Molecularly imprinted electrochemical sensor, preparation method thereof and CAP detection method

Abstract

The invention discloses a preparation method of a molecularly imprinted electrochemical sensor. The preparation method comprises the following steps: S1, pretreating the surface of a GCE electrode; step S2, modifying the surface of the pretreated GCE electrode with an MXene suspension to obtain an MXene / GCE electrode; step S3, the MXene / GCE electrode is immersed in a PBS buffer solution containing CAP and OPD; step S4, on the surface of the MXene / GCE electrode, carrying out electropolymerization on the OPD by adopting CV to obtain a CAP-MIPs / MXene / GCE electrode of a molecularly imprinted polymer containing CAP; s5, the prepared CAP-MIPs / MXene / GCE electrode is subjected to elution, and the molecularly imprinted electrochemical sensor containing the MIPs / MXene / GCE electrode is prepared. By modifying MXene on the surface of a GCE electrode and constructing a molecularly imprinted membrane with specific recognition capability, the sensor can recognize and detect chloramphenicol with high selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of sensors, and in particular to a molecular imprinting electrochemical sensor and a preparation method thereof, and a CAP detection method. Background Art

[0002] Chloramphenicol (CAP) is a broad-spectrum antibiotic with significant antibacterial activity, particularly against infections caused by Gram-negative bacteria (such as Salmonella typhi and other Salmonella species). CAP's highly effective antibacterial properties and low production cost have made it not only used in human medicine to treat a variety of bacterial infections, but also very popular in animal husbandry and aquaculture as an additive to promote growth and prevent disease. However, the use of CAP also poses serious environmental and health concerns. While CAP is highly effective in treating infections, its potential toxic side effects cannot be ignored. Studies have shown that excessive use of CAP can lead to its residues in animal-derived foods (such as meat, dairy products, and aquatic products), which can then enter the human body through the food chain, posing a threat to human health. Long-term ingestion of CAP residues can lead to more serious symptoms such as bone marrow suppression, aplastic anemia, and thrombocytopenia, and can even adversely affect the development of infants and young children. CAP residues in the environment can also disrupt ecological balance and induce the development of drug-resistant bacteria, further exacerbating public health risks. Due to the toxic side effects of CAP and its potential harm to the environment and health, many countries and regions have banned its use in aquaculture and animal husbandry. However, due to its high economic benefits and relatively low acquisition cost, illegal use of CAP still occurs, making it difficult to completely eliminate the problem of CAP residues in animal-derived foods. Therefore, the development of a rapid, accurate, and sensitive method for the detection of trace CAP is of great value for monitoring CAP residues in aquaculture water, ensuring fishery safety, and protecting the environment and human health.

[0003] Currently, the main methods for detecting CAP include liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, enzyme-linked immunosorbent assay (ELISA), capillary zone electrophoresis (CAP), and electrochemical methods. Although some methods can detect multiple targets simultaneously, they generally require complex sample pretreatment, are costly, and involve complex procedures. Therefore, electrochemical detection methods are gaining increasing attention due to their low cost, rapid response, high sensitivity, and simple operation.

[0004] Molecularly imprinted electrochemical sensors (MIP-ECS), fabricated by combining molecular imprinting technology with electrochemical sensing, are highly selective and sensitive detection technologies. MIP-ECS can specifically recognize target molecules and detect trace amounts in complex matrices, finding widespread application in environmental monitoring, food safety, and biomedicine. Molecularly imprinted polymers (MIPs) are functional materials fabricated by mimicking biomolecular recognition systems. Their internal structure contains three-dimensional cavities that closely match the spatial configuration and chemical interactions of template molecules, enabling specific recognition of target molecules. During the preparation of MIPs, molecular imprinting technology (MIT) achieves targeted synthesis by combining template molecules with functional monomers. After removing the target molecule from the resulting product, the MIP and template molecules complement each other in terms of space, shape, and functional groups, enhancing the MIP's selectivity for recognizing the imprinted molecule in complex samples. MIPs offer advantages such as resistance to harsh environments, low cost, excellent physical and chemical stability, and reusability. Figure 1 The construction process of molecular imprinting electrochemical sensors is demonstrated. Although molecular imprinting electrochemical sensors have outstanding performance in terms of selectivity and sensitivity, their limited conductivity may affect the performance of the sensor, especially in terms of signal transmission and detection sensitivity. Compared with common reinforcing materials such as reduced graphene oxide (10 -4 –1S·cm -1 ), carbon nanotubes (10500S·cm -1 ) and quantum dots (extremely low conductivity, 0.01S·cm -1 ), etc. The electrical conductivity of MXene is as high as 15100±160S·cm -1 Therefore, molecular imprinting electrochemical sensors choose to introduce MXene to improve the conductivity of the sensor.

[0005] The general chemical formula of MXene is Mn+1XnTx (n=1–3), where M represents a pre-transition metal element, X represents carbon (C) and / or nitrogen (N), and Tx refers to surface functional groups generated during the etching synthesis process, such as oxygen (O), hydroxyl (–OH), or fluorine (–F). MXene is a novel two-dimensional nanomaterial developed at Drexel University in the United States. Its unique layered structure provides abundant channels for ion transport, significantly improving ion migration rates. MXene is composed of a few atomically thick layers of transition metal carbides, nitrides, or carbonitrides. Surface hydroxyl groups or terminal oxygen groups impart metallic conductivity to transition metal carbides. Therefore, MXene materials possess dual properties: on the one hand, they retain the excellent metallic conductivity of transition metal carbides, and on the other hand, they possess the modifiability brought about by surface functional groups, showing great potential for application in electrochemistry. In addition, MXene has advantages such as excellent electron conductivity, significant affinity for water molecules, outstanding chemical stability, large specific surface area, environmentally friendly properties and non-toxicity, so it shows broad application potential in the field of analytical chemistry, especially in electrochemical sensing.

[0006] Therefore, how to develop a molecular imprinting electrochemical sensor based on MXene nanomaterials and a new detection method for analyzing CAP in aquaculture seawater is an urgent problem to be solved. Summary of the Invention

[0007] The present embodiments provide a molecularly imprinted electrochemical sensor, a method for preparing the sensor, and a CAP detection method to address the limited conductivity of molecularly imprinted electrochemical sensors in the prior art. To provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is provided below. This summary is not intended to be a comprehensive review, identify key or important components, or delineate the scope of these embodiments. Its sole purpose is to present some concepts in a simplified form as a prelude to the detailed description that follows.

[0008] According to a first aspect of an embodiment of the present invention, a method for preparing a molecularly imprinted electrochemical sensor is provided.

[0009] In one embodiment, a method for preparing a molecularly imprinted electrochemical sensor comprises:

[0010] Step S1, pre-treating the surface of the GCE electrode;

[0011] Step S2, modifying the MXene suspension on the surface of the pretreated GCE electrode to obtain a MXene / GCE electrode;

[0012] Step S3, immersing the MXene / GCE electrode in PBS buffer containing CAP and OPD;

[0013] Step S4, electropolymerizing OPD on the surface of the MXene / GCE electrode using CV to obtain a CAP-MIPs / MXene / GCE electrode containing a molecularly imprinted polymer of CAP;

[0014] In step S5, the prepared CAP–MIPs / MXene / GCE electrode is eluted to prepare a molecularly imprinted electrochemical sensor containing the MIPs / MXene / GCE electrode.

[0015] Optionally, the step of pre-treating the surface of the GCE electrode comprises:

[0016] Step S11, taking 0.5 μm Al2O3 powder, adding deionized water to make a paste, and evenly applying it on the surface of the suede;

[0017] Step S12, holding the GCE electrode vertically, and lightly pressing and polishing it in an "∞"-shaped trajectory;

[0018] Step S13, cleaning the GCE electrode and suede, replacing 0.3 μm Al2O3 powder, and repeating the above steps until the GCE electrode has a matte finish;

[0019] Step S14: Apply 0.05 μm Al2O3 slurry and gently polish until the surface of the GCE electrode presents a mirror effect;

[0020] Step S15, rinsing the surface of the GCE electrode with ultrapure water, and then ultrasonically cleaning it in anhydrous ethanol and ultrapure water respectively;

[0021] In step S16, after the GCE electrode is dried with nitrogen, cyclic voltammetry is used to scan in a 5 mM potassium ferrocyanide solution, and the difference in redox peak potential is always maintained within the range of 0.1 V.

[0022] Optionally, the step of modifying the MXene suspension on the surface of the GCE electrode comprises:

[0023] 6 μL of MXene suspension was modified on the surface of the GCE electrode and naturally dried at room temperature to obtain a MXene / GCE electrode.

[0024] Optionally, the preparation steps of the MXene suspension include:

[0025] First, 0.2 g of chitosan was added to 50 mL of 1% acetic acid solution and mixed by magnetic stirring to obtain a chitosan solution;

[0026] Then, 20 mL of chitosan solution was added to 100 mg of MXene and ultrasonically treated for 60 min to obtain a uniform MXene suspension, which was then stored in a refrigerator at 4 °C.

[0027] Optionally, the steps of preparing the PBS buffer solution include:

[0028] First, 7.164 g of Na2HPO4·12H2O and 3.121 g of NaH2PO4·2H2O were dissolved in 100 mL of deionized water and ultrasonicated for 15 min to obtain homogeneous Na2HPO4 solution and NaH2PO4 solution respectively.

[0029] Then, 12.3 mL of 0.2 M Na2HPO4 solution and 87.7 mL of 0.2 M NaH2PO4 solution were mixed and ultrasonicated for 15 min to obtain a uniform 0.2 M Na2HPO4 / NaH2PO4 buffer solution.

[0030] Optionally, the pH value of the Na2HPO4 / NaH2PO4 buffer solution is 6.0.

[0031] Optionally, the prepared CAP-MIPs / MXene / GCE electrode is subjected to an elution step, and cyclic voltammetry is used for elution, and the template molecule CAP is washed away 20 times in the eluent to prepare a molecularly imprinted electrochemical sensor containing a MIPs / MXene / GCE electrode.

[0032] Optionally, the steps of preparing the PBS buffer solution containing CAP and OPD include:

[0033] Take 10 mL of PBS buffer solution, add 6.5 mg of OPD and 6.5 mg of CAP, disperse it by ultrasonic for 30 min, and then slowly inject nitrogen for 5 min.

[0034] According to a second aspect of the embodiments of the present invention, a molecular imprinting electrochemical sensor is provided.

[0035] In one embodiment, the molecularly imprinted electrochemical sensor is prepared by the method of any one of the above embodiments.

[0036] According to a third aspect of an embodiment of the present invention, a CAP detection method is provided.

[0037] In one embodiment, the CAP detection method is based on the molecular imprinting electrochemical sensor of any of the above embodiments, comprising the following steps:

[0038] A three-electrode system was constructed, with the working electrode being the MIPs / MXene / GCE electrode, the counter electrode being the platinum wire electrode, and the reference electrode being the Ag / AgCl electrode;

[0039] Quantitative analysis of the CAP electrochemical properties of the MIPs / MXene / GCE electrode by DPV, including:

[0040] The DPV current value I0 of the MIPs / MXene / GCE electrode was recorded in the electrolyte;

[0041] The MIPs / MXene / GCE electrode was immersed in CAP standard solutions of different concentrations and incubated for 10 minutes. DPV detection was then performed and the current value I was recorded. t ;

[0042] Take I0–I t The peak current difference ΔI was used as the standard, a standard curve was drawn and a linear regression equation was established.

[0043] The technical solution provided by the embodiment of the present invention may have the following beneficial effects:

[0044] (1) Using GCE as the sensor body, MXene material as the sensitizing material, CAP as the template molecule, and OPD as the functional monomer, a molecular imprinting modified electrode (MIPs / MXene / GCE) was prepared by the drop coating method. By optimizing the detection conditions, a new detection method suitable for analyzing CAP in aquaculture seawater was established.

[0045] (2) The molecular imprinting electrochemical sensor was systematically characterized using two electrochemical characterization techniques, CV and DPV, and the key experimental parameters were optimized. The experimental results showed that the electrode activity was significantly enhanced after modification with MXene and MIP, and it had specific recognition for CAP. The optimal detection conditions were: the optimal modification amount of MXene suspension was 6 μL; the optimal pH of PBS buffer solution was 6.0; the optimal number of scan cycles was 20; the optimal molar ratio of template molecule to functional monomer was 1:3; the optimal scan rate was 50 mV / s; the optimal number of elution cycles was 20; and the optimal adsorption time was 10 min.

[0046] (3) A molecularly imprinted electrochemical sensor was constructed, which exhibited excellent linear response characteristics (R 2 >0.99), the detection limit was 0.017 μM (S / N=3), and RSD was <10%.

[0047] (4) The established molecular imprinting electrochemical sensor was applied to the quantitative detection of CAP in actual aquaculture seawater samples. The results showed that the spike recovery rate was in the range of 90.4%–105.2%, and the relative standard deviation was less than 6%, which confirmed that the sensor has good anti-interference ability, excellent detection sensitivity and reliable stability in aquaculture seawater, providing a reliable analytical means for the accurate determination of trace CAP in complex environmental samples.

[0048] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0050] Figure 1 Schematic diagram of the molecular imprinting preparation process, signal enhancement method, and detection mechanism in electrochemical sensors;

[0051] Figure 2 are the CV plots of different modified electrodes;

[0052] Figure 3 are the SEM images of CAP–MIPs / MXene / GCE and MIPs / MXene / GCE;

[0053] Figure 4a This is the cyclic voltammetry scanning curve of modified MXene with different amounts;

[0054] Figure 4b It is a dot-line graph of modified MXene with different amounts;

[0055] Figure 5a It is a dotted line graph of the DPV of elution and adsorption in potassium ferricyanide solution after the same number of polymerization cycles of MXene / GCE in polymerization solutions with different pH values ​​(solid line is elution, dashed line is adsorption);

[0056] Figure 5b It is a dot-line graph of ΔI in potassium ferricyanide solution after MXene / GCE is polymerized for the same number of cycles in polymerization solutions with different pH values;

[0057] Figure 6a It is a dotted line graph of the DPV of elution and adsorption in potassium ferricyanide solution after the same number of polymerization cycles of MXene / GCE in polymerization solutions with different molar ratios (solid line is elution, dotted line is adsorption);

[0058] Figure 6b It is a dot-line graph of ΔI in potassium ferricyanide solution after polymerization of MXene / GCE in polymerization solutions with different molar ratios for the same number of cycles;

[0059] Figure 7a It is a dotted line graph of the DPV of MXene / GCE eluted and adsorbed in potassium ferricyanide solution after polymerization at different scan rates for the same number of cycles (solid line is elution, dashed line is adsorption);

[0060] Figure 7bIt is a dot-line graph of ΔI in potassium ferricyanide solution after MXene / GCE is polymerized at different scan rates for the same number of cycles in the polymerization solution;

[0061] Figure 8a It is a dot-line graph of the DPV of elution and adsorption in potassium ferricyanide solution after different cycles of polymerization of MXene / GCE in the same concentration polymerization solution (solid line is elution, dotted line is adsorption);

[0062] Figure 8b It is a dot-line graph of ΔI of MXene / GCE in potassium ferricyanide solution after different polymerization cycles in the same concentration polymerization solution;

[0063] Figure 9a It is a dot-line graph of the eluted and adsorbed DPV in potassium ferricyanide solution after different elution cycles (solid line is elution, dotted line is adsorption);

[0064] Figure 9b It is a dot-line graph of ΔI in potassium ferricyanide solution after different elution cycles;

[0065] Figure 10a is a dotted line graph of the DPV of MIPs / MXene / GCE in potassium ferricyanide solution after adsorption in 50 μM CAP solution for different times (solid line represents elution, dashed line represents adsorption);

[0066] Figure 10b is a dot-line plot of ΔI of MIPs / MXene / GCE in potassium ferricyanide solution after adsorption in 50 μM CAP solution for different times;

[0067] Figure 11a is the DPV graph of MIPs / MXene / GCE in CAP standard solutions with different concentrations;

[0068] Figure 11b is the linear relationship diagram of MIPs / MXene / GCE in different concentrations of CAP standard solution (0.1–300 μM);

[0069] Figure 11c is the DPV graph of MIPs / MXene / GCE in CAP standard solutions with different concentrations;

[0070] Figure 11d is the linear relationship diagram of MIPs / MXene / GCE in different concentrations of CAP standard solution (0.1–300 μM);

[0071] Figure 12a is the DPV graph of CAP detected by MIPs / MXene / GCE for 5 consecutive times;

[0072] Figure 12bis the response current diagram of MIPs / MXene / GCE detecting CAP for 5 consecutive times;

[0073] Figure 13 is the response current of the sensor to and other interfering compounds. DETAILED DESCRIPTION

[0074] The following description and accompanying drawings sufficiently illustrate the specific embodiments herein to enable those skilled in the art to practice them. Portions and features of some embodiments may be included in or substituted for portions and features of other embodiments. The scope of the embodiments herein includes the entire scope of the claims, including all available equivalents thereof. Herein, the terms "first," "second," and the like are used solely to distinguish one element from another and do not require or imply any actual relationship or order between these elements. In practice, the first element can also be referred to as the second element, and vice versa. Furthermore, the terms "comprise," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a structure, device, or apparatus comprising a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such structure, device, or apparatus. Without further limitation, an element defined by the phrase "comprising a..." does not preclude the presence of other identical elements in the structure, device, or apparatus comprising the element. The various embodiments herein are described in a progressive manner, with each embodiment focusing on its differences from the other embodiments. Similar or identical parts between the various embodiments can be referenced to each other.

[0075] The terms "longitudinal", "transverse", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like used herein to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are intended only to facilitate the description of this document and simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the present invention. In the description herein, unless otherwise specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, they can be mechanical or electrical connections, or they can be internal connections between two elements, they can be directly connected, or they can be indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to the specific circumstances.

[0076] As used herein, unless otherwise specified, the term "plurality" means two or more.

[0077] In this document, the character " / " indicates that the preceding and following objects are in an "or" relationship. For example, A / B means: A or B.

[0078] In this article, the term "and / or" is used to describe the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, A and B.

[0079] It should be understood that, although the various steps in the flowchart are shown in sequence as indicated by the arrows, these steps are not necessarily performed in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and these steps may be performed in other orders. Moreover, at least a portion of the steps in the figure may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily performed at the same time, but may be performed at different times. The execution order of these sub-steps or stages is not necessarily to be performed in sequence, but may be performed in turn or alternately with other steps or at least a portion of the sub-steps or stages of other steps.

[0080] Each module in the device or system of the present application can be implemented in whole or in part by software, hardware, or a combination thereof. The above modules can be embedded in or independent of the processor in the computer device in the form of hardware, or can be stored in the memory of the computer device in the form of software so that the processor can call and execute the operations corresponding to the above modules.

[0081] In the absence of conflict, the embodiments of the present invention and the features thereof may be combined with each other.

[0082] The present invention successfully developed a high-performance molecular imprinting electrochemical sensor based on MXene nanomaterials by combining molecular imprinting technology with electrochemical sensing technology. As a new two-dimensional material, MXene significantly improves the detection sensitivity and signal response intensity of glassy carbon electrode (GCE) by virtue of its excellent conductivity and large specific surface area. By modifying MXene on the surface of GCE electrode and constructing a molecular imprinting membrane with specific recognition ability, the sensor can highly selectively identify and detect chloramphenicol. Chloramphenicol was quantitatively analyzed using differential pulse voltammetry (DPV), and a corresponding standard curve was established. The molecular imprinting electrochemical sensor of the present invention exhibited excellent performance in chloramphenicol detection, providing a new technical means for trace antibiotic detection in environmental monitoring, food safety and aquaculture.

[0083] The materials and reagents used in the experiment are as follows:

[0084] Chloramphenicol (CAP, 98%), furazolidone (FZD, 98%), and o-phenylenediamine (OPD) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; concentrated sulfuric acid (H2SO4) was of analytical grade and purchased from Yantai Sanhe Chemical Reagent Co., Ltd.; florfenicol (FFC, 98%) and sulfadiazine (SDM, 98%) were of analytical grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol, anhydrous ethanol, acetic acid, acetic acid, and phosphoric acid were of analytical grade and purchased from Tianjin Komiou Chemical Reagent Co., Ltd.

[0085] The instruments used in the experiment are as follows:

[0086] Scanning electron microscope (Hitachi SU8010, Hitachi, Japan), ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), multi-channel potentiostat (CHI1040, Shanghai Chenhua Instrument Co., Ltd.), saturated silver chloride electrode (CHI111, Shanghai Chenhua Instrument Co., Ltd.), platinum wire electrode (CHI115, Shanghai Chenhua Instrument Co., Ltd.), glassy carbon electrode (CHI104, Shanghai Chenhua Instrument Co., Ltd.), electronic analytical balance (BS224S, Sartorius Scientific Instrument Beijing Co., Ltd.), pH meter (FE28, Mettler–Toledo), disposable syringe (1 mL, Henan Shuguang Jianshi Medical Instrument Co., Ltd.), and Milli–Qchao ultrapure water device (Mettler–Toledo, Sainz Instruments).

[0087] Solution preparation

[0088] 0.2M Phosphate Buffer (PBS): First, prepare 0.2M Na2HPO4 / NaH2PO4 buffer (pH 6.0). Dissolve 7.164g of Na2HPO4·12H2O and 3.121g of NaH2PO4·2H2O in 100mL of deionized water and sonicate for 15 minutes to obtain a homogeneous Na2HPO4 solution and NaH2PO4 solution. Mix 12.3mL of the 0.2M Na2HPO4 solution and 87.7mL of the 0.2M NaH2PO4 solution and sonicate for 15 minutes to obtain a homogeneous 0.2M Na2HPO4 / NaH2PO4 buffer (pH 6.0). Store in a cool, dark place.

[0089] Electrolyte: Mix 5 mM K3Fe(CN)6 / K4Fe(CN)6 with 0.1 M KCl to prepare the required solution.

[0090] 1 mM CAP standard solution: Weigh 32.3 mg of CAP standard, use ultrapure water as solvent, sonicate to dissolve, and then dilute to 100 mL to obtain a 1 mM CAP standard solution.

[0091] Preparation of Mxene suspension

[0092] Weigh 0.2 g of chitosan and add 50 mL of 1% acetic acid solution. Mix under magnetic stirring to obtain a chitosan solution. Add 100 mg of MXene to 20 mL of this solution and sonicate for 60 minutes to obtain a uniform MXene suspension. Store in a refrigerator at 4°C.

[0093] Pre-treat the surface of the GCE electrode: Use Al2O3 powder of different particle sizes (0.5, 0.3, and 0.05μm) to mechanically polish the glassy carbon electrode on suede. First, take a small amount of 0.5μm Al2O3 powder, add deionized water to make a paste, and evenly apply it to the suede surface. Hold the electrode vertically and polish it lightly with an "∞"-shaped trajectory to avoid tilting. Then thoroughly clean the electrode and suede and replace it with 0.3μm Al2O3 powder. The polishing process is the same as the previous steps until the surface is matte but without coarse scratches. Finally, use a new area of ​​suede or a clean pad, apply 0.05μm slurry, and gently polish until the electrode surface shows a mirror-like gloss. Rinse the electrode surface with ultrapure water, and then ultrasonically clean it in anhydrous ethanol and ultrapure water for 2–3 minutes respectively to remove residual impurities. After the electrode was dried with nitrogen, cyclic voltammetry (CV) was used to scan in a 5 mM potassium ferricyanide solution. The difference in redox peak potential was always kept within 0.1 V, ensuring that the electrode surface was electrochemically clean.

[0094] Modifying the MXene suspension on the surface of the pretreated GCE electrode: Modifying the surface of the pretreated GCE with 6 μL of the prepared MXene suspension was carried out and naturally dried at room temperature to obtain a MXene / GCE modified electrode.

[0095] A specific example of the preparation process of a molecular imprinting electrochemical sensor is given below.

[0096] To 10 mL of PBS buffer solution, 6.5 mg of OPD and 6.5 mg of CAP were added. After ultrasonic dispersion for 30 minutes, nitrogen was slowly injected for 5 minutes to remove dissolved oxygen from the solution. The MXene-modified GCE was then immersed in a PBS buffer solution containing CAP and OPD. OPD was electropolymerized on the MXene / GCE surface using CV to produce molecularly imprinted polymers (CAP–MIPs) containing CAP. The prepared CAP–MIPs / MXene / GCE was then subjected to cyclic voltammetry and eluted 20 times with the eluent (dilute sulfuric acid:ethanol = 4:1, v / v) to remove the template molecule CAP, thus preparing a molecularly imprinted electrochemical sensor MIPs / MXene / GCE.

[0097] A non-imprinted electrochemical sensor was prepared as a control following the same steps without using CAP template molecules, resulting in NIPs / MXene / GCE.

[0098] A specific example of CAP detection using the molecular imprinting electrochemical sensor of the present invention is given below.

[0099] In this example, a three-electrode system was used for electrochemical performance testing. The working electrode was MIPs / MXene / GCE, the counter electrode was a platinum wire electrode, and the reference electrode was an Ag / AgCl electrode. The electrochemical properties of the modified electrode were evaluated by CV and DPV. The DPV current value I0 of MIPs / MXene / GCE was recorded in the electrolyte. The electrode was then immersed in CAP standard solutions of different concentrations and incubated for 10 minutes. DPV detection was then performed, and the current value I t . Take I0–I t The peak current difference ΔI was used as the standard, a standard curve was drawn and a linear regression equation was established.

[0100] In order to evaluate the performance of the molecular imprinting electrochemical sensor of the present invention, samples were taken from the breeding pond of the grouper breeding workshop. The water samples were filtered through a 0.22μm filter membrane and stored in an environment of -20°C. The real water samples were measured by performing a spike test. 0.0323g of CAP was weighed and added to the seawater sample to obtain a seawater sample with a concentration of 1mM. The sample was diluted with PBS solution (pH 6.0) for analysis and detection, and the DPV current response in the water sample was detected using MIPs / MXene / GCE. Figure 2As shown, CV electrochemical characterization of various modified electrodes (GCE (a), MXene / GCE (b), CAP–MIPsMXene / GCE (c), MIPs / MXene / GCE (d), and NIPs / MXene / GCE (e)) was performed. The redox reaction of the potassium ferricyanide redox probe results in a pair of reversible redox peaks on the GCE. Curve b shows that the current response is significantly enhanced after MXene modification of the GCE surface. This is because the high conductivity of the MXene material facilitates rapid electron transfer between the redox probe and the electrode. Electrochemical characterization results (curve c) show that the electrochemical signal is weakened after molecularly imprinted polymer modification of the electrode surface. This is primarily because the dense imprinted film hinders direct contact between the redox probe and the electrode surface, inhibiting the electrochemical reaction. After elution of the CAP–MIPsMXene / GCE (curve d), the current response is significantly restored. This is because after the template molecule CAP is removed, a large number of specific recognition holes are formed in the molecularly imprinted film, allowing the redox probe to contact the electrode surface and react through these channels. The response current of MIPs / MXene / GCE is still lower than that of MXene / GCE. This may be because the presence of the molecular imprinting membrane partially blocks the electron transfer path. At the same time, the size selectivity of the imprinted holes also limits the passage of some probe molecules. Because there are no holes on the surface of the NIPs / MXene / GCE electrode that match the CAP, it still hinders the potassium ferricyanide redox probe from entering the electrode surface to react.

[0101] like Figure 3 As shown in the CAP–MIPs / MXene / GCE image (a) and the SEM image of MIPs / MXene / GCE (b), MIPs / MXene / GCE has a clear layered structure. After elution, imprinted molecular cavities are formed on the surface of the MIPs / MXene / GCE electrode, which has a larger surface area, can provide more imprinting sites, and improve the performance of the imprinted sensor. By comparing MIPs / MXene / GCE with CAP–MIPs / MXene / GCE, it can be inferred that CAP has been successfully eluted.

[0102] Optimization of MXene modification amount:

[0103] After the glassy carbon electrode was modified with different volumes of MXene suspension and dried naturally, the MXene / GCE was tested in the electrolyte using the CV method and the response current value was recorded. Figure 4a and Figure 4bAs shown in the figure, when 6μL of MXene suspension is modified on the GCE surface, the peak current response of the modified electrode reaches its maximum value. When the modification amount is insufficient (<6μL), the MXene suspension cannot completely cover the electrode surface, resulting in a reduction in the effective reaction area, limiting the electrochemical reaction efficiency of the redox probe, and making the current response improvement effect insignificant. Excessive volume of modification material (>6μL) will lead to an increase in the thickness of the modification layer, hindering the electron transfer process, while reducing the effective contact area between the probe and the electrode surface and reducing the current response. Therefore, 6μL is determined to be the optimal modification amount. At this time, a uniform MXene modification layer can be formed on the electrode surface, achieving the best current response performance.

[0104] Optimization of pH of electropolymerization solution:

[0105] In order to obtain the optimal pH for dissolving CAP and OPD, the pH range of 4.5–7.0 was tested. When the pH was 6, the difference ΔI of the DPV current between elution and incubation was the largest, as shown in Figure 5a and Figure 5b As shown, this phenomenon can be explained by the electrochemical polymerization mechanism: under moderately acidic conditions, OPD monomers are more likely to undergo orderly electrochemical oxidative polymerization, forming a structurally regular, dense, and uniform polymer film. When the solution pH is too low (strongly acidic) or too high (strongly alkaline), the protonation state and reactivity of the monomers are altered, making the polymerization process difficult to control and unfavorable for the formation of an ideal polymer structure. A moderately acidic environment is key to achieving controllable OPD electropolymerization, and 6 was selected as the optimal pH for the solution of dissolved CAP and OPD.

[0106] Optimization of the molar ratio of template molecules to functional monomers:

[0107] During electropolymerization, the molar ratio of CAP to OPD is very important for the recognition of MIP. Figure 6a and Figure 6b As shown in the figure, the optimal molar ratio of CAP to OPD was explored by analyzing the difference in DPV currents after elution and incubation. The results showed that the ΔI reached its maximum value when the CAP:OPD ratio was 1:3, indicating that the sensor prepared at this ratio had the highest sensitivity. When the molar ratio was too low, the ΔI decreased, possibly due to insufficient CAP, resulting in a lack of CAP-specific recognition sites in the MIP. When the molar ratio was too high, the ΔI also decreased, possibly due to insufficient functional monomers, resulting in fewer CAP-MIPs formed during the electropolymerization process. Consequently, there were insufficient sites in the MIP for CAP-specific recognition. Therefore, the optimal molar ratio of the template molecule CAP to the functional monomer OPD is 1:3.

[0108] Scan rate optimization:

[0109] The electrochemical performance of the sensor is correlated with the scan rate parameter. The present invention systematically investigated the influence of the scan rate on the sensor performance in the range of 35–60 mV / s. The experimental results are as follows: Figure 7a and Figure 7b As shown in the figure, the difference in current response, ΔI, reaches its peak at a scan rate of 50 mV / s. This phenomenon can be explained from a kinetic perspective: when the scan rate is lower than 50 mV / s, the binding process between CAP and OPD is very slow, resulting in an excessively high density of the molecularly imprinted membrane, which reduces the recognition efficiency. When the scan rate is higher than 50 mV / s, the formation process of the molecularly imprinted membrane is too fast, making the molecularly imprinted membrane structure too loose and the binding between CAP and OPD unstable. Therefore, considering the formation kinetics and structural stability of the molecularly imprinted membrane, 50 mV / s is determined to be the optimal scan rate parameter.

[0110] Optimization of the number of polymerization cycles of the polymerization liquid:

[0111] The thickness of the CAP-MIPs membrane affects the sensitivity of the sensor and can be optimized by regulating the number of scan cycles during the electropolymerization process. When the CAP-MIPs membrane is too thin, it may be destroyed by elution, resulting in insufficient recognition sites on the MIPs; when the CAP-MIPs membrane is too thick, the template molecules are difficult to completely remove, and the electron transfer process will be hindered. Figure 8a and Figure 8b As shown in the figure, the effect of the number of scan cycles on the sensor sensitivity shows a trend of first increasing and then decreasing: as the number of scan cycles increases from 5 to 20, the difference in DPV current between elution and incubation, ΔI, gradually increases as more and more specific recognition sites are fixed. However, when the number of scan cycles exceeds 20, ΔI begins to decrease, which may be due to the decrease in electron transfer rate caused by excessive film thickness. Therefore, 20 cycles are determined to be the optimal number of electropolymerization cycles, at which the best film thickness and sensor performance can be obtained.

[0112] Optimization of elution cycles:

[0113] The number of elution cycles is one of the key conditions for regulating sensor performance, and indirectly determines the recognition characteristics of the sensor by affecting the removal efficiency of template molecules. Figure 9a and Figure 9b As shown in the figure, as the number of elution cycles increases from 5 to 20, the current response difference ΔI shows a significant upward trend, reaching a peak at 20 cycles. This phenomenon can be attributed to the fact that as the elution intensity increases, a large number of template molecules are effectively removed from the molecularly imprinted polymer (MIP), and a large number of imprinted cavities with specific recognition capabilities are formed on the electrode surface, significantly improving the recognition performance of the sensor. However, excessive elution may lead to the destruction of the imprinted cavity structure, so 20 cycles of elution may be the optimal condition.

[0114] Optimization of adsorption time:

[0115] Adsorption time is one of the key factors affecting sensor performance. Figure 10a and Figure 10b As shown, with increasing adsorption time, the difference in DPV current response, ΔI, between the elution and adsorption processes, initially increases and then decreases. Within 0–10 minutes, the current difference gradually increases with adsorption time, reaching a peak at 10 minutes. After adsorption time exceeds 10 minutes, the current difference begins to decrease and eventually stabilizes. As adsorption time increases, more CAP molecules block the cavities on the sensor, resulting in a decrease in current and an increase in ΔI. When adsorption time is too long, some of the MIP membrane swells or relaxes after adsorption, potentially increasing membrane permeability and facilitating electron transfer or the transport of electroactive species, leading to an increase in current and a decrease in ΔI. This phenomenon may be related to the saturation adsorption effect of molecular imprinting sites. While 20 minutes may be the optimal time point for reaching adsorption equilibrium, the maximum number of identifiable cavities is observed at 10 minutes, so 10 minutes is the optimal adsorption time.

[0116] Under optimized experimental conditions, the prepared molecularly imprinted polymer sensor was used for the detection of CAP. Figure 11a-Figure 11d As shown in the figure, the experimental data show that there is a good linear relationship between the CAP response difference ΔI and the target concentration: as the CAP concentration increases, the ΔI value shows a regular growth trend. In the two concentration ranges of 0.1–5μM and 5–300μM, there is a significant linear correlation between ΔI and CAP concentration, with a correlation coefficient R 2 They reach 0.996 and 0.998 respectively, indicating that the sensor has a wide linear detection range. The linear equation is I p =11.524C(μA)+19.983,I p =0.0658C(μA)+24.637, the calculated LOD is 0.017 μM, and the RSD is less than 10%, indicating that the method established based on MIPs / MXene / GCE has good performance for analyzing CAP.

[0117] In order to evaluate the reproducibility and stability of the sensor of the present invention, the following experimental investigation was carried out. The used electrode was eluted by CV for 20 cycles (voltage -0.4V–1.4V, scan rate 50mV / s) in 10mL of 0.5M sulfuric acid / ethanol (4 / 1, V / V) solution to wash off the attached template molecules and reuse them. The recovered modified electrode was adsorbed in 1mM CAP standard solution for 10min and then detected by DPV in potassium ferricyanide solution. The reusability of the MIPs / MXene / GCE was evaluated by systematically examining the current response of the electrode. The experimental results are shown in Figure 2. Figure 12a and Figure 12b As shown in the results, the molecularly imprinted electrochemical sensor can still maintain more than 95% of the initial current response value after five consecutive reuses, with a relative standard deviation of only 3.86%. This result fully confirms that the prepared modified electrode has good operational stability and reusability, providing reliable guarantees for its long-term application in actual sample analysis.

[0118] In order to evaluate the anti-interference ability of MIPs / MXene / GCE and its specific recognition of CAP, the selectivity of the electrode was studied. Florfenicol (FFC), furazolidone (FZD), and sulfadiazine (SD) were used as interfering antibiotics, and PBS buffer solution (pH 6.0) was selected as the solvent. A mixed standard solution of CAP and interfering substances at 5 μM was prepared. After adsorption in the mixed solution for 10 minutes, DPV detection was performed in potassium ferricyanide. The results are shown in Figure 2. Figure 13 The MIPs / MXene / GCE exhibited highly consistent current response characteristics (I CAP =11.45μA, I CAP-FFC =10.64μA, I CAP-FZD =11.19μA, I CAP-SD =12μA, I All =10.11 μA, with a relative standard deviation within 8.78%), the addition of competitive substances did not significantly affect the electrode's specific recognition ability for CAP, and the current response signal remained stable, fully demonstrating that the modified electrode has excellent selective recognition performance for the target molecule CAP.

[0119] Testing of actual samples:

[0120] A 1 mM CAP standard solution was diluted to 200 μM with PBS (pH 6). After filtering aquaculture seawater through a 0.22 μm filter, 0.25 mL of the 200 μM CAP solution was added to 9.75 mL of the water sample. The concentration of the test solution was adjusted to 5 μM. Analysis was performed using DPV, and the CAP concentration in the test solution was calculated using a linear regression equation. As shown in Table 2, the experimental results showed that the spiked recoveries ranged from 90.4% to 105.2%, with relative standard deviations (RSDs) below 10%. This demonstrates that the molecularly imprinted electrochemical sensor has good accuracy for the quantitative detection of CAP in actual aquaculture seawater samples and can meet the requirements for the detection of trace amounts of CAP in complex matrices.

[0121] Table 2 Analysis and determination of CAP in actual aquaculture seawater

[0122]

[0123] The present invention is not limited to the structures described above and shown in the drawings, and various modifications and changes can be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor, characterized in that: The following steps are involved: Step S1, pre-treating the surface of the GCE electrode; Step S2, modifying the MXene suspension on the surface of the pretreated GCE electrode to obtain a MXene / GCE electrode; Step S3, immersing the MXene / GCE electrode in PBS buffer containing CAP and OPD; Step S4, electropolymerizing OPD on the surface of the MXene / GCE electrode using CV to obtain a CAP-MIPs / MXene / GCE electrode containing a molecularly imprinted polymer of CAP; In step S5, the prepared CAP–MIPs / MXene / GCE electrode is eluted to prepare a molecularly imprinted electrochemical sensor containing the MIPs / MXene / GCE electrode.

2. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1, wherein: The step of pre-treating the surface of the GCE electrode comprises: Step S11, taking 0.5 μm Al2O3 powder, adding deionized water to make a paste, and evenly applying it on the surface of the suede; Step S12, holding the GCE electrode vertically, and lightly pressing and polishing it in an "∞"-shaped trajectory; Step S13, cleaning the GCE electrode and suede, replacing 0.3 μm Al2O3 powder, and repeating the above steps until the GCE electrode has a matte finish; Step S14: Apply 0.05 μm Al2O3 slurry and gently polish until the surface of the GCE electrode presents a mirror effect; Step S15, rinsing the surface of the GCE electrode with ultrapure water, and then ultrasonically cleaning it in anhydrous ethanol and ultrapure water respectively; In step S16, after the GCE electrode is dried with nitrogen, cyclic voltammetry is used to scan in a 5 mM potassium ferrocyanide solution, and the difference in redox peak potential is always maintained within the range of 0.1 V.

3. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1, wherein: The step of modifying the MXene suspension on the surface of the GCE electrode comprises: 6 μL of MXene suspension was modified on the surface of the GCE electrode and naturally dried at room temperature to obtain a MXene / GCE electrode.

4. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1 or 3, wherein: The preparation steps of the MXene suspension include: First, 0.2 g of chitosan was added to 50 mL of 1% acetic acid solution and mixed by magnetic stirring to obtain a chitosan solution; Then, 20 mL of chitosan solution was added to 100 mg of MXene and ultrasonically treated for 60 min to obtain a uniform MXene suspension, which was then stored in a refrigerator at 4 °C.

5. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1, wherein: The preparation steps of the PBS buffer solution include: First, 7.164 g of Na2HPO4·12H2O and 3.121 g of NaH2PO4·2H2O were dissolved in 100 mL of deionized water and ultrasonicated for 15 min to obtain homogeneous Na2HPO4 solution and NaH2PO4 solution respectively. Then, 12.3 mL of 0.2 M Na2HPO4 solution and 87.7 mL of 0.2 M NaH2PO4 solution were mixed and ultrasonicated for 15 min to obtain a uniform 0.2 M Na2HPO4 / NaH2PO4 buffer solution.

6. The method for preparing a molecularly imprinted electrochemical sensor according to claim 5, wherein: The pH value of the Na2HPO4 / NaH2PO4 buffer solution is 6.

0.

7. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1, wherein: The prepared CAP-MIPs / MXene / GCE electrode is subjected to an elution step, and cyclic voltammetry is used for elution. The template molecule CAP is washed away 20 times in the eluent to prepare a molecularly imprinted electrochemical sensor containing a MIPs / MXene / GCE electrode.

8. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1 or 5, wherein: The preparation steps of the PBS buffer solution containing CAP and OPD include: Take 10 mL of PBS buffer solution, add 6.5 mg of OPD and 6.5 mg of CAP, disperse it by ultrasonic for 30 min, and then slowly inject nitrogen for 5 min.

9. A molecularly imprinted electrochemical sensor prepared by the method according to any one of claims 1 to 8.

10. A CAP detection method, characterized in that: Detection based on the molecular imprinting electrochemical sensor according to claim 8 comprises the following steps: A three-electrode system was constructed, with the working electrode being the MIPs / MXene / GCE electrode, the counter electrode being the platinum wire electrode, and the reference electrode being the Ag / AgCl electrode; Quantitative analysis of the CAP electrochemical properties of the MIPs / MXene / GCE electrode by DPV, including: The DPV current value I0 of the MIPs / MXene / GCE electrode was recorded in the electrolyte; The MIPs / MXene / GCE electrode was immersed in CAP standard solutions of different concentrations and incubated for 10 minutes. DPV detection was then performed and the current value I was recorded. t ; Take I0–I t The peak current difference ΔI was used as the standard, a standard curve was drawn and a linear regression equation was established.

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