Preparation method of molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate

Abstract

The invention discloses a preparation method of a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate, which comprises the following steps: by taking reduced graphene oxide and gold nanoparticles as modification materials, dispensing the reduced graphene oxide on the surface of a glassy carbon electrode, drying to obtain a modified electrode RGO / GCE, putting the RGO / GCE into a sulfuric acid deposition solution containing chloroauric acid, washing with water, and drying to obtain the molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate. Gold nanoparticles are modified on the surface of RGO / GCE by adopting a potentiostatic method to prepare a gold substrate, a deposition electrode AuNPs / RGO / GCE is obtained, the AuNPs / RGO / GCE is placed in an electrolyte polymerization solution containing a template substance cyclic adenosine monophosphate and a functional monomer o-phenylenediamine for electropolymerization, a layer of compact and insulating polymer film is obtained, the polymerized electrode is immersed in an elution solution, and the gold substrate and the deposition electrode AuNPs / RGO / GCE are obtained. Eluting by adopting a cyclic voltammetry method to obtain MIP / AuNPs / RGO / GCE; the sensor has specific recognition selectivity on cyclic adenosine monophosphate, high detection sensitivity, lower detection limit and good reusability and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive substance detection, and relates to a method for preparing a molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate. Background Art

[0002] Cyclic adenosine monophosphate (cAMP) was first discovered by Sutherland in 1971. It is the first intracellular second messenger discovered and is widely present in animal, plant and microbial cells. cAMP is a white or off-white powder with a relative molecular mass of 329.21, a melting point close to 220°C, a pH close to neutral, slightly soluble in water, insoluble in organic solvents, and a chemical formula of C 10 H 12 N5O6P is composed of one adenine molecule, one ribose molecule and one phosphate group. cAMP is a biologically active component that is ubiquitous in the biological world and widely regulates the physiological and biochemical processes of organisms. At least 40 human diseases (including myocardial infarction, coronary heart disease, cardiogenic shock, hypertension and other major diseases) are related to the metabolic regulation of cAMP. The application fields of deep processing of functional foods based on cAMP, health care and high-purity extraction for medical use have high requirements for real-time and rapid quantitative detection of cAMP. Therefore, the detection and development and utilization of cyclic adenosine monophosphate have good development prospects.

[0003] At present, my country has no national standard for the detection of cAMP, only corporate standards for cAMP, and all of them use high-performance liquid chromatography. The literature mainly uses high-performance liquid chromatography, protein binding method, thin-layer chromatography and enzyme-linked immunosorbent assay. Among them, the chromatography method has the disadvantages of expensive instruments, complex and time-consuming pre-treatment, and difficulty in rapid detection; the protein binding method is relatively complicated to operate; the thin-layer chromatography method requires the concentration of the substance to be detected to reach a certain dose before it can be used; the immunoassay method has the disadvantages of being acid-base intolerant, easy to inactivate, and the result may be false positive.

[0004] Molecular imprinting technology is a technology for artificially synthesizing polymers that have specific binding to template molecules, and has the characteristics of high efficiency and selectivity. Molecular imprinting technology is used to prepare molecular imprinting polymers that have matching binding sites with the template molecules in structure, shape, size and functional groups. Molecular imprinting electrochemical sensors using molecular imprinting polymers as recognition elements have the advantages of strong specificity, high sensitivity, simple equipment operation and easy miniaturization. However, there are few studies on the detection of cAMP in jujube peel. So far, this technology has been mostly used in the determination of drugs, environment and biological substances.

[0005] Therefore, it is necessary to develop a cyclic AMP detection technology that is simple to operate, has strong specificity and high sensitivity to meet the needs of actual detection of jujube peel and other samples. Summary of the invention

[0006] In view of the above technical problems, the present invention aims to provide a method for preparing a molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate. The method uses cyclic adenosine monophosphate as a template substance and o-phenylenediamine as a functional monomer, and sequentially prepares the molecular imprinting electrochemical sensor through the steps of electrode modification, polymer preparation, elution, etc. The preparation process of the present invention is simple and low in cost. The prepared sensor has the advantages of high selectivity and high sensitivity, and is used to detect the content of cyclic adenosine monophosphate in a sample.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for preparing a molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate is carried out in the following order:

[0009] (1) Preparation of RGO / GCE

[0010] The reduced graphene oxide was added to dimethylformamide, and a light black dispersion was obtained after stirring and ultrasonic treatment, which was recorded as A; chitosan was added to a 1% acetic acid solution, and a solution obtained after magnetic stirring and ultrasonic treatment was recorded as B; under stirring, B was added dropwise to A, and the two were mixed to obtain a reduced graphene oxide modifier;

[0011] 5 μL of reduced graphene oxide modifier was dropped onto the surface of the glassy carbon electrode and dried to obtain the modified electrode RGO / GCE;

[0012] (2) Preparation of AuNPs / RGO / GCE

[0013] The RGO / GCE was placed in a sulfuric acid deposition solution containing chloroauric acid, and a constant potential of (-0.5)-(-0.1) V was used for deposition for 100-500 seconds to modify the gold nanoparticles on the surface of the RGO / GCE to prepare a gold substrate, thereby obtaining a deposited electrode AuNPs / RGO / GCE;

[0014] (3) Preparation of MIP / AuNPs / RGO / GCE

[0015] AuNPs / RGO / GCE was placed in an electrolyte polymerization solution containing a template substance, cyclic adenosine monophosphate, and a functional monomer, o-phenylenediamine, for electropolymerization. The initial polymerization potential was 0.1-0.5 V, the termination potential was 0.8-1.6 V, and the polymerization was repeated for 20 cycles to obtain a dense and insulating polymer film.

[0016] The polymerized electrode was immersed in an elution solution and eluted by cyclic voltammetry for 22-26 cycles to obtain MIP / AuNPs / RGO / GCE.

[0017] As a limitation of the preparation method of the present invention, in step (1), the mass volume ratio of the reduced graphene oxide to dimethylformamide is (0.5-2.5): 1 g / mL.

[0018] As another limitation of the preparation method of the present invention, in step (1), the mass volume ratio of the chitosan to the acetic acid solution is (2.5-12.5): 1 g / mL.

[0019] As a third limitation of the preparation method of the present invention, in step (1), the volume ratio of A to B is 1:1.

[0020] As a fourth limitation of the preparation method of the present invention, in step (2), the mass concentration of chloroauric acid in the deposition solution is 0.2-1.0 g / L, the molar concentration of sulfuric acid is 0.034-0.167 mol / L, and the volume ratio therebetween is 1:1.

[0021] As a fifth limitation of the preparation method of the present invention, in step (3), the molar ratio of the template substance to the functional monomer is 1:(1-5).

[0022] As a sixth limitation of the preparation method of the present invention, in step (3), the electrolyte is PBS, and the molar ratio of the template substance to the electrolyte is 1:(2-6).

[0023] As the seventh limitation of the preparation method of the present invention, in step (3), the elution solution is H2SO4 with a concentration of 0.2-0.6 mol / L.

[0024] like Figure 1 As shown, the NBO charge structure diagram of each atom, cyclic adenosine monophosphate ( Figure 1 In Figure A, the NBO charges of the two O atoms on the two hydroxyl groups, one oxygen atom, and the N atom on the amino group are O5 (-0.762), O6 (-0.946), O7 (-0.920), and N12 (-0.798), respectively. It is obvious that the electronegativity of the four atoms is strong and they are easy to accept H protons to form hydrogen bonds. In addition, the NBO charges of the four H atoms contained in the three groups of cyclic adenosine monophosphate are H29 (0.503), H31 (0.526), ​​H33 (0.433), and H34 (0.436), respectively. The more positive the H atom, the easier it is to form hydrogen bonds with atoms with strong electronegativity. The selected functional monomer, o-phenylenediamine ( Figure 1The NBO charges of the two amino groups on the template (Figure B) are N4 (-0.874) and N5 (-0.874), and the NBO charges of the four H atoms are H10 (0.408), H11 (0.407), H13 (0.408), and H15 (0.407). The results show that o-phenylenediamine can be used as both a proton donor and a proton acceptor to form hydrogen bonds with cyclic adenosine monophosphate. Among them, the strength of the interaction between the template molecule and the functional monomer complex is not only related to the charge distribution of the functional group, but also to the spatial structure of the molecule and the size of the steric hindrance of the atoms at the binding site. For a binding site with large steric hindrance, even if the charge density is large, it will be affected by the steric hindrance and it will be difficult to form hydrogen bonds and other interactions.

[0025] The above technical solution of the present invention is taken as a whole, and each step is closely related and influences each other, which jointly determine the morphological characteristics and performance of the product.

[0026] The above technical solution has the following advantages or beneficial effects:

[0027] 1. The preparation method of the present invention is simple, the process is easy to control, and it is easy to promote and apply industrialization.

[0028] 2. The sensor prepared by the present invention has specific recognition selectivity for cyclic adenosine monophosphate, high detection sensitivity, low detection limit, good reusability and stability.

[0029] The invention is suitable for detecting the content of cyclic adenosine monophosphate in jujube peel and other samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the NBO charge structure diagram of each atom in cyclic adenosine monophosphate and o-phenylenediamine, where: A is the NBO charge structure diagram of each atom in cyclic adenosine monophosphate, and B is the NBO charge structure diagram of each atom in o-phenylenediamine.

[0031] Figure 2 are configuration diagrams of complexes of cyclic adenosine monophosphate and different functional monomers at different ratios, wherein: A is a configuration diagram of a complex of cyclic adenosine monophosphate and o-aminophenol, B is a configuration diagram of a complex of cyclic adenosine monophosphate and resorcinol, C is a configuration diagram of a complex of cyclic adenosine monophosphate and o-phenylenediamine, and D is a configuration diagram of a complex of cyclic adenosine monophosphate and acrylamide;

[0032] Figure 3 The electrochemical characterization diagrams of different electrodes by different electrochemical methods, where A is the CV characterization diagram, B is the SWV characterization diagram, and C is the EIS characterization diagram;

[0033] Figure 4 It is the selectivity analysis diagram of the sensor template molecule;

[0034] Figure 5 The figure is a curve diagram of the sensor adsorbing different concentrations of cyclic adenosine monophosphate standard solution, wherein: A is the peak current curve diagram of the adsorption of different concentrations of cyclic adenosine monophosphate, and B is the linear relationship diagram between the peak current value and different concentrations of cyclic adenosine monophosphate.

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. DETAILED DESCRIPTION

[0036] Unless otherwise specified, the test methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial channels.

[0038] Example 1

[0039] In this embodiment, a molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate is prepared by following the following steps in sequence:

[0040] (1) Preparation of RGO / GCE

[0041] 0.005 g of reduced graphene oxide was added to 10 mL of dimethylformamide, and the mixture was stirred and ultrasonically treated to obtain a light black dispersion, which was recorded as A. 0.025 g of chitosan was added to 10 mL of 1% acetic acid solution, and the solution obtained was recorded as B by magnetic stirring and ultrasonic treatment. Under stirring, 5 mL of B was added dropwise to 5 mL of L-A, and the two were mixed to obtain a reduced graphene oxide modifier.

[0042] 5 μL of reduced graphene oxide modifier was dropped onto the surface of the glassy carbon electrode and dried to obtain the modified electrode RGO / GCE;

[0043] (2) Preparation of AuNPs / RGO / GCE

[0044] The RGO / GCE was placed in a 0.034 mol / L sulfuric acid deposition solution containing 0.2 g / L chloroauric acid (the volume ratio of the chloroauric acid solution to the sulfuric acid solution was 1:1), and a constant potential of -0.3 V was used for deposition for 300 s to modify the gold nanoparticles on the surface of the RGO / GCE to form a gold substrate, thereby obtaining a deposited electrode AuNPs / RGO / GCE;

[0045] (3) MIP / AuNPs / RGO / GCE

[0046] AuNPs / RGO / GCE was placed in a PBS polymerization solution containing a template substance cyclic adenosine monophosphate and a functional monomer o-phenylenediamine (molar ratio of 1:4) (the molar ratio of cyclic adenosine monophosphate to PBS polymerization solution was 1:2) for electropolymerization. The initial polymerization potential was 0.2 V, the termination potential was 1.0 V, and the polymerization was repeated for 20 cycles to obtain a dense and insulating polymer film.

[0047] The polymerized electrode was immersed in 0.4 mol / L H2SO4 and eluted by cyclic voltammetry for 24 cycles to obtain MIP / AuNPs / RGO / GCE.

[0048] The preparation method of this embodiment is simple and the process is easy to operate.

[0049] Embodiment 2-5

[0050] In Examples 2-5, a molecular imprinting electrochemical sensor for detecting cyclic adenosine monophosphate was prepared respectively. The preparation process was the same as that in Example 1. The only difference was that the corresponding technical parameters were different. The specific parameters are shown in the following table:

[0051]

[0052] The sensor prepared in Example 2-5 detects cyclic adenosine monophosphate quickly, sensitively and accurately, with mild reaction conditions and simple preparation process.

[0053] Test example

[0054] Experimental Example 1 Selection of functional monomers and determination of the ratio of template molecules to functional monomers

[0055] This test example screens the selection of functional monomers and the ratio of template material to functional monomers during the polymerization process, and tests the ratio of cyclic adenosine monophosphate to four functional monomers (o-aminophenol, resorcinol, o-phenylenediamine, and acrylamide).

[0056] Based on the possible binding sites, different proportions of complex configurations were constructed. The B3LYP / 6-311+G(d,p) algorithm was used to optimize the complex model formed by cyclic adenosine monophosphate and the four selected functional monomers in different proportions and calculate the vibration frequency to obtain thermodynamic data. The spatial structure, bond length, action site and binding energy of the stable configuration of the complexes with different proportions were analyzed to study the strength of the interaction between the template molecule and the four functional monomers. The principle that the more hydrogen bonds and action sites in the complex and the lower the binding energy, the more stable the structure of the formed complex is was used to screen the best functional monomer and optimize the best ratio of the complex.

[0057] like Figure 2As shown in the figure, the stable complex configuration formed by cyclic adenosine monophosphate and four functional monomers o-aminophenol, resorcinol, o-phenylenediamine, and acrylamide in a ratio of (1:1 to 1:4) is shown, and the action site and bond length are indicated in the figure. It can be seen from the figure that the template molecule cyclic adenosine monophosphate and the four functional monomers are mainly combined through the action of hydrogen bonds, and the hydrogen bond lengths all meet the specified lengths. Figure 2 It can be seen that through the comparison of the size of the binding energy, the four functional monomers o-aminophenol, resorcinol, o-phenylenediamine, acrylamide and cyclic adenosine monophosphate are combined in different proportions. The order of their binding energy (absolute value) is o-phenylenediamine>resorcinol>o-aminophenol>acrylamide. Among them, o-phenylenediamine has the smallest binding energy with cyclic adenosine monophosphate, the largest absolute value, and the largest heat released by the reaction. Therefore, when cyclic adenosine monophosphate is combined with o-phenylenediamine for imprinting, the energy of the complex system is lower, and the interaction force between the template molecule and the functional monomer is the strongest. Therefore, o-phenylenediamine is finally selected as the best functional monomer.

[0058] like Figure 2 C shows that as the proportion of imprinting increases, the amount of o-phenylenediamine in the complex increases, and the heat released also increases. The heat released by the formation of cyclic adenosine monophosphate-4-o-phenylenediamine complexes is 93.74 kJ·mol higher than that released by the formation of cyclic adenosine monophosphate-3-o-phenylenediamine complexes. -1 ; The heat released by the formation of cyclic adenosine monophosphate-5-o-phenylenediamine complexes is only 44.29 kJ·mol more than that released by the formation of cyclic adenosine monophosphate-4-o-phenylenediamine complexes. -1 , which is much smaller than the previous increase, indicating that at this time, the ability of o-phenylenediamine to bind to cyclic adenosine monophosphate has reached saturation, the active sites of the two are completely occupied, and the heat released reaches the maximum. If o-phenylenediamine is further increased, it will only lead to the formation of excess nonspecific forces and reduce the effective action sites. Therefore, when the ratio of the complex formed by cyclic adenosine monophosphate and o-phenylenediamine is 1:4, the interaction between the two is the strongest and the stability of the formed complex is optimal.

[0059] Experimental Example 2 Electrochemical Characterization of Different Electrodes

[0060] The three electrochemical methods of CV, SWV and EIS were used to analyze the -1 KCl and 5.0mmol·L -1 0.2 mol·L of K3[Fe(CN)6] -1 The electrochemical behaviors of different electrodes were characterized in PBS solution. Figure 3As shown in Figure A, CV was used to characterize the electrochemical behavior of different electrodes. Symmetrical and reversible redox peaks can be observed in the CV curve of the bare electrode (curve a). After the bare electrode was modified with RGO (curve b), the redox peak current increased. This is because the larger specific surface area and higher electroactivity of RGO facilitate the diffusion of probe ions on the electrode surface and improve the catalytic activity of the electrode. After further deposition of AuNPs on the surface of RGO / GCE (curve c), the redox peak current increased significantly, which was much higher than the response peak current after RGO modification. First, the modification of AuNPs further improved the conductivity and surface area of ​​the electrode and promoted electron transfer. Second, the synergistic effect of RGO and AuNPs can significantly improve the sensitivity of the electrode. After the polymer film was formed by electropolymerization (curve d), the redox peak current basically disappeared, indicating that the polymer film has been prepared and the transfer of probe ions is hindered. After the template molecule was eluted, an obvious current response appeared again (curve e). This is because a large number of imprinted holes were formed on the molecular imprinted membrane after elution, and electron transfer can be achieved on the electrode surface. After re-adsorbing cyclic AMP, the peak current of the probe ion decreases (curve f), and the template molecule occupies part of the imprinted holes, resulting in the obstruction of electron transfer. In addition, Figure 3 B is the SWV characterization curve of different electrodes. Figure 3 As can be seen in B, the current change degree of CV and SWV of different electrodes is consistent, which further proves that the modification effect and imprinting effect of the electrode are good.

[0061] EIS is an electrochemical method that can monitor the changes in interfacial properties during the assembly of modified electrodes through electron transfer ability. The larger the semicircle diameter at high frequency, the greater the interfacial impedance. Figure 3 As shown in Figure C, the interfacial impedance of GCE (curve a) is further reduced after being modified by RGO and AuNPs (curve b), which indicates that the conductivity of GCE is poor, and RGO and AuNPs increase the conductive activity of the electrode; when the surface of the AuNPs / RGO / GCE electrode is electropolymerized to form a nearly insulating polymer film, its impedance value increases sharply (curve c); after the template molecules are eluted, a large number of imprinted holes are formed on the imprinted membrane, which enables the probe ions to transfer electrons on the electrode surface, so the impedance value is significantly reduced (curve d). After re-adsorbing cyclic adenosine monophosphate, the template molecules occupy part of the imprinted holes, resulting in the obstruction of electron transfer, and the electron transfer impedance value is significantly increased (curve e). By comparison, the results of the three electrochemical characterization methods are consistent, indicating that the cyclic adenosine monophosphate-molecular imprinting electrochemical sensor has been successfully prepared.

[0062] Test Example 3 Selectivity Analysis

[0063] In this experiment, 5'-adenylic acid and 5'-adenosine triphosphate, which are similar to the template molecule cyclic adenosine monophosphate, were selected as structural analogs, and miglitol, L-ascorbic acid, and oxytetracycline, which are structurally different and water-soluble, were selected as interfering substances to study the selectivity of the sensor. -4 mol·L -1 Under the conditions of , the changes of peak current before and after adsorption of cyclic AMP, 5'-adenylic acid, 5'-adenosine triphosphate, miglitol, L-ascorbic acid, and oxytetracycline standard solutions. Figure 4 As shown in the figure, it can be seen from ΔI that the molecular imprinting electrochemical sensor has the strongest adsorption capacity for cyclic adenosine monophosphate, the adsorption capacity for structural analogues has decreased, and the adsorption of interfering substances is poor. The physical adsorption plays a major role in the adsorption process. The results show that this sensor has good selectivity.

[0064] Experimental Example 4 Linear Relationship and Detection Limit

[0065] In this test example, SWV was used to determine the adsorption of different concentrations of cyclic adenosine monophosphate standard solutions (1×10 -8 mol·L -1 ~1×10 -4 mol·L -1 ) of the peak current value ( Figure 5 A), the corresponding peak current values ​​are respectively related to the concentration of cyclic AMP at 1.0×10 -8 mol·L -1 ~1.0×10 -4 mol·L -1 In the range of linear relationship ( Figure 5 B), with -lgC corresponding to different concentrations of cyclic AMP as the horizontal axis and the peak current difference (ΔI) before and after adsorption as the vertical axis, a standard curve was drawn. The detection limit of the template molecule was determined based on S / N=3. The results are shown in Figure 5 As shown, the linear equations are ΔI=4.264lgC+36.574(R 2 =0.9952), the detection limit is 2.46×10 -9 mol·L -1 (S / N=3).

[0066] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate, characterized in that: Follow the steps below in order: (1) Preparation of RGO / GCE The reduced graphene oxide was added to dimethylformamide, and a light black dispersion was obtained after stirring and ultrasonic treatment, which was recorded as A; chitosan was added to a 1% acetic acid solution, and a solution obtained after magnetic stirring and ultrasonic treatment was recorded as B; under stirring, B was added dropwise to A, and the two were mixed to obtain a reduced graphene oxide modifier; 5 μL of reduced graphene oxide modifier was dropped onto the surface of the glassy carbon electrode and dried to obtain the modified electrode RGO / GCE; (2) Preparation of AuNPs / RGO / GCE The RGO / GCE was placed in a sulfuric acid deposition solution containing chloroauric acid, and a constant potential of (-0.5)-(-0.1) V was used for deposition for 100-500 seconds to modify the gold nanoparticles on the surface of the RGO / GCE to prepare a gold substrate, thereby obtaining a deposited electrode AuNPs / RGO / GCE; (3) Preparation of MIP / AuNPs / RGO / GCE AuNPs / RGO / GCE was placed in an electrolyte polymerization solution containing a template substance, cyclic adenosine monophosphate, and a functional monomer, o-phenylenediamine, for electropolymerization. The initial polymerization potential was 0.1-0.5 V, the termination potential was 0.8-1.6 V, and the polymerization was repeated for 20 cycles to obtain a dense and insulating polymer film. The polymerized electrode was immersed in an elution solution and eluted by cyclic voltammetry for 22-26 cycles to obtain MIP / AuNPs / RGO / GCE.

2. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (1), the mass-to-volume ratio of the reduced graphene oxide to dimethylformamide is (0.5-2.5): 1 g / L.

3. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (1), the mass-to-volume ratio of the chitosan to the acetic acid solution is (2.5-12.5): 1 g / L.

4. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (1), the volume ratio of A to B is 1:

1.

5. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (2), the mass concentration of chloroauric acid in the deposition solution is 0.2-1.0 g / L, the molar concentration of sulfuric acid is 0.034-0.167 mol / L, and the volume ratio between the two is 1:

1.

6. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (3), the molar ratio of the template material to the functional monomer is 1:(1-5).

7. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (3), the electrolyte is PBS, and the molar ratio of the template substance to the electrolyte is 1:(2-6).

8. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate according to claim 1, characterized in that: In step (3), the elution solution is H2SO4 with a concentration of 0.2-0.6 mol / L.

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