An electrochemiluminescent immunosensor and its application in detecting OTA

Abstract

The present invention discloses an electrochemiluminescent immunosensor and its application in detecting OTA, the electrochemiluminescent immunosensor comprises an electrochemiluminescent probe and an electrochemiluminescent electrode, wherein the electrochemiluminescent probe is prepared by the following method: PCN-224 and a manganese salt are dissolved in an organic solvent, PCN-224-Mn is obtained after reaction, the PCN-224-Mn is activated and then incubated with an OTA antibody to obtain the electrochemiluminescent immunosensor; the electrochemiluminescent electrode is prepared by the following method: AuPt NFs are modified on an electrode, the electrode is incubated with an OTA-coated antigen to obtain the electrochemiluminescent electrode. The present invention also provides the above-mentioned electrochemiluminescent immunosensor using an electrochemiluminescent immunoassay to detect OTA, which has strong controllability in terms of time, space and luminescence intensity compared with the existing method for detecting OTA, and it also has the advantages of simple operation, cost-effectiveness, high sensitivity, low background signal and low reagent consumption.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemiluminescence immunosensors, and in particular to an electrochemiluminescence immunosensor and an application thereof in detecting OTA. Background Art

[0002] Ochratoxins are a group of mycotoxins with similar structures. There are four compounds, A, B, C, and D. They mainly endanger the kidneys of humans and animals. Among them, ochratoxin A (OTA) is the most toxic, most closely related to human health, has the highest toxin production, is the most polluting to crops, and is the most widely distributed. Ochratoxin is a mycotoxin produced by Aspergillus ochraceus and Penicillium viridiflorum. It can be divided into two types, A and B, with A being more toxic. OTA is seriously contaminated in animal feed. When animals eat feed contaminated with OTA, OTA accumulates in their bodies and is not easily metabolized and degraded. Therefore, OTA is often detected in animal foods, especially pig kidneys, livers, muscles, blood, as well as milk and dairy products. Among the discovered mycotoxin families, OTA is considered to be second only to aflatoxin in terms of importance and harmfulness. Therefore, the detection of OTA in food, feed, and pig kidneys is crucial.

[0003] At present, the main methods for detecting ochratoxin A residues in food include high performance liquid chromatography, gas chromatography-mass spectrometry, mass spectrometry, etc. These methods usually require derivatization of samples, and the detection efficiency is not ideal; although they have the advantages of relatively accurate quantification, good linear relationship, stable peak time, and no impurity interference, they are difficult to popularize due to expensive instruments, cumbersome operations, and high costs. Although the above methods can solve some problems, they also have some shortcomings and deficiencies in terms of detection time, cost, and flexibility, and can no longer meet people's needs. Most of these traditional detection methods have limitations such as high cost, complex sample pretreatment steps, and cumbersome operations. Therefore, it is of certain practical significance to establish a method that is rapid, sensitive, and can accurately detect OTA.

[0004] Electrochemiluminescence (ECL) is a combination of chemiluminescence and electrochemistry. Compared with chemiluminescence (CL), ECL has stronger controllability and selectivity; compared with photoluminescence (PL), ECL does not require a light source; compared with electrochemical methods, ECL has stronger selectivity and less electrode contamination. Therefore, ECL not only solves problems such as scattered light and luminescent impurities, but also has many advantages such as good applicability, simple instrumentation, low background signal, wide linear working range, and high sensitivity, making ECL widely used in analytical chemistry and biosensing. At present, ECL has become a relatively mature analytical technology and is widely used in clinical testing, immunoassay, food and water quality testing, biological warfare agent detection, and drug analysis. Electrochemiluminescence immunoassay (ECLIA) is an effective combination of electrochemiluminescence and immunoassay, combining the advantages of both, with the advantages of simple operation, good specificity, and high sensitivity, and has become a powerful means of food safety analysis. Therefore, it is necessary to develop an ECLIA detection method for OTA to improve its detection sensitivity and reduce the difficulty of operation. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an electrochemiluminescence immunosensor and its application in detecting OTA. The present invention develops a competitive electrochemiluminescence immunosensor based on electrochemiluminescence, PCN-224-Mn is used as an electrochemiluminescence probe, and AuPt NFs nanocomposite modified electrode is used for ultrasensitive quantitative detection of OTA.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] An electrochemiluminescent immunosensor comprises an electrochemiluminescent probe and an electrochemiluminescent electrode, wherein the electrochemiluminescent probe is prepared by the following method: PCN-224 and a manganese salt are dissolved in an organic solvent, and PCN-224-Mn is obtained after reaction, and the PCN-224-Mn is activated and then incubated with an OTA antibody (OTA-Ab) to obtain the electrochemiluminescent immunosensor;

[0008] The electrochemiluminescent electrode is prepared by the following method: AuPt NFs are modified on the electrode, and the electrode is incubated with OTA-coated antigen (OTA-Ag) to obtain the electrochemiluminescent electrode.

[0009] In the electrochemiluminescent immunosensor of the present invention, OTA-Ab is connected to PCN-224-Mn through an amide bond (-CO-NH-) to form a PCN-224-Mn / OTA-Ab probe, Mn(II) can improve the interaction efficiency between PCN-224 and a co-reactant persulfate, and Mn(II) is directly coordinated to PCN-224, thereby improving the ECL performance of the porphyrin-based MOFs luminophore in a simple, convenient and effective endogenous method. After being modified with Mn(II), the ECL intensity and stability of PCN-224-Mn are significantly improved; in the electrochemiluminescent electrode, Pt nanowires are in situ grown on Au nanoparticles to obtain AuPt NFs nanocomposite materials, which not only improves the electron transfer efficiency, but also provides a larger surface area for fixing antigens, and combines with OTA-Ag through Au-N bonds. The electrochemiluminescent immunosensor prepared by the present invention uses PCN-224-Mn as an electrochemiluminescent probe and uses AuPt NFs nanocomposite material to modify the electrode, which has higher sensitivity (more than 100 times) than the traditional ELISA method.

[0010] In a specific embodiment, PCN-224 is prepared by the following method: zirconium oxychloride octahydrate (ZrOCl8·H2O), benzoic acid and tetrakis(4-carboxyphenyl)porphyrin (TCPP, CAS: 14609-54-2) are subjected to heating reaction in an organic solvent to obtain PCN-224.

[0011] Furthermore, the temperature of the heating reaction is 80-100° C., and the reaction time is 4-6 hours.

[0012] Furthermore, the manganese salt is manganese dichloride tetrahydrate (MnCl2·4H2O), and the organic solvent is N,N-dimethylformamide (DMF).

[0013] Furthermore, the reaction temperature of the PCN-224 and the manganese salt is 110-130° C., and the reaction time is 11-13 hours.

[0014] Furthermore, the activation method is to add a cross-linking agent, wherein the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0015] In a specific embodiment, the electrochemiluminescent probe is prepared by the following method: PCN-224 and manganese dichloride tetrahydrate are subjected to a heating reaction in DMF to obtain PCN-224-Mn; the PCN-224-Mn is activated with a cross-linking agent, and then OTA-Ab is added for incubation to obtain the electrochemiluminescent immunoprobe.

[0016] Furthermore, the mass ratio of PCN-224-Mn to OTA-Ab is (100-200):1, preferably (150-180):1, and more preferably 166:1.

[0017] Furthermore, the incubation temperature is 3-5°C, and the incubation time is 10-14h.

[0018] Furthermore, after incubation with OTA-Ab, bovine serum albumin (BSA) was added for blocking.

[0019] Furthermore, the blocking temperature is 3-5°C and the blocking time is 0.5-1.5h.

[0020] Furthermore, the concentration of the BSA is 4wt% to 6wt%.

[0021] In a specific embodiment, the AuPt NFs are prepared by the following method: heating a chloroauric acid solution to boiling and then quickly adding a sodium citrate solution to obtain Au NPs; adding ascorbic acid to the boiling solution of Au NPs, then adding a chloroplatinic acid solution, and continuing to heat to obtain the AuPt NFs.

[0022] Furthermore, the reaction temperature for preparing Au NPs is 100-110° C., and the time is 5-10 min.

[0023] Furthermore, the reaction temperature for preparing AuPt NFs is 100-110° C., and the time is 20-30 min.

[0024] Furthermore, the electrode may be a glassy carbon electrode (GCE).

[0025] Furthermore, the mass ratio of the AuPt NFs to the OTA-coated antigen is 1:(20-100), preferably 1:(40-60), and more preferably 1:50.

[0026] In a specific embodiment, the electrochemiluminescent electrode is prepared by the following method: dropping AuPt NFs on a polished electrode surface, and then dropping OTA-Ag on the electrode surface for incubation after drying to obtain the electrochemiluminescent electrode.

[0027] Furthermore, the incubation temperature is 3-5°C, and the incubation time is 10-14h.

[0028] Furthermore, after incubation with OTA-Ag, bovine serum albumin (BSA) was added for blocking.

[0029] Furthermore, the blocking temperature is 3-5°C and the blocking time is 0.5-1.5h.

[0030] Furthermore, the concentration of the BSA is 4wt% to 6wt%.

[0031] The present invention also protects the application of the electrochemiluminescence immunosensor in detecting OTA.

[0032] Further, detecting OTA includes the following steps:

[0033] S1. The electrochemiluminescent probe and OTA standard solutions of different concentrations are mixed and incubated, and then the mixed solution is contacted with an electrochemiluminescent electrode to react the OTA antibody with the OTA-coated antigen to obtain an assembled electrochemiluminescent immunosensor;

[0034] S2. The assembled electrochemiluminescent immunosensor is used as a working electrode, and a cyclic voltammetry scan is performed using a three-electrode system to record the luminescence intensity-time curve, establish the relationship between the ECL luminescence intensity and the OTA concentration, and obtain a linear regression equation;

[0035] S3. Repeat step S1 for the test solution, use the assembled electrochemiluminescent immunosensor as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, and obtain the concentration of OTA in the test solution based on the measured luminescence intensity-time curve combined with the linear regression equation.

[0036] When ochratoxin A is detected using the electrochemiluminescent immunosensor, the quantitative basis is the competition between OTA and OTA-Ag for the limited OTA-Ab.

[0037] Furthermore, the detection buffer of the three-electrode system in S2 is a persulfate buffer solution, the persulfate is a co-reactant, and the persulfate is preferably potassium persulfate (K2S2O8).

[0038] Furthermore, the concentration of persulfate in the persulfate buffer solution is 0.08-0.12M, preferably 0.1M.

[0039] Beneficial effects of the present invention:

[0040] The present invention develops a competitive ECL immunosensor based on electrochemiluminescence, PCN-224-Mn is used as an electrochemiluminescent probe, and AuPt NFs nanocomposite modified electrode is used for ultrasensitive quantitative detection of OTA. The present invention also provides the above-mentioned electrochemiluminescent immunosensor using electrochemiluminescent immunoassay to detect OTA, which has strong controllability in time, space and luminescence intensity compared with the existing method for detecting OTA, and it also has the advantages of simple operation, cost-effectiveness, high sensitivity, low background signal and low reagent consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is a schematic diagram of the construction of the electrochemiluminescence immunosensor provided by the present invention and its principle for detecting OTA.

[0042] Figure 2 TEM images of PCN-224-Mn and AuPt NFs; wherein A is the TEM image of PCN-224-Mn, and B is the TEM image of AuPtNFs.

[0043] Figure 3 The ECL intensity-time curve and linear regression curve of OTA standard solution with different concentrations.

[0044] Figure 4 Concentration optimization diagram of OTA-Ag, OTA-Ab, AuPt NFs and co-reactant K2S2O8.

[0045] Figure 5 The luminescence intensity-time curves of the ligands TCPP, PCN-224, and PCN-224-Mn after 15 consecutive cycles of scanning under the same conditions. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0048] In the following examples of the present invention, OTA coated antigen and OTA antibody were donated by Professor Deng Anping's research group at Soochow University. Among them, OTA antibody is a monoclonal antibody secreted by OTA hybridoma cell line OTA-8B10 (Hybridoma cell line 0TA-8B10) (deposited in China Center for Type Culture Collection, with a deposit number of CCTCC No. C202384, deposited at Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, deposit date: 2023-4-11), and OTA hybridoma cell line is prepared from OTA antigen. OTA-Ab has extremely high detection sensitivity for OTA (ELISA method IC 50 0.035 ng·mL -1 ) and a higher working titer. This OTA-coated antigen is prepared by the carbodiimide method, which is prepared by dissolving ochratoxin A (hapten), N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide in anhydrous tetrahydrofuran, reacting, drying with nitrogen, dissolving in N,N-dimethylformamide for activation, and then coupling with a carrier protein.

[0049] The present invention provides an electrochemiluminescent immunosensor, comprising an electrochemiluminescent probe and an electrochemiluminescent electrode. The electrochemiluminescent probe is prepared by the following method: PCN-224 and a manganese salt are dissolved in an organic solvent, and PCN-224-Mn is obtained after reaction, and the PCN-224-Mn is activated and then incubated with an OTA antibody (OTA-Ab) to obtain the electrochemiluminescent immunosensor;

[0050] The electrochemiluminescent electrode is prepared by the following method: AuPt NFs are modified on the electrode, and the electrode is incubated with OTA-coated antigen (OTA-Ag) to obtain the electrochemiluminescent electrode.

[0051] In one embodiment of the present invention, the electrochemiluminescent probe is prepared by the following method: ZrOCl8·H2O, benzoic acid and TCPP are dissolved in DMF, mixed and heated in a water bath at 90°C for 5h. The precipitate is centrifuged and washed three times, and vacuum dried to obtain PCN-224; MnCl2·4H2O and PCN-224 are dissolved in DMF, heated in an oil bath at 120°C for 12h, the precipitate is centrifuged and washed three times, and vacuum dried to obtain PCN-224-Mn; the carboxyl group in PCN-224-Mn is activated with a crosslinking agent, and then the activated PCN-224-Mn is incubated with OTA-Ab at 4°C for 12h, so that the OTA-Ab is connected to the activated PCN-224-Mn through an amide bond, and 5wt% BSA is added at 4°C for blocking for 1h to obtain the electrochemiluminescent probe.

[0052] In one embodiment of the present invention, the mass ratio of PCN-224-Mn to OTA-Ab is (100-200):1, preferably (150-180):1, and more preferably 166:1.

[0053] In one embodiment of the present invention, the electrochemiluminescent electrode is prepared by the following method: adding chloroauric acid solution to ultrapure water, stirring thoroughly and heating to boiling, then quickly adding sodium citrate solution, heating at 100°C for 5 minutes, a golden red solution appears, and Au NPs are formed; adding ascorbic acid to the boiling Au NPs solution, then adding chloroplatinic acid, heating at 100°C for 25 minutes, centrifuging and washing the precipitate three times, and vacuum freeze-drying to obtain AuPt NFs; redispersing the obtained AuPtNFs in ultrapure water, and then modifying them on the electrode, and then incubating the electrode in OTA-Ag at 4°C for 12 hours, so that the OTA-Ag is connected to the AuPt NFs through the Au-N bond, adding 5wt% BSA at 4°C for blocking for 1 hour, and obtaining the electrochemiluminescent electrode.

[0054] In one embodiment of the present invention, the mass ratio of the AuPt NFs to the OTA-coated antigen is 1:(20-100), preferably 1:(40-60), and more preferably 1:50.

[0055] The present invention also provides the use of the electrochemiluminescent immunosensor in detecting OTA, and the detection of OTA includes the following steps:

[0056] S1. The electrochemiluminescent probe and OTA standard solutions of different concentrations are mixed and incubated, and then the mixed solution is contacted with an electrochemiluminescent electrode to react the OTA antibody with the OTA-coated antigen to obtain an assembled electrochemiluminescent immunosensor;

[0057] S2. The assembled electrochemiluminescent immunosensor is used as a working electrode, and a cyclic voltammetry scan is performed using a three-electrode system to record the luminescence intensity-time curve, establish the relationship between the ECL luminescence intensity and the OTA concentration, and obtain a linear regression equation;

[0058] S3. Repeat step S1 for the test solution, use the assembled electrochemiluminescent immunosensor as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, and obtain the concentration of OTA in the test solution based on the measured luminescence intensity-time curve combined with the linear regression equation.

[0059] In one embodiment of the present invention, detecting OTA includes the following steps:

[0060] S1. The electrochemiluminescent probe solution is centrifuged to remove the supernatant, and the precipitate is evenly mixed with OTA standard solutions of different concentrations, and the obtained mixed solution is incubated with an electrochemiluminescent electrode. After the incubation is complete, the electrochemiluminescent probe and OTA that are not bound to the electrochemiluminescent electrode are washed with a buffer solution to obtain an assembled electrochemiluminescent immunosensor;

[0061] S2. The assembled electrochemiluminescent immunosensor is used as a working electrode, and a cyclic voltammetry scan is performed using a three-electrode system to record the luminescence intensity-time curve, establish a linear relationship between the ECL luminescence intensity and the logarithmic value of the OTA concentration, and obtain the corresponding linear regression equation;

[0062] S3. Add an electrochemiluminescent probe to the solution to be tested, incubate the obtained mixed solution with an electrochemiluminescent electrode, and after complete incubation, wash the electrochemiluminescent electrode with a buffer solution to obtain an assembled electrochemiluminescent immunosensor; use the assembled electrochemiluminescent immunosensor as a working electrode, perform cyclic voltammetry scanning using a three-electrode system, and obtain the concentration of OTA in the solution to be tested based on the measured luminescence intensity-time curve combined with the linear regression equation.

[0063] In one embodiment of the present invention, in S1, the concentration of the OTA standard solution is 0.0002-1000 ng·mL -1 .

[0064] In one embodiment of the present invention, in S1, the concentration of the electrochemiluminescent probe solution is 2 mg·mL -1 The volume ratio of the electrochemiluminescent probe solution to the OTA standard solution is 1:1.

[0065] In one embodiment of the present invention, the specific operation of cyclic voltammetry scanning using a three-electrode system is as follows: within the electrochemical window range of -1.6 to 0 V, the photomultiplier tube high voltage is -780 V, the amplification level is 4, the scan rate is 0.3 V / s, and cyclic voltammetry scanning is performed. The ECL signal will change with the change of the concentration of the OTA standard solution.

[0066] Figure 1 The schematic diagram of the construction of the electrochemiluminescent immunosensor provided by the present invention and the principle of OTA detection is as follows: Figure 1 As shown, Figure 1 A is a schematic diagram of the construction of the electrochemiluminescent probe. Figure 1 B is a schematic diagram of the construction of the electrochemiluminescent electrode, the detection of OTA and the ECL intensity-time curve. It can be seen from the figure that the OTA solution competes with OTA-Ag for limited OTA-Ab. When the OTA solution is not added, there is no competition between the OTA solution and OTA-Ag for the binding sites on the OTA-Ab, so OTA-Ag can bind well to OTA-Ab, thereby showing a strong ECL intensity. When the concentration of the OTA solution increases, the number of electrochemiluminescent probes connected to the electrochemiluminescent electrode decreases, and the ECL intensity weakens.

[0067] Figure 2 TEM images of PCN-224-Mn and AuPt NFs; wherein A is the TEM image of PCN-224-Mn, and B is the TEM image of AuPtNFs.

[0068] Figure 3 The ECL intensity-time curve and linear regression curve of OTA standard solution with different concentrations.

[0069] Figure 4 The concentration optimization diagrams of OTA-Ag, OTA-Ab, AuPt NFs and co-reactant K2S2O8 are shown in Figure 1. A is the concentration optimization diagram of OTA-Ag, B is the concentration optimization diagram of OTA-Ab, C is the concentration optimization diagram of AuPt NFs, and D is the concentration optimization diagram of K2S2O8. Figure 4 As can be seen in A, the OTA-Ag concentration is 12 μg mL -1When the concentration of OTA-Ab was 6 μg·mL, the ΔECL intensity reached a peak and then decreased. This is due to the limitation of the electrode surface area and the poor conductivity of biomacromolecules, which reduced the competitive efficiency of the immunosensor. -1 The optimal concentration is ( Figure 4 B). The amount of AuPt NFs is also an important factor affecting the efficiency of ECL immunosensor. AuPt NFs provide binding sites to fix coated antigens. The more AuPt NFs there are, the more coated antigens are connected, and the stronger the ECL signal is. However, when the fixed amount of AuPt NFs coated antigen reaches saturation, the overly thick modification layer may hinder electron transfer and reduce the ECL signal. Figure 4 As shown in C, the concentration of AuPt NFs is 0.6 μg mL -1 In addition, the concentration of the co-reactant is also very important. Figure 4 D shows that the optimal concentration of K2S2O8 in the system is 0.1 M. Too high a K2S2O8 concentration will result in a strong background signal and is not conducive to detection. Therefore, 12 μg mL -1 OTA-Ag, 6 μg mL -1 OTA-Ab, 0.6 μg mL -1 AuPt NFs and 0.1 M K2S2O8 were the optimal conditions for the ECL immunosensor.

[0070] Figure 5 The luminescence intensity-time curves of the ligands TCPP, PCN-224, and PCN-224-Mn under the same conditions for 15 consecutive cycles. A is the luminescence intensity-time curve of the ligand TCPP, B is the luminescence intensity-time curve of PCN-224, and C is the luminescence intensity-time curve of PCN-224-Mn. It can be seen from the figure that under the same conditions, the stability of the TCPP ligand alone is poor, the luminescence signal is not strong, and it shows a messy peak shape ( Figure 4 A). After constructing PCN-224 with Zr6 node and TCPP ligand, the stability was significantly improved, the peak shape became regular and a stronger ECL signal was generated ( Figure 5 B). After the incorporation of Mn(II), we can see that the ECL intensity has no significant change after 15 consecutive cycles of scanning, the stability has been further improved, and the value of the luminescence signal has also been significantly improved ( Figure 5 C).

[0071] Example 1

[0072] An electrochemiluminescent immunosensor comprises an electrochemiluminescent probe (PCN-224-Mn / OTA-Ab probe) and an electrochemiluminescent electrode, wherein the electrochemiluminescent probe is prepared by the following method:

[0073] 180 mg ZrOCl8·H2O, 1.68 g benzoic acid and 60 mg TCPP were dissolved in 60 mL DMF, and then the mixture was heated in a water bath at 90°C for 5 h. The purple solid particles were washed with DMF and ultrapure water for 3 times and vacuum dried to obtain PCN-224 solid; 100 mg MnCl2·4H2O and 50 mg PCN-224 were dissolved in 20 mL DMF and stirred in an oil bath at 120°C for 12 h. After the reaction was completed, the obtained suspension was centrifuged at 10000 rpm for 10 min to obtain a precipitate. After removing the supernatant, the precipitate was washed 3 times with DMF and ultrapure water, and the washed product was vacuum dried at 60°C to obtain PCN-224-Mn.

[0074] Take 150 μL of 1 mg mL -1 PCN-224-Mn solution was prepared. 150 μL NHS and 150 μL EDC were added to the solution to activate the carboxyl groups. The solution was then placed in the dark for 1 hour. The activated PCN-224-Mn solution was centrifuged and then reconstituted with 150 μL ultrapure water. 10 μL of the activated PCN-224-Mn solution and 10 μL of 6 μg mL -1 The OTA-Ab was mixed evenly and placed in a 4°C environment overnight (about 12 hours). After the reaction was complete, 10 μL of 5% BSA was added to the centrifuge tube and placed in a 4°C environment for blocking for 1 hour (blocking nonspecific binding sites to avoid nonspecific binding), and the PCN-224-Mn / OTA-Ab probe was obtained.

[0075] The electrochemical luminescent electrode is prepared by the following method:

[0076] 0.5 mL of chloroauric acid solution (wt. 1%) was added to 50 mL of aqueous solution, and the solution was heated to boiling under stirring. 0.8 mL of sodium citrate solution (wt. 1%) was quickly added to the above solution. After heating for a few minutes, a golden red solution appeared, indicating that AuNPs were formed. Subsequently, 1 mL of 0.1 M ascorbic acid was added to the boiling AuNPs solution, and then 1.25 mL of chloroplatinic acid solution (wt. 1%) was added. After heating for 25 minutes, an AuPt NFs suspension was obtained. After the reaction was completed, the obtained suspension was centrifuged at 12000 rpm for 10 minutes to obtain a precipitate. After removing the supernatant, the precipitate was washed 3 times with ultrapure water. Finally, the washed product was vacuum freeze-dried to obtain an AuPt NFs nanocomposite material, which was properly stored for later use.

[0077] The glassy carbon electrode was polished with α-Al2O3 powder on the suede to make it mirror-like, and then rinsed with ethanol and ultrapure water in turn, and dried with nitrogen, and then 10 μL of 0.6 mg mL -1 The AuPt NFs suspension was then dried in air. 10 μL of 12 μg mL -1 OTA-Ag was added and incubated overnight at 4°C to fix OTA-Ag through Au-N bonds. Then 10 μL of 5% BSA was added to block the remaining active sites and eliminate nonspecific binding. After blocking for 1 h, the electrochemiluminescent electrode was rinsed with PBS and stored in a 4°C refrigerator.

[0078] Example 2

[0079] The electrochemiluminescent immunosensor of Example 1 is used to detect OTA by electrochemiluminescent immunoassay, and the detection includes the following steps:

[0080] S1. After centrifuging 10 μL of PCN-224-Mn / OTA-Ab probe solution and removing the supernatant, the lower precipitate was added to 10 μL of different concentrations (0.0002 ng mL -1 , 0.002ng·mL -1 , 0.02ng·mL -1 , 0.2ng·mL -1 , 1ng·mL -1 , 10ng·mL -1 , 100ng·mL -1 , 1000ng·mL -1 ) in a standard OTA solution, the two were mixed evenly and the mixture was coated on an electrochemiluminescent electrode. In this process, OTA competed with OTA-Ag for limited OTA-Ab. After 1 hour, the PCN-224-Mn / OTA-Ab that was not bound to OTA-Ag and the excess OTA were washed away with PBS solution to obtain the assembled electrochemiluminescent immunosensor;

[0081] S2. The electrochemiluminescence immunosensor assembled as above was used as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. In the ECL detection buffer solution (0.1M potassium persulfate (K2S2O8) in PBS buffer with pH=7.4), within the electrochemical window range of -1.6 to 0V, the photomultiplier tube high voltage was -780V, the amplification level was 4, and the scan rate was 0.3V / s. Cyclic voltammetry was performed and the luminescence intensity-time curve was recorded. A linear relationship between the ECL intensity and the logarithm of the OTA concentration was established, and the corresponding linear regression equation was obtained: I=8930.8-1712.4lgC, and the regression coefficient was 0.9986.

[0082] The linear detection range of the electrochemiluminescent immunosensor provided by the present invention is 0.0002-1000 ng·mL -1 , based on a signal-to-noise ratio of 3, the detection limit is 0.67 pg·mL -1 .

[0083] Figure 3 The ECL intensity-time curve and linear regression curve of OTA standard solution with different concentrations; wherein, A is the ECL intensity-time curve of OTA standard solution with different concentrations, and B is the linear regression curve of ECL intensity of OTA standard solution with different concentrations and the logarithm of OTA concentration.

[0084] Test Example 1

[0085] In order to test the applicability of electrochemiluminescent immunoassay, two different grains (corn flour and wheat flour) randomly collected from the vegetable market were used for spike recovery experiments. 2g of sample powder was placed in a 50mL centrifuge tube, 10mL of extract (methanol: water = 7:3) was added, and vortexed for 10min to mix. After filtering with a filter membrane, it was mixed with dilutions of standard solutions of different concentrations to obtain solutions to be tested with different OTA addition amounts. 10μL of PCN-224-Mn / OTA-Ab probe solution was centrifuged to remove the supernatant, 10μL of the clear solution obtained through the filter membrane was added and mixed evenly, and then coated on the electrochemiluminescent electrode in Example 1 to obtain the assembled electrochemiluminescent immunosensor for detection. The results are shown in Table 1. The sensor has a recovery rate of OTA in spiked samples in the range of 98.9% to 110.5%, and a relative standard deviation (RSD) in the range of 2.2% to 9.8% (n = 3), indicating that this method can be used to detect OTA more accurately in actual samples.

[0086] Table 1 ECL immunosensor determination results of OTA spiked in actual samples

[0087]

[0088] ND = not detected.

[0089] Therefore, according to the above results, when detecting the OTA content in an unknown sample, it is only necessary to mix the sample with the PCN-224-Mn / OTA-Ab probe solution, and then coat the mixed solution on the electrochemiluminescent electrode to obtain the assembled electrochemiluminescent immunosensor for testing and then compare it with the corresponding standard curve.

[0090] The present invention develops a competitive ECL immunosensor based on electrochemiluminescence, PCN-224-Mn is used as an electrochemiluminescent probe, and AuPt NFs nanocomposite modified electrode is used for ultrasensitive quantitative detection of OTA. The present invention also provides the above-mentioned electrochemiluminescent immunosensor using electrochemiluminescent immunoassay to detect OTA, which has strong controllability in time, space and luminescence intensity compared with the existing method for detecting OTA, and it also has the advantages of simple operation, cost-effectiveness, high sensitivity, low background signal and low reagent consumption.

[0091] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An electrochemiluminescent immunosensor, comprising an electrochemiluminescent probe and an electrochemiluminescent electrode, characterized in that: The electrochemiluminescent probe is prepared by the following method: dissolving PCN-224 and manganese salt in an organic solvent, reacting to obtain PCN-224-Mn, activating the PCN-224-Mn and incubating with OTA antibody to obtain the electrochemiluminescent probe; the method for activating PCN-224-Mn is to add a cross-linking agent, and the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The electrochemiluminescent electrode is prepared by the following method: AuPt NFs are modified on the electrode, and the electrode is incubated with OTA-coated antigen to obtain the electrochemiluminescent electrode.

2. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The manganese salt is manganese dichloride tetrahydrate, and the organic solvent is N,N-dimethylformamide.

3. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The reaction temperature of the PCN-224 and the manganese salt is 110-130°C.

4. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The mass ratio of the PCN-224-Mn to the OTA antibody is (100~200):

1.

5. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The mass ratio of the AuPt NFs to the OTA-coated antigen is 1:(20-100).

6. Use of the electrochemiluminescence immunosensor according to any one of claims 1 to 5 in detecting OTA.

7. The use according to claim 6, characterized in that: Detecting OTA involves the following steps: S1. The electrochemiluminescent probe and OTA standard solutions of different concentrations are mixed and incubated, and then the mixed solution is contacted with an electrochemiluminescent electrode to react the OTA antibody with the OTA-coated antigen to obtain an assembled electrochemiluminescent immunosensor; S2. The assembled electrochemiluminescent immunosensor was used as the working electrode, and cyclic voltammetry was performed using a three-electrode system to record the luminescence intensity-time curve, establish the relationship between the luminescence intensity and the OTA concentration, and obtain a linear regression equation; S3. The electrochemiluminescent probe and the solution to be tested are mixed and incubated, and then the mixed solution is contacted with the electrochemiluminescent electrode to react the OTA antibody with the OTA-coated antigen to obtain an assembled electrochemiluminescent immunosensor. The electrochemiluminescent immunosensor assembled in this step is used as a working electrode, and a cyclic voltammetry scan is performed using a three-electrode system. The concentration of OTA in the solution to be tested is obtained based on the measured luminescence intensity-time curve combined with the linear regression equation.

8. The use according to claim 7, characterized in that: The detection buffer of the three-electrode system in S2 is a persulfate buffer solution.

9. The use according to claim 8, characterized in that The concentration of persulfate in the persulfate buffer solution is 0.08~0.12 M.

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