Fluorescence sensor for detecting thromboregulatory protein as well as preparation method and use method of fluorescence sensor

Abstract

The invention discloses a fluorescence sensor for detecting thromboregulatory protein as well as a preparation method and a use method of the fluorescence sensor. The preparation method comprises the following steps: S1, adding a capture antibody of the thromboregulatory protein into an elisa plate; s2, adding the prepared bovine serum albumin; s3, adding prepared thrombus regulatory protein solutions with different concentrations; s4, adding a prepared detection antibody modified with alkaline phosphatase and used for detecting thromboregulatory protein; s5, adding a p-nitrophenyl disodium phosphate solution, and fully reacting; s6, a NO2-Eu-MOF nanosheet solution is prepared; s7, preparing a 1, 4-benzene diphosphonic acid solution; s8, adding a 1, 4-benzene diphosphonic acid solution into the NO2-Eu-MOF nanosheet solution, and detecting a fluorescence signal; and S9, adding the solution obtained in the step S8 into the solution obtained in the step S5, detecting the fluorescence intensity, and constructing a fluorescence intensity-concentration working curve. According to the invention, the concentration of thromboregulatory protein in the sample can be quantitatively analyzed.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection of disease markers using rare earth metal organic framework materials, and specifically relates to a fluorescent sensor for detecting thrombomodulin and a preparation method thereof. Background Art

[0002] Thrombomodulin (TM) is a type 1 transmembrane protein that is continuously expressed on the surface of vascular endothelial cells. Thrombomodulin is a high-affinity receptor for all forms of thrombin and serves as a cofactor for thrombin-dependent protein C activation. Endothelial cell protein C receptor (EPCR) provides cell-specific binding sites for protein C and activated protein C (APC). When thrombin binds to thrombomodulin, the procoagulant activity of thrombin (e.g., the ability to generate fibrin and activate factor V, factor VIII, factor XI, and platelets) is neutralized, and the rate of thrombin inactivation by antithrombin is increased. The APC generated by the thrombin-thrombomodulin complex inhibits the formation of thrombin by inactivating the procoagulant cofactors factor Va and factor VIIIa. The thrombin-thrombomodulin complex, or "proteasome" complex, also has an antifibrinolytic effect by activating fibrinolytic inhibitors (i.e., thrombin-activated fibrinolytic inhibitor, TAFI). Therefore, it is very necessary to develop a sensitive and stable method for detecting TM. The current detection methods of TM mainly use enzyme-linked immunosorbent assay (ELISA) and monoclonal antibody method, which are relatively complicated, require professional operators, are time-consuming and require large instruments, etc., prompting researchers to develop convenient and accurate TM detection methods.

[0003] Metal-organic framework materials are a hot topic in current material research. Detection of TM based on metal-organic framework materials basically utilizes the simulated enzyme activity or fluorescence properties of metal-organic framework materials synthesized at high temperature and high pressure. For example, Fe / C nanosheets catalyze H2O2 while further promoting the reaction of phenol produced by TM hydrolysis of phenyl phosphate with 4-aminoantipyrine to generate red quinone imine (ACS Appl. Nano Mater. 2021, 4, 7888-7896). The excitation spectrum of UIO-TCPP MOFs effectively overlaps with the p-nitrophenol produced by TM hydrolysis of disodium p-nitrophenyl phosphate, resulting in fluorescence quenching of UIO-TCPPMOFs under 405nm excitation (Chem Comm 2020, 56, 2431-2434). However, there are almost no articles that use metal-organic framework materials (MOFs) that do not have fluorescence properties to quickly carry out ligand competitive coordination at room temperature to make them have good fluorescence properties to detect TM.

[0004] After searching, Chinese patent document CN116643042A discloses a thrombomodulin chemiluminescence detection kit. The invention specifically provides a method for preparing carboxyl magnetic beads and their application. The carboxyl magnetic beads prepared by the method have a high surface carboxyl content and a high coupling rate with antigens or antibodies. The thrombomodulin chemiluminescence detection kit prepared by the invention using carboxyl magnetic beads has stable properties, good measurement accuracy and repeatability, and can be used in clinical practice.

[0005] Chinese patent document CN110196334A discloses a thrombomodulin chemiluminescent immunoassay quantitative detection kit and its preparation method, which belongs to the field of immunodiagnosis technology. It solves the technical problems that the methods of thrombomodulin in the prior art are either low in accuracy, or complicated in operation and unstable in test results. The thrombomodulin chemiluminescent immunoassay quantitative detection kit of the present invention includes R1 reagent, R2 reagent and R3 reagent; wherein R1 reagent includes streptavidin magnetic particles and buffer I, R2 reagent includes thrombomodulin monoclonal antibody labeled with chemiluminescent marker and buffer I, and R3 reagent includes conjugated thrombomodulin monoclonal antibody and buffer I. The kit uses chemiluminescent immunoassay to detect thrombomodulin, and has the advantages of high detection sensitivity, accurate quantitative detection, simple operation, no radioactive risk and short detection time, and can detect antigen and antibody target substances.

[0006] Chinese patent document CN116183586A discloses a method and kit for detecting thrombomodulin, which has fast detection speed, high sensitivity, good specificity, small coefficient of variation, good stability of each reagent component, and can meet existing detection needs. The characteristics are as follows: the detection method comprises the following steps:

[0007] Step 1, add 30 μL of the sample to be tested and 100 μL of biotin-labeled thrombomodulin antibody to the coated plate, mix and react; after the reaction, wash 5 times with 300 μL of washing solution; Step 2, add 100 μL of horseradish peroxidase-labeled thrombomodulin antibody to the coated plate, mix and react; after the reaction, wash 5 times with 300 μL of washing solution; Step 3, add 50 μL of substrate A and 50 μL of substrate B to the coated plate in sequence, mix, and measure the luminescence intensity in a chemiluminescence analyzer; Step 4, use a calibrator of known concentration to draw a luminescence intensity standard curve, and compare the luminescence intensity obtained in step 3 with the luminescence intensity standard curve to calculate the content of thrombomodulin in the sample to be tested.

[0008] Although there are methods for detecting thrombomodulin in the prior art, they are different from the invention concept used in this application and use different key technical means. The prior art does not disclose the rapid ligand competitive coordination of metal organic framework materials that do not have fluorescence properties at room temperature to make them have good fluorescence properties to detect TM. Summary of the invention

[0009] In view of this, in order to solve the above technical problems, the present invention is to obtain a new two-dimensional rare earth metal organic framework material that exerts an "antenna effect" by quickly conducting ligand competitive coordination on a synthesized two-dimensional rare earth metal organic framework material at room temperature, and to construct a detection method for TM concentration based on the new two-dimensional rare earth metal organic framework material.

[0010] Therefore, the purpose of the present invention is to address the deficiencies in the prior art and provide a fluorescent sensor for detecting thrombomodulin and a preparation method and a use method thereof. The present invention first synthesizes a two-dimensional metal organic framework material that does not have fluorescence, and then uses ligand competitive coordination to replace the ligands in the material structure. The obtained product exerts an "antenna effect" and exhibits fluorescent properties. Combined with the specific binding between thrombomodulin and antibodies, a biosensor is constructed for quantitative analysis and detection of thrombomodulin.

[0011] The technical solutions adopted are:

[0012] A method for preparing a fluorescent sensor for detecting thrombomodulin comprises the following steps:

[0013] S1. Add the capture antibody of thrombomodulin to the ELISA plate;

[0014] S2. Add the prepared bovine serum albumin and incubate;

[0015] S3. Add the prepared thrombomodulin solution of different concentrations and incubate;

[0016] S4. adding the prepared detection antibody for detecting thrombomodulin modified with alkaline phosphatase and incubating;

[0017] S5. Add the prepared solution of p-nitrophenyl phosphate disodium dissolved in Tris-HCl buffer solution to fully react;

[0018] S6. dispersing the non-fluorescent two-dimensional metal organic framework material NO2-Eu-MOF nanosheets in ultrapure water by ultrasonic dispersion to prepare a NO2-Eu-MOF nanosheet solution;

[0019] S7. Dissolve 1,4-phenylenediphosphonic acid in Tris-HCl buffer to obtain a 1,4-phenylenediphosphonic acid solution, which is stored for later use;

[0020] S8. Adding 1,4-phenylenediphosphonic acid solution to the NO2-Eu-MOF nanosheet solution, placing at room temperature, detecting the fluorescence signal, and obtaining the fluorescence signal of the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system;

[0021] S9. Add the solution of S8 to S5, mix well at room temperature, use a fluorescence meter to detect the fluorescence intensity in the presence of different concentrations of antigen thrombomodulin, construct a fluorescence intensity-concentration working curve, and thus construct a fluorescent biosensor.

[0022] Furthermore, in S6, the NO2-Eu-MOF nanosheet solution is prepared by the following method: NO2-Eu-MOF nanosheets are dispersed in ultrapure water by ultrasonic dispersion to obtain a NO2-Eu-MOF nanosheet solution.

[0023] Further, in S6, the NO2-Eu-MOF nanosheets are prepared by the following steps:

[0024] S11. dissolving europium (III) nitrate hexahydrate in N,N-dimethylformamide solvent, sonicating and vortexing;

[0025] S12. dissolving 2-nitroterephthalic acid and 2-fluorobenzoic acid in N,N-dimethylformamide solvent, sonicating and vortexing;

[0026] S13. The solution in step S11 and step S12 is mixed, and then concentrated nitric acid is added;

[0027] S14. The solution in step S13 is transferred to a stainless steel reactor lined with polytetrafluoroethylene and reacted at 180-200°C;

[0028] S15. The white product was obtained, and the unreacted raw materials N,N-dimethylformamide and concentrated nitric acid were removed by filtration, and dried in a vacuum oven overnight to obtain NO2-Eu-MOF nanosheets.

[0029] Furthermore, in S1, 5 μg mL -1 100 μL of thrombomodulin capture antibody was added to a 96-well ELISA plate, incubated at 4°C overnight, and washed three times with PBST to remove the thrombomodulin capture antibody not fixed on the ELISA plate.

[0030] Furthermore, 100 μL of the prepared 1 vol% bovine serum albumin was added to S2, incubated at 37° C. for 1 h, and washed three times with PBST to remove excess bovine serum albumin.

[0031] Furthermore, in S3, 100 μL of the prepared antigen thrombomodulin solution of different concentrations was added, incubated at 37° C. for 1 h, and washed three times with PBST to remove the unbound antigen thrombomodulin.

[0032] Furthermore, 40 μg mL -1 100 μL of the prepared thrombomodulin detection antibody Ab2-ALP modified with alkaline phosphatase was incubated at 37° C. for 1 h, and washed three times with 50 mM Tris-HCl buffer solution, pH=8.5, to remove unbound Ab2-ALP.

[0033] Furthermore, in S5, 145 μL of a 0.1 mM solution of disodium p-nitrophenyl phosphate dissolved in a Tris-HCl buffer solution was added and reacted at 37° C. for 30 min.

[0034] Furthermore, a method for preparing a fluorescent sensor for detecting thrombomodulin comprises the following steps:

[0035] S1. Prepare thrombomodulin solutions of different concentrations;

[0036] S2. Disperse the non-fluorescent NO2-Eu-MOF nanosheets in ultrapure water and oscillate for 30 min. The concentration of NO2-Eu-MOF nanosheets in the solution is 200 μg mL -1 ;

[0037] S3. Dissolve 1,4-phenylenediphosphonic acid in Tris-HCl buffer solution to prepare 1,4-phenylenediphosphonic acid solution, and store at 4°C for later use;

[0038] S4. Disodium p-nitrophenyl phosphate hexahydrate was dissolved in tris(hydroxymethylaminomethane)-hydrochloride buffer solution to prepare disodium p-nitrophenyl phosphate solution, which was stored at 4°C for later use;

[0039] S5. Add 20 μL of 10.0 mM 1,4-phenylenediphosphonic acid solution to 50 μL of 200 μg mL -1 The NO2-Eu-MOF nanosheet solution was placed at room temperature for 10 minutes, and the fluorescence signal was detected to obtain the fluorescence signal of the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system;

[0040] S6. Add 20 μL of 25 mM disodium p-nitrophenyl phosphate solution to step 5), detect the fluorescence signal, and obtain the fluorescence intensity of the system in the absence of thrombomodulin;

[0041] S7. Incubate 10 μL of thrombomodulin solutions of different concentrations with 20 μL of 25 mM disodium p-nitrophenyl phosphate solution at 37 °C for 20 min, then add them to the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system, detect the fluorescence signal in the presence of different concentrations of thrombomodulin, and construct the fluorescence signal-concentration working curve.

[0042] Furthermore, in step S2, the method for preparing the two-dimensional metal organic framework material NO2-Eu-MOF nanosheets comprises the following steps:

[0043] (1) Dissolve 100.0-300.0 mg of europium (III) nitrate hexahydrate in 0.5-3.0 mL of ultrapure water, sonicate, and vortex;

[0044] (2) dissolving 100.0-300.0 mg of nitroterephthalic acid and 10.0-40.0 mg of 2-fluorobenzoic acid in 5.0-25.0 mL of N,N-dimethylformamide, sonicating and vortexing;

[0045] (3) mixing the solutions in step (1) and step (2), and then adding 20.0-200.0 μL of concentrated nitric acid;

[0046] (4) transferring the solution in step (3) into a stainless steel reactor lined with polytetrafluoroethylene and reacting at 100.0-200.0° C. for 12-48 hours;

[0047] (5) The white product was obtained, and the unreacted raw materials N,N-dimethylformamide and concentrated nitric acid were removed by filtration, and then dried in a vacuum oven at 60-100° C. to obtain NO2-Eu-MOF nanosheets.

[0048] The fluorescent sensor for detecting thrombomodulin of the present invention is prepared by the preparation method described in any one of the above schemes.

[0049] A method for using the fluorescence sensor of the present invention comprises the following steps:

[0050] S16. Add a solution of disodium p-nitrophenyl phosphate dissolved in a Tris-HCl buffer solution to the sample to allow for sufficient reaction;

[0051] S17. adding NO2-Eu-MOF nanosheet solution and 1,4-phenylenediphosphonic acid solution, reacting at room temperature, and detecting the fluorescence intensity with a fluorescence meter;

[0052] S18. Substitute the measured fluorescence intensity into the fluorescence intensity-concentration working curve of the fluorescence sensor to calculate the corresponding concentration of thrombomodulin, thereby achieving quantitative analysis of the thrombomodulin in the sample.

[0053] The beneficial technical effects of the present invention are:

[0054] The detection method of the present invention overcomes the shortcomings of existing thrombomodulin detection technology such as poor stability. The ligands of the synthesized two-dimensional metal-organic framework material are used to compete with 1,4-phenylenediphosphonic acid to obtain a new two-dimensional metal-organic framework material. The new two-dimensional metal-organic framework material has good fluorescence properties due to the "antenna effect". Combined with the specificity of thrombomodulin, a biosensor is constructed for quantitative analysis and detection of thrombomodulin.

[0055] In the presence of different concentrations of thrombomodulin, the capture antibody captures the antigen thrombomodulin, and the detection antibody modified with alkaline phosphatase can specifically bind to the thrombus detection protein, and then the thrombomodulin participates in the subsequent reaction; the product after the reaction of NO2-Eu-MOF nanosheets and 1,4-phenylenediphosphonic acid has a fluorescence emission unique to rare earth elements, which is quenched by disodium p-nitrophenyl phosphate. Thrombomodulin hydrolyzes disodium p-nitrophenyl phosphate to generate p-nitrophenol, which restores the fluorescence of the system. The fluorescence recovery intensity is positively correlated with the activity of thrombomodulin, and the activity of thrombomodulin is proportional to the content of thrombomodulin present. Based on this, the fluorescence signal in the presence of different concentrations of thrombomodulin in the solution can be detected, and the fluorescence signal-concentration working curve can be constructed to prepare a fluorescence sensor. First, the fluorescence signal of thrombomodulin in the sample is detected using a fluorescence sensor, and then the concentration of thrombomodulin in the sample is quantitatively analyzed by substituting it into the working curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the fluorescent sensor constructed based on NO2-Eu-MOF nanosheets for detecting thrombomodulin;

[0057] Figure 2 This is a feasibility study diagram of detecting different concentrations of thrombomodulin using a fluorescent sensor built based on NO2-Eu-MOF nanosheets;

[0058] Figure 3 Feasibility study diagram of the fluorescent sensor constructed based on NO2-Eu-MOF nanosheets to detect different concentrations of thrombomodulin (M: NO2-Eu-MOF; PEPP: 1,4-phenylenediphosphonic acid; PNPP: disodium p-nitrophenyl phosphate). Figure 3 a is the process of fluorescence enhancement after adding PEPP to M and fluorescence quenching after adding PNPP; Figure 3 b is a feasibility study diagram for detecting different concentrations of thrombomodulin.

[0059] Figure 4 The fluorescence spectra of different concentrations of thrombomodulin detected by the fluorescence sensor constructed based on NO2-Eu-MOF nanosheets (a) and the working curve of the sensor detecting thrombomodulin (b). DETAILED DESCRIPTION

[0060] The present invention is described in detail below through specific examples, but the use and purpose of these exemplary embodiments are only used to illustrate the present invention, and do not constitute any form of limitation on the actual protection scope of the present invention, and do not limit the protection scope of the present invention to these.

[0061] Among them, Tris-HCl buffer solution is tris-hydrochloride buffer solution; PBST is the full name of phosphate buffered saline Tween, which is a commonly used biochemical experimental reagent, which is mainly composed of phosphate buffer added with Tween-20. ALP is alkaline phosphatase; Ab2 is anti-idiotypic antibody, recorded as Ab2.

[0062] PEPP is 1,4-phenylenediphosphonic acid; NO2BDC is 2-nitroterephthalic acid.

[0063] The working principle of the fluorescence sensor of the present invention is:

[0064] In the present invention, 1,4-phenylenediphosphonic acid competes with the ligands in the NO2-Eu-MOF nanosheets to coordinate, causing the coordination environment of Eu3+ to change, and NO2-Eu-MOF nanosheets are transformed into Eu x (PEPP) y (NO2BDC) z ,Eu x (PEPP) y (NO2BDC) z PEPP has good fluorescence properties due to its "antenna effect". x (PEPP) y (NO2BDC) z The excitation spectra of Eu x (PEPP) y (NO2BDC) z The fluorescence of TM is quenched. When TM is present, the alkaline phosphatase modified on the TM detection antibody hydrolyzes disodium p-nitrophenyl phosphate to p-nitrophenol. Under alkaline conditions, p-nitrophenol loses protons to form a stable negative ion. The UV absorption spectrum of sodium p-nitrophenol is similar to that of Eu x (PEPP) y (NO2BDC) z The excitation or emission spectra do not overlap, so the fluorescence of the system is restored.

[0065] The presence or absence of thrombomodulin and the concentration of thrombomodulin in the system are determined by the corresponding fluorescence signal intensity to achieve the analysis and detection of thrombomodulin. Figure 1As shown, the feasibility of detection is Figure 2 shown.

[0066] In the above-mentioned method for detecting thrombomodulin with a fluorescent sensor, the two-dimensional metal organic framework material NO2-Eu-MOF nanosheet does not have fluorescent properties, but its ligand can quickly compete with 1,4-phenylenediphosphonic acid at room temperature to obtain Eu x (PEPP) y (NO2BDC) z , the Eu x (PEPP) y (NO2BDC) z PEPP has good fluorescence properties because of its "antenna effect".

[0067] Example 1

[0068] A method for preparing a fluorescent sensor for detecting thrombomodulin, the specific steps are as follows:

[0069] 1. Prepare thrombomodulin solutions of different concentrations;

[0070] 2. First synthesize NO2-Eu-MOF nanosheet material, the steps are as follows:

[0071] 1) Dissolve 178.0 mg of europium (III) nitrate hexahydrate in 0.95 mL of ultrapure water, sonicate and vortex;

[0072] 2) Dissolve 105.5 mg of nitroterephthalic acid and 28.0 mg of 2-fluorobenzoic acid in 9.0 mL of N,N-dimethylformamide, sonicate and vortex;

[0073] 3) Mix the solutions in step (1) and step (2), and then add 50.0 μL of concentrated nitric acid;

[0074] 4) transferring the solution in step (3) into a stainless steel reactor lined with polytetrafluoroethylene and reacting at 180° C. for 12 hours;

[0075] 5) A white product was obtained, and the raw materials N,N-dimethylformamide and concentrated nitric acid that did not participate in the reaction were filtered and washed away, and the product was dried in a vacuum oven at 60° C. to obtain NO2-Eu-MOF nanosheets.

[0076] 3. Disperse the non-fluorescent two-dimensional metal organic framework material NO2-Eu-MOF nanosheets in ultrapure water and oscillate for 30 minutes. The concentration of NO2-Eu-MOF nanosheets in the solution is 200 μg mL -1 ;

[0077] 4. Dissolve 1,4-phenylenediphosphonic acid in tris(hydroxymethylaminomethane)-hydrochloride buffer to prepare 1,4-phenylenediphosphonic acid solution, and store it at 4°C for later use;

[0078] 5. Dissolve p-nitrophenyl phosphate disodium hexahydrate in tris(hydroxymethylaminomethane)-hydrochloride buffer to prepare p-nitrophenyl phosphate disodium solution, and store it at 4°C for later use;

[0079] 6. Add 20 μL of 10.0 mM 1,4-phenylenediphosphonic acid solution to 50 μL of 200 μg mL -1 The NO2-Eu-MOF nanosheet solution was placed at room temperature for 10 minutes, and the fluorescence signal was detected to obtain the fluorescence signal of the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system;

[0080] 7. Add 20 μL of 25 mM disodium p-nitrophenyl phosphate solution to step 5), detect the fluorescence signal, and obtain the fluorescence intensity of the system in the absence of thrombomodulin;

[0081] 8. Incubate 10 μL of thrombomodulin solutions of different concentrations with 20 μL of 25 mM disodium p-nitrophenyl phosphate solution at 37 °C for 20 min, then add them to the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system, detect the fluorescence signal in the presence of different concentrations of thrombomodulin, and construct the fluorescence signal-concentration working curve.

[0082] Example 2

[0083] A method for using the fluorescence sensor of embodiment 1 comprises the following steps:

[0084] 1) Add the sample solution to a solution of disodium p-nitrophenyl phosphate dissolved in Tris-HCl buffer and incubate at 37°C for 20 minutes;

[0085] 2) Take NO2-Eu-MOF nanosheets and 1,4-phenylenediphosphonic acid solution and react at room temperature for 10 minutes;

[0086] 3) mixing the solution in step 2) with the solution in step 1), and detecting the fluorescence signal using a fluorescence instrument;

[0087] 4) Substituting the fluorescence signal into the fluorescence signal-concentration working curve correspondence, and performing quantitative analysis of thrombomodulin in the sample.

[0088] The results showed that when the concentration of thrombomodulin was 50-1000 pg mL -1 In the concentration range, the fluorescence intensity of the system is linearly related to the concentration (ΔF = 51.71C TM +53.77, R2=0.998), the detection limit was 17.3pg mL-1 ,like Figure 3 Shown and Figure 4 shown.

[0089] The series of detailed descriptions listed above are only specific descriptions of feasible embodiments of the present invention. They are not intended to limit the scope of protection of the present invention. All equivalent embodiments or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent sensor for detecting thrombomodulin, characterized in that: The steps include: S1. Add the capture antibody of thrombomodulin to the ELISA plate; S2. Add the prepared bovine serum albumin and incubate; S3. Add the prepared thrombomodulin solution of different concentrations and incubate; S4. adding the prepared detection antibody for detecting thrombomodulin modified with alkaline phosphatase and incubating; S5. Add the prepared disodium p-nitrophenyl phosphate solution dissolved in Tris-HCl buffer solution to fully react; S6. dispersing the non-fluorescent two-dimensional metal organic framework material NO2-Eu-MOF nanosheets in ultrapure water by ultrasonic dispersion to prepare a NO2-Eu-MOF nanosheet solution; S7. Dissolve 1,4-phenylenediphosphonic acid in a Tris-HCl buffer solution to obtain a 1,4-phenylenediphosphonic acid solution, which is stored for later use; S8. adding the 1,4-phenylenediphosphonic acid solution to the NO2-Eu-MOF nanosheet solution, reacting at room temperature, detecting the fluorescence signal, and obtaining the fluorescence signal of the NO2-Eu-MOF+1,4-phenylenediphosphonic acid system; S9. Add the solution of S8 to S5, mix well at room temperature, use a fluorescence meter to detect the fluorescence intensity in the presence of different concentrations of antigen thrombomodulin, construct a fluorescence intensity-concentration working curve, and thus construct a fluorescent biosensor.

2. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 1, characterized in that: In S6, the NO2-Eu-MOF nanosheet solution is prepared by the following method: NO2-Eu-MOF nanosheets are dispersed in ultrapure water by ultrasonic dispersion to obtain the NO2-Eu-MOF nanosheet solution.

3. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 2, characterized in that: In S6, the NO2-Eu-MOF nanosheets are prepared by the following steps: S11. dissolving europium (III) nitrate hexahydrate in N,N-dimethylformamide solvent, sonicating and vortexing; S12. dissolving 2-nitroterephthalic acid and 2-fluorobenzoic acid in N,N-dimethylformamide solvent, sonicating and vortexing; S13. The solution in step S11 and step S12 is mixed, and then concentrated nitric acid is added; S14. The solution in step S13 is transferred to a stainless steel reactor lined with polytetrafluoroethylene and reacted at 180-200°C; S15. The white product was obtained, and the unreacted raw materials N,N-dimethylformamide and concentrated nitric acid were removed by filtration, and dried in a vacuum oven overnight to obtain NO2-Eu-MOF nanosheets.

4. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 1, characterized in that: In S1, 5 μg mL -1 100 μL of thrombomodulin capture antibody was added to a 96-well ELISA plate, incubated at 4°C overnight, and washed three times with PBST to remove the thrombomodulin capture antibody not fixed on the ELISA plate.

5. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 4, characterized in that: In S2, 100 μL of the prepared 1 vol% bovine serum albumin was added, incubated at 37°C for 1 h, and washed three times with PBST to remove excess bovine serum albumin.

6. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 5, characterized in that: In S3, 100 μL of the prepared antigen thrombomodulin solution of different concentrations was added, incubated at 37°C for 1 h, and washed three times with PBST to remove the unbound antigen thrombomodulin.

7. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 6, characterized in that: S4, add 40 μg mL -1 100 μL of the prepared thrombomodulin detection antibody Ab2-ALP modified with alkaline phosphatase was incubated at 37° C. for 1 h, and washed three times with 50 mM Tris-HCl buffer solution, pH=8.5, to remove unbound Ab2-ALP.

8. The method for preparing a fluorescent sensor for detecting thrombomodulin according to claim 7, characterized in that: In S5, 145 μL of a 0.1 mM solution of disodium p-nitrophenyl phosphate dissolved in a Tris-HCl buffer solution was added and reacted at 37° C. for 30 min.

9. A fluorescent sensor for detecting thrombomodulin, which is prepared by the preparation method according to any one of claims 1 to 8.

10. A method for using the fluorescence sensor according to claim 9, characterized in that: The steps include: S16. Add a solution of disodium p-nitrophenyl phosphate dissolved in a Tris-HCl buffer solution to the sample for full reaction; S17. adding NO2-Eu-MOF nanosheet solution and 1,4-phenylenediphosphonic acid solution, reacting at room temperature, and detecting the fluorescence intensity with a fluorescence meter; S18. Substitute the measured fluorescence intensity into the fluorescence intensity-concentration working curve of the fluorescence sensor to calculate the corresponding concentration of thrombomodulin, thereby achieving quantitative analysis of the thrombomodulin in the sample.

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