Construction and application of activated quinoline nitrile aggregation-induced emission luminogen probe

Abstract

The application discloses an activated quinoline nitrile aggregation-induced emission probe, and a structure is shown in the following formula I: The AIE probe provided by the application can realize supersensitive detection of acetylcholinesterase and ROS, has fast linear response, and simultaneously realizes detection of intracellular markers, and detection and distribution drawing of intracranial acetylcholinesterase of a senile mouse. The AIE probe is simple in preparation method, does not need harsh reaction conditions, and is beneficial to subsequent scale-up test or production preparation technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fine chemical industry, and particularly relates to a method for constructing a plurality of biomarker detection probes with pyridine-substituted quinoline nitrile as a mother nucleus and application thereof. BACKGROUND

[0002] Acetylcholinesterase, reactive oxygen species, alkaline phosphatase, beta-galactosidase, and esterase, as specific markers of neurodegenerative diseases (such as Alzheimer's disease and depression) (J. Am. Chem. Soc. 2019, 141, 2061-2068. Nat. Commun., 2022, 13, 998) and malignant tumors (such as pancreatic cancer, liver cancer, and ovarian cancer) (Angew. Chem. Int. Ed. 2020, 59, 2-12, Chem. Sci., 2019, 10, 398-405), are crucial for specific diagnosis and treatment of diseases.

[0003] Near-infrared (NIR) small molecule aggregation-induced emission (AIE) dyes are the preferred materials for developing activatable fluorescent probes (Angew. Chem. Int. Ed., 2020, 59, 9812-9825.). However, due to the poor water solubility of AIEgens, it still faces great challenges to achieve activated fluorescent response in water or biological systems (Adv. Mater., 2022, 34, 2107444.). The currently reported enzyme-responsive AIE probes are mainly based on improving the water solubility of the probes and achieving the "off-on" specific fluorescent response by adjusting their aggregation behavior (J. Am. Chem. Soc., 2019, 141, 3171-3177.). Specifically, it can be divided into targeted and enzyme-cleavage-responsive types, which mainly improve the initial solubility of the probes in water through water-soluble units, and then cause aggregation after targeted aggregation or enzyme cleavage, producing AIE signals and realizing the response to specific substances. This method is effective for in vitro detection in solution (Bioconjugate Chem., 2020, 31, 276-292.). However, the biological system is not pure water environment, and many biological interferents can easily cause the undesirable aggregation of the probes, producing false positive signals (ACS Nano, 2023, 17, 15, 14347-14405.). Therefore, it is very important to improve the water solubility of the probes and to jointly quench the initial fluorescence of the probes by manipulating the excitation energy flow for constructing AIE controllable activated fluorescent probes applied to specific biomarker detection. SUMMARY

[0004] In view of the above-mentioned deficiencies of the existing fluorescent probes, the present application aims to overcome the difficulties encountered in the application of the existing AIE fluorescent probes in biological environments, improve the biocompatibility and reduce the fluorescence quantum yield by regulating the aggregation state and excitation state energy, and realize the visual detection of related biomarkers.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0006] In the first aspect of the present application, an activated quinoline nitrile aggregation-induced emission probe is provided, which has the following structure shown in formula I:

[0007]

[0008] In formula I,

[0009] wherein R1 is selected from one of ;

[0010] R2 is selected from one of hydrogen, carbon chain, halogen or its isotope, hydroxyl, mercapto, amino, carboxyl, sulfonic acid group, phosphoric acid group, dimethylamino, trimethylamine group, pyridine or its derivative, ester group or its derivative, or polyethylene chain;

[0011] R3 is selected from one of triphenylamine, N,N-diphenyl-4-(thiophene-2-yl) aniline, N,N-dimethyl-4-(thiophene-2-yl) aniline, 1-ethyl-4-phenyl pyridinium, N,N-dimethyl aniline, 9-(thiophene-2-yl)-9H-carbazole, 2-(9H-fluorene-9-yl) thiophene;

[0012] R4 is selected from any one of the following formulae:

[0013]

[0014] wherein x is O or N.

[0015] Preferably, the structure of the probe is selected from any one of the following:

[0016]

[0017] In the second aspect of the present application, a preparation method of the above-mentioned activated quinoline nitrile aggregation-induced emission probe is provided, which comprises the following steps: taking pyridine-substituted quinoline nitrile as a raw material, connecting R3 through Knoevenage condensation reaction, and then connecting R4 group through substitution reaction, and the reaction route is as follows:

[0018]

[0019] The specific preparation steps are as follows:

[0020] A. Synthesis of dye AChE-QM

[0021]

[0022] In the reaction container, Py-QM and AChE-I are added in a molar ratio of 5:6, and an organic solvent (preferably acetonitrile) is added for dissolution, and the reaction is heated to reflux under the protection of an inert gas (such as nitrogen) for 10-14 hours (preferably 12 hours). After the reaction is completed, the solvent is rotary evaporated, and the product AChE-QM is separated by DCM:MeOH = 20:1 silica gel column chromatography;

[0023] B, product synthesis

[0024] In the reaction container, AChE-QM, an organic solvent, R4 and piperidine are sequentially added, wherein the molar ratio between AChE-QM and R4 is 3:4-4:5, the volume ratio between the organic solvent and piperidine is 30:1, and the molar concentration of AChE-QM is 0.02-0.03 mol / L.

[0025] The reaction is sufficiently stirred to reflux at 100℃ under the protection of an inert gas (such as nitrogen) for 8 hours. After the reaction is completed, the reaction solution is removed, and the product AChE-QM-TPE is separated by DCM:MeOH = 10-20:1 silica gel chromatography.

[0026] In a third aspect, the application provides a use of the activated quinoline nitrile aggregation-induced emission probe described above in the preparation of an enzyme or reactive oxygen detection reagent, wherein the enzyme is acetylcholinesterase, alkaline phosphatase, beta-galactosidase or esterase.

[0027] The detection step is as follows: a probe stock solution is prepared, the probe stock solution is diluted to a certain concentration, and the diluted probe is incubated, injected or detected with the to-be-detected substance, cells or living body.

[0028] Preferably, the detection concentration is 0.01-100 μM; the stock solution solvent is selected from any one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, toluene, dichloromethane, chloroform, acetone, acetonitrile, water and a buffer; and the buffer is selected from any one or more of 3-hydroxymethyl aminomethane solution, phosphate buffer, hydroxyethyl piperazine ethanesulfonic acid solution, acetate buffer, Tris buffer, carbonate buffer, citrate buffer and HEPES buffer.

[0029] In specific application aspects, the application can realize real-time linear response of acetylcholinesterase and reactive oxygen in vitro, can realize visualization imaging of acetylcholinesterase in neuron cells, can draw intracranial acetylcholinesterase distribution, and can realize visualization imaging of reactive oxygen species in pancreatic cancer.

[0030] Enzyme response test: add different equivalents (can be described as concentration, enzyme activity) of specific biomarkers in the above probe solution, test the ultraviolet absorption and fluorescence response at different time points.

[0031] Cell imaging: transfer the cells to a NEST confocal small dish with 1.0 mL of culture medium for 12 h. Add the required concentration of the probe, incubate at 37℃ for the corresponding time, then add PBS for washing, and remove the unincorporated probe. Then use Leica TCS SP8 laser microscope for confocal fluorescence imaging. The fluorescence signal of the probe cultured cells is collected at the excitation and emission wavelengths of the probe.

[0032] In vivo imaging: for different mouse models, adopt tail vein, abdominal cavity or in situ injection for administration, and collect in vivo fluorescence imaging at different time points.

[0033] Tissue section and complete tissue ex vivo imaging: after the above in vivo imaging is completed, the mouse is sacrificed, and different tissue organs are obtained through dissection. Tissue section imaging: the specific tissue or organ is sectioned (including freezing and paraffin), the corresponding section is obtained, and the section imaging method is the same as the cell imaging. Tissue ex vivo imaging: the tissue is transparentized, then according to the wavelength of the specific probe, the data is collected through light sheet microscopy imaging, and the data is processed through registration and three-dimensional reconstruction, so that the complete tissue visualization imaging is realized.

[0034] In a fourth aspect, the present application provides an enzyme or active oxygen detection reagent, comprising an activated quinoline nitrile aggregation-induced emission probe and a solvent. The enzyme is acetylcholinesterase, alkaline phosphatase, beta-galactosidase or esterase; the activated quinoline nitrile aggregation-induced emission probe and the solvent are as described above.

[0035] The beneficial technical effects of the present application are as follows:

[0036] In terms of effects, the AIE probe provided by the present application can realize the ultra-sensitive detection of acetylcholinesterase and ROS, has a rapid linear response; at the same time, the AIE probe also realizes the detection of intracellular markers, and the detection and distribution drawing of intracranial acetylcholinesterase of aging mice.

[0037] In terms of preparation technology, the preparation method of the AIE probe of the present application is simple, does not require harsh reaction conditions, and is conducive to subsequent scale-up tests or production. DETAILED DESCRIPTION

[0038] Figure 1 The nuclear magnetic hydrogen spectrum of the dye AChE-QM-1 is shown.

[0039] Figure 2 The nuclear magnetic hydrogen spectrum of the dye AChE-QM-2 is shown.

[0040] Figure 3 1H-1H COSY spectrum of dye AChE-QM-2 is shown.

[0041] Figure 4 UV absorption spectrum of dye AChE-QM-2 (10 -5 mol / L) in different volume fraction ratio mixed solvents of ethanol and water is shown.

[0042] Figure 5 Fluorescence emission spectrum of dye AChE-QM-2 (10 -5 mol / L) in different volume fraction ratio mixed solvents of ethanol and water is shown.

[0043] Figure 6 Variation of fluorescence intensity of dye AChE-QM-2 (10 -5 mol / L) in different volume fraction ratio mixed solvents of ethanol and water at 725 nm (10-5mol / L), I0is the fluorescence intensity of AChE-QM-2 at 725 nm in pure water is shown.

[0044] Figure 7 Spectrum of variation of fluorescence intensity of dye AChE-QM-2 (10 -5 mol / L) in PBS with time in response to AChE (20 U) is shown.

[0045] Figure 8 Variation curve of fluorescence intensity of dye AChE-QM-2 (10 -5 mol / L) at 725 nm with time in PBS with and without AChE (20 U) is shown.

[0046] Figure 9 Fluorescence spectrum of dye AChE-QM-2 (10 -5 mol / L) after incubation with different equivalent AChE for 30 min in PBS is shown.

[0047] Figure 10 Variation curve of fluorescence intensity of dye AChE-QM-2 (10 -5 mol / L) at 720 nm with AChE equivalent after incubation with different equivalent AChE for 30 min in PBS is shown.

[0048] Figure 11 Confocal microscope imaging diagram of PC12 cells after incubation with Py-QM-2 (10 -5 mol / L) and AChE-QM-2 (10 -5 mol / L) for 1 h (scale bar is 25 nm) is shown.

[0049] Figure 12 A schematic diagram showing the procedure of intracranial imaging of AChE-QM-2 in normal and aged mice.

[0050] Figure 13 AChE-QM-2 for intracranial acetylcholinesterase imaging in normal and aged mice.

[0051] Figure 14 A schematic diagram showing the procedure of intracranial imaging of AChE-QM-2 in normal and aged mice.

[0052] Figure 15 A schematic diagram showing the procedure of intracranial imaging of AChE-QM-2 in normal and aged mice. DETAILED DESCRIPTION

[0053] In order to make the present application clearer, further description will be made in conjunction with preferred embodiments. Those skilled in the art should understand that the following description is only intended to explain the present application and not intended to limit the scope of the present application.

[0054] Example 1

[0055] (1) Synthesis of dye AChE-QM:

[0056]

[0057] The synthesis of AChE-I has been reported more, which will not be described here. In a 25 mL round-bottom flask, Py-QM (300.0 mg, 1.0 mmol) and AChE-I (366.1 mg, 1.2 mmol) were added and dissolved in 8 mL of acetonitrile, and heated to reflux under nitrogen protection for 12 h. After the reaction was completed, the solvent was spin-dried, and the product was separated by silica gel column chromatography (DCM:MeOH=20:1) to obtain 363.5 mg of AChE-QM (yield 58.9%). 1H NMR (400 MHz, DMSO-d6, ppm), δ: 9.49 (m, 1H, J = 2.0 Hz, quinoline-H), 9.34 (d, 2H, J = 6.8 Hz, pyridine-H), 8.51 (m, 3H, pyridine-H, quinoline-H), 8.27 (d, 1H, quinoline-H), 7.63 (d, 2H, J = 8.6 Hz, Ph-H), 7.22 (d, 2H, J = 8.5 Hz, Ph-H), 6.91 (s, 1H, quinoline-H), 5.83 (s, 2H, N-CH2), 4.52 (q, 2H, J = 7.2 Hz, N-CH2-CH3), 3.03 (s, 3H, N-CH3), 2.90 (s, 3H, N-CH3), 2.71 (s, 3H, -CH3), 1.36 (t, 3H, J = 7.2 Hz, N-CH2-CH3). Mass spectrometry (ESI positive ion mode for [M] + ): Calcd. for C 30 H 28 N5O2: 490.2243, found: 490.2235.

[0058] (2) Synthesis of dye AChE-QM-1:

[0059]

[0060] In a 50 mL round-bottom flask, AChE-QM (200.3 mg, 0.32 mmol), acetonitrile (15 mL), 4-formyltriphenylamine (106.4 mg, 0.40 mmol) and piperidine (0.5 mL) were added successively under nitrogen protection, and heated to 100 °C with stirring for 8 h. The reaction solution was removed by distillation under reduced pressure, and separated by silica gel chromatography (DCM:MeOH = 20:1) to obtain the product AChE-QM-TPE (78.5 mg, 0.10 mmol) with a yield of 31.3%. 1H NMR (400 MHz, DMSO-d6, ppm), δ: 9.49 (m, 1H, Ar-H), 9.35 (d, 2H, J = 6.9 Hz, pyridine-H), 8.52 (m, 3H, pyridine-H, quinoline-H), 8.29 (d, 1H, J = 9.5 Hz, quinoline-H), 7.74 (d, 2H, J = 8.8 Hz, Ph-H), 7.64 (d, 2H, J = 8.6 Hz, Ph-H), 7.47 (d, 1H, J = 15.5 Hz, alkene-H), 7.38 (m, 5H, 4H for Ph-H, 1H for alkene-H), 7.23 (d, 2H, J = 8.6 Hz, Ph-H), 7.13 (m, 7H, Ar-H), 6.96 (d, 2H, J = 8.7 Hz, Ph-H), 5.83 (s, 2H, pyridine-N-CH2), 4.63 (d, 2H, J = 7.3 Hz, N-CH2-CH3), 3.34 (s, 3H, N-CH3), 3.03 (s, 3H, N-CH3), 1.41 (t, 3H, J = 6.9 Hz, N-CH2-CH3). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 153.70, 153.09, 152.10, 152.02, 149.93, 149.01, 146.32, 146.15, 145.05, 140.00, 139.63, 131.03, 130.05, 129.77, 129.00, 128.28, 125.08, 124.92, 124.25, 120.62, 54.91, 48.45, 48.37, 13.65. Mass spectrometry (ESI positive ion mode for [M]+): Calcd. for C49H41N6O2+: 745.3286, found: 745.3286. See Figure 1 .

[0061] (3) Synthesis of AChE-QM-2

[0062]

[0063] In a 50 mL round-bottom flask, AChE-QM (205.0 mg, 0.3 mmol), acetonitrile (15 mL), 5-triphenylamine-2-carboxaldehyde (141.5 mg, 0.4 mmol) and piperidine (0.5 mL) were added successively, and heated to 100 °C under nitrogen protection, and stirred for 8 h. After the reaction solution was distilled under reduced pressure, the product was obtained by silica gel chromatography (DCM:MeOH = 10:1), 93.4 mg, yield 32.6%. 1H NMR (400 MHz, DMSO-d6, ppm), δ: 9.48 (m, 1H, quinoline-H), 9.34 (d, 2H, J = 6.0 Hz, pyridine-H), 8.52 (m, 3H, pyridine-H, quinoline-H), 8.28 (d, 1H, J = 9.1 Hz, quinoline-H), 7.76 (d, 1H, J = 15.4 Hz, alkene-H), 7.63 (m, 5H, Ar-H), 7.5 (s, 1H, quinoline-H), 7.36 (t, 4H, J = 7.2 Hz, Ph-H), 7.22 (d, 2H, J = 7.7 Hz, Ph-H), 7.10 (m, 8H, Ar-H), 6.98 (d, 2H, J = 8.0 Hz, Ph-H), 5.80 (m, 2H, benzyl-H), 4.61 (d, 2H, J = 6.3 Hz, N-CH2-CH3), 3.03 (s, 3H, N-CH3), 2.89 (s, 3H, N-CH3), 1.42 (t, 3H, J = 6.4 Hz, N-CH2-CH3). Mass spectrometry (ESI positive ion mode for [M]+): Calcd. for C53H43N6O2S+: 827.3163, found: 827.3164.

[0064] AChE-QM-2 NMR hydrogen spectrum is shown in Figure 2 , 1H-1H COSY spectrum is shown in Figure 3 .

[0065] Absorption and fluorescence spectra of the dye in ethanol-water system

[0066] The prepared AChE-QM-2 probe was dissolved in analytical grade dimethyl sulfoxide to prepare 1.0 mM stock solutions. In EP tubes, 2970 μL of ethanol-water mixtures with different volume ratios (ethanol volume fractions of 0, 10, 20, 30, 40, 50, 60, 70, 80, and 99%) were added, followed by 30 μL of dye stock solution, resulting in a final test volume of 3.0 mL. The UV absorption and fluorescence spectra of the target probe were measured using a 10 × 10 mm optical quartz cuvette. The results are shown below. Figures 4-6 The fluorescence intensity of each test system was the highest at 725 nm. Flat analysis of each test system showed that the fluorescence emission intensity was the highest when the volume fraction of ethanol was 20%, and the fluorescence emission intensity was the lowest when the volume fraction of ethanol was 50%.

[0067] Example 3: In vitro fluorescence response of dye to acetylcholinesterase

[0068] First, prepare a concentration of 10. -5 mol·L -1 The AChE-QM-2 probe solution was prepared, and then different equivalents of acetylcholinesterase (0-20U) were added. After mixing thoroughly, the solution was incubated at 37°C for 30 minutes, and the fluorescence response was tested. Alternatively, a constant acetylcholinesterase concentration (20U) was maintained, and fluorescence changes were measured at different time intervals.

[0069] Figure 7 The dye AChE-QM-2(10) was shown. -5 The fluorescence intensity of the response to AChE(20U) in PBS (mol / L) as a function of time was measured. The results showed that the fluorescence intensity gradually increased with time in the first 30 min, but after 30 min, the fluorescence intensity hardly changed with time.

[0070] Figure 8 The dye AChE-QM-2(10) was shown. -5 The fluorescence intensity at 725 nm was measured as a function of time in PBS containing AChE (20 U) at a concentration of mol / L. The results showed that the fluorescence intensity increased rapidly within 5 min after the addition of AChE (20 U), and then the rate of increase gradually leveled off over time.

[0071] Figure 9 The dye AChE-QM-2(10) was shown. -5 Fluorescence spectra of (mol / L) AChE after incubation in PBS with different equivalents of AChE for 30 minutes; Figure 10 The dye AChE-QM-2(10) was shown. -5The fluorescence intensity at 720 nm after incubation with different equivalents of AChE in PBS for 30 minutes (mol / L) showed a concentration-dependent relationship, with the fluorescence intensity gradually increasing as the concentration of AChE increased.

[0072] Example 4: Fluorescence imaging of intracellular acetylcholinesterase using dyes

[0073] P12 cells were spaced at a density of approximately 1 × 10⁻⁶. 5 Cells were seeded into NEST confocal microplates and cultured for 12 hours. The desired concentration of AChE-QM-2 probe was added and incubated for the corresponding time. Cells were then washed three times with PBS to remove any untaken probe. Confocal fluorescence imaging was then performed using a Leica TCS SP8 confocal laser microscope (63× oil immersion). Cells were excited at 488 nm, and fluorescence signals from probe-cultured cells were acquired at 650-800 nm.

[0074] like Figure 11 As shown, the probe Py-QM-2 exhibits poor solubility and thus aggregates in the culture medium. Due to its large particle size, it cannot be taken up by cells, resulting in large extracellular particles and a strong AIE fluorescence signal. In contrast, the probe AChE-QM-2 has good solubility due to the ionization of pyridine. Furthermore, because the cell membrane surface carries a negative charge, AChE-QM-2 can more easily enter cells through electrostatic interactions. When the probe interacts with AChE after entering the cell, its solubility decreases, and it aggregates. At the same time, the fluorescence quantum yield is increased, enabling in-situ detection of endogenous AChE.

[0075] Example 5: Imaging of intracranial acetylcholinesterase in mice with dyes

[0076] A 3.3M AChE-QM-2 probe solution was prepared, and 6 μL of the probe was injected into the lateral ventricle of the brain. In vivo fluorescence imaging was performed at different time points, and the samples were then used to create images using an in vivo imaging system at different time points. Figure 12 After imaging, the mice were perfused, their brains were removed, transparentized, and finally imaged using light-sheet microscopy. 3D reconstruction was then used to visualize the distribution of intracranial acetylcholinesterase. Figure 13 ).

[0077] Example 6: Synthesis of ROS-QM-2 and Detection of Reactive Oxygen Species

[0078] ROS-QM-2 was synthesized according to the method in Example 1, and its 1H NMR spectrum is shown in [reference needed]. Figure 14 The principle of ROS-QM-2 for in vivo oxygen detection is explained in [link to relevant documentation]. Figure 15 A; Probe ROS-QM-2 solution (10 -5The fluorescence emission spectrum of the probe ROS-QM-2 (10 μmol / L) in PBS at 725 nm within 0-2 h is shown in Figure 15 B, which shows time dependence, and the fluorescence emission intensity gradually increases with time; the probe ROS-QM-2 solution (10 μmol / L) in PBS with the addition of AChE (20 U) at 725 nm within 0-2 h is shown in -5 The fluorescence emission spectrum of the probe ROS-QM-2 (10 μmol / L) in PBS at 725 nm within 0-2 h is shown in Figure 15 C, which also shows time dependence.

[0079] The pancreatic cancer cells were inoculated into the NEST confocal dish for culture, and the required concentration of the probe ROS-QM-2 was added for incubation for a corresponding time, and then PBS was added for washing three times to remove the unincorporated probe. Then, confocal fluorescence imaging was performed using a confocal laser microscope Leica TCS SP8 (63x oil lens), and the results are shown in Figure 15 D.

[0080] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above-mentioned technical content with slight changes or equivalent embodiments without departing from the technical solution of the present application. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not depart from the technical solution of the present application, still belongs to the scope of the present application.

Claims

1. An activated quinoline nitrile aggregation-induced emission probe, characterized in that, The structure is shown in the following formula I: In formula I, wherein R1is selected from R2 is selected from hydrogen; R3 is selected from one of triphenylamine, N,N-diphenyl-4-(thiophene-2-yl) aniline, N,N-dimethyl-4-(thiophene-2-yl) aniline, N,N-dimethyl aniline, 9-(thiophene-2-yl)-9H-carbazole, 2-(9H-fluorene-9-yl) thiophene; R4 is selected from any one of the following formulae: wherein x is O or N.

2. The activated quinolinic nitrile aggregation-induced emission probe according to claim 1, wherein, The structure of the probe is selected from any one of the following:

3. The method of preparing an activated quinolinic nitrile aggregation-induced emission probe according to claim 1 or 2, characterized in that, The method comprises the following steps: A. Synthesis of dye AChE-QM In a reaction vessel, Py-QM and AChE-I are added in a molar ratio of 5:6, dissolved with an organic solvent, heated to reflux under inert gas protection for 10-14 h, after the reaction is completed, the solvent is spin-dried, and the product AChE-QM is separated by DCM:MeOH=20:1 silica gel column chromatography; B. Product synthesis In a reaction vessel, AChE-QM, organic solvent, and piperidine are sequentially added, wherein the molar ratio between AChE-QM and is 3:4-4:5, the volume ratio between the organic solvent and piperidine is 30:1, and the molar concentration of AChE-QM is 0.02-0.03 mol / L; After the reaction is sufficiently stirred and refluxed under heating to 100℃, the reaction solution is removed, and the product AChE-QM-TPE is separated by DCM:MeOH=10-20:1 silica gel chromatography.

4. The preparation method of the activated quinoline nitrile aggregation-induced emission probe according to claim 3, characterized in that: wherein In steps A and B, the organic solvent is selected from acetonitrile, and the inert gas is selected from nitrogen; The heating reflux time in step A is 12 h, and the heating reflux time in step B is 8 h.

5. Use of the activated quinolinic nitrile aggregation-induced emission probe according to claim 1 or 2 for the preparation of an enzyme or reactive oxygen species detection reagent, characterized in that, The enzyme is acetylcholinesterase.

6. Use according to claim 5, characterized in that, The method comprises the following steps: Preparation of a probe stock solution, dilution of the probe stock solution to a certain concentration, incubation, injection, and detection of the probe with a to-be-detected substance, cells, or a living body.

7. Use according to claim 5, characterized in that, The detection concentration is 0.01-100 μM; the stock solution solvent is selected from any one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, toluene, dichloromethane, chloroform, acetone, acetonitrile, water, and a buffer solution, The buffer solution is selected from any one or more of 3-hydroxymethyl aminomethane solution, phosphate buffer solution, hydroxyethyl piperazine ethanesulfonic acid solution, acetate buffer solution, Tris buffer solution, carbonate buffer solution, citrate buffer solution, and HEPES buffer solution.

8. An enzyme or active oxygen detection reagent, characterized by, The method comprises the following steps: The enzyme is acetylcholinesterase. The activated quinoline nitrile aggregation-induced emission probe is as described in claim 1 or 2.

9. The enzyme or reactive oxygen species detection reagent according to claim 8, wherein The detection concentration of the probe is 0.01-100 μM; the solvent is selected from any one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, toluene, dichloromethane, chloroform, acetone, acetonitrile, water, and a buffer solution, The buffer solution is selected from any one or more of 3-hydroxymethyl aminomethane solution, phosphate buffer solution, hydroxyethyl piperazine ethanesulfonic acid solution, acetate buffer solution, Tris buffer solution, carbonate buffer solution, citrate buffer solution, and HEPES buffer solution.

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