Preparation method and application of anti-fouling electrochemical sensor

Abstract

The invention discloses a preparation method and application of an anti-fouling electrochemical sensor. According to the anti-fouling electrochemical sensor, platinum nanoparticles are adopted as a substrate to provide modification sites for an original recognition element and a zwitterionic copolymer; wherein zwitterionic monomers SBMA and CBMA are subjected to free radical polymerization to synthesize a zwitterionic copolymer, carboxyl in CBMA is connected with amino of CYS, and then the zwitterionic copolymer is modified on the surface of an electrode through sulfydryl to form an anti-fouling surface. The anti-fouling electrochemical sensor constructed by the invention can achieve the purpose of quantitatively detecting a target object, and has the advantages of low detection limit, good selectivity, high sensitivity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of anti-fouling electrochemistry, in particular to a preparation method of an anti-fouling electrochemical sensor and application thereof. BACKGROUND

[0002] Chloramphenicol (CAP) as a broad-spectrum antibiotic has been widely used to combat infections caused by gram-positive and negative bacteria. However, regardless of intake through food or drugs, excessive use of CAP can accumulate in the food chain, causing serious side effects on human health, such as aplastic anemia, bone marrow suppression, genetic toxicity and other diseases. Therefore, the analysis and detection of chloramphenicol has always been the focus of research in the field of food safety.

[0003] Various analysis techniques for CAP residue detection have been widely reported. Among them, electrochemical sensors have shown great potential in the field of food safety detection due to their rapid analysis, high sensitivity and small device size. However, when detecting complex food matrices, such sensors still have pollution problems. Usually, pollutants (such as proteins, carbohydrates and other non-target substances) will non-specifically adhere to the sensor surface during the detection process through hydrogen bonding, hydrophobic interaction, electrostatic adsorption and other ways. This non-specific adsorption not only interferes with the capture of target substances, but also significantly affects the accuracy and stability of the detection. Although sample pretreatment can effectively remove non-target substances to reduce matrix interference, mature electrochemical sensors should not rely on complex pretreatment procedures. Improving the anti-pollution performance of electrochemical sensors to reduce matrix interference has become an effective solution to replace traditional pretreatment. Therefore, introducing anti-fouling materials to the sensor surface to improve the anti-pollution performance of electrochemical sensors is the key to achieving anti-matrix interference. Common anti-fouling materials include polyethylene glycol and its derivatives, polysaccharides, zwitterionic polymers and polypeptides. The above anti-fouling materials have strong hydrophilicity and electrical neutrality. The hydrophilic surface formed by their modification can combine with water molecules through electrostatic interaction, hydrogen bonding and other ways to form a dense hydration layer, thereby reducing non-specific adsorption. PEG and polypeptides are often used in the preparation of anti-fouling electrochemical sensors, but PEG is easily oxidized and broken, and polypeptides are easily enzymatically degraded in biological media and have high cost. Zwitterionic materials are considered to be a promising anti-fouling material due to their hydrophilicity, electrical neutrality, adjustable structure and good biocompatibility. In addition, due to the differences in properties between different zwitterionic monomers, multi-block copolymers have shown significant potential. Compared with single zwitterionic polymers, multi-block copolymers have the advantages of easier anchoring and the ability to fine-tune copolymer properties by adjusting components. Therefore, a preparation method of an anti-fouling electrochemical sensor and application thereof are proposed. SUMMARY

[0004] The application adopts platinum nanoparticles as a base for a recognition element and provides a modification site by synthesizing a zwitterionic copolymer through radical polymerization of a zwitterionic monomer SBMA and CBMA, and the zwitterionic copolymer is modified on the electrode surface through the connection of the carboxyl in CBMA and the amino in CYS, and then the zwitterionic copolymer is modified on the electrode surface through a thiol group to form an anti-fouling surface.

[0005] In order to achieve the above technical effects, the application is implemented by the following technical scheme: a preparation method of an anti-fouling electrochemical sensor, characterized by comprising the following steps:

[0006] S1, first polish a glassy carbon electrode GCE with 0.3 mu M aluminum oxide powder, then ultrasonic clean with ultrapure water and anhydrous ethanol, and finally dry with high-purity nitrogen gas to form a mirror surface;

[0007] S2, deposit platinum gold alloy on the GCE treated in S1 by adopting constant potential technology with a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, set a constant potential to 0.1 V, and the deposition time is 200 s, then take out the working electrode and wash with ultrapure water after the deposition is completed, complete the modification of platinum gold nanoparticles, and obtain a platinum gold nanoparticle modified glassy carbon electrode Pt-Au NPs / GCE;

[0008] S3, take SBMA and CBMA according to different molar ratios, then dissolve SBMA and CBMA in each ratio in 30 mL ultrapure water, then add 0.2 mmol ammonium persulfate (APS) and 10 mu L mercaptoacetic acid (TGA) and mix, so that a mixture is prepared; the prepared mixture is placed at 75 DEG C overnight, and is magnetically stirred at a speed of 350 revolutions per minute in a nitrogen atmosphere to perform a polymerization reaction; after 12 h of reaction, perform dialysis with ultrapure water to remove unreacted monomers or oligomers, then freeze-dry to obtain initial bodies of different numbers;

[0009] S4, take 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with a concentration of 10 mg mL −1 and N-hydroxysuccinimide (NHS) with a concentration of 10 mg mL −1 at a volume ratio of 1:1 and mix; an auxiliary solution is obtained;

[0010] S5, dissolve the initial body obtained in S3 in the auxiliary solution obtained in S4 to prepare a zwitterionic copolymer material PSB-CBMA mixture with a concentration of 2 mg mL −1 ;

[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 to CYS solution of 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 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 Schematic diagram for the construction of the anti-fouling electrochemical sensor of the present application;

[0026] Figure 2 Synthetic route of PSB-CBMA of the present application;

[0027] Figure 3 1H NMR spectrum for the characterization of PSBMA and PSB-CBMA of the present application;

[0028] Figure 4 Optimization of the ratio of zwitterionic monomers a is casein (CS) and b is whey protein (WP);

[0029] Figure 5 Comparison of the anti-fouling ability of Pt-Au NPs / GCE and Apt-PSB-CBMA / Pt-Au NPs / GCE in different concentrations of bovine serum albumin (BSA), bovine hemoglobin (HB), lysozyme (Lys), glucose (Glu), fructose (Fru) and lactose (Lac);

[0030] Figure 6 Change of the fouling of different modified electrodes in real samples with time;

[0031] Figure 7 Optimization of the concentration and time of aptamer of the present application;

[0032] Figure 8 DPV response signal of the anti-fouling electrochemical sensor of the present application combined with different concentrations of chloramphenicol;

[0033] Figure 9 Relationship curve between the concentration of chloramphenicol and the signal inhibition rate of the present application; the inset represents the corresponding standard curve;

[0034] Figure 10 Selectivity of the anti-fouling electrochemical sensor of the present application;

[0035] Figure 11 Stability of the anti-fouling electrochemical sensor of the present application;

[0036] Figure 12 Repeatability of the anti-fouling electrochemical sensor of the present application. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[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 was immersed in the electrochemical probe solution, scanned by DPV in the potential range of -0.2 V~0.6 V, and the signal inhibition rate was calculated by recording the DPV signals before and after detection; the amount of CAP combined with Apt was reflected by the signal inhibition rate; the amount of CAP combined with Apt was calculated by the following formula: Figure 7 It can be seen that the signal inhibition rate is the highest and tends to be stable when the aptamer concentration is 2 µmol L −1 , so 2 µmol L −1 is selected as the working concentration of the anti-fouling sensor. In addition, the incubation time is also optimized under the same conditions, and the experimental results show that 50 min is the best incubation time.

[0045] (6) Establishment of sensing method: the anti-fouling electrochemical sensor constructed in the above step was immersed in 200 μL of chloramphenicol standard solution with a concentration of 0.01, 0.05, 0.1, 1, 10, 50, 100 ng mL −1 , and after incubation for 60 min, the working electrode to be detected was washed with ultrapure water to remove the chloramphenicol that was not firmly combined; then the working electrode to be detected was immersed in the electrochemical probe solution, scanned by differential pulse voltammetry (DPV) in the potential range of -0.2 V~0.6 V, and the signal inhibition rate (SIR) was calculated by recording the current signals before and after detection. Figure 9 According to the signal inhibition rate and the logarithmic value of the chloramphenicol concentration, the standard curve is y = 3.22 x + 10.87, (R 2 = 0.999, S / N = 3).

[0046] (7) 100 ng mL −1 of chloramphenicol and 1 μg mL −1 of amoxicillin, kanamycin, clindamycin, erythromycin, penicillin and their mixtures were used as detection objects, and the prepared anti-fouling electrochemical aptamer sensor was used for detection. The results are shown in Figure 10 . The electric signal change values of amoxicillin, kanamycin, clindamycin, erythromycin and penicillin are very low, which are 4.2%, 5.3%, 1.9%, 5.8% and 3.3% respectively; while the signal inhibition rates of chloramphenicol and their mixtures are 15.9% and 16.8% respectively, indicating that the sensor has excellent selectivity.

[0047] Example 2

[0048] Construction and application of an anti-fouling electrochemical sensor surface of a zwitterionic copolymer anti-fouling surface are as follows:

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

[0050] (2) Modification of platinum-gold nanoparticles: The GCE was polished with alumina powder and 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 4 mL containing 1 mmol L −1 HAuCl4 and 1 mmol L −1 H2PtCl6. Constant potential deposition of platinum-gold alloy was performed using constant potential technology, with a constant potential of 0.1 V and a deposition time of 200 s. After deposition, the working electrode was removed; it was then washed with ultrapure water to obtain a working electrode modified with platinum-gold nanoparticles (Pt-Au NPs / GCE).

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

[0052] (4) Anti-fouling performance: The prepared different zwitterionic copolymer anti-fouling surfaces and Pt-Au NPs / GCE were incubated with 10.0 mg mL −1 of whey protein and casein for 30 min. The working electrodes to be tested were immersed in the electrochemical probe solution and scanned by DPV in the potential range of -0.2 V to -0.6 V. The signal inhibition rate was calculated by recording the current response values before and after incubation, and the anti-fouling ability of the anti-fouling sensor interface with different proportions of zwitterionic copolymer was compared more intuitively. Figure 4 (a, b) It can be concluded that the signal inhibition rate of PSB-CBMA2 / Pt-Au NPs / GCE is always the lowest, indicating that it has the best anti-fouling ability. It will be used for the construction of the subsequent anti-fouling electrochemical sensor.

[0053] The prepared different modified GCEs were immersed in 0.1, 1.0, 10.0 mg mL −1 of bovine serum albumin, lysozyme, hemoglobin, glucose, lactose and fructose solutions for 30 min. The anti-fouling sensors to be tested were immersed in the electrochemical probe solution and scanned by DPV in the potential range of -0.2 V to -0.6 V. The signal inhibition rate was calculated by recording the current response values before and after incubation, and the anti-fouling ability of the different anti-fouling electrochemical sensors was compared more intuitively. Figure 5 The milk, egg and honey were diluted 100 times with pH 7.4 10.0 mmol L −1 of PBS buffer solution. The prepared different modified GCEs were immersed in the above solutions for 0.5 h, 1 h, 1.5 h, 2 h and 2.5 h, respectively, and scanned by DPV in the potential range of -0.2 V to -0.6 V. The signal inhibition rate was calculated by recording the current response values before and after incubation. As shown in Figure 6 , the results show that the signal inhibition rate of Apt-PSB-CBMA / Pt-Au NPs / GCE in milk, honey and egg is as low as 2.5%, 1.2% and 1.6% at an incubation time of 0.5 h, and the signal inhibition rate is only 4.3%, 1.6% and 4.9% at an incubation time of 2.5 h, which is much lower than that of Pt-Au NPs / GCE (97.4%, 57.5% and 95.5%).

[0054] Anti-fouling principle of zwitterionic copolymer anti-fouling surface: The zwitterionic copolymer is connected with the electrochemical sensor through Au-S / Pt-S to construct an anti-fouling surface. The zwitterionic copolymer has excellent hydrophilicity and electrical neutrality, and can establish a stable hydration layer with water molecules through hydrogen bonding or ionic solvation, which acts as a physical and energy barrier to hinder the adhesion of dirt, effectively reducing the non-specific adsorption of non-detection substances during detection.

[0055] Example 3

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

[0057] The electrochemical experiments were all performed on IVIUM electrochemical workstation.

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

[0059] The preparation method of PBS was as follows: 35.8 g of sodium hydrogen phosphate (Na2HPO4·12H2O) and 15.6 g of sodium dihydrogen phosphate (NaH2PO4·2H2O) were accurately weighed and placed in a 500 mL volumetric flask, and then ultrapure water was added to the mark to obtain a 0.2 mol L −1 Na2HPO4 solution and a 0.2 mol L −1 NaH2PO4 solution; 324 mL of 0.2 M Na2HPO4 solution and 76 mL of 0.2 mol L −1 NaH2PO4 solution were measured by a measuring cylinder, respectively, and then 3.6 g of sodium chloride (NaCl) was added, and after mixing, a PBS solution with a pH of 7.4 and a concentration of 0.2 mol L −1 was obtained, and it was placed at room temperature.

[0060] The preparation method of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]) was as follows: 164.62 mg of potassium ferricyanide (K3[Fe(CN)6]), 184.17 mg of potassium ferrocyanide (K4[Fe(CN)6]) and 1491.02 mg of potassium chloride (KCl) were accurately weighed. The 0.2 mol L −1 pH 7.4 PBS solution was diluted to 0.01 mol L −1 , and then the above-mentioned drugs were dissolved in 0.01 mol L −1 pH 7.4 PBS solution as a solvent, and finally the volume was made up to a 100 mL volumetric flask to obtain a bright yellow solution, i.e. 5 mmol L −1 The electrochemical probe (K3[Fe(CN)6] / K4[Fe(CN)6] containing 0.2 mol / L KCl) solution was wrapped with tin foil paper and stored in a 4 ℃ refrigerator for standby use.

[0061] In the present application, the current response values before and after the incubation of the modified electrode are recorded by DPV (the electric signal before incubation is I0, and the electric signal after incubation is I), and the electric signal change value (ΔI=I0-I) and the electric signal suppression rate (Signal Suppression (%) = [(I0-I) / I0]×100) are calculated by the values.

[0062] Example 4

[0063] The application of the anti-fouling electrochemical sensor in the actual chloramphenicol detection sample is as follows:

[0064] (1) Milk, honey and egg are selected as the to-be-detected samples, and the standard addition recovery experiment is used to verify the actual application performance of the constructed sensor in the to-be-detected samples.

[0065] (2) The milk, honey and egg are diluted by 1% in a pH 7.4 0.01 mol L −1 PBS buffer solution: different concentrations of chloramphenicol standard solution are added into the diluted three food samples, so that the solution after standard addition contains 0.5 ng mL −1 , 5 ng mL −1 and 50.0 ng mL −1 chloramphenicol, and then the anti-fouling electrochemical sensor prepared in Example 2 is used for detection to obtain the standard addition recovery rate in the sample. The standard-added sample is detected by the enzyme-linked immunoassay method to verify the results obtained by the constructed sensor to ensure the accuracy of the analysis; the specific results are shown in Table 1 below:

[0066] Sample Spiked concentration (ng mL−1) Limit of detection (ng mL−1) Recovery rate (mean ± RSD, n=3, %) Recovery rate by ELISA (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 anti-fouling electrochemical sensor prepared in the present application can sensitively detect chloramphenicol in the to-be-detected sample, and the method has a broad application prospect in food matrices.

[0069] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined 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 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; 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 CYS solution at a volume ratio of PSB-CBMA mixture to CYS solution of 18:

1. −1 The cysteine ​​(CYS) solution was mixed to obtain a PSB-CBMA / CYS solution; S7. Place the Pt-Au NPs / GCE obtained in S2 into 200 μL of 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.

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) = 10:0, 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 3, characterized in that, 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.

6. The method for preparing an antifouling electrochemical sensor according to claim 3, characterized in that, In S7, the sequence of the CAP aptamer is 5'-SH-(CH2)6-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTAG-3'.

7. The 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 an antifouling electrochemical sensor.

Images