Method for detecting CREG1 by using novel electrochemical sensor based on surface molecular imprinting technology and preparation method of novel electrochemical sensor

Abstract

The invention provides a novel electrochemical sensor for detecting CREG1 based on a surface molecular imprinting technology and a preparation method of the novel electrochemical sensor. Comprising the following steps: step 1, preparing a reagent solution required in a manufacturing process; step 2, polishing the glassy carbon electrode (GCE); 3, AuNPs / GCE is prepared; step 4, preparing CREG1 / GA (at) CS / AuNPs / GCE; step 5, preparing MIP / GA (at) CS / AuNPs / GCE; and step 6, scanning in the redox probe solution by adopting a differential pulse voltammetry (DPV), and determining the content of the CREG1. According to the invention, CREG1 protein is selected as a detection template, and a molecularly imprinted polymer is combined with gold nanoparticles through a surface polymerization method to prepare a corresponding western blot material. Gold nanoparticles are used as a matrix, CREG1 is used as a template, GA (at) CS is used as a protein fixing agent, and polyphenol is used as an imprinted polymer to prepare a surface imprinted material, and the surface imprinted material is measured by measuring the change of sensor current before and after the combination of a molecularly imprinted membrane (MIP) and CREG1. According to the present invention, the surface molecular imprinting technology and the electrochemical sensing technology are combined to construct the high-sensitivity and high-specificity CREG1 sensor, and the high-sensitivity and high-specificity CREG1 sensor is the novel surface molecular imprinting electrochemical sensor with good biocompatibility, can simply, conveniently and rapidly recognize the CREG1 protein at a low cost, and has characteristics of high sensitivity and high sensitivity. In addition, the method can also be used as a universal sensing platform to be suitable for other proteins.

Description

Technical Field

[0001] The invention relates to the fields of polymer material science, medical analysis and electrochemical sensing, and in particular to a novel electrochemical sensor for detecting CREG1 based on surface molecular imprinting technology and a preparation method thereof. Background Art

[0002] Initially considered a transcriptional repressor, CREG was later found to be a glycoprotein that can be secreted into the cell culture medium and is divided into CREG1 and CREG2. CREG1 is essential for embryonic development and normal physiology, and its dysregulation is associated with various diseases, including inflammation, neurodegeneration, diabetes, and cancer. CREG1 protein is highly expressed in the spleen, liver, lung, white adipose tissue, as well as in leukocytes and bone marrow. Experiments have shown that CREG1 can inhibit oncogene E1A transcription and cell transformation, attenuate NTERA-2 (human teratocarcinoma cell) proliferation, induce neuronal differentiation, and mediate glucocorticoid-induced proliferation of ileal epithelial cells. Therefore, accurate and selective detection of CREG1 with important biological functions in complex biological samples is an important topic in proteomics research, biomedical applications, and clinical diagnosis.

[0003] At present, the commonly used protein specific detection method is still the immunoassay analysis method, such as enzyme-linked immunosorbent assay (ELISA), which relies on the specific recognition between antibodies and antigens to detect the target analyte, that is, the known antigen or antibody is coated on a solid support, and the enzyme-labeled antibody is added to specifically bind to the antigen or antibody adsorbed on the solid support. The enzyme is colored by the substrate to determine the degree of immune occurrence to achieve the purpose of detection. However, the enzyme immunoassay technology that relies on monoclonal antibodies has problems such as difficulty in antibody preparation, high production cost, poor stability, harsh environmental requirements, and short service life. Therefore, establishing a rapid, simple, specific, and high-throughput CREG1 detection method has become an urgent problem to be solved.

[0004] Molecular imprinting technology is a method that combines materials chemistry, biochemistry and macromolecular chemistry, and has the specific ability to recognize target analytes. It can be used to synthesize molecularly imprinted polymers (MIPs), whose recognition cavities are completely complementary to the template molecules in shape, size and functional groups, thereby achieving specific recognition and detection of the template molecules without interference from analogs and coexisting substances. MIPs not only have highly selective recognition capabilities comparable to natural biomolecules (such as enzymes and substrates, antigens and antibodies, receptors and hormones), but also have the advantages of good stability, low cost and easy synthesis. However, surface molecular imprinting technology not only has all the advantages of ordinary molecular imprinting technology, but also has the characteristics of extremely small protein dosage, lower cost, higher sensitivity and better specificity. Electrochemical sensors are widely used due to their high sensitivity, good controllability, rapid response and real-time monitoring. Therefore, electrochemical sensors based on surface molecular imprinting polymers with the advantages of these methods are ideal platforms for the detection of biomacromolecule targets in recent years, especially protein detection. In this study, we combined electrochemical sensors with surface MIPs and modified the surface of the working electrode with signal amplification nanomaterials, which not only enhanced the conductivity of the electrode but also provided a larger specific surface area to increase its sensitivity and reduce interference from other factors such as the environment during detection.

[0005] Gold nanoparticles (AuNPs) are obtained by electrodeposition of chloroauric acid on the surface of glassy carbon electrode. AuNPs are highly biocompatible and are essential for the functionalization of sensor surfaces. They can promote rapid electron transfer rates between electrodes and redox probes to reduce resistance. They are favored in the preparation of MIECS due to their high specific surface area to volume ratio, excellent electrocatalytic activity and conductivity, and resistance to complex environments. In addition, AuNPs can be easily deposited on the electrode surface by simple electrochemical techniques, which not only improves the corresponding electrical signal of the sensor, increases the attachment amount of selective elements, but also reduces the detection limit. The provided Au interface can also bind to molecules containing free thiol groups through "S-Au" and thus be fixed to the modified electrode. As a supporting material for MIP, AuNPs can increase the imprinting sites of the modified electrode, thereby improving the performance of the sensor.

[0006] Chitosan (CS) is a linear β-1,4-linked polysaccharide (similar to cellulose) obtained by partial deacetylation of chitin, which is the main component of the shells of crustaceans (such as crabs and shrimps). It contains a large amount of -OH and -NH3, has the common adhesiveness of sugars, and is easy to be physically and chemically modified. It can fix protein molecules through the hydrogen bond interaction between -COOH and -OH or -NH3 of proteins. In addition, it has excellent film-forming ability and is often used as an adhesive to fix materials to the electrode surface, and make the modified electrode surface rich in -OH and -NH3, thereby improving the hydrophilicity of the electrode surface. This is conducive to the adsorption and transmission of electrolyte ions on the electrode surface, promotes the charge transfer between the modified electrode and the electrochemical probe in the solution, promotes the electrochemical reaction, and improves the electrochemical activity and electrical signal of the modified electrode. The amino group on the CS molecule can introduce glutaraldehyde (GA) to connect with it, and then cross-link with the protein through GA, and cooperate with CS to fix the protein on the surface of the modified electrode. GA is a commonly used chemical cross-linking agent containing two aldehyde groups, which can react with molecules containing amino groups (such as proteins, peptides or other amino compounds) to form stable methylene bridges. In electrochemically modified electrodes, the fixation of GA-cross-linked biological molecules (such as proteins, enzymes and antibodies, etc.) on the electrode surface can promote the transfer of electrons while maintaining their biological activity. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a novel electrochemical sensor based on surface molecular imprinting technology for detecting CREG1 and a preparation method thereof. The present invention provides a novel electrochemical sensor based on surface molecular imprinting technology for detecting CREG1 and a preparation method thereof, which has strong specificity, simple preparation, convenient detection, high sensitivity and low cost. The prepared novel surface molecular imprinting sensing electrode has good reproducibility and strong stability, and can be used for rapid and sensitive detection of CREG1.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The present invention discloses a method for preparing a novel electrochemical sensor based on surface molecular imprinting technology, which is characterized by comprising the following steps: Step 1, preparing a glassy carbon electrode modified with a signal amplification material (AuNPs / GCE); Step 2, preparation of CREG1 / GA@CS / AuNPs / GCE; Step 3, preparation of MIP / GA@CS / AuNPs / GCE; Step 4: Electrochemical detection of CREG1 was performed using the successfully prepared MIP / GA@CS / AuNPs / GCE sensor modified electrode.

[0010] Furthermore, the preparation method comprises the following steps: Step 1: Preparation of glassy carbon electrode modified with signal amplification material (AuNPs / GCE): Using an in-situ electrochemical deposition method, HAuCl4 is dissolved in H2SO4 and diluted to obtain a HAuCl4 solution; the HAuCl4 solution is placed in a small electrolytic cell, and a three-electrode system is used to prepare AuNPs / GCE by constant potential deposition; Step 2, preparation of CREG1 / GA@CS / AuNPs / GCE: Using the AuNPs / GCE modified electrode prepared in step 1, drop-coat the surface of the electrode with chitosan (CS) solution, incubate at room temperature, and rinse it lightly with ultrapure water after drying to remove the residual CS that is not attached to the AuNPs / GCE modified electrode, thereby obtaining a CS / AuNPs / GCE modified electrode; Glutaraldehyde (GA) was placed in a centrifuge tube, and the CS / AuNPs / GCE modified electrode was placed in the centrifuge tube. The mixture was incubated at room temperature and in the dark. Since the aldehyde group on GA can bond with the amino group, GA will cross-link with the CS on CS / AuNPs / GCE, thereby obtaining GA@CS / AuNPs / GCE. CREG1 was drop-coated on the surface of GA@CS / AuNPs / GCE and placed in a refrigerator to complete the cross-linking of the amino groups on the protein and GA. The amino groups in CS can generate hydrogen bonds with the carboxyl groups of the amino acids in the protein, so that CS@GA can synergistically fix the protein. After the protein was fixed, the modified electrode was taken out and washed with ultrapure water to remove the residual CREG1 that was not fixed on the electrode surface, and CREG1 / GA@CS / AuNPs / GCE was obtained. Step 3, preparation of MIP / GA@CS / AuNPs / GCE: The CREG1 / GA@CS / AuNPs / GCE prepared in step 2 was immersed in a PBS buffer containing monomer phenol, and electropolymerized by cyclic voltammetry (CV) at different scan rates. After a corresponding number of cycles of polymerization, MIP / CREG1 / GA@CS / AuNPs / GCE was prepared. The template protein CREG1 was eluted in sodium hydroxide solution (NaOH), and CV scanning was used to remove the CREG1 template protein in the imprinted polymer to obtain the sensor modified electrode MIP / GA@CS / AuNPs / GCE.

[0011] Furthermore, in step 1, the working electrode in the three-electrode system comprises a dropping mercury electrode, a solid electrode, or a rotating disk electrode; the HAuCl4 is dissolved in 0.5 M H2SO4 to obtain a HAuCl4 solution with a concentration of 2 to 10 mM; the constant potential deposition method in step 1 is a current-time curve (IT curve); the constant potential is -0.2 V; and the constant potential deposition time is 50 to 400 s.

[0012] Furthermore, the concentration of CS in step 2 is 0.01~0.05 mg / mL; the incubation time of CS is 10~60 min; the content of GA in step 2 is 10~50%; the incubation time of GA is 1~3 h; the content of the fixed CREG1 protein is 10~59 μg / mL; and the incubation time of the fixed CREG1 protein is 0.5~2 h.

[0013] Furthermore, in step 3, the concentration of monomer phenol is 0.5~2 mM; the concentration of the PBS buffer solution is 0.01 M; the pH of the PBS buffer solution is 5.0~7.4; the MIP polymerization potential range is -0.2~1.0 V; the MIP polymerization scan rate is 30~100 mV / s; the MIP polymerization scan number is 5~20 circles; the concentration of sodium hydroxide in the elution solution is 0.01~0.05 M; the elution potential is -1.0 ~ 1.0 V; and the elution CV scan rate is 10~50 mV / s.

[0014] The present invention also discloses a detection method for detecting CREG1 using a novel electrochemical sensor based on surface molecular imprinting technology as described in any one of the above: The prepared MIP / GA@CS / AuNPs / GCE modified electrode was used as the working electrode of the sensor, the Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and an electrochemical probe was used to perform differential pulse voltammetry (DPV) scanning using an electrochemical workstation three-electrode system. The change in current (ΔI) before and after the binding of the MIP membrane to CREG1 was measured.

[0015] Furthermore, the electrochemical probe comprises [Fe(CN)6] 3- / 4- 、[Co(phen)3] 3+ / 2+ , KCl, KNO3 or PBS buffer solution (0.01 M, pH=7.4).

[0016] The present invention also discloses the use of an electrochemical sensor prepared by any one of the above-mentioned novel electrochemical sensor preparation methods based on surface molecular imprinting technology in the preparation of a product for detecting CREG1 in a sample.

[0017] Furthermore, a CREG1 molecular imprinting electrochemical sensor prepared by any of the above-mentioned novel electrochemical sensor preparation methods based on surface molecular imprinting technology.

[0018] Furthermore, the CREG1 molecular imprinting electrochemical sensor is used in the preparation of a device for detecting CREG1.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0020] (1) The novel surface molecular imprinting electrochemical sensor for detecting CREG1 described in the present invention is simple to prepare and easy to operate, and can achieve rapid, sensitive, and highly selective detection of samples. It is also low in cost and can be applied to portable detection, thus having market development prospects.

[0021] (2) The present invention provides a method based on metal nanoparticles (taking AuNPs as an example) for improving the conductivity of sensors, increasing the amount of biomacromolecules such as proteins that can be fixed, and assisting in the fixation of proteins, etc. Through the good conductivity and large specific surface area of ​​metal nanoparticles, some thiol-containing proteins can interact with metal nanoparticles through binding forces such as "S-Au", thereby assisting in the fixation of proteins on metal nanoparticles.

[0022] (3) The present invention also introduces CS and GA, which have good capabilities in fixing biomacromolecules such as proteins. Through the physical adhesion and ability to promote electron transfer of CS and the properties of polyamino groups, CS is connected to GA, and then proteins and the like are cross-linked with GA, ultimately enabling the metal nanoparticles, CS and GA to stably and synergistically fix biomacromolecules such as proteins.

[0023] (4) The present invention combines an electrochemical sensor with MIP. After fixing the protein on the surface of the working electrode, MIP is obtained after electropolymerization and elution. While giving the electrode specific selectivity, it also greatly reduces the amount of template molecules used and reduces the preparation cost.

[0024] (5) The surface molecular imprinting new electrochemical sensor prepared by the present invention was successfully used for the detection of CREG1, and the recovery rate was between 91.33% and 109.41%. At the same time, the surface molecular imprinting new electrochemical sensor prepared by the present invention had a specific response to CREG1. The results showed that common metal ions such as K + 、Na + Mg 2+ , Ca 2+ NH 4+When the concentration of CREG1 is 250 times that of CREG1, it does not interfere with the determination. Some commonly used excipients, such as propylene glycol, starch, aluminum oxide, glucose, etc., have no effect on the current response of CREG1 when the concentration exceeds 100 times that of CREG1 for analysis. The current values ​​of other substances, such as bovine serum albumin, are small, indicating that the surface molecular imprinting novel electrochemical sensor prepared by the present invention has strong anti-interference. Therefore, the surface molecular imprinting novel electrochemical sensor prepared by the present invention can be widely used in biomedical applications and clinical diagnosis and other related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] (The following nanomaterials use AuNPs and the working electrode uses a glassy carbon electrode).

[0026] Figure 1 This is a schematic diagram of the synthesis and preparation of the surface molecular imprinting polymer and the combined electrochemical sensor provided in Example 1.

[0027] Figure 2 The DPV detection results and linear relationship diagram after polymerization, elution, and incubation with different concentrations of CREG1.

[0028] Figure 3 This is the CV characterization result diagram of each step in the sensor preparation process.

[0029] Figure 4 This is the electrochemical impedance spectroscopy (EIS) characterization result of each step in the sensor preparation process.

[0030] Figure 5 The DPV characterization results of each step in the sensor preparation process are shown in the figure.

[0031] Figure 6 Selectivity evaluation diagram of MIP / GA@CS / AuNPs / GCE. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0033] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0034] (The nanomaterial in this example is AuNPs, and the probe is [Fe(CN)6] 3- / 4- ).

[0035] [Example 1] This example provides a novel electrochemical sensor for detecting CREG1 based on surface molecular imprinting technology and a preparation method thereof. Figure 1 Shown: Includes the following steps.

[0036] Step 1, the specific steps for preparing AuNPs / GCE are as follows: the glassy carbon electrode (3 mm, GCE) is polished on suede polishing paper with an appropriate amount of Al2O3 powder of different particle sizes (1.0, 0.3, 0.05 μm) to remove surface particles. The polishing technique is to circle in an "8" shape and keep the electrode vertical for about 80 to 100 circles. The polished glassy carbon electrode is ultrasonically washed with ultrapure water, ethanol, and ultrapure water for 1 min each. The three-electrode system is placed in a probe solution (containing 10 mM [Fe(CN)6] 3- / 4- CV scan in PBS solution (0.1M KCl and KNO3) showed a symmetrical and reversible redox peak, and 64 mV ≤ ΔE ≤ 80mV. The electrode was clean and usable, and was blown dry with nitrogen (N2) for later use. In-situ electrochemical deposition was used to electro-deposit on the clean GCE surface. 1 g of HAuCl4 solid was dissolved in 0.5 M H2SO4 and diluted to obtain a 10 mM HAuCl4 solution. 2 mL of HAuCl4 solution was taken in a small electrolytic cell, and a three-electrode system (working electrode: GCE, reference electrode: Ag / AgCl electrode, counter electrode: platinum wire electrode) was used. Constant potential deposition was used, the constant potential was -0.2 V, and the deposition time was 400 s to prepare AuNPs / GCE.

[0037] Step 2, the specific steps of preparing CREG1 / GA@CS / AuNPs / GCE are as follows: using the prepared AuNPs / GCE modified electrode, drop-coating 5 μL of chitosan (CS) solution on its surface, and incubating it at room temperature for a certain time (1 h), and after it is dried, gently rinse it with ultrapure water to remove the residual CS that is not attached to the AuNPs / GCE modified electrode, and after N2 drying, obtain the CS / AuNPs / GCE modified electrode. Take 2 mL of glutaraldehyde (GA) and place it in a centrifuge tube, place the CS / AuNPs / GCE modified electrode in the centrifuge tube, and incubate it at room temperature and dark conditions for 3 h. Since the aldehyde group on GA can bond with the amino group. Therefore, GA will cross-link with the CS on CS / AuNPs / GCE, thereby obtaining GA@CS / AuNPs / GCE. 5 μL of 59 μg / mL CREG1 was drop-coated on the surface of GA@CS / AuNPs / GCE and placed in a 4°C refrigerator for 2 h to complete the cross-linking of the amino groups on the protein and GA. The amino groups in CS can produce hydrogen bonds with the carboxyl groups of the amino acids in the protein, so that CS@GA can synergistically fix the protein. After the protein is fixed, the modified electrode is taken out and washed with ultrapure water to remove the residual CREG1 that is not fixed on the electrode surface, and CREG1 / GA@CS / AuNPs / GCE is obtained.

[0038] Step 3, the specific steps of preparing MIP / GA@CS / AuNPs / GCE are as follows: immersing CREG1 / GA@CS / AuNPs / GCE in PBS buffer (0.01 M, pH=7.4) containing 2 mM phenol, using a scan rate of 100 mV / s and a potential range of -0.2~+1.0 V, and electropolymerizing by CV method. After 20 cycles of polymerization, MIP / CREG1 / GA@CS / AuNPs / GCE is prepared. The template protein CREG1 is eluted in a sodium hydroxide solution (0.05 M, NaOH) at a scan rate of 50 mV / s and a potential range of -1.0~+1.0 V. CV scanning is used for 11 cycles to remove the CREG1 template protein in the imprinted polymer. The sensor modified electrode MIP / GA@CS / AuNPs / GCE is obtained. Correspondingly, NIP / GA@CS / AuNPs / GCE is prepared by the same procedure, but without the template protein CREG1.

[0039] Step 4: Using the prepared MIP / GA@CS / AuNPs / GCE electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, an electrochemical redox probe ([Fe(CN)6] 3- / 4- ), DPV scanning was performed using an electrochemical workstation three-electrode system, and the molecular imprinting membrane was measured by measuring the change in current before and after binding with CREG1. The electrodes of all preparation steps (GCE, AuNPs / GCE, CS / AuNPs / GCE, GA@CS / AuNPs / GCE, CREG1 / GA@CS / AuNPs / GCE, MIP / CREG1 / GA@CS / AuNPs / GCE, MIP / GA@CS / AuNPs / GCE) were placed in the same concentration of electrolyte probe solution and the same scanning conditions to obtain CV, EIS and DPV characterization diagrams, and then compared and analyzed to illustrate the generation process of surface molecular imprinting. See Figures 3-5 shown.

[0040] (The nanomaterial in this example is AuNPs, and the probe is [Co(phen)3] 3+ / 2+ ).

[0041] [Example 2] This example provides a novel electrochemical sensor for detecting CREG1 based on surface molecular imprinting technology and a preparation method thereof. The overall preparation process is similar to Figure 1 Similar, but with some changes to the electrochemical probe, the following steps are included.

[0042] Step 1, the specific steps for preparing AuNPs / GCE are as follows: the glassy carbon electrode (3 mm, GCE) is polished on suede polishing paper with an appropriate amount of Al2O3 powder of different particle sizes (1.0, 0.3, 0.05 μm) to remove surface particles. The polishing technique is to circle in an "8" shape and keep the electrode vertical for about 80 to 100 circles. The polished glassy carbon electrode is ultrasonically washed with ultrapure water, ethanol, and ultrapure water for 1 min each. The three-electrode system is placed in a probe solution (containing 10 mM [Fe(CN)6] 3- / 4- CV scan in PBS solution (0.1M KCl and KNO3), symmetrical and reversible redox peaks appeared, and 64 mV≤ΔE≤80 mV, the electrode was clean and usable, and it was blown dry with nitrogen (N2) for later use. In-situ electrochemical deposition was used to electro-deposit on the clean GCE surface. 1 g HAuCl4 solid was dissolved in 0.5 M H2SO4, and a 10 mM HAuCl4 solution was obtained after dilution. 2 mL HAuCl4 solution was taken in a small electrolytic cell, and a three-electrode system (working electrode: GCE, reference electrode: Ag / AgCl electrode, counter electrode: platinum wire electrode) was used. Constant potential deposition was used, the constant potential was -0.2 V, and the deposition time was 400 s to prepare AuNPs / GCE.

[0043] Step 2, the specific steps of preparing CREG1 / GA@CS / AuNPs / GCE are as follows: using the prepared AuNPs / GCE modified electrode, drop-coating 5 μL of chitosan (CS) solution on its surface, and incubating it at room temperature for a certain time (1 h), and after it is dried, gently rinse it with ultrapure water to remove the residual CS that is not attached to the AuNPs / GCE modified electrode, and after N2 drying, obtain the CS / AuNPs / GCE modified electrode. Take 2 mL of glutaraldehyde (GA) and place it in a centrifuge tube, place the CS / AuNPs / GCE modified electrode in the centrifuge tube, and incubate it at room temperature and dark conditions for 3 hours. Since the aldehyde group on GA can bond with the amino group. Therefore, GA will cross-link with the CS on CS / AuNPs / GCE, thereby obtaining GA@CS / AuNPs / GCE. 5 μL of 59 μg / mL CREG1 was drop-coated on the surface of GA@CS / AuNPs / GCE and placed in a 4°C refrigerator for 2 h to complete the cross-linking of the amino groups on the protein and GA. The amino groups in CS can produce hydrogen bonds with the carboxyl groups of the amino acids in the protein, so that CS@GA can synergistically fix the protein. After the protein is fixed, the modified electrode is taken out and washed with ultrapure water to remove the residual CREG1 that is not fixed on the electrode surface, and CREG1 / GA@CS / AuNPs / GCE is obtained.

[0044] Step 3, the specific steps of preparing MIP / GA@CS / AuNPs / GCE are as follows: immersing CREG1 / GA@CS / AuNPs / GCE in PBS buffer (0.01 M, pH=7.4) containing 2 mM phenol, using a scan rate of 100 mV / s and a potential range of -0.2~+1.0 V, and electropolymerizing by CV method. After 20 cycles of polymerization, MIP / CREG1 / GA@CS / AuNPs / GCE is prepared. The template protein CREG1 is eluted in a sodium hydroxide solution (0.05 M, NaOH) at a scan rate of 50 mV / s and a potential range of -1.0~+1.0 V. CV scanning is used for 11 cycles to remove the CREG1 template protein in the imprinted polymer. The sensor modified electrode MIP / GA@CS / AuNPs / GCE is obtained. Correspondingly, NIP / GA@CS / AuNPs / GCE is prepared by the same procedure, but without the template protein CREG1.

[0045] Step 4: Using the prepared MIP / GA@CS / AuNPs / GCE electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, an electrochemical redox probe ([Co(phen)3] 3+ / 2+ ), DPV scanning was performed using an electrochemical workstation three-electrode system, and the current change before and after the molecular imprinting membrane bound to CREG1 was measured.

[0046] Electrochemical performance test characterization: GCE, AuNPs / GCE, CS / AuNPs / GCE, GA@CS / AuNPs / GCE, CREG1 / GA@CS / AuNPs / GCE, MIP / CREG1 / GA@CS / AuNPs / GCE, MIP / GA@CS / AuNPs / GCE. CV ( ) of GCE, AuNPs / GCE (electrode prepared in step 1 of Example 1), and MIP / GA@CS / AuNPs / GCE (electrode prepared in step 4 of Example 1) in potassium ferrocyanide solution Figure 3 )、EIS( Figure 4 ) and DPV ( Figure 5 ) to confirm the electrochemical performance and preparation process of the above steps. Figure 2 It can be seen that in the subsequent test results, the DPV current value of the modified electrode incubated with different concentrations of CREG1 gradually decreased, indicating that after incubation in a certain concentration of template molecule solution, the holes were occupied, the resistance value increased, and the current decreased, proving the specific recognition of CREG1 by the surface molecular imprinting electrochemical sensor. Figure 3It can be seen that the resistance value of CS / AuNPs / GCE is the smallest and greater than that of GCE and AuNPs / GCE. After GA and CREG1 are on the electrode, the resistance value increases further, indicating that AuNPs, CS, GA and CREG1 have been modified. With the synthesis of MIP (CREG1), the impedance value reaches the maximum. After eluting the modified electrode and removing the template CREG1, the impedance value is greatly reduced, proving the successful preparation of MIP. The characterization results of CV and DPV are consistent with EIS.

[0047] Test result analysis: The MIP / GA@CS / AuNPs / GCE obtained in Example 1 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the auxiliary electrode. 3- / 4- As an electrochemical indicator, the electrochemical signal of CREG1 was detected by DPV method. The measurement potential range was -0.2~0.6 V and the scanning rate was 0.1 V / s. The results are shown in Table 1.

[0048] Table 1 Electrochemical signals of CREG1 detected by DPV method .

[0049] It can be seen from the results in Table 1 that the novel surface molecular imprinting electrochemical sensor for detecting CREG1 prepared by the method of the present invention has a relatively good response performance.

[0050] Selective Assessment: [Fe(CN)6] 3- / 4- As an electrochemical indicator, the surface molecular imprinting novel electrochemical sensor prepared in Example 1 was used as a working electrode, the Ag / AgCl electrode was used as a reference electrode, and the platinum wire electrode was used as an auxiliary electrode. The electrochemical signal was detected by differential pulse voltammetry, and the electrochemical response signals of dopamine hydrochloride, ascorbic acid, uric acid, bovine serum albumin and bovine hemoglobin were detected, and the anti-interference performance of the method was evaluated by the current change value. Figure 6 For the anti-interference performance graph, by measuring the electrochemical signals of CREG1 and dopamine hydrochloride, ascorbic acid, uric acid, bovine serum albumin, and bovine hemoglobin with 100 times the concentration of CREG1, there was no obvious difference in the response from the mixture compared with the presence of CREG1 alone, and the ΔI signal change was less than 5%. These evaluation results confirmed that the sensor has good selectivity for CREG1 in actual samples.

[0051] For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a novel electrochemical sensor based on surface molecular imprinting technology, characterized in that: The following steps are involved: Step 1, preparing a glassy carbon electrode modified with a signal amplification material (AuNPs / GCE); Step 2, preparation of CREG1 / GA@CS / AuNPs / GCE; Step 3, preparation of MIP / GA@CS / AuNPs / GCE; Step 4: Electrochemical detection of CREG1 was performed using the successfully prepared MIP / GA@CS / AuNPs / GCE sensor modified electrode.

2. The method for preparing a novel electrochemical sensor based on surface molecular imprinting technology according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1: Preparation of glassy carbon electrode modified with signal amplification material (AuNPs / GCE): Using an in-situ electrochemical deposition method, HAuCl4 is dissolved in H2SO4 and diluted to obtain a HAuCl4 solution; the HAuCl4 solution is placed in a small electrolytic cell, and a three-electrode system is used to prepare AuNPs / GCE by constant potential deposition; Step 2, preparation of CREG1 / GA@CS / AuNPs / GCE: Using the AuNPs / GCE modified electrode prepared in step 1, drop-coat the surface of the electrode with chitosan (CS) solution, incubate at room temperature, and rinse it lightly with ultrapure water after drying to remove the residual CS that is not attached to the AuNPs / GCE modified electrode, thereby obtaining a CS / AuNPs / GCE modified electrode; Glutaraldehyde (GA) was placed in a centrifuge tube, and the CS / AuNPs / GCE modified electrode was placed in the centrifuge tube. The mixture was incubated at room temperature and in the dark. Since the aldehyde group on GA can bond with the amino group, GA will cross-link with the CS on CS / AuNPs / GCE, thereby obtaining GA@CS / AuNPs / GCE. CREG1 was drop-coated on the surface of GA@CS / AuNPs / GCE and placed in a refrigerator to complete the cross-linking of the amino groups on the protein and GA. The amino groups in CS can generate hydrogen bonds with the carboxyl groups of the amino acids in the protein, so that CS@GA can synergistically fix the protein. After the protein was fixed, the modified electrode was taken out and washed with ultrapure water to remove the residual CREG1 that was not fixed on the electrode surface, and CREG1 / GA@CS / AuNPs / GCE was obtained. Step 3, preparation of MIP / GA@CS / AuNPs / GCE: The CREG1 / GA@CS / AuNPs / GCE prepared in step 2 was immersed in a PBS buffer containing monomer phenol, and electropolymerized by cyclic voltammetry (CV) at different scan rates. After a corresponding number of cycles of polymerization, MIP / CREG1 / GA@CS / AuNPs / GCE was prepared. The template protein CREG1 was eluted in sodium hydroxide solution (NaOH), and CV scanning was used to remove the CREG1 template protein in the imprinted polymer to obtain the sensor modified electrode MIP / GA@CS / AuNPs / GCE.

3. The preparation method according to claim 2, characterized in that: The working electrode in the three-electrode system of step 1 includes a dropping mercury electrode, a solid electrode, or a rotating disk electrode; the HAuCl4 is dissolved in 0.5 M H2SO4 to obtain a HAuCl4 solution with a concentration of 2 to 10 mM; the constant potential deposition method of step 1 is a current-time curve (IT curve); the constant potential is -0.2 V; The constant potential deposition time is 50 to 400 s.

4. The preparation method according to claim 2, characterized in that: The concentration of CS in step 2 is 0.01-0.05 mg / mL; the incubation time of CS is 10-60 min; the content of GA in step 2 is 10-50%; the incubation time of GA is 1-3 h; the content of the fixed CREG1 protein is 10-59 μg / mL; and the incubation time of the fixed CREG1 protein is 0.5-2 h.

5. The preparation method according to claim 2, characterized in that: In step 3, the concentration of monomer phenol is 0.5~2 mM; the concentration of the PBS buffer solution is 0.01 M; the pH of the PBS buffer solution is 5.0~7.4; the MIP polymerization potential range is -0.2~1.0 V; the MIP polymerization scan rate is 30~100 mV / s; the number of MIP polymerization scans is 5~20 circles; the concentration of sodium hydroxide in the elution solution is 0.01~0.05 M; the elution potential is -1.0 ~ 1.0 V; the elution CV scan rate is 10~50 mV / s.

6. A method for detecting CREG1 using a novel electrochemical sensor based on surface molecular imprinting technology according to any one of claims 1 to 5: The prepared MIP / GA@CS / AuNPs / GCE modified electrode was used as the working electrode of the sensor, the Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and an electrochemical probe was used to perform differential pulse voltammetry (DPV) scanning using an electrochemical workstation three-electrode system. The change in current (ΔI) before and after the binding of the MIP membrane to CREG1 was measured.

7. The detection method according to claim 6, characterized in that: The electrochemical probe includes [Fe(CN)6] 3- / 4- 、[Co(phen)3] 3+ / 2+ , KCl, KNO3 or PBS buffer solution (0.01 M, pH=7.4).

8. Use of an electrochemical sensor prepared by the novel electrochemical sensor preparation method based on surface molecular imprinting technology according to any one of claims 1 to 5 in preparing a product for detecting CREG1 in a sample.

9. A CREG1 molecular imprinting electrochemical sensor prepared by the novel electrochemical sensor preparation method based on surface molecular imprinting technology as described in any one of 1 to 5.

10. Use of the CREG1 molecular imprinting electrochemical sensor according to claim 9 in preparing a device for detecting CREG1.

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