A method for detecting ochratoxin a in milk based on a dual recognition electrochemical sensor

Abstract

The application discloses a method for detecting ochratoxin A in milk based on a double-identification electrochemical sensor, and compared with other OTA detection methods, the sensor has a wider linear range (0-300nM) and a lower LOD (0.09nM) under optimal conditions; the sensor has high repeatability, stability and specificity, and the sensor has good feasibility in detection of different types of milk samples, and a good recovery rate of 95.4%-105.6% is obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of food detection, and particularly relates to a method for detecting ochratoxin A in milk based on a double-identification electrochemical sensor. BACKGROUND

[0002] Mycotoxin contamination in food and agricultural products is becoming a serious worldwide problem. Ochratoxin is a group of structurally related isocoumarin-derived mycotoxins, mainly produced by some fungi of the genus Aspergillus and Penicillium. According to its chemical structure, ochratoxin can be mainly divided into three types, namely ochratoxin A (OTA), ochratoxin B (OTB) and ochratoxin C (OTC). Among them, OTA is considered to be the most abundant and toxic, and OTA is detected in various agricultural products, including grains, coffee, cocoa, beer, wine, and dairy products and meat products of animals that eat OTA-contaminated grains.

[0003] After people and animals eat food contaminated by OTA, OTA accumulates in the body, causing slow metabolism; OTA mainly endangers the kidneys of people and animals, has a high carcinogenic, teratogenic and mutagenic effect, and is believed to be related to human Balkan nephropathy and urinary system tumors, and the International Cancer Research Agency has positioned it as a class 2B carcinogen. Due to its harmful effects on human health and its impact on economic losses, OTA contamination has received increasing attention worldwide.

[0004] Currently, many countries have set limits on the concentration of OTA in agricultural food, and the detection of OTA is very important, which is the key to preventing and reducing food safety incidents.

[0005] Currently, existing OTA detection and identification methods include microbial culture method, immunological detection method, molecular biology method, etc.; among them, in the detection process of the microbial culture method, the amplification of the signal needs to be realized by the growth of individual cells into colonies, so the detection period is relatively long, and viable strains in the environment can enter a dormant state and become unculturable. In the immunological detection method of OTA, such as latex agglutination method, immunodiffusion method, enzyme-linked immunosorbent assay (ELISA), immunomagnetic bead method and immunoprecipitation method, among them, ELISA is the most commonly used and mature technology, but the preparation of antibodies is relatively cumbersome, and the acquisition of antibodies has limitations. For those molecules that are too small to have binding sites or have poor immunogenicity and high toxicity, the acquisition of antibodies is difficult to achieve, and the affinity of antibodies to the measured toxin is low, and the detection often has limitations.

[0006] Polymerase chain reaction (PCR) is a molecular biology technique developed in the 1980s for the rapid in vitro amplification of specific nucleic acid fragments. In PCR detection, as the DNA template amplification products grow exponentially, the target fragment of the analyte can be amplified millions of times, greatly improving detection sensitivity. At the same time, the development of rapid PCR technology can shorten the detection time to 20 minutes. However, PCR technology is prone to contamination, is cumbersome to operate, and can only perform qualitative analysis. Furthermore, dairy samples may contain some inhibitory compounds that may affect PCR amplification and produce false positive results. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting ochratoxin A in milk based on a dual-recognition electrochemical sensor.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for detecting ochratoxin A in milk based on a dual-recognition electrochemical sensor, comprising,

[0011] V2AlC sample was compounded with diethylene glycol diacrylate (PDDA) to prepare PDDA@V2CTx dispersion;

[0012] The glassy carbon GCE electrode was polished to a mirror finish using Al2O3 powder. PDDA@V2CTx dispersion was then dropped onto the GCE surface and dried to obtain a PDDA@V2CTx / GCE surface.

[0013] AgNPs dispersion was dropwise added to the surface of PDDA@V2CTx / GCE to obtain AgNPs / PDDA@V2CTx / GCE;

[0014] A complex solution was prepared by mixing and incubating the amino aptamer of OTA with OTA, and then dropwise onto AgNPs / PDDA@V2CTx / GCE to obtain Apt / AgNPs / PDDA@V2CTx / GCE.

[0015] Catechol was electropolymerized on the surface of Apt / AgNPs / PDDA@V2CTx / GCE to obtain Ct-Apt / AgNPs / PDDA@V2CTx / GCE, which was then eluted with anhydrous methanol to obtain MIP-Apt / AgNPs / PDDA@V2CTx / GCE.

[0016] Different concentrations of OTA standard solution were dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated. The solutions were then tested in an electrochemical workstation, and the linear standard curve of current response difference ΔI versus OTA concentration was calculated.

[0017] After pretreatment, the milk was dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated. The current response difference ΔI was obtained in an electrochemical workstation, and the concentration of OTA in the milk was obtained by substituting it into the above linear standard curve.

[0018] In a preferred embodiment of the method described in this invention, the preparation method of the PDDA@V2CTx dispersion includes,

[0019] The V2AlC sample of the MAX phase was added to a solution containing HF and HCl;

[0020] Stir at room temperature, then heat the system to 45–50°C and stir for 12–24 hours.

[0021] The powder was obtained by centrifugation and repeatedly washed with distilled water until the pH value was 6. It was then washed with anhydrous ethanol and centrifuged. The precipitate was then vacuum dried to obtain V2CTx precipitate.

[0022] The V2CTx precipitate was dispersed in a methanol solution of PDDA, placed in an autoclave, and reacted at 100–110 °C for 6–8 h. After washing with water and drying, PDDA@V2CTx was obtained.

[0023] In a preferred embodiment of the method described in this invention, the amino aptamer of OTA is mixed with OTA and incubated to obtain a complex solution, wherein the concentration of the amino aptamer of OTA is 10 μM, the concentration of OTA is 10 μM, the ratio of the amino aptamer of OTA to OTA is 30 μL:30 μL, the incubation temperature is 37°C, and the incubation time is 30–70 min.

[0024] As a preferred embodiment of the method described in this invention, the amino aptamer of the OTA has the nucleotide sequence shown in SEQ ID NO.1.

[0025] In a preferred embodiment of the method described in this invention, the incubation time is 60 minutes.

[0026] As a preferred embodiment of the method described in this invention, the step of electropolymerizing catechins on the surface of Apt / AgNPs / PDDA@V2CTx / GCE is carried out in an electrochemical workstation in CV mode with the following parameters: scan potential of -0.2 to 0.6 V, scan rate of 80 mV / s, and 10 scans.

[0027] In a preferred embodiment of the method described in this invention, MIP-Apt / AgNPs / PDDA@V2CTx / GCE is obtained by elution with anhydrous methanol, wherein the elution time is 20–60 min.

[0028] As a preferred embodiment of the method described in this invention, the step of dropping OTA standard solutions of different concentrations onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode for incubation is wherein the incubation temperature is 37°C and the incubation time is 30–70 min.

[0029] The concentrations of the OTA standard solution were 0, 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 nM.

[0030] As a preferred embodiment of the method described in this invention, the tests are conducted in an electrochemical workstation, wherein the electrochemical workstation includes a three-electrode system consisting of an Ag / AgCl reference electrode, a platinum wire counter electrode, a MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode, and an electrolyte.

[0031] The electrolyte includes potassium ferricyanide solution;

[0032] The working scanning mode is DPV mode, with parameters of -0.2V to 0.6V, frequency of 0.05mV / s, and 1 scan.

[0033] In a preferred embodiment of the method described in this invention, the pretreatment of the milk includes,

[0034] The milk sample was heated, centrifuged, and the middle layer of skim milk was recovered.

[0035] The skim milk in the middle layer was filtered through a sterile membrane, and the resulting sample was diluted with PBS to obtain the sample to be tested.

[0036] The milk samples included goat milk, sheep milk, and cow milk.

[0037] Beneficial effects of this invention:

[0038] (1) The present invention provides an electrochemical sensor with dual recognition elements for detecting OTA in milk. Compared with other OTA detection methods, the method of the present invention has a wider linear range (0-300 nM) and a lower LOD (0.09 nM).

[0039] (2) The method of the present invention has high repeatability, stability and specificity. The feasibility of detection in different types of dairy samples was investigated by sample spike recovery experiment, and a good recovery rate (95.4% to 105.6%) was obtained. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the 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. Wherein:

[0041] Figure 1 The images show the CV and EIS characterization diagrams measured during the electrochemical sensor construction process in Example 1 of this invention; where A is the CV diagram of the electrochemical sensor construction process, B is the EIS characterization diagram, a, a' represents GCE, b, b' represents PDDA@V2CTx / GCE, c, c' represents AgNPs / PDDA@V2CTx / GCE, d, d' represents Apt / AgNPs / PDDA@V2CTx / GCE, e, e'Ct-Apt / AgNPs / PDDA@V2CTx / GCE, f, f' represents MIP-Apt / AgNPs / PDDA@V2CTx / GCE, g, g' represents OTA / MIP-Apt / AgNPs / PDDA@V2CTx / GCE, and C is a magnified view of a portion of the EIS diagram.

[0042] Figure 2 The figures show the DPV response diagrams for different OTA concentrations and the calibration curves for measuring OTA in this embodiment of the invention; wherein, A represents the DPV response of different OTA concentrations from a to k: 0, 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, 300 nM, and B is the calibration curve for measuring OTA (S / N = 3).

[0043] Figure 3 This is a stability comparison chart of the sensor in the embodiment of the present invention; where A is the peak response intensity of the 6 electrodes modified under the same conditions, B is the DPV current response of the sensor to OTA and its interfering substances (S / N=3), and C is a stability comparison chart of the sensor.

[0044] Figure 4This is a preferred scanning rate diagram for the preparation of Apt / AgNPs / PDDA@V2CTx / GCE in an embodiment of the present invention. Detailed Implementation

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0046] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0048] Instruments and reagents in this invention:

[0049] (1) Reagents

[0050] OTA amino aptamer (synthesized by Shanghai Sangon Biotech Co., Ltd.):

[0051] 5'-NH2-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA (see SEQ ID NO. 1)-3;

[0052] Silver nitrate standard solution, trisodium citrate, V2AlC powder, HF, concentrated hydrochloric acid, anhydrous methanol, anhydrous ethanol, ultrapure water, OTA standard solution (provided by Tianjin Alta Technology Co., Ltd.), catechol, and polydiallyl dimethyl ammonium chloride (PDDA) are all commercially available products.

[0053] (2) Equipment

[0054] The sensor device is a CHI660E electrochemical workstation (Shanghai, China), which uses a three-electrode system, with the Ag / AgCl electrode as the reference electrode, the platinum wire as the counter electrode, and the GCE as the working electrode.

[0055] Example 1

[0056] (1) Preparation of AgNPs dispersion:

[0057] 100 mL of ultrapure water was used to prepare a 0.01% AgNO3 solution, which was then placed in an Erlenmeyer flask. A magnetic stir bar was added, and the flask was heated on a constant temperature electromagnetic stirrer. After boiling for 2 minutes, 2.5 mL of 1% trisodium citrate solution was quickly added. The mixture was stirred and heated until the solution turned pale yellow, thus generating silver nanoparticles (AgNPs).

[0058] Continue heating for 10 minutes, then stop heating, stir and cool to room temperature, and store at 4°C to obtain AgNPs dispersion.

[0059] (2) Preparation of PDDA@V2CTx:

[0060] A 5.0 g sample of V2AlC in the MAX phase was added to a solution containing 12 mL of HF (50%) and 8 mL of HCl (36%).

[0061] After stirring at room temperature for 30 minutes, the system was heated to 45°C and stirred under these conditions for 24 hours.

[0062] The powder was obtained by centrifugation and washed repeatedly with distilled water until the pH value was 6. Then it was washed three times with anhydrous ethanol and centrifuged again. The precipitate was dried under vacuum at 100°C for 6 hours.

[0063] 50 mg of V2CTx precipitate was further dispersed in 50 mL of methanol solution containing a predetermined amount of PDDA (1% by mass). The above mixture was placed in a 100 mL autoclave lined with polytetrafluoroethylene and reacted at 110 °C for 8 h. After several water washings and drying, PDDA@V2CTx was finally obtained.

[0064] (3) Constructing sensors

[0065] First, the glassy carbon (GCE) electrode (Shanghai Chenhua CHI1043mm glassy carbon disk electrode) was polished to a mirror finish using Al2O3 powder of (0.3 and 0.05 μm) respectively. Then, it was ultrasonically washed in ethanol / water solution (volume ratio 1:1) for 5 minutes. Finally, the electrode surface was dried with N2.

[0066] The dispersion of PDDA@V2CTx (10 μL 1 mg / mL, ultrapure water as solvent) was then dropped onto the GCE surface and dried in air to obtain the PDDA@V2CTx / GCE surface.

[0067] A complex solution was formed by incubating 30 μL of 10 μM OTA amino aptamer with 30 μL of 10 μM OTA at 37 °C for 1 h.

[0068] AgNPs dispersion (10 μL) was dropped onto the surface of PDDA@V2CTx / GCE to prepare AgNPs / PDDA@V2CTx / GCE;

[0069] Then, 10 μL of the complex solution was dropped onto AgNPs / PDDA@V2CTx / GCE to fix it on the surface, thus obtaining Apt / AgNPs / PDDA@V2CTx / GCE;

[0070] Electropolymerization of 1 mM catechol was performed on the surface of Apt / AgNPs / PDDA@V2CTx / GCE (using CV mode in an electrochemical workstation, parameters: scan potential -0.2 to 0.6 V, scan rate 80 mV / s, 10 scans) to obtain Ct-Apt / AgNPs / PDDA@V2CTx / GCE.

[0071] Then, the chamber was eluted with anhydrous methanol for 40 min to obtain the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode;

[0072] OTA standard solution (10 μM) was dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated for 1 h to obtain the OTA / MIP-Apt / AgNPs / PDDA@V2CTx / GCE sensor.

[0073] (4) Electrochemical characterization of the sensor construction process

[0074] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are two techniques for monitoring dynamic interfacial processes of electrode reactions and sensor construction processes.

[0075] A. Immerse the bare electrode and the modified electrode separately in 5.0 mM [Fe(CN)6] of a three-electrode system. 3- / 4- The interfacial characteristics of different electrodes were studied in the electrolyte. All electrochemical measurements were performed on a CHI660E electrochemical workstation using a three-electrode system consisting of an Ag / AgCl (saturated potassium chloride) reference electrode, a platinum wire counter electrode, and a GCE (or modified electrode). The electrolyte was a potassium ferricyanide solution.

[0076] The scanning mode is CV mode, with parameters of -0.2V to 0.6V, frequency of 0.05mV / s, and 2 scans.

[0077] Test results are as follows Figure 1 As shown in Figure A, the peak current of PDDA@V2CTx / GCE (Figure 1 Ab) was significantly higher than that of the bare electrode ( Figure 1 Aa) indicates that the metal carbide has good electrical conductivity;

[0078] For AgNPs / PDDA@V2CTx / GCE( Figure 1 Ac), because AgNPs and PDDA@V2CTx together increase the conductivity of the electrode, the peak value increases again;

[0079] For Apt / AgNPs / PDDA@V2CTx / GCE( Figure 1 A d), because Apt hinders electron transport on the electrode surface, it increases the surface resistance of the electrode, and the current value is greatly reduced;

[0080] For Ct-Apt / AgNPs / PDDA@V2CTx / GCE( Figure 1 (A e) After the electropolymerization of catechol, the current value further decreases due to the high impedance of the polymer layer;

[0081] For MIP-Apt / AgNPs / PDDA@V2CTx / GCE ( Figure 1 As shown in Figure A f), after removing the target substance (OTA) with the eluent, the cavity of the polymer membrane is exposed, and the current value increases, but the current value is lower than that of Apt / AgNPs / PDDA@V2CTx / GCE.

[0082] For OTA / MIP-Apt / AgNPs / PDDA@V2CTx / GCE ( Figure 1 As the OTA template molecules re-enter the cavity, the current value is greatly reduced (A g).

[0083] B. Adjust the mode to EIS (voltage: 0.23V), and measure the impedance curve, such as... Figure 1 As shown in B and 1C;

[0084] As can be seen, the results of EIS and CV are consistent, which indicates that the construction of the dual-recognition electrochemical sensor was successful.

[0085] Example 2

[0086] This embodiment provides a process for constructing a linear relationship between the current response difference and OTA concentration in a method for detecting ochratoxin A. The main steps are as follows:

[0087] (1) Test conditions: All electrochemical measurements were performed on a CHI660E electrochemical workstation using a three-electrode system consisting of an Ag / AgCl (saturated potassium chloride) reference electrode, a platinum wire counter electrode, and a GCE; the electrolyte was [Fe(CN)6]. 3- / 4- Solution;

[0088] The scanning mode is DPV mode, with parameters of -0.2V to 0.6V, frequency of 0.05mV / s, and 1 scan.

[0089] (2) The MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrodes prepared in Example 1 were placed in OTA solutions of different concentrations and incubated for 1 h.

[0090] After the electrode surface has dried, immerse the electrode in the [Fe(CN)6] three-electrode system. 3- / 4- The test was conducted in solution;

[0091] The OTA concentrations were 0, 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 nM, respectively.

[0092] (3) Test as follows Figure 2 As shown in Figure A, the DPV signal decreases as the OTA concentration increases from 0 to 300 nM;

[0093] Figure 2 B shows the linear relationship between the current response difference and the OTA concentration (ΔI = I0 - I, where I is the sensor's current signal to the STR, and I0 is the background signal): ΔI = 0.454C OTA +26.43, R 2 =0.9964.

[0094] Based on S / N=3, the detection limit (LOD) is calculated to be 0.09 nM. The method of the present invention has a wider detection range and a lower detection limit.

[0095] After pretreatment, the sample was dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated. The current response difference ΔI was obtained in an electrochemical workstation and then substituted into the above linear relationship to obtain the OTA concentration in the milk.

[0096] Example 3

[0097] Repeatability, specificity, and stability:

[0098] (1) Repeatability test:

[0099] Six duplicate electrodes were constructed according to the method for constructing the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode in Example 1 to verify the repeatability of the construction method.

[0100] Six MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrodes were immersed in a 5.0 mM [Fe(CN)6] three-electrode system.3- / 4- The interfacial characteristics of the electrodes were studied in the electrolyte. All electrochemical measurements were performed on a CHI660E electrochemical workstation using a three-electrode system consisting of an Ag / AgCl (saturated potassium chloride) reference electrode, a platinum wire counter electrode, and a GCE (or modified electrode). The electrolyte was a potassium ferricyanide solution.

[0101] The scanning mode is DPV mode, with parameters of -0.2V to 0.6V, frequency of 0.05mV / s, and 1 scan.

[0102] like Figure 3 As shown in Figure A, the repeatability of the sensor was evaluated using six different electrodes under the same experimental conditions, and the calculated relative standard deviation was 2.62% (n=3), which indicates that the sensor has good repeatability.

[0103] (2) Specificity assay:

[0104] Different types of solutions of the same concentration (10mM) (OTA solution, AFB1 aflatoxin B1 solution, Penicillin solution, AFM1 aflatoxin M1 solution, and STR streptomycin solution) were dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode prepared in Example 1, incubated for 1 h, and then dried before being measured on a CHI660E electrochemical workstation.

[0105] A three-electrode system consisting of an Ag / AgCl (saturated potassium chloride) reference electrode, a platinum wire counter electrode, and a GCE (or modified electrode) was used. The electrolyte was a potassium ferricyanide solution. The workstation was set to DPV mode, with the voltage set to -0.2V to 0.6V, the frequency to 0.05mV / s, and the number of scans to 1.

[0106] The measurement results are as follows Figure 3 As shown in Figure B, except for OTA, the peak currents of several interfering substances are very strong and basically the same as the blank, indicating that the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode has good specificity.

[0107] (3) Stability determination:

[0108] Given that sensor stability is crucial in practical applications, the current signal is periodically measured using DPV every three days.

[0109] The MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode prepared in Example 1 was placed in a refrigerator at 4°C and stored in the dark with a dust cover for 21 days, and measured every three days.

[0110] Set the workstation to DPV mode, set the voltage to -0.2V to 0.6V, the frequency to 0.05mV / s, and the number of scans to 1.

[0111] See results Figure 3 As shown in Figure C, after 3, 6, 9, 12, 15, 18 and 21 days, the peak current remained at 98.05%, 95.8%, 94.5%, 92.59%, 91.32%, 90.25% and 88.99% of the initial current value, respectively, indicating that the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode has good stability.

[0112] Example 4

[0113] This example provides a detection method for a real sample:

[0114] To verify the applicability of the sensor in the safety testing of dairy products, a spiked recovery method was used to detect OTA in goat milk, sheep milk, and cow milk.

[0115] (1) Take 25 ml of goat milk, goat milk and cow milk samples respectively, heat at 70℃ for 1 hour, and then centrifuge at 3500 rpm for 30 minutes;

[0116] (2) The obtained sample included the top layer of fat, the middle layer of skim milk and protein precipitate. The skim milk in the middle layer was carefully recovered and filtered through a sterile membrane. The obtained sample was diluted 10 times with PBS.

[0117] (3) The standard OTA solution was dropped into the experimental sample, and the final concentrations of OTA in the sample were 1, 10 and 50 nM.

[0118] (4) The experimental sample was immersed in the surface of the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode of Example 1. The change in the current value of the electrode surface was measured according to the method in Example 2. The concentration was calculated by plugging it into the linear relationship.

[0119] This embodiment uses the spiked recovery method, and the results are shown in Table 1.

[0120] Table 1

[0121]

[0122] As shown in Table 1 below, the recovery rate is between 96.13% and 103.21%, and the RSD is between 0.6% and 1.3%, indicating that the sensor has great application potential in the rapid detection of OTA in real samples.

[0123] Example 5

[0124] Under the experimental conditions for preparing Apt / AgNPs / PDDA@V2CTx / GCE in Example 1, the preferred scan rate during surface electropolymerization is provided as follows:

[0125] Electropolymerization of 1 mM catechol was performed on the surface of Apt / AgNPs / PDDA@V2CTx / GCE using CV mode on an electrochemical workstation with the following parameters: scan potential -0.2 to 0.6 V, 10 scans. Then the workstation was switched to DPV mode with voltage set to -0.2 V to 0.6 V, frequency 0.05 mV / s, and 1 scan. The results were then measured on the polymerized sensor.

[0126] The results are as follows Figure 4 As shown, ΔI increases with increasing scan speed, reaching its maximum value at 80 mv / s. Therefore, 80 mv / s is selected as the optimal scan rate.

[0127] It can be seen that the scan rate affects the sensitivity of the MIP sensor by influencing the mass transfer of template molecules embedded in the MIP membrane. At too low a scan rate, the resulting membrane is too tight, making it difficult to completely remove the template from the polymer, resulting in poor sensitivity. However, at too high a scan rate, the resulting loose and coarse polymer leads to low recognition ability and weak detection signal.

[0128] It should be noted that the above 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for detecting ochratoxin A in milk based on a dual-recognition electrochemical sensor, characterized in that: include, V2AlC sample was compounded with diethylene glycol diacrylate (PDDA) to prepare PDDA@V2CTx dispersion; The glassy carbon GCE electrode was polished to a mirror finish using Al2O3 powder. PDDA@V2CTx dispersion was then dropped onto the GCE surface and dried to obtain a PDDA@V2CTx / GCE surface. AgNPs dispersion was dropwise added to the surface of PDDA@V2CTx / GCE to obtain AgNPs / PDDA@V2CTx / GCE; A complex solution was prepared by mixing and incubating the amino aptamer of OTA with OTA, and then dropwise onto AgNPs / PDDA@V2CTx / GCE to obtain Apt / AgNPs / PDDA@V2CTx / GCE. Catechol was electropolymerized on the surface of Apt / AgNPs / PDDA@V2CTx / GCE to obtain Ct-Apt / AgNPs / PDDA@V2CTx / GCE, which was then eluted with anhydrous methanol to obtain MIP-Apt / AgNPs / PDDA@V2CTx / GCE. Different concentrations of OTA standard solution were dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated. The solutions were then tested in an electrochemical workstation, and the linear standard curve of current response difference ΔI versus OTA concentration was calculated. After pretreatment, the milk was dropped onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubated. The current response difference ΔI was obtained in an electrochemical workstation, and the concentration of OTA in the milk was obtained by substituting it into the above linear standard curve.

2. The method as described in claim 1, characterized in that: The preparation method of the PDDA@V2CTx dispersion includes, The V2AlC sample of the MAX phase was added to a solution containing HF and HCl; Stir at room temperature, then heat the system to 45–50°C and stir for 12–24 hours. The powder was obtained by centrifugation and repeatedly washed with distilled water until the pH value was 6. It was then washed with anhydrous ethanol and centrifuged. The precipitate was then vacuum dried to obtain V2CTx precipitate. The V2CTx precipitate was dispersed in a methanol solution of PDDA, placed in an autoclave, and reacted at 100–110 °C for 6–8 h. After washing with water and drying, PDDA@V2CTx was obtained.

3. The method as described in claim 1 or 2, characterized in that: The complex solution is prepared by mixing and incubating the amino aptamer of OTA with OTA, wherein the concentration of the amino aptamer of OTA is 10 μM, the concentration of OTA is 10 μM, the ratio of the amino aptamer of OTA to OTA is 30 μL:30 μL, the incubation temperature is 37℃, and the incubation time is 30 to 70 min.

4. The method as described in claim 3, characterized in that: The amino aptamer of the OTA has the nucleotide sequence shown in SEQ ID NO.

1.

5. The method as described in claim 3, characterized in that: The incubation time is 60 minutes.

6. The method as described in claim 1, characterized in that: The method involves electropolymerizing catechins on the surface of Apt / AgNPs / PDDA@V2CTx / GCE. The electropolymerization is carried out in an electrochemical workstation using CV mode with the following parameters: scan potential of -0.2 to 0.6 V, scan rate of 80 mV / s, and 10 scans.

7. The method as described in claim 1, characterized in that: The MIP-Apt / AgNPs / PDDA@V2CTx / GCE was obtained by elution with anhydrous methanol, wherein the elution time was 20–60 min.

8. The method as described in claim 1, characterized in that: The step involves dropping OTA standard solutions of different concentrations onto the MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode and incubating them at a temperature of 37°C for 30–70 min. The concentrations of the OTA standard solution were 0, 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 nM.

9. The method as described in claim 1, characterized in that: The tests were conducted in an electrochemical workstation, which included a three-electrode system consisting of an Ag / AgCl reference electrode, a platinum wire counter electrode, a MIP-Apt / AgNPs / PDDA@V2CTx / GCE electrode, and an electrolyte. The electrolyte includes potassium ferricyanide solution; The working scanning mode is DPV mode, with parameters of -0.2V to 0.6V, frequency of 0.05mV / s, and 1 scan.

10. The method according to any one of claims 1, 2, 4 to 9, characterized in that: The pretreatment of the milk includes, The milk sample was heated, centrifuged, and the middle layer of skim milk was recovered. The skim milk in the middle layer was filtered through a sterile membrane, and the resulting sample was diluted with PBS to obtain the sample to be tested. The milk samples included goat milk, sheep milk, and cow milk.

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