Preparation method and application of a carbon dioxide-responsive imprinted modified electrode for detecting myoglobin

Abstract

The present invention belongs to the field of electrochemical technology and discloses a method for preparing a carbon dioxide-responsive imprinted modified electrode for detecting myoglobin and its application. An electrode modified with gold nanoparticles, toluidine blue, and platinum nanoparticles is used as a catalytic electrode, and an electrode modified with an initiator is used as a working electrode. Constant current polymerization is performed for 1.5 hours to prepare an Au / AuNPs / Polymer electrode. The prepared electrode is then immersed in an SDS eluent to remove the template molecules, thereby preparing an Au / AuNPs / MIP electrode. The Au / AuNPs / MIP imprinted modified electrode of the present invention can effectively identify myoglobin and can be used as an electrochemical sensor for detecting myoglobin, enabling rapid and highly sensitive detection of myoglobin.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemistry, and relates to a preparation method and application of a carbon dioxide (CO2) responsive imprinted modified electrode for detecting myoglobin. Background Art

[0002] Myoglobin (Mb), a single-chain heme protein found primarily in cardiac and skeletal muscle, plays a role in oxygen storage and transport during life. Several common methods for detecting Mb include colorimetry, surface plasmon resonance, fluorescence analysis, and blood glucose meter detection. However, each has limitations. For example, colorimetry is difficult to quantify; surface plasmon resonance, while highly sensitive and reproducible, is complex and expensive to perform; and fluorescence analysis and blood glucose meter detection, while highly sensitive and fast, require complex experimental conditions, making them difficult to widely use. Summary of the Invention

[0003] In order to overcome the shortcomings of the existing technology, the present invention provides a preparation method and application of a CO2-responsive imprinted modified electrode for detecting myoglobin. A CO2-responsive imprinted modified electrode that can be used to detect myoglobin is prepared by using the electrochemically induced ATRP method. The modified electrode can achieve rapid and highly sensitive detection of myoglobin.

[0004] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0005] A CO2-responsive imprinted modified electrode for detecting myoglobin is disclosed. The electrode modified with nano-gold, toluidine blue, and nano-platinum is used as a catalytic electrode, and an initiator-modified electrode is used as a working electrode. An Au / AuNPs / Polymer electrode is prepared by constant current polymerization for 1.5 hours. The prepared electrode is then immersed in SDS eluent to remove the template molecules to obtain an Au / AuNPs / MIP electrode.

[0006] The preparation method of the CO2-responsive imprinted modified electrode for detecting myoglobin comprises the following specific steps:

[0007] Step a. Insert a clean gold wire electrode (Φ = 0.4 mm) into a 0.25-4% chloroauric acid solution (the solvent is ultrapure water) (the height of the electrode immersed in the solution is controlled to be 2 mm). Electrodeposition is performed at a constant potential of -0.9 V for 400 s using a chronoamperometry method. The electrode is then washed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs modified electrode. The Au / AuNPs electrode is placed in a 1.25×10 -5 ~2.5×10 -4mol / L toluidine blue (TB, prepared by 0.1mol / L pH 5.0ABS) solution, the height of the electrode immersed in the solution was controlled to be 1mm, and then cyclic voltammetry was scanned for 50 cycles; the parameters were set as follows: the potential range was -0.8~1.3V, the scan rate was 0.05V / s, and after the scan, it was rinsed with PBS to obtain the Au / AuNPs / PTB modified electrode, and the Au / AuNPs / PTB electrode was placed in a 0.0025~0.04mol / L HPtCl6 (prepared by 0.1mol / L pH 5.0ABS) solution, and nanoplatinum was electrodeposited for 100s at a potential of -0.7V using chronoamperometry (the height of the electrode immersed in the solution was controlled to be 1mm). After the deposition was completed, the Au / AuNPs / PTB / nPt catalytic electrode was obtained;

[0008] Step b. Self-assembly of an initiator (4-mercaptophenyl 2-bromo-2-methylpropanoate, 4-HTP-Br) on the surface of the Au / AuNPs modified electrode; accurately weigh 0.0231 to 0.3696 g N,N-methylenebisacrylamide (MBA) was dissolved in 12 ml of PBS (0.1 mol / L) as a cross-linking agent, 20.1-321.6 μL of 2-Diethylaminoethylmethacrylate (DEAEMA) was taken as a functional monomer (CO2-responsive functional monomer), and 0.001-0.016 g of myoglobin was used as a template. The mixture was ultrasonically shaken for 10 min to completely dissolve it to prepare an imprinting mixed solution. At room temperature, an initiator-modified electrode was inserted into the above mixed solution and polymerized under constant current for 1.5 h to obtain an Au / AuNPs / Polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 2.5-40% SDS to remove the template myoglobin. After washing the electrode with PBS three times, a CO2-responsive myoglobin imprinted polymer modified electrode Au / AuNPs / MIP was obtained.

[0009] The CO2-responsive imprinted modified electrode Au / AuNPs / MIP prepared by the above-mentioned preparation method was applied to the detection of myoglobin. Specifically, a three-electrode system was established using Au / AuNPs / MIP as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a clean platinum wire electrode (Φ = 0.5 mm) as the auxiliary electrode. Differential pulse voltammetry was used to construct a linear regression equation based on the relationship between peak current and myoglobin concentration. The detection limit was calculated based on the linear regression equation.

[0010] The linear regression equation for the relationship between myoglobin concentration and peak current change is ΔI (μA) = 7.344logC (mg / mL) + 104.826, where the correlation coefficient is R 2 =0.992, the linear range of myoglobin detection is 10 -13 ~10 - 3 mg / mL, and the detection limit was 1.96×10 -14 mg / mL.

[0011] The beneficial effects of the present invention compared with the prior art are:

[0012] The Au / AuNPs / MIP imprinted modified electrode provided by the present invention is simple to prepare and requires simple instruments and equipment. The Au / AuNPs / MIP imprinted modified electrode of the present invention can well identify myoglobin, that is, the modified electrode can be used as an electrochemical sensor for detecting myoglobin. The experimental results show that Figure 3 The results showed that the sensor can detect myoglobin rapidly and with high sensitivity, thus providing an efficient, inexpensive and convenient analytical sensor device for the field of myoglobin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The different modified electrodes in the electrode preparation process of Example 1 of the present invention are prepared in the presence of 5mM [Fe(CN)6] 3- / 4- Cyclic voltammogram in 0.1 M KCl (pH 7.0 PBS) solution.

[0014] Figure 2 The modified electrodes at each stage of Example 1 of the present invention are 5mM [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy in 0.1 M KCl (pH = 7.0 PBS) solution.

[0015] Figure 3 Figures 1 and 2 show the selectivity experimental results of the Au / AuNPs / MIP imprinted modified electrode of Example 1 of the present invention. Figure (a) shows the DPV curves of the Au / AuNPs / MIP modified electrode used as an electrochemical biosensor to detect a series of different concentrations of myoglobin; Figure (b) shows the working curve of the Au / AuNPs / MIP modified electrode for detecting myoglobin.

[0016] Figure 4 Schematic diagram of the CO2 selective responsiveness of Au / AuNPs / MIP to myoglobin detection in Example 1 of the present invention. DETAILED DESCRIPTION

[0017] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels. Example 1 in the following examples is the most preferred embodiment.

[0018] Example 1

[0019] A method for preparing a CO2-responsive imprinted modified electrode for detecting myoglobin is as follows:

[0020] a. Insert a clean gold wire electrode (Φ = 0.4 mm) into a 1% chloroauric acid solution (solvent: ultrapure water) (control the electrode immersion height in the solution to be 2 mm), and perform electrodeposition at a constant potential of -0.9 V for 400 s using the chronoamperometry method. After that, rinse with ultrapure water and blow dry with nitrogen to obtain an Au / AuNPs modified electrode. Place the Au / AuNPs electrode in a 5×10 -5 The Au / AuNPs / PTB modified electrode was prepared in a 0.1 mol / L toluidine blue (TB) solution (prepared from 0.1 mol / L pH 5.0 ABS) with the electrode immersed at a height of 1 mm. Cyclic voltammetry was then performed for 50 cycles. The parameters were set as follows: potential range -0.8–1.3 V, scan rate 0.05 V / s, and PBS rinse after the scan to obtain the Au / AuNPs / PTB modified electrode. The Au / AuNPs / PTB electrode was placed in a 0.01 mol / L HPtCl₆ solution (prepared from 0.1 mol / L pH 5.0 ABS) and nanoplatinum was electrodeposited at a potential of -0.7 V for 100 s using chronoamperometry (with the electrode immersed at a height of 1 mm). After deposition, the Au / AuNPs / PTB / nPt catalytic electrode was obtained.

[0021] b. Self-assembly of an initiator (4-thiophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) on the surface of the Au / AuNPs-modified electrode. Accurately weigh 0.0924 g of N,N-methylenebisacrylamide (MBA) as a crosslinker and dissolve it in 12 ml of 0.1 mol / L PBS. Add 80.4 μL of 2-diethylaminoethyl methacrylate (DEAEMA) as a CO2-responsive functional monomer and 0.004 g of myoglobin as a template. Ultrasonicate for 10 minutes to dissolve the components to prepare an imprinting mixture. Insert the initiator-modified electrode into the mixture at room temperature and perform galvanostatic polymerization for 1.5 hours to obtain an Au / AuNPs / polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 10% SDS to remove the template myoglobin. The electrode was washed three times with PBS to obtain a CO2-responsive myoglobin-imprinted polymer-modified electrode (Au / AuNPs / MIP).

[0022] Figure 1 The modified electrodes at different stages were exposed to 0.1mol / L KCl+5mmol / L[Fe(CN)6] 3- / 4- CV curves in PBS (0.1 mol / L pH = 7.0) electrolyte solution. Curve 1 is the bare gold wire electrode before polymer modification, and curve 2 is the CV curve of the Au / AuNPs / PTB / nPt modified gold wire electrode. The peak current is significantly higher than that of curve 1. The reason is that Au / AuNPs / PTB / nPt increases the electrode surface area and electron transfer capacity, resulting in a significant increase in the peak current. Curve 3 is the CV curve of the Au / AuNPs / MIP modified electrode without eluting myoglobin. The peak current is significantly lower than that of curve 2. This is because the polymer film is an inert substance, and the polymerized myoglobin on the electrode surface hinders the Fe(CN)6 3- / 4- The probe ions reach the electrode surface, causing the peak current to decrease. Curve 4 shows the CV curve after elution of the template myoglobin. The peak current is significantly higher than that of Curve 3. This is mainly due to the presence of imprinted holes on the electrode surface after elution of the template myoglobin. This makes it easier for the probe ions to diffuse to the electrode surface, resulting in an increase in the peak current.

[0023] Figure 2 The modified electrodes at different stages were exposed to 0.1mol / L KCl+5mmol / L[Fe(CN)6] 3- / 4-EIS curve in electrolyte solution (PBS with 0.1mol / L pH=7.0). The bare gold wire electrode shows a larger Rct (curve 1). When the gold wire electrode is modified with nano-gold (curve 2), the diameter of the semicircle decreases, proving that the electron transfer ability of the electrode modified with nano-gold is improved. When the polymer is successfully constructed on the electrode surface (curve 3), the diameter of the semicircle becomes larger. The main reason for this phenomenon may be that the polymer is an inert substance and does not have good electron transfer ability, which makes Fe(CN)6 3- / 4- When the template molecules are eluted from the polymer (curve 4), it can be observed that the semicircle diameter of curve 4 is smaller than that of curve 3. This is because a certain number of imprinted cavities are formed after the template molecules leave the polymer. The imprinted cavities are Fe(CN)6 3- / 4- The results of CV and EIS were consistent, indicating that the imprinted polymer modified electrode Au / AuNPs / MIP has been successfully constructed.

[0024] Example 2:

[0025] a. A clean gold wire electrode (Φ = 0.4 mm) was inserted into a 0.5% chloroauric acid solution (solvent: ultrapure water) (the height of the electrode immersed in the solution was controlled to be 2 mm). Electrodeposition was performed at a constant potential of -0.9 V for 400 s using the chronoamperometry method. The electrode was then washed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs modified electrode. The Au / AuNPs electrode was placed in a 2.5×10 - 5 The Au / AuNPs / PTB modified electrode was prepared in a 0.1 mol / L toluidine blue (TB) solution (prepared from 0.1 mol / L pH 5.0 ABS) with the electrode immersed at a height of 1 mm. Cyclic voltammetry was then performed for 50 cycles. The parameters were set as follows: potential range -0.8–1.3 V, scan rate 0.05 V / s, and PBS rinse after the scan to obtain the Au / AuNPs / PTB modified electrode. The Au / AuNPs / PTB electrode was placed in a 0.005 mol / L HPtCl₆ solution (prepared from 0.1 mol / L pH 5.0 ABS) and nanoplatinum was electrodeposited using chronoamperometry at a potential of -0.7 V for 100 s (with the electrode immersed at a height of 1 mm). After deposition, the Au / AuNPs / PTB / nPt catalytic electrode was obtained.

[0026] b. Self-assembly of an initiator (4-HTP-Br) onto the surface of an Au / AuNPs-modified electrode. Accurately weigh 0.0462 g of N,N-methylenebisacrylamide (MBA) as a crosslinker and dissolve it in 12 ml of 0.1 mol / L PBS. Add 40.2 μL of 2-diethylaminoethyl methacrylate (DEAEMA) as a CO2-responsive functional monomer and 0.002 g of myoglobin as a template. Ultrasonicate for 10 minutes to dissolve the components to prepare an imprinting mixture. Insert the initiator-modified electrode into this mixture at room temperature and perform galvanostatic polymerization for 1.5 hours to obtain an Au / AuNPs / polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 5% SDS to remove the template myoglobin. The electrode was washed three times with PBS to obtain a CO2-responsive myoglobin-imprinted polymer-modified electrode (Au / AuNPs / MIP).

[0027] Example 3:

[0028] a. A clean gold wire electrode (Φ = 0.4 mm) was inserted into a 0.25% chloroauric acid solution (solvent: ultrapure water) (the height of the electrode immersed in the solution was controlled to be 2 mm). Electrodeposition was performed at a constant potential of -0.9 V for 400 s using the chronoamperometry method. The electrode was then washed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs modified electrode. The Au / AuNPs electrode was placed in a 1.25×10 -5 The Au / AuNPs / PTB modified electrode was prepared in a 0.1 mol / L toluidine blue (TB) solution (prepared from 0.1 mol / L pH 5.0 ABS) with the electrode immersed at a height of 1 mm. Cyclic voltammetry was then performed for 50 cycles. The parameters were set as follows: potential range -0.8–1.3 V, scan rate 0.05 V / s, and PBS rinse after the scan to obtain the Au / AuNPs / PTB modified electrode. The Au / AuNPs / PTB electrode was placed in a 0.0025 mol / L HPtCl₆ solution (prepared from 0.1 mol / L pH 5.0 ABS) and nanoplatinum was electrodeposited using chronoamperometry at a potential of -0.7 V for 100 s (with the electrode immersed at a height of 1 mm). After deposition, the Au / AuNPs / PTB / nPt catalytic electrode was obtained.

[0029] b. Self-assembly of an initiator (4-thiophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) on the surface of the Au / AuNPs-modified electrode. Accurately weigh 0.0231 g of N,N-methylenebisacrylamide (MBA) as a crosslinker and dissolve it in 12 ml of 0.1 mol / L PBS. Add 20.1 μL of 2-diethylaminoethyl methacrylate (DEAEMA) as a CO2-responsive functional monomer and 0.001 g of myoglobin as a template. Ultrasonicate for 10 minutes to dissolve the components to prepare an imprinting mixture. Insert the initiator-modified electrode into the mixture at room temperature and perform galvanostatic polymerization for 1.5 hours to obtain an Au / AuNPs / polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 2.5% SDS to remove the template myoglobin. The electrode was washed three times with PBS to obtain a CO2-responsive myoglobin-imprinted polymer-modified electrode (Au / AuNPs / MIP).

[0030] Example 4:

[0031] a. Insert a clean gold wire electrode (Φ = 0.4 mm) into a 2% chloroauric acid solution (solvent: ultrapure water) (control the electrode immersion height in the solution to be 2 mm). Using the chronoamperometry, perform electrodeposition at a constant potential of -0.9 V for 400 s. After that, rinse with ultrapure water and blow dry with nitrogen to obtain an Au / AuNPs modified electrode. Place the Au / AuNPs electrode in a 1.25×10 - 4 The Au / AuNPs / PTB modified electrode was prepared in a 0.1 mol / L toluidine blue (TB) solution (prepared from 0.1 mol / L pH 5.0 ABS) with the electrode immersed at a height of 1 mm. Cyclic voltammetry was then performed for 50 cycles. The parameters were set as follows: potential range -0.8–1.3 V, scan rate 0.05 V / s, and PBS rinse after the scan to obtain the Au / AuNPs / PTB modified electrode. The Au / AuNPs / PTB electrode was placed in a 0.02 mol / L HPtCl₆ solution (prepared from 0.1 mol / L pH 5.0 ABS) and nanoplatinum was electrodeposited at a potential of -0.7 V for 100 s using chronoamperometry (with the electrode immersed at a height of 1 mm). After deposition, the Au / AuNPs / PTB / nPt catalytic electrode was obtained.

[0032] b. Self-assembly of an initiator (4-HTP-Br) on the surface of an Au / AuNPs-modified electrode. Accurately weigh 0.1848 g of N,N-methylenebisacrylamide (MBA) as a crosslinker and dissolve it in 12 ml of 0.1 mol / L PBS. Add 160.8 μL of 2-diethylaminoethyl methacrylate (DEAEMA) as a CO2-responsive functional monomer and 0.008 g of myoglobin as a template. Ultrasonicate for 10 minutes to dissolve the components to prepare an imprinting mixture. Insert the initiator-modified electrode into the mixture at room temperature and perform galvanostatic polymerization for 1.5 hours to obtain an Au / AuNPs / polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 20% SDS to remove the template myoglobin. The electrode was washed three times with PBS to obtain a CO2-responsive myoglobin-imprinted polymer-modified electrode (Au / AuNPs / MIP).

[0033] Example 5:

[0034] a. Insert a clean gold wire electrode (Φ = 0.4 mm) into a 4% chloroauric acid solution (solvent: ultrapure water) (control the electrode immersion height in the solution to be 2 mm). Using the chronoamperometry, perform electrodeposition at a constant potential of -0.9 V for 400 s. After that, rinse with ultrapure water and blow dry with nitrogen to obtain an Au / AuNPs modified electrode. Place the Au / AuNPs electrode in a 2.5×10 - 4 The Au / AuNPs / PTB modified electrode was prepared in a 0.1 mol / L toluidine blue (TB) solution (prepared from 0.1 mol / L pH 5.0 ABS) with the electrode immersed at a height of 1 mm. Cyclic voltammetry was then performed for 50 cycles. The parameters were set as follows: potential range -0.8–1.3 V, scan rate 0.05 V / s, and PBS rinse after the scan to obtain the Au / AuNPs / PTB modified electrode. The Au / AuNPs / PTB electrode was placed in a 0.04 mol / L HPtCl₆ solution (prepared from 0.1 mol / L pH 5.0 ABS) and nanoplatinum was electrodeposited using chronoamperometry at a potential of -0.7 V for 100 s (with the electrode immersed at a height of 1 mm). After deposition, the Au / AuNPs / PTB / nPt catalytic electrode was obtained.

[0035] b. Self-assembly of an initiator (4-mercaptophenyl 2-bromo-2-methylpropanoate, 4-HTP-Br) onto the surface of the Au / AuNPs-modified electrode. Accurately weigh 0.3696 g of N,N-methylenebisacrylamide (MBA) as a crosslinker and dissolve it in 12 ml of 0.1 mol / L PBS. Add 321.6 μL of 2-diethylaminoethyl methacrylate (DEAEMA) as a CO2-responsive functional monomer and 0.016 g of myoglobin as a template. Ultrasonicate for 10 minutes to dissolve the components to prepare an imprinting mixture. Insert the initiator-modified electrode into the mixture at room temperature and perform galvanostatic polymerization for 1.5 hours to obtain an Au / AuNPs / polymer electrode. The prepared Au / AuNPs / Polymer electrode was immersed in a 0.1 mol / L CH3COOH solution containing 40% SDS to remove the template myoglobin. The electrode was washed three times with PBS to obtain a CO2-responsive myoglobin-imprinted polymer-modified electrode (Au / AuNPs / MIP).

[0036] Application Example 1

[0037] The electrode prepared in Example 1 is used as an electrochemical biosensor for detecting myoglobin, comprising the following steps:

[0038] Using Au / AuNPs / MIP as the working electrode, a saturated calomel electrode (SCE) and a clean platinum wire electrode (Φ=0.5mm) as the reference electrode and auxiliary electrode, respectively, the three electrodes were placed in myoglobin solutions of different concentrations, and the differential pulse voltammetry curves were recorded. A linear regression equation for detection was constructed based on the change in myoglobin concentration and peak current. The detection limit can be calculated based on the regression equation.

[0039] The linear regression equation for the relationship between myoglobin concentration and peak current change in Example 1 is ΔI (μA) = 7.344logC (mg / mL) + 104.826, where the correlation coefficient is R 2 =0.992, the linear range of myoglobin detection is 10 -13 ~10 -3 mg / mL, and the detection limit was 1.96×10 -14 mg / mL.

[0040] Figure 3(a) DPV curves of Au / AuNPs / MIP modified electrode as electrochemical biosensor for detecting a series of myoglobin concentrations (myoglobin concentrations were 0, 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 mg / mL). As can be seen from the figure, as the concentration of myoglobin increases, the DPV peak current decreases. This is because after the imprinted polymer recognizes myoglobin, myoglobin occupies the imprinted holes, blocking the diffusion of probe ions to the electrode surface, resulting in a decrease in peak current. By plotting the DPV peak current difference (response signal, ΔI) against the logarithm of myoglobin concentration, the working curve of the modified electrode for detecting myoglobin is obtained ( Figure 3 As shown in (b), the linear regression equation of myoglobin detection by Au / AuNPs / MIP modified electrode is ΔI(μA)=7.344logC(mg / mL)+104.826, the correlation coefficient is 0.992, and the linear response range is 10 -13 ~10 -3 mg / mL.

[0041] In order to further explore the responsiveness of the electrochemical sensor constructed by Au / AuNPs / MIP electrode to different gases, CO2 and N2 were used to treat the SOD test solution (10 -9 The Au / AuNPs / MIP electrode was treated with Mb solution for 3 min, then incubated in the Mb test solution for 5 min, rinsed with PBS 3 times, and then subjected to DPV test. Figure 4 As shown, it can be observed that compared with the detection signal of the Au / AuNPs / MIP electrode for the CO2-treated Mb test solution (curve 3), the Au / AuNPs / MIP electrode exhibits a smaller signal response after incubation with the N2-treated Mb test solution (curve 2). The main reason for this phenomenon is that the tertiary amine group in the functional monomer (DEAEMA) can react with the CO2 in the solution to form a protonated form. The protonated form of DEAEMA makes the imprinted polymer highly hydrophilic, which is conducive to the recognition of target molecules. Therefore, the prepared Au / AuNPs / MIP electrode has better responsiveness to the CO2-treated Mb test solution.

[0042] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a carbon dioxide-responsive imprinted modified electrode for detecting myoglobin, characterized in that: The specific steps are as follows: Step a. Insert a clean gold wire electrode into a 0.25-4% chloroauric acid solution and perform electrodeposition at a constant potential of -0.9 V for 400 s using chronoamperometry. Then, rinse with ultrapure water and dry with nitrogen to obtain an Au / AuNPs modified electrode. Place the Au / AuNPs electrode in a 1.25×10 -5 ~2.5×10 -4 mol / L toluidine blue solution, the electrode was immersed in the solution to a height of 1 mm, and then cyclic voltammetry was performed for 50 cycles; the parameters were set as follows: the potential range was -0.8~1.3 V, the scan rate was 0.05 V / s, and after the scan, the Au / AuNPs / PTB modified electrode was rinsed with PBS. The Au / AuNPs / PTB electrode was placed in a 0.0025~0.04 mol / L HPtCl6 solution and nanoplatinum was electrodeposited at a potential of -0.7 V for 100 s using chronoamperometry. After the deposition was completed, an Au / AuNPs / PTB / nPt catalytic electrode was obtained. Step b. Self-assemble the initiator 4-thiophenyl-2-bromo-2-methylpropionate on the surface of the Au / AuNPs modified electrode; accurately weigh 0.0231~0.3696 g N,N-methylenebisacrylamide as a cross-linking agent and dissolve it in 12 ml of PBS, measure 20.1~321.6 μL diethylaminoethyl methacrylate as a functional monomer, and 0.001~0.016 g myoglobin as a template, and ultrasonically vibrate for 10 minutes to completely dissolve them to prepare an imprinting mixed solution; at room temperature, insert the initiator-modified electrode into the above mixed solution, and use the Au / AuNPs / PTB / nPt catalytic electrode prepared in step a as the catalytic electrode, and perform constant current polymerization for 1.5 hours to obtain an Au / AuNPs / Polymer electrode; soak the prepared Au / AuNPs / Polymer electrode in 0.1 mol / L containing 2.5~40% SDS. The template myoglobin was removed in CH3COOH solution; after the electrode was washed with PBS three times, a CO2-responsive myoglobin imprinted polymer modified electrode Au / AuNPs / MIP was obtained.

2. Use of the carbon dioxide-responsive imprinted modified electrode Au / AuNPs / MIP prepared by the preparation method according to claim 1 in detecting myoglobin.

3. The use of the carbon dioxide responsive imprinted modified electrode Au / AuNPs / MIP in detecting myoglobin as claimed in claim 2, characterized in that: A three-electrode system was established using Au / AuNPs / MIP as the working electrode, a saturated calomel electrode and a clean platinum wire electrode as the reference electrode and auxiliary electrode, respectively. Differential pulse voltammetry was used, and a linear regression equation was constructed based on the relationship between peak current and myoglobin concentration. The detection limit was calculated based on the linear regression equation. The linear regression equation for the relationship between myoglobin concentration and peak current change is: ΔI (μA)=7.344logC (mg / mL) + 104.826, where the correlation coefficient is R 2 =0.992, the linear range of myoglobin detection is 10 -13 ~10 -3 mg / mL, and the detection limit was 1.96×10 -14 mg / mL.

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