A biosensor for detecting protein and glycosylation thereof and a method for using the same

Abstract

The present invention discloses a biosensor for detecting proteins and their glycosylation and a method for using the same, comprising: a gold electrode connected to a capture antibody for the protein to be detected, a Cu 2+ electrode connected to a detection antibody for the antigen to be detected, and a 2+ The sensor, which includes SiO2 signal probe nanoparticles and a wheat germ agglutinin probe modified with horseradish peroxidase, can detect proteins and their glycosylation levels. It offers advantages such as simplicity, high specificity, and high sensitivity, making it a promising candidate for widespread production and application.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and particularly relates to a high-sensitivity electrochemical biosensor for simultaneously detecting protein and its glycosylation level and a method for using the biosensor. Background Art

[0002] Glycosylation is one of the most common post-translational modifications of proteins and plays an important role in various pathophysiological conditions. The specificity of the sugar chains on the surface of glycosylated proteins determines different protein functions. For example, O-acetylglucose (O-GlcNAc) modification is closely associated with neurodegenerative diseases. Studies have shown that O-GlcNAc modification can improve neurodegenerative lesions and have a neuroprotective effect. Furthermore, the glycosylation level of neurofilament light chain (NFL) is closely associated with disease. O-glycosylation and phosphorylation of NFL compete with each other during neurofilament formation, jointly regulating the aggregation and dissociation of neurofilaments. Glycosylated NFL (O-NFL) may constitute a new generation of therapeutic biomarkers, and its glycosylation form can add more predictive information.

[0003] Currently, there are few reports on glycosylation level detection. Methods for detecting abnormal glycosylation levels of glycoproteins mainly include lectin blotting, glycoprotein electrophoresis, mass spectrometry, lectin capture chemiluminescence immunoassay, and lectin enzyme-linked immunosorbent assay. However, no method has been developed that can simultaneously detect both a protein and its glycosylation markers.

[0004] Enzyme-linked immunosorbent assay (ELISA) is currently the primary method for detecting NFL, with a detection limit of approximately 10 pg / mL. In recent years, emerging single-molecule array technology has reduced the detection limit of NFL to 1 pg / mL. However, the complex and expensive equipment required for this technique limits its clinical application. Surface plasmon resonance (SPR) and electrochemiluminescence (ECL) detection, which combines ECL with immunoassay, also have applications in NFL detection, but the relatively expensive equipment and complex procedures make their clinical application difficult to promote. Few reports exist on the detection of NFL glycosylation, and the detection limit of mass spectrometry separation is approximately 1 pg / mL.

[0005] Therefore, it is urgent to establish simple, rapid, sensitive and practical detection methods in the prediction, clinical diagnosis and treatment, and drug development of certain diseases that use proteins and their glycosylation levels as markers. Summary of the Invention

[0006] In view of the fact that there is no method for simultaneously detecting proteins and their glycosylation in the prior art, the first object of the present invention is to provide a biosensor for electrochemical detection of proteins and their glycosylation with high sensitivity, strong signal and specific recognition.

[0007] A biosensor for detecting proteins and their glycosylation, comprising: a gold electrode with a surface connected to a capture antibody for the protein to be detected, a Cu 2+ electrode connected to a detection antibody for the antigen to be detected, 2+ -SiO2 signal probe nanoparticles, wheat germ agglutinin probe modified with horseradish peroxidase.

[0008] Nanoparticles with low density and low nonspecific adsorption can absorb complexes of other electrical signal molecules and replace Cu 2+ -SiO2.

[0009] Other substances that can recognize GlcNAc sugar groups can also replace wheat germ agglutinin probes.

[0010] The biosensor for detecting proteins and their glycosylation, wherein the gold electrode with the detection protein capture antibody connected to the surface is obtained by introducing carboxyl groups on the surface of the gold electrode, activating the carboxyl groups, and then subjecting the detection protein capture antibody to an amidation reaction with the activated carboxyl groups. After the reaction is completed, the remaining unreacted activation sites are blocked.

[0011] Preferably, a gold electrode is reacted with a thiol containing a carboxylic acid to introduce carboxyl groups on the surface of the gold electrode, a mixed solution of NHS / EDC is used to activate the carboxyl groups on the surface of the gold electrode, and then the detection protein capture antibody is subjected to an amidation reaction with the activated carboxyl groups. After the reaction is completed, bovine serum albumin is used to block the remaining unreacted activation sites.

[0012] Furthermore, the gold electrode is immersed in a solution of a thiol compound containing a carboxylic acid group to introduce a carboxyl group. The reaction conditions are 10 to 14 hours at room temperature. The thiol compound containing a carboxyl group is a saturated fatty acid with a thiol terminal group, such as mercaptoundecanoic acid.

[0013] The concentration ratio of NHS / EDC is 0.1M:0.2-0.4M, and the condition for carboxyl activation is to react at room temperature for 20-40 minutes.

[0014] The amidation reaction between the capture antibody of the protein to be tested and the activated carboxyl group is carried out at room temperature for 1 to 3 hours; the blocking condition is to use bovine serum albumin and react at room temperature for 0.5 to 1.5 hours.

[0015] The biosensor for detecting protein and glycosylation thereof, the Cu2+ connected to the antigen detection antibody to be detected 2+ -SiO2 signal probe nanoparticle preparation process: Amino-modified SiO2 nanoparticles react with copper salt solution to obtain Cu 2+ -SiO2;Cu 2+ -SiO2 and NHS / EDC mixed solution were mixed, and the antigen detection antibody to be tested was added, stirred for reaction, washed, and then blocked.

[0016] The mass ratio of the amino-modified SiO2 to the soluble copper salt solution is 1:10-50, preferably 1:10-15, preferably any one of copper sulfate and copper chloride, and the reaction is carried out for at least 8 hours, preferably 8-14 hours, and the Cu is obtained by washing. 2+ -SiO2; the Cu 2+ -After mixing the mixed solution of SiO2 and NHS / EDC, add the detection antibody for the antigen to be tested, stir for at least 1 hour, preferably 1-3 hours, wash, and then block;

[0017] Further preferred: SiO2 nanoparticles are dispersed in a mixed solution of an inorganic acid and a small molecule alcohol in a ratio of 1:5 to 9, preferably a mixed solution of hydrochloric acid and methanol, and stirred to react at a temperature of not less than 40°C, preferably 40-60°C for at least 3 hours, preferably 3-8 hours, and 3-aminopropyltrimethoxysilane (ATPMS) is added to the resulting reaction solution, with a mass ratio of SiO2 nanoparticles to ATPMS of 1:3 to 20, preferably 1:3 to 8, and stirred to react at a temperature of not less than 40°C, preferably 40-60°C for at least 3 hours, preferably 3-8 hours, and washed to obtain amino-modified SiO2 nanoparticles.

[0018] The biosensor for detecting proteins and glycosylation thereof comprises dispersing hexadecyltrimethylammonium bromide and triethanolamine in ultrapure water, stirring and reacting at a temperature not lower than 40°C, preferably 40-60°C, for at least 20 minutes, preferably 20-50 minutes, adding a mixed solution of ethyl orthosilicate and n-hexane to the obtained reaction solution, stirring and reacting at a temperature not lower than 40°C, preferably 40-60°C, for at least 1 hour, preferably 1-3 hours, and washing to obtain SiO2 nanoparticles.

[0019] The biosensor for detecting proteins and their glycosylation, and the wheat germ agglutinin probe modified with horseradish peroxidase, are prepared by dispersing horseradish peroxidase in ultrapure water, adding sodium periodate and stirring for reaction, and dialyzing the resulting reaction solution against sodium acetate buffer overnight; adding sodium carbonate buffer to the resulting reaction solution and adjusting the pH to 8-10, then adding wheat germ agglutinin and stirring for reaction, adding sodium borohydride solution to the resulting reaction solution, and dialyzing the resulting reaction solution against PBS buffer overnight.

[0020] Preferably, horseradish peroxidase is dispersed in ultrapure water, sodium periodate is added at a mass ratio of horseradish peroxidase to horseradish peroxidase of no more than 1:20, preferably 1:1-5, and the mixture is stirred for reaction for at least 20 minutes, preferably 20-50 minutes, and the resulting reaction solution is dialyzed overnight against a 1 mM sodium acetate buffer having a pH of 4.4; 0.2 M sodium carbonate buffer having a pH of 9.6 is added to the resulting reaction solution, and the pH is adjusted to 8-10, and wheat germ agglutinin is added at a mass ratio of 1:2-5 to horseradish peroxidase, and the mixture is stirred for reaction at a temperature not higher than 8° C., preferably 2-8° C., for at least 1 hour, at least 1-3 hours, and a sodium borohydride solution is added to the resulting reaction solution to a concentration of no less than 0.5 mg / mL in the reaction solution after addition, and the resulting reaction solution is dialyzed overnight against a 10 mM PBS buffer having a pH of 7.4.

[0021] The purpose of adding sodium borohydride in the above method is to reduce the generated unstable carbon-nitrogen double bond to a stable carbon-nitrogen single bond.

[0022] The second object of the present invention is to provide a method for using the biosensor for detecting proteins and glycosylation thereof:

[0023] 1) The gold electrode with the capture antibody of the protein to be tested is first reacted with the antigen to be tested, and then reacted with the Cu electrode with the detection antibody of the antigen to be tested. 2+ -SiO2 signal probe nanoparticles for antigen-antibody reaction;

[0024] 2) The gold electrode obtained in step 1) was reacted with a wheat germ agglutinin probe modified with horseradish peroxidase.

[0025] Furthermore,

[0026] The antigen-antibody reaction time in step 1) is at least 1 hour, preferably 1-3 hours;

[0027] The reaction time of the gold electrode and the horseradish peroxidase-modified wheat germ agglutinin probe in step 2) is at least 0.5 h, preferably 0.5-1.5 h.

[0028] Furthermore, after the reaction in step 1 and step 2), differential pulse voltammetry scanning is performed in a 0.2-0.05 M phosphate buffer system with a pH of 4-7 to record the response current.

[0029] In all the steps of the present invention, bovine serum albumin is preferably used to block unreacted activation sites.

[0030] The present invention prepares PBS solutions containing proteins of different concentrations and different glycosylation levels as standard solutions, and uses a standard solution with a 0 content as a blank sample. The blank standard sample is tested, and 3 times the standard deviation of the measured signal value is used as the detection limit of the sample.

[0031] Preparation of PBS solutions of proteins with different concentrations: After glycosylation treatment, the purchased protein was diluted to a concentration of 1ug / mL, and then diluted stepwise with 10mM PBS (pH 7.4) to concentrations such as 100ng / mL, 1ng / mL, 100pg / mL, 50pg / mL, 20pg / mL, 10pg / mL, and 1pg / mL. Preparation of PBS solutions of proteins with different glycosylation levels: First, prepare a certain concentration of unglycosylated protein and glycosylated protein solutions, take different volume ratios and mix the two protein solutions to prepare a series of solutions with different glycosylation levels such as 5%, 10%, 20%, 30%, and 50%, but the same total protein concentration.

[0032] When the biosensor of the present invention detects a target protein, the target protein is fixed on the surface of the gold electrode, which effectively reduces background interference during the test and has higher selectivity and sensitivity.

[0033] The present invention uses a Cu2+ antibody to connect the antigen to be detected. 2+ -SiO2 signal probe nanoparticles, the role of the antigen detection antibody is to specifically recognize the antigen to be tested, Cu 2+ As a signal molecule, SiO2 acts as a carrier of the two, and there is an obvious reduction peak near 0.1V when measuring differential pulse voltammetry.

[0034] In the horseradish peroxidase-modified wheat germ agglutinin (WGA-HRP probe) probe used in the present invention, the wheat germ agglutinin (WGA) functions to specifically recognize sugar groups on proteins, and the horseradish peroxidase HRP serves as a signal molecule. When measuring differential pulse voltammetry, the test system contains a corresponding substrate of HRP, such as hydrogen peroxide, and there is a clear reduction peak near -0.2V.

[0035] Compared with the existing technology, the technical solution of the present invention has the following advantages: the present invention provides a convenient, rapid, low-cost, and highly sensitive biosensor for the simultaneous detection of proteins and their glycosylation levels, with a low detection limit (0.2-0.1 pg / mL) and a wide linear range (0.2-100 pg / mL).

[0036] The nano-mesoporous silica used in this invention is synthesized using the inverse microemulsion method. The synthesized silica has a particle size of 30-40 nm, a 2-3 nm mesoporous structure, a rough surface, and a uniform particle size. Compared to conventional synthetic nano-silica, it has a smaller particle size, better dispersibility and stability, and is more suitable for surface modification.

[0037] The sensor's selectivity was tested using a series of proteins, including 10 pg / mL NFL, 10 pg / mL L-GlcNFL, 1 ng / mL L-Aβ40, 1 ng / mL BDNF, 1 ng / mL IF-17, 1 ng / mL IgG, and 1 ng / mL L-Myo. Peak DPV signals were read near 0.08 V and -0.2 V. The sensor exhibited excellent selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the experimental principle of the present invention for detecting neurofilament light chain and its glycosylation.

[0039] Figure 2 This is the SEM image of the prepared nano-silica.

[0040] Figure 3 is the gel electrophoresis image of the synthesized WGA-HRP probe;

[0041] From left to right: WGA, standard protein, synthetic WGA-HRP, and HRP.

[0042] Figure 4 This is the differential pulse voltammetry curve for detecting different concentrations of neurofilament light chain.

[0043] Figure 5 This is the differential pulse voltammetry curve for detecting different concentrations of glycosylated neurofilament light chain.

[0044] Figure 6 The figure shows the standard curve of glycosylated and unglycosylated neurofilament light chain within a certain concentration range and the corresponding peak current (0.1V) of differential pulse voltammetry test.

[0045] Figure 7 This is a standard curve diagram of glycosylated neurofilament light chain and the corresponding differential pulse voltammetry test peak current (-0.2V) within a certain concentration range.

[0046] Figure 8 This is the differential pulse voltammetry curve for detecting different glycosylation levels of neurofilament light chain at a concentration of 20 pg / mL.

[0047] Figure 9 The sensor selectivity is 1 (-0.2V differential pulse voltammetry test peak current plot).

[0048] Figure 10 Sensor selectivity 2 (0.1V differential pulse voltammetry test peak current plot). DETAILED DESCRIPTION

[0049] The following examples are intended to further illustrate the present invention, but are not intended to limit the scope of protection of the claims of the present invention.

[0050] NFL Ab1 and Ab2 antibodies were purchased from Sangon Biotechnology and Immunoway, respectively, and both were modified with amino and carboxyl groups.

[0051] 1)Cu 2+ -Preparation method of SiO2-Ab2 nanoprobe

[0052] First, 3.0g hexadecyltrimethylammonium bromide and 0.1g triethanolamine were dispersed in 30mL ultrapure water and stirred at 60℃ for 30min. Then, a mixed solution containing 1.6mL ethyl orthosilicate and 4.8mL n-hexane was added dropwise to the obtained reaction solution, and the mixture was stirred at 60℃ for 2h, and then centrifuged to obtain SiO2 nanoparticles. The synthesized SiO2 nanoparticles were then dispersed in a mixed solution of 40mL methanol and hydrochloric acid (methanol: hydrochloric acid volume ratio of 40:8), stirred at 60℃ for 6h, and then 0.2mL 3-aminopropyltrimethoxysilane was added to the obtained reaction solution, stirred at 60℃ for 4h, and centrifuged to obtain amino-modified SiO2 nanoparticles. 1mg of the synthesized amino-modified nano-SiO2 was dispersed in 1mL of 100mM copper chloride solution and allowed to stand at room temperature for 12h. The nano-Cu adsorbed copper ions were obtained by centrifugation. 2+ -SiO2; then 30μLCu 2+ -SiO2 solution (1 mg / mL) was mixed with 30 μL of NHS / EDC (0.2M / 0.8M) solution, and then 10 μL of neurofilament protein Ab2 (100 μg / mL) was added. The mixture was stirred at room temperature for 3 h and centrifuged for washing. Bovine serum albumin (1%) was used for blocking. The reaction conditions were stirring at room temperature for 2 h and centrifuging to obtain Cu 2+ -SiO2-Ab2 nanoprobe.

[0053] 2) Preparation of WGA-HRP probe

[0054] 5 mg of horseradish peroxidase was dispersed in 1 mL of ultrapure water, 50 μL of 0.1 M sodium periodate was added, and the mixture was stirred at room temperature in the dark for 20 minutes. The resulting reaction solution was then dialyzed overnight in 1 mM sodium acetate buffer at pH 4.4. 5 μL of 0.2 M sodium carbonate buffer at pH 9.6 was then added to the resulting reaction solution to adjust the pH to approximately 9. 4 mg of wheat germ agglutinin was immediately added and the mixture was reacted at 4°C for 4 hours. 25 μL of 4 mg / mL sodium borohydride solution was then added to the resulting reaction solution. The resulting reaction solution was then dialyzed overnight in 10 mM PBS buffer at pH 7.4 to obtain WGA-HRP.

[0055] 3) Preparation of an electrochemical sensor for simultaneous detection of neurofilament light chain and its glycosylation

[0056] A gold electrode was immersed in 100 μL of a 4 mM 1-mercaptoundecanoic acid ethanol solution, completely submerged in the solution, and allowed to stand at room temperature for 12 hours to introduce carboxyl groups onto the surface of the gold electrode. The carboxyl groups of the gold electrode modified with the carboxyl groups were activated in 40 μL of an equal volume mixed solution of NHS / EDC (0.1 M / 0.4 M) for 30 minutes. After activation, 5 μL of 1 ug / mL NFL antibody Ab1 was added dropwise to the electrode surface and allowed to react at room temperature for 3 hours to allow the NFL antibody Ab1 to attach to the electrode surface. After the reaction was complete, 5 μL of 1% bovine serum albumin was added dropwise to the electrode surface and allowed to stand at room temperature for 1 hour to block unreacted activation sites.

[0057] 4) Establish a standard curve

[0058] Different concentrations of NFL (1 pg / mL, 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 1000 pg / mL) and different concentrations of O-GlcNFL (glycosylated NFL) (0.25 pg / mL, 1 pg / mL, 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 1000 pg / mL) were added dropwise to the surface of the gold electrode that was connected to the antibody Ab1 and blocked, and then reacted with the Cu prepared above in turn. 2+ After the SiO2-Ab2 nanoprobe and WGA-HRP probe reacted for 1 h, differential pulse voltammetry was measured in PBS buffer (0.1 M, pH 6.0, containing 10 μM H2O2). Different electrochemical DPV response signals were obtained at different concentrations, and a standard curve was established.

[0059] To prepare PBS solutions of proteins with different glycosylation levels, first prepare a certain concentration of unglycosylated protein and glycosylated protein solutions, take different volume ratios and mix the two protein solutions to prepare a series of solutions with different glycosylation levels but the same total protein concentration.

[0060] The present invention constructs an electrochemical sensor that uses copper ions and horseradish peroxidase electrochemical reduction as electrical signals to simultaneously detect NFL content and its glycosylation level. This electrochemical sensor modifies the surface of a gold electrode with an NFL antibody Ab1 to capture NFL on the electrode surface. Mesoporous nanosilica, linked to the NFL antibody Ab2, is then adsorbed onto the electrode surface. The copper ions adsorbed on the mesoporous nanosilica serve as a signal for measuring NFL content. Wheat germ agglutinin (WGA) is used to identify NFL glycosylation sites, and the wheat germ agglutinin-linked horseradish peroxidase catalyzes hydrogen peroxide reduction to reflect the glycosylation level of NFL.

[0061] The present invention uses a Cu2+ antibody to connect the antigen to be detected. 2+ -SiO2 signal probe nanoparticles, the role of the antigen detection antibody is to specifically recognize the antigen to be tested, Cu 2+ As a signal molecule, SiO2 acts as a carrier of the two, and there is an obvious reduction peak near 0.1V when measuring differential pulse voltammetry.

[0062] In the wheat germ agglutinin (WGA-HRP probe) probe modified with horseradish peroxidase used in the present invention, the wheat germ agglutinin functions to specifically recognize sugar groups on proteins, and HRP serves as a signal molecule. When measuring differential pulse voltammetry, the test system contains hydrogen peroxide, the corresponding substrate of HRP, and there is a clear reduction peak near -0.2V.

[0063] Table 1 Comparison of the detection method of NFL using the present invention with other methods (no relevant literature on the detection of glycosylated NFL has been found so far)

[0064]

[0065] Table 2 Test results of NFL and its glycosylation content in ten-fold diluted serum using the detection method of the present invention (n=3)

[0066]

[0067] AD refers to Alzheimer's disease patients

[0068] Table 3: Recovery test results of NFL in serum after ten-fold dilution using the detection method of the present invention (n=3)

[0069]

[0070] Table 4: Recovery test results of O-GlcNFL in serum using the detection method of the present invention (n=3)

[0071]

[0072]

Claims

1. A biosensor for detecting NFL protein and its glycosylation, characterized in that: include: The surface is connected to the gold electrode with the capture antibody of the protein to be tested, and the surface is connected to the Cu electrode with the detection antibody of the antigen to be tested. 2+ -SiO2 signal probe nanoparticles, wheat germ agglutinin probe modified with horseradish peroxidase.

2. The biosensor for detecting NFL protein and its glycosylation according to claim 1, characterized in that: The gold electrode with the test protein capture antibody connected to the surface is obtained by introducing carboxyl groups on the gold electrode surface, activating the carboxyl groups, and then subjecting the test protein capture antibody to an amidation reaction with the activated carboxyl groups. After the reaction is completed, the remaining unreacted activation sites are blocked.

3. The biosensor for detecting NFL protein and its glycosylation according to claim 2, characterized in that: The gold electrode is reacted with a thiol containing carboxylic acid to introduce carboxyl groups on the surface of the gold electrode. The carboxyl groups on the surface of the gold electrode are activated using a mixed solution of NHS / EDC. The protein capture antibody to be tested is then subjected to an amidation reaction with the activated carboxyl groups. After the reaction is completed, bovine serum albumin is used to block the remaining unreacted activation sites.

4. The biosensor for detecting NFL protein and its glycosylation according to claim 1, characterized in that: The Cu2+ connected to the antigen detection antibody to be detected 2+ -SiO2 signal probe nanoparticle preparation process: Amino-modified SiO2 nanoparticles react with copper salt solution to obtain Cu 2+ -SiO2;Cu 2+ After mixing the mixed solution of -SiO2 and NHS / EDC, add the antigen detection antibody to be tested, stir the reaction, wash and block.

5. The biosensor for detecting NFL protein and its glycosylation according to claim 4, characterized in that: The mass ratio of the amino-modified SiO2 to the soluble copper salt solution is 1:10-50, and the reaction is carried out for at least 8 hours, and the Cu 2+ -SiO2; the Cu 2+ After mixing the mixed solution of -SiO2 and NHS / EDC, add the detection antibody for the antigen to be tested, stir for at least 1 hour, wash, and then block.

6. The biosensor for detecting NFL protein and its glycosylation according to claim 4, characterized in that: The SiO2 nanoparticles are dispersed in a mixed solution of an inorganic acid and a small molecule alcohol in a volume ratio of 1:5~9, and the mixture is stirred and reacted at a temperature not lower than 40°C for at least 3 hours. 3-aminopropyltrimethoxysilane is added to the resulting reaction solution, and the mass ratio of SiO2 nanoparticles to ATPMS is 1:3~20. The mixture is stirred and reacted at a temperature not lower than 40°C for at least 3 hours, and washed to obtain amino-modified SiO2 nanoparticles.

7. The biosensor for detecting NFL protein and its glycosylation according to claim 4, characterized in that: Hexadecyltrimethylammonium bromide and triethanolamine are dispersed in ultrapure water, stirred and reacted at a temperature not lower than 40°C for at least 20 minutes, and then a mixed solution of ethyl orthosilicate and n-hexane is added to the resulting reaction solution. After stirring and reacting at a temperature not lower than 40°C for at least 1 hour, SiO2 nanoparticles are washed to obtain.

8. The biosensor for detecting NFL protein and its glycosylation according to claim 1, characterized in that: The wheat germ agglutinin probe modified with horseradish peroxidase is prepared by dispersing horseradish peroxidase in ultrapure water, adding sodium periodate and stirring for reaction, and dialyzing the resulting reaction solution against sodium acetate buffer overnight; adjusting the pH of the resulting reaction solution to 8-10 by adding sodium carbonate buffer, and then adding wheat germ agglutinin and stirring for reaction, adding sodium borohydride solution to the resulting reaction solution, and dialyzing the resulting reaction solution against PBS buffer overnight to obtain the product.

9. The biosensor for detecting NFL protein and its glycosylation according to claim 8, characterized in that: The horseradish peroxidase is dispersed in ultrapure water, sodium periodate is added in a mass ratio of horseradish peroxidase to the horseradish peroxidase of not more than 1:20, and the reaction is stirred for at least 20 minutes. The resulting reaction solution is dialyzed overnight in a 1mM sodium acetate buffer having a pH of 4.

4. The pH of the resulting reaction solution is adjusted to 8-10 by adding 0.2M sodium carbonate buffer having a pH of 9.

6. Wheat germ agglutinin is added in a mass ratio of 1:2-5 to horseradish peroxidase. The reaction is stirred at a temperature not higher than 8°C for at least 1 hour. Sodium borohydride solution is added to the resulting reaction solution to a concentration of not less than 0.5 mg / mL in the reaction solution after addition. The resulting reaction solution is dialyzed overnight in a 10mM PBS buffer having a pH of 7.

4.

10. The method for using the biosensor for detecting NFL protein and its glycosylation according to any one of claims 1 to 9, characterized in that: 1) The gold electrode with the capture antibody of the protein to be tested is first reacted with the antigen to be tested, and then reacted with the Cu electrode with the detection antibody of the antigen to be tested. 2+ -SiO2 signal probe nanoparticles for antigen-antibody reaction; 2) The gold electrode prepared in step 1) was reacted with a wheat germ agglutinin probe modified with horseradish peroxidase.

11. The method of use according to claim 10, characterized in that: The antigen-antibody reaction time in step 1) is at least 1 hour; The reaction time of the gold electrode and the horseradish peroxidase-modified wheat germ agglutinin probe in step 2) is at least 0.5 h.

12. The method of use according to claim 10, characterized in that: After the reaction in step 1 and step 2), differential pulse voltammetry was performed in a 0.2-0.05 M phosphate buffer system with a pH of 4-7 to record the response current.

13. The method of use according to claim 12, characterized in that: The Cu2+ antibody used to detect the antigen to be detected 2+ -SiO2 signal probe nanoparticles, the role of the antigen detection antibody is to specifically recognize the antigen to be tested, Cu 2+ As a signal molecule, SiO2 acts as a carrier of the two, and there is a reduction peak at 0.1V when measuring differential pulse voltammetry.

14. The method of use according to claim 12, wherein: In the wheat germ agglutinin probe modified with horseradish peroxidase, the wheat germ agglutinin specifically recognizes the sugar groups on the protein. HRP is used as a signal molecule. When measuring differential pulse voltammetry, the test system contains hydrogen peroxide, the substrate corresponding to HRP, and there is a reduction peak at -0.2V.

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