Electrochemical luminescence sensor for citrinin detection and detection method thereof

Abstract

The invention relates to the technical field of mycotoxin rapid detection, in particular to an electrochemical luminescence sensor for citrinin detection and a detection method of the citrinin. A sensing substrate of the sensor is prepared by loading platinum nanoclusters (Pt NCs) on a NiCo-LDH / MXene composite substrate through an in-situ growth strategy; the high conductivity of MXene and the LDH confinement effect are utilized to synergistically promote three-dimensional exposure of Pt active sites, the efficiency of generating active oxygen through electro-catalysis is remarkably enhanced, and a light-emitting signal is improved. And a CRISPR / Cas12a system is further integrated, so that a regulation mechanism of recognition-cutting-signal de-suppression is realized, and food matrix interference is effectively avoided. And a new technology is provided for monitoring the fungaltoxin by combining a dual enhancement mechanism of material catalytic optimization and enzymatic signal amplification.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid detection of mycotoxins, in particular to an electrochemiluminescence sensor for detecting citrinin and a detection method thereof. Background Art

[0002] Citrinin (CIT) is a polyketide mycotoxin produced primarily by Penicillium and Monascus fungi. It is commonly found during the production, storage, and transportation of cereals, dairy products, and brewed foods (such as red yeast rice). The International Agency for Research on Cancer (IARC) classifies it as a Group 3 carcinogen. Its toxicity primarily affects the kidneys, with potential effects on the liver, bone marrow, and immune system. Its nephrotoxicity is significantly enhanced when it interacts with ochratoxin A (OTA). To ensure food safety, the European Union sets a maximum limit of 100 μg / kg for CIT in red yeast rice, while the Chinese national standard (GB5009.222–2016) requires a limit of quantification of 25 μg / kg and a limit of detection of 8 μg / kg for CIT in chili powder, corn, and rice products. However, despite the high incidence of CIT contamination in food, relevant research data remain insufficient, leading the European Food Safety Authority (EFSA) to call for strengthened validation of risk assessments.

[0003] Currently, CIT detection primarily relies on technologies such as high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and thin-layer chromatography (TLC). However, these methods rely on expensive equipment and specialized personnel, making rapid on-site screening difficult. Although immunoassays (such as ELISA) are relatively low-cost, their sensitivity and anti-interference capabilities are insufficient. Therefore, the development of portable sensors or rapid detection technologies based on nucleic acid aptamers has become a current research hotspot, balancing the requirements of detection speed, cost, and accuracy.

[0004] Electrochemiluminescence (ECL), a combination of electrochemical and chemiluminescence techniques, offers high sensitivity and efficiency, meeting the needs of real-world sample monitoring. Therefore, it has attracted widespread attention in the field of food safety analysis and testing. Therefore, a highly efficient method for detecting CIT based on an ECL sensor has been developed to prevent mycotoxin infections caused by CIT and thus ensure food safety. Summary of the Invention

[0005] (1) Technical issues

[0006] The goal of this invention is to meet the demand for immediate detection of CIT while maintaining detection sensitivity. This solution utilizes a Pt NCs@NiCo-LDH@MXenes composite as a sensing substrate, and based on a specific aptamer control strategy, it is designed into a sensor for accurate and rapid CIT detection.

[0007] (2) Technical content

[0008] This solution provides a Pt NCs@NiCo-LDH@MXenes composite material. The composite material is obtained by reducing a platinum source to Pt NCs under the action of a reducing agent and dispersing and loading the Pt NCs on NiCo-LDH@MXenes with a layered structure, wherein the particle size of the Pt NCs is 2 to 5 nm.

[0009] This solution also provides a method for preparing the above-mentioned Pt NCs@NiCo-LDH@MXenes composite material, comprising the following steps:

[0010] Ti3AlC2 was etched using LiF / HCl. Specifically, 1 g of LiF was slowly dissolved in 10 mL of 12 M HCl solution to obtain an HCl / LiF etching solution. 0.1 g of Ti3AlC2 powder was then slowly added to the solution and magnetically stirred at room temperature for 24 hours. After the reaction, the residual acid was removed by multiple centrifugations (until the pH of the supernatant reached 6), and the precipitate was collected by vacuum filtration. Finally, a single layer of MXenes was obtained by centrifugation and ultrasonic dispersion.

[0011] 6.25 mL of a single-layer MXenes colloidal solution was diluted to 50 mL with deionized water and then sonicated. Ni(NO3)2·6H2O and Co(NO3)2·6H2O (2 mmol each) were added to dissolve the solution. 10 mL of a 10 mg / mL NaBH4 aqueous solution was slowly added dropwise to grow NiCo-LDH nanosheets. The product was centrifuged, washed with ethanol, and vacuum-dried to obtain a NiCo-LDH@MXenes composite material.

[0012] Using the impregnation reduction method, 0.1 g of NiCo-LDH@MXenes support was dispersed in 30 mL of deionized water and ultrasonicated for 5 min. 100 uL of 19.3 mM H2PtCl6·6H2O solution was added dropwise and stirred for 30 min. Subsequently, 1.5 mL of a reducing agent solution containing 0.1 M NaBH4 and 0.2 M NaOH was quickly injected and stirred for 1 h to load Pt NCs onto NiCo-LDH@MXenes to obtain the sensor substrate Pt NCs@NiCo-LDH@MXenes.

[0013] Optimal technical solution: The mass molar ratio of NiCo-LDH@MXenes and Pt NCs in the Pt NCs@NiCo-LDH@MXenes composite material is 60mg~100mg: 1.2×10 -3 mmol~2×10 -3 The reaction ratio of mmol was obtained.

[0014] Optimal technical solution: the molar ratio of the reducing agent to the platinum source is 78-80:1-3, the platinum source is chloroplatinic acid, the reducing agent is NaBH4, and the immersion reduction time is 10 min to 60 min.

[0015] This solution also provides the use of the above-mentioned Pt NCs@NiCo-LDH@MXenes composite material in the preparation of a sensor for detecting citrinin, wherein the sensor is an electrochemiluminescence sensor;

[0016] The sensor includes: a substrate, and an electrode layer attached to the substrate, wherein the electrode layer includes a working electrode layer, and a Pt NCs@NiCo-LDH@MXenes composite material, TDNs-DA, and MCH are sequentially coated on the surface of the working electrode layer.

[0017] The Apt nucleotide sequence of citrinin is:

[0018] 5'-GGCCAGGCGGGGCCTGTTCGTGGGCCGTGTCTTCGGCTCGCTCGGTTG-3'.

[0019] The method for using the sensor to detect CIT comprises the following steps:

[0020] 10 μL of gradient toxin solution was mixed with 20 μL of aptamer functional solution and incubated at 37°C for 40 min to allow the aptamer to specifically bind to the toxin and release the pre-blocked activator DNA;

[0021] The above reaction solution is mixed with the pre-prepared Cas12a-crRNA complex, and the Cas12a / crRNA / activator ternary complex is formed by guiding the activator DNA to activate the trans-cleavage activity of the Cas12a enzyme;

[0022] The activated Cas12a enzyme droplets were applied to the surface of the MCH / TDNs-DA / Pt NCs@NiCo-LDH@MXenes modified electrode;

[0023] In 5 mL PBS (pH 7.4) containing 100 μM Luminol, a scanning voltage of -0.2 to 0.6 V (50 mV / s) was applied, and the ECL signal intensity was detected at a photomultiplier tube voltage of 600 V to establish a quantitative relationship between citrinin concentration and ECL intensity.

[0024] Preferred technical solution: The detection range of the sensor is 0.05~1×10 4 ng mL -1 , the detection limit was 11 pg mL -1 .

[0025] (3) Technical effects

[0026] The above structure enables this solution to have the following beneficial effects:

[0027] 1. This invention, based on the design of a NiCo-LDH@MXenes heterostructure, achieves high-density three-dimensional loading of Pt NCs through the synergistic effect of the highly conductive network of MXenes and the confinement effect of NiCo-LDH nanosheets. Electrochemical impedance spectroscopy system verified that this structure significantly optimizes the interfacial electron transport pathway and promotes the efficient electronic reduction of dissolved oxygen (O2), thereby increasing the ROS generation rate of the luminol system and achieving an electrochemiluminescence intensity of 14,000 a.u.

[0028] 2. By integrating high-affinity nucleic acid aptamers with the CRISPR / Cas12a cascade signal amplification mechanism, a dynamic regulatory system of "recognition-cleavage-signal desuppression" was constructed. Aptamers capture citrinin, triggering the trans-cleavage activity of Cas12a, specifically cleaving single-stranded DNA in the TDNs-DA complex on the electrode surface, leading to the dissociation of the quencher DA molecule and an ECL signal recovery rate of >86%. This design, through the dual effects of the excellent performance of the composite material-modified electrode and the enzyme cleavage specificity, solves the problem of biorecognition being susceptible to interference and simultaneously improves the detection sensitivity of the sensor.

[0029] 3. The sensor is in the range of 0.05-1×10 4 Excellent linear response in the ng / mL range (R 2 =0.9987), with a detection limit as low as 11 pg / mL (S / N=3), accurately meeting the limits of both the EU (100 μg / kg) and the Chinese national standard (25 μg / kg). Compared to traditional chromatographic methods, this method is simpler to operate and does not require large instrumentation, providing a highly effective solution for rapid on-site screening and precise monitoring of citrinin in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 This is a transmission electron microscope image of a single-layer MXenes nanosheet prepared in Example 1 of this scheme;

[0032] Figure 2 This is a transmission electron microscope image of NiCo-LDH prepared in Example 1 of this scheme;

[0033] Figure 3This is a transmission electron microscope image of NiCo-LDH@MXenes prepared in Example 1 of this scheme;

[0034] Figure 4 This is a transmission electron microscope image of the Pt NCs@NiCo-LDH@MXenes composite material prepared in Example 1 of this scheme;

[0035] Figure 5 EIS graphs of bare GCE, NiCo-LDH, NiCo-LDH@MXenes, and Pt NCs@NiCo-LDH@MXenes modified GCE electrodes;

[0036] Figure 6 Electrochemiluminescence intensity-time curve of the sensor construction process;

[0037] Figure 7 This is the electrochemiluminescence intensity-time test diagram of the sensor corresponding to different concentrations of CIT in this scheme;

[0038] Figure 8 This is a linear fitting curve diagram of the logarithmic value of CIT at different concentrations and the electrochemiluminescence intensity in this scheme;

[0039] Figure 9 Figure 3. Specificity test results of the electrochemiluminescence sensor provided for this protocol. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] This solution provides a Pt NCs@NiCo-LDH@MXenes composite material. The composite material is obtained by reducing a platinum source to Pt NCs under the action of a reducing agent and dispersively loading the Pt NCs on NiCo-LDH@MXenes with a layered surface structure, wherein the particle size of the Pt NCs is 2 to 5 nm.

[0042] In some specific embodiments, the mass molar ratio of NiCo-LDH@MXenes to Pt NCs in the Pt NCs@NiCo-LDH@MXenes composite material is 60 mg to 100 mg: 1.2×10 -3 mmol~2×10 -3mmol of the reaction ratio was obtained; as a preferred solution of this scheme, the composite material was prepared by NiCo-LDH@MXenes and Pt NCs at a reaction ratio of 100 mg: 1.93×10 -3 The reaction ratio of mmol was obtained.

[0043] It should be noted that in this scheme, Pt NCs@NiCo-LDH@MXenes are solid powders. The source of Pt NCs is not limited and can be prepared using platinum acid or salt solution as a raw material; in some specific embodiments, the platinum source is chloroplatinic acid.

[0044] The Pt NCs@NiCo-LDH@MXenes composite material provided in this solution has the following advantages compared with other sensing substrates:

[0045] (1) Pt NCs@NiCo-LDH@MXenes exhibits excellent catalytic properties, effectively catalyzing the reaction between luminol and dissolved oxygen, significantly enhancing the electrochemiluminescence signal. This catalytic effect reduces the time required for detection and improves the efficiency of the detection process.

[0046] (2) The structure of NiCo-LDH@MXenes effectively inhibits the self-stacking and oxidation of monolayer MXenes, while promoting charge transfer through the heterogeneous interface, providing a larger surface area and more active sites, allowing Pt NCs to be evenly distributed on it, which can significantly improve the electron transfer rate at the electrode interface;

[0047] (3) The Pt NCs@NiCo-LDH@MXenes composite material exhibits stable behavior in an electrochemical environment and can maintain its chemical and physical properties during long-term operation, thereby ensuring the stability and reliability of the sensor. Therefore, the novel co-reaction catalyst Pt NCs@NiCo-LDH@MXenes provided in this scheme as a sensing substrate can significantly improve the sensitivity of the sensor.

[0048] On the other hand, this scheme provides a method for preparing a Pt NCs@NiCo-LDH@MXenes composite material, comprising the following steps:

[0049] Ti3AlC2 was etched using LiF / HCl, and after exfoliation, clay-like monolayer MXenes were obtained by centrifugation and ultrasonic dispersion.

[0050] A monolayer MXenes colloidal solution was diluted and ultrasonically treated, then dissolved with Ni(NO₃)₂·6H₂O and Co(NO₃)₂·6H₂O. Then, a NaBH₄ solution was slowly added dropwise to reduce the solution, resulting in the growth of NiCo-LDH nanosheets. The product was centrifuged, washed with ethanol, and vacuum-dried to obtain a NiCo-LDH@MXenes composite.

[0051] Using the impregnation reduction method and NaBH4 as the reducing agent, Pt NCs were loaded onto NiCo-LDH@MXenes to obtain the platinum-based catalyst Pt NCs@NiCo-LDH@MXenes.

[0052] In this scheme, Pt NCs@NiCo-LDH@MXenes composite materials were prepared by impregnation reduction, which were used to modify the sensing interface, improve the electron transfer rate on the electrode surface, catalyze the electrochemiluminescence system, and enhance the ECL response.

[0053] In some specific embodiments, the molar ratio of the reducing agent to the platinum source is 78-80:1-3, in which case the platinum source is chloroplatinic acid, and the reducing agent is NaBH4. Specifically, the platinum source is a chloroplatinic acid solution with a molar concentration of 19.3 mM, and the reducing agent is a sodium borohydride solution with a molar concentration of 0.1 M. The solvent is water, more preferably ultrapure water. In this scheme, the purpose of the solvent is to dissolve NiCo-LDH@MXenes, and the platinum source is impregnated and reduced under the action of the reducing agent.

[0054] In addition, this solution provides an application of the above-mentioned Pt NCs@NiCo-LDH@MXenes composite material in the preparation of a sensor for detecting citrinin, wherein the sensor is an electrochemiluminescence sensor.

[0055] In some specific embodiments, the sensor includes: a substrate, and an electrode layer attached to the substrate, the electrode layer including a working electrode layer, and the surface of the working electrode layer is coated with a Pt NCs@NiCo-LDH@MXenes composite material, TDNs-DA, and MCH in sequence; as a preferred embodiment of this scheme, the working electrode layer is a glassy carbon electrode, and the PtNCs@NiCo-LDH@MXenes composite material, TDNs-DA, and MCH are coated in sequence on the surface of the glassy carbon electrode, and the electrode layer also includes a counter electrode and a reference electrode, the counter electrode can be a platinum wire electrode, and the reference electrode can be Ag / AgCl.

[0056] In some specific embodiments, the nucleotide sequence of Apt of citrinin is: 5'-GGCCAGGCGGGGCCTGTTCGTGGGCCGTGTCTTCGGCTCGCTCGGTTG-3'.

[0057] In addition, this solution also provides a method for quantitatively detecting citrinin, which is detected using the sensor. In some specific embodiments, the method for using the sensor to detect citrinin includes the following steps:

[0058] 10 μL of gradient toxin solution was mixed with 20 μL of aptamer functional solution and incubated at 37°C for 40 min to allow the aptamer to specifically bind to the toxin and release the pre-blocked activator DNA;

[0059] The above reaction solution is mixed with the pre-prepared Cas12a-crRNA complex, and the Cas12a / crRNA / activator ternary complex is formed by guiding the activator DNA to activate the trans-cleavage activity of the Cas12a enzyme;

[0060] The activated Cas12a enzyme droplets were applied to the surface of the MCH / TDNs-DA / Pt NCs@NiCo-LDH@MXenes modified electrode;

[0061] In 5 mL PBS (pH 7.4) containing 100 μM Luminol, a scanning voltage of -0.2 to 0.6 V (50 mV / s) was applied, and the ECL signal intensity was detected at a photomultiplier tube voltage of 600 V to establish a quantitative relationship between citrinin concentration and ECL intensity.

[0062] The detection range of the sensor is 0.05~1×10 4 ng mL -1 , with a detection limit of 11 pg mL -1 .

[0063] The electrochemiluminescence sensor provided in this solution has the following mechanism for detecting CIT:

[0064] By rationally designing the melting temperature, Apt can partially hybridize with the activator to form a thermodynamically stable "lock-activator" system. At this time, the Cas12a / crRNA complex is inactivated, and the DA connected to TDNs is fixed to the electrode surface, inhibiting the electrochemiluminescence signal, and the sensor is in an "off" state; when CIT exists in the system, the aptamer preferentially binds to CIT, resulting in the release of the activator; the free activator activates the trans-cleavage activity of Cas12a through base complementary pairing, and then non-specifically cuts the single-stranded DA on the electrode surface. The DA molecule is away from the electrode surface, resulting in a significant enhancement of the electrochemical signal, ultimately achieving trace detection of CIT.

[0065] The Pt NCs@NiCo-LDH@MXenes composite material prepared in this scheme can show good catalytic performance and can be used as a sensing substrate for sensors, which is beneficial to improving detection sensitivity. When the analyte CIT is present, CIT binds to the aptamer modified with DA, causing DA to detach from the electrode surface, causing a change in the electrochemiluminescence signal. Based on the specific recognition between the target and the nucleic acid aptamer and the dopamine quenching regulation strategy, quantitative detection of CIT is achieved by comparing the relationship between the change in the electrochemiluminescence signal and the concentration of the target.

[0066] In this scheme, the sensor is made of Pt NCs@NiCo-LDH@MXenes composite material. On the one hand, the constructed catalyst has excellent catalytic performance for the electrochemiluminescence system, which can effectively enhance the ECL intensity and shorten the detection time.

[0067] The Pt NCs@NiCo-LDH@MXenes electrochemiluminescence sensor constructed in this scheme for detecting CIT has a detection range of 0.05 to 1×10 4 ng mL -1 , showing a low detection limit and has good application prospects in food safety testing.

[0068] The following is further described through specific examples.

[0069] Example 1

[0070] A method for preparing a Pt NCs@NiCo-LDH@MXenes composite material comprises the following steps:

[0071] S1. Preparation of monolayer MXenes: 1 g of LiF was slowly dissolved in 10 mL of 12 M HCl solution to obtain an HCl / LiF etching solution. 0.1 g of Ti3AlC2 powder was then slowly added to the solution and magnetically stirred at room temperature for 24 h. After the reaction, the residual acid was removed by multiple centrifugation (until the pH of the supernatant reached 6), and the precipitate was collected by vacuum filtration. The precipitate was vacuum dried at 60 ° C for 12 h to obtain monolayer MXenes. Finally, the multilayer MXenes were ultrasonically treated in an ice-water bath for 1 h and then centrifuged at 3500 rpm for 30 min to obtain a dark green supernatant, which is the monolayer MXenes.

[0072] S2. Preparation of NiCo-LDH@MXenes: 6.25 mL of monolayer MXenes colloidal solution was diluted to 50 mL with deionized water, and ultrasonic treatment (150 W, 20 min) was performed to ensure uniform dispersion; then equimolar amounts of Ni(NO3)2·6H2O and Co(NO3)2·6H2O (2 mmol each) were added and stirred until completely dissolved; under continuous stirring, 10 mL of 10 mg / mL NaBH4 aqueous solution was slowly added dropwise to promote Ni 2+ and Co 2+ In situ reduction and growth of NiCo-LDH nanosheets; after the reaction is completed, the product is collected by high-speed centrifugation (10,000 rpm, 5 min), washed with ethanol multiple times to remove impurities, and finally dried under vacuum at room temperature for 2 days to obtain NiCo-LDH@MXenes;

[0073] S3. Preparation of Pt NCs@NiCo-LDH@MXenes: 0.1 g NiCo-LDH@MXenes carrier was dispersed in 30 mL deionized water and ultrasonicated for 5 min. 100 uL of 19.3 mM H2PtCl6·6H2O solution was added dropwise and stirred for 30 min. Then, 1.5 mL of a reducing agent solution containing 0.1 M NaBH4 and 0.2 M NaOH was quickly injected and stirred for 1 h. After centrifugal washing, the mixture was freeze-dried for 24 h to obtain the Pt NCs@NiCo-LDH@MXenes composite material.

[0074] The single-layer MXenes, NiCo-LDH, NiCo-LDH@MXenes layered and Pt NCs@NiCo-LDH@MXenes composites prepared in Example 1 were observed by transmission electron microscopy, and the results were as follows: Figure 1 、 Figure 2 、 Figure 3 and Figure 4 As shown, where:

[0075] Figure 1 The transmission electron microscopy image of the single-layer Mxenes prepared in Example 1 of this scheme shows a typical ultra-thin layer structure with a smooth surface without defects;

[0076] Figure 3 This is a transmission electron microscope image of NiCo-LDH@MXenes prepared in Example 1 of this scheme. Figure 3 As shown in the figure, the MXenes sheets are bent and the thickness increases significantly, and the surface is evenly loaded with NiCo-LDH nanosheets to form an open multi-level structure. The nanosheets are stacked disorderly to form a dense block of pure NiCo-LDH ( Figure 2), the MXene support effectively inhibits the stacking of LDH nanosheets, exposes more active sites, and significantly increases the specific surface area;

[0077] Figure 4 This is a transmission electron microscope image of the Pt NCs@NiCo-LDH@MXenes composite material prepared in Example 1 of this scheme; Figure 4 As shown in the figure, the particle size of Pt NCs is about 3 nm and is evenly distributed on the layered surface of NiCo-LDH@MXenes without obvious agglomeration. The prepared NiCo-LDH@MXenes layered structure provides a good loading for Pt nanoclusters, which can effectively improve the overall catalytic performance of the composite material.

[0078] Electrochemical impedance spectroscopy (EIS) was used to study the conductivity of the prepared composite materials. Figure 5 This is the AC impedance spectrum of the sensor construction process of this scheme, such as Figure 5 As shown in the figure, the semicircle diameter of the material represents the charge transfer resistance (Ret), which is related to the modification of the electrode surface. After NiCo-LDH is deposited, its poor conductivity hinders electron transfer and the charge transfer resistance increases. When NiCo-LDH@MXenes modifies the electrode, MXenes as a substrate material improves the conductivity of NiCo-LDH / GCE. Furthermore, Pt NCs@NiCo-LDH@MXenes exhibits the smallest semicircle diameter, indicating that the prepared composite material has higher conductivity, further demonstrating the successful preparation of Pt NCs@NiCo-LDH@MXenes composite material.

[0079] Example 2

[0080] A method for preparing an electrochemiluminescence sensor based on a Pt NCs@NiCo-LDH@MXenes composite material comprises the following steps:

[0081] S1, polishing the GCE to a mirror finish using alumina powder (0.3 μm and 0.05 μm) and then ultrasonic cleaning to remove residues;

[0082] S2. Take 6 μL of Pt NCs@NiCo-LDH@MXenes suspension and evenly drop-coat it on the pretreated GCE surface and dry it naturally at room temperature.

[0083] S3, add 6 μL of dopamine-modified tetrahedral DNA (TDNs-DA) to the electrode surface and let it stand for 1 h to achieve covalent binding through Pt-S bonds;

[0084] S4, add 6 μL of 1 mM mercaptohexanol (MCH) solution and incubate at room temperature for 1 h to block the remaining active sites on the electrode surface;

[0085] S5, 10 μL of aptamer (1.50 μM) and 10 μL of activator (1.0 μM) were mixed to final concentrations of 0.75 μM and 0.5 μM, respectively. The DNA was denatured by heating at 95°C for 10 min and then slowly cooled to room temperature (to promote specific hybridization). Finally, the mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was used for subsequent experiments.

[0086] The sequences used and their names are shown in Table 1 below:

[0087] Table 1 Sequence information used

[0088]

[0089] The construction process of the electrochemiluminescence sensor prepared by the above method was tested as follows:

[0090] Figure 6 The dynamic changes of the electrochemiluminescence signal during the sensor construction process are demonstrated. When the electrode is modified with Pt NCs@NiCo-LDH@MXenes (curve b), the composite material significantly enhances the ECL signal of luminol under the condition of dissolved oxygen as a co-reactant, which is due to the excellent catalytic activity and conductive properties of the composite material. When TDNs-DA is subsequently introduced (curve c), the ECL signal decreases sharply, confirming the strong quenching effect of dopamine molecules on the luminol luminescence system. After blocking the nonspecific sites with MCH (curve d), the signal continues to decay, indicating that the sensing interface is effectively optimized. Subsequently, in the presence of CIT, the self-assembly of the Cas12a / crRNA / activator ternary complex activates its trans-cleavage activity, relieves the quenching effect of the ECL signal through nonspecific cleavage reactions, and ultimately achieves a significant increase in the signal (curve e). This series of orderly ECL responses verifies the precise assembly and functional regulation mechanism based on the Pt NCs@NiCo-LDH@MXenes sensing platform, providing a reliable signal conversion strategy for CIT detection.

[0091] Example 3

[0092] An electrochemiluminescence sensor based on a Pt NCs@NiCo-LDH@MXenes composite material is used to detect citrinin, comprising the following steps:

[0093] S1. Mix 10 μL of gradient toxin solution with 20 μL of aptamer functional solution and incubate at 37°C for 40 min to allow the aptamer to specifically bind to the toxin and release the pre-blocked activator DNA.

[0094] S2, the above reaction solution is mixed with the pre-prepared Cas12a-crRNA complex, and the Cas12a / crRNA / activator ternary complex is formed by activator DNA guidance to activate the trans-cleavage activity of the Cas12a enzyme;

[0095] S3, take the activated Cas12a enzyme droplet and apply it on the MCH / TDNs-DA / Pt NCs@NiCo-LDH@MXene modified electrode surface;

[0096] S4. In 5 mL PBS (pH 7.4) containing 100 μM Luminol, a scanning voltage of -0.2 to 0.6 V (50 mV / s) was applied, and the ECL signal intensity was detected at a photomultiplier voltage of 600 V to establish a quantitative relationship between citrinin concentration and ECL intensity; the results are shown in FIG. Figure 7 and Figure 8 shown.

[0097] Figure 7 This is the time-current curve of the sensor corresponding to different concentrations of CIT in this scheme; Figure 8 This is the linear fitting curve of the logarithmic value of different concentrations of CIT and current in this scheme; Figure 7 and Figure 8 As shown, the concentration of citrinin was between 0.05 and 1×10 4 ng·mL -1 There is a good linear correlation within the range. With the increase of citrinin concentration, the ECL signal increases continuously. The linear regression equation is: ECL =2010.43LgC+3850.48, correlation coefficient R 2 is 0.9987; where I ECL is the ECL intensity change, C is the target concentration, and the LOD is calculated to be 11 pg·mL -1 (where S / N=3).

[0098] Figure 9 Figure 1 shows the specificity test results of the sensor provided by this protocol. The sensor's specificity was evaluated by measuring the ECL signal values ​​of a detection system containing citrinin and other interfering mycotoxins: ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin B1 (AFB1). As shown, the ECL signals of the four interfering toxins were significantly lower than that of the citrinin sample. Furthermore, there was no significant difference in the ECL signals between citrinin alone and when it was mixed with the aforementioned interfering toxins, indicating that the impact of the interfering toxins on citrinin detection was negligible. Therefore, the proposed sensor exhibits good specificity for citrinin detection.

[0099] It should be noted that when numerical ranges are mentioned in this solution, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, to avoid redundancy, this solution describes the preferred embodiments. Although preferred embodiments of this solution have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of this solution.

[0100] Although embodiments of the present scheme have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present scheme, and the scope of the present scheme is defined by the appended claims and their equivalents.

Claims

1. A Pt NCs@NiCo-LDH@MXenes composite material, characterized in that: The Pt NCs@NiCo-LDH@MXenes composite material is obtained by reducing a platinum source into Pt NCs under the action of a reducing agent and dispersing and loading the Pt NCs on NiCo-LDH@MXenes with a layered structure.

2. A method for preparing the Pt NCs@NiCo-LDH@MXenes composite material according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of monolayer MXenes: 1 g of LiF was slowly dissolved in 10 mL of 12 M HCl solution to obtain an HCl / LiF etching solution. Then, 0.1 g of Ti3AlC2 powder was slowly added to the HCl / LiF etching solution and magnetically stirred at room temperature for 24 h; After the reaction is completed, the supernatant is centrifuged several times until the pH value is 6, the residual acid solution is removed, and the precipitate is collected by vacuum filtration; Finally, a single-layer MXenes colloidal solution was obtained by centrifugation and ultrasonic dispersion; Step 2: Preparation of NiCo-LDH@MXenes. Specifically, 6.25 mL of a single-layer MXenes colloidal solution was diluted to 50 mL with deionized water and then ultrasonicated. 2 mmol each of Ni(NO3)2·6H2O and Co(NO3)2·6H2O was added and dissolved. 10 mL of a 10 mg / mL NaBH4 aqueous solution was slowly added dropwise to grow NiCo-LDH nanosheets. The product was centrifuged, washed with ethanol, and dried in vacuum to obtain NiCo-LDH@MXenes composite material; Step 3: Preparation of Pt NCs@NiCo-LDH@MXenes. Specifically, 0.1 g of NiCo-LDH@MXenes carrier was dispersed in 30 mL of deionized water and ultrasonicated for 5 min. 100 μL of 19.3 mM H2PtCl6·6H2O solution was added dropwise and stirred for 30 min. Subsequently, 1.5 mL of a reducing agent solution containing 0.1 M NaBH4 and 0.2 M NaOH was quickly injected and stirred for 1 h to load Pt NCs onto NiCo-LDH@MXenes to obtain the sensor substrate Pt NCs@NiCo-LDH@MXenes.

3. The method for preparing a Pt NCs@NiCo-LDH@MXenes composite material according to claim 2, characterized in that: The mass molar ratio of NiCo-LDH@MXenes to Pt NCs in the Pt NCs@NiCo-LDH@MXenes composite material is 60 mg to 100 mg: 1.2×10 -3 mmol~2×10 -3 mmol.

4. The method for preparing a Pt NCs@NiCo-LDH@MXenes composite material according to claim 2, characterized in that: The platinum source is H2PtCl6, the reducing agent is NaBH4, the molar ratio of the reducing agent to the platinum source is 78-80:1-3, and the immersion reduction time is 10 min to 60 min.

5. Use of the Pt NCs@NiCo-LDH@MXenes composite material according to claim 1 in preparing a sensor for detecting citrinin, characterized in that: The sensor is an electrochemiluminescent sensor.

6. Use of a Pt NCs@NiCo-LDH@MXenes composite material according to claim 5 in preparing a sensor for detecting citrinin, characterized in that: The sensor includes: a substrate, and an electrode layer attached to the substrate, wherein the electrode layer includes a working electrode layer, and a Pt NCs@NiCo-LDH@MXenes composite material, TDNs-DA, and MCH are sequentially coated on the surface of the working electrode layer.

7. Use of a Pt NCs@NiCo-LDH@MXenes composite material according to claim 6 in preparing a sensor for detecting citrinin, characterized in that: The sensor is used to detect citrinin, and a sensing system is constructed using nucleic acid aptamers as specific recognition units, and detection is achieved through the cascade signal amplification mechanism of the CRISPR / Cas12a system; After the aptamer captures citrinin, it triggers the activation mechanism of the Cas12a enzyme. The activated Cas12a enzyme then exhibits trans-cleavage activity, non-specifically cleaving the single-stranded structure in the TDNs-DA complex modified on the electrode surface. This cutting action destroys the luminescence inhibition system originally established by DA, prompting the DA molecules to dissociate from the electrode surface. As the electron transfer efficiency of the electrode surface recovers, the electrochemiluminescence signal is significantly enhanced. Accurate quantitative analysis of citrinin can be achieved by detecting changes in signal intensity.

8. Use of a Pt NCs@NiCo-LDH@MXenes composite material according to claim 7 in preparing a sensor for detecting citrinin, characterized in that: The Apt nucleotide sequence of citrinin is: 5'-GGCCAGGCGGGGCCTGTTCGTGGGCCGTGTCTTCGGCTCGCTCGGTTG-3'.

9. Use of a Pt NCs@NiCo-LDH@MXenes composite material according to claim 7 in preparing a sensor for detecting citrinin, characterized in that: The method for using the sensor to detect citrinin comprises the following steps: 10 μL of gradient toxin solution was mixed with 20 μL of aptamer functional solution and incubated at 37°C for 40 min to allow the aptamer to specifically bind to the toxin and release the pre-blocked activator DNA; The above reaction solution is mixed with the pre-prepared Cas12a-crRNA complex, and the Cas12a / crRNA / activator ternary complex is formed by guiding the activator DNA to activate the trans-cleavage activity of the Cas12a enzyme; The activated Cas12a enzyme droplets were applied to the surface of the MCH / TDNs-DA / Pt NCs@NiCo-LDH@MXenes modified electrode; In 5 mL of PBS (pH 7.4) containing 100 μM luminol, a scanning voltage of 50 mV / s from -0.2 to 0.6 V was applied, and the ECL signal intensity was detected at a photomultiplier tube voltage of 600 V to establish a quantitative relationship between citrinin concentration and ECL intensity.

10. Use of a Pt NCs@NiCo-LDH@MXenes composite material according to claim 9 in preparing a sensor for detecting citrinin, characterized in that: The detection range of the sensor is 0.05~1×10 4 ng mL -1 , with a detection limit of 11 pg mL -1 .

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