Electrochemical immunosensor for detection of subgroup i avian adenovirus

Abstract

The application discloses a kind of multi-walled carbon nanotube prepared by multi-walled carbon nanotube Shell chitosan-palladium / silver (MWCNT-Chi-Pt / Ag) Nanocomposite, and the way of adopting double antibody sandwich method and electrochemical immunosensor is combined to detect subgroup I avian adenovirus (FAdV-I), the preparation method of multi-walled carbon nanotube-bimetallic nanocomposite and working electrode is optimized, and the mass ratio of core component in MWCNT-Chi-Pt / Ag nanocomposite and the detection condition of electrochemical immunosensor are further screened and optimized, and finally the minimum detection limit of FAdV-I virus content is 10 0.67 EID 50 / mL, sensitivity, versatility and specificity are good, and batch and batch repeatability of preparation process are good.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials and veterinary biological products, specifically to a nanocomposite prepared from bimetallic materials and multi-walled carbon nanotubes, particularly for the detection of subgroup I avian adenovirus by an electrochemical immunosensor, and especially to an electrochemical immunosensor for the detection of subgroup I avian adenovirus prepared from the nanocomposite. Background Technology

[0002] Fowladenovirus group I (FAdV-I) is widely found in the respiratory and digestive tracts of various poultry, including chickens, ducks, and geese. FAdV-I belongs to the genus *Avianadenovirus* within the family Adenoviridae. Based on serocross-neutralization experiments, FAdV-I is divided into 12 different serotypes. Further, based on restriction endonuclease fragment maps and nucleic acid sequences, these 12 known serotypes are classified into five groups: FAdV-A (serotype: FAdV-1), FAdV-B (serotype: FAdV-5), FAdV-C (serotypes: FAdV-4, FAdV-10), FAdV-D (serotypes: FAdV-2, FAdV-3, FAdV-9, FAdV-11), and FAdV-E (serotypes: FAdV-6, FAdV-7, FAdV-8a, FAdV-8b). Okuda et al. found that FAdV-I can remain latent in chicken flocks for up to one generation. When chickens are subsequently infected with other viruses, FAdV-I acts as a secondary virus, leading to inclusion body hepatitis, pericardial effusion syndrome, and gizzard erosion. Previous studies have shown that co-infection with chicken infectious anemia virus (AIV) can significantly enhance inclusion body hepatitis caused by certain serotypes of FAdV-I, leading to increased mortality. Furthermore, FAdV-I can be transmitted vertically via eggs and horizontally via excrement. However, the pathogenicity of most subgroup I adenoviruses in poultry remains incompletely determined. In recent years, this disease has become widespread in Shandong, Hebei, Henan, Jiangsu, and Shaanxi provinces in my country, causing significant economic losses to the poultry industry. Therefore, establishing a simple, rapid, sensitive, and universal method for detecting FAdV-I is of great significance for promoting the healthy development of the poultry industry.

[0003] Currently, various methods exist for detecting FAdV-I, including virus isolation and identification, PCR-based nucleic acid detection methods, enzyme-linked immunosorbent assay (ELISA), and agarose gel diffusion assay (AGP). However, these methods all have certain limitations: virus isolation and identification have high accuracy, but require virus culture and observation of viral morphology using transmission electron microscopy, which is time-consuming. Although PCR-based nucleic acid detection methods have high sensitivity, the operation is cumbersome, requiring steps such as viral DNA extraction, PCR amplification, and electrophoresis detection. It also heavily relies on the selection of universal detection primers for different serotypes and specialized laboratory equipment and professional operators, resulting in high detection costs. ELISA methods have the drawback of low sensitivity; Okuda et al. found that the existing AGP detection technology cannot detect latent FAdV-I infection. Therefore, developing faster, more accurate, sensitive, and efficient detection technologies for FAdV-I detection remains a very important issue in veterinary science.

[0004] Electrochemical immunosensors are a novel detection technology that combines electrochemical and immunoassay methods. They offer advantages such as rapid detection, simple operation, high sensitivity, good specificity, low cost, and miniaturizable instruments. Currently, research on electrochemical immunosensors focuses on developing novel nanomaterials and signal amplification methods to improve sensitivity and specificity. Carbon nanotubes are a relatively new type of nanomaterial. Multiwalled carbon nanotubes (MWCNTs), due to their excellent conductivity, electromagnetic properties, and adsorption capabilities, can be used as sensor electrode modification materials for preparing electrochemical sensors. However, they still face challenges such as complex preparation, low sensitivity, high detection limits, and the inability to directly immobilize antigens, antibodies, and other protein molecules. Chitosan, with its abundant amino groups, can significantly improve the biocompatibility and water solubility of carbon nanotubes after modification, effectively immobilizing biorecognition elements. Nanogold and nanosilver, with their excellent conductivity, stability, optical properties, and antibacterial properties, are often used to create core / shell nanocomposites. However, the preparation and compounding of these nanomaterials are crucial for the development of bimetallic nanomaterials. Common signal amplification strategies include enzyme-catalyzed amplification and nanomaterial amplification. Enzymes have limitations due to their high cost and susceptibility to inactivation. Nanomaterials, with their unique physicochemical properties, can be used not only to immobilize biomolecules but also as signal amplification materials. Nanomaterial amplification typically operates in three ways: ① using nanomaterial-modified electrodes as a substrate to improve the performance of the electrochemical immunosensor's sensing interface; ② using nanomaterials as carriers to immobilize large quantities of biorecognition elements, enzymes, and electroactive substances to enhance electrochemical signals; ③ some nanomaterials themselves, through electrochemical oxidation or reduction, can act as electrochemically active substances to provide output signals, enabling highly sensitive detection of analytes by the electrochemical immunosensor.

[0005] Therefore, improving the sensitivity of electrochemical immunosensors and their ability to detect subgroup I avian adenoviruses universally and rapidly, especially in cases of low viral load in latent FAdV-I infections, is a key research direction for the future. Summary of the Invention

[0006] The purpose of this invention is to screen or prepare relatively superior nanocomposites and overcome the existing defects of FAdV-I due to the difficulty in universal detection of multiple serotypes, so as to provide a new electrochemical immunosensor for rapid on-site detection of subgroup I avian adenovirus (FAdV-I) that is fast, simple to operate, highly sensitive, has good versatility and specificity, low cost, and can be miniaturized.

[0007] To achieve the above-mentioned objectives or one of them, the present invention provides the following technical solution:

[0008] This invention claims protection for a multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite, the preparation of which includes the following steps: 1) Chitosan Chi solution: Chitosan Chi is taken into acetic acid solution, stirred at room temperature for 0.5 h and then thoroughly mixed and dissolved to obtain a chitosan Chi solution; wherein, 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 Chi solution is 0.1 wt%; 2) Multi-walled carbon nanotube-chitosan MWCNT-Chi nanocomposite: Multi-walled carbon nanotubes (MWCNTs) are added to the chitosan Chi solution, and after ultrasonic treatment for 2 h, a stable multi-walled carbon nanotube-chitosan MWCNT-Chi nanocomposite is obtained. The suspension of hi nanocomposites; wherein, the concentration of the suspension of multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposites is 1 mg / mL; 3) Multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposites: Take 30 mL of the suspension of the multi-walled carbon nanotube-chitosan (MWCNT-Chi), first add 10 mmol / L K2PtCl4 solution and stir at room temperature for 3 h, then add 10 mmol / L AgNO3 solution and continue stirring at room temperature for 2 h, and then place it in a 90℃ water bath for a one-step reduction reaction for 0.5 h, which yields a stable solution of multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposites.

[0009] According to a preferred embodiment of the present invention, in the multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite, the mass ratio of MWCNT:Ag:Pt is preferably 30:1:2.

[0010] This invention claims protection for an electrochemical immunosensor for detecting subgroup I avian adenovirus, comprising a working electrode made of a multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite and a signal marker made of a multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposite.

[0011] According to a preferred embodiment of the present invention, the preparation method of the working electrode includes the following steps: 1) Electrode treatment: the electrode is polished with Al2O3 polishing powder, washed with water, ultrasonically cleaned, scanned with cyclic voltammetry until stable, and then dried with N2; 2) Electrode modification with multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite: a suspension of the multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite is dropped onto the surface of the electrode and allowed to air dry at room temperature; 3) Monoclonal antibody fixation: the monoclonal antibody FAdV / MAb of subgroup I avian adenovirus is then dropped onto the electrode and fixed at 4°C for at least 8 hours; 4) Blocking: after antibody fixation, BSA solution is dropped onto the electrode and blocked at 37°C for 1 hour, then removed and washed with water to obtain the working electrode BSA-FAdV / MAb-MWCNT-Chi-GCE for detecting subgroup I avian adenovirus; wherein, the electrode is a glassy carbon electrode.

[0012] According to a preferred embodiment of the present invention, the signal marker is a polyclonal antibody-labeled nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb, which is prepared by reacting a multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite with subgroup I avian adenovirus polyclonal antibody FADV / PAb and 5wt% BSA at 4°C for at least 8 hours.

[0013] According to a preferred embodiment of the present invention, the sensor is a three-electrode system, which includes a reference electrode and a counter electrode in addition to the working electrode. The working electrode is a BSA-FAdV / MAb-MWCNT-Chi-GCE electrode, which is directly sealed after being modified by a multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite. The reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.

[0014] This invention claims protection for a detection method for subgroup I avian adenovirus, which uses an electrochemical immunosensor. The sample to be tested is dropped onto its working electrode and incubated at 37°C for a certain period of time. After the signal marker is dropped onto the electrode, the sample is placed in a sealed humidified box and incubated at 37°C for a certain period of time. The sample is then washed with water at least three times and placed in an electrolyte for chronocurrent scanning detection. After 50 seconds, H2O2 is added as the test base solution.

[0015] According to a preferred embodiment of the present invention, the incubation time after the sample to be tested is 30 min; the incubation time after the signal marker is applied is 40 min.

[0016] According to a preferred embodiment of the present invention, the electrolyte is a 0.01M PBS buffer containing 0.1M KCl with a pH of 7.4, and the concentration of H2O2 in the test substrate is 5mM.

[0017] This invention claims protection for a multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite, an electrochemical immunosensor, and a method for detecting subgroup I avian adenovirus, and its application in the preparation of products for detecting subgroup I avian adenovirus.

[0018] The beneficial effects of this invention are:

[0019] This invention first provides a novel nanomaterial: carbon nanotubes loaded with silver and platinum bimetallic nanoparticles. This nanocomposite can bind well to FAdV-I monoclonal or polyclonal antibodies, exhibits high biocompatibility with organic materials, and can also serve as an electrochemically active substance to provide amplified output signals for sensors. Based on the bimetallic nanocomposite, this invention constructs an ultrasensitive electrochemical immunosensor based on a double-antibody sandwich method for detecting FAdV-I, providing a new detection approach for FAdV-I. It develops a faster, more sensitive, and more efficient FAdV-I detection method than existing technologies. On the one hand, it overcomes the difficulties in broad-spectrum detection of FAdV-I caused by differences in multiple serotypes, genomes, and biological characteristics of subgroup I avian adenovirus; on the other hand, it overcomes the deficiency of false negatives in common clinical samples of avian adenovirus due to low viral load. This improves the sensitivity of broad-spectrum FAdV-I detection and provides strong technical support for the effective prevention and control of FAdV-I. Simultaneously, it also provides a new approach for future research on rapid detection and diagnostic technologies for other animal diseases.

[0020] The applicant of this invention has been dedicated to in-depth research on electrochemical sensor platforms. Based on extensive preliminary research and optimization of a prior patent (CN115524385A, publication date December 27, 2022), the applicant obtained the multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nanocomposite described in this invention. This nanocomposite was applied to an electrochemical sensor for the detection of subgroup I avian adenovirus. Although chitosan-modified multi-walled carbon nanotube monometallic nanocomposites have been successfully used for the detection of a single serotype of virus FAdV-4 (e.g., CN 115524385A), this invention cannot be directly applied to the broad-spectrum detection of FAdV-I due to low sensitivity and poor specificity. This invention utilizes the biocompatibility and water solubility of chitosan-modified multi-walled carbon nanotubes to immobilize platinum / silver bimetals, forming a novel nanomaterial—multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag. This nanocomposite can be used to prepare signal markers and, when combined with a multi-walled carbon nanotube-chitosan MWCNT-Chi modified working electrode, forms a double-antibody sandwich method, successfully achieving broad-spectrum detection of FAdV-I.

[0021] Conventional electrochemical immunosensors utilize the advantages of nanomaterials, such as good biocompatibility, large specific surface area, and strong conductivity, to achieve effective signal amplification. This invention creatively discovers that the bimetallic nanomaterial—multi-walled carbon nanotubes-chitosan-platinum / silver MWCNT-Chi-Pt / Ag—can be electrochemically oxidized or reduced by H2O2 to provide an output signal as an electrochemically active substance. Therefore, its application in sensors can amplify the signal in electrochemical immunosensors, further enhancing their high sensitivity for analytes and significantly improving the performance of the sensing interface.

[0022] The double-antibody sandwich method is generally used for enzyme-linked immunosorbent assay (ELISA). However, ELISA is usually not very sensitive and cannot amplify the results of specific immune reactions, thus failing to overcome the technical problem of low viral vector levels and undetectable levels caused by latent infection of FAdV-I in chicken flocks. This invention creatively applies the double-antibody sandwich method to the field of electrochemical immunosensors. The entire detection operation is convenient, requiring no complex and expensive instruments. Unlike high-sensitivity nucleic acid detection, it does not require pretreatment processes such as RNA / DNA extraction from the sample, nor does it require screening universal primers like PCR methods. Moreover, the detection time is short, including 30 minutes of sample incubation and 40 minutes of secondary antibody incubation, the entire detection takes about 70 minutes.

[0023] In this invention, a double-antibody sandwich method is used to achieve broad-spectrum detection of FAdV-I. The electrode GCE is modified with a MWCNT-Chi nanocomposite, which then adsorbs FAdV-I monoclonal antibody (FAdV / MAb) to form the working electrode. The signal marker is prepared by combining a bimetallic MWCNT-Chi-Pt / Ag nanocomposite with FAdV-I polyclonal antibody (FAdV / PAb). This monoclonal antibody-virus-biclonal antibody sandwich method achieves both specific binding to FAdV-I and broad-spectrum recognition of FAdV-I through the polyclonal antibody. Furthermore, the signal amplification effect of the polyclonal antibody-loaded MWCNT-Chi-Pt / Ag nanocomposite enhances the detection of FAdV-I in the test sample. The signal amplification process of trace FADV-I improves sensitivity. During the research and modification process, this invention unexpectedly discovered a signal marker prepared from a bimetallic MWCNT-Chi-Pt / Ag nanocomposite, combined with a working electrode modified from a multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite, forming a sensor for detecting multiple serotypes of FADV-I. This method is particularly suitable for detecting subgroup I avian adenovirus. Compared to traditional ELISA and PCR methods, it not only has higher sensitivity, stronger specificity, and better repeatability, but also surpasses the single-serotype PCR method (CN115524385A, where the detection sensitivity for FADV-4 is 10).1.89 EID 50 / mL), the detection limit of the immunosensor of the present invention for FAdV-I is 10. 0.67 EID 50 The detection sensitivity of the electrochemical immunosensor described in this invention is improved by at least 10 times per mL. Verification using 100 clinical samples showed that the positive concordance rate between the electrochemical immunosensor described in this invention and the results of traditional sequencing methods reached 100%.

[0024] This invention forms a novel MWCNT-Chi-Pt / Ag nanocomposite with good biocompatibility and excellent conductivity, and can further amplify the detection of electrochemical signals. In this invention, multi-walled carbon nanotubes (MWCNTs) are first uniformly dispersed in a chitosan (Chi) solution, then K₂PtCl₄ is added first, followed by AgNO₃, and Pt is amplified at 90°C. 2+ Ag + A one-step reduction process yielded platinum (Pt / Ag) nanoparticles, ultimately resulting in a multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposite. Unexpectedly, the study revealed that the stirring time was crucial during the reduction of the Pt / Ag bimetallic material. Insufficient stirring time or excessively long solution addition sequences made it difficult to obtain a MWCNT-Chi-Pt / Ag nanocomposite with uniform particle size and good loading. Therefore, the stirring time was optimized. Stirring times of 3 and 4 hours resulted in low Pt / Ag nanoparticle loading on MWCNT-Chi. A total stirring time of 5 hours not only resulted in abundant Pt / Ag nanoparticle loading on MWCNT-Chi but also ensured uniform Pt / Ag particle size on MWCNT-Chi, reducing the likelihood of agglomeration. Therefore, 5 hours was the optimal stirring time for Pt / Ag nanoparticle loading. 2+ Ag + The optimal total stirring time during the reduction process, and for Pt 2+ Ag + The optimal stirring times before reduction are 3h and 2h, respectively. It can be said that in the preparation method of MWCNT-Chi-Pt / Ag nanocomposite, the preparation step of stirring separately before reduction is a technical difficulty that was unexpectedly overcome by the present invention, and it also lays the foundation for improving the sensitivity and specificity of the sensor.

[0025] This invention combines and optimizes multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposites with bimetallic (Pt / Ag) nanocomposites to prepare an electrochemical immunosensor for detecting FAdV-I. A dual-antibody sandwich detection platform for the FAdV-I electrochemical immunosensor was established. On one hand, the incubation time of the test sample and the incubation time of the polyclonal antibody-labeled nanocomposites were optimized. Ultimately, only 30 minutes of incubation for the test sample and 40 minutes for the polyclonal antibody-labeled nanocomposites are required, effectively shortening the detection time compared to traditional nucleic acid detection. On the other hand, the optimal pH of the test solution was found to be 7.4 and the optimal concentration of the substrate H2O2 was 5 mM, further improving detection sensitivity while ensuring broad spectrum and specificity. This invention provides a novel and rapid detection technology for the prevention and control of FAdV-I, contributing to enhanced scientific prevention and control in poultry farming.

[0026] This invention, for the first time, proposes the preparation of a novel nanomaterial, MWCNT-Chi-Pt / Ag, by combining multi-walled carbon nanotubes (MWCNTs), chitosan, and bimetallic compounds. The working electrode and signal marker for an electrochemical immunosensor targeting subgroup I avian adenovirus (FAdV) are prepared using a double-antibody sandwich method. The preparation method is simple, establishing a novel electrochemical immunosensor detection platform. The detection method provides standardized and easily controllable results, and offers advantages such as rapid detection, high sensitivity, good specificity, low cost, and miniaturizable instrumentation. It is suitable for on-site prevention and control detection in poultry farming. By optimizing the detection conditions for subgroup I avian adenovirus, this invention constructs a novel and rapid current-driven electrochemical immunosensor detection method for broad-spectrum and specific detection of FAdV-I, providing strong technical support for the prevention and control of avian adenovirus. Furthermore, it offers a new approach for future research on rapid detection and diagnostic technologies for other avian infectious diseases and animal diseases. Attached Figure Description

[0027] Figure 1 The images show TEM images of the Chi series nanocomposites and EDS images of the bimetallic nanocomposites MWCNT-Chi-Pt / Ag of this invention; where A is the TEM image of MWCNT-Chi, B is the TEM image of MWCNT-Chi-Pt, C is the TEM image of MWCNT-Chi-Pt / Ag, and D is the EDS image of C, Pt, and Ag elements in MWCNT-Chi-Pt / Ag. The horizontal axis represents X-ray energy (KeV), and the vertical axis represents the X-ray energy dispersive spectrometer counts (au).

[0028] Figure 2The figures show electrochemical characterization diagrams of different electrodes or Chi-series nanocomposites of this invention; A to D are electrochemical change curves and corresponding bar charts of different electrode surfaces during sensor fabrication; E to F are CA diagrams and bar charts of response current values ​​for different Chi-series nanocomposites; wherein, A is the electrochemical impedance spectroscopy (EIS) diagram of different electrode surfaces, B is the bar chart of Ret values ​​corresponding to different electrode surfaces, C is the CV diagram of different electrode surfaces, and D is the bar chart of redox peaks of different electrodes; in A to D, a is GCE, b is MWCNT-Chi-GCE, c is FADV / MAb-MWCNT-Chi-GCE, and d is BSA-FAdV / MAb-MWCNT-Chi-GCE; in E to F, a is the blank control, b is MWCNT-Chi-Pt-FAdV / PAb, and c is MWCNT-Chi i-Ag-FAdV / PAb, d is Chi-Pt / Ag-FAdV / PAb, e is MWCNT-Chi-Pt / Ag-FAdV / PAb, f is the electrode BSA-MWCNT-Chi-GCE without monoclonal antibody immobilization, and g is the electrode BSA-MWCNT-Chi-GCE(+FAdV-4+MWCNT-Chi-Pt / Ag-FAdV / PAb) without monoclonal antibody immobilization.

[0029] Figure 3 The diagrams show the CA (Carbon Calibration) plots of different mass ratios of multi-walled nanotubes and bimetals in the Chi series nanocomposites of the present invention, and their corresponding current peak values. A is a CA plot of different mass ratios of multi-walled nanotubes and bimetals in the Chi series nanocomposites, and B is a bar chart of the measured response current values ​​of different mass ratios of multi-walled nanotubes and bimetals in the Chi series nanocomposites.

[0030] Figure 4 This is a graph showing the relationship between the peak response current and time of the simulated test sample and working electrode under different incubation times in this invention;

[0031] Figure 5 This is a graph showing the relationship between the peak response current and time for the multi-antibody labeled nanocomposite of the present invention at different incubation times;

[0032] Figure 6 This is a bar chart showing the CA curve and corresponding current peak values ​​measured by the working electrode of this invention in test substrates at different pH values.

[0033] Figure 7 This is a bar chart showing the CA curve and corresponding current peak values ​​measured in test base solutions of H2O2 with different concentrations of the working electrode of this invention.

[0034] Figure 8This is a sensitivity result graph of the electrochemical immunosensor for avian adenovirus subgroup I of the present invention. A is the CA graph of simulated test sample FAdV-4 virus solution at different concentrations; B is the linear relationship between the corresponding current value of CA detection and the concentration of FAdV-4 virus solution at different concentrations of simulated test sample; in a~h, a is the negative control (i.e., FAdV-4 concentration is 0), b~h represent different concentrations of FAdV-4 virus solution, and b is 10 0.93 EID 50 / mL, c is 10 1.43 EID 50 / mL, d is 10 1.93 EID 50 / mL, e is 10 2.43 EID 50 / mL, f is 10 2.93 EID 50 / mL, g is 10 3.43 EID 50 / mL, h is 10 3.93 EID 50 / mL;

[0035] Figure 9 This is a graph showing the specificity results of the electrochemical immunosensor for subgroup I avian adenovirus of the present invention. The horizontal axis represents the 24 test samples selected in the present invention, and the vertical axis represents the bar graph of the corresponding current values ​​of CA detection for the 24 test samples in the present invention.

[0036] Figure 10 This is a graph showing the repeatability results of the electrochemical immunosensor for avian adenovirus subgroup I of this invention. Detailed Implementation

[0037] The exemplary embodiments of the present invention are described in detail below with reference to practical applications. In the following detailed description, many specific details are set forth for ease of explanation to provide a full understanding of the disclosed embodiments. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. However, it is apparent that one or more embodiments may be implemented without these specific details. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Multi-walled carbon nanotubes (MWCNTs), AgNO3, K2PtCl4, bovine serum albumin (BSA), glucose (1.0 μg / mL), and vitamin C (1.0 μg / mL) were purchased from Sigma-Aldrich. KMnO4, K4Fe(CN)6, K3Fe(CN)6, H2SO4, CH3CH2OH, chitosan (Chi, also known as chitosan amine), and trisodium citrate dodecahydrate were all domestically produced analytical grade reagents. PremixTaq (ExTaq Version 2.0 Plus) and 100 bp DNA Ladder were purchased from TaKaRa, and deionized water was used in the experiments.

[0039] Anti-FAdV-I polyclonal antibody (FAdV / PAb) was routinely prepared by the Guangxi Veterinary Research Institute: it was prepared by immunizing SPF chickens with FADV-4 stored in our laboratory after inactivation with 0.2% formaldehyde. Anti-FAdV-I monoclonal antibody (FAdV / MAb) was prepared by the Guangxi Veterinary Research Institute (see the literature: Truncated expression of Fiber-2 protein of avian adenovirus type 4 and preparation of monoclonal antibody, Wei You, Xie Zhixun, Deng Xianwen, Li Xiaofeng, Xie Zhiqin, Fan Qing, Zhang Yanfang, Huang Jiaoling, Wang Sheng; Southern Agriculture Journal. 2022, 53: 2341-2349). Inactivated viruses, including avian infectious bronchitis virus (IBV), chicken infectious anemia virus (CIAV), Newcastle disease virus (NDV), egg drop syndrome virus (EDSV), avian reovirus (ARV), avian influenza virus H9 (AIV H9), and FAdV (FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, and FAdV-11), were all preserved in the form of virus preservation solutions by the Biotechnology Laboratory of Guangxi Veterinary Research Institute.

[0040] The instruments used in the following examples are: a transmission electron microscope (TEM) and an energy dispersive spectrometer (EDS), which were purchased from FIE (Made in the USA, Model: Tecnai G2 F30 S-TWIN); The immunosensor described in this invention uses a conventional three-electrode electrochemical cell for electrochemical measurements, with a modified glassy carbon electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum electrode as the counter electrode. The immunosensor is connected to the electrochemical workstation via wires.

[0041] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0042] The viral strains involved in this invention belong to the field of viral preservation solutions in the microbiology field. All are known viruses and, under biosafety conditions, can be obtained from the applicant by the public. They can only be used to repeat the experiments of this invention and may not be used for other purposes. One hundred clinical samples were collected from a large-scale chicken farm in Guangxi, all from chicken samples suspected of being infected with FAdV-4 (including cloacal swabs, liver, and spleen). These samples were tested using PCR methods in our laboratory, and the target bands were recovered, cloned, and then positive colonies were selected for sequencing. The positive concordance rate between the two methods reached 100%. The viral preservation solutions and clinical samples involved in this invention are all ex vivo, non-living biological samples. The direct purpose of the detection is not to obtain disease diagnosis results or health status, but rather to enable industrial production of in vitro diagnostic kits or related products for virus detection. Therefore, this invention does not fall under the category of disease diagnosis methods and meets the basic requirements for patent protection subject matter under the Patent Law.

[0043] According to the overall concept of the present invention, a novel multi-walled carbon nanotube (MWCNT)-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposite and its preparation method are provided. The method involves uniformly dispersing MWCNTs in a chitosan (Chi) solution, adding K2PtCl4 and AgNO3 solutions, and then heating the Pt nanocomposite at 90°C. 2+ Ag +A multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposite material was obtained by reducing it to platinum / silver nanoparticles (Pt / Ag), a simple preparation method. The prepared MWCNT-Chi-Pt / Ag nanocomposite was successfully used in an electrochemical immunosensor detection platform for subgroup I avian adenovirus. Specifically, a glassy carbon electrode was modified with MWCNT-Chi to adsorb monoclonal antibodies against subgroup I avian adenovirus. A polyclonal antibody against subgroup I avian adenovirus labeled with MWCNT-Chi-Pt / Ag was used as an electrochemical signal amplifier (i.e., a signal marker). An electrochemical immunosensor and detection method for subgroup I avian adenovirus were constructed. The incubation time of the test sample and the signal marker, as well as the pH value and H2O2 concentration of the electrolyte, were optimized. Specifically, the test sample was incubated for 30 minutes, followed by incubation with the MWCNT-Chi-Pt / Ag signal marker immobilized with the polyclonal antibody against subgroup I avian adenovirus for 40 minutes, which was then used for chronoamperometry detection.

[0044] The following describes an embodiment of the establishment of the electrochemical immunosensor detection platform for the MWCNT-Chi-Pt / Ag nanocomposite and subgroup I avian adenovirus described in this invention. The specific details are as follows:

[0045] Example 1: Preparation and chemical characterization of Chi-series nanocomposites

[0046] 1.1 Preparation of Chi-series nanocomposites

[0047] 1) Chitosan (Chi) solution:

[0048] Dissolve 0.4 g of chitosan (Chi) in 400 mL of acetic acid solution (1.0% (V / V)). Stir magnetically for 0.5 h at room temperature to obtain 400 mL of 0.1 wt% chitosan (Chi) solution.

[0049] 2) Multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite:

[0050] Take 400 mg of multi-walled carbon nanotubes (MWCNTs) and add them to 400 mL of the chitosan (Chi) solution obtained above. After sonication for 4 h, a stable suspension of multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite with a concentration of 1 mg / mL and about 400 mL is obtained.

[0051] 3) Single-metal nanocomposites:

[0052] Take 30 mL of the prepared suspension of multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite with a concentration of 1 mg / mL, add 10 mmol / L K2PtCl4 solution, and stir at room temperature, preferably for 6 hours; then place the stirred mixture in a 90℃ water bath to simultaneously reduce Pt metal, preferably for 1 hour, to obtain a stable solution of multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-Pt) nanocomposite; wherein, by adjusting the amount of K2PtCl4 solution, different mass ratios of multi-walled nanotubes and bimetals in the nanocomposite are screened: the amounts of K2PtCl4 solution are 1 mL, 2 mL, and 3 mL, respectively, corresponding to the mass ratios of MWCNT:Ag:Pt in the multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-Pt) nanocomposite of 30:0:1, 30:0:2, and 30:0:3.

[0053] Take 30 mL of the prepared suspension of multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite with a concentration of 1 mg / mL, add 10 mmol / L AgNO3 solution, and stir at room temperature, preferably for 6 hours; then place the stirred mixture in a 90℃ water bath to simultaneously reduce Ag metal, preferably for 1 hour, to obtain a stable solution of multi-walled carbon nanotube-chitosan-silver (MWCNT-Chi-Ag) nanocomposite; wherein, the mass ratio of multi-walled nanotubes and bimetals in the nanocomposite is optimized by adjusting the amount of AgNO3 solution: the amounts of AgNO3 solution used are 1 mL, 2 mL, and 3 mL, corresponding to the mass ratios of MWCNT:Ag:Pt in the multi-walled carbon nanotube-chitosan-silver (MWCNT-Chi-Ag) nanocomposite of 30:1:0, 30:2:0, and 30:3:0, respectively.

[0054] 4) Bimetallic nanocomposites:

[0055] Take 30 mL each of the prepared 0.1 wt% chitosan (Chi) solution and the suspension of multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite with a concentration of 1 mg / mL. First, add 10 mmol / L K2PtCl4 solution and stir at room temperature, preferably for 3 hours. Then, add 10 mmol / L AgNO3 solution and continue stirring at room temperature, preferably for 2 hours. Then, place the stirred mixture in a 90℃ water bath to simultaneously carry out a one-step reduction reaction of Pt and Ag bimetals, preferably for 0.5 hours, to obtain stable solutions of chitosan-platinum / silver (Chi-Pt / Ag) and multi-walled carbon nanotube-chitosan-platinum / silver (MWCNT-Chi-Pt / Ag) nanocomposite, respectively.

[0056] The different mass ratios of multi-walled carbon nanotubes and bimetals in the nanocomposite were optimized by adjusting the amounts of K2PtCl4 solution and AgNO3 solution respectively: the amounts of K2PtCl4 solution:AgNO3 solution were selected as 2mL:1mL, 1mL:1mL, 1mL:2mL and 2mL:2mL respectively, which correspond to the mass ratios of MWCNT:Ag:Pt in the nanocomposite (MWCNT-Chi-Pt / Ag) as 30:1:1, 30:2:1, 30:1:2 and 30:2:2 respectively.

[0057] The preparation of bimetallic nanocomposites also requires addressing the order in which K2PtCl4 and AgNO3 are added. Previous studies investigated three methods: adding K2PtCl4 first and then AgNO3, adding K2PtCl4 and AgNO3 simultaneously, and adding AgNO3 first and then K2PtCl4. The results showed that when adding K2PtCl4 and AgNO3 solutions simultaneously, or when adding AgNO3 solution first and then K2PtCl4 solution, almost no metal Pt could be adsorbed onto the surface of chitosan-modified multi-walled carbon nanotubes (MWCNT-Chi). Only when K2PtCl4 solution was added first and then AgNO3 solution could both bimetallic Ag and Pt be adsorbed onto the surface of MWCNT-Chi.

[0058] Regarding the reduction reaction of bimetals, the aforementioned bimetallic nanocomposite employed a one-step reduction reaction. However, preliminary experiments, under the condition of adding K₂PtCl₄ first and then AgNO₃, simultaneously investigated both two-step and one-step reduction methods. If a two-step reduction is used—that is, first adding K₂PtCl₄ solution, stirring for 3 hours, and then heating to reduce to Pt, followed by adding AgNO₃ solution, stirring for 2 hours, and then heating to reduce Ag a second time—the resulting Pt / Ag bimetallic nanocomposite is prone to agglomeration, and the nanoparticles exhibit uneven particle size and unstable performance, making subsequent experiments impossible. Therefore, future research will preferentially employ a one-step reduction method to prepare the bimetallic nanocomposite MWCNT-Chi-Pt / Ag.

[0059] 5) Multi-antibody labeled nanocomposites:

[0060] Take 10 mL each of the prepared monometallic nanocomposites (MWCNT-Chi-Ag, MWCNT-Chi-Pt) and bimetallic nanocomposites (Chi-Pt / Ag and MWCNT-Chi-Pt / Ag), add 500 μL each of subgroup I avian adenovirus polyclonal antibody (FAdV / PAb) and 400 μL each of 5 wt% BSA solution, and react at 4°C for at least 8 hours (preferably overnight at 4°C). The next day, wash repeatedly by centrifugation with deionized water (10000 r / min, 10 min). Three experiments were conducted to obtain nanocomposites labeled with polyclonal antibodies at different mass ratios of MWCNT:Ag:Pt:MWCNT-Chi-Pt-FAdV / PAb, MWCNT-Chi-Ag-FAdV / PAb, Chi-Pt / Ag-FAdV / PAb, and MWCNT-Chi-Pt / Ag-FAdV / PAb. These polyclonal antibody-labeled nanocomposites were used as signal markers, and their amplification strength of the detection signal of the electrochemical immunosensor was compared to screen out the nanosignal amplification material with the best sensitivity.

[0061] 1.2 Morphology and Chemical Characterization of Chi Series Nanocomposites

[0062] The morphology of Chi series nanocomposites (including MWCNT-Chi, monometallic nanocomposites MWCNT-Chi-Pt and MWCNT-Chi-Ag, and bimetallic nanocomposites MWCNT-Chi-Pt / Ag) was analyzed by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS), and the elemental composition of the bimetallic nanocomposite MWCNT-Chi-Pt / Ag was analyzed by energy dispersive X-ray spectroscopy (EDS). Figure 1 The images show TEM images of Chi-series nanocomposites and EDS images of the bimetallic nanocomposites MWCNT-Chi-Pt / Ag, respectively. Figure 1 As can be seen from A, MWCNT-Chi has a tubular structure; Figure 1 B shows that the monometallic nanocomposite MWCNT-Chi-Pt is composed of Pt nanoparticles uniformly distributed on the surface of MWCNT-Chi. Figure 1 As can be seen from C, a large number of Pt / Ag nanoparticles are uniformly distributed on the surface of the MWCNT-Chi bimetallic nanocomposite MWCNT-Chi-Pt / Ag. The elemental composition of the MWCNT-Chi-Pt / Ag bimetallic nanocomposite was analyzed by energy dispersive X-ray spectroscopy (EDS), and the results are as follows: Figure 1 As shown in Figure D, EDS can detect three elements, C, Pt, and Ag, in MWCNT-Chi-Pt / Ag, proving that the bimetallic Pt and Ag have been successfully assembled onto the MWCNT-Chi surface.

[0063] Example 2: Preparation and electrochemical characterization of the working electrode in the avian adenovirus detection sensor for subgroup I.

[0064] 2.1 Preparation of the working electrode

[0065] 1) Processing electrode:

[0066] Glass carbon electrode (GCE) After polishing to a mirror finish with 0.05 μm Al2O3 polishing powder, the electrode was washed with distilled water, then ultrasonically cleaned in water, anhydrous ethanol, and water for 5 min each, and dried with N2. Cyclic voltammetry was then performed in a 0.5 mol / L H2SO4 solution (oxygen was removed by bubbling with N2 for 15 min before scanning) at a scan rate of 50 mV / s and a voltage range of -0.3 to +1.5 V. The scanning continued until the cyclic voltammogram stabilized. The electrode was then removed, washed with distilled water, and dried with N2 for later use, yielding an unmodified glassy carbon electrode (GCE).

[0067] 2) Electrode modified with multi-walled carbon nanotube-chitosan (MWCNT-Chi) nanocomposite:

[0068] Using a pipette, take an optimal amount of 10 μL of the MWCNT-Chi nanocomposite prepared above and coat it onto the surface of GCE. Let it air dry naturally at 4°C to obtain the electrode MWCNT-Chi-GCE.

[0069] 3) Monoclonal antibody fixation:

[0070] On the surface of the modified electrode MWCNT-Chi-GCE, 10 μL of 1 μg / mL subgroup I avian adenovirus monoclonal antibody (FAdV / MAb) was dropped onto the electrode and reacted at 4°C for at least 8 hours to obtain the monoclonal antibody modified electrode FADV / MAb-MWCNT-Chi-GCE.

[0071] 4) Enclosure:

[0072] Electrodes MWCNT-Chi-GCE and FADV / MAb-MWCNT-Chi-GCE, before and after monoclonal antibody immobilization, were immersed in 200 μL of 1 wt% BSA solution and sealed at 37°C for 1 h. They were then removed and washed with deionized water to obtain electrode BSA-MWCNT-Chi-GCE (i.e., electrode without monoclonal antibody immobilization) for preliminary evaluation of sensor specificity and working electrode BSA-FAdV / MAb-MWCNT-Chi-GCE for detection of subgroup I avian adenovirus.

[0073] 5) Double-antibody sandwich test:

[0074] First, 10 μL of the sample to be tested was dropped onto the working electrode BSA-FAdV / MAb-MWCNT-Chi-GCE and incubated for a sufficient time in a 37°C oven to obtain FADV-Ⅰ-BSA-FAdV / MAb-MWCNT-Chi-GCE; then, 10 μL of the above-prepared "multi-antibody labeled nanocomposite" (preferably MWCNT-Chi-Pt / Ag-FAdV / PAb) was dropped onto each nanocomposite as a signal marker and placed in a sealed humidified box. The reaction was carried out in an oven at 37℃ for 40 min. The sample was washed repeatedly with deionized water at least 3 times to remove unbound test samples and polyclonal antibody-labeled signal nanocomposite, yielding MWCNT-Chi-Pt / Ag-FAdV / PAb-FAdV-Ⅰ-BSA-FAdV / MAb-MWCNT-Chi-GCE. The sample was placed in an electrolyte for chronoamperometry (CA) scanning detection. After 50 s, 10 μL of H2O2 (10 M) was added.

[0075] The counter electrode was a platinum wire electrode, the reference electrode was a saturated calomel electrode, and the water used for cleaning was deionized water. The electrolyte was a PBS buffer containing 0.1M KCl, with a concentration of 0.01M and a volume of 10mL.

[0076] 2.2 Electrochemical characterization during sensor fabrication

[0077] Electrochemical impedance spectroscopy (EIS), also known as AC impedance spectroscopy, characterizes the impedance spectrum of current (voltage) signals generated by sinusoidal AC signal perturbations at a series of frequencies. It is commonly used in sensors to characterize the electrochemical signals on the surface of electrodes or nanocomposites. Specifically, when a semicircle appears in the high-frequency region of the EIS, the diameter of the semicircle reflects the electron-transfer resistance (Ret) of the electrode surface; a larger diameter indicates a larger Ret value, and vice versa. A straight line appears in the low-frequency region, reflecting the diffusion process on the electrode surface. Cyclic voltammetry (CV) characterization involves controlling different electrodes at different rates and repeatedly scanning the surface over time with a triangular waveform. This process generates current-voltage curves due to alternating reduction and oxidation reactions on the electrodes. The curves show a pair of redox peaks; the peak height and symmetry can characterize the adsorption activity and reversibility of different modifications on the electrode surface. Chronoamperometry (CA) is mainly used to characterize the relationship between current and time on the surface of different Chi series nanocomposites. Compared with CV, CA measures higher current peaks and has higher sensitivity.

[0078] Figure 2 Electrochemical characterization diagrams for different electrodes or Chi-series nanocomposites. Figure 2 Figures A through D show the electrochemical change curves and corresponding bar charts of different electrode surfaces during the sensor fabrication process.

[0079] in, Figure 2 The electrolyte used for the AC impedance diagrams A and B was a PBS buffer containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6 and 0.1 mM Cl. The concentration of the PBS buffer was 0.01 M and the pH was 7.4. Figure 2 In A, a small semicircle appears in the AC impedance diagram of the glassy carbon electrode (GCE) shown in a; when the GCE modified with MWCNT-Chi shown in b has a smaller diameter of the semicircle in its AC impedance diagram, the resistance of the modified electrode surface decreases due to the excellent conductivity of MWCNT-Chi, which improves the electron transport ability of the GCE surface after MWCNT-Chi modification, i.e., the Ret value decreases; when the monoclonal antibody (FAdV / MAb) is modified in c, the BSA is blocked in d, and the monoclonal antibody (FAdV-4 / MAb, 10) is modified in e, the resistance of the modified electrode surface decreases, i.e., the Ret value decreases; 3.43 EID 50 The EIS image after incubation ( / mL) shows that: since FAdV / MAb, BSA, and FAdV-4 have all been successfully modified on the electrode surface and hinder electron transfer on the electrode surface, the resistance increases sequentially, the Ret value increases sequentially, and the diameter of the c-e semicircle increases accordingly; correspondingly, Figure 2 In the bar chart of Ret values ​​in B, the Ret value shown in b is less than the Ret value shown in a, and the Ret values ​​from c to e increase sequentially.

[0080] Figure 2 The electrolyte used in the C and D cyclic voltammetry was a PBS buffer containing 5 mgM 4Fe(CN)6, 5 mgM 3Fe(CN)6 and 0.1 mgCl. The concentration of the PBS buffer was 0.01 M and the pH was 7.4. Figure 2 In C, the CV curve of GCE shown in a exhibits a pair of reversible redox peaks; GCE modified with MWCNT-Chi, as shown in b, also shows an increase in the current value of the corresponding redox peak due to the excellent conductivity of MWCNT-Chi, which enhances the electron transport ability of the GCE surface. When modified with monoclonal antibody (FAdV / MAb) as shown in c, blocked with BSA as shown in d, and with serum avian adenovirus type 4 (FAdV-4, 10... 3.43 EID 50The CV chromatogram after incubation ( / mL) shows that: since FAdV / MAb, BSA, and FAdV-4 have all been successfully modified on the electrode surface and hinder electron transfer on the electrode surface, the corresponding current values ​​decrease sequentially, causing the current values ​​of the redox peaks corresponding to c to e to also decrease sequentially; accordingly, Figure 2 In the bar chart of redox peaks in D, the current value shown in b is greater than the current value shown in a, and the current values ​​from c to e decrease sequentially.

[0081] therefore, Figure 2 The changes in Ret and current values ​​from A to D demonstrate that monoclonal antibodies FAdV / MAb, BSA, and FAdV-4 can all be successfully modified onto the surface of the electrode GCE.

[0082] Figure 2 E to F represent the working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE) prepared using the same batch of immunosensor prepared during CA characterization, along with 10... 3.43 EID 50 After incubating with FADV-4 at a concentration of / mL for a certain period of time, the nanocomplexes were then incubated with a blank control (100μL PBS buffer) and Chi series nanocomplexes labeled with polyclonal antibodies for a certain period of time: monometallic MWCNT-Chi-Pt-FAdV / PAb, MWCNT-Chi-Ag-FAdV / PAb, bimetallic Chi-Pt / Ag-FAdV / PAb, and MWCNT-Chi-Pt / Ag-FAdV / PAb. After each incubation, the nanocomplexes were placed in 10mL of PBS buffer (0.01M, pH 7.4) containing 0.1MKCl for chronoamperometry scanning. At approximately 50 seconds, 5μL of H2O2 (10mM) was added as the test substrate. The CA test results are as follows: Figure 2 E and Figure 2As shown in F: When the working electrode of the sensor shown in a was incubated with the blank control, the current showed only a slight change upon the addition of H2O2, which can be considered as a background signal; when the subsequent portions of the working electrodes of the sensors shown in b to e were incubated with the multi-antibody-labeled nanocomposites MWCNT-Chi-Pt-FAdV / PAb, MWCNT-Chi-Ag-FAdV / PAb, Chi-Pt / Ag-FAdV / PAb, and MWCNT-Chi-Pt / Ag-FAdV / PAb, the current showed a significant increase upon the addition of H2O2; among them, especially The bimetallic nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb (i.e., bimetallic nanoparticles loaded on chitosan-modified multi-walled carbon nanotubes) shown in e, used as a signal marker, exhibited the highest response current value in the CA diagram after incubation with the sensor's working electrode. Furthermore, the response current value shown in e was 1.91, 1.57, and 1.30 times higher than those shown in b, c, and d, respectively. Therefore, this invention preferably uses the MWCNT-Chi-Pt / Ag-FAdV / PAb nanocomposite shown in e as the signal marker for the double-antibody sandwich method in the electrochemical immunosensor. Additionally, the electrode BSA-MWCNT-Chi-GCE, which was not immobilized with monoclonal antibody FADV / MAb, and 10... 3.43 EID 50 After incubation with / mLFAdV-4, subsequent incubation with the preferred signal marker MWCNT-Chi-Pt / Ag-FAdV / PAb containing polyclonal antibodies is not performed. Electrode BSA-MWCNT-Chi-GCE, as shown in g, is first incubated with 10 3.43 EID 50 After incubation with / mLFAdV-4, it was further incubated with the preferred signal marker MWCNT-Chi-Pt / Ag-FAdV / PAb for a certain period of time. After treatment, the electrodes shown in f and g were placed in 10mL PBS (0.01M, pH 7.4) test solution for chronoamperometry scanning. At about 50 seconds, 5μL H2O2 (10mM) was added. Figure 2 E and Figure 2 The CA test results in F show that the current values ​​shown in f and g are similar to the response current value shown in a. The response current value is so small that it can be ignored, indicating that the sensor has good specificity.

[0083] Example 3: Optimization of conditions for electrochemical immunosensor detection method of subgroup I avian adenovirus

[0084] Condition optimization is an essential research process for any detection reagent. Based on the results of previous complex multi-factor research and analysis, this paper briefly describes the verification experiments of single-factor condition optimization, which are mainly based on the following representative optimization conditions, in the electrochemical immunosensor detection method for subgroup I avian adenovirus of this invention.

[0085] 3.1 Optimization of different mass ratios of multi-walled nanotubes and bimetals

[0086] Using the same batch of prepared working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE), and 10 3.43 EID 50 / mLFAdV-4 was incubated for 30 min; the nanocomposites MWCNT-Chi-Pt-FAdV / PAb, MWCNT-Chi-Ag-FAdV / PAb, and MWCNT-Chi-Pt / Ag-FAdV / PAb prepared in step 5) of Example 1 above with different mass ratios (MWCNT:Ag:Pt = 30:0:1, 30:0:2, 30:0:3, 30:1:0, 30:2:0 and 30:3:0, 30:1:1, 30:2:1, 30:1:2 and 30:2:2) were used as signal markers and incubated for 40 min. Then, they were placed in 10 mL of PBS (0.01 M, pH 7.4) test solution for chronoamperometry scanning, and 5 μL of H2O2 (10 mM) was added at approximately 50 sec. The results are as follows: Figure 3 As shown, when monometallic MWCNT-Chi-Pt-FAdV / PAb and MWCNT-Chi-Ag-FAdV / PAb are used as signal markers, the electrochemical signal increases with increasing Pt and Ag content. Under the same metal content, the current value of the bimetallic MWCNT-Chi-Pt / Ag-FAdV / PAb response is significantly higher than that of monometallic MWCNT-Chi-Pt-FAdV / PAb and MWCNT-Chi-Ag-FAdV / PAb responses, proving that the catalytic ability of bimetallic (Pt / Ag) for H2O2 is much higher than that of monometallic (Pt or Ag). Furthermore, from... Figure 3 It can be seen that among the different mass ratios of MWCNT:Ag:Pt, the highest response current value is MWCNT:Ag:Pt=30:1:2, that is, the optimal mass ratio in the preferred nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb is MWCNT:Ag:Pt=30:1:2.

[0087] 3.2 Optimization of incubation time for test samples

[0088] Using the same batch of prepared working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE), with 10 3.43 EID 50 / mLFAdV-4 simulates the test sample, and FAdV-4 (10) is dropped onto the working electrode. 3.43 EID 50After incubating at 37°C for 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min respectively, the sample was then incubated with the preferred MWCNT:Ag:Pt = 30:1:2 nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb at 37°C for 60 min. The sample was then placed in 10 mL of the preferred pH 7.4 PBS for CA testing. 5 μL of H2O2 (10 mol / L) was added at 50 s to bring the H2O2 concentration to 5 mM. The relationship between the peak response current and time for different incubation times was obtained as follows: Figure 4 As shown: when the incubation time of the sample to be tested is in the range of 5 min to 30 min, the peak value of the response current measured by the working electrode of the sensor increases continuously with the extension of time; when the incubation time of the sample to be tested exceeds 30 min, the peak value of the response current measured by the working electrode of the sensor basically reaches a stable value, and the response current value does not change much.

[0089] 3.3 Optimization of incubation time for multi-antibody-labeled nanocomposites

[0090] Using the same batch of prepared working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE), and 10 3.43 EID 50 The sample was incubated with FADV-4 at a concentration of 1 / mL for 30 min. Then, it was incubated with the aforementioned preferred MWCNT:Ag:Pt = 30:1:2 nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb, which served as both the secondary antibody in the double antibody sandwich and a signal marker, at 37°C for 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. After incubation, the sample was placed in 10 mL of the aforementioned preferred pH 7.4 PBS for CA testing. At 50 s, 5 μL of H2O2 (10 mol / L) was added to bring the H2O2 concentration to 5 mM. The resulting curves showing the relationship between the peak response current and time for different incubation times are shown below. Figure 5 As shown: when the incubation time of the secondary antibody signal marker is in the range of 5 min to 40 min, the peak value of the response current increases continuously with the extension of time; when the incubation time of the sample exceeds 40 min, the peak value of the response current measured by the working electrode of the sensor basically reaches a stable value, and the response current value does not change much.

[0091] In the sensor detection process of this invention, the incubation time of the test sample (such as the positive test sample FAdV-Ⅰ or the simulated sample FAdV-4 in the optimization experiment), the working electrode, and the MWCNT-Chi-Pt / Ag-FAdV / PAb nanocomposite as a secondary antibody and signal marker all have a significant impact on the subsequent signal amplification of the sensor. Therefore, in the subsequent performance verification experiments, the incubation time of the test sample is preferably 30 min, and the incubation time of the signal marker (MWCNT-Chi-Pt / Ag-FAdV / PAb nanocomposite) is preferably 40 min.

[0092] 3.4 Optimization of pH value of the test solution

[0093] Using the same batch of prepared working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE), and 10 3.43 EID 50 Incubate with FADV-4 for 30 min; then incubate with the above-mentioned preferred MWCNT:Ag:Pt = 30:1:2 nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb for 40 min. Finally, place the mixture in 10 mL of 0.01 M PBS test solution containing 5 mL of H₂O₂ at different pH values ​​(pH = 6.0–8.0) for CA testing. The results are as follows: Figure 6 As shown: when the pH value of the PBS test solution is 6.0 to 7.4, the response current value increases with increasing pH value; when the pH value of the PBS test solution is 7.4 to 8.0, the response current value decreases with increasing pH value. Therefore, the pH value of the PBS test solution is preferably 7.4. In the subsequent performance verification experiment, the PBS test solution with pH value of 7.4 was used.

[0094] 3.5 Optimization of H2O2 concentration in the test substrate

[0095] Using the same batch of prepared working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE), and 10 3.43 EID 50 Incubate with 10 mL of FADV-4 for 30 min; then incubate with the above-mentioned preferred MWCNT:Ag:Pt = 30:1:2 nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb for 40 min. First, place it in 10 mL of the above-mentioned preferred pH 7.4 0.01 MPa BS test solution for CA testing. Add 1 μL of H2O2 (10 mol / L) to the test solution every 100 s, increasing the H2O2 concentration from 1 mM to 8 mM. The CA graph and the bar graph of the response current values ​​corresponding to different H2O2 concentrations are shown below. Figure 7As shown, when the concentration of H2O2 increases from 1 mM to 5 mM, the corresponding response current value also gradually increases with the increase of H2O2 concentration; when the concentration of H2O2 reaches 5 mM, its response current value reaches a basically constant high value. Therefore, it is preferable that the concentration of H2O2 in the test substrate is 5 mM.

[0096] Example 4: Performance evaluation of the electrochemical immunosensor for subgroup I avian adenovirus

[0097] 4.1 Sensitivity

[0098] The sensor sensitivity experiment was conducted as follows: Based on the optimized electrochemical immunosensor detection platform for subgroup I avian adenovirus established above, the sample preparation was as follows: a known concentration of 10... 4.43 EID 50 FAdV-4 virus solution of / mL was serially diluted 5-fold, and 10μL of each dilution was taken as a simulated test sample. These different concentrations of FAdV-4 virus solutions (10 μL / mL) were then used to simulate the test sample. 0.93 EID 50 / mL, 10 1.43 EID 50 / mL, 10 1.93 EID 50 / mL, 10 2.43 EID 50 / mL, 10 2.93 EID 50 / mL, 10 3.43 EID 50 / mL, 10 3.93 EID 50 The negative control sample was PBS. Simulated detection was performed using the sensor working electrode (BSA-FAdV / MAb-MWCNT-Chi-GCE) prepared in the same batch and the optimized detection method described above, to verify the sensor's sensitivity. The detection results are as follows: Figure 8 As shown: The linear equation between the CA plots of different concentrations of FAdV-4 virus solution and their corresponding response current values ​​is I(mA) = 2.1521lgEID 50 / mL-1.1852, R=0.9916, the limit of detection is 10 0.67 EID 50 .

[0099] 4.2 Specificity

[0100] Twenty-four samples were selected for testing, including six virus preservation solutions (IBV, CIA) for non-subgroup I avian adenovirus. The following samples were used as test samples: 12 avian adenoviruses classified as subgroup I (FAdV, NDV, EDSV, ARV, and AIVH9), common components in three virus culture media (1.0 μg / mL BSA, 1.0 μg / mL glucose, and 1.0 μg / mL vitamin C), preservation media of FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, and FAdV-11), and three mixed samples containing FADV-4 (FAdV-4+NDV, FADV-4+ARV, and FADV-4+BSA). PBS buffer was used as the negative control. The same batch of working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE) and the optimized detection method were used to verify the sensor's specificity. The results are as follows: Figure 9 As shown: Compared with the negative control, when the sensor working electrode (BSA-FAdV / MAb-MWCNT-Chi-GCE) was incubated with 9 non-FAdV-I test samples, the response current value of CA detection remained almost unchanged; when the sensor working electrode (BSA-FAdV / MAb-MWCNT-Chi-GCE) was incubated with 12 FAdV-I test samples, the response current value of CA detection increased significantly, indicating that the sensor has good specificity.

[0101] 4.3 Repeatability

[0102] Five batches of the immunosensor working electrodes (BSA-FAdV / MAb-MWCNT-Chi-GCE) of this invention were prepared. Six working electrodes from each batch were randomly selected for repeated detection experiments. The test samples were selected with a known concentration of 10. 4.43 EID 50 The working electrode and the test sample were incubated with FAdV-4 virus solution ( / mL) and PBS buffer (PBS buffer) as negative controls for 30 min each. This process was used to verify the repeatability of the sensor. The results are as follows: Figure 10 As shown, compared with the negative control, the standard deviation of both inter-batch and intra-batch repeatability is less than 5%, indicating that the sensor has good repeatability.

[0103] 4.4. Clinical Sample Consistency Verification

[0104] Using the electrochemical immunosensor detection platform for subgroup I avian adenovirus established in this study, 100 samples from suspected sick chickens collected in Guangxi were tested. The results showed that 7 samples were FAdV-I positive and 93 samples were FAdV-I negative. Simultaneously, nucleic acid was extracted from the 7 positive samples using a nucleic acid detection system (PCR method) and sequenced for analysis, revealing 45 samples to be FAdV-I and 2 samples to be FAdV-8a. The positive results of this invention are consistent with the sequencing results, proving that the positive concordance rate between the electrochemical immunosensor for subgroup I avian adenovirus described in this invention and the traditional nucleic acid test results can reach 100%.

[0105] This invention specifically provides a method for detecting avian adenovirus subgroup I (FAdV-I) using a double-antibody sandwich electrochemical immunosensor, where a multi-walled carbon nanotube-chitosan-platinum-silver (MWCNT-Chi-Pt / Ag) nanocomposite serves as both a secondary antibody and a nano-signal amplification material. The working electrode also utilizes MWCNT-Chi nanotubes; a glassy carbon electrode is first modified with MWCNT-Chi, followed by adsorption of the FADV-I monoclonal antibody to construct a working electrode containing the antibody. The invention optimizes the mass ratio of the three components in the MWCNT-Chi nanotubes and bimetallic compound, the incubation time of the sample / signal-labeled nanocomposite containing the secondary antibody, the pH of the test solution, and the H2O2 concentration. Under optimized conditions, the limit of detection for FADV-I virus is 10⁻⁶. 0.67 EID 50 The sensor exhibits high sensitivity, versatility, and specificity, with good batch-to-batch and intra-batch reproducibility in its preparation process. Furthermore, sequencing results from positive clinical samples demonstrate that the electrochemical immunosensor described in this invention possesses good versatility and specificity, with a 100% positive concordance rate between positive results and nucleic acid sequencing results.

[0106] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, similar modifications or substitutions can be made to the specific details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An electrochemical immunosensor, characterized by, The sensor is a three-electrode system, comprising a reference electrode, a counter electrode and a working electrode, and further comprising a signal marker prepared from a multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nano composite; the working electrode is BSA-FAdV / MAb-MWCNT-Chi-GCE, and the sensor is used for detecting subgroup I avian adenovirus; wherein the working electrode is prepared from a multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite, and the preparation method of the working electrode comprises the following steps: 1) electrode treatment: polishing the glassy carbon electrode with polishing powder Al2O3, washing with water, ultrasonic cleaning, scanning with cyclic voltammetry until stable, and then blowing dry with N2; 2) multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite modified electrode: dropping the suspension of the multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite on the surface of the electrode, and naturally air-drying at room temperature; 3) monoclonal antibody fixation: again dropping and coating subgroup I avian adenovirus monoclonal antibody FAdV / MAb, and fixing the reaction at 4 DEG C for at least 8 hours; 4) blocking: after antibody fixation, dropping and coating BSA solution at 37 DEG C for 1 hour, then taking out and washing with water, to obtain the working electrode BSA-FAdV / MAb-MWCNT-Chi-GCE; The preparation of the multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nano composite comprises the following steps: 1) chitosan Chi solution: taking chitosan Chi in acetic acid solution, stirring at room temperature for 0.5 hours, then fully mixing and dissolving to obtain chitosan Chi solution; wherein 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 Chi solution is 0.1wt%; 2) multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite: taking multi-walled carbon nanotube MWCNT and adding to the chitosan Chi solution, and after ultrasonic treatment for 2 hours, a stable suspension of multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite is obtained; wherein the concentration of the suspension of the multi-walled carbon nanotube-chitosan MWCNT-Chi nano composite is 1mg / mL; 3) multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nano composite: taking 30mL of the suspension of the multi-walled carbon nanotube-chitosan MWCNT-Chi, first adding 10mmol / L K2PtCl4 solution, stirring at room temperature for 3 hours, then adding 10mmol / L AgNO3 solution, continuing to stir at room temperature for 2 hours, and then placing in a 90 DEG C water bath for one-step reduction reaction for 0.5 hours, to obtain a stable solution of the multi-walled carbon nanotube-chitosan-platinum / silver MWCNT-Chi-Pt / Ag nano composite; The signal marker is a multi-antibody labeled nanocomposite MWCNT-Chi-Pt / Ag-FAdV / PAb, which is prepared by fixing reaction of the multi-wall carbon nanotube-chitosan-platinum / silver nanocomposite MWCNT-Chi-Pt / Ag, the multi-antibody FAdV / PAb of subgroup I avian adenovirus and 5wt% BSA at 4℃ for at least 8 hours.

2. The electrochemical immunosensor according to claim 1, wherein: In the multi-wall carbon nanotube-chitosan-platinum / silver nanocomposite MWCNT-Chi-Pt / Ag, the mass ratio of MWCNT:Ag:Pt is 30:1:

2.

3. The electrochemical immunosensor according to claim 1, wherein: The reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.

4. A method for the detection of a Group I avian adenovirus, characterised in that: The electrochemical immunosensor described in any one of claims 1-3 is used, the sample to be measured is drop-coated on the working electrode and incubated at 37℃ for 30 minutes, the signal marker is drop-coated and placed in a closed humidifying box, and then incubated at 37℃ for 40 minutes, and then washed with water for at least 3 times, placed in an electrolyte and detected by chronoamperometric scanning, and H2O2 is added as a test substrate after 50 seconds; The detection method does not include diagnosis and treatment methods of diseases.

5. A method for the detection of a Group I avian adenovirus according to claim 4 characterised in that: The electrolyte is 0.01M PBS buffer containing 0.1MKCl, the pH value is 7.4, and the concentration of H2O2 in the test substrate is 5mM.

6. The electrochemical immunosensor described in any one of claims 1-3 is applied in detection of subgroup I avian adenovirus product preparation, and the application does not include diagnosis and treatment methods of diseases.

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