Enzyme-Responsive Bioprobes Based on the Mechanism of

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Review

Enzyme responsive bioprobes based on the mechanism of aggregation- induced emission Jie Shi, Ya Li, Qianqian Li, and Zhen Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14943 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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ACS Applied Materials & Interfaces

Enzyme responsive bioprobes based on the mechanism of aggregation-induced emission Jie Shi#, Ya Li#, Qianqian Li† and Zhen Li*† #

Hubei Key Laboratory of Lipid Chemistry and Nutrition, Oil Crops and Lipids Process Technology

National & Local Joint Engineering Laboratory, Key Laboratory of Oilseeds Processing, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China †

Department of Chemistry, Hubei Key Lab on Organic and Polymeric Opto-Electronic Materials,

Wuhan University, Wuhan 430072, China. Abstract Enzymes play an indispensable role in maintaining the normal life activities. The abnormalities of content and activity in specific enzymes are usually associated with the occurrence and closely related the development of major diseases. Correspondingly, fluorescent bioprobes with distinctive sensing mechanisms and different functionalities have attracted growing attention, as convenient tools for optical probing and monitoring the activity of enzyme. Ideally and excitedly, the recently emerged luminogens with an aggregation-induced emission (AIE) feature could perfectly overcome the aggregation-caused quenching (ACQ) effect of conventional bioprobes. Based on the fantastic characteristics of AIE luminogens (AIEgens), specific enzyme bioprobes have been designed through the integration with recognition units, demonstrating many advantages including low background interference, a high signal to noise ratio (SNR) and superior photostability. In this review, by presenting some typical examples, we summarize the working principle and structural design of specific AIEgen-based bioprobes that triggered by enzymes, and discuss their great potential in biomedical applications, with the aim to promote the future research of fluorescent bioprobes involving enzymes. Keywords: aggregation-induced emission (AIE), enzymes, bioprobes, fluorescence, biomarkers, turn1

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on/light-up

Introduction As a special kind of biomacromolecule with catalytic effect, enzyme plays an extremely essential role in maintaining homeostasis and the normal life activities in biological systems.1 The abnormality of some specific enzymes activity is closely relevant to the occurrence and development of related diseases.2 Generally, the occurrence of certain cancers is concerning with the overexpression of βgalactosidase.3 For another instance, the nitro reductase concentrations in hypoxic neoplasms are significantly higher,4 and neurodegenerative diseases occurs usually accompanying with high levels of monoamine oxidase expression.5 Thus, to gain deep insights into metabolic mechanisms and diagnose the possible disease, it is badly required to explore the functions of enzymes and monitor their activities, including the detection of the activity of a specific biomarker enzyme and their possible visualization in vivo with an in-situ and real-time method. Unlike the colorimetric method,6 electrochemistry method,7 and enzyme-linked immunosorbent assay,8 requiring the invasive detection of biomarker enzymes, fluorescent probes are definitely much more promising for real-time, non-invasive and on-site visual investigation of biological species and specific routings in live cells, tissues and organisms. Also, in addition to the understanding of physiological alterations in pathological settings, fluorescent probes can provide important information for the researches of special interested therapies and personalized medicine for individuals.9 However, as a fly in the ointment, many reported enzyme fluorescent probes suffer from a common phenomenon: they are highly emissive in dilute solution, but the fluorescent emissions are often quenched at high concentrations or in solid state or as nanoparticles, owing to the formation of 2


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detrimental species of excimers and exciplexes etc., exhibiting the aggregation-caused quenching (ACQ) effect. As a result, the labelling degree of probe molecules to bioanalytes was overly limited, accompanying with the compromised low sensitivity for sensory applications inherited from the utilization of their dilute solutions. Thus, alternative strategies have been developed, especially those with "turn-on" modes, for example, the frequently utilized photo induced electron transfer (PET) in addition to fluorescence resonance energy transfer (FRET). In 2001, Ben Zhong Tang coined the concept of aggregation-induced emission (AIE), describing an abnormal phenomenon of silole, which displays non-ﬂuorescence in solutions, but strong emission once aggregated. This novel AIE phenomenon opened up the followed hot research field of AIE with huge potential practical application, especially as biopobes with the "turn-on" or "light-up"signals.10

Scheme 1. Schematic illustration of the restricted intramolecular motion (RIM) process and AIE phenomenon using TPE as an example. Taking tetraphenylethene (TPE, Scheme 1), the star AIEgen, as the typical example, the working mechanism is ascribed to the restriction of intramolecular motions (including vibration, rotation, twisting, etc.). The central C=C bond in TPE is surrounded by four phenyl rings, which could rotate in a dilute solution to dissipate the exciton energy, directly leading to no emission. Once aggregated, the emission of TPE is induced by the synergistic effects of the restricted intramolecular motion (RIM) and the hindered intermolecular π-π stacking interaction derived from its highly twisted molecular 3



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conformation. Thus, once the free rotation is limited, the emission will appear.11 So far, many approaches that can activate the RIM process were utilized to develop new AIE bioprobing systems, in which, a diversity of AIEgens have been designed and applied to monitor the enzyme activity in living systems.12-16 In this review article, we present the recent research advances of AIE bioprobes, which were triggered by enzyme to show fluorescence response. Actually, there are too many good papers published in this area, thus, to avoid an excessive number of references, we only selected some representative AIE bioprobes working in turn-on/light-up mode and focusing on biomarker enzymes involved in some major diseases or cancer. Based on the various applications or examples of specific AIE-based bioprobes, we discussed their different RIM approaches (electrostatic attraction, hydrogen bonding, hydrophobic effect and solubility change) for enzyme probing. With the expectation of AIEgens being utilized as versatile means for enzyme involved in disease therapeutic applications and clinical diagnostic but not just limited for the probe of the enzyme presence, some outlooks are proposed for the further development of this continually expanding area of research. Probing principles and applications According to the mechanism of restricted intramolecular motion, many distinct tactics, including chemical reactions or physical interactions, have been made use of design various sorts of AIE-active bioprobes. And in some cases, more than one principles work simultaneously for better performance. However, to simply the presentation, in the upcomging division, we try to describe each mechanism respectively and discuss their applications in detail.

Scheme 2. Schematic illustration of the aggregation prompted by electrostatic attraction. 4


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Electrostatic interaction Electrostatic interaction is a kind of force, which exists in two opposite charge species and makes them binding (Scheme 2). Many typical biomolecules, for example, phospholipids, DNA and polysaccharides, are naturally charged. By utilizing this point, Liu and Tang et al. synthesized a negative charged phosphorylated tetraphenylethene derivative (TPE-TEG-PA),17 which was watersoluble with weakly emission in aqueous buffer (Fig. 1a) because of the hydrophilic phosphate (PA) groups and tetraethylene glycol (TEG), for targeting alkaline phosphatase (ALP) and protamine. Protamine is widely applied in reverse the anticoagulant heparin activity after the surgery of cardiovascular, and the activity of ALP in human serum is often considered to be a key clinical diagnostic indicator of some major diseases like cretinism, achondroplasia, osteoporosis, hepatic diseases and myelogenous leukemia. Then, positive charged protamine and the negative charged TPETEG-PA could form micelles via electrostatic interaction. That is to say, aggregated state was formed, accompanying with the 12-fold increased fluorescence intensity (Fig. 1b). And the LOD (limit of detection) value of the protamine detection was determined to be 12 ng mL-1. Also, the end group phosphate of the probe molecule could be enzymatic hydrolyzed by ALP and transformed into hydroxyl unit, to yield TPE-TEG-OH. Due to its poor water solubility, TPE-TEG-OH aggregated in aqueous solution to activate the AIE effect (Fig. 1c). The linear light-up response of TPE-TEG-PA made the quantification of ALP possible in the range of 10-200 mU mL-1, which exactly covered the physiological concentration of ALP in human biological samples. Unlike monofunctional bioprobes, this dual-mode bioprobe offered the advantages of material saving, cost-effectiveness, less synthetic work and the potential for multiplex screening.

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Fig.1 (a) Chemical structure and reaction scheme of TPA-TEG-PA. (b) Photoluminescence spectra of TPE-TEG-PA (10 mM) upon the addition of different amount of protamine. (c) Photoluminescence spectra of TPE-TEG-PA (10 mM) in buffer solution (10 mM, pH 9.6) in the presence of ALP (200 mU mL-1) at 25 °C after the incubation of 40 min.; inset: the corresponding photographs of solutions under UV light (365 nm) in the (a) absence and (b) presence of ALP after the 30 min incubation.

Fig.2 Schematic illustration of the working mechanism of probe K(Ac)PS-TPE towards HDAC (Sirt 1). The level and activity of histone deacetylases (HDAC) are closely related to the incidence of various types of cancers, in addition to its association with the increased proliferation of tumor cells. In 2012, Kikuchi et al. designed a TPE-based fluorescent probe of K(Ac)PS-TPE,18 in which, N-α-t6


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butoxycarbonyl-N-ε-acetyl-L-lysine acted as recognized unit and propane sulphonic acid units provided a negative terminal charge (Fig. 2). Upon the addition of HDAC, K(Ac)PS-TPE could be deacetylated to KPS-TPE with the primary aliphatic ε-amine of lysine. Under physiological conditions, the produced amine groups was protonated, which could trigger the aggregation of KPSTPE through the probable electrostatic self-assembly between the sulphonate unit and the cationic ammonium ions, with the “turn on” fluorescent signal. Really, in the presence of Sirt1 (500 nM), one sort of HDAC, the fluorescence intensity was significantly increased regardless of the long test time (3 hours), while only slight one in the control experiment without the enzyme. According to the similar idea, Wu and Zeng

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intelligently designed a hyperbranched polyester bearing the amide groups,

which could be converted to the amine ones with the aid of Sirt1 (Fig. 3). Consequently, the AIEgen of TPE-2SO3- with negative ions could form the nanoaggregates with the positively charged hyperbranched polymer through the electrostatic interaction, resulting in a “turn-on” signal of blue fluorescence. Thanks to the plenty of end amide groups in the hyperbranched polymer, the test time was 2 hours, much shorter than that of K(Ac)PS-TPE. Furthermore, this sensing system has been successfully applied to the detection of endogenous Sirt1 levels in human serum, with the LOD value of 25 ng mL-1. Compared to the former system, the introduction of the globular polymer as the supporter for the test system can ensure the low LOD, making the system act as a one-pot HDAC straightforward fluorescent analysis.

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Fig. 3 Schematic illustration of the detection mechanism towards HDAC (Sirt 1).

Fig. 4 Schematic drawing of the ratiometric color changes of fibers. Protamine was adsorbed on fibers owing to the electrostatic forces between negatively charged surface of fibers and positively charged protamine, which induced static quenching of PhB and AIE effect of TPE derivative.

Another interesting work was performed by Li and co-workers,20 they combined the electrospun technique into the design of bioprobe, and prepared the fibers bonded with phloxine B (PhB) and tetraphenylethene (TPE) derivative. Then, protamine was adsorbed to the fibers to induce static 8


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quenching of phloxine B and AIE effect of the TPE derivative. In the presence of heparin or trypsin, protamine could be removed, as a result, the fluorescence of phloxine B at 574 nm was restored while the emission of the TPE derivative at 472 nm was relieved largely (Fig. 4). Therefore, the fluorescence-intensity ratio of 574 nm and 472 nm can be utilized for the ratiometric detection of heparin or trypsin, with a low LOD value of 0.02 U mL-1 toward heparin selectively. As shown in Figure 4, under UV illumination, the emission of fiber changed from cyan to green then bright, in response to the presence of heparin at the concentrations of 0.4 and 0.8 U mL-1 respectively, which is feasible in sensing therapeutic heparin levels. Also, the protamine tryptic digestion could make similar color changes at the increased trypsin levels of up to 8 μg mL-1, exhibiting the potential application in monitoring urine trypsin contents of patients. This solid-state, fibrous strips-based test system for the real-time and naked-eye detection of heparin and trypsin can serve as a self-test device for high-risk individuals.

Fig. 5 Schematic illustration of this probing system and its ratiometric probing mechanism to carboxylesterase (CaE). Wu and co-workers

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introduced the fluorescence resonance energy transfer (FRET) into the

design of AIE bioprobes, and fabricated a FRET-AIE-based ratiometric fluorescent probing system for the detection of carboxylesterase (CaE), by utilizing the efficient energy transfer between fluorescein and the nanoaggregate of tetraphenylethene derivative (TPE-N+) (Fig. 5). TPE-N+ has 9



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emission band at 430-480 nm, whereas fluorescein exhibits absorption band at 460-500 nm. Obviously, a great spectral overlap between the emission of TPE-N+ and the absorption of fluorescein , well meeting the requirement of FRET. Fluorescein diacetate (FDA), the precursor of fluorescein, was selected as substrate for the assay of esterase activity. Since there were no interactions between TPEN+ particles and FDA, only blue emissive TPE-N+ particles at 460 nm could be observed. After the 30 min incubation of 20 U L-1 CaE, at pH=7.4, fluorescein diacetate was converted to fluorescein with two negative charges, through the catalytic hydrolysis. Then, electrostatic interaction between the anionic fluorescein and cationic particles of TPE-N+ made them move close to each other, to assure the resultant FRET progress. As the result, the emission of fluorescein at 520 nm appeared, and the ratio of the two emission peaks could report the enzyme activity/level as sensitive signals, with the detection limit of 0.26 U L-1 as realized for the sensing of CaE levels in real human serum samples in aqueous media. The AIE dots involved nanoparticle-based FRET systems exhibited strong fluorescence when aggregated and showed superb photostability, which is beneficial for its applications for a longer time.

Fig. 6 (a) Schematic illustration of one-pot method for telomerase activity assay based on AIEgen. (b) Plot of telomerase activity assay by using AIEgen-based light up technology. As the most common biomarkers for cancers, telomerase has gained increasing interest recently. 10


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Still according to the electrostatic interaction, Lou and Xia have contributed much to the detection of telomerase. In 2015, they chose TPE-Z,22 a simple positively charged and water soluble AIE compound, to screen the activity of telomerase in some cell extracts (Fig 6a). Driven by the electrostatic force, the negatively charged DNA backbone can promote the positively charged TPE-Z bind spontaneously. Once bound to the backbone of the DNA, the process of the restricted intramolecular rotation (RIM) occurred to turn on its fluorescence. In the presence of short DNA oligonucleotides such as Ex-0 (18-nt), TPE-Z remained emits weakly, however, while longer singlestranded DNA exists such as Ex-6 (54-nt), strong emission appeared with the peak at 478 nm because of the larger binding amounts of TPE-Z. Therefore, the elongation of the DNA strand caused by the presence of telomerase could turn on the telomere elongation process. Moreover, this probing system has been favorably applied to the quantification of the telomerase activity intracellularly in different cell lines (HeLa, HLF, E-J and MCF-7) and the clinical test in clear or bloody urine specimens from normal people and bladder cancer patients in different cities (Fig. 6b).

Fig. 7 (a) Schematic illustration of quencher group involved specificity fluorescence method for evaluation of telomerase activity. (b) The signal increase percentage of fluorescence intensity of two systems. 11



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With the relatively high detection background, the signal-to-background ratio of the former testing system was bad, directly restricting the selectivity. Afterwards, they optimized the above method by introducing the FRET mechanism. Fortunately, after molecularly labeled a quencher group of dabcyl (4-(4-(dimethylamino)phenylazo)benzoic acid) to produce QP (quencher group-labeled TS primer), a different positively charged AIEgen, Silole-R, 23 could spontaneously bind to the negatively charged QP as driven by the electrostatic force, to quench its emission peaked at 478 nm for the good overlap of the Silole-R emission and the Dabcyl absorption (λmax = 480 nm). Then, once telomerase was involved, Silole-R bound to extension repeat units, resulting in a long distance away from the quencher. Thus, the fluorescent intensity lighted up with a signal increase percentage of 1424%, while the same experiments using TS primer without quencher group gave only 586% (Fig.7b).

Fig. 8 (a) Schematic illustration of the in situ telomerase activity detection based the AIEgen of TPEPy. (b) In situ tracking of intracellular telomerase activity in MCF-7 cells. (1, 2), cells were transfected with QP and then incubated with TPE-Py. (3, 4) Cells were incubated with TPE-Py and then transfected with QP. MCF-7 cells were pretreated without (1, 3) or with (2, 4) AZT before transfection or dyeing. In order to provide the information of telomerase in living cells, intracellular in situ detection and imaging were greatly needed, hence, they further developed a simple and rapid-responsive bioprobe for telomerase activity assay. This time, they selected a positively charged yellow-emissive AIEgen, 12


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TPE-Py, 24 which possessed a high fluorescence quantum yield and long-wavelength emissions. The sensing mechanism was as same as the two above cases, by using the electrostatic interaction between TPE-Py and the quencher group labeled TS primer (Fig. 8a). After being transferred into living cells, telomerase triggered the extension of the substrate and generated a long DNA sequence with repeat units of TTAGGG, which could bind AIE-active dye to give a dramatically fluorescence signal. By incorporating positive AIE-active TPE-Py and oligonucleotides substrate, they can achieve the detection of intracellular telomerase activity and the in situ light-up imaging (Fig 8b). An obvious “turn-on” fluorescence appeared (Fig. 8b1), while the fluorescence outputs from the cells of MCF-7 (AZT-treated) was faint (Fig. 8b2). Both of the orders for TPE-Py and QP going inside to the cells could be used to screen the activity of telomerase (Fig. 8b 3, 4), in some living cells. This assay showed great potential in clinical analysis and telomerase-involved drug monitoring because of the advantages of high stability and excellent photostability.

Fig. 9 (a) Schematic illustration of two fluorescent signal bioprobes for detection. (b) Telomerase detection in living cells by this two fluorescent signals based detection strategy. At last, a new positively charged AIEgen, with the name of TPE-H, was introduced, which combined with Silole-R and could be utilized for the telomerase activity detection.25 Silole-R is a blue emissive compound (478 nm) but highly sensitive for telomerase detection, TPE-H shows relatively 13



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low sensitivity while its red emission (605 nm) escapes from blue autofluorescence interference. This method was established under the energy transferring between Silole-R and TPE-H, hence, making up the defects of two dyes when used alone. When different concentrations of telomerase, which were extracted from 0-20000 MCF-7 cells, were added, the value of I478+605 gradually increased. In this process, Silole-R and TPE-H aggregated and closed to each other because more AIE dyes were adsorbed by static electricity interaction as the telomerase-triggered QP elongated. Subsequently, the intensities of Silole-R and TPE-H increased separately, and at the same time partial energy transferred from Silole-R to TPE-H. By using this bioprobe with two fluorescent signals, the LOD was as few as 250 cells, with an obviously wider linear range. Thanks to the long wavelength emission of TPE-H, the monitor of the telomerase activity in some living cells could be achieved according to this method, by bioimaging. As shown in Fig. 9b, 1 and 2 lines were telomerase-positive and AZT-treated HeLa cells dyed by sole Silole-R, respectively. The fluorescence images showed a very weak fluorescence change (Fig.9b 1, 2, 5 lines). Then the mixture of Silole-R and TPE-H was added similarly after transfection. As shown in the Fig. 9b (3 and 4 line), in comparison with the fluorescence intensity in AZT-treated inactivated HeLa cells, the blue emission in telomerase-positive HeLa cells was still almost indistinguishable and undiversified (1.965-fold enhancement, Fig. 9b6), but the red emission intensity apparently increased (8.081-fold enhancement). The different activity of telomerase in living cells accounted for this phenomenon, proving the bioimaging feasibility of TPE-H in the detection of telomerase activity. Apparently, Lou and Xia et al conducted the systematic work regarding the the detection of the telomerase activity, and successfully applied it to urine samples for the in vitro and intracellular in situ tracking, indicating the potential applications of those methods in cancer cells bioimaging and clinical cancer diagnosis.

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Scheme 3 Schematic illustration of the AIE mechanism based solubility change. Solubility Change To change the solubility is another commonly used strategy to design AIE bioprobes. In the solution or single molecular state, the fluorescence of AIEgens is faint but much bright when aggregated. Hence, to alternate the solubility of AIEgen in the presence of a bioanalyte could trigger the appearance of AIE nanoaggregates, to readily develop bioprobes with the turn-on fluorescent signal (Scheme 3). Actually, there were quite a few enzyme assay systems built on the solubility change strategy. The most representative ones of this type will be elucidated in the following discussion. For example, many phosphate-containing TPE compounds have been synthesized for the detection of ALP (Fig. 10). In 2013, Zhang et al. synthesized a water soluble AIEgen of 2CH3O-TPE-PA,

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which contained two

methoxy units and one phosphate unit with weakly emission. In the presence of ALP, the ALPcatalyzed hydrolysis of the phosphate group converted 2CH3O-TPE-PA into TPE-OH with much lower solubility in aqueous solutions. Thus, the resultant aggregation of TPE-OH in aqueous solutions turned on the fluorescence, and the detection limit of ALP was measured to be as low as 18 mU mL-1. Due to the electron-donating effect of the two methoxy groups, the probe exhibited a green emission, contributing toits application for the detection of ALP in living HeLa cells. Liu and co-workers27 devised an AIE fluorescent probe by functionalizing TPE with two phosphate groups (TPE-2PA). The detection limit was tested to be 0.2 U L-1 or 11.4 pM with a linear range of 3-526 U L-1. The test was further excellently applied in diluted human serum samples with a linear range up to 175 U L-1, illustrating its potential application in pathological analysis of ALP activity in some real biological 15



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samples. In 2016, Zhang’s group 28 synthesized three other phosphate substituted tetraphenylethylene (TPE) probes (TPE-PA, TPE-2PA and TPE-4PA) with different quantities of -PO3H2 groups for monitoring the ALP activity. Both TPE-PA and TPE-2PA were sensitive enough towards ALP and only TPE-2PA exhibited high fluorescence SNR and fine cell penetrability during the progress osteogenic differentiation in living cells. Unfortunately, we have not found other non-TPE based AIE luminogens linked to phosphate groups as ALP bioprobes, thereby, non-TPE based long-wave emissive probes should thus be developed.

Fig. 10 Schematic illustration of a series of AIE-based bioprobes towards ALP.

Fig. 11 (a) Schematic illustration of carboxylesterase fluorometric detection. Bright field (b) and fluorescence (c) images of molecular aggregates observed after the addition of carboxylesterase into TPE-CaE solution. Li and Chen et al. 29 reported a novel tetraphenylethylene derivative (TPE-CaE), in which, there were four carboxylic ester units responsive to the carboxylesterase 16


for the enzymatic hydrolysis

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reaction (Fig. 11a). Thanks to its four carboxylic acid groups, TPE-CaE was well soluble in aqueous medium with very weak fluorescence at 475 nm (emission quantum yield: 0.02) at pH 7.4. After the enzyme induced hydrolysis of the carboxylate unit by esterase, TPE-CaE was enzymatically converted into TPE-CaER, a relatively more hydrophobic entity, accompanying with a turn-on fluorescent signal. With the aid of fluorescence microscopy, the aggregates of TPE-CaER, generated by its self-assembly, were one-dimensional rod shape microfibers with the length of longer than 50 μm (Fig. 11b, c). In this way, TPE-CaE could be applied in the fluorescent light up assay for carboxylesterase with a LOD value of 29 pM, however, the test time is a little longer (up to 2 hours).

Fig. 12 (a,c) Schematic illustration of detection of lipase levels by probe P1 and S1. (b) Plot of I/I0 value of probe P1 in the presence of a fixed amount of commercial lipase samples. (d) The fluorescence photograph of probe S1 after the addition of corresponding diluted human serum samples. Similar to carboxylesterase, lipase, also known as triacylglycerol ester hydrolase, is a subclass of esterase. Also, lipase always serves as a critical biomarker of some pancreas-related disease. Recently, Shi and co-workers synthesized a very simple TPE derivative of P1 (Fig. 12a),30 which could be easily 17



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converted into TPE-COOH through the catalytic hydrolysis by lipase. For its much poor solubility in comparison with that of P1, aggregations of TPE-COOH appeared and the fluorescence turned on. The linear assay range of lipase concentration was located in 0.1-1.3 mg mL-1. Also, the selective identification of lipase and preliminary commercially lipase activity filtering was achieved too (Fig. 12b). As we all know, the chemical structure and spatial structure of a probe play a decisive role in its sensing/probing performance. In other words, the number of the substituted recognizing moieties, the substituted position of these recognizing units as well as the geometric configuration can greatly affect the probe’s workability, sensitivity, and specificity/selectivity, etc. Usually, even a minor alteration in the probe’s structure would lead to big changes in its performance. Thence, they further introduced glutamate to TPE as recognition units, to yield S1 as a new “light-up” fluorescent probe for lipase levels detection (Fig. 12c).31 In some heterogeneous medias, the hydrophilicity of amino and carboxyl units in the molecule could facilitate its full access to lipase at the oil-water interface and thus achieved an interfacial controlled AIE principle. Excitedly, only 7 min was needed, and the detection limit was determined to be as low as 0.13 U L-1. It is worth mentioning that the fluorescence linear response ranged from 0 to 80 U L-1, full covering the lipase level in human serum. Michaelis-Menten constant (Km) was calculated to be 4.23 μM, disclosing the splendidly affinity present in the probe molecule and lipase. Compared to the former probe P1, the LOD of S1 decreased by 338-fold. Once applied for visual diagnosis of acute pancreatitis (Fig. 12d), for serum samples obtained from healthy people (no.1-no.6), very weak fluorescence was displayed, while for samples of acute pancreatitis patient (no.7-no.12), significantly enhanced fluorescence emission could be seen by naked eyes. For the first time, these efforts focused on the fluorescence analysis of lipase, which could not only provide a beneficial pathway for further clarifying the physiological functions of lipase but also guiding lipase involved diseases pathological analysis. 18


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Fig. 13 (a) Schematic illustration of the fluorescent sensing mechanism towards GGT. (b) Timedependent of emission spectra for the probe in buffer upon addition of GGT. γ-Glutamyltranspeptidase (GGT) is crucial in glutathione metabolism and homeostasis. The evaluation of its level is used to diagnose diseases of the liver, biliary system, and pancreas. Wu et al.32 designed a fluorescent turn-on probe for the detection of γ-glutamyltranspeptidase (GGT), and the two γ-glutamyl amide groups in this TPE derivative acted as hydrophilic units and recognition groups. Through the GGT enzymatic reaction, the γ-glutamyl amide units were hydrolyzed, thus, the insoluble residues readily formed, accompanying with the correspondingly strong blue emissive signals. As shown in Fig. 13b, after the incubation with GGT, the probe solution with the concentration of 20 μM displayed almost no fluorescence. Upon the addition of GGT (60 U L-1), an emission at about 472 nm emerged. Also, the GGT levels in some biological samples could be detected by the probe, so didthe imaging endogenous GGT in living A2780 cells. The LOD was calculated to be 0.59 U L-1, which shows enough sensitivity for conducting pathological analysis for diseases involving GGT.

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Fig. 14 Schematic illustration of the working mechanism of the probe TPETH-2 (CFTERD3). Liu et al. designed a light-up red emissive bioprobe of TPETH-2 (CFTERD3) for the detection of chymase, an important enzyme closely related to inflammatory and immunoregulatory functions (Fig. 14). In TPETH-2,33 the tetraphenylethenethiophene (TPETH) part could ensure the red emission in aggregates, and the peptide sequence of Cys-Phe-Thr-Glu-Arg (CFTER) could be hydrolyzed by chymase as recognized unit. TPETH-2 was nearly nonemissive in aqueous media, however, once the hydrophilic peptide sequences were cleaved by chymase, strong fluorescence appeared as the result of dramatically changed solubility. More importantly, the probe could selectively identify chymase and effectively differentiate chymase from other enzymes in the same family (E.C. 3.4.21). The LOD is determined to be 0.1 ng mL-1 with a linear range of 0-9.0 ng mL-1.

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Fig. 15 (a) Schematic illustration of the sensing mechanism of probe TPE-KFPE for DPP-4 detection. (b) Confocal images of 3T3-L1 cells stained by probe in the absence and presence of diprotin A and EGCG. (c) Confocal images of zebrafish. (A) Zebrafish not treated with the probe. (B) Zebrafish stained by probe. (C) Zebrafish pretreated with diprotin A then stained by probe. Bright-field (BF), fluorescence (FL), overlay images (Merge) One type II transmembrane glycoprotein, dipeptidyl peptidase-4 (DPP-4), is widely distributed in tissues and participated in circulates. On the other hand, it is one of the most promising and significant targets for the treatment of type 2 diabetes mellitus (T2DM). Zhao’s group functionalized TPE with a short peptide sequence of Lys-Phe-Pro-Glu (KFPE) for the specific DPP-4 cleavage (Fig. 15a).34 Really, after treating with DPP-4, the non-emissive TPE-KFPE gave obviously switched on fluorescence for the poor solubility of the TPE-residue. Thanks to its good penetrating property in the phospholipid layer of cell membrane, after incubating 3T3-L1 preadipocyte cells with probe, the dosedependent relationship could be sensitively measured between DPP-4 levels in 3T3-L1 cells and the fluorescence intensity (Fig. 15b). On the other hand, fluorescence images of 3T3-L1 cells pretreated by diprotin A (DPP-4 inhibitor) and the (-)-epigallocatechin-3-gallate (EGCG) (DPP-4 inhibitor) indicated that only very weak fluorescence signal was monitored. The in vivo tracking DPP21



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4 of TPE-KFPE was also evaluated in zebrafish (Fig. 15c), showing that the distribution of DPP-4 mainly located in the blood vessels and yolk sac of zebrafish. The obtained information was useful for the creation of new anti-diabetes drugs. According to the same sensing mechanism,35 a different hydrophilic Ser-Asp-Lys-Pro (SDKP) peptide sequence has been bonded to TPE, for screening angiotensin converting enzyme (ACE) activity with good performance.

Fig. 16 (a) Schematic illustration of TPE-NO2 for NTR assay. (b) Bright field (a, c) and fluorescence (b, d) microphotographs of HeLa cells incubated with TPE-NO2 (10 M). The left row was taken at normoxic conditions (a) and (b), the right row was taken at hypoxic conditions (c) and (d). Nitroreductase (NTR), often involved in the reduction of nitrogen-containing compounds, would be overexpressed in the hypoxic environment, thus, deemed as a biomarker for hypoxic cells. By manipulating the aggregation and deaggregation of TPE-NO2 (Fig. 16a), Zhang and co-workers proposed a new method for the detection of NTR.36 In TPE-NO2, the salt form of pyridinium moiety ensured its good solubility in water, accompanying with very weak emission, while 5-nitrofuran group acted as a good target for NTR. It was expected that NTR could catalyze the reduction of nitro group in TPE-NO2 to amino one, followed by 2, 5-rearrangement elimination, and thereby the yielded pyridine substituted TPE emitted much strongly due to its poor solubility. Really, TPE-NO2 exhibited high sensitivity with the detection limit of 5 ng mL-1 and selectivity toward NTR over other 22


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biologically relevant species. In HeLa cells imaging, the fluorescence was switched on upon binding with cells, as the result of the inhibited internal rotations of the TPE framework. Interestingly, under the normoxic condition (Fig. 16b), the presence of pyridinium in TPE-NO2 could induce intramolecular charge-transfer to give a red-shifting emission (the orange-red fluorescence image) during the cell growth. However, the high level of NTR in hypoxic tumor cell caused the transformation of TPE-NO2 to TPE-PY, giving the blue-green fluorescence image much different from that under the normoxic condition. With the same strategy, Wang, Wu and Tang37 designed another TPE derivative as a galactosidase probe, in which, a D-galactose residue was linked to the terminal of the pendant positively charged pyridinium as the substrate of -galactosidase. In the presence of -galactosidase, the -galactopyranoside group in TPE-Gal was cleaved to yield a phenolate intermediate, which then spontaneously underwent 1, 6-elimination of p-quinone-methidem to generate pyridinium substituted TPE with bad solubility, accompanying with the turn-on fluorescent signal.

Fig. 17 (a) Schematic illustration of probing mechanism of TPECM-GFLGD3-cRGD. (b) Confocal images of A) cell MDA-MB-231, B) cell MCF-7, C) cell 293T, D) free cRGD pretreated cell MDAMB-231, E) CA-074-Me pretreated cell MDA-MB-231, and F) both cRGD and CA-074-Me pretreated cell MDA-MB-231 after 4 h probe incubated. The commercial cell nucleus dye DAPI (4’,6-diamidino23



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2-phenylindole) emits blue, the probe emits red. It is well known that many types of tumors are accompanied with cathepsin B overexpression, which can selectively cleave a specific -Gly-Phe-Leu-Gly- peptide sequence, and has been utilized for enzyme-activation drug delivery. Liu et al. developed a unique bioprobe involving cathepsin B based on a new AIEgen, TPECM-GFLGD3-cRGD,38 that could be applied in the cancer cells, such as targetable light-up imaging and activatable photodynamic ablation. In TPECM-GFLGD3-cRGD, there were four parts: 1) an orange emissive AIEgen TPECM as a photosensitizer and imaging dye, 2) a cathepsin B substrate GFLG peptide that was specificly responsive to it, 3) three Asp (D) moieties as hydrophilic spacer to enhance the solubility of the probe molecule, and 4) dual cRGD-targeting moieties led to the highly selective ablation of cancer cells. Obviously, each part of the probe molecule can cooperate well with each other. In aqueous solution, TPECM-GFLGD3-cRGD was nearly nonemissive with low reactive oxygen species (ROS)-generation ability, due to the depletion of excitonic energy by free rotations of molecules themselves. Upon uptaken by cancer cells, the cathepsin B induced cleavage of the GFLG peptide substrate led to the release of unsoluble dicyanovinylcontaining AIEgen, which interacted with biological thiol and generated ROS under light. Thus, the signal output of enhanced fluorescence accompanied by activated photoactivity was very appropriate for image-guided PDT, with the detection limit of cathepsin B determined to be 0.1 μM in solution. Furthermore, the cell-specific light-up imaging could be achieved (Fig. 17b), and the red emission in MDA-MB-231 cells increased gradually after incubated with the probe, much stronger than those in negative control cells of 293T and MCF-7 under the same factors. Nevertheless, when MDA-MB-231 cells were not pretreated with cRGD (target marker) and/or CA-074-Me (cathepsin B inhibitor) during the probe molecule incubation, dramatically decreased fluorescence intensity was displayed, which might attribute to the non-activate probe molecule or inefficient cellular uptake. This powerful strategy 24


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enabled the light-up and real-time cancer cell monitoring with a high SNR and also resulted in the high selectively ablation of cancer cells, possibly opening up a new horizon for photodynamic therapy with targeted and image-guided.

Fig. 18 (a) Schematic illustration of the working mechanism of this AIE turn-on apoptosis probe TPSDEVD-Pt-cRGD for the therapeutic effects with in situ assessment. (b) Apoptotic process of TPSDEVD-Pt-cRGD stained U87-MG cells (A-D) and MCF-7 (E) and 293T (F) cells after the incubation with TPS-DEVD-Pt-cRGD for 6 h. So far, it is still a key challenge to make drugs delivery effectively and precisely to specific tissues with tiny systemic toxicity, in the development of theranostic probes. Simultaneously, an ideal probe should bring the function of tracking drugs real-time besides monitoring the drug efficacy in situ, which could assist the initial assessment of therapeutic treatment and lower the effectiveness of drug action process. Caspase-3/7 acted as a critical regulator in the apoptosis pathway, and also has been served as essential indicators for the detection of cell apoptosis. Since caspase-3/7 can cleave the structure of Asp-Glu-Val-Asp (DEVD), a number of AIE light-up probes have been designed according to the cleave-type mechanism. For example, Liu’s group designed a prodrug with the targetable theranostic function, focusing on in situ monitoring drug induced apoptosis (Fig. 18a).39 25



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This sensing system has several components: a chemotherapeutic Pt (IV) prodrug which could be reduced inside the cell to active Pt (II), an AIE-active tetraphenylsilole (TPS) based apoptosis probe (TPS-DEVD), and the targeting ligand, a cyclic peptide (RGD) (Fig. 18a). The prodrug could preferentially accumulate in αvβ3 integrin overexpressed cancer cells, release apoptosis probe TPSDEVD and the active drug Pt(II) upon the intracellularly reduction reaction of the Pt(IV) prodrug. After that, the released Pt(II) drug could induce the process of cell apoptosis, then caspase-3 was activated to hydrolyze the short peptide DEVD in the apoptosis probe of TPS-DEVD, and generate the insoluble TPS residue, which tended to form aggregates with the restricted intramolecular motions of the phenyl rings and ultimately resultant fluorescence enhancement. Apparently, noninvasive and real-time imaging of the therapeutic responses of a particular anticancer drug can be achieved through this fluorescent turn-on response, accompanying with

good linear response towards caspase-3 levels

and the 1 pM LOD value. As shown in Fig. 18b, the U87-MG cell (overexpressed αvβ3 integrin on the cellular membrane) images demonstrated the progressive light-up of the cells after TPS-DEVD-PtcRGD incubated over 6 h. In contrast, much lower fluorescence intensity was shown from the low level of integrin expression cells of MCF-7 or 293T even after 6 h. Moreover, the viability of U87MG cells decreased obviously with the increased amount of TPS-DEVD-Pt-cRGD , while nearly no signal was recorded for MCF-7 cells. The results revealed that U87-MG cells could specifically uptake the probe molecule,

and the processes of drug release as well as its therapeutic effect were well

visually monitored by confocal imaging of the apoptotic process.

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Fig. 19 (a) Schematic illustration of Ac-DEVD-PyTPE for caspase-3/7 detection. (b) Fluorescence in vivo images of C6 tumor-positive mice after Ac-DEVD-PyTPE intratumoral injection and pretreated with STS or free STS for 12 h before the probe injection. (c) Fluorescence intensity analysis of the probe-treated tissues as a function of time Generally, the long wavelength emission is beneficial for the applications in in vivo studies. Accordingly, Liu et al. modified tetraphenylethene pyridinium (PyTPE),40 an AIEgen with orange emission, with the peptide moiety of Asp-GluVal-Asp through the Click Chemistry reaction (Fig. 19a). Once the C-terminal of Asp (D) was specifically cleaved by active caspase-3/7, Ac-DEVD-PyTPE could be easily quantified based on the varieties in fluorescence intensity with a linear fluorescence increase at 610 nm, owing to the self-aggregation of the insoluble PyTPE residues in the aqueous environment. Then, Ac-DEVD-PyTPE probe was injected into C6 tumor positive mice that treated by apoptosis inducer staurosporine (STS) or free STS(Fig. 19b), to evaluate its possible application in living animals. Excitedly, after being treated with STS, much stronger fluorescent outputs were recorded in the tumor region, rather than those in non-STS mice, as evidently demonstrated by the stronger fluorescence intensity in tumor and normal tissues at different times (Fig. 19c). Compared to normal tissues, after injection, in five minutes, 3-fold increased fluorescence was observed in the apoptotic tumor tissues. Furthermore, Ac-DEVD-PyTPE has been successfully applied for ex vivo 27



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evaluation of drug efficacy that was induced by cell apoptosis.

Fig. 20 (a) Schematic illustration shows that probe DFP for the tracking of delivery and release of the drug in MMP-2 positive living cells. (b) Real-time CLSM images displaying the responsive progress of MCF-7 cells treated with DFP during 120 min. Insets are the magnified views of single cell chosen in CLSM imaging. Channel 1 = PyTPE fluorescence: excitation wavelength, 405 nm; emission collected, 495-575 nm. Channel 2 = DOX fluorescence. Also by modifying PyTPE, Xia and Lou designed a proteolytic enzyme-responsive prodrug, DOX-FCPPsPyTPE (DFP), for rapid delivery and accurately track release of drugs in some living cells (Fig. 20a).41 Three components were selected to assemble the probe molecule DFP: a classic TPE derivative, AIE-active PyTPE, linked with cell penetrating peptides (FCPPs), which contained a cancer-related enzyme matrix metalloproteinase-2 (MMP-2) responsive peptide (LGLAG) and a cell penetrating peptide (CPP), end with a therapeutic unit (doxorubicin, DOX). This prodrug can not easily enter into the cells in the absence of MMP-2. However, when MMP-2 appears, probe DFP can be hydrolyzed to two assemblies of DOX functionalized cell penetrating peptides (CPPs) and the 28


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self-aggregate of PyTPE modified peptide.With the assistant of CPPs, DOX can easily enter the cell via its interaction with the cell membrane. Also, owing to the changed solubility, the self-aggregate of PyTPE modified peptide will be triggered thus light up the yellow fluorescence, indicating the release of the therapeutic unit to the cells. Using the inherent red emissive of DOX, the selective delivery of the drug to the MMP-2 positive cells was also powerfully proved (Fig. 20b). This method opened a novel and promising insight for controlled drug delivery and real-time monitoring of drug release in some MMP-2 overexpression cells.

Scheme 4 Schematic illustration of the AIE mechanism based hydrophobic interaction. Hydrophobic interaction

Fig. 21 (a) Structure of t-TPEM and proposed sensing mechanism of t-TPEM to MAO-A. (b) Confocal fluorescence images of MCF-7 cells upon treatment with t-TPEM (1 μM, 2 h) in the absence (d-f) or presence (g-i) of MAOs inhibitor clorgyline (200 μM, 1 h). Green = t-TPEM. Red = MitoTrackerRed. Blue = Nuclei stain. (a-c) Cells only treated with DMSO and MitoTrackerRed (50 nM). 29



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In order to minimize the tendency of exposing their surface area to polar water molecules in aqueous solutions as much as possible, nonpolar molecules are liable to forming aggregates. This is named as the hydrophobic interaction (Scheme 4). Thus, it is easily expected that driven by hydrophobic interactions in aqueous medium, amphiphilic organic luminogens would like to enter into hydrophobic domains or holes in the folded structure of the proteins if there are. Then, due to the limited volume of the domains or holes, the entered luminogens would form aggregates with restricted intramolecular motions. This case seems specially suitable for the design of AIEgen-based bioprobe, thanks to the RIM mechanism of AIE. Thus, specific proteins with abundant hydrophobic domains could be detected by AIEgens, with the exciting fluorescent light-up signals. Also, their changes of conformation could be observed clearly. Liu and Zhu et al. designed and synthesized a series of watersoluble TPE derivatives (t-TPEM).42 Because of hydrophobic interactions between proteins and the probe, the specific detection of monoamine oxidases (MAOs) could be realized. t-TPEM was composed of an N-methyl phenylpyridium, a TPE fluorogen, and a C-C double bond linker. The Nmethyl phenylpyridium and its analogues were unique inhibitors toward both MAO-A and MAO-B. The positively charged pyridium moiety was hydrophilic, which could render the probe dispersible in aqueous buffer. In the absence of MAO-A, the strong intramolecular charge transfer and the free rotation of phenyl ring caused the weak emission of t-TPEM in polar solvent. Then, when the probe formed a complex after entering the active site of MAO-A (hydrophobic pocket) under physiological conditions, the reduced environmental hydrophilicity and restricted intramolecular rotations turned on the probe fluorescence. As the result, t-TPEM displayed a 21-fold higher fluorescent intensity upon the addition of MAO. In the experiment of cell image, ss shown in Fig. 21b, no background fluorescence was observed for cells in the absence of t-TPEM (a), then upon incubated with MAOs, 30


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strong fluorescence from the cells appeared (d), when the cells were pretreated with clorgyline (MAOA inhibitor), the cell fluorescence was almost quenched. Thus, cell imaging experiments revealed that t-TPEM was able to efficiently and selectively detect the activity of MAO-A localized in mitochondria of cells, offering a new opportunity for convenient high throughput MAO related drug screening. Hydrogen bonding

Scheme 5 Schematic illustration of the AIE mechanism based hydrogen bonding. As a kind of relatively strong intermolecular interaction, hydrogen bonding is normally stronger than van der Waals forces but weaker than covalent or ionic bonds (Scheme 5). In AIE-based bioprobes, the formation of intramolecular hydrogen bond can not only inhibit the RIM motion, but also produce the effect of excited-state intramolecular proton transfer (ESIPT). Genearlly, luminogens with the ESIPT characteristic depend on intramolecular hydrogen bonding to create the fluorescence change. Thus, in excited-state, if the proton shift process from donor (amino group or hydroxyl) to acceptor (nitrogen or oxygen) was blocked, the emission wavelengths of luminogens can be alternated lightly. The introduction of hydroxyl or amino groups to a specific enzyme substrate thus could create the turn-on or ratiometric bioprobes after the cleavage of hydroxyl units. In this regard, either hydrophilic nor hydrophobic substrates

could be engaged for the sensing of enzyme levels, as the fluorescence

change could be overmastered by the formation of hydrogen bonding instead of the solubility in aqueous buffer. It should be pointed out that

the ESIPT emission could be suppressed by the

hydrogen-bond donor ability and high polarity of solvents with a large Stokes shift. However, unlikely, thanks to the formation of nanoaggregates, the ESIPT emission could be favored byyet the AIE effects , thus benefiting the improvement of the high SNR detecting sensitivity. 31



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Fig. 22 Schematic illustration of probe AIE-Lyso-1 for lysosomal esterase specific detection. Lysosomal enzymes are closely correlated with the intracellular digestion of various carbohydrates, proteins and lipids, the functional deficiencies of these enzymes would bring about numerous inherited lysosomal storage disorders (LSDs). A superb “AIE + ESIPT” probe of AIE-Lyso1 was reported for lysosomal esterase activity assay and in situ visualization by Liu and co-workers (Fig. 22),43 which was composed of a salicyladazine luminogen, esterase recognizable acetoxyl groups, and specific morpholine unit for lysosome-targeting. The absence of acetyl groups instead of hydroxyl groups would quench the salicyladazine fluorescence, because the hydrogen bonding and the intramolecular motion of the N-N bond were destroyed in a large degree. After the cleavage of acetyl groups by esterase, the fluorescence was lighted up, as the result of the activated ESIPT process by the formed intramolecular hydrogen bond and the activated AIE process upon aggregation. A good linearity could be obtained in the esterase concentration range of 0.10-0.50 U mL-1, and the LOD was determined to be 2.4×10-3 U mL-1. After incubation with this new probe of MCF-7 cells, apparent emissive signals could be recorded in lysosome, almost overlapping with the outputs of commercial LysoTracker Red. Furthermore, in situ monitoring of lysosomal esterase activity and thereby tracking lysosomal behaviors in some living cells were also performed by the probe of AIE-Lyso-1, demonstrating promising potential for the early diagnosis of Wolman disease caused by lysosomal esterase.

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Fig. 23 (a) Schematic illustration of probe DEAM for the detection of lysosomal esterase. (b) Image of esterase in MCF-7 cells. Cells a) without or b) with incubation of DEAM for 10 min; c) cells were preincubated with inhibitor for 20 min and then treated with DEAM (50 μM) for 10 min; “L”: bright field; “R”: red fluorescence image of DEAM. Tong and Liu et al. also designed a red fluorophore (DEAM) with the characteristics of AIE and ESIPT by binding an electron-acceptor maleonitrile group and a rich electron unit diethylamine to salicyladazine (Fig. 23a).44 The fluorescence of DEAM could only be turned on when the rotation around the C-C bond was restricted and the intramolecular hydrogen bonds were formed. In the presence of esterase, the acetoxyl group in DEAM was hydrolyzed to -OH, accompanying with the regenerated AIE and ESIPT characteristics. Thus, the detection limit for in vitro quantification of esterase was calculated to be 0.005 U mL-1, with a linear range of 0.01-0.15 U mL-1. Furthermore, the screening of esterase activity in living cells mitochondria was performed successfully by DEAM (Fig. 23b). These four sensing mechanisms discussed above have provided a great number of examples to prove the extraordinary charm of AIE as a unique approach for sensitive and specific monitoring of enzyme activity, including the behaviors in cell components (e.g., membrane, mitochondria, 33



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lysosomes, nuclei), intracellular environments, cellular events (e.g., expression of tumor markers and apoptosis). The distinct sensing strategy is highly flexible, relying on the design in molecular structure of bioprobes and experimental conditions. The AIE effect is also universal applicable, since it can be employed to almost all biogenic species, no matter what kind of form they exist, active or inactive, charged or neutral, small or large. The probing systems built on AIE effect are usually easy-to-prepare, simple-to-operate, highly sensitive and fast responsive. The last but not the least, AIE probing systems can be multi-mode and multifunctional, they can recognize, quantify, monitor, and visualize the analyte efficiently and conveniently. Conclusion and outlook AIEgens are an emerging group of photoluminescent molecules which are nonemissive at the soluble state, butwill produce intense luminescence uppon aggregation. For this fantastic photophysical property, their biological applications are attracting increasing attention. In this review, we summarized various working mechanism, such as electrostatic interaction, solubility change, hydrogen bonding and hydrophobic effect, for the design and synthesis of enzyme triggered bioprobes based on AIEgens and discussed typical examples of various AIEgens with high signal-noise-ratio for activity assays, imaging and imageguided therapy. Normally, RIM is the main origin for the AIE effect, however, in some sensing systems, there may be two or multiple mechanism working simultaneously, like “AIE+ESPIT” or “AIE+FRET”, which provides an effective remedy to improve the brightness, sensitivity and specifcity of the bioprobes, further expanding the probe design. Highly hydrophilic recognition elements, prodrugs, targeting ligand and other stimuli responsive elements have also been introduced to AIEgens with the functions of visualized drug delivery and release in desired locations of cells. However, some drawbacks still exist for formulating advanced AIEgens-based enzyme probes. In view of the fact that enzyme plays significant role in in maintaining homeostasis and normal 34


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life activities, real-time in situ detection and visualization of specific enzymes in vivo are of great needed. In this review, most sensing system mainly used shorter wavelength emissive AIEgens like blue or green, which have restricted the further practical applications involving in vivo researches that demand low autofluorescence and deep penetration. To greatly expand the scope of AIE-based probes’ in vivo applications, one alternative strategy is to devise more red and FR/NIR fluorescent AIE-based bioprobes with multiple functionalities. Secondly, there are a lot of proteolytic enzymes in our body, only specific peptide sequences contained probe as a recognition unit could accomplish their detection, however, this probe molecules are prone to degradation and thus require special storage condition. Hence, low molecular-weight nonpeptide-based small molecule AIE bioprobes as virtual substrates for the fluorogenic detection of proteolytic enzyme should be fully taken into account. Thirdly, to facilitate the presentation, we have divided the selected AIEgens by their sensing mechanisms, mainly including electrostatic interaction, solubility change, hydrophobic interaction, and hydrogen bonding. Although we could simplify the sensing mode in a large degree for better understanding and easy summary for the possible guidance, the actual cases are really much complicated. For example, the hydrophobic interaction between AIEgens and the enzyme analytes have been nearly ignored in most cases, which could surely cause some influence. Also, in the above discussed cases, we could partially expect the sensing behavior of the AIE bioprobe at the design stage, but not accurately customize the property, due to the lack of well-built theoretical modes and the related theories. To further the development of AIE bioprobe and deepen the present understanding, the tight cooperation with theoretical scientists is badly needed. Similarly, the practical applications in disease diagnosis and treatment, the support and help of medical scientists are also required. So far, the present researches have already demonstrated the advantages of AIEgens for the design 35



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of bioprobes over others. Upon addressing some tissues encountered, huge potential applications and unexpected exciting surprise could be explored. Perhaps, this review, as a collective of important information in this area, could inspire more and more endeavors to further promote AIEgens to emerging enzyme based biomedical applications. AUTHOR INFORMATION Corresponding Author Corresponding author. Phone: 86-27-68755767; Fax: 86-27-68756757; E-mail: [email protected] or [email protected] (Z. Li). Funding Sources We are grateful to the National Science Foundation of China (no. 31501423, and 21325416), and Director Fund of Oil Crops Research Institute, CAAS (no. 1610172015006) for ﬁnancial support. Notes The authors declare no competing financial interest. REFERENCES (1) Wijdeven, R. H.; Neefjes, J.; Ovaa, H. How chemistry supports cell biology: the chemical toolbox at your service. Trends Cell Biol. 2014, 24, 751-760. (2) Joo, J. H.; Wang, B.; Frankel, E.; Ge, L.; Xu, L.; Iyengar, R.; Li-Harms, X.; Wright, C.; Shaw, T. I.; Lindsten, T.; Green, D. R.; Peng, J. M.; Hendershot, L. M.; Kilic, F.; Sze, J. Y.; Audhya, A.; Kundu, M. The noncanonical role of ULK/ATG1 in ER-to-Golgi trafficking is essential for cellular homeostasis. Mol. Cell 2016, 62, 491-506. (3) Xue, C.; Lei, Y.; Zhang, S.; Sha, Y. A cyanine-derived "turn-on" fluorescent probe for imaging nitroreductase in hypoxic tumor cells. Anal. Methods 2015, 7, 10125-10128. (4) Yuan, J.; Xu, Y.; Zhou, N.; Wang, R.; Qian, X.; Xu, Y. A highly selective turn-on fluorescent 36


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Enzyme-Responsive Bioprobes Based on the Mechanism of

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