A beta-cyclodextrin modified electrochemiluminescence gold cerium bimetallic nanocluster, a preparation method and application thereof in electrochemiluminescence-impedance dual-mode detection of malathion

Abstract

The application provides a beta-cyclodextrin modified electrochemiluminescence gold cerium bimetallic nanocluster, a preparation method and application thereof in electrochemiluminescence-impedance dual-mode detection of malathion, and relates to the field of malathion detection. The preparation method of the beta-cyclodextrin modified electrochemiluminescence gold cerium bimetallic nanocluster comprises the following steps: after gold source and cerium source are incubated with methionine for the first time in an alkaline environment, the gold source and the cerium source are dispersed in an ammonia solution to be incubated for the second time; and finally, the gold source and the cerium source are incubated with NH2-beta-cyclodextrin for the third time to obtain the beta-cyclodextrin modified electrochemiluminescence gold cerium bimetallic nanocluster. The beta-cyclodextrin modified electrochemiluminescence gold cerium bimetallic nanocluster is used as an ECL luminophore, and a sensor using triethylamine as a co-reaction agent is applied to the detection of malathion, and the sensor has high sensitivity, selectivity, stability, realizes ECL and EIS dual-mode cooperative detection and mutual verification, and has the advantages of simple operation and good repeatability.

Description

Technical Field

[0001] This invention relates to the field of malathion detection, and in particular to a β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster, its preparation method, and its application in the electrochemiluminescence-impedance dual-mode detection of malathion. Background Technology

[0002] Organophosphorus pesticides play a vital role in modern agricultural production; however, their excessive use and resulting residue problems pose a serious threat to the ecological environment and food safety. Malathion, as a typical organophosphorus pesticide, has made residue detection in agricultural products and environmental media an important issue in the field of public health. Currently, while conventional detection methods such as chromatography and mass spectrometry have high sensitivity, they suffer from limitations such as expensive equipment, complex operation, and long analysis cycles, making it difficult to meet the needs of rapid on-site detection.

[0003] Electrochemical sensing technology has shown broad application prospects in the field of pesticide residue detection due to its advantages such as simple operation, rapid response, and low cost. Among them, electrochemiluminescence (ECL) and electrochemical impedance spectroscopy (EIS) are two typical sensing strategies, which rely on the luminescence behavior of electrogenerated excited-state substances and changes in interfacial electron transport resistance to achieve detection functions, respectively. However, traditional electrochemiluminescence (ECL) materials suffer from problems such as low luminescence efficiency and poor stability, and are easily interfered with in the analysis of complex samples, resulting in insufficient selectivity. At the same time, traditional electrochemical impedance spectroscopy (EIS) materials still have significant shortcomings in terms of sensitivity, selectivity, detection speed, and stability, specifically: they are easily interfered with in complex samples, resulting in poor selectivity; they have a weak response to low concentrations of target substances, resulting in limited sensitivity; the testing time is long, resulting in poor detection speed; and the interface is prone to contamination, leading to poor reproducibility.

[0004] Nanomaterials, as carriers of sensing elements, directly affect detection performance. Gold nanoclusters (AuNCs) have been extensively studied due to their unique electrical properties and biocompatibility. However, the inventors have found that their single metal composition limits the diversity of catalytic activity and signal response types. Chinese patent CN121108973A discloses a microwave-assisted synthesis method for near-infrared electrochemiluminescent gold nanoclusters and their application in miRNA detection. This method uses HAuCl4·3H2O and methionine to prepare near-infrared electrochemiluminescent gold nanoclusters (Met-AuNCs). However, due to their single metal composition and surface chemical characteristics, they suffer from low electrochemiluminescence quantum yield, insufficient photophysical stability, lack of endogenous molecular recognition sites, limited signal amplification, and susceptibility to non-specific adsorption interference in complex matrices.

[0005] Studies have shown that constructing bimetallic nanostructures can synergistically enhance the photoelectric properties and catalytic activity of materials. Meanwhile, β-cyclodextrin (β-CD), as a natural macromolecule with hydrophobic cavities, can improve selective recognition capabilities through inclusion of target analytes; however, its application in the modification of bimetallic nanoclusters has not been fully explored. Therefore, providing a nanosensor material that combines high sensitivity, excellent selectivity, high stability, and enables synergistic detection and mutual verification of ECL and EIS modes, while integrating the advantages of ECL and EIS dual-mode detection, further improves its electrochemiluminescence quantum yield, enhances photophysical stability, constructs endogenous molecular recognition sites, optimizes signal amplification capabilities, and effectively avoids the problem of susceptibility to non-specific adsorption interference in complex matrices, is of great significance and research value for advancing the development of organophosphorus pesticide residue detection technology. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention provides a β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters and its preparation method. This nanosensor material combines high sensitivity, excellent selectivity, high stability, and enables synergistic detection and mutual verification of ECL and EIS dual modes. While integrating the advantages of ECL and EIS dual-mode detection, it further improves its electrochemiluminescence quantum yield, enhances photophysical stability, constructs endogenous molecular recognition sites, optimizes signal amplification capabilities, and effectively avoids the problem of susceptibility to non-specific adsorption interference in complex matrices. This invention also provides the application of the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters in the electrochemiluminescence-impedance impedance dual-mode detection of malathion.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] The first objective of this invention is to provide a method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters, comprising the following steps: a first incubation, a second incubation, and a third incubation;

[0009] The first incubation method is as follows: in an alkaline environment, gold source, cerium source and methionine are contacted for the first incubation; then the pH is adjusted to acidic, the precipitate is collected, and bimetallic nanoclusters are obtained.

[0010] The second incubation method involves dispersing the bimetallic nanoclusters in an ammonia solution and performing a second incubation; then separating and collecting the solids to obtain methionine-protected electrochemiluminescent gold-cerium nanoclusters.

[0011] The third incubation method involves mixing a mixed solution of methionine-protected electrochemiluminescent gold-cerium nanoclusters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide with an NH2-β-cyclodextrin solution for a third incubation; then separating and collecting the solids to obtain β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters.

[0012] Preferably, in the first incubation, the gold source includes tetrachloroauric acid; the cerium source includes cerium nitrate; and the molar ratio of the gold source, methionine and cerium source is 1:64-65:7.7-8.0.

[0013] Preferably, in the second incubation, the concentration of the bimetallic nanoclusters in the ammonia solution is 1-1.2 mg / mL; the concentration of the ammonia solution is 2-2.3 wt%.

[0014] Preferably, in the third incubation, the concentration of methionine-protected electrochemiluminescent gold-cerium nanoclusters in the mixed solution is 1-1.2 mg / mL; the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.85-0.9 mg / mL; and the concentration of N-hydroxysuccinimide is 0.4-0.45 mg / mL.

[0015] The concentration of the NH2-β-cyclodextrin solution is 1-1.2 mg / mL;

[0016] The molar ratio of NH2-β-cyclodextrin used in the third incubation to that used in the first incubation was 1:110-120.

[0017] Preferably, in the first incubation, the incubation temperature is 35-37℃ and the incubation time is 10-12h;

[0018] In the second incubation, the incubation temperature is 75-80℃ and the incubation time is 20-30 minutes;

[0019] In the third incubation, the incubation temperature is 22-26℃ and the incubation time is 4-5 hours.

[0020] The second objective of this invention is to provide an electrochemiluminescent gold-cerium bimetallic nanocluster modified with β-cyclodextrin prepared by the aforementioned method.

[0021] The third objective of this invention is to provide an electrochemiluminescence-impedance dual-mode sensor, comprising: an MB / dsDNA probe and a pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode.

[0022] The pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode is prepared by coating the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters onto the surface of the GCE electrode and then pre-oxidizing it.

[0023] The MB / dsDNA probe includes: dsDNA and magnetic beads;

[0024] The dsDNA is obtained by hybridization of the capture probe cDNA and its complementary azobenzene aptamer Azo-aptamer; the biotin of the capture probe cDNA is linked to streptavidin-modified magnetic beads.

[0025] The nucleotide sequence of the cDNA is shown in SEQ ID No. 1; the nucleotide sequence of the Azo-aptamer is shown in SEQ ID No. 2.

[0026] The fourth objective of this invention is to provide a method for preparing the aforementioned electrochemiluminescence-impedance dual-mode sensor, comprising the following steps: preparing an MB / dsDNA probe and preparing an electrode;

[0027] The method for preparing the MB / dsDNA probe is as follows: the capture probe cDNA is added to streptavidin-modified magnetic beads and incubated to obtain cDNA-modified magnetic beads; the cDNA-modified magnetic beads are dispersed in Azo-aptamer solution for DNA hybridization to obtain the MB / dsDNA probe.

[0028] The method for preparing the electrode is as follows: a solution of β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters is coated onto the surface of a GCE electrode to obtain a β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode. The electrode is then placed in a phosphate buffer solution containing triethylamine and pre-oxidized for 50-70 seconds under a continuous potential pulse of 0.9-1.1V.

[0029] Preferably, in the preparation of the MB / dsDNA probe, the incubation temperature of the capture probe cDNA with the streptavidin-modified magnetic beads is 30-37°C, and the incubation time is 50-70 min.

[0030] The temperature for DNA hybridization is 35-38℃, and the time for DNA hybridization is 25-35 minutes.

[0031] In the electrode preparation, the concentration of the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster solution is 1-1.5 mg / mL;

[0032] The triethylamine concentration in the phosphate buffer containing triethylamine is 0.1-0.15 mol / L, the concentration of the phosphate buffer is 0.1-0.15 mol / L, and the pH of the phosphate buffer is 7.2-7.6.

[0033] The fifth objective of this invention is to provide an application of the aforementioned electrochemiluminescence-impedance dual-mode sensor in the detection of malathion, comprising the following steps: incubating an MB / dsDNA probe with a test solution containing malathion for the first time; after magnetic separation, adding the supernatant to a pre-oxidized β-cyclodextrin-modified electrochemiluminescence gold-cerium bimetallic nanoclusters / GCE electrode for the second incubation; performing ECL measurement after pre-oxidation under continuous potential pulse conditions; and measuring the impedance signal in a potassium ferricyanide solution.

[0034] Preferably, the temperature for the first incubation is 32-37℃, and the time is 1-2 hours;

[0035] The second incubation should be carried out at a temperature of 32-37℃ for 40-60 minutes.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1) The method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters of the present invention involves introducing the rare earth element cerium (Ce) to construct a bimetallic nanocluster core, synthesizing it with methionine as a ligand, and utilizing the supramolecular inclusion function of β-cyclodextrin to obtain water-soluble bimetallic nanoclusters. The preparation process is green and environmentally friendly, simple and rapid, and has good reproducibility. Furthermore, the water-soluble methionine-protected electrochemiluminescent β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters prepared by this method exhibit good biocompatibility, high electrochemiluminescence efficiency, and fully passivated nanomaterial properties, effectively optimizing the electrode interface. Combined with a multi-mode signal fusion and rapid detection strategy, it effectively overcomes the shortcomings of traditional electrochemiluminescence (ECL) and electrochemical impedance spectroscopy (EIS) in terms of sensitivity, selectivity, detection speed, and stability, achieving highly sensitive, highly selective, rapid, and stable detection of malathion in both electrochemiluminescence and impedance modes, significantly improving the reliability of complex real-world sample analysis.

[0038] Furthermore, addressing the issues of low electrochemiluminescence quantum yield, insufficient photophysical stability, lack of endogenous molecular recognition sites, limited signal amplification, and susceptibility to non-specific adsorption interference in complex matrices found in existing gold nanoclusters, this invention introduces the rare earth element cerium (Ce) to construct a bimetallic nanocluster core, significantly enhancing the electrocatalytic activity and luminescence efficiency of the material. Simultaneously, leveraging the supramolecular inclusion function of β-cyclodextrin, the material is endowed with the ability to specifically recognize and pre-enrich organophosphorus pesticide molecules. This composite structure not only significantly improves the stability and anti-fouling properties of the sensing interface but also integrates ECL and EIS dual-mode detection mechanisms, enabling synergistic detection and mutual verification of ECL and EIS modes. This allows for highly sensitive, selective, and stable analysis of trace amounts of malathion in agricultural and environmental samples.

[0039] 2) The preparation method of the electrochemiluminescence-impedance dual-mode sensor of the present invention, on the one hand, uses specific β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters as ECL emitters and triethylamine (TEA) as a co-reactant to achieve excellent near-infrared ECL emission performance; on the other hand, the ECL efficiency of the gold-cerium bimetallic nanoclusters is further improved by using a pre-oxidation strategy; in modern bioanalysis, this provides a new perspective for constructing AuCeNCs-β-CD / TEA systems to improve ECL response.

[0040] 3) The electrochemiluminescence-impedance dual-mode sensor constructed from β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters of this invention, when applied to the detection of malathion, exhibits excellent sensing potential: the electrochemiluminescence mode can sensitively detect malathion in the range of 0.0005-100 ng / mL, with a detection limit of 1.2 × 10⁻⁶. −4 ng / mL; impedance mode can sensitively detect malathion in the range of 0.0005-100 ng / mL, with a detection limit of 8.6 × 10⁻⁶ ng / mL. −4 ng / mL.

[0041] 4) The electrochemiluminescence-impedance dual-mode sensor of this invention has good recyclability. Under ultraviolet irradiation, the configuration of azobenzene changes from trans to cis, causing Azo-aptamer to desorb from the electrode surface, thus effectively enabling electrode recycling. In addition, this sensor is simple to operate, has good repeatability, and has significant scientific and application value for monitoring malathion in agricultural products and the environment. Attached Figure Description

[0042] Figure 1 This is a high-magnification transmission electron microscope image of AuCeNCs-β-CD prepared in the embodiments of the present invention.

[0043] Figure 2This is a histogram showing the size distribution of AuCeNCs-β-CD prepared in this invention.

[0044] Figure 3 The image shows the UV absorption and fluorescence spectra of AuCeNCs-β-CD prepared in this invention.

[0045] Figure 4 This is a fluorescence lifetime curve of AuCeNCs-β-CD prepared in the embodiments of the present invention.

[0046] Figure 5 This is the electrochemiluminescence spectrum of AuCeNCs-β-CD prepared in this invention.

[0047] Figure 6 The image shows the FT-IR spectrum of AuCeNCs-β-CD prepared in this invention.

[0048] Figure 7 The image shows the XPS spectrum of AuCeNCs-β-CD prepared in this invention.

[0049] Figure 8 The image shows the CV diagram of AuCeNCs-β-CD prepared in this invention.

[0050] Figure 9 This is the energy level diagram calculated by TEA DFT in this embodiment of the invention.

[0051] Figure 10 This is a diagram showing the energy level positions of AuCeNCs-β-CD and triethylamine TEA prepared in this invention.

[0052] Figure 11 This is the EPR spectrum of AuCeNCs-β-CD prepared in this invention.

[0053] Figure 12 TEA generated by electrolysis in AuCeNCs-β-CD prepared in this invention •+ EPR spectrum of the signal.

[0054] Figure 13 The images show the 1H NMR spectra of AuCeNCs-β-CD and AuCeNCs-β-CD / TEA in D2O prepared in this invention.

[0055] Figure 14 The diagram shows the enhanced electrochemiluminescence performance of AuCeNCs-β-CD (A) after pre-oxidation treatment and the dual-mode sensing mechanism (B) diagram prepared in this invention.

[0056] Figure 15 This is the electrochemiluminescence response curve during the sensor assembly process in this invention.

[0057] Figure 16 This is an electrochemical impedance spectroscopy curve of the sensor assembly process in this invention.

[0058] Figure 17 The electrochemiluminescence response curves of the electrochemiluminescence-impedance dual-mode sensor prepared in this invention to 0.0005-100 ng / mL malathion are shown.

[0059] Figure 18 The linear calibration curves of electrochemiluminescence-impedance dual-mode sensor prepared in this invention for 0.0005-100 ng / mL malathion electrochemiluminescence are shown.

[0060] Figure 19 The impedance response curves of the electrochemiluminescence-impedance dual-mode sensor prepared in this invention to 0.0005-100 ng / mL malathion are shown.

[0061] Figure 20 The image shows the impedance linear calibration curves of the electrochemiluminescence-impedance dual-mode sensor prepared in this invention for 0.0005-100 ng / mL malathion.

[0062] Figure 21 This is a selectivity test diagram of the electrochemiluminescence-impedance dual-mode sensor prepared in the embodiments of the present invention.

[0063] Figure 22 This is a stability test diagram of the electrochemiluminescence-impedance dual-mode sensor prepared in the embodiments of the present invention.

[0064] Figure 23 This is a repeatability test diagram of the electrochemiluminescence-impedance dual-mode sensor prepared in the embodiments of the present invention.

[0065] Figure 24 This is a process flow diagram illustrating the fabrication process of the electrochemiluminescence-impedance dual-mode sensor implemented in this invention. Detailed Implementation

[0066] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0067] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, "first," "second," etc., are used to distinguish similar objects and are not used to describe a particular order or sequence. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0068] This invention provides a method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters, comprising the following steps: a first incubation, a second incubation, and a third incubation.

[0069] The first incubation method is as follows: in an alkaline environment, gold source, cerium source and methionine are contacted for the first incubation; after the incubation is completed, the pH of the system is adjusted to acidic by acid solution to induce cluster precipitation, the precipitate is collected and bimetallic nanoclusters are obtained.

[0070] In the first incubation, the gold source included: HAuCl4·3H2O;

[0071] Cerium sources include: cerium nitrate hexahydrate;

[0072] The molar ratio of gold source, methionine and cerium source is 1:64-65:7.7-8.0.

[0073] In the first incubation, the temperature is 35-37℃ and the incubation time is 10-12 hours.

[0074] During the first incubation, the alkaline environment was provided by a sodium hydroxide solution; the concentration of the sodium hydroxide solution used was 20-25 mg / mL.

[0075] Acid solutions include: sulfuric acid solution; the concentration of sulfuric acid solution is 0.9-1.2 mol / L.

[0076] Furthermore, the first incubation method is as follows: under stirring conditions, sodium hydroxide solution is added to a mixed aqueous solution of gold source, cerium source and methionine to adjust the pH of the system to 10-12 for the first incubation; after the incubation is completed, sulfuric acid solution is used to adjust the pH of the system to 2-3 to induce cluster precipitation. After the precipitation is complete, the precipitate is collected to obtain bimetallic nanoclusters, namely gold-cerium nanoclusters.

[0077] In the first incubation, the concentration of the gold source in the mixed aqueous solution of gold source, cerium source and methionine was 1.2-1.3 mmol / L.

[0078] The second incubation method involves dispersing the bimetallic nanoclusters in an ammonia solution and performing a second incubation; after the incubation is completed, the solid is separated and collected to obtain methionine-protected electrochemiluminescent gold-cerium nanoclusters.

[0079] During the second incubation, the concentration of bimetallic nanoclusters in the ammonia solution was 1-1.2 mg / mL;

[0080] The concentration of the ammonia solution is 2-2.3 wt%.

[0081] In the second incubation, the temperature is 75-80℃ and the incubation time is 20-30 minutes.

[0082] The third incubation method involves mixing the methionine-protected electrochemiluminescent gold-cerium nanoclusters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide with an NH2-β-cyclodextrin solution for a third incubation; after incubation, the solid is separated and collected to obtain β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters.

[0083] In the third incubation, the concentration of methionine-protected electrochemiluminescent gold-cerium nanoclusters in the mixed solution was 1-1.2 mg / mL;

[0084] The concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in the mixed solution was 0.85-0.9 mg / mL;

[0085] The concentration of N-hydroxysuccinimide in the mixed solution is 0.4-0.45 mg / mL.

[0086] In the third incubation, the concentration of the NH2-β-cyclodextrin solution was 1-1.2 mg / mL;

[0087] The molar ratio of NH2-β-cyclodextrin used in the third incubation to that used in the first incubation was 1:110-120.

[0088] In the third incubation, the temperature is 22-26℃ and the incubation time is 4-5 hours.

[0089] Furthermore, the third incubation method involves mixing and activating the methionine-protected electrochemiluminescent gold-cerium nanoclusters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and N-hydroxysuccinimide solution for 20-30 minutes to obtain a mixed solution; mixing the mixed solution with NH2-β-cyclodextrin solution for a third incubation; after incubation, separating and collecting the solids to obtain β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters.

[0090] In the third incubation, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution was 9-12 mg / mL;

[0091] The concentration of the N-hydroxysuccinimide solution is 9-12 mg / mL.

[0092] This invention also provides β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters prepared using the aforementioned method. Specifically, a bimetallic nanocluster core is constructed by introducing the rare earth element cerium (Ce), synthesized using methionine as a ligand, and the supramolecular inclusion function of β-cyclodextrin is utilized to create β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters. These nanoclusters not only exhibit good water solubility and biocompatibility but also possess high electrochemiluminescence efficiency and fully passivated nanomaterial properties, effectively optimizing the electrode interface. Combined with a multi-mode signal fusion and rapid detection strategy, this effectively overcomes the limitations of traditional electrochemiluminescence (ECL) and electrochemical impedance spectroscopy (EIS) in terms of sensitivity, selectivity, detection speed, and stability. It achieves highly sensitive, highly selective, rapid, and stable detection of malathion in both electrochemiluminescence and impedance modes, significantly improving the reliability of complex real-world sample analysis.

[0093] Meanwhile, addressing the problems of low electrochemiluminescence quantum yield, insufficient photophysical stability, lack of endogenous molecular recognition sites, limited signal amplification, and susceptibility to non-specific adsorption interference in complex matrices found in existing gold nanoclusters, this invention introduces the rare earth element cerium (Ce) to construct a bimetallic nanocluster core, significantly enhancing the electrocatalytic activity and luminescence efficiency of the material. Simultaneously, leveraging the supramolecular inclusion function of β-cyclodextrin, the material is endowed with the ability to specifically recognize and pre-enrich organophosphorus pesticide molecules. This composite structure not only significantly improves the stability and anti-fouling properties of the sensing interface but also supports both ECL and EIS dual-mode detection mechanisms, enabling synergistic detection and mutual verification of ECL and EIS modes. This allows for highly sensitive, selective, and stable analysis of trace amounts of malathion in agricultural and environmental samples.

[0094] This invention also provides an electrochemiluminescence-impedance dual-mode sensor, comprising: an MB / dsDNA probe and a pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode;

[0095] The pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode is prepared by coating β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters onto the surface of the GCE electrode and then pre-oxidizing them.

[0096] The MB / dsDNA probe includes: dsDNA and magnetic beads;

[0097] The dsDNA is obtained by hybridization of the capture probe cDNA and its complementary azobenzene aptamer Azo-aptamer; the biotin of the capture probe cDNA is linked to streptavidin-modified magnetic beads.

[0098] The nucleotide sequence of the cDNA is shown in SEQ ID No. 1; the nucleotide sequence of the Azo-aptamer is shown in SEQ ID No. 2.

[0099] Preferably, the pre-oxidation involves coating β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters onto the surface of a GCE electrode, then placing it in a phosphate-PBS buffer containing 0.1-0.15 mol / L TEA, and applying a continuous potential pulse of 0.9-1.1 V for 50-70 s.

[0100] The concentration of the phosphate-buffered saline (PBS) buffer is 0.1-0.15 mol / L, and the pH is 7.2-7.6.

[0101] Preferably, the capture probe cDNA and its complementary strand azo-aptamer hybridization are performed in PBS buffer at pH 7.2-7.6 and a concentration of 0.1-0.15 mol / L; the hybridization temperature is 35-38℃ and the hybridization time is 25-35 min.

[0102] Preferably, the molar ratio of the capture probe cDNA to its complementary strand azo-aptamer is 1:1.2-1.3.

[0103] Preferably, the volume ratio of the capture probe cDNA to the streptavidin-modified magnetic beads is 1:18-22; the reaction temperature for linking the biotin of the capture probe cDNA to the streptavidin-modified magnetic beads is 30-37℃, and the time is 50-70 min.

[0104] This invention also provides a method for preparing an electrochemiluminescence-impedance dual-mode sensor, comprising the following steps: preparing an MB / dsDNA probe and preparing an electrode;

[0105] The method for preparing the MB / dsDNA probe is as follows: cDNA is added to streptavidin-modified magnetic beads and incubated. The biotin in the cDNA interacts with streptavidin to obtain cDNA-modified magnetic beads (MB / cDNA); the MB / cDNA is dispersed in Azo-aptamer solution and incubated for DNA hybridization to obtain the MB / dsDNA probe.

[0106] The method for preparing the electrode is as follows: after surface pretreatment of the GCE electrode, a solution of β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters is coated onto the surface of the GCE electrode to obtain a β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode; then the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode is placed in a PBS buffer containing 0.1-0.15 mol / L TEA and pre-oxidized for 50-70 s under continuous potential pulse conditions of 0.9-1.1 V.

[0107] Preferably, the concentration of the Azo-aptamer solution in the preparation of the MB / dsDNA probe is 1-2 μmol / L.

[0108] Preferably, in the preparation of the electrode, the concentration of the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster solution is 1-1.5 mg / mL.

[0109] Preferably, in the preparation of the electrode, the surface pretreatment includes the following operations: after polishing the GCE electrode with Al2O3 slurry, it is ultrasonically treated with distilled water and ethanol in sequence.

[0110] This invention also provides the application of the electrochemiluminescence-impedance dual-mode sensor in the detection of malathion, including the following steps: after the MB / dsDNA probe is incubated with the test solution containing malathion for the first time, after magnetic separation, the supernatant is added to the pre-oxidized β-cyclodextrin modified electrochemiluminescence gold-cerium bimetallic nanoclusters / GCE electrode for the second incubation, and after pre-oxidation under continuous potential pulse conditions, ECL measurement and impedance measurement are performed.

[0111] Preferably, in the application of the electrochemiluminescence-impedance dual-mode sensor for detecting malathion, the first incubation temperature is 32-37℃ and the time is 1-2h;

[0112] The second incubation should be carried out at a temperature of 32-37℃ for 40-60 minutes.

[0113] Preferably, in the application of the electrochemiluminescence-impedance dual-mode sensor for the detection of malathion, the pre-oxidation under continuous potential pulse conditions is carried out in a PBS buffer containing 0.1-0.15 mol / L TEA, and the concentration of the PBS buffer is 0.1-0.15 mol / L, and the pH is 7.2-7.6.

[0114] Furthermore, during the ECL measurement process, the test conditions for the ECL signal are: 5 seconds at an initial potential of 0V and 1 second at a final potential of 1.2V.

[0115] ECL measurements were performed in PBS buffer containing 0.1–0.15 mol / L TEA (pH 7.2–7.6); impedance signal measurements were performed in 5 mmol / L [Fe(CN)6] buffer containing 0.1 mol / L KCl. 3− / 4− It is carried out in solution.

[0116] The electrochemiluminescence-impedance dual-mode sensor provided in this invention uses methionine-protected electrochemiluminescent β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters (AuCeNCs-β-CD) as the ECL luminescent agent and TEA as the co-reactant. AuCeNCs-β-CD can generate a significant ECL emission signal. In the presence of malathion, the Azo-aptamer in the MB / dsDNA probe will be released; β-CD (i.e., β-cyclodextrin) can specifically recognize the released Azo through guest-host interactions, leading to quenching of the ECL signal and an increase in the impedance signal. The constructed sensing platform can sensitively and selectively detect malathion by monitoring changes in ECL and impedance signals (sensitive detection of malathion is crucial for environmental monitoring).

[0117] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with some specific embodiments.

[0118] For subsequent embodiments where specific experimental steps or conditions are not specified, the procedures or conditions should be performed according to the conventional experimental steps described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0119] The streptavidin-modified magnetic beads used in subsequent embodiments were purchased from New England Biolabs. Oligonucleotides (i.e., cDNA and Azo-aptamer) were synthesized using Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the cDNA is shown in SEQ ID No. 1, and the nucleotide sequence of its complementary Azo-aptamer is shown in SEQ ID No. 2.

[0120] Example 1

[0121] This embodiment provides an electrochemiluminescence-impedance impedance dual-mode sensor, the fabrication process of which is shown in the flowchart below. Figure 24 As shown, the specific steps are as follows:

[0122] I. Preparation of β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters:

[0123] 1. First incubation

[0124] Under stirring conditions, 0.5 mL of 12.5 mmol / L HAuCl4·3H2O solution, 2.5 mL of 20 mmol / L Ce(NO3)3·6H2O solution, and 2 mL of 30 mg / mL methionine solution were mixed. Then, 22 mg / mL sodium hydroxide solution was added to adjust the pH to 11. The mixture was then incubated for 10 h in an oven at 37 °C. After that, 1 mol / L sulfuric acid solution was added to adjust the pH to 3 to induce cluster precipitation. After the precipitation was complete, the precipitate was collected to obtain gold-cerium nanoclusters.

[0125] 2. Second incubation

[0126] The above-mentioned gold-cerium nanoclusters were dispersed in 3 mL of 2 wt% ammonia solution, and the concentration of gold-cerium nanoclusters in the ammonia solution was controlled at 1 mg / mL. Then, a second incubation was carried out in an oven at 80 °C for 20 min. After incubation, the mixture was centrifuged at 10,000 rpm for 5 min, and the solid was collected to obtain methionine-protected electrochemiluminescent gold-cerium nanoclusters (AuCeNCs). The methionine-protected electrochemiluminescent gold-cerium nanoclusters were dispersed in PBS buffer, and the concentration of methionine-protected electrochemiluminescent gold-cerium nanoclusters was controlled at 1 mg / mL to obtain a methionine-protected electrochemiluminescent gold-cerium nanoclusters dispersion (AuCeNCs dispersion), which was ready for use.

[0127] 3. Third incubation

[0128] The above 1.0 mL AuCeNCs dispersion was mixed with 100 μL EDC solution (10 mg / mL, dissolved in PBS) and 50 μL NHS solution (10 mg / mL, dissolved in PBS) to activate the carboxyl groups on the surface of AuCeNCs. After activation for 30 min, 60 μL NH2-β-cyclodextrin solution (1 mg / mL, dissolved in PBS) was added, and the mixture was stirred at room temperature for a third incubation for 4 h. After incubation, the mixture was centrifuged at 10000 rpm for 5 min, and the solid was collected to obtain methionine-protected β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters (AuCeNCs-β-CD).

[0129] II. Structural Characterization:

[0130] High-magnification transmission electron microscope (TEM) images obtained using an HT7700 transmission electron microscope are shown below. Figure 1 As shown, by Figure 1 It can be seen that AuCeNCs-β-CD has a uniform particle size;

[0131] by Figure 1Based on the experimental data, a histogram of the size distribution of gold nanoclusters was plotted using size as the x-axis and the proportion of the corresponding size as the y-axis (see [reference]). Figure 2 Data shows that AuCeNCs-β-CD exhibits uniform monodisperse particle size characteristics, with a particle size of 2.8±0.5 nm.

[0132] Figure 3 The UV absorption and fluorescence emission spectra of AuCeNCs-β-CD are shown. The UV absorption curve reveals that the absorption intensity of this material decreases significantly with increasing incident wavelength in the UV region, and a shoulder peak appears near 300 nm. The fluorescence emission spectrum shows a strong emission peak at 610 nm and a weak emission peak at 825 nm.

[0133] Figure 4 The fluorescence lifetime curves of AuCeNCs-β-CD were obtained by fitting an exponential model. The results show that when the emission wavelength is set to 611 nm, the fluorescence lifetime of the nanoclusters reaches 132.02 ns.

[0134] Figure 5 The electrochemiluminescence spectrum of AuCeNCs-β-CD is shown. The electrochemiluminescence spectrum at 830 nm is similar to that of [other materials / processes]. Figure 3 Compared to the fluorescence spectrum of AuCeNCs-β-CD, AuCeNCs-β-CD exhibits a redshift of 210 nm. Figure 5 The electrochemiluminescence spectrum of AuCeNCs-β-CD is given, with the electrochemiluminescence peak located at 830 nm; compared with... Figure 3 Compared to the fluorescence emission spectrum in the previous study, the electrochemiluminescence signal of this material exhibited a 210 nm redshift.

[0135] To further verify the successful synthesis of AuCeNCs-β-CD, it was characterized by FT-IR spectroscopy. Figure 6 Characterization results show that the -SH group in the original system is at 2062 cm⁻¹. −1 The complete disappearance of the weak stretching vibration absorption peak at the point confirms the formation of Au-S covalent bonds, indirectly corroborating the formation of the target product.

[0136] Figure 7 XPS spectra of AuCeNCs-β-CD, from Figure 7 It can be seen that AuCeNCs-β-CD is mainly composed of Au, Ce, S, C, N and O. High-resolution XPS of Au is observed at Au4f... 7 / 2 (84.1eV) and Au4f 5 / 2 (87.8eV) Figure 7The inset shows two strong peaks, indicating the simultaneous presence of Au(I) and Au(0) metallic states within AuNCs. The fitted peaks of Ce3d spectrum at 906.8, 900.8, 888.4, and 882.2 eV correspond to Ce... 4+ The peaks at 903.8 and 885.5 eV belong to Ce. 3+ ( Figure 7 illustration).

[0137] The above Figures 1-7 The results show that this embodiment successfully prepared methionine-protected β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters (AuCeNCs-β-CD).

[0138] III. The influence of energy levels of AuNCs on their electrochemiluminescence performance:

[0139] The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of AuNCs and co-reactants were evaluated. First, the optical band gap (E0) of AuCeNCs-β-CD was calculated based on the UV-Vis absorption spectra (Figure 3). g The value is 2.97 eV, which is obtained through the formula E. g =1240 / λ cut-off (λ) cut-off The wavelength (in nm) is the spectral cutoff wavelength. Secondly, the cyclic voltammetry (CV) method ( Figure 8 This indicates that the HOMO level of AuCeNCs-β-CD is -5.06 eV. For the co-reactant TEA... •+ Its HOMO level (E HOMO ) and LUMO level (E LUMO Density functional theory (DFT) calculations yielded values ​​of -5.52 eV and 0.96 eV, respectively. Figure 9 Finally, according to the energy level calculation formula E... LUMO =E g +E HOMO The band structure of nanoclusters can be plotted. Figure 10 The study found that the band gap value of AuCeNCs-β-CD was significantly lower than that of Met-AuNCs compared to previously published literature (Anal. Chem. 2025, 97, 22380-22389). This reduced E g The values ​​indicate that dual-ligand regulation promotes electron excitation in AuNCs, making radiative transitions more likely. Furthermore, the narrower E... g The value also indicates that the electron transport capability has been enhanced.

[0140] The internal electronic states of AuCeNCs-β-CD, particularly its paramagnetic properties, were investigated using electron magnetic resonance (EPR) spectroscopy. Figure 11 As shown, the EPR spectrum of AuCeNCs-β-CD under dark conditions exhibits a distinct peak with a g value of 2.0028, which is related to the unpaired endogenous free electrons generated by sulfur vacancies within AuNCs.

[0141] Under normal circumstances, unprotected active TEA when exposed to aqueous media... • It is easily inhibited due to competition with dissolved oxygen, water, etc., but the TEA in the hydrophobic cavity • The formation of [the group] can be protected, thereby improving the stability of the co-reactive groups. This is achieved through EPR ([…]. Figure 12 It can be seen that using AuCeNCs-β-CD, TEA •+ The signal strength is very significant, which is attributed to the shielding effect of AuCeNCs-β-CD, which not only accelerates the electrochemical oxidation of TEA but also enhances the TEA's performance. •+ Stability.

[0142] With TEA 1 Compared to HNMR signals, AuCeNCs-β-CD signals are shifted to higher fields. Figure 13 Furthermore, in the presence of TEA, the H-3 and H-5 protons located within the AuCeNCs-β-CD cavity shift significantly towards the higher field compared to the absence of TEA. Based on these results, it is speculated that TEA can be chemically encapsulated into the ligand cavity through host-guest chemistry.

[0143] IV. Fabrication of an electrochemiluminescence-impedance dual-mode sensor:

[0144] 1. Probe preparation:

[0145] cDNA with the sequence 5′-AATTCACCAGCAGTCAAGTCCACCCA-biotin-3′ was added to streptavidin-modified magnetic beads and incubated. The biotin in the cDNA interacted with streptavidin at 37°C for 1 hour to form cDNA-modified magnetic beads (MB / cDNA).

[0146] Then, 20 μL of MB / cDNA with a concentration of 10 mg / mL was dispersed in 200 μL of Azo-aptamer solution with a concentration of 1 μmol / L and the sequence 5′-Azo-dT10-ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTGACTGCTGGTGTTGGCTCCCGTAT-3′, and DNA hybridization was performed at 37 °C for 30 min to form MB / dsDNA probes.

[0147] 2. The electrode coating process is as follows: First, the glassy carbon electrode (GCE) with a diameter of 5 mm was polished using Al2O3 slurry. Then, it was ultrasonically cleaned in distilled water and ethanol for 3 min each to complete the electrode surface pretreatment. 10 μL of a 1 mg / mL β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster solution (this solution was prepared by dissolving methionine-protected β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters in ultrapure water) was uniformly coated onto the pretreated GCE surface to finally obtain the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster / GCE electrode.

[0148] 3. Pre-oxidation: The β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE were placed in 0.1 mol / L PBS buffer (containing 0.12 mol / L TEA) at pH 7.4 and pre-oxidized for 60 s under a continuous potential pulse of 1.0 V to obtain an electrochemiluminescence-impedance dual-mode sensor.

[0149] 4. ECL and impedance mechanism: Figure 14 A demonstrates the ECL enhancement mechanism of AuCeNCs-β-CD. Without extensive chemical modification, the β-CD ligand-based shielding method not only improves the stability of the co-reactant radicals by reducing side reactions with dissolved oxygen and water, but also enhances the electrochemical oxidation kinetics of the TEA due to the shortened charge transfer path caused by pre-oxidation. Therefore, the obtained large number of stable co-reactant radicals directly react with the electrogenerated AuCeNCs-β-CD. •+ The reaction produces a large number of excited-state AuCeNCs-β-CD*, which significantly enhances ECL emission, and AuCeNCs-β-CD* returns to the ground state.

[0150] Impedance detection mechanism such as Figure 14 As shown in Figure B, azobenzene enters the cavity of β-CD and hinders the entry of TEA. Due to this interference, the length of the charge transfer path increases, thus significantly reducing the intensity of the ECL signal and significantly increasing the impedance signal.

[0151] V. Using the above-mentioned electrochemiluminescence-impedance dual-mode sensor, malathion was detected according to the following method:

[0152] After incubating 10 μL of the above-mentioned MB / dsDNA probe with the test solution containing malathion at 37 °C for 2 h, and then separating it by secondary magnetic separation, the supernatant was added to the pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode for a second incubation of 50 min. Then, it was placed in 0.1 mol / L PBS buffer (containing 0.12 mol / L TEA) at pH 7.4 and pre-oxidized for 60 s under a continuous potential pulse of 1.0 V before ECL and impedance measurements were performed.

[0153] ECL measurements were performed in PBS buffer (0.1 mol / L, pH 7.4) containing 0.12 mol / L TEA. The ECL signal was measured under the following conditions: an initial potential of 0V for 5 seconds, followed by a final potential of 1.2V for 1 second. Impedance signals were measured in 5 mmol / L [Fe(CN)6] buffer containing 0.1 mol / L KCl. 3− / 4− It is carried out in solution.

[0154] In the above detection process, cDNA / Azo-aptamer consists of two complementary dsDNA strands. The cDNA assembles onto streptavidin-modified magnetic beads (MBs) via a specific streptavidin-biotin interaction, and then hybridizes with Azo-aptamer to form a dsDNA probe containing a specific recognition sequence for malathion. In the presence of the target malathion, the MB / dsDNA probe releases Azo-aptamer. After magnetic separation, β-CD can recognize the azobenzene (Azo) released from the MB via guest-host interaction, causing Azo to quench ECL emission and increase impedance signal.

[0155] Figure 15The figures show the electrochemiluminescence response curves corresponding to the ECL sensor assembly process. Data shows that the bare GCE did not exhibit a significant electrochemiluminescence signal. Compared to AuCeNCs-β-CD / GCE, the ECL intensity of Ox-AuCeNCs-β-CD / GCE after pre-oxidation treatment was significantly improved. When the supernatant from the reaction with malathion was incubated with AuCeNCs-β-CD / GCE and pre-oxidized, the ECL signal of the resulting Azo / Ox-AuCeNCs-β-CD / GCE was significantly lower than that of Ox-AuCeNCs-β-CD / GCE. However, under UV irradiation, azobenzene (Azo) was successfully removed, and the ECL emission intensity of the pre-oxidized Ox-AuCeNCs-β-CD / GCE (with Azo removed) essentially recovered to its original level. These dynamic changes in the ECL signal perfectly match the assembly and reaction process of the biosensor, confirming the successful fabrication of the target biosensor.

[0156] Figure 16 This is the electrochemical impedance spectroscopy (EIS) curve corresponding to the assembly process of the impedance sensor. The data shows that the charge transfer resistance (Rc) of AuCeNCs-β-CD / GCE is... ct The Ω is 140Ω (curve b), which is the same as the R of the bare GCE. ct The values ​​(105 Ω, curve a) are quite close, indicating that AuCeNCs-β-CD possesses excellent electrical conductivity and a large specific surface area. After pre-oxidation treatment, the Rx of Ox / AuCeNCs-β-CD / GCE (curve c) is... ct The value showed a slight increase; however, after the formation of Ox-Azo / AuCeNCs-β-CD / GCE, its R... ct The value significantly increased to 7.93 kΩ (curve d). Under UV irradiation, azobenzene (Azo) was successfully removed, at which point the Ro of Ox-AuCeNCs-β-CD / GCE increased significantly. ct The value dropped to 200Ω (curve e). The dynamic change in electrode impedance value showed a good correlation with the assembly process of the biosensor, confirming the successful implementation of the electrode assembly process.

[0157] Figure 17 The figure shows the electrochemiluminescence response curves of the ECL sensor for malathion in the concentration range of 0.0005-100 ng / mL. As can be seen from the figure, within the concentration range of 0.0005 ng / mL to 100 ng / mL, the response signal of the ECL sensor gradually weakens as the concentration of malathion increases.

[0158] Figure 18 Linear calibration curves for the ECL sensor at concentrations of 0.0005–100 ng / mL malathion are shown below. Figure 18 As shown, the ECL signal exhibits a very strong linear correlation with the logarithm of malathion concentration, and the corresponding linear regression equation is I. ECL =0.4889-0.1452lgC(R) 2 =0.9965), the calculated detection limit (LOD) is 1.2 × 10⁻⁶. −4 The resulting biosensor, at ng / mL, enabled sensitive detection of malathion.

[0159] Figure 19 The impedance response curves of the impedance sensor to malathion in the range of 0.0005-100 ng / mL are shown below. Figure 19 As shown, from 0.0005 ng / mL to 100 ng / mL, the redox reaction of [Fe(CN)6] on the electrode surface increased with increasing malathion concentration. 3- / [Fe(CN)6] 4- Electron transfer between them slows down due to the increase in the amount of non-conductive bioactive substances, thus the impedance value gradually increases.

[0160] Figure 20 Linear calibration curves of the impedance sensor for malathion in the concentration range of 0.0005-100 ng / mL are shown. Data indicates a good linear correlation between the sensor's impedance signal and the logarithm of the malathion concentration, with the corresponding linear regression equation being I. EIS =4.6542–1.2362lgC(R) 2 =0.9932). The calculated limit of detection (LOD) of this sensor is 8.6 × 10⁻⁶. −4 The result, ng / mL, indicates that the constructed biosensor can achieve highly sensitive detection of malathion.

[0161] Figure 21 The results presented show the selectivity test results of the ECL sensor. The test data indicate that the ECL intensity decreases minimally for interfering substances and blank samples, while the target analyte, malathion, causes a significant decrease in ECL intensity. Furthermore, the response signal of the ECL sensor to malathion alone exhibits good consistency with the response signal in the mixture of malathion and potential interfering substances, further confirming the excellent selectivity of this biosensor.

[0162] Figure 22 The results presented show the stability test results of the constructed ECL sensor. The test data shows that when the sensor performs 12 consecutive potential step operations within the 0-1V potential range, the relative standard deviation (RSD) for detecting malathion at concentrations of 0.001 ng / mL and 10 ng / mL are 3.65% and 3.25%, respectively. This result indicates that the ECL sensor has excellent operational stability. Figure 23 To demonstrate the repeatability of the constructed ECL sensor, the reproducibility performance was evaluated by detecting a 0.1 ng / mL malathion standard solution. Data showed that the response signals of the seven parallel prepared electrodes under the same detection conditions were not significantly different, with an RSD of only 3.32% at a concentration of 0.1 ng / mL. This result indicates that the biosensor designed in this study possesses excellent reproducibility.

[0163] VI. Electrode recycling:

[0164] Under ultraviolet irradiation, the configuration of azobenzene changes from trans to cis, causing the azo-aptamer to desorb and be removed from the electrode surface, thus allowing the electrode to be recycled. Figure 24 C). For example Figure 15 and Figure 16 As shown in curve e, when the Azo-aptamer is removed from the electrode surface, its ECL signal and electron transfer impedance both return to their initial levels.

[0165] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters, characterized in that, The process includes the following steps: first incubation, second incubation, and third incubation; The first incubation method is as follows: in an alkaline environment, gold source, cerium source and methionine are contacted for the first incubation; then the pH is adjusted to acidic, the precipitate is collected, and bimetallic nanoclusters are obtained. The second incubation method involves dispersing bimetallic nanoclusters in an ammonia solution and then performing a second incubation. The solids were then separated and collected to obtain methionine-protected electrochemiluminescent gold-cerium nanoclusters. The third incubation method involves mixing a mixed solution of methionine-protected electrochemiluminescent gold-cerium nanoclusters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide with an NH2-β-cyclodextrin solution for a third incubation; then separating and collecting the solids to obtain β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters.

2. The method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters according to claim 1, characterized in that, In the first incubation, the gold source includes tetrachloroauric acid; the cerium source includes cerium nitrate; and the molar ratio of the gold source, methionine, and cerium source is 1:64-65:7.7-8.

0. In the second incubation, the concentration of bimetallic nanoclusters in the ammonia solution was 1-1.2 mg / mL; the concentration of the ammonia solution was 2-2.3 wt%.

3. The method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters according to claim 1, characterized in that, In the third incubation, the concentration of methionine-protected electrochemiluminescent gold-cerium nanoclusters in the mixed solution was 1-1.2 mg / mL; the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was 0.85-0.9 mg / mL; and the concentration of N-hydroxysuccinimide was 0.4-0.45 mg / mL. The concentration of the NH2-β-cyclodextrin solution is 1-1.2 mg / mL; The molar ratio of NH2-β-cyclodextrin used in the third incubation to that used in the first incubation was 1:110-120.

4. The method for preparing β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters according to claim 1, characterized in that, In the first incubation, the incubation temperature is 35-37℃ and the incubation time is 10-12h; In the second incubation, the incubation temperature is 75-80℃ and the incubation time is 20-30 minutes; In the third incubation, the incubation temperature is 22-26℃ and the incubation time is 4-5 hours.

5. A β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster prepared by the method according to any one of claims 1-4.

6. An electrochemiluminescence-impedance dual-mode sensor, characterized in that, Includes: MB / dsDNA probe, and electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode modified with pre-oxidized β-cyclodextrin; The pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode is prepared by coating the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters of claim 5 onto the surface of the GCE electrode and then pre-oxidizing them. The MB / dsDNA probe includes: dsDNA and magnetic beads; The dsDNA is obtained by hybridization of the capture probe cDNA and its complementary azobenzene aptamer Azo-aptamer; the biotin of the capture probe cDNA is linked to streptavidin-modified magnetic beads. The nucleotide sequence of the cDNA is shown in SEQ ID No. 1; the nucleotide sequence of the Azo-aptamer is shown in SEQ ID No.

2.

7. A method for fabricating an electrochemiluminescence-impedance dual-mode sensor as described in claim 6, characterized in that, The process includes the following steps: preparing MB / dsDNA probes and preparing electrodes; The method for preparing the MB / dsDNA probe is as follows: the capture probe cDNA is added to streptavidin-modified magnetic beads and incubated to obtain cDNA-modified magnetic beads; the cDNA-modified magnetic beads are dispersed in Azo-aptamer solution for DNA hybridization to obtain the MB / dsDNA probe. The method for preparing the electrode is as follows: a solution of β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters is coated onto the surface of a GCE electrode to obtain a β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode. The electrode is then placed in a phosphate buffer solution containing triethylamine and pre-oxidized for 50-70 seconds under a continuous potential pulse of 0.9-1.1V.

8. The method for fabricating the electrochemiluminescence-impedance dual-mode sensor according to claim 7, characterized in that, In the preparation of the MB / dsDNA probe, the incubation temperature of the capture probe cDNA with the streptavidin-modified magnetic beads is 30-37℃, and the incubation time is 50-70 min. The temperature for DNA hybridization is 35-38℃, and the time for DNA hybridization is 25-35 minutes. In the electrode preparation, the concentration of the β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanocluster solution is 1-1.5 mg / mL; The triethylamine concentration in the phosphate buffer containing triethylamine is 0.1-0.15 mol / L, the concentration of the phosphate buffer is 0.1-0.15 mol / L, and the pH of the phosphate buffer is 7.2-7.

6.

9. The application of the electrochemiluminescence-impedance dual-mode sensor as described in claim 6 in the detection of malathion, characterized in that, The process includes the following steps: first incubation of the MB / dsDNA probe with the test solution containing malathion; magnetic separation followed by adding the supernatant to the pre-oxidized β-cyclodextrin-modified electrochemiluminescent gold-cerium bimetallic nanoclusters / GCE electrode for a second incubation; pre-oxidation under continuous potential pulse conditions followed by ECL measurement; and impedance signal measurement in potassium ferricyanide solution.

10. The application of the electrochemiluminescence-impedance dual-mode sensor according to claim 9 in the detection of malathion, characterized in that, The first incubation should be carried out at a temperature of 32-37℃ for 1-2 hours. The second incubation should be carried out at a temperature of 32-37℃ for 40-60 minutes.

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