Method for detecting DON based on molecularly imprinted cell electrochemical sensor

Abstract

The invention discloses a method for detecting DON based on a molecularly imprinted cell electrochemical sensor, and belongs to the field of electrochemical detection. The preparation method comprises the following steps: taking an apoptotic substance Caspase-3 as a template, and preparing a molecularly imprinted electrode through electro-polymerization; then incubating the molecularly imprinted electrode and the cells together to form a molecularly imprinted cell electrochemical sensor; and finally, detecting deoxynivalenol (DON) by using the probe. The DON is detected by adopting a molecular imprinting technology, the mass production can be realized, the detection limit is low, and the sensitivity is high.

Description

Technical Field

[0001] The invention relates to a method for detecting DON based on a molecular imprinted cell electrochemical sensor, and belongs to the field of electrochemical detection. Background Art

[0002] Mycotoxins are naturally toxic secondary metabolites produced by various fungi growing on food. Mycotoxins can proliferate on food under appropriate conditions (especially high temperature and humidity) and can enter the food chain through contaminated food, thereby increasing potential health risks to humans and animals. Among them, deoxynivalenol (DON), also known as vomitoxin, is the most representative of the trichothecenes. Therefore, there is a need to develop a reliable analytical tool to detect deoxynivalenol (DON) in food in a sensitive and effective manner to properly safeguard human health.

[0003] Currently, a variety of methods have been developed to analyze and detect deoxynivalenol (DON) with high accuracy and precision; the most commonly used are chromatography and mass spectrometry, but they require complex and laborious extraction processes to maintain the desired specificity and sensitivity, and these methods also require expensive instruments and professional operation. The method of constructing an electrochemical sensor has fast detection speed, high sensitivity, good specificity, and strong stability, and shows great potential in detecting low-level target substances (deoxynivalenol (DON)).

[0004] The literature (Fe-MOF / AuNP-based ratiometric electrochemical immunosensor for the detection of deoxynivalenol in grain products) is based on antigen-antibody specific reaction, DON antibody is fixed on the electrode surface, when DON binds to the antibody, the detection is achieved through the change of electrochemical signal;

[0005] The literature (Li Tiantian, Wang Yifan, Liu Zhenhua. Research progress of electrochemical sensors in domestic food safety [J]. Progress in Analytical Chemistry, 2019, 9(1): 23-29. https: / / doi.org / 10.12677 / AAC.2019.91004) uses nucleic acid aptamers that specifically bind to DON to achieve detection through changes in electrochemical signals;

[0006] However, the chemical structure of DON is complex and similar to that of other mycotoxins (such as D3G), which can easily lead to cross-reactions in some sensors and poor detection accuracy. The detection stability of electrochemical sensors is poor, and the sensitivity cannot meet actual needs. Summary of the invention

[0007] [Technical issues]

[0008] Conventional electrochemical methods for detecting DON have problems such as poor accuracy, poor stability and low sensitivity.

[0009] [Technical solution]

[0010] In order to solve the above problems, the present invention uses apoptotic substance Caspase-3 as a template to prepare a molecular imprinting electrode by electropolymerization; then the molecular imprinting electrode and cells are co-incubated to form a molecular imprinting cell electrochemical sensor; finally, it is used for the detection of deoxynivalenol (DON). The present invention uses molecular imprinting technology to detect DON, which can be mass-produced, has a low detection limit and high sensitivity.

[0011] The first object of the present invention is to provide a method for detecting DON based on a molecular imprinted cell electrochemical sensor, comprising the following steps:

[0012] (1) Preparation of molecular imprinted electrodes:

[0013] o-phenylenediamine (o-PD) is used as a functional monomer, polyethylene glycol diacrylate (PEGDA) is used as a cross-linking agent, azobisisobutyronitrile (AIBN) is used as an initiator, and apoptotic substance Caspase-3 is used as a template molecule. The four are mixed evenly to obtain a mixture;

[0014] The mixture was electropolymerized on the surface of the screen-printed electrode by cyclic voltammetry (CV), dried, and eluted to obtain a molecularly imprinted electrode;

[0015] (2) Preparation of molecular imprinted electrochemical sensors:

[0016] Using the molecular imprinted electrode as the working electrode, a three-electrode system was constructed and connected to an electrochemical workstation to obtain a molecular imprinted (MIP) electrochemical sensor.

[0017] (3) Detection of DON:

[0018] The analyte containing DON, liver cancer cells (Hep G2), and culture medium were mixed evenly, incubated, and the supernatant was extracted;

[0019] The clear solution was added dropwise to the surface of the molecular imprinted electrode, incubated, and detected by differential pulse voltammetry (DPV) to obtain the current value at the peak position of 0.02 V, and the concentration of DON was obtained according to the standard curve.

[0020] In one embodiment of the present invention, in step (1), the dosage ratio of o-phenylenediamine (o-PD), polyethylene glycol diacrylate (PEGDA), azobisisobutyronitrile (AIBN), and the apoptotic substance Caspase-3 is 10mM:50mg:100μL:3.33mM, wherein the concentrations of o-phenylenediamine (o-PD) and the apoptotic substance Caspase-3 are relative to azobisisobutyronitrile (AIBN).

[0021] In one embodiment of the present invention, the uniform mixing in step (1) is ultrasonic mixing.

[0022] In one embodiment of the present invention, the screen-printed electrode in step (1) needs to be pre-treated before use, specifically:

[0023] 100 μL of 3 mM [Fe(CN)6] 4- / 3- The electrolyte solution was scanned by cyclic voltammetry (CV) to remove possible impurities on the electrode.

[0024] In one embodiment of the present invention, the size of the screen-printed electrode in step (1) is as follows:

[0025] Electrode size: 12*34mm, electrode thickness: 0.3mm, working electrode diameter: 4mm.

[0026] In one embodiment of the present invention, the amount of the mixture in step (1) is 8-12 μL / 4πmm 2 , more preferably 10μL / 4πmm 2 .

[0027] In one embodiment of the present invention, the parameters of cyclic voltammetry (CV) in step (1) are as follows:

[0028] The voltage was 0.0 to +1.1 V, the scan rate was 0.1 V / s, the step potential was 0.01 V, the calibration time was 5 s, and the number of cycle scans was 7.

[0029] In one embodiment of the present invention, the drying in step (1) is carried out under an infrared lamp for 1 hour.

[0030] In one embodiment of the present invention, the eluent used for elution in step (1) is a mixed solution of acetonitrile, acetic acid and ultrapure water, the volume ratio of acetonitrile, acetic acid and ultrapure water is 50:1:49, and the elution time is 60 minutes.

[0031] In one embodiment of the present invention, the three-electrode system in step (2) uses a molecular imprinting electrode as a working electrode, silver / silver chloride (Ag / AgCl) as a reference electrode, and a platinum wire (Pt) electrode as a counter electrode.

[0032] In one embodiment of the present invention, the culture medium in step (3) is DMEM culture medium; and the concentration of DON in the DON-containing test substance ranges from 0.2 to 100 pg / mL.

[0033] In one embodiment of the present invention, in step (3), the concentration of liver cancer cells (Hep G2) in the culture medium is 1*10 6 cells / mL.

[0034] In one embodiment of the present invention, the supernatant in step (3) is prepared by centrifugation at 4°C and 13,000 rpm for 15 min.

[0035] In one embodiment of the present invention, the amount of supernatant added in step (3) is 8-12 μL / 4πmm 2 , more preferably 10μL / 4πmm 2 .

[0036] In one embodiment of the present invention, the incubation on the electrode in step (3) is carried out at 37° C. for 15 minutes.

[0037] In one embodiment of the present invention, the parameters of differential pulse voltammetry (DPV) in step (3) are set as follows:

[0038] The voltage range was -0.4 to 0.5 V, the step potential was 0.01 V, and the scan rate was 0.1 V / s.

[0039] In one embodiment of the present invention, the standard curve in step (3) is: DPV (mA)=0.11243+8.96495 -4 C DON(pg / mL) , when the linear range is 0.2-100pg / mL, the correlation coefficient R 2 =0.9977 (n=3), and the lowest detection limit (LOD) was 1.93 pg / mL.

[0040] The second object of the present invention is the application of the method according to the present invention in the field of food testing.

[0041] In one embodiment of the present invention, the method can be used for the quantitative detection of DON in foods such as wheat, corn, and flour.

[0042] [Beneficial Effects]

[0043] (1) The present invention generates a molecular imprinting layer by electropolymerization on the electrode surface, which has the advantages of simple and rapid preparation, high adhesion to the electrode surface, easy control of film thickness, and high reproducibility.

[0044] (2) The present invention utilizes molecular imprinting technology to overcome the shortcomings of traditional biosensors, such as being unsuitable for field applications, bioreceptors being easily inactivated in the presence of potential inhibitors, and being limited to specific working conditions. The cavity within the MIP architecture can perfectly mimic the specific shape of the target molecule obtained after removing the template molecule, and has enhanced affinity and specificity.

[0045] (3) The present invention utilizes molecular imprinting to detect DON, while conventional methods generally focus on mass spectrometry and spectral analysis methods, which require complex and laborious extraction processes to maintain the expected specificity and sensitivity. In addition, these methods face the problem of insufficient sensitivity due to their own technical limitations.

[0046] (4) The present invention utilizes the stimulation of deoxynivalenol (DON) on cells to cause cell apoptosis, and Caspase-3 can be used as a unique biomarker for cell apoptosis detection, which can timely reflect the dynamic process of monitoring cell apoptosis and perform real-time imaging of the cell apoptosis process.

[0047] (5) The present invention combines the molecular imprinting sensor with an electrochemical workstation. When the molecular imprinting sensor is stimulated by DON, the changes in cell response are directly converted by the transducer into analyzable electrical signals, thereby achieving the purpose of detection and analysis.

[0048] (6) The molecular imprinting sensor used in the present invention has the advantages of simple electropolymerization, high-throughput production, low detection limit and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a detection flow chart of the present invention.

[0050] Figure 2 These are DPV optimization diagrams of different functional monomers, where A is before elution, B is after elution, and C is the functional monomer incubation diagram.

[0051] Figure 3 These are optimization diagrams of the functional monomer o-phenylenediamine (o-PD) of Example 1 before elution, after elution, and after re-incubation, wherein A is a DPV diagram and B is a CV diagram.

[0052] Figure 4 DPV diagram for optimizing the ratio of template molecule to functional monomer, where A is after elution and B is after incubation.

[0053] Figure 5 Optimization diagram for the number of MIP membrane cycles.

[0054] Figure 6 Optimization diagram for eluent.

[0055] Figure 7 Optimize the plot for elution time.

[0056] Figure 8 These are scanning electron microscope (SEM) characterization images of MIP and NIP, where A is before MIP elution, B is after MIP elution, C is before NIP elution, and D is after NIP elution.

[0057] Fig. 9 DPV diagrams of electrochemical detection of MIP and NIP.

[0058] Fig.10 For the construction of standard curve.

[0059] Fig.11 This is a diagram for optimizing cell dosage.

[0060] Fig.12 Optimize the incubation time.

[0061] Fig.13 The results of the detection of different mycotoxins.

[0062] Fig.14 Figure 2 is the detection accuracy diagram of different mycotoxins.

[0063] Fig.15 The effective area characterization diagram (CV) of the bare electrode, where A is the electrode scanning rate of 25-500mv s -1 Electrochemical CV diagram, B is the scan rate and current linearity diagram.

[0064] Fig.16 The effective area characterization diagram (CV) of the NIP electrode, where A is the electrode scanning rate of 25-500mv s -1 Electrochemical CV diagram, B is the scan rate and current linearity diagram. DETAILED DESCRIPTION

[0065] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0066] The raw materials and equipment in the embodiments are funded by the National Key R&D Program (2023YFF1104602).

[0067] The raw materials used in the embodiment:

[0068] Screen-printed electrode: electrode size: 12*34mm, electrode thickness: 0.3mm, working electrode diameter: 4mm.

[0069] o-Phenylenediamine, polyethylene glycol diacrylate, and azobisisobutyronitrile were from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0070] Caspase-3: Caspase-3 / CASP3 protein, Human, from Shanghai Haoyuan Biotechnology Co., Ltd.

[0071] Screen printed electrodes need to be pre-treated before use, specifically:

[0072] 100 μL of 3 mM [Fe(CN)6] 4- / 3- The electrolyte solution was scanned by cyclic voltammetry (CV) (voltage from -0.6 to +0.8 V, scan rate of 0.1 V / s, step potential of 0.01 V, equilibrium time of 5 s, and scan number of 20 circles) to remove possible impurities on the electrode.

[0073] Culture medium: DMEM medium from Thermo Fisher Scientific.

[0074] Cells: Hep G2, from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0075] Example 1

[0076] The preparation method of the molecular imprinting electrochemical sensor comprises the following steps:

[0077] (1) Preparation of molecular imprinted electrodes:

[0078] o-phenylenediamine (o-PD) was used as a functional monomer, polyethylene glycol diacrylate (PEGDA) was used as a cross-linking agent, azobisisobutyronitrile (AIBN) was used as an initiator, and the apoptotic substance Caspase-3 was used as a template molecule. The dosage ratio of o-phenylenediamine (o-PD), polyethylene glycol diacrylate (PEGDA), azobisisobutyronitrile (AIBN), and apoptotic substance Caspase-3 was 10mM:50mg:100μL:3.33mM, and ultrasonic mixing was performed for 30min to obtain a mixture.

[0079] 10 μL of the mixture was electropolymerized on the surface of the screen-printed electrode using cyclic voltammetry (CV) (voltage 0.0 to +1.1 V, scan rate 0.1 V / s, step potential 0.01 V, calibration time 5 s, 7 cycles), dried under an infrared lamp for 1 h, and eluted for 60 min using a mixed solution of acetonitrile, acetic acid, and ultrapure water in a volume ratio of 50:1:49 as an eluent to obtain a molecularly imprinted electrode;

[0080] (2) Preparation of molecular imprinted electrochemical sensors:

[0081] A molecular imprinted electrode was used as the working electrode, silver / silver chloride (Ag / AgCl) was used as the reference electrode, and a platinum wire (Pt) electrode was used as the counter electrode. A three-electrode system was constructed and connected to an electrochemical workstation to obtain a molecular imprinted (MIP) electrochemical sensor.

[0082] Afterwards, 3 mM [Fe(CN)6] 4- / 3- DPV (differential pulse voltammetry) was used as an electrochemical indicator to detect the electrochemical reaction of DON stimulating Hep G2 to release Caspase-3, thereby achieving ultra-sensitive detection of DON. The specific detection flow chart is as follows Figure 1 shown.

[0083] Example 2

[0084] (1) Optimization of different functional monomers

[0085] The functional monomer o-phenylenediamine (o-PD) in Example 1 was adjusted to 4-aminobenzoic acid (4-aminophenyl) ester (4-APA), L-cysteine ​​(L-Cys), L-arginine (L-Arg), and L-glycine (L-Gly), and the others remained consistent with Example 1.

[0086] The test results are as follows:

[0087] Figure 2 The DPV optimization diagrams of different functional monomers, where A is before elution, B is after elution, and C is the functional monomer incubation diagram. Figure 2 It can be seen that o-phenylenediamine has the highest current response and the best binding effect, so o-phenylenediamine is selected as the functional monomer.

[0088] Figure 3 The functional monomer o-phenylenediamine (o-PD) of Example 1 is optimized before elution, after elution, and after re-incubation, wherein A is a DPV graph and B is a CV graph. Figure 3 It can be seen that: before elution, the peak current is significantly lower due to the presence of non-conductive template molecules. After elution, an enhanced redox peak is observed due to the removal of the template molecules, because the removal of the template generates holes and the redox probe can easily diffuse and reach the electrode surface. However, after re-incubation in the template molecule solution, the current response decreases, which is due to the occupation of the imprinting cavity by the template molecule, resulting in the obstruction of the electron transfer channel and reducing the access sites of the redox probe to the electrode surface.

[0089] (2) Optimization of the dosage of functional monomers and template molecules

[0090] The amount of the template molecule in Example 1 was maintained, and the amount of the functional monomer was adjusted so that the molar ratio of the template molecule to the functional monomer was 1:1, 1:3, 1:5, 1:7, and 1:9, and the rest was the same as in Example 1.

[0091] The test results are as follows:

[0092] Figure 4 DPV diagram for optimizing the ratio of template molecule to functional monomer, where A is after elution and B is after incubation. Figure 4 It can be seen that: initially, the ΔI value increases with the increase in the proportion of functional monomers. When the ratio of template molecules to functional monomers reaches 1:3, ΔI reaches the maximum value, but as the content of functional monomers further increases, ΔI decreases. This is because when the content of functional monomers is low, as the content of functional monomers increases, the thickness of the poly-o-PD film increases, the number of effective holes increases, and the corresponding ΔI value also increases. However, when the content of functional monomers is too high, the relative content of template molecules is too low, and it is difficult to form enough holes, resulting in a decrease in the ΔI value. Insufficient functional monomers will result in fewer binding sites, and too many monomers will result in sparse distribution of binding sites, and the binding sites will be buried in the polymer matrix. Therefore, a molar ratio of 1:3 was selected as the optimal ratio for preparing MIP.

[0093] (3) Optimization of cyclic voltammetry (CV) parameters

[0094] The number of cyclic voltammetry (CV) cycles in Example 1 was adjusted to 3, 4, 5, 6, and 8; the others were kept consistent with Example 1.

[0095] The test results are as follows:

[0096] Figure 5 The optimization diagram of the number of cycles of MIP membrane. The number of cycles of o-PD electropolymerization affects the thickness of the MIP membrane. The number of imprinting sites in the polymer film increases with the thickness, which in turn affects the stability, sensitivity and specificity of the sensor. Figure 5 It can be seen that the optimal cycle is 7 cycles, at which time ΔI reaches a stable value. In other words, the adsorption capacity of molecular imprinting polymer has reached the optimal value. Further increasing the number of polymerization cycles will lead to the formation of an overly thick MIP layer on the electrode, hindering the elution of the internal template molecules.

[0097] (4) Eluent optimization

[0098] The eluent in Example 1 was adjusted to methanol, acetic acid, and methanol / acetic acid (volume ratio 1:1); the rest remained the same as in Example 1.

[0099] The test results are as follows:

[0100] Figure 6 This is the optimization diagram of the eluent. Whether the template molecule in the molecular imprinting polymer can be completely eluted has a great influence on the number of specific holes, which in turn affects the specific adsorption amount of the template molecule. Figure 6It can be seen that ΔI is the largest when the mixture of acetonitrile: acetic acid: ultrapure water with a volume ratio of 50:1:49 is used for elution, indicating that the eluent can completely elute the template molecules.

[0101] (5) Optimization of elution time

[0102] The eluent time in Example 1 was adjusted to 20, 40, 80, and 100 min; the rest remained the same as in Example 1.

[0103] The test results are as follows:

[0104] Figure 7 Optimize the elution time. Figure 7 It can be seen that it takes 60 min to completely elute the template molecules, but as the elution time is further extended, the ΔI value begins to decrease, which is mainly related to the damage of the MIP structure caused by the extended elution time, which in turn causes the imprinted cavity to be unable to specifically recognize and adsorb the template molecules. Therefore, 60 min was selected as the optimal elution time.

[0105] Comparative Example 1

[0106] The template molecule in step (1) of Example 1 was omitted, and the rest was kept the same as Example 1 to obtain a non-molecular imprinting (NIP) electrochemical sensor.

[0107] The molecular imprinted (MIP) electrochemical sensor prepared in Example 1 and the non-molecular imprinted (NIP) electrochemical sensor prepared in Comparative Example 1 were placed under ultraviolet light for drying, and then placed under a scanning electron microscope (SEM) for detection and characterization, and electrochemical detection was performed.

[0108] The results are as follows:

[0109] Figure 8 The following are the scanning electron microscope (SEM) characterization images of MIP and NIP, where A is before MIP elution, B is after MIP elution, C is before NIP elution, and D is after NIP elution. Figure 8 It can be seen that the surfaces of MIP and NIP were slightly flat and smooth before elution, forming a molecular imprinting film, indicating that o-PD was successfully electrodeposited on the SPCE surface. After elution, a large number of holes and protrusions appeared, which was attributed to the formation of imprinting cavities of template molecules.

[0110] Fig. 9 DPV diagrams for electrochemical detection of MIP and NIP. Fig. 9It can be seen that after the template molecule is removed, an increase in the peak is observed, which is due to the creation of holes by the template removal, resulting in the redox probe being able to diffuse easily at the electrode. After the template molecule is re-bound, the current intensity decreases, which is due to the occupation of the imprinted cavity by the template molecule, which reduces the access sites of the redox probe to the electrode surface. For the NIP, the redox peak is greatly reduced and almost unaffected when exposed to the same conditions as the MIP electrode.

[0111] Example 3 Preparation of detection standard curve

[0112] 0.01-200 pg / mL of deoxynivalenol (DON) was taken and acted on 1*10 6 cells / mL cells for 3 h, and after extracting the supernatant, the electrochemical workstation was connected to 3 mM [Fe(CN)6] 4- / 3- As an electrochemical indicator, the DPV method was used for detection. The stimulation of cells by different concentrations of fungal toxins caused changes in current, and a linear regression model was established between the peak current and the concentration of fungal toxins.

[0113] Fig.10 For the construction of the standard curve. Fig.10 It can be seen that the change in DON concentration is linearly correlated with the change in peak current value. When the linear range is 0.2-100 pg / mL, the standard curve equation obtained by linear fitting is I DPV (mA)=0.11243+8.96495 -4 C DON(pg / mL) , correlation coefficient R 2 =0.9977 (n=3), and the lowest detection limit (LOD) was 1.93 pg / mL. The above results show the feasibility of the constructed molecular imprinted cell electrochemical sensor for detecting DON.

[0114] Example 4

[0115] A method for detecting DON based on molecular imprinted cell electrochemical sensor comprises the following steps:

[0116] The DON test substance, liver cancer cells (Hep G2) and culture medium were mixed evenly, centrifuged at 4°C and 13000 rpm for 15 min, and the supernatant was extracted; the concentration of liver cancer cells (Hep G2) in the culture medium was 1*10 6 cells / mL;

[0117] 10 μL of supernatant was added to the surface of the molecular imprinting electrode and incubated at 37°C for 15 min. The electrode was detected by differential pulse voltammetry (DPV) (voltage range -0.4 to 0.5 V, step potential 0.01 V, scan rate 0.1 V / s) to obtain the current value at the peak position of 0.02 V. The concentration of DON was obtained according to the standard curve.

[0118] The error between the DON concentration in the DON test object and the actual detected concentration is within 10%.

[0119] Example 5

[0120] (1) Cell concentration optimization

[0121] Cells in logarithmic growth phase were taken, treated with trypsin-EDTA digestion solution, counted and plated, washed with phosphate buffered saline (PBS) after inducing cell apoptosis with DON, and lysed in lysis buffer. At the same time, ultrasonic treatment was performed in ice water for 15 minutes. The lysate was centrifuged at 13000 rpm for 15 minutes at 4°C, and the supernatant was collected and immediately used for cell apoptosis determination.

[0122] Fig.11 This is a cell dosage optimization diagram. Fig.11 It can be seen that as the number of cells increases, the semicircle diameter of the Nyquist curve gradually increases, and the corresponding impedance value also increases continuously. However, when the cell concentration reaches 1*10 7 cells / mL, the impedance value decreases, indicating that the cells on the electrode have reached saturation. Therefore, 1*10 6 cells / mL is the optimal cell concentration for subsequent detection of molecular imprinted cell electrochemical sensors.

[0123] (2) Optimization of incubation time:

[0124] After collecting the cell supernatant, it was dropped on the electrode for different incubation times before cell apoptosis assay.

[0125] Fig.12 Optimize the incubation time. Fig.12 It can be seen that ΔI increases with the increase of incubation time and reaches the maximum value at 15 min, indicating that the template molecules have been saturatedly adsorbed by the imprinting cavity. Therefore, 15 min was selected as the optimal incubation time.

[0126] Example 6 Specificity Test

[0127] Take 0.15pg / mL of DON, ZEN, and AFB1 respectively and act on 1*10 6cells / mL cells for 3 h, and after extracting the supernatant, the electrochemical workstation was connected to 3 mM [Fe(CN)6] 4- / 3- As an electrochemical indicator, the DPV method was used for detection. Under the same experimental conditions, the peak current change response was compared when different fungal toxins stimulated cell apoptosis.

[0128] The test results are as follows:

[0129] Fig.13 The test results of different mycotoxins are shown in Figure 2. Fig.13 It can be seen that the maximum peak positions of DON, ZEN and AFB1 detected by DPV are 0.02V, 0.11V and 0.15V respectively. It can be concluded that the method of the present invention can make a good distinction between the above three common fungal toxins, which confirms the specificity of this method.

[0130] Fig.14 The detection accuracy of different mycotoxins is shown in Figure 2. Fig.14 It can be seen that the RSDs of ZEN, DON and AFB1 are 1.37%, 0.23% and 5.82% respectively.

[0131] Example 7 Characterization of electrode effective area

[0132] Fig.15 A is a bare electrode with a current of 25-500mv s -1 Cyclic voltammetry scans were performed at a scan rate of Fig.15 In B, it can be seen that there is a good linear relationship between the scan rate and the current, y = 7.334x-1.115, R 2 = 0.9997, and the effective area A is 0.329 cm 2 , The cyclic voltammetry (CV) parameters were set as: voltage from -0.6 to +0.8 V, step potential of 0.05 V, and calibration time of 6 s.

[0133] Fig.16 A is a NIP electrode with a current of 25-500mv s -1 Cyclic voltammetry scans were performed at a scan rate of Fig.16 In B, it can be seen that there is a good linear relationship between the scan rate and the current, y = 1.555x + 0.437, R 2 = 0.9879, and its effective area A is 0.070 cm 2 , which is smaller than the effective area of ​​the bare electrode, indicating that a molecular imprinting film is formed on its surface, which hinders the transfer of electrons. The cyclic voltammetry (CV) parameters are set as follows: voltage of -0.6 to +0.8 V, step potential of 0.05 V, and calibration time of 6 s.

[0134] The effective area of ​​the NIP electrode is lower than that of the bare electrode, mainly because the prepared NIP electrode has successfully electropolymerized the imprinted material, forming a polymer film on the electrode surface, which has a certain hindering effect on the transmission of current, further confirming the synthesis of molecular imprinting electrochemical sensors.

[0135] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for detecting DON based on molecular imprinted cell electrochemical sensor, characterized in that: The steps include: (1) Preparation of molecular imprinted electrodes: o-phenylenediamine (o-PD) is used as a functional monomer, polyethylene glycol diacrylate (PEGDA) is used as a cross-linking agent, azobisisobutyronitrile (AIBN) is used as an initiator, and apoptotic substance Caspase-3 is used as a template molecule. The four are mixed evenly to obtain a mixture; The mixture was electropolymerized on the surface of the screen-printed electrode by cyclic voltammetry (CV), dried, and eluted to obtain a molecularly imprinted electrode; (2) Preparation of molecular imprinted electrochemical sensors: Using the molecular imprinted electrode as the working electrode, a three-electrode system was constructed and connected to an electrochemical workstation to obtain a molecular imprinted (MIP) electrochemical sensor. (3) Detection of DON: The analyte containing DON, liver cancer cells (Hep G2), and culture medium were mixed evenly, incubated, and the supernatant was extracted; The clear solution was added dropwise to the surface of the molecular imprinted electrode, incubated, and detected by differential pulse voltammetry (DPV) to obtain the current value at the peak position of 0.02 V, and the concentration of DON was obtained according to the standard curve.

2. The method according to claim 1, characterized in that In step (1), the dosage ratio of o-phenylenediamine (o-PD), polyethylene glycol diacrylate (PEGDA), azobisisobutyronitrile (AIBN), and apoptotic substance Caspase-3 is 10mM:50mg:100μL:3.33mM.

3. The method according to claim 1, characterized in that The amount of the mixture in step (1) is 8-12 μL / 4πmm 2 .

4. The method according to claim 1, characterized in that: The parameters of cyclic voltammetry (CV) in step (1) are as follows: The voltage was 0.0 to +1.1 V, the scan rate was 0.1 V / s, the step potential was 0.01 V, the calibration time was 5 s, and the number of cycle scans was 7.

5. The method according to claim 1, characterized in that The eluent used for elution in step (1) is a mixed solution of acetonitrile, acetic acid and ultrapure water, the volume ratio of acetonitrile, acetic acid and ultrapure water is 50:1:49, and the elution time is 60 minutes.

6. The method according to claim 1, characterized in that The incubation on the electrode in step (3) is carried out at 37° C. for 15 min.

7. The method according to claim 1, characterized in that The parameters of differential pulse voltammetry (DPV) in step (3) are set as follows: The voltage range was -0.4 to 0.5 V, the step potential was 0.01 V, and the scan rate was 0.1 V / s.

8. The method according to claim 1, characterized in that The standard curve in step (3) is: DPV (mA)=0.11243+8.96495 -4 C DON(pg / mL) , when the linear range is 0.2-100pg / mL, the correlation coefficient R 2 =0.9977 (n=3), and the limit of detection (LOD) was 1.93 pg / mL.

9. The method according to claim 1, characterized in that: In step (2), the three-electrode system uses a molecular imprinting electrode as a working electrode, silver / silver chloride (Ag / AgCl) as a reference electrode, and a platinum wire (Pt) electrode as a counter electrode.

10. Application of the method according to any one of claims 1 to 9 in the field of food testing.

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