A universal peptide detection fluorescent probe and its preparation and application

Abstract

The present invention discloses a universal peptide detection fluorescent probe and its preparation and application. The fluorescent probe is a covalent organic porous framework nanosheet (CONs) prepared by a top-down strategy using a bulk covalent organic porous framework (COFs) with fluorescent properties as a raw material. The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophobic peptides does not require sample pretreatment processes such as chemical labeling and desalting of the target peptide, and has the characteristics of being fast, convenient and resistant to matrix interference. The fluorescence data can be read within five minutes after the target peptide and the probe are mixed, and the practicability is strong. The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophilic peptides and proteins effectively expands the application scope of the hydrophobic peptide-responsive fluorescent probe through the strategy of enzyme cleavage before detection, and promotes the probe as a universal peptide detection method.

Description

Technical Field

[0001] The present invention belongs to the field of bioluminescence analysis, and specifically relates to a fluorescent probe for identifying peptides, and its preparation and application. The fluorescent probe is a covalent organic porous framework nanosheet with fluorescent properties. The hydrophilic-hydrophobic interaction between the fluorescent probe and the hydrophobic peptide molecules causes the change of its luminescence intensity to achieve highly sensitive quantitative analysis of hydrophobic peptides in the solution, and can also achieve quantitative analysis of hydrophilic peptides or proteins through relative quantification of hydrophobic peptides in enzymatic hydrolysis products. The present invention has potential application prospects in the rapid detection of peptide drugs and diagnostic markers. Background Art

[0002] Bioactive peptides are widely present in organisms and participate in many important physiological activities and pathological processes, including cell differentiation, neurotransmitter regulation, and the occurrence of tumor diseases. They are often used as clinical therapeutic drugs and disease diagnostic molecules. Endogenous peptide biomarkers and exogenous peptide drugs are almost all low-abundance in highly complex biological environments, so highly sensitive and rapid peptide detection technologies are required. Fluorescence-based analytical methods are highly sensitive, but most of them require destructive pre-treatment processes such as fluorescent labeling and chemical derivatization, which will change the molecular structure of the target peptide and are incompatible with subsequent characterization techniques. Therefore, there is still a lack of non-destructive and universal peptide fluorescence detection methods.

[0003] Covalent organic frameworks (COFs) are a class of crystalline porous materials constructed entirely of strong covalent bonds. The combination of aggregation-induced emission (AIE) and the planar extended π-π conjugated skeleton endows some COFs with excellent luminescent activity. In recent years, detection platforms based on the luminescent response of fluorescent COFs to target molecules have begun to flourish and have been applied to the quantitative analysis of heavy metal ions, pH, small molecule disease markers, reactive oxygen species, and antibiotics, with the characteristics of high sensitivity, rapidity, and convenience. There have been no reports on the fluorescent sensing of peptides using direct interactions between peptides and fluorescent COFs materials. Summary of the invention

[0004] In view of the above defects and needs of the prior art, the present invention aims to provide a fluorescent probe for quantitative detection of peptides and its preparation and application.

[0005] The present invention prepares CONs containing aggregation-induced emission (AIE) groups through a top-down strategy and uniformly disperses them in a solution to obtain a switch-type fluorescent probe. The fluorescent CONs probe in the present invention can be used for rapid quantitative analysis of hydrophobic peptides in a solution, and can also achieve quantitative analysis of hydrophilic peptides or proteins through relative quantification of hydrophobic peptides in enzymatic hydrolysis products. The invention has universal applicability for rapid and sensitive quantitative detection of peptides.

[0006] To achieve the above objectives, one aspect of the present invention provides a fluorescent probe.

[0007] The fluorescent probe provided by the present invention is: a covalent organic porous framework nanosheet (CONs) prepared by a top-down strategy using a bulk covalent organic porous framework (COFs) with fluorescent properties as a raw material;

[0008] Wherein, the bulk covalent organic porous framework (COFs) with fluorescent properties is a bulk covalent organic porous framework (COFs) with cyano-substituted stilbene structure as an organic building block connecting group.

[0009] Specifically, the covalent organic porous framework nanosheet CONs also contains 2,4,6-triphenyl-1,3,5-triazine structural units, and the structure is shown in the following figure:

[0010]

[0011] Wherein, n=1, 2 or 3.

[0012] More specifically, the covalent organic porous framework nanosheets (CONs) contain the structural units shown below:

[0013]

[0014] Another aspect of the present invention provides a method for preparing the fluorescent probe.

[0015] The preparation method of the fluorescent probe provided by the present invention comprises the following steps: treating bulk covalent organic porous frameworks (COFs) with fluorescent properties from top to bottom, separating CONs from the product, and drying to obtain the fluorescent probe.

[0016] The fluorescence property of the COFs raw material with fluorescence property originates from the aggregation-induced fluorescence chromophore formed by the bonding between the organic modules constructing the COFs, specifically the cyano-substituted stilbene structure.

[0017] The top-down processing method is a liquid phase exfoliation method, specifically a solvent-assisted ultrasonic exfoliation method.

[0018] The top-down processing operation is: disperse the bulk covalent organic porous framework (COFs) with fluorescent properties into a solvent, and perform ultrasonic treatment.

[0019] Wherein, the solvent is water and an organic solvent such as methanol, ethanol, acetonitrile, isopropanol, etc.;

[0020] The concentration of the bulk covalent organic porous framework (COFs) in the solvent may be 1-100 mg / mL;

[0021] The power of the ultrasonic treatment may be 100-300 W, specifically 140 W, the frequency may be 20-40 kHz, specifically 20 kHz, and the time may be 0.5-12 h, specifically 6 h.

[0022] The operation of separating CONs from the product can be performed by taking the upper layer after standing at room temperature for 3-12 hours or taking the upper layer after centrifugation at 1000-5000 rpm for 5-10 minutes.

[0023] Another aspect of the present invention provides the use of the above-mentioned covalent organic porous framework nanosheets (CONs) as fluorescent probes in the detection of hydrophobic peptides, hydrophilic peptides and proteins.

[0024] The present invention also provides a method for quantitatively detecting hydrophobic peptides.

[0025] The method for quantitatively detecting hydrophobic peptides provided by the present invention comprises the following steps: uniformly dispersing a CONs fluorescent probe in a solvent to obtain a fluorescent probe solution, respectively mixing a known target hydrophobic peptide solution of different concentrations and a blank control with the fluorescent probe solution, allowing the mixture to stand, detecting the fluorescence intensity of the obtained mixed solution, calculating the fluorescence quenching rate, establishing a concentration-fluorescence quenching rate linear standard curve, mixing a hydrophobic peptide solution to be tested with the fluorescent probe solution, allowing the mixture to stand, detecting the fluorescence intensity of the obtained solution, calculating the fluorescence quenching rate, and calculating the concentration of the hydrophobic peptide solution according to the concentration-fluorescence quenching rate linear standard curve to achieve quantitative analysis of the hydrophobic peptide.

[0026] The fluorescence quenching rate is calculated by the following formula: 0 -F x ) / F 0

[0027] F x Represents the fluorescence intensity of the experimental group, F 0 represents the fluorescence intensity of the blank control group.

[0028] In the above method, the solvent can specifically be water or ethanol solutions of different concentrations.

[0029] The target hydrophobic peptide solution can use water, ethanol in different proportions, PBS, and physiological saline as solvent.

[0030] The standing time may be 3-60 min, specifically 5 min.

[0031] The concentration of the CONs fluorescent probe in the mixed solution may be 1-100 μg / mL.

[0032] The linear range of the CONs fluorescent probe for quantification of hydrophobic peptides is 5-1000 ng / mL.

[0033] Collect the fluorescence intensity at the maximum emission wavelength; for example, if the excitation wavelength is set to 360 nm, collect the fluorescence intensity value at 530 nm.

[0034] The last aspect of the present invention provides a method for quantitatively detecting hydrophilic peptides and proteins.

[0035] The method for quantitatively detecting hydrophilic peptides and proteins provided by the present invention comprises the following steps: uniformly dispersing a CONs fluorescent probe in a solvent to obtain a fluorescent probe solution, enzymatically hydrolyzing known hydrophilic target peptides or proteins of different concentrations using a decomposing enzyme to obtain an enzymatic hydrolysis product containing a small molecule peptide segment mixture; mixing the enzymatic hydrolysis product of a target hydrophilic peptide or protein standard and a blank control with the fluorescent probe solution, standing, detecting the fluorescence intensity, and calculating the fluorescence quenching rate; establishing a concentration-fluorescence quenching rate linear standard curve, enzymatically hydrolyzing the hydrophilic target peptide or protein to be detected, mixing the obtained enzymatic hydrolysis product with the fluorescent probe solution, standing, detecting the fluorescence intensity of the obtained solution, and calculating the fluorescence quenching rate; and calculating the concentration of the hydrophilic target peptide or protein according to the concentration-fluorescence quenching rate linear standard curve to achieve quantitative analysis of the target hydrophilic peptide or protein.

[0036] In the above method, the solvent can specifically be water or ethanol solutions of different concentrations.

[0037] The hydrophilic target peptide or protein is dissolved in a phosphate buffer or an ammonium bicarbonate solution before enzymatic hydrolysis.

[0038] The degrading enzyme can cleave peptides and proteins into small molecule peptide fragments, including but not limited to trypsin, chymotrypsin, pepsin, preferably trypsin.

[0039] The enzymolysis time is 0-72 hours, and the enzymolysis time and the amount of enzyme used need to be optimized.

[0040] The standing time may be 3-60 min, specifically 5 min.

[0041] The fluorescence intensity was collected at the wavelength of maximum emission.

[0042] The covalent organic porous framework nanosheet (CONs) of the present invention is a two-dimensional nanomaterial. In addition to the characteristics of COFs such as stable structure, large specific surface area, and high structural diversity, it also has adjustable optical and electronic properties and good dispersibility, showing good application prospects in the fields of adsorption separation, fluorescence and sensing. The present invention simultaneously exerts the luminescence characteristics and adsorption capacity of CONs, and can achieve rapid, non-destructive, and highly sensitive peptide determination. This is of great significance for the clinical detection and related research of peptide drugs and biomarkers.

[0043] The covalent organic porous framework nanosheet (CONs) peptide detection probe and its preparation method and application method provided by the present invention can achieve the following beneficial results:

[0044] (1) The fluorescent probe provided by the present invention has a unique nanosheet structure, good stability and optical properties, and the preparation method is simple and versatile. COFs have diverse structures, and the preparation method can prepare various types of fluorescent COFs raw materials into hydrophobic peptide-responsive fluorescent probes.

[0045] (2) The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophobic peptides does not require sample pretreatment processes such as chemical labeling and desalting of the target peptides. It is fast, convenient and resistant to matrix interference. The fluorescence data can be read within five minutes after the target peptide and the probe are mixed, and it is highly practical.

[0046] (3) The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophobic peptides has a quantitative linear range of 5-1000 ng / mL, has the characteristics of high sensitivity, and is expected to be used for the rapid detection of trace peptides.

[0047] (4) The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophobic peptides adopts a non-destructive fluorescence sensing strategy, does not rely on the intrinsic fluorescence of the peptide analyte or any chemical derivatives, and has good versatility.

[0048] (5) The application method of the CONs fluorescent probe provided by the present invention in the detection of hydrophilic peptides and proteins effectively expands the application scope of the hydrophobic peptide-responsive fluorescent probe through the strategy of enzyme cleavage before detection, and promotes the probe as a universal peptide detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the chemical structure of CONs in Example 1 of the present invention.

[0050] Figure 2 This is a low-pressure transmission electron microscopy image of the COFs raw material in Example 1 of the present invention.

[0051] Figure 3 This is a low-voltage transmission electron microscopy image of CONs prepared in Example 1 of the present invention.

[0052] Figure 4 This is the fluorescence spectrum of CONs prepared in Example 1 of the present invention.

[0053] Figure 5 The selectivity experimental results of the CONs fluorescent probe in Example 2 of the present invention. The blank in the abscissa represents the blank control group without hydrophobic peptides and interfering substances, and 1-8 respectively contain 5 μg / mL of FGFGF, HGHGH, EGEGE, GSH, GSSG, Na+ , K + , Ca 2+ of sample solution.

[0054] Figure 6 The interference experiment results of the CONs fluorescent probe in Example 2 of the present invention are shown in Figure 1. The blank in the abscissa represents the blank control group without hydrophobic peptides and interfering substances, 1 is a sample solution containing 5 μg / mL of FGFGF, and 2-8 are sample solutions containing 5 μg / mL of HGHGH, EGEGE, GSH, GSSG, Na + , K + , Ca 2+ and 5 μg / mL FGFGF mixed solution.

[0055] Figure 7 The response of the fluorescence quenching rate of CONs in the aqueous solution to different concentrations of somatostatin in Example 3 of the present invention.

[0056] Figure 8 This is the response of the fluorescence quenching rate of CONs in PBS solution to different concentrations of somatostatin in Example 3 of the present invention.

[0057] Fig. 9 This is the response of the fluorescence quenching rate of CONs in Example 4 of the present invention to the enzymatic hydrolysis products of ProGRP (31-98) at different concentrations. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] Example 1

[0061] The fluorescent probe preparation method of this embodiment includes the following steps:

[0062] References Yang, Y.; Luo, N.; Lin, S.; Yao, H.; Cai, Y., Cyano Substituent on the Olefin Linkage: Promoting Rather than Inhibiting the Performance of Covalent Organic Frameworks. ACS Catalysis 2022, 12 (17), 10718-10726. reported that 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (TFPT) and 2,4,6-tris(4-benzylcyanophenyl)-1,3,5-triazine (TCPT) were used as raw materials to prepare a large amount of acrylonitrile-linked bulk TTAN-COF as a raw material for the preparation of CONs. The bulk TTAN-COF synthesis route is as follows:

[0063]

[0064] TFPT and TCPT were purchased from Jilin Zhongkeshen Technology Co., Ltd. TTAN-COF raw materials were dispersed in 100 mL of water at a concentration of 10 mg / mL, and the mixture was ultrasonically treated for 6 h (140 W, 20 kHz) in an ice bath, and then allowed to stand for 6 h, and the upper layer was taken and nitrogen-blown to dryness at 60 °C to obtain CONs nanosheets, which were peptide-responsive fluorescent probes.

[0065] Figure 1 Schematic diagram of the chemical structure of the fluorescent probe CONs. Liquid phase erosion does not change the molecular structure, so the structure of CONs is consistent with the bulk TTAN-COF synthesized in large quantities. As can be seen from the figure, CONs contain cyano-substituted stilbene structures, which are typical AIE chromophores and are the basis for the fluorescent properties of CONs.

[0066] Figure 2 This is a low-voltage transmission electron microscopy image of the mass-synthesized TTAN-COF raw material. It can be seen that the bulk TTAN-COF synthesized in large quantities has a rod-like morphology and is micron-sized.

[0067] Figure 3 This is the low-pressure transmission electron microscopy image of the prepared CONs. It can be seen that the solvent-assisted ultrasonic ablation changes the morphology of the raw materials, and the CONs present a ductile thin layer morphology.

[0068] Figure 4The fluorescence spectrum of CONs in solid state is shown in Figure 2. The maximum excitation wavelength of CONs nanosheets is 372nm, and the maximum emission wavelength is 518nm. The fluorescence quantum yield of CONs is 24.45%. The above results show that the prepared CONs material has good fluorescence luminescence performance.

[0069] Example 2

[0070] This example investigates the selectivity and anti-matrix interference ability of the CONs fluorescent probe prepared in Example 1.

[0071] A series of hydrophobic peptides were selected to detect related interferences, including chemically synthesized hydrophilic peptide molecules (HGHGH, EGEGE), common hydrophilic bioactive peptides (GSH and GSSG) and electrolyte metal ions (Na + , K + , Ca 2+ ). First, the selectivity of CONs fluorescent probe for hydrophobic peptide detection was investigated. The chemically synthesized hydrophobic peptide FGFGF and each interfering substance were mixed with 3 μg / mL CONs fluorescent probe in equal volumes, and the concentrations of FGFGF and interfering substances in the obtained single component test solution were both 5 μg / mL. Figure 5 As shown in the figure, except for the hydrophobic peptide FGFGF, the other interfering substances could not quench the fluorescence intensity of the CONs solution, proving that the CONs fluorescent probe has a specific response to the hydrophobic peptide. Secondly, an anti-interference experiment was carried out. 50μg / mL interfering substance, 10μg / mL FGFGF and 3μg / mL CONs fluorescent probe were mixed in a volume ratio of 2:10:8. The concentrations of interfering substance and hydrophobic peptide FGFGF in the obtained mixed solution were equal, which was 5μg / mL. Figure 6 As shown, the introduction of interfering ions cannot change the fluorescence quenching effect of hydrophobic peptide FGFGF on CONs. The results of this example prove that the CONs fluorescent probe has good hydrophobic peptide selectivity and resistance to matrix interference, and is expected to be used to detect hydrophobic peptides in complex biological samples.

[0072] Example 3

[0073] The application of the fluorescent probe of this example in the detection of the hydrophobic peptide somatostatin can realize the rapid quantitative analysis of somatostatin using the CONs fluorescent probe prepared in Example 1. The specific operation steps are as follows:

[0074] Take 30 μg CONs and add them to 10 mL water. After ultrasonication for 10 minutes, a 3 μg / mL CONs dispersion was obtained. First, add 100 μL of 3 μg / mL CONs aqueous solution to a 96-well plate, then add 100 μL of somatostatin aqueous solution of different concentrations, mix well and let stand for 3 minutes. Use an ELISA reader for fluorescence measurement. After optimization, set the excitation wavelength to 360 nm, collect the fluorescence intensity value at 530 nm, and calculate the fluorescence quenching rate. Figure 7 As shown in the figure, within the concentration range of 5-1000 ng / mL, the fluorescence quenching rate of CONs is linearly positively correlated with the concentration of somatostatin, with a correlation coefficient of 0.997, indicating that the CONs fluorescent probe has high sensitivity and can be used to detect the concentration of hydrophobic peptide somatostatin. In addition, different concentrations of somatostatin PBS solutions were prepared, and the samples were pretreated using the same method as above. The fluorescence was measured using an enzyme reader, with the excitation wavelength unchanged, and the fluorescence intensity value at 525 nm was collected. Figure 8 As shown, with PBS as the target hydrophobic peptide solvent, in the concentration range of 5-1000 ng / mL, the fluorescence quenching rate of CONs is still linearly positively correlated with the somatostatin concentration, with a correlation coefficient of 0.992, indicating that the CONs fluorescent probe has the ability to resist matrix interference and is expected to be used for the rapid detection of hydrophobic peptides in complex matrices.

[0075] Example 4

[0076] The fluorescent probe of this embodiment is used in the detection of hydrophilic gastrin-releasing peptide precursor fragment ProGRP (31-98). CONs only has a highly sensitive response to hydrophobic peptides, and hydrophilic peptides and proteins cannot be directly quantitatively analyzed using the CONs fluorescent probe. The present invention proposes a strategy of first enzyme cleavage and then detection, which can achieve quantitative analysis of hydrophilic peptides using the CONs fluorescent probe prepared in Example 1. This embodiment selects hydrophilic ProGRP (31-98) as the target analyte and uses the CONs fluorescent probe to quantitatively detect it. The specific operating steps are as follows:

[0077] First, 100 μg of ProGRP (31-98) was dissolved in 1 mL of PBS buffer to prepare a 100 μg / mL stock solution. ProGRP (31-98) was diluted to the required concentration with PBS buffer. The sample solution needs to be prepared as needed. Take 100 μL of ProGRP (31-98) solutions of different concentrations (4, 6, 8, 10, 12, 14, 16, 18 μg / mL), add 10 μL of 20 μg / mL trypsin solution, and dilute to a total volume of 150 μL with PBS buffer. Incubate at 37 °C for 12 hours to obtain an enzymatic product solution. Take 60 μg of CONs and add it to 10 mL of water. After ultrasonication for 10 minutes, a CONs dispersion with a concentration of 6 μg / mL is obtained. First, add 50 μL of 6 μg / mL CONs aqueous solution to a 96-well plate, then add 150 μL of the above enzymatic product solution, mix well and let stand for 3 minutes. The fluorescence was measured using an ELISA instrument. After optimization, the excitation wavelength was set to 360 nm, the fluorescence intensity value at 530 nm was collected, and the fluorescence quenching rate was calculated. Fig. 9 As shown in the figure, the fluorescence quenching rate of the enzymatic hydrolysis product to CONs is linearly positively correlated with the concentration of ProGRP (31-98), and the correlation coefficient is 0.989, indicating that the CONs fluorescent probe can be used for the detection of ProGRP (31-98) concentration. It can be seen from this example that the CONs fluorescent probe provided by the present invention can realize the quantitative detection of hydrophilic peptides and proteins.

[0078] As can be seen from the above examples, the present invention provides a CONs fluorescent probe with a unique nanosheet structure and good optical properties, which has a simple preparation method and strong versatility. Highly sensitive and rapid quantitative detection of hydrophobic peptides can be achieved without chemical labeling, desalting and other sample pretreatment processes for the target peptide; the strategy of enzyme cleavage followed by detection further expands the application range of hydrophobic peptide-responsive fluorescent probes, which can be used for quantitative detection of hydrophilic peptides and proteins. The CONs probe provided by the present invention is expected to become a universal peptide detection method.

[0079] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A fluorescent probe, which is a covalent organic porous framework nanosheet CONs prepared by a top-down strategy using bulk covalent organic porous framework COFs with fluorescent properties as raw materials; The covalent organic porous framework nanosheet CONs contains 2,4,6-triphenyl-1,3,5-triazine structural units, and the structure is shown below: in, n=1, 2 or 3.

2. The fluorescent probe according to claim 1, characterized in that: The covalent organic porous framework nanosheet CONs contains the structural units shown below: 。 3. A method for preparing the fluorescent probe according to claim 1 or 2, comprising the following steps: treating the bulk covalent organic porous framework COFs with fluorescent properties from top to bottom, separating CONs from the product, and drying to obtain the product.

4. The method according to claim 3, characterized in that: The top-down processing method is a liquid phase exfoliation method, specifically a solvent-assisted ultrasonic exfoliation method; The top-down processing operation is: dispersing the bulk covalent organic porous framework COFs with fluorescent properties into a solvent, and ultrasonically treating the solvent. Wherein, the solvent is water and at least one of methanol, ethanol, acetonitrile and isopropanol; The concentration of the bulk covalent organic porous framework COFs in the solvent is 1-1000 μg / mL; The ultrasonic treatment has a power of 100-300 W, a frequency of 20-40 kHz, and a time of 0.5-12 h. The operation of separating CONs from the product is to take the upper layer liquid after standing at room temperature for 3-12 hours or to take the upper layer liquid after centrifugation at 1000-5000 rpm for 5-10 minutes.

5. Use of the fluorescent probe according to claim 1 or 2 in the detection of hydrophobic peptides, hydrophilic peptides and proteins.

6. A method for quantitatively detecting hydrophobic peptides, comprising the following steps: dispersing the fluorescent probe according to claim 1 or 2 in a solvent to obtain a fluorescent probe solution, mixing known target hydrophobic peptide solutions of different concentrations and blank controls with the fluorescent probe solution, allowing the solution to stand, detecting the fluorescence intensity, calculating the fluorescence quenching rate, establishing a concentration-fluorescence quenching rate linear standard curve, mixing the hydrophobic peptide solution to be tested with the fluorescent probe solution, allowing the solution to stand, detecting the fluorescence intensity of the resulting solution, calculating the fluorescence quenching rate, and calculating the concentration of the hydrophobic peptide solution according to the concentration-fluorescence quenching rate linear standard curve to achieve quantitative analysis of the hydrophobic peptide.

7. A method for quantitatively detecting hydrophilic peptides and proteins, comprising the following steps: dispersing the fluorescent probe described in claim 1 or 2 in a solvent to obtain a fluorescent probe solution, using a decomposing enzyme to enzymatically hydrolyze known hydrophilic target peptides or proteins of different concentrations to obtain an enzymatic hydrolysis product containing a mixture of small molecule peptide segments; mixing the enzymatic hydrolysis product of the target hydrophilic peptide or protein standard and a blank control with the fluorescent probe solution, allowing the mixture to stand, detecting the fluorescence intensity, calculating the fluorescence quenching rate, and establishing a concentration-fluorescence quenching rate linear standard curve; enzymatically hydrolyzing the hydrophilic target peptide or protein to be tested, mixing the obtained enzymatic hydrolysis product with the fluorescent probe solution, allowing the mixture to stand, detecting the fluorescence intensity of the obtained solution, calculating the fluorescence quenching rate, and calculating the concentration of the hydrophilic target peptide or protein according to the concentration-fluorescence quenching rate linear standard curve to achieve quantitative analysis of the target hydrophilic peptide or protein.

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