for biosensing in aqueous media. .

9.3 Luminescent Probes for Lectins Glycoconjugates, such as glycoproteins and glycolipids, generally located on the cell surface, play a critical role in the process of cell adhesion with proteins of pathogens [26]. It is well known that the early stage of cell adhesion involves carbohydrate-mediated specific recognition of pathogens. A combination of the specific recognition and the carbohydrate cluster effect has been applied for the molecular design of artificial inhibitors and neutralizing agents of pathogens [6].

196 Aggregation-Induced Emission: Applications

NaN3

Br

O O

Br

O

N3

34

35

AcO

O O

OAc O

AcO AcO

N3

O

DMF

AcO AcO AcO

O

O

Water-THF S

36 S

AcO AcO AcO

Cu(I) Water-THF

30

Cu(I)

OAc O

OAc O

N3

N N N S

N N N

O O

AcO AcO AcO

OAc O

O

N N

O

N

37

S

RO RO RO

N N

OR O

OR O

N N

S N

RO RO RO

OR O S

N

N O

O N

N N

O OAc AcO

38 R = Ac NaOMe

S O OR

39 OR1 O

O R1O R1O

AcO OAc

O

OAc

OAc

O

O OAc AcO 40

O

O

OAc

OR1 O

O

OR1

N N N

O O

N N N

R1O O

O R1O

41 R1 = Ac, R2 = H, R3 = OAc 42 R1 = Ac, R2 = OAc, R3 = H

OR OR OR

OR OR OR

O OR

CTPE-1 R = H

AcO

O

OR OR OR

S

OAc

O

R2

N N N

O

O O

R3 R1O

O OR

N N N

OAc

Cu(I) Water-THF

O

N

N N N

39 or 40

O O

AcO AcO

35

S N N N

O

O

S RO RO RO

N

NaOMe

OR1OR1 O O R1O

OR1 R3 R2

CTPE-3 R1 = H, R2 = H, R3 = OH CTPE-4 R1 = H, R2 = OH, R3 = H

Scheme 9.6 Preparation of carbohydrate-bearing TPE derivatives CTPE-1, CTPE-3, and CTPE-4.

Carbohydrate
protein interactions, which are found in interactions of proteins, viruses, and bacteria, are among the most important events or mechanisms in biological systems. Lectins, which are a series of carbohydrate-binding proteins and are located on cell surfaces, mediate the initial recognition processes in biological systems by interaction with saccharide receptors. There is a high demand for the development of a rapid, simple, sensitive, and selective sensing method for lectins.

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 197

Scheme 9.7 Preparation of mannose-modified TPEs.

In order to study carbohydrate
protein interactions, Con A, a member of the lectin family, was usually chosen as a target protein since it is a well-known a-mannose- and a-glucose-binding protein, which exists predominantly as a tetramer of four identical subunits and possesses four binding sites to interact with four a-mannose or a-glucose units simultaneously [27]. Through aggregation derived from carbohydrate
lectin binding, multivalent mannosyl-bearing TPE shows a good selectivity and sensitivity towards Con A by switching on the fluorescence of water-soluble tetraphenylethene-based glycoconjugates in aqueous solution [19]. In dilute aqueous solution, as expected, CTPE-1 shows almost no luminescence. After treatment with

198 Aggregation-Induced Emission: Applications

Fluorescence Intensity (a.u.)

350 300

CTPE-1 CTPE-1 + Con A

250

CTPE-2 CTPE-2 + Con A

200 150 100 50 0 350

400

450

500

550

600

Wavelength/nm

Figure 9.2 Fluorescence spectra of CTPE-1 and CTPE-2 [20 mM in phosphate-buffered saline (PBS) (10 mM) buffer solution, pH ¼ 7.6] in the absence and presence of Con A (30 mM). The inset shows photographs of the corresponding buffer solutions of CTPE-1 (20 mM) in the absence (A) and presence (B) of Con A (30 mM) under UV illumination (365 nm).

Con A (30 mM), the aqueous mixture is highly emissive, due to the formation of a CTPE-1–Con A ensemble. From the molecular solution in water to the aggregated state with Con A, the fluorescence intensity of CTPE-1 at 469 nm increases by 11-fold (Figure 9.2). The nonemissive nature of the molecular state and the emissive nature of the aggregates were clearly indicated in the photographs shown in the insets in Figure 9.2. To evaluate the influence of the glycoside cluster effect, CTPE-2 was also treated with Con A (30 mM) under the same conditions. In presence of Con A (30 mM), the fluorescence intensity of CTPE-2 at 469 nm only increased by threefold compared with that in absence of Con A, which means that the TPE motif containing a glycocluster residue possesses a higher affinity to lectin. Fluorescence spectra of CTPE-1 [20 mM in phosphate-buffered saline (PBS) (10 mM) buffer solution, pH ¼ 7.6] in the presence of different concentrations of Con A are shown in Figure 9.3, indicating that the emission enhancement of CTPE-1 is

Figure 9.3 Fluorescence spectra of CTPE-1 [20 mM in PBS (10 mM) buffer solution, pH ¼ 7.6] in the presence of Con A at concentrations of 0, 5, 10, 15, 20, 25, and 30 mM from bottom to top.

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 199

Figure 9.4 Illustration of studies of carbohydrate–protein interactions using TPE-based artificial glycoconjugates as fluorescent probes based on the AIE effect.

observed at a concentration of Con A as low as 1.0 mM. To investigate the selectivity of fluorimetric detection to Con A, CTPE-1 was also treated with bovine serum albumin (BSA) protein under the same conditions and no significant change in PL intensity was observed [19]. Using a similar method as shown in Figure 9.4, some other sugar-bearing silole [17], phosphole oxide [18], and TPE [20] derivatives were also reported for lectin sensing, which demonstrates that light-up sensors with fluorescence turn-on responses as signal readout possess high efficiency and increased sensitivity. It should be noted that the ‘turn-on’ fluorescence for sugar-carrying AIE molecules upon addition of proteins cannot be completely ascribed to aggregation, because insertion of AIE chromophores into the hydrophobic cavities of proteins may also restrict the respective internal rotations and thus lead to fluorescence enhancement. More and more strategies based on the AIE principle have been introduced to design sensitive ‘turn-on’ fluorescent sensors for potential applications in the fields of chemical, biological, medical, and environmental science.

9.4 Luminescent Probes for Enzymes b-Glucosidase with specificity for a variety of b-D-glycoside substrates catalyzes the hydrolysis of terminal nonreducing residues in b-D-glucosides with release of glucose [28]. b-Cellobiosyl-carrying TPE (CTPE-3), as a certain substrate for b-glucosidase, can be hydrolyzed by the enzyme in aqueous buffer and lose glucose units one by one. Carbohydrate-bearing TPEs show almost no luminescence in dilute aqueous solution when existing in the molecular state. However, aggregation derived from selective glycosidase-induced hydrolysis can ‘turn on’ the PL due to the TPE moiety with AIE feature, which can be used to detect glycosidase [19]. It can be expected that the water-soluble CTPE-3 will be gradually transformed into water-insoluble TPE derivatives after enzymatic hydrolysis. In that case, the fluorescence intensity of the TPE moiety with AIE properties will increase significantly owing to glycosidase-induced aggregation and the aim of detecting the glycosidase can be achieved. As shown in Figure 9.5, CTPE-3 (20 mM) displays weak fluorescence in aqueous buffer solution. After treatment with b-glucosidase (1.5 U), the resulting mixture becomes turbid, which indicates the formation of the aggregate. The fluorescence intensity of the resulting suspension at 460 nm increases

200 Aggregation-Induced Emission: Applications

Figure 9.5 Fluorescence spectra of CTPE-3 (20 mM in citric acid–Na2HPO4 buffer solution, pH ¼ 5.8) in the absence and presence of b-glucosidase at concentrations of 0, 0.5, 1.0, 1.2, and 1.5 U from bottom to top. The inset displays photographs of the corresponding buffer solutions of CTPE-3 (20 mM) in the absence (A) and presence (B) of b-glucosidase (1.5 U) under UV illumination (365 nm).

ninefold compared with CTPE-3 in the absence of b-glucosidase. Photographs of solutions of CTPE-3 and the resulting suspension in buffer taken under UV irradiation are displayed in the inset in Figure 9.5. The enzymatic hydrolysis process can be further confirmed by electrospray ionization mass spectrometry (ESI-MS). Figure 9.6 shows the mass spectrum of a CTPE-3 sample digested with b-glucosidase for 1 h.

Figure 9.6 Mass spectrum of CTPE-3 solution digested with b-glucosidase for 1 h.

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 201

Figure 9.7 Illustration of fluorimetric assay for alkaline phosphatase using GH-TPE as a fluorescent probe based on the AIE effect.

The ion at m/z 1264.64 corresponds to [CTPE-3 þ H]þ. The peaks at m/z 1102.82, 939.33, 775.85, and 614.54 indicate the glucose losses, which clearly shows that the glucose residues in CTPE-3 are released one by one. To investigate the selectivity of fluorimetric detection towards b-glucosidase, lactosyl-carrying TPE (CTPE-4) was also treated with b-glucosidase under the same conditions and no significant change in PL intensity was observed. The terminal nonreducing sugar residue in CTPE-4 is b-galactose, which cannot be easily hydrolyzed by b-glucosidase. It can be inferred from these results that functionalization of TPE with a certain glycosyl residue provided a unique platform for selective glycosidase detection. In clinical practice, enzymatic reactions are usually used in the diagnosis of disease. Alkaline phosphatase (ALP, EC 3.1.3.1), as one of the most commonly assayed enzymes, is capable of catalyzing the hydrolysis of a wide variety of phosphate compounds and has broad substrate specificity in vitro [29]. Several diseases, such as bone disease, liver dysfunction, breast and prostatic cancer, and diabetes, can be preliminarily diagnosed based on an abnormal level of ALP in serum.[30] Among various methods for ALP assays, fluorescence-based techniques have attracted much attention owing to their convenient and cost-effective screening format. A novel cationic water-soluble tetraphenylethene derivative, GH-TPE, was reported as a fluorescent probe for ALP assay based on the AIE effects [14]. The design rationale for ALP assay is illustrated schematically in Figure 9.7. GH-TPE, possessing two units of glucosamine hydrochloride, is expected to display weak PL in aqueous media. According to previous studies [31], monododecylphosphate, as an amphiphilic compound, can form a heteroaggregated complex with positively charged GH-TPE based on the electrostatic binding when the concentration of monododecylphosphate is lower than its critical micelle concentration [32]. As a result, the fluorescence of the ensemble would increase significantly, which is induced by the aggregation of GH-TPE. When treated

202 Aggregation-Induced Emission: Applications

Figure 9.8 Fluorescence spectra of GH-TPE [20 mM in PBS (10 mM) buffer solution, pH ¼ 8.0] in the presence of different amounts of monododecylphosphate (from 0 to 30 mM). The inset displays photographs of the corresponding buffer solutions of GH-TPE (20 mM) in the absence (A) and presence (B) of monododecylphosphate (30 mM) under UV illumination (365 nm).

with ALP, the aggregated complex would be disassembled after loss of phosphate groups. Thus, the fluorescence of the ensemble could be reduced and aim of using GH-TPE as a fluorescent probe for ALP detection would be achieved. Heteroaggregate complexation of the water-soluble TPE salt with monododecylphosphate was studied by spectrometric titration in aqueous phosphate buffer solution (PBS, 10 mM, pH ¼ 8.0) at 25  C. With addition of monododecylphosphate (the concentration of monododecylphosphate is lower than its critical micelle concentration, nearly 2.0 mM), the fluorescence intensity of GH-TPE gradually increased 11-fold (Figure 9.8). Further, a red shift as large as 90 nm (from 377 to 467 nm) of its emission maximum was observed. The absorption band around 312 nm was also red shifted by 16 nm in the presence of monododecylphosphate (30 mM). These results are in agreement with the formation of a heteroaggregate complex. Photographs of solutions of GH-TPE and the GH-TPE–monododecylphosphate ensemble in PBS taken under UV irradiation are displayed in Figure 9.8. To prove that the NH3þ group of glucosamine is critical for the fluorescence change caused by the electrostatic interaction, Glu-TPE was also treated with monododecylphosphate (30 mM) under the same conditions and no significant fluorescence change was observed [14]. As mentioned, ALP is able to catalyze the hydrolysis of monododecylphosphate to dodecyl alcohol and phosphoric acid. Therefore, it is expected that the fluorescence of the GH-TPE–monododecylphosphate ensemble would be reduced after addition of ALP, because the interaction between GH-TPE and monododecylphosphate was weakened after the loss of a phosphate group, leading to disassembly of GH-TPE– monododecylphosphate heteroaggregate complexes and a decrease in fluorescence. Figure 9.9 shows the fluorescence spectra of the ensemble of GH-TPE [20 mM in PBS (10 mM), pH ¼ 8.0] and monododecylphosphate (30 mM) in the presence of ALP (1.5 U ml
1) after different reaction periods. Apparently, the fluorescence intensity of the ensemble started to decrease gradually upon treatment with ALP. On prolonging the hydrolysis reaction time, a significant decrease in the fluorescence intensity was observed. The fluorescence variation of the ensemble solution and the disassembled solution upon addition of ALP can be distinguished with the naked eye under UV illumination, as shown in the inset of Figure 9.9. Therefore, GH-TPE could be a potentially useful fluorescent probe for the detection of ALP.

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 203

Figure 9.9 Fluorescence spectra of the ensemble of GH-TPE [20 mM in PBS (10 mM), pH ¼ 8.0] and monododecylphosphate (30 mM) in the presence of ALP (1.5 U ml
1) incubated at 25  C for different periods. The inset displays photographs of the corresponding solutions of GH-TPE and monododecylphosphate in the absence (A) and presence (B) of ALP (1.5 U ml
1) after 30 min of incubation at 25  C under UV illumination (365 nm).

9.5 Luminescent Probes for Viruses and Toxins Cell surface oligosaccharides act as receptors for bacteria, viruses, toxins, and other cells (such as white blood cells and cancer cells). The sialyllactosyl/sialylneolactosyl series sugar chains in glycoproteins and glycolipids are the functional receptor sugar chains for influenza A virus of humans and animals [33, 34], Human influenza viruses bind preferentially to sialic acids linked to galactose by an a-2,6-linkage. Kato et al. examined whether influenza virus could be specifically detected by the fluorescence oligosaccharide probe, because the influenza virus possesses HA molecules at a high density (about 1000 molecules per virus) on its surface [13]. Based on the fluorescence spectrum of lactosyl-bearing TPE (22) and a-2,6-SLTPE (23) in the presence of different concentrations of influenza virus A/WSN/33 in 10 mM Tris–HCl (pH 7.6), enhancement of the fluorescence of a-2,6-SL-TPE was observed in the presence of influenza virus. On the other hand, the fluorescence of lactosyl-bearing TPE was not enhanced in the presence of influenza virus. These results suggest that AIE may have been caused by binding of influenza virus to the 60 -sialyllactose moiety ligated to a-2,6-SL-TPE. The a-2,6-SL-TPE may bind to the HA molecules on the surface of an influenza virus and freeze its intramolecular rotation. Kato et al. also examined the amount of influenza virus detectable. The fluorescence intensity of a-2,6-SL-TPE at 460 nm increased significantly with increase in the amount of influenza virus. Influenza virus at concentrations higher than 105 pfu per 100 ml could be detected. Cholera continues to represent a major threat to both humans and animals, especially in developing countries. Cholera is an acute intestinal infection, characterized by profuse watery diarrhea and vomiting, which can lead to severe dehydration, electrolyte imbalance, and even death if treatment is not given appropriately [35]. Cholera toxin (CT), the primary virulence factor of cholera, is secreted by the bacterium Vibrio cholerae and possesses an AB5 hexameric architecture. The five identical B-subunits of the toxin bind selectively to the glycolipid ganglioside GM1 that contains a pentasaccharide. It is the recognition of this pentasaccharide that facilitates the initial attachment of cholera toxin B-subunit (CTB) to the intestinal cells, which is the first step towards the disease cholera. CTB is known to bind to lactose through the recognition of the terminal galactose portion of the molecule [36, 37], which is also the terminal sugar in the pentasaccharide headgroup of GM1 ganglioside. CT is a pentamer composed of five identical monomers each with a binding site for the GM1 ganglioside [38]. These binding sites, matching with the galactose moieties of the GM1 ganglioside, make it possible for

204 Aggregation-Induced Emission: Applications

Figure 9.10 Illustration of fluorescence ‘turn on’ assay for CT with Lac-TPE based on the AIE effect.

CTB to bind to various types of molecules that have the same terminal galactose. Lactose, as a disaccharide consisting of glucose and a terminal galactose, was therefore used in the design of CT sensors. The design rationale for CT assay is illustrated in Figure 9.10. Lactosyl-attached TPE (Lac-TPE, 24), possessing four lactosyl units, is expected to display weak PL in aqueous media. When CT was added to a dilute aqueous solution of Lac-TPE, multiple binding events occurred between the toxin and the multivalent lactosylattached TPE, leading to aggregation of Lac-TPE. As a result, the fluorescence of the ensemble would be increased significantly, which is induced by the aggregation of Lac-TPE. As shown in Figure 9.11, CTB-induced aggregation of Lac-TPE results in enhanced fluorescence intensity at 475 nm. The fluorescence intensity increases significantly when the concentration of CTB

Figure 9.11 Fluorescence spectra of Lac-TPE [2.0 mM in PBS (50 mM) solution, pH ¼ 7.3] in the presence of different amounts of CTB (from 0 to 5.0 mM). The inset displays photographs of the corresponding solutions of Lac-TPE in the absence (A) and presence (B) of CTB (5.0 mM) under UV illumination (365 nm).

Carbohydrate-Functionalized AIE-Active Molecules as Luminescent Probes for Biosensing 205 reaches 5.0 mM. Photographs of solutions of Lac-TPE and the Lac-TPE–CTB ensemble in PBS buffer solution taken under UV irradiation are displayed in the inset of Figure 9.11. The observed fluorescence enhancement is mainly caused by restricted intramolecular rotation of phenyl groups in the aggregated state, which blocks the nonradiative decay of Lac-TPE and makes it highly luminescent. The change in fluorescence intensity became larger with increasing amount of the toxin owing to the formation of larger and more aggregates. When the concentration of CTB is as low as 1.0 mM, the fluorescence emission of Lac-TPE also increases to up to three times that of Lac-TPE solution in the absence of CTB. Compared with previous reports on CT sensors [39, 40], Lac-TPE has the advantages of rapidity and simplicity, without the need for complex instrumentation, although with lower sensitivity. Furthermore, the sensitivity is considered to increase when more lactosyl moieties are introduced to TPE owing to the multivalent interaction. To investigate the selectivity of fluorimetric detection for CT, and also the specificity of the carbohydrate–protein interaction, Lac-TPE was treated with another protein, BSA, under the same conditions and no significant change in PL intensity was observed. Furthermore, as a control study, Cel-TPE (25) was also used to investigate the effect of sugar ligands on the selectivity of CT detection. Compared with the high fluorescence intensity induced by the Lac-TPE–CTB ensemble, the CTB can not ‘turn on’ the fluorescence of Cel-TPE, which implies that there is no specific interaction between Cel-TPE and CTB. Both sets of results substantiate the specificity of the interaction between Lac-TPE and CTB.

9.6 Conclusion Considering the crucial role of carbohydrate-mediated biological interactions in numerous biological processes and the unique optical properties of AIE-active materials, carbohydrate-functionalized AIE-active molecules, possessing both a fluorescent dye and reporting carbohydrate ligands, are attractive for use in carbohydrate-mediated interaction studies and biosensor applications owing to their intrinsic optical properties, high sensitivities to minor stimuli, and good biocompatibility. Depending on the target biomacromolecule, various carbohydrate ligands such as monosugars, disaccharides, and oligosaccharides were introduced into AIE-active core molecules (silole, phosphole oxide, and TPE) to form carbohydrate-bearing AIE-active molecules, which generally contain three parts: AIE-active core structure, linker, and carbohydrate ligands. These probes with good water solubilities, unique optical properties, and specific binding selectivities, have been studied for biosensing lectins, enzymes, viruses, and toxins. We believe that the method inspired by this novel concept to investigate carbohydrate-mediated biological interactions will have promising applications in biomacromolecule detection and glycobiology studies.

Acknowledgments This work was supported by the Ministry of Science and Technology of China (National Basic Research Program, Grant 2011CB932500) and the National Science Foundation of China (Grants 20972035, 21002017, and 21274033).

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10 Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging Jun Qian, Dan Wang and Sailing He Center for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, China

10.1 Introduction Due to the characteristics of aggregation-induced emission (AIE), AIE dyes have already been utilized in many chemical and biological sensing applications, for example, explosives detection, metal ion sensing, pH sensing, DNA/RNA sensing, and ATP sensing [1–4]. Since AIE dyes show a high quantum yield in the formation of nanoaggregates due to restriction of intermolecular vibrational and rotational motions, and also good biocompatibility, they can also be used as a new type of fluorescent nanoprobe for biomedical imaging. In 2007, Kim et al. used organically modified silica (ormosil) nanoparticles to encapsulate a kind of AIE dye, which was called BDSA [5]. The nanoparticles showed a high quantum yield as the doped AIE dyes with high concentrations showed the AIE effect, which is not possessed by common fluorophores. By virtue of this unique advantage, they further applied the nanoplatform in two-photon cell imaging, intraparticle fluorescence resonance energy transfer (FRET), and photodynamic therapy (PDT) [6, 7]. Since then, many other groups have attempted to extend the biomedical imaging applications of AIE dyes in other directions,for example, in vitro tumor cell targeting, ex vivo tumor imaging, and in vivo animal imaging [8–12]. It was found that the nanoaggregation of AIE dyes could be achieved by mixing different kinds of solvents, encapsulated with silica nanoparticles, polymeric nanoparticles, or even proteins. Early in 2009, Lim et al. subcutaneously injected AIE nanoparticles into the side of a live mouse, and preliminarily illustrated the near-infrared (NIR) in vivo imaging potential of AIE materials [13]. However, reports concerning in vivo functional imaging of AIE dyes are still very rare so far. On the other hand, although the two-photon fluorescence properties of AIE dyes [14], and also their applications in in vitro cell imaging and ex vivo tissue imaging [10, 12], have been systematically investigated, no groups appear to have applied this phenomenon in in vivo animal imaging. Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

210 Aggregation-Induced Emission: Applications This chapter focuses on the in vivo functional applications of AIE dyes. We introduce several examples of AIE dye-encapsulated nanomicelles for in vivo functional animal imaging [12, 15, 16]. Other types of AIE nanoparticles [11, 17], and also their in vivo bioimaging applications, are also demonstrated. We further introduce multiphoton-induced fluorescence from AIE dyes and its applications in in vivo functional microscopic imaging.

10.2 AIE Dyes for Macro In Vivo Functional Bioimaging 10.2.1 AIE dye-encapsulated phospholipid–PEG nanomicelles AIE dyes show great potential in the construction of fluorescent nanoprobes with high quantum yields owing to restriction of intermolecular vibrational and rotational motions in the formation of nanoaggregates. In recent years, various strategies have been developed to fabricate AIE-encapsulated nanoparticles for cellular imaging applications [5, 9, 12]. Phospholipid–poly(ethylene glycol) (PEG) nanomicelles can be utilized as an ideal platform to obtain nanoaggregates of the encapsulated AIE dyes, as a large hydrophobic core in the nanomicelle, which arises due to the presence of long acyl chains of phospholipids, can facilitate the loading of high concentrations of hydrophobic AIE dyes into the micelles. Phospholipid–PEG nanomicelles also have many advantages in bioimaging applications: (1) they show good biocompatibility and possess no cytotoxicity; (2) the preparation process of phospholipid–PEG nanomicelles is much simpler than that of other nanocarriers, such as silica nanoparticles [18, 19] and protein nanoparticles [11]; (3) the long PEG chains in nanomicelles can improve the long-term circulation of nanomicelles in an animal body and help to avoid capture/degradation by reticuloendothelial systems, which is very important for in vivo animal experiments [20, 21]; (4) the end of PEG chains in nanomicelles can be grafted with various functional groups (e.g. carboxyl, amino, maleimide), and can achieve the bioconjugation of nanomicelles with different targeting biomolecules (such as proteins and antibodies) and additional functionalities (such as probes for magnetic resonance and radio imaging); (5) nanomicelles are transparent to visible/NIR light, which is helpful for the excitation and emission of fluorescence signals. Recently, AIE dye-encapsulated nanomicelles have been successively applied to in vitro cell imaging. However, in vivo bioimaging examples have rarely been reported. In Section 10.2.2 and Section 10.2.3, we introduce the applications of AIE dye-encapsulated nanomicelles in macro in vivo sentinel lymph node (SLN) mapping and tumor targeting of mice [15]. 10.2.2 AIE dye-encapsulated nanomicelles for SLN mapping of mice SLNs are the first group of lymph nodes receiving metastatic cancer cells by direct lymphatic drainage from a primary tumor. Accurate identification and biopsy of SLNs can enable clinicians to focus on certain lymph nodes and perform more detailed tracking of cancer cell diffusion. SLN imaging utilizing fluorophores as labeling agents has become a research area attracting intense interest. AIE dye-encapsulated phospholipid– PEG nanomicelles, which have excellent chemical stability and high fluorescence quantum yields, have motivated researchers to investigate their applicability in in vivo SLN mapping. (Z)-2,3-Bis[4-(N-4-(diphenylamino)styryl)phenyl]acrylonitrile (StCN), a donor–acceptor–donor (D–A– D)-type stilbene derivative with a cyano group (as fluorescence acceptor group) in the center and vinyl and triphenylamine (as fluorescence donor groups) at both ends, was chosen for doping in the nanomicelles. In the molecular structure of StCN, the intermolecular orientation is interrupted by the bulky cyano group during the formation of aggregates, which prevents the parallel overlap of fluorophores from becoming face-to-face oriented H-aggregates, but facilitates the formation of head-to-tail oriented J-aggregates [22]. Park et al. reported that the nanoaggregates of StCN in 50% water–N,N-dimethylformamide (DMF)

Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging 211

Figure 10.1 Schematic illustration of the preparation of StCN nanomicelles. Reproduced with permission from [15], # 2011 Elsevier.

exhibited increased fluorescence intensity and a blue shift of the emission maximum wavelength with respect to DMF solution [14]. The synthesis protocol (as shown in Figure 10.1) of StCN-encapsulated phospholipid–PEG nanomicelles (abbreviated to StCN nanomicelles) was described by Wang et al. [15]. The loading density ([StCN]/[StCN þ mPEG-DSPE] in wt%) of StCN in StCN nanomicelles can be calculated from the weight ratio of the materials utilized in the reaction. By varying the quantities of StCN and mPEG-DSPE solution added, nanomicelles with different StCN loading densities can be prepared by the same method (as shown in Table 10.1). Precursor mPEG-DSPE is a type of well-established surfactant with stable properties in aqueous solution [23, 24]. The presence of PEG chains not only assisted the water solubility of StCN nanomicelles, but also provided a feasible bioconjugation method owing to the existence of functional groups at the end of its molecule. Figure 10.2 shows a representative transmission electron microscopy (TEM) image of the StCN nanomicelles with a 60 wt% loading density, which appear uniform in size and have an average diameter of less than 30 nm. Figure 10.3 shows the UV–vis absorption and photoluminescence (PL) spectra of StCN in chloroform and StCN nanomicelles in aqueous dispersion (both of them contain the same quantity of StCN). After the Table 10.1 Synthetic conditions for reactions to prepare StCN nanomicelles with different loading densities Loading density (wt%) 20 40 60

1 mg ml1 StCN solution in chloroform (ml)

10 mg ml1 mPEG–DSPE solution in chloroform (ml)

250 670 1500

100 100 100

212 Aggregation-Induced Emission: Applications

Figure 10.2 Representative TEM image of StCN nanomicelles. Reproduced with permission from [15], # 2011 Elsevier.

encapsulation of nanomicelles, the optical properties of StCN were still retained very well, with only a slight red shift of the PL spectra. The percentage leakage of the encapsulated StCN from StCN nanomicelles in Tween-20 surfactant solution can be used as a crucial characteristic to evaluate the stability of nanomicelles, which is of great importance for optical imaging. By measuring the absorbance intensity at 435 nm to calibrate the StCN concentration, Wang et al. [15] found that the percentage release of StCN from StCN nanomicelles was less than 5% after incubation with 1% of Tween-20 at 40  C during the overall time of their experiment (even after 12 h), indicating that no severe release of StCN from StCN nanomicelles occurred in aqueous solutions, as shown in Figure 10.4. Furthermore, as a kind of efficient fluorescent probe for biological applications, StCN nanomicelles should be stable in various bioenvironments and over a wide range of pH values. Otherwise, monodispersed nanoparticles may be transformed into clusters composed of large numbers of particles, which are too big to be taken up by cells, and cannot circulate smoothly in the vessels for in vivo experimental applications. Wang et al. [15] systematically studied the fluorescence intensity changes of these nanomicelles under

Figure 10.3 UV–vis absorption (a) and PL spectra (b) of StCN in chloroform and StCN nanomicelles in aqueous dispersion. Insets: photographs of StCN in chloroform (left) and StCN nanomicelles in aqueous dispersion (right) under visible light (a) and UV light excitation (325 nm) (b). Reproduced with permission from [15], # 2011 Elsevier.

Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging 213

Figure 10.4 Release kinetics of StCN nanomicelles (60 wt% StCN loading) in 1% Tween-20 suspension at 40  C. Reproduced with permission from {15], # 2011 Elsevier.

different treatments [e.g. phosphate-buffered saline (PBS), serum, and solutions of pH 4–10] for 12 h. As shown in Figure 10.5, the fluorescence peak intensities of StCN nanomicelles changed by less than 10% under all experimental conditions, indicating that the StCN nanomicelles were chemically stable in those solutions, which is very positive for various bioapplications. In order to investigate the characteristic of the AIE of the fluorescence of StCN nanoprobes, Wang et al. optically characterized nanomicelles solutions with various StCN loading densities (20, 40, and 60 wt%) [15]. For an accurate quantitative comparison of PL emission, the absorption intensities of the excitation wavelength for all the samples were kept the same by utilizing different amounts of nanomicelles to encapsulate the same amount of StCN molecules (Table 10.2). The total PL intensity increased significantly, without any distinct wavelength shift of the emission peak (Figure 10.6), as the StCN loading density

Figure 10.5 Stability comparison of the StCN nanomicelles treated with PBS, serum, and solutions of pH 4–10. The PL intensities of mPEG–DSPE solutions in water were also measured as control. The inset shows the corresponding fluorescence images of StCN nanomicelles dispersed in various solutions and the control solutions. The excitation source was blue broadband light with a peak wavelength of 455 nm. Reproduced with permission from [15], # 2011 Elsevier.

214 Aggregation-Induced Emission: Applications Table 10.2 Concentrations of various StCN nanomicelles in water and corresponding absorbance and PL intensity Loading density of StCN nanomicelles (wt%) 20 40 60

Concentration of nanomicelles in DI water (mg ml1)

Absorbance at 431 nm

PL intensity at 577 nm

0.30 0.15 0.10

0.229 0.226 0.225

24.91 31.48 34.57

Figure 10.6 Aggregation-enhanced fluorescence analysis of StCN nanomicelles. (a) UV–vis absorption and PL spectra of the StCN nanomicelles with various StCN loading densities (20, 40, and 60 wt%). (b) Normalized fluorescence intensities of hydrophobic dyes (StCN, DTDC and Nile Red) doped in phospholipid–PEG nanomicelles with various loading densities (20, 40, and 60 wt%), respectively. Reproduced with permission from [15], # 2011 Elsevier.

Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging 215 increased in the nanomicelles. Furthermore, two common fluorophores [DTDC (3,3g-diethylthiadicarbocyanine iodide) and Nile Red) were selected as controls. As shown in Figure 10.6, the PL intensities (normalized by the PL intensity under the condition of loading density ¼ 20 wt%) of DTDC/Nile Red-doped nanomicelles decreased significantly as the loading density of DTDC/Nile Red in the nanomicelles increased, which was due to the property of aggregation-caused quenching (ACQ) of fluorescence. It can be concluded that StCN nanomicelles exhibit an aggregation-enhanced fluorescence emission effect, due to the unique chemical structure of the StCN molecules. To perform SLN mapping of mice with AIE dyes, StCN nanomicelles were injected intradermally into the forepaw pads of an experimental group of anesthetized nude mice (60 wt% of StCN nanomicelles, 1 mg ml1 in 10 mM PBS, 10 ml of solution per mouse). As a control experiment, the other group of nude mice were injected intradermally with mPEG-DSPE solution (0.4 mg ml1 in 10 mM PBS, 10 ml of solution per mouse). The in vivo fluorescence imaging of the experimental group and the control group was performed by utilizing a Maestro in vivo optical imaging system. Figure 10.7 shows the diffusion and accumulation process of nanomicelles at the SLNs over time. After injection, the nanomicelles diffused rapidly from the injection site into the lymphatics; 5 min later, fluorescence signals were detected at an axillary node, giving a clear SLN mapping of the mouse (shown in Figure 10.7b). In contrast, mice injected with mPEG-DSPE solution (Figure 10.7a) showed no signal in the

Figure 10.7 Pseudo-color fluorescence images of mice with mPEG–DSPE (a) and StCN nanomicelles (b) injected into the right paw at various time points (5, 20, 40 and 60 min). Arrows indicate the SLN sites. (c) Fluorescence spectra obtained from the SLN (red) and skin (green) of the mice. (d) Variations of fluorescence peak intensities in the SLN sites of the mice that had been injected with StCN nanomicelles. Peak wavelength of excitation light: 455 nm. Reproduced with permission from [15], # 2011 Elsevier.

216 Aggregation-Induced Emission: Applications

Figure 10.8 Bright field (a) and fluorescence (b) images of the dissected SLN of mice 10 min after injection of nanomicelles. (c) Fluorescence spectra obtained from the dissected SLN. Reproduced with permission from [15], # 2011 Elsevier.

area of SLNs. Fluorescence spectra acquired from the SLNs and the skin of the mouse are shown in Figure 10.7c. With passage of time, the nanomicelles gradually migrated from the SLNs and the fluorescence signal intensity at the SLNs decreased (as shown in Figure 10.7d). Furthermore, mice treated with StCN nanomicelles were sacrificed and their SLNs were removed and imaged. As shown in Figure 10.8, the dissected SLNs emitted red fluorescence, and the spectrum was consistent with that of StCN nanomicelles in aqueous solution. The ex vivo imaging confirmed the in vivo experimental results yet again, illustrating that StCN nanomicelles could be used as optical probes for SLN mapping of live animals. 10.2.3 AIE dye-encapsulated nanomicelles for tumor targeting of mice During the past few years, many kinds of nanoparticles (e.g. quantum dots, gold nanoparticles) have been applied in in vitro cancer cell imaging and in vivo tumor targeting [25–28]. Since 2007, some groups also began to report that novel AIE dye-encapsulated nanoprobes could be used for cancer cell targeting [5]. However, the first report concerning the in vivo tumor targeting capacity of AIE dye-encapsulated nanoprobes did not appear until 2009 [15]. Here, we introduce several examples of AIE dye-encapsulated nanomicelles for targeting of mice [11, 12, 15]. Wang et al. adopted two kinds of AIE nanoprobes for in vivo tumor imaging [15]. One is the aforementioned StCN nanomicelles and the other is RGD (arginine–glycine–aspartic acid peptides)-conjugated StCN nanomicelles. The protocol for synthesizing RGD-conjugated StCN nanomicelles is illustrated in

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Figure 10.9 Schematic illustration of the preparation of RGD-conjugated StCN nanomicelles.

Figure 10.9. Long-chain PEG molecules can improve the long-term circulation of nanomicelles in the animal body, and due to the EPR (enhanced permeability and retention) effect in the tumor tissues, nanomicelles could be preferentially taken up by tumors. Hence it is expected that StCN nanomicelles can passively target tumors of mice. Furthermore, triplet peptide RGD can specifically target (bind to) avb3 integrins [29–31], which are overexpressed at the endothelium of growing blood vessels (vasculature) associated with tumor growth (angiogenesis) [32, 33]. Hence RGD-conjugated StCN nanomicelles can potentially play a critical role in the diagnosis of developing tumors in vivo via noninvasive optical imaging. In the experiment, mPEG-DSPE (0.4 mg ml1 in PBS, 100 ml of solution per mouse, as control), StCN nanomicelles (with 60 wt% loading density, 1 mg ml1 in PBS, 100 ml of solution per mouse), and RGDconjugated StCN nanomicelles (with 60 wt% loading density, 1 mg ml1 in PBS, 100 ml of solution per mouse) were separately injected intravenously into nude mice bearing subcutaneous lung tumor xenografts. In vivo fluorescence imaging of the tumor-bearing nude mice was performed with the in vivo imaging system at various time points post-injection. For control mice, no fluorescence contrast could be observed from the tumors and surrounding skin after injection of mPEG-DSPE. Figure 10.10 shows representative whole-body in vivo optical imaging results for mice injected with StCN nanomicelles and RGD-conjugated StCN nanomicelles. The spectral signatures from the tumor sites and the auto-fluorescence of skin sites are also shown in Figure 10.10. It is obvious that the fluorescence spectra were consistent with those of StCN, and it could easily be differentiated from the auto-fluorescence of skin sites. For the mice injected with StCN nanomicelles, there were fluorescence signals from the tumors at 48 h post-injection (Figure 10.10f and l, top), whereas the images taken 1 and 24 h post-injection only showed bright signals at the tail injection sites (Figure 10.10d, e, j, and k, top). These results indicate that the accumulation of StCN nanomicelles in the tumor sites requires a long time (e.g. 48 h post-injection) to take place, which could be attributed to the slow process of the EPR effect [34, 35]. For the mice injected with RGD-conjugated StCN nanomicelles, there were no signals in the tumor sites 1 h post-injection (Figure 10.10d and j, bottom), but intense fluorescence signals could be observed in the tumor sites at 24 h post-injection (Figure 10.10e and k, bottom), and also at 48 h post-injection (Figure 10.10f and l, bottom). The accumulation of the RGD-conjugated StCN nanomicelles in the tumor sites was much faster than that of the StCN nanomicelles, and one possible explanation is that the

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Figure 10.10 In vivo imaging of mice bearing subcutaneous lung tumor xenografts, injected with StCN nanomicelles (top images) and RGD-conjugated StCN nanomicelles (bottom images). The concentrations of two StCN nanomicelle samples were the same (with 60 wt% loading density, 1 mg ml1 in PBS, 100 ml of solution per mouse). The spectral profiles in (d)–(i) were used to unmix images. Reproduced with permission from [15], # 2011 Elsevier.

high binding affinity of RGD peptides to the avb3 integrin receptor contributed more than the EPR effect to tumor targeting of nanomicelles, making the RGD-conjugated StCN nanomicelles exhibit higher efficiency of targeting to the subcutaneous lung tumor xenografts in mice. It is worth noting that the StCN dye emits orange fluorescence that is adequate for superficial in vivo imaging of mice, but still useless for deep-tissue imaging. To overcome this obstacle, a far-red/NIR fluorescent AIE dye should be synthesized and adopted. Compared with visible light, far-red/NIR light (700–900 nm) is less absorbed and scattered by biological tissue and therefore its excitation can greatly increase the penetration depth and emission. Furthermore, far-red/NIR light has lower energy than ultraviolet and visible light and less fluorophores in tissue can be stimulated if far-red/NIR excitation is applied, and it can therefore efficiently restrain the generation of autofluorescence and improve the contrast of the image.

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Figure 10.11 Structures of BTPEPBI, DSPE-PEG2000 and DSPE-PEG5000–folate. Reproduced with permission from [16], # 2012 The Royal Society of Chemistry.

One type of far-red/NIR AIE dye, which was named BTPEPBI (Figure 10.11), was synthesized by Tang’s group [16]. The PL spectrum of BTPEPBI ranges from 600 to 850 nm, covering a fairly large area in the NIR region. They studied the PL behavior of BTPEPBI in water–tetrahydrofuran (THF) mixtures. As shown in Figure 10.12a, in dilute THF solution BTPEPBI shows very weak fluorescence. When fw (volume fraction of water) varies from 0 to 50%, the PL spectra of the solutions show very small changes. When fw is 50% or higher, the PL intensity increase strongly. This enhancement is ascribed to aggregate formation, which is induced by the addition of water. In addition, I/I0  1 data show that the PL intensity is enhanced over 233-fold when fw changes from 0 to 90% (Figure 10.12b). These results indicate that BTPEPBI is a typical AIE molecule. In contrast to Wang et al.’s work, Tang and co-workers fabricated BTPEPBI-doped nanomicelles through a modified nano-precipitation method [16], using a mixture of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and DSPE-PEG5000-folate (Figure 10.11) as the encapsulation matrix with good biocompatibility and different surface folic acid densities.

Figure 10.12 (a) PL spectra of BTPEPBI in THF–H2O mixtures with different fw values, excited at 538 nm. (b) Plot of I/I0  1 versus fw, where I and I0 are the PL intensity of BTPEPBI in THF–H2O mixtures in the presence and absence of H2O, respectively. The inset shows PL photographs of BTPEPBI in pure THF and THF–H2O mixture ( fw ¼ 90%). [BTPEPBI] ¼ 1  105 mol l1. Reproduced with permission from [16], # 2012 The Royal Society of Chemistry.

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Figure 10.13 (a) Particle size distribution of BTPEPBI-NP50 in water studied via laser light scattering. Inset: HRTEM image of BTPEPBI-NP50. (b) UV–vis absorption and fluorescence spectra of BTPEPBI-NP50 in water at room temperature (excited at 543 nm). Reproduced with permission from [16], # 2012 The Royal Society of Chemistry.

BTPEPBI-NP0 and BTPEPBI-NP50 represent BTPEPBI-doped nanomicelles that were formulated with polymers containing a feed ratio of 0% and 50% for DSPE-PEG5000-folate in the polymer matrix. During the formation of nanomicelles, the hydrophobic DSPE segments tend to be embedded into the hydrophobic core whereas the hydrophilic PEG-folate chains extend into the aqueous phase. The morphology of BTPEPBI-NP50 was studied by high -resolution transmission electron microscopy (HR-TEM) (Figure 10.13a, inset). The spherical shape of BTPEPBI-NP50 can be clearly distinguished from the black dots due to the high electron density of BTPEPBI molecules. Laser light scattering results suggest a narrow particle size distribution for BTPEPBI-NP50 (Figure 10.13a), and the volume average hydrodynamic diameter of BTPEPBI-NP50 is 57  1 nm. The absorption and fluorescence spectra of BTPEPBI-NP50 in water are presented in Figure 10.13b. In vivo imaging based on BTPEPBI-NP50 and BTPEPBI-NP0 was studied on a tumor-bearing mouse model. The animal model was established by inoculating murine hepatic H22 cancer cells subcutaneously into the left axillary space of each mouse. When the tumor volume reached about 300 mm3, the mice were injected intravenously with BTPEPBI-NP50 and BTPEPBI-NP0. The mice were subsequently imaged by an in vivo fluorescence imaging system. Figure 10.14a shows the in vivo distribution of BTPEPBI-NP0 in the

Figure 10.14 In vivo PL imaging of H22 tumor-bearing mice after intravenous injection of BTPEPBI-NP0 (a) and BTPEPBI-NP50 (b). The red circle indicates the tumor site. Reproduced with permission from [16], # 2012 The Royal Society of Chemistry.

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Figure 10.15 Structure of TPE-TPA-DCM.

tumor-bearing mouse at 1 and 3 h post-injection. The different PL intensities are shown by different colors, and the order red, orange, yellow, green, and blue refers to a successive decrease in intensity. Obvious PL is observed in the area of tumor tissue at 1 and 3 h, suggesting that BTPEPBI-NP0 has efficiently accumulated in the tumor through an enhanced permeability and retention (EPR) effect. This result is consistent with Wang et al.’s result. In addition, owing to the deep-tissue imaging capacity of far-red/NIR excitation, strong PL from the liver region is also observed, which implies that some nanomicelles in the blood circulation tend to be enriched in the liver. This phenomenon could not be observed in Wang et al.’s work since the orange PL signal from StCN nanomicelles was highly scattered in biological tissues. The specific tumortargeting ability of BTPEPBI-NP50 was also evaluated on the same tumor-bearing mouse model, as displayed in Figure 10.14b. Much higher PL intensity is found in the tumor tissue of a BTPEPBI-NP50-treated mouse compared with a BTPEPBI-NP0-treated mouse at both 1 and 3 h post-injection, demonstrating that BTPEPBI-NP50 has a specific targeting ability to the tumor that contains folate receptor-overexpressed cancer cells in a living body. Tang et al. also utilized DSPE-PEG2000 and DSPE-PEG5000-folate to encapsulate another type of far-red/ NIR AIE dye, named TPE-TPA-DCM (Figure 10.15), and achieved similar in vivo imaging results to those for BTPEPBI-doped nanomicelles (Figure 10.16a) [12]. These results illustrate that far-red/NIR AIE dyedoped nanomicelles can be used as effective probes for in vivo tumor diagnosis with high specificity and good optical contrast. In addition, TPE-TPA-DCM dye possesses a large two-photon absorption cross-section. Ex vivo twophoton excited imaging of a tumor in a mouse that was injected intratumorally with TPE-TPA-DCM-doped nanomicelles was further performed. The results revealed that they can be easily detected in the tumor mass at a depth of 400 mm (Figure 10.16b) [12]. We discuss the two-photon in vivo imaging of TPE-TPA-DCM doped nanomicelles further in Section 10.3. 10.2.4 Other types of AIE-nanoparticles for in vivo functional bioimaging In addition to phospholipid–PEG nanomicelles, other nanoparticles can also encapsulate AIE dyes and be applied in in vivo functional bioimaging. Zhang and co-workers synthesized poly(maleic anhydride-alt-1octadecene)-poly(ethylene glycol) (C18PMH-PEG) and folic acid-conjugated C18PMH-PEG (C18PMHPEG-FA) molecules, and used them to encapsulate a type of far-red/NIR aggregation-induced emission enhancement (AIEE) dye named NPAPF [17]. The modified nanoparticles possess favorable biocompatibility, robust pH stability covering a wide pH range of 4–10, high brightness with a high quantum yield of up to 14.9%, and much more superior photostability than conventional dyes. In addition to in vivo

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Figure 10.16 One-photon excited in vivo fluorescence imaging of an H22 tumor-bearing mouse after intravenous injection of (a) FTTDNPs and (b) TTDNPs. The white circles mark the tumor sites. (c-i) 3D twophoton fluorescence image of a C6 tumor from a mouse that was injected intratumorally with FTTDNPs. (c-ii) 100 mm, (c-iii) 300 mm, and (c-iv) 400 mm deep images from the C6 tumor. The images were recorded upon 800 nm excitation with a 600–780 nm bandpass filter. Reproduced with permission from [12], # 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

tumor targeting, they also investigated the blood circulation and biodistribution of NPAPF-PEG NPs in live mice. Tang and co-workers selected bovine serum albumin (BSA) as the polymer matrix for formulating NPs loaded with TPE-TPA-DCM (Figure 10.17) because albumin is biocompatible, nonantigenic, and utilized clinically [11]. The TPE-TPA-DCM-loaded BSA NPs show a prominent tumor-targeting capability by the EPR effect on the H22-tumor-bearing mouse model, and the biodistribution of NPs in live mice was also investigated (Figure 10.18).

Figure 10.17 Schematic illustration of the fabrication of BSA NPs loaded with TPE-TPA-DCM. Reproduced with permission from [11], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figure 10.18 Ex vivo fluorescence imaging on tumor tissue and major organs of mice treated with TPE-TPADCM-loaded BSA NPs, which were sacrificed at 24 h post-injection. The tissue autofluorescence was removed by spectral unmixing software. Reproduced with permission from [11], # 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

10.3 Multiphoton-Induced Fluorescence from AIE Dyes and Applications in In Vivo Functional Microscopic Imaging [36] 10.3.1 Two- and three-photon-induced fluorescence of AIE dyes As discussed in previous chapters, AIE dyes possess two-photon-induced fluorescence characteristic, just like many common fluorophores. The up-converted fluorescence of AIE dyes can be widely applied in various in vitro cell imaging and ex vivo tissue imaging [5–7, 10, 12]. In this section, we introduce the two- and threephoton-induced photoluminescence of AIE dye-encapsulated nanomicelles under femtosecond laser excitation. TPE-TPA-DCM (Figure 10.15) is a kind of red/NIR fluorescent AIE dye, and it was synthesized by Tang’s group [11]. Qian and co-workers used phospholipid–PEG nanomicelles to encapsulate TPE-TPADCM molecules and investigated the two-photon-induced fluorescence characteristic of the nanomicelles. The synthesis protocol of TPE-TPA-DCM-encapsulated phospholipid–PEG nanomicelles (abbreviated to TTD-nanomicelles) is similar to that of StCN nanomicelles. By varying the quantities of TPE-TPA-DCM and mPEG-DSPE solution added, nanomicelles with different TPE-TPA-DCM loading densities can be prepared. In order to investigate the characteristics of the AIE of fluorescence of TTD-nanoprobes, Qian and coworkers optically characterized nanomicelle solutions with various TPE-TPA-DCM loading densities (10, 15, and 20 wt%). For an accurate quantitative comparison of PL emission, the absorption intensities of the excitation wavelength (512 nm) for all samples should be kept the same, by utilizing different amounts of nanomicelles to encapsulate the same amount of TPE-TPA-DCM molecules. As shown in Figure 10.19, the envelopes of absorption and PL spectra of TTD-nanomicelles accord well with those of TPE-TPA-DCM dyes in water, but with a slight red shift of the absorption maximum and a slight blue shift of the emission maximum [11]. The total PL intensity increased significantly, without any distinct wavelength shift of the emission peak, as the TPE-TPA-DCM loading density increased in the nanomicelles. This indicates that TTD-nanomicelles exhibit an aggregation-enhanced fluorescence emission effect, due to the unique chemical structure of TPE-TPA-DCM molecules.

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Figure 10.19 Aggregation-enhanced fluorescence analysis of TTD-nanomicelles: UV–vis absorption and PL spectra of the TTD-nanomicelles with various TPE-TPA-DCM loading densities (10, 15, 20 wt%).

According to the absorption spectrum of TTD-nanomicelles in aqueous dispersion (Figure 10.19), they have negligible one-photon attenuation in the 700–900 nm range, and a femtosecond laser with its wavelength in this range can be used for two-photon excitation of the nanomicelles. Hence Qian and co-workers utilized an 850 nm, 150 fs Ti:sapphire pulsed laser (Mira HP, Coherent Inc.) at a repetition rate of 80 MHz for the two-photon study of an aqueous dispersion of TTD-nanomicelles in a cuvette. The two-photoninduced fluorescence spectrum was recorded with an optical fiber spectrometer (PG2000, Ideaoptics Instruments). As shown in Figure 10.20, the two-photon luminescence spectrum of TTD-nanomicelles is in the range 550–800 nm (with the maximum located at 655 nm), and its envelope is very similar to that under

Figure 10.20 Two-photon-induced fluorescence spectrum of TTD-nanomicelles.

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Figure 10.21 Dependence of the two-photon-induced fluorescence of TTD-nanomicelles on the excitation intensity of a femtosecond laser.

one-photon excitation (Figure 10.19). That means that in both the one- and two-photon processes, the excited TTD-nanomicelles are finally relaxed to the same lowest excited electronic-vibrational state(s), from which the luminescence emission occurs. Therefore, when the TTD-nanomicelle sample is stimulated by 850 nm laser pulses, it needs to absorb at least two photons at the same time to become excited [37]. To verify that the observed luminescence was induced by two-photon excitation, a series of emission intensities of TTD-nanomicelles under femtosecond laser excitation (at 850 nm) were measured with various average powers. The dependence of the luminescence intensity on the excitation intensity is plotted in Figure 10.21, where it can be seen that the two-photon-induced emission intensity is proportional to the square of the femtosecond excitation intensity, confirming the characterization of a two-photon process. The two-photon-induced luminescence feature of TTD-nanomicelle facilitates its wide application in two-photon microscopy-based bioimaging, which can achieve higher spatial resolution, a deeper imaging range, and less photobleaching (this is discussed in detail in the next section). Furthermore, TTDnanomicelles with two-photon absorption properties also have wide potential applications in other fields, such as laser lithography, power limiting, optical stabilization, and optical reshaping [38–40]. In addition to TPE-TPA-DCM, other AIE dyes also possess the feature of two-photon-induced fluorescence. 1,1,2,3,4,5-Hexaphenylsilole (HPS), with which the AIE phenomenon was first observed [41], shows its two-photon and (further) three-photon fluorescence under femtosecond laser excitation. Qian and co-workers synthesized HPS-encapsulated phospholipid–PEG nanomicelles; the protocol is similar to that for preparing StCN nanomicelles and TTD-nanomicelles. As shown in Figure 10.22, when the absorption intensities of the excitation wavelength (367 nm) for all the samples were kept the same, the total (one-photon) PL intensity of HPS-nanomicelles increased significantly without any distinct wavelength shift in the emission peak, as the HPS loading density increased in the nanomicelles (from 5 to 40 wt%). The results confirmed the aggregation-enhanced fluorescence effect of HPS-nanomicelles. Figure 10.23 shows the one-photon (367 nm continuous-wave light excited) and two-photon (690/735 nm femtosecond laser excited) induced fluorescence spectra of HPS-nanomicelles. The two-photon

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Figure 10.22 Aggregation-enhanced fluorescence analysis of HPS-nanomicelles: UV–vis absorption and PL spectra of the HPS-nanomicelles with various HPS loading densities (5, 20, 40 wt%).

fluorescence spectra accord well with those under one-photon excitation, but with a slight red shift of the emission maximum. Qian and co-workers further measured the three-photon-induced fluorescence of an HPS-nanomicelle film on a glass slide with a femtosecond laser with a wavelength of 1040 nm. The femtosecond laser source (80 MHz, 100 fs) was the amplified output of a large-mode-area ytterbium-doped PCF oscillator. The femtosecond laser beam was introduced into an objective [40, numerical aperture (NA) ¼ 0.6] to achieve a tight focus and high excitation intensity towards the nanomicelle film, which was smeared on a glass slide. The

Figure 10.23 One- and two-photon-induced fluorescence spectra of HPS-nanomicelles.

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Figure 10.24 Three-photon excited behaviors of HPS-nanomicelles. (a) Three-photon-induced luminescence spectrum; (b) cubic dependence of three-photon-induced luminescence on the excitation intensity of the femtosecond laser.

excited signals were collected by the same objective and transmitted to an optical fiber spectrometer for recording. As shown in Figure 10.24a, the envelope of the three-photon-induced fluorescence spectrum is very similar to those under one-photon and two-photon excitations. Similarly to the principle of two-photon luminescence, when the HPS-nanomicelle sample is stimulated by 1040 nm laser pulses, it needs to absorb at least three-photons at the same time to become excited and then relax to the same lowest excited electronic-vibrational state(s) to produce luminescence emission [37]. Various three-photon-induced emission intensities of HPS-nanomicelles were then obtained under femtosecond laser excitation with different average powers, and the dependence of the intensity on the pump pulse energy is shown in Figure 10.24b. It can be seen that the three-photon-induced emission intensity is proportional to the cube of the femtosecond excitation intensity, which verifies the characterization of a three-photon process. Although a number of papers have been published on the three-photon luminescence behaviors of several materials (e.g., noble metal nanoparticles, quantum dots, and organic dyes) [42–44], this is the first time that three-photon-induced fluorescence performance from AIE dye-nanomicelles was observed. This feature has great potential in bioimaging, military protection, and optical signal processing applications. Owing to the higher order dependence on the excitation laser intensity, three-photon-induced auto-fluorescence of cells and tissue can be largely eliminated, which cannot be achieved with either (one-photon) confocal or twophoton microscopy [42]. Considering this advantage, HPS-nanomicelle-assisted three-photon microscopy can be a potential method for low-background and high-contrast bioimaging. Furthermore, the 1040 nm femtosecond laser has low absorption and scattering in biological tissues, which may facilitate HPSnanomicelle-based three-photon microscopy for potential ex vivo tissue and in vivo animal imaging. The three-photon absorption properties of HPS-nanomicelles can also be applied in power limiting, optical stabilization, and optical reshaping, and the effects can be more distinct compared with two-photon excitation as it requires a higher excitation laser intensity to achieve a higher order nonlinear effect [45]. 10.3.2 AIE dye-encapsulated nanomicelles for two-photon blood vessel imaging of live mice NIR fluorescence and Raman spectroscopy have been used to investigate the circulation of carbon nanotubes in animals [46, 47]. Inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) are widely used to measure the biodistribution of gold nanostructures [48, 49]. In such

228 Aggregation-Induced Emission: Applications studies, however, blood has to be extracted at each time point post-injection for in vitro measurements, which is cumbersome, time consuming, and incapable of in vivo monitoring the clearance in the same animal. Qian et al. functionalized gold nanorods (GNRs) with both NIR fluorescence and surface-enhanced Raman scattering (SERS) [28]. This approach has the combined advantages of high real timing, high imaging contrast, and deep detection ability, and the distribution and excretion of intravenously injected GNRs in deep tissues of live mice were observed in vivo for the first time through purely optical imaging. However, this method lacks the spatial resolution to locate nanoparticles in the blood and organs, since the imaging was performed in a macro in vivo imaging system. Therefore, new methods that are able to track nanostructures directly in the live animals with high spatial resolution are needed to elucidate the real-time circulation and clearance behavior of nanoparticles. Two-photon excitation-induced bioimaging has many unique advantages [50]. Owing to a square dependence of two-photon absorption on laser intensity, the sample region outside the beam focus cannot be excited, and it could reduce the possibility of photobleaching of the sample signal. The nonlinear excitation mode is also helpful for improving the spatial resolution of imaging, since only the site where the laser beam is focused can be efficiently excited. More importantly, two-photon excitation has great potential for deep-range tissue imaging. For one-photon bioimaging, photosensitizers usually absorb light in the visible spectral region below 700 nm, where light penetration into the tissue is limited. On the other hand, twophoton excitation wavelengths are usually in the range 700–900 or 1000–1350 nm. The 700–900 nm wavelength range is typically considered as the main optically transparent window for tissues since water shows very low light absorption in this range [51]. The 1000–1350 nm wavelength range is considered another window for in vivo imaging owing to the relatively low light absorption, very low light scattering and low (one-photon absorption-induced) auto-fluorescence, and consequently the penetration of two-photon excitation light can be efficiently improved [52, 53]. Furthermore, a femtosecond pulsed laser is a promising light source for multiphoton excitation. With low average power, high peak power, and relatively low repetition period, a femtosecond laser can be properly operated to produce mimimal thermal effects during its interaction with biosamples, which can effectively avoid certain thermal damage. As mentioned, AIE dyes, which possess the two-photon-induced fluorescence feature and can be loaded with nanoparticles, have been widely applied in various two-photon in vitro cell imaging and ex vivo tissue imaging. In addition, AIE dyes have also been utilized in various types of in vivo research, such as distribution monitoring, tumor targeting, and SLN mapping. However, no research group has ever extended their applications to two-photon microscopic in vivo imaging. In order to observe more detailed and deeper information, a scanning microscopic imaging system rather than a macro in vivo imaging system should be adopted. In traditional one-photon confocal microscopy, visible excitation/emission light is prone to be absorbed by water in the tissue, and scattered due to the Rayleigh scattering effect, so the imaging depth cannot reach 100 mm. By virtue of less absorption/scattering of an NIR femtosecond excitation source and nonlinear excitation mode, two-photon scanning microscopy can achieve a deeper tissue imaging capability, and also higher spatial resolution of imaging, rather than one-photon confocal microscopy. Considering the good performance of two-photon fluorescence of AIE dye-doped nanomicelles, together with the advantages of two-photon scanning microscopy, Wang and co-workers applied them in blood vessel imaging of live mice to observe the real-time circulation and clearance behavior of nanoparticles. In their experiment, 8-week-old female BALB/c mice were used. TTD-nanomicelles were selected as the optical agent, and their dispersion (in 1 PBS) was filtered twice and then exposed to UV light overnight for sterilization. After anesthetized the mice with pentobarbital, TTD-nanomicelles were administrated via the tail vein. The mice were immediately placed on a Petri dish with one ear attached to the coverslip. An upright two-photon scanning microscope (Olympus FV1000 femtosecond laser at 850 nm) was used for two-photon microscopy, in which a long working distance (2 mm) water-immersion objective lens (25, NA ¼ 1.05) was then used to focus the femtosecond laser beam on to the water-immersed earlobe, and an

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Figure 10.25 In vivo two-photon scanning fluorescence imaging of intravenously injected TTD-nanomicelles in the ear blood vessel of mice at various time points after sample treatment. Scale bar: 50 mm.

external photomultiplier tube (PMT) was used to collect two-photon-induced emission signals via non-descanned detection (NDD). Another group of mice without treatment with nanomicelles were used for the control experiment. Figure 10.25 shows the in vivo two-photon blood vessel imaging results for mice at various time points after TTD-nanomicelle treatment. At 1 h after sample injection, bright two-photon fluorescence was observed in the ear blood vessel of mice, whereas almost no two-photon fluorescence signals could be obtained from the ear blood vessel of the control mice. This illustrates that intense luminescence was emitted from femtosecond laser-excited TTD-nanomicelles. At 2 h post-treatment, the two-photon fluorescence was still very distinct. By 6 h post-treatment, the two-photon luminescence began to decrease in the blood vessel due to the clearance of TTD-nanomicelles over time. At 15 h after sample injection, only very weak two-photon fluorescence could be observed, indicating the distribution of TTD-nanomicelles in the blood vessel was very diffuse, and by 24 h after sample injection almost no TTD-nanomicelles could be detected any longer. Since the two-photon fluorescence of TTD-nanomicelles was very distinct, intravenously injected nanomicelles can reveal the vascular architecture. Figure 10.26 shows a three-dimensional (3D) reconstructed picture of the blood vessel of a mouse (1 h post-treatment), with the assistance of two-photon scanning

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Figure 10.26 A reconstructed 3D image illustrating the distribution of TTD-nanomicelles in the blood vessel of a mouse (1 h post-treatment) via two-photon confocal microscopy.

microscopy. The two-photon fluorescence of TTD-nanomicelles can also reveal individual RBCs (red blood cells), which appeared as shadows flowing with the two-photon fluorescence signals. A rapid line scan along the capillary axis, which is one of the functions of the commercial confocal microscope software, was then used to determine the RBC flow parameters. RBCs moving through the capillary appeared as oblique shadows from which the instantaneous velocity (dx/dt) of each RBC can be determined, as shown in Figure 10.27. 10.3.3 AIE dye-encapsulated nanomicelles for two-photon brain imaging of live mice Owing to the advantages of its deep tissue imaging capability (to 1 mm or more), high spatial resolution, and low thermal damage towards biosamples, two-photon fluorescence microscopy is also a very useful tool for brain imaging of small animals. During the past few years, two-photon brain imaging has been

Figure 10.27 A line scan along the capillaries of the mouse was used to observe the RBC flow and determine its instantaneous velocity (dx/dt) via two-photon confocal microscopy.

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Figure 10.28 Scheme illustrating the two-photon fluorescence microscopy of a mouse brain.

widely applied in various biological studies, such as neurobehavioral analysis/regulation, gene therapy in the brain, and blood– brain barrier penetration [54, 55]. Usually, the fluorophores adopted for brain imaging are gene transfected or commercially available. The former approach is very cumbersome and time consuming, and commercial dyes are easily limited by the ACQ effect when their concentration becomes high. Since AIE dyes show a higher quantum yield in the construction of nanomicelles due to restriction of intermolecular vibrational and rotational motions in the formation of nanoaggregates, and also facile biosample staining operation, they have the potential to be a new type of fluorescent probe for in vivo brain imaging. Wang and co-workers transfected mice with YFP (yellow fluorescent protein) via transgenic technology. After the transfection, the neurons and dendritic in mice brains were stained with YFP. TTD-nanomicelles were filtered twice and then exposed to UV light overnight for sterilization. After anesthetizing the mice with pentobarbital, TTD-nanomicelles were administered via the tail vein. The skulls of the mice were then opened up through microsurgery and imaged under an upright two-photon scanning microscope (Olympus FV1000 femtosecond laser at 850 nm) (Figure 10.28). The detailed method for the immobilization of the heads of the mice, and their contact with the objective of the upright two-photon scanning microscope, can be found in a recent paper [56]. Figure 10.29 shows a 3D reconstructed mixed image of TTD-nanomicelles in the blood vessel of a mouse brain. The imaged region (254  254  50 mm) was at a depth of about 300 mm in the mouse brain. For twophoton excitation, an 850 nm femtosecond laser was applied, and a grating filter ranging from 600 to 690 nm was used to extract the two-photon-induced emission signals from the TTD-nanomicelles. Since the neurons and dendrites in the mouse brain was stained with YFP through gene transfection, a wavelength filter ranging from 500 to 575 nm was used to extract the fluorescence signals of YFP. As can be seen, the two-photon fluorescence of the nanomicelles (red), together that of YFP (green, spot shape), could be observed under the background (weak green, which was from the brain tissue) and could easily be discriminated from each other. Since the two-photon fluorescence of TTD-nanomicelles was very distinct, intravenously injected nanomicelles can reveal the vascular architecture in the mouse brain. In contrast, the activities (e.g. flowing, penetrating) of nanomicelles in/near a blood vessel can also be illustrated via the two-photon fluorescence signals. Figure 10.30 shows the in vivo two-photon brain imaging results for a mouse at various time points. The two-photon fluorescence signal dynamically and vividly illustrates the flow process of TTD-nanomicelles in the blood vessel. Usually, the cerebellum and cerebral cortex blood vessels of small mice distribute at a depth not beyond 800 mm in the brain. If the fluorescence spectra of encapsulated AIE dyes are well tuned to the NIR region (e.g. 700–900 nm) and the femtosecond laser excitation conditions (e.g. repetition rate, peak power of pulse) can be further optimized, AIE nanomicelle-assisted two-photon microscopy can achieve such an imaging

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Figure 10.29 A reconstructed 3D image illustrating the distribution of TTD-nanomicelles in the blood vessel of a mouse brain (1 h post-treatment) via two-photon confocal microscopy.

depth or more, which will be very helpful towards more potential in vivo applications (e.g. neurobehavioral analysis/regulation, gene therapy in the brain, and blood–brain barrier penetration).

10.4 Summary and Perspectives In this chapter, the first examples of AIE dye-encapsulated nanomicelles for in vivo SLN mapping and tumor targeting were introduced. Two- and three-photon-induced fluorescence of AIE dyes, and the application of two-photon microscopic in vivo blood vessel and brain imaging of mice, were also systematically investigated. In the future, AIE dye-encapsulated nanoparticles could be used as highly efficient fluorescent nanoprobes for more potential in vivo biological applications. To achieve this, various well-designed and improved technologies are still need, which are discussed briefly in the following. Materials: First, the fluorescence quantum yield of AIE dyes should be as high as possible. For macro (one-photon) in vivo imaging, the excitation and emission spectra of AIE dyes can be optimized and tuned to 700–900 nm, which is an optically transparent window for tissues, owing to the small light absorption of water in this range. If possible, the excitation and emission spectra of AIE dyes can also be tuned to 1000–1350 nm, which is considered another window for in vivo imaging owing to the relatively low light absorption, very low light scattering, and low auto-fluorescence, and consequently the penetration of excitation/emission light, the spatial resolution of imaging, and the signal-to-noise ratio can be efficiently improved. For multiphoton in vivo imaging, the cross-section of two-photon (three-photon) absorption of AIE dyes should be improved and increased. It is better that the two-photon (three-photon) excitation and emission spectra of AIE dyes are optimized in the 700–900 or 1000–1350 nm region. Furthermore, new nanoplatforms with high biocompatibility should be designed to achieve a high loading efficiency of AIE dyes.

Aggregation-Induced Emission Dyes for In Vivo Functional Bioimaging 233

Figure 10.30 In vivo two-photon scanning fluorescence imaging of intravenously injected TTD-nanomicelles in a blood vessel of a mouse brain at various time points. Scale bar: 50 mm.

Optical signals: So far, only one-photon/two-photon fluorescence intensity/spectrum was adopted as the imaging signal. However, many other optical signals of AIE dyes, such as lifetime, polarization, and threephoton fluorescence, also have wide potential applications. As discussed in Section 10.3.1, three-photoninduced auto-fluorescence of cells and tissue can be largely eliminated, owing to the higher order dependence on excitation laser intensity. This restriction cannot be overcome in either (one-photon) confocal or two-photon microscopy. Hence TTD-nanomicelle-assisted three-photon microscopy can be a potential method for low-background and high-contrast bioimaging. Furthermore, optical imaging systems also need to be optimized to achieve clearer and deeper tissue imaging.

234 Aggregation-Induced Emission: Applications Biomedical applications: New biomedical applications should be explored. For example, two-photon in vivo blood vessel imaging can measure the flow velocity of RBCs, which is very helpful for evaluating the effect/influence of drugs. AIE dye-encapsulated nanoparticles may penetrate the blood–brain barrier of small animals and achieve inner-brain drug delivery, which can also be dynamically monitored with twophoton in vivo imaging. Furthermore, the femtosecond laser used for multiphoton excitation of AIE dyes can assist in the manipulation of cellular/tissue behaviors, which can also be under the guidance of twophoton fluorescence imaging. We believe that there is still plenty of room for in vivo biomedical applications of AIE dyes in the future.

Acknowledgments This work was partially supported by the Science and Technology Department of Zhejiang Province, the National Basic Research Program (973) of China, a Special Financial Grant from the China Postdoctoral Science Foundation (No. 201104741), the National Natural Science Foundation of China (61275190, 61008052, 61178062, and 60990322), and the Fundamental Research Funds for the Central Universities. We are grateful to Miss S.S. Liu for assistance with two-photon laser scanning confocal microscopy. We also express our deepest gratitude to Dr W. Xi for his help with brain imaging of mice.

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11 Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics for Protein Sensing Jing Liang1, Haibin Shi1, Ben Zhong Tang2 and Bin Liu1 1

Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Department of Chemistry, Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology, Hong Kong, and Institute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, China 2

11.1 Introduction There is a ubiquitous phenomenon observed from most organic and polymeric fluorophores, aggregationcaused quenching (ACQ), which is the tendency to show quenched fluorescence in aggregated states. The fluorophores at high concentration or in the solid state experience strong p–p interactions, leading to the formation of aggregates whose excited states are dissipated through nonradiative pathways [1]. As aggregation can frequently occur in aqueous solution or in the presence of hydrophobic species, such fluorophores often show quenched emission in biological media, which greatly hampers their application for biosensing. Although various strategies have been attempted to alleviate the ACQ effect by minimizing aggregation, many of them provide only a temporary or less effective remedy [2]. The development of fluorogens with aggregation-induced emission (AIE) characteristics, in contrast, offers a facile and cost-effective solution. In an AIE system, the nonemissive fluorogens in dilute solutions become highly fluorescent when aggregated, giving rise to emission efficiencies of up to unity [3]. These unique characteristics make AIE fluorogens ideal sensory materials, as aggregation favors signal transduction in aqueous sensing media, leading to superior sensitivity. The AIE phenomenon is commonly attributed to the restriction of intramolecular rotation (RIR) in the aggregated state. This effect has been observed in several types of structures, such as silole derivatives – hexaphenylsilole (HPS) and tetraphenylsilole (TPS) – and tetraphenylethene (TPE) and its derivatives (Scheme 11.1). Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

240 Aggregation-Induced Emission: Applications

Scheme 11.1 Structures of representative fluorogens with AIE characteristics.

The attractive properties of AIE fluorogens have prompted the design and synthesis of their water-soluble counterparts for sensing applications. Specific identification of biologically significant targets plays a vital role in the early diagnosis of diseases, study of biological processes, drug screening, and so on. With proper structural modifications, these water-soluble AIE fluorogens were found to show fluorescence turn-on responses to a wide range of biomolecules, such as nucleic acids [4, 5], proteins [6, 7], sugars [8, 9], and ATP [10] upon nonspecific interactions. However, as these interactions are largely electrostatic or hydrophobic in nature, these probes are suitable for the detection of only a limited number of targets. In addition, the interfering substances present in the media, such as proteins, electrolytes, and amino acids, may also interact with the probe through similar interactions, giving rise to poor bioselectivity. To apply the bioassays to a wider range of targets and achieve specific target recognition, the preparation of water-soluble AIE probes with targeting ability is greatly in demand. In this chapter, we present a general strategy of developing target-specific AIE probes through conjugation of hydrophobic AIE molecules with a hydrophilic peptide that is specific to biomolecules or biological events. They are not only able to detect target proteins but are also useful in monitoring biological processes.

11.2 In Vitro Detection of Integrin avb3 Using a TPS-Based Probe Integrins are transmembrane glycoproteins that mediate the adhesion of cells to neighboring cells or the matrix that surrounds them – the extracellular matrix (ECM) [11]. Integrins are composed of a set of noncovalently associated a and b subunits and each subunit consists of a large extracellular domain, a transmembrane helix, and a short cytoplasmic tail [12]. There are at least 24 different heterodimers, each of which can bind to a specific component in the ECM, such as collagens, fibronectin, and laminins driven by the interaction of the cytoplasmic tail with signaling proteins and cytoskeletal proteins [13]. By facilitating the communication between intracellular and extracellular environments, integrins play a vital role in the maintenance of cell integrity and regulation of cellular functions, such as cell migration, proliferation, and death [14]. There has been increasing evidence suggesting that integrin expression and function are linked to tumor progression and metastasis [15–17]. Among the many heterodimers within the integrin family, integrin avb3 is well identified as a biomarker for cancer diagnosis and therapy and its expression is correlated with the cancer progression [18]. The detection of integrin avb3 is therefore of practical significance for the early detection and treatment of tumors [19]. As integrin avb3 is found to bind with the exposed arginine–

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 241

Scheme 11.2 Schematic illustration of integrin avb3 detection with the light-up probe TPS-2cRGD.

glycine–aspartic acid (RGD) motif of fibronectin in the ECM, a large number of radiolabeled and optically labeled probes with RGD-based ligands have been reported [20, 21]. While the radiolabeled probes introduce potential radioactive hazards to patients, the probes labeled with fluorogens such as organic dyes and quantum dots may suffer from the ACQ effect or high cytotoxicity. We therefore developed a bioprobe by integrating an AIE dye, TPS, with two highly specific cyclic RGD tripeptide (cRGD) units for sensing and imaging of integrin avb3. 11.2.1 Detection mechanisms The mechanism for the detection of integrin avb3 is illustrated in Scheme 11.2. The TPS dye is functionalized with two units of cRGD which are specific to integrin avb3. As the TPS-2cRGD conjugate is water soluble, its fluorescence is in the ‘off’ state in water due to annihilation of excited states by active intermolecular rotations [22]. Upon addition of integrin avb3, the specific binding between cRGD and integrin avb3 strongly restricts the rotations of the aromatic rotors of TPS, and the fluorescence is switched ‘on.’ In the presence of nonspecific proteins, however, the RIR process cannot be activated and no fluorescence turn-on can be observed. The distinct fluorescence responses can be used to differentiate integrin avb3 from other proteins and the correlation between the amount of integrin avb3 present and the extent of fluorescence light-up is useful for the quantification of integrin avb3. 11.2.2 Synthesis and characterization of the TPS-2cRGD probe The TPS-2cRGD probe was synthesized by a copper-catalyzed ‘click’ reaction between a bisazido-functionalized TPS (BATPS) and an ethylene-terminated cRGD (E-cRGD) (Scheme 11.3) in a mixture of dimethyl sulfoxide (DMSO) and H2O in the presence of sodium ascorbate and CuSO4. Further purification with reversed-phase high-performance liquid chromatography (HPLC) yielded the purified probe in over 80% yield. BATPS and TPS-2cRGD display similar absorption profiles (Figure 11.1), with the absorption maximum at
360 nm and tails extending to 450 nm. In contrast, the photoluminescence (PL) intensity of BATPS is significantly higher than that of TPS-2cRGD in a DMSO–water mixture (1:199 v/v), which is attributed to their difference in solubility in the medium. As BATPS is poorly soluble in the DMSO–water mixture, it emits strong fluorescence due to AIE. The aggregate formation was confirmed by laser light scattering (LLS) measurements, indicating the presence of nanoaggregates with an average size of 103 nm. In contrast, TPS-2cRGD is highly soluble in the same medium and no emission was observed. The LLS results also indicated the absence of any aggregates with measurable sizes.

242 Aggregation-Induced Emission: Applications

Scheme 11.3 Click synthesis of the TPS-2cRGD.

Figure 11.1 UV–vis absorption (left) and PL (right) spectra of BATPS (dashed line) and TPS-2cRGD (solid line) in DMSO–water (1:199 v/v). lex ¼ 356 nm.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 243 It was found that the probe shows high resistance to ionic strength. There is little change in the PL spectra of the probe (10 mM) in sodium chloride solution with NaCl concentrations up to 960 mM. The PL properties are also not altered in Dulbecco’s Modified Eagle Medium (DMEM), a complex medium containing salts, amino acids, glucose, and vitamins. These experiments confirm that the probe remains in the off state in biological media, giving rise to minimal background interference. 11.2.3 Detection of integrin in solutions To test the specific response of the TPS-2cRGD probe to integrin avb3, titration experiments were carried out by addition of various amounts of integrin to the TPS-2cRGD probe. Figure 11.2 shows the PL spectra of the probe (10 mM) incubated with integrin at different concentrations (0–100 mg ml1). Addition of increasing amounts of integrin avb3 leads to progressive fluorescence light-up of the probe. As can be seen in the photographs shown in the inset, this assay also permits visual detection of integrin avb3, providing an even faster and simpler route for signal output. Compared with the probe in the buffer solution, the fluorescence of the probe is intensified 182-fold when incubated with 100 mg ml1 of integrin avb3. The plot of change in PL intensity versus protein concentration below 50 mg ml1 yields a perfect straight line, indicating the feasibility of the assay for integrin avb3 quantification. The limit of detection is estimated to be 0.5 mg ml1 based on the plot. The selectivity of the assay was assessed by incubation of the TPS-2cRGD probe with DNA and a group of interference proteins under the same experimental conditions as for integrin. Proteins with different isoelectric points (pI) were selected: bovine serum albumin (BSA) (pI ¼ 4.9), heparinase (pI ¼ 7.9), concanavalin A (Con A) (pI ¼ 8.4), papain (pI ¼ 9.6), trypsin (pI ¼ 10.1), and lysozyme (pI ¼ 11.0). It was found that integrin avb3 induces fluorescence changes that are
10–182-fold larger than with the other six macromolecules, indicating that the assay is highly selective to integrin avb3.

Figure 11.2 PL spectra of TPS-2cRGD in the presence of different amounts of integrin avb3 (0–100 mg ml1) at lex ¼ 356 nm. Inset: photographs taken under UV illumination at lex ¼ 365 nm.

244 Aggregation-Induced Emission: Applications 11.2.4 In vitro sensing of integrin in cancer cells To demonstrate the ability of TPS-2cRGD for in vitro detection of integrin, its receptor-mediated binding with integrin was explored in mammalian cells. HT29 cancer cells are known to overexpress integrin on cellular membranes and were chosen as integrin-positive cancer cells for the study. Breast cancer cell MCF-7 with a low level of expression of integrin was selected as the negative control [23]. Both cell lines were precultured at 37  C in DMEM containing 10% fetal bovine serum (FBS) to reach 80% confluence. then TPS-2cRGD solution was added. After incubation for 30 min at 4  C, a commercial deep-red plasma membrane tracker (CellMask, Invitrogen) was co-stained to visualize the location of cell membranes. In contrast to TPS-2cRGD probe, which relies on interaction with integrin, the membrane tracker stains the membrane by locking its lipophilic moiety and hydrophilic dye in the membrane lipid bilayer. Figure 11.3 shows confocal laser scanning microscopy (CLSM) images of MCF-7 (a–c) and HT29 (d–f) live cells after incubation of the TPS-2cRGD probe and membrane tracker. The images were taken under excitation at 405 nm with a 505–525 nm bandpass filter for the probe (Figure 11.3a and d) and at 543 nm with a 575–635 nm bandpass filter for the membrane tracker (Figure 11.3b and e). As shown in Figure 11.3a, no fluorescence signal of the MCF-7 cells can be observed under excitation at 405 nm, and the membrane is only visible after staining with the membrane tracker upon excitation at 543 nm (Figure 11.3b). In contrast, the fluorescence signal of HT29 cell line incubated with the probe is

Figure 11.3 CLSM images of live cells after incubation with TPS-2cRGD (2 mM) in the absence and presence of a membrane tracker at 4  C for 30 min. Fluorescence images of (a–c) MCF-7 and (d–f) HT29 cells stained with (a and d) TPS-2cRGD and (b and e) membrane tracker, with panels (c) and (f) showing their overlay images. The images were taken under excitation at (a and d) 405 nm and (b and e) 543 nm using optical filters with bandpasses of (a and d) 505–525 nm and (b and e) 575–635 nm at 5% laser power. The scale bar of 10 mm applies to all images. Reproduced with permission from Shi, H., et al., Detection of Integrin avb3 by Light-Up Bioprobe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc., 2012, 134 (23), 9569–9572. # 2012, American Chemical Society.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 245 clearly visible (Figure 11.3d) and overlaps well with that of the membrane tracker (Figure 11.3f). These results provide direct evidence of specific binding between TPS-2cRGD and integrin which is localized at the cell membranes. The distinct fluorescence response of HT29 and MCF-7 cells (Figure 11.3a and d) resulting from the specific interaction with the probe can be utilized for the unambiguous discrimination of the integrin-positive and -negative cells. The cytotoxicity of TPS-2cRGD probe was examined using the conventional assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as the indicator. The HT29 cells were incubated with TPS-2cRGD suspensions at concentrations of 2, 5, and 10 mM for 12, 24 and 48 h, followed by incubation of MTT. The viability under all of the above conditions was measured to be
100%, indicating excellent biocompatibility of the probe.

11.3 Real-Time Monitoring of Cell Apoptosis and Drug Screening with a TPE-Based Probe Apoptosis, also known as programmed cell death, plays an essential role in the development of multicellular organisms and the regulation of immune response [24]. Dysregulation of apoptosis pathways may lead to a host of diseases, such as cancer, autoimmune disease, immunodeficiencies, atherosclerosis, and lymphoid malignancies [25, 26]. Understanding the apoptosis process therefore has practical implications for early detection of diseases, tracking of disease progression, and therapy development. A series of morphological and biochemical changes occur during the apoptosis process, for example, cell blebbing and shrinkage, chromosomal DNA fragmentation, and protease activation [27]. A number of strategies have been developed to detect apoptosis. Conventional methods include electron microscopy to monitor morphological changes [28], detection of DNA fragmentation using a DNA ladder assay [29], and Western blotting assay of cytochrome c which is released after induction of apoptosis [30]. The major disadvantage of these methods is their invasive nature, and the first two can only detect apoptosis in the last stage [31]. Annexin V can also be applied to identify mid- to late-stage apoptotic cells by staining the phosphatidylserine exposed on the surface of the plasma membrane during apoptosis [32]. However, this method is liable to produce false-positive results. Caspases, a group of cysteine proteases that universally exist in animal cells, act as central mediators of apoptosis through a cascade of cleavage reactions [33, 34]. Caspases can effect apoptotic cell death by cleaving downstream caspase precursors called procaspases or other cellular proteins after their aspartate residue [35]. Among the 14 caspases that have been identified so far, caspase-3 is the most critical one and it has become an appealing target for apoptosis imaging. A few types of activatable optical probes have been developed to monitor caspase-3 activity. The optical probes may consist of a caspase-specific peptide substrate such as Asp–Glu–Val–Asp (DEVD) labeled with a luciferin or latent fluorophore, which can be released to generate fluorescence upon cleavage by caspase-3 [36, 37]; the peptide substrate may also be dual labeled with donor–quencher pairs [38, 39] or fluorescence resonance energy transfer (FRET) pairs [40], the separation of which can lead to changes in fluorescence intensity or wavelength in response to caspase cleavage. However, these probes are generally suboptimal for apoptosis imaging owing to limitations such as high background signal, poor cell permeability, or complicated synthetic steps. In view of the above considerations, we designed a simple, noninvasive strategy for monitoring caspase activity with a novel fluorescent light-up probe. The probe consists of an AIE dye conjugated with the caspase-specific peptide substrate DEVD. It is capable of detecting caspase-3 and -7 in both solution and living cells, and is useful for real-time monitoring of cell apoptosis and screening of apoptosis-related drugs. 11.3.1 Design principles As illustrated in Scheme 11.4, the acetyl-protective N-terminal Asp–Glu–Val–Asp–Lys–TPE (Ac-DEVDKTPE) probe contains three components: (1) a peptide with a sequence of DEVD, which is used as a substrate

246 Aggregation-Induced Emission: Applications

Scheme 11.4 Illustration of caspase activity study using Ac-DEVDK-TPE.

cleavable by caspase-3/7 and to endow the probe with water solubility; (2) an azido-functionalized TPE (TPE-N3) fluorescence reporter that is activatable upon external stimuli; and (3) an alkynyl-functionalized lysine (K) linker that bridges the TPE and DEVD moieties. The water-soluble Ac-DEVDK-TPE probe is non-fluorescent in aqueous solution owing to free intramolecular rotations [6]. As caspase-3 can specifically cleave on the C-terminal of aspartic acid residue, the DEVDK-containing probe is cleaved via the Asp (D)– Lys (K) linkage by activated caspase-3 once it is internalized by cells and upon cell apoptosis. The released TPE-K residue is hydrophobic and becomes highly fluorescent according to the AIE mechanism. 11.3.2 Synthesis and characterization of Ac-DEVEK-TPE probe The synthesis of the Ac-DEVDK-TPE probe was carried out in both solution and solid phases. The alkynebearing Ac-DEVDK peptide was prepared via the conventional Fmoc solid-phase peptide synthesis using Rink amide resin as the solid support [41]. The Ac-DEVDK-A product obtained was then coupled with TPE-N3 using click chemistry catalyzed by sodium ascorbate and CuSO4 in DMSO–H2O to yield the AcDEVDK-TPE probe (Scheme 11.5). The synthetic procedures are similar to those introduced in the first section. The probe was purified by reversed-phase HPLC in 70% yield.

Scheme 11.5 Click synthesis of Ac-DEVDK-TPE.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 247

Figure 11.4 Absorption (left) and emission (right) spectra of TPE-N3 (dashed line) and Ac-DEVDK-TPE (solid line) in DMSO–water (1:199 v/v). lex ¼ 312 nm.

The absorption and emission properties of the probe before and after peptide cleavage were examined and compared. As shown in Figure 11.4, the UV–vis absorption spectra of TPE-N3 and Ac-DEVDK-TPE show similar profiles and intensities in the absorption range 300–350 nm. The emission spectra of the two compounds, however, are significantly different. The hydrophobic TPE-N3 is highly fluorescent in DMSO– water (1:199 v/v) mixture with a quantum yield (w) of 0.2 whereas the water-soluble Ac-DEVDK-TPE is virtually nonfluorescent in the same medium (w ¼ 0.001). This is in accordance with the AIE effect that an AIE fluorogen emits weakly in a good solvent and strongly in a poor solvent owing to aggregate formation. The LLS results also confirmed the existence of aggregates in TPE-N3 with an average diameter of 126 nm and the absence of aggregation in Ac-DEVDK-TPE solution. To prepare the probe for caspase sensing in cell media, the effect of ionic strength on the probe stability was studied. Similarly to TPS-2cRGD discussed previously, the fluorescence properties of the Ac-DEVDKTPE probe are not sensitive to electrolytes or DMEM, which ensures the low fluorescence background required for real applications. 11.3.3 Detection of caspase and kinetic study of caspase activities in solutions To explore the effectiveness of the Ac-DEVDK-TPE probe for the detection of caspase, in vitro enzymatic assays were carried out with recombinant caspase-3 and caspase-7. The probe and caspases were mixed and incubated in piperazine-N,N0 -bis(2-ethanesulfonic acid) (PIPES) buffer {50 mM PIPES, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% w/v 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, and 25% w/v sucrose, pH ¼ 7.2} at 37  C. PL measurements showed that fluorescence turn-on was recorded for both enzymes. However, upon pretreatment of the enzymes with 5-[(S)-(þ)-2-(methoxymethyl) pyrrolidino]sulfonylisatin (MPS), a potent inhibitor, the fluorescence was substantially suppressed, indicating that a specific enzymatic reaction took place. The inhibitor works selectively toward caspase-3/7 through interaction with the hydrophobic S2 pockets proximal to the catalytic cysteine residue of the enzymes [42]. To study the specificity of caspase-3/7 towards the probe, Ac-DEVDK-TPE (10 mM) was incubated withcaspase-3/7 and a group of other proteins, namely lysozyme, trypsin, BSA, and pepsin. The PL measurements showed that caspase-3 and -7 can induce fluorescence intensity changes 45- and 28-fold higher than other proteins, indicating that Ac-DEVDK-TPE is highly selective for caspase-3/7.

248 Aggregation-Induced Emission: Applications

Figure 11.5 Time-dependent PL spectra of Ac-DEVDK-TPE upon addition of caspase-3 and caspase-7 from 0 to 120 min. [Caspase-3] ¼ [caspase-7] ¼ 100 pM. Reproduced with permission from Shi, H., et al., Detection of Integrin avb3 by Light-Up Bioprobe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc., 2012, 134 (23), 9569–9572. # 2012, American Chemical Society.

Enzyme kinetic studies of caspase-3/7 were then performed by monitoring the fluorescence response of the probe to the enzymes in a time-dependent manner. Figure 11.5 shows the variation of the fluorescence intensity of Ac-DEVDK-TPE incubated with caspases in PIPES buffer at 37  C over a time period of 2 h. In contrast to the consistently low fluorescence observed for the probe alone, the addition of caspases induces considerable fluorescence enhancement within the first 30 min, which is followed by saturation. To determine the kinetic parameters for caspase-3, the fluorescence increase of the probe in the presence of caspase-3 (I  I0) to that in the absence of the enzyme (I0) was plotted against probe concentration (0–100 mM) and fitted with the Michaelis–Menten equation, V0 ¼ Kcat[E]0[S]/Km þ [S], which describes the initial enzymatic rate (V0) in terms of substrate concentration ([S]) [43]. Kcat, the turnover number, refers to the maximum number of substrate molecules that can be converted per unit catalytic site per unit time. Km, the Michaelis constant, is defined as the substrate concentration at which half of the maximum rate is achieved and is a measure of the affinity of substrate to enzyme. The Km and Kcat values were obtained as 5.38  0.03 mM and 17.1  0.2 s1, respectively, which are better than those reported for most commercial substrates [44, 45]. The smaller Km and larger Kcat indicate stronger affinity of the probe and higher substrate conversion efficiency, which are desirable for enzymatic studies. 11.3.4 Imaging of cell apoptosis and screening of apoptosis-inducing agents Live-cell imaging of caspase-3 activation was performed using the MCF-7 cell line. Cytotoxicity of the probes was first assessed using the standard MTT assay. The viability of cells was found to be close to 100% after incubation with Ac-DEVDK-TPE at concentrations of 5, 10 or 25 mM for 48 h, indicating good cyto-compatibility of the probe. Both normal and apoptotic MCF-7 cells were treated with Ac-DEVDKTPE probe for fluorescence imaging. Figure 11.6a shows the CLSM image of normal MCF-7 cells incubated with Ac-DEVDK-TPE and almost no fluorescence is observable. Apoptosis of cells was induced by treatment of the normal cells with staurosporine (STS) before incubation with the probe. STS is a protein

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 249

Figure 11.6 CLSM images: (a–c) normal MCF-7 cells treated with Ac-DEVDK-TPE; (d–f) apoptotic MCF-7 cells treated with Ac-DEVDK-TPE (5 mM, 1% DMSO); (g  i) apoptotic MCF-7 cells treated with Ac-DEVDK-TPE (5 mM, 1% DMSO), inhibitor (10 mM), and caspase-3 antibody. STS (1 mM) was used to induce cell apoptosis. Blue ¼ probe fluorescence; red ¼ immunofluorescence signal generated from anti-caspase-3 primary antibody and a TR-labeled secondary antibody. The images were acquired using a fluorescence microscope (Nikon) equipped with 40 ,6-diamidino-2-phenylindole (DAPI) and TR. The scale bar of 10 mm applies to all images. Reproduced with permission from Shi, H., et al., Detection of Integrin avb3 by Light-Up Bioprobe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc., 2012, 134 (23), 9569–9572. # 2012, American Chemical Society.

kinase inhibitor that may induce cell death via intrinsic apoptotic pathways, although its mechanism remains unclear [46]. Compared with the uninduced cells, the induced cells displayed an intense fluorescence signal, suggesting the activation of caspase during apoptosis. As expected, the fluorescence of the apoptotic cells was greatly suppressed with pretreatment of the inhibitor MPS (Figure 11.6g). The cells were also colocalized with anti-caspase-3 antibody and Texas Red (TR)-labeled secondary antibody (mouse anti-rabbit IgG) to confirm the specificity of the probe (Figure 11.6b, e, and h). As shown in Figure 11.6c, f, and i, there is excellent overlap between the signals from the probe and TR. Evidently, the probe has been efficiently internalized to initiate the caspase-specific enzymatic reaction. Real-time imaging of apoptosis in MCF-7 cells was examined by incubation of the cells with Ac-DEVDKTPE solution for 2 h at 37  C followed by treatment with the apoptosis inducer STS. Figure 11.7 shows CLSM images of the cell undergoing apoptosis, taken using the DAPI channel every 15 min. The dark background at 5 min indicates that the probe is nonfluorescent in the cell medium. As time elapses, the fluorescence of the cells gradually lights up, reaching a maximum at 90 min. This experiment demonstrates that the probe is not only useful for detection of caspase detection, but can also be applied for real-time imaging of cell apoptosis.

250 Aggregation-Induced Emission: Applications

Figure 11.7 Fluorescence images showing the real-time cell apoptotic process of MCF-7 cells with Ac-DEVDK-TPE at room temperature. STS (1 mM) was used to induce cell apoptosis. The images were acquired with a DAPI filter. The scale bar of 20 mm applies to all images. Reproduced with permission from Shi, H., et al., Real-Time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-Up Probe with Aggregation-Induced Emission Characteristics, J. Am. Chem. Soc., 2012, 134 (43), 17972–17981. # 2012, American Chemical Society.

As apoptosis-inducing agents can potentially be used as active anticancer drugs, the capability of the AcDEVDK-TPE probe for in situ screening of compounds that can induce apoptosis was further studied using sodium ascorbate [47], cisplatin, and STS [48] as examples. The cells were first incubated with Ac-DEVDKTPE (10 mM) at 37  C for 2 h, and then treated with 100 ml of DMSO, sodium ascorbate, cisplatin, and STS in DMEM at different concentrations. After incubation for 2 h, the cell fluorescence was monitored with a

Figure 11.8 PL spectra of Ac-DEVDK-TPE preincubated MCF-7 cells upon treatment with different amounts of DMSO, sodium ascorbate (Na asb), cisplatin, and STS. lex ¼ 312 nm; lem ¼ 470 nm.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 251 T-CAN microplate reader. The PL intensity of cells treated with different inducers and DMSO are plotted against drug concentration in Figure 11.8. It can be observed that the fluorescence intensity increases with increase in concentration of all three inducers (sodium ascorbate, cisplatin, and STS). Compared with DMSO-treated cells, sodium ascorbate and cisplatin show better performance with similar efficacy and STS achieves the highest efficacy among the four. Evidently, the results demonstrate that the Ac-DEVDK-TPE probe is highly promising for quantitative analysis of the efficacy of apoptosis-related drugs in living cells.

11.4 In Vivo Monitoring of Cell Apoptosis and Drug Screening with PyTPE-Based Probe Apoptosis induction has been identified as the primary mechanism through which radiation and chemotherapies cause tumor cell death [49]. There is a pressing need to develop noninvasive methods for in vivo monitoring of the apoptosis process in cancer patients undergoing such treatment and also for screening of new drugs. Although the conventional probe based on radiolabeled annexin V has been widely studied both in vitro and in vivo [50, 51], the high background activity and poor specificity have greatly hindered its clinical application. Alternative strategies for in vivo applications have been developed based on fluorescent proteins [52], luciferin [53], quantum dots [54], and fluorescent polymeric nanoparticles [55], but each suffers from particular drawbacks. In addition to substrate-based probes, radiolabeled small-molecule inhibitors have also been reported for targeting of caspase-3 activation, such as 18 F-labeled isatin sulfonamide analogs (18 F-WC-II-89) and M808 [56]. However, these probes may prove problematic for in vivo imaging owing to a lack of in vivo selectivity. An 18 F-labeled g-carboxyglutamic acid analog (18 F-ML-10), a member of the aposense family, proved to be efficient for in vivo apoptosis imaging and was the first apoptosis tracer to be advanced into clinical trials for neurovascular cells [57]. However, the limited understanding of the mechanism makes it difficult to assess the overall applicability to cells of other origins [58]. A competent probe for in vivo apoptosis imaging should satisfy several criteria: noninvasive, selective and specific to apoptotic cells, efficient cell internalization, adequate biodistribution, and little interference from the background [59]. In the previous section, we demonstrated the successful application of the AcDEVDK-TPE probe for the in vitro detection of caspase-3 activity and real-time imaging of cell apoptosis. This probe is not suitable, however, for in vivo applications because of the blue-emitting TPE fluorogen. To overcome this limitation, we further developed an azido-functionalized pyridine tetraphenylethene derivative (N3-PyTPE) bearing a pyridine moiety. This AIE fluorogen exhibited emission at longer wavelength with a large Stokes shift. The subsequent conjugation with a caspase-specific peptide (DEVD) afforded an AIE light-up probe with modulated on–off fluorescence. 11.4.1 Working principles As illustrated in Scheme 11.6, the Ac-DEVD-PyTPE probe is designed with two major components, namely the azido-functionalized PyTPE (N3-PyTPE) with AIE characteristics and the alkyne-bearing peptide DEVD (Ac-DEVD-A) that can be specifically cleaved by caspase-3/7. The water solubility of the probe

Scheme 11.6 Illustration of caspase detection using Ac-DEVD-PyTPE.

252 Aggregation-Induced Emission: Applications

Figure 11.9 UV–vis absorption (left) and PL (right) spectra of N3-PyTPE (dashed line) and DEVD-PyTPE (solid line) in DMSO–water (1:199 v/v). [N3-PyTPE] ¼ [Ac-DEVD-PyTPE] ¼ 10 mM. lex ¼ 405 nm.

Ac-DEVD-PyTPE endowed by DEVD peptide makes the probe weakly fluorescent in aqueous media. However, upon cleavage of the probe at the C-terminus of Asp (D) in response to caspase-3 activation, the probe residue emits strongly in the same medium owing to aggregate formation. The long-wavelength emission, good water solubility, switchable fluorescence, and specificity towards caspase render the probe a promising candidate for in vivo apoptosis imaging. 11.4.2 Synthesis and characterization of DEVD-PyTPE probe The AIE fluorogen N3-PyTPE has an absorption maximum at 405 nm and an emission maximum at 636 nm with bright fluorescence in aqueous media (Figure 11.9). The Ac-DEVD-PyTPE probe was synthesized via the click reaction between N3-PyTPE and Ac-DEVD-A in DMSO–water mixture under the catalysis of CuSO4 and sodium ascorbate, similarly to the procedures mentioned earlier (Scheme 11.7). Although

Scheme 11.7 Click synthesis of Ac-DEVD-PyTPE.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 253

Figure 11.10 PL spectra of Ac-DEVD-PyTPE in the presence of different amounts of caspase-3 (0, 0.2, 1, 5, 10, and 20 mg ml1). [Ac-DEVD-PyTPE] ¼ 10 mM; lex ¼ 405 nm.

similar absorption profiles are observed from N3-PyTPE and Ac-DEVD-PyTPE in DMSO–water (1:199 v/ v), the former emits much more strongly in the same medium as a result of aggregate formation whereas the latter is highly water soluble and remains very weakly fluorescent. 11.4.3 Monitoring of caspase activities in solutions The distinct optical properties of N3-PyTPE and Ac-DEVD-PyTPE prompted us to investigate the fluorescence response of the probe to caspase in buffers. An in vitro enzymatic assay for recombinant caspase-3 was performed using Ac-DEVD-PyTPE probe. Figure 11.10 shows the titration results for Ac-DEVDPyTPE after incubation with caspase-3 at different concentrations. The fluorescence gradually lights up as the concentration of caspase-3 increases and a 25-fold increase is observed at 20 mg ml1. It was found that the co-incubation of Ac-DEVD-PyTPE and caspase-3 with the inhibitor MPS does not lead to pronounced fluorescence enhancement as in the case with no inhibitor. This is an indication of the inhibition of the specific interaction between the probe and caspase-3. The selectivity of the probe over caspase-3 was further tested by monitoring the fluorescence intensity of Ac-DEVD-PyTPE in the presence of several other proteins. The results showed that caspase-3 and -7 induce 43- and 36-fold higher changes, respectively, in (I  I0)/I0 than other four proteins. 11.4.4 In vitro and in vivo imaging of cell apoptosis The in vitro imaging of cell apoptosis was first performed using the MCF-7 cell line. Before application to the cells, the cytotoxicity of Ac-DEVD-PyTPE in different doses was assessed using an MTT assay. the results showed that cells incubated with the probe at 5, 10, and 20 mM all retain high viability (
100%) after incubation for 12, 24, and 48 h. For cell imaging of apoptosis activation, the MCF-7 cells were first incubated with Ac-DEVD-PyTPE in DMEM for 2 h at 37  C and then co-stained with anti-caspase-3

254 Aggregation-Induced Emission: Applications

Figure 11.11 CLSM images of live cell apoptosis: (a–c) normal MCF-7 cells treated with Ac-DEVD-PyTPE (5 mM, 1% DMSO) for 2 h; (d–f) apoptotic MCF-7 cells treated with Ac-DEVD-PyTPE (5 M, 1% DMSO) and caspase-3 antibody. STS (3 mM) was used to induce cell apoptosis. Red ¼ probe fluorescence; green ¼ immunofluorescence signal generated from anti-caspase-3 primary antibody and a FITC-labeled secondary antibody. The scale bar of 20 mm applies to all images.

primary antibody and a TR-labeled secondary antibody. To initiate apoptosis, the cells were treated with the apoptosis-inducing agent STS, and the CLSM images are shown in Figure 11.11d, e, and f . Cells that were incubated only with Ac-DEVD-PyTPE probe were used as a control and their fluorescence images are shown in Figure 11.11d, e and f. It is evident that the normal cells exhibit extremely low fluorescence (Figure 11.11b) whereas the apoptotic cells after STS treatment show a significantly enhanced fluorescence signal (Figure 11.11e). In addition, there is good overlap between the fluorescence collected from Ac-DEVD-PyTPE and that from fluorescein isothiocyanate (FITC)-labeled secondary antibody (Figure 11.11f), indicative of caspase-specific activation of the probe. To explore the potential of the Ac-DEVD-PyTPE probe for in vivo imaging of apoptosis, subcutaneous C6 tumor-bearing mice were selected for apoptosis studies in an animal model. Three mice were used for each study. One of the tumor-bearing mice was preinjected with STS to induce apoptosis and incubated for 12 h. Ac-DEVD-PyTPE was then injected into the tumor-bearing mice with and without STS treatment and also into the normal tissue of a healthy mouse. The mice were imaged at designated time intervals under anesthesia. Figure 11.12 shows the fluorescence images taken for all three mice at 1, 15, and 30 min with a 1 s exposure using a 610/20 nm filter upon excitation at 405 nm. The fluorescence signal from tumor pretreated with STS gradually intensifies with time. Compared with normal tissue, around three-fold fluorescence enhancement was observed for apoptotic tumoral tissues as early as 5 min after probe injection. In contrast, the fluorescence of the tumor without apoptosis induction remained weak throughout the process, similar to that of the normal tissues. The results are consistent with the findings in Figure 11.11, demonstrating the feasibility of using the probe for in vivo apoptosis studies.

Specific Light-Up Bioprobes with Aggregation-Induced Emission Characteristics 255

Figure 11.12 In vivo fluorescence images of subcutaneous C6 tumor-bearing mice after intratumoral injection of Ac-DEVD-PyTPE with or without pretreatment with STS (12 h before the probe injection). lex ¼ 405 nm.

11.5 Conclusion In the light of the intriguing properties displayed by AIE fluorogens, extensive research attention has been focused on the development of novel chemo- and biosensing platforms using AIE systems. We are among the first to exploit the target-specific AIE bioprobes for biodetection and bioimaging. In this chapter, we have presented a few examples using peptide-functionalized AIE fluorogens for advanced specific applications. They have been proved successful for the in vitro detection of integrin avb3, real-time monitoring of cell apoptosis both in vitro and in vivo, and in situ drug screening. The peptides not only serve as a targetrecognized affinity ligand or substrate, but also render the AIE probe highly water soluble in detection media, delivering a low-background signal. The significant fluorescence light-up response upon target recognition offers high sensitivity and excellent image contrast. The strategy presented here can be readily generalized to accomplish a diversity of tasks by simply replacing the peptide with other biorecognition elements, such as antibodies, aptamers, and other ligands. The emission wavelength of the AIE fluorogen can be further tuned to the red or near-infrared range to cater for a wider range of in vivo applications.

Acknowledgments We thank the Singapore National Research Foundation (R-279-000-323-281), the Temasek Defence Systems Institute of Singapore (R279-000-305-592/422/232), the Defense Research Innovative Program (R279-000-340-232), and the Research Grants Council of Hong Kong (HKUST2/CRF/10 and N_HKUST620/11) for financial support.

256 Aggregation-Induced Emission: Applications

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258 Aggregation-Induced Emission: Applications 47. Kang, J.S., Cho, D., Kim, Y.I., Hahm, E., Kim, Y.S., Jin, S.N., Kim, H.N., Kim, D., Hur, D., Par, H., Wang, Y.I., and Lee, W.J. (2005) Sodium ascorbate (vitamin C) induces apoptosis in melanoma cells via the down-regulation of transferrin receptor dependent iron uptake. J. Cell. Physiol., 204(1), 192–197. 48. Herrmann, R., Fayad, W., Schwarz, S., Berndtsson, M., and Linder, S. (2008) Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids. J. Biomol. Screen., 13(1), 1–8. 49. Niu, G., and Chen, X. (2010) Apoptosis imaging: beyond annexin V. J. Nucl. Med. 51(11), 1659–1662. 50. Ntziachristos, V., Schellenberger, E.A., Ripoll, J., Yessayan, D., Graves, E., Bogdanov, A., Josephson, L., and Weissleder, R. (2004) Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V–Cy5.5 conjugate. Proc. Natl. Acad. Sci. U. S. A., 101(33), 12294–12299. 51. Belhocine, T., Steinmetz, N., Hustinx, R., Bartsch, P., Jerusalem, G., Seidel, L., Rigo, P., and Green, A. (2002) Increased uptake of the apoptosis-imaging agent 99m Tc recombinant human annexin V in human tumors after one course of chemotherapy as a predictor of tumor response and patient prognosis. Clin. Cancer Res., 8(9), 2766–2774. 52. Ray, P., De, A., Patel, M., and Gambhir, S.S. (2008) Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects. Clin. Cancer Res., 14(18), 5801–5809. 53. Laxman, B., Hall, D.E., Bhojani, M.S., Hamstra, D.A., Chenevert, T.L., Ross, B.D., and Rehemtulla, A. (2002) Noninvasive real-time imaging of apoptosis. Proc. Natl. Acad. Sci. U. S. A., 99(26), 16551–16555. 54. Li, Z.-G., Yang, K., Cao, Y.-A., Zheng, G., Sun, D.-P., Zhao, C., and Yang, J. (2010) In vivo study of the effects of peptide-conjugated near-infrared fluorescent quantum dots on the tumorigenic and lymphatic metastatic capacities of squamous cell carcinoma cell line Tca8113 and U14. Int. J. Mol. Sci., 11(4), 1413–1422. 55. Lee, S., Choi, K.Y., Chung, H., Ryu, J.H., Lee, A., Koo, H., Youn, I.-C., Park, J.H., Kim, I.-S., Kim, S.Y., Chen, X., Jeong, S.Y., Kwon, I.C., Kim, K., and Choi, K. (2011) Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe. Bioconjug. Chem., 22(2), 125–131. 56. Zhou, D., Chu, W., Rothfuss, J., Zeng, C., Xu, J., Jones, L., Welch, M.J., and Mach, R.H. (2006) Synthesis, radiolabeling, and in vivo evaluation of an 18 F-labeled isatin analog for imaging caspase-3 activation in apoptosis. Bioorg. Med. Chem. Lett., 16(19), 5041–5046. 57. Cohen, A., Shirvan, A., Levin, G., Grimberg, H., Reshef, A., and Ziv, I. (2009) From the Gla domain to a novel small-molecule detector of apoptosis. Cell Res., 19(5), 625–637. 58. Edgington, L.E., Berger, A.B., Blum, G., Albrow, V.E., Paulick, M.G., Lineberry, N., and Bogyo, M. (2009) Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes. Nat. Med. 15(8), 967–973. 59. Reshef, A., Shirvan, A., Akselrod-Ballin, A., Wall, A., and Ziv, I. (2010) Small-molecule biomarkers for clinical PET imaging of apoptosis. J. Nucl. Med. 51(6), 837–840.

12 Applications of Aggregation-Induced Emission Materials in Biotechnology Yuning Hong, Jacky W.Y. Lam and Ben Zhong Tang Department of Chemistry, The Hong Kong University of Science and Technology, China

12.1 Introduction Fluorescence-based techniques offer high sensitivity, low background noise, and a broad dynamic range [1–3]. Many fluorescence probes have been investigated and used in biotechnology, for microscopic studies, and in clinical assays [4–6]. Most of them show favorable spectral properties of visible absorption and emission wavelengths, high extinction coefficients, and reasonable quantum yields in solution [7]. These fluorophores are therefore incorporated into biological and chemical systems as labels via chemical attachment or probes via physical mixing [8, 9]. Owing to the aggregation-caused quenching (ACQ) problem, most of the labels/ probes are initially dissolved and hence emissive (on) but become nonemissive (off) when they are induced to aggregate by changes in the analytes [10, 11]. The aggregation-induced emission (AIE) luminogens, on the other hand, are faintly emissive when molecularly dissolved but highly luminescent when aggregated [12]. Through experimental and theoretical approaches, restriction of intramolecular motion (RIR) has been proposed as the main cause of the AIE effect [13]. To endow them with water solubility, hydrophilic functional groups, such as hydroxyl, amino, ammonium, sulfonate, and boronate, are incorporated into the structures of the AIE dyes [14–17]. The luminogens are nonemissive (off) in aqueous buffers but become emissive (on) when bound to biological molecules. Such turn-on biosensors are advantageous over their turn-off counterparts [18–22]. They are more sensitive and faster and less likely to generate false-positive signals. This is particularly helpful for on-site trials, field screening, and household testing, in which constraints on space, transportation, and so on prevent the use of sophisticated instruments. In this chapter, we introduce the use of water-soluble AIE luminogens for the quantification and visualization of biomolecules, such as DNA and proteins. Since the intramolecular motions can be modulated by

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

260 Aggregation-Induced Emission: Applications changes of the microenvironment, AIE molecules can also be used to monitor conformational changes of macromolecules. Moreover, the AIE attribute allows the use of high chromophore loadings in nanoparticles and high fluorophore-to-protein ratios in nanocrystals, which are useful for cell imaging and immunoassays.

12.2 AIE Materials for Nucleic Acid Studies The quantification and visualization of nucleic acids, such as DNA and RNA, are of great importance in genetic engineering, forensics, and bioinformatics [23, 24]. Traditional nucleic acid stains are mainly intercalators, which are potential mutagens, or groove binders, which show a high preference only to double-stranded DNA [25, 26]. Cationic AIE luminogens can bind to negatively charged nucleic acids via electrostatic attraction, which triggers the emission of the chromophores [15]. Such a ‘light-up’ property permits the quantitation and visualization of nucleic acids in aqueous solution and electrophoretic gel, respectively [27]. The AIE features also allow real-time monitoring of the folding process of a single-stranded DNA (ssDNA) into a G-quadruplex structure in the absence of any preattached fluorophores on the DNA strand [27, 28]. 12.2.1 Quantitation and gel visualization of DNA and RNA Several probes for nucleic acid detection based on fluorescence enhancement have been developed, including ethidium bromide (EB), Hoechst dyes, acridizinium salts, cyanine derivatives, and ruthenium complexes [29–33]. Through intercalating between two adjacent base pairs or binding to the minor grooves of the DNA chains, their fluorescence can be triggered. Among them, EB is a universal nucleic acid stain in molecular biology laboratories, especially for gel electrophoresis, because of its sensitivity and low cost. However, EB is suspected to be a very strong mutagen or carcinogen. There are alternatives to EB in the laboratory, such as SYBR-based dyes [34]. These dyes have been found to be less carcinogenic. However, they are lipophilic and have to be suspended in an organic solvent such as dimethyl sulfoxide (DMSO), which can rapidly pass through skin. Hence it is desirable to develop a ‘safe’ probe for the quantitation and visualization of nucleic acids. To serve this purpose, cationic AIE luminogens, TTAPE-Me and TTAPE-Et, were designed and synthesized (Figure 12.1). A dilute solution of TTAPE-Me in phosphate-buffered saline (PBS) solution (pH 7.0) is virtually nonluminescent, but addition of a small amount of calf-thymus DNA (ctDNA) into the aqueous solution turns on its emission. Increasing the DNA concentration further enhances the fluorescence intensity but causes no change in the emission wavelength. The fluorescence intensity recorded at 470 nm increases

Figure 12.1 Structures of AIE luminogens used for nucleic acid detection. Reproduced with permission from [28], # 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figure 12.2 Fluorimetric titration of (a) ctDNA and (b) RNA in an aqueous solution of TTAPE-Me in PBS solution (pH 7.0). The inset in (b) shows the linear region of the binding isotherm.

rapidly at low ctDNA concentration and gradually becomes saturated as the ctDNA concentration increases (Figure 12.2A). The emission of TTAPE-Me is also turned on in the presence of RNA. With increase in RNA concentration, the fluorescence intensity of TTAPE-Me continues to rise without reaching a maximum, indicating a wide dynamic range for RNA detection (Figure 12.2b). In the RNA concentration range 0–125 mg ml
1, a good linear relationship between fluorescence increase and RNA concentration is established with an R2 value of 0.997. These results demonstrate the utility of TTAPEs for the detection and quantitation of nucleic acids in aqueous media. In buffer solutions, the cationic dye molecules are molecularly dissolved and thus faintly luminescent. Driven by electrostatic attractions, TTAPEs spontaneously bind to the negatively charged DNA and RNA to form TTAPE–nucleic acid complexes. When docked on the surfaces of the biopolymers, the intramolecular motions of the TPE moiety are restricted, which impedes their radiationless transitions and activates their fluorescence processes. TTAPE-Et exhibits a similar fluorescence ‘turn-on’ property when bound to DNA and RNA. The ‘turn-on’ switching implies that TTAPEs can be used as a DNA marker in gel electrophoresis. After running polyacrylamide gel electrophoresis (PAGE) of oligonucleotides, the gel plate is stained with a solution of TTAPEs for 5 min. Upon UV illumination, the stained PAGE plate displays emissive bands of oligonucleotides with different lengths (Figure 12.3). Close inspection reveals that TTAPE-Me gives a better performance than TTAPE-Et in terms of contrast and background noise. This is understandable because the trimethylammonium groups in TTAPE-Me are less bulky than the triethylammonium functionalities in TTAPE-Et. This shields slightly the positively charged nitrogen atoms and hence increases the solubility of the fluorophores in the aqueous solution and also strengthens its interaction with the negatively charged DNA strands. Although EB is a widely used visualization agent in PAGE assays, it takes at least 30 min to start to function [27]. The visualization of bands by EB is realized by its intercalation into the hydrophobic region

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Figure 12.3 Staining of ssDNA in native PAGE by (a) TTAPE-Me and (b) TTAPE-Et. [ssDNA] ¼ 2 mg; [TTAPE] ¼ 10 mM.

of the DNA strands, which makes EB staining a slow process. On the other hand, the emissions of TTAPEs are activated by their spontaneous electrostatic interaction with the charged surfaces of DNA, which is therefore a fast process. The detection limit can be tuned by optimizing the concentration of the luminogens. The visualization system by AIE luminogens thus has advantages of fast response and high sensitivity, in addition to their excellent miscibility with aqueous media. 12.2.2 Specific probing of G-quadruplex DNA formation An ssDNA with a guanine (G)-rich repeat sequence is known to fold into a secondary structure named G-quadruplex [35]. This structure is stabilized by the monovalent cations (e.g. Kþ) located in the centers of the G-quadruplex plates. The quadruplex formation in human telomeric DNA can affect gene expression and inhibit telomerase activity in cancer cells [36]. It is envisaged that quadruplex-targeting drugs may permit artificial regulation of gene expression and control of cancel cell proliferation. The detection of the G-quadruplex structure therefore has medicinal implications. Because of its water miscibility, TTAPE-Et is nonemissive in an aqueous buffer. When its molecules are bound to DNA strand HG21, the intramolecular rotation is restricted and the fluorescence is thus turned on (Figure 12.4, step 1). Addition of Kþ ion induces HG21 to fold into a G-quadruplex structure, resulting in a red shift (from 470 to 492 nm) in the photoluminescence (PL) spectrum (step 2). Hybridization with a complementary DNA (C21) unfolds the quadruplex and gives a duplex (ds) structure. The Kþ ions in the solution compete with the molecules of TTAPE-Et for binding with the dsDNA structure. The overwhelmingly large quantity of Kþ ions helps them win the competition. As a result, the molecules of TTAPE-Et are released back to the solution and the emission is thus diminished (step 4). The spectral red shift diagnostically manifests the presence of the quadruplex structure, allowing a visual distinction of the G-quadruplex from other DNA conformations, especially the double-helix structure [27]. The AIE characteristics of TTAPE-Et permit real-time monitoring of the folding process of HG21 in the absence of any preattached luminogen labels on the DNA strand. These results are useful for biomedical investigations, especially high-throughput quadruplex-targeting anticancer drug screening [28]. Further, TTAPE-Et can be used as a Kþ biosensor because of its high specificity to the Kþ-stabilized G-quadruplex structure over those stabilized by other cationic species, such as Naþ, Liþ, NH4þ, Mg2þ, and Ca2þ.

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Figure 12.4 Fluorescent bioprobing processes of TTAPE. Reproduced with permission from [27], # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

12.3 AIE Materials for Protein Studies Protein analysis is of fundamental importance to proteome research that aims to decipher biological processes at the protein level. Absorption-based protein assays, for example, are inherently limited in sensitivity and effective range (e.g. Bradford and Lowry assays) [37, 38]. Fluorimetric methods have been proven useful for the assay of proteins in solution and helped researchers to probe the concentration, distributions, and even structures of proteins by means of fluorescence microscopy, flow cytometry, and spectrofluorimetry [39]. In this section, TPE derivatives with hydrophilic units as noncovalent probes and bioactive groups as covalent probes for protein quantitation and visualization are introduced [16]. Upon binding to the hydrophobic region of the proteins, these probes can be used as reporters to monitor the structural integrity and conformational changes of the proteins [40, 41]. On the other hand, a novel class of biofunctional silole nanocrystals is used for immunoassays which offer a sensitivity 100-fold higher than that of a state-of-theart immunoassay directly using fluorophore-labeled antibodies [42]. 12.3.1 Quantitation and PAGE staining of proteins The sulfonated TPE derivative BSPOTPE (Figure 12.5) is nonluminescent in buffer solution. Addition of bovine serum albumin (BSA) to the buffer can switch on its emission. As shown in Figure 12.5, the PL intensity at [BSA] ¼ 500 mg l
1 is 240-fold higher than that in the absence of BSA. This high sensitivity allows the detection of BSA at a concentration as low as 0.05 mg l
1 or 50 ppb. The plot of the emission intensity as a

264 Aggregation-Induced Emission: Applications

Figure 12.5 Structures of AIE luminogens used for protein analysis.

function of BSA concentration gives a linear calibration curve at BSA concentrations up to 100 mg l
1, indicating that the AIE luminogen can be used for BSA quantitation over a wide concentration range. Traditional fluorophores for the detection and quantitation of proteins often involve lengthy procedures with carefully timed steps. Some of them show small Stokes shifts and nonlinear curves, and others are even environmentally unstable (e.g. fluorescamine). In contrast, BSPOTPE gives a large Stokes shift (>100 nm) and a linear calibration curve over a wide range of concentration (up to 100 mg l
1). It is environmentally stable, with no changes in its PL spectra observed after its solution has been stored under ambient conditions without protection from light for more than 2 months. Moreover, conventional fluorophores suffer from self-quenching problems at high dye concentrations. The PL of BSPOTPE, however, is intensified with increase in concentration, owing to its unique AIE attribute (Figure 12.6). To improve the selectivity of BSPOTPE to serum albumin, graphene oxide is used as a detection platform [43, 44]. Owing to the TPE moiety, BSPOTPE is able to bind to graphene oxide through p–p interactions, accompanied by the fluorescence quenching effect. In the presence of BSA, the binding affinity of BSPOTPE to BSA is so high that the dye molecules are dragged out from graphene oxide platform. Upon binding to BSA, the intramolecular motions of BSPOTPE are restricted, resulting in strong fluorescence (Figure 12.7). However, if the binding of BSPOTPE and the analytes is not strong enough, the fluorescence of BSPOTPE cannot be turned on. In this way, both the selectivity and sensitivity of AIE molecules are improved.

Figure 12.6 (a) PL spectra of BSPOTPE in PBS (pH 7.0) containing different amounts of BSA. (b) Plot of [BSA] versus I/ I0
1, where I0 ¼ intensity at [BSA] ¼ 0 mg l
1. (c) Linear region of the binding isotherm of BSPOTPE to BSA. Reproduced with permission from [12], # 2009 The Royal Society of Chemistry.

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Figure 12.7 Schematic representation of the use of graphene oxide as a platform for the detection of BSA. Reproduced with permission from [44], # 2011 The Royal Society of Chemistry.

The ‘lighting up’ of BSPOTPE by BSA in PBS buffer implies the possibility of using the AIE luminogens as a protein staining reagent in PAGE assays. Figure 12.8a shows the gel images of electrophoresed human serum albumin (HSA) after staining with BSPOTPE solution for 5 min. The protein bands become visible under UV illumination. The detection limit can be down to 50 ng per lane (lane 4). Coomassie Brilliant Blue (CBB) is one of the most commonly used protein stains in bioassays. A gel stained with CBB is shown in Figure 12.8b for comparison. Only a few protein bands are discernible in the gel even when higher CBB concentrations are applied. Another advantage of using BSPOTPE as a gel stain is its simplicity and promptness, in contrast to conventional methods. Neither careful timing nor a destaining step is necessary with BSPOTPE staining. The protein bands can be visualized after staining for 5 min. Soaking the gel in the luminogen solution for a longer time does not cause overstaining. As the resolution of the protein band is appreciably high, no washing step is required, which greatly shortens the time required and lowers the workload. Post- and prestaining methods are two major approaches for staining proteins in PAGE. The above BSPOTPE is useful for poststaining. For prestaining, proteins are labeled prior to loading on to the gel where electrophoresis takes place. To meet this requirement, amine-reactive TPE-NCS and thiol-reactive TPE-CBr were designed and synthesized. The experimental results demonstrated the suitability of these two dyes for the detection of a low concentrations of proteins in sodium dodecyl sulfate (SDS) PAGE gel.

Figure 12.8 PAGE analyses of HSA using (a) BSPOTPE and (b) Coomassie Blue R-250 as staining reagents. Lanes correspond to the protein bands with amounts of HSA of (1) 1000, (2) 500, (3) 100, (4) 50 and (5) 2.5 ng. [BSPOTPE] ¼ 1 mg per 100 ml; staining time 5 min. Reproduced with permission from [41], # 2010 American Chemical Society.

266 Aggregation-Induced Emission: Applications

Figure 12.9 (a) Schematic illustration of a sandwich-type immunoassay process using polyelectrolyteencapsulated, antibody-functionalized HPS nanocrystals as a fluorescent bioprobe. (b) Plot of PL intensity of the HPS nanocrystals functionalized by goat anti-mouse immunoglobulin G versus the concentration of mouse immunoglobulin G (M IgG). Data for the system using fluorescein isothiocyanate (FITC) as a bioprobe are shown for comparison. Reproduced with permission from [12], # 2009 The Royal Society of Chemistry.

12.3.2 Fluorescence immunoassay by AIE materials In traditional fluorescence immunoassay (FIA) systems, the fluorophore to protein (F : P) ratios are often low because of the problems caused by the notorious self-quenching effect. When several fluorophoric labels are attached to one antibody, the fluorophores are located in close vicinity, which activates energy transfer and decreases the luminescence intensity and efficiency. The AIE effect permits the use of high F:P ratios and the AIE luminogens can thus serve as powerful immunosensors. Biofunctionalized hexaphenylsilole (HPS) nanocrystals were used in our FIA study, and were prepared by the procedures shown in Figure 12.9a. The HPS crystals are ball-milled in a mixture of hydroxypropylcellulose and SDS in water (step 1). After encapsulation with polyelectrolyte multilayers (step 2), specific immunoreagents such as antibodies are attached to the nanocrystals (step 3). The nanocrystal core is comprised of a huge number of HPS molecules, and the encapsulated crystal surface is decorated with biomolecules. This configuration confers an extremely high F : P ratio on the AIE immunosensor. The nanocrystalline HPS biomarkers are used for sandwich-type immunoassays. The analyte is first immobilized by the capture antibody preadsorbed on a solid substrate and then exposed to the antibodylabeled bioprobe (step 4). The PL intensity increases with increase in analyte concentration (Figure 12.9b). The sensitivity of the HPS bioprobes is 140-fold higher than that of fluorescein isothiocyanate (FITC)–antibody conjugate. The signals of the nanocrystalline bioprobes are dramatically amplified, thanks to the extremely high F : P ratios in the FIA system. This manifests the great value of the AIE effect to the development of ultrasensitive FIA systems. 12.3.3 Monitoring of the unfolding/refolding process of human serum albumin For a protein to perform its biological functions, it must assume a specific chain conformation [39]. The development of luminescent tools for studying protein conformations is therefore of obvious importance.

Applications of Aggregation-Induced Emission Materials in Biotechnology 267

Figure 12.10 Dependence of FRET ratio (I470 / I350) between HSA and BSPOTPE on the concentration of GndHCl and illustration of fluorescence tracking of the GndHCl-induced HSA unfolding process. Reproduced with permission from [13], # 2011 The Royal Society of Chemistry.

The investigation of the global stability of protein chains in the presence of denaturants has attracted much interest, as it offers mechanistic insights into protein (un)folding processes. Although intermediate states are often involved in the (un)folding processes of many proteins, they are difficult to detect owing to the lack of appropriate probes. We developed an emission ‘turn-on’ sensor for HSA using a sulfonated TPE (BSPOTPE) as probe [40]. The emission of BSPOTPE in an aqueous buffer was switched on in the presence of HSA. The HSA-triggered emission allowed BSPOTPE to be used as a visualization agent in PAGE assays (Figure 12.8). HSA is a major protein component of blood plasma. Its hydrophobic regions are capable of binding with insoluble endo- and exogenous compounds such as fatty acids and drugs. The hydrophobicity of the phenyl rings of BSPOTPE prompted the luminogenic molecules to enter into and aggregate inside the hydrophobic cavities or pockets of the folding structures of HSA chains (Figure 12.10). Utilizing the AIE nature of BSPOTPE and the F€ orster resonance energy transfer (FRET) from the intrinsic fluorescence of HSA at 350 nm to the AIE emission of BSPOTPE at 470 nm, the unfolding trajectory of HSA chains induced by the denaturant guanidine hydrochloride (GndHCl) was traced, which revealed a three-step transition with a stable molten-globule intermediate involved in the unfolding process. 12.3.4 Monitoring and inhibition of amyloid fibrillation of insulin Amyloids are insoluble fibrous aggregates of proteins, an excess accumulation of which in organs and tissues can lead to biological dysfunction and pathological symptoms [45]. An abnormal deposit of amyloids in the brain, for example, causes neurodegenerative problems (e.g. Alzheimer’s, Parkinson’s and Huntington’s diseases). The development of new molecules that can monitor amyloidosis kinetics and inhibit fibril formation is of great diagnostic and therapeutic value. In our recent work, a fluorescent probing system for tracing protein aggregation process was developed [41]. Insulin is a biopolymer that readily undergoes fibrosis under acidic conditions and was therefore chosen as a model protein. An aqueous mixture of native insulin and BSPOTPE is virtually nonfluorescent. In the presence of fibrillar insulin, however, BSPOTPE becomes emissive, as its interaction with the insulin aggregates activates the process of restriction of intramolecular motion (Figure 12.11a). The distinct contrast in

268 Aggregation-Induced Emission: Applications

Figure 12.11 (a) Emission spectra of BSPOTPE in the presence of native and fibrillar forms of bovine insulin. (b) Process of insulin fibrosis monitored by the change in the luminescence of BSPOTPE. Reproduced with permission from [13], # 2011 The Royal Society of Chemistry.

the emission efficiency permits clear discrimination between the native and denatured forms of insulin and enables its fibrosis process to be tracked. The trajectory of the variation in the fluorescence intensity implies that the fibrosis process proceeds through three steps: induction (I), exponential (II), and plateau stages (III) (Figure 12.11b). The amyloid fibrils stained by BSPOTPE can be readily observed under a fluorescence microscope, thanks to the bright light emitted from the bound aggregates of BSPOTPE. Owing to the excellent water miscibility of the unbound free molecules of BSPOTPE, virtually no background emission is observed in the fluorescence image. In addition, premixing of BSPOTPE with insulin inhibits the nucleation process and impedes protofibril formation. Increasing the dose of BSPOTPE boosts its inhibitory potency. Theoretical modeling using molecular dynamics simulation and docking revealed that BSPOTPE is prone to binding to partially unfolded insulin through the hydrophobic interaction of the phenyl rings of BSPOTPE with the exposed hydrophobic residues of insulin (Figure 12.12). Such binding is assumed to have stabilized the partially unfolded insulin and obstructed the formation of the critical oligomeric species in the protein fibrillogenesis process.

Figure 12.12 Schematic representation of the dual functions of BSPOTPE as ex situ monitor and in situ inhibitor in the process of insulin amyloidogenesis. Reproduced with permission from [41], # 2012 American Chemical Society.

Applications of Aggregation-Induced Emission Materials in Biotechnology 269

12.4 AIE Materials for Live Cell Imaging Fluorescent probes are routinely used for the visualization and tracking of various cellular events [5]. Typical materials used for this purpose include natural polymers, inorganic nanoparticles, and organic dyes. Green fluorescent protein (GFP), for example, has been used as a reporter of expression for morphological differentiation [46]. The visualization process, however, is realized through complicated and time-consuming transfection procedures. Inorganic nanoparticles, such as quantum dots (QDs), are highly luminescent and resistant to photobleaching but limited in variety and inherently toxic to living cells because they are commonly made of heavy metals and chalcogens (e.g. CdSe and PbS) [47, 48]. Organic dyes are rich in variety and easy to use. Most of these organic dyes contain large fused polynuclear cores which are prone to aggregate when dispersed in aqueous media. To avoid the notorious ACQ problem, very dilute solutions of dye molecules are used. The use of dilute solutions causes other problems in the imaging process. The small numbers of dye molecules in dilute solutions can be quickly photobleached when a harsh laser beam is used as the excitation light source. The photostability cannot be improved by using high fluorophore concentrations because of the ACQ effect. AIE materials, on the other hand, can solve this problem. Nanoaggregates of silole derivatives have been successfully applied for long-term cell tracking [49]. AIE dye-hybridized silica nanoparticles with both efficient fluorescence and strong magnetization have been fabricated and used for live cell imaging [50, 51]. 12.4.1 AIE bioprobes for long-term cell tracking Ionic luminophores have often been used for cell imaging applications because of their good water miscibility. However, at high concentrations, their charges can perturb the membrane potential and cellular physiology. On the other hand, at low concentrations, the small numbers of the luminophores that have entered into the cellular interiors can be easily photobleached in the imaging process. During the cell division process and when the confluent cells are passaged, the intracellular dyes often diffuse back to the extracellular media owing to the existence of a concentration gradient. This leads to a decrease in the emission of the stained cells and a concurrent increase in the solution (or background) emission, together with random staining in a cell co-culture system. Aminated silole derivatives silole-1 and
2 are nonionic luminogens that spontaneously aggregate into nanoparticles in aqueous media (Figure 12.13) [49]. The nanoparticles are cytocompatible and can be internalized via an endocytosis process. Owing to their AIE nature and electrical neutrality, the luminogens can be used in high concentrations in cell imaging processes. The nanoaggregates indelibly stain HeLa cells because it is difficult for the internalized particles to escape from the cellular compartments. The strong retention of the nanoparticles in the HeLa cells prevents cross-staining between different cell lines and allows fluorescence cell differentiation (Figure 12.13b). The internalized particles are held inside the HeLa cells so robustly that they remain visible for a long period (up to four passages), permitting visual monitoring of the growth of a specific type of cell line (Figure 12.13c). 12.4.2 AIE nanoparticles for cell staining As mentioned above, fluorophoric molecules suffer from the photobleaching problem when they are exposed to harsh laser light in the imaging systems. Silica nanoparticles are cytocompatible and optically transparent but fluorescently inactive. These attributes make them ideal host materials for the synthesis of fluorescent silica nanoparticles (FSNPs) for cell imaging applications. The silica matrix can serve as a protective shield, reducing the likelihood of penetration of oxygenic and other reactive species that may accelerate the photobleaching processes of fluorophores.

270 Aggregation-Induced Emission: Applications

Figure 12.13 (a) Scanning electron microscope image of nanoparticles of silole-1 formed in a tetrahydrofuran– water mixture with 70% water content. (b) Phase contrast and fluorescence images of silole-2-stained HeLa cells co-cultured with unstained HEK 293T cells. (c) Fluorescence images of living HeLa cells stained with 5 mM of silole-2 at the first and fourth passages. Reproduced with permission from [13], # 2011 The Royal Society of Chemistry.

FSNPs have been prepared by using fluorophores such as FITC. However, high loadings of FITC elicit the ACQ problem, and low loadings afford only weak emission. AIE luminogens become stronger emitters with increasing concentration and are therefore well suitable for FSNP fabrication. Using a triethoxysilylated TPE derivative (TPE-TEOS) as starting material, emissive FSNPs with a core–shell structure were prepared by a one-pot, surfactant-free, sol–gel process, as shown in Figure 12.14a [51]. The FSNPs are uniformly sized, surface charged and colloidally stable. Their sizes are tunable in the range 45–295 nm by varying the reaction conditions. The FSNPs emit strong blue light, owing to the AIE effect of their TPE components (Figure 12.14b). The FSNPs pose no toxicity to living cells and can be used for the selective staining of the cytoplasms of HeLa cells.

Figure 12.14 (a) Fabrication, (b) light emission, and (c) cell staining of core–shell FSNPs consisting of fluorogenic cores and silica shells. Reproduced with permission from [13], # 2011 The Royal Society of Chemistry.

Applications of Aggregation-Induced Emission Materials in Biotechnology 271

Figure 12.15 (a) Plot of magnetization (M) versus applied magnetic field (H) for silole-containing magnetic fluorescent nanoparticles at 300 K. (b) Transmission electron microscope images of the nanoparticles and (c) fluorescence images of the HeLa cells stained by the nanoparticles. Reproduced with permission from [13], # 2011 The Royal Society of Chemistry.

The development of multifunctional nanomaterials is of great interest, as such materials are expected to serve as versatile building blocks for the construction of advanced nanodevices [50]. Mixtures of magnetic and fluorescent components formed via sol–gel procedures are envisaged to generate nanomaterials with dual functionalities, such as magnetic and fluorescent silica nanoparticles (MFSNPs), which may find biotechnological applications in fluorescent cell imaging and PAGE visualization and also magnetic cell separation and tumor therapy. The fabrication of MFSNPs with strong fluorescence and high magnetizability has been challenging, however, owing to the incompatibility between magnetic and fluorescent units: light emission is often quenched when inorganic magnets are hybridized with organic fluorophores. In AIE systems, the luminescence is not from isolated molecules but from assembled aggregates, which should be more robust than individual molecules of the traditional luminophores and thus more resistant to magnet quenching. Indeed, nanoparticles consisting of magnetite cores and silolylsilica shells are highly emissive. The MFSNPs are uniform in diameter (50 nm), have a magnetizability of 6 emu g
1, and can be readily modulated by a magnet bar (Figure 12.15a). They are readily internalized by HeLa cells, enabling cytoplasmic staining (Figure 12.15c). Structural modifications of their surfaces by target-specific ligands such as folates and antibodies will yield nanoparticles that may be used for in vivo imaging of specific cells by magnetic resonance imaging and for intracellular hyperthermia treatment based on the magnetocaloric effect.

12.5 Conclusion AIE luminogens are a group of molecules whose emissions are induced by aggregate formation. RIR is proposed as the main cause of the AIE effect. Based on the mechanistic understanding, the AIE luminogens have been successfully utilized for the quantitation of nucleic acids and proteins, gel visualization, real-time monitoring of conformational transitions, and cell imaging. Compared with conventional luminescent

272 Aggregation-Induced Emission: Applications materials, these materials are more attractive for high-tech applications because of their facile synthesis, ready functionalization, good photostability, high fluorescence quantum yields, and negligible cytotoxicity. As a final remark, more and more outstanding work on the use of AIE materials for biotechnological applications has been emerging during the preparation of this book chapter. Owing to length and time limitations, we could not incorporate all of them in this chapter. We are enthusiastically anticipating new advances in this exciting area of research.

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Index

Acetonitrile-water mixture 10 ACQ 1, 44, 62, 102, 131, 165, 239 AEE 122 Aggregate formation 44 Aggregated particles 115 Aggregated state 88, 108, 132 Aggregation-caused quenching 1, 44, 62, 102, 131, 239 Aggregation-enhanced emission 122 Aggregation-induced circular dichroism 108 Aggregation-induced circularly polarized luminescence 113 Aggregation-induced emission 1, 44, 62, 87, 107, 131, 157, 165, 190, 209, 239, 259 AIE 1, 44, 62, 88, 131, 165, 190, 209, 239, 259 AIE-active liquid crystalline polymers 54 AIE-active materials 107 AIE dye-encapsulated nanomicelles 210, 227 AIE-nanoparticles 221 AIE nanoprobes 216 Aluminum ion 136 Amphiphilic molecules 176 Amyloid fibrillation 267 Aromatic ketone 44 Aryl-substituted pyrrole 131 Benzophenone 44 Biogenic amines 157 Bioimaging 209 Biomolecules 259 Biosensors 185, 190 Biotechnology 259 Carbohydrate-mediated biological interactions Cation and anion sensors 166

189

Cell staining 269 Chemosensors 62, 136, 168, 185 Chiral acids 91 Chiral AIE receptors 95 Chiral amines 88 Chiral fluorescence receptors 88 Chiral recognition 87 Circularly polarized luminescence, 107 Click synthesis 242 Concentration-quenching effect 1, 44 CPL 107 Crystalline state 44 Crystallization-induced emission 44 Crystallization-induced emission enhancements 76 Crystallization-induced phosphorescence Current efficiency 5 Cyano-substituted stilbenoid derivatives 14

43

DCM-hexane mixture 110 Detection of explosives 183 Dissymmetry factors 107 Distyrylbenzene derivatives 14 Duschinsky rotation 113 Dynamic quenching 125 EL efficiency 5 Electroluminescence 1, 64 Electron mobility 7 Electron-transporting layer 2 Emission dynamics 113 Emissive core-shell silica nanoparticles 178 Enantiomeric excess determination 87

Aggregation-Induced Emission: Applications, First Edition. Edited by Anjun Qin and Ben Zhong Tang. # 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

276 Index Enantioselectivity 96 Energy/charge-transfer interactions

122

F€ orster resonance energy transfer 171 Fluorescence-based assays 189 Fluorescence immunoassay 266 Fluorescence turn-on detection 166 Fluorescent aggregates 173 Fluorescent nanoprobes 210 Fluorimetric assays 173 Fluorimetric biosensors 169 Fluorimetric sensing 158 Fluorimetric titration 261 G-quadruplex 169, 262 Helical assembly 108 Heteroaggregate complexation 202 Hierarchical helical structures 108 Hole-transporting layer 2 Hydrogen bonds 45, 150 Hydrophobic interactions 171 In vitro imaging 253 In vitro sensing 244 In vivo monitoring 251 Intermolecular interactions 48, 117 Intersystem crossing 44 Intramolecular charge-transfer 136, 165 Intramolecular rotations 115 Label-free fluorimetric enzymatic assays 176 Lifetime 113 Light-emitting layer 2 Light-up bioprobes 239 Linear discriminant analysis 160 Live cell imaging 269 Long-term cell tracking 269 Luminescent liquid crystals 52 Luminescent materials 131 Luminescent probes 195 LUMO 3, 61, 151, 191 Maleimide fluorophore 10 Mechanism 101 Mechanofluorochromic materials 61 Mesoporous material-based sensor 127 Miscellaneous sensors 183 Molecular organogels 98 Molecular packing 68

Molecular packing structures 65 Multiphoton-induced fluorescence

223

Nanoparticles 216 Nondopant device 10 Nonradiative decay 151 OLEDs 1 Organic chiral p-conjugated molecules Organic lasing 107, 118 Organic light-emitting devices 62 Organic light-emitting diodes 1

108

PAGE analyses 265 Particle size distribution 138 Phase transition 65 Phosphole 192 Phosphole oxide 190 Phosphorescence 43 Photoinduced electron transfer 165 Photoluminescence 1, 64, 110, 132, 190, 211 Piezofluorochromic AIE compounds 66 Polycyclic aromatic hydrocarbons 27 Polymer nanoaggregates 124 Power efficiency 5 Propeller-like molecular structure 13 Protein amyloid fibrillation 171 Protein sensing 239 Pseudo-color fluorescence images 215 Purely organic phosphors, 44 Pyrroles 13 Quantum efficiency 108 Quantum yield 1, 45, 132, 210 Ratiometric fluorescence detection 173 Real-time monitoring 245 Reorganization energy 113 Restriction of intramolecular rotation 44, 101, 132, 239, 259 RIR 44, 101, 132, 239, 259 Room temperature phosphorescence 44 Selectivity 243 Silole derivatives 2, 108, 131, 165, 173, 191 Single crystals 150 Singlet excited state 44 Solid-state fluorescence 7 Solid-state PL quantum yield 5 Spin-orbit coupling 47 Static quenching 125

Index 277 Statistical analysis 160 Stimuli-responsive materials 64 Structural modeling 115 Superamplified detection of explosives 107 Supramolecular assembly 115 Target-specific AIE probes 240 Tetraphenylethene 17, 53, 108, 157, 165, 190 THF-water mixture 122 3D Topological structure 122 Three-photon-induced fluorescence 226 Time-resolved photoluminescence 45 Titration 138 TPE 21, 53 Triarylamine derivatives 17 Triphenylethene derivatives 17 Triphenylpyrrole derivatives 132

Triplet excited state 44 Tumor targeting 216 Turn on fluorescence 158 Twisted conformation 150 Two-color fluorescence switching behavior 71 Two-photon blood vessel imaging 227 Two-photon brain imaging 230 Two-photon-induced fluorescence spectrum 224 Unfolding/refolding process Vapochromic effect

266

77

Water fraction 122, 149 Water-soluble AIE luminogens White OLEDs 31

259