Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor

An avian influenza virus, electrochemical technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of destroying the graphite SP2 hybrid system, and the conductivity of graphene oxide is much poorer, achieving poor conductivity, high sensitivity, good water solubility

Active Publication Date: 2013-03-27
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, graphene oxide has good compatibility due to the abundant oxygen-containing groups such as carboxyl, hydroxyl and epoxy groups on its surface, but it destroys the original SP2 hybrid system of graphite, resulting in the formation of graphene oxide. The electrical conductivity is much worse than that of graphite, thus largely limiting its application in the field of electrochemical sensor research

Method used

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  • Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor
  • Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor
  • Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, the preparation of antibody A-graphene oxide-BSA

[0060] 1) Preparation of graphene oxide (GO)

[0061] Graphene oxide was prepared according to the improved Hummer method, and the specific steps were as follows: under ice-water bath conditions, 1 g of graphite powder, 2.5 g of potassium nitrate, and 100 mL of H were added to a 200 mL beaker. 2 SO 4 , after stirring well, slowly add 5g KMnO 4 , then put the beaker in a 35°C water bath for 2 hours, gradually add 100mL of deionized water, the temperature rises to 95°C and continue the reaction for 1h, the mixture turns from brown to bright yellow, after cooling to room temperature (25°C), add 300mL of water to dilute, and adding H with a mass fraction of 30% 2 o 2 To neutralize unreacted potassium permanganate, first wash with 0.5 mol / L hydrochloric acid aqueous solution, then repeatedly centrifuge and wash with water, and dry in vacuum to obtain graphite oxide.

[0062] Weigh 10 mg of the graphite oxi...

Embodiment 2

[0066] Embodiment 2, Preparation of H5N1 Subtype Avian Influenza Virus Electrochemical Immunosensor and Kit

[0067] 1. Preparation of H5N1 Subtype Avian Influenza Virus Electrochemical Immunosensor

[0068] 1. Preparation of immune electrode (working electrode)

[0069] gold electrode With 0.05μm Al 2 o 3 After the polishing powder is polished to the mirror surface, wash it with distilled water, and then ultrasonically clean it in secondary deionized water, absolute ethanol, and secondary deionized water for 5 minutes, and use N 2 blow dry. Then at 0.5mol·L -1 h 2 SO 4 solution (into N before scanning 2 Scanning with cyclic voltammetry during deoxygenation for 15min) with a scanning speed of 50mV / s and a voltage range of -0.3 ～ +1.5V, keep scanning until the cyclic voltammogram is stable, take it out and wash it with distilled water, N 2 Blow dry for later use, and obtain the gold electrode to be modified;

[0070] Then immerse the gold electrode to be modified i...

Embodiment 3

[0076] Example 3, Application of H5N1 Subtype Avian Influenza Virus Electrochemical Immunosensor and Kit

[0077] 1. Electrochemical Immunosensor Electrode Characterization of H5N1 Subtype Avian Influenza Virus

[0078] 1. Preparation of H5N1 Subtype Avian Influenza Virus Test Solution

[0079] The inactivated H5N1 subtype avian influenza virus was diluted to a concentration of 2 with PBS buffer solution with a concentration of 0.01mol / L and a pH value of 7. -4HA unit / 50 μL to obtain the H5N1 subtype avian influenza virus test solution.

[0080] 2. Methods and Results

[0081] The electrode surface modification process was characterized by alternating current impedance spectroscopy (EIS), and the quantitative detection of CEA was realized by differential pulse voltammetry (DVP). The electrochemical performance of the immunosensor was studied using a conventional three-electrode system. The prepared BSA-blocked antibody B-gold nanoparticles-thiourea-gold electrode (gold ele...

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Abstract

The invention discloses an electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and a kit. An antibody A-graphene oxide-BSA (Bovine Serum Albumin) compound for detecting the H5N1 avian influenza viruses provided by the invention is formed by an antibody A of the H5N1 avian influenza viruses and bovine serum albumin which are connected with graphene oxide through amido bonds. Proofed by an experiment provided by the invention, the nanometer antibody A-graphene oxide-BSA compound has an amplifying function, and the detection kit of the super-sensitive electrochemical immunosensor is established on the basis of a signal amplification property of the nanometer compound; and when the content of the H5N1subtype avian influenza viruses in a sample is small, a signal is amplified by the nanometer antibody A-graphene oxide-BSA compound, thereby fulfilling the purpose of effective analysis and detection. The method provided by the invention has the advantages of simple operation, high sensitivity and high specificity. According to the characteristics of the electrochemical immunosensor, if other antibodies are selected in experiments, the method can be used for detecting other pathogenic microorganisms.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an electrochemical immunosensor for detecting H5N1 subtype avian influenza virus and a preparation method thereof. Background technique [0002] Avian influenza (Avian influenza, AI) not only seriously affects the development of poultry industry, but also endangers human health. According to the World Health Organization (WHO), from November 2003 to November 2012, there were 606 cases of H5N1 infection in humans, 357 cases died, and the mortality rate was as high as 58.9%. Therefore, it is of great significance to establish a rapid, sensitive and specific method for detecting H5N1 to prevent and control the spread of H5N1 virus. [0003] At present, the methods used to detect AIV include virus isolation and identification, enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA) and hemagglutination inhibition (HI), reverse transcription polymerase chain reaction (RT-PCR), n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N27/26G01N27/327
Inventor 谢芝勋黄娇玲罗思思谢志勤谢丽基刘加波庞耀珊范晴
Owner GUANGXI VETERINARY RES INST
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