Theranostic applications of biomolecule‐responsive aggregation‐induced emission luminogens

Highly sensitive and high‐accuracy detection of biomolecular changes is vital for the early diagnosis and treatment of diverse diseases. Due to their unique advantages and the broad application prospects in biomolecular detection, aggregation‐induced emission luminogens (AIEgens) have especially attracted the attention of related researchers. In recent years, the development of AIEgens has been increasingly rapid and a myriad of related AIEgens have been reported. This review thus presents a systematic overview on the theranostic applications of biomolecule‐responsive AIEgens based on four representative types, including enzymes, reactive nitrogen species, reactive oxygen species and biological thiols. In each type, we provide detailed elucidation of the associated design strategies, discuss the pertinent responsive mechanisms and present the corresponding diagnostic and therapeutic applications in cancer, inflammation and organic injury. Additionally, the main findings are summarized and the existing limitations and promising directions for future research in this field are also outlined. Through this review, we hope to provide inspiration for researchers in related fields to design new biomolecule‐responsive fluorescent materials for clinical diagnosis and therapy of diseases.

Disease is a major threat to human health and seriously impacts the quality of life.According to the analysis from the Global Burden of Disease Study, the gap between overall life expectancy and healthy life expectancy (HALE) has widened in most countries, meaning that people are spending more years in ill health. 1 Researchers have been devoting continuous efforts to achieving the effective diagnosis and treatment of various diseases.The disease progression is always accompanied by the changes in the internal environment, cellular status, and the levels of certain biomolecules.In turn, these biologically environmental or molecular alterations can signal the progress of diseases.For example, the level of alphafetoprotein (AFP) have been applied to diagnose hepatocellular carcinoma (HCC), and to guide and monitor the corresponding treatment. 2Likewise, the level of procalcitonin (PCT) can be used as an indicator for the early diagnosis of bacterial infection, and the detection of creatine kinase MB (CK-MB) level is very important for the evaluation of myocardial infarction, etc. [3][4][5][6] Accordingly, highly sensitive and high-accuracy detection of disease biomarkers is essential for the early diagnosis and treatment of diverse diseases (Scheme 1).
Fluorescence techniques have attracted considerable interests due to their advantages, such as selectivity, visualization, high sensitivity, versatility and noninvasiveness.It is noteworthy that the utilization of fluorescent probes allows us to facilely detect different kinds of biomolecules via the specific interactions and fluorescent responses.][9][10][11][12][13][14] Among them, there are many conventional fluorescent systems that face unavoidable aggregation-induced quenching (ACQ) problems, which seriously limit their practical applications in biological detection due to photobleaching and low signal-to-noise ratio. 15,16In sharp contrast to ACQ systems, Tang's group discovered a special type of fluorophore in 2001, that is, aggregation-induced emission luminogens (AIEgens), which are non-emissive in the monodisperse state but emit intense fluorescence in the aggregate state. 17It is noteworthy that AIEgens could provide higher photobleaching thresholds, better photostability and higher signal reliability than those of the conventional ACQ fluorophores. 15,18,19][26][27][28] Herein, we make a systematic summarization and give a clear classification of the recent progress on the biomolecule-responsive AIEgens according to the type of biomolecules, including enzymes, reactive nitrogen species (RNS), reactive oxygen species (ROS) and biological thiols.We illustrate the design strategies of biomolecule-responsive AIEgens with representative examples in each section, and explain how they respond to various biomolecules and how they can be utilized for diagnosis and treatment of diseases.We also give our perspective on the future of this research area.This review aims to inspire researchers in related fields to create more easily synthesized and multifunction AIEgens that can be used in the theranostics of more diseases.

| ENZYME
][35] Fluorescence imaging techniques based on various types of fluorescent probes can not only detect enzyme activity in situ and in real-time but also have the advantage of high sensitivity and rapidity. 36,37Therefore, designing new fluorescent materials that can detect different enzymes is of great significance for clinical applications.In this section, we summarize the design and detection principles of AIEgens and their detections for several critical enzymes like alkaline phosphatase, β-galactosidase, reductase, acetylcholinesterase, telomerase and caspase.

S C H E M E 1
Schematic illustration of different types of biomolecules responded by AIEgens for diagnosis and therapy.

| Alkaline phosphatase
Alkaline phosphatase (ALP) is a crucial enzyme that can catalyze the hydrolysis of phosphate monoesters and thus remove the phosphate group of substrate molecules. 38As an essential biomarker, the abnormal levels of ALP are correlated with a variety of diseases including liver diseases (hepatitis, Hepatic neoplasia, and hepatic necrosis), bone disease (Paget's disease, malignant bone tumors, and osteomalacia), endocrine disease (hyperadrenocorticism, hyperparathyroidism, and so on). 39Besides, the expression level of ALP in peripheral blood is markedly increased in tumorous patients, so it is also an important clinical indicator associated with the occurrence, development and deterioration of cancer. 40e design strategy of ALP-responsive AIEgens is commonly based on the fact that ALP could effectively hydrolyze the phosphate group and cause the hydrophilic-hydrophobic transition of organic molecules. 38One typical example is the following AIEgen, DQM-ALP (Figure 1A). 41DQM-ALP had an AIE-active core, a hydrophobic quinoline-malononitrile (QM) derivative, and an ALP-recognition unit, a hydrophilic phosphate group.The hydrophilic phosphate group endowed water solubility with DQM-ALP, so it showed no fluorescence in vitro.In contrast, when ALP was present, the phosphate group of DQM-ALP was hydrolyzed, which allowed the rapid release of hydrophobic DQM-OH and exhibited bright fluorescence due to the AIE effect.Based on this light-up characteristic, DQM- -3 of 25 ALP could be applied for long-term monitoring of endogenous ALP produced by tumor cells (Figure 1B) and monitoring the changes of ALP level in the druginduced acute liver injury (DIALI) caused by an overdose of acetaminophen (APAP) (Figure 1C).From Figure 1D, we could see an obvious fluorescence signal in the liver tissue of APAP-administered mice realized the early prediction of DIALI.In addition, DQM-ALP could be utilized to distinguish between normal and tumor tissue due to the difference in ALP level, so DQM-ALP could be a valuable tool to assist surgeons during tumor resection in the future.This work demonstrates that the ALP-responsive DQM-ALP is promising for the long-term monitoring of cellular ALP and can be applied as a practical tool for the diagnosis of DIALI and cancer as well as for assisting the excision of cancerous tissues.
Based on the same "light-up" strategy, Zhu and coworkers also designed an ALP-responsive AIEgen, TPEQHA. 42TPEQHA could detect ALP in tumor cells in situ and distinguish tumor cells from normal cells by adding the phosphate group onto the phenolic hydroxyl group of TPEQH.TPEQHA possessed higher resolution and accuracy than the ACQ fluorescent probe.In the same way, Zhao's group developed a new AIEgen, TPEQN-P, which also exhibited a "turn-on" fluorescent response in the presence of ALP. 43The low detection limit of ALP and good applicability in fetal bovine serum samples demonstrated the great potential of TPEQHA in clinical diagnostic and biomedical applications.

| β-galactosidase
β-galactosidase (β-gal) is a glycoside hydrolase.The activity of β-gal is usually elevated in senescent cells, 44 and its expression is also higher in primary ovarian cancer than that in the normal ovaries. 45Thus, β-gal is considered as an important biomarker for the detection of senescent cells as well as primary ovarian cancer.Since ACQ probes cannot constantly provide in situ signal at the molecular level to achieve in situ sensing and long-term tracing of β-gal, Guo and coworkers thus designed an enzyme-activatable AIEgen, QM-βgal, to overcome this problem. 46s shown in Figure 2A, QM-βgal consisted of two parts, an AIE signal reporter and a β-gal responsive unit, which also conferred water solubility to QM-βgal.When QM-βgal was hydrolyzed by β-gal, QM-OH was released and aggregated in situ to form nanoaggregates with strong fluorescence emission.As a biological probe, QMβgal possessed good selectivity towards β-gal (Figure 2B) and showed high photostability and biocompatibility, making it suitable for long-term tracking and imaging.
Accordingly, intracellular β-gal levels were successfully imaged by QM-βgal.As shown in Figures 2C,D, there was no emission in 293T cells, but a clear fluorescence signal could be observed in SKOV-3 cells.As reverse verification, when the β-gal inhibitor was added to SKOV-3 cells, no fluorescent signal could be observed.Furthermore, the fluorescence of the cells could be seen from the enlarged images located locally rather than throughout the cytoplasm, indicating that QM-βgal could generate nanoaggregates in situ to achieve the on-site detection of β-gal activity.Moreover, QM-βgal could be employed for longterm imaging of overexpressed β-gal cells for up to 12 h.This work offers an excellent tool which enables on-site imaging and long-term tracking of β-gal and also inspires the design of enzyme-activated AIEgens in preclinical applications.
Recently, another novel AIEgen, TPh-PyBz-β-gal, was reported by Jia et al. 47 In this molecule, β-gal could break the glycosidic bond on it, where the release of hydrophobic and AIE-active fluorophore, TPh-PyBz, would aggregate in aqueous solution and show turn-on fluorescence.Thus, the AIEgen, TPh-PyBz-β-gal, could be exploited for β-gal imaging in live cells.Based on these two examples, it is not difficult to see that we can construct diverse β-gal-responsive AIEgens by introducing the units containing galactosidic bonds into the AIE framework by making use of the hydrolysis ability of β-gal for galactosidic bonds.The water solubility of AIEgens can also be solved.These examples provide inspiration for designing β-gal responsive AIEgens, and these new AIEgens have great potential for clinical applications as well as helping us to explore the biological functions of β-gal.

| Reductase
When the oxygen supply is not enough, hypoxia occurs, and it is a feature of various diseases such as cancer, heart disease and vascular disease. 48Besides, it is an important factor contributing to cellular diversity in intra-and intertumor. 49Hypoxia can lead to cellular dysfunction and changes in the levels of some reductases, such as the elevated azoreductase and nitroreductase (NTR). 50,51herefore, detecting these enzymes via responsive probes can reflect the hypoxic state in cells.Typically, Tang's group developed an azo-containing dark pro-AIEgen, TPE-Azo, that could respond to azoreductase in living cells. 52TPE-Azo remained in a non-fluorescent dormant state because the azo group move actively in cells.However, in the presence of azoreductase in hypoxic cells, TPE-Azo was reduced, and TPE-Am was generated, which emitted intense fluorescence to visualize the hypoxic state of cells (Figure 3A).From the imaging experiments of HeLa cells, it could be seen that the fluorescence intensity gradually increased with the gradual decrease in oxygen concentration, indicating an increase in the expression level of azoreductase under hypoxic conditions.Furthermore, when HeLa cells were cocultured with the cellular azoreductase inhibitor diphenyliodonium chloride (DPI) in a hypoxic environment, a significant reduction in the fluorescence signal could be observed, indicating that the release of TPE-Am was indeed regulated by the intracellular azoreductase (Figure 3B).Lin et al. synthesized another AIE fluorescent probe, TPE-HY, with nitro as the recognition site, and successfully employed it to detect NTR activity in cells. 53Resulted from the intramolecular charge transfer (ICT) effect, TPE-HY exhibited a strong fluorescence.In hypoxia, the reductive NTR in the cell could gradually reduce the nitro group on TPE-HY to the amino group and release the reduction product TPE-NH.The electronic structure of the molecule changed from "D-π-A" to "D-π-D", and the ICT process was inhibited, causing fluorescence quenching.TPE-HY held better selectivity, higher sensitivity, and faster response than the previously reported probes, and it was also applied for the activating monitoring of NTR in cells.Acetylcholinesterase (AChE) has a very essential function in the body.By breaking down the neurotransmitter acetylcholine into choline, it can take part in the processes of neurotransmission. 546][57][58] To understand the related diseases' causes and development better, it is important to detecting AChE accurately, in real time, and at the site of action.Herein, AIE fluorescent probes can achieve this aim.For instance, Zhang and coworkers designed an AIE-active fluorescent probe, TCFPB-AChE, to monitor the activity of AChE in vivo and in vitro. 59Different from the conventional ACQ probes based on polycyclic aromatic fluorophores, TCFPB-AChE was synthesized with AIEactive TCFIS as the core and dimethyl carbamate as the responsive group (Figure 4A).TCFPB-AChE was nonemissive in the aqueous solution due to the weak ICT effect, but AChE could induce the production of hydrophobic TCFIS that could release strong fluorescence due to the strong ICT effect.In the AChE inhibition assay, the addition of the AChE inhibitor donepezil significantly reduced the fluorescence, indicating the excellent selectivity of TCFPB-AChE for AChE (Figure 4B).And in the Alzheimer's disease (AD) model, the TCFPB-AChE was also successfully activated by the AChE existed in the brain tissue of AD mice, emitting a significant fluorescent signal (Figure 4C).More importantly, we could visualize AChE within the mouse brain when employing TCFPB-AChE.As shown in Figure 4D, we could observe a weak fluorescence signal in the phosphate buffered saline (PBS) group.In the experimental group, a remarkable enhanced fluorescence signal could be seen (Figure 4E), demonstrating that this AIEgen could realize real-time monitoring of AChE activity in vivo.

| Telomerase
Telomerase is a kind of ribonucleoprotein that is necessary for the synthesis of telomeric DNA.The activation of telomerase is usually associated with the cellular immortality. 60The ability to express telomerase and maintain telomeric DNA is very critical in carcinogenesis.The activity of telomerase is relatively high in most cancer cells but is virtually undetectable in normal cells.For the early detection of cancer, telomerase is a significant biomarker. 61,62There are various methods to detect telomerase, but fluorescence imaging has especially appealed to researchers due to the merits of simplicity, rapidity and sensitivity.
In 2021, Qu's group developed a novel nucleic aciddriven AIEgen based on Au nanoclusters (AuNCs) for the detection of telomerase activity, where the AuNCs were modified by strands A and B, respectively. 63Strand A contained a hairpin structure.Under the action of telomerase, the TS primer on strand A was extended and the hairpin structure was gradually opened.After the hairpin was opened, a toehold domain at the 5'end of strand A was released and hybridized with strand B,

F I G U R E 3 (A) Schematic illustration of TPE-Azo for hypoxia detection in living cells. (B) Fluorescent images of HeLa cells cultured with 200 μM TPE-Azo at various oxygen concentrations for 3 h in the absence or presence of DPI (an inhibitor of azoreductase).
Reproduced with permission. 52Copyright 2021, John Wiley and Sons.
causing the AuNCs to aggregate and emit fluorescence.This system exhibited high selectivity, low cytotoxicity as well as cell entry capability.It could distinguish cancer cells from normal cells based on telomerase activity.Successfully, it imaged telomerase in live cells and visualized solid tumors in mice.Due to the advantages mentioned above, this AIEgen could realize the early diagnosis of tumor formation.In 2020, Lou and coworkers reported an AIE fluorescence detecting system, which could analyze the change of TERT mRNA levels and telomerase levels in cancer cells during different cell cycles (G0/G1, G1/S, S and G2/M phases). 64During the detection process, PyTPA-DNA could complement hybridization with TERT mRNA and release the fluorogen PyTPA-N 3 by the action of exonuclease III.Furthermore, in the presence of active telomerase in the cell, the template strand primer (TP) could be extended to form a DNA strand, which was negatively charged and could bind to the positively charged Silole-R, showing a "turnon" fluorescent signal.The fluorescence imaging showed significant differences for different telomerase activity in different cell cycles.This fluorescence-responsive system possessed excellent sensitivity and accuracy, which had also been successfully applied in clinical malignant tissue samples.In 2022, Lou et al. reported another new cellcycle synchronization telomerase detection system based on the AIEgen, SSNB, which was capable of outputting dual signals of fluorescence and reactive oxygen species. 65As shown in Figure 5A, SSNB comprised an AIE molecular backbone and dual positive charge moieties.Under the action of telomerase, TP was extended, and DNA could interact with SSNB through different interactions causing the aggregation of SSNB and releasing an intensive red fluorescent signal.Because of the difference in the telomerase activity in various cell cycle phases, HeLa cells were arrested in different cell cycles separately (Figure 5B).When adding SSNB, both in vitro experiments (Figure 5C) and in situ imaging (Figure 5D,E) displayed significant differences in fluorescence signals of different cell phases, and the results were consistent.The brightest fluorescence was observed in HeLa cells arrested in the S phase, which signified the strongest telomerase activity in this period.In addition to the fluorescence signal, the SSNB assay system could indirectly indicate the activity of telomerase via the level of ROS.Under light irradiation, the aggregated SSNB released ROS, causing cell death, and the apoptosis rate of cells could also reflect the telomerase activity.The results were consistent with those of the fluorescence signal (Figure 5F,G), and the dual signal output significantly improved the accuracy of the assay.It should be noted that Lou et al. detected cells with different cell cycles separately to prevent the effect of cell cycle on the results and provided ideas for the accurate detection of other cell cycle-dependent biomarkers.Taken together, the above three works all take advantage of the ability of telomerase that can synthesize telomeric DNA to design the AIE-responsive systems.

| Caspase
Apoptosis, a vital physiological process that plays a crucial role in the development of tissue homeostasis, is a common way of cell death regulated by a series of signaling cascades under certain conditions. 66,67poptosis has been widely utilized in anti-cancer treatment, and some drugs achieve anti-cancer effects by inducing apoptosis. 68Caspases belong to a conserved family of cysteine aspartate proteases involved in apoptosis. 69They are valuable as key factors of apoptosis in diagnosing and treating diseases as well as for screening anti-cancer drugs and assessing anti-cancer efficacy.Reproduced with permission. 65Copyright 2022, American Chemical Society.
Jin et al. designed a prodrug system that could respond to caspase-3 by combining an anticancer drug (gemcitabine, GEM) with a caspase-3-targeted AIEgen (TPE-DEVD-RGD). 70In pancreatic cancer cells, overexpressed histone protease B could hydrolyze the prodrug to release GEM and TPE-DEVD-RGD, where GEM induced apoptosis by activating caspase-3.Then, the DEVD peptide of the AIEgen was cleaved, releasing the AIE characteristic tetraphenylethylene (TPE), which emitted a strong blue fluorescence.Based on the fluorescence intensity, therapeutic monitoring and efficacy assessment of the drug could be performed.This theranostic GEM prodrug avoided the problem of GEM inactivation and enabled realtime, non-invasive and in situ imaging of therapeutic response, so it held great potential for clinical application.Liu et al. also developed a novel AIEgen TPETH-DVEDIETH-TPS to detect apoptosis-generated caspases. 71It was successfully utilized to monitor the caspase cascade activation during apoptosis in real-time and evaluate the therapeutic anti-cancer effect drugs.
Moreover, fluorescent imaging could be combined with other imaging modalities to realize more accurate detection, Meade and coworkers designed a bimodal fluorescence-magnetic-resonance (FL-MR) AIEgen, Caspase Probe 1 (CP1), which exhibited a concurrent FL-MR turn-on response to caspase-3/7 (Figure 6). 72CP1 had three main components: a caspase-3/7 substrate DEVD peptide, a Gd-DOTA MR agent that shortened T1 and an AIE core tetraphenylethylene (TPE) (Figure 6A).As shown in Figure 6B, when caspase-3/7 specifically cleaved the DEVD, the residual Gd-AIE would aggregate together, causing the FL and MR signals to increase significantly.CP1 exhibited a large dynamic range, good sensitivity, selectivity and biocompatibility, so it was successfully applied for in vitro fluorescence imaging of apoptotic cells.As shown in Figure 6C, time-lapse fluorescence imaging was obtained after incubating HeLa cells with CP1 and staurosporine (STS).And an incremental increase in fluorescence intensity over time was monitored in HeLa cells, suggesting an increase in caspase-3/7 activity during the process of apoptosis.Similarly, CP1 exhibited an excellent MR response for caspase-3/7.Exploiting T 2 -weighted magnetic resonance imaging at 7T, it could be employed to detect the level of caspase-3 (Figure 6D).Furthermore, the FL signal of CP1 could be exploited to measure the concentration of active probes during caspase-3 assays, thus precisely predicting in vitro MR responses and greatly improving probe accuracy.Therefore, this example provides a universal structural platform where the DEVD peptide fraction could be replaced with a different biologically responsive fraction to generate various bimodal molecular probes.

| REACTIVE NITROGEN SPECIES (RNS)
RNS are a kind of species derived from nitric oxide (NO) whose production is closely linked to nitric oxide synthase (NOS). 73RNS plays crucial roles in the physiological regulation of many living cells. 74However, excessive levels of RNS can lead to cellular damage and death through the induction of nitric stress. 757][78] Therefore, the development of RNS-responsive AIEgens enables us to visualize and monitor many diseases.). 79ONOO − can damage cellular biomolecules such as DNA, thiols, phospholipids, and proteins, affecting signal transduction, inflammatory responses, and promoting tissue damage and even cell death. 80The formation and reactions of peroxynitrite are proposed to be closely related to the pathogenesis of many diseases, including acute and chronic inflammatory processes, ischemia-reperfusion and neurodegenerative diseases. 81- 85ONOO − possesses a short lifetime and complex reaction, and its detection process is also susceptible to interference from other reactive oxygen and nitrogen species (RONS). 80The detection of ONOO − by AIEgens with fast response and high sensitivity have been achieved.Tang and coworkers reported a ratiometric ONOO − -responsive AIEgen, ATV-PPB. 86As seen in Figure 7A, the boronated ester group on the ATV-PPB could be oxidatively cleaved by ONOO − , releasing the hydrophobic AIE residue ATV-Py, which showed a redto-green fluorescence color change (Figure 7B).ATV-PPB possessed good selectivity for ONOO − , so it could not be affected by other RONS.ATV-PPB has been applied to monitor ONOO − in APAP-induced cellular hepatotoxicity (Figure 7C).ATV-PPB could be utilized as a probe for ONOO − sensing but also reduced the production of ONOO − .As shown in Figure 7D, in cells treated with the same dose of APAP, fainter fluorescence from DCFH-DA (RONS indicator) was observed in cells that had been treated with ATV-PPB, indicating that ATV-PPB could neutralize redundant ONOO − .In addition, the cation on ATV-PPB could drive the AIEgen to target mitochondria, reduce ONOO − induced tyrosine nitration, instantly shield ONOO − induced liver damage and mitigate the hepatotoxicity of excess APAP.][89] Except borate groups, ONOO − -responsive AIEgens have been developed by employing a diphenylphosphinate group as the reactive site.For instance, Shen's group developed a "turn-on" AIEgen, HPP, with excited state intramolecular proton transfer (ESIPT) property to detect ONOO − in cells. 90HPP consisted of a fluorophore of salicylaldehyde azine and a recognition site of the diphenylphosphinate group.Original HPP displayed a weak emission in aqueous solution, but ONOO − could release the salicylaldehyde azine from HPP by stripping the diphenylphosphinate group, which resulted in an obvious fluorescent signal due to the AIE-ESIPT mechanism.Additionally, HPP worked well with good selectivity and sensitivity for ONOO − at physiological pH, and it was utilized in ONOO − imaging in living cells successfully.In 2020, Wang et al. synthesized another ratiometric AIEgen, BTCV-PN, based on the response site of diphenylphosphinate group, 91 where the benzothiazolyl derivative served as the AIE fluorophore.After adding ONOO − , the emission at 632 nm attributed to BTCV-PN gradually increased, and the emission at 525 nm from the newly generated BTIC gradually increased simultaneously, leading to ratiometric emission changes.This ratiometric AIEgen exhibited outstanding sensitivity and remarkable specificity towards ONOO − .Copyright 2019, American Chemical Society.

| Nitroxyl (HNO)
Nitroxyl (HNO) is the one-electron reduction product, also the protonation form of NO. 92 HNO is essential in many biological and pharmacological activities and is involved in many signal transduction processes. 93,94][97][98][99] However, the dimeric instability of HNO molecules and their short lifetime make their detection challenging. 100Traditionally, the levels of HNO are evaluated by detecting their by-products using high-performance liquid-chromatography mass spectrometry (HPLC-MS), electron paramagnetic resonance (EPR) spectroscopy, etc. [100][101][102] In addition, these traditional methods require specific and expensive instruments and are difficult to operate and costly.The development of reliable methods to directly detect HNO is significant for understanding its critical signaling property and pathophysiological role.
In 2020, Tang's group reported the first ratiometric HNO-responsive AIEgen, TCFPB-HNO, composed of a luminescent TCFPB-HNO part and a 2-(diphenylphosphino)-benzoyl reaction site, which could detect HNO in vivo. 103Due to the weak ICT effect, the fluorescent signal of TCPFB-HNO at 670 nm in phosphate buffered saline (PBS) was low.While TCFPB-HNO reacted with HNO, the newly generated tricyanofuranyl iminosalicylaldehyde (TCFIS) showed strong ICT effects and exhibited a typical ratio fluorescence change (I 618 /I 670 ) in PBS solution (Figure 8A).As illustrated in Figure 8B, the I green /I red ratio increased markedly as the HNO concentration rose, demonstrating that TCFPB-HNO could be utilized for cell imaging.Besides, TCFPB-HNO successfully achieved the visual imaging of HNO in vivo.Likewise, , the mice injected with AS solution exhibited significantly enhanced fluorescence compared to that of the control group treated by TCFPB-HNO only (Figure 8C).Thanks to its high sensitivity, excellent anti-interference ability and wonderful selectivity for HNO, TCFPB-HNO was able to visualize HNO in situ and in vitro with a high signal-tonoise ratio.However, up to now, there are few reports on the AIEgens for HNO detection, so more new systems need to be explored.Reproduced with permission. 86Copyright 2021, Royal Society of Chemistry.

| Nitric oxide (NO)
NO is known as a crucial molecule in the regulation of endothelial function in the body. 104,105[110][111] Compared to normal brain tissue, the concentration of NO is usually elevated at the site of encephalitis, and its level can reflect different stages of encephalitis.A sensitive "turn-on" photoacoustic (PA) probe, OTTAB, with AIE property was developed by Tang and coworkers to detect NO in encephalitis. 112By exploiting the reactive ability of phenylenediamine with NO, diaminesubstituted benzothiadiazole (AB) was employed as the reaction unit of OTTAB, which underwent a reaction with NO to form a more electron-deficient triazole structure, OTTTB.OTTTB possessed enhanced donoracceptor (D-A) interaction and ICT effect, which enabled a significant turn-on of the NIR-PA signal (Fig- ures 9A,B).In the imaging of brain inflammation (Fig- ures 9C-E), the PA intensity of the inflamed ventricle increased significantly (Figure 9E) after injecting LPS compared with the other contrast groups, indicating the successful monitoring of endogenous NO production in brain inflammation by OTTAB nanoparticles.The level upregulation of inducible nitric oxide synthase (iNOS) is the main contributor to NO production.As illustrated in Figure 9F, the level of iNOS mRNA in the brain tissue significantly increased as the LPS concentration increased, indicating that OTTAB has some reactivity to the severity of encephalitis.This work demonstrates that there is great potential for exploring and applying PA probes with AIE characteristics, which contributes to the understanding of the progression of related diseases.
Based on the same responsive mechanism, Tian et al. developed an AIEgen, TPE-2NH 2 and its silica nanoparticles in 2017 for the ratiometric detection of NO in vitro. 113NO could react with o-phenylenediamine to form benzotriazole, producing TPEBZT with a typical ICT effect, so the fluorescence shifted from 519 nm (green emission) to 655 nm (red emission) to achieve the ratiometric detection.Therefore, endogenous NO detection in live cells was successfully achieved in vitro using TPE-2NH2@SiO 2 NPs.In 2019, Costero and coworkers developed another new TPE derivative (probe 1) for detecting NO through a "click" reaction. 114NO was able to trigger the steric hindrance change of the side arms for TPE by reducing Cu(II) to Cu(I), thus contributing to an increase in fluorescence intensity and causing an AIE effect.The AIEgen was able to selectively detect gaseous NO at a low detection limit of 15 ppm and was employed to control inhaled nitric oxide (iNO) in clinical applications.

| REACTIVE OXYGEN SPECIES (ROS)
ROS are mainly produced in mitochondria as a byproduct of cellular metabolism 115 and are involved in various physiological processes. 116,1179][120][121] Thus, it is of significance to design AIEgens capable of detecting ROS in vivo to indicate the progression of the diseases.In this part, we present some typical instances of ROS-responsive AIE systems.

| Hydrogen peroxide (H 2 O 2 )
Hydrogen peroxide (H 2 O 2 ), as a type of ROS, has a significant regulatory effect on signal transduction. 122xcessive level of H 2 O 2 will lead to oxidative stress and cellular damage, which is closely connected with the process of some diseases, such as atherosclerosis, diabetes and neurodegenerative diseases. 122,123In addition, the levels of H 2 O 2 in inflammatory and cancerous tissues are usually overexpressed.Therefore, the development of H 2 O 2 -activated AIEgens is highly important for biological study of many diseases and clinical diagnoses. 124,125In 2020, a ROS-responsive theranostic nanoplatform (TPP@PMM) was constructed by Wang and coworkers, it had two-photon bioimaging function, could diagnose and treat inflammation. 126The structure of TPP with diagnostic property was shown in Figure 10A, where the antiinflammatory glucocorticoid prednisolone (Pred) group and AIE-active fluorescent unit (TP) with two-photon absorption were connected by a ROS-sensitive ester bond.TPP was further encapsulated into polymer micelles (TPP@PMM) that could be responsive to ROS accompanied by a switch from hydrophobicity to hydrophilicity and light-up emission.Thus, the excessive ROS in inflammation could react with TPP@PMM, causing the gradual release of Pred to exert an anti-inflammatory effect.The pertinent experimental results demonstrated that TPP@PMM showed desirable ROS responsiveness, which successfully achieved in vitro fluorescence imaging and effective treatments of arthritis and atherosclerosis as well as acute lung injury (Figure 10).
As H 2 O 2 is usually overexpressed in many cancer tissues, many H 2 O 2 -responsive drug delivery systems (DDSs) have been developed recently. 127,128In 2018, Tang and colleagues reported a H 2 O 2 -sensitive therapeutic drug delivery system, ABD-system. 129This system consisted of three components, that is, carboxylated TPE (AIEgen), benzyl borate, BBE (trigger) and doxorubicin (DOX) prodrug.The original ABD system exhibited weak emission in both solution and aggregate states.However, after being activated by H 2 O 2 , it could emit fluorescence with the release of TPE and DOX.Additionally, the ABD system possessed desirable selectivity for H 2 O 2 and thus exhibited effective therapeutic ability.After carefully analyze the related H 2 O 2 -responsive AIE systems, the incorporation of borate is a general strategy to achieve the sensitive response to H 2 O 2 , and these developed AIEgens present great potentials in imaging and therapeutic applications.
Glucose and cholesterol can generate H 2 O 2 catalyzed by specific oxidases by which some H 2 O 2 -responsive AIEgens were developed. 130,131For example, Qu and coworkers reported glutathione-capped Cu nanoclusters (GSH-CuNCs) with AIE properties for detecting glucose and cholesterol. 132Metal ions can trigger the AIE behavior of GSH-CuNCs, thus the GSH-CuNCs-Pb 2þ -Zr 4þ system exhibited strong emission.However, adding H 2 O 2 could significantly quench the fluorescence, so the fluorescence intensity decreased with the increased H 2 O 2 concentration.Ultimately, utilizing this new GSH-CuNCs-Pb 2þ -Zr 4þ system, we could monitor glucose and cholesterol in the samples of human serum successfully.Therefore, except for "turn-on" fluorescent AIEgens, the systems that undergo fluorescence  ) is a precursor of other ROS species in cells and is capable of causing DNA damage. 133t has been proved that the production of O 2 •− is excessive in cancer cells and is strongly associated with carcinogenesis. 134,135Therefore, the development of fluorescent probes capable of in situ monitoring for O 2 , the CLA unit containing the imidazopyrazinone fragment could be oxidized to a dioxetanone that further decomposed to generate a singlet-excited amide, thus decaying to the ground state and releasing CL.At the same time, hydrophobic TPE-PZA was generated, and the fluorescence signal was turned on because of aggregation (Figure 11A).TPE-CLA had high sensitivity and specificity towards O 2 •− and low cytotoxicity, so it successfully detected endogenous stimulated O 2 •− and could also monitor original O 2 •− in living cells (Figure 11B).In addition, the detection of endogenously produced O 2 •− in inflamed mice (Figure 11C) and real-time detecting of O 2 •− levels in APAP overdose cells (Figure 11D) were realized.This work demonstrates a good example that achieves FL/CL dual detection, which will provide inspiration for the construction of new systems.(F) Two-photon CLSM images of the plaques at various imaging depths.The scale bars were all 200 μm.Reproduced with permission. 126Copyright 2020, American Chemical Society.
Tang et al. reported another two-channel responsive AIEgen, Probe I, that enabled selective imaging of O 2 •− in living cells. 137In this probe, a pyridine-modified TPE derivative was utilized as the fluorescent reporter and a diphenyl-phosphine group served as the superoxide anion reactive site, which were connected via a cresol-like structure.Probe I possessed low solubility in water and exhibited red fluorescence.Once the active phenylphosphine group reacted with, leading to the break of the linker and the release of pyridine-modified TPE, Probe II., thus providing inspiration for the detection of bioactive small molecules.

| Hypochlorous acid (HClO)
Hypochlorous acid (HClO) is a strong oxidant produced by intracellular H 2 O 2 with Cl − catalyzed by myeloperoxidase (MPO). 139HClO possesses powerful antibacterial properties and is essential to the human immune system. 140,1413][144][145][146][147] In turn, the diagnosis of related diseases could be realized by the detection of HClO.In 2020, Wang and coworkers reported a water-miscible AIEgen, HOTN, and successfully detected HClO in inflammation and cancer. 148The response mechanism is shown in Figure 12A.HClO could oxidize the double bond of nonemissive HOTN, leading to the formation of OTOH with an enol structure that further transformed into the hydrophobic product HOT with AIE property.Due to the generation of strong fluorescence after interacting with HClO, HOTN successfully detected endogenous and exogenous HClO in living cells (Figure 12B).Moreover, HOTN could be employed to image HClO in inflammatory mouse models (Figures 12C,D) and hepatocellular carcinoma, thus presenting great potential for the diagnosis and monitoring of diseases.
In the construction of probes for detecting HClO, ratiometric probes are widely used. 149For instance, Zhang et al. reported two ratiometric and lysosometargeting AIE systems, PNRFN and PDAM-Lyso, and realized the imaging of HClO in cellular and in vivo levels, respectively. 150,151In addition, the effect of dark through-bond energy transfer (DTBET) is also commonly used in the designing of HClO-responsive ratiometric AIEgens. 152,153In 2020, Tang and coworkers developed a ratiometric AIEgen based on DTBET effect, TPE-RNS, for detecting HClO in cells. 152TPE-RNS could be divided into TPE units and rhodamine B units.Rhodamine was connected to a lactam group containing a phenyl isothiocyanate moiety, which could be selectively oxidized by HClO, resulting in structural changes and increased water solubility.Due to the DTBET effect, the energy of the TPE donor was quickly and totally transferred to the rhodamine acceptor, leading to the fluorescence change from green-blue to orange-red, achieving the ratiometric detection of HClO.Singlet oxygen ( 1 O 2 ) is the electronically excited state of molecular oxygen and is reactive as well as toxic. 154In photodynamic therapy (PDT), singlet oxygen can be cytotoxic to cancerous tissues and is associated with the defense mechanisms of phagocytes against viruses and bacteria. 155,156It is involved in lipid oxidation processes, has a destructive effect on genetic material and is associated with many diseases. 154,156,157In 2020, Tang et al. developed an NIR AIEgen (TBL) with CL and used F127 as the surfactant to construct the more stable TBLdots, which achieved the imaging of 1 O 2 in vitro and in vivo. 158he CL mechanism has been illustrated in Figure 13A.The luminol unit of TBL as a CL emitter could be oxidized by 1 O 2 to produce AIE-active TBLCOOH, which produced near-infrared emission (Figure 13B).TBL dots showed good stability, biocompatibility, and ROS selectivity and were far more reactive to 1 O 2 than other ROS.Due to the great advantage of CL the CL signal of the TBL dots could penetrate through 30 mm of tissue, demonstrating superior deep-tissue imaging capability.In addition, owing to the high ROS levels in the tumor microenvironment, tumor tissues as well as healthy tissues could be easily identified using TBL dots, thus showing great potential for CL-guided cancer theranostic applications.

| BIOLOGICAL THIOLS
Thiols are organic sulfur derivatives containing sulfhydryl residues (-SH) at their active sites. 159Thiols play a crucial role in enzymatic reactions, apoptosis, detoxification, and antioxidant protection in vivo. 160ence, AIEgens with thiol responsiveness may be beneficial for diagnosing oxidative stress-related diseases.In addition, by ingeniously designing AIEgens, we can exploit the thiol expression level to treat these diseases.

| Glutathione (GSH)
Glutathione is a tripeptide (γ-glutamyl-cysteinyl-glycine) composed of glutamic acid, cysteine, and glycine. 161GSH regulates numerous critical biological functions in cells, such as gene regulation, maintenance of redox state, intracellular signal transduction, growth and metabolism. 162,163In addition, the expression of GSH is usually elevated in cancer and it has a close relationship with tumorigenesis, development and metastasis. 164,165Utilizing this feature and the reducing effect of GSH, Tang's group reported a GSH-responsive polymeric prodrug micelle, TB@PMPT, consisted of two AIE photosensitizers for enhanced chemo-photodynamic therapy. 166As illustrated in Figure 14A, the AIE photosensitizer PyTPE underwent a reaction with the anticancer drug paclitaxel (PTX) to form a polymer prodrug (PMPT) that was sensitive to reduction.PMPT spontaneously formed micelles, and AIEgen TPA-BDTP (TB) was encapsulated because of its hydrophobic core, resulting in TB@PMPT that displayed yellow and red fluorescence.Remarkably, the disulfide bond of the PMPT prodrug was broken by GSH that was overexpressed at the tumor site, releasing PTX.Simultaneously, the increased hydrophilicity of the residual polymer caused PyTPE to disperse, diminishing the yellow fluorescence emission.TB@PMPT was increased in the tumor tissue of mice, and we were able to monitor the drug metabolism by the alteration of fluorescence signal (Figure 14B).Moreover, TB@PMPT exhibited good tumor targeting, selectively accumulating in the tumor, and having less adverse effects to other organs (Figure 14C,D) and had a strong inhibitory effect on tumor.
Another AIE-based GSH-activatable photosensitizer, TPEPY-S-Fc, was reported by Kim et al. for imaging-F I G U R E 1 3 (A) The proposed CL generation mechanism of TBL oxidized by 1 O 2 .(B) Schematic illustration of the preparation of TBL dots and the generation of CL.Reproduced with permission. 158Copyright 2020, John Wiley and Sons.guided PDT of tumor cells. 167TPEPY-S-Fc consisted of ferrocene and vinyl pyridinium-substituted tetraphenylethylene (TPEPY) linked by disulfide bonds.In the presence of GSH, the disulfide bond of TPEPY-S-Fc was cleaved, releasing TPEPY-SH with significantly enhanced red fluorescence signal.In addition, TPEPY-SH generated singlet oxygen under light irradiation and induced apoptosis of cancer cells.TPEPY-S-Fc showed good biocompatibility in the dark environment, could discriminate cancer cells from normal cells and guided PDT of tumor cells.Incorporating disulfide bonds for cleavage was the most common principle for designing ion-responsive AIEgens, which utilized the powerful reducibility of GSH, and through this ingenious design we could selectively image and treat cancer cells with overexpressed GSH.
In 2018, Wang et al. reported another AIEgen, TPE-DPP, capable of detecting GSH in both turn-on and ratiometric fluorescence signal modes. 168TPE was connected to diketopyrrolopyrrole (DPP) by imine, forming TPE-DPP.The imine part served as the recognition site for glutathione and prevented its release.In the low concentration range of GSH, TPE-DPP emitted fluorescence signal by turn-on mode.Conversely, a high concentration of GSH disrupted the binding of TPE-DPP, forming a ratiometric fluorescence mode.TPE-DPP had good selectivity for GSH and could detect GSH in living cells.Similarly, Yin's group also achieved selective detection of GSH by TPE derived from naphthalimide. 169he use of imine bond was another strategy for designing GSH-responsive AIEgens.
Cu-tpMOF, is an AIEgen-based, non-emitting metalorganic framework (MOF) nanoprobe with Cu(II) as a node and quencher was reported by Lei and coworkers to distinguish GSH in different subcellular localizations. 170Two-channel ratio analysis by dual-color emission of Cu-tpMOF allowed real-time monitoring of subcellular GSH in living cells.Huang developed AIE reverse-process-based turn-off AIEgens, CM-Au NCs, to monitor GSH in living cells. 171The addition of GSH caused the size of chitosan micelles to increase, leading to the quenching of AIE fluorescence.CM-Au NCs had good selectivity and sensitivity for GSH, providing a reliable tool for the identification of tumor cells and the long-term tracking of GSH in different growth stages of cells.Reproduced with permission. 166Copyright 2021, American Chemical Society.
GSH possesses excellent reducing ability and using this property, GSH can selectively hydrolyze AIEgen designed to be connected by disulfide or imine bonds to achieve GSH response.Due to the high expression of GSH in tumors, many GSH-activated AIE photosensitizers or drug delivery systems have been developed to kill cancer cells and suppress tumor growth with precision and low side effects.In conclusion, GSH-activated AIEgen has a very high potential for clinical applications.

| Cysteine (Cys)
Cysteine is a sulfur-containing molecule involved in many essential biological processes and plays a critical role in biological systems. 172Cysteine deficiency can cause edema, slow growth, liver injury, muscle and fat loss, skin damage, hair loss and weakness. 173Abnormally elevated cysteine is also related to the pathophysiology of diseases, such as motor neuron disease, coronary heart disease, Parkinson's disease and AD. 174,175[169] In 2017, Wang et al. reported a TPE-based AIEgen, TPENNO 2 , that could specifically image Cys but not Hcy or GSH in cells. 176In this molecule, 2, 4dinitrophenylsulfonyl could burst the fluorescence and it was also a recognition site for Cys.After the thiol group of Cys promoted the nucleophilic aromatic substitution reaction, TPENNO 2 was converted to TPENH 2 and the fluorescence was light up.However, the thiol residues of Hcy and GSH with 2, 4-dinitrophenylsulfonyl had different reaction kinetics and the fluorescence response exhibited different time courses and intensity enhancement as a way to distinguish different biological thiols.TPENNO 2 was able to enter cells and image biological thiols in living cells.In 2019, a Cys-responsive fluorescent probe, hydroxy-1naphthal-4-aminoantipyrine, was developed based on AIE and ESIPT effects by Wu and coworkers. 177Because of the AIE property and the activation of ESIPT, this probe aggregated and released fluorescence in water, while Cu 2þ ion could block the ESIPT process leading to the quenching of fluorescence.When Cu 2þ ion was exposed to Cys, the combination of Cys and Cu 2þ was able to restore the fluorescence and achieve selective imaging.As a result, it exhibited good selectivity for Cys and enabled quantification and imaging of Cys in living cells.

| Homocysteine (Hcy)
Homocysteine is an intermediate product in the metabolism of methionine to cysteine and plays an essential role in metabolism and other physiological functions. 1780][181][182][183][184] Therefore, the monitoring of Hcy concentration in the body and the development of AIEgens with Hcy-responsive properties are important for studying related diseases.However, due to the structural similarity between Hcy and Cys, there are relatively few AIEgens developed that can effectively distinguish Hcy from Cys or can specifically detect Hcy. 185Therefore, efficient and simple Hcy-active AIEgens remain to be developed.In 2019, Feng's group reported an AIEgen, APTC, with an ICT effect and high selectivity for Hcy in cells.Because of its D-π-A structure, APTC exhibited ICT effect as well as AIE enhance properties. 186Additionally, the authors linked an electron-rich anthracene that could more effectively weaken the fluorescence of the fluorophore through a "stronger ICT" effect, reducing the fluorescence of APTC.After the addition of Hcy, the ICT process was blocked due to the cyclization reaction between Hcy and the formyl group of APTC, so APTC emitted an enhanced fluorescence signal.The AIEgen APTC was sensitive, rapid, highly selective and specific for detecting Hcy, and the imaging ability for Hcy was verified in living cells.Employing similar strategy, another Hcy-responsive AIEgen, DBTC, was also developed by Feng and coworkers, where the framework of triphenylamine and 2phenyl-1,2,3-triazole served as the luminescent unit and the aldehyde group played as the reaction unit. 187The aldehyde and thiol groups on DBTC underwent nucleophilic addition reactions, so five or six-membered rings would form and the corresponding electron transfer would change, thus causing changes in fluorescence intensity to achieve a specific response to Hcy.And the experimental results indicated that DBTC possessed good biocompatibility and could detect Hcy in cells.

| CONCLUSION AND PERSPECTIVES
In this review, we systematically summarize the design ideas of AIEgens that can achieve specific responses to enzymes, RNS, ROS and biological thiols, and how these AIEgens achieve selective responses to specific substances.For each biological substance or analyte, we have elaborately selected several representative examples and illustrated the response mechanisms behind the AIEgens as well as the experimental validations.We can find that most of the AIEgen fluorescent probes for bioimaging are derived from compounds such as TPE and TPA.A common strategy for designing biomolecule-responsive AIEgen is to link the compounds with cleavable chemical bonds or attach functional groups that can alter the water solubility of AIEgen upon specific reactions.These special modifications enable AIEgen to selectively image biological substances.
Research on AIEgens has made significant progress in bioimaging and applications for the diagnosis and therapy of diseases, but there is still much room for their further explorations in clinical applications.Here, we present some perspectives for the design of future AIEgens related to the responses of biomolecules: 1) In terms of the development of AIEgens, we propose the following aspects: (i) Currently, AIEgens are mainly composed of fluorescent units, which are short-lived and cannot realize long-term imaging in vivo.Therefore, some long-lifetime AIEgens can be developed for long-term tracking of bioimaging.(ii) If further research is needed in in vivo theranostics, it would be meaningful to develop AIEgens with long wavelengths.Compared with short-wavelength materials, AIEgens with long wavelength possess higher imaging resolution.In addition, short-wavelength AIEgens have limited penetration in vivo imaging, and increasing their wavelength can help us achieve deep-tissue or organ imaging.(iii) If we can use chemiluminescent materials to replace the fluorescent AIEgens, then we can achieve luminescence without an external excitation light source, avoiding the possible damage caused by excitation light, and also solving the issue of insufficient penetration depth.(iv) Some AIEgens still have some limitations in sensitivity, selectivity and detection efficiency, which may be further improved.9][190] EVs have attracted considerable research attention in recent years.AIEgens that can selectively interact with EVs or be encapsulated by EVs for disease diagnosis and therapy are emerging as a promising research direction.(ii) The development of multifunctional AIEgens with multimodual imaging or therapeutic properties will greatly improve the accuracy of diagnosis, such as photoacoustic probes, fluorescence-magnetic resonance probes, etc., and promote the therapeutic efficiency.(iii) AIEgens that enable the diagnosis and therapy of viruses also remain to be developed.(iv) Although many cell imaging AIEgens have been reported, there is still a need to develop some AIE fluorescent materials that can specifically target different organelles and achieve subcellular imaging.
illustration of the assembly of DQM-ALP and process of AIE activation in vitro and in cell.(B) Long-term fluorescence imaging of HeLa cells treated with DQM-ALP.a-l) λ ex = 473 nm, λ em = 490-590 nm.(C) Process of APAP administered to Balb/c mice and fluorescence imaging of tissue sections.(D) Mice treated with PBS (a-e) or APAP (f-j) for 12 h, followed by incubation with DQM-ALP (10 μm).λ ex = 488 nm, λ em = 520-620 nm.(A-D) Reproduced with permission. 41Copyright 2020, John Wiley and Sons.LIAO ET AL.

F
I G U R E 5 (A) Scheme Illustration of the Process of Detecting Telomerase Activities after Cell-Cycle Synchronization by the SSNB-Based Detection System.(B) Cells were synchronized at the phases of G 0 , G 1 /S, S, and G 2 /M.(C) Relative fluorescence intensity (I/I 0 − 1) of SSNB þ TP at different phases.(D) CLSM images of HeLa cells after synchronized and incubated with the SSNB-based detection system.E x = 488 nm, E m = 550−650 nm.Scale bar: 20 μm.(E) The relative fluorescence intensity (I/I 0 − 1) in (D).(F) The viability of cells at different phases was affected by ROS generation of SSNB þ TP.Scale bar: 100 μm.(G) The relative apoptosis rate according to Figure 5F.

F
I G U R E 6 (A) Chemical structures of CP1 and Gad-AIE.(B) Caspase-3/7 sensing mechanism of CP1.(C) Time-lapse fluorescence imaging of apoptotic HeLa cells incubated with CP1 (50 μM) prior to apoptosis induction with STS (1 μM).(D) Top row: T 1 -weighted MR images of CP1 and CP1-ctrl solution incubated with and without caspase-3 overnight.Bottom row: T 2 -weighted MR images of CP1 and CP1-ctrl solution incubated with and without caspase-3 overnight.Reproduced with permission.

F
I G U R E 8 (A) Recognition mechanism of ratiometric AIE-active fluorescent probe TCFPB-HNO toward HNO.(B) Cell imaging of probe TCFPB-HNO with different concentrations of AS. λ ex = 543 nm.(C) Time-dependent fluorescence mages of probe TCFPB-HNO in live mice.Fluorescence emissions were collected from 580 to 900 nm.λ ex = 523 nm.Reproduced with permission. 103Copyright 2021, Royal Society of Chemistry.
in response to biomolecules are also promising in clinical applications.

4. 2 |
Superoxide studying oncogenic and pathogenic mechanisms.In 2017, Tang's group developed an AIEgen, TPE-CLA, that enabled to turn on fluorescence and chemiluminescent (CL) signals simultaneously and realized the monitoring of endogenous O 2 •− . 136TPE-CLA was composed of a TPE backbone with AIE characteristic and two CLA units for specific responsiveness of O 2 •− .The designed TPE-CLA was hydrophilic and could be dispersed in aqueous solution without luminescence.After recognizing O 2 •−

F I G U R E 1 0
(A) Illustration of Developing a Nanoplatform with Two-Photon Imaging and Serial ROS Sensitivity.(B) Ex vivo fluorescent images of the joint from arthritic mice injected with TPP@PMM.(C) Photographs of the right hind limbs from arthritic mice with different formulations on day 27.(D) Ex vivo fluorescence images and quantitative result of TPP@PMM accumulation in aortas.(E) Two-photon confocal image of the atherosclerotic plaques.

2 •−
The hydrophobic Probe II aggregated in water and emitted green fluorescence, thus showing a ratiometric change to evaluate the concentration of O 2 •− .The pertinent results demonstrated that Probe I could image endogenous O 2 •− in living cells via a two-channel response and also successfully differentiated the apoptotic and inflammatory cells from normal cells.In 2018, Lei and coworkers synthesized two AIEgens using a triphenylamine unit (TPA), TPA-DHP-1,2,3 and TPA-PPA-1,2,3, for the imaging of O in cells. 138Dihydropyridine (DHP) and pyridine (PPA) could react with O 2 •− to generate products with a significant wavelength shift and increased fluorescence intensity, thus achieving a two-channel response to O 2 •−.Using them, we were able to monitor O2 •− in living cells and in vitro quickly and accurately.Collectively, this work takes full advantage of the oxidative property of O 2 •− to develop AIEgens that can specifically respond to O 2 •−

F
I G U R E 1 4 (A) Mechanism of PTX release from polymeric prodrug PMPT in the reducing environment.(B) Optical imaging of HeLa tumor-bearing mice after the intravenous injection of TB@PMPT micelles at different times.(C) Fluorescence images and (D) relative mean fluorescence intensity (MFI) of the major organs and tumor of the TB@PMPT micelles treated mice at 24 h post injection.