Sensing detection system based on aptamer antibody double-recognition sandwich model and detection method thereof

Abstract

The invention relates to the technical field of biosensing, in particular to a sandwich model sensing system based on aptamer and antibody double recognition and a detection method. The system comprises an aptamer, an antibody, a DNA tetrahedron, a glassy carbon electrode and a detection system of the glassy carbon electrode, the aptamer is a single-stranded DNA (deoxyribonucleic acid) and is used for specifically capturing the mycotoxin ZEN; the antibody specifically recognizes ZEN and is connected to the top of a DNA tetrahedron, the tetrahedron is formed by self-assembly of nucleic acid with a specific sequence, the top end is modified with amino and connected with the antibody, and the bottom is connected with a glutathione copper nano-cluster on the surface of a glassy carbon electrode through an SH group. The system disclosed by the invention realizes high-sensitivity detection of ZEN, has the advantages of strong specificity, good stability, simplicity and convenience in operation and the like, and is suitable for the field of food safety detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to a sensing detection system based on an aptamer-antibody double recognition sandwich model and a detection method thereof. Background Art

[0002] Mycotoxins, as a class of naturally occurring biotoxins, have long been a major threat to global livestock and poultry feed and human food safety. It is estimated that about 25% of the world's grains are contaminated by mycotoxins to varying degrees each year. Zearalenone (ZEN), as a secondary metabolite produced by Fusarium fungi, is widely present in corn, barley, wheat, sorghum and other grains, becoming one of the most common mycotoxins.

[0003] ZEN is a heat-stable, non-steroidal toxic metabolite with estrogen-like effects. Its chemical structure is stable and difficult to decompose even under high temperature conditions. This toxin not only pollutes a wide range of areas, but also poses great harm, posing a serious threat to the health of humans and animals, mainly manifested in reproductive toxicity, immunotoxicity, and hepato-kidney toxicity. ZEN and its metabolites can competitively bind to estrogen receptors, causing reproductive problems such as vulvar edema, false estrus, infertility, and miscarriage in sows, and have similar effects on the male reproductive system. At the same time, ZEN is also carcinogenic and can cause DNA damage, posing a potential risk to the long-term health of humans and animals.

[0004] It is particularly noteworthy that infants and children are more sensitive to ZEN and more susceptible to its effects because their physiological development is not yet fully mature. In order to protect the health of infants and young children, the European Commission has clearly stipulated that the maximum content of ZEN in cereal-based foods produced for children is 20μg / kg, which is much lower than the maximum amount allowed in adult cereals (100μg / kg).

[0005] In terms of mycotoxin detection technology, traditional national standard detection methods include high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and enzyme-linked immunosorbent assay (ELISA). Although high performance liquid chromatography (HPLC) has high accuracy and can separate and detect a variety of mycotoxins, it has disadvantages such as high equipment cost, complex pre-treatment steps, and high operation requirements, which limit its application in large-scale detection; thin layer chromatography (TLC) is easy to operate and low cost, but the accuracy is relatively low, the operator's experience requirements are high, and it is not suitable for large-scale detection. In recent years, enzyme-linked immunosorbent assay (ELISA) has been widely used as a rapid detection technology due to its advantages such as simple operation, short time and low cost. However, the ELISA method has the problems of false negatives caused by low antibody titer and false positives caused by complex sample environment, and its detection limit is difficult to meet the current detection needs for low-concentration mycotoxins. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a sandwich model sensing system based on dual recognition of aptamers and antibodies, which realizes high sensitivity and high specificity detection of fungal toxins (ZEN) by combining the high specificity of aptamers and the strong dual recognition binding force of antibodies. The sensing system uses DNA tetrahedron as a signal amplification platform and combines electrochemiluminescence (ECL) detection technology to achieve qualitative and quantitative detection of ZEN.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention constructs a sandwich model sensing system based on dual recognition of aptamers and antibodies, comprising aptamers and antibodies, DNA tetrahedrons, and glassy carbon electrodes. The aptamer AP2 is a single-stranded DNA, and its nucleic acid sequence is shown in SEQ ID NO: 6, which can specifically capture fungal toxins (ZEN), and the antibody can specifically recognize fungal toxins (ZEN); the antibody is connected to the top of the DNA tetrahedron, and the DNA tetrahedron is anchored on the surface of a glassy carbon electrode (GCE), and the surface of the GCE is coated with glutathione copper nanoclusters (GSH-Cu NCs);

[0009] Further, the DNA tetrahedron is composed of four nucleic acid single strands S1-S4, which are hybridized with a four-fold amount of a single-stranded nucleic acid S0 modified with ferrocene (Fc). The nucleic acid single strands S1-S4 self-assemble to form a DNA tetrahedron, the tetrahedron top is modified with an amino group, the tetrahedron edge is modified with Fc, the 3' end of the S1 nucleic acid single strand is modified with SH, the 5' end of the S2 nucleic acid single strand is modified with SH, the 5' end of the S3 nucleic acid single strand is modified with NH2, the 3' end of the S4 nucleic acid single strand is modified with SH, the 5' end and the 3' end of the single-stranded nucleic acid S0 are modified with Fc, the DNA sequences of the four nucleic acid single strands S1-S4 are shown in SEQ ID NOs: 1-4, and the DNA sequence of the single-stranded nucleic acid S0 is shown in SEQ ID NO: 5;

[0010] Furthermore, the DNA tetrahedron apex is connected by NH 2 Connected with antifungal toxin antibodies, the bottom of the DNA tetrahedron is connected to the GSH-Cu NCs on the surface of the glassy carbon electrode through the SH group;

[0011] The sensing system also includes a detection system for measuring electrochemiluminescence (ECL) signals to quantitatively detect fungal toxins (ZEN);

[0012] The present invention also constructs a method for detecting fungal toxins (ZEN) using a sensor system, comprising the following steps:

[0013] The sample containing ZEN and aptamer Ap2 were mixed and incubated at a volume ratio of 4:3 to allow ZEN to bind to the aptamer;

[0014] The aptamer complex bound to ZEN and H 1 The DNA chain was introduced into the surface of the sensing system, and the aptamer Ap2 and H 1 The molar ratio of DNA chains is 1:1, which allows ZEN to bind to the antibody to form a sandwich structure. 1 The DNA sequence of the DNA strand is shown in SEQ ID NO: 7;

[0015] The ECL signal was measured by an electrochemiluminescence detector, and the concentration of ZEN was determined based on the signal intensity;

[0016] The invention is applied in qualitative and quantitative detection of ZEN.

[0017] The beneficial effects of the present invention are:

[0018] The present invention constructs a sensing system for rapid detection of mycotoxins in crops, which combines the high specificity of aptamers and the strong binding force of antibodies to achieve high-sensitivity and high-specificity detection of mycotoxins (ZEN); the DNA tetrahedron structure is stable and has a fixed size, which improves the stability and sensitivity of detection; the ECL technology has the advantages of low background signal, high sensitivity and wide dynamic range, so that the sensing system can accurately measure the content of ZEN in a wide concentration range, and achieves fast and simple detection operation, which helps to reduce detection costs, improve detection efficiency, and make the detection of mycotoxins more popular and feasible.

[0019] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0021] Figure 1 It is the polyacrylamide gel electrophoresis diagram of biotin-modified TDN (Fc);

[0022] Figure 2 The technical roadmap of the present invention is as follows: construction of biosensor platform and response mechanism;

[0023] Figure 3 (A) Dissociation constant analysis and (B) Molecular docking and molecular dynamics simulation of aptamer-antibody and diabody sandwich models;

[0024] Figure 4 A. ECL response curves of biosensors incubated with different concentrations of ZEN (from ag: 1fg / mL, 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL and 1ng / mL); B. Calibration curve between ECL intensity and logarithm of ZEN concentration; C. Specificity of ECL biosensor tested under different interfering toxin small molecules (error bars, SD, n=3); D. Inter-batch and intra-batch differences of the biosensor of the present invention when the ZEN concentration is 1pg / mL. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0026] Embodiment 1

[0027] 1. Materials and Reagents

[0028] Anti-fungal toxin monoclonal antibody (mAb-ZEN): used for target incubation and specific recognition of ZEN.

[0029] Random ssDNA library: contains a sequence of 71 nucleotides in length, of which 35 nucleotides are random sequences and 18 nucleotides on both sides are constant sequences.

[0030] Antibody cocktail: including mAb-FB1 (fumonisin B1), mAb-AFB1 (aflatoxin B1) and mAb-DON (deoxynivalenol), used for the counter-selection step.

[0031] Buffer and washing solution: PBST (phosphate buffered saline + Tween 20) was used for washing steps; 3% bovine serum albumin (BSA) was used for blocking steps.

[0032] PCR amplification reagents: including Taq enzyme, dNTPs, symmetric and asymmetric PCR primers, etc.

[0033] Elution buffer: used to elute bound ssDNA from the antibody.

[0034] ELISA kit: used for preliminary detection of the binding affinity between aptamer and mAb-ZEN.

[0035] TM buffer: contains 10 mM Tris-HCl, 35 mM MgCl 2 , pH 8.0, used for DNA hybridization reactions.

[0036] Cross-linking agents: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the COOH groups in antibodies.

[0037] Antibody (mAb-ZEN): used to couple with DNA tetrahedron to improve recognition specificity.

[0038] Ultrafiltration tube (120Kd): used for purification and concentration of TDN (Fc).

[0039] Polyacrylamide gel electrophoresis system: used for the characterization and analysis of TDN (Fc).

[0040] Electrode preparation materials: NaOH solution, GSH solution, Cu(NO 3 ) 2 Solution, glassy carbon electrode, alumina powder MPI-EII electrochemiluminescence detector

[0041] Other conventional experimental equipment: such as PCR instrument, water bath, pipette, centrifuge, etc.

[0042] Single-stranded DNA (S1, S2, S3, S4, Fc-labeled S0, nucleic acid sequences are shown in Table 1): used for self-assembly of DNA tetrahedron (TDN).

[0043] Table 1: DNA oligonucleotide sequences

[0044]

[0045] 2. Methods and steps

[0046] 1. Screening of aptamer Ap2

[0047] (1) Antibody coating

[0048] Anti-fungal toxin monoclonal antibody (mAb-ZEN) was diluted to 10 μg / mL.

[0049] Use a micropipette to evenly coat the antibody solution in each well of a 96-well microplate, approximately 100 μL per well.

[0050] Place the microplate in a 4°C refrigerator and incubate overnight to ensure that the antibody is firmly bound to the bottom of the plate.

[0051] (2) Washing and sealing

[0052] Wash the microplate three times with PBST buffer, soaking for 3-5 minutes each time to remove unbound antibodies and impurities.

[0053] Add 200 μL of 3% BSA solution to each well and place in a 37°C incubator for 2 hours to block to reduce nonspecific binding. Wash again with PBST buffer for 3 times and prepare for the next step.

[0054] (3) ssDNA library preparation and counter-selection

[0055] Denaturation of ssDNA library: The random ssDNA library is subjected to heat denaturation treatment and then quickly cooled to room temperature to make the DNA in a single-stranded state.

[0056] For non-specific binding removal, the denatured ssDNA library was incubated with blank wells coated with BSA only, and the ssDNA non-specifically bound to BSA was removed by washing.

[0057] For counter-selection, the treated ssDNA was incubated with mixed mAb-FB1, mAb-AFB1, and mAb-DON antibodies, and the ssDNA non-specifically bound to these antibodies was removed by washing to enhance target specificity.

[0058] (4) Target incubation and elution

[0059] The counter-selected ssDNA was incubated with mAb-ZEN antibody to ensure that the ssDNA specifically bound to ZEN was effectively captured. Unbound ssDNA was removed by washing with PBST buffer.

[0060] Elution buffer was added to elute the ssDNA bound to mAb-ZEN, and the eluate was collected for later use.

[0061] (5) PCR amplification and condition optimization

[0062] Symmetrical PCR: PCR amplification of the eluted ssDNA is performed using symmetrical primers, and the optimal number of cycles (preferably 20 cycles) and annealing temperature (a temperature with no significant effect in the range of 50-60°C) are determined by gradient experiments.

[0063] Asymmetric PCR: Further optimize PCR conditions, adjust the ratio of forward and reverse primers to obtain the maximum amount of ssDNA product (optimal ratio is 80:1 forward primer:reverse primer), and determine the optimal number of cycles for asymmetric PCR (preferably 30-35 cycles).

[0064] (6) Aptamer selection and sequence analysis

[0065] After multiple rounds (preferably 15 rounds) of screening, the final eluted ssDNA was cloned into the pMD-18T vector.

[0066] Multiple clones were randomly selected for sequencing, and the homology and secondary structure of the sequences were analyzed using software such as DNAMAN to screen out candidate aptamer sequences with potential high affinity and specificity.

[0067] (7) ELISA affinity test

[0068] The binding affinity of ssDNA clones to ZEN was detected by ELISA.

[0069] The aptamer with the highest affinity and specificity (such as Ap2) is selected as the recognition element for subsequent applications. Ap2 is a single-stranded DNA, and its nucleic acid sequence is shown in SEQ ID NO:6.

[0070] 2. Preparation of highly specific nano-recognition element Ab-TDN (Fc). A highly specific nano-recognition element Ab-TDN (Fc) was prepared. This element was modified with a DNA tetrahedron structure and combined with antibodies to achieve efficient capture and recognition of target molecules (such as ZEN and other fungal toxins). This element combines DNA nanotechnology and bioconjugation technology to improve the specificity and sensitivity of recognition, providing a basis for subsequent sensor development.

[0071] (1) Preparation of Tetrahedron DNA (TDN)

[0072] DNA strand preparation: four single-stranded DNAs S1, S2, S3, and S4 and four times the amount of Fc-labeled S0 strand were mixed in TM buffer at an equal molar ratio to ensure that the concentration of each strand was consistent. The 3' end of the S1 nucleic acid strand was modified with SH, the 5' end of the S2 nucleic acid strand was modified with SH, and the 5' end of the S3 nucleic acid strand was modified with NH. 2 The 3' end of the S4 nucleic acid single strand is modified with SH, the 5' and 3' ends of the single strand nucleic acid S0 are modified with Fc, the DNA sequences of the four nucleic acid single strands S1-S4 are shown in SEQ ID NOs: 1-4, and the DNA sequence of the single strand nucleic acid S0 is shown in SEQ ID NO: 5.

[0073] DNA denaturation: Place the mixture in a PCR instrument and heat at 95°C for 5 minutes to fully denature the DNA chain into a single-stranded state. DNA self-assembly: Move the denatured DNA mixture quickly to an ice-water bath to cool, and then let it stand at room temperature for more than 2 hours to allow the DNA chain to self-assemble into a tetrahedral structure (TDN) through complementary base pairing.

[0074] Purification: Use a 120Kd ultrafiltration tube to ultrafilter the TDN (Fc) solution to remove unassembled single-stranded DNA and other impurities, and collect the purified TDN (Fc) solution for later use.

[0075] (2) Preparation of Ab-TDN(Fc)

[0076] For antibody activation, take an appropriate amount of anti-ZEN mAb solution, add EDC and NHS as cross-linking agents, and activate the COOH groups in the antibody at room temperature according to the conventional ratio (such as the molar ratio of EDC:NHS:COOH group is 1:1:1). The reaction time is determined according to the instructions of the cross-linking agent.

[0077] In the coupling reaction, the activated antibody solution is mixed with the purified TDN (Fc) solution to ensure that the molar ratio of the antibody to TDN (Fc) is appropriate (such as between 1:1 and 1:10), and the mixture is reacted at 4°C for 6 hours to allow the antibody to be firmly coupled to TDN (Fc) through an amidation reaction.

[0078] Purification and characterization: Use ultrafiltration tube to remove uncoupled antibodies and reaction byproducts, and collect Ab-TDN (Fc) solution. Ab-TDN (Fc) can be characterized by polyacrylamide gel electrophoresis and other methods ( Figure 2 ) to confirm its structure and purity.

[0079] 3. Sensor system assembly

[0080] (1) Synthesis of GSH-Cu NCs

[0081] 200 μL of 1 M NaOH solution was slowly added dropwise to 36 mL of 4 mM GSH solution with stirring until the pH of the solution was adjusted to 10.

[0082] Add 4 mL of 20 mM Cu(NO 3 ) 2 Solution, mix thoroughly.

[0083] The mixed solution was placed in a shaker at 60°C and gently shaken for 24 hours. The color of the solution was observed to gradually change from blue-green to yellow-green, indicating the formation of GSH-Cu NCs.

[0084] The reaction solution was centrifuged at 12,000 rpm for 10 minutes to remove unreacted precipitates.

[0085] The supernatant (i.e., GSH-Cu NCs solution) was transferred to a light-proof container and refrigerated for future use.

[0086] (2) Construction of the sensing system

[0087] 1) Glassy carbon electrode pretreatment

[0088] GCE was polished on chamois leather using 0.3 and 0.05 μm aluminum oxide powder until the surface was smooth.

[0089] The polished GCE was placed in ultrapure water and ethanol for alternating ultrasonic cleaning for 5 min and then dried with nitrogen.

[0090] 2) Sensing layer construction

[0091] 5 μL of GSH-Cu NCs solution was evenly coated on the pretreated GCE surface and placed in a 37°C incubator to dry into a film. 10 μL of Ab-TDN(Fc) nanorecognition element with thiol (-SH) was added to the GSH-Cu NCs film and incubated overnight at 4°C to ensure that Ab-TDN(Fc) was firmly bound to the electrode surface. The electrode surface was rinsed with deionized water to remove unbound Ab-TDN(Fc). 10 μL of 5 mM hexanethiol solution was added to the electrode surface and incubated at 4°C for 1 hour to block nonspecific binding sites. The electrode was rinsed with deionized water again and dried with nitrogen to obtain a spare biosensor. The modified electrode was placed in air-saturated 0.1 M pH 7.4 PBS, and the ECL signal of the modified electrode was measured using an MPI-EII electrochemiluminescence detector. The scanning potential was -1 V to 1.3 V, the scanning rate was 0.3 V / s, and the amplifier gain was set to 10×4.

[0092] The aptamer-antibody dual recognition sandwich designed in the present invention is calculated by the dissociation constant ( Figure 3 A) and molecular docking and molecular dynamics simulation ( Figure 3 B) It is clear that this model can achieve high specificity in identifying toxin small molecules.

[0093] 4. Sample testing

[0094] (1) 4 μL of sample treatment solution containing toxin molecules, 3 μL of 10 μM Ap2 DNA chain, 3 μL of 10 μM H 1 The DNA strands were simultaneously added to the biosensor surface and incubated at 4°C for 1 hour. After the incubation, the electrode surface was gently rinsed with deionized water to remove unbound samples and aptamers. 1 The DNA sequence of the DNA chain is shown in SEQ ID NO:7.

[0095] (2) The modified GCE was placed in air-saturated 0.1 M pH 7.4 PBS, and the ECL signal was measured using an MPI-EII electrochemiluminescence detector.

[0096] (3) Set the scanning potential to -1 V to 1.3 V, the scanning rate to 0.3 V / s, and the amplifier gain to 10×4.

[0097] Observe and record the changes in ECL signals. In the absence of ZEN, the ECL signal is weak (signal off) due to the quenching effect of Fc; when fungal toxins are present, the aptamer and ZEN complex is recognized by the Ab-TDN (Fc) pattern, one of the antibodies is anchored at the vertex of TDN, and the aptamer acts as a DNA walker, undergoing an intramolecular chain displacement reaction at the adjacent position, displacing the DNA S labeled with Fc. 0 (Fc-S 0 ). Furthermore, the exposed sequence at the edge of TDN acts as a pivot point to connect with the hairpin H 1 Hybridization releases the swing arm. Therefore, by the hairpin H 1 The triggered strand displacement reaction will drive the DNA walker to move autonomously along the TDN until all Fc-S 0 is eliminated and the ECL signal is restored (signal on).

[0098] (4) ECL response curves of biosensors incubated with different concentrations of ZEN ( Figure 4 ), A: from ag: 1fg / mL, 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL and 1ng / mL; B: calibration curve between ECL intensity and the logarithm of ZEN concentration; C: specificity of ECL biosensor tested under different interfering toxin small molecules (error bars, SD, n=3); D: inter-batch difference and intra-batch difference of the biosensor of the present invention when the ZEN concentration is 1pg / mL.

[0099] As shown in Table 2, among 60 positive samples, 40 were detected positive using ELISA, and 57 were found positive using the method of the present invention. Among 60 negative samples, 54 were detected negative using ELISA, and 59 were found negative using the method of the present invention. Compared with the classic rapid detection method, ELISA showed high positive and negative predictive values, and low false positive and false negative rates (Table 3).

[0100] Table 2

[0101]

[0102] Table 3

[0103]

[0104] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0105] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A sandwich model sensing system based on dual recognition of aptamers and antibodies, comprising aptamers and antibodies, DNA tetrahedrons, and glassy carbon electrodes, characterized in that: The aptamer Ap2 is a single-stranded DNA, and its nucleic acid sequence is shown in SEQ ID NO: 6, which can specifically capture the fungal toxin (ZEN), and the antibody can specifically recognize the fungal toxin (ZEN); the antibody is connected to the top of the DNA tetrahedron, and the DNA tetrahedron is anchored on the surface of the glassy carbon electrode (GCE), and its surface is coated with glutathione copper nanoclusters (GSH-Cu NCs).

2. The sensor system according to claim 1, characterized in that: The DNA tetrahedron is composed of four nucleic acid single strands S1-S4, which are hybridized with a four-fold amount of a single-stranded nucleic acid S0 modified with ferrocene (Fc), the 3' end of the S1 nucleic acid single strand is modified with SH, the 5' end of the S2 nucleic acid single strand is modified with SH, the 5' end of the S3 nucleic acid single strand is modified with NH2, the 3' end of the S4 nucleic acid single strand is modified with SH, the 5' end and the 3' end of the single-stranded nucleic acid S0 are modified with Fc, the nucleic acid single strands S1-S4 self-assemble to form a DNA tetrahedron, the top of the tetrahedron is modified with amino groups, and the edges of the tetrahedron are modified with Fc, the DNA sequences of the four nucleic acid single strands S1-S4 are shown in SEQ ID NOs: 1-4, and the DNA sequence of the single-stranded nucleic acid S0 is shown in SEQ ID NO:

5.

3. The sensor system according to claim 2, characterized in that: The top of the DNA tetrahedron is connected to the anti-fungal toxin antibody (mAb-ZEN) through NH2, and the bottom of the DNA tetrahedron is connected to the GSH-Cu NCs on the surface of the glassy carbon electrode through the SH group.

4. The sensor system according to claim 3, characterized in that: The system also includes a detection system for measuring electrochemiluminescence (ECL) signals to quantitatively detect the fungal toxin (ZEN).

5. The method for detecting mycotoxin (ZEN) using a sensor system according to any one of claims 1 to 4, comprising the following steps: Step 1: Mix the sample containing ZEN and the aptamer Ap2 at a volume ratio of 4:3 and incubate to allow ZEN to bind to the aptamer; Step 2: Introduce the aptamer complex bound to ZEN and the H1 DNA chain to the surface of the sensor system, the molar ratio of the aptamer Ap2 to the H1 DNA chain is 1:1, so that ZEN binds to the antibody to form a sandwich structure, and the DNA sequence of the H1 DNA chain is shown in SEQ ID NO: 7; Step 3: Measure the ECL signal using an electrochemiluminescence detector and determine the concentration of ZEN based on the signal intensity.

6. The detection method according to claim 5, characterized in that: The ECL signal without ZEN was set as the baseline signal (signal off), and the ECL signal with ZEN was significantly enhanced (signal on), so that ZEN was qualitatively and quantitatively detected.

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