A two-photon fluorescent probe selectively recognizing cysteine and its preparation and application

Abstract

This invention discloses a two-photon fluorescent probe that selectively recognizes cysteine, its preparation, and its application. The molecular formula of the two-photon fluorescent probe is C2. 23 H 19 The N3O2 probe, with a structure as shown in formula (I), features low background, good selectivity, and a rapid reaction rate with cysteine. After interaction with cysteine, the fluorescence intensity at 580 nm gradually increases from weak to strong, thus classifying it as a fluorescence-enhanced probe. This fluorescent probe exhibits a good linear response to cysteine ​​within a certain concentration range, enabling qualitative and quantitative detection of cysteine. Furthermore, this probe can detect cysteine ​​content in water, plants, and animals (such as zebrafish), showing promising applications in plant genetics and molecular biology. Additionally, this probe can be used for protein staining in SDS-PAGE, offering a faster, simpler, and more sensitive method compared to traditional staining techniques.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis and detection technology, specifically relating to a fluorescence-enhanced molecular probe that can rapidly identify and detect cysteine, and the application of this probe in animal imaging detection, plant imaging detection, and protein staining in SDS-PAGE (SDS polyacrylamide gel electrophoresis). Background Technology

[0002] Cysteine ​​(Cys) is a small-molecule biothiol that participates in various physiological processes and plays a crucial role in plant growth. Cysteine ​​is an important precursor for the synthesis of sulfur metabolites and also possesses significant antioxidant activity, protecting plants from free radicals and other harmful compounds. However, due to its strong reducing properties, excessive concentrations of Cys can easily cause oxidative damage in plants, leading to cytotoxicity.

[0003] Currently, methods for detecting cysteine ​​include high-performance liquid chromatography (HPLC), gas chromatography (GC), electrochemical methods, ultraviolet spectrophotometry, and fluorescent probe analysis. Among these, fluorescent probe analysis has attracted widespread attention due to its advantages such as simple operation, high sensitivity, low detection limit, fast response, strong anti-interference ability, and the ability to directly detect cysteine ​​in living cells, tissues, or organisms. Furthermore, fluorescent probes for detecting cysteine ​​need to be selective to eliminate interference from other thiols. Therefore, designing and developing fluorescent probes that selectively respond to cysteine ​​and can detect it in organisms has significant practical implications. Summary of the Invention

[0004] In view of this, the present invention aims to provide a fluorescence-enhanced probe that can rapidly and selectively respond to cysteine, providing a new tool for the specific detection of cysteine.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of this invention provides a two-photon fluorescent probe that selectively recognizes cysteine, the molecular formula of which is C2. 23 H 19 N3O2, and its structural formula is shown in formula (I):

[0006] (I).

[0007] The two-photon fluorescent probe of this invention reacts with cysteine ​​to generate a product with the molecular formula C. 26 H 26 The product of N4O4S exhibits a maximum emission wavelength of 580 nm and displays yellow fluorescence. The structural formula of this product is shown below:

[0008] (II).

[0009] A second aspect of the present invention provides a method for preparing the two-photon fluorescent probe of the present invention, comprising the following steps: S1. Dissolve isoflurone (1,1,3-trimethylcyclohexenone) and malononitrile in an organic solvent, add piperidine and acetic acid, and react to generate intermediate a; S2. Dissolve intermediate a in an organic solvent, add 4-acetaminobenzaldehyde, and react to generate intermediate b. S3. Dissolve intermediate b in an organic solvent, add concentrated hydrochloric acid, and react to obtain intermediate c. S4. Dissolve intermediate c and maleic anhydride in an organic solvent, and after the reaction, a two-photon fluorescent probe is obtained. The structural formulas of intermediates a, b, and c are shown below: .

[0010] Preferably, in the method of the present invention, step S1 specifically involves dissolving 1 molar equivalent of isoflurane and 1-5 molar equivalents of malononitrile in anhydrous ethanol, adding piperidine and acetic acid, reacting at high temperature for 20-30 hours, and separating and purifying to obtain intermediate a.

[0011] More preferably, the separation and purification operation in step S1 is as follows: after the reaction is completed, the solvent is removed by evaporation, and the obtained product is purified by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent.

[0012] Preferably, in the method of the present invention, step S2 specifically involves: dissolving 1 molar equivalent of intermediate a and 1-3 molar equivalents of 4-acetaminobenzaldehyde in acetonitrile, adding piperidine, reacting at high temperature, and then separating and purifying the reaction solution to obtain intermediate b.

[0013] More preferably, the separation and purification operation in step S2 is as follows: after the reaction is completed, the reaction solution is purified by vacuum distillation to obtain intermediate b, or the reaction solution is cooled to precipitate solid, filtered and washed with anhydrous ethanol to obtain intermediate b, wherein intermediate b is an orange solid.

[0014] Preferably, in the method of the present invention, step S3 specifically involves: reacting intermediate b in a mixed solution of concentrated hydrochloric acid and ethanol at high temperature for 10-20 hours, and after the reaction is completed, separating and purifying the reaction solution to obtain intermediate c.

[0015] More preferably, the separation and purification operation in step S2 is as follows: after the reaction is complete, the pH of the reaction solution is adjusted to neutral or weakly alkaline, extracted with ethyl acetate and dried, and the crude product is purified by silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain intermediate c; the intermediate c is a reddish-black solid.

[0016] Preferably, in the method of the present invention, step S4 specifically involves: adding 1 molar equivalent of intermediate c and 1-3 molar equivalents of maleic anhydride to acetic acid and reacting for 5-10 hours; after the reaction is completed, evaporating the solvent; then adding 1-3 molar equivalents of sodium acetate and reacting at high temperature in acetic anhydride for several hours; and finally obtaining a two-photon fluorescent probe by separation and purification.

[0017] A third aspect of this invention provides the application of the two-photon fluorescent probe described herein in the qualitative and / or quantitative detection of cysteine. Experimental data from this invention show that the two-photon fluorescent probe of this invention has low background, high selectivity for cysteine, and a fast response speed and high intensity. Moreover, within a certain concentration range, the fluorescence intensity of the reaction system containing the fluorescent probe and cysteine ​​at 580 nm increases with increasing cysteine ​​concentration. This demonstrates that the two-photon fluorescent probe described herein can achieve qualitative and quantitative detection of cysteine.

[0018] In view of the fact that the two-photon fluorescent probe described in this invention can be used to detect cysteine ​​in plant and animal samples, a fourth aspect of this invention provides the application of the two-photon fluorescent probe in visual qualitative detection and / or imaging detection of plants and animals. The plants include, but are not limited to, Arabidopsis thaliana, and the animals include, but are not limited to, zebrafish.

[0019] The fifth aspect of this invention provides the application of the two-photon fluorescent probe of this invention in protein staining of SDS-PAGE. That is, the two-photon fluorescent probe of this invention can be used as a staining agent for proteins in SDS-PAGE. Compared with the traditional Coomassie Brilliant Blue staining agent, the two-photon fluorescent probe has the characteristics of simple operation, short staining time, no need for destaining, and high sensitivity.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a novel fluorescent probe for the specific detection of cysteine. The probe has a novel structure, is simple to synthesize, has a fast response speed, and has a large Stokes shift (180 nm), resulting in good selectivity for cysteine. The fluorescent probe provided by the present invention can not only detect cysteine ​​in aquatic environments and biological samples (including plants and animals), but can also be used as a novel protein fluorescent staining agent in SDS-PAGE. Attached Figure Description

[0021] Figure 1This is a schematic diagram illustrating the recognition mechanism of cysteine ​​by the two-photon fluorescent probe of this invention.

[0022] Figure 2 This is a synthesis route diagram for the two-photon fluorescent probe of the present invention.

[0023] Figure 3 The two-photon fluorescent probe prepared in Example 1 of this invention 1 H NMR spectrum.

[0024] Figure 4 The two-photon fluorescent probe prepared in Example 1 of this invention 13 C10 NMR spectrum.

[0025] Figure 5 The fluorescence spectra of the two-photon fluorescent probe before and after reacting with cysteine ​​and other biothiols in Example 2 of this invention are shown.

[0026] Figure 6 The image shows the fluorescence spectra of the two-photon fluorescent probe reacting with different concentrations of cysteine ​​in Example 3 of this invention.

[0027] Figure 7 The images show the fluorescence spectra of the two-photon fluorescent probe reacting with cysteine ​​at different times in Example 4 of this invention.

[0028] Figure 8 This is a bar graph showing the fluorescence intensity at 580 nm after the two-photon fluorescent probe in Example 5 of this invention reacts with cations, amino acids, anions, and different thiol compounds.

[0029] Figure 9 (a) is a schematic diagram of the two-photon fluorescent probe after protein staining in SDS-PAGE in Example 6 of the present invention, and (b) is a schematic diagram of staining with Coomassie Brilliant Blue.

[0030] Figure 10 This is a two-photon fluorescence imaging of a two-photon fluorescent probe in a plant, as shown in Example 7 of the present invention.

[0031] Figure 11 This is a fluorescence imaging of a two-photon fluorescent probe in a live zebrafish in Example 8 of the present invention. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples and cannot be used to limit the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art; the term "comprising" and any variations thereof in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0034] This invention provides a two-photon fluorescent probe that selectively recognizes cysteine, with the molecular formula C. 23 H 19 N3O2, the structural formula is shown in formula (I). Figure 1 As shown, the working principle of the two-photon fluorescent probe and cysteine ​​in this invention is as follows: the two-photon fluorescent probe (I) contains an electron-withdrawing maleimide recognition group, which puts the two-photon fluorescent probe in a fluorescence-quenched state; when the two-photon fluorescent probe detects cysteine, cysteine ​​first nucleophilically attacks the recognition group and undergoes a Michael addition reaction to obtain a weakly fluorescent intermediate, which is then rapidly converted into a strongly fluorescent compound (II).

[0035] Compared with previously reported maleimide-containing cysteine ​​fluorescent probes, the two-photon fluorescent probe of the present invention has the following advantages: ① It has two-photon properties, thus the imaging has the characteristics of high resolution, high throughput (high speed), non-invasiveness, and large imaging depth; ② The response to cysteine ​​is selective and is not affected by other biological thiols; ③ It has application prospects in animal imaging, plant imaging, and protein staining (see Table 1).

[0036] Table 1

[0037] The two-photon fluorescent probe provided by this invention can be used not only to detect cysteine ​​in aquatic environments and biological samples, but also to stain proteins in SDS-PAGE. Compared with traditional Cos Brilliant Blue G250 staining, the two-photon fluorescent probe of this invention has the following advantages: simple operation, short staining time, no need for destaining, and high sensitivity (see Table 2).

[0038] Table 2

[0039] Unless otherwise specified, the techniques or conditions described in the following examples are based on those described in the literature or the product instructions. Unless otherwise specified, the reagents or instruments used are all commercially available products.

[0040] Example 1 The technical route for synthesizing the two-photon fluorescent probe in this example is as follows: Figure 1 As shown, the specific steps include: (1) Synthesis of intermediate a.

[0041] To an anhydrous ethanol solution (10 mL) of isophorone (599.9 g, 4.3405 mmol) and malononitrile (717.5 mg, 10.8613 mmol), 4 drops of piperidine and 4 drops of glacial acetic acid were added sequentially. The reaction mixture was refluxed overnight at 80 °C. After the reaction was complete, the solvent was evaporated, and the crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (10 / 1, v / v) as eluent to give intermediate a. Intermediate a was a white solid with a yield of 651.1 mg, representing a yield of 80.6%. 1 H NMR (400 MHz, DMSO- d 6) 6.55 (s,1H), 2.53 (s,2H), 2.23 (s,2H), 2.04 (s,3H), 0.95 (s,6H).

[0042] (2) Synthesis of intermediate b.

[0043] Four drops of piperidine were added to an acetonitrile solution (8 mL) of intermediate a (306.4 mg, 1.6450 mmol), and the mixture was stirred at room temperature for 10 min. Then, 4-acetamidobenzaldehyde (277.7 mg, 1.7018 mmol) was added. The mixture was refluxed at 80 °C for 8 h. After the reaction was complete, the solid precipitated upon cooling. The solid was filtered and the filter cake was washed with anhydrous ethanol to obtain intermediate b. The yield of intermediate b was 396.5 mg, with a yield of 72.7%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.08 (s, 1H), 7.64-7.55 (m,4H), 7.28-7.17 (m, 2H), 6.80 (s, 1H), 2.57 (s, 2H), 2.47 (p, J = 1.9 Hz, 2H), 2.03 (s, 3H), 0.98 (s, 6H).

[0044] (3) Synthesis of intermediate c.

[0045] 20 mL of concentrated hydrochloric acid was added to 20 mL of anhydrous ethanol solution of intermediate b (505.9 mg, 1.5265 mmol). The reaction mixture was refluxed overnight at 80 °C. The reaction mixture was neutralized by adding 15% sodium hydroxide aqueous solution until neutral, and the reaction mixture was extracted with ethyl acetate. The organic layer was then dried over Na₂SO₄, filtered, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as eluent to give intermediate c. The yield of intermediate c was 335.1 mg, with a yield of 75.9%.1 H NMR (400 MHz, CDCl3) δ 7.39-7.33(m, 2H), 7.01 (d, J = 16.0 Hz, 1H), 6.86-6.75 (m, 2H), 6.72-6.66 (m, 2H), 4.05(s, 2H), 2.59 (s, 2H), 2.46 (s, 2H), 1.08 (s, 6H).

[0046] (4) Synthesis of fluorescent probe d.

[0047] Intermediate c (512.6 mg, 1.7714 mmol) and maleic anhydride (298.6 mg, 3.0450 mmol) were dissolved in 20 mL of acetic acid solution and stirred at room temperature for 2 h. The solvent was then evaporated under vacuum. The remaining solid was dissolved in 20 mL of acetic anhydride, and then sodium acetate (220.7 mg, 2.6902 mmol) was added to the solution. The mixture was stirred at 80 °C for 2 h, and then the solvent was evaporated under vacuum. The crude product was purified by column chromatography (PE:EA = 2:1, v / v) to obtain fluorescent probe d, with a yield of 524.0 mg, representing a yield of 80.1%.

[0048] fluorescent probe d 1 H NMR spectrum and 13 The C NMR spectra are as follows: Figure 3 and Figure 4 As shown, its structure is as shown in equation (I), where, 1 H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5Hz, 2H), 7.10-6.94 (m, 2H), 6.87 (d, J = 7.1 Hz, 3H), 2.61 (s, 2H), 2.48 (s, 2H), 1.09 (s, 6H).

[0049] Example 2 This example demonstrates the effectiveness of the two-photon fluorescent probe obtained in Example 1 in reacting with different biothiols. The measurement process is as follows: A 10 mM solution of compound d was prepared using acetone. 10 mM solutions of cysteine, glutathione, and homocysteine ​​were prepared using deionized water. 1 μL of the above compound d solution and 999 μL of PBS (pH=7.4) buffer were placed in a cuvette, and the fluorescence spectrum under 400 nm excitation was detected. Similarly, 1 μL of the above compound d solution, 989 μL of PBS (pH=7.4) buffer, and 10 μL of cysteine, glutathione, or homocysteine ​​solution were placed in a cuvette, and the fluorescence spectrum under 400 nm excitation was detected.

[0050] Test results as follows Figure 5 As shown, from Figure 5 It can be seen that this fluorescent probe has a low background and a strong response only to cysteine, making it a fluorescently enhanced probe selective for cysteine.

[0051] Example 3 This example measured the reaction of the two-photon fluorescent probe obtained in Example 1 with different concentrations of cysteine. The specific process is as follows: Add 1 μL of a solution of compound d (same as in Example 2) and PBS (pH=7.4) buffer to a cuvette, and then add cysteine ​​at working concentrations of 0, 2, 4, 6, 8, 10, 20, 40, 60, 80, 90, and 100 μM to prepare a 1 mL reaction system. Measure the fluorescence spectra of different concentrations of cysteine ​​reacting with the fluorescent probe for 20 min.

[0052] Test results as follows Figure 6 As shown, from Figure 6 It can be seen that within a certain concentration range, the fluorescence intensity of the reaction system at 580 nm increases with the increase of cysteine ​​concentration, indicating that the probe can detect cysteine ​​at different concentrations.

[0053] Example 4 This example measures the change in the reaction intensity of the two-photon fluorescent probe obtained in Example 1 with cysteine ​​over time. The detection process is as follows: Add 1 μL of compound d solution, 989 μL of PBS (pH=7.4) buffer, and 10 μL of cysteine ​​solution to a cuvette, ensuring that the working concentration of the fluorescent probe is 10 μM and the working concentration of cysteine ​​is 100 μM. Detect the fluorescence spectrum of this reaction system under 400 nm excitation every few minutes.

[0054] The results are as follows Figure 7 As shown, from Figure 7It can be seen that within 10 minutes, the fluorescence intensity of the reaction system at 580 nm is directly proportional to the reaction time of cysteine, indicating that the probe can quickly detect cysteine ​​and has a fast response speed.

[0055] Example 5 This example demonstrates the selectivity of the two-photon fluorescent probe obtained in Example 1 for detecting cysteine. The specific procedure is as follows: Add 1 μL of a solution of compound d to a cuvette (same as in Example 2), then add 100 μM of different analytes, such as Fe. 3+ Zn 2+ , K + Na + Mg 2+ Ca 2+ NO3 - HCO3 - NO2 - SO4 2- The fluorescence intensity of the reaction system at 580 nm was measured after adding different analytes, including H2O2, Cly, Ala, Arg, Glu, Try, His, Leu, Hcy, GSH, and Cys.

[0056] The results are as follows Figure 8 As shown, from Figure 8 It can be seen that the fluorescent probe is selective for cysteine, and the selectivity of this probe is high.

[0057] Example 6 This example demonstrates the staining effect of the two-photon fluorescent probe obtained in Example 1 on proteins in SDS-PAGE. The specific procedure is as follows: Dithiothreitol was added to HPPD samples containing fluorescent probes, and the proteins were denatured at high temperature. During this process, the three-dimensional structure of the proteins was destroyed, the disulfide bonds were broken, and the fluorescent probes covalently bound to the thiol groups to achieve the staining purpose. Sodium dodecyl sulfate was then added to make the proteins negatively charged. Finally, these samples were analyzed by SDS-PAGE.

[0058] from Figure 9 As can be seen from this, using fluorescent probes to stain proteins ( Figure 9 a) and the use of Coomassie Brilliant Blue ( Figure 9 The experimental results in b) are consistent, but the fluorescent probe staining is faster and easier to observe, indicating that the method of staining proteins in SDSD-PAGE with fluorescent probes has practical significance.

[0059] Example 7 This example demonstrates the imaging detection of cysteine ​​in plants using the two-photon fluorescent probe obtained in Example 1. The specific procedure is as follows: Arabidopsis thaliana was placed in centrifuge tubes, and experimental and control groups were set up. In the control group, a solution of compound d (same as in Example 2) was added to achieve a working concentration of 10 μM for the fluorescent probe, and fluorescence imaging of the green channel was performed directly using a two-photon laser scanning microscope. In the experimental group, N-ethylmaleimide was first added to achieve a working concentration of 500 μM, and after incubation for 30 min, the mixture was washed. Then, Arabidopsis thaliana was incubated in different concentrations of cysteine ​​(0, 25, 50 μM) for 30 min, followed by washing. Finally, Arabidopsis thaliana was incubated in the fluorescent probe solution for 30 min to achieve a working concentration of 10 μM for the fluorescent probe.

[0060] Green channel fluorescence imaging was performed on Arabidopsis root tips incubated with different concentrations of cysteine. The results are as follows: Figure 10 As shown. From Figure 10 The results show that the higher the probe concentration, the greater the fluorescence intensity at the root tip, indicating that the probe can detect cysteine ​​in plants.

[0061] Example 8 This example demonstrates the imaging detection of cysteine ​​in live zebrafish using the two-photon fluorescent probe obtained in Example 1. The specific process is as follows: Zebrafish larvae aged 0-7 days were cultured in 6-well plates and divided into three groups of 5 larvae per group. NEM was first added to a working concentration of 500 μM, and after incubation for 30 min, the larvae were washed. Then, different concentrations of cysteine ​​(0, 25, 50 μM) were added to each group of zebrafish culture medium to achieve a working concentration of 10 μM for the fluorescent probe. After incubation for 30 min, bright-field imaging and yellow-channel fluorescence imaging were performed on the zebrafish larvae incubated with different concentrations of cysteine. The results are shown below. Figure 11 As shown.

[0062] from Figure 11 As can be seen, the higher the concentration of cysteine ​​during incubation, the stronger the fluorescence intensity in zebrafish, indicating that the probe can detect cysteine ​​in zebrafish and perform fluorescence imaging detection.

[0063] Comparative Example 1 Reference Figure 1 In this example, compounds g and j, which have structures similar to the two-photon fluorescent probe obtained in Example 1, were synthesized.

[0064] Compound g can be prepared by the following steps: (1.1) Synthesize intermediate a according to step (1) of Example 1.

[0065] (1.2) Add 1 molar equivalent of intermediate a and 1-3 molar equivalents of 3-nitrobenzaldehyde to acetonitrile and stir to dissolve. Then add 2-4 drops of piperidine and react for several hours. After separation and purification, intermediate e is obtained.

[0066] (1.3) Dissolve 1 molar equivalent of intermediate e and 2-5 molar equivalents of tin chloride dihydrate in ethyl acetate solution, then add a few drops of concentrated hydrochloric acid and react at high temperature for several hours. After the reaction is complete, separate and purify the reaction solution to obtain intermediate f.

[0067] (1.4) Add 1 molar equivalent of intermediate f and 1-3 molar equivalents of maleic anhydride to acetic acid and react for 5-10 hours. After the reaction is complete, evaporate the solvent. Then add 1-3 molar equivalents of sodium acetate and react at high temperature in acetic anhydride for several hours. After separation and purification, obtain fluorescent probe g.

[0068] Compound j can be prepared by the following steps: (2.1) Synthesize intermediate a according to step (1) of Example 1.

[0069] (2.2) Add 1 molar equivalent of intermediate a and 1-3 molar equivalents of 2-nitrobenzaldehyde to acetonitrile and stir to dissolve. Then add 2-4 drops of piperidine and react for several hours. After separation and purification, intermediate h is obtained.

[0070] (2.3) Dissolve 1 molar equivalent of intermediate h and 2-5 molar equivalents of tin chloride dihydrate in ethyl acetate solution, then add a few drops of concentrated hydrochloric acid and react at high temperature for several hours. After the reaction is complete, separate and purify the reaction solution to obtain intermediate i.

[0071] (2.4) Add 1 molar equivalent of intermediate i and 1-3 molar equivalents of maleic anhydride to acetic acid and react for 5-10 hours. After the reaction is complete, evaporate the solvent. Then add 1-3 molar equivalents of sodium acetate and react at high temperature in acetic anhydride for several hours. After separation and purification, obtain compound j.

[0072] Fluorescence experiments showed that compounds g and j only underwent Michael addition reactions with Cys, resulting in products with only weak fluorescence, which could not achieve selective detection of cysteine.

[0073] In summary, the two-photon fluorescent probe provided by this invention has the advantages of good selectivity and strong anti-interference ability. It can not only detect cysteine ​​in aquatic environments, plants and animals, but also be used as a novel protein fluorescent staining agent in SDS-PAGE.

[0074] It should be noted that the above embodiments are only some embodiments of the present invention and not all embodiments, and are only used to illustrate the technical solutions of the present invention and not to limit it; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A two-photon fluorescent probe that selectively recognizes cysteine, characterized in that, The molecular formula of the two-photon fluorescent probe is C 23 H 19 N3O2, and its structural formula is shown below: 。 2. A method for preparing the two-photon fluorescent probe as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve isoflurone and malononitrile in an organic solvent, add piperidine and acetic acid, and react to generate intermediate a; S2. Dissolve intermediate a in an organic solvent, add 4-acetaminobenzaldehyde, and react to generate intermediate b. S3. Dissolve intermediate b in an organic solvent, add concentrated hydrochloric acid, and react to obtain intermediate c. S4. Dissolve intermediate c and maleic anhydride in an organic solvent, and after the reaction, a two-photon fluorescent probe is obtained. The structural formulas of intermediates a, b, and c are shown below: 。 3. The method according to claim 2, characterized in that, Step S1 is as follows: Dissolve 1 molar equivalent of isoflurane and 1-5 molar equivalents of malononitrile in anhydrous ethanol, add piperidine and acetic acid, react at 60-100℃ for 20-30 hours, and separate and purify to obtain intermediate a.

4. The method according to claim 2, characterized in that, Step S2 specifically involves dissolving 1 molar equivalent of intermediate a and 1-3 molar equivalents of 4-acetaminobenzaldehyde in acetonitrile, adding piperidine, reacting at 60-100°C, and then separating and purifying the reaction solution to obtain intermediate b.

5. The method according to claim 2, characterized in that, Step S3 specifically involves reacting intermediate b in a mixed solution of concentrated hydrochloric acid and ethanol at 60-100°C for 10-20 hours. After the reaction is complete, the reaction solution is separated and purified to obtain intermediate c.

6. The method according to claim 2, characterized in that, Step S4 is as follows: 1 molar equivalent of intermediate c and 1-3 molar equivalents of maleic anhydride are added to acetic acid and reacted for 1-5 hours. After the reaction is completed, the solvent is evaporated. Then, 1-3 molar equivalents of sodium acetate are added and reacted in acetic anhydride at 60-100℃ for several hours. The two-photon fluorescent probe is obtained by separation and purification.

7. The application of the two-photon fluorescent probe as described in claim 1, characterized in that, At least one of the following: a) Application in the preparation of reagents for qualitative and / or quantitative detection of cysteine; b) Application in the preparation of animal imaging detection reagents or plant imaging detection reagents; c) Application in the preparation of protein staining reagents for SDS-PAGE.

8. The application according to claim 7, characterized in that, The two-photon fluorescent probe, in response to cysteine, generates a product with the molecular formula C. 26 H 26 The product of N4O4S, which has a maximum emission wavelength at 580 nm and exhibits yellow fluorescence, has the following structural formula: 。

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