Multi-walled carbon nanotubes and their applications in electrochemical immunosensors

Abstract

The present application discloses a multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite prepared from multi-walled carbon nanotubes, and used for the detection of fowl adenovirus type 4 (FAdV-4) electrochemical immunosensor. The gold electrode is modified with MWCNT-Chi-PtNP, and then anti-FA dV-4 monoclonal antibody is adsorbed to construct an electrochemical immunosensor for FAdV-4. The incubation time of the sample to be tested and the pH value of the electrolyte are optimized. The sample to be tested can be incubated for only 30 minutes before the electrochemical impedance spectroscopy (EIS) scanning detection is performed. The final minimum detection limit of the FAdV-4 virus content is 10 1.89 EID 50 / mL, detection range is 10 1.46 EID 50 / mL~10 5.46 EID 50 / mL, the detection sensitivity was increased by 37 times, the specificity was stronger, and the preparation process had good inter-batch and intra-batch repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials and veterinary biological products, and in particular to a nanocomposite prepared from multi-walled carbon nanotubes and precious metal platinum, which is particularly applicable to electrochemical immunosensors for detecting avian adenovirus, and in particular to a resistive electrochemical immunosensor prepared from multi-walled carbon nanotubes. Background Art

[0002] Fowl adenovirus (FAdV) is a non-enveloped double-stranded DNA virus belonging to the genus Adenovirus of the family Adenoviridae. According to the molecular structure of the serotype and the results of serum cross-neutralization experiments, FAdV can be subdivided into 12 serotypes (FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11). Different serotypes have different abilities to cause morbidity and mortality / or respiratory diseases. Among them, FAdV-4 is one of the dominant serotypes prevalent in chickens in my country. It is also the main pathogen causing hydropericardium-hepatitis syndrome, mainly infecting broilers aged 3 to 6 weeks, and is highly contagious and pathogenic. In recent years, FAdV-4 has been prevalent in Shandong, Hebei, Henan, Jiangsu, Shaanxi and other places in my country, causing huge economic losses to the poultry farming industry.

[0003] At present, the diagnostic detection methods for FAdV-4 include virus isolation and identification, PCR-based nucleic acid detection methods, enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion test (AGP), etc. These methods all have certain limitations: the accuracy of virus isolation and identification is relatively high, but it requires the virus to be cultured and the virus morphology to be observed by transmission electron microscopy, which is time-consuming; the PCR-based nucleic acid detection method has a high sensitivity, but the operation is relatively cumbersome (it requires extraction of viral nucleic acid, PCR amplification, electrophoresis detection and other steps), requires close laboratory equipment and professional operators, and the detection cost and dependence on specialized instruments and equipment are very high; although serological diagnostic methods such as ELISA and AGP do not rely on expensive instruments, they have the disadvantages of low sensitivity, long time consumption, difficulty in standardization, and inability to quickly and timely judge the negative / positive results of FAdV-4 detection.

[0004] Electrochemical immunosensor is a new detection technology that combines electrochemical analysis method and immunoanalysis method. In recent years, electrochemical immunosensors have been successfully applied in the fields of national defense security, environmental analysis, food analysis and clinical medicine. The principle of electrochemical immunosensor is: through physical and chemical methods, the sensitive element 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. Electrochemical immunosensor is a product of the intersection of chemistry, materials science and life sciences. It has the advantages of fast detection, simple operation, high sensitivity, good specificity, low cost, and miniaturization of instruments.

[0005] Based on the advantages of electrochemical immunosensor technology itself, researchers have also used various signal amplification technologies to continuously improve the sensitivity of electrochemical immunosensors. Among them, "nanomaterial amplification technology" is one of the common signal amplification strategies for electrochemical immunosensors. Nanomaterials, due to their unique physical and chemical properties (such as small size effect, quantum size effect, surface effect, macroscopic quantum tunneling effect, etc.), can not only be used to fix biological molecules, but also as a signal amplification material, providing a good platform for building electrochemical immunosensors with excellent performance. Usually, electrochemical immunosensors use a series of advantages of nanomaterials such as good biocompatibility, large specific surface area, and strong conductivity to achieve an effective amplification detection signal strategy and significantly improve the various properties of the electrochemical immunosensor sensing interface. Multi-walled carbon nanotubes (MWCNT) can be used as sensor electrode modification materials for the preparation of electrochemical sensors because of their good conductivity, electromagnetic properties, and adsorption properties. However, there are still a series of problems such as complex preparation, low sensitivity, and excessively high detection limits. Summary of the invention

[0006] The object of the present invention is to at least partially overcome the defects of the prior art and provide a sensor that is fast in detection, simple in operation, high in sensitivity, good in specificity, low in cost, and miniaturizable in instrument, and is conducive to the on-site rapid detection of avian adenovirus serotype 4.

[0007] The present invention also aims to improve the application of multi-walled carbon nanotubes in the field of electrochemical immunosensors, provide a new nanocomposite prepared from multi-walled carbon nanotubes, and improve its sensitivity and detection limit in electrochemical sensor detection.

[0008] In order to achieve the above purpose or one of the purposes, the present invention provides the following technical solutions:

[0009] A multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite, the preparation method of which comprises the following steps: taking chitosan (Chi) in an acetic acid solution, stirring at room temperature for 0.5h and fully mixing and dissolving to obtain a chitosan (Chi) solution; wherein the acetic acid solution is a mixed solution of glacial acetic acid and water in a volume ratio of 1.0%, and the concentration of the obtained chitosan (Chi) solution is 0.1wt%. Taking multi-walled carbon nanotubes (MWCNT) and adding them to the chitosan (Chi) solution, and ultrasonicating for 2h to obtain a stable multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension; wherein the concentration of the multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension is 1mg / mL. Take 10mmol / L K2PtCl4 solution and add it to the above-mentioned multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension. After stirring at room temperature for 6 hours, heat it to 90°C in a water bath and continue to react for 1 hour to obtain a stable solution of multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite; wherein the volume ratio of K2PtCl4 solution to multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension is 1:10.

[0010] The present invention claims protection for an electrochemical immunosensor, which is a three-electrode system, including a working electrode, a reference electrode and a counter electrode, wherein the working electrode is prepared by a multi-walled carbon nanotube-chitosan-platinum nanocomposite (MWCNT-Chi-PtNP), the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.

[0011] According to a preferred embodiment of the present invention, the electrochemical immunosensor is used to detect fowl adenovirus type 4, and the preparation method of the working electrode thereof comprises the following steps: polishing the electrode with polishing powder Al2O3, washing with water, and ultrasonically cleaning, scanning with cyclic voltammetry until stable, and then drying with N2; dripping a stable solution of multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite on the surface of the electrode, and drying naturally at room temperature; then dripping a monoclonal antibody (MAb / FAdV-4) of fowl adenovirus type 4, and fixing and reacting at 4°C for 8h; after the antibody fixation reaction, dripping a BSA solution, sealing in a 37°C incubator for 1h, taking out and washing with water, and obtaining a working electrode that can be used to detect fowl adenovirus type 4.

[0012] According to a preferred embodiment of the present invention, the electrode is a gold electrode.

[0013] The present invention claims protection for a method for detecting avian adenovirus type 4, using the electrochemical immunosensor prepared as above, drop-coating the sample to be tested on the working electrode, incubating the reaction at 37°C for a certain period of time, then washing with water at least 3 times, and placing it in an electrolyte for electrochemical impedance spectroscopy (EIS) scanning detection.

[0014] According to a preferred embodiment of the present invention, in the detection method, the incubation reaction time at 37° C. is 30 minutes.

[0015] According to a preferred embodiment of the present invention, in the detection method, the electrolyte is a PBS buffer containing 5mM K4Fe(CN)6, 5mM K3Fe(CN)6 and 0.1M KCl, and the concentration of the PBS buffer is 10mM and the pH value is 7.0.

[0016] The present invention claims a detection kit for avian adenovirus type 4, comprising an electrolyte and the above-mentioned electrochemical immunosensor, wherein the detection linear range of the detection kit for avian adenovirus type 4 is 10 1.46 EID 50 / mL~10 5.46 EID 50 / mL, the minimum detection limit is 10 1.89 EID 50 / mL.

[0017] According to a preferred embodiment of the present invention, in the detection kit for avian adenovirus type 4, the electrolyte is a PBS buffer containing 5mM K4Fe(CN)6, 5mM K3Fe(CN)6 and 0.1M KCl, the concentration of the PBS buffer is 10mM and the pH value is 7.0. During the detection, the electrochemical immunosensor and the sample to be tested are incubated at 37°C for 30 minutes.

[0018] The present invention claims protection for the use of a multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite, an electrochemical immunosensor, and a detection kit for avian adenovirus type 4 in preparing a product for detecting avian adenovirus type 4.

[0019] The beneficial effects of the present invention are:

[0020] The applicant of the present invention is committed to continuously deepening and refining the electrochemical sensor platform. After a lot of preliminary research, he unexpectedly obtained the multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite described in this application, applied it to electrochemical sensors, and used it to detect avian adenovirus type 4.

[0021] 1) The present invention also establishes a methodological comparison and verification platform: a set of primers is designed, that is, a primer set that can specifically amplify positive FAdV-4, and the PCR reaction conditions are optimized, such as the annealing temperature and the amount of primers, thereby establishing a nucleic acid detection platform that can be used for parallel comparison and evaluation of the multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomplex described in the present application in detecting avian adenovirus type 4.

[0022] 2) Based on the above objectives, the present invention provides a multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite prepared from multi-walled carbon nanotubes (MWCNT) and precious metal platinum (Pt), and optimizes the preparation method of the multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite; for the first time, the electrode GE is modified with the MWCNT-Chi-PtNP nanocomposite to adsorb the monoclonal antibody MAb / FAdV-4, and a sensor prepared from the MWCNT-Chi-PtNP nanocomposite, a detection method and a detection kit for detecting serotype 4 of avian adenovirus are provided, and unexpectedly, it is found that the electrochemical immunosensor is particularly suitable for identifying serotype 4 of avian adenovirus, and has higher detection sensitivity, stronger specificity and good repeatability than the traditional PCR method; compared with the conventional PCR method (sensitivity of 10 3.46 EID 50 / mL), the minimum detection limit of the immunosensor for FAdV-4 is 10 1.89 EID 50 / mL, detection range is 10 1.46 EID 50 / mL~10 4.46 EID 50 / mL, compared with the traditional PCR detection, the detection sensitivity of the electrochemical immunosensor of the present invention is relatively improved by 37 times. The electrochemical immunosensor of the present invention and the traditional PCR method test results were verified with 36 clinical samples, and the positive and negative coincidence rate of the two methods can reach 100%.

[0023] 3) The present invention optimizes the preparation method of multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite. On the basis of using multi-walled carbon nanotubes (MWCNT), it further combines the precious metal platinum (Pt), and finally forms a new MWCNT-Chi-PtNP nanocomposite with good biocompatibility and excellent conductivity, and can further realize the amplification of the detection electrochemical signal. The present invention first disperses the multi-walled carbon nanotubes (MWCNT) evenly in the chitosan (Chi) solution, adds K2PtCl4, and heats the Pt at 90°C. 2+ Reduced to platinum nanoparticles (PtNP), and finally obtained multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite. The study unexpectedly found that Pt 2+The stirring time is the most important in the reduction process. If the stirring time is too short or too long, it is difficult to obtain a MWCNT-Chi-PtNP nanocomposite with uniform particle size and good loading. Therefore, the stirring time was optimized. When the stirring time was 2h and 4h, the loading of Pt nanoparticles on MWCNT-Chi was small. When the stirring time was 6h, the loading of Pt nanoparticles on MWCNT-Chi was rich. When the stirring time was 8h, the particle size of Pt nanoparticles on MWCNT-Chi was uneven. Therefore, 6h was preferred for Pt 2+ The optimal stirring time during the reduction process. It can be said that in the preparation method of the MWCNT-Chi-PtNP nanocomposite, the final stirring and combining step is an important technical point of the present invention, and also lays the foundation for the subsequent improvement of the sensitivity and specificity of the sensor.

[0024] 4) The present invention applies multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomplexes to prepare electrochemical immunosensors for detecting avian adenovirus type 4, and establishes an electrochemical immunosensor detection platform for avian adenovirus type 4. On the one hand, the incubation time of the sample to be tested is optimized, and the sample to be tested only needs to be incubated for 30 minutes, which effectively shortens the detection time; on the other hand, it is found that the optimal pH value of the electrolyte is 7.0, which is conducive to further improving the detection sensitivity and specificity. The present invention is conducive to providing a new type of rapid detection technology for the prevention and control of avian adenovirus type 4 (FAdV-4), and is conducive to enhancing the scientific prevention and control level for the healthy development of poultry farming.

[0025] 5) The present invention further determines the negative and positive judgment criteria of the electrochemical immunosensor for detecting FAdV-4 by avian adenovirus type 4 through a large number of experiments. The present invention detects 15 FAdV-4 negative samples and obtains that the positive and negative critical point of the electrochemical immunosensor for detecting avian adenovirus type 4 described in the present invention is 2070Ω, which is conducive to the promotion of electrochemical immunosensors in the standardization of avian virus detection and further improves the standardization of avian virus detection.

[0026] 6) The present invention proposes for the first time to use multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposites for the preparation of electrochemical immunosensors for avian adenovirus type 4. Not only is the preparation method simple, the judgment criteria are established, and the results of the detection method are standardized and easy to control, but it also has the advantages of rapid detection, high sensitivity, good specificity, low cost, and miniaturization of instruments, and is suitable for on-site prevention and control detection in poultry farming. By optimizing the detection method of avian adenovirus type 4, the novel and rapid resistive electrochemical immunosensor constructed by the present invention is used for the specific detection of FAdV-4, which can provide strong technical support for the prevention and control of FAdV-4. At the same time, it also provides a new idea for the research of rapid detection and diagnosis technologies for other avian infectious diseases and animal diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph of agarose gel electrophoresis results of the nucleic acid detection system, wherein A is a graph of annealing temperature optimization results, and the annealing temperatures are: 1 is 60.0°C, 2 is 59.2°C, 3 is 58.0°C, 4 is 56.1°C, 5 is 53.8°C, 6 is 51.9°C, 7 is 50.7°C, and 8 is 50.0°C; B is a graph of optimization results of primer volume usage; the primer volumes are: 1 is 0.2μL, 2 is 0.4μL, 3 is 0.6μL, 4 is 0.8μL, 5 is 1.0μL, 6 is 1.2μL, 7 is 1.4μL, 8 is 1.6μL, 9 is 1.8μL, and 10 is 2.0μL; and in A and B: N is a negative control, and M is a DNA ladder;

[0028] Figure 2 Electron microscope images of different stirring times during the preparation of MWCNT-Chi-PtNP nanocomposites, where the stirring times are A for 0 h, B for 2 h, C for 4 h, D for 6 h, and E for 8 h;

[0029] Figure 3 The bar graphs are the electrochemical impedance spectra and Ret values ​​of the electrodes or electrodes modified with different nanocomposites, where a is GE, b is GE-MWCNT-Chi-PtNP, c is GE-MWCNT-Chi-PtNP-MAb / FAdV-4, d is GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA, and e is GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA-FAdV-4;

[0030] Figure 4 The Ret value variation trend diagram of the electrochemical impedance spectrum of electrolytes with different pH values;

[0031] Figure 5 The Ret value variation trend diagram of the electrochemical impedance spectrum measured at different incubation times of the tested samples;

[0032] Figure 6 The electrochemical immunosensor of the present invention for fowl adenovirus type 4 and the comparison of the sensitivity test results of nucleic acid detection are shown in FIG. A and B are the electrochemical impedance spectrum of the electrochemical immunosensor for fowl adenovirus type 4 of the present invention and the linear relationship between the detection Ret value and the FAdV-4 content, respectively. a to f represent different concentration values ​​of the positive virus liquid FAdV-4: a is 0, b is 10 1.46 EID 50 / mL, c is 10 2.46 EID 50 / mL, d is 10 3.46EID 50 / mL, e is 10 4.46 EID 50 / mL, f is 10 5.46 EID 50 / mL; C is the agarose gel electrophoresis diagram of the nucleic acid detection system, and the sensitivity of the nucleic acid detection system is compared in parallel. 1 to 5 represent different concentration values ​​of the positive virus liquid FAdV-4: 1 is 10 5.46 EID 50 / mL, 2 is 10 4.46 EID 50 / mL, 3 is 10 3.46 EID 50 / mL, 4 is 10 2.46 EID 50 / mL, 5 is 10 1.46 EID 50 / mL, N is negative control, M is DNA ladder;

[0033] Figure 7 A bar graph showing the detection results of a specificity experiment of the electrochemical immunosensor for avian adenovirus type 4 of the present invention;

[0034] Figure 8 This is a bar graph showing the test results of the repeatability experiment of the electrochemical immunosensor for avian adenovirus type 4 of the present invention, wherein 1 to 5 represent sensors produced in 5 batches respectively. DETAILED DESCRIPTION

[0035] Below in conjunction with practical application, exemplary embodiments of the present invention are described in detail. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the disclosed embodiments. The following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. However, it is obvious that one or more embodiments may also be implemented without these specific details. In the embodiment, those without specifying specific conditions are carried out according to the conditions recommended by normal conditions or manufacturers.

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified. Among them, multi-walled carbon nanotubes (MWCNT), K2PtCl4 and bovine serum albumin (BSA) were purchased from Sigma; KMnO4, K4Fe(CN)6, K3Fe(CN)6, H2SO4, CH3CH2OH, chitosan (Chi, also known as chitosan) and the like were all domestic analytical grade, Premix Taq (Ex TaqVersion 2.0Plus dye), 100bp DNA Ladder were purchased from TaKaRa, EasyPure Genomic DNA Kit (DNA / RNA extraction kit) was purchased from Quanshijin Biotechnology Co., Ltd. (Beijing), and the reverse transcription kit was purchased from Bao Bioengineering Co., Ltd. (Dalian). The experimental water was secondary deionized water. The primers were synthesized by BGI Biotechnology (Shenzhen) Co., Ltd.

[0037] Anti-FAdV-4 monoclonal antibody (MAb / FAdV-4) was routinely prepared by Guangxi Veterinary Research Institute. Viruses FAdV-1, FAdV-2, FAdV-3, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11, Chicken infectious anemia virus (CIAV), Egg drop syndrome virus (EDSV), Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Avian reovirus (ARV), Avian influenza virus H9 (AIV H9) and Fowl adenovirus group 4 (FAdV-4) were all preserved in the form of virus preservation solution by the Biotechnology Laboratory of Guangxi Veterinary Research Institute. Among them, 13 virus preservation solutions, including FAdV-1, FAdV-2, FAdV-3, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11, CIAV and EDSV, were used to extract the corresponding DNA according to the operating instructions of EasyPure Genomic DNA Kit, and stored in a -70°C refrigerator for future use; 4 virus preservation solutions, including IBV, NDV, ARV and AIV H9, were used to extract the corresponding RNA according to the operating instructions of EasyPureGenomic RNA Kit, and then the RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription reagent, and stored in a -70°C refrigerator for future use.

[0038] The instruments used in the following examples are: Electrochemical workstation (Princeton Applied Research), the immunosensor of the present invention uses a conventional three-electrode electrochemical cell for electrochemical measurement, a modified gold electrode is used as a working electrode, a saturated calomel electrode (SCE) is used as a reference electrode, a platinum electrode is used as a counter electrode, and the immunosensor is connected to the electrochemical workstation via a wire.

[0039] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0040] The virus strains involved in the present invention belong to the virus preservation liquid in the field of microorganisms, which are all known viruses. Under the condition of complying with biosafety operation, the public can obtain them from the applicant, which can only be used for repeating the test of the present invention and cannot be used for other purposes. 36 clinical samples were collected from a large-scale chicken farm in Guangxi, all from chicken disease samples suspected of FAdV-4 infection (including cloacal cotton swabs, liver and spleen), and were tested and verified in parallel by the nucleic acid detection system established by the laboratory comparison verification platform. The virus preservation fluid and clinical samples involved in the application of the present invention are in vitro inanimate biological samples, and only involve biological samples collected from tissues, body fluids or excrement of human or animal bodies, including blood samples (whole blood / serum / plasma) and body fluid samples (urine / feces / cerebrospinal fluid / serous cavity effusion / semen / prostatic fluid / vaginal secretions / gastric juice / duodenal drainage fluid and bile / sputum) that have been separated from living human or animal bodies, and do not belong to living human or animal bodies; that is, the method and application of the present invention are not directly implemented on living human or animal bodies, nor do they include the steps of comparing the detected biological information with the benchmark data, and the steps of drawing specific diagnostic conclusions based on the biological detection information; the direct purpose of the detection is not to obtain the diagnosis results of the disease or the health status, but to be able to be used in the industry to produce in vitro detection kits or related products for virus detection. Therefore, the present invention does not belong to the diagnosis method of the disease and meets the basic requirements of the Patent Law for the subject matter of patent protection.

[0041] According to the general concept of the present invention, the present invention provides a novel multi-walled carbon nanotube (MWCNT)-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite prepared from multi-walled carbon nanotubes (MWCNT) and a preparation method thereof, wherein MWCNT is uniformly dispersed in a chitosan (Chi) solution, a K2PtCl4 solution is added, and Pt 2+ Reduced to platinum nanoparticles (PtNP), a multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite material is obtained, and the preparation method is simple. The prepared MWCNT-Chi-PtNP nanocomposite is used in the electrochemical immunosensor detection platform of avian adenovirus type 4. The gold electrode is modified with MWCNT-Chi-PtNP, and the monoclonal antibody against avian adenovirus type 4 is adsorbed to construct an electrochemical immunosensor for avian adenovirus type 4, and the incubation time of the sample to be tested and the pH value of the electrolyte are optimized. The sample to be tested can be used for electrochemical impedance spectroscopy (EIS) scanning detection after incubation for 30 minutes.

[0042] The following describes an embodiment of the electrochemical immunosensor detection platform for the MWCNT-Chi-PtNP nanocomposite and avian adenovirus type 4 of the present invention, and the specific contents are as follows:

[0043] Example 1: Comparative verification platform: nucleic acid detection system

[0044] Design and synthesis of PCR primers: According to the FAdV-4 gene sequence published on Genbank, primers were designed for the FAdV-4 hexon gene using primer5.0 software, and compared using DNAStar software and NCBI's Blast. The primer sequences screened and optimized were: upstream primer (FAdv-4-253F): 5′-AGGACCTCCAACAGTTCATTT-3′ (92—112); downstream primer (FAdv-4-253R): 5′-CCTCGGTCTAGGATTCCCT-3′ (326—344); the target fragment size was 253 bp. The primers synthesized by entrustment were diluted to 10 μmol / L with ultrapure water and frozen for later use.

[0045] Optimization of PCR reaction conditions: In the first step, the gradient PCR instrument was used to set 8 temperature gradients from 50°C to 60°C to explore the best annealing temperature. The reaction system and other conditions were the same. The results are shown in Figure 1 As shown in A: When the annealing temperature is 56.1°C, the non-specific amplification fragments are minimal while the specific amplification bands are most clearly visible, that is, the optimal annealing temperature is 56.1°C.

[0046] In the second step, the volume and dosage of the primers were optimized. Ten gradients from 0.2 to 2.0 μL were designed, with each gradient interval of 0.2 μL, and optimized screening was performed. The optimized screening results are shown in Figure 1 As shown in B: When the dosage of the upstream primer / downstream primer (10 μmol / L) is 0.8 μL, the specific amplification band can be guaranteed to be the clearest, and the content of primer dimers formed is relatively minimal, that is, 0.8 μL is the dosage of the primer volume for the best amplification effect.

[0047] The optimized annealing temperature was followed, and the amplified product was verified by 1.0% agarose gel electrophoresis. The final optimized annealing temperature was 56.1°C, and the optimized primer volume was 0.8 μL.

[0048] PCR amplification reaction system: TaKaRa Ex Taq 12.5μL, upstream primer / downstream primer (10μmol / L) 0.8μL, FAdv-4 DNA 1.0μL, and finally supplemented to 25μL with sterile ddH2O; at the same time, RNAase-free water was added as a template to the reaction tube as a negative control.

[0049] Reaction conditions and detection: 95°C for 5 min; 95°C for 30 s, 56.1°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min. The amplified product was detected by 1.0% agarose gel electrophoresis.

[0050] Among them, the FAdV-4 virus preservation fluid preserved by the Biotechnology Laboratory of Guangxi Veterinary Research Institute was used to extract FAdV-4 DNA according to the operating instructions of the EasyPure Genomic DNA Kit and stored at -70°C for future use.

[0051] Example 2: Preparation of MWCNT-Chi-PtNP Nanocomposite

[0052] 1) Chitosan (Chi) solution: Take 0.05 g of chitosan (Chi) and dissolve it in 50 mL of acetic acid solution (1.0% (V / V)). At room temperature, stir it with a magnetic stirrer for 0.5 h and mix it thoroughly to obtain 50 mL of 0.1 wt% chitosan (Chi) solution;

[0053] 2) Multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension: 50 mg of multi-walled carbon nanotubes (MWCNT) were added to the 50 mL chitosan Chi solution obtained above, and a stable multi-walled carbon nanotube-chitosan MWCNT-Chi suspension (concentration of 1 mg / mL) was obtained after ultrasonic treatment for 2 h.

[0054] 3) Nanocomposite: Take 5 mL of 10 mmol / L K2PtCl4 solution and add it to 50 mL of the prepared multi-walled carbon nanotube-chitosan MWCNT-Chi suspension, stir it at room temperature for a certain period of time, and then place it in a 90°C water bath to continue reacting for 1 hour to obtain a stable solution containing multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite; wherein the volume ratio of the K2PtCl4 solution to the multi-walled carbon nanotube-chitosan (MWCNT-Chi) suspension is 1:10.

[0055] Regarding step 3) "stirring at room temperature for a certain period of time", the stirring time was further optimized and compared, and the electron microscopy observation results of stirring at room temperature for 2h, 4h, 6h and 8h were compared in parallel. Through the comparative experiment, it was unexpectedly found that the key to the combination of multi-walled carbon nanotubes (MWCNT) and precious metal platinum (Pt) to form MWCNT-Chi-PtNP nanocomposites is the stirring time in the last step. The electron microscopy images of multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposites formed at different stirring times are detailed in Figure 2As shown: before mixing and stirring, that is, the stirring time of 0h was used as a negative control. When the stirring time was 2h and 4h, the loading amount of Pt nanoparticles on MWCNT-Chi was relatively small; when the stirring time was 6h, the loading amount of Pt nanoparticles on MWCNT-Chi was relatively uniform and abundant; when the stirring time was 8h, the particle size of the loading amount of Pt nanoparticles on MWCNT-Chi became uneven and agglomerated in some places; the experiment proved that 6h of stirring was the best stirring time. It can be seen that: if the stirring time is too short or too long, it is impossible to obtain a MWCNT-Chi-PtNP nanocomposite with uniform particle size and good loading amount.

[0056] Example 3: Establishment of an electrochemical immunosensor detection platform for avian adenovirus type 4

[0057] 3.1 Preparation of working electrode

[0058] 1) Electrode treatment: Gold electrode (GE), also known as glassy carbon electrode After polishing to a mirror surface with 0.05μm polishing powder Al2O3, wash with distilled water, then ultrasonically clean in water, anhydrous ethanol, and water for 5 minutes, and blow dry with N2. Then scan with cyclic voltammetry in 0.5mol / L H2SO4 solution (N2 was introduced for deoxygenation for 15 minutes before scanning), with a scanning speed of 50mV / s and a voltage range of -0.3 to +1.5V. Continue scanning until the cyclic voltammogram is stable, take it out and wash it with distilled water, blow dry it with N2 for standby use, and obtain an unmodified gold electrode (GE).

[0059] 2) Multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite modified electrode: Use a pipette to take 8 μL of MWCNT-Chi-PtNP nanocomposite and apply it to the surface of the unmodified gold electrode (GE). Dry it naturally at 4°C to obtain the electrode GE-MWCNT-Chi-PtNP modified by a gold-platinum nanocomposite combined with multi-walled carbon nanotubes (MWCNT) and precious metal platinum (Pt).

[0060] 3) Antibody fixation: On the surface of the modified electrode GE-MWCNT-Chi-PtNP, 10 μL of monoclonal antibody MAb / FAdV-4 (concentration of 1 μg / mL) against avian adenovirus type 4 was drop-coated and fixed at 4°C for 8 h to obtain the antibody-modified electrode GE-MWCNT-Chi-PtNP-MAb / FAdV.

[0061] 4) Blocking: After the monoclonal antibody is fixed, 10 μL of 1 wt% BSA solution is dropped and blocked in a 37°C incubator for 1 hour, then taken out and washed with water to obtain the working electrode GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA that can be used to detect avian adenovirus type 4.

[0062] 5) Detection: 10 μL of the sample to be tested was dropped onto the working electrode GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA, and the reaction was incubated in a 37°C incubator for a sufficient time to obtain GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA-FAdV-4, and then washed with water at least 3 times to remove the unbound sample to be tested, and then placed in an electrolyte for electrochemical impedance spectroscopy (EIS) scanning detection; wherein the reference electrode was a saturated calomel electrode and the counter electrode was a platinum wire electrode.

[0063] The water used for cleaning is secondary deionized water; the electrolyte is a PBS buffer solution containing 5mM K4Fe(CN)6, 5mM K3Fe(CN)6 and 0.1M KCl, and the concentration of the PBS buffer solution is 10mM.

[0064] 3.2 Electrochemical characterization during sensor preparation

[0065] The obtained complexes formed in the above steps are respectively subjected to electrochemical impedance spectroscopy (EIS) detection, which can be used to characterize the electrochemical information of the surface of the electrode or complex obtained in each step. The results are as follows Figure 3 As shown: a is the Nyquist plot of electrochemical impedance spectroscopy (EIS) of the gold electrode in the electrolyte, in which a small semicircle appears (Ret value = 974); but when the gold electrode is modified and covered with a layer of MWCNT-Chi-PtNP nanocomposite, the diameter of the semicircle decreases (Ret value = 409), corresponding to Figure 3 b in this figure, this is because the MWCNT-Chi-PtNP nanocomposite has good electrical conductivity, which improves the electronic conductivity of the electrode surface. Subsequently, MAb / FAdV-4, BSA and FAdV-4 (10 4.46 EID 50 / mL), the radius of the obtained Nyquist curve increases successively, corresponding to Figure 3c, d and e in the figure, this is because MAb / FAdV-4, BSA and FAdV-4 are protein molecules that are not conductive, which hinders the electron transfer on the electrode surface. The results show that MWCNT-Chi-PtNP nanocomposites, MAb / FAdV-4, BSA and FAdV-4 can be successfully fixed and modified on the surface of the gold electrode, indicating that the immunosensor was successfully prepared.

[0066] 3.3 Optimization of electrolyte pH

[0067] Regarding step 5), based on the electrolyte composition and concentration determined in the previous experiment, the detection effects of electrolytes with different pH values ​​were further compared in parallel. The working electrode GE-MWCNT-Chi-PtNP-MAb / FAdV-4-BSA obtained in step 4) was compared with 10 4.46 EID 50 After incubation with virus solution FAdV-4 / mL, the samples were placed in electrolytes of pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0 for electrochemical impedance spectroscopy (EIS) detection. The results are shown in Figure 4 As shown in the figure, when the pH value is 6.0-7.0, the Ret value of the sensor increases with the increase of the pH value. When the pH value continues to increase, the Ret value decreases. Therefore, the test verifies that the optimal pH value of the electrolyte is 7.0, which is the best test base solution.

[0068] 3.4. Optimization of incubation time

[0069] Regarding step 5) "Fully incubate the sample in a 37°C incubator", the incubation time of the sample and the working electrode was further optimized and compared, and the changes in resistance after incubation in a 37°C incubator for 10, 20, 30, 40, 50 and 60 minutes were compared. The results are as follows: Figure 5 As shown in the figure, the Ret value of the sensor increases with time within 30 minutes, and the Ret value of the sensor remains basically unchanged after 30 minutes, indicating that the immune response has ended. Therefore, 30 minutes is the best incubation time for the sample to be tested. In the subsequent performance verification experiment, it was verified by the experiment that the best incubation time for the sample to be tested and the working electrode in the 37°C incubator is 30 minutes.

[0070] 3.4 Determination of negative and positive criteria

[0071] Experimental determination of FAdV-4 negative and positive judgment criteria: It is well known in the art that when the Ret value of a sample is greater than the negative sample Ret mean (X) + 3×SD, the test result can be judged as positive at a statistical level (99.9%). The above-prepared immunosensor working electrode was used to detect 15 FAdV-4 negative samples, and the Ret mean was calculated to be 1749Ω, and the SD was 107, and the result was: negative mean + 3×SD = 1749 + 3×107 = 2070, so the positive and negative critical point was preliminarily determined to be 2070Ω.

[0072] Example 4: Performance study of electrochemical immunosensor detection platform for avian adenovirus type 4

[0073] 4.1 Sensitivity

[0074] The sensitivity experiment of the sensor was performed as follows: under the optimal reaction conditions of the electrochemical immunosensor detection platform for fowl adenovirus type 4 established above, the concentration of FAdV-4 virus was 10 5.46 EID 50 / mL of positive virus solution was diluted 10 times, and 10 μL of each dilution was taken. The prepared sensor working electrode was used to measure the positive virus solution FAdV-4 (10 1.46 EID 50 / mL~10 5.46 EID 50 / mL) for testing, and the test results are as follows Figure 6 A. Figure 6 As shown in B.

[0075] Figure 6 It can be clearly seen in A that with the increase of FAdV-4 concentration, the Ret value of the sensor surface increases accordingly, which is equivalent to the increase of viruses fixed on the sensor surface, and the Ret value of the sensor surface increases accordingly; compared with the negative control (the virus of the negative control is 0), when the FAdV-4 virus content is 10 1.46 EID 50 / mL, the corresponding Ret value did not change much. When the FAdV-4 virus content was 10 2.46 EID 50 / mL~10 5.46 EID 50 / mL, the corresponding Ret value increases with the increase of virus content, but when the FAdV-4 virus content is 10 4.46 EID 50 / mL and 10 5.46 EID 50 / mL, the Ret values ​​of the two were not much different; further, for FAdV-4 virus content of 10 1.46 EID 50 / mL~10 4.46 EID 50 / mL to construct a standard curve, such as Figure 6 As shown in B, it can be seen that the linear regression equation is: Ret value (Ω) = 961lgEID 50 / mL+257, the minimum detection limit of the electrochemical immunosensor for fowl adenovirus type 4 of the present invention for FAdV-4 virus content is 10 1.89 EID 50 / mL (S / N=3), and finally the detection range of the electrochemical immunosensor for avian adenovirus type 4 of the present invention is 10 1.46 EID 50 / mL~10 5 . 46 EID 50 / mL.

[0076] In order to compare the sensitivity of the immunosensor, the nucleic acid detection system established by the comparison verification platform was used for sensitivity comparison verification. The specific operation of the nucleic acid detection sensitivity experiment is as follows: Take the FAdV-4 virus with a known concentration of 10 5.46 EID 50 / mL of positive virus solution, and dilute it 10 times to obtain FAdV-4 virus content of 10 5.46 EID 50 / mL, 10 4.46 EID 50 / mL, 10 3.46 EID 50 / mL, 10 2.46 EID 50 / mL and 10 1.46 EID 50 / mL, respectively, extracted DNA, amplified by PCR, and the amplified products were detected by 1.0% agarose gel electrophoresis. The experiment was repeated three times, and the results were consistent. The test results are as follows Figure 6 As shown in C.

[0077] Figure 6 C, the nucleic acid detection system parallelly detects different concentrations of positive virus liquid FAdV-4 (10 1.46 EID 50 / mL~10 5.46 EID 50 / mL), when the FAdV-4 virus content was 10 5.46 EID 50 / mL, bright specific amplification bands were visible. When the FAdV-4 virus content was 10 4.46 EID 50 / mL, the brightness of the specific amplification band decreased significantly. When the FAdV-4 virus content was 10 3.46 EID 50 When the concentration of FAdV-4 was 0.14477 / mL, although the specific amplification band was visible, the brightness was very weak, that is, the minimum detection limit of the nucleic acid detection system for FAdV-4 virus content was 10 3.46 EID 50 / mL.

[0078] It can be seen that compared with conventional nucleic acid detection systems, the sensitivity of the electrochemical immunosensor for avian adenovirus type 4 of the present invention is 37 times higher.

[0079] 4.2 Specificity

[0080] Selection of samples to be tested: The positive control sample is the known concentration of 10 5.46 EID 50 / mL of the positive virus liquid of FAdV-4 virus was diluted 10 times for testing; the other 17 common chicken disease pathogens prevalent in chickens were used as interfering virus samples for specific experiments, including FAdV-1, FAdV-2, FAdV-3, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11, CIAV, EDSV, IBV, NDV, ARV, and AIV H9, and the negative control sample was PBS.

[0081] The electrochemical immunosensor of avian adenovirus type 4 manufactured in the same batch was selected and incubated with the above 19 samples for electrochemical impedance spectroscopy (EIS) scanning detection. The results are as follows: Figure 7 As shown in the figure, compared with the negative control (Blank), the Ret value of the sensor hardly changed when detecting 17 non-FAdV-4 virus samples; when detecting the positive control sample FAdV-4 (10 4.46 EID 50 / mL), the Ret value increased significantly, indicating that the sensor can only specifically detect positive results for the sample containing FAdV-4 virus, that is, the electrochemical immunosensor for fowl adenovirus type 4 has good detection specificity.

[0082] 4.3 Repeatability

[0083] According to the preparation steps described in Example 3, 5 batches of electrochemical immunosensors for avian adenovirus type 4 of the present invention were prepared, and 6 sensors from each batch were randomly selected for parallel repetitive experiments. 5.46 EID 50 / mL of FAdV-4 virus positive virus solution, and dilute it 10 times to a concentration of 10 4.46 EID 50 / mL FAdV-4 virus liquid was used as the positive sample to be tested. According to the detection steps described in Example 3, 6 sensors from each of the 5 batches were incubated with the prepared positive samples to be tested in a 37°C incubator for 30 minutes, washed, and then subjected to electrochemical impedance spectroscopy (EIS) scanning detection. The results are as follows Figure 8 As shown, the detection results of 5 batches and 6 repeated experiments in the same batch are similar, and the standard deviations are all less than 5%. It can be seen that the electrochemical immunosensor for avian adenovirus type 4 described in the present invention has good repeatability (including inter-batch repeatability and intra-batch repeatability).

[0084] 4.4. Clinical Sample Consistency Verification

[0085] The electrochemical immunosensor for fowl adenovirus type 4 established in this institute was used to detect 36 samples of suspected sick chickens collected in Guangxi. The results showed that 4 samples were FAdV-4 positive and 32 samples were FAdV-4 negative. At the same time, parallel experimental detection was carried out using the nucleic acid detection system (PCR method) established by the comparative verification platform. The results showed that the positive and negative results were consistent, proving that the electrochemical immunosensor for fowl adenovirus type 4 described in the present invention and the traditional PCR method have a positive and negative consistency rate of up to 100%, as shown in Table 1 below.

[0086] Table 1. Comparison of detection results between electrochemical immunosensor and nucleic acid detection system

[0087]

[0088] The present invention specifically provides a multi-walled carbon nanotube-chitosan-platinum (MWCNT-Chi-PtNP) nanocomposite prepared from multi-walled carbon nanotubes and precious metal platinum, and used for the detection of fowl adenovirus type 4 (FAdV-4) by an electrochemical immunosensor; a gold electrode is modified with MWCNT-Chi-PtNP, and then an anti-FAdV-4 monoclonal antibody is adsorbed to construct an electrochemical immunosensor for FAdV-4, and the incubation time of the sample to be tested and the pH value of the electrolyte are optimized. The sample to be tested can be incubated for only 30 minutes before an electrochemical impedance spectroscopy (EIS) scanning detection can be performed, and the final minimum detection limit of the FAdV-4 virus content is 10 1.89 EID 50 / mL, detection range is 10 1.46 EID 50 / mL~10 5.46 EID 50 / mL, compared with traditional PCR detection, the detection sensitivity of the electrochemical immunosensor of the present invention is increased by 37 times, the specificity is stronger, and the inter-batch and intra-batch repeatability of the preparation process are good. In addition, the verification results of 36 clinical samples show that the positive and negative coincidence rates of the electrochemical immunosensor of the present invention and the traditional PCR method are both 100%, and the consistency results are relatively good.

[0089] The specific embodiments of the present invention have been described in detail so that those skilled in the art will be easily understood. However, based on all the descriptions disclosed, similar modifications or replacements can be made to the specific details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An electrochemical immunosensor, characterized in that: The sensor is a three-electrode system, including a working electrode, a reference electrode and a counter electrode, wherein the working electrode is prepared by using a multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite modified gold electrode, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode; the sensor is used to detect avian adenovirus type 4, and the preparation method of the working electrode comprises the following steps: (1) Electrode treatment: polish the electrode with Al2O3 polishing powder, wash with water, clean with ultrasound, scan with cyclic voltammetry until stable, and then blow dry with N2; (2) Multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite modified electrode: a multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite solution was dripped onto the surface of the electrode and dried naturally at room temperature; (3) Antibody fixation: Apply the monoclonal antibody MAb / FAdV-4 against avian adenovirus type 4 and fix it at 4°C for 8 h. (4) Blocking: After the antibody is fixed, apply a drop of BSA solution and block at 37°C for 1 hour, then take it out and wash it with water to obtain a working electrode that can be used to detect avian adenovirus type 4 FAdV-4; The method for preparing the multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite comprises the following steps: 1) Chitosan Chi solution: 0.05 g of chitosan Chi was added to 50 mL of acetic acid solution, stirred at room temperature for 0.5 h, and then fully mixed and dissolved to obtain 50 mL of chitosan Chi solution; wherein the acetic acid solution was a mixed solution of glacial acetic acid and water in a volume ratio of 1.0%, and the concentration of the obtained chitosan Chi solution was 0.1 wt%; 2) Multi-walled carbon nanotube-chitosan MWCNT-Chi suspension: 50 mg of multi-walled carbon nanotube MWCNT was added to the above 50 mL of chitosan Chi solution, and a stable 50 mL multi-walled carbon nanotube-chitosan MWCNT-Chi suspension was obtained after ultrasonic treatment for 2 hours; wherein the concentration of the multi-walled carbon nanotube-chitosan MWCNT-Chi suspension was 1 mg / mL; 3) Nanocomposite: Take 5 mL of 10 mmol / L K2PtCl4 solution and add it to the above 50 mL multi-walled carbon nanotube-chitosan MWCNT-Chi suspension. After stirring at room temperature for 6 hours, heat it to 90°C in a water bath and continue to react for 1 hour to obtain a stable solution of multi-walled carbon nanotube-chitosan-platinum MWCNT-Chi-PtNP nanocomposite; wherein the volume ratio of the K2PtCl4 solution to the multi-walled carbon nanotube-chitosan MWCNT-Chi suspension is 1:

10.

2. A method for detecting avian adenovirus type 4 for non-diagnostic purposes, characterized in that: Using the electrochemical immunosensor according to claim 1, the sample to be tested is dripped on the working electrode, incubated at 37° C. for a certain period of time, then washed with water at least 3 times, and placed in an electrolyte for electrochemical impedance spectroscopy (EIS) scanning detection.

3. The method for detecting avian adenovirus type 4 for non-diagnostic purposes according to claim 2, characterized in that: The incubation reaction time is 30 min.

4. A detection kit for avian adenovirus type 4, characterized in that: The electrochemical immunosensor according to claim 1 is included, wherein the linear range of the detection kit for avian adenovirus type 4 is 10 1.46 EID 50 / mL~10 5.46 EID 50 / mL.

5. The detection kit for avian adenovirus type 4 according to claim 4, characterized in that: When the detection kit is used for detection, the electrochemical immunosensor and the sample to be detected are incubated at 37° C. for 30 minutes.

6. Use of the electrochemical immunosensor according to claim 1 and the detection kit for avian adenovirus type 4 according to any one of claims 4 to 5 in the preparation of a product for detecting avian adenovirus type 4.

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