Fluorescence immunosensor for detecting thromboregulatory protein and preparation method of fluorescence immunosensor

Abstract

The invention discloses a fluorescence immunosensor for detecting thromboregulatory protein as well as a preparation method and a use method of the fluorescence immunosensor. The preparation method comprises the following steps: S5, adding a prepared buffer solution dissolved in Tris-HCl and a PNPP solution, and incubating; s6, dispersing nanoflowers stacked by fluorescent two-dimensional metal organic framework material Eu-MOF nanosheets into a Tris-HCl buffer solution, and carrying out ultrasonic treatment and oscillation to prepare an Eu-MOF nanosheet solution; and S7, adding the Eu-MOF nanosheet solution to ensure the total volume for testing, directly starting detection without any treatment, detecting the fluorescence intensity in the presence of different concentrations of thromboregulatory proteins by using a luminoscope, and constructing a fluorescence intensity-concentration working curve. According to the invention, the synthesized two-dimensional metal organic framework material Eu-MOF nanosheet has the characteristic of red fluorescence, so that the concentration of thromboregulatory protein in a sample is quantitatively analyzed.

Description

Technical Field

[0001] This invention belongs to the field of lanthanide metal-organic framework materials for the analysis and detection of cardiovascular disease biomarkers, specifically relating to a fluorescent immunosensor for detecting thrombotic regulatory proteins and its preparation method. Background Technology

[0002] Thrombomomodulin (TM) is a transmembrane glycoprotein primarily expressed on the surface of vascular endothelial cells. It exerts its anticoagulant, anti-inflammatory, and endothelial protective functions mainly by binding to thrombin and activating the protein C system. In cardiovascular disease, endothelial injury leads to the detachment of thrombomodulin from the endothelial cell surface, releasing it into the bloodstream to form soluble thrombomodulin (sTM). Elevated plasma concentrations of sTM sensitively reflect vascular risk and the severity of endothelial injury. Studies have shown that TM (or sTM) levels are closely related to cardiovascular events such as atherosclerosis, disseminated intravascular coagulation, and cardiogenic stroke. It can serve as a biomarker for assessing endothelial dysfunction, cardiovascular disease progression, and prognosis, and has potential clinical value, especially in the early diagnosis of high-risk cardiovascular patients and in guiding anticoagulation therapy.

[0003] Metal-organic frameworks (MOFs) are a class of porous crystalline materials composed of metal ions or metal clusters and organic ligands self-assembled through coordination bonds. Lanthanide metal-organic frameworks (Ln-MOFs) are a class of nanomaterials with even more unique luminescent properties. Ln-MOFs possess both the unique metal-ligand structure of MOFs and the unique photoluminescence (PL) characteristics of lanthanide metal ions, laying the foundation for their applications in multiple fields. Furthermore, Ln-MOFs are considered potential fluorescent probes for analytical detection due to their broad emission spectrum from blue to infrared. Fluorescence spectroscopy is a sensitive optical detection technique that achieves qualitative identification or quantitative analysis of substances by measuring the characteristic fluorescence spectrum emitted after excitation by light of a specific wavelength. The optical properties of Ln-MOFs are highly compatible with the principles of fluorescence detection. The internal filtration effect (IFE) refers to the phenomenon where light-absorbing substances in a sensing system absorb the excitation or emission light of fluorescent materials, leading to fluorescence quenching; it is a special non-radiative energy transfer model in fluorescence spectroscopy. Compared to fluorescence analysis techniques such as fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET), the IFE mechanism is easier to implement, has extremely high regulatory flexibility, and does not require establishing a complicated chemical connection between two substances. Therefore, it is increasingly being used in the field of fluorescence sensing.

[0004] Chinese patent document CN119716036A discloses a method for preparing a fluorescent sensor for detecting thrombomodulin. This method utilizes the reaction of non-fluorescent two-dimensional metal-organic framework material NO2-Eu-MOF nanosheets with 1,4-phenylenediamine. The reaction involves competitive coordination between 1,4-phenylenediamine and ligands in the NO2-Eu-MOF nanosheets, thereby enabling the detection of thrombomodulin. 3+ The coordination environment changes, which is different from the principle of the present invention; and CN119716036A provides the opposite technical inspiration to those skilled in the art by using NO2-Eu-MOF nanosheets, which are two-dimensional metal-organic framework materials without fluorescence, so that those skilled in the art will not have the technical inspiration to directly construct an immunosensor to detect proteins in serum based on the internal filtration effect of Ln-MOF nanosheets, but instead choose to compete for coordination with 1,4-phenylenediamine.

[0005] To our knowledge, there are currently no reports on constructing an immunosensor based on the internal filtration effect of Ln-MOF nanosheets for detecting proteins in serum. We synthesized Eu-MOF nanosheets emitting characteristic red fluorescence using the antenna ligand H2bpydb via a solvothermal method and characterized the prepared Eu-MOF nanosheets. When the analyte TM is present in the system, ALP is introduced through a sandwich structure of capture antibody / TM / detection antibody formed by an enzyme-linked immunosorbent assay (ELISA). 4-Nitrophenyl phosphate (PNPP) serves as the substrate for ALP and can be enzymatically hydrolyzed to 4-nitrophenol (4-NP). 4-NP triggers the strong internal filtration effect of the Eu-MOF nanosheets, quenching its red fluorescence. The degree of quenching is proportional to the concentration of the analyte TM. Therefore, a fluorescent immunosensor was constructed based on this to achieve specific and sensitive detection of TM in serum. Summary of the Invention

[0006] Therefore, the purpose of this invention is to address the shortcomings of existing technologies by providing a fluorescent immunosensor for detecting thrombomodulin and its preparation method. This invention utilizes the red fluorescence properties of the synthesized two-dimensional metal-organic framework material Eu-MOF nanosheets and constructs an analytical detection method for thrombomodulin based on an enzyme-linked immunosorbent assay (ELISA) induced by alkaline phosphatase, thereby achieving quantitative analysis of the concentration of thrombomodulin in samples.

[0007] The technical solution adopted is as follows:

[0008] A method for preparing a fluorescent immunosensor for detecting thrombomodulin, comprising the following steps:

[0009] S1. Add the capture antibody for thrombomodulin to the ELISA plate;

[0010] S2. Add the prepared bovine serum albumin and incubate to block non-specific binding sites;

[0011] S3. Add the prepared thrombomodulin solutions of different concentrations and incubate;

[0012] S4. Add the detection antibody of thrombomodulin modified with alkaline phosphatase and incubate;

[0013] S5. Add the prepared Tris-HCl buffer solution and PNPP solution, and incubate.

[0014] S6. Disperse the nanoflowers formed by stacking fluorescent two-dimensional metal-organic framework material Eu-MOF nanosheets in Tris-HCl buffer solution and sonicate and vibrate to prepare Eu-MOF nanosheet solution;

[0015] S7. After adding Eu-MOF nanosheet solution to ensure the total volume available for testing, no further treatment is required to start the detection directly. Use a fluorescence spectrometer to detect the fluorescence intensity in the presence of different concentrations of thrombomodulin and construct a fluorescence intensity-concentration working curve.

[0016] Further, in S6, the Eu-MOF nanosheet solution is prepared by the following method: nanoflowers formed by stacking fluorescent two-dimensional metal-organic framework material Eu-MOF nanosheets are dispersed in a 50 mM Tris-HCl buffer solution with pH=9, sonicated for 30 min, and oscillated to make the concentration of Eu-MOF nanosheet material in the solution 400 μg / mL.

[0017] Further, in S6, the two-dimensional metal-organic framework material Eu-MOF nanosheets are prepared by the following method, including the following steps:

[0018] S11. Dissolve 10.0-300.0 mg europium(III) nitrate hexahydrate in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex.

[0019] S12. Dissolve 10.0-300.0 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex.

[0020] S13. Mix the solutions from S11 and S12, add 20-100 μL of concentrated nitric acid to adjust the pH of the solution, transfer it to a stainless steel reactor with a polytetrafluoroethylene liner, and react at 50.0-200.0 ℃ for 6-48 hours;

[0021] S14. A pale yellow product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60-100 °C to obtain ultrathin Eu-MOF nanosheets.

[0022] Further, in S1, 100 μL of thrombomodulin capture antibody at a concentration of 5 μg / mL was added to a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with TBST to remove any thrombomodulin capture antibody that was not immobilized on the microplate.

[0023] Further, in S2, 100 µL of prepared 1 vol% bovine serum albumin was added and incubated at 37°C for 1 h to block non-specific binding sites. The mixture was then washed three times with TBST to remove excess bovine serum albumin.

[0024] Further, in S3, 100 µL of prepared thrombomodulin at different concentrations was added, and the mixture was incubated at 37°C for 1 hour. The mixture was then washed three times with TBST to thoroughly remove unbound thrombomodulin.

[0025] Further, in S4, 100 μL of the prepared detection antibody modified with alkaline phosphatase and thrombomodulin (80 μg / mL) was added, and the mixture was incubated at 37 °C for 1 h. The mixture was then washed three times with 50 mM Tris-HCl buffer solution (pH=9.0) to remove non-specifically bound detection antibody.

[0026] Further, in S5, 140 μL of 50 mM Tris-HCl buffer solution (pH=9) and 10 μL of 6 mM PNPP solution were added, and the mixture was incubated at 37°C for 30 min.

[0027] Furthermore, in S7, 50 μL of Eu-MOF nanosheet solution at 400 μg / mL was added to ensure a total volume of 200 µL.

[0028] The present invention provides a fluorescent immunosensor for detecting thrombomodulin, which is prepared by the preparation method described above.

[0029] The beneficial technical effects of this invention are as follows:

[0030] The detection method of this invention overcomes the shortcomings of existing single detection methods for thrombomodulin detection technology. It utilizes the fluorescent properties of the synthesized two-dimensional metal-organic framework material Eu-MOF and constructs an enzyme-linked immunosorbent assay (ELISA) based on the internal filtration effect of Eu-MOF nanosheets to build a fluorescent immunosensor for qualitative and quantitative analysis and detection of thrombomodulin.

[0031] This invention utilizes an enzyme-linked immunosorbent assay (ELISA) triggered by alkaline phosphatase to detect the fluorescence intensity of thrombomodulin at different concentrations, constructing a fluorescence intensity-concentration working curve to create a fluorescent immunosensor. First, a fluorescence spectrometer is used to detect the fluorescence intensity of Eu-MOF nanosheets in the presence of different concentrations of thrombomodulin in the sample. Then, the fluorescence intensity is substituted into the working curve to achieve quantitative analysis of the concentration of thrombomodulin in the sample. Attached Figure Description

[0032] Figure 1 This diagram illustrates the synthesis strategy of Eu-MOF NS and a schematic diagram of a fluorescent immunosensor constructed based on its internal filtering effect for TM detection.

[0033] Figure 2 This is a scanning electron microscope (SEM) characterization image of Eu-MOF nanosheets.

[0034] Figure 3 Figure showing a feasibility study of a fluorescent immunosensor based on Eu-MOF nanosheets for detecting different concentrations of blood circulation regulatory proteins.

[0035] Figure 4 Fluorescence spectra of different concentrations of thrombomodulin detected by a fluorescence immunosensor based on Eu-MOF nanosheets, and working curves of fluorescence detection of thrombomodulin (a, b). Detailed Implementation

[0036] The present invention will be described in detail below through specific embodiments. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual protection scope of the present invention, nor are they intended to limit the protection scope of the present invention to this.

[0037] Among them, Tris-HCl buffer solution is tris(hydroxymethyl)aminomethane-hydrochloride buffer solution; PNPP solution is disodium p-nitrophenyl phosphate solution; TBST is Tris buffer containing Tween 20, which is a mixture of Tris buffer and Tween-20; Eu-MOF NS is europium metal-organic framework material nanosheet, NS is nanosheet; ALP is alkaline phosphatase; Ab is antibody.

[0038] The working principle of the fluorescent immunosensor of the present invention is as follows:

[0039] In this invention, a TM fluorescent immunosensor is constructed based on the internal filtration effect of Eu-MOF nanosheets, using an alkaline phosphatase-induced enzyme-linked immunosorbent assay (ELISA). When only the substrate PNPP is present, Eu-MOF NS retains its red fluorescence. When the analyte TM is introduced, due to the specific binding of the antigen and antibody, a sandwich-like structure of Ab1-TM-Ab2-ALP is formed. The ALP coupled to Ab2 catalyzes the decomposition of the substrate PNPP into 4-NP, which quenches the red fluorescence of Eu-MOF NS. The quenching effect increases with increasing TM concentration. Based on the fluorescence intensity change detected under 375 nm excitation light, a fluorescent immunosensor capable of detecting TM concentration is constructed. The detection principle is as follows: Figure 1 As shown.

[0040] The presence or absence of thrombomodulin and its concentration in the system correspond to specific fluorescence intensity values, enabling the analysis and detection of thrombomodulin. The feasibility of this detection is as follows: Figure 3 As shown.

[0041] Example 1

[0042] A method for preparing a fluorescent immunosensor for detecting thrombomodulin, comprising the following steps:

[0043] 1. First, synthesize Eu-MOF nanosheet materials, following these steps:

[0044] (1) Dissolve 44.606 mg europium(III) nitrate hexahydrate in 5 mL of N,N-dimethylformamide solvent, and sonicate and vortex.

[0045] (2) Dissolve 19.82 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 5 mL of N,N-dimethylformamide solvent, and sonicate and vortex.

[0046] (3) Mix the solutions from steps (1) and (2), add 50 μL of concentrated nitric acid to adjust the pH of the solution, transfer it to a stainless steel reactor with a polytetrafluoroethylene liner, and react at 180 °C for 12 hours.

[0047] (4) A pale yellow product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60 °C to obtain ultrathin Eu-MOF nanosheets, such as... Figure 2 As shown.

[0048] 2. Add the thrombomodulin capture antibody (5 μg / mL, 100 μL) to a 96-well microplate, incubate overnight at 4°C, and wash three times with TBST to remove the thrombomodulin capture antibody that is not immobilized on the microplate.

[0049] 3. Add the prepared bovine serum albumin (1%, 100 µL) and incubate at 37°C for 1 h to block non-specific binding sites. Wash three times with TBST to remove excess bovine serum albumin.

[0050] 4. Add different concentrations of prepared thrombomodulin (100 µL), incubate at 37°C for 1 h, and wash three times with TBST to thoroughly remove unbound thrombomodulin;

[0051] 5. Add the prepared detection antibody (80 μg / mL, 100 μL) modified with alkaline phosphatase to the thrombomodulin, incubate at 37 °C for 1 h, and wash three times with Tris-HCl buffer (50 mM, pH=9.0) to remove non-specifically bound detection antibody.

[0052] 6. Add 140 μL Tris-HCl buffer solution (50 mM, pH=9) and 10 μL PNPP (6 mM) solution and incubate at 37℃ for 30 min;

[0053] 7. The nanoflowers formed by stacking fluorescent two-dimensional metal-organic framework material Eu-MOF nanosheets were dispersed in Tris-HCl buffer solution (50 mM, pH=9) and sonicated for 30 min, followed by oscillation. The concentration of Eu-MOF nanosheet material in the solution was 400 μg / mL.

[0054] 8. After adding 50 μL of 400 μg / mL Eu-MOF nanosheet solution to ensure a total volume of 200 µL, no further treatment is required to start the detection. Use a fluorescence spectrometer to detect the fluorescence intensity in the presence of different concentrations of thrombomodulin and construct a fluorescence intensity-concentration working curve.

[0055] The results showed that when the concentration of thrombomodulin was 100 pg / mL -1 Up to 10 μg mL -1 Within the specified concentration range, the fluorescence intensity of the system exhibits a linear relationship with the concentration (FL = -38.25lgC). TM +295.67, R 2 = 0.994), with a detection limit of 36.4 pg / mL. -1 ,like Figure 4 As shown.

[0056] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent immunosensor for detecting thrombomodulin, characterized in that, Includes the following steps: S1. Add the capture antibody of thrombomodulin to the ELISA plate; S2. Add the prepared bovine serum albumin and incubate to block non-specific binding sites; S3. Add the prepared thrombomodulin solutions of different concentrations and incubate; S4. Add the detection antibody of thrombomodulin modified with alkaline phosphatase and incubate; S5. Add the prepared Tris-HCl buffer solution and PNPP solution, and incubate. S6. Disperse the nanoflowers formed by stacking fluorescent two-dimensional metal-organic framework material Eu-MOF nanosheets in Tris-HCl buffer solution and sonicate and vibrate to prepare Eu-MOF nanosheet solution; S7. After adding Eu-MOF nanosheet solution to ensure the total volume available for testing, no further treatment is required to start the detection directly. Use a fluorescence spectrometer to detect the fluorescence intensity in the presence of different concentrations of thrombomodulin and construct a fluorescence intensity-concentration working curve.

2. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 1, characterized in that, In S6, the Eu-MOF nanosheet solution is prepared by the following method: nanoflowers formed by stacking fluorescent two-dimensional metal-organic framework material Eu-MOF nanosheets are dispersed in a 50 mM Tris-HCl buffer solution with pH=9, sonicated for 30 min, and oscillated to make the concentration of Eu-MOF nanosheet material in the solution 400 μg / mL.

3. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 2, characterized in that, In S6, the two-dimensional metal-organic framework material Eu-MOF nanosheets are prepared by the following method, including the following steps: S11. Dissolve 10.0-300.0 mg europium(III) nitrate hexahydrate in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex. S12. Dissolve 10.0-300.0 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex. S13. Mix the solutions from S11 and S12, add 20-100 μL of concentrated nitric acid to adjust the pH of the solution, transfer it to a stainless steel reactor with a polytetrafluoroethylene liner, and react at 50.0-200.0 ℃ for 6-48 hours; S14. A pale yellow product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60-100 °C to obtain ultrathin Eu-MOF nanosheets.

4. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 1, characterized in that, In S1, 100 μL of thrombomodulin capture antibody (5 μg / mL) was added to a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with TBST to remove any thrombomodulin capture antibody that was not immobilized on the microplate.

5. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 4, characterized in that, In S2, add 100 µL of the prepared 1 vol% bovine serum albumin and incubate at 37°C for 1 h to block non-specific binding sites. Wash three times with TBST to remove excess bovine serum albumin.

6. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 5, characterized in that, In S3, add 100 µL of prepared thrombomodulin at different concentrations, incubate at 37°C for 1 h, and wash three times with TBST to thoroughly remove unbound thrombomodulin.

7. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 6, characterized in that, In step S4, add 100 μL of the prepared detection antibody modified with alkaline phosphatase (80 μg / mL) to the thrombomodulin, incubate at 37 °C for 1 h, and wash three times with 50 mM Tris-HCl buffer (pH 9.0) to remove non-specifically bound detection antibody.

8. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 7, characterized in that, Add 140 μL of 50 mM Tris-HCl buffer solution (pH=9) and 10 μL of 6 mM PNPP solution to S5, and incubate at 37°C for 30 min.

9. The method for preparing a fluorescent immunosensor for detecting thrombomodulin according to claim 7, characterized in that, In S7, 50 μL of Eu-MOF nanosheet solution with a concentration of 400 μg / mL was added to ensure a total volume of 200 µL.

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

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