Preparation method of switch-on type electrochemiluminescence aptamer sensor for detecting ochratoxin a in cereals

Abstract

The application discloses a preparation and use method of a switch-on and switch-off type electrochemiluminescence aptamer sensor for detecting ochratoxin A in grains, characterized in that: a new self-enhanced luminophore is prepared by combining black phosphorus quantum dots and ruthenium bipyridine in a nanoparticle through electrostatic action; a composite nanomaterial is synthesized by using multi-walled carbon nanotubes, polyethyleneimine and gold; the strong catalysis of the black phosphorus quantum dots on the ruthenium bipyridine, the high initial electrochemiluminescence intensity provided by the self-enhanced luminophore, the specific recognition of the aptamer to the ochratoxin A, the quenching of the luminous signal of the ruthenium bipyridine by the ferrocene and the recovery of the luminous signal after the target is added; and a switch-on and switch-off type electrochemiluminescence aptamer sensor for detecting ochratoxin A in grains is constructed, which has good sensitivity, stability, reproducibility and a low detection limit, and provides a general analysis tool for the detection of other mycotoxins and small molecule substances.

Description

TECHNICAL FIELD

[0001] The present application relates to a switch-on electrochemiluminescence aptamer sensor and a preparation method and use method of the sensor, and belongs to the technical field of electrochemiluminescence aptamer sensors. BACKGROUND

[0002] Fungal toxin pollution is a global problem that poses a significant threat to food and feed safety. Among various types of fungal toxins, ochratoxin A (OTA) is a representative toxin that is abundant in reserves, contaminates food, and is associated with serious health problems such as nephrotoxicity, hepatotoxicity, neurotoxicity, teratogenicity, carcinogenicity, and immunotoxicity in humans. The European Union has established maximum concentrations in cereals (5 g / kg), wine (2 g / kg), and coffee products (5 mg / kg). It is essential to develop a rapid and sensitive detection technology to enable accurate OTA detection.

[0003] Methods for detecting ochratoxin A: Current methods for detecting ochratoxin A include gas chromatography, enzyme-linked immunosorbent assay, chromatography, capillary electrophoresis, colorimetry, fluorescence analysis, and surface-enhanced Raman spectroscopy. Although these methods provide quantitative and qualitative results, they are time-consuming, require expensive equipment, involve complex sample preparation steps, and require well-trained personnel. Therefore, a reliable and sensitive method for detecting ochratoxin A is crucial for ensuring food safety and human life safety.

[0004] Electrochemiluminescence (ECL) technology, as a powerful analytical method developed on the basis of chemiluminescence, does not require the introduction of an external light source. With the aid of optical instruments such as photomultiplier tubes, the luminescence intensity spectrum is collected, and its relationship with the analyte is established to achieve microanalysis. ECL combines the high controllability of electrochemical technology and the high sensitivity of chemiluminescence technology, and has the advantages of strong controllability, low background signal, good selectivity, short response time, and simple operation. Aptamers are a new type of recognition element selected by systematic evolution of ligands by exponential enrichment (SELEX), which has high affinity and high sensitivity comparable to antibodies. Aptamers are simple and fast to synthesize, low in cost, good in selectivity, high in stability, and easy to modify and label. They can rapidly and specifically bind to target substances with high affinity. By cross-linking the ends of aptamers to solid carriers as capture molecules, target substances in the sample can be captured. Therefore, aptamer detection of fungal toxins has also developed.

[0005] The selection of luminophores is crucial in the construction of ECL non-radioactive sensors. Among various ECL luminophores, ruthenium (II) tris-2,2'-bipyridine (Ru(bpy)3 2+The ECL system of the application has high luminous efficiency and chemical stability, making it an ideal choice for sensing applications in complex environments. Quantum dots (QDs) have high luminescence quality, strong biocompatibility, good stability, and low toxicity, and with the rapid development of nanotechnology, quantum dots have become a hot topic in the field of ECL research. In traditional ECL systems, inter-particle interactions between the emitter and its co-reactant occur, leading to long-range electron transfer and energy loss, while electrochemiluminescence sensors based on self-enhanced emitters can effectively solve this problem. The switch-based design also improves the stability and sensitivity of the biosensor, as the signal difference between different states is significant. In a switch system, obtaining high "on" signals and low "off" signals is crucial for achieving high sensitivity, which is also a key point in the structural design of switch systems.

[0006] The application aims to provide a switch-on type electrochemiluminescence aptamer sensor that overcomes the above problems and has simple preparation, flexible operation, high sensitivity, high accuracy, selectivity, and good stability. The application combines black phosphorus quantum dots and ruthenium bipyridine in a nanoparticle through electrostatic interaction to prepare a new type of self-enhanced emitter. Since electron transfer occurs within the nanoparticle in the self-enhanced emitter, the electron transfer distance is greatly shortened, thereby improving the electrochemiluminescence efficiency. The constructed sensor does not require a co-reactant and can provide high initial luminescence intensity by relying on the self-enhanced emitter itself. Without the catalytic action of enzymes, the sensor directly detects the target substance through specific binding of the aptamer, and the preparation method of the electrochemiluminescence aptamer sensor for specific detection of ochratoxin A in grains. SUMMARY

[0007] The technical solution is a preparation method of an aptamer sensor for detecting ochratoxin A, characterized in that the sensitive interface of the aptamer sensor is composed of an (AuNPs-PEI-MWCNTs) composite material of multi-walled carbon nanotubes, polyethyleneimine, and gold, and a self-enhanced emitter (Ru@SiO2-BPQDs) composite material, and then an aptamer for ochratoxin A is fixed, thereby introducing ferrocene (Fc) to the sensor interface to achieve quenching of Fc on Ru(bpy)3 2+ and recovery of the luminescence signal after release of the target substance, so that the target substance can be sensitively detected according to the luminescence intensity of the sensor at different stages.

[0008] The preparation method of the aptamer sensor for detecting ochratoxin A is characterized by cleaning of the glassy carbon electrode (d = 3 mm), construction of the sensitive interface of the aptamer sensor and process characterization (preparation of AuNPs-PEI-MWCNTs), synthesis of the self-enhanced luminophore (Ru@SiO2-BPQDs), establishment of the working curve of the aptamer sensor, detection of the performance of the aptamer sensor, and detection of the actual sample by the aptamer sensor.

[0009] The preparation method of the aptamer sensor for detecting ochratoxin A is characterized by optimization of experimental conditions, mainly including testing of the PH of the base solution, the concentration of the aptamer, the hybridization time of the aptamer and the complementary strand, and the incubation time of the aptamer and the target substance; the working curve of the prepared aptamer sensor is y = 6728.17 + 2460.33lgx (R = 0.995); the performance detection of the aptamer sensor includes specific selectivity, reproducibility, stability, and determination of the recovery rate of the grain sample by the aptamer sensor. 2

[0010] Electrochemiluminescence detection of target substances: The three-electrode system is used to detect the grain sample under the optimal experimental conditions by the electrochemiluminescence detection method, and the electrochemiluminescence intensity value in the detection result is analyzed to obtain that the electrochemiluminescence intensity peak value is proportional to the concentration of the ochratoxin A, and thus the concentration information of the ochratoxin A can be obtained.

[0011] ​The preparation principle of the application is: the glassy carbon electrode is modified by AuNPs-PEI-MWCNTs and Ru@SiO2-BPQDs, due to the high specific surface area and high conductivity of AuNPs-PEI-MWCNTs, the electrode surface electron transmission capacity and stability are increased, so that more Ru@SiO2-BPQDs can be fixed on the electrode surface to generate a higher initial luminescence intensity. The ability of Ru@SiO2-BPQDs to directionally fix the OTA aptamer complementary chain is used, so as to provide a good sensor interface for the fixation of the OTA aptamer complementary chain, maintain the effective fixation amount of the OTA aptamer complementary chain, and then ensure that the ferrocene on the aptamer effectively quenches the ruthenium bipyridine, thereby increasing the detection accuracy of the sensor. The prepared on-off type sensor has the characteristics of high sensitivity, low detection limit and wide detection range, which is due to the self-enhanced luminescent body and the electron transfer quenching strategy. The first "on" state is provided by Ru@SiO2-BPQDs, which produces the highest background signal. The "off" state is caused by the quenching of ferrocene on the ruthenium bipyridine, thereby producing the lowest background signal. After the addition of OTA, the aptamer modified with ferrocene is released from the surface of the sensor, resulting in the recovery of the luminescence intensity, thereby achieving the second "on" state. Through the above-mentioned steps, the aptamer sensor prepared is simple and sensitive, has good stability and specificity, has good reproducibility, and the recovery rate meets the requirements.

[0012] In order to achieve the above purpose, the following technical scheme is adopted: a preparation method of an aptamer sensor for detecting ochratoxin A, characterized in that: (1) the bare glassy carbon electrode is cleaned, activated and performance tested before the preparation of the aptamer sensor, if the peak potential difference in the test cyclic voltammetry curve is below 100 mV, and the oxidation peak and the reduction peak are symmetrical, the glassy carbon electrode can be used, otherwise it needs to return to the cleaning step until it meets the requirements; (2) the cleaned glassy carbon electrode surface is drop-coated with uniformly dispersed AuNPs-PEI-MWCNTs, then modified with Ru@SiO2-BPQDs, and then the aptamer complementary chain is fixed on the Ru@SiO2-BPQDs through carbamido reaction, non-specific sites are blocked by drop-coating BSA, then the aptamer modified with ferrocene is added, and finally the target material is added. After the preparation of the aptamer sensor is completed, it is stored in a 4℃ refrigerator for standby use.

[0013] To achieve the above object, the following technical scheme is adopted: a preparation method of an aptamer sensor for detecting ochratoxin A, characterized in that: (1) the prepared aptamer sensor is detected in a working base solution by an electrochemiluminescence method to obtain the relationship between the peak value and the target substance; (2) a series of OTA concentration standard solutions are configured, and the electrochemiluminescence method is used for determination to further obtain the working curve, detection range and detection limit of the prepared aptamer sensor; (3) a series of common mycotoxin solutions are configured to detect the selectivity of the prepared aptamer sensor; (4) the stability and repeatability of the aptamer sensor are verified by the electrochemiluminescence method multiple times; and (5) actual grain samples are analyzed to obtain the recovery rate of the aptamer sensor.

[0014] The present application uses black phosphorus quantum dots (BPQDs) as a co-reactant of ruthenium bipyridine, encapsulates the ruthenium bipyridine in silica nanoparticles, and combines the BPQDs through electrostatic interaction, to obtain a self-enhanced luminophore (Ru@SiO2-BPQDs). Due to the electron transfer path in the nanoparticles of the Ru@SiO2-BPQDs, the ECL efficiency is improved, a higher initial electrochemical luminescence intensity is provided, the change range of the peak value is enhanced, and thus the detection sensitivity of the aptamer sensor is increased. Through the introduction of ferrocene by the aptamer and the release of the ferrocene after the addition of the target, the on-off state of the sensor can be realized. Through the ECL intensity in different states, the OTA in the target solution can be sensitively detected. In addition, the sensor has a simple preparation method, high stability and specificity, and can be used for the detection of OTA in grain samples, and lays a foundation for the development of a rapid detection portable device for mycotoxin pollution in agricultural products.

[0015] The preparation process of the aptamer sensor is as follows: 5 μL of 5 mg / mL AuNPs-PEI-MWCNTs is added on the treated glassy carbon electrode (GCE) and dried at room temperature to obtain AuNPs-PEI-MWCNTs / GCE, then 5 μL of Ru@SiO2-BPQDs is added on the electrode and placed at room temperature for half an hour, and the electrode is naturally dried to obtain Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE. 5 μL of 200 nM aptamer complementary chain is added on the electrode and placed at room temperature for one hour to obtain cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE. Then 5 μL of BSA solution is added on the electrode, the electrode is naturally dried, and then 5 μL of 200 nM aptamer chain is added, which is placed at room temperature for one hour, and the aptamer sensor Apt-Fc / BSA / cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE is obtained after natural drying, which is stored at 4°C for standby. The sequence of the amino-modified aptamer complementary chain used is: 5'-NH2-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3'; the sequence of the ferrocene-modified aptamer used is: 5'-Fc-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Characterization of composite nanomaterials of electrochemiluminescence sensor.

[0017] Figure 2 Electrochemical characterization of electrochemiluminescence sensor. (a: bare GCE; b: AuNPs-PEI-MWCNTs / GCE; c: Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE; d: cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE; e: BSA / cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE; f: Apt-Fc / BSA / cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE; g: OTA / Apt-Fc / BSA / cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE)

[0018] Figure 3Optimization of experimental parameters of electrochemiluminescence sensor. (A: pH value; B: aptamer concentration; C: hybridization time of cDNA and Apt-Fc; D: incubation time of Apt-Fc and OTA)

[0019] Figure 4 Standard curve of electrochemiluminescence sensor for measuring different concentrations of ochratoxin A.

[0020] Figure 5 Specificity study of electrochemiluminescence sensor.

[0021] Figure 6 Reproducibility study of electrochemiluminescence sensor.

[0022] Figure 7 Stability study of electrochemiluminescence sensor. DETAILED DESCRIPTION

[0023] Example:

[0024] (1) Preparation of gold nanoparticles solution: All glass containers that might be used in the experiment were immersed in aqua regia (volume ratio of nitric acid to hydrochloric acid = 1:3) for 24 hours in advance, washed with ultrapure water for 5 times, and dried for use. 200 mL of ultrapure water was measured, 2 mL of chloroauric acid solution was added, the solution was mixed by shaking, and was heated in a microwave oven until the solution boiled. Remove quickly, shake the bottle in the same direction to make the solution in the bottle rotate, quickly add 1% trisodium citrate, continue to shake and mix, put back into the microwave oven and heat for 4 minutes, take out the container, at this time the solution is wine red. Cool to room temperature, add ultrapure water to 200 ml, which is (0.1 mg / mL) gold nanoparticle solution.

[0025] (2) Preparation of AuNPs-PEI-MWCNTs: 2.0 mg of carboxylated MWCNTs was added to 4 mL of PEI solution, then the mixture was ultrasonically dispersed, then the obtained mixture was centrifuged and redispersed to obtain PEI-MWCNTs. Then, 1 mL of synthesized AuNPs was gradually added to 1 mL of prepared PEI-MWCNTs, and then stirred in the dark overnight. After centrifugation, the black solid was redispersed in 2 mL of 0.01M phosphate buffered saline with pH 7.4, and AuNPs-PEI-MWCNTs was successfully synthesized.

[0026] (3) Preparation of BPQDs: 4 mL of N-methyl pyrrolidone (NMP) and 5 mg of black phosphorus powder were ultrasonically dispersed in an ice bath at 100 W for 8 hours. The obtained dispersion was centrifuged at 7000 rpm for 20 minutes, and then centrifuged at 10000 rpm for 20 minutes. The supernatant containing 75% of the top of the solution was collected each time.

[0027] (4) Preparation of Ru@SiO2: First, 7.08 mL Triton solution, 30 mL cyclohexane, 7.2 mL n-hexanol and 1.36 mL water were stirred for 5 minutes to form a water-in-oil reverse microemulsion. Then 320 μL of 0.1M ruthenium bipyridine solution was added, and the solution was stirred for 30 minutes until it became transparent. Then 400 μL of tetraethyl orthosilicate and 240 μL of ammonia water were quickly injected to initiate polymerization. The mixture was stirred in the dark for 24 hours. The final product was separated with 40 mL of acetone, centrifuged at 10000 rpm for 10 minutes, and then washed with a mixture of ethanol and water three times. Finally, Ru@SiO2 was dried in a vacuum freeze dryer and stored at 4°C for future use.

[0028] (5) Preparation of Ru@SiO2-BPQDs: First, 10 mg of Ru@SiO2 was dispersed in 5 mL of ethanol, 200 μL of aminopropyltriethoxysilane was added, and after stirring for 4 hours, it was washed with a mixture of ethanol and water. Then Ru@SiO2-NH2 was obtained by drying in a vacuum freeze dryer. 170 μL of 10 mg / ml Ru@SiO2-NH2 and 850 μL of 3 mg / ml BPQDs were added to a beaker and stirred in the dark for 12 hours. Since the BPQDs and Ru@SiO2-NH2 have opposite charges, they are combined by electrostatic interaction to form a self-enhanced luminescent Ru@SiO2-BPQDs.

[0029] (6) Cleaning of glassy carbon electrode: Before modification, the glassy carbon electrode was polished to a mirror surface with 0.3 μm Al2O3 on a chamois leather, and then washed with deionized water to remove surface dirt. Then it was moved into an ultrasonic water bath for cleaning, 5 minutes each time, repeated twice. It was then ultrasonically cleaned with anhydrous ethanol and deionized water, and dried in a nitrogen environment.

[0030] (7) Test of glassy carbon electrode: Run cyclic voltammetry curves in a 0.01M PBS solution containing 0.1M KCl and 5mM [Fe(CN)6] 3- / 4- , with a scan rate of 50 mV / s and a scan potential of -0.1-0.6V to test the performance of the glassy carbon electrode. When the redox peak potential difference in the cyclic voltammetry curve is below 100 mV, the glassy carbon electrode can be further used, otherwise it needs to be returned to step (6) for further processing of the glassy carbon electrode until it meets the requirements.

[0031] (8) After pretreatment, 5 uL of AuNPs-PEI-MWCNTs and Ru@SiO2-BPQDs solution were dropped on the glassy carbon electrode in turn, and naturally dried at room temperature. 5 uL of 200 nM complementary strand of aptamer was added, and placed for one hour at room temperature. Then 5 uL of BSA solution was added, and the electrode was naturally dried. After that, 5 uL of 200 nM aptamer strand was added, and placed for one hour at room temperature. The preparation of aptamer sensor was completed, and stored at 4℃ for standby.

[0032] (9) The test solution pH, aptamer concentration, hybridization time, incubation time and other experimental factors were optimized by electrochemiluminescence method in a three-electrode system. The test solution pH was in the range of 5.5-9.3, the aptamer concentration was in the range of 50 nM-400 nM, and the hybridization time and incubation time were in the range of 15-90 minutes.

[0033] (10) Under the optimal test conditions, the concentration gradient quantitative analysis test was carried out by electrochemiluminescence method. The aptamer concentration was 200 nM, the test solution pH was 7.7, the hybridization time and incubation time were 60 minutes, and the concentration range of ochratoxin A used for testing was 0.1-320 ng / mL. The minimum detection limit of ochratoxin A was 0.03 ng / mL, and the regression equation was I ECL = 6728.17 + 2460.33 lgc OTA , R 2 = 0.995, see the attached Figure 4 .

[0034] (11) In the test system, 50 ng / mL of aflatoxin (B1, B2, M1), deoxynivalenol, T-2 toxin and zearalenone interfering ions were added respectively, and the anti-interference was tested by electrochemiluminescence method. Four prepared electrodes were selected to measure the ochratoxin A concentration of 50 ng / mL to test the repeatability. Three groups of prepared electrodes were selected to measure the ochratoxin A concentration of 50 ng / mL on the same day, 7 days and 14 days to test the stability.

[0035] (12) After grinding, filtering, ultrasonicating and centrifuging the grain sample, ochratoxin A solution was added for actual sample detection and analysis.

[0036] The switch-on electrochemiluminescence aptamer sensor can detect ochratoxin A in grains, and has low cost, simple operation, short detection time, strong anti-interference ability, high sensitivity, selectivity, stability and repeatability, high detection sensitivity, and meets the requirements of rapid detection technology development and internationalization of ochratoxin A in grains in China.

Claims

1. A switch-on electrochemiluminescence aptamer sensor for detecting ochratoxin A in grain samples, characterized in that: Black phosphorus quantum dots (BPQDs) were used as co-reactants for ruthenium bipyridine (RBB), encapsulating RBB in silica nanoparticles and binding it to BPQDs via electrostatic interactions to obtain self-enhanced luminescent materials Ru@SiO2-BPQDs. A glassy carbon electrode was modified with AuNPs-PEI-MWCNTs and Ru@SiO2-BPQDs, and the complementary chain of the ochratoxin A aptamer was directionally immobilized using Ru@SiO2-BPQDs. The specific binding of the aptamer to ochratoxin A was then utilized for the targeted detection of ochratoxin A. Ferrocene is introduced via aptamers to quench the luminescence signal. After the target substance is introduced, the release of ferrocene leads to the recovery of the luminescence signal. The target substance is sensitively detected based on the luminescence intensity of the sensor at different stages. The first "on" state is provided by Ru@SiO2-BPQDs, which generates the highest background signal. The "off" state is caused by the quenching of ferrocene with ruthenium bipyridine, which generates the lowest background signal. The addition of OTA causes the ferrocene-modified aptamer to be released from the sensor surface, resulting in the recovery of the luminescence intensity, thus achieving the second "on" state. The preparation steps of the aptamer sensor are as follows: 5 μL of 5 mg / mL AuNPs-PEI-MWCNTs is added to the treated glassy carbon electrode GCE and dried at room temperature to obtain AuNPs-PEI-MWCNTs / GCE. Then, 5 μL of Ru@SiO2-BPQDs is added to the electrode and allowed to air dry at room temperature to obtain Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE. Next, 5 μL of 200 nM aptamer complementary strand is added to the electrode and allowed to air dry at room temperature for one hour to obtain cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE. Finally, 5 μL of BSA solution is added to the electrode and allowed to air dry, followed by another 5 μL of... The aptamer strand of 200 nM was placed at room temperature for one hour to prepare the aptamer sensor Apt-Fc / BSA / cDNA / Ru@SiO2-BPQDs / AuNPs-PEI-MWCNTs / GCE, and was stored at 4°C for later use.

2. The on / off electrochemiluminescence aptamer sensor for detecting ochratoxin A in grain samples according to claim 1, characterized in that: The complementary chain sequence of the amino-modified aptamer is: 5′-NH2-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3′; the sequence of the ferrocene-modified aptamer is: 5′-Fc-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′.

3. The on / off electrochemiluminescence aptamer sensor for detecting ochratoxin A in grain samples according to claim 1, characterized in that: The preparation steps of AuNPs-PEI-MWCNTs and Ru@SiO2-BPQDs are as follows: (1) Preparation of AuNPs-PEI-MWCNTs: 2.0 mg of carboxylated MWCNTs were added to 4 mL of PEI solution, and the mixture was then ultrasonically dispersed. The resulting mixture was then centrifuged and redispersed to obtain PEI-MWCNTs. Subsequently, 1 mL of synthesized AuNPs was gradually added dropwise to 1 mL of prepared PEI-MWCNTs. The mixture was then stirred overnight in the dark. After centrifugation, the black solid was redistributed in 2 mL of 0.01 M phosphate buffered saline with pH 7.

4. AuNPs-PEI-MWCNTs were successfully synthesized. (2) Preparation of BPQDs: 4 mL of N-methylpyrrolidone (NMP) and 5 mg of black phosphorus powder were sonicated in an ice bath at 100 W for 8 hours. The resulting dispersion was centrifuged at 7000 rpm for 20 minutes, and then centrifuged again at 10000 rpm for 20 minutes. The supernatant containing 75% of the solution top was collected each time. (3) Preparation of Ru@SiO2: First, 7.08 mL of Triton solution, 30 mL of cyclohexane, 7.2 mL of n-hexanol and 1.36 mL of water were stirred for 5 minutes to form a water-in-oil reverse microemulsion. Then, 320 μL of 0.1 M ruthenium bipyridine solution was added and stirred for 30 minutes until the solution was clear. Then, 400 μL of tetraethyl orthosilicate and 240 μL of ammonia were quickly injected to initiate polymerization. The mixture was stirred in the dark for 24 hours. The final product was separated with 40 mL of acetone, centrifuged at 10,000 rpm for 10 minutes, and then washed three times with a mixture of ethanol and water. Finally, Ru@SiO2 was dried in a vacuum freeze dryer and stored at 4 °C for future use. (4) Preparation of Ru@SiO2-BPQDs: First, 10 mg Ru@SiO2 was dispersed in 5 mL of ethanol, and 200 μL of aminopropyltriethoxysilane was added. After stirring for 4 hours, it was washed with a mixture of ethanol and water, and then dried in a vacuum freeze dryer to obtain Ru@SiO2-NH2. 170 μL of 10 mg / mL Ru@SiO2-NH2 and 850 μL of 3 mg / mL BPQDs were added to a beaker and stirred in the dark for 12 hours. Since BPQDs and Ru@SiO2-NH2 have opposite charges, they combine through electrostatic interaction to form self-enhanced luminescent material Ru@SiO2-BPQDs.

4. A switch-on electrochemiluminescence aptamer sensor for detecting ochratoxin A in grain samples according to any one of claims 1-3, characterized in that: Using the electrochemiluminescence aptamer sensor as the working electrode, saturated calomel as the reference electrode, and platinum wire electrode as the counter electrode, a three-electrode system is formed. Ochratoxin A in the sample is quantitatively captured onto the surface of the sensor, and the generated luminescence signal is used to detect ochratoxin A.

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