Label-free quantum dot electrochemical luminescence sensing electrode for detecting mycotoxin as well as preparation method and application of label-free quantum dot electrochemical luminescence sensing electrode

Abstract

The invention discloses an unmarked quantum dot electrochemical luminescence sensing electrode for detecting mycotoxin as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing an interdigital electrode pair with two groups of independent detection channels on a glass substrate; self-assembling cysteamine on the surface of the gold working electrode to form an amination interface; sequentially carrying out covalent immobilization on the ZnCdS (at) ZnS quantum dots and different fungaltoxin aptamers through amidation reaction; and finally, sealing by using bovine serum albumin. The prepared sensing electrode has a unique complementary segmented interdigital structure, comb tooth parts of the gold working electrode and the platinum counter electrode are arranged in a staggered and non-contact manner, and annular parts are arranged in a concentric circle manner, so that electric field interference between channels is effectively avoided. The invention further provides application of the electrode, chemical labeling is not needed in the whole process of the method, high-sensitivity, high-specificity and parallel in-situ detection of high-harm mycotoxins such as ochratoxin A and zearalenone is achieved, the integration level is high, and the electrode is suitable for on-site rapid screening of food safety.

Description

Technical Field

[0001] This invention relates to the field of electrochemical detection technology, and more specifically, to a label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins, its preparation method, and its application. Background Technology

[0002] Mycotoxins are low-molecular-weight secondary metabolites produced by fungi such as Aspergillus, Fusarium, Penicillium, and Alternaria during their growth. There are over 350 species, widely contaminating agricultural products, food, pharmaceuticals, and traditional Chinese medicine. Among them, ochratoxin A (OTA), zearalenone (ZEN), aflatoxins (AFs), fumonisins (FMs), and vomitoxin (DON) have become key contaminants threatening food safety due to their high toxicity and wide range of contamination. These toxins generally possess carcinogenic, teratogenic, and mutagenic properties, and can cause liver and kidney damage, reproductive system disorders, and immunosuppression in humans. They enter the food chain through contaminated plant-based foods and animal feed, posing a threat to human and animal health. In actual agricultural production and storage, mycotoxin contamination is highly likely to occur due to factors such as temperature and humidity fluctuations and high-humidity environments. Studies show that approximately one-quarter of global grain and oil crops and their by-products are contaminated to varying degrees. Of particular note is the fact that the suitable growth conditions for different toxin-producing fungi often overlap. For example, in environments with 25–30°C and relative humidity above 75%, Aspergillus species producing OTA and Fusarium species producing ZEN may proliferate simultaneously, leading to co-contamination of OTA and ZEN in major grain and oil crops such as corn and wheat. This co-contamination not only manifests as the coexistence of toxins but may also trigger toxicity enhancement effects. For instance, when aflatoxin B1 and fumonisin B1 act together, the oxidative damage to hepatocytes is significantly higher than that of a single toxin, further exacerbating food safety risks. Therefore, the Codex Alimentarius Commission (CAC), the European Union, and the US Food and Drug Administration (FDA) have all established strict limits for mycotoxins in food. However, due to limitations in production control conditions and the concealment of contamination, mycotoxin contamination remains difficult to completely eliminate. Therefore, strengthening on-site rapid testing capabilities at all stages of the food supply chain has become an urgent need to ensure food safety.

[0003] Currently, mycotoxin detection technologies are mainly divided into two categories: traditional detection methods and rapid detection methods. Traditional methods are represented by biological analysis techniques and chromatographic analysis techniques. Biological analysis techniques rely on the specific response of organisms or their components to toxins. Although they are simple to operate and have low cost, their sensitivity and detection efficiency are often insufficient. Chromatographic analysis techniques (such as high performance liquid chromatography, gas chromatography and their coupling with mass spectrometry) achieve accurate qualitative and quantitative analysis through physical separation. They have high sensitivity and high selectivity, but usually rely on complex sample pretreatment, expensive large instruments and professional operators, making them difficult to apply to rapid on-site detection scenarios.

[0004] With the increasing demand for detection, rapid detection technologies have gradually developed, mainly including liquid chromatography-tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), surface-enhanced Raman scattering (SERS), and electrochemiluminescence (ECL) detection. While LC-MS / MS offers precise separation and high sensitivity, its instruments are expensive and lack portability. ELISA has a wide range of applications but is susceptible to cross-reaction interference, affecting its specificity. SERS can achieve ultrasensitive detection, but its signal stability depends on the plasmon resonance effect of metal nanostructures, and the consistency and long-term stability of batch preparations still face challenges. In contrast, ECL detection, with its advantages of simple operation, rapid response, high sensitivity, and no need for an external excitation light source, has shown significant application potential in the field of rapid on-site food safety detection and has become an important technical direction for the quantitative analysis of mycotoxins.

[0005] In the specific research and application of ECL detection of mycotoxins, many technical solutions have been proposed, but these solutions still generally have limitations. For example, one existing technical solution: Shandong University of Technology's patent application with publication number CN118731134A, which discloses a method for preparing a switch-type electrochemiluminescence aptamer sensor for detecting ochratoxin A in grains, discloses a method using Ti3C2T x A switch-type ECL sensor, using MXene as the sensitive interface and CdS quantum dot-modified aptamers as the recognition element, detects OTA in grains through the difference in "on-off" signals. While it boasts high sensitivity, ease of operation, and compatibility with real samples, it can only detect a single toxin, relies on a traditional three-electrode system, and lacks integration and portability. Another existing technology, patent application CN120594625A from Dalian Minzu University, discloses an ECL sensor for detecting penicillin (CIT) using Pt NCs@NiCo-LDH@MXenes as the sensing substrate and integrating a CRISPR / Cas12a system with nucleic acid aptamers. This sensor detects CIT through a recognition-cleavage-signal desuppression mechanism. Although it has a low detection limit, wide linear range, and strong anti-interference ability, it also suffers from the same limitations: single-toxin detection and insufficient integration and portability. These examples reflect a common limitation of current ECL sensing technology: the difficulty in achieving efficient, parallel detection of multiple mycotoxins on a single platform.

[0006] In summary, existing mycotoxin ECL detection technologies mainly follow the following paths, accompanied by corresponding drawbacks: Most schemes require chemical labeling of recognition molecules (such as antibodies or aptamers) or target toxins. Labeling reagents may interfere with the stability of ECL signals, while increasing operational steps, detection costs, and preparation cycles. Furthermore, common detection electrodes often employ traditional disc electrodes or screen-printed electrodes, which are relatively large and have poor compatibility with aptamer immobilization processes, hindering the miniaturization and integration of sensing systems. Using multiple independent electrodes for multi-toxin detection can easily lead to structural dispersion and electric field cross-interference between channels, and the differentiated labeling process may further affect detection specificity. While a few label-free ECL schemes avoid labeling-related problems, the binding between the luminescent group and the electrode, and between the recognition element and the luminescent group, largely relies on physical adsorption, making them prone to detachment. Non-specific adsorption is not effectively suppressed, potentially reducing detection accuracy. In addition, some schemes suffer from insufficient ECL signal response sensitivity due to the large distance between the luminescent group and the electrode surface, or the formation of a thick film layer after modification of the recognition element, which hinders electron transfer.

[0007] Therefore, current mycotoxin ECL detection technologies still have significant shortcomings in terms of label-free modification, compatibility with multi-toxin detection, system integration, and detection stability. There is an urgent need to develop a new ECL sensing method that can achieve parallel detection of multiple toxins (especially common synergistic contaminants such as OTA and ZEN) without relying on chemical labeling, with a high degree of integration and stable and reliable signals, in order to meet the actual needs of rapid on-site detection in the food supply chain and improve the overall monitoring capability of mycotoxin contamination. Summary of the Invention

[0008] The first technical problem to be solved by the present invention is to provide a method for preparing a label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins, so as to solve the problems of cumbersome modification process, poor stability of sensing interface, and long preparation cycle and high detection cost due to reliance on chemical labeling in the prior art.

[0009] To overcome the shortcomings of the prior art, the present invention provides a method for preparing a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, comprising the following steps: S1: Electrode substrate fabrication: Two independent detection channels are fabricated on a glass substrate. Each detection channel includes a gold working electrode and a platinum counter electrode. The two platinum counter electrodes are connected to a platinum pad on the glass substrate through a platinum lead. The two gold working electrodes are connected to two independent gold pads through independent gold leads. S2: Construction of self-assembled monolayer: The gold working electrode prepared in step S1 is immersed in a cysteamine solution, so that the cysteamine molecules form Au-S covalent bonds with the surface of the gold working electrode through thiol groups, thereby constructing a cysteamine self-assembled monolayer. S3: Quantum dot fixation: ZnCdS@ZnS quantum dots with carboxyl groups on the surface are activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then covalently fixed to the surface of the cysteine ​​self-assembled monolayer after step S2 by amidation reaction. S4: Aptamer immobilization: Different types of fungal toxin aptamers with terminal amino groups are covalently immobilized onto the residual carboxyl groups of quantum dots on the surface of the gold working electrode of different detection channels through an amidation reaction. S5: Blocking treatment: Treat the surface of the gold working electrode after aptamer immobilization with bovine serum albumin solution to block non-specific binding sites.

[0010] Compared with existing technologies, the preparation method of the label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins of the present invention has the following advantages: The present invention provides a novel label-free modification method. Through a triple covalent bonding system consisting of an Au-S covalent bond formed between cysteine ​​thiol groups and a gold working electrode, an amide bond formed between quantum dot carboxyl groups and cysteine ​​amino groups, and an amide bond formed between the mycotoxin aptamer amino group and the quantum dot residual carboxyl group, a continuous and stable sensing interface structure is constructed on the electrode surface. This triple covalent bonding system completely eliminates the need for complex chemical labeling of recognition molecules or target toxins to construct detection signals, significantly simplifying the electrode preparation process and shortening the preparation cycle. Simultaneously, since the functional components are sequentially connected by covalent bonds, the problem of easy detachment of luminescent groups and recognition elements in traditional physical adsorption methods is avoided, fundamentally improving the stability and reliability of the sensing interface and effectively suppressing the interference of non-specific adsorption on the electrochemiluminescence signal. Furthermore, the present invention combines the above-mentioned label-free triple covalent modification strategy with the electrode substrate structure design, by setting the electrode on the same glass substrate… Two independent detection channels are designed with platinum counter electrodes electrically connected at the edge of the glass substrate and sharing a common output terminal. This design ensures independent acquisition of signals from the gold working electrodes in each detection channel while reducing the risk of electric field cross-interference during multi-channel detection. This provides a structural basis for the parallel detection of various mycotoxins. Combined with the complementary interdigitated structure of the platinum counter electrodes on the electrode substrate, which share a common output terminal, electric field cross-interference during multi-channel detection is effectively eliminated. The synergistic effect of the above chemical modification strategy and electrode structure design enables the present invention to achieve high stability, high specificity, and high integration of the sensing electrode without the need for any chemical labeling. This effectively solves the technical bottlenecks in the prior art, such as cumbersome modification process, poor interface stability, easy fluctuation of detection signal, and difficulty in integrating multi-toxin detection. It is suitable for highly sensitive, stable, and multi-channel electrochemiluminescence detection of mycotoxins.

[0011] In one possible implementation, after step S1 and before S2, step S1.5 is further included: electrode activation: the gold working electrode and the platinum counter electrode are immersed in a 0.4-0.6M dilute sulfuric acid solution and activated by cyclic voltammetry, wherein the potential scan range is 0V to 1.4-1.6V, the scan rate is 50mV / s, and the number of scan cycles is 5-20 cycles, after which they are cleaned and dried.

[0012] Compared with existing technologies, the above-mentioned technical solution removes the oxide layer and organic contaminants from the surfaces of the gold working electrode and the platinum counter electrode under electrochemical conditions, exposing more active sites on the gold surface. Simultaneously, it restores the conductivity and catalytic activity of the platinum counter electrode. Under cyclic voltammetry scans from 0V to 1.4-1.6V, a reversible redox process can be induced on the gold surface, thereby achieving surface reconstruction and activation. This facilitates the subsequent formation of stable Au–S covalent bonds between cysteine ​​molecules and the gold surface via thiol groups. Furthermore, the activated platinum counter electrode provides a more stable auxiliary reaction environment during detection, improving the uniformity and compactness of subsequent self-assembled monolayer construction, reducing interface defects, and ultimately enhancing the consistency of the electrochemical response and the stability of the electrochemiluminescence signal of the sensing electrode.

[0013] In one possible implementation, in step S2, the cysteamine solution is an aqueous solution of cysteamine hydrochloride with a concentration of 1 mM, and the reaction conditions are: carried out at 2-8°C under light-protected conditions, with an incubation time of 0.5-2 hours.

[0014] Compared with existing technologies, the above-mentioned technical solution can form a more uniform, dense, and stable cysteamine self-assembled monolayer on the surface of the gold working electrode. By controlling the concentration of the cysteamine solution within the range of 1 mM, it is beneficial for molecules to arrange themselves in an orderly manner in the form of a monolayer on the gold surface, avoiding multilayer stacking or disordered adsorption between molecules caused by excessive concentration. Incubation at low temperature and in the dark at 2–8 ℃ reduces the probability of molecular thermal motion and side reactions, promotes the formation of stable Au–S covalent bonds between thiol groups and the gold surface, and reduces adverse reactions such as cysteamine oxidation. By reasonably controlling the incubation time, the self-assembly process is fully completed, ensuring the effective exposure of amino functional sites on the gold surface. This provides a stable and uniform reaction interface for the subsequent covalent fixation of quantum dots, improving the repeatability and long-term stability of the sensing interface.

[0015] In one possible implementation, step S3 includes: preparing ZnCdS@ZnS quantum dots into an aqueous solution with a concentration of 0.5-2 mg / mL; activating the carboxyl groups on the surface of the quantum dots using a mixed solution composed of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; dripping 20 μL of the activated quantum dot solution onto the electrode surface and incubating it at 2-8°C in the dark for 1-4 hours.

[0016] Compared with existing technologies, the above-mentioned technical solution, by controlling the quantum dot concentration within the range of 0.5–2 mg / mL, facilitates the formation of a uniformly distributed and moderately dense luminescent layer on the electrode surface, avoiding electron transfer obstruction caused by excessive quantum dot accumulation. Furthermore, the activation of the carboxyl groups on the quantum dot surface using the EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide) activation system significantly improves the efficiency of the amidation reaction with the amino groups in the cysteine ​​self-assembled monolayer, forming stable covalent bonds. Incubation under low-temperature and light-protected conditions helps maintain the optical and electrochemical stability of the quantum dots, reducing photodegradation and side reactions. These parameters further work synergistically to firmly fix the quantum dots on the electrode surface and maintain good electrochemiluminescence performance, providing a stable carrier for subsequent covalent modification of the aptamer, thereby ultimately improving the luminescence signal intensity, stability, and detection sensitivity of the sensing electrode.

[0017] In one possible implementation, step S4 includes: first, activating the carboxyl groups on the electrode surface immobilized with quantum dots, then immersing the electrodes in different aptamer solutions with a concentration of 20-30 μM, and incubating at 35-39°C for 0.5-2 hours; wherein the aptamer is a nucleic acid aptamer with an amino group modified at the 5' end, comprising: Ochratoxin A aptamer has the base sequence shown in SEQ ID NO: 1; Zearalenone aptamer has the base sequence shown in SEQ ID NO: 2; The aflatoxin B1 aptamer has the base sequence shown in SEQ ID NO: 3; The deoxynivalenol aptamer has the base sequence shown in SEQ ID NO: 4.

[0018] Compared with existing technologies, the above-mentioned technical solution utilizes aptamers with the sequences shown above, all of which are single-stranded nucleic acid molecules obtained through screening and capable of folding into specific spatial conformations in solution. Their spatial conformations are highly complementary to the corresponding mycotoxin molecules in terms of shape, charge distribution, and action sites. This allows for high-affinity and specific binding to ochratoxin A and zearalenone through non-covalent interactions such as hydrogen bonds, π–π interactions, and electrostatic interactions. Furthermore, by reactivating the residual carboxyl groups on the quantum dot surface, the reaction efficiency of the amidation reaction with the terminal amino groups of the aptamer is significantly improved, enabling the aptamer to be firmly connected to the quantum dot surface via covalent bonds, completing the third layer of fixation in the triple covalent bonding system. Further controlling the aptamer concentration within the range of 20–30 μM helps to ensure sufficient reaction while avoiding steric hindrance or disordered accumulation caused by excessive aptamer, thus maintaining effective exposure of the recognition site. The reaction is carried out at 35–39 °C. Incubation under these conditions can accelerate the amidation reaction kinetics without damaging the spatial conformation and recognition activity of the aptamers. In this embodiment, by synergistically setting the above parameters, different types of fungal toxin aptamers can be stably fixed on the surface of the gold working electrode of the corresponding detection channel. This not only ensures the recognition activity and specificity of the aptamers, but also provides a reliable interface basis for the parallel detection of multiple fungal toxins, thereby improving the detection sensitivity and accuracy of the sensing electrode for the target toxin.

[0019] In one possible implementation, in step S5, the bovine serum albumin solution has a mass concentration of 4%-6%, and the incubation conditions are 35-39°C for 0.5-2 hours, followed by washing.

[0020] Compared with existing technologies, the above-mentioned technical solution utilizes bovine serum albumin solutions within this concentration range and incubates them at temperatures close to the optimal operating temperature of the aptamer (35-39℃). This allows BSA molecules to efficiently and saturately adsorb and cover the active sites and potential non-specific adsorption sites on the electrode surface that are not occupied by the aptamer after the aforementioned modifications. Furthermore, subsequent washing removes physically loosely adsorbed BSA molecules, enabling the construction of a stable and dense inert protein sealing layer at the electrode interface. This prevents non-specific binding of non-target molecules (such as extraneous proteins and lipids) within complex sample matrices to the electrode surface during subsequent detection, effectively reducing interference.

[0021] The second technical problem to be solved by the present invention is to provide a label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins, so as to solve the problems of dispersed detection electrode structure, low degree of miniaturization integration, easy generation of electric field cross-interference in multi-channel detection, and difficulty in balancing detection sensitivity and structural stability in the prior art.

[0022] To overcome the shortcomings of the prior art, the present invention also provides a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, wherein the electrode is prepared by the above-described preparation method, and the electrode comprises: Glass substrate; Two independent detection channels are set on the glass substrate; Each detection channel group includes a gold working electrode and a platinum counter electrode; The gold working electrode includes an integrally formed annular portion and comb-tooth portions extending from the annular portion in a direction away from the gold working counter electrode; the platinum counter electrode includes an integrally formed annular portion and comb-tooth portions extending from the annular portion in a direction toward the annular portion of the gold working electrode; the comb teeth of the gold working electrode and the comb teeth of the platinum counter electrode are arranged in a one-to-one correspondence and spaced apart, with an insulating gap between each comb tooth; the annular portion of the gold working electrode and the annular portion of the platinum counter electrode are arranged concentrically on the glass substrate. The two platinum counter electrodes are connected together on the glass substrate by platinum leads and connected to a common platinum pad; The two gold working electrodes are each connected to two independent gold pads via independent gold leads; The surface of the gold working electrode is sequentially modified with a cysteine ​​self-assembled monolayer, a ZnCdS@ZnS quantum dot layer, and a fungal toxin aptamer layer.

[0023] Compared with existing technologies, the sensing electrode of this invention has the following advantages: This invention replaces the existing structure of using a single disk electrode or multiple independent electrodes for multi-toxin detection with an integrated structure of two sets of independent interdigitated detection channels on the same glass substrate. It employs a coaxial ring-interdigitated integrated design of the gold working electrode and platinum counter electrode, achieving a high degree of integration of the detection unit at the structural level. The staggered arrangement of the interdigitated portions of the gold working electrode and platinum counter electrode significantly increases the effective reaction interface within a limited area, improving the electron transfer efficiency at the electrode-solution interface and thus enhancing the electrochemiluminescence signal intensity. Furthermore, the coaxially arranged ring shape facilitates the formation of a relatively uniform electric field distribution, reducing... The impact of local electric field distortion on detection stability; and by connecting two sets of platinum counter electrodes at the edge of the glass substrate and sharing the same output terminal, this invention effectively reduces electric field cross-interference and circuit complexity that may occur during multi-channel detection, while ensuring that the detection signals of each gold working electrode are independent. The interdigitated electrode structure design, multi-channel integrated layout, and shared output of the counter electrodes are interconnected and synergistic, enabling the sensing electrode of this invention to achieve parallel detection of multiple fungal toxins while taking into account high sensitivity, high stability, and high integration. This effectively solves the problems of large electrode volume, dispersed structure, severe inter-channel interference, and difficulty in meeting the needs of rapid on-site detection in the background technology.

[0024] In one possible implementation, the annular portion of the gold working electrode has a diameter of 2.80-3.00 mm and an outer diameter of 3.30-3.40 mm; the comb portion of the gold working electrode includes 9 interdigitated fingers, wherein the width of a single interdigitated finger is 80-120 μm, and the center-to-center distance between adjacent interdigitated fingers is 300-500 μm; the annular portion of the platinum counter electrode has the same dimensions as the annular portion of the gold working electrode; the comb portion of the platinum counter electrode also includes 9 interdigitated fingers, wherein the width of a single interdigitated finger is 160-240 μm, and the center-to-center distance between adjacent interdigitated fingers is 200-400 μm; the width of the insulating gap between the comb portion of the gold working electrode and the comb portion of the platinum counter electrode is 80-120 μm.

[0025] Compared with existing technologies, the above-mentioned technical solution can achieve a reasonable balance between electrode reaction area and electric field distribution while ensuring the manufacturability of the electrode structure. By coordinating the cross-index, width, and spacing of the gold working electrode and the platinum counter electrode, the contact area of ​​the electrode solution interface can be effectively increased per unit area, thereby increasing the probability of the target reactant participating on the electrode surface. At the same time, by reasonably setting the insulation gap width to 80-120 μm, it helps to avoid the problem of short circuit or excessive electric field coupling between the gold working electrode and the platinum counter electrode. The limitation of the above-mentioned size parameters in this embodiment enables the electrode to obtain a high signal response intensity and maintain good signal stability and repeatability during electrochemiluminescence detection, ultimately improving the detection sensitivity and reliability of the sensing electrode.

[0026] In one possible implementation, the glass substrate is BF33 glass with an external length of 18-22 mm and a width of 9-12 mm; the width of the gold lead and platinum wire is 180-220 μm; and the size of the gold pad and platinum pad is (1.0-1.2) mm × (1.0-1.2) mm.

[0027] Compared with existing technologies, the above-mentioned technical solution can balance the mechanical stability of the electrodes, the reliability of electrical connections, and the requirements of system integration. By selecting BF33 glass as the substrate material, it provides good electrical insulation performance and dimensional stability while ensuring the overall strength and chemical stability of the electrodes. Controlling the size of the glass substrate within the above-mentioned range is beneficial for integration with PDMS microfluidic chips or detection modules. Furthermore, by reasonably limiting the size of gold leads, platinum wires, and pads, the wire resistance is reduced and the welding reliability is improved, avoiding additional problems caused by poor contact during the detection process.

[0028] The third technical problem to be solved by this invention is to provide an application of a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, so as to solve the problems of low integration of detection systems, complex detection processes, poor adaptability to on-site detection, and difficulty in achieving stable and synchronous detection of multiple mycotoxins in the existing technology.

[0029] To overcome the shortcomings of the prior art, the present invention also provides an application of the sensing electrode, the application including applying the sensing electrode to a mycotoxin detection system, the mycotoxin detection system including the sensing electrode and: A PDMS microfluidic chip bonded to the sensing electrode to form a closed detection chamber, the PDMS microfluidic chip having an inlet, an outlet and a microchannel; A photomultiplier tube is positioned above the detection chamber to detect electrochemiluminescence signals; And an electrochemical workstation connected to the sensing electrode and photomultiplier tube for providing constant potential excitation and acquiring and analyzing signals.

[0030] Compared with existing technologies, the application of a sensing electrode according to the present invention has the following advantages: The present invention replaces the existing electrochemiluminescence detection device with a dispersed structure, independent detection unit and signal acquisition unit, and difficulty in achieving systematic integration. Instead, it integrates a label-free quantum dot electrochemiluminescence sensing electrode with a PDMS microfluidic chip, a photomultiplier tube, and an electrochemical workstation into a single system. By bonding the PDMS microfluidic chip to the sensing electrode to form a closed detection chamber, the spatial limitation and controllability of sample introduction, reaction, and detection processes are achieved. In the application of the present invention, the introduction of microfluidic channels allows the sample to uniformly contact the electrode reaction interface under low-volume conditions, improving the repeatability and stability of the detection process. The photomultiplier tube, positioned above the detection chamber, enables real-time, non-contact acquisition of the electrochemiluminescence signal generated by the sensing electrode under constant potential excitation, avoiding… The system eliminates background interference caused by external light sources, while the electrochemical workstation provides stable constant potential excitation conditions for the sensing electrodes and simultaneously completes data acquisition and analysis of electrical and optical signals. The synergistic effect of the microfluidic module, optical detection module, and electrochemical control module enables the detection system of this invention to achieve high sensitivity, low background, and stable detection of mycotoxins without the need for chemical labeling of the identified molecules or target toxins. Furthermore, by applying the aforementioned sensing electrodes with multi-detection channel structures to the same detection system, multiple mycotoxins can be detected in parallel under the same detection environment, avoiding the error accumulation problem caused by multiple sampling or multi-system detection. Thus, at the application level, it effectively solves the technical problems of low integration of detection systems, complex operation, poor on-site applicability, and difficulty in simultaneous detection of multiple toxins in the background technology, making it suitable for rapid and accurate detection of mycotoxins in the field of food safety. Attached Figure Description

[0031] Figure 1 A schematic diagram of the surface modification steps for a gold working electrode; Figure 2 Transmission electron microscopy image of ZnCdS@ZnS quantum dots; Figure 3 CV and EIS plots for bare electrode, CS, QDs / CS, Apt / QDs / CS, BSA / Apt / QDs / CS and BSA / Apt / QDs / CS modified electrode; Figure 4 The graph shows the change in electrochemiluminescence intensity after each modification step in the sensor construction process. Figure 5 A schematic diagram of the structure of a label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins; Figure 6 A schematic diagram of the bonding structure between the PDMS microfluidic chip and the sensing electrode. Figure 7 This is a schematic diagram of a sensing electrode used in a fungal toxin detection system. Figure 8 This is an enlarged structural diagram of the sensing electrode; Figure 9 This is a flowchart illustrating the fabrication process of the sensing electrode of the present invention.

[0032] Explanation of reference numerals in the attached figures: 1. Glass substrate; 2. Gold working electrode; 3. Platinum counter electrode; 4. Sensing electrode chip; 5. PDMS microfluidic chip; 6. Gold pad; 7. Platinum pad; 8. Detection chamber; 9. Sample inlet; 10. Sample outlet; 11. Electrochemical workstation; 12. Light shield; 13. Microchannel; 14. Photomultiplier tube; 15. Insulation gap. Detailed Implementation

[0033] First, those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0034] In the description of the embodiments of the present invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention based on the specific circumstances.

[0035] In embodiments of the present invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0036] In the following embodiments of the present invention, "sensing electrode chip" refers to the "label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins" of the present invention.

[0037] This invention provides a method for preparing a label-free quantum dot electrochemiluminescence sensing electrode for detecting fungal toxins, comprising the following steps: S1: Electrode substrate preparation: Gold (Au) was selected as the working electrode material and platinum (Pt) as the counter electrode material; both electrodes were immersed in dilute sulfuric acid solution and electrochemical activation was performed by cyclic voltammetry to remove the surface oxide layer and impurities; after the scan was completed, they were rinsed with deionized water and dried with nitrogen gas in sequence; after the pretreatment was completed, the subsequent surface modification steps were only performed on the gold working electrode 2, and the platinum counter electrode 3 was not modified; S2: Construction of cysteamine self-assembled monolayers: The pretreated gold working electrode 2 is immersed in an aqueous solution of cysteamine hydrochloride and incubated under light-protected conditions. During this process, cysteamine molecules form strong Au-S covalent bonds with the surface of the gold working electrode 2 through their terminal thiol groups (-SH), thereby constructing a dense and ordered cysteamine self-assembled monolayer (SAMs) on the electrode surface. After incubation, the electrode is cleaned and dried.

[0038] S3: Quantum Dot Immobilization: First, prepare an aqueous solution of ZnCdS@ZnS quantum dots; separately prepare a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and mix it with the quantum dot solution in a certain proportion. The mixture is then shaken at room temperature in the dark to activate the carboxyl groups on the quantum dot surface. Subsequently, a certain amount of the activated quantum dot solution is transferred and uniformly dropped onto the surface of the gold working electrode 2, which has been modified with cysteine ​​SAMs, and incubated in a dark environment. The activated carboxyl groups on the quantum dot surface undergo a specific amidation reaction with the exposed amino groups of the cysteine ​​SAMs, achieving covalent and stable immobilization of the quantum dots on the electrode surface. After incubation, the electrode is cleaned.

[0039] S4: Aptamer Immobilization: Two different mycotoxin-specific aptamers were selected from nucleic acid aptamers modified with an amino group (-NH2) at the 5' end. The aptamers were selected from the group including ochratoxin A (OTA) aptamer (SEQ ID NO: 1), zearalenone (ZEN) aptamer (SEQ ID NO: 2), aflatoxin B1 (AFB1) aptamer (SEQ ID NO: 3), and vomitoxin (DON) aptamer (SEQ ID NO: 4). The two selected aptamers were prepared into solutions using TE buffer. After carboxyl activation of the surface of the quantum dot-immobilized gold working electrode 2, the corresponding channels of the gold working electrode 2 were immersed in different aptamer solutions and incubated under constant temperature conditions. During incubation, the amino group at the apse end of the aptamer reacted with the activated carboxyl group remaining on the quantum dots on the electrode surface through an amidation reaction, thereby achieving directional and covalent immobilization of the two different aptamers on two independent sets of gold working electrodes 2. After incubation, rinse the electrode with PBS buffer.

[0040] It should be noted that the present invention has demonstrated a complete process for immobilizing two specific aptamers on a dual-channel electrode. The label-free covalent immobilization method constructed by the present invention is a general platform applicable to a variety of functional nucleic acid aptamers with amino modifications. In practical applications, the corresponding aptamers can be selected from the aptamer library according to the type of toxin to be tested, and immobilized on the two channels respectively through the above steps, thereby customizing the construction of sensing electrodes for detecting different combinations of target substances.

[0041] S5: Blocking treatment: Bovine serum albumin (BSA) solution is added to the electrode surface immobilized with aptamers and incubated in a constant temperature environment; BSA molecules can effectively block the remaining active sites on the electrode surface that are not occupied by aptamers, thereby inhibiting non-specific adsorption; after incubation, the electrode is rinsed with PBS buffer and dried to obtain the label-free electrochemiluminescence sensing electrode.

[0042] As a preferred embodiment, in step S1, the electrode activation includes: immersing the gold working electrode 2 and the platinum counter electrode 3 in a 0.4-0.6M dilute sulfuric acid solution, and performing activation treatment using cyclic voltammetry, wherein the potential scan range is 0V to 1.4-1.6V, the scan rate is 50mV / s, and the number of scan cycles is 5-20, followed by cleaning and drying.

[0043] As a preferred embodiment, in step S2, the concentration of the cysteamine hydrochloride aqueous solution is 1 mM, and the reaction conditions are: carried out at 2-8°C under light-protected conditions, with an incubation time of 0.5-2 hours.

[0044] As a preferred embodiment, in step S3, preparing the ZnCdS@ZnS quantum dot aqueous solution includes: preparing the ZnCdS@ZnS quantum dots into an aqueous solution with a concentration of 0.5-2 mg / mL; The activation conditions are as follows: the carboxyl groups on the surface of the quantum dots are activated using a mixed solution composed of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; 20 μL of the activated quantum dot solution is dropped onto the electrode surface and incubated at 2-8°C in the dark for 1-4 hours.

[0045] As a preferred embodiment, in step S4, carboxyl activation includes: first, activating the carboxyl groups on the electrode surface immobilized with quantum dots, and then immersing the electrodes in different aptamer solutions with a concentration of 20-30 μM, and incubating at 35-39°C for 0.5-2 hours; wherein the aptamer is a nucleic acid aptamer with an amino group modified at the 5' end, including: Ochratoxin A aptamer has the base sequence shown in SEQ ID NO: 1; Zearalenone aptamer has the base sequence shown in SEQ ID NO: 2; The aflatoxin B1 aptamer has the base sequence shown in SEQ ID NO: 3; The deoxynivalenol aptamer has the base sequence shown in SEQ ID NO: 4.

[0046] The specific sequence is as follows: SEQ ID NO: 1: 5'-NH2-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3' SEQ ID NO: 2: 5'-NH2-TCA TCT ATC TAT GGT ACA TTA CTA TCT GTA ATG TGATATG-3' SEQ ID NO: 3: 5'-NH2-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCTTCG CTA GGC CCA CA--3' SEQ ID NO: 4: 5'-NH2-GCA TCA CTA CAG TCA TTA CGC ATC GTA GGG GGG ATCGTT AAG GAA GTG CCC GGA GGC GGT ATC GTG TGA AGT GCT GTC CC-3'.

[0047] As a preferred embodiment, in step S5, the mass concentration of the bovine serum albumin solution is 4%-6%, the incubation conditions are 35-39°C for 0.5-2 hours, and then washing is performed.

[0048] The label-free electrochemiluminescence sensing electrode provided by this invention achieves quantitative analysis of target mycotoxins through the following detection procedure: First, the prepared sensing electrode (sealed with BSA) is placed in a detection buffer system containing co-reactants, and its initial electrochemiluminescence signal (denoted as ECL1) is measured. Then, a series of standard toxin solutions (taking ochratoxin A (OTA) and zearalenone (ZEN) as examples) are applied to the electrode surface and incubated to allow the toxin to specifically bind to the aptamer. The electrochemiluminescence signal after binding is measured again (denoted as ECL2). The signal change value ΔECL (ΔECL = ECL1 - ECL2) is calculated, and a standard working curve of ΔECL versus toxin concentration is established accordingly. For the sample to be tested, ECL1 is measured first, then ECL2 is measured after incubation. The ΔECL value is calculated and substituted into the standard curve to obtain the accurate concentration of the target toxin in the sample. This method, through explicit signal difference quantification, integrates specific identification, signal conversion, and analysis, achieving highly sensitive and specific label-free detection of mycotoxins.

[0049] Compared with existing technologies, this invention completely avoids chemical labeling of recognition molecules or target toxins by adopting a label-free assembly strategy, thereby simplifying the modification process, shortening the preparation cycle, and reducing costs. By constructing a triple covalent fixation system with "Au-S covalent bond + double amidation covalent bond" as the core, it replaces the traditional easily detachable physical adsorption method, significantly enhancing the binding strength and interfacial stability of luminescent groups and recognition molecules on the electrode surface. Furthermore, by utilizing the selective blocking effect of bovine serum albumin (BSA) molecules, it effectively shields unbound sites on the electrode surface, greatly reducing non-specific adsorption interference. This systematically solves the problems of cumbersome modification steps, insufficient sensing interface stability, and severe non-specific adsorption in existing technologies, achieving efficient simplification of the sensing electrode modification process, long-term stable operation of the interface, and simultaneous improvement of detection specificity and signal-to-noise ratio.

[0050] This invention also provides a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, the electrode being prepared by the above-described method, and the electrode comprising: Glass substrate 1; Two independent detection channels are set on the glass substrate 1; Each detection channel group includes a gold working electrode 2 and a platinum counter electrode 3; The gold working electrode 2 includes an integrally formed annular portion and comb-tooth portions extending from the annular portion in a direction away from the gold working electrode; the platinum electrode 3 includes an integrally formed annular portion and comb-tooth portions extending from the annular portion in a direction toward the annular portion of the gold working electrode 2; the comb teeth of the gold working electrode 2 and the comb teeth of the platinum electrode 3 are arranged in a one-to-one correspondence, and an insulating gap 15 is provided between each comb tooth, specifically as follows: Figure 8 As shown; the annular portion of the gold working electrode 2 and the annular portion of the platinum counter electrode 3 are arranged concentrically on the glass substrate 1; The two platinum counter electrodes 3 are connected together on the glass substrate 1 by platinum leads and connected to a common platinum pad 7; The two gold working electrodes 2 are respectively connected to two independent gold pads 6 through independent gold leads; The surface of the gold working electrode 2 is sequentially modified with a cysteine ​​self-assembled monolayer, a ZnCdS@ZnS quantum dot layer, and a fungal toxin aptamer layer.

[0051] More specifically, the electrode described above in this invention employs a highly integrated dual-independent detection channel complementary segmented interdigital configuration. The sensing electrode uses glass as a substrate, and a single sensing electrode chip 4 integrates two completely independent detection channels, which can simultaneously detect two different fungal toxins (such as OTA and ZEN). Each channel group includes a gold (Au) working electrode and a platinum (Pt) counter electrode, with the specific configuration as follows: Gold working electrode 2: It adopts an integrated segmented design of an upper annular section and a lower comb-tooth section. The upper annular section serves as an electric field confinement region, with an inner diameter of approximately 2.80-3.00 mm and an outer diameter of approximately 3.30-3.40 mm, used to optimize the electric field distribution in the detection area and suppress edge effects. The lower comb-tooth section contains multiple parallel interdigitated fingers, each with a width of 80-120 μm and a center-to-center distance of 300-500 μm between adjacent interdigitated fingers. The ends of the interdigitated fingers seamlessly connect to the annular section, and the other end extends to the edge of the substrate via an independent gold lead with a width of 180-220 μm, connecting to a gold pad 6 with dimensions of 1.0-1.2 mm × 1.0-1.2 mm, enabling independent signal extraction and acquisition.

[0052] Platinum counter electrode 3: It adopts an "upper comb section + lower annular section" configuration that is spatially complementary to the gold working electrode 2. The interdigitated fingers of the upper comb section and the lower interdigitated fingers of the gold working electrode 2 are non-contactly interlocked, with an insulating gap 15 of 80-120 μm in width between them to ensure efficient electric field coupling and avoid short circuits. The width of a single interdigitated finger of the comb section is 160-240 μm, and the center-to-center distance between adjacent interdigitated fingers is 200-400 μm. The dimensions of the lower annular section are exactly the same as those of the upper annular section of the gold working electrode 2 to achieve electric field matching. The two platinum counter electrodes 3 are connected at the edge of the substrate by platinum wires (180-220 μm wide) and finally converge to a common platinum pad 7 (1.0-1.2 mm × 1.0-1.2 mm), thereby simplifying the external circuit connection.

[0053] The electrodes are mainly fabricated using microelectromechanical systems (MEMS) technology, and the fabrication method steps are as follows: A1: Substrate pretreatment: 4-inch BF33 glass was selected as the substrate, and ultrasonically cleaned with concentrated sulfuric acid and deionized water in sequence to remove organic matter and impurities from the surface. After drying with nitrogen, it was ready for use. A2: Fabrication of Platinum Counter Electrode 3: Negative photoresist was spin-coated onto a clean substrate. After soft baking, mask bonding, UV exposure, and development, a patterned photoresist layer for platinum counter electrode 3 was formed. Subsequently, a chromium (Cr) adhesion layer and a platinum (Pt) functional layer were sequentially deposited using a magnetron sputtering process. The Cr layer was approximately 50 nm thick, and the Pt layer was approximately 200 nm thick. Finally, the photoresist and excess metal were removed using a lift-off process to form the platinum counter electrode 3 structure. (The thickness data above and below can be adjusted.) A3: Preparation of gold working electrode 2: On the substrate of the prepared platinum counter electrode 3, repeat the spin coating and soft baking steps, replace the mask with the pattern of gold working electrode 2 and accurately align it with the existing pattern of platinum counter electrode 3, and obtain the patterned adhesive layer of gold working electrode 2 after exposure and development; similarly, use magnetron sputtering to sequentially deposit a Cr adhesion layer of about 50 nm thick and an Au functional layer of about 200 nm thick, and then form gold working electrode 2 through a lift-off process. During this process, the alignment accuracy must be strictly controlled to ensure that the interdigitation of gold working electrode 2 and platinum counter electrode 3 is arranged in a preset non-contact staggered manner. A4: Post-processing and cutting: After the metallization preparation is completed, the sensing electrode chip 4 is cleaned and dried. Finally, the entire substrate is cut into individual sensing electrode chips 4 that meet the design size (e.g., 20 mm × 10.9 mm) using a dicing machine, and the edges are polished.

[0054] Through the above design and fabrication scheme, the unique complementary segmented interdigitated layout combined with the ring-shaped electric field confinement structure of this invention effectively isolates the electric field between the two detection channels in physical space, eliminates signal crosstalk, and achieves truly parallel and interference-free detection of two toxins. Furthermore, relying on standard MEMS planar technology, high-density integration of dual-channel electrodes is achieved on a single glass substrate 1, eliminating the need for multi-chip assembly, significantly improving the system's integration, reliability, and portability, and making it more suitable for rapid on-site detection scenarios. This invention also selects platinum, with its high catalytic activity and stability, as the counter electrode material, and combines it with high-precision microfabrication technology to ensure the uniformity and electrochemical stability of the electrode interface structure, thereby significantly reducing background fluctuations in the electrochemiluminescence (ECL) signal and improving the repeatability and accuracy of detection. In summary, this scheme systematically solves the technical defects of existing interdigitated electrodes in multi-channel detection, such as significant electric field interference, low integration, and insufficient signal stability.

[0055] The present invention also provides an application of the sensing electrode, the application comprising using the sensing electrode in a mycotoxin detection system, the mycotoxin detection system comprising the sensing electrode and: A PDMS microfluidic chip 5 is bonded to the sensing electrode to form a closed detection chamber 8. The PDMS microfluidic chip 5 is provided with an inlet 9, an outlet 10 and a microchannel 13. A photomultiplier tube 14 is positioned above the detection chamber 8 to detect electrochemiluminescence signals; And an electrochemical workstation 11 connected to the sensing electrode and photomultiplier tube 14 for providing constant potential excitation and acquiring and analyzing signals.

[0056] The following example illustrates the application of this electrode in a simultaneous detection system for ochratoxin A (OTA) and zearalenone (ZEN).

[0057] The detection system, as described above, mainly consists of a sensing electrode, a PDMS microfluidic chip 5, a photomultiplier tube 14 (PMT), and an electrochemical workstation 11. Its assembly and integration method is as follows: Fabrication of PDMS microfluidic chip 5: The chip was fabricated using soft photolithography. First, a silicon-based patterned mold was prepared: a 4-inch silicon wafer was selected and, after plasma cleaning (90 s), SU8-3025 negative photoresist was spin-coated (2 mL drop volume, low speed 500 rpm / 10 s, high speed 1200 rpm / 30 s, photoresist thickness approximately 80 μm). Pre-baking was then performed (90℃ hot plate, 12 h). Ultraviolet exposure was then performed using the microchannel 13 cavity pattern as a mask (45 s), followed by staged post-baking (in 3℃ increments from 44℃ to 65℃, then in 5℃ increments from 65℃ to 95℃ and held for 3 min). Development (SU8 dedicated developer, manually shaken for 3 min) and rinsing were then performed to obtain the silicon-based mold. PDMS molding is then performed: PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1, vacuum degassing (1 h) is performed, and then poured into a mold. The mixture is cured on a hot plate at 80°C (1 h). After peeling, holes are punched (the diameter of the inlet and outlet ports 10 is 1 mm), thus obtaining a PDMS microfluidic chip 5 with microchannels 13 (width 100 μm) and detection chambers 8. It should be noted that the above process parameters (such as cleaning time, adhesive thickness, temperature, time, etc.) can be adjusted within a certain range to adapt to specific process conditions without affecting the chip function.

[0058] Detection system assembly: The PDMS microfluidic chip 5 and the sensing electrode chip 4 are respectively subjected to plasma treatment, precisely aligned using pre-reserved alignment marks, and bonded to form a sealed detection chamber 8. After bonding, the entire assembly can be heated on a 90°C hot plate for 1 hour to enhance the bonding strength. Then, leads are soldered onto the pads of the sensing electrode and connected to the signal input terminal of the photomultiplier tube 14 and the electrochemical workstation 11, respectively. A light shield 12 is then installed outside the photomultiplier tube 14 to isolate ambient light interference, thus completing the system integration.

[0059] Compared with existing technologies, this system achieves the following technical effects through the above-mentioned integrated design and assembly: The precise and sealed bonding of the PDMS microfluidic chip 5 and the sensing electrode chip 4 forms an independent sample reaction and detection space, effectively avoiding sample cross-contamination and crosstalk between signal channels; relying on microfluidic technology to precisely control fluid and interface reactions, combined with the electrochemical workstation 11 providing stable electrical signal excitation and acquisition, significantly improving the repeatability and accuracy of detection; the entire system is compact, easy to operate, and consumes less sample, effectively overcoming the shortcomings of traditional detection equipment being complex and difficult to carry, providing a feasible integrated solution for the on-site, rapid, and highly sensitive detection of fungal toxins such as OTA and ZEN.

[0060] The following provides more detailed embodiments, based on specific data and actual operating methods, to further elaborate on the technical solution of the present invention: Example 1: Preparation of a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins This embodiment details a specific implementation process of the preparation method in this invention, taking the preparation of a sensing electrode that can be used to simultaneously detect ochratoxin A (OTA) and zearalenone (ZEN) as an example.

[0061] S1: Electrode substrate preparation: This step corresponds to the microfabrication of the sensing electrode in this invention, such as... Figure 9 As shown, the specific operation is as follows: (1) Substrate pretreatment: Take a 4-inch BF33 glass substrate 1, ultrasonically clean it with concentrated sulfuric acid solution, and then ultrasonically clean it with deionized water to thoroughly remove organic impurities and oxide layer on the surface. After cleaning, place the substrate in a nitrogen dryer to dry it to obtain a clean substrate.

[0062] (2) Fabrication of Platinum Counter Electrode 3: A clean substrate was placed in a spin coater and ROL7133 negative photoresist was spin-coated to form a uniform film with a thickness of approximately 3 μm. It was then soft-baked on a hot plate at 110°C for 90 seconds. Using a hard contact method, the coated substrate was tightly bonded to a mask containing the complementary interdigitated pattern of the platinum counter electrode 3, and exposed under ultraviolet light for 10 seconds. After exposure, it was placed in NMP developer and slowly shaken for 45 seconds to remove the photoresist outside the pattern, forming a patterned photoresist layer. After rinsing with deionized water and drying, a 50 nm thick chromium (Cr) adhesion layer and a 200 nm thick platinum (Pt) functional layer were sequentially deposited using magnetron sputtering (deposition temperature 25±2°C). Finally, the substrate was ultrasonically treated in 30°C acetone solution for 15 minutes, and the photoresist and metal in the non-electrode areas were removed using a lift-off process to obtain the platinum counter electrode 3 structure. It was rinsed with deionized water and dried with nitrogen. (3) Preparation of gold working electrode 2: On the substrate of the prepared platinum counter electrode 3, repeat the above spin coating of photoresist and soft baking at 110°C for 90 seconds; replace the mask containing the interdigitated pattern of gold working electrode 2, and achieve precise spatial alignment with the pattern of the lower platinum counter electrode 3 by alignment marks; perform ultraviolet exposure (10 seconds) and development (45 seconds) under the same conditions; then use magnetron sputtering to deposit a 50 nm thick Cr adhesion layer and a 200 nm thick Au functional layer in sequence, and then use acetone ultrasonic stripping to remove excess photoresist and metal, thereby forming a gold working electrode 2 that is non-contactly staggered with the platinum counter electrode 3, together forming a complete dual independent detection channel interdigitated array; (4) Post-processing: The substrate carrying the complete electrode array is cut into individual sensing electrode chips 4 with a specification of 20 mm × 10.9 mm using a dicing machine, and the edges are polished to remove burrs, thus obtaining the electrode substrate. The substrate contains two sets of independent detection channels, each set of channels containing a gold working electrode 2 and a platinum counter electrode 3, and the two platinum counter electrodes 3 are connected together at the edge of the substrate by a wire.

[0063] S1.5: Electrode activation: The gold working electrode 2 and the platinum counter electrode 3 were immersed in a 0.5 M dilute sulfuric acid solution and activated using cyclic voltammetry. The potential scan range was set from 0 V to 1.5 V, the scan rate was 50 mV / s, and 10 consecutive scans were performed. After completion, the electrode surfaces were thoroughly rinsed with deionized water and dried with nitrogen gas.

[0064] S2: Self-assembled monolayer construction: The electrode substrate prepared above (with only the gold working electrode region 2 modified) was immersed in a 1 mM aqueous solution of cysteamine hydrochloride. The system was incubated at 4°C in the dark for 1 hour. During this period, cysteamine molecules formed strong Au-S covalent bonds with the Au electrode surface through their terminal thiol groups (-SH), thereby constructing dense and ordered cysteamine self-assembled monolayers (SAMs). After incubation, the electrode surface was gently rinsed with deionized water and dried with nitrogen gas.

[0065] S3: Quantum dot fixation: ZnCdS@ZnS quantum dots with carboxyl groups on their surface were prepared into an aqueous solution with a concentration of 1 mg / mL. Separately, an activation mixture solution composed of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was prepared. The EDC / NHS mixture solution was mixed with the quantum dot aqueous solution at a volume ratio of 1:1 and ultrasonically treated at room temperature in the dark for 15 minutes to fully activate the carboxyl groups on the surface of the quantum dots. Then, 20 μL of the activated quantum dot solution was transferred and uniformly dropped onto the surface of a gold working electrode 2 modified with cysteine ​​SAMs. The electrode was incubated at 4°C in the dark for 2 hours to allow the activated carboxyl groups on the surface of the quantum dots to undergo an amidation reaction with the amino groups on the SAMs, thereby achieving covalent fixation of the quantum dots. After incubation, the electrodes were rinsed with deionized water and dried with nitrogen.

[0066] S4: Aptamer fixation: Taking the detection of ochratoxin A (OTA) and zearalenone (ZEN) as an example, OTA aptamer (SEQ ID NO: 1) and ZEN aptamer (SEQ ID NO: 2) were selected from the aptamer group, and the two aptamers were prepared into solutions with a concentration of 25 μM using TE buffer. Subsequently, the residual carboxyl groups on the surface of the quantum dot-immobilized electrodes were reactivated using EDC / NHS. After activation, the electrodes were rinsed with 1× phosphate-buffered saline (PBS, pH 7.4). The gold working electrodes 2 of the two channels were then immersed in the corresponding OTA aptamer solutions or ZEN aptamer solutions and incubated at 37°C for 1 hour. During this process, the amino group at the 5' end of the aptamer undergoes a specific amidation reaction with the activated carboxyl groups of the quantum dots on the electrode surface, thereby achieving directional and covalent immobilization of the two different aptamers on two independent sets of gold working electrodes 2. After incubation, the electrodes were rinsed with 1× PBS buffer to remove unbound aptamers and dried with nitrogen gas.

[0067] S5: Sealing treatment: 10 μL of a 5% bovine serum albumin (BSA) solution was dropped onto the surface of the gold working electrode 2, which had already undergone aptamer immobilization, ensuring complete coverage of the modified area. The electrode was then incubated at a constant temperature of 37°C for 1 hour. The BSA molecules effectively blocked the remaining active sites on the electrode surface not occupied by the aptamer, thereby minimizing non-specific adsorption. After incubation, the electrode surface was thoroughly rinsed three times with 1×PBS buffer and dried with nitrogen gas to obtain the label-free quantum dot electrochemiluminescence sensing electrode.

[0068] Through the above steps S1-S5, a sensing electrode with a highly stable and highly specific interface was successfully prepared. This electrode can be directly used for detection system integration and toxin analysis in the subsequent Example 2.

[0069] The schematic diagram of the surface modification steps of the gold working electrode 2 involved in Example 1, the transmission electron microscope (TEM) characterization image of the ZnCdS@ZnS quantum dots, and the characterization images of the electrode after each modification step are shown below. Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown, where: Figure 2 TEM image of the ZnCdS@ZnS quantum dots used in Example 1 of this scheme: Figure 2 As can be seen, the quantum dots have a smooth and uniform surface, good dispersion, and no obvious agglomeration, with an average particle size of approximately 10 nm. Their large specific surface area provides ample active sites for subsequent aptamer immobilization, which is beneficial for improving the efficiency of subsequent toxin recognition.

[0070] Figure 3The cyclic voltammetry (CV) curves (Figure a) and electrochemical impedance spectroscopy (EIS) curves (Figure b) for the bare Au electrode, cysteamine-modified electrode (CS / Au), quantum dot-modified electrode (QDs / CS / Au), aptamer-modified electrode (Apt / QDs / CS / Au), and BSA-blocked electrode (BSA / Apt / QDs / CS / Au) in Example 1 of this scheme are shown below: In Figure a, the CV peak current of the bare Au electrode is 275 μA. After modification with cysteine, the peak current increases to 280 μA, indicating that the introduction of cysteine ​​molecules can improve the conductivity of the electrode surface and promote electron transfer. After further modification with semiconductor quantum dots, the CV peak current drops significantly to 192 μA, mainly because the quantum dot layer has certain resistance characteristics, which hinders electron transport at the electrode / solution interface. Subsequently, after immobilizing the aptamer, the CV peak current further decreases to 163 μA. This is because the aptamer molecules are negatively charged and have electrostatic repulsion with the [Fe(CN)6]3- / 4- redox probe in the electrolyte, thereby inhibiting interfacial electron transfer. After BSA sealing, the CV peak current continues to decrease to 152 μA, indicating that BSA has successfully sealed the unoccupied active sites on the electrode surface, and its steric hindrance effect further blocks the electron transfer path. The trend of the current changing step by step with the modification steps indicates that cysteine ​​SAMs, quantum dots, aptamers, and BSA have been successfully loaded onto the Au electrode surface in sequence. In Figure b, the electron transfer impedance (Ret) changes systematically with the modification process: Ret is approximately 25 Ω for the bare Au electrode; it decreases to 13 Ω after cysteine ​​modification; it increases significantly to 243 Ω after quantum dot modification; it increases to 479 Ω after aptamer fixation; and it further increases to 640 Ω after BSA sealing. The dynamic changes in impedance correspond to the gradual loading process of each modification layer, further demonstrating the successful construction and structural integrity of the sensing interface.

[0071] Figure 4The following are the electrochemiluminescence (ECL) intensity change curves after each modification step in the sensor construction process of Example 1 of this scheme: The ECL intensity of the bare Au electrode is only 2308 au, mainly due to the lack of efficient luminescent groups on its surface, resulting in low ECL activity. After cysteine ​​modification, the ECL intensity increases to 3423 au, indicating that cysteine ​​self-assembled monolayers (SAMs) improve the electron transport microenvironment on the electrode surface, providing a basis for luminescent group fixation and enhancing the ECL signal to a certain extent. After quantum dot modification, the ECL intensity significantly increases to 29460 au, fully demonstrating the excellent ECL activity of quantum dots, which, as efficient luminescent groups, can effectively participate in the ECL reaction and release strong photon signals. After aptamer fixation, the ECL intensity decreases to 13335 au. This is because the thin film formed by the aptamer on the electrode surface introduces steric hindrance, inhibiting interfacial electron transfer and ECL reaction efficiency. After further blocking with BSA, the ECL intensity stabilized at approximately 11647 au, indicating that BSA blocked the active sites on the electrode surface that were not bound to the aptamer.

[0072] In summary, the experimental results show that the label-free electrochemiluminescence sensing electrode can be successfully prepared through steps S1–S5 in this embodiment. Cysteine ​​SAMs, quantum dots, aptamers and BSA modification layers are sequentially formed on the surface of the electrode. The sensing interface structure is complete and has good stability, and it can be used for the specific detection of ochratoxin A (OTA) and zearalenone (ZEN).

[0073] Example 2: Based on Example 1, this embodiment further bonds and assembles the sensor electrode chip 4 and PDMS microfluidic chip 5 constructed in the example to prepare a microfluidic integrated label-free electrochemiluminescence interdigitated sensing detection system for parallel, rapid, and on-site detection of ochratoxin A (OTA) and zearalenone (ZEN). The specific steps include: S6: Fabrication of PDMS microfluidic chip 5 (1) Preparation of silicon-based patterned mold: A 4-inch silicon wafer was selected and plasma cleaned for 90 s; the silicon wafer was fixed on a spin coater, and 2 mL of SU8-3025 negative photoresist was added. Spin coating was completed at a low speed of 500 rpm for 10 s and a high speed of 1200 rpm for 30 s; then it was placed in front of a 90 ℃ horizontal hot plate for 12 h. The microchannel 13 cavity pattern was used as a photomask and exposed to ultraviolet light for 45 s; then it was baked in stages (3 ℃ step from 44 ℃ to 65 ℃, 5 ℃ step from 65 ℃ to 95 ℃, and held for 3 min); finally, it was manually shaken and developed in SU8 special developer for 3 min, rinsed twice with deionized water and dried to obtain the silicon-based patterned mold; (2) Molding of PDMS microfluidic chip 5: Prepare a mixture of PDMS matrix and curing agent at a mass ratio of 10:1, stir thoroughly and then defoam under vacuum for 1 h; wrap the silicon mold with tin foil into a bowl shape, pour in the PDMS mixture, and cure on a horizontal hot plate at 80 ℃ for 1 h; after curing, peel it off from the mold and cut it into individual chips, punch holes to form sample inlet and outlet (inlet 9 with a diameter of 1 mm and outlet 10 with a diameter of 1 mm), and obtain PDMS microfluidic chip 5.

[0074] S7: Assembly of the detection system (1) Plasma bonding: Plasma treatment was performed on the PDMS microfluidic chip 5 and the sensing electrode chip 4 respectively; then the bonding machine was used to accurately align them according to the reserved alignment marks and complete the initial bonding. The whole assembly was then placed on a 90℃ hot plate for 1 h to enhance the bonding strength and sealing stability. (2) Electrical connection and optical shielding: Solder leads are used to lead wires at the pads of the sensing electrode chip 4. The other end of the wire is connected to the signal input terminal of the photomultiplier tube 14, and the photomultiplier tube 14 is connected to the electrochemical workstation 11 through the wire to realize the linkage of signal acquisition and electrochemical excitation. A light shield 12 is put on the outside of the photomultiplier tube 14 and the gap is sealed with tape to avoid ambient light interference, thus completing the assembly of the detection system.

[0075] Fungal toxin detection: (1) Prepare a series of OTA and ZEN standard solutions (100 pM to 1 μM) and prepare a detection co-reaction solution (1×PBS buffer containing 0.1 M potassium persulfate and 0.1 M potassium chloride). (2) Inject 200 μL of standard solution through the injection port 9 of the PDMS microfluidic chip 5 and incubate at 37 °C for 1 h.

[0076] (3) Connect the detection system to the electrochemical workstation 11 and set the constant voltage excitation condition; (4) The ECL signal was acquired by the chronoamperometry method, and ΔECL (ΔECL = initial ECL signal − ECL signal after binding toxin) was calculated. The corresponding toxin concentration was obtained by substituting ΔECL into the standard curve.

[0077] Figure 5 This is a schematic diagram of the label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins prepared in Example 2 of this scheme: (See diagram) Figure 5 As shown, the sensing electrode chip 4 uses glass as a substrate and integrates two sets of independent detection channels. Each channel includes one gold working electrode 2 and one platinum counter electrode 3. The gold working electrode 2 adopts a structure design of "upper section ring-shaped + lower section comb-shaped interdigitated" and the platinum counter electrode 3 adopts a complementary structure of "upper section comb-shaped interdigitated + lower section ring-shaped". Each electrode is connected to the corresponding gold pad 6 or platinum pad 7. The electrode arrangement does not overlap, which effectively reduces electric field cross-coupling and channel interference at the structural level, ensuring parallel and non-interfering detection of OTA and ZEN.

[0078] Figure 6 This is a schematic diagram of the bonding structure between the PDMS microfluidic chip 5 and the sensing electrode in Embodiment 2 of this scheme: Figure 6 As shown, the PDMS microfluidic chip 5 and the sensing electrode chip 4 are plasma-bonded to form a closed detection chamber 8, and are equipped with an inlet 9, a microchannel 13 and an outlet 10. PDMS has good sealing and biocompatibility, and the closed reaction space can reduce the risk of sample cross-contamination. The microchannel 13 can achieve precise control of the sample transport path and residence time, ensuring that the toxin and the aptamer on the electrode surface fully contact and bind, thereby improving the repeatability and stability of detection.

[0079] Figure 7 This is a schematic diagram of the sensor electrode applied to the mycotoxin detection system in Embodiment 2 of this scheme: (See diagram below) Figure 7 As shown, the PDMS microfluidic chip 5 is precisely aligned with the sensing electrode chip 4 through plasma thermo-press bonding to form a stable and closed reaction space; the photomultiplier tube 14 is located directly above the bonding structure, with its photosensitive window aligned with the detection chamber 8 and equipped with a light shield 12 to suppress ambient light; the lead wires from the pads of the sensing electrode chip 4 are connected to the photomultiplier tube 14, and the electrochemical workstation 11 is connected to the photomultiplier tube 14 through wires and provides constant voltage excitation for the sensing electrode, realizing an integrated detection link of electrochemical excitation—ECL luminescence—photoelectric conversion—signal acquisition.

[0080] Experimental results show that this embodiment successfully prepared label-free electrochemiluminescence sensing electrode chip 4 and completed system integration through steps S1 to S7. The system features high integration, small size, and convenient operation, with a single injection volume not exceeding 200 μL and a total detection time controllable within 60 minutes. For OTA and ZEN, the two-channel detection exhibits good linear response and low detection limits, meeting the requirements for trace toxin detection. Compared with existing technologies, this system effectively reduces electric field cross-interference, improves system integration level and field adaptability, and can be used for efficient, accurate, and rapid on-site detection of OTA and ZEN in the food supply chain.

[0081] This invention employs a label-free assembly strategy and a multiple covalent bond immobilization system, eliminating the need for any chemical labeling of the target toxin, recognition molecule, or signal luminescent group during the preparation process, thereby simplifying the modification process and shortening the preparation cycle. First, a dense and stable cysteine ​​self-assembled monolayer (SAMs) is constructed on the electrode surface by forming a stable Au–S covalent bond between the thiol group (–SH) at one end of the cysteine ​​molecule and the surface of the gold working electrode 2. Then, an activation system composed of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used to activate the carboxyl groups (–COOH) on the surface of ZnCdS@ZnS quantum dots. This allows the activated quantum dots to specifically couple with the exposed amino groups of the cysteine ​​SAMs through amide bonds (–CO–NH–), thus achieving stable fixation of the quantum dots on the electrode surface. Finally, the remaining unreacted carboxyl groups on the surface of the quantum dots are activated again by EDC / NHS, causing the aptamer molecules with amino groups (–NH2) to undergo a further amidation reaction with the remaining carboxyl groups of the quantum dots, thereby achieving covalent fixation of the recognition molecules on the electrode surface. The above-mentioned triple covalent bonding design not only avoids the cumbersome steps in traditional methods and reduces the complexity of operation, but also, relying on the high stability of covalent bonds, effectively reduces the risk of luminescent groups or recognition elements falling off due to physical adsorption, thereby improving the structural stability and repeatability of the sensing interface. Furthermore, because the overall modification process is simpler, the preparation cycle is further shortened, which is conducive to the construction of a stable and repeatable sensing interface.

[0082] In summary, this invention provides a label-free quantum dot electrochemiluminescence sensing electrode, detection system, and fabrication method for detecting mycotoxins. This invention utilizes MEMS technology to fabricate a miniaturized sensing electrode chip 4 with dual independent detection channels and complementary segmented interdigitated structures. On the surface of the gold working electrode 2, ZnCdS@ZnS quantum dots are covalently immobilized with corresponding toxin aptamers via an amidation reaction mediated by cysteine ​​self-assembled monolayers (SAMs), and further immobilization is achieved using BSA. By sealing non-specific active sites, a highly specific and stable label-free electrochemiluminescence sensing interface is constructed. Combined with the precise bonding of the PDMS microfluidic chip 5 to the sensing electrode, and the electrical connection and collaborative configuration of the photomultiplier tube 14 and the electrochemical workstation 11, this invention enables parallel detection and rapid analysis of multiple mycotoxins without the need for chemical labeling of the recognition molecule or target toxin. This embodiment uses ochratoxin A (OTA) and zearalenone (ZEN) as representative targets, but by changing or combining suitable ligands, this invention can be extended to the detection of various mycotoxins and further expanded to the detection of other mycotoxins or small molecule pollutants. This invention targets existing mycotoxin ECL... Common problems in detection technologies, such as signal interference introduced by chemical labeling, cross-coupling interference from multi-channel electric fields, low system integration, and difficulty in adapting to rapid on-site detection, are addressed by proposing a chip-based and integrated system solution. This solution integrates sample transmission, specific identification, and signal acquisition and detection, significantly improving detection repeatability and efficiency. It also offers advantages such as low sample volume, fast detection speed, and ease of operation. Furthermore, the detection system described in this invention can be widely applied to rapid on-site screening of mycotoxins in grain and oil crops, agricultural products, and food in the food supply chain. It provides an efficient, accurate, and portable technical platform for food safety risk management, agricultural product quality monitoring, and regulatory enforcement, which is of great significance for ensuring food safety and human and animal health. In the description of the embodiments of this invention, it should be noted that the terms "inner" and "outer," etc., indicating directional or positional relationships, are based on the directional or positional relationships shown in the accompanying drawings. This is merely for ease of description and does not indicate or imply that the device or component must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of the invention.

[0083] In the description of this invention, the references to "one embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0084] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, characterized in that, Includes the following steps: S1: Electrode substrate fabrication: Two independent detection channels are fabricated on a glass substrate. Each detection channel includes a gold working electrode and a platinum counter electrode. The two platinum counter electrodes are connected to a platinum pad on the glass substrate through a platinum lead. The two gold working electrodes are connected to two independent gold pads through independent gold leads. S2: Construction of self-assembled monolayer: The gold working electrode prepared in step S1 is immersed in a cysteamine solution, so that the cysteamine molecules form Au-S covalent bonds with the surface of the gold working electrode through thiol groups, thereby constructing a cysteamine self-assembled monolayer. S3: Quantum dot fixation: ZnCdS@ZnS quantum dots with carboxyl groups on the surface are activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then covalently fixed to the surface of the cysteine ​​self-assembled monolayer after step S2 by amidation reaction. S4: Aptamer immobilization: Different types of fungal toxin aptamers with terminal amino groups are covalently immobilized onto the residual carboxyl groups of quantum dots on the surface of the gold working electrode of different detection channels through an amidation reaction. S5: Blocking treatment: Treat the surface of the gold working electrode after aptamer immobilization with bovine serum albumin solution to block non-specific binding sites.

2. The preparation method according to claim 1, characterized in that, After step S1 and before step S2, step S1.5 is also included: electrode activation: the gold working electrode and the platinum counter electrode are immersed in a 0.4-0.6M dilute sulfuric acid solution and activated by cyclic voltammetry, wherein the potential scan range is 0V to 1.4-1.6V, the scan rate is 50mV / s, and the number of scan cycles is 5-20. After that, the electrode is cleaned and dried.

3. The preparation method according to claim 1, characterized in that, In step S2, the cysteamine solution is a 1 mM aqueous solution of cysteamine hydrochloride, and the reaction conditions are: carried out at 2-8°C in the dark, and the incubation time is 0.5-2 hours.

4. The preparation method according to claim 1, characterized in that, Step S3 includes: preparing ZnCdS@ZnS quantum dots into an aqueous solution with a concentration of 0.5-2 mg / mL; activating the carboxyl groups on the surface of the quantum dots using a mixed solution composed of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; dripping 20 μL of the activated quantum dot solution onto the electrode surface and incubating it at 2-8°C in the dark for 1-4 hours.

5. The preparation method according to claim 1, characterized in that, Step S4 includes: first, activating the carboxyl groups on the electrode surface with immobilized quantum dots; then, immersing the electrodes in different aptamer solutions with a concentration of 20-30 μM and incubating them at 35-39°C for 0.5-2 hours; wherein the aptamer is a nucleic acid aptamer with an amino group modified at the 5' end, including: Ochratoxin A aptamer has the base sequence shown in SEQ ID NO: 1; Zearalenone aptamer has the base sequence shown in SEQ ID NO: 2; The aflatoxin B1 aptamer has the base sequence shown in SEQ ID NO: 3; The deoxynivalenol aptamer has the base sequence shown in SEQ ID NO:

4.

6. The preparation method according to claim 1, characterized in that, In step S5, the bovine serum albumin solution has a mass concentration of 4%-6%, and the incubation conditions are 35-39°C for 0.5-2 hours, followed by washing.

7. A label-free quantum dot electrochemiluminescence sensing electrode for detecting mycotoxins, characterized in that, The electrode is prepared by the preparation method according to any one of claims 1-6, and the electrode comprises: Glass substrate (1); Two independent detection channels are set on the glass substrate (1); Each detection channel includes a gold working electrode (2) and a platinum counter electrode (3); The gold working electrode (2) includes an integrally formed annular portion and a comb-tooth portion extending from the annular portion in a direction away from the gold working electrode; the platinum electrode (3) includes an integrally formed annular portion and a comb-tooth portion extending from the annular portion in a direction toward the annular portion of the gold working electrode (2); the comb teeth of the gold working electrode (2) and the comb teeth of the platinum electrode (3) are arranged in a one-to-one correspondence and spaced apart, and an insulating gap (15) is provided between each comb tooth; the annular portion of the gold working electrode (2) and the annular portion of the platinum electrode (3) are arranged in concentric circles on the glass substrate (1). The two platinum counter electrodes (3) are connected together on the glass substrate (1) by platinum leads and connected to a common platinum pad (7). The two gold working electrodes (2) are respectively connected to two independent gold pads (6) through independent gold leads; The surface of the gold working electrode (2) is sequentially modified with a cysteine ​​self-assembled monolayer, a ZnCdS@ZnS quantum dot layer, and a fungal toxin aptamer layer.

8. The sensing electrode according to claim 7, characterized in that, The annular portion of the gold working electrode (2) has a diameter of 2.80-3.00 mm and an outer diameter of 3.30-3.40 mm; the comb portion of the gold working electrode (2) includes 9 interdigitated fingers, wherein the width of a single interdigitated finger is 80-120 μm and the center-to-center distance between adjacent interdigitated fingers is 300-500 μm. The size of the annular portion of the platinum counter electrode (3) is the same as that of the annular portion of the gold working electrode (2); the comb portion of the platinum counter electrode (3) also includes 9 interdigitated fingers, wherein the width of a single interdigitated finger is 160-240 μm, and the center-to-center distance between adjacent interdigitated fingers is 200-400 μm; the width of the insulating gap (15) between the comb portion of the gold working electrode (2) and the comb portion of the platinum counter electrode (3) is 80-120 μm.

9. The sensing electrode according to claim 7, characterized in that, The glass substrate (1) is BF33 glass with an external length of 18-22 mm and a width of 9-12 mm; the width of the gold lead and the platinum wire is 180-220 μm; the dimensions of the gold pad (6) and the platinum pad (7) are both (1.0-1.2) mm × (1.0-1.2) mm.

10. An application of the sensing electrode according to any one of claims 7-9, characterized in that, The application includes using the sensing electrode in a mycotoxin detection system, the mycotoxin detection system comprising the sensing electrode and: A PDMS microfluidic chip (5) is bonded to the sensing electrode to form a closed detection chamber (8). The PDMS microfluidic chip (5) is provided with an inlet (9), an outlet (10) and a microchannel (13). A photomultiplier tube (14) is installed above the detection chamber (8) for detecting electrochemiluminescence signals. And an electrochemical workstation (11) connected to the sensing electrode and photomultiplier tube (14) for providing constant potential excitation and acquiring and analyzing signals.

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