CN114032087A - Enzyme-activated aggregation-induced emission fluorescent probe, and preparation method and application thereof - Google Patents
Enzyme-activated aggregation-induced emission fluorescent probe, and preparation method and application thereof Download PDFInfo
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- CN114032087A CN114032087A CN202110823009.2A CN202110823009A CN114032087A CN 114032087 A CN114032087 A CN 114032087A CN 202110823009 A CN202110823009 A CN 202110823009A CN 114032087 A CN114032087 A CN 114032087A
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Abstract
The invention belongs to the technical field of fine chemical engineering, and particularly relates to an enzyme-activated aggregation-induced emission fluorescent probe, a preparation method and application thereof, which can be applied to autophagy detection for detecting various living cells or isolated tissues and has a chemical structural formula shown in formula I. Tests show that the fluorescent probe provided by the invention has high-selectivity fluorescent response to an autophagy key enzyme Atg4b, and can realize accurate fluorescent imaging on live cells and mouse tissues for inducing autophagy, and the probe has high signal-to-noise ratio because the probe does not have fluorescence in aqueous solution and organic solvent and emits bright fluorescence only when the autophagy is activated, so that the fluorescent probe can conveniently and accurately detect the autophagy imaging.
Description
Technical Field
The invention belongs to the technical field of fine chemical engineering, and particularly relates to an enzyme-activated fluorescent probe constructed based on a fluorescence parent quinacrilonitrile induced by aggregation to detect autophagy in cell tissues and realize quick, sensitive and accurate response.
Technical Field
Autophagy is an important and indispensable degradation process that provides material and energy for the survival of damaged or unnecessary components in cells, mainly by encapsulating them and degrading them by fusion with lysosomes, to maintain cellular homeostasis. Autophagy is involved in the renewal of organelles and in the regulation of various biological activities in cells, and is closely associated with a variety of human diseases. Therefore, detection of the autophagy process is of great importance for the regulation of autophagy and the realization of a treatment.
To date, the research methods for detecting autophagy mainly include Transmission Electron Microscopy (TEM), immunoblotting and immunofluorescence staining based on detecting LC3-II/I protein conversion, etc., but these methods are time consuming, expensive and have limitations in applications that cannot be applied to living cells. To overcome these bottlenecks, fluorescent probes developed based on organic dyes have become an effective method for detecting autophagy levels due to their ease of use, non-invasiveness and real-time detection. In particular, the fluorescent probes that have been reported to respond to environmental sensitivity can be used to measure changes in pH, polarity, and viscosity during autophagy. However, it is noteworthy that most organic dyes have significant photobleaching phenomena and are susceptible to quenching of aggregate fluorescence at high concentrations or in the aggregate state, thereby resulting in susceptibility to "false positive" responses that reduce detection sensitivity, further limiting their utility.
Fortunately, the concept of aggregation-induced emission (AIE) was proposed by the down-council in 2001, and the AIE fluorophore exhibits a luminescent property that is diametrically opposite to that of the conventional fluorophore, can emit fluorescence under aggregation, and has advantages of high stability, good light stability, and large stokes shift. Therefore, the AIE fluorescent probe is widely applied to the fields of organelle imaging, biological process monitoring and the like. However, the current AIE probes for detecting autophagy still suffer from the problems of poor targeting property and difficulty in realizing accurate positioning. And because enzymes widely existing in organisms have high specificity and high catalytic performance, the peptide-based AIE biological probe has great application prospect. Therefore, the construction of the enzyme-activated probe with aggregation-induced emission characteristics has important significance for accurately and effectively detecting the autophagy process, and the signal-to-noise ratio can be effectively improved, so that accurate imaging of cell autophagy is successfully realized.
Disclosure of Invention
Aiming at the problems that the existing detection method has poor targeting property and cannot be applied to living cells, the invention provides an enzyme activated aggregation-induced emission probe which selectively responds to an autophagy key enzyme Atg4b, wherein the probe does not have fluorescence in water or an organic solvent, but can be effectively cut off by enzyme to emit remarkable fluorescence when autophagy is activated and Atg4b enzyme is released. Due to the specificity of enzyme digestion reaction and the 'on-off' property of probe fluorescence, the probe can realize high selectivity and high signal-to-noise ratio imaging of autophagy process.
One of the objectives of the present invention is to provide an enzyme-activated aggregation-induced emission fluorescent probe, which is composed of a fluorescent parent molecule with aggregation-induced emission characteristics and a polypeptide chain responding to the autophagy key enzyme Atg4b, and the chemical structural formula of the probe is shown in fig. I:
in formula I, AIE Fluorophore is an AIE fluorescent molecule with carboxyl functional group, and Peptide is a polypeptide chain responding to the autophagy key enzyme Atg4 b.
Further, AIE fluorphores are AIE fluorescent molecules having carboxyl functional groups as shown in formula II below, such as AIE derivatives developed with Quinolinecarbonitrile (QM) precursor, tricyano-methylene-pyridine (TCM) precursor, Tetraphenylethylene (TPE) precursor, etc.:
further, Peptide is the polypeptide chain GFTN or GFTE or GYVS (where the curve label is a substitution site) as shown in formula III below in response to the autophagy critical enzyme Atg4 b:
the invention also aims to provide a preparation method of the enzyme-activated aggregation-induced emission fluorescent probe, which takes fluorescent molecules with AIE characteristics as a matrix, obtains AIE-COOH modified by carboxyl functional groups through a series of reactions such as condensation, esterification and hydrolysis, and finally obtains the enzyme-activated aggregation-induced emission fluorescent probe AIE-Peptide through reaction with polypeptide amino acid chains. The AIE-Peptide has good water solubility due to the action of the polypeptide, so that the AIE-Peptide is subjected to fluorescence quenching in water or an organic solvent, and the polypeptide chain can be cut off by an autophagy key enzyme Atg4b to release fluorescent AIE-COOH, so that efficient and accurate lighting type fluorescence detection of an autophagy process is realized.
According to the preparation method, a series of enzyme-activated aggregation-induced luminescence fluorescent probes capable of realizing rapid autophagy detection are prepared, and the chemical structural formula of the probes is shown in figure IV:
the synthetic route of the enzyme-activated AIE probe of the present invention is as follows (QM-GFTN as an example):
the invention also provides application of the enzyme activated aggregation-induced emission fluorescent probe, which is applied to autophagy imaging for detecting various living cells or isolated tissues through high-selectivity isolated fluorescent response to the autophagy key enzyme Atg4 b.
Further, the cells used for autophagy imaging were Hela cells, PC12 cells and SW1990 cells.
The enzyme activated AIE fluorescent probe has no fluorescence in aqueous solution or tetrahydrofuran organic solvent, and only after the autophagy key enzyme Atg4b is added into a probe test system, the probe emits remarkable fluorescence.
In a test system of a meta-acidic PBS buffer solution, the probe has no obvious fluorescent response to various interference substances (such as biological enzyme, biological macromolecules and inorganic salt) and has obvious fluorescent change only to autophagy key enzyme.
The enzyme-activated AIE fluorescent probe AIE-Peptide overcomes the limitation that a commercial autophagy probe is aggregated in water for fluorescence quenching, and compared with a reported autophagy detection fluorescent probe, the problem of poor background fluorescence intensity due to poor targeting property is solved. The probe disclosed by the invention has no fluorescence in both a water-soluble environment and a fat-soluble environment, only responds to the fluorescence of autophagy key enzyme, and realizes high signal-to-noise ratio fluorescence imaging of autophagy. The probe provided by the invention adopts an enzyme-cutting response mechanism, so that the accuracy of autophagy detection is improved, and the autophagy key enzyme is mainly aggregated and activated in the early autophagy stage, so that the probe provided by the invention can realize fluorescence imaging in the early autophagy stage and is expected to realize early autophagy imaging. In addition, the probe can realize simple, convenient and accurate detection of isolated tissues, and provides possibility for rapid and convenient autophagy detection.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectra of probe QM-GFTN.
FIG. 2 shows an ultraviolet absorption spectrum and a fluorescence emission spectrum (10) of a probe QM-GFTN with increasing water content in a mixed solvent of tetrahydrofuran and water-5mol/L)。
FIG. 3 shows an ultraviolet absorption spectrum and a fluorescence emission spectrum (10) of a probe cleavage product QM-COOH, which shows an increasing water content in a mixed solvent of tetrahydrofuran and water-5mol/L)。
FIG. 4 shows probe QM-GFTN (10)-5mol/L) UV absorption spectra and fluorescence emission spectra as a function of time were incubated with the autophagy critical enzyme Atg4b (10U/mL) in PBS.
FIG. 5, the digestion mechanism of the probe QM-GFTN is verified: the mass spectrum of the reaction solution after the probe QM-GFTN (10-5mol/L) and the autophagy key enzyme Atg4b (10U/mL) are incubated for 1 h.
FIG. 6 Selective detection of Probe QM-GFTN: and (3) respectively incubating the probe with the target enzyme Atg4b, other biological enzymes and the macromolecular interferent for 1h to obtain the ratio of the change of the fluorescence intensity.
FIG. 7 fluorescence imaging of autophagic Hela cells by probe QM-GFTN.
FIG. 8 autophagy fluorescence imaging of probes QM-GFTN on PC12 cells and SW1990 cells.
FIG. 9 shows the autophagy fluorescence imaging of mouse heart and liver tissues by the probe QM-GFTN.
FIG. 10 is a scheme of synthesizing an enzyme-activated AIE probe exemplified by QM-GFTN in the examples.
Detailed Description
Example 1: synthesis of QM-OH
QM (1.2g, 5.1mmol) and 4-hydroxybenzaldehyde (671mg, 5.5mmol) were dissolved in acetonitrile (30.0mL) containing 1mL piperidine under nitrogen and refluxed for 10 hours. After the reaction system is cooled to room temperature, a large amount of orange-red solid is separated out. Filtration and purification of the crude product by column chromatography on silica gel (DCM/MeOH ═ 20: 1, v/v) gave QM-OH (985mg, 2.9mmol) as an orange-red solid in 56.9% yield.1H NMR(400MHz，DMSO-d6，ppm)：δ＝10.01(s，1H，-OH)，8.92(d，J＝8.4 Hz，1H，phenyl-H)，8.07(d，J＝8.8Hz，1H，phenyl-H)，7.92(t，J＝7.2 Hz，1H，phenyl-H)，7.67(d，J＝8.4Hz，2H，pheny1-H)，7.60(t，J＝8.0 Hz，1H，phenyl-H)，7.38-7.28(m，2H，alkene-H)，7.00(s，1H，quinoline-H)， 6.85(d，J＝8.8Hz，2H，phenyl-H)，4.56(q，J＝6.8Hz，2H，-CH2CH3)，1.40 (t，J＝6.8Hz，3H，-CH2CH3).
Example 2: synthesis of QM-COOH (II-1)
QM-OH (500mg, 1.47mmol), ethyl bromoacetate (251mg, 1.50mmol) and potassium carbonate (310mg, 2.25mmol) were dissolved in acetonitrile (30.0mL) under nitrogen, heated to 90 ℃ and stirred for 15 hours. After completion of the reaction, the crude product (1.1g) was filtered, dissolved in THF (20.0mL), and 5mL of NaOH solution (90mg, 2.25mmol) was gradually added thereto, followed by stirring at room temperature for 12 hours. After adjusting the pH, it was extracted with saturated brine and dichloromethane, dried over anhydrous sodium sulfate and finally purified by silica gel column chromatography (DCM/PE ═ 5: 1, v/v) to give QM-COOH (452mg, 1.14mmol) as an orange solid in 77.3% yield.1H-NMR(400MHz，DMSO-d6，ppm)：δ＝13.09(s，1H，-COOH)，8.93(d，J＝ 8.4Hz，1H，Ph-H)，8.10(d，J＝8.8Hz，1H，Ph-H)，7.94(t，J＝7.6Hz， 1H，Ph-H)，7.79(d，J＝8.4Hz，1H，Ph-H)，7.63(t，J＝7.6Hz，2H，Ph-H)， 7.40(s，1H，Ph-H)，7.01(d，3H，J＝6.0Hz，alkene-H and quinoline-H)， 4.77(s，2H，0-CH2-)，4.58(d，2H，J＝6.7Hz，-CH2CH3)，1.42(t，3H，J ＝6.7Hz，-CH2CH3).
Example 3: synthesis of QM-N-COOH (II-2)
Under the protection of nitrogen, 6-methylquinoline (21.6g, 150.85mmol) and 2-bromoethanol (22.6g, 181.02mmol) were dissolved in o-dichlorobenzene (50mL), heated to 140 ℃ and stirred for 8 hours, cooled to room temperature and filtered with suction to obtain 27.8g of crude product (about 103.7mmol), which was used directly in the next reaction and malononitrile (13.7g, 207.4mmol) were dissolved in ethanol (50mL), sodium alkoxide (9.5 g sodium in 250mL ethanol) was slowly added dropwise with stirring in ice bath, and stirred in ice bath. The resulting crystals were filtered and recrystallized to give QM-N-COOH (II-2) (11.2g, 44% yield).1H-NMR(400MHz，DMSO-d6，ppm)：δ＝8.9(d，J ＝8.4Hz，1H，Ph-H)，8.0(d，J＝9.2Hz，1H，Ph-H)，7.8-7.9(t，J＝7.8Hz，1H，Ph-H)，7.5-7.6(t，J＝7.8Hz，1H，Ph-H)，6.8(s，1H， Ar-H)，4.5(t，J＝5.4Hz，2H，CH2CH2OH)，3.7-3.8(m，2H，CH2CH2OH)， 2.7(s，3H，CH3).
Example 4: synthesis of QM-GFTN
The compound QM-COOH reacts with the polypeptide amino acid chain, and the final product is purified by HPLC to obtain the final target product QM-GFTN. As shown in figure 1 of the drawings, in which,1H-NMR(400MHz，DMSO-d6，ppm)：δ＝12.62 (s，1H，-COOH)，8.95(d，J＝8.4Hz，1H，Ph-H)，8.26(t，J＝5.7Hz，1H， Ph-H)，8.17-8.09(m，3H，Ph-H)，8.03(d，J＝8.0Hz，1H，Ph-H)，7.93(t， J＝7.9Hz，1H，Ph-H)，7.81(d，J＝8.8Hz，2H，Ph-H)，7.62(t，J＝7.6 Hz，1H，Ph-H)，7.42(s，3H，-CONH-)，7.29-7.22(m，4H，Ph-H)，7.19(d， J＝7.2Hz，1H，Ph-H)，7.05(t，J＝4.4Hz，3H，-NH2 and-OH)，6.95(s， 1H，Ph-H)，4.91(s，1H，-CH-)，4.67(d，J＝2.4Hz，1H，-CH-)，4.58(m， 4H，-CH2CH3and-CH2-)，4.24(t，J＝4.4Hz，1H，-CH-)，3.99(d，J＝5.2 Hz，1H，-CH-)，3.78-3.69(m，2H，-CH2-)，3.08-2.56(m，3H)，2.00(m，1H)， 1.40(t，J＝7.0Hz，3H，-CH2CH3)，1.07(d，J＝6.4Hz，3H，-CHCH3)，0.85 (t，1H，J＝6.8Hz).
example 5: the concentration of the prepared probe QM-GFTN in a tetrahydrofuran-water system is 1 x 10 in ultraviolet absorption and fluorescence spectrum test-3M of QM-GFTN. Tetrahydrofuran solutions (0%, 10%, 30%, 50%, 70%, 90%, 95%) with different water volume fractions were placed in 10mL volumetric flasks to give a final test solution concentration of 10-5And M. 3mL of each test solution was transferred to an optical quartz cuvette (10X 10mm) and the UV absorption and fluorescence spectra were measured. As shown in FIG. 2, the probe has a broad set of double absorption peaks (355 and 434nm) at 300nm-550nm, and the fluorescence always shows a quenching state with the increase of water content, because the polypeptide amino acid chain with water solubility improves the solubility of the probe.
Example 6: fluorescent response of Probe QM-GFTN to Atg4b, a key enzyme for autophagy
Since the environment during autophagy is acidic, the spectral response of probes QM-GFTN to Atg4b enzyme was selected to be studied in PBS buffer (10.0. mu.M, pH 5.5) at 37 ℃. As shown in FIG. 4, the absorbance of the test system gradually increased with time, and the fluorescence intensity at around 590nm also significantly increased, and after about 120min, the reaction was substantially complete. This indicates that the probe QM-GFTN reacted with Atg4b enzyme to generate QM-COOH and aggregated, resulting in fluorescence, which provides a solid foundation for the detection of autophagy in cells.
Example 7: enzyme activation mechanism verification of probe QM-GFTN
QM-GFTN was reacted with Atg4b enzyme at 37 ℃ for 1 hour at pH 5.5, and ESI-MS test was carried out after pretreatment of the reaction mixture. As shown in fig. 5, in the negative ion mode, it was observed that a [ QM-GFTN-H ] -signal peak was present at m/z 815.3986 (theoretical value of 815.3153), and a [ QM-COOH-H ] -signal peak was present at m/z 396.3155 (theoretical value of 396.1354). The above tests demonstrated that the target probe QM-GFTN specifically reacted with Atg4b enzyme, and the product was QM-COOH.
Example 8: atopic selection of Atg4b by Probe QM-GFTN
The selectivity of probe QM-GFTN over Atg4b was examined under different interfering substances. The interfering substances include biological enzymes (tyrosinase, acetylcholinesterase, butyrylcholinesterase, lipase, cathepsin), large biological molecules (human serum albumin, chondroitin sulfate, fucose, heparin sodium, protamine, hyaluronic acid, glutamic acid) and inorganic salts (ferric sulfate, potassium chloride, ammonium chloride). As shown in FIG. 6, the fluorescence intensity ratio gave the maximum response only when QM-GFTN was reacted with Atg4 b; and when the fluorescent material is acted on other substances, no obvious fluorescent response exists. The results prove that the target probe has good selectivity to Atg4b due to the existence of the specific polypeptide amino acid chain related to autophagy, and also indicate that the target probe has the potential application of tracing Atg4b in living cells.
Example 9: culture of Hela cells, PC12 cells and SW1990 cells
Hela cells, PC12 cells and SW1990 cell line were cultured in high-glucose DMEM medium containing 12% fetal bovine serum and 1% diabody, and the culture dish was placed in a 5% CO2 incubator at 37 ℃. Passage is performed when the cell grows to be full: firstly, washing cells in a culture dish for 3 times by PBS, adding 1mL of 0.25% trypsin for digestion, sucking out the trypsin after digestion is finished, adding 2mL of culture medium to blow the cells uniformly, counting the cells by the cells, leaving the cells with proper density, and finally adding 10mL of culture medium to put into an incubator for continuous culture.
Example 10: detection of autophagy Hela cells by probe QM-GFTN
Human cervical cancer cell lines (HeLa cells) were selected as cell models for testing. An autophagy inducer rapamycin (Rapa) was added to the Hela cells and incubated for 2h to construct an autophagy Hela cell model, and the untreated Hela cells were set as a control group. Untreated HeLa cells and autophagy-inducing HeLa cells were placed in a petri dish for adherent growth, followed by addition of a medium solution of probe (10. mu.M concentration) and incubation at 37 ℃ for 1h in an incubator containing humidified 5% C02. The medium was aspirated off, washed three times with PBS buffer, and then confocal imaging was performed. A confocal laser scanning microscope (CLSM, Leica TCS SP8, Germany) used for shooting selects 458nm laser as an excitation light source, and the fluorescence receiving range is 550-700 nm. As shown in FIG. 7, after the probe was incubated with the Hela cells inducing autophagy for 1h, a significant fluorescent signal was generated in the cells. Whereas no fluorescent signal was observed in both untreated Hela cells and unstained autophagy cells. The results prove that the probe can effectively detect the autophagic Hela cells, and the generated product QM-COOH has AIE characteristics and is expected to realize more stable and long-term fluorescence imaging.
Example 11: construction of rapamycin-induced autophagy mouse model
After 12 males were acclimatized for 7 days, they were randomly divided into a control group (4) and an autophagy group (8). All mice were placed in sterile cages in special laminar flow hood under 12h light/12 h dark cycle conditions and fed autoclaved food and water. Mice in the autophagy group were intraperitoneally injected with a Rapa solution (4.0 mg/kg) once a day for 14 days, while the control group was injected with an equal amount of physiological saline.
Example 12: imaging of staining of autophagic mouse tissue with Probe QM-GFTN
48h after the last dose, the mice in example 10 were sacrificed by cervical dislocation and frozen sections (3 tissue pieces per mouse) were prepared from the heart and liver tissues of the mice. The sections of mice in the autophagy group were divided into autophagy staining group and autophagy blank group, and the section of the tissues of mice in the control group was set as the control group. 10 μ M probe in PBS was incubated with sections from autophagy and control for 1h at 37 deg.C, stained and washed three times with PBS buffer for confocal imaging. As shown in fig. 8 and 9, the probe can effectively distinguish the mouse tissues of the autophagy staining group and the autophagy blank group by fluorescence, indicating that the probe is successfully applied to the detection of the autophagy isolated tissue.
Experimental results prove that the enzyme-activated AIE fluorescent probe QM-Peptide has no fluorescence in a water-tetrahydrofuran system all the time, and an enzyme digestion product QM-COOH emits obvious fluorescence in the same system. After the QM-Peptide and the autophagy key enzyme are incubated together, the fluorescence of the system is gradually enhanced along with time to realize the fluorescent response to the enzyme, so that the probe can be effectively activated by the autophagy key enzyme and realize the fluorescent lighting type response to the enzyme, and the background fluorescence can be effectively avoided. Enzyme digestion mechanism verification and selective experiments prove the correctness of the probe enzyme activation principle, and can effectively overcome the influence of interferents to realize high-selectivity fluorescent response to target enzymes. The enzyme-activated AIE fluorescent probe QM-Peptide is co-stained with a Hela cell, a PC12 cell and a SW1990 cell which induce autophagy respectively, and the QM-Peptide can be found by laser confocal imaging to effectively distinguish whether the autophagy occurs in the cells through fluorescence and avoid false response signals to realize high signal-to-noise ratio imaging. Meanwhile, tissue fluorescence imaging experiments also prove that QM-Peptide can stain and image autophagy detection of mouse tissues through simple experimental operation, so that QM-Peptide is expected to be applied to convenient and accurate autophagy detection of living cells and three-dimensional tissues, and a simple method is provided for medical diagnosis and biological research related to autophagy.
Claims (8)
1. An aggregation-induced emission fluorescent probe for enzyme-activated autophagy detection is characterized by consisting of a fluorescent parent molecule with aggregation-induced emission AIE characteristics and a polypeptide chain responding to an autophagy key enzyme Atg4b, and the chemical structural formula of the probe is shown as formula I:
in formula I, AIE Fluorophore is an AIE fluorescent molecule with carboxyl functional group, and Peptide is a polypeptide chain responding to the autophagy key enzyme Atg4 b.
2. The fluorescence probe for aggregation-induced emission in an enzyme-activated autophagy assay as claimed in claim 1, wherein said AIE fluorescent molecule with carboxyl functional group is an AIE derivative synthesized by modification of AIE fluorescent precursor, and includes AIE derivatives represented by formula II including quinolinecarbonitrile QM precursor, tricyano-methylene-pyridine TCM precursor, and tetraphenylethylene TPE precursor:
in the formula II, the reaction mixture is shown in the specification,
R1-R7 are respectively and independently selected from one or none of dimethylamino aniline, diethylamino aniline, hydroxyl, carboxyl, halogen, dimethylamino, trimethylamine, sulfonic acid group, N-dimethylaniline, triphenylamine, pyridine or carboxyl.
3. The enzyme-activated autophagy-detecting aggregation-induced emission fluorescent probe of claim 2, wherein said AIE fluorescent parent derivative comprises any one of:
4. the enzyme-activated autophagy assay aggregation-induced luminescent fluorescent probe according to claim 1, wherein Peptide is the polypeptide chain GFTN or GFTE or GYVS shown in formula III and responding to the autophagy key enzyme Atg4b, wherein the curve label is the substitution position:
5. the enzyme-activated autophagy assay aggregation-induced emission fluorescent probe of claim 1, wherein the enzyme-activated fluorescent probe comprises any one of:
6. a preparation method of an enzyme-activated aggregation-induced emission fluorescent probe is characterized in that fluorescent molecules with AIE characteristics are used as a matrix, condensation, esterification and hydrolysis reactions are sequentially carried out to obtain AIE-COOH modified by carboxyl functional groups, and finally the AIE-Peptide reacts with polypeptide amino acid chains to obtain the enzyme-activated aggregation-induced emission fluorescent probe AIE-Peptide.
7. The method for preparing the enzyme-activated aggregation-induced emission fluorescent probe according to claim 6, wherein QM and GFTN are used as the AIE fluorescent precursor and the responsive polypeptide amino acid chain, respectively, and the synthesis reaction is as follows:
8. an application of an aggregation-induced emission fluorescent probe for enzyme-activated autophagy detection is applied to autophagy imaging for detecting a plurality of living cells or isolated tissues through selective in-vitro fluorescent response to an autophagy key enzyme Atg4 b.
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