An anti-fouling molecularly imprinted electrochemical sensor and a preparation method and application thereof

Abstract

The application belongs to the technical field of electrochemical sensors, and discloses an anti-fouling molecular imprinting electrochemical sensor, a preparation method and application thereof. The sensor comprises a conductive substrate and a cobalt-diferrocene metal organic framework calcined material layer, a gold nanoparticle layer, an amphoteric ion polypeptide layer and a molecular imprinting polymer layer which are sequentially modified on the surface of the conductive substrate. Through synergistic enhancement of the multi-level composite structure, high sensitivity, high specificity and anti-fouling detection of mycotoxins are realized, and the sensor is suitable for rapid screening of trace mycotoxins in complex matrices.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, and particularly relates to an antifouling molecularly imprinted electrochemical sensor, its preparation method, and its application. Background Technology

[0002] my country is a major producer and consumer of oilseeds. Oilseed crops such as corn, peanuts, and grains are highly susceptible to fungal infection during planting, harvesting, storage, transportation, and processing, leading to the production of aflatoxin B1 (AFB1). AFB1 is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), possessing high toxicity and significant harmfulness, seriously threatening food safety and human health. Therefore, establishing a rapid, sensitive, and low-cost detection technology for AFB1 in oilseeds is of significant practical importance.

[0003] Currently, conventional detection methods for AFB1 mainly rely on large-scale, sophisticated instruments such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and nuclear magnetic resonance spectroscopy (NMR). Although these methods offer high accuracy, they generally suffer from drawbacks such as expensive instruments, high operating costs, complex sample pretreatment, high requirements for operator expertise, and long detection cycles, making them unsuitable for rapid, large-scale on-site screening.

[0004] Electrochemical sensors have gained widespread attention in the field of mycotoxin detection in food and oilseeds due to their advantages such as high sensitivity, low cost, portable equipment, easy miniaturization, and fast detection speed. Molecularly imprinted polymers (MIPs) have the advantages of high specificity, good stability, and low cost, and are often used as recognition elements of sensors. Metal-organic frameworks (MOFs) have the characteristics of large specific surface area and tunable pore structure. After high-temperature calcination, the porous structure can be further optimized to obtain an even larger specific surface area and more electrochemical active sites. Gold nanoparticles have excellent conductivity, which can amplify electrochemical signals, and can also fix peptide molecules through gold-sulfur bonds (Au-S). Zwitterionic peptides can form a dense hydration layer on the electrode surface, effectively inhibiting the non-specific adsorption of proteins and impurities, and giving the sensing interface excellent anti-fouling ability.

[0005] However, existing conventional electrochemical sensors still have significant drawbacks: on the one hand, most existing sensors only use a single material for modification and lack a hierarchical composite structure. The sensing interface does not have a dedicated anti-fouling design, and proteins are easily adsorbed and scaled in complex oil matrices, leading to decreased sensitivity and poorer specificity. On the other hand, existing technologies do not simultaneously achieve high sensitivity, high specificity, and anti-fouling performance, and have not systematically optimized the preparation and detection processes, making it difficult to meet the detection needs of trace fungal toxins in actual oil samples.

[0006] For example, existing conventional electrochemical sensors typically use bare glassy carbon electrodes or electrodes modified with single carbon materials and gold nanoparticles as substrates, combined with molecularly imprinted polymers (MIPs) to construct a detection platform. They rely solely on the MIP's own structure to identify target analytes, without a specially designed antifouling modification layer. In complex oil environments, these sensors are prone to forming biofouling layers due to the non-specific adsorption of impurities such as proteins and lipids, leading to decreased sensitivity and deteriorated stability. Furthermore, the limited active sites and low electron transport efficiency of single-material modifications result in high detection limits for trace AFB1.

[0007] Other sensing studies have introduced zwitterionic peptides as antifouling layers or utilized the porous structure of MOF materials to enhance the enrichment of target analytes. However, these approaches only modify a single function, lack synergistic matching between materials, and result in simple composite structures with unreasonable hierarchical design. Such approaches cannot simultaneously achieve high sensitivity, high specificity, and antifouling properties, and they fail to optimize key process parameters such as material concentration, electrodeposition time, polymerization solution pH, and adsorption-elution time for the AFB1 detection system. Consequently, the overall detection performance does not meet the requirements for trace detection.

[0008] In summary, existing electrochemical sensors still have shortcomings in terms of antifouling ability, sensitivity, specificity, and process systematization. There is an urgent need to develop an electrochemical sensor that can simultaneously meet the triple requirements of high sensitivity, high specificity, and antifouling, and has a simple preparation process that is suitable for the detection of trace AFB1 in complex oil matrices. Summary of the Invention

[0009] To achieve the above objectives, the first technical solution of this application discloses an antifouling molecularly imprinted electrochemical sensor, comprising a conductive substrate, and a cobalt-ferrocene metal-organic framework calcined material layer, a gold nanoparticle layer, a zwitterionic peptide layer and a molecularly imprinted polymer layer sequentially modified on the surface of the conductive substrate.

[0010] The amino acid sequence of the zwitterionic polypeptide contains positively charged residues and negatively charged residues, and the C-terminus is modified by amidation.

[0011] The molecularly imprinted polymer layer contains a molecularly imprinted cavity that matches the target template molecule and an acrylamide functional monomer.

[0012] Furthermore, the conductive substrate is a glassy carbon electrode, carbon nanotube paper, or flexible carbon composite paper.

[0013] Furthermore, the calcined material of the cobalt-ferrocene metal-organic framework calcined material layer is obtained by calcining the cobalt-ferrocene metal-organic framework in an inert gas atmosphere at 300-500°C.

[0014] Furthermore, the amino acid sequence of the zwitterionic polypeptide is NH2-CPPPPEKDQDK.

[0015] Furthermore, the target template molecule of the molecularly imprinted polymer layer is a fungal toxin.

[0016] Preferably, the fungal toxin is aflatoxin B1.

[0017] Preferably, the target template molecule may also include small food molecule contaminants.

[0018] The second technical solution of this application discloses a method for fabricating a sensor as described in the first technical solution, comprising:

[0019] A dispersion of cobalt-ferrocene metal-organic framework calcined material was modified onto the surface of a conductive substrate.

[0020] Gold nanoparticles were electrochemically deposited on the modified electrode surface;

[0021] The electrode after electrodeposition was placed in a zwitterionic peptide solution and incubated, so that the peptide self-assembled onto the surface of gold nanoparticles through gold-sulfur bonds.

[0022] Using the incubated electrode as the working electrode, electrochemical polymerization is carried out in a polymerization solution containing target template molecules and functional monomers to form a molecularly imprinted polymer film.

[0023] The template molecules are removed by elution to obtain the sensor.

[0024] Furthermore, the electrochemical deposition is carried out in an acidic solution containing chloroauric acid, with a deposition voltage of -0.1 to -0.3 V and a deposition time of 100-300 s.

[0025] Furthermore, the electrochemical polymerization is carried out using cyclic voltammetry, with 3-7 polymerization cycles and a polymerization solution pH of 5.8-8.2.

[0026] The third technical solution of this application discloses the application of the sensor described in the first technical solution in detecting analytes corresponding to target template molecules.

[0027] Preferably, the analytes include mycotoxins and minor food contaminants.

[0028] Furthermore, the target template molecule is aflatoxin B1.

[0029] Beneficial effects

[0030] 1. This invention employs a four-level composite hierarchical structure consisting of a metal-organic framework calcined material layer, a gold nanoparticle layer, a zwitterionic peptide layer, and a molecularly imprinted polymer layer. Through the synergistic effect of multiple components, it simultaneously achieves high sensitivity response, enrichment of multiple active sites, construction of antifouling interfaces, and high specificity molecular recognition in a single sensor.

[0031] 2. By forming a dense hydrated layer on the electrode surface through zwitterionic peptides, the non-specific adsorption of biological proteins such as bovine serum albumin, lysozyme, and myoglobin, as well as oil impurities, is effectively suppressed, significantly reducing the signal suppression rate and making the sensor suitable for direct detection of complex oil matrices.

[0032] 3. After high-temperature calcination, the cobalt-ferrocene metal-organic framework has a larger specific surface area and more electrochemical active sites. The gold nanoparticles further improve the electron transmission efficiency and signal amplification capability, making the sensor highly sensitive, with a wide linear range and a low detection limit for aflatoxin B1.

[0033] 4. The sensor has a simple manufacturing process, mild conditions, and low cost. It has good stability and repeatability, is easy to operate and does not require large precision instruments. It is suitable for rapid on-site screening of food safety in grains and oils, and also meets the needs of precise laboratory testing.

[0034] 5. The composite modification system of the present invention can be extended to the antifouling electrochemical detection of other mycotoxins and small molecule contaminants in food by changing the template molecule, with wide applicability and strong scalability. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The concentration is 0.2 mg / mL. -1 CD diagram of zwitterionic peptides in phosphate-buffered saline (PBS) solution at pH 7.4, where red represents NH2-CPPPPEKDQDK and black represents CKDQDK;

[0037] Figure 2 The results are as follows: (A) is the SEM image of Co-Fc MOF; (B) is the SEM image of Co-Fc MOF-C; and (C) is the EDS image of Co-Fc MOF-C.

[0038] Figure 3 The results show the structural and compositional characterization results; where (A) is the FT-IR plot; (B) is the XRD plot; (C) is the BET plot of Co-Fc MOF; and (D) is the BET plot of Co-Fc MOF-C.

[0039] Figure 4The results show the changes in current response; where (AB) represents the LSV response curves of the MIP sensor after incubation in AFB1 solutions of different concentrations and the linear fitting results of the corresponding current values ​​with the logarithm of concentration (lgC); and (CD) represents the LSV response curves of the control NIP electrode after incubation in AFB1 solutions of the same concentration gradient and the linear fitting results of the corresponding current values ​​with the logarithm of concentration (lgC).

[0040] Figure 5 The results show the electrochemical characterization and key process optimization of the electrode modification process, where (A) is the optimized result of Co-Fc MOF-C concentration; (B) is the optimized result of Au NPs electrodeposition time; (C) is the optimized result of Z-peptide concentration; and (D) is the optimized result of Z-peptide concentration (characterized by signal suppression rate).

[0041] Figure 6 The results of the optimization of key process conditions for sensor fabrication are as follows: (A) Optimization results of AFB1 and AM complexation time; (B) Optimization results of electrochemical polymerization cycle number; (C) Optimization results of scan rate; (D) Optimization results of polymerization solution pH; (E) Optimization results of elution time; (F) Optimization results of absorption time.

[0042] Figure 7 The water contact angle test results for electrodes at different modification stages;

[0043] Figure 8 LSV response curves of different modified electrodes in solutions of different concentrations of interfering proteins (BSA, LYS, Mb); where (AC)Au NPs / Co-Fc MOF-C / GCE, (DF)C-peptide / Au NPs / Co-Fc MOF-C / GCE, and (GI)Z-peptide / Au NPs / Co-Fc MOF-C / GCE are shown.

[0044] Figure 9 The signal inhibition rates of the two electrodes in different concentrations of interfering protein solutions are shown; where (A) is the Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrode, and (B) is the C-peptide / Au NPs / Co-Fc MOF-C / GCE electrode.

[0045] Figure 10 Comparison of current response and imprinting factor of MIP and NIP electrodes to different fungal toxins (AFB2, AFG1, OTA, OTB, FB1, AFB1);

[0046] Figure 11The change in current response ΔI of 7 electrodes prepared in the same batch (pink column) and different batches (blue column) to the target material;

[0047] Figure 12 The sensor's current response change value ΔI to the target object was stored at 4 ℃ for 0–5 days.

[0048] Figure 13 A bar chart comparing the signal suppression rates of two electrodes, Au NPs / Co-Fc MOF-C / GCE and Z-peptide / Au NPs / Co-Fc MOF-C / GCE, at different incubation times;

[0049] Figure 14 A bar chart comparing the signal suppression rates of the three electrodes in peanut sample solutions of different concentrations. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other. Parameters, conditions, or materials indicated by "exemplarily" or "preferredly" in the embodiments of this application are only used to illustrate preferred implementations of the technical solutions of this application and do not constitute a limitation on the scope of protection of the claims.

[0051] I. Overall Structure and Technical Principle of the Sensor

[0052] This application provides an antifouling molecularly imprinted electrochemical sensor, the overall structure of which consists of a four-level composite hierarchical structure formed by sequentially modifying a conductive substrate surface with a cobalt-ferrocene metal-organic framework calcined material (Co-Fc MOF-C) layer, a gold nanoparticle (Au NPs) layer, a zwitterionic peptide (Z-peptide) layer, and a molecularly imprinted polymer (MIP) layer. This structure, through the synergistic matching between the functional layers, simultaneously achieves highly sensitive electrochemical response, antifouling properties in complex matrices, and highly specific molecular recognition within a single sensing interface.

[0053] 1. Conductive substrate

[0054] The conductive substrate can be a glassy carbon electrode (GCE), carbon nanotube paper, or flexible carbon composite paper. Exemplarily, this embodiment uses a glassy carbon electrode as the substrate, but those skilled in the art should understand that conductive flexible substrates such as carbon nanotube paper or flexible carbon composite paper are also applicable and can be used to construct portable, flexible, and antifouling electrochemical sensors.

[0055] 2. Cobalt-ferrocene metal-organic framework calcined material layer (Co-Fc MOF-C)

[0056] The Co-Fc MOF-C layer is formed by high-temperature calcination of a cobalt-ferrocene metal-organic framework (Co-Fc MOF). The Co-Fc MOF can be synthesized using a hydrothermal method. The synthesized Co-Fc MOF powder is placed in an inert gas atmosphere (such as nitrogen or argon) and calcined at 300-500°C, causing the organic ligands to carbonize and decompose, and the metal components to be uniformly dispersed in the carbon matrix, transforming it into a porous carbon-metal composite active material. For example, the calcination temperature can be 400°C, and the calcination time can be 2 hours.

[0057] After calcination, the ordered MOF transforms into a porous carbon composite material, significantly increasing its specific surface area and pore volume, forming abundant microporous and mesoporous structures. Simultaneously, the bimetallic compounds Co and Fe are uniformly dispersed within the carbon matrix, providing ample active sites for electrochemical sensing and improving the material's conductivity and electron transport properties. Optionally, the metal-organic framework can also be selected from other transition metal MOF materials such as Ni-Fe and Zn-Co, which, after high-temperature calcination, serve as conductive modification materials to achieve equivalent sensing effects.

[0058] 3. Gold nanoparticle layer (Au NPs)

[0059] The Au NPs layer is formed on the surface of the Co-Fc MOF-C layer by electrochemical deposition. Specifically, the Co-Fc MOF-C modified electrode is placed in an acidic solution containing chloroauric acid (e.g., 0.25 mM chloroauric acid / 0.1 M sulfuric acid) and electrochemically deposited for 100-300 s at a constant voltage of -0.1 to -0.3 V. For example, the deposition voltage can be -0.2 V and the deposition time can be 200 s.

[0060] Au NPs form a uniform conductive network on the electrode surface, which significantly improves the interfacial electron transfer efficiency and amplifies the electrochemical signal. At the same time, the surface of Au NPs can provide stable chemical binding sites for the subsequent fixation of peptide molecules through gold-sulfur bonds (Au-S).

[0061] 4. Z-peptide layer

[0062] The amino acid sequence of the zwitterionic peptide includes positively charged residues (such as lysine K) and negatively charged residues (such as glutamic acid E and aspartic acid D), and the C-terminus is modified with amidation to achieve zwitterionic charge balance. For example, the amino acid sequence of the zwitterionic peptide is NH2-CPPPPEKDQDK, with C-terminal amidation.

[0063] The electrode after electrodeposition of Au NPs was placed in the peptide solution (e.g., 0.01 M PBS at pH 7.4, concentration 0.05-0.50 mg / mL). -1In this process, the peptide molecules are incubated at a low temperature (e.g., 4°C) for at least 2 hours, allowing them to spontaneously and orderly self-assemble on the surface of gold nanoparticles via gold-sulfur bonds (Au-S). For example, the peptide concentration can be 0.20 mg / mL. -1 .

[0064] The peptide itself possesses strong hydrophilicity and electroneutrality, enabling it to form a dense hydrated layer on the electrode surface, constructing a biofouling interface that effectively inhibits the non-specific adsorption of proteins, lipids, and oil impurities. Optionally, other zwitterionic antifouling peptides with gold-sulfur bond self-assembly capabilities and hydrophilic antifouling properties can also be used to replace this sequence.

[0065] 5. Molecularly Imprinted Polymer Layer (MIP)

[0066] The molecularly imprinted polymer layer uses acrylamide (AM) as the functional monomer and is formed in situ on the surface of the polypeptide layer through electrochemical polymerization. The polymerization solution contains a target template molecule, which can be a fungal toxin, including but not limited to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), ochratoxin A (OTA), ochratoxin B (OTB), fumonisin B1 (FB1), etc. Furthermore, those skilled in the art should understand that by changing the template molecule, this composite modification system can also be extended to the antifouling electrochemical detection of small molecule contaminants in food (such as pesticide residues, veterinary drug residues, etc.).

[0067] During electrochemical polymerization, the incubated electrode serves as the working electrode. Polymerization is carried out using cyclic voltammetry (CV) in a polymerization solution containing the target template molecule and functional monomer. The number of polymerization cycles can be 3-7, and the pH of the polymerization solution can be 5.8-8.2. For example, the number of polymerization cycles can be 5, and the pH of the polymerization solution can be 6.6. After polymerization, a suitable elution solvent is used for continuous immersion and elution to completely remove the template molecules embedded in the polymer membrane, leaving a specific recognition cavity inside the MIP membrane that perfectly matches the spatial structure, size, and action site of the target template molecule. After elution, the membrane is washed with PBS buffer until the electrochemical signal stabilizes, thus obtaining an antifouling molecularly imprinted electrochemical sensor with high sensitivity, high specificity, and antifouling capabilities.

[0068] 6. Overall technical effect

[0069] The aforementioned four-tiered composite structure achieves the following technical effects through the synergistic effect of its multiple components: the Co-Fc MOF-C layer provides a large specific surface area and abundant electrochemical active sites; the Au NPs layer enhances conductivity and signal amplification; the Z-peptide layer constructs a dense, hydrated, and antifouling interface, suppressing interference from complex matrices; and the MIP layer provides a specific molecular recognition cavity. This synergistic matching of the four layers enables the sensor to simultaneously achieve high sensitivity, high specificity, and excellent antifouling performance. Furthermore, the fabrication process is simple, the conditions are mild, and the cost is low, making it suitable for the rapid detection of trace fungal toxins in practical, complex samples (such as oil samples).

[0070] The complete technical means and screening process of this application will be described in detail below.

[0071] This invention provides an antifouling molecularly imprinted electrochemical sensor based on calcined Co-Fc MOF material, gold nanoparticles, and zwitterionic peptide composite modification. The overall structure of the sensor is: MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE.

[0072] Example 1: Design and Screening of Zwitterionic Peptides

[0073] A novel antifouling zwitterionic peptide (Z-peptide, sequence: NH2-CPPPPEKDQDK, C'-N) was designed and synthesized. , ) and control bioactive peptide (C-peptide, sequence: CKDQDK, C'-N) , To achieve zwitterionic charge balance, the C-terminus of the Z-peptide was amidated. Both peptides were custom-synthesized by Hefei Bank Peptide Biotechnology Co., Ltd., and their purity was verified by a combination of high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS), showing that the purity of both was greater than 95%.

[0074] To determine the secondary structure information of the designed peptide, circular dichroism (CD) was used to characterize the zwitterionic peptide (Z-peptide, sequence: NH2-CPPPPEKDQDK) and the control peptide (C-peptide, sequence: CKDQDK). Both peptides were prepared to a concentration of 0.2 mg / mL using 0.02 M PBS solution (pH=7.4). -1 CD spectra were collected for the solution in the wavelength range of 190–260 nm.

[0075] The results are as follows Figure 1As shown, Z-peptide exhibits a significant strong positive absorption band near 190–200 nm, and a relatively weak positive absorption band in the 220–230 nm region, indicating that this zwitterionic peptide has formed a specific ordered secondary structure. This ordered secondary structure is beneficial for achieving higher packing density on the electrode surface, providing a structural basis for constructing a stable and dense antifouling interface. In contrast, C-peptide only exhibits a single negative absorption signal near 190–195 nm, and no obvious positive absorption feature near 220–230 nm, indicating that its secondary structure is more inclined towards a disordered conformation and lacks a supporting rigid structure.

[0076] In summary, this study screened and selected Z-peptide for the subsequent construction of electrochemical antifouling interfaces and sensing platforms.

[0077] Example 2 Preparation and characterization of Co-Fc MOF and Co-Fc MOF-C

[0078] Cobalt-ferrocene metal-organic frameworks (Co-Fc MOFs) were synthesized by a hydrothermal method. The prepared Co-Fc MOF powder was placed in a small porcelain boat and calcined at 400 °C for 2 hours in a tube furnace under a nitrogen atmosphere. After calcination, the material was naturally cooled to room temperature to obtain Co-Fc MOF-C material.

[0079] High-temperature calcination can transform ordered MOFs into porous carbon composite materials, increasing the specific surface area, adding electrochemical active sites, and improving the material's electrical conductivity and electron transport properties, thus providing a highly active substrate material for electrode modification.

[0080] (1) Material characterization

[0081] The morphology, elemental composition, crystal structure, and pore structure of the material were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption-desorption isotherm (BET), and energy dispersive spectroscopy (EDS). Figure 2 , 3 As shown.

[0082] (a) Morphological and elemental distribution characterization (SEM and EDS Mapping)

[0083] Figure 2 (A) is a SEM image of Co-Fc MOF. The material has a regular morphology and uniform size microsphere structure, which is assembled from nanoparticles. It has good dispersibility, no obvious agglomeration, and has a typical MOF framework structure.

[0084] Figure 2(B) is Co-Fc MOF-C after calcination. The original microsphere skeleton is maintained as a whole, and the surface becomes rough and loose, forming a porous carbon-based structure. High-temperature calcination causes the organic ligands to carbonize and decompose, and the metal components are uniformly dispersed in the carbon matrix to construct a carbon-metal composite active structure.

[0085] Figure 2 (C) shows the EDS elemental distribution of Co-Fc MOF-C. C, N, O, Fe, and Co elements are uniformly distributed in the material without segregation or agglomeration. The N element signal is weak, indicating that the nitrogen-containing organic ligands have basically decomposed and escaped at high temperature. The bimetallic Co and Fe are uniformly dispersed, providing sufficient active sites for electrochemical sensing.

[0086] (b) Structural and compositional characterization (FT-IR and XRD)

[0087] Figure 3 (A) FT-IR shows that the Co-Fc MOF is at 1400-1600 cm⁻¹ -1 The presence of characteristic absorption peaks for organic ligands indicates complete coordination between the metal and ligands and a stable MOF framework; after calcination, the organic characteristic peaks largely disappear, with only the 3400 cm⁻¹ peak remaining. -1 The broad hydroxyl peaks and low wavenumber metal-oxygen bond vibration peaks in the vicinity indicate that the organic matter is completely carbonized and transformed into a carbon-based metal oxide composite structure.

[0088] Figure 3 (B) XRD results show that the Co-Fc MOF precursor has sharp diffraction peaks and good crystallinity; after calcination, the characteristic peaks of MOF disappear, and diffraction peaks of Co and Fe-based phases appear in the 2θ range of 30°-50°. In the 2θ = 20°-30° range, the curve baseline is flat, indicating that the generated carbon material has a very low degree of graphitization, proving that after calcination, it transforms into a composite system of metal active components and amorphous carbon.

[0089] (c) Pore structure characterization (BET)

[0090] Figure 3 (C) and (D) are nitrogen adsorption-desorption curves for Co-Fc MOF and Co-Fc MOF-C, respectively. Both are type IV isotherms with hysteresis loops, indicating that both materials possess mesoporous structures. The specific surface area and pore volume of calcined Co-Fc MOF-C are significantly increased. High-temperature decomposition of organic ligands constructs abundant micropores and mesopores, effectively increasing active sites, accelerating electron mass transfer, and improving electrochemical sensing sensitivity and response performance.

[0091] Example 3: Construction of a Stepwise Modified Electrode (Preparation of an Antifouling Molecularly Imprinted Electrochemical Sensor)

[0092] (1)Pretreatment of glassy carbon electrode (GCE) (This sensor uses a glassy carbon electrode as the conductive substrate).

[0093] Select a bare glassy carbon electrode and polish it repeatedly in a circular motion on alumina powder with a particle size of 0.02 - 0.05 μm until the electrode surface appears mirror - bright, without scratches and visible impurities to the naked eye. Then, place it in anhydrous ethanol and ultrapure water respectively and ultrasonic clean for 2 min to remove the residual polishing powder and organic impurities on the surface, and air - dry it naturally at room temperature for standby. Using a 5 mM potassium ferricyanide / ferrocyanide buffer solution containing 0.2 M potassium chloride as the test system, electrochemical characterization is carried out by cyclic voltammetry (CV). Set the potential range to - 0.4 - 0.8 V and the scanning rate to 0.05 V / s. When the potential difference between the oxidation - reduction peak potentials is less than 80 mV, it is determined that the electrode pretreatment is qualified, and it is sealed and left standing for standby.

[0094] (2)Co - Fc MOF - C modified electrode:

[0095] Accurately weigh a certain amount of Co - Fc MOF - C powder, add 0.2% chitosan acetate to prepare a uniformly dispersed solution with a concentration of 0.10 mg mL -1 . Take an appropriate amount and evenly drop - coat it on the surface of the pretreated glassy carbon electrode, and air - dry it naturally at room temperature to make the material firmly adhere to the electrode surface, obtaining Co - Fc MOF - C / GCE.

[0096] (3)Electrodeposition modification of gold nanoparticles (Au NPs):

[0097] Place Co - Fc MOF - C / GCE in a 0.25 mM chloroauric acid / 0.1 M H2SO4 deposition solution system for electrochemical deposition. The voltage is - 0.2 V and the electrodeposition time is 200 s to obtain Au NPs / Co - Fc MOF - C / GCE. Gold nanoparticles improve the conductivity of the electrode and amplify the electrochemical signal, and at the same time provide a fixation site for subsequent polypeptides through gold - sulfur bonds (Au - S).

[0098] (4)Modification of zwitterionic peptide Z - peptide anti - fouling layer:

[0099] Prepare a Z - peptide zwitterionic polypeptide solution using 0.01 M phosphate - buffered saline (PBS) with pH = 7.4 as the solvent, and prepare a Z - peptide zwitterionic peptide solution with a concentration of 0.2 mg mL -1 .

[0100] Take an appropriate amount of the prepared Z-peptide solution and place it on Au NPs / Co-Fc MOF-C / GCE. Soak and incubate at 4°C for more than 2 hours to allow the peptide molecules to spontaneously and orderly self-assemble on the surface of gold nanoparticles through gold-sulfur bonds (Au-S). After incubation, remove the electrode, slowly rinse off any excess peptides that have not been physically adsorbed with PBS buffer, and air dry at room temperature.

[0101] Peptides possess strong hydrophilicity and electroneutrality, forming a dense hydration layer on the electrode surface and constructing a biofouling-resistant interface that effectively inhibits the non-specific adsorption of proteins and impurity molecules. Thus, a Z-peptide / Au NPs / Co-Fc MOF-C / GCE composite modified electrode is prepared.

[0102] (5) Preparation of molecularly imprinted polymers (MIPs) and template elution:

[0103] (a) Preparation of polymerization solution: AFB1 was selected as the template molecule and acrylamide (AM) as the functional monomer, and the polymerization solution was prepared by dissolving them in methanol after pH adjustment.

[0104] (b) Electrochemical polymerization: Z-peptide / Au NPs / Co-Fc MOF-C / GCE was used as the working electrode and placed in the prepared MIP polymerization solution. Electrochemical polymerization was carried out by cyclic voltammetry to form a uniform and dense molecularly imprinted polymer film in situ on the electrode surface.

[0105] (c) Template elution: After polymerization, the elution electrode is continuously immersed in a suitable elution solvent to completely remove the AFB1 template molecules embedded in the polymer membrane, leaving a specific recognition cavity inside the MIP membrane that perfectly matches the spatial structure, size, and action site of the AFB1 molecule; after elution, the electrode is rinsed with PBS until the electrochemical signal is stable, and then air-dried at room temperature to finally obtain a highly sensitive, highly specific, and antifouling MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE antifouling electrochemical sensor, which can be used for sensitive electrochemical detection of AFB1 in oil.

[0106] Example 4: Establishment of a detection method for the AFB1 electrochemical antifouling sensor

[0107] (1) Electrochemical detection conditions

[0108] Linear sweep voltammetry (LSV) detection conditions: LSV detection was performed in 5 mM potassium ferricyanide and potassium ferrocyanide phosphate buffer solution (PBS, pH 7.4, 0.01 M) containing 0.2 M potassium chloride. The detection potential range was 0-1 V, the amplitude was 50 mV, and the scan rate was 0.1 V / s.

[0109] Cyclic voltammetry (CV) detection conditions: CV detection was performed in 5 mM potassium ferricyanide and potassium ferrocyanide phosphate buffer solution (PBS, pH 7.4, 0.01 M) containing 0.2 M potassium chloride. The detection potential range was -0.4 to 0.8 V, and the scan rate was 0.05 V / s.

[0110] (2) AFB1 standard test method

[0111] The prepared MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrochemical antifouling sensor and the control NIP (non-imprinted polymer) electrode were placed in a series of AFB1 solutions with concentration gradients (concentration range of 10). -5 -10 3 ngmL -1 The sample was incubated at room temperature for 10 min, followed by washing with pH 7.4 PBS buffer to remove non-specifically adsorbed AFB1. The current response changes before and after incubation were recorded using linear sweep voltammetry (LSV), and the results are shown below. Figure 4 As shown.

[0112] As the concentration of AFB1 increases, the specific recognition cavity on the surface of the MIP sensor is occupied by the target, hindering electron transfer, and the peak value of the LSV current gradually decreases; while after the NIP electrode is incubated with different concentrations of AFB1, the current signal does not show obvious regular changes, proving that the sensor's response originates from the specific recognition of the MIP cavity, rather than non-specific adsorption.

[0113] A linear regression equation was obtained by fitting the current response of the MIP sensor to the logarithm of AFB1 concentration (LgC): y = -1.78 lgC (ng mL) -1 ) + 91.55, correlation coefficient R 2 =0.9978. Where y is the LSV current value measured after incubation, and C is the concentration of the AFB1 solution (ng / mL). -1 The limit of detection (LOD) for AFB1 by this sensor was measured to be 3.2 fg / mL. -1 (S / N=3).

[0114] Example 5: Optimization of Process Parameters

[0115] This invention optimizes key parameters throughout the entire process of sensor modification, preparation, and detection using single-factor optimization to determine the optimal process conditions.

[0116] (1) Electrochemical characterization and key process optimization of electrode modification process

[0117] This invention employs cyclic voltammetry (CV) to electrochemically characterize the stepwise modification process of the glassy carbon electrode, and optimizes key process parameters such as Co-Fc MOF-C concentration, Au NPs electrodeposition time, and Z-peptide concentration. The results are as follows: Figure 5 As shown:

[0118] (a) Optimization of Co-Fc MOF-C modification concentration

[0119] Using bare glassy carbon electrode (bare GCE) as a substrate, different concentrations (0.05, 0.10, 0.15, 0.20, 0.25 mg / mL) were drop-coated. -1 Co-Fc MOF-C aqueous dispersion was used to prepare Co-Fc MOF-C / GCE. The results are as follows: Figure 5 As shown in Figure A, compared with bare GCE, the redox peak current of the electrode after modification with Co-Fc MOF-C significantly increased, indicating that the material effectively promoted electron transfer; when the concentration of Co-Fc MOF-C was 0.10 mg / mL -1 At this point, the current reaches its maximum value, and the current increase slows down with further increases in concentration; therefore, 0.10 mg / mL is preferred. -1 The optimal modification concentration is [value missing].

[0120] (b) Optimization of AuNPs electrodeposition time

[0121] Using the optimized Co-Fc MOF-C / GCE as the working electrode, Au NPs / Co-Fc MOF-C / GCE were prepared by electrodeposition in chloroauric acid solution for 100, 200, 300, 400, 500, and 600 s, respectively. The results are as follows: Figure 5 As shown in Figure B, the bare GCE exhibits the lowest redox peak current, indicating a significant resistance to electron transport in the bare electrode. Modification with Co-Fc MOF-C significantly increases the current, demonstrating successful modification and improved electrode conductivity. As the Au NPs electrodeposition time increases from 100 s to 200 s, the electrode current response further increases, indicating that gold nanoparticles form a uniform conductive network on the electrode surface, significantly enhancing electron transport efficiency. When the deposition time exceeds 200 s, the current tends to decrease, as prolonged deposition can lead to gold nanoparticle aggregation; therefore, a deposition time of 200 s is preferred.

[0122] (c) Optimization of Z-peptide modification concentration

[0123] Using Au NPs / Co-Fc MOF-C / GCE as the working electrode, the electrode was immersed in different concentrations (0.05, 0.10, 0.20, 0.30, 0.40, 0.50 mg / mL). -1Z-peptide / Au NPs / Co-FcMOF-C / GCE was prepared by self-assembly in a Z-peptide solution.

[0124] like Figure 5 As shown in Figure C, compared with Au NPs / Co-Fc MOF-C / GCE, the redox peak current of the electrode modified with Z-peptide was significantly reduced, and the decrease in current gradually increased with the increase of peptide concentration, indicating that the peptide successfully formed a dense interface layer on the electrode surface, which hindered electron transfer to a certain extent.

[0125] Further integration Figure 5 The signal inhibition curve of D shows that as the concentration of Z-peptide increases, the non-specific adsorption inhibition rate of the electrode on the protein gradually increases, reaching a maximum when the concentration reaches 0.20 mg / mL. -1 Subsequently, the inhibition rate stabilized, indicating that the self-assembly of the peptides on the electrode surface had reached saturation. Further increasing the concentration would not improve antifouling performance but would instead excessively hinder electron transfer. Therefore, considering both antifouling performance and electrochemical response efficiency, a Z-peptide concentration of 0.20 mg / mL was optimal. -1 .

[0126] (2) Optimization of key process conditions for sensor fabrication

[0127] This invention optimizes key parameters for preparation and detection using single-factor methods, including: AFB1 complexation time with AM, number of electrochemical polymerization cycles, polymerization solution pH, template elution time, and target adsorption time, to determine the optimal preparation and testing process parameters. The results are as follows: Figure 6 As shown in the figure. The blue bar (washed) represents the current response (I) after template elution and before the target analyte is adsorbed. o The purple bar (adsorbed) represents the current response (I) after adsorption of the target analyte; the red curve represents the peak current difference ΔI between the two. p (ΔI) p =I o -I).

[0128] (a) Optimization of AFB1 complexation time with functional monomer (AM)

[0129] The prepared sensor was placed in a mixed solution of AFB1 and AM and complexed for 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. The changes in LSV current before and after complexation were compared, and the electrical signal before complexation was recorded as I. o The electrical signal after complexation is I. Calculate the peak current change ΔI. p (I) o -I). The result is as follows: Figure 6As shown in Figure A, ΔI increases with increasing complexation time. p The complexation time initially increases and then stabilizes, reaching a peak at 30 min. This indicates that the binding of AFB1 to the functional monomer is close to saturation at this point, resulting in the optimal imprinting effect. Therefore, the preferred complexation time is 30 min.

[0130] (b) Optimization of electrochemical polymerization cycle number

[0131] The prepared sensor was placed in the polymerization solution and subjected to cyclic voltammetric polymerization for 1, 3, 5, 7, and 9 cycles, respectively. The changes in LSV current before and after polymerization were compared. The electrical signal before polymerization was recorded as I0, and the electrical signal after polymerization was recorded as I. The peak current change value ΔI was calculated. p (I) o -I). The result is as follows: Figure 6 As shown in B, ΔI increases with the number of cycles. p The number of polymerization cycles initially increases and then stabilizes, reaching a peak at 5 cycles. Continuing to increase the number of cycles can easily cause the imprint film to become too thick, hindering electron mass transfer and reducing the imprint recognition effect. Therefore, the preferred number of polymerization cycles is 5.

[0132] (c) Optimization of electrochemical test scan rate

[0133] The prepared sensor was placed in the test substrate, and scan rates of 30 mV / s, 40 mV / s, 50 mV / s, 60 mV / s, and 70 mV / s were set respectively. The changes in LSV current at different scan rates were compared, and the electrical signal at different scan rates was recorded as I. o Calculate the peak current change ΔI. p (I) o -I). The result is as follows: Figure 6 As shown in Figure C, ΔI increases with increasing scan rate. p The value first increases and then decreases, reaching a peak at 50 mV / s. At this point, the electrode electron transfer efficiency and detection sensitivity are optimal, so the preferred test scan rate is 50 mV / s.

[0134] (d) Optimization of polymerization solution pH

[0135] The prepared sensor was placed in polymerization solutions with different pH values ​​for fabrication. The pH values ​​were set to 5.8, 6.6, 7.4, 8.2, and 9.0, respectively. The changes in LSV current under different pH conditions were compared, and the electrical signal at different pH values ​​was recorded as I0. o Calculate the peak current change ΔI. p (I) o -I). The result is as follows: Figure 6 As shown in Figure D, ΔI increases with increasing pH. pThe pH value first increases and then decreases, reaching a peak at pH 6.6. At this point, the hydrogen bonding and electrostatic interaction between the template and the functional monomer are strongest, resulting in the best imprinting and recombination effect. Therefore, the optimal pH value for the polymerization solution is 6.6.

[0136] (e) Template elution time optimization

[0137] After polymerization, the sensor was eluted with a suitable elution solvent for 20 min, 25 min, 30 min, 35 min, and 40 min, respectively. The changes in LSV current before and after elution were compared, and the electrical signal before elution was recorded as I. o The electrical signal after elution is I. Calculate the peak current change ΔI. p (I) o -I). The result is as follows: Figure 6 As shown in E, ΔI increases with elution time. p The elution time gradually increases and tends to stabilize, reaching a peak at 35 min. At this point, the template molecules are basically completely eluted, and the imprint cavity is fully exposed. Therefore, the preferred template elution time is 35 min.

[0138] (f) Optimization of target adsorption time

[0139] The prepared sensor was placed in a standard solution of AFB1 of the same concentration and incubated for 5 min, 10 min, 15 min, 20 min, and 25 min, respectively. The changes in LSV current before and after adsorption were compared, and the electrical signal before adsorption was recorded as I. o The electrical signal after adsorption is I. Calculate the peak current change ΔI. p (I) o -I). The result is as follows: Figure 6 As shown in Figure F, when the adsorption time increases from 5 min to 10 min, ΔI p The significant increase indicates that the adsorption capacity increases with time; while in the 10-25 min range, ΔI... p The adsorption was basically stable, indicating that the adsorption was approaching saturation. Further extending the time would not significantly improve the signal. Therefore, the preferred adsorption time for the target substance was 10 min.

[0140] 2.2.7 Performance Verification of Electrochemical Antifouling Sensor MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE

[0141] (1) Electrode performance testing

[0142] The water contact angle of the electrode surface at different modification stages was measured using the static drop method to investigate the effect of each modification step on the hydrophilicity of the electrode surface. The results are as follows: Figure 7As shown, the contact angle of the bare GCE surface was approximately 78.7°; after modification with Co-Fc MOF-C, the contact angle decreased to 46.4°, indicating that the material imparts a certain degree of hydrophilicity to the electrode surface; after further electrodeposition of Au NPs, the contact angle rebounded to approximately 74.5°, indicating that the introduction of Au NPs enhanced the hydrophobicity of the interface; after immobilization of C-peptide, the contact angle decreased to approximately 60.0°; finally, after modification with Z-peptide, the contact angle significantly decreased to 35.2°, indicating that this zwitterionic peptide has good hydrophilicity.

[0143] (2) Electrode antifouling effect test

[0144] The prepared Au NPs / Co-Fc MOF-C / GCE, C-peptide / Au NPs / Co-Fc MOF-C / GCE, and Z-peptide / Au NPs / Co-Fc MOF-C / GCE were placed in different concentrations (0.0, 0.1, 1.0, 10.0 mg / mL) respectively. -1 The electrodes were incubated for 30 min in solutions containing positively charged bovine serum albumin (BSA), negatively charged lysozyme (LYS), and neutral myoglobin (Mb). The current response of each electrode before and after incubation was recorded using an LSV (Low Current Sensor). The electrical signal before incubation was defined as I0, and the electrical signal after incubation as I. The signal inhibition rate was calculated. The results are as follows: Figure 8 , 9 As shown, in protein solutions of different concentrations and with different charge properties, the signal inhibition rate of the Z-peptide-modified electrode was significantly lower than that of the C-peptide-modified electrode and the electrode modified only with Au NPs / Co-Fc MOF-C, demonstrating that Z-peptide / Au NPs / Co-Fc MOF-C / GCE has excellent antifouling properties.

[0145] Signal Suppression (%) = (Electrical signal I0 - Electrical signal I) / Electrical signal I0 * 100%

[0146] (3) Specificity

[0147] The MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrochemical sensor and the corresponding non-imprinted NIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrode were used to detect aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), ochratoxin A (OTA), ochratoxin B (OTB), fumonisin B1 (FB1), and the target aflatoxin B1 (AFB1) at concentrations of 0.5 μM, respectively, to verify the sensor's specificity. The current changes of the sensor in detecting different substances were recorded using LSV, combined with the imprinting factor (IF=ΔI). MIP / ΔI NIP The specificity of the sensor was evaluated, and the results are as follows: Figure 10 As shown, the MIP electrode exhibits a significant current response only to the target analyte AFB1, while its response to other interfering toxins is similar to that of the NIP electrode with no significant difference. In contrast, the NIP electrode shows no significant selective response to any of the test substances, and the imprinting factor (IF) results further confirm the sensor's specific recognition ability for AFB1. This indicates that the MIP-modified electrochemical sensor prepared in this invention possesses excellent specific recognition performance.

[0148] (4) Repeatability

[0149] To examine the repeatability of the sensors, seven sensors from the same batch were prepared in parallel under identical experimental conditions and tested for the same concentration of target analyte. The ΔI signal showed small fluctuations and minimal numerical differences. Simultaneously, parallel comparative tests were conducted on sensors prepared from different batches. The ΔI signal fluctuations of each batch were small, the curves showed high overlap, and the deviation between batches was controllable. The results are as follows: Figure 11 As shown, the sensor constructed in this invention not only has excellent batch-to-batch repeatability but also good batch-to-batch reproducibility. The fabrication process is stable and controllable, meeting the application requirements for batch detection of actual samples.

[0150] (5) Stability

[0151] The prepared MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrochemical sensor was stored at 4 ℃. AFB1 was measured every day, and the peak current change ΔI was recorded. The test was conducted continuously for 5 days, and the relative standard deviation (RSD) was 0.54%. The results are as follows: Figure 12 As shown, the sensor's current response change value did not significantly decrease over 5 days, and the current change value of the AFB1 solution measured on the 5th day was 98.57% of the initial detection value of the AFB1 solution current change value, indicating that the electrochemical sensor prepared in this invention has good stability.

[0152] (6) Long-term

[0153] Au NPs / Co-Fc MOF-C / GCE and Z-peptide / Au NPs / Co-Fc MOF-C / GCE electrodes were placed in a 1% peanut sample solution and incubated for 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h, respectively. The changes in current signals before and after incubation were recorded, and the signal suppression rate was calculated. The results are as follows: Figure 13 As shown, with prolonged incubation time, the signal inhibition rate of Au NPs / Co-Fc MOF-C / GCE continuously increased from approximately 55% to approximately 81%, indicating that protein adsorption on the electrode surface significantly intensified over time. In contrast, the signal inhibition rate of the Z-peptide-modified electrode remained below 5% throughout the 4-h incubation period, showing no significant upward trend. This indicates that Z-peptide modification endows the electrode with excellent long-term antifouling properties, effectively inhibiting non-specific adsorption even after prolonged exposure to complex protein environments.

[0154] (7) Detection in real samples

[0155] Three electrodes—Au NPs / Co-Fc MOF-C / GCE, Z-peptide / Au NPs / Co-Fc MOF-C / GCE, and MIP / Z-peptide / Au NPs / Co-Fc MOF-C / GCE—were incubated in peanut sample solutions with volume fractions of 1%, 3%, 5%, 10%, 15%, and 25%, respectively. Changes in current signals before and after incubation were recorded, and the signal suppression rate was calculated. The results are as follows: Figure 14 As shown, with increasing peanut sample concentration, the signal inhibition rate of Au NPs / Co-Fc MOF-C / GCE remained above 75%, indicating severe matrix interference. The signal inhibition rate of the Z-peptide-modified electrode decreased significantly, remaining below 22% even at a 25% concentration. The signal inhibition rate of the MIP / Z-peptide-modified electrode further decreased, demonstrating excellent resistance to matrix interference. This indicates that the MIP / Z-peptide-modified electrode constructed in this invention can effectively resist interference from complex sample matrices and is suitable for the detection of target analytes in actual peanut samples (oilseed samples).

[0156] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A fouling-resistant molecularly imprinted electrochemical sensor, characterized in that, It includes a conductive substrate, and a cobalt-ferrocene metal-organic framework calcined material layer, a gold nanoparticle layer, a zwitterionic peptide layer and a molecularly imprinted polymer layer sequentially modified on the surface of the conductive substrate; The amino acid sequence of the zwitterionic polypeptide contains positively charged residues and negatively charged residues, and the C-terminus is modified by amidation. The molecularly imprinted polymer layer contains a molecularly imprinted cavity that matches the target template molecule and an acrylamide functional monomer.

2. The sensor according to claim 1, characterized in that, The conductive substrate is a glassy carbon electrode, carbon nanotube paper, or flexible carbon composite paper.

3. The sensor according to claim 1, characterized in that, The calcined material of the cobalt-ferrocene metal-organic framework calcined material layer is obtained by calcining the cobalt-ferrocene metal-organic framework in an inert gas atmosphere at 300-500°C.

4. The sensor according to claim 1, characterized in that, The amino acid sequence of the zwitterionic polypeptide is NH2-CPPPPEKDQDK.

5. The sensor according to claim 1, characterized in that, The target template molecule of the molecularly imprinted polymer layer is a fungal toxin.

6. A method for manufacturing the sensor according to any one of claims 1-5, characterized in that, include: A dispersion of cobalt-ferrocene metal-organic framework calcined material was modified onto the surface of a conductive substrate. Gold nanoparticles were electrochemically deposited on the modified electrode surface; The electrode after electrodeposition was placed in a zwitterionic peptide solution and incubated, so that the peptide self-assembled onto the surface of gold nanoparticles through gold-sulfur bonds. Using the incubated electrode as the working electrode, electrochemical polymerization is carried out in a polymerization solution containing target template molecules and functional monomers to form a molecularly imprinted polymer film. The template molecules are removed by elution to obtain the sensor.

7. The preparation method according to claim 6, characterized in that, The electrochemical deposition was carried out in an acidic solution containing chloroauric acid, with a deposition voltage of -0.1 to -0.3 V and a deposition time of 100-300 s.

8. The preparation method according to claim 7, characterized in that, The electrochemical polymerization was carried out using cyclic voltammetry, with 3-7 polymerization cycles and a polymerization solution pH of 5.8-8.

2.

9. The use of the sensor according to any one of claims 1-5 in detecting analytes corresponding to target template molecules.

10. The application according to claim 9, characterized in that, The target template molecule is aflatoxin B1.

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