G-C3N4 (at) Au NCs composite material, sensor and method for detecting aflatoxin B1

Abstract

The invention relates to the technical field of food safety detection, in particular to a g-C3N4 (at) Au NCs composite material, a sensor and a method for detecting aflatoxin B1. The composite material is prepared by anchoring Au NCs on the surface of g-C3N4 through a chemical reduction method, and can serve as a sensing substrate for modification, an electrochemical luminescence (ECL) sensor for detecting AFB1 based on CRISPR / Cas12a system regulation and control is constructed, and the composite material can be used for detecting AFB1 of 0.2 pg / mL-100 ng / mL. The sensor with the substrate modified by the g-C3N4 (at) Au NCs composite material, provided by the invention, also shows good selectivity and reproducibility, and has a wide application value.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and specifically to a g-C3N4@Au NCs composite material, a sensor, and a method for detecting aflatoxin B1. Background Art

[0002] Aflatoxins are a class of mycotoxins produced primarily by Aspergillus flavus and Aspergillus parasiticus, consisting of a coumarin and furan rings. Among all aflatoxins, AFB1 is the most toxic and is classified as a Class I carcinogen by the International Agency for Research on Cancer. It is widely found in cereals, peanuts, and dried fruits. Therefore, to ensure food, drug, and public health safety, a rapid, accurate, and sensitive AFB1 detection method is urgently needed.

[0003] Currently, traditional methods for detecting AFB1 mainly include thin-layer chromatography (TLC), liquid chromatography (LC), and liquid chromatography-mass spectrometry (LC-MS). Although these methods offer the advantage of quantitative detection, they suffer from disadvantages such as high cost, complex operation, and long detection times, severely limiting the demand for rapid on-site detection. To address these challenges, biosensors, with their advantages of high sensitivity, ease of operation, and rapid analysis, have attracted widespread attention from analysts. Currently reported AFB1 biosensors generally utilize an antibody-antigen competitive reaction mechanism to achieve signal conversion. However, these traditional competitive sensors are susceptible to false positives caused by nonspecific adsorption or label leakage in actual detection. This work, by incorporating the unique cascade signal amplification and regulation mechanism of the CRISPR / Cas12a system, constructs a novel non-competitive sensing system. On the one hand, the precise recognition of crRNA significantly reduces background signal interference. On the other hand, leveraging the high-fidelity characteristics of the CRISPR system, the risk of false positives caused by label desorption in traditional competitive strategies is effectively avoided, thereby improving the accuracy and reliability of detection results. Summary of the Invention

[0004] (1) Technical issues

[0005] The present invention aims to at least solve the problem of insufficient precision and accuracy in AFB1 detection in the prior art.

[0006] This application aims to provide a self-enhanced electrochemiluminescent composite material for use as a sensing substrate modification, based on the CRISPR / Cas12a system regulation strategy, and then designed into an electrochemiluminescent sensor.

[0007] (2) Technical content

[0008] This solution provides a g-C3N4@Au NCs composite material, which is achieved by the following specific technical means: the composite material is a structure in which Au NCs with an average particle size of 3 nm are uniformly anchored on the surface of g-C3N4;

[0009] Specifically, the g-C3N4@Au NCs composite material is prepared by anchoring Au NCs on the surface of g-C3N4 through a chemical reduction method.

[0010] Preferred technical solution 1: wherein g-C3N4 is solid powder, and Au NCs are prepared using chloroauric acid (HAuCl4) as raw material.

[0011] Preferred technical solution 2: The preparation method of the composite material comprises: dissolving g-C3N4 solid in deionized water to prepare a g-C3N4 suspension, mixing the g-C3N4 suspension with a gold source under stirring, then ultrasonically treating for 10 minutes, adding a reducing agent to react for 10 minutes, and centrifuging and drying to obtain a solid product;

[0012] The gold source is tetrachloroauric acid (HAuCl4), preferably, the gold source is a tetrachloroauric acid solution with a molar concentration of 10 mM; the reducing agent is sodium borohydride (NaBH4), and the reducing agent is a solution with a molar concentration of 10 mM sodium borohydride and 10 mM sodium citrate;

[0013] More specifically:

[0014] 5 mL of the prepared g-C3N4 suspension (1.5 mg / mL) was added with 20 μL of 10 mM HAuCl4 solution under stirring;

[0015] Then, the suspension was sonicated for 10 min, and 10 μL of freshly prepared 10 mM NaBH4 solution was quickly added to reduce AuCl4 under stirring. - , react for 10 min under stirring;

[0016] Finally, 10 μL of 10 mM sodium citrate solution was added dropwise to the above suspension and reacted for 10 min under stirring;

[0017] In order to remove excess NaBH4, sodium citrate and unbound gold nanoclusters, the g-C3N4@Au NCs composite material was obtained by centrifugal separation and drying.

[0018] The g-C3N4@Au NCs composite material provided in this application has the following advantages compared with other sensing substrates: (1) Au NCs has a strong affinity for S2O8 2- The efficient electrocatalytic reduction of sulfuric acid SO4 ·-The results show that the self-enhanced electrochemiluminescence composite material g-C3N4@Au NCs can significantly enhance the ECL signal intensity of g-C3N4; (2) by optimizing the loading density of Au NCs on the g-C3N4 surface, more binding sites of DNA tetrahedral structure complexes can be constructed, thereby significantly improving the detection sensitivity of the sensor; (3) Mechanism studies have shown that Au NCs effectively suppress the rapid decay of the ECL signal in the potential range of 0 to -1.5V by capturing and storing electrons from the conduction band of g-C3N4, thereby avoiding the occurrence of electrode surface passivation problems; therefore, the self-enhanced electrochemiluminescence composite material g-C3N4@Au NCs provided in this application can be used as a sensing substrate to significantly improve the sensitivity of the electrochemiluminescence sensor.

[0019] Preferred technical solution three: The g-C3N4 is obtained by calcining urea in a muffle furnace at a temperature of 550°C for 2 hours.

[0020] The present application also provides an application of the g-C3N4@Au NCs composite material in preparing a sensor for detecting AFB1.

[0021] Preferred Technical Solution 1: The sensor is an electrochemiluminescent sensor for detecting AFB1 based on the regulation of the CRISPR / Cas12a system, and the sensor comprises:

[0022] A substrate, and an electrode layer attached to the substrate, wherein the electrode layer includes a working electrode layer;

[0023] The surface of the working electrode layer is coated with the g-C3N4@Au NCs, TDNs, and ssDNA-Au NPs@PDA in sequence.

[0024] Preferred technical solution 2: The working electrode layer is the substrate, and the g-C3N4@Au NCs composite material, TDNs, and ssDNA-AuNPs@PDA are coated on its surface in sequence;

[0025] Preferably, the electrode layer further comprises a platinum wire as a counter electrode and silver / silver chloride as a reference electrode.

[0026] The present application also provides a method for quantitatively detecting AFB1, the method comprising the steps of detecting using the sensor, specifically comprising:

[0027] The sample to be tested is dropped onto the surface of the working electrode layer and reacted at room temperature for 40 to 60 minutes. Under the detection of the constructed sensor, the corresponding electrochemical signal is measured and recorded, and the electrochemiluminescence intensity and time curve is plotted to achieve quantitative detection of AFB1.

[0028] Preferred technical solution 1: The detection range of the electrochemiluminescence sensor is 0.2 pg / mL to 100 ng / mL.

[0029] The electrochemiluminescence sensor provided by the present application detects AFB1 by the following mechanism: Figure 1 As shown, the details are as follows:

[0030] When AFB1 is absent, ssDNA-Au NPs@PDA binds to TDNs through complementary ligand pairing to form a double-stranded structure, bringing Au NPs@PDA close to the electrode surface and generating an energy resonance transfer effect with the g-C3N4@Au NCs composite material on the electrode surface, thereby suppressing the electrochemiluminescence signal of the self-reinforced composite material on the electrode surface and causing the sensor to present a "signal-off" state.

[0031] When AFB1 is present, the trans-cleavage activity of CRISPR / Cas12a, based on the target-responsive regulation system, is activated to cut the ssDNA-Au NPs@PDA on the electrode surface, causing the Au NPs@PDA molecules to detach from the electrode interface and the sensor to switch to the "signal-on" state.

[0032] Therefore, the qualitative and quantitative detection of AFB1 is achieved based on the relationship between the intensity change of the electrochemiluminescence signal of the self-reinforced composite material sensor and the concentration of AFB1.

[0033] The g-C3N4@Au NCs composite material prepared in this application can exhibit good electrochemical properties and can be used as an electrochemiluminescence sensing substrate, which is beneficial to improving detection sensitivity; and based on the CRISPR / Cas12a system regulated by target response, it causes changes in the electrochemiluminescence signal, realizing qualitative and quantitative detection of AFB1.

[0034] The g-C3N4@Au NCs-based electrochemiluminescence sensor constructed in this application for detecting AFB1 has a detection range of 0.2 pg / mL to 100 ng / mL, showing a low detection limit and has good application prospects in food safety testing.

[0035] (3) Technical effects

[0036] The above structure enables this solution to have the following beneficial effects:

[0037] 1. In this application, g-C3N4 was used as a carrier, and Au NCs were anchored on the surface of g-C3N4 by chemical reduction to prepare a g-C3N4@Au NCs composite material, which was used to modify the sensing interface, increase the electron transfer rate on the electrode surface, and enhance the electrochemiluminescence signal response;

[0038] 2. This application uses a target-responsive regulation strategy based on the CRISPR / Cas12a system, which achieves highly sensitive and reliable detection of AFB1 by combining a "signal off / on" signal conversion mechanism through a linear positive correlation between AFB1 concentration and electrochemiluminescence signal intensity.

[0039] 3. This application is based on the electrochemiluminescence sensor, which has the characteristics of low cost, simple operation, rapidity and accuracy, and meets the needs of on-site detection;

[0040] 4. This application successfully constructed an AFB1 electrochemiluminescence sensor based on the CRISPR / Cas12a system regulation strategy, with a detection range of 0.2pg / mL to 100ng / mL of AFB1 and a detection limit of 0.08pg / mL;

[0041] In addition, the sensor exhibited high specificity, selectivity, and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0043] In the attached figure:

[0044] Figure 1 Schematic diagram of the principle of detecting AFB1 based on the sensor regulated by the CRISPR / Cas12a system provided in this application;

[0045] Figure 2 Figure 1 is a transmission electron microscopy image of the g-C3N4@Au NCs composite material prepared in Example 1, wherein Figure A is a transmission electron microscopy image of g-C3N4, Figure B is a transmission electron microscopy image of the g-C3N4@Au NCs composite material, and Figure C is a high-resolution transmission electron microscopy image of Au NCs;

[0046] Figure 3 Comparison of electrochemiluminescence signal intensities of g-C3N4, g-C3N4 / Au NCs and g-C3N4@Au NCs;

[0047] Figure 4 Schematic diagram of the stability of the electrochemiluminescence signal intensity of g-C3N4 / Au NCs;

[0048] Figure 5 Schematic diagram of the stability of the electrochemiluminescence signal intensity of g-C3N4@Au NCs;

[0049] Figure 6 Schematic diagram of electrochemiluminescence signal intensity detection of the sensor corresponding to different concentrations of AFB1;

[0050] Figure 7 is the linear fitting curve of the logarithm of AFB1 at different concentrations and the electrochemiluminescence intensity;

[0051] Figure 8 Selectivity test results of the sensors provided for this application;

[0052] Figure 9 Reproducibility test results of the sensor provided for this application;

[0053] Figure 10 The long-term stability test results of the sensor provided in this application. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0055] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0056] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0057] Example 1

[0058] This embodiment provides a g-C3N4@Au NCs composite material and an Au NPs@PDA material, the preparation method of which includes the following steps:

[0059] (1) Preparation of g-C3N4

[0060] 10 g of urea was added to an alumina crucible, which was placed in a muffle furnace;

[0061] It was heated to 550 °C at a rate of 15 °C / min and reacted for 2 h;

[0062] After cooling to room temperature, g-C3N4 nanosheets were obtained;

[0063] (2) Preparation of g-C3N4@Au NCs

[0064] 5 mL of g-C3N4 suspension (1.5 mg / mL) was stirred and added with 20 μL of 10 mM HAuCl4 solution. The suspension was ultrasonicated for 10 min and 10 μL of freshly prepared 10 mM NaBH4 solution was quickly added to the suspension under stirring to reduce AuCl4. - , react for 10 min under stirring;

[0065] Finally, 10 μL of 10 mM sodium citrate solution was added dropwise to the above suspension and reacted for 10 min under stirring;

[0066] To remove excess NaBH4, sodium citrate, and unbound gold nanoclusters, the obtained composites were separated by centrifugation, washed thoroughly with distilled water, and finally redispersed in 1 mL of water for further use and characterization;

[0067] (3) Preparation of Au NPs

[0068] 50 mL of 10 mM HAuCl4 solution was added to a 100 mL round-bottom flask equipped with a condenser;

[0069] The solution was heated to a boil with vigorous stirring, and then 5 mL of 38.8 mM sodium citrate solution was quickly added to the vortex; the addition of sodium citrate changed the color of the solution from light yellow to red.

[0070] The solution was heated for another 20 min, after which the heating mantle was removed and stirred for an additional 20 min;

[0071] Finally, the prepared gold nanoparticle solution was stored in a brown bottle and kept at 4 °C;

[0072] (4) Preparation of Au NPs@PDA

[0073] 1 mL of the prepared Au NPs was added to 1 mL of Tris buffer (10 mM) containing 0.05 mg / mL dopamine;

[0074] The reaction mixture was gently stirred for 6 h, and then the Au NPs@PDA nanoparticles were separated by centrifugation and redissolved in 1 mL of ultrapure water.

[0075] The composite material prepared by the above method was observed by transmission electron microscopy. The results are as follows: Figure 2 As shown;

[0076] Depend on Figure 2 It can be seen that Au NCs with an average particle size of 3 nm are uniformly dispersed on the surface of g-C3N4, and no obvious agglomeration occurs.

[0077] Furthermore, the effect of electrochemiluminescence intensity-voltage curve on the electrochemiluminescence performance of the prepared g-C3N4@Au NCs composite material was investigated. The results are as follows Figure 3 As shown;

[0078] Depend on Figure 3 It can be seen that the g-C3N4@Au NCs composite material prepared by the chemical reduction method of Au NCs exhibits significantly enhanced electrochemiluminescence performance. Its luminescence intensity is not only significantly better than that of a single g-C3N4 material, but is also about 2.73 times higher than that of the g-C3N4@Au NCs composite material prepared by the physical mixing method. This significant performance improvement can be attributed to the heterogeneous structure formed by chemical bonding, which enables the composite material to exhibit excellent electrochemiluminescence activity by enhancing electron transfer efficiency and synergistic luminescence effect.

[0079] Depend on Figure 4 It can be seen that the electrochemiluminescence signal stability of physically mixed g-C3N4 / Au NCs is poor. Figure 5 The in-situ reduced g-C3N4@Au NCs composite material prepared in the embodiment showed a stronger and more stable electrochemiluminescence signal, which is due to the presence of S2O8 2- In the system, Au NCs has a great influence on the S2O8 2- Highly efficient electrocatalytic reduction, thereby generating more SO4 ·- Free radicals further promote the recombination efficiency of electron-hole pairs, and ultimately enhance the signal intensity of electrochemiluminescence.

[0080] Example 2

[0081] This example provides an electrochemiluminescent sensor based on the g-C3N4@Au NCs composite material regulated by the CRISPR / Cas12a system in Example 1. The preparation method of the sensor is as follows:

[0082] Step a: Preparation of electrochemiluminescence sensor of g-C3N4@Au NCs composite material

[0083] (b-1) 6 μL of 2.00 mg / mL ultrapure aqueous solution of g-C3N4@Au NCs was drop-coated on the surface of the glassy carbon electrode and allowed to dry at room temperature. The electrode surface was then rinsed with ultrapure water and allowed to dry.

[0084] (b-2) 6 μL of 1.0 μg / mL TDNs solution was added dropwise onto the surface of the g-C3N4@Au NCs-modified working electrode layer;

[0085] (b-3) 3 μL of 6-mercaptohexanol (MCH) solution was added dropwise to the surface of the working electrode layer to block the nonspecific active sites on the electrode surface.

[0086] (b-4) 6 μL of 1.0 μg / mL ssDNA-Au NPs@PDA solution was added dropwise to the surface of the working electrode layer to bind to the TDNs through the complementary pairing principle;

[0087] (b-5) Drying to obtain an electrochemiluminescence sensor for detecting AFB1.

[0088] The detection effect of the electrochemiluminescence sensor prepared by the above method on AFB1 was tested as follows:

[0089] 10 μL, 0.2 pg / mL to 100 ng / mL of AFB1 solution at different concentrations were mixed with 20 μL of nucleic acid aptamer / activator 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 was mixed with the pre-prepared Cas12a-crRNA binary complex, and the Cas12a / crRNA / activator ternary complex was formed under the guidance of crRNA to activate the trans-cleavage activity of the Cas12a enzyme. The activated Cas12a enzyme droplet was applied to the g-C3N4@Au NCs / TDNs / MCH / ssDNA-Au NPs@PDA modified electrode surface and reacted at room temperature for 40 min.

[0090] Then electrochemiluminescence detection was performed, and the results were as follows Figure 6 and Figure 7 shown.

[0091] The nucleotide sequence required above is as follows:

[0092] Table 1 Nucleotide sequence

[0093]

[0094]

[0095] For AFB1 solutions with concentrations ranging from 0.2 pg / mL to 100 ng / mL, a fitting curve of electrochemiluminescence signal intensity and AFB1 concentration was drawn under the detection of the constructed electrochemiluminescence sensor. Figure 6 and Figure 7 As shown, the electrochemiluminescence sensor provided by this application has a detection range of 0.2pg / mL to 100ng / mL, and its linear equation is I ECL =8091.4+1644.9logC AFB1 (R 2 =0.998), and the detection limit was 0.08 pg / mL.

[0096] Interfering fungal toxins, including deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), and patulin, which may be present in actual samples, were used to test the selectivity of the electrochemiluminescence sensor. The results are shown in Figure 2. Figure 8 As shown;

[0097] Depend on Figure 8 The results show that the signal response of the interfering fungal toxin sample is consistent with that of the blank sample. When the target compound AFB1 is present in the sample, the sensor shows a significant signal change, indicating that the sensor has good selectivity.

[0098] In addition, the reproducibility of the electrochemiluminescence sensor was tested, and the results were as follows Figure 9 As shown;

[0099] Depend on Figure 9 The results show that the relative standard deviation of the electrochemiluminescence signal response is 1.13%, indicating that the electrochemiluminescence sensor has good reproducibility.

[0100] In addition, the long-term stability of the electrochemiluminescence sensor was tested, and the results were as follows Figure 10 As shown;

[0101] Depend on Figure 10 The results show that the electrochemiluminescence sensor has good long-term stability.

[0102] While embodiments of the present invention have been shown and described, it will be appreciated 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 invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A g-C3N4@Au NCs composite material, characterized in that The composite material is obtained by uniformly dispersing Au NCs with an average particle size of 3 nm on the surface of the g-C3N4 structure.

2. A g-C3N4@Au NCs composite material according to claim 1, characterized in that The composite material is prepared by anchoring AuNCs on the surface of g-C3N4 through a chemical reduction method.

3. A g-C3N4@Au NCs composite material according to claim 2, characterized in that The preparation method of the composite material comprises: The g-C3N4 suspension was mixed with the gold source under stirring, ultrasonically treated for 10 min, a reducing agent was added to react for 10 min, and a solid product was obtained by centrifugation and drying.

4. A g-C3N4@Au NCs composite material according to claim 3, characterized in that The g-C3N4 is obtained by calcining urea at 550°C in a muffle furnace for 2 hours.

5. The g-C3N4@Au NCs composite material according to claim 3, characterized in that: The gold source is chloroauric acid, and the reducing agent is sodium borohydride.

6. Use of the g-C3N4@Au NCs composite material according to any one of claims 1 to 5 in preparing a sensor for detecting AFB1.

7. Use of a g-C3N4@Au NCs composite material according to claim 6 in preparing a sensor for detecting AFB1, characterized in that: The sensor is an electrochemiluminescence sensor regulated by the CRISPR / Cas12a system.

8. The electrochemiluminescence sensor for detecting AFB1 according to claim 7, characterized in that: The sensor comprises: A substrate and an electrode layer attached to the substrate, wherein the electrode layer includes a working electrode layer; The surface of the working electrode layer was coated with 6 μL g-C 3 N 4 @Au NCs, 6 μL TDNs, and 6 μL ssDNA-Au NPs@PDA (ssDNA-AP) in sequence.

9. A method for quantitatively detecting AFB1, characterized in that: The step of detecting with the sensor according to claim 7 specifically comprises: The sample to be tested is dropped onto the surface of the working electrode layer and reacted at room temperature for 40 to 60 minutes. Under the detection of the constructed sensor, the corresponding electrochemical signal is measured and recorded, and a fitting curve of the electrochemical signal intensity response and the target concentration is drawn to achieve quantitative detection of AFB1.

10. The method for quantitative detection of AFB1 according to claim 9, characterized in that: The detection range of the sensor in electrochemiluminescence mode is 0.2 pg / mL to 100 ng / mL.

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