A water-soluble environmentally sensitive fluorescent probe and its preparation method and application

Abstract

The present invention relates to a water-soluble environmentally sensitive fluorescent probe, which is obtained by connecting a fluorophore to a benzoxadiazole ring through nucleophilic substitution of L-glutamic acid. The fluorescent probe of the present invention retains the characteristics of environmental sensitivity, and the introduced carboxyl group makes it water-soluble and convenient for subsequent functional modification. After the fluorescent probe of the present invention is combined with a protein through hydrophobic and electrostatic interactions, rapid and sensitive detection of the protein can be achieved by measuring the change in fluorescence intensity.

Description

Technical Field

[0001] The invention relates to a water-soluble environment-sensitive fluorescent probe for protein analysis and a preparation method and application thereof, belonging to the technical field of protein analysis. Background Art

[0002] Protein is the basic substance of human life activities. It is closely related to the structure of cells, the transmission of genetic material and biological evolution. The quantitative analysis of protein has always been an important topic in the research fields of chemistry, biochemistry, medicine, etc., and has great significance for disease diagnosis, food and drug analysis and clinical analysis.

[0003] At present, the analytical techniques used for quantitative determination of proteins are relatively mature, including spectrophotometry, resonance Rayleigh scattering, immunoassay, high performance liquid chromatography, capillary electrophoresis analysis, etc. Among them, fluorescence analysis is favored in the detection of biomacromolecules due to its advantages of high sensitivity, high selectivity, and wide dynamic response range. Fluorescence analysis is to achieve the purpose of detecting proteins by affecting the fluorescence signal of the system through the adsorption or covalent binding of fluorescent probes to proteins. However, most fluorescent probes contain aromatic or heterocyclic structures, have poor water solubility, and low fluorescence response, and cannot achieve the purpose of detecting proteins in aqueous solutions. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art, to propose a water-soluble environmentally sensitive fluorescent probe that can be used for a wide range of protein analysis, and to provide a preparation method and application thereof.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] A water-soluble environmentally sensitive fluorescent probe for protein analysis, the general chemical structure of which is shown below:

[0007]

[0008] The present invention provides an organic small molecule fluorescent probe, which uses a benzoxadiazole ring as a basic skeleton, and L-glutamic acid (L-Glu) is connected to a fluorophore through nucleophilic substitution to obtain a water-soluble environmentally sensitive fluorescent probe DBD-L-Glu. The fluorescent probe DBD-L-Glu not only retains the characteristics of environmental sensitivity, but also has the characteristics of water solubility. After combining with proteins through hydrophobic and electrostatic interactions, rapid and sensitive detection of proteins can be achieved by measuring changes in fluorescence intensity. Therefore, the fluorescent probe of the present invention can be applied to labeling macromolecular proteins, quantitative analysis of proteins, and exploration of the hydrophobic index of protein surfaces, and the synthesis process is simple and easy to implement, and easy to promote.

[0009] The fluorescent probe DBD-L-Glu of the present invention not only retains the characteristics of environmental sensitivity, but also exhibits very weak fluorescence in aqueous solution and strong fluorescence in organic solution. At the same time, the dye has hydrophilic characteristics. Most of the ligand binding sites in proteins are hydrophobic. The combination of the probe and the hydrophobic ligand binding domain of the target protein makes the environmentally sensitive fluorophore closer to the hydrophobic pocket of the protein, so that the fluorophore emits stronger light. On the contrary, in the absence of the target protein, the fluorescent probe will be retained in the aqueous buffer and show weak fluorescence. Different proteins have different isoelectric points, and there will be electrostatic adsorption or repulsion on the surface of the fluorescent dye, so that the dye is in close contact with or away from the dye. Based on the hydrophobic effect and the electrostatic effect, the rapid and sensitive detection of the protein can be achieved by measuring the change in fluorescence intensity.

[0010] The present invention provides a method for preparing a water-soluble environmentally sensitive fluorescent probe, comprising the following steps:

[0011] Step 1, dissolving 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in acetonitrile to obtain solution A;

[0012] Step 2, adding tetraethylammonium iodide aqueous solution to solution A, stirring thoroughly at room temperature to obtain solution B;

[0013] Step 3, dissolving L-glutamic acid and sodium carbonate in water to obtain solution C;

[0014] Step 4, mixing solution B and solution C, and stirring them fully at room temperature to obtain solution D;

[0015] Step 5: Solution D is extracted, acidified, extracted again, dried, and filtered to obtain a solid, namely, the target probe compound.

[0016] The present invention combines small molecule amino acids without changing the probe's parent fluorescent group, effectively improving the luminescent properties and chemical properties of the fluorescent probe, making it water-soluble, and broadening the application of the fluorescent probe in the detection of macromolecular proteins.

[0017] The technical solution further refined by the present invention is as follows:

[0018] The method for preparing the water-soluble environmentally sensitive fluorescent probe of the present invention specifically comprises the following steps:

[0019] Step 1, dissolve 22 mg of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) in 1.2 mL of acetonitrile, and vortex to completely dissolve to obtain a mixed solution A;

[0020] Step 2, dissolve 69 mg of tetraethylammonium iodide (TEAI) in 200 μL of deionized water, vortex to completely dissolve it, add mixed solution A thereto, stir well at room temperature for 3 h, and obtain a yellow mixed solution B;

[0021] Step 3, dissolve 80 mg of L-glutamic acid (L-Glu) and 86 mg of sodium carbonate in 2.2 mL of water, and vortex to completely dissolve them to obtain a mixed solution C;

[0022] Step 4, mixing the mixed solution B and the mixed solution C, and stirring them at room temperature for 12 hours to obtain a dark yellow solution D;

[0023] Step 5. Add ethyl acetate to solution D, extract twice, take the water layer, and acidify with 50% hydrochloric acid; after acidification, extract twice with ethyl acetate, take the ethyl acetate layer, dry with anhydrous magnesium sulfate, filter, use methanol as solvent, purify by thin layer chromatography, distill, and finally obtain an orange-yellow solid, i.e., the target probe compound.

[0024] The present invention also provides a water-soluble environment-sensitive fluorescent probe for use as a fluorescent indicator in protein analysis. The fluorescent probe has universal applicability to protein detection.

[0025] In the above application, the fluorescent probe has good water solubility and environmental sensitivity, and has different fluorescence response values ​​in buffer solutions or / solvents with different pH values.

[0026] The above solvents include, but are not limited to, water, ether, n-octane, formic acid, dimethyl sulfoxide, methanol, ethanol, tetrahydrofuran, and N'N-dimethylformamide.

[0027] The above-mentioned buffer solutions include strongly acidic solutions, strongly alkaline solutions, weakly acidic solutions and weakly alkaline solutions.

[0028] In the above application, a PBS buffer solution with a pH of 7.4 is used as a protein detection solvent, and the detection conditions during the detection and evaluation are: an excitation wavelength of 430 nm, and fluorescence emission spectrum detection is performed between 480-660 nm.

[0029] The fluorescent probe of the present invention retains the characteristics of environmental sensitivity, and the introduced carboxyl group makes it water-soluble and convenient for subsequent functional modification. After the fluorescent probe of the present invention is combined with the protein through hydrophobic interaction and electrostatic interaction, it can achieve rapid and sensitive detection of the protein by measuring the change of fluorescence intensity.

[0030] The fluorescent probe of the present invention has the characteristics of environmental sensitivity, and its specific characteristics include:

[0031] (1) The fluorescent probe of the present invention exhibits different ultraviolet absorption and fluorescence intensities in different solvents such as water, ether, n-octane, formic acid, dimethyl sulfoxide, methanol, ethanol, tetrahydrofuran, and N'N-dimethylformamide.

[0032] (2) The fluorescence intensity of the fluorescent probe of the present invention is relatively high in a strongly acidic buffer solution, the fluorescence intensity has little difference in weakly acidic to weakly alkaline buffer solutions, and the fluorescence intensity is relatively weak in a strongly alkaline buffer solution.

[0033] (3) The fluorescent probe of the present invention is not affected by temperature changes.

[0034] (4) The fluorescent probe of the present invention has almost no fluorescence intensity in the aqueous solution when there is no protein. When there is protein, electrostatic and hydrophobic interactions exist between the protein and the fluorescent probe, which increases the fluorescence intensity.

[0035] (5) When the fluorescent probe of the present invention is used for protein analysis and evaluation, the specific determination method is: adding the sample to be tested into a PBS buffer solution with a pH of 7.4 containing the fluorescent probe, and determining the fluorescence intensity of the solution as an evaluation index of the protein concentration.

[0036] (6) The novel fluorescent probe for protein analysis can be applied to the analysis and evaluation of various proteins. The evaluation conditions are: PBS buffer solution with pH = 7.4 as solvent, excitation wavelength of 430 nm, and fluorescence emission spectrum detection between 480-660 nm.

[0037] The fluorescent probe of the present invention has environmental sensitivity and water solubility, and has good fluorescence emission spectrum characteristics under the condition of 480-660nm; the fluorescent probe used for detecting proteins can be obtained through a chemical synthesis process, and the synthesis process is simple and easy to promote. In short, the fluorescent probe of the present invention can be universally applied to the detection of macromolecular proteins, especially the detection of non-enzymatic proteins, and realizes the quantitative and qualitative analysis and labeling of multiple proteins in aqueous solution for the first time, which improves the application prospects of benzothiazole dyes in life analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be further described below in conjunction with the accompanying drawings.

[0039] Figure 1 is a synthetic route map of the fluorescent probe of the present invention;

[0040] Figure 2 is the infrared spectrum of the fluorescent probe of the present invention;

[0041] Figure 3 is the mass spectrum of the fluorescent probe of the present invention;

[0042] Figure 4The figures are the ultraviolet spectra and fluorescence spectra of the fluorescent probe of the present invention in several organic solvents; A in the figure is the ultraviolet spectra of the fluorescent probe in several organic solvents, and B is the fluorescence spectra of the fluorescent probe in several organic solvents;

[0043] Figure 5 is a fluorescence intensity diagram of the fluorescent probe of the present invention in buffer solutions of different pH values;

[0044] Figure 6 It is the fluorescence spectrum of the fluorescent probe of the present invention under heating and non-heating conditions;

[0045] Figure 7 It is a fluorescence spectrum of the fluorescent probe of the present invention combined with bovine serum albumin (BSA), bovine immunoglobulin (bIgG), and lysozyme; in the figure, A is a fluorescence spectrum of the fluorescent probe combined with bovine serum albumin, B is a fluorescence spectrum of the fluorescent probe combined with bovine immunoglobulin, and C is a fluorescence spectrum of the fluorescent probe combined with lysozyme;

[0046] Figure 8 It is a standard curve diagram of the fluorescence spectrum of the conjugate in tetrahydrofuran after the fluorescent probe of the present invention is combined with BSA and the uncombined fluorescent probe is removed;

[0047] Fig. 9 The present invention is a graph of the use of the fluorescent dye of the present invention for exploring the surface hydrophobic index of proteins at different pH values, wherein A is a graph for exploring the surface hydrophobic index of bovine serum albumin at pH=3.2, B is a graph for exploring the surface hydrophobic index of bovine serum albumin at pH=5.3, C is a graph for exploring the surface hydrophobic index of bovine serum albumin at pH=7.4, D is a graph for exploring the surface hydrophobic index of bovine serum albumin at pH=9.2, E is a graph for exploring the surface hydrophobic index of bovine serum albumin at pH=11, and F refers to a graph of the surface hydrophobic index of bovine serum albumin under different pH conditions. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. Unless otherwise specified, the "solution" involved in the present invention refers to an aqueous solution formed by dissolving various substances in deionized water.

[0049] Example 1 Preparation of fluorescent probe for protein detection

[0050] (1) Take a 5 mL brown reaction bottle, dissolve 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F; 22 mg; CAS: 98358-90-8) in 1.2 mL of acetonitrile, and vortex to completely dissolve to obtain solution A;

[0051] (2) Tetraethylammonium iodide (TEAI; 69 mg; CAS: 68-05-3) was dissolved in 200 μL of deionized water, vortexed to completely dissolve, and then solution A was added thereto. The mixture was stirred at room temperature for 3 h to obtain a yellow mixed solution B.

[0052] (3) L-glutamic acid (L-Glu; 80 mg; CAS: 56-86-0) and sodium carbonate (Na2CO3; 86 mg; CAS: 497-19-8) were dissolved in 2.2 mL of water and vortexed to completely dissolve to obtain a mixed solution C;

[0053] (4) Mixing solution B and solution C, stirring thoroughly at room temperature for 12 h, and obtaining a dark yellow solution D;

[0054] (5) Ethyl acetate was added to the mixed solution D, and the mixture was extracted twice. The aqueous layer was taken and acidified with 50% hydrochloric acid. After acidification, the mixture was extracted twice with ethyl acetate. The ethyl acetate layer was taken and dried with anhydrous magnesium sulfate, filtered, and purified by thin layer chromatography using methanol as a solvent. The mixture was distilled to obtain an orange-yellow solid, i.e., the target probe compound DBD-L-Glu. The specific synthetic route is shown in Figure 1 The general chemical structure of the probe is shown below:

[0055]

[0056] Example 2 Infrared spectroscopy characterization of fluorescent probes for protein detection

[0057] KBr (potassium bromide) was selected as a solid dispersion medium, which was fully ground to obtain powdered KBr for later use. The fluorescent probe powder prepared in Example 1 was ground and dispersed in powdered KBr, and then pressed into transparent sheets using a tablet press and characterized by infrared spectra. The most obvious change of the fluorescent probe synthesized in Example 1 is the formation of secondary amine groups, such as Figure 2 As shown, at 3328cm -1 There is a single peak nearby, indicating the presence of secondary amine groups.

[0058] Example 3 Mass Spectrometry Characterization of Fluorescent Probes

[0059] The fluorescent probe was characterized by mass spectrometry using the negative ion source mode of HRMS. Since the theoretical value of the amount of substance of the fluorescent probe is 372.07392, in the negative ion source mode, after subtracting the theoretical measured value of hydrogen ions, it is 371.0669, and the precise molecular weight of the fluorescent probe detected is 371.0694, which is equivalent to the theoretical value. Its mass spectrum is shown in Figure 3 .

[0060] Example 4 UV spectra and fluorescence spectra of fluorescent probes in different solvents

[0061] The fluorescent probe was dissolved in water, ether, n-octane, formic acid, dimethyl sulfoxide, methanol, ethanol, tetrahydrofuran, N'N-dimethylformamide and other solutions respectively, so that the final concentration of the fluorescent probe was 167.1 μM, and then fluorescence measurement was performed. The results are shown in Table 1.

[0062] Table 1 Fluorescence response in different solvents

[0063]

[0064] Figure 4 A in the figure indicates that DBD-FL-Glu has different intensities of ultraviolet absorption in different solvents, and the corresponding wavelengths are different. Figure 4 B indicates that DBD-FL-Glu exhibits different fluorescence intensities in different solvents. In tetrahydrofuran solution, DBD-FL-Glu has the strongest emission. In aqueous solution, the ultraviolet absorption peak position of DBD-FL-Glu is around 430nm, the excitation wavelength of DBD-FL-Glu is around 430.5nm, and its emission wavelength is about 596nm.

[0065] Example 5 Fluorescence spectra of fluorescent probes in buffer solutions of different pH values

[0066] Prepare buffer solutions of different pH values ​​in advance (including citric acid / sodium citrate solution of pH=3.2, citric acid / sodium citrate solution of pH=5.3, PBS buffer solution of pH=7.4, Tris:HCl solution of pH=9.2, NaHCO3 / Na2CO3 solution of pH=11), and dissolve the fluorescent probes in the buffer solutions of different pH values, respectively. The concentration of the fixed fluorescent probes is 50 μM. Figure 5 As shown, under strong acid conditions, the fluorescence intensity of the fluorescent probe increases. Under weak acid to alkaline conditions, the pH condition has little effect on the fluorescence intensity of the fluorescent probe. Under strong alkaline conditions, the fluorescence intensity of the fluorescent probe is weak. The linear relationship between the pH of the buffer solution and the fluorescence response value of the probe is y = -6.05638x + 120.79585, where y refers to the fluorescence response value and x refers to the pH value. Therefore, the subsequent application of the fluorescent probe is suitable for PBS buffer solution with a pH of 7.4.

[0067] Example 6 Fluorescence spectrum of fluorescent probe before and after heating

[0068] The fluorescent probe was dissolved in a PBS buffer solution with a pH of 7.4, and the concentration of the fluorescent probe was fixed at 50 μM. The sample was then placed in a 25°C room temperature and an 80°C water bath for 15 min, and the fluorescence intensity was measured. Figure 6 As shown, temperature has no effect on the fluorescent probe.

[0069] Example 7 Fluorescence spectra of fluorescent probes combined with bovine serum albumin, bovine immunoglobulin, and lysozyme

[0070] In combination with the environmental sensitivity and hydrophilicity of the fluorescent probe itself, a PBS buffer solution with pH = 7.4 was prepared in advance to analyze bovine serum albumin, bovine immunoglobulin, and lysozyme. The concentration of bovine serum albumin was fixed at 2.82μM, and the concentration of DBD-L-Glu was changed from 2μM to 240μM; the concentration of bovine immunoglobulin was fixed at 6.67μM, and the concentration of DBD-L-Glu was changed from 2μM to 300μM; the concentration of lysozyme was fixed at 357μM, and the concentration of DBD-L-Glu was changed from 4μM to 290μM, and the fluorescence intensity of different concentrations of DBD-L-Glu binding to bovine serum albumin, bovine immunoglobulin, and lysozyme was measured. The results are shown in Figure 7 , Figure 7 A in the figure shows the curve of the fluorescence intensity at the emission wavelength of 570 nm changing with the concentration of the fluorescent probe when DBD-L-Glu binds to bovine serum albumin. Figure 7 B in the figure shows the curve of the fluorescence intensity at the emission wavelength of 590 nm changing with the concentration of the fluorescent probe when DBD-L-Glu binds to bovine immunoglobulin. Figure 7 C in the figure shows the curve of the fluorescence intensity at 585nm as a function of the concentration of the fluorescent probe when DBD-L-Glu binds to lysozyme. Figure 7 It can be seen that with the increase of DBD-L-Glu concentration, the fluorescence value gradually increases. After the DBD-L-Glu concentration reaches a certain value, the fluorescence intensity remains stable. When binding to bovine serum albumin, DBD-L-Glu reaches saturation at 140μM; when binding to bovine immunoglobulin, DBD-L-Glu reaches saturation at 210μM; when binding to lysozyme, DBD-L-Glu reaches saturation at 195μM. Continuing to increase the DBD-L-Glu concentration, the aggregation of the fluorescent probe will cause the fluorescence intensity to decay. The property of enhancing fluorescence after the fluorescent probe binds to the protein can be used to quantitatively analyze the protein.

[0071] Example 8: Fluorescent probe for detecting protein for quantitative detection of bovine serum albumin

[0072] The concentration of the fixed fluorescent probe is 50 μM. In a PBS buffer solution at pH = 7.4, bovine serum albumin is added. The final concentration of bovine serum albumin is 0 nM, 0.5 nM, 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM. Prepare 14 30Kd ultrafiltration tubes, ultrafilter at 14000G for 8 minutes, and wash twice with a PBS buffer solution at pH = 7.4. Finally, the conjugate of the fluorescent probe and bovine serum albumin is redispersed in a tetrahydrofuran solution, and the fluorescence intensity is measured to obtain a standard curve for bovine serum albumin. The results are as follows: Figure 8 As shown, the detection limit for BSA is 50 nM.

[0073] In combination with Example 7 and Example 8, the fluorescent probe of the present invention has the following characteristics:

[0074] (1) When the chemical and physical environments are stable, the fluorescence signal of the emitted light is proportional to the concentration of macromolecular proteins. Therefore, DBD-L-Glu can be used for the quantitative analysis of macromolecular proteins such as lysozyme and bovine serum albumin.

[0075] (2) The hydrophobic cavity of large molecular proteins may encapsulate fluorescent dyes and show stronger fluorescence signals. Therefore, DBD-L-Glu can be used to label proteins in aqueous solutions.

[0076] Example 9: Fluorescent probe for protein detection for investigation of protein surface hydrophobicity index (So) at different pH values

[0077] The presence of two carboxyl groups in DBD-L-Glu determines that the fluorescent probe is an anionic fluorescent probe. When using this probe to explore the hydrophobic index of the protein surface, the effect of pH should be considered first to determine the appropriate pH system. In buffer solutions of different pH values ​​(pH = 3.2, 5.3, 7.4, 9.2, 11.0), the concentration of DBD-L-Glu was fixed at 160 μM, and the concentration of bovine serum albumin in the solution was changed. In a buffer solution of pH=3.2, the concentration of BSA changes from 1μM to 5μM; in a buffer solution of pH=5.3, the concentration of BSA changes from 0.5μM to 11.5μM; in a buffer solution of pH=7.4, the concentration of BSA changes from 0.5μM to 10.5μM; in a buffer solution of pH=9.2, the concentration of BSA changes from 0.5μM to 15μM; in a buffer solution of pH=11, the concentration of BSA changes from 0.5μM to 73μM. Then the fluorescence intensity of BSA at different concentrations under different pH conditions was measured under excitation at 430nm. The results are as follows: Fig. 9As shown in the figure, as the concentration of bovine serum albumin increases, the fluorescence intensity increases. The emission light intensity at 570nm is plotted against the concentration of bovine serum albumin to obtain a curve of the fluorescence intensity changing with the concentration of bovine serum albumin under different pH conditions. The slope of each curve is the So of bovine serum albumin under different pH conditions. Fig. 9 As shown in F, at pH = 3.2, the So of bovine serum albumin = 104; at pH = 5.3, the So of bovine serum albumin = 57; at pH = 7.4, the So of bovine serum albumin = 63; at pH = 9.2, the So of bovine serum albumin = 36; and at pH = 11, the So of bovine serum albumin = 1.

[0078] The experimental results show that pH is a key parameter for determining the surface hydrophobic index of proteins using DBD-L-Glu, which has a great influence on the conformation of proteins. The isoelectric point of bovine serum albumin is 4.7. When the protein is in a buffer solution with a pH of 3.2, the surface of bovine serum albumin is positively charged. In addition to the hydrophobic binding between DBD-L-Glu and bovine serum albumin, the electrostatic interaction is enhanced, so the fluorescence intensity is large and So is large; when the protein is in a buffer solution with a pH of 5 to 9, the change of So is not obvious; when the protein is in a buffer solution with a pH of 11, the negative charge on the surface of BSA increases, and an electrostatic repulsion is generated with DBD-L-Glu, and So drops sharply. Therefore, the structure of BSA is relatively stable under neutral conditions.

[0079] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A water-soluble environmentally sensitive fluorescent probe, characterized in that: The chemical structure formula of the fluorescent probe is as follows: 。 2. A method for preparing a water-soluble environmentally sensitive fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: Step 1, dissolving 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in acetonitrile to obtain solution A; Step 2, adding tetraethylammonium iodide aqueous solution to solution A, stirring thoroughly at room temperature to obtain solution B; Step 3, dissolving L-glutamic acid and sodium carbonate in water to obtain solution C; Step 4, mixing solution B and solution C, and stirring them fully at room temperature to obtain solution D; Step 5: Solution D is extracted, acidified, extracted again, dried, and filtered to obtain a solid, namely, the target probe compound.

3. The method for preparing a water-soluble environmentally sensitive fluorescent probe according to claim 2, characterized in that: The specific steps include: Step 1, dissolve 22 mg of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in 1.2 mL of acetonitrile and vortex to completely dissolve to obtain a mixed solution A; Step 2: Dissolve 69 mg of tetraethylammonium iodide in 200 µL of deionized water, vortex to completely dissolve it, add mixed solution A, and stir well at room temperature for 3 h to obtain a yellow mixed solution B; Step 3, dissolve 80 mg of L-glutamic acid and 86 mg of sodium carbonate in 2.2 mL of water, and vortex to completely dissolve them to obtain a mixed solution C; Step 4, mixing the mixed solution B and the mixed solution C, and stirring them at room temperature for 12 h to obtain a dark yellow solution D; Step 5, add ethyl acetate to solution D, extract twice, take the water layer, and acidify with 50% hydrochloric acid; after acidification, extract twice with ethyl acetate, take the ethyl acetate layer, dry with anhydrous magnesium sulfate, filter, use methanol as solvent, purify by thin layer chromatography, distill, and finally obtain an orange-yellow solid, i.e., the target probe compound.

4. Use of the water-soluble environmentally sensitive fluorescent probe as claimed in claim 1 as a fluorescent indicator in protein analysis.

5. The use according to claim 4, characterized in that: The fluorescent probe has different fluorescence response values ​​in buffer solutions or solvents with different pH values.

6. The use according to claim 5, characterized in that: The solvent includes, but is not limited to, water, ethyl ether, n-octane, formic acid, dimethyl sulfoxide, methanol, ethanol, tetrahydrofuran, and N'N-dimethylformamide.

7. The use according to claim 5, characterized in that: The buffer solution includes a strong acid solution, a strong alkaline solution, a weak acid solution and a weak alkaline solution.

8. The use according to claim 7, characterized in that: The buffer solution is a PBS buffer solution with a pH of 7.

4. The PBS buffer solution with a pH of 7.4 is used as a protein detection solvent. The detection conditions during the detection and evaluation are: the excitation wavelength is 430 nm, and the fluorescence emission spectrum is detected between 480-660 nm.

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