Progress and Trends in AIE-Based Bioprobes: A Brief Overview

Review Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

www.acsami.org

Progress and Trends in AIE-Based Bioprobes: A Brief Overview Ju Mei,† Youhong Huang,† and He Tian*,† †

Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science & Technology, No. 130 Meilong Road, Shanghai 200237, China

ABSTRACT: Luminescent bioprobes are powerful analytical means for biosensing and optical imaging. Luminogens featured with aggregation-induced emission (AIE) attributes have emerged as ideal building blocks for high-performance bioprobes. Bioprobes constructed with AIE luminogens have been identified to be a novel class of FL light-up probing tools. In contrast to conventional bioprobes based on the luminophores with aggregation-caused quenching (ACQ) effect, the AIE-based bioprobes enjoy diverse superiorities, such as lower background, higher signal-to-noise ratio and sensitivity, better accuracy, and more outstanding resistance to photobleaching. AIE-based bioprobes have been tailored for a vast variety of purposes ranging from biospecies sensing to bioimaging to theranostics (i.e., image-guided therapies). In this review, recent five years’ advances in AIEbased bioprobes are briefly overviewed in a perspective distinct from other reviews, focusing on the most appealing trends and progresses in this flourishing research field. There are altogether 11 trends outlined, which have been classified into four aspects: the probe composition and form (bioconjugtes, nanoprobes), the output signal of probe (far-red/near-infrared luminescence, two/three-photon excited fluorescence, phosphorescence), the modality and functionality of probing system (dual-modality, dual/multifunctionality), the probing object and application outlet (specific organelles, cancer cells, bacteria, real samples). Typical examples of each trend are presented and specifically demonstrated. Some important prospects and challenges are pointed out as well in the hope of intriguing more interests from researchers working in diverse areas into this exciting research field. KEYWORDS: aggregation-induced emission (AIE), bioconjugates, nanoprobes, far-red/near-infrared luminescence, two/three-photon excited fluorescence, phosphorescence, dual-modality, dual/multifunctionality conversion NPs, 8−10 metallic NPs, 11,12 and fluorescent proteins,13 have been developed and utilized as bioprobes. Among them, the organic dyes and organic NPs are more advantageous in view of their better biocompatibility and easier preparation processes. However, it was believed that most organic luminophores usually suffer from the ACQ effect that once the luminophoric molecules aggregate, the luminescence would be partially or completely quenched due to π−π stacking.14,15 Such an effect greatly limits their application as bioprobes. With multiple aromatic rings, organic dyes are hydrophobic and immiscible with water, making them intrinsically form aggregates in the

1. INTRODUCTION Biological and life areas are appealing, boundless, and full of opportunities. There are countless biogenic species need to be analyzed and innumerable biological/physiological processes need to be unveiled, monitored, or even regulated. Luminescence technique has been made a perfect choice for biological applications by the merits such as superb sensitivity, simplicity, rapidity, visibility, real-time and on-site responsiveness, economic applicability, high temporal-spatial resolution, noninvasiveness, diversity of luminophores, and so forth. In light of this, luminescence could be applied as a useful tool for the facile array of bioanalytes and direct visualization of biological structures and processes. Nowadays, luminescent probes have been a favorite in the field of contemporary bioscience. A vast variety of luminescent materials, including organic dyes,1,2 organic nanoparticles (NPs),3,4 inorganic NPs such as quantum dots,5−7 rare-earth metal ion-doped up© XXXX American Chemical Society

Special Issue: AIE Materials Received: September 21, 2017 Accepted: November 15, 2017 Published: November 15, 2017 A

DOI: 10.1021/acsami.7b14343 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Review

ACS Applied Materials & Interfaces

terms of their working mechanisms or the analyte type, the review presented here will focus on the latest and most representative AIE-based bioprobing systems and organize them in the following concise and ingenious fashion. Taking a panoramic view of the flourishing situation, we will comb the main trends and progress of this vigorous research area and clarify them from the following points of view: the probe composition and form (bioconjugates, nanoprobes), the output signal of probe (red/far-red/NIR luminescence, two/threephoton excited fluorescence, phosphorescence), the modality and functionality of probing systems (dual-modality, dual/ multifunctionality), and the probing object and application outlet (specific organelles, cancer cells, bacteria, real samples). We will first introduce and discuss these trends and progresses sequentially with highlights on some representative works. At the end of this review article, we will briefly prospect the future development within this field.

bioenvironments that are aqueous or hydrophilic and consequently leading to severe self-quenching. Thus, ACQ dyes always cannot be used at high concentrations, which for one thing gives rise to the compromised sensitivity, and for another results in unsatisfactory photostability because the small quantity of molecules could be easily photobleached by the high-energy excitation light. Moreover, the ACQ effect also imposes difficulty on the fabrication of organic luminophoredoped NPs, as the NPs’ brightness cannot be improved by simply increasing the loading ratio or aggregation degree of dyes in each NP.16,17 AIE, a concept coined by Tang et al. in 2001,18 originated from the phenomena that a class of luminogens are weakly or nonemissive as molecular species, while highly luminescent as aggregates/clusters. Evidently, AIE is a diametric opposite of the ACQ effect. The AIE mechanism has been rationalized to be the restriction of intramolecular motions (RIM).19−22 When the AIE-active luminogen (AIEgen) 21,22 is molecularly dispersed, the active intramolecular motions including rotations and vibrations dissipate the excited-state energy, boosting the nonradiative decay. Upon aggregation, the RIM process is activated, blocking the radiationless consumption of the excitation energy and populating the radiative decay channels. The twisted three-dimensional (3D) conformations of AIEgens effectively impede π−π stacking, which also contributes to the brightened aggregated-state luminescence. In this regard, lightup/turn-on bioprobes could be easily constructed by taking advantage of AIE, or in other words, AIEgens are born FL lightup bioprobes. Under appropriate conditions, AIE-based bioprobe luminesces dimly with a low background noise. Once interacted with biospecies or influenced by the surrounding bioenvironment, the AIEgen’s intramolecular motions are restricted, the lighted-up luminescence can thereby detect the bioanalytes and reveal certain biological status in a sensitive manner.23−26 Furthermore, the high brightness of nanoaggregates or NPs formed by AIEgens can visualize the organelles, cells, bacteria, or tissues, etc., with high contrast and superb spatial resolution and without the need for washing.27−33 In addition, these AIE NPs are fairly photostable under laser excitation, enabling reliable long-term tracking of dynamic biological processes.16,17,32−34 In the past few years, AIE-based bioprobes are springing up at an incredible speed and shining in the field of life science and biomedical engineering. A myriad of thrilling AIE-based probes have been designed for the detection of biogenic molecules ranging from amino acids35,36 to carbohydrates37 to DNA/ RNAs38 and to proteins/enzymes,39,40 the monitoring of biological processes such as protein fibrillation,41,42 cell apoptosis,40,43 osteogenic differentiation,34 autophagy/mitophagy,44,45 and the imaging of specific organelles,46 cells,47,48 microorganisms,49−51 tissues,52,53 and even animals.54−56 Multifunctional systems have also been constructed with AIEgens for bioimaging, diagnosis, and therapy.57−61 The annual publications and citations on AIE-based bioprobes in recent five years have undergone steady and sustained growths as indicated by the data provided by Web of Science. There have been more than 560 papers published since 2012, when searching with “aggregation-induced emission” as the keywords and refining the results with “BIOCHEMISTRY MOLECULAR BIOLOGY” on July 18th, 2017. As a result, it is high time to sort out the papers and outline the main advances and trends in the area of AIE-based bioprobes. Different from the previous reviews, which summarized the AIE bioprobes in

2. TRENDS AND PROGRESS 2.1. Trends and Progress in Probe Composition and Form. 2.1.1. AIE Bioconjugates. Selectivity and sensitivity are the utmost important parameters as for bioprobes used for optical sensing and imaging. The identification of specific bioanalytes and the study of biological processes rely on the sensitivity and selectivity of the probes. The selectivity is closely associated with the recognition ability, and the sensitivity is dependent on the signal-to-noise ratio (SNR). In the pursuit of high-performance AIE-based bioprobes, there have come out a new generation of bioprobes, namely AIE bioconjugates. Herein, AIE bioconjugates referrs to the conjugates that are generated by connecting biomolecule(s) and AIEgen(s) together through stable covalent link(s) via chemical strategies. The incorporation of biomolecule(s) into AIE systems is undoubtedly a powerful combination, which not only opens up new avenues to the efficient bioprobes but also adds a string of extra advantages to the probing systems. In comparison to the AIEgens without biocomponents, AIE bioconjugates are more favorable for biological applications. For example, to enlarge the SNR and improve the sensitivity, one of the most effective ways is to reduce the background noise. Hence, the AIEgens usually need to be made cationic or anionic in order to ensure the water-solubility or hydrophilicity. Although these AIE compounds could be engineered into light-up probes for a variety of analytes, the probe−analyte interactions are often based on electrostatic or hydrophobic forces which are neither directional nor specific. Given this, high selectivity and specificity of these AIEgens is hard to be counted on. In contrast, the conjugation of AIEgens with biological elements that hold specific affinity to the species of interest could significantly improve the selectivity of the AIE-based bioprobes. It is noteworthy that the biomolecule(s) used for bioconjugates are always highly hydrophilic, which could confer satisfactory water-solubility onto the AIE bioconjugates and consequently guarantee the low fluorescence (FL) background and high sensitivity in bioassays. Moreover, the biological origins of these ligands inherit their biocompatibility to the resultant AIE bioconjugates. Intrigued by the above benefits, considerable attention has been drawn by the AIE bioconjugates. Accordingly, a large quantity of fantastic AIE bioconjugates that could serve diverse probing purposes have been constructed.62 In this regard, a new trend in AIE-based probes has come into being. In this section, the discussion is organized in terms of the type of biological B


Review


Chart 1. Molecular Structures of AIEgen-Peptide Bioconjugates involving cRGD, KDVED, DVED, IETD, and D5 Peptide Sequencea

a

The scissors indicate the cleaving sites when the bioconjugates interact with analytes.

elements conjugated to AIEgens, which include peptides, nucleic acids, and carbohydrates. 2.1.1.1. AIEgen-Peptide Bioconjugates. Peptides, as the building blocks of proteins, have become more and more popular for the design of fluorometric probes, because of their proper size, facile preparation, and convenient modification.

Moreover, peptides have a relatively long shelf life and high activity per mass thanks to their low molecular weight. The peptide library has been enriched by the phage display technology, enabling scientists to find peptides that interact with biotargets with high affinity and specificity. With proper sequences, peptides can not only function as recognition C


Review

ACS Applied Materials & Interfaces Chart 2. Molecular Structures of Other Representative AIEgen-Peptide Bioconjugates

D


Review

ACS Applied Materials & Interfaces ligands but also as versatile moieties for the fine-tuning of probe’s water-solubility. The representative AIEgen-peptide bioconjugates are listed in Charts 1 and 2. The first successful example of the AIEgen-peptide conjugate was reported by Liu and Tang et al. in 2012, for the specific detection of integrin αvβ3.39 Integrin is an important protein biomarker of many kinds of tumors and also a receptor for the cyclic-arginine-glycine-aspartic acids (cRGD) peptides derived from extracellular matrix protein. As shown in Chart 1, the probe TPS-2cRGD (1) was generated by the copper(I)catalyzed azide−alkyne cycloaddition (CuAAC) between an AIE-active tetraphenylsilole (TPS) unit and two integrintargeting cRGD peptides. Thanks to the hydrophilic cRGD sequences, the TPS-2cRGD showed negligible background and FL light-up response to integrin αvβ3, with excellent specificity and high sensitivity. The detection limit was as low as 0.5 μg mL−1. Besides quantitative detection of integrin in aqueous solution, this AIE-active silole-cRGD bioconjugate is also capable of imaging the binding process between cRGD and integrin on cell membrane, tracing integrin αvβ3, and identifying integrin-positive cancer cells. Also in 2012, Liu and Tang et al. cooperatively utilized the CuAAC method to create another AIEgen-peptide bioconjugate, namely TPE-KDVED-Ac (2 in Chart 1), for the specific light-up detection of caspase and monitoring cell apoptosis in real time.40 TPE-KDVED-Ac is composed of a hydrophobic AIE-active tetraphenylethene (TPE) group and a hydrophilic caspase-specific Lys-Asp-Glu-Val-Asp (KDVED) peptide. The probe in aqueous solution faintly fluoresced but showed dramatic enhancement in FL when responded to caspase-3/-7, which were activated by cell apoptosis and could cut off the DVED-Ac sequence from TPE-KDVED-Ac. Such an FL “switch-on” response is attributed to the aggregation of the cleaved lysine-conjugated TPE (TPE-K) residues, which restricts the intramolecular motions of TPE moieties and promotes the radiative decay. The light-up feature of TPEKDVED-Ac enables real-time monitoring of caspase-3/-7 activities both in solutions and in living cells with a high SNR, offering a new approach to screen and evaluate the apoptosis-associated drugs in situ. With a very similar structure to bioconjugate 2, TPE-DVED (3) obtained by integrating TPE with DVED peptide via an amidation reaction was exploited for the rapid screening of gefitinib-sensitive non-small cell lung carcinoma (NSCLC).63 Because TPE-DVED could respond to caspase-3 and confirm the apoptosis of NSCLC cells in an FL “turn-on” mode, it could indicate that gefitinib exhibits higher efficacy against NSCLC HCC827 cells in comparison to A549 and H1650 cells. It has been recognized that enzyme− substrate interactions rely heavily on the conformation, rendering the reaction kinetics sensitive to subtle variation in binding affinities. Dual DVED-labeled AIE bioconjugates, i.e., TPE-2(DVED-Ac) (4), were therefore designed and synthesized by Liu and Tang et al. through CuAAC to investigate the stereoisomeric effect on the probing performance.43 It has been found that both the E- and Z-isomers of TPE-2(DVED-Ac) were able to work as light-up probes for caspase-3 assay. However, the Z-isomer had a more remarkable light-up response with a higher SNR, whereas the E-isomer displayed a faster response rate, because the cleaved residue of the Zisomer is more hydrophobic and the E-isomer binds better with caspase-3 and favors faster cleavage of DVED. The aforesaid AIEgen-peptide bioconjugates 2−4 are based on the TPE module whose emission maximum situates at

around 470 nm, making them unsuitable for in vivo caspase detection and apoptosis study. In this respect, orange-emissive AIEgen tetraphenylethene pyridinium (TPEPy) was conjugated with the DVED peptide via CuAAC, affording TPEPy-DVEDAc (5).64 This bioconjugate specifically responded to caspase3/7 with a high SNR. With large Stokes shift, long-wavelength absorption (λabs = 405 nm) and emission (λabs = 636 nm), good water-solubility, and superior biocompatibility, this probe can be applied for in vivo real-time monitoring of caspase activation and cell apoptosis as well as in situ screening of apoptosisinducing drugs. Tethering a target-specific peptide to the above apoptosis probes can transform them into versatile tools for apoptosis imaging in specific cells. Monitoring and imaging the apoptosis of targeted cancer cells in real time has great significance in providing insights into cancer diagnosis and offering evaluation of the anticancer drugs. In view of this, the cancer-targeting cRGD peptide and caspase-specific DVED sequence were facilely coattached onto the TPS core by means of CuAAC, generating an asymmetric and FL light-up bioprobe cRGDTPS-DVED-Ac (6).65 As anticipated, it displayed specific targeting ability to U87MG glioblastoma cells with the help of the efficient binding between cRGD and integrin αvβ3, and was capable of real-time monitoring and imaging the cancer cell apoptosis with high specificity and sensitivity. With elaborate molecular design, AIEgen-peptide bioconjugates could even fulfill more complicated tasks. For example, a self-validated probe for accurate caspase detection with dual signal turn-on effect was developed by Liu et al. via a simple and smart Förster resonance energy transfer (FRET) strategy.66 The AIE bioconjugate TPETP-DVED-Cou (7 in Chart 1) is made of a red-fluorescent AIEgen tetraphenylethenethiophene (TPETP) as the energy acceptor, a green-emissive coumarin unit as the energy donor, and a caspase-3-specific peptide linker. This bioconjugate itself is nonemissive due to the energy transfer as well as the nonradiative energy consumption of the FRET acceptor TPETP through the free intramolecular motions, whereas intense green and red FL signals are synchronously displayed upon the interaction with caspase-3, as a result of the separation of the energy donor and acceptor as well as the aggregation of released TPETP residues. Probe 7 is quite different from the widely studied traditional FRET probes which only exhibit a single FL light-up when interacting with the analytes. Such an FL switch-on response and dual signal amplification permitted the self-indicated detection of caspase-3 with high SNR, and offered an opportunity to accurately monitor the cell apoptosis process in real time. Soon after the work on bioconjugate 7, Liu’s team further reported another dual signal turn-on probe, namely TPETPDVED-IETD-TPS (8).67 Unlike 7, AIE bioconjugate 8 is comprised of two AIEgens, one is the red-emissive TPETP and the other is green-fluorescent TPS, which are linked together by a hydrophilic peptide DVED-Ile-Glu-Thr-Asp (IETD). IETD and DVED are the substrates of apoptosis initiator caspase-8 and effector caspase-3, respectively. The bioconjugate 8 is hardly fluorescent in aqueous media. In early apoptotic HeLa cells induced by H2O2, when the peptide DVED-IETD was cleaved by the cascade activation of caspase-8 and caspase-3, the green and red FL could be sequentially switched on. Such a sequential FL turn-on feature enabled real-time monitoring of the cascade caspase activation during the apoptosis course and the assessment of the cancer therapy efficacy. This innovative E


Review


peptide bioconjugates, e.g., TPS-DVED-Pt-cRGD (12)72 and cRGD-TPE-Pt-DOX (13).73 Bioconjugate 12 could be exclusively up-taken by αvβ3-overexpressed tumor cells, and then the prodrug could be reduced into active Pt(II) drug and simultaneously release the TPS-DVED segment. The cell apoptosis induced by the Pt(II) drug activated caspase-3 to cleave the DVED peptide and the resultant TPS residues tended to aggregate and the FL was lit up because of the RIM process. With such a built-in apoptosis sensor, 12 could be employed as an indicator for early evaluation of the therapeutic efficacy of a specific anticancer drug.72 Bioconjugate 13 is a traceable delivery system containing two anticancer drugs, i.e., cisplatin prodrug and doxorubicin (DOX).73 DOX in this prodrug system performed multiple roles: the energy acceptor of FRET, antitumor drug, and one of the FL trackers. The FRET between TPE and DOX rendered the probe redemissive. With the aid of cRGD, 13 could target cancer cells, and the prodrug could be traced by the red FL of DOX. After the cellular internalization and reduction, the active Pt(II) drug and DOX are simultaneously released. The TPE and DOX are consequently separated, eliminating the FRET effect and leading to the FL recovery of TPE, which can monitor the activation of drugs in real time. Such a prodrug-peptide AIE bioconjugate has the capacity of tracking the location and distribution of prodrug, monitoring dual-drug activation, visualizing the cancer cell ablation, as well as enhancing the anticancer efficacy. Recently, the prodrug-peptide AIE bioconjugates are capturing interest from other groups. For example, a protease-responsive prodrug conjugated with an AIEgen was constructed by Xia and Lou et al. for controlled delivery and release tracking of DOX in living cells.74 The prodrug is constituted by AIEgen TPEPy, a cell penetrating peptide (CPP), a short protease-responsive peptide LGLAG that is selectively cleavable by the cancer-associated enzyme matrix metalloproteinase-2 (MMP-2), and DOX. The peptide motif in this probe functioned an important role in regulating the FL of TPEPy and DOX by MMP-2 cleavage as well as facilitating the drug delivery process. The probe system was highly watermiscible with faint FL at 595 nm in aqueous solution, whereas upon cleavage by MMP-2, two distinct FL signals at 565 and 595 nm were shown. In addition to the AIEgen-peptide bioconjugates for cisplatin prodrug and doxorubicin, a multifunctional prodrug for the delivery and tracking of a novel antitumor drug gemcitabine was developed by Ji and Jin et al.75 The prodrug was afforded via the conjugation of a TPE unit, a cathepsin B-cleavable Gly-Phe-Leu-Gly (GFLG) peptide, a hydrophilic D5 peptide, a cancer-targeting RGD moiety, and a reduction-cleavable disulfide (−S−S−) bond with gemcitabine. Such a bioconjugate was successfully utilized for intracellular FL light-up imaging and GSH-responsive release of gemcitabine for the suppression of pancreatic cancer cells. As can be learnt from the above-discussed apoptosis probes, functionalizing the AIEgen with hydrophilic peptides which could be selectively cut off by a particular protein (usually an enzyme) to release the hydrophobic AIE residues into the aqueous media could serve as an effective strategy for the construction of enzyme-specific FL light-up AIE bioprobes. This design rationale works on the basis of the solubility change of the probing system before and after the interaction with enzyme. The bioconjugates 14−18 shown in Chart 2 verified the universality and feasibility of such a design principle. AIEgen-peptide bioconjugate TPE-SDKP (14) was intended

work opened up a new avenue for the direct monitoring and multiplexed imaging of multiple cellular enzyme activities. With two fluorogens in one bionconjugate could not only generate dual turn-on signal but also can readily afford dualtargeted probe for real-time and in situ monitoring of specific cellular events and processes. Bioconjugate cRGD-TPS-DVEDSS-TPETP (9) shown in Chart 1 is such a typical example.68 In this probe, TPETP is both a red emitter and a photosensitizer (PS) that can generate reactive oxygen species (ROS) under light irradiation and hence can be used for photodynamic therapy (PDT), while TPS is a green-emissive archetypal AIEgen. When excited with a 405 nm laser, distinct emission peaks could be shown. The TPS core was asymmetrically functionalized with a cancer cell-specific cRGD peptide and a DVED spaced −S−S−TPETP unit. Upon cRGD-mediated cellular uptake, the −S−S− linker between DVED and TPETP was cleaved by the glutathione (GSH), lighting up the red FL of TPETP meanwhile releasing the apoptosis probe cRGDTPS-DVED. Upon light irradiation, the caspases activated by the PDT-induced cell apoptosis cut off the DVED peptide from the probe and released the hydrophobic TPS fragments with strong green FL, reporting the therapeutic effect of TPETP on cancer. In this way, a smart multifunctional probe capable of targeted imaging, activatable and trackable cancer cell PDT, as well as real-time self-reporting of PS activation and therapeutic responses under a single excitation wavelength was established. Besides the peptides with special affinity to some biospecies, some peptides without recognition ability have also been used for a certain purpose. Take Asp-Asp-Asp-Asp-Asp (D5) for example. D5 is a highly hydrophilic peptide comprised of five aspartic acids and hence is often used to improve the watersolubility of AIE-based bioprobes.69−71 For instance, in the bioconjugate TPE-SS-D5-cRGD (10), D5 acts as a hydrophilic unit to minimize the FL background and enhance the SNR.69 As mentioned above, the −S−S− bond is GSH-cleavable and the cRGD is integrin αvβ3-specific, TPE-SS-D5-cRGD therefore possesses the capability of monitoring free thiols in both aqueous solution and targeted cancer cells in a FL light-up fashion with a high SNR. Given the excellent hydrophilicity of D5 and integrin affinity of cRGD, the AIE-active prodrugpeptide bioconjugate TPEPy-Pt-D5-cRGD (11) was synthesized by Liu and Tang et al. for in situ monitoring of the platinum(IV) prodrug activation in targeted cancer cells.70 TPEPy-Pt-D5-cRGD consists of four components: the AIEgen TPEPy, the platinum(IV) prodrug, the hydrophilic D5 peptide, and a targeting ligand cRGD. Bioconjugate 11 was practically nonfluorescent in aqueous media with the intense orange FL turned on by the reduction of the prodrug inside the αvβ3overexpressed cancer cells. This design established a versatile protocol for efficient cisplatin drug delivery and real-time monitoring of the drug activation in a high contrast mode. It is worth mentioning that when the TPEPy in TPEPy-Pt-D5cRGD was replaced by the PS TPETP, an AIE peptide-prodrug bioconjugate for combinational PDT-chemotherapy would be readily obtained.71 Such a probe was applied for the monitoring of cisplatin activation and image-guided combinational PDT and chemotherapy for the ablation of Pt(II)-resistant cancer cells. It means that altering the AIEgen could change the functionality of the AIEgen-peptide bioconjugates. Considering it is highly important for personalized medicine to delivery drugs into targeted cancer cells with minimized side effects and to monitor the drug efficacy in situ and in real time, Liu and Tang et al. further developed several other prodrugF


Review


a more hydrophobic dimer which tended to form nanoaggregates at the site of activated furin (e.g., Golgi bodies). The FL response signal was thus further enhanced. By virtue of this dual AIE strategy, furin activity in cancer cells could be detected or imaged in a more sensitive way. Human chymases are important proteases extensively distributed in mast cell granules, the increased level of chymases and other serine proteases is closely connected to inflammatory and immune regulatory functions. For the assay of chymase, two Cys-Phe-Thr-Glu-Arg-Asp-Asp-Asp (CFTERD3) peptide sequences hydrolyzable by chymase were integrated with the red-luminescent AIEgen TPETP to yield the AIEgen-peptide bioconjugate 18.80 By itself, 18 is practically nonemissive in aqueous media with a low background. When coexisting with chymase, the CFTERD3 moieties were hydrolyzed, leading to the decrease in water-solubility. Significant FL light-up response with a high SNR was hence achieved. The probe held an excellent selectivity to chymase over other proteins and can effectively discriminate chymase from other enzymes in the same family. Furthermore, with a detection limit as low as 0.1 ng mL−1 in PBS buffer and a linear response range of 0−9.0 ng mL−1, 18 could function as a simple light-up probe for real-time chymase assay, with direct readout and superb sensitivity and selectivity. Aside from the peptides selectively reacting with analytes, those specifically interact but do not react with the species of interest have also been introduced into AIE systems to construct highly performed AIE-based probes.42,81−91 Representatives (19−26) are depicted in Chart 2. As threadlike protein aggregates with crossed β-sheet secondary structures, amyloid fibrils are related to a wide range of neurodegenerative and other diseases such as the well-known Alzheimers and Parkinsons. The efficient assay and monitoring of amyloid protein fibrillation can significantly promote early diagnosis and therapy. Bioconjugate 19, made of a TPE group and a RGKLVFFGR peptide sequence, was devised as an FL turn-on probe for the amyloid fibrillation detection and monitoring.42 The peptide ensured the selective binding with the amyloid structure, and the AIE-active TPE moiety guaranteed the lightup FL response upon binding with amyloid fibrils. In comparison to the conventionally applied thioflavin T, this AIE bioconjugate offered a higher SNR, exempted from being affected by the quencher ions or nanoparticles. Apart from detecting proteins in solutions, the assay of proteins in living cells can also been realized by means of AIEgen-peptide bioconjugates. By grafting a four-arginine (Arg)-constituted peptide and a lipophilic tail to the TPE module, bionconjugate TR4 (20) was successfully obtained.81 The short tetra-peptide sequence was employed as a positively charged hydrophilic moiety for cell membrane targeting, and the TPE and long alkyl chain functioned as lipophilic motifs for the membrane insertion. As a consequence, luminescence of TPE was turned on upon the embedment in the membrane. The as-designed bioconjugate was able to bind with the lipid bilayers of liposomes and hence lit up the cell membrane with high contrast. Meanwhile, the low toxicity, excellent specificity, and remarkable photostability made 20 promising for long-term and real-time cell membrane tracking. Moreover, inspired by viral structures, Liang et al. further developed bioconjugate 20 into a small-molecule gene vector.82 Nanofibers with low cytotoxicity, excellent stability and high transfection efficiency were formed via the self-assembly of 20-based vector with plasmid DNA in solution. The nanofibers can transfect various

for the assay of angiotensin converting enzyme (ACE) and the screening of ACE inhibitor.76 ACE is a zinc-dependent peptidase that could hydrolyze angiotensin I into the vasoconstrictor angiotensin II and acts crucial roles in many physiological processes such as regulating blood pressure, hematopoiesis, immune response, renal development, etc. The Ser-Asp-Lys-Pro (SDKP) peptide conjugated to the TPE moiety imparts the hydrophilicity to the bioconjugate, making it nonemissive in aqueous media. When the SDKP sequences were cleaved by the N-terminal active site of ACE, the TPE-SD residues were released and aggregated due to the reduced water-solubility. The AIE feature of TPE gave a light-up FL response to ACE, rendering the bioconjugate able to monitor the ACE activity in both solutions and living cells and to screen ACE inhibitors in vitro. Dipeptidyl peptidase-4 (DPP-4), a type II transmembrane glycoprotein abundant in circulates and tissues, could cleave N-terminal amino acids from peptides that play important parts in regulating glucose-induced insulin secretion. Inhibiting the function of DPP-4 is hence a key issue in the therapy of type II diabetes mellitus. To this end, TPE was conjugated with a hydrophilic Lys-Phe-Pro-Glu (KFPE) peptide which is cleavable by DPP-4, producing AIE peptidebioconjugate TPE-KFPE (15) which hardly fluoresced in aqueous solution.77 Light-up FL signal was induced when 15 was incubated with DPP-4. Benefiting from its good cell membrane penetration capability and AIE characteristics, 15 was capable of imaging DPP-4 activity and screening DPP-4 inhibitors in living cells and organisms. TPE-GK(Ac)YDD (16)78 was specially designed for the modulator screening of Sirtuin type 1 (SIRT1), an important member of evolutionarily conserved intracellular protein deacetylases called Sirtuins (SIRTs). As SIRT1 could catalyze the nicotinamide adenine dinucleotide (NAD)+-dependent deacetylation reaction, it is considered as a novel target for the treatment of metabolic disorders and aging-associated diseases. The peptide sequence Gly-Lys(Ac)-Tyr-Asp-Asp (GK(Ac)YDD) endows the resultant bioconjugate with high hydrophilicity and specificity to SIRT1. Bioconjugate 16 was almost nonluminescent in aqueous media. When the acetyl group of lysine was deacetylated by SIRT1, the GKYDD peptide was cleaved by the lysyl endopeptidase, and the released TPE-G residues aggregated to switch on the FL. In this manner, the activation and inhibition of SIRT1 in its deacetylation ability could be evaluated both in solution and living cells.78 Furin, a kind of trans-Golgi protein convertase intimately related to many diseases such as Ebola fever, Alzheimer’s, and cancers, was reported to overexpress in quite a few cancers. AIEgen-peptide bioconjugate 17 constituted by a TPE unit and the 2-cyanobenzothiazole (CBT)-functionalized Lys-Cys(StBu)-Arg-Arg-Val-Arg(Ac) peptide sequence was explored to sense furin activity.79 The peptide consists of three parts: an Arg-Arg-Val-Arg (RVRR) sequence that facilitates the cellular uptake and serves as the substrate for furin cleavage, a CBT motif, and a disulfide cysteine (Cys) moiety for CBT-Cys condensation. Upon internalization into the furin-overexpressed cancer cells, the disulfide bond on the Cys was reduced by intracellular GSH and the RVRR peptide was cleaved by furin, generating the reactive intermediate. The reactive intermediate is more hydrophobic than bionconjugate 17, resulting in an FL turn-on signal. Immediately, the free 1,2aminothiol and cyano groups on the CBT motif of the intermediate underwent a biocompatible condensation to afford G


Review


acid, is a representative among the diverse carbohydrates which is overexpressed in ovarian, breast, pancreatic and hepatic cancers. Rapid and reliable detection of salic acid is extremely important to the cancer diagnosis and therapy. Intrigued by the light-up nature of AIE-based probes, Chang and Xu et al. tagged the TPE unit with a GKGKGKK peptide sequence together with a phenylboronic acid group to yield the bioconjugate TPBA (24) for the targeted recognition of cancer-related sialic acid and specific imaging of cancer cells.88 TPBA exhibited fairly low FL when dissolved in aqueous solution but highly fluoresced in the aggregated state or when anchored onto cells, resulting from the binding between the peptidyl boronic acid group and the sialic acids on the cell surface. With peptide and boronic acid that can selectively recognize sialic acid, the bioconjugate 24 displayed superb specificity toward targeted biomarker and hence can image tumor cells. Apart from monosaccharides (e.g., sialic acid), polysaccharides like heparin (Hep) could also been assayed by AIEgen-peptide bioconjugates.89 Hep, a highly sulfated glycosaminoglycan and most negatively charged biomacromolecule, has been extensively applied in clinics as prophylactic and therapeutic agents especially an anticoagulant in surgery. Although a myriad of probes have been designed for Hep detection, highly selective and sensitive turn-on FL assay of Hep remains challenging. Bioconjugate 25 was designed for this purpose. 8 9 A Hep-specific peptide, namely AG73 (RKRLQVQLSIRT), was introduced to the TPE system to enhance the selectivity. 25 has the capacity of detecting Hep in a wide pH range from 3 to 10 without being interfered by other tested anions and biomolecules. The detection limit was estimated to be as low as 3.8 ng mL−1, far below the clinical level of Hep. In particular, this AIE bioconjugate enabled the development of a heparinase-assisted approach for turn-on sensing of oversulfated chondroitin sulfate with a detection limit of 0.001 wt %, which is a major contaminant in Hep and may cause serious adverse effects including death. The bacteria identification, imaging, and killing are receiving more and more attention. Bacteria-related study becomes a new research branch in the area of AIE-based bioprobes, which will be discussed later in details in Section 2.4.3. AIEgen-peptide bioconjugates have taken an important part in the bacteriaassociated research. For instance, with specific binding affinity to the peptidoglycan sequence (N-acyl-D-Ala-D-Ala) existing on the walls of Gram-positive bacterial cells, the glycopeptide antibiotic vancomycin (Van) was conjugated to TPEPM, a redfluorescent AIEgen and a PS generating ROS.90 The obtained bioconjugate TPEPM-2Van (26) was able to selectively recognize bacteria and kill Gram-positive bacteria through image-guided photodynamic inactivation. Most recently, the antimicrobial peptide (AMP) CysHHC10 (Cys-Lys-Arg-TrpTrp-Lys-Trp-Ile-Arg-Trp) which provides immediate and effective defense against infections was attached to the TPE unit via a simple and fast thiol−ene conjugation, producing the AIEgen-peptide bioconjugate TPE-AMP.91 As CysHHC10 possesses remarkable antimicrobial properties against both Gram-negative and Gram-positive bacteria, the resulting TPEAMP inherited the antimicrobial feature with the minimum inhibitory concentration against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus and S. epidermidis of 15.8, 31.8, 15.8, and 7.9 μM, respectively. Moreover, the AIE nature of TPE offers a light-up FL technique to investigate the bacterial membrane interactions and antibacterial activities of AMPs.

cell lines including stem cells. The intense FL from TPE induced by the self-assembly process rendered the nanofibers visible under confocal laser scanning microscopy (CLSM) and hence enabled the tracing of gene delivery process. With this amphiphilic AIEgen-peptide bioconjugate, transferrin-dressed virus-like ternary AIE NPs were also constructed and applied for targeted delivery and rapid cytosolic release of siRNA.83 Detecting and monitoring cancer-related biomolecular interactions in living cells in real time are of critical importance to disease diagnostics and drug screening. Mdm2 protein is the vital negative regulator of the p53 tumor suppressor protein. For the intracellular detection of Mdm2, a target-specific FL light-up probe was created through conjugating a specially designed AIEgen TPECM to an Mdm2-specific p53-derived peptide (MPRFMDYWEGLS, 12.1Pep).84 The as-developed probe TPECM-12.1Pep (21) is cell permeable and could image Mdm2 in real time in live cells with switch-on FL. Bioconjugate 21 hardly luminesced in the isolated state but became highly fluorescent upon binding to Mdm2, allowing quantification of Mdm2 and the demonstration of the specific interaction between the bioconjugate and Mdm2 protein. Another redemissive AIEgen TPEPM was fabricated into a light-up bioprobe by capping two AP2H (IHGHHIISVG) peptide sequences on it.85 AP2H can exclusively bind the hydrophilic extracellular loop of lysosomal protein transmembrane 4 beta (LAPTM4B), a tumor-associated protein overexpressed by the majority of solid tumors. Benefiting from the AIE nature of TPEPM as well as the cancer-specificity of AP2H, TPEPM2(AP2H) (22) could respond to the target protein and acidic microenvironment of cancer cells in a light-up manner with high SNR. TPEPM is a PS that generates ROS upon visible light irradiation. The as-prepared bioconjugate was thus used for targeted PDT. By identifying the expression level of LAPTM4B, this dual-functional bioprobe can reveal the progression status of tumors. Moreover, by tethering both ends of TPECM with the GFLG-D3-cRGD peptide sequences, a dual-targeted enzyme-activatable bioprobe and PS was established.86 Since cRGD is highly affinitive to cancer cells overexpressing integrin αvβ3 and the GFLG is cleavable by cathepsin B, a lysosomal protease overexpressed in various tumors, simultaneous turn-on FL imaging and activated PDT for specific cancer cells has been achieved. Precisely and targetedly transporting a long-term tracker into a nucleus with low toxicity is one of the most challenging issues in revealing cancer cell behaviors. Xia and Lou et al. designed and synthesized a dual-targeted bioconjugate featured with AIE characteristics.87 The developed bioconjugate RGD-NLSTPEPy-CPP-cNGR (23) is constituted by two targeting peptides RGD and cNGR (CNGRC), a nuclear localization signaler NLS (RRRRK), a CPP (RRRR), and an AIEgen TPEpy, which is an FL imaging agent. By virtue of cNGR and RGD, bioconjugate 23 can bind to aminopeptidase N (CD13) and integrin αvβ3 with switch-on yellow FL response. Because of CPP, 23 can be effectively internalized into the cytoplasm and afterward be delivered into the nucleus with the assistance of NLS. The integrin αvβ3-overexpressed cells and the nucleus of CD13 were lit up by the strong yellow FL of 23. Moreover, 23 can be employed for long-term tracking in live tumor cells in more than ten passages without influencing normal cells and with very low toxicity. Besides cancer-associated proteins, specific carbohydrates overexpressed on cell surface are also closely related to cancers’ developing and progression. Sialic acid, or N-acetyl-neuraminic H


Review


two-armed TPE-DNA bioconjugate was achieved.92 In comparison to 27, the two-armed AIEgen-DNA bioconjugate (TPE-2DNA, 28, Chart 3) displayed a lower background in the absence of analyte and a higher SNR of 6.1 to the perfect target single-stranded DNA. 28 could differentiate target DNA sequence from that with even one-base mutation. Abnormal expression of microRNAs has been recognized as a hallmark of many critical disease states. Detecting microRNAs is thus vital to the early diagnosis of diseases including cancers. On the basis of the simple TPE-DNA bioconjugate 29 (TPEPhos-DNA, Chart 3), Xia and Lou et al. developed an ultrasensitive one-pot assay approach for microRNA, without introducing any quencher.93 It outperformed the commonly used microRNA detection methods that require complicated and troublesome steps to accurately control the relative distance between the fluorogens and quenching groups. The as-prepared TPE-DNA bioconjugate is water-soluble and merely weakly emissive in aqueous solution. MiR-21, one of the first identified mammalian microRNAs, was chosen as a model analyte. Hybridization of the miR-21 and the TPE-DNA bioconjugate caused conformational change from random coil single-stranded DNA to duplex DNAs with a blunt 3′ terminus. Consequently, the stepwise removal of mononucleotides from 3′ terminus was catalyzed by exonuclease III and the TPE residue was released. The liberated miR-21 then hybridized with the second TPE-DNA molecule, initiating a new cycle. In this way, a single copy of the miR-21 produced many TPE residues, which aggregated together to activate the RIM process and arouse the FL. Because of the temperature-dependence of the exonuclease III’s activity, the detection limits of this probing system can be fine-tuned. To be more specific, at 37 °C, 1 pM miR-21 can be discriminated in 40 min, whereas at 4 °C, 10 aM (approximately 300 molecules in 50 μL) miR-21 can be recognized in 7 days. The superb specificity allowed the real 21 urine samples from the bladder cancer patients to be successfully analyzed by this assay method. As special single-stranded DNA or RNA oligonucleotides, aptamers are able to specifically and tightly bind to various targets including metal ions, small molecules, proteins, entire viruses or cells. As compared to other recognition ligands such as antibodies, aptamers are more stable, cheaper, and more easily chemically synthesized and labeled; as such, aptamerbased bioprobes become increasingly popular. Lately, the first AIEgen-aptamer bioconjugate, i.e., 2TPE-aptamer (30, Chart 3), was created by capping the both ends of the Ramos cellspecific aptamer with TPE units for the specific detection of cancer cells.94 Only faint FL of the TPE-aptamer bioconjugate 30 was observed in the aqueous buffer solution. When coexisting with Ramos cells, specific binding between the Ramos cells and the aptamer moiety induced the RIM process and turned on the FL, making the cancer cells be detected in a light-up fashion. Such an assay enjoyed several important advantages. First, this “mix-and-detect” approach was simple and fast. Second, this turn-on probe could greatly reduce the possibility of false-positive signals associated with turn-off probes. Third, this assay had no requirement for signal amplification but sensitive cancer cells detection was still achieved. Fourth, such an assay could be carried out in complicated sample mixtures and be applicable for the specific cancer cell imaging. Therefore, it can be envisaged that the design principle of this assay would provide guidance for the future development of AIE bioprobes on the basis of AIEgens and aptamers.

2.1.1.2. AIEgen-Nucleic Acid Bioconjugates. As negatively charged biomacromolecules, nucleic acids including oligonucleotides (DNAs) and ribonucleic acid (RNAs) carry vital genetic information on all living organisms and viruses. The probes constructed with cationic AIEgens exhibited light-up response to DNA or RNA as a result of the complexationinduced aggregation but the responses were hardly specific. Since selectively probing the DNA or RNA sequence is of paramount importance to genetic mapping, disease diagnosis, oncology studies and so on, specific DNA/RNA probes based on AIE were created by making use of the hybridization between complementary single-stranded DNAs or the recognition ability of aptamers (Chart 3). These AIEgen-nucleic acid Chart 3. Molecular Structures of Representative AIEgenNucleic Acid Bioconjugates

bioconjugates are easy-to-prepare, low-costed, highly tolerant to environment with high ionic strength, and widely applicable to a broad range of DNA/RNA sequences. Liu and Tang et al. reported the first light-up and homogeneous DNA-specific probe based on an AIEgen-DNA bioconjugate in 2013.38 TPE-DNA (27, Chart 3) owns two constituents: one is the AIE motif (i.e., TPE) and the other is a 20-mer single-stranded DNA sequence. The TPE unit was employed to show modulated switch-on FL upon analyte binding. The DNA sequence played dual roles: the specific hybridization to its complementary strand, and to bestow the probe with high specificity and water-solubility. Once hybridizing with the complementary DNA strand, the FL of the probing system was boosted by around 3.6 times, because of the RIM process of TPE induced by the increased rigidity and molecular weight. The detection limit of the probe was calculated to be 0.3 μM. As revealed by further investigations, the FL intensity of the perfectly hybridized duplex was much higher than that of the probe complexed with one or two mismatched strands. By taking advantage of this selective probe, single nucleotide polymorphism could be detected. Because the SNR achieved by the above monosubstituted TPEDNA bioconjugate is only 3.6, to further improve the SNR and realize more sensitive and specific DNA detection, two oligonucleotides were tagged to one TPE moiety and the I


Review

ACS Applied Materials & Interfaces Chart 4. Molecular Structures of Representative AIEgen-Carbohydrate Bioconjugates

2.1.1.3. AIEgen-Carbohydrate Bioconjugates. Carbohydrates are referred to as a class of biomolecules that are comprised of carbon, hydrogen and oxygen, and are also synonyms of saccharides, which include sugars (monosaccharides and disaccharides), starch, and cellulose (polysaccharides). Carbohydrates play numerous important parts in living organisms. Because of their biological nature and some special biofunctions, various AIEgen-carbohydrate bioconjugates varying both in AIE core and carbohydrate moieties have been constructed and applied for the specific detection of certain biospecies and/or for bioimaging.95−103 There are mainly two pathways to activate the AIE process of the AIEgencarbohydrate bioconjugates: one is utilizing the carbohydrate−protein interaction to draw the bioconjugate and analyte molecules together, and the other is employing the glycosidase (enzyme) to induce hydrolysis of the bioconjugates, which leads to the reduction in water-solubility. The famous carbohydrate−protein interactions that modulate diverse biological events including cell recognition and differentiation, cancer metastasis, inflammation, etc., were made full use of in the development of AIE-based bioprobes.95−100 The specificity of carbohydrate−protein interactions facilitates the AIE-based bioassays and guarantees the selectivity, and in turn, the carbohydrate−protein interaction could be evaluated by the AIE effect.95 Sanji and Tanaka et al. pioneered in this area by designing and synthesizing two phosphole oxide−sugar bioconjugates for the FL turn-on detection of lectins such as concanavalin A (Con A) and peanut agglutinin in 2009.96 In 2010, by means of TPE-mannose bioconjugates, Sanji and Tanaka et al. further achieved the highly sensitive turn-on detection of lectins with a detection limit of 20 nM Con A.97 On the basis of the alliance of carbohydrate−protein interactions and AIE effect, disease-related protein species such as cholera toxin98 and influenza virus99 have also been recognized in a light-up, highly sensitive, and selective manner. As sugar-recognizing proteins, lectins are widely distributed in natural plants, mammalian cells, and tissues. Specific sugarlectin interactions are one type of the aforementioned carbohydrate−protein interactions and mediate a variety of

physiological and pathological events. Sensitively and selectively probing sugar-lectin interactions can provide insights into the advancement of life science as well as medical engineering. To this end, the AIEgen-carbohydrate bioconjugates 31a and 31b (Chart 4) were particularly designed and synthesized by CuAAC reactions.100 Both the mannosyl-substituted and galactosyl-conjugated diketopyrrolopyrrole (DPP) derivatives merely showed inconspicuous FL in the DMSO/Tris-HCl buffer (1/99, v/v). In the presence of Con A or lentil lectin, dramatic and concentration-dependent increase of the FL of bioconjugates 31a in the near-infrared (NIR) region was observed. Similar response was given by the coexistence of 31b and peanut agglutinin. The FL signal was rationalized to have originated from the AIE effect of the DPP core activated by the complexation of the divalent glycodyes with the polymeric lectins. These two probes displayed FL intensity−concentration plots with satisfactory linearity in a low concentration region (0.25−2.0 μM). The detection limits for Con A, lentil lectin, and peanut agglutinin were determined to be 12, 6, and 11 nM, respectively. It is worth mentioning that benefiting from its high sensitivity and NIR light-up FL response, bioconjugate was able to accurately quantify Con A in a serum sample. Recently, β-galactosidase, an enzyme often used as a reporter examining transcription and transfection efficiencies, and a crucial biomarker for primary ovarian cancers and cell senescence, has aroused considerable research interest. A series of AIE-based bioprobes were designed based on the second turn-on strategy that takes advantage of the solubility difference between the AIEgen-carbohydrate bioconjugate and the βgalactosidase-cleaved AIEgen residue.101,102 In the light of the great importance of β-galactosidase, the TPE-Gal bioconjugate 32 (Chart 4) was designed and synthesized to detect this enzyme.101 Bearing a positively charged pyridinium pendant and a D-galactose moiety, 32 is water-soluble with very weak emission in aqueous media. As D-galactose is the substrate of βgalactosidase, the bioconjugate 32 showed specific FL response to β-galactosidase. Upon interaction with β-galactosidase, the βgalactopyranoside group was cleaved off from 32, generating a phenolate intermediate that spontaneously underwent a 1,6J


Review

ACS Applied Materials & Interfaces elimination of p-quinone-methide to yield a neutral pyridinesubstituted TPE derivative. With poor water-solubility, the resultant molecules tended to aggregate and the FL was turned on because of the activated RIM process. As a consequence, βgalactosidase was assayed by the intensified and blue-shifted FL. As revealed by the plot of FL intensity at 512 nm versus the βgalactosidase concentration, the FL response was well linearly correlated with the β-galactosidase concentration in the range of 0.8−4.8 U mL−1 with a detection limit as low as 0.33 U mL−1. Besides detecting β-galactosidase with light-up response and high selectivity in aqueous solution, bioconjugate 32 could also readily image the endogenous β-galactosidase activity in living cells. Aside from the typical AIEgens such as TPE derivatives, AIEgens characterized with the excited-state intramolecular proton transfer (ESIPT) property were also employed for the construction of efficient AIEgen-carbohydrate bioconjugates. The SA-Gal (33) shown in Chart 4 is such an example.102 In SA-Gal, β-galactopyranosides were conjugated to the orthopositions of the benzene rings of the salicylaldehyde azine (SA), a prototypal ESIPT fluorogen. SA-Gal was faintly fluorescent in aqueous buffer. In sharp contrast, intense greenish yellow FL was exhibited by the mixture of SA-Gal and β-galactosidase. Such an FL turn-on response could be attributed to the cleavage of β-galactopyranoside groups from the bioconjugate 33, which resulted in the activated ESIPT and AIE processes. The linear relationship between the FL response and the βgalactosidase concentration allowed the quantification of βgalactosidase ranging from 0 to 0.1 U mL−1, with a detection limit of 0.014 U mL−1. This bioconjugate possessed various advantages, such as large Stokes shift up to 190 nm, high specificity to β-galactosidase and excellent SNR of 820, good cellular retention, and capability of imaging intracellular βgalactosidase with high contrast. Besides the above AIEgen-sugar bioconjugates based on the carbohydrate−protein binding or enzyme−substrate interaction for biosensing, bioconjugates established with an AIEgen and a polysaccharide moiety particularly for bioimaging have also been reported.103 Take TPE-CS (34 in Chart 4), which can be used for long-term cell tracing, for instance.103 Long-lasting cellular trackers are scientifically valuable and practically important, for they allow people to follow cellular events and processes in a systematic and continuous manner. Bioconjugate 34 was obtained by conjugating a large number of TPE units with a chitosan (CS) chain via the reaction between the isothiocyanate and primary amine groups. 34 is characterized with AIE feature. It is practically nonluminescent in the isolated state but brightly emissive when its molecules aggregate. The aggregates of TPE-CS bioconjugate were easily up-taken by HeLa cells and retained in the cells for as long as 15 passages. The internalized AIE aggregates preferred to reside in one cell instead of being equally separated to two daughter cells in each cell division cycle, resulting in the extraordinary long-term cell tracing capability. The AIE aggregates located in the cellular compartments without contaminating other cocultured cell lines, enabling the discrimination of specific cancerous cells from normal cells. It can be easily learnt from the above discussions, AIE bioconjugates are usually constructed by the bioconjugation methods such as click reaction (e.g., CuAAC), the amidation reaction between carboxyl group or N-hydroxysuccinimide (NHS) ester and primary amine, the reaction between isothiocyanate and primary amine which yields a thiourea, the

thiol−ene reaction between maleimide and thiol which forms a thioester bond, as well as the Michael addition reaction. The AIE bioconjugates can be applied for the sensing of a wide variety of bioelements such as biomarkers, proteins (e.g., enzymes, lectins), nucleic acids, the real-time monitoring/ imaging of specific biological/physiological processes such as apoptosis, the image-guided therapy including chemotherapy and PDT. It also can be concluded that the great majority of specific light-up bioprobes based on AIE bioconjugates operate through direct binding with a specific target or site-specific reaction in the presence of the target analyte. In the former mechanism, an AIEgen is functionalized with a specific biological ligand that can exclusively bind to the target analyte, which triggers the RIM process of the AIE motif to switch on the FL. The recognition bioligands attached to the AIEgens are preferably biomolecules with relatively low molecular weight, because conjugating very large molecules may impede the intramolecular motions of the bioconjugate to give an unignorable background signal and lead to insensitive response to the analyte of interest with low SNR. In the latter mechanism, an AIEgen is coupled with a bioligand that can undergo such reaction as enzymatic cleavage by the target analyte to afford a resultant that would form highly fluorescent aggregates. In a word, because of the unique AIE feature, the AIE bionconjugates excelled their ACQ counterparts for no self-quenching at high concentration, wash-free sensing, high SNR and contrast, superior photostability. Moreover, because of the particularity of bioligands, the AIE bionconjugates show many advantages over the pure chemically constituted AIE probes, such as excellent specificity toward target analytes, good cell permeability and biocompatibility, low toxicity, and high applicability in living cells. Therefore, with rational design, the AIE bioconjugates are competent for any task by integrating the merits of AIEgens and the functionality of bioligands. Despite the considerable difficulty in the synthesis, purification, and characterization, developing probes based on AIE bioconjugates overwhelmingly becomes a popular trend. 2.1.2. AIE Nanoprobes. Bioimaging has been recognized to be a fairly powerful tool in current biological research, because it offers visual information on cells, organelles, tissues, and biological processes in living systems. Among all biological imaging techniques, FL imaging is the most promising, because it is rapid, noninvasive, sensitive, and inexpensive, with outstanding temporal and spatial resolution as well as high SNR. The properties of imaging agents are decisive to the performance of FL imaging. Although discrete small molecules such as organic dyes and fluorescent proteins that possess good biocompatibility and small size are ideal for intracellular target recognition, fluorescent NPs own advantages like superior photostability, tunable size, ease of surface functionalization, and multifunctional potentials, which are more favorable in biomedical research. As aforementioned, in comparison to the NPs based on conventional fluorophores, AIE NPs integrate the advantages of AIEgens and NPs, and thus are free of selfquenching and photoblinking, are more photostable, and featured with higher luminosity which is directly proportional to the concentration of fluorogen. In this way, AIE NPs are attracting more and more research interest, rendering the utilization of AIE NPs for bioimaging becoming a new trend in AIE-based bioprobes as well as a new territory of bioimaging. Such a rapidly expanding area has been extensively reviewed from different viewpoints in recent years.16,27−30,32,33,104−109 To avoid repetitive illustration, only a few representative AIE NP K


Review


Figure 1. Representatives of newly developed AIE nanoprobes based on small AIEgens. (A) Schematic illustration of the general preparation protocol of AIE & AIE-Pep dots. Adapted and modified with permission from ref 118. Copyright 2013 Nature Publishing Group. (B) Structures of TPE-EP (35) and 35-Tat dots, and the 3D reconstruction and sectional images of the bone marrow stromal cells incubated with 35-Tat dots. The cell nucleus was costained with Hoechst dye. Adapted and modified with permission from ref 128. Copyright 2016 Wiley−VCH. (C) Structure of TPE-IPB (36), and the performance of 36-PEG after being incubated with ONOO−. (D) Schematic representation of the application of 36-PEG for specific in vivo inflammation imaging, and the time-dependent in vivo FL images of inflammation-bearing mice before and after injecting 36-PEG. (E) Confocal images of infected and uninfected skin tissues of 36-PEG-treated mice, where the red FL represents blood vessels. (F) In vivo FL images of both MRSA- and E. coli-infected mice before and after vancomycin and penicillin treatment for 14 days. Adapted and modified with permission from ref 136. Copyright 2016 Wiley−VCH.

polymer matrix to encapsulate a far-red (FR)/NIR-emissive AIEgen.54 These AIEgen-loaded BSA NPs owned high brightness and low cytotoxicity and when applied to image MCF-7 breast cancer cells and a murine hepatoma-22-tumorbearing mouse, they exhibited superb cancer cell uptake and good tumor-targeting ability in vivo as a result of the enhanced permeability and retention (EPR) effect. In the light of that the AIE NPs formed by direct encapsulation of AIEgens into polymers showed similar optical features and cellular retention time as quantum dots, the term of AIE dots was formally put forward in 2013.118 Specifically, AIE dots are referred to a class of NPs where AIEgens are noncovalently incorporated into biocompatible polymer matrices through physical encapsulation.16,22,33,54,118 Theoretically, uncountable AIE dots could be derived by varying the AIEgens and the biocompatible polymers. By far, although a diversity of AIEgens have been employed for the formulation of AIE dots, the most frequently used encapsulation matrix is lipid-PEG, namely 1,2-distearoyl-

systems developed lately would be discussed in this review article. Broadly speaking, nanosized imaging agents based on AIEgens can be classified into two categories in terms of their chemical nature and likely behaviors in bioenvironments: soft NPs (e.g., AIE dots and polymeric AIE NPs) and hard ones (e.g., AIEgen-doped silica NPs). Although the hard AIE NPs have been widely utilized for FL imaging,110−117 the inorganic nature and potential unsatisfactory biocompatibility and nonbiodegradability impede their further practical application. In contrast, soft AIE NPs fabricated by physically encapsulating AIEgens into polymeric matrices or through the self-assembly of polymeric AIEgens are believed to be easier to prepare, more biocompatible, less toxic, and potentially more biodegradable. Therefore, we would only highlight some soft AIE NPs in this section. In 2012, Tang and Liu et al. invented the uniformly sized protein AIE NPs by using bovine serum albumin (BSA) as the L


Review

ACS Applied Materials & Interfaces sn-glycero-3-phosphoethanolamine-N-[methoxyl-(polyethylene glycol)] (DSPE-PEG). Liu’s group and Tang’s team pioneered in the development and application of AIE dots, and have developed the majority of AIE dots reported so far. On the basis of DSPE-PEG, Liu and Tang et al. collaboratively established a nanoprecipitation method for the preparation of AIE dots,118,119 and the general fabrication protocol is schematically presented in Figure 1A. To prepare the surface-functionalized AIE dots which could undergo chemical modification or biological conjugation, DSPE-PEGx and its derivatives (DSPE-PEGx-FG) with reactive groups such as maleimide (Mal), amine, carboxyl, etc., or recognition ligands like folic acid or biotin were utilized as polymer matrices, where x stands for the molecular weight of the PEG part. The homogeneous THF solution of AIEgen, DSPE-PEGx, and DSPE-PEGx-FG was added into Milli-Q water, and the resulting mixture was then subjected to the ultrasound sonication by a microtip probe sonicator. In the course of THF/water mixing, the hydrophobic DSPE chains and the AIE molecules intertwined with each other to form the core while the hydrophilic PEG domains stretched toward the aqueous phase to keep the AIE NPs from aggregation and endow the dots with abundant surface reactive sites. To enhance the biocompatibility, cell permeability, and/or targeting ability, the resultant AIE dots were usually further conjugated with a particularly selected peptide via the reaction between the surface reactive groups and the specific reactive moieties of peptides. The free peptides were get rid of by dialysis against Milli-Q water and the resulting AIE dots suspension was depurated by filtration through a 0.2 μm syringe driven filter. The obtained AIE-Pep dots were then stored at 4 °C for further use. Prior to the biological use, the AIE and AIE-Pep dots were fully characterized by a string of experiments including dynamic light scattering and high resolution-transmission electron microscopy measurements, colloidal stability evaluation, photophysical properties studies, and single-dot imaging, etc. Given its facility and universality, such a lipid-PEG formulation has been extensively employed to fabricate highly performed AIE dots for FL imaging, dual-modal imaging, theranostic applications, and multifunctional systems.52−54,59,118−138 In this part, the imaging utilities of the AIE dots would be primarily discussed and their dual-modal imaging and multifunctionality will be set aside for discussion in Section 2.3.2. As reported, AIE dots enjoy ultrahigh brightness, satisfactory cellular uptake efficiency, high intracellular retention, good biocompatibility, excellent photostability, making them ideal contrast agents for long-term cell imaging.118,119,122,124,126,128,129 Stem cells are a special kind of biological cells, which are capable of differentiating into specialized cells. The development of stem cell-based therapy calls for probes that can trace the fate and regenerative ability of the administrated stem cells over a long period of time. To this end, AIE dots have been recently developed as long-term, continuous, and precise in vivo trackers for stem cells such as adipose-derived stem cells (ADSCs)126 and bone marrow stromal cells (BMSCs).34,128 ADSCs are promising cell-based regenerative medicine, for they can be facilely separated from the stromal vascular and possess differentiation potential with easy expanding capacity. By encapsulating the FR/NIRfluorescent AIEgen 2,3-bis(4-(phenyl-(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile (TPE-TPA-FN) into the DSPE-PEG2000 matrix through the above-introduced nano-

precipitation method, spherical AIE dots with a mean diameter of ∼35 nm have been attained.126 The intense FR/NIR FL, large Stokes shift (∼160 nm), outstanding biological and photophysical stabilities, low cytotoxicity, and excellent retention allowed the AIE dots precisely and quantitatively reporting the long-term fate of ADSCs and their regenerative capability for as long as 42 days in the mouse model bearing an ischemic hind limb. Such AIE dots are superior to the most popular commercial cell tracers PKH26 and Qtracker 655 in cell tracking ability. Because of the regenerative ability and longer therapeutic time window, stem-cell based therapy becomes a promising alternative scheme for acute stroke treatment. Benefiting from the easy gaining from patients and multipotentiality of differentiation, self-renewal as well as regeneration, BMSCs, which are commonly used as a cell source for stem cell therapy, exhibit promising therapeutic outcomes for stroke treatment. Establishing reliable tracking strategy for the monitoring of the BMSCs’ fate and evaluation of their therapeutic effects in pursuit of improved success rate of stroke treatment remains a challenge. Regarding this, Liu and Liao et al. created the NIR-emissive AIEgen TPE-EP (35)128 and fabricated them into AIE dots functionalized with a CPP derived from HIV-1 transactivator of transcription protein (Tat) through the route shown in Figure 1A. The obtained 35Tat dots displayed an FL maximum at 690 nm with a high quantum yield (ΦF) of 35 ± 1% in water. As can be seen from Figure 1B, 35-Tat dots lit up the BMSCs with high contrast and resolution by even distribution in the cell cytoplasm, suggesting the efficient cellular uptake and the excellent imaging capacity of 35-Tat dots. In vitro and in vivo BMSCs tracking studies have further demonstrated the superb long-term tracking performance of the 35-Tat dots. The migration of 35-Tat dots-labeled BMSCs to the stroke lesion site in a rat photothrombotic ischemia model was revealed by the intense red FL signal of 35-Tat dots. Immunofluorescence staining further validated that the 35-Tat dots labeling did not interfere the normal function of BMSCs, verifying their good in vivo biocompatibility. Together with low cytotoxicity and good physical and photo stabilities, these advantages render 35-Tat dots a promising cell tracer to assess the fate of BMSCs in clinical cell therapy. In addition to the long-term cell tracking, AIE dots in principle could fulfill any task with ingenious design. For instance, lately, Tang and Ding et al. have creatively constructed a type of activatable AIE dots with integrated merits of AIEgen, FL turn-on mode, and nanotechnology, for the selective imaging of elevated peroxynitrite (ONOO−) generation.136 The novel imine-decorated TPE derivative TPE-IPB (36) was specially designed, which consists of the AIE-active TPE moiety, the emission mediating imine group, and the ONOO− recognition site phenylboronate (Figure 1C). The AIE dots based on 36 were formulated by using the lipid-PEG as matrix. The resulting nanoprobe 36-PEG showed no FL in aqueous media, because of the quenching effect of the iminephenylboronate exerting on the TPE unit. Upon reaction with ONOO− at pH 7.4, intensive yellow FL was switched on, as a result of the cleavage of the phenylboronic ester via oxidative reaction and the subsequent release of p-quinonemethide and the AIE-active TPE-phenol residue. The detection limit was calculated to be about 100 μM, which lies in the pathological level range. Since the blood vessels in the inflammatory region are leaky and permeable, 36-PEG would preferentially accumulate to the inflammation area driven by M


Review


Figure 2. Representatives of newly developed AIE nanoprobes based on polymeric AIEgens. (A) Schematic demonstration of the 37/38 integrated binary nanoprobes and their photoswitchable response to UV/visible light illumination. (B) True color images of a 37b/38 nanoprobe-injected mouse. The images were taken at 5 min postinjection under excitation by a 365 nm hand-held UV lamp before (left) and after (right) red laser irradiation (655 nm, 5 min). Circles indicate the laser illuminated position. (C) 3D reconstructed images taken after selectively turning on the 37b/ 38 nanoprobe FL at the lymph nodes. Green and yellow arrows indicate axillary and brachial nodes, respectively. And the lymph nodes resected from the switched-on mouse body. (D) Histological micrographs and FL images for a section of the switched-on lymph node. The 37b/38 nanoprobe FL (red) was spectrally unmixed from the tissue autofluorescence (green). (E) Reversible photoswitching modulation of 37b/38 nanoprobe embedded in the resected lymph node. Adapted and modified with permission from ref 153. Copyright 2013 Wiley−VCH.

EPR effect. Due to the elevated production of ONOO−, FL was turned on exclusively in the inflammatory region (Figure 1D). The in vivo inflammation imaging capacity has been clearly validated by the results shown in Figure 1D, E. The FL signal was aroused by inflammation and increased over time. The blood vessel staining and CLSM pictures of the slices of infected and uninfected skin are depicted in Figure 1E. Yellowfluorescent dots were clearly visible around the blood vessels presented in red FL in the bacterial infected area, while no FL signal from the activated 36-PEG was observed in the normal skin slice. This further verified the excellent selectivity of the nanoprobe to the in vivo inflammation. As displayed in Figure 1F, 36-PEG dots also hold the capacity to visualize the in vivo therapeutic efficacy of anti-inflammation drugs. Inflammationbearing mouse model were established by the subcutaneous inoculation of Staphylococcus aureus (MRSA) and E. coli at the left and right back of nude mice, respectively. Before antibiotic treatments, both sides of the mice backs exhibited intense FL. After being treated by antibiotics for 14 days, the 36-PEG was injected into these mice and the FL signals from MRSAinfected foci in the vancomycin-treated mice and E. coli-infected foci in the penicillin-treated mice almost disappeared. On the contrary, strong FL signals from the inflammatory sites induced by the bacteria resistant to the injected antibiotics were preserved even after 14 days of treatments. Therefore, the 36PEG is an activatable nanoprobe that can sense in vivo inflammation and monitor treatment efficacy of anti-inflammatory agents in a precise and noninvasive manner. Besides the AIE dots where the AIEgens are physically encased by polymeric matrices, AIE NPs with the AIE motifs covalently bonded to the polymer chains have also been

frequently reported in the past few years.139−153 The AIEchitosan bioconjugate 34 is such an example. As discussed in Section 2.1.1.3, the spontaneously formed aggregates of 34 in cell culture media were microsized and nonuniform. However, the NPs of 34 fabricated by taking advantage of a simple ionic gelation strategy under mild conditions are uniform nanospheres with positive charges on the surface, which can be readily up-taken by living cells and retain in the cytoplasm to realize the long-term cell tracking.139 In this manner, a general approach for the fabrication of AIE NPs based on polymeric AIEgens has been established. Directly attaching AIEgens to biocompatible polymers yields the polymeric AIEgens first, and then the AIE NPs could be easily generated by virtue of the NP synthesis methodologies. Another method to afford covalently bound AIE polymers which could form NPs under proper conditions is to in situ polymerize the AIEgens into polymer chains.140−152 Wei’s group contributes most to this subject. They have developed a large quantity of AIE NPs with various polymerizable AIEgens and diverse polymerization methods including free radical polymerization,144,145 reversible addition−fragmentation chain transfer polymerization,146,147 emulsion polymerization,148,149 ring-opening polymerization,150,151 and redox polymerization.152 The self-assembly of the resultant amphiphilic polymeric AIEgens could facilely give rise to the AIE NPs with compact structures, appreciable bio/cytocompatibility, and high cellular internalization efficiency. Very interestingly, sometimes in situ polymerizing appropriate non-AIEgens in the micelles could not only generate polymeric AIEgens but also simultaneously yield high-performance AIE NPs. Such a work has been reported by Kim and Park et al.153 Unlike the aforementioned AIE NPs constructed with N


Review


Figure 3. (A) Schematic depiction of the design strategy for FR/NIR AIEgens 39−47. (B, D) Molecular structures of FR/NIR AIEgens 39−46, the data in parentheses are λem and ΦF values. (C) FL images of 41-injected mouse taken at different time points postinjection (upper panel), and the 3D FL imaging of the mouse bearing tumor after intravenous injection of 41 for 24 h (lower panel). Adapted and modified with permission from ref 55. Copyright 2015 Wiley−VCH. (E) CLSM images of A549 cells cocultured with 45, the nuclei were stained with DAPI. (F) In vivo FL imaging of mice with F127 and 45-F127 NPs at different time points. Adapted and modified with permission from ref 156. Copyright 2016 Wiley−VCH.

could be switched on and off by the alternate 254 nm UV and 650 nm visible light illumination, owing to the photochromism of the cointegrated 38. Combing the intense NIR FL, highcontrast on/off photoswitching, and tiny colloidal size, the composite 37b/38 NPs are well suited for in-vivo bioimaging. When the switched off 37b/38 NPs was injected into the mouse, there was no observable FL signal. Whereas the FL of 37b was immediately turned on, when employing an intense 655 nm laser. A fluorescent spot emerged close to the laserirradiated site, precisely locating a subcutaneous sentinel lymph node (SLN) with brilliant and naked-eye recognizable signal (Figure 2B). As can be learnt from Figure 2C, the administrated NPs were enriched at the axillary and brachial nodes as soon as being injected for 5 min. Histological analysis of the resected nodes shown in Figure 2D elaborated that the NPs were internalized by cells into the sinusoidal region where the NIR FL spectrum of the turned-on NPs was evidently distinguishable from the background autofluorescence. More importantly, the NPs delivered to the SLN maintained the reversible photoswitching capacity, as demonstrated by the alternate irradiation of UV and visible light (Figure 2E). The consequent on/off contrast of as high as ∼15 suggested that the physically integrated 37/38 NPs retained their initial structural integrity

premade AIEgens, the AIE NPs shown in Figure 2A were made from materials without AIE activity. The AIE motif 37a or 37b in these NPs was originated from the Knoevenagelpolycondensed 4,4′-biphenyldicarboxaldehyde or bis(octyloxy)terephthalaldehyde and p-xylylene dicyanide. Considering that the switchable signals in FL imaging can surmount the limitations of the photon-based modality including interferences with light absorption and scattering, and autofluorescence from biological species and therefore improve the detectability as well as spatial resolution of FL imaging, photoswitchable 1,2bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-hexafluoro-1cyclopentene (BTE, 38) was incorporated into the AIE NPs systems. As such, the photomodulatable binary composite NPs were smartly fabricated through the in situ and one-pot colloidal polymerization of the dialdehyde and dicyanide monomers in the presence of 38 within the hydrophobic interior of the Tween 80 micelles dispersed in water. These fine composite NPs are quite small (1 μM) in the inflammatory process, sensitively detecting and imaging in vivo H2O2 is of great significance to the early diagnosis of inflammation-related diseases. H2O2-specific peroxalate chemiluminescence (POCL) in a form of NPs coloading peroxalates as the chemical energy source and fluorogens as the emitting constituent has been applied to image the inflammation in vivo. Apart from the extraordinary specificity to H2O2, POCL is potentially able to provide outstanding in vivo sensitivity due to the noninvolvement of background noises from autofluorescence and stray excitation light, distinct from the FL imaging by photoexcitation. Integrating the benefits from POCL with the multimerits of 47, the POCL nanoprobe CLNP-PPV/BDP (47/BDP NP) was conveniently fabricated via the coaggregation of 47 with peroxalates, wherein the energy gap between the emitter and POCL was bridged by the codoped photonic molecule BODIY (Figure 4A). The BODIY effectively accepted the chemically produced energy and relayed it to the aggregates of AIE polymer 47 through the consecutive intraparticle energy transfer, and thus boosted the POCL energy matching. In this manner, sensitive imaging of deep-tissue inflammation was realized by the 47-based NIR POCL nanoprobe. 47/BDP NPs showed a reliable NIR CL response to H2O2 with the intensity

characteristics, as well as large Stokes shift. Such a design rationale can be generally interpreted as that the existence of electron donor(s) and acceptor(s), for one thing, decreases the energy gap of the π conjugated system and consequently facilitates the long-wavelength emission, and for another, provides sufficient motional elements and nonplanar units that can ensure the AIE effect. More specifically, Zhu and Guo et al. reported a series of fantastic quinolone-malononitrile (QM)-based FR/NIR AIEgens, some of which have been further applied for tumortargeted bioimaging.55,117,157 As displayed in Figure 3B, each of the fluorogens 39−42 is composed of an electron-donating triphenylamine (TPA) unit, a thiophene π-bridge, and an electron-accepting QM block.55 Although all these AIEgens emitted in the region of FR/NIR, the alteration in the substituents on the TPA group and/or the thiophene moiety gave rise to the changes in the photophysical properties as well as the nanostructure morphologies of these AIEgens. The archetypal TPA-thiophene-QM adduct 39 displayed a solidstate FL peaked at 655 nm. When the epoxyethyl unit was attached to the thiophene moiety in 39, the AIEgen 40 fluorescing at 675 nm in the solid state was derived. As the substituents on the TPA unit changed from hydrogen (40) to methoxyl (41) to ethoxy (42) groups, the solid-state emission maximum accordingly red-shifted from 675 to 708 and finally to 738 nm, as a result of the increased D−A effect. The aggregates of 39 formed via a solution evaporation approach were uniform 1D microrods, whereas those of 40−42 were predominately spherical-shaped NPs in a diameter of around 80−200 nm, indicating the introduced epoxyethyl in the thiophene unit played a crucial role in the morphology modulation of these AIE emitters. Considering 39 and 41 respectively showed distinct rod-like and spherical morphology, they were chosen to conduct imaging experiments to evaluate the shape effects of the bioprobes on their bioimaging performance. Both 39 and 41 exhibited negligible toxicity to cells and were able to track the HeLa cells with bright FR/NIR FL for as long as four passages. When applied for in vivo imaging, the aggregates of 39 rapidly distributed all over the mouse body through blood circulation. In stark contrast, the naturally formed NPs of 41 could specifically light up the tumor site with NIR FL within 0.5 h (Figure 3C). Furthermore, the 41 NPs could exclusively retain in the tumor tissue for more than 24 h, making 41 a promising probe for tumor tracking. Such a passive tumor-targeting ability of 41 was ascribed to the EPR effect. The distinct in vivo imaging behaviors of the analogues 39 and 41 offered useful clues for the rational molecular design and morphology modulation of high-performance bioprobes for tumor-targeted imaging. Aside from the linear D−π−A structured FR/NIR AIEgens, those with branched or even starburst-shaped D−π−A, A−π− D−π−A, and D−π−A−π−D architectures have also been constructed.53,156 For instance, by means of using the diphenylamine (DPA) and cyano groups as electron donors and acceptors, respectively, the fluorogens 43−46 (Figure 3D), each of which owns a cross-shaped/cruciform D−π−A molecular framework, have been obtained.156 Luminogens 43−46 showed intense aggregated-state FL, which lied in the FR/NIR region and could be fine-tuned from 644 to 686 nm by the variation of substituents on the electron donors and acceptors. Thanks to the multiple motional moieties and the highly distorted 3D conformation resulted from the steric hindrance, 43−46 are featured with AIE attributes. It is Q


Review


Figure 5. Representative AIE-based bioprobes excitable by two-photon light source. (A) Structures of TPE-Cy (48a) and its base form 48b. (B) One-photon (left) and two-photon (right) excited FL images of 48-stained HeLa cells. (C) Comparison of FL signals from cell autofluorescence and 48-stained cells upon two-photon excitation. (D) Fluorescence lifetime distribution histogram of 48-stained HeLa cells. Adapted and modified with permission from ref 158. Copyright 2015 Wiley−VCH. (E) Structures of 49 and its dots, stacked and 3D reconstructed two-photon excited FL images of brain blood vessels stained by 49 dots from a depth of 0−1200 μm. Adapted and modified with permission from ref 159. Copyright 2017 Elsevier.

linearly correlated to the H2O2 concentration. The detection limit of 10−9 M is far below the normal in vivo H2O2 concentration (10−7 M), which along with the deep-tissue penetrating-ability (depth >12 mm) allowed for the sensitive in vivo imaging of inflammation-responsive H2O2. More importantly, comparative deep imaging of peritonitis in hair shaved and unshaved mouse models (Figure 4B) has demonstrated the benefits of the rationally designed NIR CL nanoprobe for imaging the inflammatory diseases in an in vivo and sensitive fashion. In this subsection, general construction strategies of FR/NIR AIEgens have been briefly summarized with an emphasis on the creation of AIE-active FR/NIR emitters via rational combination of AIE-inactive electron acceptor(s) and donor(s). Then the representative examples ranging from small molecules with linear D−π−A or cross-shaped D−π−A structures to conjugated D−π−A polymer have been detailedly introduced focusing on their structure−property relationships and bioimaging application. Given their excellent performance in in vitro and in vivo imaging, AIE-based FR/NIR probes have been and will continue to be the goal pursued by researchers working in the biology-related areas. 2.2.2. Two/Three-Photon Excitable AIE Bioprobes. Nowadays, the rapid advancement of FL imaging demands excellent bioprobes with better tissue-penetrating capacity, lower damage to biospecies, higher SNR ratio, and superior spatiotemporal resolution. However, the commonly used one-photon FL

imaging bioprobes can hardly satisfy these requirements, because their excitation lights lie in the UV−vis region (350− 500 nm). Such short and high-energy lights not only restrict the tissue penetration depth to generally lower than 100 μm, but also exert unignorable photodamage or toxicity to the biosubstances. Moreover, the absorption of such excitation light could also excite the autofluorescence of biosystems, which will interfere with the real FL signals and lower the SNR. In this context, the two/three-photon excitable emitters emerged and are receiving fast growing interest. Such a fluorophore could absorb two or multiple NIR photons (700− 1500 nm) and emit at the same wavelength as excited by one photon. Evidently, two/three-photon excited FL imaging possess various superiorities over the one-photon technique including reduced photodamage to biosubstrate, deeper penetration depth, smaller autofluorescence interference, improved spatial resolution, and better photostability. Moreover, since water and blood are nearly transparent to NIR light, the FL signal perturbation by biomedia is negligible in the two/ three-photon FL imaging. In consequence, two/three-photon FL technique has been broadly applied for imaging of diverse biospecies. To achieve vivid images with excellent spatial resolution, the bioprobes for two/three-photon FL imaging are required to possess large two/three-photon absorption section as well as high ΦF, which could lower the demand on excitation power, minimize photodamage, and reduce the photobleaching.33,52,53 That is why the two/three-photon excitable AIER


Review


Figure 6. Representative AIE-based bioprobes excitable by three-photon light source. (A) Structures of 50 and its dots, bright-field and three-photon FL images of 50 dots-labeled zebrafish and the blood vessels. Adapted and modified with permission from ref 134. Copyright 2016 Tsinghua University Press and Springer-Verlag. (B) Structures of 51 and its biotin-decorated dots, comparative one-photon FL images of normal kidney cells and HeLa cells, two-photon excited FL image of HeLa cells (λex = 980 nm), and diagrammatic illustration of the proposed excitation mechanism of third harmonic generation (THG) and THG-induced FL. Adapted and modified with permission from ref 138. Copyright 2017 Royal Society of Chemistry.

excitation and emission would be more conducive. Hence, the two-photon excitable AIEgens with FR/NIR FL would be even more favorable in bioimaging, because of the combined merits of AIE, two-photon excitation, and FR/NIR emission. Fortunately, there have come out a few of outstanding twophoton excitable AIE-active FR/NIR bioprobes.53,116,118,121,159 The NIR FL imaging and 2PF imaging are two important techniques for deep-tissue imaging, because they could refrain from tissue absorption and scattering to some degree. The most significant feature of such bioprobes thus lies in their deeptissue penetration ability. The AIEgen TPABDFN (49) presented in Figure 5E is an example that fully demonstrated this.159 The large π-conjugation and D−A structure collectively endued 49 with remarkable NIR FL (λem = 710 nm) and large 2PA cross-section. 49 was encased in the poly(styrene-comaleic anhydride) to generate AIE dots, which were further grafted with PEG to increase the biocompatibility and circulation time in live animals. With a FR/NIR FL (λem = 678 nm) and a 2PA cross-section up to 5.56 × 105 GM, the obtained 49 dots were used as contrast agents for the NIR 2PF imaging under a 1040 nm femtosecond (fs) excitation. The major blood vessels and small capillaries in the mice ear and brain could be clearly visualized with the aid of 49 dots (Figure 5E). Because of the excellent deep-tissue penetrating ability of both 1040 nm excitation and NIR FL, ultra-deep in vivo imaging with a depth up to ∼1.2 mm was achieved. Similar to two-photon excitable FR/NIR AIEgens, the AIEactive fluorogens that can simultaneously absorb three or more NIR photons and emit in the FR/NIR region also hold the capability to penetrate deep into biological tissues.134,138,160,161 For instance, TPE-TPA-FN (50) is an AIEgen that emits threephoton excited FL (3PF) in the FR region and can be used for

based bioprobes are so hot and popular these days. The examples showcased in Figure 5 would provide a strong proof to the trend of exploring AIE-based bioprobes outputting two/ three-photon excited FL signals. Comprised of a TPE core and a hemicyanine pendant, the TPE-Cy (48a) exhibited in Figure 5A is AIE-active and pHsensitive. The FL color could change from red to blue when the environment varies from acidic to basic, due to the transformation from 48a to 48b. As reported, 48a could diffuse into cells and majorly stain the cytoplasmic region with blue FL (λem = 490 nm).155 Since 48a is cell-permeable and biocompatible, it was further utilized to probe the extracellular and intracellular viscosity.158 The FL intensity was enhanced and its lifetime elongated with the increase in viscosity, owing to the reinforced RIM effect. Benefiting from its two-photon absorption (2PA) at 600 nm, the FL signals of 48a could be completely distinguished from the cell autofluorescnece (λem = 350 nm), avoiding the intrinsic interference and producing FL images with much higher resolution and contrast as compared to the ones excited by one-photon light (Figures 5B and 5C). In the cells, the FL lifetime of the probe obtained by two-photon excitation was widely distributed in a range from 300 to 1500 ps. The lifetime of 48 in the tubular mitochondria was about 1 ns, which was much longer than that of 48 in the lipid droplets (∼500 ps). Thus, these two populations could be easily differentiated by 48, as indicated by the distinct two peaks in the FL lifetime histogram (Figure 5D). In other words, the heterogeneity of the intracellular viscosity and complexity of the intracellular microenvironment could be facilely revealed by this two-photon excited FL (2PF) lifetime imaging. As aforementioned, the FR/NIR emission is highly helpful to the FL imaging. Actually, the probes with both long-wavelength S


Review


Figure 7. Typical examples of phosphorescent AIE-based bioprobes. (A) Molecular structures of AIE-active phosphors 52 and 53. (B) Real-time phosphorescence images of HeLa cells incubated with 5 μM 52 at 37 °C for 1 h and subsequently treated with 20 μM carbonyl cyanide mchlorophenylhydrazone (CCCP) with increasing scan time (upper panel), and merged photographs of the phosphorescence and bright-field images (lower pannel). Adapted and modified with permission from ref 163. Copyright 2013 Royal Society of Chemistry. (C) Phosphorescence images of HeLa cells stained with 0.5 μM 53 (lane 1) or 0.1 μM MTR (lane 2), bright-field images (lane 3), the merged lanes 1, 2, and 3, and the Pearson’s colocalization coefficient R. (D) Phosphorescence images of 10 μM CCCP-treated living HeLa cells stained with 0.5 μM 53. (E) Confocal images of HeLa cells simultaneously incubated with 0.5 μM 53 and 0.1 μM LysoTracker Green in the presence of 10 μM CCCP. Adapted and modified with permission from ref 45. Copyright 2016 Nature Publishing Group.

the zebrafish imaging.134 The AIE dots of 50 were prepared via the nanoprecipitation method with the DSPE-PEG2000 as the polymer matrix (Figure 6A), which were chemically stable and exhibited no in vivo toxicity to zebrafish. Bright FR 3PF (λem = 660 nm) of 50 dots under the 1560 nm-fs laser excitation allowed the in vivo imaging of zebrafish at different stages of growth as well as the achievement of a 3PF angiogram of zebrafish (Figure 6A). The 50 dots were able to retain inside the zebrafish body for as long as 120 h. The strong resistance of these AIE dots to photobleaching under 1560 nm-fs excitation enabled the long-term bioimaging. More recently, the nanoaggregates of 50 were encapsulated with nanographene oxide (NGO), which could greatly improve the stability of NPs in aqueous media. The NGO-modified 50 NPs were capable of imaging the architecture of mice ear blood vessels and mapping the NP distribution in zebrafish in a high-resolution manner.160 Most recently, Qian and Hua et al. collaboratively developed another AIEgen possessing 3PF feature, a D−A−D structure and a large three-photon absorption (3PA) cross-section of 6.33 × 10−78 cm6 S2, and employed its FR-emissive (λem = 654 nm) AIE dots for in vivo mouse brain vasculature imaging under a 1550 nm-fs laser excitation. An imaging depth up to 500 μm as well as a fine 3D reconstruction image of the mouse brain blood vessels was achieved.161 Very shortly after this work, Tang’s group reported a new AIEgen (51) featured with FR 3PF.138 51 was created by tethering a tetrad pair of TPE units on to the ACQ-active 2-(2,6-bis((E)-4-(diphenylamino)styryl)-4H-pyran-4-ylidene)malononitrile (TPA-DCM) core (Figure 6B), following the “AIE plus ACQ” principle. With a starburst D−π−A−π−D architecture and multiple rotatable conjugated rings, 51 exhibited a combination of both AIE and

TICT properties, a favorable long-wavelength absorption (λex = 500 nm), and a FR emission in the aggregated state (λem = 675 nm). Biotin-functionalized dots of 51 were fabricated utilizing a modified nanoprecipitation method as shown in Figure 1A, to facilitate the targeted cellular internalization. 51 dots were efficiently and selectively taken up by the cancer cells through the avidin−biotin pathway, and preferentially located in the mitochondrial region, demonstrating the excellent cancer cell selectivity and mitochondria specificity. As expected, these 51 dots were biocompatible, photostable, and two-photon excitable with a 2PA cross-section as large as 313 MG at 830 nm. More importantly, in addition to imaging cells with 2PF upon 980 nm excitation, 51 dots were found to display rich nonlinear optical properties, such as aggregation-induced third harmonic generation (THG) and FR 3PF. Tang et al. has proposed the mechanism for THG and the THG-induced FL, and thought that the aggregation benefited the THG and 3PF. Such a work not only provided a multifunctional FR-fluorescent two/three-photon excitable AIE bioprobe, but also offered some insights into the aggregated-state THG as well as the THG-induced FL. Several AIE bioprobes with 2PF/3PF have been discussed in this small section. No matter been used to map the intracellular parameters such as viscosity or stain cells or image the tissues like blood vessels, the 2PF and 3PF techniques exhibited superb spatial resolution, high SNR, low phototoxicity, and outstanding photostability. Integrated with FR or NIR emission, extra benefits such as ultra-deep tissue penetration ability were gained. In view of these attractive advantages, the family of two/three-photon excitable AIE bioprobes especially those with FR/NIR emissions should be vastly expanded and enriched. T


Review

ACS Applied Materials & Interfaces 2.2.3. Phosphorescent AIE Bioprobes. In general, the output signal of luminescent bioprobes is mostly FL, which is easily subjected to the interferences from biological background autofluorescence and scattered light. Besides the aboveintroduced long-wavelength luminescence and 2PF/3PF, there is another effective way to solve this problem. As can be learnt from the discussion on 2PF lifetime imaging of the AIE bioprobe 48a that time-resolved luminescence assays have their own advantages such as lower background and higher SNR, in compared to those only rely on the luminescence intensity. In this means, developing AIE bioprobes with phosphorescent output signal is of great significance, since the luminescence lifetimes of phosphors are in the microsecond to millisecond range which is much longer than those of the fluorophores (several to dozens of nanoseconds). Theoretically, the interference from short-lived background FL and scattered light could be minimized to a negligible level by the phosphorescence, giving a satisfactorily high contrast and SNR. Although such examples are not many, yet do exist.45,162,163 Huang and Zhao et al. are the first to demonstrate the promising application of luminophores characterized with aggregation-induced phosphorescence (AIP) feature for time-resolved luminescence assay using the long-lived phosphorescence signal.162 Targeted luminescence imaging of cancer cells were realized by the folic acid-decorated NPs of the AIP-active Pt(II) complex. Regretfully, the luminescence lifetimes of this Pt(II) complex in the aggregated state were measured to be only τ1 = 0.157 μs (44%) and τ2 = 0.429 μs (44%), leaving room for improvement. Soon after the above work, an iridium(III) complex-based AIP-active luminogen with a solid-state luminescence lifetime as long as 1.228 μs was developed by Chao’s group.163 As depicted in Figure 7A, 52 were constituted of multiple conjugated rings which are distorted to each other, allowing for twisting and vibrational intramolecular motions and a highly twisted 3D conformation, and ultimately resulting in the marked AIP effect. When incubated with HeLa cells, 52 exhibited almost no cytotoxicity and high specificity to mitochondria. The colocalization experiments with the commercially available mitochondria tracker MitoTrackerGreen (MTG) manifested that the FL images of MTG and 52 were well overlapped with a Pearson’s colocalization coefficient of up to 0.94. The mitochondrial-specific staining by 52 was not limited to cancer cell lines, for the primary cell line FLS could also been visualized by the red phosphorescence of 52. The profound AIP feature contributed to the superb photostability of 52 nanoaggregates. The mitochondrial shape changes are closely associated with various cellular functions such as apoptosis. 52 was highly photostable and very tolerant to the reduction of mitochondrial potential (ΔΨm), and hence it could work as a mitochondrial tracer for apoptosis studies. The mitochondrial morphology changes induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) were monitored by 52 in real time (Figure 7B). As revealed by the phosphorescence images, when exposed to CCCP, the intact reticulum structure of mitochondria was changed into dispersed small fragments little by little, which is in good accordance with the mitochondria morphological changes evidenced in the early stages of apoptosis. 52 is the first example of an AIP-active complex to be employed as a phosphorescent bioprobe for mitochondrial imaging and tracing. In the light of their outstanding physicochemical characteristics favorable for bioimaging including long luminescence

lifetime, large Stokes shift up to hundreds of nanometers, and minimal photobleaching, Chao’s team further explored another series of new cyclometalated iridium(III) complexes and utilized them for mitophagy monitoring.45 The luminogen 53 exhibited in Figure 7A is a representative example out of them, which was generated via coupling TPA unit with 2phenylimidazo[4,5-f ][1,10]phenanthroline. With very low toxicity to cells and notable AIP feature, 53 was verified to be suitable for bioimaging. The results shown in Figure 7C manifested that the orange phosphorescence of the cells stained with 53 agreed well with the green FL from the MTG, indicative of the selective accumulation of 53 in mitochondria. The good mitochondrial specificity was further validated by the large Pearson’s coefficient of 0.90. With the help of bright and long-lived luminescence, 53 was proven to be internalized into the cells via a nonendocytic energy dependent active transport. When compared to MTG, 53 possessed a superior resistance to photobleaching and good chemical stability in the physiological pH range. Mitophagy is a unique autophagy, which eliminates defective mitochondria to retain sufficient healthy mitochondria and overcomes the shortage generated by cellular loss in necrosis and apoptosis. Given the importance of mitophagy study and the satisfactory mitochondria selectivity of 53, 53 was utilized to monitor the mitophagy process in a real-time and high contrast mode. The mitophagy of HeLa cells was triggered by CCCP while the mitochondria and lysosome were localized by 53 and LysoTracker Green (LTG), respectively. The green luminescence signal from LTG was dramatically boosted along with the change of mitochondria from reticulum to small dispersed debris upon exposure to CCCP, suggestive of the mitophagy’s occurrence (Figure 7D). A new green fluorescent spot emerged and overlaid with the 53-stained mitochondria at the time point of 20 min, indicating the acidic autophagosome was formed and the mitophagy process in this area was initiated. The completion of the mitophagy process in this region was suggested by the disappearance of green FL signal at 26 min (Figure 7E). By virtue of this facile and competent AIPactive bioprobe, the problems that always occurred in mitophagy tracking, including short-time dynamics variation, pH fluctuation, drastic morphology change, and membrane potential decrease, were able to be tackled. As can be learnt from the above elucidation, although AIP-based bioprobes enjoy various advantages over FL bioprobes, their development is still in the infancy stage but has already become a new trend. More efforts need to be spent on this active and promising topic. 2.3. Trends and Progress in Modality and Functionality of Probing System. The new trends in the development of AIE-based bioprobes, i.e., from short-wavelength emission to FR/NIR luminescence, from one-photon excited fluorescence to two/three-photon excited fluorescence, and from FL to phosphorescence (or from short-lived to long-lived luminescence), are rendered in Section 2.2 in terms of the output signals. In this section, the newly emerged advancing trends of the AIE-based bioprobes, namely from monomodal FL imaging agents to dual-modal imaging systems, and from monofunctional AIE probes to dual/multifunctional AIE probing systems, will be revealed according to the modality and functionality of the probing system. 2.3.1. Dual-Modal AIE Bioprobes. Up until now, various imaging techniques, including FL, magnetic, and radionuclide imaging, have been developed to offer physiological and pathological information for sensitive and specific disease U


Review


Figure 8. Representative AIE-based bioprobes with dual modalities. (A) Molecular structure of TPE-2Gd (54). (B) FL images of HeLa cells stained by 54 or costained by 54 and propidium iodide (PI). (C) Axial T1-weighted magnetic resonance images through the rat liver injected with 54 or Magnevist. Adapted and modified with permission from ref 165. Copyright 2014 American Chemical Society. (D) Molecular structure of 55 in 55Au NPs. (E) Photos and corresponding computed tomography (CT) images of 55-Au NPs samples with increasing concentration (μg/mL), and standard curve of CT values versus concentration of 55-Au NPs. (F) FL and (G) CT images of transplanted CT26 tumor-bearing mice before or after intravenous injection of 55-Au NPs. Adapted and modified with permission from ref 166. Copyright 2014 Elsevier Ltd.

of TPE-TPA-FN (50) with a DSPE-PEG matrix via the method shown in Figure 1A, then attached the diethylenetriaminepentaacetic (DTPA) moieties onto the dots surface, subsequently incorporated the MRI reagent gadolinium(III) via the chelation of DTPA and Gd(III), and finally modified the Gd(III)chelated dots with a HIV-1 Tat peptide. The obtained fluorescent-magnetic AIE dots exhibited a high ΦF of 25% as well as a T1 relaxivity of 7.91 mM−1 s−1, and could be efficiently taken up by living cancerous cells. As a result, the cancer cell biodistribution was accurately quantified with the help of Gd(III), while the engraftment information on cancer cells at single cellular level was offered by the bright FL. Such dualmodal AIE dots displayed collective goodness of both MRI and FL imaging. It was regrettable that although these AIE dots were proven to be efficient T1 contrasts, the cells labeled by them could not be detected by MRI, which might be owing to the insufficient amount of Gd(III) in the injected cells. Such a pioneering work opened up a generic platform for the

diagnosis. However, each imaging modality has its own merits and demerits, or in other words, only limited information which is sometimes inadequate for accurate imaging diagnosis could be provided by a single imaging technique. In view of this limitation, dual-modal bioprobes that combine the superiorities of each imaging modality and show complementary and synergistic benefits are highly demanded and attracting growing attention in recent years. As stated above, FL imaging based on AIE possesses a variety of advantages over that on the basis of ACQ. Dual-modal AIE bioprobes are therefore supposed to perfectly interpret the “1 + 1 > 2” principle.164−167 Liu’s group made the first attempt to couple the AIE-based FL imaging with magnetic resonance imaging (MRI) in 2013.164 The MRI technique, supplying desirable tissue penetration depth and free of radiation harm, is a noninvasive tool that has been broadly applied in clinical diagnosis in the past decades. However, MRI is lack of high sensitivity and resolution at single cellular level which are possessed by the FL imaging technique. Therefore, Liu et al. first made the AIE dots V


Review


existence of Au NPs, guaranteeing the efficient FL imaging. Moreover, the resulted 55-Au NPs owned the combined advanced features of large Stokes shift (∼120 nm), proper size (65−120 nm) particularly favorable for tumor-targeted imaging ascribable to the EPR effect, and superior biocompatibility. The samples of 55-Au NPs with a higher concentration exhibited a deeper color and were more radiodense compared to the more dilute samples (Figure 8E). The X-ray CT attenuation intensity (HU) of 55-Au NPs was well linearly correlated with the concentration (Figure 8E), indicating that 55-Au NPs were an ideal positive CT imaging nanoprobe. After being intravenously injected, the FL signal of 55-Au NPs at the tumor site gradually got more intense and was evidently distinguishable from the mice autofluorescence as the time elapsed (Figure 8F). At 24 h postinjection, the tumor area was much brighter than the other body part, attributed to the passive accumulation of 55-Au NPs in tumor tissue by the EPR effect. As depicted in Figure 8G, the average preinjection and postinjection HU values of 73 and 94 (6 h), 110 (12 h), 149 (24 h) shown by the CT26 tumor bearing mice injected with 55-Au NPs corresponded well with the multispectral FL imaging data, clearly demonstrating the capabilities of tumor-targeting and CT imaging. In a word, such a composite AIE nanoprobe has a variety of advantages including satisfactory cyto- and hemo-compatibility, long blood circulation half-life, superb tumor targeting ability, negligible in vivo toxicity, superior FL and CT imaging capacities, revealing the significant potential to be used as a dual-modal FL/X-ray CT nanoprobe for noninvasive in vivo tumor-targeted imaging and diagnosis. Although there are not many examples of AIE bioprobes with dual imaging modalities, it does not mean developing such systems is unimportant; on the contrary, it is of great significance to both fundamental research and clinical applications. The reason for the relatively insufficient advancement of this topic is the difficulty to compatibly fuse the two imaging modalities into a single probe without impairing their original advantages. Therefore, the design principles and strategies of the already reported dual-modal AIE bioprobes were carefully elucidated in this subsection in hope of stimulating more and better work in this promising research direction. 2.3.2. Dual/Multifunctional AIE Bioprobes. The examples discussed in the above subsections are mostly showcasing the monofunctional AIEgens and their applications in the biological area. Accompanying with the rapid development of AIE research and the progress in biomedical areas, recently, there have come out a large string of fabulous AIE bioprobes that enjoy dual or even multiple functions and can be employed for disease diagnosis and therapies.57−59,133,137,168,169 Integrating two or more functions in a single probe would largely reduce the complexity of probing system. Nowadays, developing AIEbased bioprobes with dual or multiple functions has become one of the mainstreams in AIE research. In this part, the most representative works would be selected out and discussed in details, with the aim of showing the charm, powerfulness, adaptability, and flexibility of AIE-based dual/multifunctional bioprobes. Integrating diagnostic imaging capacity with therapy efficacy could enable the drug to be tracked and monitored, which allows tracing drug distribution and release and assessing drug response and efficacy in a real-time and in situ manner. Exploring theranostic drugs that combine both therapeutic and diagnostic functionalities is among the most popular research

construction of more dual/multimodal imaging systems based on AIEgens. Delightfully, in 2014, Tang and Zheng et al. cooperatively developed the first AIEgen that truly held the capability of MRI and FL imaging.165 TPE-2Gd (54) shown in Figure 8A was constructed by the conjugation of a hydrophobic TPE unit with two hydrophilic Gd(III)-DTPA groups. The resultant amphiphilic 54 was AIE-active, could aggregate spontaneously into highly fluorescent nanomicelles, and functioned as a fluorescent agent for cell imaging with almost noncytotoxicity and superb photostability. 54 can be efficiently internalized into living HeLa cells and exclusively light up their cytoplasm with strong blue FL (Figure 8B). Notably, 54 was also able to work as a MRI contrast agent and showed a longitudinal relaxivity in water (R1 = 3.36 ± 0.01 s−1 per mM of Gd3+) comparable to those commercial agents such as Magnevist (R1 = 3.70 ± 0.02 s−1 per mM of Gd3+). In comparison to Magnevist, the circulation lifetime of 54 nanoaggregates in living rats was prolonged from 10 min to 1 h. The higher contrast and longer circulation lifetime demonstrated by 54 were ascribed to its ability to form nanoaggregates and enter the cells. The MRI maintained hyperintense in liver even after 150 min post administration due to the relatively high specificity to liver, excelling the Magnevist (45 min, Figure 8C). As a consequence, 54 could be employed to differentiate normal and lesion tissues. 54 could be gradually eliminated from body via the renal filtration thanks to the disintegration of its nanoaggregates into small particles or molecules during circulation. Thus, 54 owns great potential to be utilized as a MRI contrast agent specific to liver in clinical diagnosis. This study represented a new generation of materials that merge the AIE feature and magnetic relaxivity to create bioprobes with dual modalities of FL imaging and MRI. Besides MRI, another clinically approved imaging modality, namely X-ray computed tomography (CT), has also been successfully integrated with AIE-based FL imaging.166 The incomparable superiorities of CT including unlimited penetration depth and excellent spatial resolution render CT a widely utilized imaging technique in clinical diagnosis. The inherent shortcoming of CT imaging modality is its low sensitivity. Given the extremely high X-ray absorption coefficient regardless of the preparation method, shape, and diameter, gold (Au) NPs were chosen as the CT contrast agent in this work. In the meantime, as mentioned in Section 2.2.1, FR/NIR FL imaging is a highly attractive noninvasive imaging modality for its high sensitivity, low consumption as well as facile operation. However, even with FR/NIR emitters, FL imaging still has its own deficiencies, including relatively low spatial resolution and limited penetration depth. Thus, the combination of FR/NIR FL imaging and CT would hopefully derive a complementary dual-modal imaging probe which owns high spatial resolution, limitless tissue penetration depth, and high sensitivity. Because the Au NPs were reported to impose sever quenching effect on the FL, it is greatly challenging to fabricate FL/CT dual-modal imaging bioprobes. Considering its efficient FL and high resistance to self-quenching in the aggregated state, the FR/NIR AIE dye NPAPF (55, Figure 8D) was selected as the FL source. Co-encapsulating 55 and Au NPs into the DSPE-PEG2000 micelles through the convenient “onepot” ultrasonic emulsification method yielded the unique dualmodal AIE nanoprobe.166 Surprisingly, 55 exhibited relatively intensified FR/NIR FL (λem ∼ 640 nm) and retained the AIE attribute in the as-prepared nanomicelles in spite of the W


Review


Figure 9. Representatives of newly developed dual/multifunctional AIE bioprobes. (A) Bright-field and FL images of MCF-7 cells taken at different time points post the injection of 56. Adapted and modified with permission from ref 57. Copyright 2016 Royal Society of Chemistry. (B) FL image of HeLa cells stained with 57, optical density change of yeast incubated with different antifungal agents with various incubation time, and the antifungal activity evaluated by disc diffusion method. Adapted and modified with permission from ref 168. Copyright 2015 Wiley−VCH. (C) FL image of 58-stained A549 cancer cells, survival curves of A549 cancer cells receiving different treatment (left) or pretreated with various radiosensitizers (right). Adapted and modified with permission from ref 58. Copyright 2017 Wiley−VCH. (D) FL image of HeLa cells incubated with 59, the colocalization scatter plot for 59 in the mitochondria, the viability of HeLa cells when treated with 59 under different durations of white light irradiation, the viability of HeLa and NIH-3T3 cells treated with different concentration of 59 under white light irradiation, and FL and overlaid FL and bright-field images of PI-stained HeLa cells after being incubated without 59 (top), or with 59 in dark for 24 h (middle), or with 59 for 3h in dark followed by washing-away of the probe and irradiation with white light for 8 min and further incubation for 24 h (bottom). Adapted and modified with permission from ref 169. Copyright 2015 Royal Society of Chemistry.

topics in clinical fields. In this regard, Tang’s group developed an AIE-active antitumor theranostic agent, inspired by the similarity in the structures between TPE and tamoxifen (TMX).57 TMX is a selective modulator of estrogen receptor

(ER) for targeting breast cancer, and a chemotherapeutic agent for curing around 70% patient with ER-positive breast cancer. By smartly replacing the ethyl group of TMX with a phenyl group, a modified TMX derivative was afforded (56, Figure X


Review


ascribed to the increased inhibition of the phosphorylation of Akt and ERK. It should be emphasized that the “58 + light irradiation” attained a SER10 (the sensitizer enhancement ratio at 10% cell survival) value of up to 1.62, which was much higher than those of the most broadly used radiosensitizers, such as Au NPs (1.19) and paclitaxel (1.32). Furthermore, the SER10 of 1.62 for lung cancer should be considered as ultrahigh among the currently available radiosensitizers. Such a work is envisioned to intrigue new insights and outshining materials in radiation therapy field. Besides 58, another mitochondria-targeted multifunctional AIE bioprobe, i.e., 59, has been reported.169 AIEgen 59 (Figure 9D) is composed of two lipophilic triphenylphosphonium (TPP) units and a propanylidenemalononitrile-decorated TPE moiety (TPECM), wherein the TPP works as a mitochondriaanchor while the TPECM serves as an AIE motif and a PS. Benefiting from its AIE signature and D−A structure, 59 specifically visualized mitochondria with intense red FL in a turn-on fashion and showed a high colocalization coefficient of 0.956. Interestingly, 59 can be preferentially taken up by cancer cells relative to normal cells. It was also able to depolarize the mitochondrial membrane potential and selectively inflict strong chemo-cytotoxicity (in dark) on the studied cancer cells with an IC50 value of 6.31 μM. Moreover, upon white light illumination, 59 offered efficient generation of reactive 1O2 with potent phototoxicity. At the same probe concentration, 59 exhibited stronger inhibition to cell viability with longer irradiation time or at higher irradiation power. The IC50 value of HeLa cells was down to 0.69 μM, which was much lower than that recorded under dark condition. Very importantly, 59 exerted much less phototoxicity on the normal NIH-3T3 cells compared to that on HeLa cells, which was in good accordance with the observation that NIH-3T3 cells internalized less amount of 59 than HeLa cells. The PI staining further validated the MTT results. PI is a cell-impermeable dye that merely stains dead cells or late apoptotic cells with damaged membrane. As exhibited in Figure 9D, only a portion of 59-treated cells incubated in dark were stained, while almost all the cells were labeled by PI after exposure to white light irradiation for 8 min. It demonstrated that the combined chemo-therapy and PDT offered by 59 synergistically resulted in an enhanced anticancer effect in comparison to the single therapeutic approach alone. In addition, unlike the existing systems with combined chemoand PDT therapeutic effects which usually require the covalent conjugation of drug and PS or coencapsulation of them into NPs, 59 was the first molecular bioprobe for image-guided combined chemotherapy and PDT without direct drug conjugation. In this regard, 59 is thus a representative of a new generation subcellular targeted theranostic agent with multiple functions, including cancer cell recognition, organelle imaging, chemotherapy, and PDT. Such a simple probe design rationale laid a basis for further development of multifunctional molecular AIE probes. Aside from the dual/multifunctional molecular AIE probes, there have also emerged a great many AIE nanoprobes with dual or multiple functions.59,137 For instance, Zheng and Liu et al. formulated a type of AIE dots that could target cancer cells and be used for image-guided PDT of cholangiocarcinoma.137 The red-fluorescent AIEgen 2-(2,6-bis((E)-4-(phenyl(4′-(1,2,2triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl)-4H-pyran-4ylidene)malononitrile (TTD) was employed as both a FL signal source and a PS producing 1O2 to achieve PDT in this work (Figure 10A). By taking advantage of the one-step nano-

9A). Structurally analogue to TMX, 56 retained the antitumor attribute of TMX. In the meantime, 56 is essentially a TPE derivative and thereby was endued with the AIE feature. Unlike the almost nonfluorescent TMX, 56 was highly emissive as aggregates. Therefore, 56 was verified to be able to image and show a highly specific therapeutic response to the ER-positive breast cancer cells, indicative of its theranostic capability for the imaging and treatment of breast cancer (Figure 9A). Furthermore, the high luminosity and good photostability conferred the long-term cell tracking ability on 56, permitting the elucidation of its working mechanism. Such a work not only provided a new theranostic probe together with an approach to visually study the working principle of TMX but also opened a new pathway for drug development. In addition to the antitumor theranostic agent 56, Tang’s group and Liu’s team collaboratively invented a novel dualfunctional AIEgen featured with mitochondria-specific imaging and antifungal activities.168 Enlightened by the fact that many commercially available antifungal drugs contain imidazole moiety, an imidazole-cored new AIEgen was developed by following the RIM principle (Figure 9B). The diphenylimidazole derivative 57 was conveniently synthesized through the one-step coupling of aldehyde-substituted diphenylimidazole with 1,2,3,3-tetramethyl-3H-indol-1-ium. Attributed to the multiple rotors, highly distorted 3D conformation, and D−A structure, 57 exhibited typical AIE behaviors with a deep-red solid-state FL (λem = 642 nm). Owing to the positive charge on the indolium unit and the hydrophobicity of 57, it tended to enrich in the mitochondrial region with a moderate specificity. More importantly, 57 showed superior antifungal activity (Figure 9B). Five μM of 57 exhibited similar inhibition effect on the proliferation of yeast to 10 μM of miconazole, whereas 5 μM of miconazole displayed only inconspicuous inhibitory activity, implying that 57 possessed a lower concentration threshold of inhibitory effect. The evaluation of the antifungal activity of 57 by disc diffusion method manifested the much greater inhibition zone around 57, demonstrating that 57 is a more effective antifungal agent compared with its pyridiniumfunctionalized analogue DPI-BP and miconazole under parallel conditions. Besides acting as a fungi inhibitor, 57 could also serve as a staining agent to both live bacteria and fungi. Most recently, a multifunctional AIE bioprobe with mitochondria-targeting ability, 1O2 generation capability, as well as the radiosensitivity boosting capacity, has been reported by Tang, Li, and Ding et al.58 58 displayed in Figure 9C was rationally designed, with α-cyanostilbene as the simple AIE building block, a DPA unit as the strong electron donor to benefit the red FL and efficient light-controlled 1O2 generation, and a pyridinium salt as the mitochondria-targeting moiety. The marked AIE characteristics bestowed the capability to specifically light up mitochondria on 58. As the experimental results indicated, 58 was a typical PS that generated ROS and offered a 1O2-rich environment in the mitochondria under white light irradiation, but did not cause apoptosis or death of cancer cells. More significantly, raising intracellular level of 1O2 in mitochondria was proven to be a universal method to synergistically boost the radiosensitivity of cancer cells with the supra-additive effect of “0 + 1 > 1”. It is worth mentioning that in spite of the negligible toxic effect, 1O2 yielded by “58 + light irradiation” could affect cell behaviors and result in the enhanced radiosensitizing effect as well as the amplified antitumor efficacy, which was also regarded as PDT effect. Such outstanding radiosensitization effect was clarified to be Y


Review


Figure 10. Representatives of latest dual/multifunctional bioprobes based on AIE dots. (A) The synthetic route of 60-Tat dots; FL images of QBC939, L-O2, and HK-2 cells after being stained with 60-Tat dots for 4 h, where the red, blue, and green FL signals are from the 60-Tat dots, nuclei labels, and cytoskeleton labels, respectively; biodistribution of 60-Tat dots in mice bearing tumors 8 h post injection of 60-Tat dots without or with blocking the receptors; antitumor efficacy of different in vivo treatments. Adapted and modified with permission from ref 137. Copyright 2017 American Chemical Society. (B) Synthesis of 61-Tat dots; ROS generation of 61-Tat dots indicated by dichlorofluorescein diacetate; and the in vivo two-photon imaging and closure of brain blood vessels with 61-Tat dots. Adapted and modified with permission from ref 59. Copyright 2017 Wiley− VCH.

precipitation method, the integrin αvβ3-specific cRGD moieties (Tat) were facilely introduced into the dots of TTD (60) to reinforce the specificity to cancer cells. The working principle of this dual-functional probe can be interpreted as follows: In brief, when intravenously injected into a tumor-bearing mouse, the 60-Tat dots could accumulate into tumor interstitial fluid with the aid of EPR effect owing to the abnormal vascular architecture. The specific interaction between the cRGDs on 60-Tat dots and the overexpressed integrin αvβ3 on the cholangiocarcinoma cells surface enables the active targeting to cancer cells, triggers the ligand−receptor-mediated endocytosis process, and facilitates the efficient internalization of 60-Tat

dots. The obtained 60-Tat dots showed an absorption band peaked at 502 nm and an emission peak centered at 660 nm. When illuminated with light, the 60-Tat dots would outline the tumor with deep-red FL. The imaging of tumor will guide the subsequent PDT to induce tumor cell death. As anticipated, cholangiocarcinoma cells-targeted imaging was achieved by the 60-Tat dots in vitro and in vivo. As exhibited by Figure 10A, strong red FL was observed in the cancer cell, i.e. QBC939 cells, whereas nearly no FL was observable in the normal L-O2 and HK-2 cells. Much brighter FL signals were shown by tumors from the mice without blocking (Figure 10A). When injected with dots followed by 530 nm laser irradiation with a Z


Review

ACS Applied Materials & Interfaces power of 250 mW cm−2, obvious tumor shrinkage was exhibited 3 days later, clearly elucidating the PDT effect of 60-Tat dots. The simple synthesis, profound AIE feature, superb tumor specificity, and excellent PDT efficacy made the 60-Tat dots promising for translational research in cancer diagnosis and therapy in future. Besides the above-discussed one-photon excitable AIE dots for image-guided PDT, two-photon excitable dual-functional AIE dots with FL imaging and PDT capacities have also been reported.59 As mentioned in Section 2.2.2, one-photon excited FL (1PF) shows smaller tissue penetration depth and lower spatial resolution as compared to 2PF. As a result, the onephoton-activated PDT is restricted to the treatment of skin or hollow organs’ cancers and hence cannot reach its full potential. In this regard, two-photon PDT (2P-PDT) where two NIR photons instead of a single visible photon are used for PS activation is supposed to gain larger penetration depth and higher spatial selectivity, allowing us to treat a tiny pathologic region without affecting the surrounding normal tissue or enabling the treatment of solid and deeper tumors. Thereby, given that 2P-PDT might fully utilize the great potential of AIE PSs for highly precise and efficient PDT, the TPE-DC (61) was tailored as a 2P-PS.59 D−A structures are believed to be conducive to ROS generation and large 2PA cross-section. 61 was therefore constructed with the TPE unit as an AIE motif, the methoxy group as the electron donor, and the dicyanovinyl group as the acceptor (Figure 10B). The 61-Tat dots were prepared via the nanoprecipitation method using DSPEPEG2000 as the matrix and the HIV transactivator as a Tat peptide. The 61-Tat dots were about 40 nm in diameter, emitted a FR FL (λem = 620 nm) with an ΦF of 16%, and had a large 2PA cross-section of 3500 GM at 850 nm. The ROS generation ability under one-photon excitation of 61-Tat dots proved by the ROS indicator was much better than the commercial PS Ce6. More importantly, the ROS generation of 61-Tat dots excited by two-photon light source was verified to be very effective. Both in vitro and in vivo image-guided 2PPDTs were achieved by 61-Tat dots, by virtue of the high FL efficiency, large 2PA cross-section, efficient ROS generation, good photostability, and excellent biocompatibility. Upon injection of 8 mg kg−1 61-Tat dots, the brain blood vessels could be vividly visualized by the FR 2PF with excellent SNR (Figure 10B). The penetration depth up to 200 μm ensured the precise PDT action to occur in deep tissues. Arteries with a diameter of 5 μm situated at the depth of 100 μm were selected to conduct the precise blood-vessel closure. As can be seen from Figure 10B, the blood vessels continuously irradiated for around 4 min showed very faint FL, suggestive of their successful closure. In sharp contrast, the blood vessels stained by the Luminicell dots, commercial AIE NPs for multiphoton imaging, did not exhibit significant change before and after irradiation, eliminating the possibility of interference stemmed from the laser irradiation itself. This is the first example that the 2P-PDT was successfully exploited to close brain blood vessels with a precision up to 5 μm, which would definitely provide guidance to the future design of two-photon activatable PSs. A variety of AIE bioprobes with dual or multiple functions have been showcased in this small section. The form of these bioprobes varies from molecules to NPs. The output signal ranges from blue to red and finally to FR/NIR, and varies from 1PF to 2PF. Moreover, the therapy mode involved in these bioprobes ranges from chemotherapy to radiotherapy and all the way to PDT. All these studies have fully demonstrated the

superiorities of AIE. There are surely a few beautiful works that have not been presented here because of the page limitation.60,133 By taking advantage of such a jigsaw puzzle, we can achieve limitless dual-functional and multifunctional systems, which will undoubtedly widen the AIE scope and diversify the AIE-based bioprobes and more importantly might provide efficient probes for clinical use. 2.4. Trends and Progress in Probing Object and Application Outlet. In addition to the probe composition and form, the output signal, and the modality and functionality, the probing object and application outlet is also an important issue regarding to the development of bioprobes. Nowadays, the AIE-based bioprobes is becoming more and more precise and practicable, with the probing objects shifting from cells to specific organelles, from all cell lines to cancer cells, and from animals to microorganisms, and the application outlet changing from lab sample test to clinical real-sample analysis. These developing trends and their corresponding progresses will be elaborated in this section. 2.4.1. AIE Bioprobes for Specific Organelle Imaging. The discussions in sections 2.1−2.3 focus on trends and progresses in the probe composition and form, output signal, and modality and functionality of AIE probing systems. From this subsection on to Section 2.4.4, we will concentrate on the introduction of the most active research objects and application outlets of the AIE-based bioprobes, including specific organelles, cancer cells, bacteria, and real samples. Organelles, including membrane, mitochondria, lipid droplets (LDs), lysosomes, Golgi apparatus, nucleus, etc., play paramount roles in cellular function. For instance, the lipidbilayer cell membranes guarantee the relative stability of the intracellular environment and regulate the transport of substances into and out of cells. LDs can regulate intracellular lipid metabolism and storage. Mitochondria are the power workhouse of cells, and also involved in events such as cell communication, differentiation and apoptosis. Lysosomes are cleaners for they clear away disabled biomacromolecules and organelles, and engulf intruding pathogens. Nucleus behaves like the brain of cells for its maintenance of gene expression. The abnormality in biological activities of these organelles releases precise signals for a great many diseases such as diabetes, angiocardiopathy, Alzheimer’s diseases, Parkinson’s diseases, cancers and so on. Therefore, visualizing specific organelles and monitoring their morphology or function variations has great implications as it provides helpful information on cell bioactivities at the subcellular and/or molecular level for disease diagnosis. FL imaging has been proven to be an effective tool for specific organelles analysis. Because the AIEgen-based FL imaging owns diverse advantages over the FL imaging based on the conventional fluorophores, a large quantity of AIE bioprobes have been developed to specifically target different organelles,46 including membranes,39,81,170 LDs,171−176 mitochondria,44,45,58,163,168,169,177−184 lysosomes,86,185,186 and nucleus.87,187 Among these organelles, the mitochondria and LDs are the most actively studied by AIE bioprobes. Thereby, in this subsection, only the bioprobes specifically designed for LDs and mitochondria would be highlighted to exemplify the great potential of AIE in organelle-specific imaging. LDs are highly dynamic lipid-rich organelles that exist mainly in adipose tissues, function as a reservoir for neutral lipid storage, regulate the lipid metabolism and stockpile, and are highly involved in membrane transfer for supplementing acylAA


Review


Figure 11. Molecular structures of AIE bioprobes designed for specific imaging of (A) lipid droplets and (F) mitochondria. FL images of HeLa cells stained with (B) 62 and (C) 63 or 64. Reproduced with permission from refs 172 and 173, respectively. Copyright 2016 Royal Society of Chemistry and 2016 American Chemical Society. (D) One-photon and two-photon FL images of HeLa cells incubated with 65. Reproduced with permission from ref 175. Copyright 2017 Royal Society of Chemistry. (E) CLSM images of live HCC827 cells stained with 66 before and after photoactivation. Reproduced with permission from ref 176. Copyright 2017 Royal Society of Chemistry. (G) FL images of HeLa cells and mouse sperm cells stained by 67, and the merged FL image of HeLa cells incubated with 67, Hoechst 33342 and the bright-field image. Reproduced with permission from ref 179. Copyright 2015 Royal Society of Chemistry. (H) FL image of HeLa cells stained with 68, and the confocal images of HeLa cells costained with 68 (yellow) and LysoTracker Red DND-99 (red) in the presence of rapamycin. Reproduced with permission from ref 44. Copyright 2015 Royal Society of Chemistry. (I) FL image of 69-stained HeLa cells (left), and CLSM images of living zebrafish embryos stained with 69 (right). Reproduced with permission from ref 182. Copyright 2016 Wiley−VCH.

The first AIE-based bioprobe for LD-specific imaging was reported by Tang’s group in 2014.171 It has been well-known that TICT dyes show polarity-dependent FL, as their emissions get intensified and blue-shifted along with the polarity decrease. Such a LD probe (TPE-AmAl) took on a TPE-cored D−A structure and was therefore endued with TICT plus AIE characteristics. The FL color of TPE-AmAl altered from orange to blue when the environment varied from polar to nonpolar. With high selectivity, good biocompatibility, and superb photostability, TPE-AmAl was successfully applied as a FL

glycerols and cholesterol. Abnormal LD activities or numbers indicate various diseases including type II diabetes, fatty liver diseases and inflammatory myopathy. Mapping the location and distribution of LDs is of great use for early diagnosis of associated diseases. The highly hydrophobic lipid environment has been considered as the key point of the LD-targeted bioprobe design. In this sense, any AIEgen that could sensitively respond to the polarity change should possess the potential for LD detection and imaging. AB


Review


with a blurred background by 1PF, a much clearer image of LDs was presented under the two-photon excitation. This was attributed to the intrinsic sectioning ability of two-photon microscopy (2PM) that allows a small layer of the fluorogens was excited at the focus plane while all of the fluorogens were excited in the light path in one-photon microscopy (1PM). The 2PM image of LDs in fixed liver tissue slice stained by 65 exhibited a clearer vision and much lower background than the 1PM ones. The FL signal of the spherical spots in the liver slice could be detectable at a depth of 45 μm, demonstrating the great potential of 65 for tissue slice-based disease diagnosis of LDs. FL probes that can be photoactivated are highly desirable for cell biological studies, as they can realize light-controlled imaging with high spatial and temporal resolution. In response to the call for highly precise probes for LD-specific imaging in live cells, Tang’s group creatively developed a series of photoactivatable AIE probes.176 66 was selected as an example in this review. The dihydro-2-azafluorenone 66 can readily undergo a photo-oxidative dehydrogenation, generating the 2azafluorenone characterized with AIE behaviors. The excellent photoactivation efficiency of 66, the AIE nature of its oxidation product, as well as the lipophilicity of 66 and its oxidation resultant collectively made 66 a superb photoactivatable probe for live cell LD imaging. 66 can be efficiently taken up by HCC827 cells and preferably located in the LDs, and a very rapid FL turn-on process in less than 2 min was observed under irradiation at 405 nm with merely 1% laser power (Figure 11E). The obtained light-up ratio was determined to be as high as 265. Such a fast FL switch-on effect was also observed in the A549 cells. Merely few LDs in normal lung HLF cells were lit up by the photoactivated 66, as compared with lung cancer cells such as HCC827 and A549. These results indicated that as a photoactivatable and LD-targeted probe, 66 can be utilized to distinguish the cancer and normal cells through the difference in expression levels of LDs. Meanwhile, since light is a valuable and facilely controllable external trigger with high spatial and temporal accuracy, sequential photoactivation of 66 in selected cells can be achieved in a multicellular environment. As it has its own DNA that can replicate and express independently, mitochondrion is referred to as a semiautonomous organelle with genetic material. It functions as a power factory for the oxidation of carbohydrates, fats, and amino acids to yield energy currency adenosine triphosphate (ATP); plays important roles in many biological processes including cell differentiation, communication, and apoptosis; and holds the capability to modulate cell growth and cell cycle. Mitochondria movement is largely associated with their morphology, as such directly visualizing mitochondrial dynamics and changes via FL technique would offer important information for the understanding of mitochondria-involved metabolism and diseases. The negative potential of mitochondria is several orders of magnitude higher than that in the other organelles, providing an opportunity to design targeting ligands for mitochondria-specific imaging. Since the first AIE bioprobe for mitochondria imaging reported by Tang’s group in 2013,177 there have sprung out a great number of excellent mitochondria-specific probes up until now, and the related research has been an active topic. As some of them have been discussed in the above subsections and some will be introduced in the following subsection, in this part, only three examples will be highlighted to reveal the importance of developing mitochondria-specific bioprobes.

probe for LD-targeted imaging and tracking in live HeLa, liver L-O2 cells, and green algae. The incubation with TPE-AmAl readily made the spherical LDs visible with intense greenish blue FL and a low background. The better photostability of TPE-AmAl originated from its AIE nature allowed the better LD imaging performance as compared with Nile red. Subsequently, Tang et al. further designed the NIR-emissive AIEgen TPE-AC (62) and utilized it for LD imaging to avoid the interference from autofluorescence of cells.172 Turning the aldehyde group on TPE-AmAl into a propanylidenemalononitrile unit afforded 62, a novel AIEgen with a stronger D−A effect (Figure 11A). 62 could efficiently enter the live HeLa cells, be prone to enriching in the LDs, and light up them with bright NIR FL (λem = 705 nm; Figure 11B). Moreover, 62 possessed high specificity to LDs, low cytotoxicity and excellent photostability, and hence was able to monitor the LD accumulation in live cells. The long-wavelength absorption (λex = 455 nm) and NIR FL made 62 a promising bioprobe for in vivo deep-tissue LD imaging, real-time monitoring of the macrophage foam cells, as well as the early diagnosis of liver diseases. Apart from the fluorogens possessing combined AIE plus TICT properties, ESIPT + AIE luminogens, namely 63 and 64, have also been reported to be able to target LDs in living and fixed cells.173 The salicylaldehyde Schiff-base structures conferred remarkable ESIPT feature on these two AIEgens, owing to which, the yellow-emissive 63 and orange-fluorescent 64 exhibited superior Stokes shifts of as large as about 200 nm. The LDs in both A549 and HeLa cells could be clearly visualized by the bright FL of 63 and 64 in a high resolution and contrast fashion (Figure 11C). The overlap ratios of 63 and 64 with the commercial LD-targeting BODIPY were up to 98% and 97%, respectively, indicative of the high specificity to LDs. Even incubated with 63 or 64 at a concentration of up to 10 μM, no evident inhibition on HeLa cell growth was observed, implying the satisfactory biocompatibility of these probes. Upon continuous light irradiation for 60 min, the FL intensities of 63 and 64 kept unchanged. In stark contrast, the FL signal loss of BODIPY arrived at 90% after merely 5 min irradiation. Moreover, there was almost no change in the FL of 63 and 64, when the pH varied from 1.0 to 11.0, suggesting their great tolerance to pH alteration. In addition to the one-photon excitable AIE-based LDtargeting probes, most recently, Tang’s group has reported a two-photon excitable AIE bioprobe that specifically imaged LDs under the excitation of 840 nm laser.175 TPA-BI (65) consisting of a benzylidene imidazolone moiety and a TPA unit was facilely synthesized in a good yield via the Suzuki coupling. The D−π−A-structured 65 was featured with AIE, TICT, solvatochromism properties, and 2PF, which resulted in the large Stokes shift of up to 202 nm and a 2PA cross-section of as large as 231 GM. With an excitation at a wavelength of 400− 440 nm, the preferential accumulation of hydrophobic 65 in the lipophilic spherical LDs resulting from the “like−like” interactons enabled the light-up of LDs by the intense greenish blue FL (Figure 11D). The LD-specific imaging ability of 65 was not limited by cell lines. With AIE attribute and good specificity to LDs, 65 exhibited a lower background as compared to BODIPY 493/503. More importantly, 65 can be employed for 2PF imaging of LDs, and showed higher 3D resolution, lesser photobleaching, lower damage to cells, smaller interference by autofluorescence, and larger penetration depth. Specifically, sharply contrasted to the clustered LDs observed AC


Review


Besides the AIE bioprobes for in vitro mitochondria imaging, AIE systems capable of in vivo specific imaging of mitochondria have also been reported. Probe TPE-PyN3 (69) is actually a precursor for 68. 69 and 68 share the same molecular framework except for the terminal group, and hence enjoy similar photophysical properties as well as bioprobing ability.182 For example, 69 also showed both AIE and TICT behaviors, strong aggregated-state yellow FL, low cytotoxicity, and the mitochondria-targeting ability. When incubated with 69 for as short as 5 min, the mitochondria of HeLa cells became fairly emissive, whereas the other cellular parts remained dark (Figure 11I), manifesting the preferential enrichment of 69 in mitochondria. The excellent specificity with a Pearson’s correlation coefficient of 0.96, high SNR and fairly good contrast enabled the morphological details to be visualized with a superb resolution. The HeLa cells were stained with 69 or 5chloromethylfluorescein diacetate (CMFDA), a noted commercial agent for long-term cell labeling due to its chemical reaction with thiols on proteins and peptides to form aldehydefixable conjugates, and subcultured every 24 h. Even after five generations, the FL signal from 69 was still well kept in live cells, which rivaled to that of the CMFDA. Without the need of chemical reaction, 69 possessed outstanding long-term cell tracking ability, making it more favorable for biological research. Moreover, 69 was much more resistant to photobleaching and photo-oxidation compared to CMFDA. The above-mentioned remarkable merits of 69 allowed it to image cell morphology in living zebrafish embryos. No obvious change of the mortality rate between the treated and contrast groups was observed, when the live zebrafish embryos at 72 h stage were incubated with 3 μM of 69 for 2 h. This indicated a low toxicity of 69 to zebrafish embryos. After vital staining, 69 spread over the whole surface of embryo with intense FL (Figure 11I). It is noteworthy that all the surface cells were intensively fluorescent, suggesting the high membrane penetrability of 69. Notably, 69 predominantly stained the mitochondria in cells, which enabled the precise visualization of the shape, boundary, and location of each cell as well as the mitochondrial and nucleus morphologies within live embryos. Besides, living zebrafish embryos labeled by 69 kept emissive and the cellular morphology were still clearly visible even after 60 h, which was in line with the in vitro results and suggested the outstanding retention and stability of 69 in vivo. Moreover, the wide spatial distribution capability and excellent photostability of 69 permitted the morphology of embryo to be reconstructed in 3D with a high resolution. As the early stages of cell apoptosis could be revealed by the disruption of mitochondrial ΔΨm and 69 was sensitive to ΔΨm variation, the in vivo cell apoptosis could be sensed by 69 in real time. During the apoptosis of cells in the living zebrafish embryo stained by 69, the reduced ΔΨm resulted in the diffusion of 69 from intracellular mitochondria to extracellular medium, leading to the marked FL “turn-off” response. Real-time imaging of cell behaviors such as population and apoptosis within living multicellular organisms is of great value to life science. As such, this work provided a promising simple and noninvasive bioprobe for cell behaviors study in living organisms. Additionally, photoactivatable mitochondria-specific probe was also been developed and the super-resolution imaging of mitochondria was successfully achieved on the basis of the photocyclodehydrogenation of the AIE probe.180 Moreover, the mitochondria-anchoring probe for the selective recognition of cancer cells were also reported, whereas some examples of

TPE-Ph-In (67) shown in Figure 11F is a cationic AIEgen with an indolium salt-decorated TPE skeleton.179 It fluoresced in the red light region in the aggregated state with a Stokes shift of more than 200 nm. Besides selectively and uniformly staining the mitochondria in live cells with a high SNR (Figure 11G), 67 also showed a photostability much superior to the commercially available red-emissive mitochondrial probe MitoTracker red FM, thanks to the AIE properties. Mitochondrial potential ΔΨm is a critical parameter indicating the mitochondrial functional status, and therefore closely associated with cell health, damage and function. Meanwhile, ΔΨm is also a main driving force for cationic hydrophobic dyes to enter and stain the mitochondria. It is worth noting that ΔΨm could be directly reflected by 67 on the basis of the positive correlation between the FL intensity and the local concentration of 67 in mitochondria, which can hardly be achieved by traditional dyes owing to the ACQ effect. Using CCCP and oligomycin as stimulants, the changes in the intracellular ΔΨm were traced by 67 in situ and in real time. As ΔΨm is an indispensable indicator for evaluating the viability and fertilization capacity of a sperm, the well biocompatible and ΔΨm-indicatable 67 was used to image sperms and assess their viability. As illustrated in Figure 11G, the midpieces of sperms exhibited various degrees of FL intensity. It was found that the brilliant red FL originated from the vital sperms, whereas the unenergetic sperms merely gave very faint red FL or even non-FL, which suggested that 67 was a promising probe for sperm activity monitoring. An isothiocyanate-bearing TPE derivative was designed and synthesized as a bioprobe for mitochondria imaging by Zheng and Tang et al.44 The covalent anchoring of the bioprobe TPEPyNCS (68) to mitochondrial proteins bestowed it with high resistance to microenvironmental variations, facilitating the real-time monitoring of mitophagy. Since mitophagy is of great importance in maintaining cell health, the development of new tools for mitophagy tracking is intriguing and urgently demanded for better understanding of the mitophagy mechanism. 68 was an AIEgen showing TICT effect, and the highly bright yellow FL of 68 was quickly observable in the mitochondrial region when incubated with HeLa cells for 15 min (Figure 11H). The high specificity to mitochondria of 68 was confirmed by the Pearson’s correlation coefficient of up to 0.97. Noteworthily, the FL from the mitochondria in fixed cells was mostly preserved even after repetitive washing with acetone or DMSO, implying the formation of chemical bonds between 68 and mitochondrial proteins. 68 was highly resistant to microenvironmental changes as well as photobleaching, and thus suitable for real-time monitoring of the mitophagy process. The rapamycin, a well-known drug, was used to trigger the mitophagy, and in the meantime, the red-fluorescent LysoTracker Red DND-99 (LTR) was utilized to label the lysosomes. HeLa cells were coincubated with 68 and LTR simultaneously. No observable changes occurred in the yellow FL of mitochondria and the red FL of lysosomes in the time range of 0 to 72 min post rapamycin addition. However, at 73.5 min, a new red FL spot overlapping with the mitochondria emerged. It implied the generation of acidic autophagosome, which moved to mitochondria to initiate the mitophagy process. The vanishing of autophagosome at 79.5 min was an indication of the completion of mitophagy. Moreover, the mitochondrial region overlaid with autophagosome exhibited relatively weaker FL with respect to the unaffected part, further verifying that the mitochondria were hydrolytically disgested by autophagosome. AD


Review


Figure 12. Representatives of AIE bioprobes for selective cancer cell recognition and killing. Molecular structures of (A) 70a and 70b, and (C) 71. (B) CLSM images of HepG2 and HEK293 cells treated with 70a (upper pannel) or 70b (lower pannel). Reproduced with permission from ref 189. Copyright 2016 Royal Society of Chemistry. (D) FL images (left) and relative FL intensities of different cell lines after the incubation with 71 (200 nM) for 20 min. (E) Change in mitochondrial morphology before (upper) and after (lower) white light irradiation. Reproduced with permission from ref 47. Copyright 2017 Royal Society of Chemistry.

bioconjugates and AIE-Tat dots, the cancer-selectivity of the bioprobes presented here are not restricted to one or two types of cancer cells, instead, they can differentiate all cancer cells from normal cell no matter which type is.47,183,188,189 For instance, Zhang and Zhao et al. reported two pyridiniumfurnished TPE salts that could specifically image and exert selective cytotoxicity toward cancer cells in vitro and in vivo.189 The resultant TPE derivatives 70a and 70b shown in Figure 12A were AIE-active and fluoresced with the maximum at 605 and 620 nm, respectively. The high FL efficiency allowed to monitor the interaction of probes with different cells. Brilliant red FL was only detected in the cancer cells including HepG2, HeLa and A549 cells after incubation with 70a or 70b, whereas no FL was observable for normal cells such as HEK293, Chang liver and L-O2 cells with the same treatment (Figure 12B). These results clearly demonstrated the high selectivity of 70a and 70b to cancer cells. The distinct responses of 70a or 70b to cancer and normal cells was attributed to the different membrane potentials of cancer and normal cells. Membranes of cancerous cells are more significantly negatively charged in comparison to those of normal cells. Thus, the postively charged 70a and 70b could interact more efficiently with cancer cells instead of with normal cells. Furthermore, the hydrophobic TPE framework in 70a and 70b also contributed to their high internalization efficiency into cancer cells through the interaction with lipid bilayers. As revealed by the FL colocaliztion assay, 70a and 70b gave high coefficients of 0.94 and 0.91 with the commerically avaliable MitoTracker Green, suggestive of the good targeting ability to mitochondria. It has been recognized that cancer cells usually possess more mitochondria and their mitochondrial membranes are unusually negatively charged, that is why 70a and 70b selectively targeted the mitochondria of cancer cells. Considering that selectively killing cancer cells without damaging healthy cells is a long-awaited aim of cancer therapy,

mitochondria-targeted cancer therapies have already been elucidated in Section 2.3.2 and another two will be discussed in the next subsection. In this small section, a series of LD-targeted AIE probes with FL color ranging from greenish blue to yellow to orange to NIR were presented. Meanwhile, two-photon excitable and photoactivatable LD probes were also detailedly discussed to show the most striking progresses in the AIE-based LD-specific imaging probes. As for mitochondria-specific AIE bioprobes, the most recent progress was showcased by three typical examples. As compared to the conventional organelle-specific imaging agents, AIE bioprobes hold comparable specificity and biocompatibility, but better photostability, longer cellular retention, higher contrast, and larger SNR. Undoubtedly, the AIE feature generally makes the probes outperform their ACQactive opponents. 2.4.2. AIE Bioprobes for Selective Cancer Cell Recognition and Killing. Cancer has been recognized as the leading cause of worldwide morbidity and mortality. Early detection of cancer before the metastasis is the most effective way to increase the survival rate of patients, but remains very challenging. In particular, the diagnosis of cancer at the cellular level is even more challenging for the great difficulty in the discrimination of cancerous cells from normal cells. FL techinique has been verified to be superior to conventional imaging tools in many aspects. As demonstrated in the above subsections, AIE-based imaging agents have been widely explored for cancer detection, making the devlopment of cancer-selective bioprobes based on AIEgens become a new and attractive trend in AIE research. Generally, the AIEgens (Section 2.1.1) or AIE NPs (Section 2.1.2) were usually conjugated with tumor-targeting bioligands, such as peptides or antibodies to achieve higher selectivity to cancer cells. In this subsection, AIEgens without being coupled with tumor-specific bioligand but highly specific to cancer cells would be discussed in details. In comparison to the AE


Review


Figure 13. Typical examples of AIE bioprobes for bacteria imaging and/or killing. (A) Molecular structures of 72−74. (B) CLSM images of living (left) and dead (right) bacteria cells incubated with 72 for 0.5 h. Reproduced with permission from ref 49. Copyright 2014 Wiley−VCH. (C) CLSM images of S. epidermidis stained with 73 for 10 min, and FL photographs of 73 in the MOPS/ethanol (8/2, v/v) solution without or with 108 CFU mL−1 of S. epidermidis. (D) Illustration of the high-throughput antibiotics screening strategy based on the FL change of 73. Reproduced with permission from ref 51. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (E) CLSM images of S. epidermidis incubated with 74 for 10 min. (F) The killing efficiency of 74 on E. coli and S. epidermidis in the absence and presence of room-light irradiation for different time periods. Reproduced with permission from ref 191. Copyright 2015 American Chemical Society. (G) CLSM images of B. subtilis, E. coli, Van A, and Van B incubated with 26 (5 μM) for 15 min. Insets are the FL photographs of bacteria suspension treated by 26 (20 μM) in the presence of graphene oxide (1.6 μg). (H) Biocidal activity of 26 toward B. subtilis, E. coli, and Van B. Reproduced with permission from ref 90. Copyright 2015 Royal Society of Chemistry.

the cytotoxicity of 70a and 70b toward both cancer and normal cells was evaluated with the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Being treated with 70a or 70b, the viability of HepG2 cells was determined to be as low as 13 and 29%, respectively. Simiarly, the viabilities were only 23 and 31% for A549 cells, and 23% and 8% for HeLa cells when underwent respective treatments with 70a and 70b. In contrast, all the normal cells subjected to the same treatments were healthy and remained almost unaffected. It means that 70a and 70b were selectively toxic to cancer cells. Further experimental results indicated that the selective cytotoxicity of 70a and 70b to cancer cells may operate through the cell apoptosis mediated by mitochondria damages. 70a and 70b can alter the membrane potential and

inhibit the oxidative phosphorylation in mitochondria, and meanwhile the reduced ATP supply in the mitochondria of cancer cells caused by 70a and 70b finally resulted in cell death. Benefiting from their cancer cell-specific uptake as well as the mitochondria-damaging cytotoxicity, 70a and 70b were suited for in vivo antitumor therapy. These probes can exclusively enrich in tumor sites and impose efficient suppression on tumor growth with negligible systemic toxicity. In this sense, 70a and 70b were also multifunctional AIE probes which act multiple roles such as cancer cell-specific imaging agent, mitochondria anchor, and chemotherapeutic drug. It has also been found that the counter anions in 70 were decisive to the emission, size, surface charge, as well as the resultant cancer cell AF


Review


the diseases caused by bacterial infections pose a grave challenge to human. The most important issues in the bacterial-related researches are the bacterial detection (e.g., imaging), bacterial viability evaluation, antibiotics screening, and bacterial killing. FL-based methods have been widely used for such purposes due to its rapidity, simplicity and sensitivity, among which the AIE-based FL bioprobes have drawn considerable attentions, making the bacterial imaging and killing become a vigorous research subject in AIE area.49−51,90,91,190−194 Such an intriguing research trend will be reflected by the examples shown in Figure 13. Tang’s group reported the first AIE probe for bacteria studies, which can discriminate dead bacteria from living ones and worked as a highly fluorescent and photostable probe for long-term monitoring of bacterial viability.49 The designed diboronic acid-decorated TPE derivative TPE-2BA (72, Figure 13A) cannot stain live bacteria but can selectively light up dead bacterial cells with bright blue FL, no matter the bacteria is Gram-positive or negative (Figure 13B). As revealed by the experimental results, 72 stained bacteria by means of binding to the DNA grooves. 72 can hardly penetrate the intact membranes of living bacteria but can pass through the damaged membrane of dead bacteria and enter the protoplasm to interact with intracellular nucleic acids. In this way, the RIM process of 72 came into play, arousing the intense emission. In other words, the light-up response of 72 to dead bacteria was ascribed to the loss of membrane integrity, which is a critical indicator of cell death. In comparison to commercial viability probes, AIEgen 72 is brightly fluorescent, highly photostable and fairly compatible to bacteria, and hence well-suitable for long-term tracking of bacterial viability. Because effective bactericides can cause bacterial death and lead to compromised membrane, 72 was suited to screen bactericide. When the bacteria were treated with effective bactericide, 72 in the system would become intensively emissive to indicate that the bacteria are killed, demonstrating the feasibility of utilizing 72 for bactericide screening. In the battle between human and harmful bacteria, antibiotics are regarded as potent weapons of human beings. Despite this, nowadays, the power of antibiotics is gradually weakened by the increasingly emerged antibiotic resistance of bacteria. With the aim to alleviate or solve the antibiotic resistance problem, endeavors should be devoted to developing efficient approaches to evaluate bacterial susceptibility and selecting out the most effective antibiotics for infection prevention and treatment. To this end, Tang et al. further rationally designed a new AIEgen, and established a facile and rapid method for bacterial susceptibility assessment and high-throughput antibiotics screening based on this AIE probe.51 73 exhibited remarkable AIE effect and emitted a bright orange FL (λem ≈ 570 nm). The long alkoxy chain together with the TPE core conferred the hydrophobicity on 73 while the positively charges amines endued it with hydrophilicity, cooperatively making 73 an amphiphilic compound (Figure 13A). It is dissolvable in water at a concentration lower than 20 μM, but form micelles at higher concentrations. As the bacterial cell envelop is negatively charged, the probe 73 bearing two amine groups and positively charged could bound to the bacteria via the electrostatic interactions, activating the RIM process and imaging/sensing the bacteria in a FL light-up mode (Figure 13C). 73 was able to stain both Gram-positive and Gram-negative bacteria. Benefiting from its AIE nature, the free 73 molecules remained nonemissive and only those bound to bacteria were switched

specificity and toxicitiy, offering valable clues for the molecular design of cancer cell-selective theranostic probes. Unlike the chemotherapeutic 70a and 70b, the novel AIE theranostic agent 71 (Figure 12C) developed by Tang’s group could selectively killing cancer cells via PDT. 47 The isoquinolinium-based fluorogen 71 displayed marked AIE effect and showed bright yellow FL in the aggregated state. HeLa cells stained with 71 exbibited intense greenish yellow FL. The correlation coefficient of the images of HeLa cells incubated by 71 and MitoTracker Red were estimated to be up to 0.98, indiative of the superb specificity of 71 to mitochondria. As reported, the mitochondria membrane potential of cancer cells is at least 60 mV higher than that of the normal cells due to their more active metabolism, which favors the interaction with oppositely charged species. It was found that all the cancerous cells, such as MCF-7, PC-9, MDA-MB-231, A549, HeLa, HCC827, and HepG2, incubated with 71 displayed bright greenish yellow FL signals (Figure 12D). Comparatively, normal cells including COS-7, LX-2, HEK-293 and MDCK-II exhibited much fainter FL. Notably, the clear and fine structures of mitochondria in HeLa cells were observable with strong greenish yellow FL, whereas nearly no FL was detectable in COS-7 cells. In addition to the targeting ability to a broad range of cancer cells, 71 could also selectively accumulate merely in the mitochondria of cancer cells in the mixture of cocultured normal and cancer cells. The ability of distinguishing cancer cells from normal cells with high contrast was originated from the difference lying in mitochondrial membrane potential as well as the mitochondria quantity. Furthermore, 71 could also function as a PS, producing ROS upon light irradiation for PDT. As depicted in Figure 12E, the mitochondria of 71-stained HeLa cells transformed from intact reticulum into irregular puncta after being illuminated with white light. It suggested that the ROS yielded by 71 could damage the mitochondria and eventually kill the cells through PDT. The white light illumination of HeLa cells untreated by 71 did not bring any change to the cell viability, while in the presence of 71 and white light irradiation, the HeLa cell viability decreased with the increase in the concentration of 71. No evident change in the cell viability of 71-treated HeLa cells was observed in dark, indicating the minimal chemotoxicity of 71 toward cancer cells. The combined cancer cell recognition, mitochondria targeting, and PDT made 71 a promising theranostic probe. What’s more, the excellent FL contrast between the cancer and normal cells incubated with 71 enabled their discrimination, rendering 71 useful in image-guided surgery. These interesting works clearly demonstrated that the smart combination of hydrophobic AIE molecular skeleton and appropriate positive charge species can give rise to new multifunctional probes that can be synchronously utilized for selective cancer cell imaging and antitumor therapies. By virtue of such a design rationale, uncountable cancer-specific theranostic AIE bioprobes could be developed by altering the AIE core and/or changing the positively charged species. 2.4.3. AIE Bioprobes for Bacteria Imaging and/or Killing. Microorganisms, e.g., bacteria, exist everywhere and play various roles in all the aspects of human life, including environmental monitoring, food and water, medical hygiene, pharmaceutical industry and so on. A majority of bacteria are benign to human health and some of them such as Escherichia coli (E. coli) are even beneficial to human beings. However, some species of bacteria are harmful or even fatal to people, and AG


Review


enhance the killing efficiency of 74 posing on both Grampositive and -negative bacteria. Their viability could be reduced to less than 1% by 1 h white-light illumination. The photosensitizing ability of 74 accounted for the photodynamic killing effect as well as the enhanced bacterial killing efficiency of 74 under light irradiation. The 74-containing agar plate can be repetitively and continuously used and remained effective, manifesting that 74 was a photostable and efficient PS capable of bacterial elimination. Besides the above-discussed chemically composed AIE probes, those comprising chemical AIE motif and biological ligand(s) have also been applied for bacteria research.90,91 For example, as mentioned in Section 2.1.1.1, Liu and Zhang et al. collaboratively designed a light-up probe based on the AIE bioconjugate TPEPM-2Van (26, Chart 2) for selective imaging, naked-eye recognition and photodynamic killing of Grampositive bacteria.90 Owing to the two hydrophilic Van moieties, 26 was highly water-soluble and merely faintly fluorescent in dilute aqueous solution or incubated with Gram-negative bacteria like E. coli. Comparatively, when interacted with Gram-positive bacteria, such as B. subtilis, strong red FL peaked at 650 nm of 26 was turned on (Figure 13G). Although not intense, the 26 incubated with the Van-resistant enterococcus (VRE) stains, such as Enterococcus faecium (Van A) and Enterococcus faecalis (Van B), did show red FL. It implied that the dual-decoration of Van groups on the TPECM core enhanced the binding affinity toward VER bacteria. Distinct from the ACQ dyes that exhibited intrinsic FL before binding to bacteria and demanded additional washing steps to reduce the background, the bioconjugate 26 could eliminate the background noise by virtue of its AIE attribute. As such, direct naked-eye identification of bacteria in solution was achieved at a probe concentration of 20 μM with the aid of a small amount of graphene oxide (GO). The insets of Figure 13G further proved that 26 could specifically stain Gram-positive bacteria regardless of Van-sensitive or VRE stains. Thanks to the dark toxicity endowed by the antibiotic Van units and the photodynamic inactivation ability conferred by the TPECM moiety, 26 displayed significant antibacterial effects toward Gram-positive bacteria including VRE strains. As can be seen from Figure 13H, 26 imposed great dark toxicity to B. subtilis, and nearly all the B. subtilis could be killed by 26 at a concentration of 10 μM. With the assistance of white light, even 2 μM of 26 was able to eliminate all B. subtilis. To the contrary, almost no toxicity was shown by 26 toward Gram-negative E. coli. Additionally, in the dark, 26 posed low killing efficiencies to VER strains, due to their resistance to Van, whereas the photodynamic treatment with light irradiation dramatically improved the killing efficiency. Apart from the small AIEgen and AIEgen-peptide bioconjugates, nanoassemblies loading TPE derivatives have also been constructed via layer-by-layer self-assembly for responsive detection and elimination of bacteria.194 Moreover, even dualmodal FL/MR probe has been developed for concomitant bacteria recognition and inhibition, based on the amphiphilic TPE-cored star polymers with Gd(III)-chelated arms.50 The FL colors of the AIE probes presented in this small part almost cover the whole visible light range from blue to orange to red. Their functions range from bacteria imaging, bacteria viability and susceptibility assessing, antibiotics screening all the way to bacteria killing. Their outstanding performances would definitely intrigue researchers to pursue more AIE-based bioprobes for bacteria-related studies.

on, enabling the bacteria clearly imaged with a high SNR and without the need for washing process. It simplified the imaging procedures, avoided the bacteria loss during washing, and consequently improved the accuracy of bacterial quantification. Moreover, the bacteria detection could be realized in bulk solution. The MOPS/ethanol solution (8/2, v/v) of 73 containing S. epidermidis was intensely orange-fluorescent, whereas that without bacteria emitted nearly no FL. Such a difference can be witnessed by naked eyes (Figure 13C). It is noteworthy that the FL intensity of 73 was found to be linearly correlated with the bacteria concentration, allowing the easy identification of bacteria amounts in the culture media containing different antibiotics. As such, because of the FL switch-on visualization of bacteria, simple imaging processes with increased accuracy, and positive linear relationship between bacteria quantity and FL intensity, high-throughput antibiotics screening was achieved by 73 within 5 h, which was significantly shortened as compared to traditional method. With effective antibiotics, the bacterial growth will be retarded or completely inhibited, leading to a reduced bacteria amount and resulted weaker FL compared to those with ineffective antibiotics (Figure 13D). With the same antibiotic, the bacteria strain showed different susceptibilities and resulted in distinct FL intensities. In this way, the bacterial susceptibility can be facilely and reliably evaluated. Exploring new antibiotics, although useful to fight against the antibiotic resistance, is always suffering from its long-term and costly development process. Recently, new disinfection pathways with which bacteria can hardly generate resistance are attracting increasing research interest. Utilizing a PS to produce ROS, PDT is not only effective for cancer treatment but also useful to eliminate pathogenic bacteria. In this regard, on the basis of an AIE-active PS, Tang’s group creatively developed a new bactericide that will not induce the emergence of resistance and can also image bacteria.191 The probe TPE-Bac (74) can be viewed as the derivative of 73, with the only difference lying in the extra alkoxy chain (Figure 13A). As a result of the enhanced D−A effect, 74 showed a much redder aggregated-state FL (λem = 641 nm) as compared to 73. The quaternary amine along with the positively charged pyridinium salt bestowed good hydrophilicity on 74. Similar to 73, 74 was amphiphilic and typically AIE-active. When the concentration of 74 in water reached 10 μM, the micelle formation took place and its FL was switched on. Incubated with 10 μM of 74 for 10 min, S. epidermidis were clearly visualized by the bright orange-red FL of 74 (Figure 13E). Thanks to the water solubility and AIE feature, the 74 molecules unbound to the bacteria were practically nonemissive, minimizing the background while maximizing the SNR. What’s more, such a washing-free imaging process simplified the procedures and reduced the bacterial loss, allowing for the high-throughput applications. Like 73, 74 stained both Gram-positive and -negative bacteria. More importantly, 74 can be employed as a bactericide to eliminate both Gram-positive and Gram-negative bacteria based on its dark toxicity as well as its 1O2 generation ability under light irradiation (Figure 13F). Specifically, the viability of E. coli treated with 74 and stored in dark for 10 min was decreased to about 70%, and the viability of S. epidermidis incubated with 74 was even as low as 50%. The dark toxicity was found to be ascribable to the two long alkoxy chains and the two positively charged amines that can intercalate into the bacterial membrane, damage the membrane integrity, and increase the membrane permeability. Light irradiation can markedly AH


Review


Figure 14. Systematic examples of using AIE-based bioprobes for telomerase assay in clinical samples. (A) Schematic illustration of simple one-pot approach for telomerase activity evaluation based on AIEgen 75. (B) Box chart representing telomerase activity detection by means of AIE-based turn-on strategy in real urine samples. Reproduced with permission from ref 200. Copyright 2015 American Chemical Society. (C) Schematically representing the FL strategy where the high specificity for telomerase activity assessment was induced by a quencher group (QP). (D) The results reflecting the improved specificity of the QP strategy to telomerase compared with that without quencher group. Reproduced with permission from ref 201. Copyright 2015 American Chemical Society. (E) Schematic illustration of GO-based platform for the assay of telomerase activity with labelfree AIE-active molecular beacon (AIE-MB). (F) Box plot representing activity analysis of telomerase extracted from urine specimens of 18 clear bladder patients (blue), 18 bloody bladder patients (red), and 15 normal sunjects (dark yellow) using AIE-MB strategy. Reproduced with permission from ref 202. Copyright 2016 Elsevier. (G) Schematic representation of the ratiometric fluorometric bioprobing system for telomerase activity assay. (H) FL intensity ratio (I478/I665) of telomerases extracted from 20 bloody bladder cancer urine specimens (left), box chart representing activity evaluation of telomerase from urine samples of 20 bladder cancer patients (red) and 10 healthy people (gray) with the aid of the ratiometric detection strategy (right). Reproduced with permission from ref 203. Copyright 2016 American Chemical Society.

2.4.4. AIE Bioprobes for Real-Sample Analysis. Probing a certain analyte in the real sample is extremely desirable for clinical diagnosis; however, it has been regarded as an extremely challenging task because of the high complexity of real specimens, multiple or even unknown interferences from nontarget species, and the individual difference. A bioprobe which is competent to the real-sample analysis should satisfy several paramount criteria, including high sensitivity, excellent

specificity, and good reproducibility. Among all the bioprobing techniques, the ones based on FL are the most attractive in view of their rapidity, simplicity, and high accuracy. Despite this, FL approaches utilizing ACQ fluorophores suffered from self-quenching and usually operate in a turn-off manner, resulting in unsatisfactory sensitivity and making them less suitable for practical use. Comparatively, the FL turn-on mode can significantly boost the sensitivity by enhancing the FL AI


Review


More importantly, the telomerase activity detection based on 75 could be carried out in clinically real samples. Urine samples from 41 bladder cancer patients (23 of them were clear while the other 18 were bloody) and 15 healthy people were collected to assess the applicability of 75 in bladder cancer diagnosis. The ratios of positive results to total tested samples were calculated to be 23/23 (100%) for clear specimens and 13/18 (72%) for bloody ones. This implied that the existence of blood would exert negative effect on the results as the interference more likely occurs in bloody specimens. Moreover, there was no marked difference in the FL intensity of probe when analyzing the same type of urine samples of the patients from different regions (Figure 14B). The FL intensities of normal urine samples were close to the control and nearly invariable. All the results confirmed the feasibility of applying this AIE strategy for bladder cancer diagnosis in untreated real urine. In the above case, the FL shown by the solution of 75 and TS primer was weak but unneglectable. In order to further reduce the detection background noise and improve the selectivity or specificity, Lou and her co-workers elaborately attached the quencher Dabcyl, namely 4-(4-(dimethylamino)phenylazo)benzoic acid, onto the 5′-end of TS primer (TP) to afford the QP, the quencher group-decorated TS primer.201 Another prototypal AIEgen, 1,1-dimethyl-2,3,4,5-tetraphenylsilole (DMTPS) was selected as the AIE motif, and was functionalized with two quaternary ammonium groups to produce the positively charged probe 76. Analogous to 75, 76 is amphiphilic and water-soluble in a certain concentration range. When it was well dispersed in aqueous media, 76 exhibited quite weak FL and hence showed a low background. Because of its positive charges, 76 can automatically bind to the negatively charged QP via the electrostatic attraction (Figure 14C). Because the emission of 76 (λem = 478 nm) is greatly overlapped with the absorption of Dabcyl (λem = 480 nm), before telomerase extension reaction, the emission of 76 induced by the interaction with the 18-nt short oligonucleotides on QP would be efficiently quenched owing to the FRET from the 76-oligonucleotides complex to the Dabcyl unit. As a result, the background was tremendously lowered. After the addition of telomerase extracted from 10000 MCF-7 cancer cells, the 75 molecules bound to the repeat units which undergo extension reaction at their 3′-ends were kept away from the quencher, thereby interrupting the FERT. In the meantime, more molecules of 76 were pulled to the extended DNA strand by the electrostatic force, and the FL was turned on with a signal increase percentage of up to 1424%. In contrast, similar experiments performed with TP (TS primer without quenching group) gave a signal increase percentage of merely 586%, much lower than that utilizing QP, proving the important role of quencher in lowering the background and increasing the SNR. Notably, the quencher also greatly improved the specificity by increasing the SNR (Figure 14D). The remarkable difference between active telomerase (extracted from E-J, MCF-7, and HeLa cancer cells) and heat-inactivated telomerase effectively certified the high specificity of this method. FL intensity increments of QP systems in the presence of active telomerase were apparently larger than those of TP systems. While for the control systems including inactive telomerase, telomerase extracted from normal cells (HLF), other proteins and so on, the FL enhancements of QP and TP systems were nearly identical. Therefore, the SNR and specificity were definitely increased by applying QP, and consequently the

signal and meanwhile providing an ultralow background. As such, turn-on bioprobes are especially favorable in real-sample assays for their reduced possibility of generating false positive/ negative artifacts as compared with their turn-off counterparts. It has been emphasized for many times that AIE-based bioprobes are featured with an inborn FL light-up response ability to the target analyte. Before binding to the targeted analyte, isolated AIEgen is almost nonemissive, providing a minimal background, whereas their binding to the targets would let the RIM process take effect and turn on the FL, showing a light-up response with high SNR. In the light of their excellent performance, AIE-based bioprobes have recently been applied for biological assays in real samples.93,195−207 On this issue, the systematic work regarding the telomerase activity detection done by Lou and Xia et al. can be taken as a typical example.200−203 Detecting cancer biomarkers in a highly sensitive and selective manner is essential for the cancer diagnosis, prevention and therapy as well as for cancer pathogenesis understanding. Telomerase is a ribonucleoprotein that can add specific sequence (TTAGGG)n to the 3′-end of a telomere when mixed with template strand primer (TS primer). As it is highly active in cancer cells while lowly active or inactive in normal cells, telomerase has been viewed as a specific and sensitive biomarker for cancer, playing significant roles in early detection and diagnosis of cancers. In 2015, Xia and Lou et al. reported the first AIE probe for real-time, quantitative, and light-up detection of telomerase in bladder cancer patients’ urines.200 Specifically, a simple modification of the easy-toprepare and ready-to-functionalize iconic AIE gen, i.e. TPE, derived the positively charged quaternized TPE salt (TPE-Z). The amphiphilic nature of TPE-Z (75) rendered it watersoluble. As a result, 75 was almost nonfluorescent in buffer solutions, offering a low background for the probing system. The FL of 75 kept weak in the presence of short oligonucleotides such as Ex-0 (18-nt). When coexisting with longer single-stranded DNA (ssDNA), such as Ex-6 (54-nt), the FL was switched on with a peak at 478 nm. The longer the ssDNA was, the stronger the FL of probe would be. In this way, both the elongation of DNA strand and the extension of TS primer under the manipulation of telomerase can be monitored by the FL change. The principle of this AIE-based simple one-pot assay of telomerase activity is illustrated in Figure 14A. Without active telomerase extracted from urine samples of bladder cancer patients, the solution containing 75 and telomerase substrate oligonucleotides (TS primer, 18-nt) was faintly emissive. Coexisting with the active telomerase, TS primer was extended with dNTPs, generating single strand of telomeric repeated sequence at the 3′-terminal. Once the DNA chain was elongated, the FL of 75 was intensified. Driven by the electrostatic force, the positive charge-bearing 75 bound to the negatively charged DNA backbone, triggering the RIM process and turning on the FL. A longer DNA strand provided more negative charges and more binding sites for 75 molecules, giving rise to more intense FL signals. On the other hand, the FL of 75 remained weak if there was no telomerase or the telomerase was inactive. As a result, the FL intensity of 75 was gradually boosted when the number of the E-J (telomerasepositive human bladder cancer) cells was increased from 0 to 10000. 75 can probe telomerase extracted from down to 10 E-J cells, with a wide detection range. Such a good sensitivity was comparable or even superior to most conventional methods. AJ


Review


urine’s complexed environment as well as its great promise for clinical cancer diagnosis. Aside from the above AIE-based FL turn-on mode assays of telomerase, with the aim to improve the reproducibility and reliability, Lou and her colleagues also explored a simple ratiometric fluorometric probing system for telomerase detection with the help of 76 and a conventional fluorophore Cy5.203 The single signal from FL turn-on system is prone to be perturbed by various experimental conditions, such as signal variations caused by dye bleaching or quenching, fluctuations in excitation light intensity, and so on. These environmental influences can be eliminated by ratiometric probes which give more accurate signals due to their self-referencing capacity by calculating the intensity ratio of two distinct emission peaks. The ratiometric probe for telomerase displayed in the lower panel of Figure 14G was constructed by using the positively charged 76 as a reporter of telomerase activity and the ACQ fluorophore Cy5 as an internal control or reference. Similar to the Dabcyl mentioned above, the Cy5 was labeled at the 5′terminal of TS primer (TP), generating the Cy5-labeled TS primer (CP). The solution containing CP and 76 exhibited dual FL bands at 478 nm (blue emission of rigidified 76) and 665 nm (red emission from the isolated Cy5) under a singlewavelength excitation. When there existed active telomerase, the blue FL got enhanced owing to the increased negatively charged sites for 76 to tightly bind which restricted the intramolecular motions. On the other hand, the red luminescence kept almost constant as a stable internal control. Ratiometric representation of the FL signal (I478/I665) was employed to eliminate the system errors. I478/I665 value of this probing system was gradually raised with the increasing amount of telomerase and hence can be used for the quantification of telomerase. When the telomerase extracted from 10000 E-J cells coexisted with CP and dNTPs, the I478/I665 value was markedly increased. As revealed by the reproducibility studies, the standard deviation (SD) in 31 individual experiments with this ratiometric probing system was down to 0.055, which was much smaller than that of the FL turn-on probe (0.305), demonstrating the excellent reproducibility of the ratiometric probe. Moreover, this probe could distinctly assay the telomerase activity as few as 5 cells in 1 h, indicating its high sensitivity and rapidity. Remarkable response differences between the active telomerase and other potential interferers manifested the high specificity of this ratiometric probe. As can be easily learnt from Figure 14H, when the telomerases extracted from 20 bloody urine specimens of bladder cancer patients, a notable increase in the I478/I665 value was obviously observed. It is worth mentioning that all the 20 bloody urine samples were found to show I478/I665 value higher than the threshold level, suggesting that the positive result rate of this method was 100% (20/20). This positive result rate obtained in bloody urine of bladder cancer patients was much higher than those FL turn-on assays (72 and 89.5%). The distributions of I478/I665 value of bloody bladder cancer and normal urine samples displayed an apparent difference between them (Figure 14H). The urine sample diagnosis with this ratiometric strategy also preserved an excellent reproducibility with a SD of 0.031, which was much lower than the SD of 0.283 obtained when utilizing the turn-on probe. Such a ratiometric bioprobe system was verified to be facile, reliable, reproducible, and thereby promising for bladder cancer diagnosis. The discussions in this part demonstrated the applicability of AIE-based bioprobes for real-sample analysis, exemplified the

active telomerase could be easily distinguished from other interferers. Moreover, the telomerase activity in the extracts of E-J, MCF-7, and HeLa cells equivalent to 5−10 000 cells can be assayed within 1 h by this protocol. In other words, with this method, the telomerase from as few as 5 cancer cells could be identified. Telomerases extracted from urine samples of 38 bladder cancer patients (a half of them were clear and the other half were bloody) and 15 normal people were tested by this low background strategy. The positive result rates were 100% (19/ 19) for clear urine and 89.5% (17/19) for bloody urine. In comparison to the results obtained with 75, which was 100% and 72% for clear and bloody urine specimens, respectively, this QP protocol preserved the outstanding accuracy in clear urine and achieved more satisfactory correct rate in bloody urine samples. It means that with a lower background and higher SNR, the QP is more specific to perform well under a complicated condition such as in blood. Besides the quencher-labeling strategy, Lou et al. smartly utilized the GO208 as a quencher to reduce the background, and a label-free molecular beacon (MB) which was comprised of a special short nucleic acid strand that can induce a comb-like DNA structure and enhance the FL signal was constructed.202 In this way, the SNR and specificity of the AIE probe can be increased, and meanwhile its sensitivity can also be improved. In this approach, 76 was also used as an AIE probe. As depicted in Figure 14E, the working principle of this strategy can be explained as follows: When the TS primer and MB were added to the solution containing 76, the aggregation complexes were formed as a result of electrostatic interactions, which accordingly enhanced the FL because of the AIE nature of 76. Upon the addition of GO, the adsorptive binding of TS primer, MB and 76 made their complexes close to GO to efficiently quench their emission, resulting in a minimal detection background. In the presence of active telomerase, telomeric repeat sequences would be continuously added onto the 3′-terminal of the primer to produce a longer ssDNA. Then its repeated sequences can bind with the loop region of the MB, opening the beacon and forming a comb-like DNA structure which will detach itself from the GO surface. In the meantime, 76 molecules were still adsorbed on the DNA strands and separated from GO, then the FL will be recovered and gradually get intensified. While, when there was no active telomerase, TS primer was not extended, and the AIEgenattached MB (AIE-MB) had no binding site and retained its original conformation to be attracted by GO. In this condition, the FL will not be switched on. The detection experiments with the extracts from telomerase-positive cancer cells including HeLa, MCF-7, and E-J confirmed that the label-free AIE-MB was superior to the conventional single-labeled MB with nonAIEgen, in terms of the SNR as well as the photostability. The FL enhancement of this AIE-MB was 13.17 while that of the single-labeled MB was only 3.59. It thus can be seen that higher SNR can be achieved by this GO-based AIE-MB through the combination of lower background and stronger FL signal. The detection limit of this method was deduced to be equivalent to 10 HeLa cells. Similarly, different telomerase expression levels of clinical cancer patients were evaluated by this protocol. Crude extracts from 36 cancer patient’s urine specimens (a half of them were clear and the other half were bloody) and 15 normal people’s urine were assayed (Figure 14F). Obviously, the cancer patient’s urines exhibited larger FL enhancements than those of the healthy people, demonstrating the capability of this GO-assisted AIE MB strategy to assay telomerase under AK


Review


three-photon FL technique thus has been a favorite for bioimaging. The output FL signal is easily subjected to the interferences from biological background autofluorescence and scattered light. In addition to the FR/NIR FL and two/threephoton excited luminescence, phosphorescence is another effective signal that can afford satisfactorily low detection background, good contrast, and high SNR, because the interference from short-lived background FL and scattered light could be minimized by the “time-gate”. When come to the modality and functionality, there are distinctively two trends, one is from monomodal FL imaging agents to dual-modal imaging systems, and one is from monofunctional AIE probes to dual/multifunctional AIE probing systems. Every single imaging modality owns its pros and cons, whereas different imaging modalities could compensate each other and combine superiorities of each imaging modality. Dual-modal AIE bioprobes are perfect interpretation of the “1 + 1 > 2” principle. The integration of two or more functions into a single probe would largely reduce the complexity of probing system. AIE bioprobes that enjoy dual or even multiple functions have been employed for disease diagnosis, therapies, as well as image-guided therapies. With respect to the study objects and the application outlets, the recent research interests are overwhelmingly concentrated on the utilization of AIE-based bioprobes for the specific organelle imaging, cancer cell-selective recognition and killing, bacteria imaging and/or killing, and the real-sample analysis. These topics are closely related to biomedical research, human healthcare, and disease diagnosis and therapy. The overall trends in the AIE-based bioprobes for these purposes are to be more precise, selective, practicable, and clinically usable. AIE-based bioprobes have showcased their charm and power in biological sensing, imaging, and theranostics. The future developing directions of AIE-based bioprobes include but are not limited to the above-mentioned 11 respects. On the basis of the thrilling progresses achieved so far, in order to promote the development of AIE-based bioprobes to a new height, more efforts should be devoted into the following issues: (1) Expanding the family of AIE-based bioprobes. For example, the most used AIE motifs are still restricted to TPE derivatives and siloles, thus besides which, more bran-new AIEgens should be explored and used as AIE elements in the construction of bioprobes. (2) Optimizing the output signal of AIE-based bioprobes or improving the optical performance of AIEgens. The ultrahigh brightness and emission efficiency, long-wavelength emission (FR/NIR or even beyond NIR), longwavelength absorption, large 2PA and 3PA cross-section, long luminescence lifetime (e.g., room-temperature phosphorescence) are the eternal pursuits of a high-performance bioprobe. AIEgens with two or more such features are favored. (3) Endowing the AIE-based bioprobes with dual/multimodality, multifunctionality, and/or controllability. The already developed AIE systems are limited to dual-modal FL imaging/MRI, FL imaging/CT and FL imaging/photoacustic. The combination of FL imaging with ultrasound or positron emission tomography (PET) imaging has not been reported yet. Also, the AIE-based multimodal imaging systems have not been touched upon. Similarly, combining different therapy modalities such as chemotherapy, PDT, photothermal therapy, and radiotherapy into one AIE-based bioprobe remains to be explored. Delightfully, there have come out a few such examples, e.g. probes 11 and 59. More facile and efficient AIE probes with dual/multiple modalities and/or function-

stepwise optimization of the AIE-based telomerase detection system. Focusing on the purpose of meeting the criteria including sensitivity, specificity, and reproducibility, ionic AIEgen was first employed to eliminate the background from itself and realize the FL light-up detection, then the quenching moiety was introduced via chemical labeling or physical blending to further reduce the background noise from the detection system and enhance the selectivity, and ultimately a ratiometric probing system was established to guarantee the reproducibility and further improve the clinical practicality. Such design rationales would undoubtedly offer guidance for further development of real-sample applicable bioprobes, wherein the probes are not restricted to AIE-based ones.

3. SUMMARY AND PERSPECTIVES This review summarizes and outlines the most appealing trends and progresses in the flourishingly developing AIE-based bioprobes. In total, 11 advancing trends classified into four aspects: the probe composition and form, the output signal of probe, the modality and functionality of probing systems, and the probing object and application outlet have been pointed out and specifically demonstrated. Briefly, from the probe composition and form viewpoint, there are mainly two trends, i.e., from pure chemically composed AIEgens to AIE bioconjugates, and from molecular probes to nanoscopic probes. Introducing biogenic ligand(s)/ element(s) into the AIE systems plays such roles as improving specificity, ensuring biocompatibility, and enhancing watersolubility, etc. As a result of the preserved AIE attribute and the extra benefits provided by the bioligand(s), both the variety and the bioapplications of them are enriched at an amazing speed since the first AIE bioconjugates was reported in 2012. Tailoring AIEgens into nanoscopic particles via physically encapsulating AIE molecules into polymeric matrices or through self-assembly of the polymeric AIEgens brings about a class of fabulous nanoprobes which integrate the merits of both AIEgens and nanomaterials. Benefiting from the AIE nature, the loading concentration of AIEgens in each NP can be very high and the luminosity is positively correlated with the loading concentration. AIE nanoprobes enjoy a vast diversity of advantages, including easy fabrication, facile functionalization which could incorporate dual-modality and/or dual/multifunctionality, intense luminescence, excellent photostability, non-photo-blinking, good biocompatibility, superior cellular retention ability, passive tumor targeting capability due to the proper size (EPR), and so on. As such, AIE NPs are theoretically capable of fulfilling any imaging task and even serving as theranostic agents. In terms of the signal output by the AIE-based bioprobes, the most notable trends are from short-wavelength emission to FR/ NIR luminescence, from one-photon excited fluorescence to two/three-photon excited fluorescence, from FL to phosphorescence (or from short-lived to long-lived luminescence). With FR/NIR luminescence, the autofluorescence of biosubstrates could be filtered out to afford a low background and high signal-to-noise ratio as well as high resolution. Its low-energy nature reduces the photodamage to biosubstances and its long wavelength favors deep-tissue penetration. Two/three-photon excited FL imaging possesses various superiorities over the onephoton FL technique, including lowered photodamage to biosubstrate, deeper penetration depth, smaller autofluorescence interference, improved spatial resolution, better photostability, and negligible perturbation from biomedia. Two/ AL


Review


Polymer Dots and Nanoparticles for Biological Imaging and Medicine. Anal. Chem. 2017, 89, 42−56. (5) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530−10574. (6) Adegoke, O.; Kato, T.; Park, E. Y. An Ultrasensitive Alloyed Near-Infrared Quinternary Quantum Dot-Molecular Beacon Nanodiagnostic Bioprobe for Influenza Virus RNA. Biosens. Bioelectron. 2016, 80, 483−490. (7) Dong, H.; Tang, S.; Hao, Y.; Yu, H.; Dai, W.; Zhao, G.; Cao, Y.; Lu, H.; Zhang, X.; Ju, H. Fluorescent MoS2 Quantum Dots: Ultrasonic Preparation, Up-Conversion and Down-Conversion Bioimaging, and Photodynamic Therapy. ACS Appl. Mater. Interfaces 2016, 8, 3107−3114. (8) Liu, C.; Gao, Z.; Zeng, J.; Hou, J.; Fang, F.; Li, Y.; Qiao, R.; Shen, L.; Lei, H.; Yang, W.; Gao, M. Magnetic/Upconversion Fluorescent NaGdF4:Yb,Er Nanoparticle-Based Dual-Modal Molecular Probes for Imaging Tiny Tumors In Vivo. ACS Nano 2013, 7, 7227−7240. (9) Zhou, B.; Shi, B.; Jin, D.; Liu, X. Controlling Upconversion Nanocrystals for Emerging Applications. Nat. Nanotechnol. 2015, 10, 924−936. (10) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for SmallAnimal Imaging. Chem. Soc. Rev. 2012, 41, 1323−1349. (11) Chen, L.-Y.; Wang, C.-W.; Yuan, Z.; Chang, H.-T. Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Anal. Chem. 2015, 87, 216−229. (12) Hu, X.-L.; Zang, Y.; Li, J.; Chen, G.-R.; James, T. D.; He, X.-P.; Tian, H. Targeted Multimodal Theranostics via Biorecognition Controlled Aggregation of Metallic Nanoparticle Composites. Chem. Sci. 2016, 7, 4004−4008. (13) Tsien, R. Y. Constructing and Exploiting the Fluorescent Protein Paintbox (Nobel Lecture). Angew. Chem., Int. Ed. 2009, 48, 5612−5626. (14) Förster, T.; Kasper, K. Ein Konzentrationsumschlag der Fluoreszenz. Z. Phys. Chem. (Muenchen, Ger.) 1954, 1, 275−277. (15) Photophysics of Aromatic Molecules; Birks, J. B., Ed.; Wiley: London, 1970. (16) Chen, S.; Wang, H.; Hong, Y.; Tang, B. Z. Fabrication of Fluorescent Nanoparticles Based on AIE Luminogens (AIE Dots) and Their Applications in Bioimaging. Mater. Horiz. 2016, 3, 283−293. (17) Qian, J.; Tang, B. Z. AIE Luminogens for Bioimaging and Theranostics: From Organelles to Animals. Chem. 2017, 3, 56−91. (18) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Tang, B. Z.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D. Aggregation-Induced Emission of 1-Methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 1740−1741. (19) Chen, J. W.; Law, C. C. W.; Lam, J. W. Y.; Dong, Y. P.; Lo, S. M. F.; Williams, I. D.; Zhu, D. B.; Tang, B. Z. Synthesis, Light Emission, Nanoaggregation, and Restricted Intramolecular Rotation of 1,1Substituted 2,3,4,5-tetraphenylsiloles. Chem. Mater. 2003, 15, 1535− 1546. (20) Leung, N. L. C.; Xie, N.; Yuan, W.; Liu, Y.; Wu, Q.; Peng, Q.; Miao, Q.; Lam, J. W. Y.; Tang, B. Z. Restriction of Intramolecular Motions: The General Mechanism Behind Aggregation-Induced Emission. Chem. - Eur. J. 2014, 20, 15349−15353. (21) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-Induced Emission: The Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429−5479. (22) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718−11940. (23) Wang, M.; Zhang, G.; Zhang, D.; Zhu, D.; Tang, B. Z. Fluorescent Bio/Chemosensors Based on Silole and Tetraphenylethene Luminogens with Aggregation-Induced Emission Feature. J. Mater. Chem. 2010, 20, 1858−1867. (24) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes Based on AIE Fluorogens. Acc. Chem. Res. 2013, 46, 2441−2453.

alities which contain fewer components or even only a single component should be pursued for practical use. Most of the existing AIE systems possessing multiple functions and/or dual modalities are usually constituted by several components via the all-in-one approach, suffering from shortcomings related to complicated molecular structure and potentially multistep synthesis. AIE probes with a simple molecular structure and dual/multiple functionality/modality will be most favored, e.g., TPE-TMX discussed in this review. In addition, more stimuliresponsive (e.g., light activatable, pH triggerable) AIE-based bioprobes should be designed to boost the controllability. (4) Enhancing the clinical practicability by guaranteeing sufficient specificity, sensitivity, reproducibility, and reliability. As for this, there are several points should be taken into consideration: (a) how to achieve high specificity and sensitivity in the real samples; (b) how to avoid fluorescence quenching caused by unknown species in complex biological systems; (c) how to ensure the universality or generality of a probing system in complex real samples from different sources (e.g., age, gender, region, etc.); (d) how to overcome the omission and false detection (reliability issues); (e) how to engineer the labdeveloped AIE-based bioprobes into clinically usable diagnostic kits and/or theranostic agents. (5) Last but not the least, clarifying the potential toxicity, distribution, clearance, and final fate of AIE bioprobes at the cellular and organism levels. Such a systematic task depends on the joint efforts of a wide spectrum of experts, including chemists, physicists, biologists, biomedical engineers, and clinicians. We hope that this review will stimulate more research interests from chemistry, materials, life science, and biomedical areas. Together, we will promote the development of AIE-based bioprobes and make full use of the unique AIE effect.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +86-021-64252756. ORCID

He Tian: 0000-0003-3547-7485 Funding

This work is financially supported by the Programme of Introducing Talents of Discipline to Universities (B16017) and National Natural Science Foundation of China (21604023), and sponsored by the Shanghai Sailing Program (16YF1402200) and the Fundamental Research Funds for the Central Universities (222201714011, 222201717003). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Li, M.; Feng, W.; Zhai, Q.; Feng, G. Selenocysteine Detection and Bioimaging in Living Cells by a Colorimetric and Near-Infrared Fluorescent Turn-On Probe with a Large Stokes Shift. Biosens. Bioelectron. 2017, 87, 894−900. (2) He, L.; Yang, X.; Xu, K.; Kong, X.; Lin, W. A Multi-Signal Fluorescent Probe for Simultaneously Distinguishing and Sequentially Sensing Cysteine/Homocysteine, Glutathione, and Hydrogen Sulfide in Living Cells. Chem. Sci. 2017, 8, 6257−6265. (3) Reisch, A.; Klymchenko, A. S. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging. Small 2016, 12, 1968−1992. (4) Yu, J.; Rong, Y.; Kuo, C.-T.; Zhou, X.-H.; Chiu, D. T. Recent Advances in the Development of Highly Luminescent Semiconducting AM


Review

ACS Applied Materials & Interfaces (25) Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. Biosensing by Luminogens with Aggregation-Induced Emission Characteristics. Chem. Soc. Rev. 2015, 44, 4228−4238. (26) Huang, L.; Dai, L. Aggregation-Induced Emission for Highly Selective and Sensitive Fluorescent Biosensing and Cell Imaging. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 653−659. (27) Li, K.; Liu, B. Polymer-Encapsulated Organic Nnanoparticles for Fluorescence and Photoacoustic Imaging. Chem. Soc. Rev. 2014, 43, 6570−6597. (28) Zhang, X.; Wang, K.; Liu, M.; Zhang, X.; Tao, L.; Chen, Y.; Wei, Y. Polymeric AIE-Bbased Nnanoprobes for Biomedical Aapplications: Recent Advances and Perspectives. Nanoscale 2015, 7, 11486−11508. (29) Yang, B.; Zhang, X.; Zhang, X.; Huang, Z.; Wei, Y.; Tao, L. Fabrication of Aggregation-Induced Emission Based Fluorescent Nanoparticles and Their Biological Imaging Application: Recent Progress and Perspectives. Mater. Today 2016, 19, 284−291. (30) Li, D.; Yu, J. AIEgens-Functionalized Inorganic-Organic Hybrid Materials: Fabrications and Applications. Small 2016, 12, 6478−6494. (31) Wang, Y.-F.; Zhang, T.; Liang, X.-J. Aggregation-Induced Emission: Lighting Up Cells, Revealing Life! Small 2016, 12, 6451− 6477. (32) Yan, L.; Zhang, Y.; Xu, B.; Tian, W. Fluorescent Nanoparticles Based on AIE Fluorogens for Bioimaging. Nanoscale 2016, 8, 2471− 2487. (33) Lou, X.; Zhao, Z.; Tang, B. Z. Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging. Small 2016, 12, 6430− 6450. (34) Gao, M.; Chen, J.; Lin, G.; Li, S.; Wang, L.; Qin, A.; Zhao, Z.; Ren, L.; Wang, Y.; Tang, B. Z. Long-Term Tracking of the Osteogenic Differentiation of Mouse BMSCs by Aggregation-Induced Emission Nanoparticles. ACS Appl. Mater. Interfaces 2016, 8, 17878−17884. (35) Zhang, M.; Saha, M. L.; Wang, M.; Zhou, Z.; Song, B.; Lu, C.; Yan, X.; Li, X.; Huang, F.; Yin, S.; Stang, P. J. Multicomponent Platinum(II) Cages with Tunable Emission and Amino Acid Sensing. J. Am. Chem. Soc. 2017, 139, 5067−5074. (36) Cui, L.; Baek, Y.; Lee, S.; Kwon, N.; Yoon, J. An AIE and ESIPT Based Kinetically Resolved Fluorescent Probe for Biothiols. J. Mater. Chem. C 2016, 4, 2909−2914. (37) Liu, Y.; Deng, C.; Tang, L.; Qin, A.; Hu, R.; Sun, J. Z.; Tang, B. Z. Specific Detection of D-Glucose by a Tetraphenylethene-Based Fluorescent Sensor. J. Am. Chem. Soc. 2011, 133, 660−663. (38) Li, Y.; Kwok, R. T. K.; Tang, B. Z.; Liu, B. Specific Nucleic Acid Detection Based on Fluorescent Light-Up Probe from Fluorogens with Aggregation-Induced Emission Characteristics. RSC Adv. 2013, 3, 10135−10138. (39) Shi, H.; Liu, J.; Geng, J.; Tang, B. Z.; Liu, B. Specific Detection of Integrin αvβ3 by Light-Up Bioprobe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 9569−9572. (40) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. 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, 17972−17981. (41) Hong, Y.; Meng, L.; Chen, S.; Leung, C. W. T.; Da, L.-T.; Faisal, M.; Silva, D.-A.; Liu, J.; Lam, J. W. Y.; Huang, X.; Tang, B. Z. Monitoring and Inhibition of Insulin Fibrillation by a Small Organic Fluorogen with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 1680−1689. (42) Pradhan, N.; Jana, D.; Ghorai, B. K.; Jana, N. R. Detection and Monitoring of Amyloid Fibrillation Using a Fluorescence “Switch-On” Probe. ACS Appl. Mater. Interfaces 2015, 7, 25813−25820. (43) Liang, J.; Shi, H.; Kwok, R. T. K.; Gao, M.; Yuan, Y.; Zhang, W.; Tang, B. Z.; Liu, B. Distinct Optical and Kinetic Responses from E/Z Isomers of Caspase Probes with Aggregation-Induced Emission Characteristics. J. Mater. Chem. B 2014, 2, 4363−4370. (44) Zhang, W.; Kwok, R. T. K.; Chen, Y.; Chen, S.; Zhao, E.; Yu, C. Y. Y.; Lam, J. W. Y.; Zheng, Q.; Tang, B. Z. Real-Time Monitoring of the Mitophagy Process by A Photostable Fluorescent MitochondrionSpecific Bioprobe with AIE Characteristics. Chem. Commun. 2015, 51, 9022−9025.

(45) Jin, C.; Liu, J.; Chen, Y.; Guan, R.; Ouyang, C.; Zhu, Y.; Ji, L.; Chao, H. Cyclometalated Iridium(III) Complexes as AIE Phosphorescent Probes for Real-Time Monitoring of Mitophagy in Living Cells. Sci. Rep. 2016, 6, 22039. (46) Hu, F.; Liu, B. Organelle-Specific Bioprobes Based on Fluorogens with Aggregation-Induced Emission (AIE) Characteristics. Org. Biomol. Chem. 2016, 14, 9931−9944. (47) Gui, C.; Zhao, E.; Kwok, R. T. K.; Leung, A. C. S.; Lam, J. W. Y.; Jiang, M.; Deng, H.; Cai, Y.; Zhang, W.; Su, H.; Tang, B. Z. AIE-Active Theranostic System: Selective Staining and Killing of Cancer Cells. Chem. Sci. 2017, 8, 1822−1830. (48) Shi, X.; Yu, C. Y. Y.; Su, H.; Kwok, R. T. K.; Jiang, M.; He, Z.; Lam, J. W. Y.; Tang, B. Z. A Red-Emissive Antibody−AIEgen Conjugate for Turn-On and Wash-Free Imaging of Specific Cancer Cells. Chem. Sci. 2017, 8, 7014−7024. (49) Zhao, E.; Hong, Y.; Chen, S.; Leung, C. W. T.; Chan, C. Y. K.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Highly Fluorescent and Photostable Probe for Long-Term Bacterial Viability Assay Based on Aggregation-Induced Emission. Adv. Healthcare Mater. 2014, 3, 88− 96. (50) Li, Y.; Yu, H.; Qian, Y.; Hu, J.; Liu, S. Amphiphilic Star Copolymer-Based Bimodal Fluorogenic/Magnetic Resonance Probes for Concomitant Bacteria Detection and Inhibition. Adv. Mater. 2014, 26, 6734−6741. (51) Zhao, E.; Chen, Y.; Chen, S.; Deng, H.; Gui, C.; Leung, C. W. T.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z. A Luminogen with Aggregation-Induced Emission Characteristics for Wash-Free Bacterial Imaging, High-Throughput Antibiotics Screening and Bacterial Susceptibility Evaluation. Adv. Mater. 2015, 27, 4931−4937. (52) Ding, D.; Goh, C. C.; Feng, G.; Zhao, Z.; Liu, J.; Liu, R.; Tomczak, N.; Geng, J.; Tang, B. Z.; Ng, L. G.; Liu, B. Ultrabright Organic Dots with Aggregation-Induced Emission Characteristics for Real-Time Two-Photon Intravital Vasculature Imaging. Adv. Mater. 2013, 25, 6083−6088. (53) Gao, Y.; Feng, G.; Jiang, T.; Goh, C.; Ng, L.; Liu, B.; Li, B.; Yang, L.; Hua, J.; Tian, H. Biocompatible Nanoparticles Based on Diketo-Pyrrolo-Pyrrole (DPP) with Aggregation-Induced Red/NIR Emission for In Vivo Two-Photon Fluorescence Imaging. Adv. Funct. Mater. 2015, 25, 2857−2866. (54) Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible Nanoparticles with Aggregation-Induced Emission Characteristics as Far-Red/Near-Infrared Fluorescent Bioprobes for In Vitro and In Vivo Imaging Applications. Adv. Funct. Mater. 2012, 22, 771−779. (55) Shao, A.; Xie, Y.; Zhu, S.; Guo, Z.; Zhu, S.; Guo, J.; Shi, P.; James, T. D.; Tian, H.; Zhu, W.-H. Far-Red and Near-IR AIE-Active Fluorescent Organic Nanoprobes with Enhanced Tumor-Targeting Efficacy: Shape-Specific Effects. Angew. Chem., Int. Ed. 2015, 54, 7275−7280. (56) Seo, Y. H.; Singh, A.; Cho, H.-J.; Kim, Y.; Heo, J.; Lim, C.-K.; Park, S. Y.; Jang, W.-D.; Kim, S. Rational Design for Enhancing Inflammation-Responsive In Vivo Chemiluminescence via Nanophotonic Energy Relay to Near-Infrared AIE-Active Conjugated Polymer. Biomaterials 2016, 84, 111−118. (57) Zhao, Y.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. A Highly Fluorescent AIE-Active Theranostic Agent with Anti-Tumor Activity to Specific Cancer Cells. Nanoscale 2016, 8, 12520−12523. (58) Yu, C. Y. Y.; Xu, H.; Ji, S.; Kwok, R. T. K.; Lam, J. W. Y.; Li, X.; Krishnan, S.; Ding, D.; Tang, B. Z. Mitochondrion-Anchoring Photosensitizer with Aggregation-Induced Emission Characteristics Synergistically Boosts the Radiosensitivity of Cancer Cells to Ionizing Radiation. Adv. Mater. 2017, 29, 1606167. (59) Gu, B.; Wu, W.; Xu, G.; Feng, G.; Yin, F.; Chong, P. H. J.; Qu, J.; Yong, K.-T.; Liu, B. Precise Two-Photon Photodynamic Therapy Using an Efficient Photosensitizer with Aggregation-Induced Emission Characteristics. Adv. Mater. 2017, 29, 1701076. (60) Feng, G.; Liu, B. Multifunctional AIEgens for Future Theranostics. Small 2016, 12, 6528−6535. AN


Review

ACS Applied Materials & Interfaces (61) Yuan, Y.; Liu, B. Visualization of Drug Delivery Processes Using AIEgens. Chem. Sci. 2017, 8, 2537−2546. (62) Liang, J.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobes Based on AIEgen Conjugates. Chem. Soc. Rev. 2015, 44, 2798−2811. (63) Hu, Y.; Shi, L.; Su, Y.; Zhang, C.; Jin, X.; Zhu, X. A Fluorescent Light-Up Aggregation-Induced Emission Probe for Screening Gefitinib-Sensitive Non-Small Cell Lung Carcinoma. Biomater. Sci. 2017, 5, 792−799. (64) Shi, H.; Zhao, N.; Ding, D.; Liang, J.; Tang, B. Z.; Liu, B. Fluorescent Light-Up Probe with Aggregation-Induced Emission Characteristics for In Vivo Imaging of Cell Apoptosis. Org. Biomol. Chem. 2013, 11, 7289−7296. (65) Ding, D.; Liang, J.; Shi, H.; Kwok, R. T. K.; Gao, M.; Feng, G.; Yuan, Y.; Tang, B. Z.; Liu, B. Light-Up Bioprobe with AggregationInduced Emission Characteristics for Real-Time Apoptosis Imaging in Target Cancer Cells. J. Mater. Chem. B 2014, 2, 231−238. (66) Yuan, Y.; Zhang, R.; Cheng, X.; Xu, S.; Liu, B. A FRET Probe with AIEgen as the Energy Quencher: Dual Signal Turn-On for SelfValidated Caspase Detection. Chem. Sci. 2016, 7, 4245−4250. (67) Yuan, Y.; Zhang, C.-J.; Kwok, R. T. K.; Mao, D.; Tang, B. Z.; Liu, B. Light-Up Probe Based on AIEgens: Dual Signal Turn-On for Caspase Cascade Activation Monitoring. Chem. Sci. 2017, 8, 2723− 2728. (68) Yuan, Y.; Zhang, C.-J.; Kwok, R. T. K.; Xu, S.; Zhang, R.; Wu, J.; Tang, B. Z.; Liu, B. Light-Up Probe for Targeted and Activatable Photodynamic Therapy with Real-Time In Situ Reporting of Sensitizer Activation and Therapeutic Responses. Adv. Funct. Mater. 2015, 25, 6586−6595. (69) Yuan, Y.; Kwok, R. T. K.; Feng, G.; Liang, J.; Geng, J.; Tang, B. Z.; Liu, B. Rational Design of Fluorescent Light-Up Probes Based on an AIE Luminogen for Targeted Intracellular Thiol Imaging. Chem. Commun. 2014, 50, 295−297. (70) Yuan, Y.; Chen, Y.; Tang, B. Z.; Liu, B. A Targeted Theranostic Platinum(IV) Prodrug Containing a Luminogen with AggregationInduced Emission (AIE) Characteristics for In Situ Monitoring of Drug Activation. Chem. Commun. 2014, 50, 3868−3870. (71) Yuan, Y.; Zhang, C.-J.; Liu, B. A Platinum Prodrug Conjugated with a Photosensitizer with Aggregation-Induced Emission (AIE) Characteristics for Drug Activation Monitoring and Combinatorial Photodynamic−Chemotherapy Against Cisplatin Resistant Cancer Cells. Chem. Commun. 2015, 51, 8626−8629. (72) Yuan, Y.; Kwok, R. T. K.; Tang, B. Z.; Liu, B. Targeted Theranostic Platinum(IV) Prodrug with a Built-In AggregationInduced Emission Light-Up Apoptosis Sensor for Noninvasive Early Evaluation of Its Therapeutic Responses. J. Am. Chem. Soc. 2014, 136, 2546−2554. (73) Yuan, Y.; Kwok, R. T. K.; Zhang, R.; Tang, B. Z.; Liu, B. Targeted Theranostic Prodrugs Based on an Aggregation-Induced Emission (AIE) Luminogen for Real-Time Dual-Drug Tracking. Chem. Commun. 2014, 50, 11465−11468. (74) Cheng, Y.; Huang, F.; Min, X.; Gao, P.; Zhang, T.; Li, X.; Liu, B.; Hong, Y.; Lou, X.; Xia, F. Protease-Responsive Prodrug with Aggregation-Induced Emission Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells. Anal. Chem. 2016, 88, 8913−8919. (75) Han, H.; Jin, Q.; Wang, Y.; Chen, Y.; Ji, J. The Rational Design of a Gemcitabine Prodrug with AIE-Based Intracellular Light-Up Characteristics for Selective Suppression of Pancreatic Cancer Cells. Chem. Commun. 2015, 51, 17435−17438. (76) Wang, H.; Huang, Y.; Zhao, X.; Gong, W.; Wang, Y.; Cheng, Y. A Novel Aggregation-Induced Emission Based Fluorescent Probe for An Angiotensin Converting Enzyme (ACE) Assay and Inhibitor Screening. Chem. Commun. 2014, 50, 15075−15078. (77) Wang, Y.; Wu, X.; Cheng, Y.; Zhao, X. A Fluorescent Switchable AIE Probe for Selective Imaging of Dipeptidyl Peptidase-4 In Vitro and In Vivo and Its Application in Screening DPP-4 Inhibitors. Chem. Commun. 2016, 52, 3478−3481. (78) Wang, Y.; Chen, Y.; Wang, H.; Cheng, Y.; Zhao, X. Specific Turn-On Fluorescent Probe with Aggregation-Induced Emission

Characteristics for SIRT1Modulator Screening and Living-Cell Imaging. Anal. Chem. 2015, 87, 5046−5049. (79) Liu, X.; Liang, G. Dual Aggregation-Induced Emission for Enhanced Fluorescence Sensing of Furin Activity In Vitro and in Living Cells. Chem. Commun. 2017, 53, 1037−1040. (80) Zhang, R.; Zhang, C.-J.; Feng, G.; Hu, F.; Wang, J.; Liu, B. Specific Light-Up Probe with Aggregation-Induced Emission for Facile Detection of Chymase. Anal. Chem. 2016, 88, 9111−9117. (81) Zhang, C.; Jin, S.; Yang, K.; Xue, X.; Li, Z.; Jiang, Y.; Chen, W.Q.; Dai, L.; Zou, G.; Liang, X. Cell Membrane Tracker Based on Restriction of Intramolecular Rotation. ACS Appl. Mater. Interfaces 2014, 6, 8971−8975. (82) Zhang, C.; Zhang, T.; Jin, S.; Xue, X.; Yang, X.; Gong, N.; Zhang, J.; Wang, P. C.; Tian, J.-H.; Xing, J.; Liang, X.-J. ACS Appl. Mater. Interfaces 2017, 9, 4425−4432. (83) Zhang, T.; Guo, W.; Zhang, C.; Yu, J.; Xu, J.; Li, S.; Tian, J.-H.; Wang, P. C.; Xing, J.-F.; Liang, X.-J. Transferrin-Dressed Virus-like Ternary Nanoparticles with Aggregation-Induced Emission for Targeted Delivery and Rapid Cytosolic Release of siRNA. ACS Appl. Mater. Interfaces 2017, 9, 16006−16014. (84) Geng, J.; Goh, W. L.; Zhang, C.; Lane, D. P.; Liu, B.; Ghadessy, F.; Tan, Y. N. A Highly Sensitive Fluorescent Light-Up Probe for RealTime Detection of the Endogenous Protein Target and Its Antagonism in Live Cells. J. Mater. Chem. B 2015, 3, 5933−5937. (85) Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Targeted Bioimaging and Photodynamic Therapy of Cancer Cells with an Activatable Red Fluorescent Bioprobe. Anal. Chem. 2014, 86, 7987−7995. (86) Yuan, Y.; Zhang, C.-J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2015, 54, 1780−1786. (87) Cheng, Y.; Sun, C.; Ou, X.; Liu, B.; Lou, X.; Xia, F. DualTargeted Peptide-Conjugated Multifunctional Fluorescent Probe with AIEgen for Efficient Nucleus-Specific Imaging and Long-Term Tracing of Cancer Cells. Chem. Sci. 2017, 8, 4571−4578. (88) Peng, N.; Xu, R.; Si, M.; Victorious, A.; Ha, E.; Chang, C.-Y.; Xu, X.-D. Fluorescent Probe with Aggregation-Induced Emission Characteristics for Targeted Labelling and Imaging of Cancer Cells. RSC Adv. 2017, 7, 11282−11285. (89) Ding, Y.; Shi, L.; Wei, H. A “Turn On” Fluorescent Probe for Heparin and Its Oversulfated Chondroitin Sulfate Contaminant. Chem. Sci. 2015, 6, 6361−6366. (90) Feng, G.; Yuan, Y.; Fang, H.; Zhang, R.; Xing, B.; Zhang, G.; Zhang, D.; Liu, B. A Light-Up Probe with Aggregation-Induced Emission Characteristics (AIE) for Selective Imaging, Naked-Eye Detection and Photodynamic Killing of Gram-Positive Bacteria. Chem. Commun. 2015, 51, 12490−12493. (91) Li, N. N.; Li, J. Z.; Liu, P.; Pranantyo, D.; Luo, L.; Chen, J. C.; Kang, E.-T.; Hu, X. F.; Li, C. M.; Xu, L. Q. An Antimicrobial Peptide with an Aggregation-Induced Emission (AIE) Luminogen for Studying Bacterial Membrane Interactions and Antibacterial Actions. Chem. Commun. 2017, 53, 3315−3318. (92) Zhang, R.; Kwok, R. T. K.; Tang, B. Z.; Liu, B. Hybridization Induced Fluorescence Turn-On of AIEgen−Oligonucleotide Conjugates for Specific DNA Detection. RSC Adv. 2015, 5, 28332−28337. (93) Min, X.; Zhuang, Y.; Zhang, Z.; Jia, Y.; Hakeem, A.; Zheng, F.; Cheng, Y.; Tang, B. Z.; Lou, X.; Xia, F. Lab in a Tube: Sensitive Detection of MicroRNAs in Urine Samples from Bladder Cancer Patients Using a Single-Label DNA Probe with AIEgens. ACS Appl. Mater. Interfaces 2015, 7, 16813−16818. (94) Chen, J.; Jiang, H.; Zhou, H.; Hu, Z.; Niu, N.; Shahzad, S. A.; Yu, C. Specific Detection of Cancer Cells through AggregationInduced Emission of a Light-Up Bioprobe. Chem. Commun. 2017, 53, 2398−2401. (95) Wang, J.-X.; Chen, Q.; Bian, N.; Yang, F.; Sun, J.; Qi, A.-D.; Yan, C.-G.; Han, B.-H. Sugar-Bearing Tetraphenylethylene: Novel Fluorescent Probe for Studies of Carbohydrate−Protein Interaction Based AO


Review

ACS Applied Materials & Interfaces on Aggregation-Induced Emission. Org. Biomol. Chem. 2011, 9, 2219− 2226. (96) Sanji, T.; Shiraishi, K.; Tanaka, M. Sugar-Phosphole Oxide Conjugates as “Turn-on” Luminescent Sensors for Lectins. ACS Appl. Mater. Interfaces 2009, 1, 270−273. (97) Sanji, T.; Shiraishi, K.; Nakamura, M.; Tanaka, M. Fluorescence Turn-On Sensing of Lectins with Mannose-Substituted Tetraphenylethenes Based on Aggregation-Induced Emission. Chem. - Asian J. 2010, 5, 817−824. (98) Hu, X.-M.; Chen, Q.; Wang, J.-X.; Cheng, Q.-Y.; Yan, C.-G.; Cao, J.; He, Y.-J.; Han, B.-H. Tetraphenylethylene-Based Glycoconjugate as a Fluorescence “Turn-On” Sensor for Cholera Toxin. Chem. Asian J. 2011, 6, 2376−2381. (99) Kato, T.; Kawaguchi, A.; Nagata, K.; Hatanaka, K. Development of Tetraphenylethylene-Based Fluorescent Oligosaccharide Probes for Detection of Influenza Virus. Biochem. Biophys. Res. Commun. 2010, 394, 200−204. (100) Hang, Y.; He, X.-P.; Yang, L.; Hua, J. Probing Sugar−Lectin Recognitions in the Near-Infrared Region Using Glyco-diketopyrrolopyrrole with Aggregation-Induced-Emission. Biosens. Bioelectron. 2015, 65, 420−426. (101) Jiang, G.; Zeng, G.; Zhu, W.; Li, Y.; Dong, X.; Zhang, G.; Fan, X.; Wang, J.; Wu, Y.; Tang, B. Z. A Selective and Light-Up Fluorescent Probe for β-Galactosidase Activity Detection and Imaging in Living Cells Based on an AIE Tetraphenylethylene Derivative. Chem. Commun. 2017, 53, 4505−4508. (102) Peng, L.; Gao, M.; Cai, X.; Zhang, R.; Li, K.; Feng, G.; Tong, A.; Liu, B. A Fluorescent Light-Up Probe Based on AIE and ESIPT Processes for β-Galactosidase Activity Detection and Visualization in Living Cells. J. Mater. Chem. B 2015, 3, 9168−9172. (103) Wang, Z.; Chen, S.; Lam, J. W. Y.; Qin, W.; Kwok, R. T. K.; Xie, N.; Hu, Q.; Tang, B. Z. Long-Term Fluorescent Cellular Tracing by the Aggregates of AIE Bioconjugates. J. Am. Chem. Soc. 2013, 135, 8238−8245. (104) Peng, H.-S.; Chiu, D. T. Soft Fluorescent Nanomaterials for Biological and Biomedical Imaging. Chem. Soc. Rev. 2015, 44, 4699− 4722. (105) Xing, P.; Zhao, Y. Multifunctional Nanoparticles SelfAssembled from Small Organic Building Blocks for Biomedicine. Adv. Mater. 2016, 28, 7304−7339. (106) Wang, L.; Yang, P.-P.; Zhao, X.-X.; Wang, H. Self-Assembled Nanomaterials for Photoacoustic Imaging. Nanoscale 2016, 8, 2488− 2509. (107) Papadimitriou, A. A.; Salinas, Y.; Resmini, M. Smart Polymeric Nanoparticles as Emerging Tools for ImagingThe Parallel Evolution of Materials. Chem. - Eur. J. 2016, 22, 3612−3620. (108) Wan, Q.; Huang, Q.; Liu, M.; Xu, D.; Huang, H.; Zhang, X.; Wei, Y. Aggregation-Induced Emission Active Luminescent Polymeric Nanoparticles: Non-Covalent Fabrication Methodologies and Biomedical Applications. Appl. Mater. Today 2017, 9, 145−160. (109) Yi, X.; Li, J.; Zhu, Z.; Liu, Q.; Xue, Q.; Ding, D. In Vivo Cancer Research Using Aggregation-Induced Emission Organic Nanoparticles. Drug Discovery Today 2017, 22, 1412−1420. (110) Mahtab, F.; Yu, Y.; Lam, J. W. Y.; Liu, J.; Zhang, B.; Lu, P.; Zhang, X.; Tang, B. Z. Fabrication of Silica Nanoparticles with Both Efficient Fluorescence and Strong Magnetization and Exploration of Their Biological Applications. Adv. Funct. Mater. 2011, 21, 1733−1740. (111) Xia, Y.; Li, M.; Peng, T.; Zhang, W.; Xiong, J.; Hu, Q.; Song, Z.; Zheng, Q. In Vitro Cytotoxicity of Fluorescent Silica Nanoparticles Hybridized with Aggregation-Induced Emission Luminogens for Living Cell Imaging. Int. J. Mol. Sci. 2013, 14, 1080−1092. (112) Li, M.; Lam, J. W. Y.; Mahtab, F.; Chen, S.; Zhang, W.; Hong, Y.; Xiong, J.; Zheng, Q.; Tang, B. Z. Biotin-Ddecorated Fluorescent Silica Nanoparticles with Aggregation-Induced Emission Characteristics: Fabrication, Cytotoxicity and Biological Applications. J. Mater. Chem. B 2013, 1, 676−684. (113) Zhu, Z.; Zhao, X.; Qin, W.; Chen, G.; Qian, J.; Xu, Z. Fluorescent AIE Dots Encapsulated Organically Modified Silica

(ORMOSIL) Nanoparticles for Two-Photon Cellular Imaging. Sci. China: Chem. 2013, 56, 1247−1252. (114) Wang, Z.; Xu, B.; Zhang, L.; Zhang, J.; Ma, T.; Zhang, J.; Fu, X.; Tian, W. Folic Acid-Functionalized Mesoporous Silica Nanospheres Hybridized with AIE Luminogens for Targeted Cancer Cell Imaging. Nanoscale 2013, 5, 2065−2072. (115) Zhang, X.; Zhang, X.; Wang, S.; Liu, M.; Zhang, Y.; Tao, L.; Wei, Y. Facile Incorporation of Aggregation-Induced Emission Materials into Mesoporous Silica Nanoparticles for Intracellular Imaging and Cancer Therapy. ACS Appl. Mater. Interfaces 2013, 5, 1943−1947. (116) Geng, J.; Goh, C. C.; Qin, W.; Liu, R.; Tomczak, N.; Ng, L. G.; Tang, B. Z.; Liu, B. Silica Shelled and Block Copolymer Encapsulated Red-Emissive AIE Nanoparticles with 50% Quantum Yield for TwoPhoton Excited Vascular Imaging. Chem. Commun. 2015, 51, 13416− 13419. (117) Li, Y.; Shao, A.; Wang, Y.; Mei, J.; Niu, D.; Gu, J.; Shi, P.; Zhu, W.; Tian, H.; Shi, J. Morphology-Tailoring of A Red AIEgen from Microsized Rods to Nanospheres for Tumor-Targeted Bioimaging. Adv. Mater. 2016, 28, 3187−3193. (118) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Photostable Fluorescent Organic Dots with Aggregation-Induced Emission (AIE Dots) for Noninvasive Long-Term Cell Tracing. Sci. Rep. 2013, 3, 1150. (119) Feng, G.; Tay, C. Y.; Chui, Q. X.; Liu, R.; Tomczak, N.; Liu, J.; Tang, B. Z.; Leong, D. T.; Liu, B. Ultrabright Organic Dots with Aggregation-Induced Emission Characteristics for Cell Tracking. Biomaterials 2014, 35, 8669−8677. (120) Zhao, Q.; Li, K.; Chen, S.; Qin, A.; Ding, D.; Zhang, S.; Liu, Y.; Liu, B.; Sun, J. Z.; Tang, B. Z. Aggregation-Induced Red-NIR Emission Organic Nanoparticles as Effective and Photostable Fluorescent Probes for Bioimaging. J. Mater. Chem. 2012, 22, 15128−15135. (121) Geng, J.; Li, K.; Ding, D.; Zhang, X.; Qin, W.; Liu, J.; Tang, B. Z.; Liu, B. Lipid-PEG-Folate Encapsulated Nanoparticles with Aggregation Induced Emission Characteristics: Cellular Uptake Mechanism and Two-Photon Fluorescence Imaging. Small 2012, 8, 3655−3663. (122) Li, K.; Zhu, Z.; Cai, P.; Liu, R.; Tomczak, N.; Ding, D.; Liu, J.; Qin, W.; Zhao, Z.; Hu, Y.; Chen, X.; Tang, B. Z.; Liu, B. Organic Dots with Aggregation-Induced Emission (AIE Dots) Characteristics for Dual-Color Cell Tracing. Chem. Mater. 2013, 25, 4181−4187. (123) Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B. Targeted and Image-Guided Photodynamic Cancer Therapy Based on Organic Nanoparticles with Aggregation-Induced Emission Characteristics. Chem. Commun. 2014, 50, 8757−8760. (124) Qin, W.; Li, K.; Feng, G.; Li, M.; Yang, Z.; Liu, B.; Tang, B. Z. Bright and Photostable Organic Fluorescent Dots with AggregationInduced Emission Characteristics for Noninvasive Long-Term Cell Imaging. Adv. Funct. Mater. 2014, 24, 635−643. (125) Geng, J.; Zhu, Z.; Qin, W.; Ma, L.; Hu, Y.; Gurzadyan, G. G.; Tang, B. Z.; Liu, B. Near-Infrared Fluorescence Amplified Organic Nanoparticles with Aggregation-Induced Emission Characteristics for In Vivo Imaging. Nanoscale 2014, 6, 939−945. (126) Ding, D.; Mao, D.; Li, K.; Wang, X.; Qin, W.; Liu, R.; Chiam, D. S.; Tomczak, N.; Yang, Z.; Tang, B. Z.; Kong, D.; Liu, B. Precise and Long-Term Tracking of Adipose-Derived Stem Cells and Their Regenerative Capacity via Superb Bright and Stable Organic Nanodots. ACS Nano 2014, 8, 12620−12631. (127) Xiang, J.; Cai, X.; Lou, X.; Feng, G.; Min, X.; Luo, W.; He, B.; Goh, C. C.; Ng, L. G.; Zhou, J.; Zhao, Z.; Liu, B.; Tang, B. Z. Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization. ACS Appl. Mater. Interfaces 2015, 7, 14965−14974. (128) Cai, X.; Zhang, C.-J.; Lim, F. T. W.; Chan, S. J.; Bandla, A.; Chuan, C. K.; Hu, F.; Xu, S.; Thakor, N. V.; Liao, L.-D.; Liu, B. Organic Nanoparticles with Aggregation-Induced Emission for Bone Marrow Stromal Cell Tracking in a Rat PTI Model. Small 2016, 12, 6576−6585. AP


Review

ACS Applied Materials & Interfaces (129) Cai, X.; Bandla, A.; Mao, D.; Feng, G.; Qin, W.; Liao, L.-D.; Thakor, N.; Tang, B. Z.; Liu, B. Biocompatible Red Fluorescent Organic Nanoparticles with Tunable Size and Aggregation-Induced Emission for Evaluation of Blood−Brain Barrier Damage. Adv. Mater. 2016, 28, 8760−8765. (130) Gao, M.; Su, H.; Lin, G.; Li, S.; Yu, X.; Qin, A.; Zhao, Z.; Zhang, Z.; Tang, B. Z. Targeted Imaging of EGFR Overexpressed Cancer Cells by Brightly Fluorescent Nanoparticles Conjugated with Cetuximab. Nanoscale 2016, 8, 15027−15032. (131) Wu, W.; Feng, G.; Xu, S.; Liu, B. A Photostable Far-Red/NearInfrared Conjugated Polymer Photosensitizer with AggregationInduced Emission for Image-Guided Cancer Cell Ablation. Macromolecules 2016, 49, 5017−5025. (132) Jin, G.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B.; Li, K. Multifunctional Organic Nanoparticles with Aggregation-Induced Emission (AIE) Characteristics for Targeted Photodynamic Therapy and RNA Interference Therapy. Chem. Commun. 2016, 52, 2752− 2755. (133) Yu, G.; Cook, T. R.; Li, Y.; Yan, X.; Wu, D.; Shao, L.; Shen, J.; Tang, G.; Huang, F.; Chen, X.; Stang, P. J. Tetraphenylethene-Based Highly Emissive Metallacage as a Component of Theranostic Supramolecular Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 13720−13725. (134) Li, D.; Zhao, X.; Qin, W.; Zhang, H.; Fei, Y.; Liu, L.; Yong, K.T.; Chen, G.; Tang, B. Z.; Qian, J. Toxicity Assessment and LongTerm Three-Photon Fluorescence Imaging of Bright AggregationInduced Emission Nanodots in Zebrafish. Nano Res. 2016, 9, 1921− 1933. (135) Wang, Y. J.; Shi, Y.; Wang, Z.; Zhu, Z.; Zhao, X.; Nie, H.; Qian, J.; Qin, A.; Sun, J. Z.; Tang, B. Z. A Red to Near-IR Fluorogen: Aggregation-Induced Emission, Large Stokes Shift, High Solid Efficiency and Application in Cell-Imaging. Chem. - Eur. J. 2016, 22, 9784−9791. (136) Song, Z.; Mao, D.; Sung, S. H. P.; Kwok, R. T. K.; Lam, J. W. Y.; Kong, D.; Ding, D.; Tang, B. Z. Activatable Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristics for Selective In Vivo Imaging of Elevated Peroxynitrite Generation. Adv. Mater. 2016, 28, 7249−7256. (137) Li, M.; Gao, Y.; Yuan, Y.; Wu, Y.; Song, Z.; Tang, B. Z.; Liu, B.; Zheng, Q. C. One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma. ACS Nano 2017, 11, 3922−3932. (138) Nicol, A.; Qin, W.; Kwok, R. T. K.; Burkhartsmeyer, J. M.; Zhu, Z.; Su, H.; Luo, W.; Lam, J. W. Y.; Qian, J.; Wong, K. S.; Tang, B. Z. Functionalized AIE Nanoparticles with Efficient Deep-Red Emission, Mitochondrial Specificity, Cancer Cell Selectivity and Multiphoton Susceptibility. Chem. Sci. 2017, 8, 4634−4643. (139) Li, M.; Hong, Y.; Wang, Z.; Chen, S.; Gao, M.; Kwok, R. T. K.; Qin, W.; Lam, J. W. Y.; Zheng, Q.; Tang, B. Z. Fabrication of Chitosan Nanoparticles with Aggregation-Induced Emission Characteristics and Their Applications in Long-Term Live Cell Imaging. Macromol. Rapid Commun. 2013, 34, 767−771. (140) Zhang, Y.; Chen, Y.; Li, X.; Zhang, J.; Chen, J.; Xu, B.; Fu, X.; Tian, W. Folic Acid-Functionalized AIE Pdots Based on Amphiphilic PCL-b-PEG for Targeted Cell Imaging. Polym. Chem. 2014, 5, 3824− 3830. (141) Mondal; Sarkar, J.; Ghosh, S. Fluorescent PEGlated Oligourethane Nanoparticles for Long-Term Cellular Tracing. Chem. - Eur. J. 2016, 22, 10930−10936. (142) Ma, H.; Qi, C.; Cheng, C.; Yang, Z.; Cao, H.; Yang, Z.; Tong, J.; Yao, X.; Lei, Z. AIE-Active Tetraphenylethylene Cross-Linked NIsopropylacrylamide Polymer: A Long-Term Fluorescent Cellular Tracker. ACS Appl. Mater. Interfaces 2016, 8, 8341−8348. (143) Cao, Z.; Liang, X.; Chen, H.; Gao, M.; Zhao, Z.; Chen, X.; Xu, C.; Qu, C.; Qi, D.; Tang, B. Z. Bright and Biocompatible AIE Polymeric Nanoparticles Prepared from Miniemulsion for Fluorescence Cell Imaging. Polym. Chem. 2016, 7, 5571−5578.

(144) Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Wei, Y. Renewable Itaconic Acid Based Cross-Linked Fluorescent Polymeric Nanoparticles for Cell Imaging. Polym. Chem. 2014, 5, 5885−5889. (145) Wang, K.; Zhang, X.; Zhang, X.; Ma, C.; Li, Z.; Huang, Z.; Zhang, Q.; Wei, Y. Preparation of Emissive Glucose-Containing Polymer Nanoparticles and Their Cell Imaging Applications. Polym. Chem. 2015, 6, 4455−4461. (146) Li, H.; Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Wei, Y. Stable Cross-Linked Fluorescent Polymeric Nanoparticles for Cell Imaging. Macromol. Rapid Commun. 2014, 35, 1661−1667. (147) Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. A Novel Method for Preparing AIE Dye Based CrossLinked Fluorescent Polymeric Nanoparticles for Cell Imaging. Polym. Chem. 2014, 5, 683−688. (148) Zhang, X.; Zhang, X.; Yang, B.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Fabrication of Aggregation Induced Emission Dye-based Fluorescent Organic Nanoparticles via Emulsion Polymerization and Their Cell Imaging Applications. Polym. Chem. 2014, 5, 399−404. (149) Liu, M.; Zhang, X.; Yang, B.; Liu, L.; Deng, F.; Zhang, X.; Wei, Y. Polylysine Crosslinked AIE Dye Based Fluorescent Organic Nanoparticles for Biological Imaging Applications. Macromol. Biosci. 2014, 14, 1260−1267. (150) Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. PEGylation and Cell Imaging Applications of AIE Based Fluorescent Organic Nanoparticles via Ring-Opening Reaction. Polym. Chem. 2014, 5, 689−693. (151) Xie, G.; Ma, C.; Zhang, X.; Liu, H.; Yang, L.; Wang, K.; Wei, Y. Chitosan-Based Cross-Linked Fluorescent Polymer Containing Aggregation-Induced Emission Fluorogen for Cell Imaging. Dyes Pigm. 2017, 143, 276−283. (152) Wan, Q.; Xu, D.; Mao, L.; He, Z.; Zeng, G.; Shi, Y.; Deng, F.; Liu, M.; Zhang, X.; Wei, Y. Facile Fabrication of AIE-Active Fluorescent Polymeric Nanoparticles with Ultra-Low Critical Micelle Concentration Based on Ce(IV) Redox Polymerization for Biological Imaging Applications. Macromol. Rapid Commun. 2017, 38, 1600752. (153) Jeong, K.; Park, S.; Lee, Y.-D.; Lim, C.-K.; Kim, J.; Chung, B. H.; Kwon, I. C.; Park, C. R.; Kim, S. Conjugated Polymer/ Photochromophore Binary Nanococktails: Bistable Photoswitching of Near-Infrared Fluorescence for In Vivo Imaging. Adv. Mater. 2013, 25, 5574−5580. (154) Zhao, Q.; Sun, J. Z. Red and Near Infrared Emission Materials with AIE Characteristics. J. Mater. Chem. C 2016, 4, 10588−10609. (155) Chen, S.; Hong, Y.; Liu, Y.; Liu, J.; Leung, C. W. T.; Li, M.; Kwok, R. T. K.; Zhao, E.; Lam, J. W. Y.; Yu, Y.; Tang, B. Z. Full-Range Intracellular pH Sensing by an Aggregation-Induced Emission-Active Two-Channel Ratiometric Fluorogen. J. Am. Chem. Soc. 2013, 135, 4926−4929. (156) Lu, H.; Zheng, Y.; Zhao, X.; Wang, L.; Ma, S.; Han, X.; Xu, B.; Tian, W.; Gao, H. Highly Efficient Far Red/Near-Infrared Solid Fluorophores: Aggregation-Induced Emission, Intramolecular Charge Transfer, Twisted Molecular Conformation, and Bioimaging Applications. Angew. Chem., Int. Ed. 2016, 55, 155−159. (157) Guo, Z.; Shao, A.; Zhu, W.-H. Long Wavelength AIEgen of Quinoline-Malononitrile. J. Mater. Chem. C 2016, 4, 2640−2646. (158) Chen, S.; Hong, Y.; Zeng, Y.; Sun, Q.; Liu, Y.; Zhao, E.; Bai, G.; Qu, J.; Hao, J.; Tang, B. Z. Mapping Live Cell Viscosity with an Aggregation-Induced Emission Fluorogen by Means of Two-Photon Fluorescence Lifetime Imaging. Chem. - Eur. J. 2015, 21, 4315−4320. (159) Alifu, N.; Yan, L.; Zhang, H.; Zebibula, A.; Zhu, Z.; Xi, W.; Roe, A. W.; Xu, B.; Tian, W.; Qian, J. Organic Dye Doped Nanoparticles with NIR Emission and Biocompatibility for UltraDeep In Vivo Two-Photon Microscopy under 1040 nm Femtosecond Excitation. Dyes Pigm. 2017, 143, 76−85. (160) Zhu, Z.; Qian, J.; Zhao, X.; Qin, W.; Hu, R.; Zhang, H.; Li, D.; Xu, Z.; Tang, B. Z.; He, S. Stable and Size-Tunable AggregationInduced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging. ACS Nano 2016, 10, 588−597. AQ


Review

ACS Applied Materials & Interfaces (161) Zhang, H.; Alifu, N.; Jiang, T.; Zhu, Z.; Wang, Y.; Hua, J.; Qian, J. Biocompatible Aggregation-Induced Emission Nanoparticles with Red Emission for In Vivo Three-Photon Brain Vascular Imaging. J. Mater. Chem. B 2017, 5, 2757−2762. (162) Liu, S.; Sun, H.; Ma, Y.; Ye, S.; Liu, X.; Zhou, X.; Mou, X.; Wang, L.; Zhao, Q.; Huang, W. Rational Design of Metallophosphors with Tunable Aggregation-Induced Phosphorescent Emission and Their Promising Applications in Time-Resolved Luminescence Assay and Targeted Luminescence Imaging of Cancer Cells. J. Mater. Chem. 2012, 22, 22167−22173. (163) Chen, Y.; Qiao, L.; Yu, B.; Li, G.; Liu, C.; Ji, L.; Chao, H. Mitochondria-Specific Phosphorescent Imaging and Tracking in Living Cells with an AIPE-Active Iridium(III) Complex. Chem. Commun. 2013, 49, 11095−11097. (164) Li, K.; Ding, D.; Prashant, C.; Qin, W.; Yang, C.-T.; Tang, B. Z.; Liu, B. Gadolinium-Functionalized Aggregation-Induced Emission Dots as Dual-Modality Probes for Cancer Metastasis Study. Adv. Healthcare Mater. 2013, 2, 1600−1605. (165) Chen, Y.; Li, M.; Hong, Y.; Lam, J. W. Y.; Zheng, Q.; Tang, B. Z. Dual-Modal MRI Contrast Agent with Aggregation-Induced Emission Characteristic for Liver Specific Imaging with Long Circulation Lifetime. ACS Appl. Mater. Interfaces 2014, 6, 10783− 10791. (166) Zhang, J.; Li, C.; Zhang, X.; Huo, S.; Jin, S.; An, F.-F.; Wang, X.; Xue, X.; Okeke, C. I.; Duan, G.; Guo, F.; Zhang, X.; Hao, J.; Wang, P. C.; Zhang, J.; Liang, X.-J. In Vivo Tumor-Targeted Dual-Modal Fluorescence/CT Imaging Using A Nanoprobe Co-loaded with an Aggregation-Induced Emission Dye and Gold Nanoparticles. Biomaterials 2015, 42, 103−111. (167) Geng, J.; Liao, L.-D.; Qin, W.; Tang, B. Z.; Thakor, N.; Liu, B. Fluorogens with Aggregation Induced Emission: Ideal Photoacoustic Contrast Reagents due to Intramolecular Rotation. J. Nanosci. Nanotechnol. 2015, 15, 1864−1868. (168) Song, Z.; Zhang, W.; Jiang, M.; Sung, H. H. Y.; Kwok, R. T. K.; Nie, H.; Williams, I. D.; Liu, B.; Tang, B. Z. Synthesis of ImidazoleBased AIEgens with Wide Color Tunability and Exploration of their Biological Applications. Adv. Funct. Mater. 2016, 26, 824−832. (169) Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B. Image-Guided Combination Chemotherapy and Photodynamic Therapy Using a Mitochondria-Targeted Molecular Probe with Aggregation-Induced Emission Characteristics. Chem. Sci. 2015, 6, 4580−4586. (170) Li, Y.; Wu, Y.; Chang, J.; Chen, M.; Liu, R.; Li, F. A Bioprobe Based on Aggregation Induced Emission (AIE) for Cell Membrane Tracking. Chem. Commun. 2013, 49, 11335−11337. (171) Wang, E.; Zhao, E.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z. A Highly Selective AIE Fluorogen for Lipid Droplet Imaging in Live Cells and Green Algae. J. Mater. Chem. B 2014, 2, 2013−2019. (172) Kang, M.; Gu, X.; Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Li, F.; Tang, B. Z. A Near-Infrared AIEgen for Specific Imaging of Lipid Droplets. Chem. Commun. 2016, 52, 5957−5960. (173) Wang, Z.; Gui, C.; Zhao, E.; Wang, J.; Li, X.; Qin, A.; Zhao, Z.; Yu, Z.; Tang, B. Z. Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens. ACS Appl. Mater. Interfaces 2016, 8, 10193− 10200. (174) Gao, M.; Su, H.; Li, S.; Lin, Y.; Ling, X.; Qin, A.; Tang, B. Z. An Easily Accessible Aggregation-Induced Emission Probe for Lipid Droplet-Specific Imaging and Movement Tracking. Chem. Commun. 2017, 53, 921−924. (175) Jiang, M.; Gu, X.; Lam, J. W. Y.; Zhang, Y.; Kwok, R. T. K.; Wong, K. S.; Tang, B. Z. Two-Photon AIE Bio-Probe with Large Stokes Shift for Specific Imaging of Lipid Droplets. Chem. Sci. 2017, 8, 5440−5446. (176) Gao, M.; Su, H.; Lin, Y.; Ling, X.; Li, S.; Qin, A.; Tang, B. Z. Photoactivatable Aggregation-Induced Emission Probes for Lipid Droplets-Specific Live Cell Imaging. Chem. Sci. 2017, 8, 1763−1768. (177) Leung, C. W. T.; Hong, Y.; Chen, S.; Zhao, E.; Lam, J. W. Y.; Tang, B. Z. A Photostable AIE Luminogen for Specific Mitochondrial Imaging and Tracking. J. Am. Chem. Soc. 2013, 135, 62−65.

(178) Gao, M.; Sim, C. K.; Leung, C. W. T.; Hu, Q.; Feng, G.; Xu, F.; Tang, B. Z.; Liu, B. A Fluorescent Light-Up Probe with AIE Characteristics for Specific Mitochondrial Imaging to Identify Differentiating Brown Adipose Cells. Chem. Commun. 2014, 50, 8312−8315. (179) Zhao, N.; Chen, S.; Hong, Y.; Tang, B. Z. A Red Emitting Mitochondria-Targeted AIE Probe as an Indicator for Membrane Potential and Mouse Sperm Activity. Chem. Commun. 2015, 51, 13599−13602. (180) Gu, X.; Zhao, E.; Lam, J. W. Y.; Peng, Q.; Xie, Y.; Zhang, Y.; Wong, K. S.; Sung, H. H. Y.; Williams, I. D.; Tang, B. Z. Mitochondrion-Specific Live-Cell Bioprobe Operated in a Fluorescence Turn-On Manner and a Well-Designed Photoactivatable Mechanism. Adv. Mater. 2015, 27, 7093−7100. (181) Yu, G.; Wu, D.; Li, Y.; Zhang, Z.; Shao, L.; Zhou, J.; Hu, Q.; Tang, G.; Huang, F. A Pillar[5]arene-Based [2]Rotaxane Lights Up Mitochondria. Chem. Sci. 2016, 7, 3017−3024. (182) Situ, B.; Chen, S.; Zhao, E.; Leung, C. W. T.; Chen, Y.; Hong, Y.; Lam, J. W. Y.; Wen, Z.; Liu, W.; Zhang, W.; Zheng, L.; Tang, B. Z. Real-Time Imaging of Cell Behaviors in Living Organisms by a Mitochondria-Targeting AIE Fluorogen. Adv. Funct. Mater. 2016, 26, 7132−7138. (183) Shin, W. S.; Lee, M.-G.; Verwilst, P.; Lee, J. H.; Chi, S.-G.; Kim, J. S. Mitochondria-Targeted Aggregation Induced Emission Theranostics: Crucial Importance of In Situ Activation. Chem. Sci. 2016, 7, 6050−6059. (184) Yang, X.; Wang, N.; Zhang, L.; Dai, L.; Shao, H.; Jiang, X. Organic Nanostructure-Based Probes for Two-Photon Imaging of Mitochondria and Microbes with Emission Between 430 nm and 640 nm. Nanoscale 2017, 9, 4770−4776. (185) Gao, M.; Hu, Q.; Feng, G.; Tang, B. Z.; Liu, B. A Fluorescent Light-Up Probe with “AIE + ESIPT” Characteristics for Specific Detection of Lysosomal Esterase. J. Mater. Chem. B 2014, 2, 3438− 3442. (186) Lou, X.; Zhang, M.; Zhao, Z.; Min, X.; Hakeem, A.; Huang, F.; Gao, P.; Xia, F.; Tang, B. Z. A Photostable AIE Fluorogen for Lysosome-Targetable Imaging of Living Cells. J. Mater. Chem. B 2016, 4, 5412−5417. (187) Yu, C. Y. Y.; Zhang, W.; Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. A Photostable AIEgen for Nucleolus and Mitochondria Imaging with Organelle-Specific Emission. J. Mater. Chem. B 2016, 4, 2614−2619. (188) Yu, G.; Tang, G.; Huang, F. Supramolecular Enhancement of Aggregation-induced Emission and Its Application in Cancer Cell Imaging. J. Mater. Chem. C 2014, 2, 6609−6617. (189) Huang, Y.; Zhang, G.; Hu, F.; Jin, Y.; Zhao, R.; Zhang, D. Emissive Nanoparticles from Pyridinium-substituted Tetraphenylethylene Salts: Imaging and Selective Cytotoxicity Towards Cancer Cells In Vitro and In Vivo by Varying Counter Anions. Chem. Sci. 2016, 7, 7013−7019. (190) Li, Y.; Hu, X.; Tian, S.; Li, Y.; Zhang, G.; Zhang, G.; Liu, S. Polyion Complex Micellar Nanoparticles for Integrated Fluorometric Detection and Bacteria Inhibition in Aqueous Media. Biomaterials 2014, 35, 1618−1626. (191) Zhao, E.; Chen, Y.; Wang, H.; Chen, S.; Lam, J. W. Y.; Leung, C. W. T.; Hong, Y.; Tang, B. Z. Light-Enhanced Bacterial Killing and Wash-Free Imaging Based on AIE Fluorogen. ACS Appl. Mater. Interfaces 2015, 7, 7180−7188. (192) Zhao, L.; Chen, Y.; Yuan, J.; Chen, M.; Zhang, H.; Li, X. Electrospun Fibrous Mats with Conjugated Tetraphenylethylene and Mannose for Sensitive Turn-On Fluorescent Sensing of Escherichia coli. ACS Appl. Mater. Interfaces 2015, 7, 5177−5186. (193) Gao, M.; Wang, L.; Chen, J.; Li, S.; Lu, G.; Wang, L.; Wang, Y.; Ren, L.; Qin, A.; Tang, B. Z. Aggregation-Induced Emission Active Probe for Light-Up Detection of Anionic Surfactants and Wash-Free Bacterial Imaging. Chem. - Eur. J. 2016, 22, 5107−5112. (194) Li, Q.; Wu, Y.; Lu, H.; Wu, X.; Chen, S.; Song, N.; Yang, Y.-W.; Gao, H. Construction of Supramolecular Nanoassembly for AR


Review

ACS Applied Materials & Interfaces Responsive Bacterial Elimination and Effective Bacterial Detection. ACS Appl. Mater. Interfaces 2017, 9, 10180−10189. (195) Hou, X.; Zeng, F.; Wu, S. A Fluorescent Assay for γGlutamyltranspeptidase via Aggregation Induced Emission and Its Applications in Real Samples. Biosens. Bioelectron. 2016, 85, 317−323. (196) Hang, Y.; Wang, J.; Jiang, T.; Lu, N.; Hua, J. Diketopyrrolopyrrole-Based Ratiometric/Turn-On Fluorescent Chemosensors for Citrate Detection in the Near-Infrared Region by an Aggregation-Induced Emission Mechanism. Anal. Chem. 2016, 88, 1696−1703. (197) Jiang, T.; Lu, N.; Hang, Y.; Yang, J.; Mei, J.; Wang, J.; Hua, J.; Tian, H. Dimethoxy Triarylamine-Derived Terpyridine−Zinc Complex: A Fluorescence Light-Up Sensor for Citrate Detection Based on Aggregation-Induced Emission. J. Mater. Chem. C 2016, 4, 10040− 10046. (198) Zhao, L.; Wang, T.; Wu, Q.; Liu, Y.; Chen, Z.; Li, X. Fluorescent Strips of Electrospun Fibers for Ratiometric Sensing of Serum Heparin and Urine Trypsin. ACS Appl. Mater. Interfaces 2017, 9, 3400−3410. (199) Zhao, L.; Xie, S.; Song, X.; Wei, J.; Zhang, Z.; Li, X. Ratiometric Fluorescent Response of Electrospun Fibrous Strips for Real-Time Sensing of Alkaline Phosphatase in Serum. Biosens. Bioelectron. 2017, 91, 217−224. (200) Lou, X.; Zhuang, Y.; Zuo, X.; Jia, Y.; Hong, Y.; Min, X.; Zhang, Z.; Xu, X.; Liu, N.; Xia, F.; Tang, B. Z. Real-Time, Quantitative Lighting-up Detection of Telomerase in Urines of Bladder Cancer Patients by AIEgens. Anal. Chem. 2015, 87, 6822−6827. (201) Zhuang, Y.; Zhang, M.; Chen, B.; Duan, R.; Min, X.; Zhang, Z.; Zheng, F.; Liang, H.; Zhao, Z.; Lou, X.; Xia, F. Quencher Group Induced High Specificity Detection of Telomerase in Clear and Bloody Urines by AIEgens. Anal. Chem. 2015, 87, 9487−9493. (202) Ou, X.; Hong, F.; Zhang, Z.; Cheng, Y.; Zhao, Z.; Gao, P.; Lou, X.; Xia, F.; Wang, S. A Highly Sensitive and Facile Graphene OxideBased Nucleic Acid Probe: Label-Free Detection of Telomerase Activity in Cancer Patient’s Urine Using AIEgens. Biosens. Bioelectron. 2017, 89, 417−421. (203) Zhuang, Y.; Xu, Q.; Huang, F.; Gao, P.; Zhao, Z.; Lou, X.; Xia, F. Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients. ACS Sens. 2016, 1, 572−578. (204) Wu, Y.; Huang, S.; Zeng, F.; Wang, J.; Yu, C.; Huang, J.; Xie, H.; Wu, S. A Ratiometric Fluorescent System for Carboxylesterase Detection with AIE Dots as FRET Donors. Chem. Commun. 2015, 51, 12791−12794. (205) Shi, J.; Deng, Q.; Wan, C.; Zheng, M.; Huang, F.; Tang, B. Fluorometric Probing of the Lipase Level as Acute Pancreatitis Biomarkers Based on Interfacially Controlled Aggregation-Induced Emission (AIE). Chem. Sci. 2017, 8, 6188−6195. (206) Jiang, G.; Wang, J.; Yang, Y.; Zhang, G.; Liu, Y.; Lin, H.; Zhang, G.; Li, Y.; Fan, X. Fluorescent Turn-On Sensing of Bacterial Lipopolysaccharide in Artificial Urine Sample with Sensitivity down to Nanomolar by Tetraphenylethylene Based Aggregation Induced Emission Molecule. Biosens. Bioelectron. 2016, 85, 62−67. (207) Yang, S.; Gao, T.; Dong, J.; Xu, H.; Gao, F.; Chen, Q.; Gu, Y.; Zeng, W. A Novel Water-Soluble AIE-Based Fluorescence Probe with Red Emission for the Sensitive Detection of Heparin in Aqueous Solution and Human Serum Samples. Tetrahedron Lett. 2017, 58, 3681−3686. (208) Li, Q.; Li, Z. AIE Probes towards Biomolecules: The Improved Selectivity with the Aid of Graphene Oxide. Sci. China: Chem. 2015, 58, 1800−1809.

AS


Progress and Trends in AIE-Based Bioprobes: A Brief Overview

Recommend Documents