A method for antibody immobilization in an avian reovirus detection sensor

Abstract

The present invention discloses a method for enhancing antibody immobilization in an avian reovirus detection sensor. The present invention provides a preparation method for an electrochemical immunosensor for detecting reovirus, comprising the following steps: (1) treating the electrode; (2) modifying the electrode with a nanocomposite; (3) pre-treating before antibody immobilization; (4) antibody immobilization; (5) working electrode and detection. The nanocomposite is a gold-platinum nanocomposite, and the pre-treatment is incubation with a cysteamine hydrochloride solution. It is experimentally proven that the detection linear range of the sensor of the present invention is from 0 to 10 5.82 EID 50 / mL, and the lowest detection limit is 10 0.46 EID 50 / mL. Compared with the method of directly immobilizing antibodies, the detection linear range is increased by 10 times, and it has high sensitivity and good specificity.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an avian reovirus detection sensor, and more particularly to a method for enhancing antibody fixation performance in the avian reovirus detection sensor. Background Art

[0002] Avian reovirus (ARV) is a member of the Reoviridae family and the Orthoreovirus genus. It can infect chickens, turkeys, ducks, geese and other wild birds, causing viral arthritis, respiratory diseases, intestinal diseases, central nervous system symptoms and immunosuppression. The main methods for detecting ARV are ELISA, PCR, semi-cased RT-PCR, nucleic acid probe technology. CN103235123A discloses a method for preparing a working electrode of an electrochemical immunosensor for detecting reovirus, which adopts a technical solution of first fixing the probe of the target substance in the sample to be tested, with the purpose of extending the effective storage time of the probe. In order to ensure the sensitivity and specificity of the sensor, the sample pre-treatment time is relatively long, thus sacrificing the advantage of rapid detection in detection time.

[0003] Electrochemical immunosensor is a new detection technology that combines electrochemical analysis method and immunoassay method. The principle is: through physical and chemical methods, the sensitive element (eg antibody) that can identify the target is fixed to the surface of the working electrode, and then specifically binds to the target to be detected. The generated biochemical energy is converted into electrical energy and output in the form of electrical signals. The detection of the target is achieved through the detection and analysis of the electrical signals. At present, most of the research on electrochemical immunosensors is still in the basic experimental stage. Among the "electrochemical immunoassay", the only Class II medical device registered by Zhongshan Hexin Biotechnology Co., Ltd. has been approved for sale by the Food and Drug Administration: Procalcitonin Detection Kit (Electrochemical Immunoassay) (Registration Certificate No.: Guangdong Medical Device No. 20202401983). The main reason is that electrochemical immunosensors cannot meet the requirements of sensitivity, stability and detection range in IVD clinical testing.

[0004] Nanomaterials have a large specific surface area, so a large number of studies have shown that using nanomaterials to modify electrodes can effectively improve the loading capacity of the electrode surface, thereby expanding the detection linear range and improving the detection sensitivity. At present, researchers are mainly concerned about how to develop various new nanomaterials and combine various signal amplification technologies to continuously improve the performance of electrochemical immunosensors. However, there is still a lack of systematic research on the application of nanomaterials in electrochemical immunosensors, and it is not yet possible to fully realize the rapid and sensitive detection of complex biological samples.

[0005] Important parameters of the electrochemical immunosensor, such as the linear range and sensitivity, are jointly determined by factors such as the modifiers on the electrode surface, the antibody immobilization method, the electrode surface loading capacity, and the signal generation method. Among them, more importantly, it is affected by the electrode modification material and the antibody immobilization method on the electrode surface. In particular, the antibody immobilization method should receive sufficient attention. However, there is still relatively little systematic research on the influence of the antibody immobilization method on the sensor.

[0006] Common antibody types include IgG, IgA, and IgM, all of which are Y-shaped proteins. Among them, IgG is widely used in immunosensors. It is a serum protein with a molecular weight of about 100 kDa, and is composed of a Y-shaped structure formed by disulfide bonds connecting two light chains and two heavy chains. The Y-shaped head containing the active site for antigen binding is composed of amino groups, while the tail contains carboxyl groups. Currently, the commonly used methods for antibody immobilization are as follows: ① Utilize the specific binding of streptavidin-biotin to immobilize biotinylated antibodies on the surface of electrodes modified with streptavidin; ② Coating the electrode with carboxymethylated dextran, and then using the glutaraldehyde (Glu) cross-linking method, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) chemical method, or direct method to immobilize antibodies. The purpose of the present invention is to find a preparation method for electrochemical immunosensors that is superior to the existing antibody immobilization methods, thereby improving the linear range and sensitivity of the sensor. Summary of the Invention

[0007] The purpose of the present invention is to at least partially overcome the defects of the prior art and provide a method that is beneficial to enhancing the antibody immobilization performance in the avian reovirus detection sensor.

[0008] The purpose of the present invention is also to provide a method that is beneficial to enhancing the linear range and sensitivity of the avian reovirus detection sensor.

[0009] To achieve the above purpose or one of the purposes, the present invention provides the following technical solutions:

[0010] A preparation method of an electrochemical immunosensor, and the prepared sensor is used to detect avian reovirus. Among them, the preparation of the working electrode of the sensor includes a pretreatment step before antibody immobilization, and is prepared according to the following steps: Treating the electrode: including polishing, washing with water, ultrasonic cleaning, soaking, scanning with cyclic voltammetry until stable and then drying with N2; Modifying the electrode with a nanocomposite: dropping a nanocomposite made of a noble metal on the surface of the electrode and naturally drying it at 4°C. The noble metal is gold, platinum or gold + platinum; Pretreatment before antibody immobilization: dropping 10 μL of incubation solution on the surface of the nanocomposite-modified electrode, and rinsing it with PBS washing solution at least 3 times after incubation; Antibody immobilization: then dropping the monoclonal antibody ARV / MAb of avian reovirus, and carrying out a fixation reaction at 4°C for 8 h under airtight conditions; Blocking and detection: after the antibody fixation reaction, rinse with PBS washing solution, block with BSA solution, and rinse again with PBS washing solution to obtain the working electrode, directly drop the sample to be detected, and detect it by electrochemical scanning.

[0011] According to a preferred embodiment of the present invention, in the preparation method of the electrochemical immunosensor, the electrode is a glassy carbon electrode, and the nanocomposite is preferably a graphene-chitosan-gold-platinum nanoparticle G-Chi-Au / Pt nanocomposite. The preparation method of the composite includes: Graphene G: Graphite powder is oxidized at high temperature, ultrasonically treated, reduced with sodium borohydride at high temperature, and washed to obtain graphene G; Chitosan solution Chi: Chitosan is dissolved in an acetic acid solution, and magnetically stirred at 25°C for 1 h to be mixed evenly to obtain a chitosan solution Chi; Among them, the acetic acid solution is a mixed solution of glacial acetic acid and water with a volume ratio of 1.0%, and the concentration of the obtained chitosan solution is 0.1 wt%; Graphene-chitosan suspension G-Chi: Mix graphene with the above chitosan solution and ultrasonically treat it to obtain a graphene-chitosan suspension G-Chi; Among them, the ultrasonic power is 250 W and the continuous ultrasonic treatment is 2 h, and the feeding ratio of graphene G to chitosan solution Chi is 1 mg: 1 mL; Nanocomposite: Adding chloroauric acid solution and potassium tetrachloroplatinate solution to the above graphene-chitosan suspension G-Chi, stirring at room temperature for 3 h, and then continuing the reaction in a water bath at 80°C for 1 h to obtain a graphene-chitosan-gold-platinum nanocomposite G-Chi-Au / Pt, that is, a gold-platinum nanocomposite. The concentrations of the chloroauric acid solution and the potassium tetrachloroplatinate solution are both 10 mmol / L, and the dosages are both 1 mL. The dosage of the graphene-chitosan suspension G-Chi is 20 mL. Among them, the feeding ratio of gold Au, platinum Pt, and graphene G is 1 mmol: 1 mmol: 2 g.

[0012] According to a preferred embodiment of the present invention, the incubation solution is a cysteamine hydrochloride solution, and the concentration of the cysteamine hydrochloride solution is 2 mg / mL. The incubation is carried out under light-shielded conditions for 4 h.

[0013] According to a preferred embodiment of the present invention, the incubation solution is a glutaraldehyde solution with a concentration of 5%, and the incubation is carried out at room temperature for 3 h.

[0014] According to a preferred embodiment of the present invention, when the incubation solution is EDC / NHS, the nano - composite modified electrode is first placed in a NaOH solution and subjected to a constant potential of +1.3 V for 40 s, and then the incubation solution is dropped on its surface; the concentration of the NaOH solution is 0.5 mol / L, the incubation solution is a MES solution containing EDC and NHS, the concentration of EDC is 50 mmol / L, the concentration of NHS is 30 mmol / L, and the pH value of the MES solution is 4.7, and the incubation is carried out at room temperature for 1 h.

[0015] According to a preferred embodiment of the present invention, the scanning detection is carried out in an electrolyte solution, which is a PBS buffer solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 M KCl, and the pH value of the PBS buffer solution is 7.0.

[0016] The present invention claims an electrochemical immunosensor for detecting avian reovirus. The sensor is a three - electrode system, including a working electrode, an auxiliary electrode and a reference electrode. The working electrode is prepared according to the preparation method of the above - mentioned electrochemical immunosensor, the auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.

[0017] The present invention claims a detection kit for avian reovirus, including an electrochemical immunosensor for detecting avian reovirus. The detection linear range of the kit for detecting avian reovirus is 0 - 10 5.82 EID 50 / mL, and the lowest detection limit is 10 0.46 EID 50 / mL.

[0018] According to a preferred embodiment of the present invention, the kit further includes a PBS washing solution, an electrolyte solution, an auxiliary electrode and a reference electrode. The auxiliary electrode is a platinum wire electrode, the reference electrode is a saturated calomel electrode, the electrolyte solution is a PBS buffer solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 M KCl, and the pH value of the PBS buffer solution is 7.0.

[0019] The present invention claims an application method for detecting reovirus, including the preparation method of an electrochemical immunosensor and its application in the preparation of products for detecting reovirus.

[0020] The beneficial effects of the present invention:

[0021] The present invention is committed to continuously delving into the field of electrochemical sensor platforms. Based on the disclosure of CN103235123A, a complete and systematic study on the same detection object (reovirus) is conducted in the field of electrochemical immunosensors. The research on nanomaterials and antibody immobilization methods is further improved to achieve the purpose of rapidly and sensitively detecting complex and variable virus-like samples, and unexpected technical effects are obtained.

[0022] 1) The present invention prepares a gold-platinum nanocomposite. The graphene-chitosan-gold / platinum nanoparticle G-Chi-Au / Pt nanocomposite is a bimetallic nanocomposite containing both gold and platinum. Using graphene-chitosan as the substrate, gold and platinum ions are adsorbed by Chi through chelation in the solution and in-situ reduced to metal nanoparticles by Chi after heating, and then combined on the surface of G-Chi to form the G-Chi-Au / Pt nanocomposite. On the one hand, it can increase the active area of the electrode surface, which is beneficial for further binding modification on the gold-platinum bimetallic surface, and the formed immune complex after immobilization has higher and more stable stability; on the other hand, the gold-platinum bimetallic has a strong charge carrier mobility, which improves the electron transfer ability on the electrode surface, has relatively better conductivity and also helps to increase the signal; furthermore, the catalytic performance of the gold-platinum bimetallic also helps to improve the sensitivity of the sensor and broaden the linear range, resulting in a lower detection limit.

[0023] 2) The present invention not only optimizes the gold-platinum nanocomposite but also further optimizes the preparation method of the nanocomposite, especially the gold-platinum bimetallic nanocomposite. Mainly, chloroauric acid solution and potassium tetrachloroplatinate solution are added to the graphene-chitosan suspension G-Chi, and the gold-platinum bimetallic particles are covalently bonded to the surface of graphene-chitosan G-Chi after stirring and heating. This method belongs to a chemical preparation method, with simple and convenient preparation steps and does not require expensive instrument equipment; compared with physical methods such as crushing method, gas phase method, arc method and irradiation, the cost of this preparation method is lower and the yield of the prepared nanoparticles is higher; compared with other chemical methods such as seed growth method, template method, microemulsion method and microwave synthesis method, the prepared graphene-chitosan-gold / platinum nanoparticle G-Chi-Au / Pt nanocomposite, that is, the gold-platinum nanocomposite has better thermal stability, the particle size distribution of the nanoparticles is 1.5 - 5.2 nm, the dispersion degree is relatively high, it is easily soluble in common solvents and is not prone to aggregation or decomposition. Therefore, it is easy to use it as a complex solution for electrode modification for subsequent modification or functionalization of organic molecules or compounds.

[0024] 3) The present invention further conducts a systematic study on the optimized gold-platinum nanocomposite and the nanocomposites (single-metal gold nanocomposite, platinum nanocomposite) used for traditional electrode modification and characterization. Unexpectedly, it is found that, compared with the commonly used single-metal nanocomposites, the conductivity of the optimized dual-metal gold-platinum nanocomposite is relatively better. Through systematic analysis of the reasons, it may be that, compared with single metals, dual metals have a larger active surface and stronger charge carrier mobility. Therefore, it is beneficial to improve the sensitivity and electrochemical catalytic activity of the sensor.

[0025] 4) The present invention uses a cysteamine hydrochloride solution (CH) as the incubation solution for pre-incubation pretreatment before antibody immobilization, and then antibody immobilization is carried out. The alternating current impedance diagram of the modified electrode shows that its Ret value is relatively large; the Ret value of the working electrode GE-G-Chi-Au / PtNP-CH-ARV / MAb-BSA-ARV obtained after blocking with BSA is also relatively the largest; through the CH incubation method, more proteins (such as BSA and antibodies, etc.) are successfully immobilized on the electrode surface. The specific reason may be that: the CH incubation method can enrich the mercapto and amino groups on the electrode surface, providing more active sites for the binding of proteins (such as BSA and antibodies, etc.); further, after the CH incubation method successfully immobilizes a relatively large amount of antibody (such as monoclonal antibody ARV / MAb) on the electrode surface, the corresponding working electrode obtained by blocking with BSA after antibody immobilization by this method can also bind a relatively large amount of ARV virus particles.

[0026] 5) The present invention further conducts a systematic comparative study on the optimized CH incubation method and traditional incubation methods (EDC / NHS incubation method, Glu incubation method), and systematically analyzes the advantages and reasons of each method: ① Using the MES solution of EDC and NHS (abbreviated as EDC / NHS) as the incubation solution, applying a constant potential in an alkaline solution (NaOH solution) before incubation, and then fixing the antibody after EDC / NHS incubation, more proteins (such as BSA and antibodies, etc.) can also be fixed and adsorbed. It may be that the EDC / NHS incubation method can enrich the carboxyl and amino groups on the electrode surface, providing more active sites for the binding of proteins (such as BSA and antibodies, etc.). Compared with the direct antibody fixation method, the amount of proteins (such as BSA and antibodies, etc.) fixed and adsorbed on the electrode surface is a little more, but compared with the CH incubation method, the amount of proteins (such as BSA and antibodies, etc.) fixed and adsorbed on the electrode surface is less, and the preparation process is more complex. Further, for the working electrode obtained by incubating and pretreating with the EDC / NHS method, fixing the antibody and then blocking with BSA, the result shows that compared with the working electrode obtained by the direct antibody fixation method, it can only bind a relatively small amount of ARV virus particles. The possible reason for the analysis is that the EDC / NHS method of incubation pretreatment connects the amino group at the Fab end of the monoclonal antibody ARV / MAb to the carboxyl group on the electrode surface, and the binding site of the monoclonal antibody ARV / MAb to the ARV virus particle is also at the Fab end. Therefore, after fixing the monoclonal antibody ARV / MAb by the EDC / NHS method, a certain degree of steric hindrance will be caused at the Fab end, hindering the binding of the monoclonal antibody ARV / MAb to the ARV virus particle; in the direct antibody fixation method, the carboxyl group at the Fc end of the monoclonal antibody ARV / MAb is connected to the surface of the modified electrode, or the thiol group on the side chain of the monoclonal antibody ARV / MAb is connected to the surface of Au / PtNP. Whether it is the carboxyl group at the Fc end or the thiol group on the side chain, they are both far from the binding site of the monoclonal antibody ARV / MAb to ARV at the Fab end, with less steric hindrance; ② Using the traditional glutaraldehyde solution (Glu) incubation method for pretreatment and then fixing the antibody. Since the Glu terminal contains two aldehyde groups, compared with directly fixing the antibody, although it can provide active sites for proteins (such as BSA and antibodies, etc.) to a certain extent, due to glutaraldehyde being a bifunctional cross-linking agent and self-linking phenomenon occurring, the amount of proteins fixed and adsorbed is relatively lower than that of the CH incubation method.

[0027] 5) The present invention systematically pairs and combines the bimetallic nanocomposite of gold and platinum with the pretreatment method before antibody fixation, obtaining an optimized combination that is beneficial to improving the sensitivity, stability, and detection range of the avian reovirus detection sensor. Finally, after modifying and characterizing the electrode with the gold-platinum nanocomposite and pretreating before antibody fixation using the CH incubation method, an electrochemical immunosensor for detecting avian reovirus is finally optimized and prepared, and the detection linear range is 0 - 10 5.82EID 50 / mL ARV, the lowest detection limit is 10 0.46 EID 50 / mL ARV. Compared with the antibody direct immobilization method (the detection linear range is 0 - 10 4.82 EID 50 / mL ARV), the detection linear range is increased by 10 times.

[0028] 6) The electrochemical immunosensor optimized in the present invention for detecting avian reovirus belongs to the direct method in electrochemical immunosensors, combines the advantages of immunoassay and electrochemical detection, adopts a three - electrode system. After the working electrode is modified with nanomaterials, the capture antibody is immobilized to form a biosensitive film. When the antigen in the liquid to be tested binds to the immobilized antibody, as a signal label, it will increase the resistance on the electrode surface and reduce the electron transfer rate. The change in the electrochemical properties of the electrode surface is used to provide an electrochemical signal, and the degree of change is related to the concentration of the antigen to be tested. The concentration information of the antigen to be tested (such as the avian reovirus described in the present invention) can also be obtained through electrochemical detection. Brief Description of the Drawings

[0029] Figure 1 are the electrochemical impedance spectra and bar graphs of Ret values of electrodes modified with different nanocomposites. A is the electrochemical impedance spectrum, B is the bar graph of Ret values. Among them, a is GE; b is GE - G - Chi - AuNP; c is GE - G - Chi - PtNP; d is GE - G - Chi - Au / PtNP;

[0030] Figure 2 are the electrochemical impedance spectra of monoclonal antibody immobilization after different pretreatments;

[0031] Figure 3 are the electrochemical impedance spectra of the protein - immobilized control group after different pretreatments;

[0032] Figure 4 are the electrochemical impedance spectra of the working electrodes prepared after different pretreatments;

[0033] Figure 5 are the electrochemical impedance spectra of the working electrodes detecting positive test solutions after different pretreatments;

[0034] Figure 6 are the electrochemical impedance spectra of the protein - immobilized control group detecting positive test solutions after different pretreatments;

[0035] Figure 7 are Figures 2 - 6 bar graphs of the Ret values corresponding to the electrochemical impedance spectra;

[0036] Figure 8The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the Glu incubation method with the ARV content;

[0037] Figure 9 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the EDC / NHS incubation method with the ARV content;

[0038] Figure 10 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the antibody direct immobilization method with the ARV content;

[0039] Figure 11 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the CH incubation method with the ARV content;

[0040] Figure 12 The bar chart of the Ret values in the sensor specificity experiments after 4 groups of different pretreatments; where A represents the Glu incubation method, B represents the EDC / NHS incubation method, C represents the antibody direct immobilization method, and D represents the CH incubation method;

[0041] Figure 13 The bar chart of the Ret values in the 5-batch preparation process stability test of the sensors after 4 groups of different pretreatments; where A represents the Glu incubation method, B represents the EDC / NHS incubation method, C represents the antibody direct immobilization method, and D represents the CH incubation method;

[0042] Figure 14 The bar chart of the Ret values in the thermal stability test of 4 groups of sensors with different pretreatments prepared in the same batch; where A represents the Glu incubation method, B represents the EDC / NHS incubation method, C represents the antibody direct immobilization method, and D represents the CH incubation method; The abscissa represents 1 week, 2 weeks, 3 weeks, and 4 weeks of storage at 4°C respectively. Detailed implementation manners

[0043] The following describes the exemplary embodiments of the present invention in detail in combination with practical applications. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the disclosed embodiments. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. However, obviously, one or more embodiments can also be implemented without these specific details. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0044] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified. Among them, HAuCl4, K2PtCl4 and bovine serum albumin (BSA) were purchased from Sigma; KMnO4, K4Fe(CN)6, K3Fe(CN)6, H2SO4, CH3CH2OH, chitosan (Chi), graphite powder (G), vitamin C, etc. are all domestic analytical pure reagents, and the experimental water is secondary deionized water; the anti-ARV monoclonal antibody (ARV / MAb) was prepared by the Guangxi Veterinary Research Institute (Reference: Xie Zhiqin, Xie Zhixun, Liu Jiabo, Pang Yaoshan, Deng Xianwen, Xie Liji, Fan Qing. Preparation of monoclonal antibodies against avian reovirus strain S1733 and establishment of sandwich ELISA detection method. China Animal Husbandry & Veterinary Medicine. 2012, 39(11): 47-51).

[0045] The instruments used in the following examples are as follows: A 4000A electrochemical workstation (Princeton Applied Research) was used for electrochemical measurements with a conventional three-electrode electrochemical cell. The modified gold electrode served as the working electrode, the saturated calomel electrode (SCE) served as the reference electrode, and the platinum electrode served as the auxiliary electrode. The immunosensor consists of a three-electrode system composed of a working electrode, a reference electrode, and an auxiliary electrode, and the immunosensor is connected to the electrochemical workstation through a wire.

[0046] For the molecular biology experimental methods not specifically described in the following examples, they were all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kit and product instructions.

[0047] The test samples involved in the content of the present invention are mainly test virus solutions: avian reovirus (ARV), avian influenza virus subtype H3 (AIV H3), avian influenza virus subtype H9 (AIV H9), Newcastle disease virus (NDV), laryngotracheitis virus (LTV), infectious bronchitis virus (IBV), and infectious bursal disease virus (IBDV) are all stored in the Biotechnology Laboratory of the Guangxi Veterinary Research Institute and belong to the virus preservation solution in the field of microorganisms; the test samples involved in the application of the present invention are in vitro inanimate biological samples, only involving biological samples such as tissues, body fluids or excreta collected from the human body or animal body, including blood samples (whole blood / serum / plasma) and body fluid samples (urine / feces / cerebrospinal fluid / serous cavity effusion / semen / prostatic fluid / vaginal secretion / gastric juice / duodenal drainage fluid and bile / sputum) that have been separated from the living human body or animal body, and do not belong to the living human body or animal body; that is, the methods and applications of the present invention are not directly livingThe present invention is not directed to the human or animal body as the object of implementation, nor does it include the steps of comparing the detected biological information with reference data and drawing specific diagnostic conclusions based on the biological detection information. The direct purpose of the detection is not to obtain a disease diagnosis result or health status, but rather to be industrially utilized for the production of an in vitro detection kit or related products for detecting avian reovirus. Therefore, the present invention does not belong to a disease diagnosis method and meets the basic requirements of the Patent Law for patent protection objects.

[0048] The present invention provides a method for preparing an electrochemical immunosensor for detecting avian reovirus, and the method includes the following steps: (1) treating the electrode; (2) modifying the electrode with a nanocomposite; (3) pre-treating before antibody immobilization; (4) antibody immobilization; (5) working electrode and detection. In the preparation method of the present invention, a pre-treatment step before antibody immobilization is added in the preparation of the working electrode, and the nanocomposite is a gold-platinum bimetallic nanocomposite, and the pre-treatment is incubation with a cysteamine hydrochloride solution. It has been experimentally proven that the detection linear range of the sensor of the present invention is 0-10 5.82 EID 50 / mL, and the lowest detection limit is 10 0.46 EID 50 / mL. Compared with the method of directly immobilizing antibodies, the detection linear range is increased by 10 times, and it has high sensitivity and good specificity.

[0049] Example 1: Preparation of gold-platinum nanocomposite and positive test solution

[0050] 1) Preparation of graphene (G)

[0051] Graphene oxide is prepared by improving the Hummer method, and the specific experimental steps are as follows: Under ice-water bath conditions, add 1 g of graphite powder, 2.5 g of potassium nitrate (KNO3), and 100 mL of H2SO4 to a beaker. After stirring evenly, slowly add 5 g of potassium permanganate (KMnO4). Then place the beaker in a 35 °C water bath and react for 2 h. Gradually add 100 mL of deionized water, and continue to react at 95 °C for 1 h. When it is observed that the mixture changes from brownish-yellow to bright yellow, cool it to room temperature (25 °C), then add 300 mL of distilled water for dilution, and add 30% hydrogen peroxide (H2O2) by mass fraction to neutralize the unreacted potassium permanganate. Then wash it first with a 0.5 mol / L hydrochloric acid (HCl) aqueous solution, and then wash it repeatedly by centrifugation with water 5 times, and dry it under vacuum to obtain graphite oxide.

[0052] Weigh 10 mg of the above-prepared graphite oxide and place it in a beaker, add 100 mL of water, and ultrasonicate (continuous ultrasonication at 250 W) for 1 h to obtain graphene oxide (GO).

[0053] Using sodium borohydride (NaBH4) as the reducing agent, graphene (G) was obtained under the condition of 95 °C as follows: Under magnetic stirring, 1 mL of 1 mg / mL sodium borohydride (NaBH4) aqueous solution was added dropwise to 10 mL of 0.1 mg / mL graphene oxide (GO). Then the temperature was raised to 95 °C and reacted for 30 min. After natural cooling at room temperature (25 °C), it was centrifuged and washed three times with secondary deionized water, and vacuum dried at 85 °C for 24 hours to obtain graphene (G).

[0054] Note: The graphene prepared above can be directly used in later experiments, or the graphene purchased commercially can be directly used to prepare the following composites.

[0055] 2) Preparation of chitosan solution (Chi)

[0056] 0.3 g of chitosan (Chi), the molecular formula of chitosan is: (C6H11NO4)n, MV: ~1.5×10 5 , Viscosity: ~100 Mpa.S) was added to 300 mL of 1.0% (volume percentage V / V) acetic acid solution (glacial acetic acid dissolved in water), and magnetically stirred at room temperature (25 °C) for 1 h to obtain 0.1 wt% (mass percentage) chitosan solution (Chi).

[0057] 3) Preparation of graphene-chitosan suspension (G-Chi)

[0058] 300 mg of the graphene (G) obtained in 1) above was added to 300 mL of 0.1 wt% (mass percentage) chitosan solution (Chi), and continuously ultrasonicated (ultrasonic power 250 W) for 2 h to obtain a stable 1 mg / mL graphene-chitosan suspension (G-Chi).

[0059] 4) Preparation of gold-platinum nanocomposite

[0060] Gold-platinum nanocomposite: 1 mL of 10 mmol / L chloroauric acid solution (HAuCl4) and 1 mL of 10 mmol / L potassium tetrachloroplatinate solution (K2PtCl4) were simultaneously added to the 20 ml graphene-chitosan suspension (G-Chi) prepared above, stirred at room temperature (25 °C) for 3 h, and then heated in a water bath to 80 °C and reacted for another 1 h to obtain graphene-chitosan-gold-platinum nanocomposite G-Chi-Au / Pt. Among them, the feeding ratio of gold Au, platinum Pt, and graphene G is 1 mmol: 1 mmol: 2 g. This graphene-chitosan-gold-platinum nanocomposite G-Chi-Au / Pt is a bimetallic nanocomposite jointly prepared by two noble metals, gold and platinum, and is also simply referred to as gold-platinum nanocomposite.

[0061] Note: Graphene-chitosan-gold-platinum nanocomposites G-Chi-Au / Pt with different feeding ratios can also be prepared by adding different volumes of HAuCl4 (10 mmol / L) and K2PtCl4 (10 mmol / L) solutions to the G-Chi (20 mL) suspension.

[0062] Nanocomposite control group: Nanocomposites containing one noble metal, gold or platinum, were prepared respectively. The preparation process is as follows:

[0063] ① Gold nanocomposite: Take 1 mL of 10 mmol / L chloroauric acid solution (HAuCl4) and add it to the prepared 20 mL graphene-chitosan suspension (G-Chi). Stir at room temperature (25 °C) for 3 h, then heat in a water bath to 80 °C and continue to react for 1 h to obtain graphene-chitosan-gold nanocomposite G-Chi-Au, abbreviated as gold nanocomposite.

[0064] ② Platinum nanocomposite: Take 1 mL of 10 mmol / L potassium tetrachloroplatinate solution (K2PtCl4) and add it to the prepared 20 mL graphene-chitosan suspension (G-Chi). Stir at room temperature (25 °C) for 3 h, then heat in a water bath to 80 °C and continue to react for 1 h to obtain graphene-chitosan-platinum nanocomposite G-Chi-Pt, abbreviated as platinum nanocomposite.

[0065] 5) Preparation of positive test solution

[0066] Preparation of positive test solution containing avian reovirus ARV: Dilute avian reovirus S1133 (purchased from the China Institute of Veterinary Drug Control, product catalog number: AV2311) with a 0.1 mol / L PBS (pH = 7.4) buffer solution to 10 6.82 EID 50 / mL to obtain a positive test solution of 10 6.82 EID 50 / mL.

[0067] 6) Electrolyte

[0068] The electrolyte used for electrochemical impedance detection is a PBS solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 M KCl, and the pH of the PBS solution is 7.0.

[0069] Example 2. Preparation of avian reovirus electrochemical immunosensor

[0070] 1) Treat the electrode:

[0071] Treat the glassy carbon electrode (Gold electrocle, GE), also known as the gold electrode After polishing with 0.05 μm Al2O3 polishing powder until it becomes mirror-like, it is washed clean with distilled water, and then ultrasonically cleaned in water, absolute ethanol, and water for 5 min successively, and dried with N2. Then, it is scanned by cyclic voltammetry in a 0.5 mol / L H2SO4 solution (deoxygenated by introducing N2 for 15 min before scanning). The scanning rate is 50 mV / s, the voltage range is -0.3 to +1.5 V, and continuous scanning is carried out until the cyclic voltammogram is stable. Then, it is taken out, washed with distilled water, and dried with N2 for standby to obtain the unmodified gold electrode GE.

[0072] 2) Nanocomposite modified electrode:

[0073] Modification with gold-platinum nanocomposite: Use a pipette to take 8 μL of graphene-chitosan-gold-platinum nanocomposite G-Chi-Au / Pt and coat it on the surface of GE, and let it dry naturally at 4 °C to obtain the modified electrode GE-G-Chi-Au / Pt containing the bimetallic gold-platinum nanocomposite.

[0074] Modification control group: Referring to the gold-platinum nanocomposite modification method, the prepared gold nanocomposite and platinum nanocomposite are used to modify the electrode respectively to obtain the modified electrodes GE-G-Chi-Au and GE-G-Chi-Pt of the control group.

[0075] 3) Pretreatment before antibody immobilization

[0076] ① Incubation with glutaraldehyde solution (Glu): Drop 10 μL of 5% glutaraldehyde solution on the surface of the above-mentioned nanocomposite modified electrode GE-G-Chi-Au / Pt, incubate at room temperature for 3 h, and wash 3 times with PBS washing solution to remove the unbound glutaraldehyde to obtain the pretreated modified electrode GE-G-Chi-Au / Pt-Glu.

[0077] ②EDC / NHS incubation: First, the above-mentioned nano-composite modified electrode GE-G-Chi-Au / Pt was placed in a 0.5 mol / L NaOH solution and anodic oxidation was carried out at a constant potential of +1.3 V for 40 s to increase the number of carboxyl groups on the surface of the modified electrode GE-G-Chi-Au / Pt. Then, 10 μL of MES solution containing 50 mmol / L of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (abbreviated as EDC) and 30 mmol / L of N-hydroxysuccinimide (abbreviated as NHS) (abbreviated as EDC / NHS) was dropped onto the anodized modified electrode GE-G-Chi-Au / Pt. The pH value of the MES solution was 4.7, and it was incubated with the incubation solution EDC / NHS at room temperature for 1 h. It was rinsed 3 times with PBS wash solution to remove unbound EDC and NHS. At this time, the carboxyl groups on the surface of the modified electrode GE-G-Chi-Au / Pt were converted into amine-reactive NHS esters, and the pretreated modified electrode GE-G-Chi-Au / Pt-EDC / NHS was obtained.

[0078] ③Cysteamine hydrochloride solution (CH) incubation: 10 μL of 2 mg / mL cysteamine hydrochloride solution was dropped onto the surface of the above-mentioned nano-composite modified electrode GE-G-Chi-Au / Pt, incubated in the dark for 4 h, and rinsed 3 times with PBS wash solution to remove unbound cysteinate, and the pretreated modified electrode GE-G-Chi-Au / Pt-CH was obtained.

[0079] ④Negative control: Also known as the antibody direct immobilization method, it does not include the pretreatment step before antibody immobilization, that is, no incubation solution is added for pretreatment, and it is still the above-mentioned nano-composite modified electrode GE-G-Chi-Au / Pt.

[0080] 4) Protein immobilization (monoclonal antibody + BSA):

[0081] On the surfaces of the above 4 groups of pretreated modified electrodes GE-G-Chi-Au / Pt-Glu, GE-G-Chi-Au / Pt-EDC / NHS, GE-G-Chi-Au / Pt-CH and negative control (i.e., the modified electrode GE-G-Chi-Au / Pt) obtained by incubating with different incubation solutions, 8 μL of 100 μg / mL avian reovirus monoclonal antibody (hereinafter referred to as ARV / MAb) was dropped, placed in a sealed small box to prevent evaporation, and allowed to act at 4 °C for 8 h to complete antibody immobilization. It was rinsed 3 times with PBS to obtain 4 groups of electrodes GE-G-Chi-Au / Pt-Glu-ARV / MAb, GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb, GE-G-Chi-Au / Pt-CH-ARV / MAb and GE-G-Chi-Au / Pt-ARV / MAb after monoclonal antibody immobilization respectively.

[0082] Protein immobilization control group: To verify the ability of the above four different pretreatment methods to immobilize proteins, in the protein immobilization control group, BSA was used instead of the monoclonal antibody ARV / MAb. That is, on the surfaces of the pretreated modified electrodes and the negative control obtained by incubating in the above four groups with different incubation solutions, 8 μL of a solution containing 1 wt% BSA was added dropwise, placed in a sealed small box to prevent evaporation, and allowed to act at 4 °C for 8 h, thus completing the immobilization of BSA protein. Four groups of electrodes after BSA protein immobilization, namely GE-G-Chi-Au / Pt-Glu-BSA, GE-G-Chi-Au / Pt-EDC / NHS-BSA, GE-G-Chi-Au / Pt-CH-BSA, and GE-G-Chi-Au / Pt-BSA, were obtained respectively.

[0083] 5) Working electrode after BSA blocking:

[0084] The electrodes obtained by immobilizing the above four groups of avian reovirus monoclonal antibodies were respectively immersed in 200 μL of a solution containing 1 wt% BSA and allowed to act at a constant temperature of 37 °C for 1 h to block the non-specific active sites on the electrode surface; then each was rinsed 3 times with PBS washing solution to remove the unbound BSA, and four groups of working electrodes after blocking, namely GE-G-Chi-Au / Pt-Glu-ARV / MAb-BSA, GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb-BSA, GE-G-Chi-Au / Pt-CH-ARV / MAb-BSA, and GE-G-Chi-Au / Pt-ARV / MAb-BSA, were obtained respectively.

[0085] 6) Detection:

[0086] On the surfaces of the working electrodes after immobilizing the above four groups of monoclonal antibodies and blocking with BSA, 8 μL of the sample to be tested was added dropwise respectively, allowed to act at a constant temperature of 37 °C for 30 min, and then rinsed 3 times with PBS washing solution respectively; finally, electrochemical impedance spectroscopy (EIS) scanning analysis was carried out in the electrolyte solution, and the scanning frequency range was 0.01 Hz to 100 kHz.

[0087] Protein immobilization control detection group: For the electrodes prepared in the protein immobilization control group with BSA immobilized after the four pretreatment groups, referring to the detection method of the above working electrodes, 8 μL of the positive sample to be tested was added dropwise directly, and EIS detection and analysis were carried out respectively after incubation at 37 °C for 30 min.

[0088] Example 3, Systematic analysis of an avian reovirus electrochemical immunosensor

[0089] Electrochemical Impedance Spectroscopy (EIS): It was called AC impedance in early electrochemical literature and can effectively characterize the electrochemical information of the modified electrode surface. A semicircle will appear in the high-frequency region: the diameter of the semicircle reflects the value of the electron-transfer resistance (Ret) on the electrode surface. The larger the diameter of the semicircle, the larger the Ret value corresponding to the electrode surface; conversely, the smaller the diameter of the semicircle, the smaller the Ret value corresponding to the electrode surface. A straight line will appear in the low-frequency region, which is used to characterize the diffusion process on the reaction electrode surface.

[0090] 1) Electrochemical characterization of electrodes modified with different nanocomposites:

[0091] Figure 1 In A, it is the electrochemical impedance spectra of the unmodified gold electrode GE and the gold electrodes modified with 3 different nanocomposites in the electrolyte. In B, it is the bar chart of the Ret values of the unmodified gold electrode GE and the gold electrodes modified with 3 different nanocomposites. a represents the unmodified gold electrode GE, and a semicircle appears in its AC impedance diagram. When modified with the gold nanocomposite G-Chi-AuNP, platinum nanocomposite G-Chi-PtNP, and gold-platinum nanocomposite G-Chi-Au / PtNP respectively, 3 modified electrodes with different nanocomposites are obtained: GE-G-Chi-AuNP, GE-G-Chi-PtNP, and GE-G-Chi-Au / PtNP, corresponding to Figure 1 b, c, d in it respectively. It can be seen that: the diameters of the semicircles of a, b, c, and d in the electrochemical impedance spectra decrease in turn, and the diameter of the semicircle of the gold-platinum nanocomposite G-Chi-Au / PtNP modified electrode GE-G-Chi-Au / PtNP is the smallest; the results of the Ret values are: GE > GE-G-Chi-AuNP, GE-G-Chi-PtNP > GE-G-Chi-Au / PtNP.

[0092] It can be seen from this that although the 3 different nanocomposites G-Chi-AuNP, G-Chi-PtNP, and G-Chi-Au / PtNP all have good conductivity and can improve the electron transfer ability on the surface of the gold electrode GE, relatively speaking, the Ret value of GE-G-Chi-Au / PtNP is the smallest, that is, the conductivity of G-Chi-Au / PtNP is relatively the best. Therefore, in this invention, the gold-platinum nanocomposite modified electrode GE-G-Chi-Au / PtNP containing bimetals is selected as the preferred modified electrode.

[0093] 2) Electrochemical characterization of monoclonal antibody immobilization after different pretreatments

[0094] Figure 2Electrochemical impedance spectra of monoclonal antibody immobilized after different pretreatments. After 4 different pretreatment steps before antibody immobilization, avian reovirus monoclonal antibody (ARV / MAb) was immobilized to obtain 4 groups of electrodes after monoclonal antibody immobilization: GE-G-Chi-Au / Pt-CH-ARV / MAb, GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb, GE-G-Chi-Au / Pt-ARV / MAb, and GE-G-Chi-Au / Pt-Glu-ARV / MAb. Figure 2 It can be seen that the semicircle diameters of the 4 groups of electrodes after monoclonal antibody immobilization decrease in sequence. It can be seen that the results of the corresponding Ret values should be: GE-G-Chi-Au / Pt-CH-ARV / MAb > GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb > GE-G-Chi-Au / Pt-ARV / MAb > GE-G-Chi-Au / Pt-Glu-ARV / MAb.

[0095] Since the monoclonal antibody ARV / MAb is a protein molecule, if a certain amount of monoclonal antibody ARV / MAb is immobilized, the conductivity of the electrode will inevitably be reduced. Thus Figure 2 it can be known that: the Ret value of the electrode GE-G-Chi-Au / PtNP-CH-ARV / MAb is relatively the largest, that is, the largest amount of monoclonal antibody ARV / MAb can be immobilized on the electrode surface by the CH incubation method, followed by the EDC / NHS incubation method, then the direct immobilization method, and the amount of monoclonal antibody ARV / MAb immobilized on the electrode surface by the Glu incubation method is the least.

[0096] 3) Electrochemical characterization of the protein immobilization control group after different pretreatments

[0097] To verify again the protein immobilization ability after these 4 different pretreatments, a protein immobilization control group experiment was set up, using BSA instead of the antibody (such as monoclonal antibody ARV / MAb). Figure 3 Electrochemical impedance spectra of the protein immobilization control group after different pretreatments. The 4 groups of electrodes of the protein immobilization control group prepared in Example 2: GE-G-Chi-Au / Pt-CH-BSA, GE-G-Chi-Au / Pt-EDC / NHS-BSA, GE-G-Chi-Au / Pt-BSA, and GE-G-Chi-Au / Pt-Glu-BSA. Figure 3It shows that the semicircle diameters of the electrodes after the fixation of 4 groups of BSA proteins decrease in turn. It can be seen that the corresponding results of the Ret values should be: GE-G-Chi-Au / Pt-CH-BSA > GE-G-Chi-Au / Pt-EDC / NHS-BSA > GE-G-Chi-Au / Pt-BSA > GE-G-Chi-Au / Pt-Glu-BSA, which is consistent with the electrochemical characterization of the fixation of monoclonal antibody ARV / MAb.

[0098] According to Figure 2 and Figure 3 By comparing the protein fixation abilities after 4 different pretreatments, relative to the pretreatment negative control group, that is, relative to the direct fixation method without pretreatment, the two methods of CH incubation and EDC / NHS incubation are beneficial to fixing more proteins (monoclonal antibody ARV / MAb and BSA). The possible reason is that the carboxyl groups and amino groups on the electrode surface are enriched after CH and EDC / NHS incubation, which can provide more active sites for the binding of proteins (monoclonal antibody ARV / MAb and BSA); however, relative to the direct fixation method without pretreatment, the protein fixation ability decreases after Glu incubation. The possible reason is that after Glu is fixed to the electrode surface after Glu incubation, Glu itself will undergo self-linkage, thus blocking some of the active sites on the electrode surface that are beneficial to the binding of proteins (monoclonal antibody ARV / MAb and BSA), so the least amount of protein is fixed.

[0099] 4) Electrochemical characterization of the working electrodes prepared after different pretreatments

[0100] The 4 groups of working electrodes sealed with BSA prepared in Example 2: GE-G-Chi-Au / Pt-CH-ARV / MAb-BSA, GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb-BSA, GE-G-Chi-Au / Pt-ARV / MAb-BSA and GE-G-Chi-Au / Pt-Glu-ARV / MAb-BSA, Figure 4 It shows that the semicircle diameters of the 4 groups of working electrodes decrease in turn. It can be seen that the corresponding results of the Ret values should be: GE-G-Chi-Au / Pt-CH-ARV / MAb-BSA > GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb-BSA > GE-G-Chi-Au / Pt-ARV / MAb-BSA > GE-G-Chi-Au / Pt-Glu-ARV / MAb-BSA. Among them, the working electrode GE-G-Chi-Au / Pt-CH-ARV / MAb-BSA pretreated by CH incubation still has the largest Ret value, while the working electrode GE-G-Chi-Au / Pt-Glu-ARV / MAb-BSA pretreated by Glu incubation has the smallest Ret value.

[0101] 5) Electrochemical Characterization of the Working Electrode after Different Pretreatments for Detecting Positive Test Solutions

[0102] The 4 groups of working electrodes prepared in Example 2 were incubated with 8 μL of 10 6.82 EID 50 / mL ARV positive test solution at 37 °C for 30 min, and then rinsed 3 times with PBS washing solution to obtain 4 groups of detection electrodes: GE-G-Chi-Au / PtNP-CH-ARV / MAb-BSA-ARV, GE-G-Chi-Au / PtNP-ARV / MAb-BSA-ARV, GE-G-Chi-Au / PtNP-EDC / NHS-ARV / MAb-BSA-ARV, and GE-G-Chi-Au / PtNP-Glu-ARV / MAb-BSA-ARV. Electrochemical impedance analysis was performed on these 4 groups of detection electrodes, and the obtained electrochemical impedance spectra are as Figure 5 shown Figure 5 The electrochemical impedance spectra of the working electrodes after different pretreatments for detecting positive test solutions. It can be seen that the semicircle diameters of these 4 groups of detection electrodes decrease in turn. It can be seen that the corresponding Ret value results should be: GE-G-Chi-Au / PtNP-CH-ARV / MAb-BSA-ARV > GE-G-Chi-Au / PtNP-ARV / MAb-BSA-ARV > GE-G-Chi-Au / PtNP-EDC / NHS-ARV / MAb-BSA-ARV > GE-G-Chi-Au / PtNP-Glu-ARV / MAb-BSA-ARV. Among them, the Ret value of the detection electrode GE-G-Chi-Au / PtNP-CH-ARV / MAb-BSA-ARV after CH incubation pretreatment is still the largest, and the Ret value of the detection electrode GE-G-Chi-Au / PtNP-Glu-ARV / MAb-BSA-ARV after Glu incubation pretreatment is still the smallest. The possible reasons for the analysis are as follows: In the early stage, the number of monoclonal antibody ARV / MAb that can be immobilized by self-linking after Glu incubation is small, and Glu as a coupling agent may connect the amino group at the Fab end of the monoclonal antibody ARV / MAb to the amino group on the electrode surface. The specific binding site of the monoclonal antibody ARV / MAb and the virus antigen ARV is also located at the Fab end. Therefore, when the working electrode prepared after Glu incubation binds to the virus antigen ARV, due to the occupation of the binding site at the Fab end, there is steric hindrance in the detection binding of the virus antigen ARV, so fewer virus particles are captured and bound. After the superposition of multiple factors, the Ret value of the detection electrode GE-G-Chi-Au / PtNP-Glu-ARV / MAb-BSA-ARV after Glu incubation is the smallest.

[0103] However, the Ret value of the working electrode pre-treated by incubation with EDC / NHS in the early stage was greater than that of the antibody direct immobilization method, that is, GE-G-Chi-Au / Pt-EDC / NHS-ARV / MAb-BSA > GE-G-Chi-Au / Pt-ARV / MAb-BSA, which is equivalent to the amount of monoclonal antibody ARV / MAb immobilized after incubation with EDC / NHS being more than that of the antibody direct immobilization method. However, after incubation with the positive test solution for detection, the Ret value showed an inversion, that is, GE-G-Chi-Au / PtNP-ARV / MAb-BSA-ARV > GE-G-Chi-Au / PtNP-EDC / NHS-ARV / MAb-BSA-ARV, which is equivalent to the detection electrode after incubation with EDC / NHS failing to capture and bind more avian reovirus ARV antigen particles. The possible reasons for the analysis are as follows: The EDC / NHS incubation method connects the amino group at the Fab end of the monoclonal antibody ARV / MAb to the carboxyl group on the electrode surface, and the specific binding site with the virus antigen ARV is also located at the Fab end. Therefore, after immobilizing the monoclonal antibody ARV / MAb by the EDC / NHS incubation method, a certain amount of the subsequent binding sites of the virus antigen ARV will be shielded, that is, the EDC / NHS incubation method (the same as the Glu incubation method) will bring a certain degree of steric hindrance to the subsequent binding of the working electrode to the virus ARV antigen. Therefore, to a certain extent, it will hinder the number of specific bindings between the monoclonal antibody ARV / MAb and the virus ARV antigen; However, when directly immobilizing the monoclonal antibody ARV / MAb by the antibody direct immobilization method, it may be directly connected to the electrode surface through the carboxyl group at the Fc end of the monoclonal antibody ARV / MAb, or connected to the gold-platinum nanocomposite Au / PtNP on the electrode surface through the thiol group on the side chain of the monoclonal antibody ARV / MAb, that is, the site of monoclonal antibody immobilization is far from the binding site between the immobilized monoclonal antibody ARV / MAb and the virus antigen ARV, and the corresponding steric hindrance is smaller. Therefore, it can capture and bind a larger amount of virus antigen ARV particles, and the Ret value of the corresponding detection electrode is larger, that is, GE-G-Chi-Au / PtNP-ARV / MAb-BSA-ARV > GE-G-Chi-Au / PtNP-EDC / NHS-ARV / MAb-BSA-ARV.

[0104] 6) Electrochemical characterization of the protein immobilization control group after different pre-treatments for detecting the positive test solution

[0105] The 4 groups of BSA protein immobilization control groups prepared in Example 2 were directly mixed with 8 μL of 10 6.82 EID 50After incubating the ARV-positive test solution at 37 °C for 30 min at a concentration of / mL, four groups of protein-fixed control test group electrodes were obtained by rinsing three times with PBS washing solution: GE-G-Chi-Au / PtNP-CH-BSA-ARV, GE-G-Chi-Au / PtNP-EDC / NHS-BSA-ARV, GE-G-Chi-Au / PtNP-BSA-ARV, and GE-G-Chi-Au / PtNP-Glu-BSA-ARV. Electrochemical impedance analysis was performed on the electrodes of these four groups of protein-fixed control test groups, and the obtained electrochemical impedance spectra are as Figure 6 shown, Figure 6 which is the electrochemical impedance spectrum of the BSA protein-fixed control group for detecting positive test solutions after different pretreatments. It can be seen that the change in the semicircle diameter of these four groups of test electrodes is almost no different from that before virus incubation, and the corresponding Ret values are still: GE-G-Chi-Au / Pt-CH-BSA > GE-G-Chi-Au / Pt-EDC / NHS-BSA > GE-G-Chi-Au / Pt-BSA > GE-G-Chi-Au / Pt-Glu-BSA. That is, since the monoclonal antibody ARV / MAb that can specifically bind to the virus ARV antigen was not immobilized, the virus ARV particles could not be bound and captured either. The Ret value basically did not change, proving that the sensor has good specificity.

[0106] 7) Comparison of Ret values of five electrochemical characterizations of four groups after different pretreatments

[0107] A column comparison chart was drawn based on the Ret values corresponding to the electrochemical impedance spectra obtained from the above analyses (1) to (6), as Figure 7 shown. First, compared with single-metal nanocomposites, the gold-platinum nanocomposite has the lowest Ret value and the best conductivity for the modified electrode, which is the preferred electrode modification composite. Second, it can be seen that the Ret value of the gold-platinum nanocomposite modified electrode GE-G-Chi-Au / PtNP without pretreatment and protein fixation is the lowest. After different pretreatments, whether or not monoclonal antibody or BSA protein is fixed, whether or not blocked with BSA, and whether or not incubated with positive test solutions, compared with the modified electrode GE-G-Chi-Au / PtNP, the electrodes after treatment will have a different degree of increase in the Ret value due to the different degrees of immobilization of a certain amount of protein on the surface, which will reduce the conductivity of the electrode. Third, it is obvious that the Ret values of the four groups of electrodes after different pretreatments, immobilized with monoclonal antibody ARV / MAb and incubated with positive test solutions, all increase abnormally, proving that this series of sensors can detect the specific binding reaction between antibody and antigen and has good specificity.

[0108] Example 4. Performance analysis of an avian reovirus electrochemical immunosensor

[0109] 1) Sensitivity

[0110] 10 prepared from Example 1 6.82 EID 50 The ARV-positive test solution at 10

[0111] a is the negative control, representing an ARV-positive test solution concentration of 0;

[0112] b represents an ARV-positive test solution concentration of 10 0.82 EID 50 / mL;

[0113] c represents an ARV-positive test solution concentration of 10 1.82 EID 50 / mL;

[0114] d represents an ARV-positive test solution concentration of 10 2.82 EID 50 / mL;

[0115] e represents an ARV-positive test solution concentration of 10 3.82 EID 50 / mL;

[0116] f represents an ARV-positive test solution concentration of 10 4.82 EID 50 / mL;

[0117] g represents an ARV-positive test solution concentration of 10 5.82 EID 50 / mL;

[0118] h represents an ARV-positive test solution concentration of 10 6.82 EID 50 / mL.

[0119] Take 10 μL of each of the above series of test solutions and drop them onto the surfaces of 4 different pre-treated working electrodes prepared in Example 2, and perform according to the detection steps described in Example 2 to obtain the electrochemical impedance spectra and linear graphs of Ret values of 4 corresponding series of detection electrodes.

[0120] Figure 8 It is a linear relationship graph of the electrochemical impedance spectrum and Ret value of the series of detection electrodes obtained by the Glu incubation method and the ARV content. It can be seen that the detection linear range of the Glu incubation method is 0-10 3.82 EID 50 / mL of the virus ARV, and the lowest detection limit is 10 0.63 EID 50 / mL of the virus ARV.

[0121] Figure 9 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the EDC / NHS incubation method with the ARV content. It can be seen that the detection linear range of the EDC / NHS incubation method is 0 - 10 3.82 EID 50 EID / mL of the virus ARV, and the lowest detection limit is 10 0.48 EID 50 EID / mL of the virus ARV.

[0122] Figure 10 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the antibody direct immobilization method with the ARV content. It can be seen that the detection linear range of the antibody direct immobilization method is 0 - 10 4.82 EID 50 EID / mL of the virus ARV, and the lowest detection limit is 10 0.37 EID 50 EID / mL of the virus ARV.

[0123] Figure 11 The linear relationship diagrams of the electrochemical impedance spectra and Ret values of a series of detection electrodes obtained by the CH incubation method with the ARV content. It can be seen that the detection linear range of the CH incubation method is 0 - 10 5.82 EID 50 EID / mL of the virus ARV, and the lowest detection limit is 10 0.46 EID 50 EID / mL of the virus ARV.

[0124] Sensitivity analysis: It can be seen that since the nanomaterials of the modified electrodes all use the gold-platinum nanocomposite GE-G-Chi-Au / PtNP, the electron transfer ability on the sensor surface is similar. During the detection process, the principle of signal change is the same, that is, specific binding occurs between antigen and antibody. The virus ARV particles, as antigens that can specifically bind to monoclonal antibody ARV / MAb, also belong to protein molecules. The complex formed by the binding of antigen and antibody hinders the electron transfer on the electrode surface. When the amount of antigen is small, the immobilized monoclonal antibody ARV / MAb on the surfaces of the 4 sensors pretreated differently can all provide sufficient active sites to bind to the virus ARV particles, and the amount of antigen fixed on the electrode surface is the same, and the signal changes are also similar. Therefore, the lowest detection limits of the 4 groups of sensors pretreated differently are not very different.

[0125] Regarding the detection linear range, among these 4 sensors pretreated differently, the detection linear ranges of the Glu incubation method and the EDC / NHS incubation method are the same, and the highest detection linear range is 10 3.82 EID 50 / mL. This may be because after incubation with Glu and EDC / NHS, the amino groups at the Fab end of the monoclonal antibody ARV / MAb are both connected to the electrode surface, and the specific binding site to the antigen ARV virus particles is also located at the Fab end of the monoclonal antibody ARV / MAb. Therefore, there is a certain steric hindrance after incubation with Glu and EDC / NHS. Relatively speaking, the highest detection linear range of the antibody direct immobilization method is 10 4.82 EID 50 / mL. Because the monoclonal antibody ARV / MAb is connected to the electrode surface through the sulfhydryl group (-SH) of the side chain or the carboxyl group (-COOH) of the Fc end, and the steric hindrance during the specific binding to the antigen ARV virus particles is relatively small, providing more active sites for binding to ARV. Therefore, the detection linear range is 10 times higher than that of the Glu incubation method and the EDC / NHS incubation method.

[0126] Finally, the highest detection linear range of the CH incubation method is 10 5.82 EID 50 / mL, which is 10 times higher than that of the antibody direct immobilization method. This is because after CH incubation, the CH modified on the electrode surface can introduce and enrich the amino groups (-NH2) on the electrode surface, and then more monoclonal antibodies ARV / MAb can be immobilized; and the monoclonal antibody ARV / MAb immobilized after CH incubation is connected to the electrode surface through the carboxyl group (-COOH) of the Fc end, and the steric hindrance during the specific binding to the antigen ARV virus particles is relatively small. Therefore, the detection linear range is further improved after the superposition effect.

[0127] 2) Specificity

[0128] Selection of the test sample: The positive sample is the 10 6.82 EID 50 / mL ARV positive test solution prepared in Example 1; the other 8 common virus, protein, and vitamin control samples are avian influenza virus subtype H3 (AIV H3, 10 4.71 EID 50 / mL), avian influenza virus subtype H9 (AIV H9, 10 3.74 EID 50 / mL), Newcastle disease virus (NDV, 10 4.53 EID 50 / mL), laryngotracheitis virus (LTV, 10 3.86 EID 50 / mL), infectious bronchitis virus (IBV, 10 4.36 EID 50 / mL), and infectious bursal disease virus (IBDV, 10 4.67 EID 50( / mL), bovine serum albumin (BSA 1.0 μg / mL), and vitamin C (vitamin C, 1.0 μg / mL). The test results are as follows Figure 12 shown. For the sensors with these 4 different pretreatments, when detecting non-ARV, the Ret values hardly changed compared with the blank control (Blank). And for these 4 sensors with different pretreatments, when detecting ARV samples mixed with NDV (10 4.53 EID 50 / mL) and BSA (1.0 μg / mL) (10 2.82 EID 50 / mL), the Ret values hardly changed compared with the samples containing only ARV (10 2.82 EID 50 / mL). The results indicate that these 4 sensors can specifically detect ARV with good specificity.

[0129] 3) Stability

[0130] Stability of the preparation process: According to the preparation method of the sensors in Example 2, 5 batches of sensors with 4 different pretreatments were prepared respectively, obtaining 5-batch series products of 4 groups of sensors. After incubating with the diluted test solution to be detected (e in the sensitivity experiment) at 10 3.82 EID 50 / mL for 30 min and then detecting the electrochemical impedance, the column chart of the obtained Ret values is as shown in Figure 13 shown. For the sensors obtained by the Glu incubation method, EDC / NHS incubation method, direct antibody immobilization method, and CH incubation method respectively among the 5-batch series products, the relative standard deviations of the calculated Ret values of the corresponding electrochemical impedance are 2.04%, 2.12%, 1.30%, and 1.92% respectively, that is, the production process stability of the preparation methods of the 4 groups of sensors with different pretreatments is good.

[0131] Storage validity period: Four groups of sensors prepared according to the preparation method of the sensors in Example 2, and 4 groups of sensors of the same production batch were randomly selected and stored at 4°C for thermal stability test. Every 1 week, after incubating with the diluted test solution to be detected (g in the sensitivity experiment) at 10 5.82 EID 50 / mL for 30 min and then detecting the electrochemical impedance, the column chart of the obtained Ret values is as shown in Figure 14 shown. After storing at 4°C for 4 weeks, the Ret values of the sensors obtained by the Glu incubation method, EDC / NHS incubation method, direct antibody immobilization method, and CH incubation method are 86.75%, 88.89%, 89.02%, and 87.92% of the newly prepared ones respectively. That is, the validity period of the 4 groups of sensors is about 4 weeks. It does not rule out that there is room for further improvement in the validity period after process optimization and material optimization configuration.

[0132] Example 5. Application of an electrochemical immunosensor for avian reovirus

[0133] A detection kit for avian reovirus, comprising a sensor of a three - electrode system for detecting avian reovirus. The three - electrode system includes a working electrode, an auxiliary electrode, and a reference electrode. The working electrode consists of 4 groups of working electrodes prepared in Example 2, preferably the working electrode after incubation with CH; the auxiliary electrode is a platinum wire electrode; the reference electrode is a saturated calomel electrode (SCE). The kit may also include a negative control, a positive control (the positive test solution prepared in Example 1), and an electrolyte solution (the electrolyte solution prepared in Example 1).

Claims

1. A preparation method of an electrochemical immunosensor, the sensor being used for detecting avian reovirus, wherein, The preparation of the working electrode of the sensor includes a pretreatment step before antibody immobilization, and the method is prepared according to the following steps: (1) Treat the electrode: including polishing, washing with water, ultrasonic cleaning, soaking, scanning with cyclic voltammetry until stable and then drying with N2; (2) Modify the electrode with the nanocomposite: Drop the nanocomposite made of noble metals on the surface of the electrode and let it dry naturally at 4°C. The noble metals are gold + platinum, and the prepared nanocomposite is a bimetallic nanocomposite jointly prepared by two noble metals, gold and platinum; the nanocomposite is a graphene-chitosan-gold / platinum nanoparticle G-Chi-Au / Pt nanocomposite, and its particle size distribution is 1.5-5.2 nm; among them, the feeding ratio of gold, platinum, and graphene is 1 mmol: 1 mmol: 2 g; (3) Pretreatment before antibody immobilization: Drop 10 μL of the incubation solution on the surface of the nanocomposite-modified electrode. The incubation solution is a cysteamine hydrochloride solution or a MES solution containing EDC and NHS; when the incubation solution is a cysteamine hydrochloride solution, the concentration of the cysteamine hydrochloride solution is 2 mg / mL, and the incubation is carried out under dark conditions for 4 h; when the incubation solution is a MES solution containing EDC and NHS, first place the nanocomposite-modified electrode in a NaOH solution and apply a constant potential of +1.3 V for 40 s, and then drop the incubation solution on its surface; the concentration of the NaOH solution is 0.5 mol / L. In the MES solution containing EDC and NHS, the concentration of EDC is 50 mmol / L, the concentration of NHS is 30 mmol / L, and the pH value of the MES solution is 4.

7. The incubation is carried out at room temperature for 1 h; After incubation, rinse with PBS washing solution at least 3 times; (4) Antibody immobilization: Then drop 8 μL of the monoclonal antibody ARV / MAb of avian reovirus, and under airtight conditions, carry out the immobilization reaction at 4°C for 8 h; (5) Blocking and detection: After the antibody immobilization reaction, rinse with PBS washing solution, block with BSA solution, and rinse with PBS washing solution again to obtain the working electrode, directly drop the sample to be tested, and detect by electrochemical scanning.

2. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that: In step (1), the electrode is a glassy carbon electrode. The preparation method of the nanocomposite in step (2) includes: 1) Graphene G: Graphite powder is oxidized at high temperature, ultrasonicated, reduced with sodium borohydride at high temperature, and washed to obtain graphene G; 2) Chitosan solution Chi: Chitosan is dissolved in an acetic acid solution, and magnetically stirred at 25°C for 1 h to mix evenly to obtain a chitosan solution Chi; among them, the acetic acid solution is a mixed solution of glacial acetic acid and water with a volume ratio of 1.0%, and the concentration of the obtained chitosan solution is 0.1 wt%; 3) Graphene-chitosan suspension G-Chi: Mix graphene with the above chitosan solution and ultrasonicate to obtain a graphene-chitosan suspension G-Chi; among them, the power of the ultrasonication is 250 W, and the continuous ultrasonication is 2 h. The feeding ratio of graphene G to chitosan solution Chi is 1 mg: 1 mL; 4) Nano - composite: Add chloroauric acid solution and potassium tetrachloroplatinate solution into the above graphene - chitosan suspension G - Chi, stir at room temperature for 3 h, and then continue to react in a water bath at 80 °C for 1 h to obtain graphene - chitosan - gold - platinum nano - composite G - Chi - Au / Pt, that is, gold - platinum nano - composite. The concentrations of the chloroauric acid solution and the potassium tetrachloroplatinate solution are both 10 mmol / L, and the dosages are both 1 mL. The dosage of the graphene - chitosan suspension G - Chi is 20 mL.

3. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that: The scanning detection in step (5) is carried out in the electrolyte. The electrolyte is a PBS buffer solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 M KCl, and the pH value of the PBS buffer solution is 7.

0.

4. An electrochemical immunosensor for detecting avian reovirus, characterized in that: The sensor is a three - electrode system, including a working electrode, an auxiliary electrode and a reference electrode. The working electrode is prepared by the preparation method of the electrochemical immunosensor according to any one of claims 1 - 2. The auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.

5. A detection kit for avian reovirus, characterized in that: An electrochemical immunosensor for detecting avian reovirus as described in claim 4, wherein the detection linear range of the kit for detecting avian reovirus is 0 to 10 5.82 EID 50 / mL, and the lowest detection limit is 10 0.46 EID 50 / mL.

6. The detection kit for avian reovirus according to claim 5, characterized in that: It also includes a PBS washing solution, an electrolyte, an auxiliary electrode and a reference electrode. The auxiliary electrode is a platinum wire electrode, the reference electrode is a saturated calomel electrode, the electrolyte is a PBS buffer solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 M KCl, and the pH value of the PBS buffer solution is 7.

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7. Use of a reagent for detecting reovirus in the preparation of an electrochemical immunosensor for detecting reovirus, and the use includes the preparation method of the electrochemical immunosensor according to any one of claims 1 - 3.

8. Use of a reagent for detecting reovirus in the preparation of a detection kit for avian reovirus, and the use includes the preparation method of the electrochemical immunosensor according to any one of claims 1 - 3.

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