A preparation method of a glycoprotein molecular imprinting electrochemical sensor

Abstract

The application provides a self-supporting electrode of transition metal doped carbon nanofiber prepared by an electrostatic spinning technology, and a method for preparing a glycoprotein molecular imprinting electrochemical sensor which can be detected. The molecular imprinting electrochemical sensor takes a carbon nanofiber sheet as a working electrode, and further selectively detects practical samples.

Description

Technical Field

[0001] This invention relates to sensor fabrication technology, specifically a method for fabricating a glycoprotein molecularly imprinted electrochemical sensor. Background Technology

[0002] Glycoproteins play crucial roles in biological processes such as molecular recognition, intercellular and intracellular signal transduction, and immune responses. Structural changes and aberrant expression of glycoproteins are closely related to the occurrence and development of diseases; therefore, glycoprotein detection is of significant value for disease diagnosis and prognosis. However, the complex composition of biological samples and the generally low abundance of protein molecules make the analysis and detection of marker glycoprotein molecules challenging. Selective detection of glycoproteins remains a challenging problem to be solved. Currently, conventional detection techniques for glycoproteins mainly focus on multidimensional instrumentation methods, such as high-performance liquid chromatography-mass spectrometry, surface plasmon resonance, quartz crystal microbalance, and resonance Rayleigh scattering. However, these methods not only require complex and expensive instruments but also necessitate complex separation and processing of the target protein, resulting in complex, time-consuming, or labor-intensive experimental procedures. Therefore, designing artificial biomimetic recognition materials with high glycoprotein affinity and selective recognition properties holds great potential in various research fields.

[0003] In recent years, the research on combining molecularly imprinted polymers (MIPs) and electrochemical sensing technology to prepare molecularly imprinted electrochemical sensors has become a major application area in sensor development. This method can produce high-sensitivity detection sensors that are low-cost, accurate, and simple to use. However, the structural properties of proteins mean that protein-template imprinting is not as simple as that of small molecules. For protein molecular imprinting, on the one hand, the large mass transfer resistance caused by the large size of proteins makes it difficult for template molecules to approach MIPs and also difficult to elute from the material; on the other hand, proteins, as biological macromolecules, have complex structures and are easily inactivated, requiring the synthesis of MIPs and the elution of templates under relatively mild natural conditions. Protein imprinting technology suffers from problems such as hole embedding, poor accessibility, uneven binding sites, and low imprinting efficiency. Furthermore, traditional molecular imprinting electrodeposition modification suffers from low imprinting efficiency and poor signal response due to the limited number of template molecules during the deposition process. How to improve the imprinting efficiency and signal response capability of electrodeposited layers has become an urgent problem to be solved in the development of molecularly imprinted biomimetic sensors. Currently, surface molecular imprinting methods have greater capabilities and wider applications in the field of protein imprinting due to their advantages such as high template utilization and simple kinetic theory. Furthermore, glycoproteins contain one or more glycosyl groups, and the cis-ortho-diol groups within these glycosyl groups can form reversible borate ester bonds with phenylboronic acid. This allows for convenient regulation of the binding and dissociation of the template glycoprotein with the functional monomer of the borate group by adjusting the pH of the environment, effectively solving the problem of difficult template removal during protein imprinting.

[0004] Therefore, addressing the problems of antibody acquisition difficulties and poor stability in the current development of glycoprotein cancer biomarker analysis systems, this invention combines electrospinning and heat treatment technologies. Utilizing the advantages of the three-dimensional nanostructure and high specific surface area of ​​electrospinned fiber materials, and employing these electrospinned carbon nanofibers with good conductivity and high loading capacity as self-supporting electrodes, this invention further combines surface-oriented molecular imprinting technology. Using horseradish peroxidase (HRP) as a glycoprotein biomarker model molecule, a surface-oriented molecular imprinted self-supporting sensing electrode for glycoprotein disease biomarkers is constructed through a dual recognition technique using boric acid affinity molecular imprinting. This establishes a highly specific and interference-resistant electrochemical immunosensing analysis method for glycoprotein detection. This technology utilizes the two-dimensional planar structure, excellent electron transfer performance, and high specific surface area of ​​electrospinned carbon nanofiber materials with a spatial network and self-supporting structure to improve the recognition efficiency and conductivity of the molecular imprinted sensor. This results in a highly sensitive and selective molecular imprinted electrochemical immunosensing electrode, enabling specific, sensitive, and rapid detection of trace amounts of the model glycoprotein molecule HRP in human serum samples. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for fabricating a molecularly imprinted electrochemical sensor capable of detecting glycoproteins, using transition metal-doped carbon nanofibers prepared by electrospinning as a self-supporting electrode. This molecularly imprinted electrochemical sensor uses carbon nanofiber sheets as the working electrode and further enables selective detection of actual samples. The specific operation is as follows:

[0006] 1. Preparation of transition metal-doped carbon nanofiber sheets (CNF) by electrospinning

[0007] (1) Dissolve polyacrylonitrile in N,N dimethylformamide (DMF) solution and stir on a magnetic stirrer until completely dissolved. Add cobalt acetate tetrahydrate and molybdenum acetylacetonate in sequence and continue stirring until a stable and homogeneous solution is formed. Then, use a syringe to draw up a certain amount of solution, clamp it and place it on a push pump, electrospin, collect the nanofiber material, and dry and store it.

[0008] (2) The above nanofiber material was cut into appropriate sizes, placed on a ceramic plate and pre-oxidized in a muffle furnace, and then carbonized in a tube furnace under an argon atmosphere. The prepared material was a flexible membrane, which was activated by soaking in concentrated nitric acid overnight, washed and dried, and then cut into 0.2×0.5cm sizes to obtain CNF as a self-supporting electrode.

[0009] 2. Fabrication of self-supported electrodes for surface-imprinted glycoprotein electrochemical sensing

[0010] (1) Gold nanoparticle modification: The cut CNF was placed in an aqueous solution of chloroauric acid, and a layer of gold nanoparticles was modified on its surface using the current-time curve method. After electroplating, it was cleaned with deionized water to obtain Au@CNF, which was then dried at room temperature.

[0011] (2) Recognition site establishment: First, the two functional monomers, 4-mercaptophenylboronic acid and L-cysteine ​​hydrochloride, were dissolved in a mixed solution of deionized water and ethanol to allow for sufficient pre-binding, forming a phenylboronic acid recognition molecule pair. Then, Au@CNF was immersed in the above mixed solution, and the phenylboronic acid recognition molecule pair was self-assembled into Au@CNF through Au-S bonds. Subsequently, the template molecule HRP was added, allowing it to covalently react with the phenylboronic acid recognition molecule pair through a cis-diol esterification covalent reaction, achieving self-assembly.

[0012] (3) Preparation of molecularly imprinted electrode: After self-assembly, an HRP molecularly imprinted electrochemical working electrode (MIPs / CNF) was prepared by electropolymerization using an L-cysteine ​​buffer solution as the electrolyte and cyclic voltammetry on the Au@CNF surface. The thickness of the polymer layer was controlled by the number of cyclic voltammetry scans.

[0013] (4) Elution to remove template molecules: MIPs / CNF were immersed in an acetic acid / acetonitrile aqueous solution and eluted by vortexing to remove the template molecule HRP, obtaining specific imprinted hole recognition sites. After complete elution of the template molecules, MIPs / CNF were washed with deionized water and dried at room temperature to obtain the surface molecularly imprinted HRP electrochemical sensor electrode. Simultaneously, a comparative experiment was conducted to prepare the corresponding non-imprinted electrochemical sensor electrode (NIPs / CNF) without the addition of template molecules, while maintaining the same steps.

[0014] The beneficial effects of this invention are:

[0015] Flexible, self-supporting carbon nanofiber sheets, prepared using electrospinning technology with polyacrylonitrile polymer as the carbon source and the addition of two transition metals, cobalt and molybdenum, possess a unique structure with a large surface area and uniform nanofiber distribution. These self-supporting carbon nanofiber sheets can be directly used as electrodes without the need for any binders, providing more active sites and facilitating rapid electron transfer, resulting in excellent electrochemical performance. Horseradish peroxidase, extracted from the plant horseradish, is one of the most common glycoproteins and is widely used as a model glycoprotein molecule in biosensing due to its low cost and easy availability. Molecularly imprinted electrochemical sensors combine the advantages of electrochemistry and molecularly imprinted polymers, exhibiting high selectivity and ease of preparation, and are widely researched and applied in numerous fields.

[0016] This invention constructs a molecularly imprinted electrochemical sensor for detecting glycoproteins based on a combination of electrospinning and molecular imprinting techniques. First, we use transition metal-doped carbon nanofiber sheets prepared by electrospinning as the support material. Their fibrous structure provides a large specific surface area, and the doping with cobalt and molybdenum inhibits nanoparticle aggregation, providing more active sites. Furthermore, the synergistic effect between cobalt and molybdenum gives the material excellent electron transfer capability, effectively solving the problem of insufficient imprinted holes and weak electron transfer due to a small imprinted layer area, thus playing a significant role in protein imprinting and detection. Second, we introduce a synergistic recognition strategy using phenylboronic acid recognition molecules. The introduced 4-mercaptophenylboronic acid and L-cysteine ​​can undergo cis-dihydroxy esterification with the target glycoprotein under neutral pH conditions, forming a stable cyclic ester structure. This enhances the material's imprinting recognition ability and overcomes the limitations of boronic acid affinity groups in glycoprotein recognition and capture, enabling good recognition performance under human environmental pH conditions, which is beneficial for the development of detection methods for real human blood samples. Furthermore, the flexible, self-supporting carbon nanosheets used in this invention possess excellent structural stability, allowing them to be directly used as working electrodes. They can also be mass-produced and stably stored, making them highly valuable for practical applications. Simultaneously, compared to existing technologies, this invention offers advantages in HRP protein recognition and detection, including a low detection limit (0.0074 pg / mL), a wide detection range (0.01 pg / mL-100 ng / mL), and a short detection time (3 min). Attached image description:

[0017] Figure 1 This is a flowchart illustrating the fabrication process of the self-supporting MIPs / CNF-HRP electrochemical sensor for detecting HRP protein molecules according to the present invention.

[0018] Figure 2 This is a comparison of CNF current response signals for doped and undoped transition metals.

[0019] Figure 3 Scanning electron microscope image of a self-supporting MIPs / CNF-HRP electrochemical sensor for detecting HRP protein molecules.

[0020] Figure 4 Cyclic voltammetry characterization of the fabrication process of the self-supporting MIPs / CNF-HRP electrochemical sensor.

[0021] Figure 5 The current signal response values ​​and corresponding fitting curves for different concentrations of HRP are shown.

[0022] Figure 6 The graph shows the selectivity test results of the self-supporting MIPs / CNF-HRP electrochemical sensor.

[0023] Figure 7 Figure showing the stability test results of the self-supporting MIPs / CNF-HRP electrochemical sensor. Detailed Implementation

[0024] To facilitate understanding of the technical solution of the present invention, the technical solution of the present invention will be further explained and described below in conjunction with specific accompanying drawings.

[0025] Example

[0026] A method for preparing a transition metal-doped electrospun molecularly imprinted electrochemical sensing electrode for detecting glycoproteins (e.g.) Figure 1 (As shown), the specific preparation steps are as follows:

[0027] (1) Fabrication of transition metal-doped carbon nanofiber (CNF) self-supporting electrodes

[0028] a. First, dissolve 1.7g of polyacrylonitrile in 15mL of N,N-dimethylformamide solution and stir on a magnetic stirrer until completely dissolved. Then, add 0.933g of cobalt acetate tetrahydrate and 0.244g of molybdenum acetylacetonate sequentially and stir until a stable and homogeneous solution is formed. Use a syringe to draw a certain volume of solution and place it on a feed pump for electrospinning. The spinning conditions are set as follows: feed speed of 1.8mL / h, voltage of 17.54kV, distance between needle and receiver of 20cm, and injection volume of 5-6mL. Finally, dry and store the collected nanofiber material.

[0029] b. The above-mentioned nanofiber material was cut into appropriate sizes, placed on a ceramic plate, and pre-oxidized in a muffle furnace. The heating rate was 1℃ / min, reaching 250℃ and holding for 90 min. After naturally cooling to room temperature, it was removed and then carbonized in a tube furnace under an argon atmosphere. The heating rate was 5℃ / min, reaching 750℃ and holding for 2 h. After cooling, it was removed. The prepared material was a flexible membrane, which was activated by soaking in concentrated nitric acid overnight. After washing and drying, it was cut into 0.2 x 0.5 cm pieces to obtain CNF. As a control experiment, carbon nanofiber sheets without transition metal doping were also prepared, and the current response signals of the two materials were compared by cyclic voltammetry (e.g., ...). Figure 2 As shown in the figure, the results indicate that doping with transition metals can significantly improve the electrochemical signal response of CNF, which in turn benefits the sensing performance of MIPs / CNF self-supporting electrodes.

[0030] (2) Fabrication of MIPs / CNF-HRP electrochemical sensor

[0031] a. Gold nanoparticle modification: The cut CNF was subjected to gold nanoparticle modification electrodeposition reaction in 0.01 g / mL chloroauric acid aqueous solution using the current-time curve method. The electrodeposition parameters were: voltage -0.2 V, electrodeposition time 650 s (optimized range 250-850 s: 250 s, 450 s, 650 s, 850 s). After electrodeposition, the bound CNF was washed with deionized water and dried at room temperature to obtain Au@CNF.

[0032] b. Recognition site establishment: 4-mercaptophenylboronic acid and L-cysteine ​​hydrochloride (1:1) were added to 4 mL of deionized water / ethanol (1:1) mixed solution, and then reacted in a vortex mixer for 2 h (optimized time range 0.5-4 h: 0.5 h, 1 h, 2 h, 4 h). After the reaction was completed, Au@CNF was immersed in the above solution and reacted in a vortex mixer for 3 h (optimized time range 1-9 h: 1 h, 3 h, 5 h, 7 h, 9 h) to carry out functional monomer self-assembly. After assembly, Au@CNF was washed with 0.1 mol / L phosphate buffer (pH 7.4) and then placed in 1 mg / mL HRP phosphate buffer (pH 7.4) (optimized HRP concentration range 0.5-3 mg / mL: 0.5, 1, 2, 3 mg / mL). Template protein pre-assembly was performed at room temperature for 5 h (optimized time range 3-9 h: 3 h, 5 h, 7 h, 9 h) to obtain HRP / Au@CNF.

[0033] c. Preparation of molecularly imprinted electrodes: An imprinted polymer layer of HRP protein was prepared on the surface of HRP / Au@CNF by electropolymerization. The electropolymerization solution was a phosphate buffer solution containing 5 mg / mL L-cysteine ​​(pH 7.4). Electropolymerization was carried out for 10 cycles at a scan rate of 100 mV / s (optimized cycle number range 6-14: 6, 8, 10, 12, 14), and the voltage range was -0.8-2V to obtain L-Cys / HRP / Au@CNF.

[0034] d. Elution to remove template molecules: After electropolymerization, L-Cys / HRP / Au@CNF was immersed in an acetic acid / acetonitrile aqueous solution, and then eluted in a vortex mixer for 3 h (optimized time range 2-5 h: 2 h, 3 h, 4 h, 5 h) to remove the embedded template protein. It was then washed with 0.1 mol / L phosphate buffer solution (pH 7.4) and dried at room temperature to obtain the MIPs / CNF electrochemical sensor capable of detecting glycoprotein HRP. The morphology of the prepared MIPs / CNF-HRP was characterized by scanning electron microscopy (e.g., ...). Figure 3 As shown), the entire preparation process was characterized by electrochemical tracking using cyclic voltammetry in a 5.0 mM [Fe(CN)6]3- / 4- solution (containing 0.1 M KCl). Figure 4 (As shown).

[0035] The testing of the aforementioned MIPs / CNF-HRP electrochemical sensor specifically includes the following steps:

[0036] (1) Electrode preparation: The MIPs / CNF-HRP electrochemical sensor prepared above is used as the self-supporting working electrode and fixed with an electrode clamp; the saturated calomel electrode is used as the reference electrode; and the platinum wire electrode is used as the counter electrode to construct a three-electrode system.

[0037] (2) Linearity Range Test: The working electrode was immersed in a certain concentration of HRP phosphate buffer solution to adsorb the template molecule HRP at pH 7.4 (optimized pH range 5.4-9.4: 5.4, 6.4, 7.4, 8.4, 9.4) for 3 min (optimized adsorption time range 1-5 min: 1, 2, 3, 4, 5 min). Interfering substances were removed by rinsing with buffer solution. The current response signal value IP (current response peak value minus background signal) in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution with different HRP solution concentrations was measured using differential pulse voltammetry (DPV). The DPV scan range was -0.2 to 0.6 V, and the scan rate was 50 mV / s. The HRP solution concentration range was 0.01 pg / mL to 100 ng / mL, and the tests were conducted in descending order of HRP solution concentration.

[0038] (3) Working curve fitting: Based on the multiple sets of different HRP solution concentrations and the corresponding IP values ​​obtained from the test in step (2), data fitting is performed to obtain the fitting curve between HRP solution concentration and IP value.

[0039] (4) Actual sample test: The working electrode was immersed in an unknown HRP concentration phosphate buffer solution with pH=7.4. After adsorption for 3 min, it was rinsed with buffer solution to remove interfering substances. The IP value of the HRP solution in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution was tested by differential pulse voltammetry (DPV). The concentration of HRP in the sample to be tested was calculated by using the fitting curve in step (3).

[0040] Application Examples

[0041] Example 1: Establishing an HRP testing method

[0042] The testing of the aforementioned MIPs / CNF-HRP electrochemical sensor specifically includes the following steps:

[0043] (1) Using the MIPs / CNF-HRP electrochemical sensor prepared above as the self-supporting working electrode and fixed with an electrode clamp; a saturated calomel electrode as the reference electrode; and a platinum wire electrode as the counter electrode, a three-electrode system is constructed.

[0044] (2) The working electrode was immersed in HRP protein buffer solution (pH=7.4) with concentrations of 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL and 100 ng / mL respectively. After adsorption for 3 min, it was rinsed with buffer solution to remove interfering substances. Then, the current response signal value Ip corresponding to different concentrations was tested by differential pulse voltammetry in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution.

[0045] (3) The test results were fitted using a model. The current response signal Ip showed a linear relationship with the HRP solution concentration in the range of 0.01 pg / mL to 100 ng / mL: Ip = 16.843 - 2.193 C, R² = 0.992 (e.g., ...). Figure 5 (As shown).

[0046] (4) Immerse the working electrode in a phosphate buffer solution of unknown HRP concentration at pH 7.4. After adsorption for 3 min, rinse with buffer solution to remove interfering substances. Measure the IP value of the HRP solution in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution by differential pulse voltammetry (DPV). Then calculate the concentration of HRP in the sample to be tested based on the current response signal value and the fitted curve, in pg / mL.

[0047] As shown in Example 1, the current response signal value Ip is linearly related to the HRP solution concentration in the range of 0.01 pg / mL to 100 ng / mL. Therefore, according to the detection limit calculation formula LOD = 3δ / S, the detection limit of the MIPs / CNF-HRP electrochemical sensor prepared by the example can reach 0.0074 pg / mL, indicating that the MIPs / CNF-HRP electrochemical sensor of the present invention has good detection sensitivity.

[0048] Example 2: Selective Testing

[0049] The MIPs / CNF-HRP electrochemical sensor prepared according to the above preparation example was used as the self-supporting working electrode, and the experimental conditions were the same as step (2) in specific example 1 above. HRP and solutions of four interfering proteins: OVA, HAS, BSA, and MUC1 (all concentrations 10 ng / mL) were detected, and the mixed solution of HRP and interfering proteins was also detected.

[0050] The ΔI values ​​of different sample solutions were obtained by differential pulse voltammetry in a 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution (ΔI = I0 – I, where I0 and I are the electrochemical signals before and after sensor detection, respectively). The results are as follows: Figure 6 As shown.

[0051] The results showed that, except for the template molecule HRP, the observed... Figure 6 No significant change in current signal value was observed, indicating that MIPs / CNF-HRP failed to recognize and bind interfering proteins. This demonstrates that the prepared MIPs / CNF-HRP possesses good specificity and detection capability.

[0052] Example 3: HPR content test in human serum samples

[0053] The MIPs / CNF-HRP electrochemical sensor prepared according to the above preparation example was used as the self-supporting working electrode, and the experimental conditions were the same as step (2) in specific example 1 above. 1 pg / mL, 100 pg / mL, and 10000 pg / mL HRP protein molecules were added to serum diluted 50 times, respectively. The working electrode was immersed in different sample solutions, and after adsorption for 3 min, it was rinsed with buffer solution to remove interfering substances. The IP value of the HRP solution in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution was tested by differential pulse voltammetry (DPV). The result was calculated using the fitting curve in example 1 to obtain the corresponding detection concentration. The spiked recovery rate and RSD value were calculated, indicating that the sensor has reliable detection performance in real sample detection.

[0054] The results showed that the sample recovery rate obtained by spiked recovery was between 89.2% and 116.7%, and the RSD value was between 1.068% and 1.376% (as shown in Table 1 below). This indicates that the MIPs / CNF-HRP electrochemical sensor has good method accuracy and precision as a working electrode and can be used to determine the HRP protein molecule content in actual human serum sample solutions.

[0055] Table 1. Spike recoveries and RSD values ​​in human serum samples

[0056]

[0057] Example 4 Stability Test

[0058] The MIPs / CNF-HRP electrochemical sensor prepared according to the above preparation example was used as the self-supporting working electrode. The experimental conditions were the same as step (2) in specific example 1 above. The working electrode was immersed in 10 ng / mL HRP buffer solution (pH = 7.4). After adsorption for 3 min, it was rinsed with buffer solution to remove unbound substances. Then, the current response signal ΔI of the HRP solution was tested by differential pulse voltammetry in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution. Thereafter, the working electrode was dried and stored at room temperature. The current response signal ΔI was tested daily in 5.0 mM [Fe(CN)6]3- / 4- (containing 0.1 M KCl) solution by differential pulse voltammetry for 7 days. The results are as follows. Figure 7 As shown. After 7 days of testing, the sensor signal showed no significant change, with an RSD value of 2.64%. The results indicate that the MIPs / CNF-HRP electrochemical sensor prepared in this invention exhibits good stability as a working electrode.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor capable of detecting glycoproteins, characterized in that: Step 1. Prepare transition metal-doped carbon nanofiber sheets (CNF) using electrospinning technology: 1.1 Dissolve polyacrylonitrile in N,N-dimethylformamide solution and stir on a magnetic stirrer until completely dissolved. Add cobalt acetate tetrahydrate and molybdenum acetylacetonate in sequence and continue stirring until a stable and homogeneous solution is formed. Then, use a syringe to draw up a certain amount of solution, clamp it and place it on a push pump, electrospin it, collect the nanofiber material, and dry and store it. 1.2 The above nanofiber material was cut into appropriate sizes, placed on a ceramic plate and placed in a muffle furnace for pre-oxidation, and then placed in a tube furnace under an argon atmosphere for carbonization. The prepared material was a flexible film. It was activated by soaking in concentrated nitric acid overnight, washed and dried, and then cut into 0.2×0.5cm sizes to obtain CNF as a self-supporting electrode. Step 2. Fabrication of a surface-imprinted glycoprotein electrochemical sensing self-supporting electrode: 2.1 Gold nanoparticle modification: The cut CNF was placed in an aqueous solution of chloroauric acid, and a layer of gold nanoparticles was modified on its surface using the current-time curve method. After electroplating, it was washed with deionized water to obtain Au@CNF, which was then dried at room temperature. 2.2 Recognition Site Establishment: First, the two functional monomers 4-mercaptophenylboronic acid and L-cysteine ​​hydrochloride were dissolved in a mixed solution of deionized water and ethanol to allow them to fully pre-bind and form a phenylboronic acid recognition molecule pair. Then, Au@CNF was immersed in the above mixed solution, and the phenylboronic acid recognition molecule pair was self-assembled into Au@CNF through Au-S bonds. Subsequently, the template molecule HRP was added, and it was covalently reacted with the phenylboronic acid recognition molecule pair through a cis-diol esterification covalent reaction to achieve self-assembly. 2.3 Preparation of molecularly imprinted electrodes: After self-assembly, electropolymerization was carried out on the Au@CNF surface after self-assembly using an L-cysteine ​​buffer solution as the electrolyte. HRP molecularly imprinted electrochemical working electrodes MIPs / CNF were prepared by cyclic voltammetry. The thickness of the polymer layer was controlled by the number of cyclic voltammetry scans. 2.4 Elution to remove template molecules: MIPs / CNF were immersed in an acetic acid / acetonitrile aqueous solution and eluted by vortexing to remove the template molecule HRP, obtaining specific imprinted hole recognition sites. After the template molecules were completely eluted, MIPs / CNF were washed with deionized water and dried at room temperature to obtain a molecularly imprinted electrochemical sensor that can detect glycoproteins.

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