Preparation method and sensor of electrochemiluminescence modified electrode for detecting ochratoxin A

Abstract

The invention discloses a method for detecting ochratoxin A (OTA) by signal-amplified electrochemiluminescent sensing based on energy resonance transfer (RET) between g-C3N4 and NU-1000 (Zr). G-C3N4 nanosheets and OTA nanoantibody heptamer (Nb28-C4bpα) are modified on the electrode surface, and then nonspecific binding sites are blocked with BSA to obtain a modified electrode. The modified electrode is used as a working electrode, and an electrochemiluminescent immunosensor is constructed by continuing to modify OTA and OTA-Apt-NU-1000 (Zr) nanocomposite materials, and OTA is quantitatively analyzed by detecting changes in electrochemiluminescent signals. The present invention uses nanoantibody polymers as recognition molecules to construct an electrochemiluminescent immunosensor, achieves high specificity and high sensitivity detection of OTA, and is used for actual detection applications of complex food matrices such as coffee, uses nanocomposites to replace toxic luminescent reagents, reduces detection costs, and reduces safety issues existing in traditional luminescent systems.

Description

Technical Field

[0001] The invention belongs to the technical field of biological toxin detection and electrochemiluminescence, and particularly relates to a preparation method of an electrochemiluminescence modified electrode for detecting ochratoxin A and a sensor. Background Art

[0002] Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium. It is widely distributed in nature and can easily contaminate foods such as grains, coffee beans and spices and their products. OTA has stable chemical properties and is not easily degraded. Long-term accumulation of OTA in humans and animals will cause irreversible damage to the liver, kidneys, nervous system, etc., and may cause teratogenicity or carcinogenesis. The International Agency for Research on Cancer (IARC) has designated it as a human Class IIB carcinogen. In order to reduce the risk of OTA contamination, it is crucial to develop sensitive and accurate OTA detection and analysis technology.

[0003] Currently, high performance liquid chromatography, fluorescence resonance energy transfer on lateral flow immunoassay and enzyme-linked immunosorbent assay have been used for OTA detection. These technologies have high sensitivity, but they have shortcomings such as expensive analyzers, high cost and weak anti-interference ability.

[0004] Electrochemiluminescence detection technology (ECL) is a viable alternative method for detecting ochratoxin A due to its high sensitivity, high stability and low background signal. In electrochemiluminescence energy resonance transfer (ECL-RET), due to the large-area overlap of the spectra of the energy donor and the acceptor, the energy transfer efficiency is high, which can significantly improve its accuracy and sensitivity. Select a suitable ECL-RET donor-acceptor pair to construct an ECL-RET sensing platform to achieve OTA quantitative analysis. Traditional antibodies (monoclonal antibodies or polyclonal antibodies) have complex structures. In order to overcome the shortcomings of traditional OTA antibodies, some researchers have developed nanoantibodies with small molecular weight (about 15kDa), strong stability and affinity, and strong resistance to matrix interference. OTA is a small molecule, and the traditional electrode modification process has little effect on the change of ECL signal, and it is impossible to achieve ultra-sensitive detection of OTA. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing an electrochemiluminescent modified electrode and a sensor for detecting ochratoxin A. The present invention uses OTA nanoantibody polymer (Nb28-C4bpα) as a specific recognition molecule, based on the energy resonance transfer between the two nanomaterials, and uses g-C3N4 as a RET energy donor and NU-1000 (Zr) as a RET energy acceptor to construct a modified electrode. When the modified electrode is used as a working electrode of an ECL immunosensor, it has the characteristics of high safety, high specificity and sensitivity, wide detection range, and good stability.

[0006] The present invention utilizes the different characteristics of nanomaterials, combines energy resonance transfer with ECL, and prepares a modified electrode for detecting OTA stably and sensitively.

[0007] The present invention uses OTA nanobody heptamer as a specific recognition molecule, graphite carbon nitride (g-C3N4) and NU-1000 (Zr) as the energy donor and acceptor of ECL-RET, respectively, to construct a signal-amplified electrochemiluminescent immunosensor, which provides a reference for the application of nanobody polymers in the field of electrochemiluminescence technology. The use of energy resonance transfer between two nanomaterials to replace the luminescent system containing toxic luminescent agents provides a new idea for the construction of electrochemiluminescent sensing. The specific technical scheme of the present invention is as follows:

[0008] A method for preparing an electrochemiluminescent modified electrode for detecting ochratoxin A, the method comprising the following steps:

[0009] (1) g-C3N4 nanosheets were carboxylated using nitric acid (HNO3), and then the carboxyl groups of the carboxylated g-C3N4 were activated using a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS);

[0010] (2) preparing NU-1000(Zr) nanoparticles, and connecting the NU-1000(Zr) nanoparticles and OTA via OTA aptamers (Apt) to obtain OTA-Apt-NU-1000(Zr) composite nanoparticles;

[0011] (3) dispersing g-C3N4 nanosheets in water, dropping the resulting suspension onto the electrode surface, and drying to obtain a g-C3N4 / GCE modified electrode;

[0012] (4) adding OTA nanoantibody heptamer Nb28-C4bpα solution to the surface of the g-C3N4 / GCE modified electrode, washing the electrode surface after incubation to obtain a Nb28-C4bpα / g-C3N4 / GCE modified electrode, and then incubating the modified electrode with a bovine serum albumin (BSA) solution;

[0013] (5) Add a certain concentration of OTA solution to the surface of the BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode, incubate, wash and dry;

[0014] (6) On the surface of the OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode, OTA-Apt-NU-1000 (Zr) suspension was added dropwise, and after washing and drying, a modified electrode for detecting ochratoxin A was obtained.

[0015] Furthermore, the electrode may be any working electrode reported in the prior art that can be used for an electrochemiluminescent immunosensor, such as a glassy carbon electrode (GCE).

[0016] Furthermore, in step (1), the concentration of HNO3 is 5 mol / L, and the dosage of g-C3N4 and HNO3 is in the following relationship: 1 g: 50-150 mL.

[0017] Furthermore, in step (1), the concentrations of EDC and NHS used are 0.4 mol / L and 0.1 mol / L, respectively.

[0018] Furthermore, in step (1), the g-C3N4 nanosheets are added to HNO3 and refluxed at 125°C for 24-30h to carboxylate the g-C3N4.

[0019] Furthermore, in step (1), the carboxylated g-C3N4 is added to a mixed solution of EDC and NHS and shaken at 200 rpm on a constant temperature shaker for 5-10 h to activate the carboxyl group.

[0020] Furthermore, in step (2), the dosage of H4PTPA, ZrOCl2·8H2O, benzoic acid and dimethyl sulfoxide (DMF) is 34 mg: 120 mg: 1.2 g: 50-100 mL; H4PTPA, ZrOCl2·8H2O and benzoic acid are dissolved in DMF and stirred at 90°C for 10-15 h to obtain NU-1000 (Zr).

[0021] Furthermore, in step (2), NU-1000 (Zr) was added to the mixed solution of EDC and NHS, and shaken at 200 rpm for 5-8 hours on a constant temperature shaker to activate the carboxyl groups. The concentrations of EDC and NHS were 0.4 mol / L and 0.1 mol / L, respectively.

[0022] Furthermore, in step (2), NU-1000(Zr) and OTA aptamer are mixed and incubated on a constant temperature shaker at 200 rpm for 10-15 h to obtain Apt-NU-1000(Zr), and the dosage relationship between NU-1000(Zr) and OTA aptamer is 3 mg:1 OD.

[0023] Furthermore, in step (2), Apt-NU-1000(Zr) and OTA are mixed and shaken at 200 rpm for 4 hours on a constant temperature shaker to obtain OTA-Apt-NU-1000(Zr). The dosage of Apt-NU-1000(Zr) and OTA is 3 mg:10 mg / mL, 1-2 mL.

[0024] Furthermore, in step (3), the concentration of g-C3N4 is 1-2 mg / mL, and 5-10 μL of the suspension is dropped onto the electrode surface.

[0025] Furthermore, in step (4), the concentration of the OTA nanobody heptamer Nb28-C4bpα solution is 5-15 mg / mL, 5-10 μL of the suspension is dropped onto the electrode surface, incubated at 37° C. for 1-2 h, and then washed.

[0026] Furthermore, in step (4), the concentration of BSA solution added to the modified electrode surface is 1-1.5 wt %, and 5-10 μL BSA solution is dropped onto the electrode surface and incubated at 37° C. for 0.5-1 h.

[0027] Furthermore, in step (5), 5-10 μL of OTA solution with a concentration of 0.0001-500 ng / mL was added to the surface of the BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode and incubated at 37° C. for 0.5-1 h.

[0028] Furthermore, in step (6), 5-10 μL of OTA-Apt-NU-1000 (Zr) solution with a concentration of 1-2 ng / mL was added to the surface of the OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode and incubated at 37°C for 0.5-1 h.

[0029] Furthermore, the modified electrode for detecting ochratoxin A prepared according to the above method is also within the protection scope of the present invention. The modified electrode of the present invention immobilizes g-C3N4 nanoparticles on the electrode surface, which can not only increase the adsorption area of ​​the electrode surface, provide more adsorption sites for OTA nanoantibody polymers, covalently bind more Nb28-C4bpα, and improve the sensitivity of the ECL sensor, but also can form an electrochemiluminescence system with potassium persulfate solution (K2S2O8) to provide ECL signals.

[0030] Furthermore, the present invention modifies Nb28-C4bpα on the electrode surface and blocks nonspecific binding sites with BSA. When the substance to be detected, OTA, is incubated on the modified electrode, the antigen-antibody specifically binds, OTA occupies part of the binding sites, and then OTA-Apt-NU-1000 (Zr) is incubated on the modified electrode and binds to the remaining sites. The ECL-RET effect between g-C3N4 and OTA-Apt-NU-1000 (Zr) causes the energy of g-C3N4 to be transferred to OTA-Apt-NU-1000 (Zr), thereby reducing the ECL signal to achieve the purpose of detecting samples of unknown concentration.

[0031] Furthermore, the present invention also provides the use of the modified electrode for detecting ochratoxin A in detecting ochratoxin A. The modified electrode can be used as a working electrode of an electrochemiluminescent immunosensor, and can be used in combination with a three-electrode system of an auxiliary electrode and a reference electrode to achieve the purpose of detecting ochratoxin A with high sensitivity and high specificity.

[0032] Furthermore, the present invention also provides an electrochemiluminescent immunosensor for detecting ochratoxin A. The electrochemiluminescent immunosensor comprises a working electrode, and the working electrode is the modified electrode for detecting ochratoxin A mentioned above.

[0033] Furthermore, the electrochemiluminescent immunosensor also includes an auxiliary electrode and a reference electrode, the auxiliary electrode is a platinum electrode, and the reference electrode is a calomel electrode.

[0034] Furthermore, the electrochemiluminescent immunosensor of the present invention can stimulate the luminescent material on the electrode surface to react with its co-reactant through an electrical signal, use the luminescence intensity as the detection signal, and use the OTA-specific nanoantibody heptamer Nb28-C4bpα as the recognition element to specifically detect OTA. The g-C3N4 / K2S2O8 luminescent test system is adopted, g-C3N4 provides the luminescent signal, and the ECL signal is detected in the K2S2O8 solution. At the same time, the g-C3N4 with large specific surface area, high stability and high safety is used to immobilize the recognition molecule, thereby improving the sensitivity and safety of the sensor. The ochratoxin A nanoantibody heptamer (Nb28-C4bpα) is used as the specific recognition element to improve the specificity of the sensor.

[0035] Furthermore, the present invention also provides a method for detecting ochratoxin A, which uses the above-mentioned modified electrode for detecting ochratoxin A as a working electrode, a platinum electrode as an auxiliary electrode, a calomel electrode as a reference electrode, and a co-reactant K2S2O8 solution as a detection solution, and jointly detects the content of ochratoxin A by cyclic voltammetry and electrochemiluminescence.

[0036] Furthermore, in the electrochemiluminescence method, the measurement type is intensity mode, the scanning potential is -0.2V to 0.7V, the scanning rate is 0.1V / s, the amplification level is 2, and the photomultiplier tube high voltage is 700V.

[0037] Furthermore, in the above electrochemiluminescence method, the concentration of K2S2O8 solution is 20-30mmol / L.

[0038] Furthermore, the above method for detecting ochratoxin A comprises the following steps:

[0039] (1) preparing ochratoxin A standard solutions of different concentrations for later use;

[0040] (2) incubating the modified electrode for detecting ochratoxin A prepared above with the above ochratoxin A standard solutions of different concentrations at 37° C. for 1 h;

[0041] (3) incubating the modified electrode for detecting ochratoxin A prepared above with OTA-Apt-NU-1000 (Zr) solution at 37° C. for 1 h;

[0042] (4) taking out the modified electrode, matching it with the auxiliary electrode and the reference electrode, using K2S2O8 solution as the detection solution, and detecting the electrochemical luminescence intensity signal;

[0043] (5) Draw a standard curve with the logarithm of OTA concentration as the abscissa and the electrochemiluminescence intensity as the ordinate;

[0044] (6) The modified electrode for detecting ochratoxin A was incubated with the sample solution at 37°C for 1 h, and then incubated with OTA-Apt-NU-1000 (Zr) solution at 37°C for 1 h. It was matched with an auxiliary electrode and a reference electrode, and K2S2O8 solution was used as the detection solution to detect the electrochemiluminescence intensity signal. The electrochemiluminescence intensity was substituted into the standard curve to obtain the OTA concentration in the sample.

[0045] In order to construct an ECL sensor with higher sensitivity, stronger specificity, higher safety and wider detection range, two nanomaterials, g-C3N4 and NU-1000(Zr), were directly used as donors and acceptors of ECL-RET. Based on the changes in the luminescent signals generated by their ECL-RET, the OTA nanoantibody polymer Nb28-C4bpα was used as a specific recognition molecule to construct an ECL immunosensor for the detection of ochratoxin A, which provides a new strategy for food safety control and meets the urgent need for rapid detection of harmful substances in food.

[0046] The present invention uses OTA nanobody heptamer as a specific recognition molecule, g-C3N4 and NU-1000 (Zr) as the energy donor and receptor of ECL-RET, respectively, to construct a signal-amplified electrochemiluminescent immunosensor, uses OTA nanobody heptamer Nb28-C4bpα as a targeting molecule, and uses K2S2O8 as a co-reactant to construct an electrochemiluminescent sensor for detecting ochratoxin A with high specificity, high sensitivity, high safety, and a wide detection range. Compared with the prior art, the present invention has the following advantages:

[0047] 1. The present invention uses g-C3N4 and K2S2O8 as the luminescent system, replacing the traditional toxic luminescent reagent system, greatly improving the safety of the immunosensor. The lamellar structure of g-C3N4 provides more adsorption and covalent binding sites for OTA nanoantibodies, greatly improving the sensitivity of the immunosensor.

[0048] 2. The electrochemiluminescent immunosensor of the present invention is based on the energy resonance transfer of g-C3N4 and NU-1000 (Zr), and uses the OTA nanoantibody heptamer Nb28-C4bpα as a specific recognition molecule to achieve double signal amplification, greatly improving the sensitivity of the immunosensor.

[0049] 3. The present invention uses the OTA nanobody heptamer Nb28-C4bpα as a specific recognition element for the first time to construct an electrochemiluminescent immunosensor, providing a reference for the development of nanoantibodies in the field of electrochemiluminescent technology. It has been verified that Nb28-C4bpα is used as a specific recognition element, g-C3N4 and K2S2O8 luminescence system are selected, and based on the energy resonance transfer of g-C3N4 and NU-1000 (Zr), OTA is quantitatively analyzed by detecting electrochemiluminescence signals, with a detection range of 0.1pg / mL-500ng / mL, and a detection limit as low as 33fg / mL, which has the advantages of low detection limit and wide detection range.

[0050] 4. The electrochemiluminescence immunosensor of the present invention can achieve high specificity and high sensitivity detection of OTA, and can be used for detection applications of actual samples such as complex matrix coffee. The samples can be used for detection without complicated processing procedures, and the detection speed is fast, which solves the problem of complex sample pre-treatment and inability to quickly detect a large number of samples at the same time in traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 : Schematic diagram of the construction process of electrochemiluminescent immunosensor based on nanobody polymers and energy resonance transfer between g-C3N4 and NU-1000(Zr);

[0052] Figure 2 : Optimization experimental results of Nb28-C4bpα concentration, Nb28-C4bpα incubation time and OTA-Apt-NU-1000(Zr) incubation time during the construction of ECL immunosensor;

[0053] Figure 3 : ECL reaction at different concentrations of OTA from 0.1 pg / mL to 500 ng / mL in the immunosensor containing 20 mM K2S2O8 and 0.1 M K2S2O8 in 0.1 M PBS (A), calibration curve of ECL-RET immunosensor for detecting OTA (B);

[0054] Figure 4 : The stability test results of ECL immunosensor;

[0055] Figure 5 : Specificity experimental results of ECL immunosensor. DETAILED DESCRIPTION

[0056] The present invention is further described below by specific examples, which are only exemplary and not intended to limit the content thereof. Unless otherwise specified, the following terms or methods are not described in detail and are all prior art.

[0057] In the following examples, the OTA nanobody heptamer Nb28-C4bpα was obtained by the method described in the literature "Nanobody multimerization strategy to enhance the sensitivity of competitive ELISA for detection of ochratoxin A in coffee samples" (Bao KL, Liu X, Xu Q, et al, Nanobody multimerization strategy to enhance the sensitivity of competitive ELISA for detection of ochratoxin A in coffee samples[J]. Food Control, 2021, 127: 108167).

[0058] Example 1

[0059] (1) Carboxylation and activation of g-C3N4

[0060] 1 g of g-C3N4 powder was added to 100 mL of 5 M HNO3, refluxed at 125 °C for 24 h, and naturally cooled to room temperature. The refluxed product was centrifuged and washed with ultrapure water. The obtained solid product was vacuum dried at 35 °C for 12 h to obtain carboxylated g-C3N4.

[0061] 1.5 mL of a mixed solution of 0.4 M EDC and 0.1 M NHS was added to 3 mg of carboxylated g-C3N4, and the mixture was kept in a constant temperature shaker at 200 rpm for 6 h. The mixture was washed and centrifuged three times, and finally the g-C3N4 was redispersed in 1.5 mL of 0.001% chitosan acetate solution and stored at 4°C for later use.

[0062] (2) Preparation of OTA-Apt-NU-1000(Zr)

[0063] 3 mg NU-1000 (Zr) was added to a mixed solution of 1.5 mL 0.4 M EDC and 0.1 M NHS, and the mixture was kept at 200 rpm on a constant temperature shaker for 6 h, washed and centrifuged three times to obtain activated NU-1000 (Zr);

[0064] 1.5 mL 1×PBS solution was added to 3 mg activated NU-1000 (Zr) and 1 OD OTA aptamer (OTA-Apt) to dissolve, and the mixture was kept on a constant temperature shaker at 200 rpm for 12 h, and centrifuged and washed 3 times. Finally, 3 mg Apt-NU-1000 (Zr) was mixed with 1 mL 10 mg / mL OTA, and the shaker was kept at 200 rpm for 4 h, and centrifuged and washed 3 times. The prepared OTA-Apt-NU-1000 (Zr) was redispersed in 1.5 mL 0.01 M PBS and stored at 4°C for future use.

[0065] (3) Construction of OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE ECL immunosensor

[0066] Before modification of the glassy carbon electrode (GCE), the electrode surface was first polished with 0.3 μm and 0.05 μm alumina powders, then ultrasonically cleaned with ethanol and ultrapure water for 2 min, respectively, and dried with nitrogen.

[0067] 5 μL of 2 mg / mL g-C3N4 nanosheets were dropped onto the pretreated electrode surface, and the prepared g-C3N4 / GCE modified electrode was naturally dried in the air.

[0068] 5 μL of 10 mg / mL Nb28-C4bpα heptamer was dropped onto the electrode surface and incubated at 37°C for 1 h. The solution was gently rinsed with ultrapure water to remove Nb28-C4bpα not adsorbed on the electrode surface and dried at room temperature.

[0069] The modified electrode was blocked with 5 μL of 1% BSA solution at room temperature for 0.5 h to block nonspecific binding sites, and then gently rinsed with ultrapure water to remove unbound BSA molecules;

[0070] The obtained BSA / Nb28-C4bpα / g-C3N4 / GCE ECL immunosensor was incubated with standard concentrations of OTA at 37 °C for 2 h, gently rinsed with ultrapure water and then dried naturally;

[0071] Finally, 5 μL OTA-Apt-NU-1000(Zr) was dropped on the electrode and incubated at 37 °C for 1 h. The electrode surface was gently rinsed with ultrapure water and dried naturally.

[0072] (4) Condition Optimization

[0073] The concentration of Nb28-C4bpα and the incubation time of Nb28-C4bpα and OTA-Apt-NU-1000(Zr) were optimized in the immune recognition experiment:

[0074] Nb28-C4bpα solutions with concentrations of 2.5 μL / mL, 5 μL / mL, 7.5 μL / mL, 10 μL / mL, 12.5 μL / mL, 15 μL / mL, 17.5 μL / mL, and 20 μL / mL were prepared and set aside.

[0075] The g-C3N4 / GCE modified electrode was incubated in different concentrations of Nb28-C4bpα solutions at 37°C for 1 h. The other steps and conditions were the same, and finally the OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was formed. Then the modified electrode was used as the working electrode to construct a three-electrode system with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode). 20mM K2S2O8 in 0.1M KCl and 0.01M PBS solution was used as the detection solution to construct an ECL immunosensor.

[0076] The working electrode, auxiliary electrode and reference electrode were immersed in the K2S2O8 detection solution, and the electrochemiluminescence method was used to detect the ECL signal (see Figure 2 ).

[0077] Similarly, other conditions were the same, and the incubation time of Nb28-C4bpα or OTA-Apt-NU-1000(Zr) was changed (0-105 min), the working electrode, auxiliary electrode and reference electrode were immersed in the K2S2O8 detection solution, and the electrochemiluminescence method was used to detect the ECL signal with a scanning potential of -0.2 V to 0.7 V, a scanning rate of 100 V / s, a photomultiplier tube high voltage of 700 V, and an amplification level of 2 (see Figure 2 ).

[0078] (5) Determination of standard curve and detection limit

[0079] Prepare OTA standard solutions with concentrations of 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 500 ng / mL for use.

[0080] The BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was used as the working electrode and was first incubated with OTA standard solutions of different concentrations at 37°C for 1 h. Then, the OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was incubated with OTA-Apt-NU-1000(Zr) at 37°C for 1 h to form the OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode. Then, the modified electrode was used as the working electrode to construct a three-electrode system with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode). 20 mM K2S2O8 in 0.1 M KCl and 0.01 M PBS solutions were used as detection solutions to construct an ECL immunosensor.

[0081] The working electrode, auxiliary electrode and reference electrode were immersed in K2S2O8 detection solution, and the electrochemiluminescence method was used to scan the potential from -0.2V to 0.7V, the scanning rate was 100V / s, the photomultiplier tube high voltage was 700V, and the amplification level was 2 to obtain the ECL signal.

[0082] The reaction principle between the working electrode and the detection solution is as follows:

[0083] g-CN+e - →g-CN ·- (1)

[0084] S2O8 2- +e - →SO4 2- +SO4 ·- (2)

[0085] g-CN ·- +SO4 ·- →g-CN * +SO4 2- (3)

[0086] g-CN * →g-CN+light (4)

[0087] The working electrode OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE reacts with the co-reactant K2S2O8 under a high voltage of 700V to generate an ECL signal. Nb28-C4bpα and BSA are incubated on the g-C3N4 / GCE modified electrode respectively. The inhibitory effect of the protein will reduce the efficiency of electron transfer, thereby reducing the ECL signal. When OTA is incubated with the BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode, the specific binding of the antigen-antibody enhances the inhibitory effect on electron transfer. However, since OTA is a small molecule, the low concentration of OTA has no obvious effect on reducing the ECL signal. Finally, when OTA-Apt-NU-1000(Zr) was incubated with the OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode, OTA-Apt-NU-1000(Zr) bound to the remaining sites of Nb28-C4bpα, and the ECL-RET effect between NU-1000(Zr) and g-C3N4 further reduced the ECL signal, and the ECL signal intensity was proportional to the OTA concentration.

[0088] At 37 °C, the optimized Nb28-C4bpα concentration, Nb28-C4bpα incubation time and OTA-Apt-NU-1000(Zr) incubation time were used to modify different concentrations of OTA standard solutions (0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 500 ng / mL), and the ECL intensity of the OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was recorded as I. The logarithm of the OTA concentration was used as the abscissa and I as the ordinate to draw a standard curve (see Figure 3 ).

[0089] From the standard curve, we can see that the ECL signal is proportional to the logarithm of the OTA concentration, and the linear relationship is I = 1277.65logC OTA +6345.32(R 2 =0.9904), the detection limit of the ECL sensor was 33 fg / mL.

[0090] (6) Stability test

[0091] The BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was incubated with any three concentrations (0.0001, 0.1 and 10 ng / mL) of OTA, and then the modified electrode was incubated with OTA-Apt-NU-1000(Zr) at 37°C for 1 h to form the OTA-Apt-NU-1000(Zr) / (toxin) / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode. The modified electrode was then used as the working electrode to construct a three-electrode system with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode), and a 20 mM K2S2O8 solution in 0.1 M KCl and 0.01 M PBS was used as the detection solution to construct an ECL immunosensor.

[0092] The working electrode, auxiliary electrode and reference electrode were immersed in the K2S2O8 detection solution, and the electrochemiluminescence method was used to detect the ECL signal by scanning the potential from -0.2V to 0.7V, scanning rate of 100V / s, photomultiplier tube high voltage of 700V, and amplification level of 2. After continuous scanning of 5 cyclic potentials, relatively stable ECL curves under different concentrations of OTA were obtained. The ECL immunosensor modified with 500ng / mL OTA was continuously scanned for 14 cyclic potentials, and the relative standard deviation (RSD) of the ECL signal was only 6.176%. The above results show that the ECL immunosensor has good stability in detecting OTA.

[0093] (7) Specificity experiment

[0094] Prepare standard solutions of OTB, OTC, AFB1, FB1, ZEN, and DON with a concentration of 10 ng / mL respectively and set aside.

[0095] The BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was used as the working electrode and incubated with different mycotoxin standard solutions at 37°C for 1h. Then the modified electrode was incubated with OTA-Apt-NU-1000(Zr) at 37°C for 1h to form the OTA-Apt-NU-1000(Zr) / (toxin) / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode. The modified electrode was then used as the working electrode to construct a three-electrode system with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode). The ECL immunosensor was constructed using 20mM K2S2O8 in 0.1M KCl and 0.01M PBS as the detection solution.

[0096] The working electrode, auxiliary electrode and reference electrode were immersed in K2S2O8 detection solution, and the electrochemiluminescence method was used to scan the potential from -0.2V to 0.7V, the scanning rate was 100V / s, the photomultiplier tube high voltage was 700V, and the amplification level was 2 to obtain the ECL signal.

[0097] At 37°C, the working electrode containing 10 ng / mL of different toxins was detected using an ECL sensor, and the ECL signal was obtained using the OTA-Apt-NU-1000(Zr) / (toxin) / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode as the detection value. The ECL signal obtained by the OTA-Apt-NU-1000(Zr) / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode was used as the blank value, and the difference between the detection value and the blank value (ΔECL) was calculated. The different toxins were used as the horizontal axis and the ECL difference was used as the vertical axis to make a bar graph (see Figure 4 ). As can be seen from the figure, only OTA has an obvious ECL signal change, and the results show that the sensor of the present invention exhibits good specificity for OTA.

[0098] Example 2

[0099] Preparation of sample test solution: Grind coffee beans into powder, weigh 4 mg of coffee powder into a centrifuge tube, add 1 mL of ultrapure water, and ultrasonicate for 30 min. Add OTA of standard concentration into the coffee suspension to make the final concentration of OTA in the coffee suspension 0.01, 1, and 100 ng / mL, then centrifuge at 10,000 rpm for 10 min, take the supernatant, and obtain the actual sample test solution.

[0100] The BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode prepared in step (3) of Example 1 was incubated with coffee sample test solutions containing different concentrations of OTA at 37°C for 1 hour, and then the modified electrode was incubated with OTA-Apt-NU-1000(Zr) at 37°C for 1 hour to form an OTA-Apt-NU-1000(Zr) / OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode. The modified electrode was then used as a working electrode to construct an ECL immunosensor together with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode).

[0101] The working electrode, auxiliary electrode and reference electrode were immersed in the K2S2O8 detection solution, and the electrochemiluminescence method was used to detect the OTA recovery concentration and the average value and standard deviation of the recovery rate by scanning potential -0.2V to 0.7V, scanning rate 100V / s, photomultiplier tube high voltage 700V, and amplification level 2. Each sample was tested three times, and the average value and standard deviation of the OTA recovery concentration and recovery rate were calculated.

[0102]

[0103] Relative standard deviation (RSD) = standard deviation of recovery rate / average value of recovery rate × 100%.

[0104] The test results are shown in Table 1 below:

[0105]

[0106] It can be seen from the results in the above table that the method of the present invention has a high recovery rate, a wide detection range, and a low detection limit, and can meet the use requirements.

Claims

1. A method for preparing an electrochemiluminescent modified electrode for detecting ochratoxin A, characterized in that: Use the following steps: (1) Activating the carboxyl groups of carboxylated g-C3N4 with a mixed solution of EDC and NHS; (2) connecting NU-1000(Zr) nanoparticles and OTA via OTA aptamers to obtain OTA-Apt-NU-1000(Zr) composite nanoparticles; (3) dispersing the g-C3N4 nanosheets prepared in step (1) in water to obtain a suspension, dropping the suspension onto the surface of a glassy carbon electrode (GCE), and drying to obtain a g-C3N4 / GCE modified electrode; (4) adding a solution of ochratoxin A nanobody heptamer Nb28-C4bpα to the surface of the g-C3N4 / GCE modified electrode prepared in step (3), incubating and cleaning the electrode surface to obtain a Nb28-C4bpα / g-C3N4 / GCE modified electrode, and then incubating the modified electrode with a BSA solution to obtain a BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode; (5) adding OTA solution to the surface of the BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode prepared in step (4), incubating, washing and drying; (6) The OTA-Apt-NU-1000(Zr) composite nanoparticles prepared in step (2) are dispersed in water to obtain a suspension, and the OTA-Apt-NU-1000(Zr) suspension is added dropwise to the surface of the OTA / BSA / Nb28-C4bpα / g-C3N4 / GCE modified electrode prepared in step (5). After washing and drying, a modified electrode for detecting ochratoxin A is obtained.

2. The preparation method according to claim 1, characterized in that: In step (1), HNO3 is used to carboxylate g-C3N4; wherein the concentration of HNO3 is 5M, the dosage relationship of g-C3N4 and HNO3 is: 1g:50-150mL, and the concentrations of EDC and NHS are 0.4mol / L and 0.1mol / L, respectively.

3. The preparation method according to claim 1, characterized in that: The treatment method of step (1) is to add g-C3N4 nanosheets to HNO3, reflux at 125°C for 24 hours to carboxylate the g-C3N4, and then add the carboxylated g-C3N4 to a mixed solution of EDC and NHS, and shake at 37°C and 200 rpm for 6 hours in a constant temperature shaker to activate the carboxyl group.

4. The preparation method according to claim 1, characterized in that: In step (2), OTA-Apt-NU-1000 (Zr) composite nanoparticles are prepared by the following method: ① Dissolve H4PTPA, ZrOCl2·8H2O and benzoic acid in DMF and stir at 90°C for 12h to obtain NU-1000(Zr); ② Add the NU-1000 (Zr) prepared in step ① to the mixed solution of EDC and NHS, shake at 37°C constant temperature shaker at 200 rpm for 6 h to activate the carboxyl group, then mix the activated carboxyl group NU-1000 (Zr) and the OTA aptamer, and incubate at 37°C constant temperature shaker at 200 rpm for 12 h to obtain Apt-NU-1000 (Zr); ③ The Apt-NU-1000 (Zr) prepared in step ② was mixed with OTA, and the mixture was shaken at 200 rpm at 37° C. for 4 h to obtain OTA-Apt-NU-1000 (Zr) composite nanoparticles.

5. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the suspension is 1-2 mg / mL, and 5-10 μL of the suspension is dropped onto the electrode surface; in step (4), the concentration of the ochratoxin A nanobody heptamer Nb28-C4bpα solution is 5-15 μg / mL, and 5-10 μL of the suspension is dropped onto the electrode surface, incubated at 37° C. for 1-2 hours, and then washed, and then the modified electrode is incubated with 1-1.5wt% BSA solution at 37° C. for 0.5-1 hour; in step (5), 5-10 μL of OTA solution is dropped onto the electrode surface; in step (6), the concentration of OTA-Apt-NU-1000 (Zr) composite nanoparticles is 1-2 mg / mL, and 5-10 μL of the suspension is dropped onto the electrode surface.

6. The preparation method according to claim 1, characterized in that: The electrode is a glassy carbon electrode.

7. An electrode prepared by the method for preparing an electrochemiluminescent modified electrode for detecting ochratoxin A according to any one of claims 1 to 6.

8. An electrochemiluminescent immunosensor for detecting ochratoxin A, characterized in that: The method comprises a working electrode, wherein the working electrode is a modified electrode prepared by any preparation method of claims 1 to 6 or a modified electrode according to claim 7.

9. A method for detecting ochratoxin A, characterized in that: The modified electrode for detecting ochratoxin A according to claim 7 is used as a working electrode, a platinum electrode is used as an auxiliary electrode, a calomel electrode is used as a reference electrode, and a co-reactant potassium persulfate (K2S2O8) solution is used as a detection solution, and the content of ochratoxin A is jointly detected by cyclic voltammetry and electrochemiluminescence.

10. The method according to claim 9, characterized in that: The scanning potential for detecting the content of ochratoxin A is -0.2V to 0.7V, the scanning rate is 0.1V / s, and the concentration of the K2S2O8 solution is 20mmol / L.

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