Cysteine fluorescent probe capable of realizing persistent imaging as well as preparation method and application of cysteine fluorescent probe

Abstract

The invention discloses a cysteine fluorescent probe capable of lasting imaging and a preparation method and application thereof, the structure of the probe is one of the following formulas 1-2, and the probe has the functions of detecting the level of cysteine (Cys) in cells and organisms and lasting imaging. The cysteine fluorescent probe capable of lasting imaging is a novel Cys small-molecule fluorescent probe, and compared with the previously reported Cys small-molecule fluorescent probe, the cysteine fluorescent probe capable of lasting imaging has the advantages that the content of Cys in cells and living bodies can be monitored for a long time; the probe has a wide application prospect in early clinical diagnosis of diseases and long-term imaging of living bodies based on small molecular probes in the future. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a cysteine ​​fluorescent probe for persistent imaging, a preparation method and an application thereof. Background Art

[0002] Cysteine ​​(Cys), a type of biothiol, plays an important role in life processes, and changes in its concentration can cause a variety of diseases. Activatable near-infrared (NIR) cysteine ​​fluorescent probes have the advantages of high selectivity, strong sensitivity, and deep imaging depth, and have great application potential in early diagnosis and therapeutic intervention of diseases. However, the application of small molecule fluorescent probes is still greatly limited due to the rapid diffusion and metabolic clearance of activated fluorophores in the body. Considering that the time to observe the fluorescent signal after the application of the probe may vary, Cys probes that can maintain the fluorescent signal for a longer time are highly desired.

[0003] Currently, most reported Cys probes can image Cys in cells and organisms, but during persistent imaging and fixation, fluorescent hydrolysis products will leak out of the cells. Through literature research, although some fluorescent probes for persistent imaging of Cys have been developed, these fluorescent probes are all nanoprobes or solid-state fluorescent probes, and there are no reports on single-molecule fluorescent probes for persistent imaging of Cys.

[0004] Therefore, the rational design of fluorescent probes for persistent imaging based on single-molecule systems for the detection and persistent imaging of Cys levels in cells and organisms is of great significance for the early diagnosis and therapeutic intervention of diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a persistent imaging cysteine ​​fluorescent probe and its preparation method and application. The probe has the function of detecting and persistently imaging the Cys level in cells and organisms, and has great advantages in early diagnosis and therapeutic intervention of diseases.

[0006] The technical solution to achieve the purpose of the present invention is:

[0007] The present invention provides a cysteine ​​fluorescent probe for persistent imaging, the structure of which is one of the following formulas 1 and 2:

[0008]

[0009] The synthetic route of the cysteine ​​fluorescent probe for persistent imaging of the present invention is as follows:

[0010]

[0011] The method for preparing the cysteine ​​fluorescent probe for persistent imaging of the present invention comprises the following steps:

[0012] (1) Anthocyanidin, 2,4-dihydroxybenzyl alcohol and sodium hydride were weighed into a round-bottom flask, N,N-dimethylformamide was added to dissolve, and after dissolution, the mixture was placed in an oil bath to react for a period of time to obtain a crude product;

[0013] (2) The crude product obtained in step (1) is cooled, poured into a beaker, and distilled water and perchloric acid are added in sequence. After stirring to precipitate a solid, it is filtered by suction, dissolved with dichloromethane, and then concentrated with a rotary evaporator, eluted with an eluent, and purified with a silica gel chromatography column to obtain a fluorescent dye having a structure represented by formula (I);

[0014] (3) weighing the fluorescent dye having the structure represented by formula (I) obtained in step (2), acryloyl chloride and triethylamine into a round-bottom flask, adding dichloromethane to dissolve, placing in an oil bath to react for a period of time to obtain a crude product;

[0015] (4) pouring the crude product obtained in step (3) into a beaker filled with water, adding dichloromethane for extraction, concentrating it with a rotary evaporator, eluting it with an eluent, and purifying it with a silica gel chromatography column to obtain a fluorescent probe having a structure represented by formula (II);

[0016] (5) weighing the fluorescent probe having the structure of formula (II) obtained in step (4), adding dichloromethane to dissolve it, adding diethylaminosulfur trifluoride (DAST) under ice bath conditions, and reacting at room temperature for a period of time;

[0017] (6) After the reaction is completed, methanol is added to quench DAST, which is then concentrated using a rotary evaporator, eluted with an eluent, and purified using a silica gel chromatography column to obtain a persistent imaging cysteine ​​fluorescent probe having a structure shown in formula (III) (same as structural formula 1).

[0018] Furthermore, in the preparation method, the equivalent ratio of anthocyanin to 2,4-dihydroxybenzyl alcohol in step (1) is 1:2, and the equivalent ratio to sodium hydride is 1:1, and the amount of N,N-dimethylformamide used is preferably sufficient to dissolve the raw materials participating in the reaction;

[0019] The temperature of the oil bath pot is 45-65° C., and the oil bath reaction is performed for 4-6 hours.

[0020] Furthermore, in the preparation method, the amount of distilled water and perchloric acid added in step (2) is 50 mL: 3-5 mL, and the amount of dichloromethane used is preferably such that it can dissolve the raw materials participating in the reaction;

[0021] The crude product was eluted with dichloromethane and methanol in a volume ratio of 100:1 as an eluent and purified using a 200-300 mesh silica gel chromatography column.

[0022] Furthermore, in the preparation method, the molar ratio of the fluorescent dye, acryloyl chloride and triethylamine in step (3) is 1:2:1, and the amount of dichloromethane used is preferably such that it can dissolve the raw materials participating in the reaction;

[0023] The bath temperature is 45-65°C, and the oil bath reaction is performed for 4-6 hours.

[0024] Furthermore, in the preparation method, the amount of water and dichloromethane added in step (4) is 150 mL:50 mL;

[0025] The eluent is dichloromethane and methanol in a volume ratio of 100:1, and purification is performed using a 200-300 mesh silica gel chromatography column.

[0026] Furthermore, in the preparation method, the molar ratio of the fluorescent probe having the structure shown in formula (II) and DAST in step (5) is 1:2, and the amount of dichloromethane used is preferably such that it can dissolve the raw materials participating in the reaction;

[0027] The ice bath is at 0°C and the reaction is carried out at room temperature for 4-6 hours.

[0028] Furthermore, in the preparation method, in step (6), methanol is added to quench DAST, and the amount of methanol used is preferably sufficient to quench DAST;

[0029] The eluent is dichloromethane and methanol in a volume ratio of 100:1, and purification is performed using a 200-300 mesh silica gel chromatography column.

[0030] In addition, the present invention also applies the probe to aspects such as in vivo Cys persistent imaging, and is specifically used for detecting changes in Cys content in cells and organisms, so as to achieve the functions of detecting and persistently imaging Cys levels in cells and organisms.

[0031] The specific application method of the cysteine ​​fluorescent probe for persistent imaging of the present invention is:

[0032] (1) dissolving the fluorescent probe having the structure represented by formula (III) in dimethyl sulfoxide (DMSO) to prepare a probe mother solution;

[0033] (2) adding the probe stock solution to the test solution and the biological sample;

[0034] (3) adding a Cys mother solution (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μL) with a total concentration of 1 mmol, mixing evenly for five minutes, and observing the changes in the fluorescence spectrum of the test solution containing the probe Cys fluorescent probe using an ultraviolet and fluorescence spectrometer;

[0035] (4) respectively adding the prepared calcium chloride (CaCl2), copper bromide (CuBr2), glutathione (GSH), homocysteine ​​(Hcy), hydrogen peroxide (H2O2), tyrosine (Tyr), hydrogen sulfide (H2S), sodium bisulfite (NaHSO3), sodium nitrite (NaNO2), sodium sulfate (Na2SO4), allylthiourea (ATU) and cysteine ​​(Cys), wherein the final concentration of these analytes after dilution in the solution is 120 μM, the solution is mixed evenly for five minutes, and the changes in the fluorescence spectra of the probe containing different analytical interferents and Cys are observed by ultraviolet and fluorescence spectrometers;

[0036] (5) The probe was applied to intracellular fluorescence imaging. The first group was incubated with the probe (5.0 μM) for 10 minutes. The other three groups were first incubated with NEM (10.0 μM) for 30 minutes, and then Cys (100.0 μM), Hcy (100.0 μM) and GSH (100.0 μM) were added for 30 minutes before adding the probe (5.0 μM). The fifth group was first incubated with NEM (10.0 μM) for 30 minutes, and then the probe (5.0 μM) was added. The images were taken and analyzed using a laser confocal microscope with an excitation wavelength of 633 nm.

[0037] (6) The probe was applied to long-term fluorescence imaging of intracellular Cys. The probe (5.0 μM) was added and the changes in fluorescence intensity were monitored in a living cell workstation from 0 to 6 hours;

[0038] (7) The probe was applied to zebrafish in vivo fluorescence imaging. The first group was incubated with the probe Cys (5.0 μM) for 10 minutes. The other three groups were first incubated with NEM (10.0 μM) for 30 minutes, and then Cys (100.0 μM), Hcy (100.0 μM) and GSH (100.0 μM) were added for 30 minutes before adding the probe (5.0 mM). The fifth group was first incubated with NEM (10.0 μM) for 30 minutes, and then the probe (5.0 mM) was added. The images were taken and analyzed using a laser confocal microscope with an excitation wavelength of 633 nm to observe the changes in fluorescence intensity.

[0039] (8) The probe was applied to long-term fluorescence imaging of Cys in zebrafish. The probe (5.0 μM) was added and the changes in the fluorescence intensity of the probe in zebrafish were monitored from 0 to 6 hours.

[0040] The change of fluorescence spectrum refers to the change of fluorescence intensity at 725nm in the fluorescence spectrum. The fluorescence spectrum is observed by fluorescence spectrometer, and the excitation wavelength of the fluorescence spectrometer is 633nm. The obtained fluorescence imaging image means that the probe can perform specific imaging and persistent imaging of Cys in cells and zebrafish.

[0041] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0042] (1) The cysteine ​​fluorescent probe provided by the present invention is a new type of small molecule fluorescent probe, which has the function of detecting and persistently imaging the Cys level in cells and organisms, and has broad application prospects in the early diagnosis and therapeutic intervention of diseases;

[0043] (2) The fluorescent probe of the present invention has simple preparation steps, convenient purification, and high yield;

[0044] (3) The present invention realizes Cys persistent imaging in cells and zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is the Cy-Cys-F prepared in Example 1 1 H NMR spectrum;

[0046] Figure 2 is the Cy-Cys-F prepared in Example 1 13 C NMR spectrum;

[0047] Figure 3 is the mass spectrum of Cy-Cys-F prepared in Example 1;

[0048] Figure 4 is a diagram showing changes in the ultraviolet spectrum and fluorescence spectrum of Cy-Cys-F prepared in Example 1 in the titration of Cys;

[0049] Figure 5 is a fluorescence ratio diagram of the Cy-Cys-F prepared in Example 1 for different analytes in fluorescence test at 724 nm;

[0050] Figure 6 This is the fluorescence image of Cys-specific imaging in cells by Cy-Cys-F prepared in Example 1;

[0051] Figure 7 This is the long-term fluorescence imaging image of Cys in cells by Cy-Cys-F prepared in Example 1;

[0052] Figure 8 This is the fluorescence image of Cys-specific imaging in zebrafish by Cy-Cys-F prepared in Example 1;

[0053] Fig. 9 This is the long-term fluorescence imaging image of Cys in zebrafish by Cy-Cys-F prepared in Example 1. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with the embodiments and drawings, but the present invention is not limited by the following embodiments.

[0055] Example 1

[0056] Preparation of cysteine ​​fluorescent probe for persistent imaging: cyanine was selected to prepare the fluorescent probe, named Cy-Cys-F;

[0057] Preparation of Cy-Cys-F:

[0058] (1) Anthocyanin (100 mg, 0.1 mmol), 2,4-dihydroxybenzyl alcohol (90 mg, 0.2 mmol) and sodium hydride (25 mg, 0.1 mmol) were weighed into a round-bottom flask, and 10 mL of N,N-dimethylformamide was added to dissolve the mixture. After dissolution, the mixture was placed in an oil bath, and the temperature was set to 45°C at the beginning. The mixture was reacted for about half an hour, and then the temperature was raised to 55°C. The reaction was monitored by a plate. The reaction took 5 hours under the protection of nitrogen to obtain the crude product.

[0059] (2) The crude product obtained in step (1) was cooled, poured into a beaker, 50 mL of distilled water and 3-5 mL of perchloric acid were added in sequence, stirred and placed in a refrigerator for standing, a large amount of solid precipitated and then filtered, dissolved with 20 mL of dichloromethane, concentrated on a rotary evaporator, eluted with dichloromethane and methanol in a volume ratio of 100:1 as an eluent, and purified with a 200-300 mesh silica gel chromatography column to obtain a fluorescent dye having a structure represented by formula (I) (molecular formula: C 28 H 30 NO3 + );

[0060] (3) Weigh the fluorescent dye having the structure represented by formula (I) obtained in step (2) (22 mg, 0.04 mmol), acryloyl chloride (0.76 mg, 0.08 mmol) and triethylamine (4 mg, 0.04 mmol) into a round-bottom flask, add 50 mL of dichloromethane to dissolve, place in an oil bath, set the temperature to 45°C at the beginning, react for about half an hour, raise the temperature to 55°C, monitor with a plate, and react for 5 hours under the protection of nitrogen to obtain a crude product;

[0061] (4) Pour the crude product obtained in step (3) into a beaker containing 50 mL of water, add 150 mL of dichloromethane for extraction, and then concentrate using a rotary evaporator, elute using dichloromethane and methanol in a volume ratio of 100:1 as an eluent, and then purify using a 200-300 mesh silica gel chromatography column to obtain a fluorescent probe with a structure represented by formula (II);

[0062] (5) Weigh the fluorescent probe having the structure of formula (II) obtained in step (4) (15 mg, 0.025 mmol), add 15 mL of dichloromethane to dissolve it, quickly add DAST (8 mg, 0.05 mmol) in an ice bath at 0°C, and react at room temperature for 5 hours under nitrogen protection;

[0063] (6) After the reaction, 2 mL of methanol was added to quench DAST, and the DAST was concentrated by a rotary evaporator, eluted with dichloromethane and methanol in a volume ratio of 100:1, and purified by a 200-300 mesh silica gel column to obtain a persistent imaging cysteine ​​fluorescent probe having a structure represented by formula (III).

[0064] The structural formula of the probe Cy-Cys-F prepared in Example 1 is:

[0065]

[0066] Reference Figure 1 , 1 H NMR(500MHz,DMSO-d6)δ8.60(d,J=15.2Hz,1H),7.82–7.74(m,2H),7.59(d,J=8.1Hz,2H ),7.53(d,J=7.8Hz,2H),6.70(d,J=15.2Hz,1H),6.62(d,J=17.2Hz,1H),6.47(dd,J=17. 2,10.4Hz,1H),6.25(d,J=10.4Hz,1H),5.75(s,2H),5.67(s,1H),4.51(q,J=7.3Hz,2H), 2.75 (dt, J = 34.8, 6.1 Hz, 4H), 1.86 (p, J = 5.9 Hz, 2H), 1.76 (s, 6H), 1.40 (t, J = 7.1 Hz, 3H).

[0067] Reference Figure 2 , 13 C NMR (126MHz, DMSO-d6) δ178.83,164.24,158.66,153.41,151.90,145.95,143.20,141.33,135.03,134.24,131.01,129.96,129.51, 128.35,127.71,127.16,123.35,118.51,114.90,114.18,111.28,106.92,80.66,55.36,51.40,28.85,27.57,22.85,20.23,13.37.

[0068] Reference Figure 3 The theoretical molecular weight is 484.2282, and the actual high-resolution results show that the molecular weight is 484.2306.

[0069] Spectral experiment of the probe Cy-Cys-F prepared in Example 1 on the titration of Cys concentration:

[0070] The DMSO stock solution of the fluorescent probe prepared in Example 1 was prepared to a concentration of 1 mM; then 1970 μL of liquid (PBS:DMSO=9:1) and 30 μL of the probe stock solution were added to the UV and fluorescent dishes respectively, and Cys was added to the dishes until the UV and absorption of the compound no longer changed.

[0071] When the concentration of Cys added was (0-120 μm), the ultraviolet spectrum of Cy-Cys-F was red-shifted by 110 nm, and the fluorescence intensity at 725 nm was enhanced by 24 times, which indicated that the probe could be used as a fluorescent probe of Cys. Figure 4 shown.

[0072] Experimental test on the selectivity of the probe Cy-Cys-F prepared in Example 1 for Cys:

[0073] The fluorescent probe DMSO stock solution prepared in Example 1 was prepared to a concentration of 1 mM.

[0074] The probe was tested for its response to 16 intracellular analytes (amino acids, metal ions, reactive oxygen species (ROS) and reactive nitrogen species (RNS). A significant change in fluorescence intensity was observed in the mixture of the probe and Cys, with a fluorescence ratio of 10. In contrast, the fluorescence intensity of the solution hardly changed after the addition of other analytes, indicating that the probe has good selectivity for Cys and can specifically detect Cys under physiological conditions. The results are shown in Figure 2. Figure 5 shown.

[0075] Experimental study on endogenous and exogenous Cys cell fluorescence imaging of the probe Cy-Cys-F prepared in Example 1:

[0076] The DMSO stock solution of the fluorescent probe prepared in Example 1 was prepared at a concentration of 1 mM. After incubation with the probe (5 μM) for 15 minutes, fluorescence imaging was performed. N-ethylmaleimide (NEM, 10 μM) was added to the cells in advance and incubated for 30 minutes to eliminate a certain amount of biothiols in the cells as a control. The cells that were first added with NEM and then with the probe group had almost no fluorescence. After the cells were pretreated with NEM, Cys (100 μM), Hcy (100 μM) and GSH (100 μM) were added and incubated respectively, and then the probe was added. The confocal fluorescence imaging results showed that only the cell group treated with Cys showed obvious fluorescence enhancement, indicating that the probe can specifically recognize Cys in the cells. The results are as follows: Figure 6 shown.

[0077] Experimental test of long-term Cys imaging in cells using the probe Cy-Cys-F prepared in Example 1:

[0078] Digested HeLa (human cervical cancer) cell suspension was added to each well at 1×10 5180 μL per cell -1 The cells were inoculated at a density of 100 μg / cm2 in a 96-well culture plate and placed in a cell culture incubator for 24 hours. When the cell density was 40-50% under microscope observation, Cy-Cys-F (5 μM) was added to each well and the cells were incubated for different time periods (0-9 hours). Figure 7 As shown, the results show that the fluorescence intensity of the probe in the cells gradually increases with the extension of incubation time between half an hour and nine hours, indicating that the probe has the ability to image Cys in cells for a long time.

[0079] Experimental study on endogenous and exogenous Cys fluorescence imaging of zebrafish using the probe Cy-Cys-F prepared in Example 1:

[0080] The fluorescent probe DMSO stock solution prepared in Example 1 was prepared at a concentration of 1 mM. After the probe (5 μM) was incubated for 3 hours, fluorescence imaging was performed. N-ethylmaleimide (NEM, 200 μM) was selected and added to the zebrafish nutrient solution in advance and incubated for 30 minutes to eliminate a certain amount of biothiols in the zebrafish as a control. The zebrafish that were added with NEM first and then the probe group had almost no fluorescence. After the zebrafish were pretreated with NEM, Cys (200 μM), Hcy (200 μM) and GSH (200 μM) were added and incubated respectively, and then the probe was added. The confocal fluorescence imaging results showed that only the zebrafish group treated with Cys showed obvious fluorescence enhancement, indicating that the probe can specifically recognize Cys in zebrafish. The results are as follows: Figure 8 shown.

[0081] Experimental test of long-term Cys imaging of the probe Cy-Cys-F prepared in Example 1 in zebrafish:

[0082] The fluorescent probe DMSO stock solution prepared in Example 1 was prepared to a concentration of 1 mM. The probe (5 μM) was incubated for 3-24 hours and fluorescence imaging was performed. The confocal experiment results are shown in Fig. 9 As shown, the results showed that the fluorescence intensity of the probe in zebrafish gradually increased with the extension of time between 3 hours and 24 hours of incubation, indicating that the probe has the ability to image Cys for a long time in zebrafish.

[0083] Experiments have shown that the fluorescent probe prepared in Example 1 has the ability to specifically detect and persistently image Cys in cells and zebrafish, which is of great significance in the early diagnosis and treatment intervention of diseases.

[0084] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. A cysteine ​​fluorescent probe for persistent imaging, the structure of which is one of the following formulas 1 and 2:

2. A method for preparing a cysteine ​​fluorescent probe for persistent imaging, characterized in that: The synthetic route is as follows: The preparation method comprises the following steps: (1) Anthocyanidin, 2,4-dihydroxybenzyl alcohol and sodium hydride were weighed into a round-bottom flask, N,N-dimethylformamide was added to dissolve, and after dissolution, the mixture was placed in an oil bath to react for a period of time to obtain a crude product; (2) The crude product obtained in step (1) is cooled, poured into a beaker, and distilled water and perchloric acid are added in sequence. After stirring to precipitate a solid, it is filtered by suction, dissolved with dichloromethane, and then concentrated with a rotary evaporator, eluted with an eluent, and purified with a silica gel chromatography column to obtain a fluorescent dye having a structure represented by formula (I); (3) weighing the fluorescent dye having the structure represented by formula (I) obtained in step (2), acryloyl chloride and triethylamine into a round-bottom flask, adding dichloromethane to dissolve, placing in an oil bath to react for a period of time to obtain a crude product; (4) pouring the crude product obtained in step (3) into a beaker filled with water, adding dichloromethane for extraction, concentrating it with a rotary evaporator, eluting it with an eluent, and purifying it with a silica gel chromatography column to obtain a fluorescent probe having a structure represented by formula (II); (5) weighing the fluorescent probe having the structure represented by formula (II) obtained in step (4), adding dichloromethane to dissolve it, adding DAST under ice bath conditions, and reacting at room temperature for a period of time; (6) After the reaction is completed, methanol is added to quench the DAST, which is then concentrated using a rotary evaporator, eluted with an eluent, and purified using a silica gel chromatography column to obtain a persistent imaging cysteine ​​fluorescent probe having a structure represented by formula (III).

3. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (1), the equivalent ratio of anthocyanin to 2,4-dihydroxybenzyl alcohol is 1:2, and the equivalent ratio of anthocyanin to sodium hydride is 1:

1. The amount of N,N-dimethylformamide used is preferably sufficient to dissolve the raw materials participating in the reaction; The temperature of the oil bath pot is 45-65° C., and the oil bath reaction is performed for 4-6 hours.

4. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: The amount of distilled water and perchloric acid added in step (2) is 50 mL: 3-5 mL, and the amount of dichloromethane used is preferably sufficient to dissolve the raw materials participating in the reaction; The crude product was eluted with dichloromethane and methanol in a volume ratio of 100:1 as an eluent and purified using a 200-300 mesh silica gel chromatography column.

5. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (3), the molar ratio of the fluorescent dye, acryloyl chloride and triethylamine is 1:2:1, and the amount of dichloromethane used is preferably such that it can dissolve the raw materials participating in the reaction; The bath temperature is 45-65°C, and the oil bath reaction is performed for 4-6 hours.

6. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: The amount of water and dichloromethane added in step (4) is 150mL:50mL; The eluent is dichloromethane and methanol in a volume ratio of 100:1, and purification is performed using a 200-300 mesh silica gel chromatography column.

7. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (5), the molar ratio of the fluorescent probe having the structure shown in formula (II) to DAST is 1:2, and the amount of dichloromethane used is preferably such that it can dissolve the raw materials participating in the reaction; The ice bath is at 0°C and the reaction is carried out at room temperature for 4-6 hours.

8. The method for preparing a cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (6), methanol is added to quench DAST, and the amount of methanol used is preferably sufficient to quench DAST; The eluent is dichloromethane and methanol in a volume ratio of 100:1, and purification is performed using a 200-300 mesh silica gel chromatography column.

9. The use of the cysteine ​​fluorescent probe for persistent imaging according to claim 1, characterized in that: The probe is used to detect changes in Cys content in cells and organisms, so as to realize the functions of detecting and persistent imaging of Cys levels in cells and organisms.

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