Preparation method and application of anti-fouling electrochemical sensor

Abstract

The application discloses a preparation method of an anti-fouling electrochemical sensor and application thereof; the anti-fouling electrochemical sensor adopts platinum gold nanoparticles as a base for a recognition element and provides a modification site for an amphoteric ion copolymer; wherein amphoteric ion monomers SBMA and CBMA are polymerized to synthesize the amphoteric ion copolymer, the carboxyl in CBMA is connected with the amino of CYS, and then the amphoteric ion copolymer is modified on the electrode surface through a thiol group to form an anti-fouling surface. The anti-fouling electrochemical sensor constructed by the application can achieve the purpose of quantitative detection of target objects, and has the advantages of low detection limit, good selectivity and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of antifouling electrochemical technology, specifically to a method for preparing an antifouling electrochemical sensor and its application. Background Technology

[0002] Chloramphenicol (CAP), a broad-spectrum antibiotic, was once widely used to combat infections caused by Gram-positive and Gram-negative bacteria. However, whether ingested through food or medication, excessive use of CAP can accumulate in the food chain, causing serious side effects on human health, such as aplastic anemia, bone marrow suppression, and genotoxicity. Therefore, the analysis and detection of chloramphenicol has always been a key focus of research in the field of food safety.

[0003] Various analytical techniques for detecting CAP residues have been widely reported. Among them, electrochemical sensors have shown great potential in the field of food safety testing due to their advantages such as rapid analysis, high sensitivity, and miniaturized equipment. However, these sensors still face contamination problems when detecting complex food matrices. Typically, contaminants (such as proteins, carbohydrates, and other non-target substances) can non-specifically adhere to the sensor surface during detection through hydrogen bonding, hydrophobic interactions, and electrostatic adsorption. This non-specific adsorption not only interferes with the capture of target substances but also significantly affects the accuracy and stability of detection. Although sample pretreatment can effectively remove non-target substances to reduce matrix interference, mature electrochemical sensors should not rely on complex pretreatment processes. Improving the anti-contamination performance of electrochemical sensors to reduce matrix interference has become an effective solution to replace traditional pretreatment. Therefore, introducing anti-contamination materials onto the sensor surface to improve the anti-contamination performance of the electrochemical sensor itself is key to achieving resistance to matrix interference. Common anti-contamination materials include polyethylene glycol and its derivatives, polysaccharides, zwitterionic polymers, and peptides. The antifouling materials mentioned above all possess strong hydrophilicity and electroneutrality. Their modified hydrophilic surfaces can bind with water molecules through electrostatic interactions and hydrogen bonds to form a dense hydration layer, thereby reducing non-specific adsorption. PEG and peptides are commonly used in the fabrication of antifouling electrochemical sensors; however, PEG is easily oxidized and broken down, and peptides are prone to enzymatic degradation in biological media and are costly. Zwitterionic materials, due to their hydrophilicity, electroneutrality, tunable structure, and good biocompatibility, are considered highly promising antifouling materials. Furthermore, multi-block copolymers have shown significant potential due to the property differences between different zwitterionic monomers. Compared to single zwitterionic polymers, multi-block copolymers offer advantages such as easier anchoring and the ability to finely tune copolymer properties through compositional adjustments. Therefore, this paper proposes a method for fabricating an antifouling electrochemical sensor and its application. Summary of the Invention

[0004] This invention uses platinum nanoparticles as a substrate to provide modification sites for the identification element and the free radical polymerization of zwitterionic monomers SBMA and CBMA to synthesize zwitterionic copolymers. The zwitterionic copolymers are then modified onto the electrode surface through the connection of carboxyl groups in CBMA and amino groups in CYS to form an anti-fouling surface.

[0005] To achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: a method for preparing an antifouling electrochemical sensor, characterized by comprising the following steps:

[0006] S1. First, polish the glassy carbon electrode GCE with 0.3μM alumina powder, then clean it ultrasonically with ultrapure water and anhydrous ethanol, and finally dry it with high-purity nitrogen to make it mirror-like.

[0007] S2. The GCE treated in S1 was placed in a mixed solution of HAuCl4 and H2PtCl6. A platinum electrode was used as the counter electrode and a saturated calomel electrode was used as the reference electrode. A constant potential deposition of platinum alloy was performed using a constant potential technique. The constant potential was set to 0.1 V and the deposition time was 200 s. After the deposition was completed, the working electrode was removed and rinsed with ultrapure water to complete the modification of platinum nanoparticles and obtain a glassy carbon electrode Pt-Au NPs / GCE modified with platinum nanoparticles.

[0008] S3. Take SBMA and CBMA in different molar ratios, then dissolve each proportion of SBMA and CBMA in 30 mL of ultrapure water, then add 0.2 mmol of ammonium persulfate (APS) and 10 μL of mercaptoacetic acid (TGA) and mix well to obtain a medium-term solution; incubate the medium-term solution at 75 °C overnight and perform a polymerization reaction by magnetic stirring at 350 rpm under a nitrogen atmosphere; after reacting for 12 h, dialyze with ultrapure water to remove unreacted monomers or oligomers, and then freeze-dry to obtain initial bodies with different numbers;

[0009] S4. Take a concentration of 10 mg / mL at a volume ratio of 1:1. −1 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and a concentration of 10 mg / mL −1 N-hydroxysuccinimide (NHS) and mixed; to obtain an auxiliary solution;

[0010] S5. Dissolve the initial sample obtained in S3 in the auxiliary solution obtained in S4 to prepare a solution with a concentration of 2 mg / mL. −1 A mixture of zwitterionic copolymer material PSB-CBMA;

[0011] S6. Take the PSB-CBMA mixture obtained in S5 and a 50 mmol / L solution of CYS solution at a volume ratio of PSB-CBMA mixture:CYS solution = 18:1. −1 The cysteine ​​(CYS) solution was mixed to obtain a PSB-CBMA / CYS solution;

[0012] S7. Place the Pt-Au NPs / GCE obtained in S2 into 200 μL of the PSB-CBMA / CYS solution obtained in S6, and then add 2 μmol / L of thiol-modified solution at the 5' end. −1 The CAP aptamer of mercaptochloramphenicol was obtained and placed in a 4°C refrigerator for 10 h to obtain the antifouling electrochemical sensor Apt-PSB-CBMA / Pt-Au NPs / GCE.

[0013] Furthermore, in S2, the concentrations of both HAuCl4 and H2PtCl6 are 1 mmol / L. −1 The mixing molar ratio is 1:1.

[0014] Furthermore, in S3, the different molar ratios of sulfomethacrylic acid betaine (SBMA) and carboxymethacrylic acid betaine (CBMA) are: sulfomethacrylic acid betaine (SBMA): carboxymethacrylic acid betaine (CBMA) = 10:0, 9:1, 8:2, 7:3.

[0015] Furthermore, in S3, the molar ratio of sulfomethacrylic acid betaine (SBMA) to carboxymethacrylic acid betaine (CBMA) is 8:2.

[0016] Furthermore, in S6, the CYS solution preparation process is as follows: Weigh 19.25 mg of CYS to obtain a concentration of 0.01 mol / L. −1 Dilute the solution to 5 mL with PBS buffer solution at pH 7.4 and store at 4°C.

[0017] Furthermore, in S7, the sequence of the CAP aptamer is 5'-SH-(CH2)6-ACT TCA GTG AGT TGT CCCACG GTC GGC GAG TCG GTG GTAG-3'.

[0018] Another objective of this invention is to provide an application of an antifouling electrochemical sensor in chloramphenicol detection, characterized in that the concentration of chloramphenicol can be obtained by inputting the electrical signals before and after detection into a constructed standard curve using the antifouling electrochemical sensor.

[0019] Furthermore, the standard curve was constructed as follows: the prepared antifouling electrochemical sensor Apt-PSB-CBMA / Pt-Au NPs / GCE was immersed in 200 μL of a solution containing 0.01 ng / mL of [a specific chemical compound]. −1 ~100 ng / mL −1 The chloramphenicol standard solution was incubated, and then the unbound chloramphenicol was rinsed off with ultrapure water to form the working electrode for detection. The working electrode was then immersed in a solution containing 5 mmol L... −1 K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2 mol L −1 In an electrochemical probe solution of KCl in PBS, a potential range of −0.2 V to 0.6 V, an amplitude of 10 mV, and a scan rate of 50 mV / s were used. −1 The differential pulse voltammetry scanning method was used to calculate the signal inhibition rate by recording the current signals before and after detection; a standard curve was obtained based on the signal inhibition rate and the logarithm of chloramphenicol concentration.

[0020] Furthermore, the specific concentration of chloramphenicol detected was as follows: the simply treated food sample was diluted 100 times, and a concentration of 0.5 ng / mL was added. −1 5ng mL −1 and 50 ng mL −1 The chloramphenicol standard solution was prepared and detected using a prepared antifouling electrochemical sensor; the concentration of chloramphenicol could be obtained by substituting the electrical signals before and after detection into the constructed standard curve.

[0021] The beneficial effects of this invention are:

[0022] (1) The antifouling layer used in this invention is a zwitterionic copolymer, which solves the problem of zwitterionic materials being difficult to anchor onto the electrode surface through the carboxyl groups on CBMA. In addition, since zwitterionic polymers have strong hydrophilicity and electroneutrality, the solvation effect and hydrogen bonding effect of charged functional groups can form a hydrated layer on the surface of zwitterionic polymers. This hydrated layer surface can effectively resist non-specific adsorption and has high antifouling performance;

[0023] (2) The antifouling electrochemical sensor described in this invention has a simple construction process and excellent sensitivity and accuracy. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments 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.

[0025] Figure 1 This is a schematic diagram illustrating the construction of the antifouling electrochemical sensor of the present invention;

[0026] Figure 2 This is a schematic diagram of the PSB-CBMA synthesis route of the present invention;

[0027] Figure 3 The above are the hydrogen nuclear magnetic resonance spectra characterized by PSBMA and PSB-CBMA in this invention;

[0028] Figure 4 To optimize the ratio of zwitterionic monomers in this invention, a is casein (CS) and b is whey protein (WP);

[0029] Figure 5 This is a comparison chart showing the antifouling capabilities of Pt-Au NPs / GCE and Apt-PSB-CBMA / Pt-Au NPs / GCE at different concentrations of bovine serum albumin (BSA), bovine hemoglobin (HB), lysozyme (Lys), glucose (Glu), fructose (Fru), and lactose (Lac) in this invention.

[0030] Figure 6 This illustrates the change in contamination levels of different modified electrodes in real samples over time.

[0031] Figure 7 The graph shows the concentration optimization and time optimization of the aptamer of this invention;

[0032] Figure 8 The present invention combines the DPV response signals of different concentrations of chloramphenicol with the antifouling electrochemical sensor.

[0033] Figure 9 The curve showing the relationship between chloramphenicol concentration and signal inhibition rate in this invention is shown; the inset represents the corresponding standard curve.

[0034] Figure 10 This invention provides the selectivity specificity of the antifouling electrochemical sensor.

[0035] Figure 11 To ensure the stability of the antifouling electrochemical sensor of this invention;

[0036] Figure 12 This is to improve the repeatability of the antifouling electrochemical sensor of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] A method for preparing an antifouling electrochemical sensor includes the following steps:

[0040] (1) Modification of platinum nanoparticles: The glassy carbon electrode (GCE) was polished with alumina powder, then ultrasonically cleaned with ultrapure water and anhydrous ethanol, and dried with high-purity nitrogen to obtain a clean glassy carbon electrode; the GCE was immersed in a mixed solution of HAuCl4 and H2PtCl6, with the platinum electrode as the counter electrode and the saturated calomel electrode as the reference electrode. The platinum alloy was deposited using a constant potential technique, with the constant potential set at 0.1 V and the deposition time at 200 s. After the deposition was completed, the working electrode was removed; it was rinsed with ultrapure water to obtain the working electrode (Pt-Au NPs / GCE) modified with platinum nanoparticles.

[0041] (2) Modification of zwitterionic copolymers with CAP aptamers; 50 mmol / L of PBS buffer solution was prepared. −1 The CYS solution; PSB-CBMA and CAP aptamer were modified on the surface of Pt-Au NPs / GCE using a one-step modification; specifically; zwitterionic copolymers synthesized at different molar ratios of 10:0, 9:1, 8:2, and 7:3 were dissolved in EDC / NHS mixed solutions, and the PSB-CBMA solution and CYS solution were miscible at a volume ratio of 18:1. The CAP aptamer (5'-SH-(CH2)6-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTAG-3') was added to the solution.

[0042] (3) The Pt-Au NPs / GCE obtained in step (1) was immersed in 200 µL of Apt / PSB-CBMA / CYS solution and incubated for 8 h to obtain a working electrode (Apt-PSB-CBMA / Pt-AuNPs / GCE) modified by zwitterionic copolymer and CAP aptamer.

[0043] (4) Immerse Apt-PSB-CBMA / Pt-Au NPs / GCE in 200 µL of 100 ng / mL solution. −1 In the CAP standard solution, after 50 min, the unbound CAP was rinsed off with ultrapure water to form the working electrode to be tested;

[0044] (5) Then, the working electrode to be detected is immersed in the electrochemical probe solution, and DPV scanning is performed in the potential range of −0.2 V to 0.6 V. The signal inhibition rate is calculated by recording the DPV signals before and after detection; the signal inhibition rate reflects the amount of CAP binding to Apt; and the signal inhibition rate reflects the amount of CAP binding to Apt. Figure 7 It can be seen that the aptamer concentration is 2 µmol L. −1 The signal suppression rate is highest and tends to stabilize at this time, therefore 2 µmol L is selected. −1 It was used to construct an anti-fouling sensor. Furthermore, the incubation time was optimized under the same conditions, and experimental results showed that 50 min was the optimal incubation time.

[0045] (6) Establishment of the sensing method: Take the antifouling electrochemical sensor constructed in the above steps and immerse it in 200 μL of solutions with concentrations of 0.01, 0.05, 0.1, 1, 10, 50, and 100 ng / mL. −1 In a chloramphenicol standard solution, after incubation for 60 min, the unbound chloramphenicol was rinsed with ultrapure water to form the working electrode to be detected. Then, the working electrode was immersed in an electrochemical probe solution, and differential pulse voltammetry (DPV) was used to scan within a potential range of −0.2 V to 0.6 V. The signal suppression rate was calculated by recording the current signals before and after detection. Figure 9 The standard curve obtained based on the logarithm of the signal inhibition rate and chloramphenicol concentration is: y = 3.22x + 10.87, (R0...). 2 = 0.999, S / N=3).

[0046] (7) with 100 ng mL −1 Chloramphenicol and 1 μg mL −1 Amoxicillin, kanamycin, clindamycin, erythromycin, penicillin, and mixtures thereof were used as detection targets, and the prepared antifouling electrochemical aptamer sensor was used for detection. The results are as follows: Figure 10 As shown, the electrical signal changes for amoxicillin, kanamycin, clindamycin, erythromycin, and penicillin were very low, at 4.2%, 5.3%, 1.9%, 5.8%, and 3.3%, respectively; while the signal inhibition rates for chloramphenicol and mixtures thereof were 15.9% and 16.8%, respectively, indicating that the sensor has excellent selectivity.

[0047] Example 2

[0048] The construction and application of an antifouling surface made of zwitterionic copolymer for an antifouling electrochemical sensor are detailed below:

[0049] (1) Preparation process of zwitterionic copolymer (PSB-CBMA): The molar ratio of SBMA to CBMA was 10:0, 9:1, 8:2, and 7:3, and the resulting copolymers were named PSB-CBMA0, PSB-CBMA1, PSB-CBMA2, and PSB-CBMA3, respectively (when the ratio was 10:0, the mass of SBMA was 2.78 g 10 mmol). Specifically, different proportions of SBMA and CBMA were dissolved in 30 mL of ultrapure water, and then 40.0 mg (0.2 mmol) of ammonium persulfate (APS) and TGA (10 μL) were added and mixed. The resulting solution was stirred overnight at 75 °C under a nitrogen atmosphere with a magnetic stirring speed of 350 rpm to complete the polymerization reaction. After 12 h of reaction, the product was dialyzed with ultrapure water to remove unreacted monomers or oligomers, and then freeze-dried to obtain zwitterionic polymer material. The characteristics of the product were determined by ¹H NMR (400 MHz, D₂O). Figure 3 As shown, compared with PSB-CBMA0, PSB-CBMA2 exhibits a characteristic peak of H on the carbon atom adjacent to the carboxyl group in CBMA at 2.74 ppm.

[0050] (2) Modification of platinum nanoparticles: GCE was polished with alumina powder, then ultrasonically cleaned with ultrapure water and anhydrous ethanol, and dried with high-purity nitrogen to obtain a clean glassy carbon electrode. GCE was then immersed in 4 mL of solution containing 1 mmol L... −1 HAuCl4 and 1 mmol L −1 The platinum alloy was deposited in a mixed solution of H2PtCl6 using a potentiostatic technique. The constant potential was set at 0.1 V and the deposition time was 200 s. After deposition, the working electrode was removed and rinsed with ultrapure water to obtain a working electrode modified with platinum nanoparticles (Pt-Au NPs / GCE).

[0051] (3) Modification with zwitterionic copolymers: using 10 mmol L −1 Prepare 50 mmol / L PBS buffer solution −1 The CYS solution was used. PSB-CBMA was modified onto the surface of Pt-Au NPs / GCE using a one-step modification. Specifically, zwitterionic copolymers synthesized at different molar ratios of 10:0, 9:1, 8:2, and 7:3 were dissolved in EDC / NHS mixed solutions, and the PSB-CBMA solution and CYS solution were miscible at a volume ratio of 18:1. The Pt-Au NPs / GCE obtained in step (2) was immersed in 200 µL of PSB-CBMA / CYS solution for incubation to obtain a working electrode modified with zwitterionic copolymer (Apt-PSB-CBMA / Pt-AuNPs / GCE).

[0052] (4) Investigation of antifouling performance: The prepared antifouling surfaces of different zwitterionic copolymers and Pt-Au NPs / GCE were immersed in 10.0 mg / mL solution. −1 The whey protein and casein were incubated for 30 min. The working electrode to be detected was immersed in the electrochemical probe solution, and DPV scanning was performed within a potential range of −0.2 V − 0.6 V. The current response values ​​before and after incubation were recorded to obtain the signal suppression rate, thereby more intuitively comparing the antifouling ability of zwitterionic copolymer antifouling sensing interfaces with different proportions. Figure 4 (a, b) show that the signal suppression rate of PSB-CBMA2 / Pt-Au NPs / GCE is consistently the lowest, indicating that it has the best antifouling ability, and can be used for the construction of subsequent antifouling electrochemical sensors.

[0053] The prepared modified GCEs were immersed in 0.1, 1.0, and 10.0 mg / mL solutions. −1 The antifouling sensor was incubated for 30 min in a solution of bovine serum albumin, lysozyme, hemoglobin, glucose, lactose, and fructose. The antifouling sensor to be tested was then immersed in the electrochemical probe solution, and DPV scanning was performed within a potential range of −0.2 V − 0.6 V. The current response values ​​before and after incubation were recorded to derive the signal suppression rate, thus providing a more intuitive comparison of the antifouling capabilities of different antifouling electrochemical sensors. Figure 5 ) Using pH 7.4 10.0 mmol L −1 Milk, eggs, and honey were diluted 100-fold with PBS buffer. Different modified GCEs were immersed in the above solutions and incubated for 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h, respectively. The signal suppression rate was determined by DPV scanning within a potential range of −0.2 V to 0.6 V and recording the current response values ​​before and after incubation. Figure 6 As shown, the results indicated that the signal inhibition rates of Apt-PSB-CBMA / Pt-Au NPs / GCE in milk, honey, and eggs were as low as 2.5%, 1.2%, and 1.6%, respectively, after an incubation time of 0.5 h. The signal inhibition rates in milk, honey, and eggs were only 4.3%, 1.6%, and 4.9%, respectively, after an incubation time of 2.5 h, which were far lower than the signal inhibition rates of Pt-Au NPs / GCE (97.4%, 57.5%, and 95.5%).

[0054] Antifouling principle of zwitterionic copolymer antifouling surface: The zwitterionic copolymer is connected to the electrochemical sensor through Au-S / Pt-S to construct an antifouling surface. The zwitterionic copolymer has excellent hydrophilicity and electroneutrality. It can establish a stable hydration layer with water molecules through hydrogen bonding or ionic dissolution, which acts as a physical and energy barrier to prevent the adhesion of dirt and effectively reduce the non-specific adsorption of non-detectable substances during the detection process.

[0055] Example 3

[0056] The electrode system used in this invention is based on the traditional three-electrode system: a glassy carbon electrode as the working electrode; a platinum electrode as the counter electrode; and a saturated calomel electrode as the reference electrode.

[0057] All electrochemical experiments were conducted on an IVIUM electrochemical workstation.

[0058] All electrochemical tests were performed in an electrochemical probe solution. The electrochemical probe solution contained 5 mmol / L. −1 K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2 mol L −1 KCl in PBS.

[0059] Preparation method of PBS: Accurately weigh 35.8 g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 15.6 g of sodium dihydrogen phosphate (NaH2PO4·2H2O), place them in a 500 mL volumetric flask, and dilute to the mark with ultrapure water to obtain 0.2 mol L⁻¹ −1 Na2HPO4 solution and 0.2 mol L −1 NaH₂PO₄ solution; measure 324 mL of 0.2 M Na₂HPO₄ solution and 0.2 mol L of NaH₂PO₄ solution using a graduated cylinder respectively. −1 76 mL of NaH₂PO₄ solution was mixed with 3.6 g of solid sodium chloride (NaCl) to obtain a solution with pH 7.4 and a concentration of 0.2 mol / L. −1 The PBS solution was incubated at room temperature.

[0060] Preparation method of electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]): Accurately weigh 164.62 mg of potassium ferrocyanide (K3[Fe(CN)6]) solid, 184.17 mg of potassium ferrocyanide (K4[Fe(CN)6]) and 1491.02 mg of potassium chloride (KCl). Add 0.2 mol L... −1 Dilute the pH 7.4 PBS solution to 0.01 mol / L. −1 Then use 0.01 mol L −1 The above-mentioned drugs were dissolved in PBS solution at pH 7.4, and the solution was finally diluted to 100 mL in a volumetric flask to obtain a bright yellow solution, which yielded 5 mmol L. −1 The electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6], containing 0.2 mol / L KCl) was wrapped in aluminum foil and stored in a refrigerator at 4 ℃ for later use.

[0061] In this invention, DPV is used to record the current response values ​​of the modified electrode before and after incubation (the electrical signal before incubation is I0, and the electrical signal after incubation is I). The change value of electrical signal (ΔI=I0–I) and the electrical signal suppression rate (SignalSuppression (%) = [(I0–I) / I0]×100) are calculated from these values.

[0062] Example 4

[0063] The application of an antifouling electrochemical sensor in actual chloramphenicol detection samples is as follows:

[0064] (1) Milk, honey and eggs were selected as the test samples, and the spiked recovery experiment was used to verify the actual application performance of the constructed sensor in the test samples.

[0065] (2) Using pH 7.4 0.01 mol L −1 Milk, honey, and eggs were diluted 1% with PBS buffer. Different concentrations of chloramphenicol standard solution were then added to the diluted food samples to ensure that the solutions contained 0.5 ng / mL of chloramphenicol standard solution. −1 5 ng mL −1 and 50.0 ng mL −1 Chloramphenicol was then detected using the antifouling electrochemical sensor prepared in Example 2, and the spiked recovery rate in the sample was obtained. The spiked sample was then analyzed using enzyme-linked immunosorbent assay (ELISA) to verify the results obtained by the constructed sensor and ensure the accuracy of the analysis; the specific results are shown in Table 1 below:

[0066] sample Spiked concentration (ng mL−1) Detected concentration (ng mL−1) Recovery rate (mean ± RSD, n=3, %) Recovery rate of enzyme-linked immunosorbent assay (mean ± RSD, n=3, %) 0.50 0.51 102.3 ± 2.5 89.4 ± 1.3 milk 5.00 5.08 101.6 ± 1.2 96.3 ± 0.9 50.00 52.29 104.58 ± 0.6 96.8 ± 0.7 0.50 0.54 108.6 ± 1.2 102.2 ± 2.2 Honey 5.00 5.09 101.7 ± 1.6 92.7 ± 2.1 50.00 51.56 103.1 ± 0.3 94.6 ± 0.7 0.50 0.54 108.9 ± 2.1 94.2 ± 4.2 egg 5.00 5.35 106.9 ± 0.3 96.3 ± 1.9 50.00 53.27 106.5 ± 0.8 96.0 ± 1.2

[0067] Table 1

[0068] As can be seen from Table 1 above, the antifouling electrochemical sensor prepared by this invention can sensitively detect chloramphenicol in the sample to be tested, and this method has broad application prospects in food matrices.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing an antifouling electrochemical sensor, characterized in that, Includes the following steps: S1. First, polish the glassy carbon electrode GCE with 0.3μM alumina powder, then clean it ultrasonically with ultrapure water and anhydrous ethanol, and finally dry it with high-purity nitrogen to make it mirror-like. S2. The GCE treated in S1 was placed in a mixed solution of HAuCl4 and H2PtCl6. A platinum electrode was used as the counter electrode and a saturated calomel electrode was used as the reference electrode. A constant potential deposition of platinum alloy was performed using a constant potential technique. The constant potential was set to 0.1 V and the deposition time was 200 s. After the deposition was completed, the working electrode was removed and rinsed with ultrapure water to complete the modification of platinum nanoparticles and obtain a glassy carbon electrode Pt-Au NPs / GCE modified with platinum nanoparticles. S3. Take sulfomethacrylic acid betaine (SBMA) and carboxymethacrylic acid betaine (CBMA) in different molar ratios, then dissolve each proportion of sulfomethacrylic acid betaine (SBMA) and carboxymethacrylic acid betaine (CBMA) in 30 mL of ultrapure water, then add 0.2 mmol of ammonium persulfate (APS) and 10 μL of mercaptoacetic acid (TGA) and mix well to obtain a intermediate solution; incubate the obtained intermediate solution at 75 °C overnight and perform a polymerization reaction by magnetic stirring at 350 rpm under a nitrogen atmosphere; after reacting for 12 h, dialyze with ultrapure water to remove unreacted monomers or oligomers, and then freeze-dry to obtain initial bodies with different numbers; S4. Take a concentration of 10 mg / mL at a volume ratio of 1:

1. −1 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and a concentration of 10 mg / mL −1 N-hydroxysuccinimide (NHS) and mixed; Auxiliary solution is obtained; S5. Dissolve the initial sample obtained in S3 in the auxiliary solution obtained in S4 to prepare a solution with a concentration of 2 mg / mL. −1 A mixture of zwitterionic copolymer material PSB-CBMA; S6. Take the PSB-CBMA mixture obtained in S5 and a 50 mmol / L solution of cysteine ​​solution at a volume ratio of PSB-CBMA mixture to cysteine ​​solution of 18:

1. −1 The cysteamine solution was mixed to obtain a PSB-CBMA / cysteamine solution; S7. Place the Pt-Au NPs / GCE obtained in S2 into a 200 μL PSB-CBMA / cysteine ​​solution obtained in S6, and then add a 5'-terminated thiol-modified solution at a concentration of 2 μmol / L. −1 The mercaptochloramphenicol aptamer was obtained and placed in a 4°C refrigerator for 10 h to obtain the antifouling electrochemical sensor Apt-PSB-CBMA / Pt-Au NPs / GCE; In S7, the sequence of the aptamer for mercaptochloramphenicol is 5'-SH-(CH2)6-ACT TCA GTG AGT TGT CCC ACG GTCGGC GAG TCG GTG GTAG-3'.

2. The method for preparing an antifouling electrochemical sensor according to claim 1, characterized in that, In S2, the concentrations of both HAuCl4 and H2PtCl6 are 1 mmol / L. −1 The mixing molar ratio is 1:

1.

3. The method for preparing an antifouling electrochemical sensor according to claim 1, characterized in that, In S3, the different molar ratios of sulfomethacrylic acid betaine (SBMA) and carboxymethacrylic acid betaine (CBMA) are: sulfomethacrylic acid betaine (SBMA): carboxymethacrylic acid betaine (CBMA) = 9:1, 8:2, 7:

3.

4. The method for preparing an antifouling electrochemical sensor according to claim 3, characterized in that, In S3, the molar ratio of sulfomethacrylic acid betaine (SBMA) to carboxymethacrylic acid betaine (CBMA) is 8:

2.

5. The method for preparing an antifouling electrochemical sensor according to claim 1, characterized in that, In S6, the cysteamine solution was prepared as follows: 19.25 mg of cysteamine was weighed, and a concentration of 0.01 mol / L was used. −1 Dilute to 5 mL with PBS buffer solution at pH 7.4 and store at 4°C.

6. The application of an antifouling electrochemical sensor prepared according to the method of claim 1 in chloramphenicol detection, characterized in that, The concentration of chloramphenicol can be obtained by inputting the electrical signals before and after detection into a constructed standard curve using an antifouling electrochemical sensor.

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