Method for preparing multi-recognition site imprinted sensor based on step-by-step self-assembly

Abstract

The application discloses a method for preparing a multi-recognition site imprinted sensor based on a step-by-step self-assembly, which comprises the following steps in sequence: pretreatment of a glassy carbon electrode, reduction of graphene oxide modification of the electrode, gold nano modification of the electrode, step-by-step self-assembly of a composite functional monomer and a template molecule, electro-polymerization of an imprinted film, elution of the template molecule and the like; the preparation method is simple and easy to control; the prepared polymer film has specific directivity; the number of recognition sites is large; the imprinted electrode has strong specific recognition; the molecular imprinted electrochemical sensor has the advantages of good stability, high sensitivity, low cost and fast response; and the sensor prepared by the application is suitable for rapid analysis and determination of zearalanol, zearalenol, zearalenone and the like residues in samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular imprinting technology, supramolecular chemistry and electrochemical analysis, and relates to a preparation method of a molecular imprinting electrochemical sensor and application thereof, in particular to a method for preparing a multi-recognition site imprinting sensor based on step-by-step self-assembly. BACKGROUND

[0002] Zeranol (ZOL) is a reduction product of zearalenone and zearalanone, which is a kind of semi-synthetic growth promoter, can improve the level of growth hormone in animals, and can effectively promote the growth and development of animals. ZOL has been widely used in food animals such as cattle and sheep. ZOL is a kind of weak estrogen mycotoxin, and long-term contact will increase the risk of breast cancer. When ZOL enters the human body through the food chain, it can cause human body function disorder, and even affect fertility through genetic toxicity. The Ministry of Agriculture of China No. 235 announced that ZOL is prohibited for all food animals. Due to the good weight gain effect and high economic return of ZOL, there are still reports of ZOL exceeding the standard in recent years. Therefore, it is necessary to establish a rapid and sensitive technology and method for determining ZOL.

[0003] At present, the methods commonly used for detecting ZOL, zearalenol and zearalanone residues are mainly chromatography and chromatography-mass spectrometry, but they have the disadvantages of long processing time, complicated operation, high running cost, poor selectivity, large amount of organic solvent, etc. The molecular imprinting electrochemical sensor overcomes the above defects, has the advantages of simple operation, low cost, strong selectivity, good stability, fast mass transfer, etc., and is expected to realize rapid and sensitive detection of ZOL, zearalenol and zearalanone residues. At present, there is no report on the preparation of a molecular imprinting electrochemical sensor using zeranol as a template molecule and ethyl mercaptoacetate and β-mercaptoethylamine as a composite functional monomer and its application in actual sample detection.

[0004] Molecular imprinting technology (MIT) is a preparation technology for obtaining a polymer that is completely matched with a target compound in spatial configuration and binding site, belongs to the category of host-guest chemistry in supramolecular chemistry, and is a frontier science of polymer chemistry, biochemistry and other disciplines. In recent years, the combination of molecular imprinting technology and electrochemical rapid analysis to prepare a molecular imprinting electrochemical sensor has become a research hotspot. The molecular imprinting polymer sensitive material has the advantages of resistance to high temperature, high pressure, acid, alkali and organic solvents, can be synthesized by chemical method, can be repeatedly used, and is easy to store. The successful fixation of the molecular imprinting polymer on the surface of the transducer is a key step in the preparation of the molecular imprinting electrochemical sensor.

[0005] The common preparation methods of the molecular imprinting electrochemical sensor include in-situ initiation polymerization method, coating film method and electrochemical polymerization method. However, the electrochemical polymerization method is the most researched method due to the advantages of rapid film formation, close combination of the film and the transducer and controllable film thickness. The electrochemical polymerization method is to place the imprinting electrode into a supporting electrolyte solution containing a template molecule and a functional monomer, the functional monomer generates positive ion or negative ion radicals on the electrode surface through oxidation or reduction, the radicals generate a polymer through condensation reaction, and meanwhile the template molecule introduces the selective recognition site into the polymer film by means of the interaction force between the functional monomer molecules, thereby preparing the molecular imprinting electrochemical sensor. The electrochemical polymerization method includes constant potential deposition method, cyclic voltammetry method and constant current deposition method. The constant potential deposition method is currently mainly applied to the deposition test of chitosan and has certain limitations; on the contrary, the cyclic voltammetry method is widely applied to the preparation process of the electrochemical sensor, and the polymer film with electrical activity and non-electrical activity can be prepared by selecting different types of functional monomers or adjusting the pH value of the electrolyte solution in the polymerization process. At present, the functional monomer and the template molecule are directly electro-polymerized on the surface of the bare electrode, which is limited by the small number and types of the polar functional groups contained in the functional monomer molecular structure, thereby affecting the stability and selectivity of the polymer film.

[0006] At present, there is no report on the preparation of zearalanol molecular imprinting electrochemical sensor by using the step-by-step self-assembly technology. SUMMARY

[0007] The present application aims to provide a method for preparing a multi-recognition site imprinting sensor based on step-by-step self-assembly, which comprises the following steps in sequence: pretreatment of a glassy carbon electrode, reduction of graphene oxide modified electrode, nano-gold modified electrode, step-by-step self-assembly of a composite functional monomer and a template molecule, electro-polymerization of an imprinting film, elution of the template molecule and the like. The preparation method is simple and easy to control, the prepared polymer film has specific directionality, the number of recognition sites is large, the specific recognition of the imprinting electrode is strong, the molecular imprinting electrochemical sensor has good stability, high sensitivity, low cost and fast response.

[0008] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] A method for preparing a multi-recognition site imprinting sensor based on step-by-step self-assembly, which is performed according to the following steps in sequence:

[0010] (1) Pretreatment of a glassy carbon electrode

[0011] The glassy carbon electrode is polished and washed, and then the electrode is placed in a 0.4-0.6 mol / L H2SO4 solution and cyclic scanning is performed; the electrode is taken out, washed, dried by nitrogen blowing, and placed in a K3[Fe(CN)6]-KCl characterization solution and cyclic scanning is performed until a stable cyclic voltammetry response is obtained, so that an active electrode with good usability is obtained, and the active electrode is recorded as A;

[0012] (2) Reduced graphene oxide modified electrode

[0013] 100 mg of CS is placed in 10 mL of 1% CH3COOH solution, and magnetic stirring and ultrasonic treatment are performed to obtain CS-1% CH3COOH; 20 mg of rGO is dissolved in 10 mL of N,N-dimethylformamide, and after ultrasonic dissolution, an equal amount of CS-1% CH3COOH is added, and magnetic stirring and ultrasonic treatment are performed to prepare rGO-CS modifier; 5-10 μL of the rGO-CS modifier is drop-casted onto the surface of the glassy carbon electrode in A by using a pipette, and the rGO modified electrode is obtained after drying under an infrared lamp, and the rGO modified electrode is recorded as B;

[0014] (3) Gold nanoparticle modified electrode

[0015] B is immersed in a 0.1-0.3 mol / L H2SO4 solution containing chloroauric acid hydrate, and after deposition for 500-700 s at a constant potential of -0.3 to -0.1 V, the electrode is taken out, washed with ultrapure water and anhydrous ethanol in sequence, and then naturally dried at room temperature to obtain the AuNPs@rGO@GCE, and the AuNPs@rGO@GCE is recorded as C;

[0016] (4) Stepwise self-assembly of composite functional monomer and template molecule

[0017] First, a solution of one functional monomer containing a mercapto group and a solution of a template molecule are mixed uniformly, and self-assembly is performed at 4°C in the dark for 4-8 h; a solution of another functional monomer containing a mercapto group is injected into the system, and self-assembly is continued at 4°C in the dark for 3-6 h; then C is immersed in the mixed solution, and self-assembly is performed at 4°C in the dark for 22-26 h; then the template molecules physically adsorbed are removed by washing with ultrapure water and anhydrous ethanol in sequence, and the self-assembled electrode is obtained after drying by nitrogen blowing, and the self-assembled electrode is recorded as D;

[0018] (5) Electro-polymerized imprinting film

[0019] D is placed in a polymerization solution, and electro-polymerization is performed in a potential range of 0-1.4 V to obtain a dense and non-conductive electro-polymerized imprinting film embedded with template molecules, and the electro-polymerized imprinting film is dried by nitrogen blowing to obtain E;

[0020] The polymerization solution is a potassium chloride buffer solution containing the template molecule and the composite functional monomer described in step (4) above, and the concentration of the potassium chloride buffer solution is 0.2 mol / L;

[0021] (6) elution of the template molecule

[0022] The E is placed in ethanol-NaOH aqueous solution to induce elution of the template molecule for 10-20 min, and repeatedly washed with water to obtain a zearalanol molecule imprinting electrochemical sensor.

[0023] As a limitation of the present application:

[0024] I. In step (1), the electroactive probe is potassium ferricyanide, and the electroactive probe solution is prepared from potassium ferricyanide and potassium chloride in a molar ratio of 10:1.

[0025] II. In step (1), the glassy carbon electrode is polished with Al2O3 powder on a polishing material, and then ultrasonically cleaned with dilute nitric acid solution, anhydrous ethanol and ultrapure water.

[0026] III. In step (3), the concentration of chloroauric acid hydrate is 0.5-1.0 g / L.

[0027] IV. In step (4), the molar ratio of the one functional monomer containing sulfydryl, the template molecule and the other functional monomer containing sulfydryl is 6:1:4.

[0028] The one functional monomer containing sulfydryl is EMAC, the template molecule is ZOL, and the other functional monomer containing sulfydryl is β-MEA.

[0029] When preparing the complex functional monomer solution and the template molecule solution respectively, the complex functional monomer and the template molecule can be dissolved in an organic solvent, such as acetonitrile.

[0030] V. In step (5), the number of cycles of cyclic scanning is 18-22 cycles.

[0031] VI. In step (5), the template molecule is ZOL, the complex functional monomer is EMAC and β-MEA, and the molar ratio of the template molecule to EMAC and β-MEA is 1:6:4.

[0032] VII. In step (6), the ethanol-NaOH aqueous solution is prepared from ethanol and 0.4 mol / L NaOH aqueous solution in a volume ratio of (2-4):1.

[0033] VIII. In step (1), the potential range during cyclic scanning in the electroactive carbon needle solution is -0.2-0.8 V, and the scanning speed is 0.05 V / s.

[0034] The present application also has another limitation, the prepared sensor can be used for analysis and determination of ZOL, zearalanol, zearalenone and other residues.

[0035] In the present application, graphene is a two-dimensional atomic crystal formed by sp 2 The two-dimensional atomic crystal formed by the hybrid form connected together due to its unique structure and properties is applied to the modification of the electrode to increase the conductivity and surface area of the modified electrode; the nano gold particles have good conductivity and stable biocompatibility. By using the distributed self-assembly technology, the active molecules in the present application are spontaneously adsorbed on the heterogeneous interface to form an ordered molecular assembly system, which is mainly based on the strong chemical bonding effect of long-chain organic molecules on the substrate surface and the interaction between organic molecule chains, and endows the polymer film with specific directionality, increases the number of recognition sites, and improves the specific recognition of the imprinted electrode. The self-assembly of the sulfur-gold system is simple, easy to form a film, and has good stability and order.

[0036] By using the step-by-step self-assembly technology in combination with the above preparation steps as a whole scheme, the association between the composite functional monomers can be effectively avoided, the yield of by-products is reduced, and the use of composite functional monomers to make a molecular imprinting film can increase the effective recognition sites and further increase the specificity of the recognition of target substances.

[0037] Compared with the prior art, the technical progress achieved by the present application is that:

[0038] ① The sensor first uses rGO and AuNPs layer-by-layer modified electrode to increase its specific surface area and conductivity; the step-by-step self-assembly technology is used to prepare a polymer film, which effectively avoids the association between the composite functional monomers and endows the polymer film with specific directionality, increases the number of recognition sites, and improves the specific recognition of the imprinted electrode.

[0039] ② The preparation method is simple, the process is easy to control, the prepared sensor has high selectivity, good stability, high sensitivity, low cost and fast response.

[0040] ③ Compared with other zearalanol detection technologies, the present application has the advantages of simple operation process, low cost, short time consumption, high selectivity, high sensitivity, good stability, fast response and easy miniaturization, which is conducive to on-site detection.

[0041] The imprinted sensor prepared by the present application is not only suitable for the analysis and determination of zearalanol, zearalenol and zearalenone residues in food, but also suitable for the analysis and determination of zearalanol, zearalenol and zearalenone residues in other samples.

[0042] The present application will be further described in detail below with reference to specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a schematic diagram of the preparation process of the zearalanol molecular imprinting electrochemical sensor in Example 1.

[0044] Figure 2 Concentration-current plot of rGO modifier;

[0045] Figure 3 Concentration-current plot of chloroauric acid in gold nanoparticles electrodeposition solution;

[0046] Figure 4 Time-current plot of gold nanoparticles electrodeposition;

[0047] Figure 5 Characterization of electrode modification effect (a-GCE; b-rGO@GCE; c-AuNPs@rGO@GCE);

[0048] Figure 6 Configuration optimization of different ratios of composites (a: ZOL-EMAC; b: ZOL-2EMAC; c: ZOL-3EMAC; d: ZOL-(β-MEA)-3EMAC; e: ZOL-2(β-MEA)-3EMAC);

[0049] Figure 7 Template molecule to complex functional monomer ratio-absorbance plot in polymerization system;

[0050] Figure 8 Different electrolytes-current plot (a-KCl; b-sodium acetate; c-ammonium chloride; d-PBS);

[0051] Figure 9 Number of polymerization cycles-current plot during electropolymerization;

[0052] Figure 10 Template elution time-current plot of imprinted sensor;

[0053] Figure 11 Differential pulse voltammogram of different electrodes in electroactive probe solution (a-adsorption electrode; b-elution electrode; c-polymerization electrode; d-AuNPs@rGO@GCE; e-rGO@GCE; f-GCE);

[0054] Figure 12 Column chart of selectivity study of zearalanol molecularly imprinted electrochemical sensor;

[0055] Figure 13 SWV response curve of imprinted sensor to different concentrations of zearalanol in electroactive probe solution;

[0056] Figure 14 Linear relationship curve of zearalanol imprinted sensor. DETAILED DESCRIPTION

[0057] The test methods used in the following examples are conventional methods unless otherwise specified.

[0058] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0059] Example 1 A method for preparing a multi-recognition site imprinting sensor based on step-by-step self-assembly

[0060] The method for preparing a multi-recognition site imprinting sensor based on step-by-step self-assembly in this example is shown in the preparation process schematic diagram as Figure 1 The following steps are performed in sequence according to the following order:

[0061] (11) Pretreatment of glassy carbon electrode

[0062] The glassy carbon electrode is polished with 0.3 μm and 0.05 μm Al2O3 powder on a buffing wheel, washed with ultrapure water, and then ultrasonically washed in anhydrous ethanol and ultrapure water, respectively. Then the electrode is placed in a 0.5 mol / L H2SO4 solution and cyclically scanned.

[0063] After the electrode is removed and washed, it is placed in an electroactive probe solution, wherein the electroactive probe is potassium ferricyanide, and the electroactive probe solution is prepared by mixing 10 mmol / L potassium ferricyanide and 1 mol / L potassium chloride solution at a volume ratio of 1:1.

[0064] Cyclically scan at a potential range of -0.2-0.8 V and a scan speed of 0.05 V / s until a stable cyclic voltammetry response is obtained, i.e. symmetrical and reversible cyclic voltammetry peaks (peak current ratio is 1:1, and peak potential difference is less than 90 mV), to obtain an active electrode, denoted as A1.

[0065] (12) Reduced graphene oxide modified electrode

[0066] Take 100 mg CS and place it in 10 mL of 1% CH3COOH solution, and magnetically stir for 30 min. Ultrasonically prepare CS-1% CH3COOH. Take 20 mg rGO and dissolve it in 10 mL of N,N-dimethylformamide, and ultrasonically dissolve for 30 min. Then take 5 mL and add an equal volume of CS-1% CH3COOH dropwise into a beaker, and magnetically stir for 30 min to prepare rGO-CS modifier. Use a pipette gun to drop 5 μL of rGO-CS modifier onto the surface of the glassy carbon electrode in A1, and dry it under an infrared lamp to obtain a rGO modified electrode, denoted as B1.

[0067] (13) Gold nanoparticle modified electrode

[0068] B1 was immersed in 0.1 mol / L H2SO4 solution containing 0.6 g / L HAuCl4·4H2O, and after 600 s of deposition at a constant potential of -0.2 V, the electrode was taken out, washed with ultrapure water and anhydrous ethanol in turn, and then naturally dried at room temperature to prepare a reduced graphene oxide and gold nanoparticle modified glassy carbon electrode (AuNPs@rGO@GCE), denoted as C1;

[0069] (14) Stepwise self-assembly of composite functional monomers and template molecules

[0070] First, 2 mL of 30 mmol / L EMAC acetonitrile solution and 1 mL of 10 mmol / L ZOL acetonitrile solution were mixed uniformly, and hydrogen bonds were formed by the action of the carbonyl group on EMAC and the hydroxyl group in ZOL molecules through self-assembly for 4.5 h at 4°C in the dark; then 2 mL of 20 mmol / L β-MEA solution was injected into the system, and the self-assembly was continued for 3.5 h at 4°C in the dark; then C1 was immersed in the mixed solution, and self-assembly was carried out at 4°C in the dark for 24 h; then the physically adsorbed ZOL was removed by washing with ultrapure water and anhydrous ethanol in turn, and nitrogen blowing to dryness, to obtain a ZOL, EMAC and β-MEA modified electrode (ZOL / EMAC / β-MEA@AuNPs@rGO@GCE), denoted as D1;

[0071] (15) Electro-polymerized imprinting film

[0072] D1 was placed in a polymerization solution, and an electro-polymerized imprinting film embedded with template molecules was obtained by electro-polymerization for 20 cycles at a potential range of 0-1.4 V, and then nitrogen blowing to dryness, to obtain E1;

[0073] The polymerization solution was a potassium chloride buffer solution containing composite functional monomers and template molecules, wherein the concentration of the potassium chloride buffer solution was 0.2 mol / L, the composite functional monomers were EMAC and β-MEA, and the template molecules were ZOL; the molar ratio of ZOL:EMAC:β-MEA was 1:6:4;

[0074] (16) Elution of template molecules

[0075] E1 was placed in ethanol-0.4 mol / L NaOH aqueous solution (volume ratio of ethanol to NaOH aqueous solution was 4:1) to induce elution of the template molecules for 10 min, and then the physically adsorbed template molecules were removed by repeated washing with water, to obtain a zearalenol molecularly imprinted electrochemical sensor.

[0076] The sensor prepared in the embodiment has the advantages of strong selectivity, good stability, high sensitivity, low cost, fast response, simple preparation process and easy control. Compared with other zearalanol detection technologies, the sensor has the advantages of simple operation process, low cost and short time consumption.

[0077] Example 2-6: One method for preparing a multi-recognition site imprinted sensor by stepwise self-assembly

[0078] The embodiment is one method for preparing a multi-recognition site imprinted sensor by stepwise self-assembly. The preparation steps and technical parameters are the same as those in Example 1, and the only difference is that the corresponding technical parameters in the preparation process are different. The specific results are shown in Table 1.

[0079] Table 1: Technical parameters of the preparation process

[0080]

[0081]

[0082] The sensor prepared in Example 2-6 has the advantages of strong selectivity, good stability, high sensitivity, low cost, fast response, simple preparation process and easy control. Compared with other zearalanol, zearalenol and zearalenone detection technologies, the sensor has the advantages of simple operation process, low cost and short time consumption.

[0083] Example 7: Reduction of graphene oxide modification condition selection experiment

[0084] In order to achieve the best modification effect of reduced graphene oxide, the dispersion effect of different dispersants on rGO and the attachment and distribution effect on the electrode surface were studied in this embodiment. Among them, the reagent DMF with good rGO dispersion effect and chitosan with good cross-linking effect were combined, the rGO-DMF dispersion liquid was mixed with the CS-1% CH3COOH solution in equal volume to obtain the rGO modifier, and the appropriate amount of rGO modifier was dropped on the electrode surface, and the rGO could be uniformly distributed.

[0085] In order to make the rGO modifier stably modify on the electrode surface and not affect the conductivity of rGO, the amount of dispersant was fixed and the amount of rGO was optimized in this embodiment. By comparing the current response of the electrodes modified by different concentrations of rGO modifier (0.25 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L), the effect of rGO amount on the modification effect was investigated, and the results are as follows Figure 2As shown in Fig. 1, with the increase of the amount of rGO, the redox peak current intensity of the CV curve obviously increases, indicating that the electrode modified by rGO can improve the electrical conductivity thereof; when the amount of rGO is 1.0 g / L, the peak current is the highest, and the ratio of the oxidation peak current to the reduction peak current is close to 1:1; when the amount of rGO exceeds 1.0 g / L, the current intensity shows a downward trend, because the amount of rGO exceeds the dispersion capacity of the dispersing agent, causing the rGO in the modifier to agglomerate and reduce the electrical conductivity thereof, and in addition, too much rGO will also increase the thickness of the modified film and affect the current response.

[0086] Example 8 Optimization experiment of gold nanoparticle modification conditions

[0087] In this example, the concentration of HAuCl4·4H2O in the electrodeposition solution is first optimized. The peak current change is investigated by differential pulse voltammetry after the electrodeposition for the same time when the concentration of HAuCl4·4H2O in the electrodeposition solution is 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L and 1.0 g / L respectively, and the results are shown in Fig. 2. Figure 3 As shown in Fig. 2, with the increase of the concentration of HAuCl4·4H2O in the electrodeposition solution, the current response intensity gradually increases, and when the concentration exceeds 0.6 g / L, the peak current intensity gradually stabilizes, therefore, 0.5-1.0 g / L is selected as the optimal concentration of HAuCl4·4H2O.

[0088] In this example, the effect of deposition time on the deposition effect is explored, and the characteristic peak size of gold element of the electrodeposited electrode with the deposition time of 100 s-800 s is investigated in 0.1 mol / L H2SO4 solution, and the results are shown in Fig. 3. Figure 4 As shown in Fig. 3, the current intensity of the characteristic peak increases with the increase of the deposition time, when the deposition time is 500-700 s, the gold nanoparticles are stably deposited on the electrode surface, uniformly distributed, and the characteristic peak current value is large, therefore, 500-700 s is determined as the optimal deposition time of HAuCl4·4H2O.

[0089] Example 9 Research on electrode modification effect

[0090] In this example, the electrode modified by reduced graphene oxide and gold nanoparticles is characterized, and the results are shown in Fig. 4. Figure 5 As shown in Fig. 4, the bare electrode is scanned by cyclic voltammetry in potassium ferricyanide solution, and the scanning curve is shown in Fig. 4a. Figure 5 As can be seen from the figure, the probe ion Fe 3+ on the electrode surface undergoes redox reaction, and presents a group of redox peaks, and the ratio of the peak current values is close to 1:1; when the reduced graphene oxide is modified on the bare electrode, the redox peak current value of the cyclic voltammetry curve obviously increases, and the peak shape is more symmetrical than that of the bare electrode, as shown in Fig. 4b. Figure 5The CV curve shown in the middle b curve indicates that the reduced graphene oxide has good conductivity and catalyzes the redox reaction of the probe ion on the electrode; after the gold nanoparticles are electrodeposited on the surface of the rGO@GCE, the CV curve is shown in the middle c curve, and the redox peak current intensity slightly increases, but it is not obvious, and its main role is to increase the binding site of the self-assembly of the complex functional monomer. Figure 5

[0091] Example 10 Computer simulation analysis

[0092] In this example, the molecular configuration of the template molecule and the complex functional monomer is optimized, and the spatial configuration of the complex with different proportions is optimized to investigate its bonding and the change of binding energy in the reaction process. The configuration optimization of the complex with different proportions is shown in Figure 6 ; the energy and binding energy of the complex with different proportions are shown in Table 2.

[0093] Table 2 Energy and binding energy of the complex with different proportions

[0094]

[0095]

[0096] From Figure 6 and Table 2, it can be seen that the binding ability of the ZOL-2 (β-MEA)-3EMAC type complex is strong, and the spatial configuration is stable.

[0097] Example 11 Influence of different concentrations of raw materials on current during electropolymerization

[0098] In this example, the influence of different molar concentration ratios of the complex functional monomer and the template substance on the current during the electropolymerization process is explored. The preparation process of the polymer film is the same as that of Example 1, and the only difference is that the molar concentration ratio of the complex functional monomer and the template substance is different. The specific test results are shown in Figure 7 .

[0099] From Figure 7 it can be seen that when the molar concentration ratio of the template substance to the complex functional monomers β-MEA and EMAC is 1:4:6 as determined in the application, the imprinting effect is the best.

[0100] Example 12 Influence of different polymer electrolytes on polymerization effect

[0101] In this example, the polymerization effects of different polymer electrolytes are compared, and the polymerization conditions are the same as those of Example 1, and the only difference is that the types of polymer electrolytes are different. The specific test results are shown in Figure 8 .

[0102] From Figure 8 ​As shown in curve a, the potassium chloride buffer solution as a polyelectrolyte has the highest response peak current, smaller peak potential and can form more stable imprint sites.

[0103] Example 13 Selection of polymerization potential and polymerization turns

[0104] In this example, the polymerization potential and polymerization turns of the composite functional monomer and the template molecule were investigated, and the preparation process of the polymerization film was the same as that in Example 1, except that the polymerization potential and polymerization turns were different. The specific test results are shown in Table 1. Figure 9

[0105] It can be seen from Table 1 that when the polymerization potential is in the range of 0V-1.4V and the polymerization turns are 18-22, the polymerization effect is the best. Figure 9

[0106] Example 14 Influence of template substance elution conditions on the current of the imprint sensor

[0107] The conditions of template substance elution have an important influence on the electrochemical performance of the multi-recognition site imprint sensor. In this example, the eluent and elution time were explored, and the elution process of the template substance in this example was the same as that in Example 1, except that the eluent and elution time during the elution process of the template substance were different. Finally, ethanol-0.4mol / L NaOH ((4-2):1, V / V) was determined as the eluent.

[0108] It can be seen from Table 2 that when the elution time is in the range of 10-20min determined by the present application, the response current value meets the detection requirements; if it is not in this range, the response current value is low, and the prepared electrochemical sensor is not suitable for the detection of actual samples. Figure 10

[0109] Example 15 Characterization of imprint effect of the multi-recognition site imprint sensor prepared by stepwise self-assembly

[0110] Differential pulse voltammetry characterization:

[0111] The multi-recognition site imprint sensor provided in Example 1 was placed in an electroactive probe solution (the electroactive probe solution and the type of probe were the same as those in Example 1) for cyclic voltammetry characterization. The electrochemical behaviors of the bare glassy carbon electrode, AuNPs@rGO@GCE, EMAC and β-MEA and ZOL modified electrode, ZOL-MIP / GCE, MIP / GCE and MIP / GCE after re-adsorption of the six kinds of electrodes were characterized, and the results are shown in Figure 4. Figure 11

[0112] It can be seen from Figure 4 that the multi-recognition site imprint sensor provided in Example 1 has good electrochemical performance, and the response current is larger than that of the bare glassy carbon electrode, AuNPs@rGO@GCE, EMAC and β-MEA and ZOL modified electrode, ZOL-MIP / GCE, MIP / GCE and MIP / GCE after re-adsorption. Figure 11 ​​​​It can be seen that the oxidation peak current of the rGO modified electrode (curve e) is larger than that of the bare electrode (curve f); when AuNPs are deposited (curve d), the response peak current is close to 200 μA, indicating that the modification of AuNPs can significantly improve the sensitivity of the molecular imprinting electrochemical sensor; after the characterization of the polymerized electrode, no oxidation peak (curve c) is shown, indicating that ZOL and EMAC and β-MEA successfully form a molecular imprinting film through hydrogen bonding; when the eluent is used to remove ZOL, the response current appears again (curve b), indicating that the probe can pass through the imprinting cavity on the molecular imprinting film to perform the redox reaction on the electrode surface to generate a response signal; after the eluted electrode is repositioned in the ZOL solution for adsorption, it is found that the current response decreases again, as shown in curve a, indicating that most of the template molecules have reoccupied the imprinting cavities and hindered the probe from passing through. Through analysis, it is found that the molecular imprinting electrochemical sensor has good imprinting effect.

[0113] Example 16 Study on the selectivity and repeatability of the sensor

[0114] (1) Study on the selectivity

[0115] In order to study the selectivity of the zearalanol film to the template material, two substances similar in structure to the template molecule, zearalenol and zearalanone, are selected in this embodiment, and enrofloxacin and thiamphenicol are selected as interfering substances for the selective adsorption test.

[0116] The imprinting electrode prepared in Example 1 is placed in an electroactive probe solution (the electroactive probe solution and the type of probe are the same as those in Example 1), and 1.0 × 10 -6 mol / L of the target substance is added for adsorption test, and the SWV method is used for characterization, and the results are shown in Figure 12 It can be seen from Figure 12 that the sensor has high response values to ZOL, zearalenol and zearalanone, and almost no response to the interfering substances enrofloxacin and thiamphenicol, thus indicating that the sensor has good selectivity to zearalanol.

[0117] (2) Study on the repeatability

[0118] In this embodiment, the repeatability of the imprinting sensor provided in Example 1 is studied, and the prepared zearalanol molecular imprinting electrochemical sensor is eluted and adsorbed with the template molecule for 20 times, wherein the elution step and the reagents used are the same as those in Example 1, and the relative standard deviation of the obtained ΔI is ≤1.93% (n=20), indicating that the sensor has good repeatability and no performance attenuation.

[0119] Example 17 Preparation of a multi-recognition site imprinting sensor based on stepwise self-assembly and its application

[0120] (1) Sample processing

[0121] ① Sample extraction

[0122] 2.00 g (2 mL for liquid sample) of sample (beef, corn) was weighed, 10 mL of acetonitrile solution was added, and oscillation, ultrasonic, and centrifugation (4000 r / min, 10 min) were sequentially performed. The supernatant was taken, 5 mL of acetonitrile was added for secondary extraction, and the two supernatants were combined and diluted with acetonitrile to 20 mL for measurement.

[0123] ② Sample detection

[0124] The treated beef and corn samples were detected at room temperature by CV and SWV methods, and the measurement parameters were set as follows: CV, scan potential -0.2 V to 0.8 V, scan rate 50 mV / s; SWV, start and end potential -0.1 V to 0.6 V, potential increment 0.005 V, pulse amplitude 0.025 V, and frequency 5 Hz. After each measurement, the electrode was eluted in ethanol-0.4 mol / L H2SO4 (3:1, V / V) solution to remove the template molecule, and the next measurement was performed.

[0125] (2) Linear relationship and detection limit

[0126] Under the optimal experimental conditions, the response current of the imprinted sensor to different concentrations of zearalanol was as shown in Figure 13 . Curves 1-6 in Figure 13 correspond to the concentrations of zearalanol (i.e., 1-6 correspond to the concentrations from high to low, respectively). The standard curve was plotted with the relative peak current ΔI as the vertical coordinate and the concentration as the horizontal coordinate, as shown in Figure 14 . It can be seen from Figure 14 that the concentration of zearalanol in the range of 1.0×10 -10 mol / L to 1.0×10 -6 mol / L has a good linear relationship with the relative peak current, the linear equation is ΔI = 5.0654lgC + 56.08242, the linear correlation coefficient is 0.9929, and the detection limit is 3.3×10 -11 mol / L.

[0127] (3) Sample standard addition and precision test of zearalanol in beef and corn

[0128] Under the optimal experimental conditions, the beef and corn samples were added with 1×10 -9 mol / L, 1×10 -8 mol / L, and 1×10 -7The sample standard addition test was carried out under 3 addition levels of 0.5, 1 and 2 mol / L, and the analysis results are shown in Table 3. The sample addition average recovery rate is between 91.65% and 99.85%, and the relative standard deviation (RSD) is between 1.19% and 4.28% (n=5). It is shown that the multi-recognition site imprinting sensor prepared by the present application has high recovery rate and good precision.

[0129] Table 3 Standard addition and precision test results of different samples

[0130]

[0131] The embodiments 1-6 are only the preferred embodiments of the present application, and are not intended to limit the other forms of the present application. Any skilled person in the art can use the above technical contents as an inspiration to make changes or modifications into equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments without departing from the technical essence of the present application still falls within the protection scope of the present application.

Claims

1. A method for fabricating a multi-recognition site imprint sensor based on stepwise self-assembly, characterized in that, Follow these steps in sequence: (1) Pretreatment of glassy carbon electrode The glassy carbon electrode was polished and washed, and then placed in a 0.4–0.6 mol / L H2SO4 solution for cyclic scanning. After rinsing the electrode, it was dried with nitrogen gas and placed in a K3[Fe(CN)6]-KCl characterization solution for cyclic scanning until a stable cyclic voltammetric response was obtained. The resulting active electrode with good usability was denoted as A. (2) Reduced graphene oxide modified electrode 100 mg of CS was placed in 10 mL of 1% CH3COOH solution, and the mixture was magnetically stirred and sonicated to prepare CS-1% CH3COOH. 20 mg of rGO was dissolved in 10 mL of N,N-dimethylformamide, and after sonication, an equal volume of CS-1% CH3COOH was added. The mixture was magnetically stirred and sonicated to prepare rGO-CS modifier. 5–10 μL of rGO-CS modifier was drop-coated onto the surface of the glassy carbon electrode in A using a pipette, and dried under an infrared lamp to obtain the rGO-modified electrode, thus obtaining B. (3) Nano-gold modified electrode B was immersed in a 0.1–0.3 mol / L H₂SO₄ solution containing chloroauric acid hydrate. After deposition at a constant potential of -0.3–0.1 V for 500–700 s, the electrode was removed, rinsed sequentially with ultrapure water and anhydrous ethanol, and then air-dried at room temperature to obtain AuNPs@rGO@GCE, thus obtaining C. (4) Stepwise self-assembly of composite functional monomers and template molecules First, a solution of a functional monomer containing thiol groups and a template molecule solution are mixed evenly and self-assembled at 4°C in the dark for 4–8 h. Then, another functional monomer solution containing thiol groups is injected into the system and self-assembled at 4°C in the dark for 3–6 h. Then, the C surface is immersed in the mixed solution and self-assembled at 4°C in the dark for 22–26 h. Then, it is taken out and the physically adsorbed template molecules are removed sequentially with ultrapure water and anhydrous ethanol. The electrode is dried with nitrogen to obtain the self-assembled electrode, D. The functional monomer containing a thiol group is EMAC, the template molecule is ZOL, and the other functional monomer containing a thiol group is β-MEA. The molar ratio of the functional monomer containing a thiol group, the template molecule, and the other functional monomer containing a thiol group is 6:1:

4. (5) Electropolymerized imprinted film D was placed in a polymerization solution and electropolymerized within a potential range of 0–1.4 V to obtain a dense, non-conductive electropolymerized imprinted film embedded with template molecules. After drying with nitrogen, E was obtained. The template molecule is ZOL, the composite functional monomers are EMAC and β-MEA, and the molar ratio of the template molecule to EMAC and β-MEA is 1:6:

4. The polymerization solution is a potassium chloride buffer solution containing the template molecule and the composite functional monomer described in step (4) above, wherein the concentration of the potassium chloride buffer solution is 0.2 mol / L; (6) Elution of template molecules E was placed in an ethanol-NaOH aqueous solution to induce elution of the template molecule for 10-20 min, and then repeatedly rinsed with water to obtain the zearalenone molecularly imprinted electrochemical sensor. The ethanol-NaOH aqueous solution is prepared by mixing ethanol and a 0.4 mol / L NaOH aqueous solution in a volume ratio of (2-4):

1.

2. The method for preparing a multi-recognition site imprint sensor based on stepwise self-assembly according to claim 1, characterized in that: In step (1), the electroactive probe is potassium ferricyanide, and the electroactive probe solution is prepared by mixing potassium ferricyanide and potassium chloride in a molar ratio of 10:

1.

3. The method for preparing a multi-recognition site imprint sensor based on stepwise self-assembly according to claim 1, characterized in that: In step (1), the glassy carbon electrode is polished on a polishing material with Al2O3 powder and then ultrasonically cleaned with dilute nitric acid solution, anhydrous ethanol, and ultrapure water in sequence.

4. The method for fabricating a multi-recognition site imprint sensor based on stepwise self-assembly according to claim 1, characterized in that: In step (3), the concentration of chloroauric acid hydrate is 0.5–1.0 g / L.

5. The method for fabricating a multi-recognition site imprint sensor based on stepwise self-assembly according to claim 1, characterized in that: In step (5), the number of cyclic scans is 18 to 22.

6. The method for preparing a multi-recognition site imprint sensor based on stepwise self-assembly according to claim 1, characterized in that: In step (1), the potential range during the cyclic scanning of the electroactivated carbon needle solution is -0.2 to 0.8 V, and the scanning speed is 0.05 V / s.

7. The method for preparing a multi-recognition site imprint sensor based on stepwise self-assembly according to any one of claims 1-6, characterized in that: The prepared sensor can be used for the analysis and determination of residues such as ZOL, zearalenone, and zearalenone.

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