A preparation method of chloramphenicol molecular imprinting sensor based on dual polymerization strategy

Abstract

The present invention provides a method for preparing a chloramphenicol molecular imprinting sensor based on a dual polymerization strategy, and relates to the field of electrochemistry and molecular imprinting technology. The method comprises the following steps: (1) oxidative prepolymerization: mixing a functional monomer and chloramphenicol in a solution, adding an oxidant, and performing prepolymerization to obtain an oligomer solution; (2) electrochemical polymerization: electrochemically polymerizing the oligomer solution to obtain a polymer modified electrode coated with chloramphenicol; (3) eluting chloramphenicol: removing chloramphenicol from the polymer modified electrode coated with chloramphenicol to obtain a chloramphenicol molecular imprinting sensor; wherein the oxidant is selected from HAuCl4, H2PtCl6 or K3[Fe(CN)6]. The chloramphenicol molecular imprinting sensor prepared by the present invention has good CAP sensing performance, good stability, and high sensitivity, and is of great significance to the research and application of CAP detection of molecular imprinting sensors in animal-derived foods.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemistry and molecular imprinting, and in particular to a preparation method of a chloramphenicol molecular imprinting sensor based on a double polymerization strategy. Background Art

[0002] Antibiotic residues are an important factor threatening food safety and human health. Chloramphenicol (CAP) antibiotics are now banned in clinical and animal husbandry because of their severe inhibitory effect on bone marrow hematopoiesis. However, CAP residues still exist in common animal foods such as milk and aquatic products, and their impact on human health cannot be ignored. Establishing efficient, accurate and rapid detection methods is the key to solving the problem of CAP abuse.

[0003] In recent years, electrochemical sensors based on molecular imprinting technology have been vigorously developed. It is mainly based on the unique specific recognition effect of the molecular imprinting polymer film on the array on the target molecule ("key" effect) to cause changes in electrical properties for detection. It has extremely high selectivity and does not require complex and numerous operation steps. It is a safe, cheap, sensitive and rapid detection method. However, for the actual application of residual CAP detection in animal food, the application performance of molecular imprinting sensors prepared by traditional single polymerization is obviously insufficient and far from meeting the large-scale detection application in the market. Its application performance is mainly reflected in the following aspects: (1) The traditional single polymerization preparation method usually uses chemical oxidation or electrochemical polymerization to synthesize polymers and embed template molecules. After the reaction is completed, the product is often transferred to the electrode surface by drop coating or direct electrodeposition. There are problems such as uncontrollable film thickness, unstable film easy to fall off, and difficult template elution. (2) The single polymerization method is a method in which a monomer is chemically or electrochemically polymerized in a solution containing a monomer and a template molecule to generate a polymer, and then modified to the electrode surface. However, there are problems such as a small number of template molecule recognition sites, easy leakage of template molecules, and low sensor sensitivity. (3) The molecular imprinting membrane has poor conductivity and a low electrochemical signal output rate, which further reduces the sensitivity of the sensor and cannot meet the market demand for trace analysis of residual CAP in animal-derived foods.

[0004] In the prior art, the commonly used method is to optimize the preparation method of molecular imprinting polymer film, enhance the stability of polymer film, and combine nanotechnology to enhance the conductivity of polymer and improve the sensitivity of sensor. For example, Cardoso et al. prepared molecular imprinting polymer by direct electropolymerization, and introduced porous multilayer graphene on molecular imprinting electrochemical sensing electrode to enhance the conductivity of polymer film electrode, thereby improving the sensitivity of sensor and realizing sensitive detection of CAP (Cardoso Ana R.et al., Biosens.Bioelectron., 2019, 124-125, 167). Geng's team prepared CAP molecular imprinting polymer by electropolymerization, and introduced chitosan-carbon nanotube complex to enhance the conductivity and stability of electrode, which was used for the detection of CAP in milk, with good selectivity, stability and reproducibility (Geng LJet al., Microchem.J., 2022, 182, 107887). Li et al. used a suspension polymerization method to prepare a bifunctional monomer molecular imprinting polymer and introduced magnetic nanomaterials Fe3O4 magnetic nanoparticles to enhance the molecular imprinting recognition sites and separate and extract CAP molecules, obtaining satisfactory CAP detection results (Li ZW et al., J. Chromatogr. B, 2018, 1100-1101, 113). However, the molecular imprinting sensing membranes prepared by these single polymerization methods still have fewer target molecule recognition sites and low sensor sensitivity; although the conductivity of the molecular imprinting membrane is improved after adding conductive nanomaterials, it still cannot achieve effective transmission of electrical signals. At present, how to achieve controllable preparation of CAP molecular imprinting sensors, improve the stability and sensitivity of sensors, and apply them to trace analysis of residual CAP in animal-derived foods is still a huge challenge. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a chloramphenicol molecular imprinting sensor based on a dual polymerization strategy to solve the problems existing in the above-mentioned prior art and achieve high-sensitivity detection of chloramphenicol.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a chloramphenicol molecular imprinting sensor, comprising the following steps:

[0008] (1) Oxidative prepolymerization: functional monomers and target molecule chloramphenicol are mixed in a solution, an oxidant is added, and prepolymerization is performed to obtain an oligomer solution;

[0009] (2) electrochemical polymerization: electrochemically polymerizing the oligomer solution to obtain a polymer modified electrode coated with chloramphenicol;

[0010] (3) eluting chloramphenicol: removing chloramphenicol from the polymer-modified electrode coated with chloramphenicol in step (2) to obtain the chloramphenicol molecular imprinting sensor;

[0011] The oxidant is any one or more of HAuCl4, H2PtCl6 or K3[Fe(CN)6].

[0012] Furthermore, the functional monomer is any one of dopamine (DA), aminophenylboronic acid (ABA) or epinephrine (EP). Dopamine (DA), aminophenylboronic acid (ABA) and epinephrine (EP) monomers can be oxidized by the above oxidant, and then undergo subsequent electrochemical polymerization.

[0013] Further, the molar ratio of the functional monomer to chloramphenicol is 1-10:10-1;

[0014] Furthermore, the prepolymerization reaction time is 1-10h.

[0015] Furthermore, in step (2), the electrochemical polymerization adopts electrochemical cyclic voltammetry; the potential width of the electrochemical cyclic voltammetry is -0.5 to 0.5 V vs SCE; the electrodeposition scan rate is 10 to 50 mV / s, preferably 20 mV / s; the number of electrodeposition cycles of the electrochemical cyclic voltammetry is 5 to 40 cycles, preferably 20 cycles.

[0016] Furthermore, in step (3), the polymer modified electrode coated with chloramphenicol is immersed in an eluent for elution, wherein the eluent is a mixed solution of anhydrous ethanol and ammonia water in a volume ratio of 19:1 to 1:19, preferably a volume ratio of ethanol to ammonia water of 19:1; the concentration of the ammonia water is 14.8 mol / L; and the elution time is 0.5 to 3 h, preferably 1 h.

[0017] The present invention also provides a chloramphenicol molecular imprinting sensor prepared by the above preparation method.

[0018] The present invention further provides application of the chloramphenicol molecular imprinting sensor in chloramphenicol detection.

[0019] Furthermore, the application comprises the following steps:

[0020] The chloramphenicol molecular imprinting sensor is used to adsorb chloramphenicol in a sample to be tested, and then electrochemical detection is performed in a solution to output an electrochemical signal to achieve the detection of chloramphenicol.

[0021] Furthermore, the electrochemical detection adopts differential pulse voltammetry (DPV) scanning, preferably with a potential width of -0.1 to 0.6 V and a scanning rate of 5 mV / s; the solution used in the detection process is a K3[Fe(CN)6] solution containing Na2SO4, preferably a 0.01 mol / L K3[Fe(CN)6] solution containing 0.2 mol / L Na2SO4.

[0022] The present invention adopts a dual polymerization strategy to prepare CAP molecular imprinting sensors, enhance the stability of the polymer film, and increase the target molecules in the polymer to increase the molecular imprinting holes, so as to improve the stability and sensitivity of the sensor. In the preparation process, metal nanoparticles are generated in situ to enhance the conductivity of the polymer, effectively improve the electron transfer efficiency, and thus further improve the sensitivity of the sensor.

[0023] The present invention discloses the following technical effects:

[0024] The chloramphenicol molecular imprinting sensor prepared by the invention has good CAP sensing performance, good stability and high sensitivity.

[0025] The present invention combines chemical oxidation prepolymerization and electrochemical polymerization dual polymerization methods to enhance the stability of the molecular imprinting membrane, and enables the polymer membrane to contain more target molecules, thereby obtaining more recognition sites and improving the sensitivity of the sensor. In the process of dual polymerization, metal nanomaterials are generated in situ, which greatly enhances the conductivity of the polymer membrane, thereby enhancing the electron transfer efficiency, further improving the sensitivity of the sensor, and obtaining a CAP molecular imprinting sensor with high selectivity, high stability and high sensitivity; at the same time, the method is simple, fast and easy to control.

[0026] The raw materials of the invention are simple and cheap, the preparation process and the processing method are simple and fast, the process is controllable, the processing time is short, the instruments and equipment are simple and efficient, and the invention has important significance for the detection research and application of residual trace CAP in animal-derived foods by molecular imprinting sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is a transmission electron microscopy (TEM) image of the oligomer obtained after the reaction in step 1) of Example 1 of the present invention;

[0029] Figure 2Preparation of CAP-AuNPs-MIP by electropolymerization of the oligomer solution on a gold electrode in Example 1 of the present invention double-p Cyclic voltammogram of modified electrode;

[0030] Figure 3 CAP-AuNPs-MIP of Example 1 of the present invention double-p Cyclic voltammetry characterization diagram of modified electrode and bare gold electrode in 0.1 mol / L PBS buffer solution;

[0031] Figure 4 The AuNPs-MIP prepared in Example 1 and Comparative Example 1 of the present invention are double-p Molecularly imprinted polymer modified electrodes and MIPs single-p The molecularly imprinted polymer modified electrode was exposed to 0.2 mol / L Na2SO4 in 0.01 mmol / L [Fe(CN)6] 3- DPV curve diagram in solution;

[0032] Figure 5 The AuNPs-MIP prepared in Example 1 of the present invention double-p Modified electrodes, CAP-AuNPs-MIP double-p Modified electrodes and CAP ads -AuNPs-MIP double-p DPV curve of the modified electrode in 0.01mmol / L K3[Fe(CN)6] solution containing 0.2mol / L Na2SO4;

[0033] Figure 6 The AuNPs-MIP prepared in Example 1 of the present invention double-p The modified electrode was immersed in different concentrations of CAP solutions to adsorb CAP for 10 minutes, and then tested in 0.2 mol / L Na2SO4 in 0.01 mmol / L K3[Fe(CN)6] solution by DPV test.

[0034] Figure 7 The AuNPs-MIP prepared in Example 1 of the present invention double-p The linear relationship between DPV peak current and CAP concentration in 0.01mmol / L K3[Fe(CN)6] solution containing 0.2mol / L Na2SO4 after the modified electrode adsorbed different concentrations of CAP. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] Preparation of chloramphenicol molecularly imprinted polymers (AuNPs-MIP) by dual polymerization strategy double-p ) The preparation method of the modified electrode comprises the following steps:

[0042] 1) Add 3.0 mmol / L HAuCl4 to 0.01 mol / L phosphate buffer solution (PBS) containing 30 mmol / L dopamine and 15 mmol / L CAP, and let stand for 5 h to obtain an oligomer solution containing the CAP target molecule and generate AuNPs in situ.

[0043] 2) Using a three-electrode system, a saturated calomel electrode as a reference electrode, a carbon rod electrode as a counter electrode, and a gold electrode as a working electrode, the low polymer solution obtained in step 1) is further electropolymerized by an electrochemical method to prepare a molecular imprinting polymer modified electrode. The specific method is: in the three-electrode system, electrochemical cyclic voltammetry is used to perform electrodeposition (potential width: -0.5-0.5V, scan rate: 20mV / s, number of polymerization cycles: 20 cycles) in the low polymer solution obtained in step 1) to obtain a polymer CAP-AuNPs-MIP containing the target molecule. double-p Modified electrode.

[0044] 3) CAP-AuNPs-MIP prepared in step 2) double-p The modified electrode was placed in a mixed solution of anhydrous ethanol / ammonia water (concentration of 14.8 mol / L) with a volume ratio of 19:1 for elution for 1 h to remove the template molecule chloramphenicol in the imprinted polymer, and then rinsed with distilled water twice to remove surface pollutants to obtain a chloramphenicol molecularly imprinted polymer modified electrode (AuNPs-MIP double-p modified electrode).

[0045] The chloramphenicol molecularly imprinted polymer modified electrode (AuNPs-MIP) prepared in Example 1 was double-p The modified electrode was immersed in a PBS solution containing 50 μmol / L chloramphenicol and adsorbed for 10 min before being taken out. It was then rinsed twice with distilled water to remove the chloramphenicol molecules that were not firmly adsorbed on the surface to obtain CAP ads -AuNPs-MIP double-p Modified electrode.

[0046] Figure 1 This is a transmission electron microscope (TEM) image of the oligomer obtained after the reaction in step 1) of Example 1 of the present invention. Figure 1 It can be seen that AuNPs were generated in situ while the polymerization reaction occurred.

[0047] Figure 2 The oligomer solution obtained in Example 1 of the present invention was electropolymerized on a gold electrode to prepare CAP-AuNPs-MIP double-p Cyclic voltammogram of modified electrode. During the preparation process, a pair of obvious redox peaks were observed on the cyclic voltammogram, which was attributed to the electro-oxidation and reduction reaction of dopamine oligomers. Dopamine oligomers were electropolymerized on the electrode surface to further generate polymers. The experimental results show that electropolymerized CAP-AuNPs-MIP double-p The modified electrode was successfully prepared.

[0048] Comparative Example 1

[0049] The difference from Example 1 is that no oxidative prepolymerization was performed, as follows:

[0050] 1) Using a three-electrode system, a saturated calomel electrode as a reference electrode, a carbon rod electrode as a counter electrode, and a gold electrode as a working electrode, a molecular imprinting polymer film modified electrode was prepared by direct electropolymerization using an electrochemical method in a 0.01 mol / L phosphate buffer solution (PBS) containing 30 mmol / L dopamine and 15 mmol / L chloramphenicol. The specific method is: in the three-electrode system, electrochemical cyclic voltammetry is used to perform electrodeposition (potential width: -0.5-0.5 V, scan rate: 20 mV / s, number of polymerization cycles: 20 cycles) in the oligomer solution obtained in step 1) to obtain a CAP-MIP containing the target molecule. single-p Modified electrode.

[0051] 2) CAP-MIP prepared in step 1) single-p The modified electrode was placed in a mixed solution of ethanol / ammonia water with a volume ratio of 19:1 and eluted for 1 h to remove the template molecule CAP in the imprinted polymer, and then rinsed with distilled water twice to remove surface pollutants. The CAP molecular imprinted polymer MIP was obtained by single polymerization. single-p Modified electrode.

[0052] The CAP-AuNPs-MIP obtained in Example 1 was tested on an electrochemical workstation using cyclic voltammetry testing technology. double-p The cyclic voltammetry characterization diagrams of the modified electrode and the bare gold electrode in 0.1 mol / L PBS buffer solution are shown in Figure 3 The results show that the bare gold electrode has no obvious redox peak in PBS buffer solution, while CAP-AuNPs-MIP double-p The modified electrode has a strong oxidation peak near 0.38 V vs SCE and an observable reduction peak near 0.02 V vs SCE, indicating that CAP-AuNPs-MIP double-p Molecularly imprinted polymers have been successfully modified on gold electrodes.

[0053] Figure 4 The AuNPs-MIP prepared in Example 1 and Comparative Example 1 of the present invention are double-p Molecularly imprinted polymer modified electrodes and MIPs single-p The molecularly imprinted polymer modified electrode was exposed to 0.2 mol / L Na2SO4 in 0.01 mmol / L [Fe(CN)6] 3- Solution DPV curve. Figure 3 It can be seen that the AuNPs-MIP prepared in Example 1 of the present invention double-p The DPV peak current of the molecularly imprinted polymer modified electrode is compared with that of the MIP prepared in Example 1. single-pThe peak current of the molecularly imprinted polymer modified electrode is large, indicating that the AuNPs-MIP prepared by the dual polymerization strategy double-p Molecularly imprinted polymers are more efficient than MIPs prepared by single polymerization. single-p Molecularly imprinted polymers have good conductivity. During the prepolymerization process, the addition of oxidants can generate AuNPs in situ, enhancing the conductivity of MIP. double-p The conductivity of molecularly imprinted polymers improves the efficiency of electron transfer and increases the current.

[0054] Figure 5 The AuNPs-MIP prepared in Example 1 double-p Molecularly imprinted polymer modified electrode, polymer without elution of CAP (CAP-AuNPs-MIP double-p ) modified electrode and molecularly imprinted polymer (CAP) adsorbed with 50 μmol / L CAP ads -AuNPs-MIP double-p ) electrode in 0.01mmol / L [Fe(CN)6] containing 0.2mol / L Na2SO4 3- Solution DPV curve. Compared with CAP-AuNPs-MIP without eluted CAP molecules double-p Peak current of DPV curve of modified electrode, AuNPs-MIP obtained after eluting CAP molecules double-p The peak current of the DPV curve of the modified electrode is greatly enhanced. After CAP is eluted, CAP imprinted holes appear on the electrode surface, and the conductivity of the electrode surface is enhanced, indicating that CAP has been successfully eluted. double-p After the modified electrode re-adsorbs CAP, the obtained CAP ads -AuNPs-MIP double-p The peak current of the DPV curve of the modified electrode is relative to that of AuNPs-MIP double-p The modified electrode decreased, indicating that CAP was successfully adsorbed on AuNPs-MIP double-p In the molecularly imprinted holes on the molecularly imprinted polymer, the conductivity of the polymer film decreases.

[0055] The AuNPs-MIP prepared in Example 1 double-p The modified electrode was immersed in a centrifuge tube containing solutions of different concentrations of CAP and taken out after adsorption for 10 minutes to obtain the CAP solution with different concentrations. ads -AuNPs-MIP double-p Modified electrode. In 0.2mol / L Na2SO4 and 0.01mmol / L [Fe(CN)6] 3- DPV test was performed in the solution. The results are as follows Figure 6 As shown in Figure 2, with the increase of CAP concentration, the CAP adsorbed by CAPads -AuNPs-MIP double-p The greater the decrease in DPV peak current of the modified electrode, the better the linear relationship between DPV peak current and CAP concentration ( Figure 7 ), when the linear range of CAP detection is 10μmol / L-100μmol / L, its linear equation is y=-0.0285x+4.259, R 2 =0.9851, detection limit is 43nM, (signal-to-noise ratio S / N=3).

[0056] AuNPs-MIP prepared by the dual polymerization strategy of the present invention double-p Compared with the MIP prepared by single polymerization, the modified electrode single-p The modified electrode has better conductivity, enhanced stability, and improved CAP detection performance.

[0057] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a chloramphenicol molecular imprinting sensor based on a dual polymerization strategy, characterized in that: The following steps are involved: (1) Oxidative prepolymerization: functional monomers and chloramphenicol are mixed in a solution, an oxidant is added, and prepolymerization is performed to obtain an oligomer solution; (2) electrochemical polymerization: electrochemically polymerizing the oligomer solution by electrochemical cyclic voltammetry to obtain a polymer modified electrode coated with chloramphenicol; (3) eluting chloramphenicol: immersing the polymer modified electrode coated with chloramphenicol in an eluent to elute and remove the chloramphenicol, thereby obtaining the chloramphenicol molecular imprinting sensor; The oxidant is any one or more of HAuCl4, H2PtCl6 or K3[Fe(CN)6]; The functional monomer is any one of dopamine, aminophenylboronic acid or adrenaline; The molar ratio of the functional monomer to chloramphenicol is 1-10:10-1; The prepolymerization time is 1-10h; The potential width of the electrochemical cyclic voltammetry is -0.5 to 0.5 V vs SCE; the electrodeposition scanning speed of the electrochemical cyclic voltammetry is 10 to 50 mV / s, and the number of electrodeposition cycles is 5 to 40 cycles; The eluent is a mixed solution of anhydrous ethanol and ammonia water in a volume ratio of 19:1 to 1:19, and the concentration of the ammonia water is 14.8 mol / L; the elution time is 0.5 to 3 hours.

2. The chloramphenicol molecular imprinting sensor prepared by the preparation method as claimed in claim 1.

3. Use of the chloramphenicol molecular imprinting sensor as claimed in claim 2 in the detection of chloramphenicol.

4. The use according to claim 3, characterized in that: The following steps are involved: The chloramphenicol molecular imprinting sensor is used to adsorb chloramphenicol in a sample to be tested, and then electrochemical detection is performed in a solution to output an electrochemical signal to achieve the detection of chloramphenicol.

5. The use according to claim 4, characterized in that: The electrochemical detection adopts differential pulse voltammetry; the solution used in the detection process is a K3[Fe(CN)6] solution containing Na2SO4.

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