Impedance visualization dual-mode sensor for citrinin detection

Abstract

The invention belongs to the technical field of mycotoxin rapid detection, and particularly discloses a novel portable sensing device which is used for impedance / visual dual-mode detection of penicillium citrinum. CIT quantitative detection can be realized through two modes of impedance change and color development length change; no professional equipment is needed for length measurement, and data can be read conveniently and quickly; furthermore, the impedance change is measured through a small electrochemical workstation, and the concentration information obtained by the two signals can be subjected to cross validation, so that the detection reliability is improved. Meanwhile, the detection sensitivity is higher in an impedance mode, and the defect that the visual detection sensitivity is low can be effectively overcome; the CIT detection range of the sensor disclosed by the invention is 0.001 to 200ng / mL.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid detection of mycotoxins, and specifically discloses a novel portable sensor device for impedance / visualization dual-mode detection of Penicillium citrinum. Background Art

[0002] Citrinin (CIT) is a mycotoxin produced by fungi such as Penicillium and Aspergillus, and is widely found in dairy products, fruits, cereals and brewed foods. As a common mycotoxin, citrinin has significant health hazards to humans and animals, may cause nervous system, liver or kidney diseases, and has potential hazards such as genotoxicity, carcinogenicity, embryotoxicity and teratogenicity. In order to ensure food safety, it is crucial to develop appropriate detection methods. High performance liquid chromatography and liquid chromatography-mass spectrometry have the advantages of high accuracy and high sensitivity, but require high equipment and technical requirements; although enzyme-linked immunosorbent assay and immunochromatography are low-cost, they have relatively low sensitivity. At the same time, detection based on a single optical signal change is prone to false positive or false negative detection errors.

[0003] Dual-mode sensing is a sensor technology that combines two different sensing mechanisms. This technology can detect target analytes simultaneously or separately through two different signal output pathways (or "modes") on a single sensor platform. Dual-mode sensing can generally provide richer and more accurate information than single-mode biosensing, detecting the same target through two different signal pathways. If one of the signal pathways is interfered with, the other pathway can still provide reliable information, thereby reducing false positive and false negative results and improving the sensitivity and accuracy of detection. Complex matrices may contain many interfering substances. Single-mode biosensors are easily affected by these interfering substances, while dual-mode biosensors can provide complementary information through different sensor modes. If one mode is interfered with, the other mode can compensate, thereby enhancing the overall anti-interference ability. At the same time, different signal pathways have different sensitivities, and dual-mode sensing can more flexibly select detection modes according to the different requirements for detection sensitivity in different application scenarios.

[0004] There is currently no dual-mode sensor designed for citrinin detection. Summary of the invention

[0005] To solve the above problems, the present invention discloses an impedance visualization dual-mode sensor for citrinin detection, which can realize CIT quantitative detection through two modes: impedance change and color development length change.

[0006] The present invention includes the following technical solutions:

[0007] An impedance visualization dual-mode sensor for citrinin detection.

[0008] The sensor is based on FTO conductive glass and includes a double-electrode structure including a detection area and a signal output area;

[0009] The detection region is modified with Cu@Zn-oxalate to form Cu@Zn-oxalate / FTO;

[0010] Optionally, a nucleic acid aptamer capable of specifically binding to citrinin CIT is immobilized on the Cu@Zn-oxalate / FTO;

[0011] The signal output region is modified with Prussian blue PB by electrodeposition.

[0012] Furthermore, in the above-mentioned impedance visualization dual-mode sensor for citrinin detection, the preparation of the Cu@Zn-oxalate comprises the following steps:

[0013] DMF was mixed with ultrapure water, and Cu(NO3)2 and Zn(NO3)2·6H2O were added in sequence and stirred. Oxalic acid solution and hydrochloric acid were then added for ultrasonic treatment. The solution was heated and refluxed to obtain a turbid solution, which was centrifuged to obtain a precipitate, which was washed and dried.

[0014] Furthermore, in the above-mentioned impedance visualization dual-mode sensor for citrinin detection, the mass ratio of Cu(NO3)2 and Zn(NO3)2·6H2O is 1:1-1:4; preferably 1:3.

[0015] Furthermore, in the above-mentioned impedance visualization dual-mode sensor for citrinin detection, the nucleic acid sequence of the nucleic acid aptamer that can specifically bind to citrinin is shown as SEQ ID NO.1.

[0016] Furthermore, in the above-mentioned impedance visualization dual-mode sensor for citrinin detection, the nucleic acid aptamer capable of specifically binding to citrinin is prepared by rolling circle amplification.

[0017] Furthermore, in the above-mentioned impedance visualization dual-mode sensor for citrinin detection, in the rolling circle amplification, the primer sequence is shown in SEQ ID NO.2; the template chain sequence is shown in SEQ ID NO.3; the lock ring probe sequence is shown in SEQ ID NO.4; and the formed circular probe sequence is shown in SEQ ID NO.5.

[0018] The present invention also discloses a method for preparing the impedance visualization dual-mode sensor for citrinin detection, comprising the following steps:

[0019] 1) Double electrode construction: Cut the FTO electrode to the required size, clean and remove surface contaminants, and dry it; Use brown insulating tape to cover the treated FTO except the signal output area, and then place it in the configured electrodeposition solution; Use constant voltage deposition method on the electrochemical workstation to deposit; After the deposition is completed, clean and dry, and use laser etching technology to obtain the required electrode pattern. The designed electrode pattern consists of a detection area and a signal output area;

[0020] 2) The Cu@Zn-oxalate composite material is fully ground, dispersed in deionized water, prepared into a suspension, dropped on the detection area of ​​the above electrode, and transferred to an oven for drying and fixing, and then a mixed solution of EDC and NHS is continued to be dropped on the detection area to activate the carboxyl groups in the composite material.

[0021] Furthermore, the above preparation method also includes 3) aspirating a nucleic acid aptamer that can specifically bind to citrinin and dropping it into the detection area for reaction at low temperature, so that COOH in Cu@Zn-oxalate and NH2 in the nucleic acid aptamer undergo a bonding reaction.

[0022] The present invention also discloses a method for using the impedance visualization dual-mode sensor for citrinin detection, which can realize CIT quantitative detection through two modes: impedance change and color development length change.

[0023] Furthermore, the quantitative detection range of the above CIT is 0.001 to 200 ng / mL.

[0024] In some embodiments, the sensor is based on FTO conductive glass and is powered by an ordinary AA battery.

[0025] Structure Figure 1 As shown in A, the bipolar electrode structure is first processed by laser etching technology, and then Prussian blue (PB) is modified to the signal output area in the middle of the bipolar electrode by electrodeposition. Cu@Zn-oxalate is prepared and added and modified to the detection area of ​​the chip. During the detection, a rolling circle amplification reaction is carried out in a centrifuge tube, and after the reaction, it is added to the detection area. The designed product is fixed on Cu@Zn-oxalate / FTO through the chemical bonding of amino-carboxyl groups, resulting in an increase in the electrode surface impedance. The designed primer contains the nucleotide sequence of the CIT aptamer. Once the aptamer binds to the target, the two form a complex and fall off the surface of the material, resulting in interference with the electronic transmission of the detection part, which in turn affects the electrochromic change of the PB in the signal output area under a certain voltage, and the length of the conversion to colorless Prussian white (PW).

[0026] Detection effect Figure 1As shown in Figure B, when only Cu@Zn-oxalate is modified, it presents a small impedance (curve a); after the amplification product is introduced on the modified electrode surface, the impedance increases significantly due to the increase in the interfacial electron transfer resistance (curve b); after the test substance is added, most of the complex falls off the electrode surface, and the impedance on the sensing platform decreases (curve c). The PW lengths in the three states a, b, and c are also significantly different, which are 23 mm, 4 mm, and 12 mm, respectively. Therefore, the sensor can effectively detect different concentrations of CIT through both impedance and visualization modes.

[0027] In some embodiments, EIS responses of different CIT concentrations in impedance sensing mode:

[0028] like Figure 2 As shown in A, within the range of 0.001 to 200 ng / mL, as the CIT concentration increases, the amplified product falls off, resulting in a decrease in the Ret value.

[0029] Figure 2 B indicates that ΔRet increases with increasing CIT concentration within the range of 0.001 to 200 ng / mL, and ΔRet indicates the difference between Ret without and with CIT addition.

[0030] Figure 2 C shows the relationship between ΔRet and lg(C CIT ng / mL), and the linear equation is ΔRet=395.5lg(C CIT ng / mL)+1612, (R 2 =0.992). The detection limit was 0.37 pg / mL (S / N=3).

[0031] Figure 2 D shows that the sensor Prussian white output length L and CIT concentration show a good linear relationship in the range of 0.1-200 ng / mL, and the relationship is L=0.1156C CIT +1.39, where L is in mm, C PEF The unit is ng / mL. 2 = =0.997, detection limit is 0.019 ng / mL (S / N = 3).

[0032] Through dual-mode detection, the sensor can achieve sensitive detection of CIT in the concentration range of 0.001 to 200 ng / mL. The detection results of the two modes can be cross-validated, effectively improving the accuracy of the detection.

[0033] The present invention has the following beneficial effects:

[0034] 1) Dual-mode detection to improve accuracy: The sensor combines impedance sensing and visual detection modes to perform dual detection of the target (CIT) at the same time. This design not only provides more detection data points, but also can significantly improve the accuracy and reliability of detection through cross-validation of the two detection results.

[0035] 2) High sensitivity and wide detection range: The sensor shows good detection performance in the CIT concentration range of 0.001 to 200 ng / mL, with a wide detection range. At the same time, its detection limit is as low as 0.37 pg / mL (in impedance sensing mode) and 0.019 ng / mL (in visual detection mode), showing extremely high sensitivity and meeting the needs for accurate detection of low-concentration CIT.

[0036] 3) Good linear relationship and easy quantification: Both the relationship between ΔRet and lg (CCIT ng / mL) in the impedance sensing mode and the relationship between the Prussian white output length L and the CIT concentration in the visual detection mode show a strong linear relationship, and the linear equation is clear, which is convenient for quantification and analysis of the test results.

[0037] 4) Easy to operate and implement: The preparation process of the sensor is relatively simple. The bipolar electrode structure can be processed and modified by laser etching technology and electrodeposition modification technology. At the same time, the detection process is also relatively simple. It only needs to perform rolling circle amplification reaction in a centrifuge tube and then drop the reaction product into the detection area. This design makes the sensor easier to operate and implement in practical applications.

[0038] 5) Broad application prospects: Due to the advantages of high sensitivity, wide detection range, and easy operation, the sensor has broad application prospects in environmental monitoring, biomedical research, food safety testing, etc. Especially in the detection of biological molecules such as CIT, the sensor is expected to become an important detection tool. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .(A) Sensor detection mechanism diagram, (B) EIS and color development length test results under different states:

[0040] (a)Cu@Zn-oxalate / FTO, (b)NH2-dsDNA / Cu@Zn-oxalate / FTO,

[0041] (c) CIT / NH2-dsDNA / Cu@Zn-oxalate / FTO, where the CIT concentration was 100 ng / mL;

[0042] Figure 2.Dual-mode detection results of the sensor chip at different CIT concentrations:

[0043] (A) Impedance spectrum of CIT concentration in the range of 0.001-200 ng / mL, (B) ΔRet value corresponding to CIT concentration in the range of 0.001-200 ng / mL, (C) Linear relationship between ΔRet value and logarithmic value of CIT concentration in the range of 0.001-200 ng / mL, (D) Linear relationship between CIT concentration and color development length in the range of 0.1-200 ng / mL;

[0044] Figure 3 . Electron microscopy images of the synthesized materials A, B: Zn-oxalate, C, D: Cu@Zn-oxalate;

[0045] Figure 4 .XRD patterns of synthesized Zn-oxalate MOF and Cu@Zn-oxalate MOF;

[0046] Figure 5 .(A) The mass ratio of Cu(NO3)2 to Zn(NO3)2·6H2O in Cu@Zn-oxalate materials (a: 1:1, b: 1:2, c: 1:4 and d: 1:3), (B) voltage optimization, (C) T4 DNA ligase ligation time optimization and (D) RCA amplification time optimization;

[0047] Figure 6 (A) Anti-interference ability of the sensor to other toxins and (B) stability investigation. The concentrations of CIT and other toxins were both 100 ng / mL. DETAILED DESCRIPTION

[0048] The technical scheme in the embodiment of the present invention is clearly and completely described below, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The reagent or instrument used in the embodiment of the present invention do not indicate the manufacturer, and are all conventional reagent products that can be obtained by commercial purchase.

[0049] Example 1

[0050] Preparation of Cu@Zn-oxalate composites

[0051] First, 30 mL of N,N-dimethylformamide (DMF) solution was mixed with 15 mL of ultrapure water. Then 30 mg of Cu(NO3)2 and 90 mg of Zn(NO3)2·6H2O were added in sequence and stirred thoroughly. Then 300 μL of 0.75 M oxalic acid solution (prepared with DMF as solvent) and 3 mL of hydrochloric acid (3 M) were added to the solution and ultrasonically treated for 5 minutes. The solution was then transferred to a 100 mL round-bottom flask, heated and stirred under reflux for 24 hours, and a light blue turbid solution was obtained at 60 ° C. After the reaction was completed, the turbid solution was centrifuged at 8000 rpm for 5 minutes to obtain a light blue precipitate. The precipitate was washed with DMF, anhydrous ethanol and ultrapure water in sequence, and finally dried overnight at 50 ° C in vacuum.

[0052] For comparison, other ratios of Cu@Zn-oxalate (Cu(NO3)2:Zn(NO3)2·6H2O mass ratios of 1:1, 1:2 and 1:4, respectively) were prepared using the same steps and in the same proportions.

[0053] Example 2

[0054] Sensor Construction

[0055] 1) Electrode construction: First, the FTO electrode was cut to the required size (61mm×16mm). Subsequently, it was placed in a 0.1M sodium hydroxide solution and boiled for 30 minutes. After cooling, it was ultrasonically cleaned in toluene, acetone, anhydrous ethanol and ultrapure water in turn to remove surface contaminants. Finally, after drying under a nitrogen (N2) flow, it was stored in a watch glass for later use. Next, an electrodeposition solution (5mM HCl, 5mM K3[Fe(CN)6] and 5mM FeCl3) was prepared. The treated FTO was covered with brown insulating tape except for the signal output area, and then placed in the prepared electrodeposition solution. Deposition was performed using a constant voltage deposition method (0.3V, 75s) on an electrochemical workstation. After deposition, it was rinsed with ultrapure water and placed in a 60°C oven for 12 hours. After being removed from the oven, the desired electrode pattern was obtained using laser etching technology. The designed electrode pattern consisted of a 1cm×1cm detection area and a 26mm×2.5mm signal output area. Finally, in order to prevent the subsequent dripping of liquid from seeping out, a customized brown insulating tape (1.3 cm × 1.3 cm × 0.15 mm) was pasted around the detection area.

[0056] 2) Preparation of nucleic acid aptamers: In this study, we used rolling circle amplification (RCA) to prepare amplification products. The DNA sequences are shown in Table 1 below.

[0057] Table 1 DNA sequence list

[0058]

[0059] First, 2.5 μL of primer, 2.5 μL of template chain and 3 μL of locked circle probe were mixed in a 1 mL centrifuge tube, annealed in a 95°C constant temperature metal bath for 5 minutes, and then cooled to room temperature to form a circular probe (after annealing, part of the Primer and P-Circle bases are complementary paired to assemble into a circular probe. Subsequently, T4 DNA ligase catalyzes adjacent 5'-P and 3'-OH to form a phosphodiester bond to obtain a circular template. That is, the main sequence of the circular probe is the same as P-Circle).

[0060] The preparation method of RCA template is as follows: 8 μL circular probe solution, 10×T4 DNA ligase reaction buffer (2 μL, 5 U / μL), 2 μL protective agent and 7 μL ultrapure water, and finally 1 μL T4 DNA ligase is added for ligation reaction (37°C, 2 hours), and then heated at 65°C for 10 minutes to inactivate T4 DNA ligase. Next, RCA amplification reaction is performed, which contains 1 μL phi29 DNA polymerase (10 U / μL), 10 μL phi29 DNA polymerase reaction buffer (10×), 14 μL dNTP (10 mM), 20 μL RCA template and 55 μL ultrapure water, and the total reaction volume is 100 μL. The mixture is incubated at 37°C for 90 minutes. At this point, the amplification process is completed, and the product (amplification product (nucleic acid aptamer) is obtained by cyclic amplification based on the circular template under the action of phi29 polymerase) is stored at 4°C for later use.

[0061] 3) Modify the detection area of ​​the electrode with Cu@Zn-oxalate:

[0062] The prepared Cu@Zn-oxalate composite material was fully ground, dispersed in deionized water, and prepared into a 2 mg / mL suspension. 20 μL was dropped on the detection area of ​​the above-mentioned electrode, and it was transferred to a 60°C oven for drying for 3 hours to fix the Cu@Zn-oxalate on the electrode. Subsequently, 20 μL of a mixed solution of EDC and NHS (containing 100 mM EDC and 25 mM NHS) was continued to be dropped in the detection area and reacted at room temperature for one hour to activate the carboxyl groups in the composite material.

[0063] 4) Nucleic acid aptamer and Cu@Zn-oxalate bonding

[0064] Then, 20 μL of the amplified product was dripped into the detection area and reacted at 4°C for 12 hours to allow the COOH in the Cu@Zn-oxalate and the NH2 in the amplified product to fully bond and react.

[0065] After the above 1)-4) sensors are constructed, CIT series concentration solutions are added dropwise to complete the quantitative detection of CIT.

[0066] Example 3

[0067] Material characterization

[0068] The morphology of the synthesized materials was characterized by SEM. Figure 3 In A and B, it can be seen that the morphology of Zn-oxalate is an irregular polyhedron with a diameter of about 5 to 10 μm, while the composite material (3C, D) Cu@Zn-oxalate is a cube with holes with a diameter of about 1 to 5 μm. The crystal structure of the material was deeply analyzed by XRD. Figure 4 It can be seen that the Cu@Zn-oxalate MOF composite material exhibits good crystal structure and crystallinity, which is consistent with the results reported in previous literature. 2+ The doping did not damage the skeleton integrity of Zn-oxalate MOF, maintaining its original morphology and structure, further verifying the stability and reliability of the composite material.

[0069] Example 4

[0070] Optimization of experimental conditions for sensor preparation

[0071] The test environment often affects the performance of the sensor. Only by adjusting to the optimal test environment can the sensor achieve the best detection effect. First, the Cu content in the Cu@Zn-oxalate composite material was optimized. As the Cu content increased, the impedance of Cu@Zn-oxalate first decreased and then increased ( Figure 5 A), so the mass ratio of Cu(NO3)2: Zn(NO3)2·6H2O is 1:3 as the best ratio. Due to the change of materials, the impedance value generated by it changes, so we also adjusted the applied voltage of the sensor and found that in the same time, if Figure 5 As shown in B, when only Cu@Zn-oxalate is dripped on the FTO electrode, voltages of -1.2V, -1.3V, -1.4V, -1.5V, -1.6V and -1.7V are applied to the electrode respectively. When the voltage is -1.6V, PB has been completely converted into PW. In order to have a clear distinction and considering the detection range and detection mechanism, it is decided to use -1.5V as the external voltage of the sensor. In order to obtain the rolling circle amplification distance readout sensing platform under the best conditions, it is necessary to optimize the relevant parameters in the amplification process. Since the T4 ligase connection time and amplification time of the rolling circle amplification reaction have a significant effect on the amplification efficiency, the reaction time of the two is optimized. Figure 5C can be seen that after adding T4 ligase, the PW length increases with the increase of reaction time. When the ligation time reaches 2h, the PW length no longer changes, which indicates that the optimal ligation time of T4 enzyme for amplification reaction is 2h. Figure 5 As shown in Figure D, with the increase of amplification time, the length of PW in the signal output region gradually shortened, and PW basically disappeared after 90 minutes of reaction, and completely disappeared after 100 minutes. Therefore, 90 minutes was selected as the optimal reaction time for subsequent experiments.

[0072] Example 5

[0073] Research on Anti-interference and Stability of Sensors

[0074] Based on the optimized detection conditions, the selectivity of the sensor was tested. The toxins involved included ochratoxin A (OTA), vomitoxin (DON), T-2 toxin (T-2), zearalenone (ZEN) and CIT, all at a concentration of 100 ng / mL. The experimental results showed that the sensor only had a significant detection response to CIT, indicating that the sensor designed in this work has the ability to selectively detect CIT ( Figure 6 A). Similarly, the stability of the sensors was analyzed. After the sensors were prepared, they were stored in a refrigerator at 4°C and tested after 4, 8, and 12 days. Figure 6 B shows that the deviation of the PW color change length value is less than 5%, indicating that the sensor can maintain stability for about 12 days. The impedance mode test results are similar to those of the visualization mode.

[0075] Example 6

[0076] Actual sample analysis

[0077] In order to prove the feasibility of the sensor in actual detection. First, 5g corn flour (purchased from a local supermarket) was weighed and dispersed in 20mL acetonitrile-water (70%, v / v) solution, and ultrasonically treated for 2min. After standing for 10min, centrifuged at 10000rpm for 15min and repeated twice, and finally diluted 100 times with PBS buffer solution to obtain the supernatant. The prepared sample was then used to dilute CIT standard solutions of different concentrations of 10-200ng / mL. The results are shown in Table 2. After detection, it was found that the recovery rate deviation was within 5%, indicating that the detection performance of the sensor was good.

[0078] Table 2 Analysis results of CIT in corn flour samples by the constructed sensor. (n=3)

[0079]

[0080] It can be seen from the results of the above embodiments that in order to achieve rapid and sensitive detection of CIT, we have successfully developed a portable impedance / visualization dual-mode sensor, which can realize quantitative detection of CIT through two modes: impedance change and color length change. High sensitivity and wide detection range: The sensor shows good detection performance in the CIT concentration range of 0.001 to 200 ng / mL, and has a wide detection range. At the same time, its detection limit is as low as 0.37pg / mL (in impedance sensing mode) and 0.019ng / mL (in visual detection mode), showing extremely high sensitivity, which can meet the needs of accurate detection of low-concentration CIT. No professional equipment is required for length measurement, and data can be read quickly and conveniently. Further, the impedance change is measured by a small electrochemical workstation, and the concentration information obtained from the two signals can be cross-validated to improve the reliability of the detection. At the same time, the detection sensitivity is higher in the impedance mode, which can effectively make up for the defect of low sensitivity of visual inspection.

[0081] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An impedance visualization dual-mode sensor for citrinin detection, characterized in that: The sensor is based on FTO conductive glass and includes a double-electrode structure including a detection area and a signal output area; The detection region is modified with Cu@Zn-oxalate to form Cu@Zn-oxalate / FTO; Optionally, a nucleic acid aptamer capable of specifically binding to citrinin CIT is immobilized on the Cu@Zn-oxalate / FTO; The signal output region is modified with Prussian blue PB by electrodeposition.

2. The impedance visualization dual-mode sensor for citrinin detection according to claim 1, characterized in that: The preparation of the Cu@Zn-oxalate comprises the following steps: DMF was mixed with ultrapure water, and Cu(NO3)2 and Zn(NO3)2·6H2O were added in sequence and stirred. Oxalic acid solution and hydrochloric acid were then added for ultrasonic treatment. The solution was heated and refluxed to obtain a turbid solution, which was centrifuged to obtain a precipitate, which was washed and dried.

3. The impedance visualization dual-mode sensor for citrinin detection according to claim 2, characterized in that: The mass ratio of Cu(NO3)2 and Zn(NO3)2·6H2O is 1:1-1:4, preferably 1:

3.

4. The impedance visualization dual-mode sensor for citrinin detection according to claim 1, characterized in that: The nucleic acid sequence of the nucleic acid aptamer capable of specifically binding to citrinin is shown in SEQ ID NO.

1.

5. The impedance visualization dual-mode sensor for citrinin detection according to claim 4, characterized in that: The nucleic acid aptamer capable of specifically binding to citrinin is prepared by rolling circle amplification.

6. The impedance visualization dual-mode sensor for citrinin detection according to claim 5, characterized in that: In the rolling circle amplification, the primer sequence is shown in SEQ ID NO.2; the template chain sequence is shown in SEQ ID NO.3; the locked circle probe sequence is shown in SEQ ID NO.4; and the formed circular probe sequence is shown in SEQ ID NO.

5.

7. The method for preparing a dual-mode sensor according to any one of claims 1 to 6, characterized in that: The following steps are involved: 1) Double electrode construction: Cut the FTO electrode to the required size, clean and remove surface contaminants, and dry it; Use brown insulating tape to cover the treated FTO except the signal output area, and then place it in the configured electrodeposition solution; Use constant voltage deposition method on the electrochemical workstation to deposit; After the deposition is completed, clean and dry, and use laser etching technology to obtain the required electrode pattern. The designed electrode pattern consists of a detection area and a signal output area; 2) The Cu@Zn-oxalate composite material is fully ground, dispersed in deionized water, prepared into a suspension, dropped on the detection area of ​​the above electrode, and transferred to an oven for drying and fixing, and then a mixed solution of EDC and NHS is continued to be dropped on the detection area to activate the carboxyl groups in the composite material.

8. The method for preparing a dual-mode sensor according to claim 7, characterized in that: Also includes: 3) The nucleic acid aptamer that can specifically bind to citrinin is aspirated and dropped into the detection area for reaction at low temperature, so that the COOH in Cu@Zn-oxalate and the NH2 in the nucleic acid aptamer can react with each other.

9. The method for using the impedance visualization dual-mode sensor for citrinin detection according to any one of claims 1 to 6, characterized in that: CIT quantitative detection is achieved through two modes: impedance change and color development length change.

10. The method of use according to claim 9, characterized in that: The CIT quantitative detection range is 0.001 to 200 ng / mL.

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