Preparation method of an electrochemical immunosensor electrode for collagen detection

Abstract

The application relates to the field of sensors, and discloses a preparation method of an electrochemical immunosensor electrode for collagen detection. The silk fabric is activated and heat treated to obtain a nitrogen-containing porous three-dimensional skeleton with multiple active sites and conductivity; a three-dimensional skeleton is used as a working electrode, a platinum sheet is used as a counter electrode, and an Ag / AgCl electrode is used as a reference electrode; a polypyrrole is formed through a constant potential electrochemical deposition method to complementarily complete preliminary modification of an electrode substrate together with a carbon material; then, gold nanoparticles and antibodies are loaded on the electrode to complete preparation of the electrode. The three-dimensional electrode skeleton with high porosity is prepared, the active material loading capacity is increased, a conductive polymer is introduced to modify the electrode to increase the electrochemical performances such as conductivity, specific capacity and specific surface area of the electrode, and the gold nanoparticles are introduced to increase the biocompatibility of the material and load the antibodies, so that the defects of a single material are supplemented.

Description

Technical Field

[0001] This invention relates to the field of sensors, and more particularly to a method for preparing an electrochemical immunosensor electrode for collagen detection. Background Technology

[0002] The main component of leather artifacts and leather products is collagen. Traditional detection methods include Fourier transform infrared spectroscopy and Raman spectroscopy, which determine the composition of substances based on absorption characteristic peaks. However, these methods suffer from poor sample recovery, the preciousness of the samples, and the need for multiple tests on different points of the same sample to ensure data reliability. Therefore, the combination of immunoassay technology and signal amplification principle can overcome these shortcomings. Electrochemical immunosensors, as a novel approach, can play a significant role in the detection of cultural relics and leather products.

[0003] The performance of electrochemical immunosensors is closely related to the characteristics of their electrodes and surface modification materials. Currently, glassy carbon electrodes, gold electrodes, screen-printed electrodes, and ITO electrodes are mainly used as base electrodes, which are then modified. These base electrodes all have different drawbacks, such as high cost and non-reusability. Furthermore, most base electrodes in current systems share a common problem: a two-dimensional planar structure and a small interface area. This means that the amount and probability of recognizing loaded biomolecules are relatively low, which is detrimental to the sensing process. There are many combinations of modification materials. Theoretically, immunosensor materials should possess good biocompatibility to maintain biomolecule activity, the ability to bind antibodies to load and immobilize antibodies to construct the reaction interface layer, and high conductivity and signal amplification capabilities to monitor the reaction. Therefore, preparing electrode materials with a three-dimensional structure, capable of loading antibodies, and with good conductivity can effectively enhance the performance of electrochemical immunosensors. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing an electrochemical immunosensor electrode for collagen detection. This invention uses silk fabric as a source, activating and heat-treating it to obtain a nitrogen-containing, porous, multi-active-site, conductive three-dimensional framework. Simultaneously, using pyrrole monomer as a raw material and sodium dodecylbenzenesulfonate as a surfactant, the treated silk fabric serves as the working electrode, a platinum sheet as the counter electrode, and an Ag / AgCl electrode as the reference electrode. A constant-potential electrochemical deposition method is used to form polypyrrole on the active sites of the silk fabric surface, complementing the carbon material to complete the initial modification of the electrode substrate. This invention also prepares a gold nanoparticle solution using a sodium citrate reduction method, and then loads the gold nanoparticles onto the electrode to complete the electrode preparation. This invention increases the loading of active materials by preparing a three-dimensional electrode framework with high porosity, and then introduces conductive polymers to modify the electrode to increase its electrochemical properties such as conductivity, specific capacity and specific surface area. The introduction of gold nanoparticles increases the biocompatibility of the material and can load antibodies, making up for the defects of single materials. It has good stability, high sensitivity, no danger or pollution, and the self-assembly process is easy to characterize, making it particularly suitable for detecting collagen and leather artifacts.

[0005] The specific technical solution of this invention is: a method for preparing an electrochemical immunosensor electrode for collagen detection, comprising the following steps:

[0006] Step 1: The silk fabric is ultrasonically cleaned and dried, then activated by immersion in a saturated KCl solution, dried a second time, and then subjected to heat treatment for carbonization. After cooling, a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional skeleton is obtained.

[0007] Step 2: After distilling the pyrrole monomer solution, add sodium dodecylbenzenesulfonate to obtain a mixture.

[0008] Step 3: Using the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in Step 1 as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the mixed solution obtained in Step 2 as the electrolyte, a C / PPy composite electrode was prepared by constant potential electrochemical deposition. The electrode was then cleaned with anhydrous ethanol and deionized water and dried.

[0009] Step 4: Heat the chloroauric acid solution to boiling, then add sodium citrate dropwise and stir until homogeneous to obtain a wine-red gold nanoparticle solution.

[0010] Step 5: Take the C / PPy composite electrode obtained in Step 3, add the gold nanoparticle solution obtained in Step 4, heat and fix to obtain C / PPy / AuNps.

[0011] Step 6: Add mercaptopropionic acid solution dropwise to C / PPy / AuNps and heat to combine.

[0012] Step 7: Add a mixed solution of EDC and NHS to the electrode obtained in Step 6 and heat to activate it.

[0013] Step 8: Take bovine serum albumin and add it to PBS buffer at a ratio of 1:90-110 to obtain a dilution solution, then mix it with the antibody to obtain a collagen monoclonal antibody dilution solution.

[0014] Step 9: Drop the collagen monoclonal antibody dilution solution onto the surface of the activation electrode, and after incubation, obtain the electrochemical immunosensor electrode for collagen detection.

[0015] The preparation principle of this invention is as follows: Silk fabric products possess a well-defined three-dimensional spatial structure, making them suitable as substrates for flexible electrode materials. This invention uses silk fabric as a carbon source for heat treatment to obtain nitrogen-rich carbon materials. These carbon materials are typical double-layer capacitor materials, storing energy through rapid adsorption and release of charges on the electrode surface. Nitrogen enhances the conductivity of the carbon materials, and the carbonized silk protein contains numerous carboxyl groups on its surface, allowing for modification as needed. After activation with a saturated KCl solution, metal ions during heat treatment etch the material, transforming its microstructure into porous carbon, thus increasing the sp2 of the material after 350°C to a certain extent. 2 The interlayer spacing of pseudo-graphite formed by carbon hexagonal hybridization promotes the formation of graphite structure, improves electrochemical performance, and increases the active sites of the material. Polypyrrole is a conductive polymer material with a very large specific capacitance, but in practical applications, its molecular chains are prone to breakage during charge and discharge, resulting in poor cycle life and stability. Furthermore, as a pseudocapacitive material, irreversible redox reactions during electrochemical detection also affect its lifespan. This invention utilizes a three-dimensional framework of C / PPy to obtain a composite electrode that is resistant to detachment, has a large specific surface area, high porosity, high conductivity, good cycling performance, and is rich in amino functional groups. The electrochemical deposition method ensures uniform material distribution, guaranteeing the reproducibility and replicability of the experiment. It complements the shortcomings of the two materials while retaining their advantages. Modifying the surface with gold nanoparticles enhances the conductivity of the material and also has excellent biocompatibility. Most importantly, it provides sites for antibody loading. At 37°C, gold nanoparticles form relatively stable gold-sulfur bonds with mercaptopropionic acid. Mercaptopropionic acid has a carboxyl terminus, which can be activated by adding EDC and NHS. The activated carboxyl group can then form strong chemical bonds with the amino groups in the antibody, thereby loading the antibody and completing self-assembly.

[0016] As a preferred option, in step 1: use anhydrous ethanol for ultrasonic cleaning for 20-40 minutes, activate at a temperature of 50-70℃, and activate for 10-14 hours.

[0017] As a preferred embodiment, in step 1, the heat treatment carbonization is performed as follows: starting from room temperature, the temperature is raised to 280-320℃ in air at a rate of 5-8℃ / min, held for 1.5-2.5h, nitrogen gas is introduced and the temperature is raised to 850-950℃ at a rate of 2-5℃ / min, held for 1.5-2h, and then cooled in the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon material.

[0018] The principle of step 1 of this invention is as follows: selecting silk fabrics rich in nitrogen as the carbon source. Further research in this invention has revealed that graphitization of silk fibroin under different high-temperature heat treatment processes yields carbon materials with different compositions and microstructures. Therefore, this invention has specifically designed a heat treatment process to obtain the desired nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework.

[0019] Specifically: Under normal circumstances, silk fibroin requires 2800℃ to generate the highly developed graphite stacked structure desired in this invention, but this structure results in decreased crystallinity and increased defects. This invention discovers that pretreatment of silk fibroin with KCl solution, upon heating, etches carbon into a honeycomb structure, creating mesopores, increasing active sites and improving conductivity. Simultaneously, the pretreated silk fibroin exhibits a graphite-like structure earlier (around 900℃) and better crystallinity. Next, this invention first heats the material in air to around 300℃, causing mild dehydration, graphitization, and ashing of the biomass material. This removes some impurities while obtaining a semi-graphitized material with a rich porous structure. Then, nitrogen gas is introduced, and the temperature is further increased to around 900℃, during which the β-sheet structure gradually transforms into a conjugated sp structure around 350℃. 2 The hybrid carbon hexagonal structure ultimately yields a pseudo-graphite crystalline layer structure. Furthermore, this invention reveals that a slower heating rate at this stage can increase the material's crystallinity and reduce defects.

[0020] In summary, this invention can obtain carbon materials with high porosity, large specific surface area and complete pseudo-graphite layer structure by pretreating silk fabrics and controlling parameters such as heating rate, temperature and holding time in stages during heat treatment. The material also contains a certain amount of nitrogen elements that form CN bonds with carbon atoms, which can enhance the conductivity of the material.

[0021] Preferably, in step 2: the concentration of the pyrrole monomer solution is 0.1-0.2M; and the concentration of sodium dodecylbenzenesulfonate is 4-6mM.

[0022] Preferably, in step 3: the potential window is set to 0.7-0.9V; and the electrochemical deposition time is 400-1000s.

[0023] Preferably, in step 4: the concentration of chloroauric acid is 0.8-1.2 mM; the concentration of sodium citrate is 0.08-0.12 M; and the stirring time is 10-20 min.

[0024] Preferably, in step 5, the heating temperature is fixed at 35-40℃ for 0.5-1.5 hours.

[0025] Preferably, in step 6: the concentration of the mercaptopropionic acid solution is 0.2-0.4M; the volume ratio of the gold nanoparticle solution to the mercaptopropionic acid solution is 1:3-5.

[0026] Preferably, in step 6, the heating temperature is 35-40℃ and the time is 0.5-1.5 seconds.

[0027] Preferably, in step 7: the molar ratio of EDC to NHS is 1.8-2.2:1; the heating activation temperature is 35-40℃, and the time is 0.5-1.5h.

[0028] Preferably, in step 8: the weight ratio of bovine serum albumin to PBS buffer is 1:80-120; the concentration of antibody in the collagen monoclonal antibody dilution solution is 2-4 mg / mL; and the pH of the PBS buffer is 7.4.

[0029] Preferably, in step 9, the incubation temperature is 35-40℃ and the incubation time is 0.5-1.5h.

[0030] Compared with the prior art, the present invention has the following technical effects:

[0031] (1) This invention obtains a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional skeleton with abundant surface active sites, high material transport rate, high conductivity, and pseudo-graphite structure by activating and heat-treating a nitrogen-containing carbon source. The heteroatoms can provide a certain pseudocapacitance, and the carbon material has double-layer capacitance.

[0032] (2) By preparing a three-dimensional electrode, the present invention has a larger interface area in the direction of immunosensor compared with glassy carbon electrodes, and greatly improves the loading of biomolecules, active substances and electrochemical activity.

[0033] (3) This invention obtains a fully distributed, uniform and robust C / PPy composite electrode by controlling the potential and electrodeposition time and introducing sodium dodecylbenzenesulfonate, a solvent with high affinity and mildness, to promote the polymerization reaction of pyrrole. The sensing interface can be modified accordingly as needed.

[0034] (4) In this invention, gold nanoparticles are introduced by increasing the overall conductivity and reaction rate of the material through metal nanoparticles, and then mercaptopropionic acid with a carboxyl group at the end that can form a gold-sulfur bond with gold is introduced, so that the gold nanoparticles modified with mercaptopropionic acid can be directly and firmly loaded with antibodies after the carboxyl group is activated.

[0035] (5) The electrode prepared by the present invention does not require the addition of any additional conductive agent or binder. Compared with traditional coating and rolling mechanical methods, the material load is more uniform and effective, simpler, and has lower resistance.

[0036] (6) The materials used in this invention have good biocompatibility, are green and pollution-free, have a simple preparation process, save time and effort, and the self-assembly process is easy to detect and can be modified. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments.

[0038] Example 1

[0039] Step 1: The natural silk fabric was ultrasonically cleaned with anhydrous ethanol and deionized water for 30 minutes each, dried at room temperature, and then activated by immersing it in a saturated KCl solution in a 60℃ constant temperature oven for 12 hours. The activated fabric was rinsed with deionized water and dried. After being cut to the required electrode size, it was transferred to a tube furnace for heat treatment. Starting from room temperature of 25℃, the temperature was increased by 6℃ per minute to 300℃ and held for 2 hours. Then, nitrogen gas was introduced and the temperature was increased to 900℃ at a rate of 5℃ / min and held for 2 hours. The fabric was then cooled down with the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework. 0.034g of the finished product was weighed and stored.

[0040] Step 2: Take 0.68g of pyrrole monomer, approximately 0.7ml, and add deionized water to 50ml to prepare a 0.2M solution. Distill the solution until it is colorless, indicating that the deoxidation is complete. Take 2.5ml and record it as solution A. Then, take 0.087g of sodium dodecylbenzenesulfonate and add it to 50ml of deionized water to prepare a 5mM solution. Take 47.5ml and record it as solution B. Mix solutions A and B to obtain solution C.

[0041] Step 3: Using the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in Step 1 as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the C solution obtained in Step 2 as the electrolyte, the constant potential deposition voltage was set to 0.8V and the deposition time was set to 700s to prepare the C / PPy composite electrode. After that, it was washed and dried multiple times with anhydrous ethanol and deionized water, and weighed to obtain 0.0388g, that is, the single electrode was loaded with 0.0048g of PPy.

[0042] The active substance mass loading was 4.8 mg / cm³. 3 The peak DPV response is 5.17mA.

[0043] Step 4: Take 0.068g of chloroauric acid and add 200ml of deionized water to prepare a 1mM solution. Heat the solution to boiling with an alcohol lamp and remove it. Take 0.258g of sodium citrate and add 10ml of deionized water to obtain a 0.1M sodium citrate aqueous solution. Then add the solution to the boiling chloroauric acid solution and stir rapidly for 15min to obtain a wine-red solution, which is the gold nanoparticle solution. Store the solution in a refrigerator at 4℃.

[0044] Step 5: Take 15 μL of the gold nanoparticle solution obtained in Step 4 and drop it evenly onto the electrode obtained in Step 3. Place it in a 37°C constant temperature oven for 1 hour to fix the gold nanoparticles, then wash with deionized water and dry.

[0045] The peak DPV response was 5.66 mA, proving that gold nanoparticles were successfully modified on the electrode and enhanced the conductivity of the material.

[0046] Step 6: Add 60 μL of 0.3 M MPA solution to the surface of the electrode obtained in Step 5, and place it in a 37 °C constant temperature oven for 1 hour to form gold-sulfur bonds.

[0047] Step 7: Take 40 μL of 0.06 M EDC and 20 μL of 0.03 M NHS and place them in a vortex mixer. After mixing evenly, drop the mixture onto the resulting electrode and place it in a 37°C constant temperature oven for 1 hour to activate the carboxyl terminus of MPA.

[0048] Step 8: Mix the prepared BSA with PBS at pH 7.4 at a ratio of 1:100 and use the mixture to dilute the collagen monoclonal antibody to 3 mg / ml.

[0049] Step 9: Drop the diluted collagen antibody onto the electrode activated in Step 7 and incubate it in a 37°C oven for 1 hour.

[0050] The peak DPV response was 2.8 mA, proving that the modified gold nanoparticles were successfully loaded with antibodies. The loading of biomolecules and the addition of insulating BSA reduced the conductivity and porosity of the material, thereby causing a decrease in the response current, which verified the self-assembly process.

[0051] Comparative Example 1 (changing pyrrole concentration)

[0052] Step 1: The natural silk fabric was ultrasonically cleaned with anhydrous ethanol and deionized water for 30 minutes each, dried at room temperature, and then activated by immersing it in a saturated KCl solution in a 60℃ constant temperature oven for 12 hours. The activated fabric was rinsed with deionized water and dried. After being cut to the required electrode size, it was transferred to a tube furnace for heat treatment. Starting from room temperature of 25℃, the temperature was increased by 6℃ per minute to 300℃ and held for 2 hours. Then, nitrogen gas was introduced and the temperature was increased to 900℃ at a rate of 5℃ / min and held for 2 hours. The fabric was then cooled down with the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework. 0.034g of the finished product was weighed and stored.

[0053] Step 2: Take 0.34g of pyrrole monomer, approximately 0.35ml, and add deionized water to 50ml to prepare a 0.1M solution. Distill the solution until it is colorless, indicating that the deoxidation is complete. Take 2.5ml and record it as solution A. Then, take 0.087g of sodium dodecylbenzenesulfonate and add it to 50ml of deionized water to prepare a 5mM solution. Take 47.5ml and record it as solution B. Mix solutions A and B to obtain solution C.

[0054] Step 3: Using the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in Step 1 as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the C solution obtained in Step 2 as the electrolyte, the constant potential deposition voltage was set to 0.8V and the deposition time was set to 700s to prepare the C / PPy composite electrode. After that, it was washed and dried multiple times with anhydrous ethanol and deionized water, and weighed to obtain 0.0379g, that is, 0.0039g of PPy was loaded on a single electrode.

[0055] The mass loading was 3.9 mg / cm³. 3 The peak DPV response was 4.53 mA, proving that when the raw material is insufficient, the polymer structure is unstable, forming short chains with low binding amount.

[0056] Comparative Example 2 (Effect of Activator)

[0057] Step 1: The natural silk fabric was ultrasonically cleaned with anhydrous ethanol and deionized water for 30 minutes each, dried at room temperature, and then activated by immersing it in a saturated KCl solution in a 60℃ constant temperature oven for 12 hours. The activated fabric was rinsed with deionized water and dried. After being cut to the required electrode size, it was transferred to a tube furnace for heat treatment. Starting from room temperature of 25℃, the temperature was increased by 6℃ per minute to 300℃ and held for 2 hours. Then, nitrogen gas was introduced and the temperature was increased to 900℃ at a rate of 5℃ / min and held for 2 hours. The fabric was then cooled down with the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework. 0.034g of the finished product was weighed and stored.

[0058] Step 2: Take 0.68g of pyrrole monomer (approximately 0.7ml) and add deionized water to 50ml to prepare a 0.2M solution. Distill the solution until it is colorless, indicating that the deoxidation is complete. Take 2.5ml and record it as solution A. Then, take 0.1g of sodium dodecylbenzenesulfonate and add it to 50ml of deionized water to prepare a 6mM solution. Take 47.5ml and record it as solution B. Mix solutions A and B to obtain solution C.

[0059] Step 3: Using the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in Step 1 as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the C solution obtained in Step 2 as the electrolyte, the constant potential deposition voltage was set to 0.8V and the deposition time was set to 700s to prepare the C / PPy composite electrode. After washing and drying with anhydrous ethanol and deionized water multiple times, 0.0389g was weighed, that is, 0.0049g of PPy was loaded on a single electrode.

[0060] The active substance mass loading was 4.9 mg / cm³. 3 The peak DPV response was 5.23 mA, indicating that the effect of active substances on the deposits is limited and will not significantly alter the material properties.

[0061] Comparative Example 3 (Influence of Deposition Time)

[0062] Step 1: The natural silk fabric was ultrasonically cleaned with anhydrous ethanol and deionized water for 30 minutes each, dried at room temperature, and then activated by immersing it in a saturated KCl solution in a 60℃ constant temperature oven for 12 hours. The activated fabric was rinsed with deionized water and dried. After being cut to the required electrode size, it was transferred to a tube furnace for heat treatment. Starting from room temperature of 25℃, the temperature was increased by 6℃ per minute to 300℃ and held for 2 hours. Then, nitrogen gas was introduced and the temperature was increased to 900℃ at a rate of 5℃ / min and held for 2 hours. The fabric was then cooled down with the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework. 0.034g of the finished product was weighed and stored.

[0063] Step 2: Take 0.68g of pyrrole monomer, approximately 0.7ml, and add deionized water to 50ml to prepare a 0.2M solution. Distill the solution until it is colorless, indicating that the deoxidation is complete. Take 2.5ml and record it as solution A. Then, take 0.087g of sodium dodecylbenzenesulfonate and add it to 50ml of deionized water to prepare a 5mM solution. Take 47.5ml and record it as solution B. Mix solutions A and B to obtain solution C.

[0064] Step 3: Using the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in Step 1 as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the C solution obtained in Step 2 as the electrolyte, the constant potential deposition voltage was set to 0.8V and the deposition time was set to 1000s to prepare the C / PPy composite electrode. After that, it was washed and dried multiple times with anhydrous ethanol and deionized water, and weighed to obtain 0.0396g, that is, the single electrode was loaded with 0.0056g of PPy.

[0065] The active substance mass loading was 5.6 mg / cm³. 3 The peak DPV response is 4.34 mA. When the deposition time is too long, more Py monomers will be deposited on the material. Excessive material will hinder electron transport, resulting in a decrease in conductivity and current response.

[0066] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor electrode for collagen detection, characterized by: The method comprises the following steps: Step 1: ultrasonic cleaning and drying of silk fabric, activation by soaking in saturated KCl solution, carbonization by heat treatment after secondary drying, cooling, and obtaining a nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework; Step 2: distillation of pyrrole monomer solution, then adding sodium dodecyl benzene sulfonate to obtain a mixed solution; Step 3: taking the nitrogen-containing porous pseudo-graphite layer structure carbon three-dimensional framework obtained in step 1 as a working electrode, a platinum sheet as a counter electrode, an Ag / AgCl electrode as a reference electrode, and the mixed solution obtained in step 2 as an electrolyte, and performing constant potential electrochemical deposition to prepare a C / PPy composite electrode, and then washing with anhydrous ethanol and deionized water and drying; Step 4: heating and boiling chloroauric acid solution, then adding sodium citrate dropwise, stirring uniformly, and obtaining a wine-red gold nanoparticle solution; Step 5: taking the C / PPy composite electrode obtained in step 3, adding the gold nanoparticle solution obtained in step 4 dropwise, heating and fixing, and obtaining C / PPy / AuNps; Step 6: adding mercaptopropionic acid solution to C / PPy / AuNps and heating and combining; Step 7: adding a mixed solution of EDC and NHS to the electrode obtained in step 6 dropwise, and heating and activating; Step 8: taking bovine serum albumin, adding to PBS buffer solution at a ratio of 1:90-110 to obtain a diluent, and then mixing with an antibody to obtain a collagen monoclonal antibody diluent; Step 9: dropping the collagen monoclonal antibody diluent on the surface of the activated electrode, incubating, and obtaining an electrochemical immunosensor electrode for collagen detection.

2. The production method according to claim 1, characterized by: In step 1: ultrasonic cleaning with anhydrous ethanol for 20-40 min, activation temperature of 50-70 DEG C, and activation time of 10-14 h; the heat treatment carbonization is as follows: taking room temperature as the starting point, increasing to 280-320 DEG C at a rate of 5-8 DEG C / min in air, keeping for 1.5-2.5 h, passing nitrogen, increasing to 850-950 DEG C at a rate of 2-5 DEG C / min, keeping for 1.5-2 h, and naturally cooling in the furnace to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon material.

3. The production method according to claim 1, wherein: In step 2: the concentration of pyrrole monomer solution is 0.1-0.2 M; and the concentration of sodium dodecyl benzene sulfonate is 4-6 mM.

4. The production method according to claim 1, wherein: In step 3: the potential window is set to 0.7-0.9 V; and the electrochemical deposition time is 400-1000 s.

5. The production method according to claim 1, wherein: In step 4: the concentration of chloroauric acid is 0.8-1.2 mM; the concentration of sodium citrate is 0.08-0.12 M; and the stirring time is 10-20 min.

6. The production method according to claim 1, wherein: In step 5: the heating and fixing temperature is 35-40 DEG C, and the time is 0.5-1.5 h.

7. The production method according to claim 1, wherein: In step 6: the concentration of mercaptopropionic acid solution is 0.2-0.4 M; the volume ratio of gold nanoparticle solution to mercaptopropionic acid solution is 1:3-5; the heating and combining temperature is 35-40 DEG C, and the time is 0.5- 1.5 h.

8. The production method according to claim 1, wherein: In step 7: the molar ratio of EDC to NHS is 1.8-2.2:1, the heating and activating temperature is 35-40 DEG C, and the time is 0.5-2 h.

9. The production method according to claim 1, wherein: In step 8: the weight ratio of bovine serum albumin to PBS buffer solution is 1:80-120; the concentration of antibody in the collagen monoclonal antibody diluent is 2-4 mg / mL; the pH of the PBS buffer solution is 7.

4.

10. The production method according to claim 1, wherein: In step 9: the incubation temperature is 35-40°C and the incubation time is 0.5-1.5h.