Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof

A technology of immunosensors and nuclear polyhedrons, applied in the field of electrochemical immunosensors based on hybrid self-assembled membranes, and electrochemical immunosensors, can solve the problems of cumbersome operations, long detection cycles, and large virus requirements

Active Publication Date: 2020-07-07
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Routine symptom observation, microscopic examination and serological methods mainly rely on experience, require a large amount of viruses, and have a long detection cycle. It is difficult to diagnose the incubation period of the virus and early virus infection, and cannot achieve early detection, early prevention and timely cut off of silkworm disease. The purpose of pathogen transmission and secondary infection route
Although molecular biology diagnostic technology has high specificity and sensitivity, it has high requirements for detection equipment and operators, cumbersome operation, relatively high detection cost, and is not suitable for popularization and application in production.

Method used

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  • Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof
  • Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof
  • Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of an electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus

[0047] The electrochemical immunosensor comprises: a working electrode, a reference electrode and an auxiliary electrode; wherein, the reference electrode is a saturated calomel electrode, and the auxiliary electrode is a platinum electrode;

[0048] The working electrode was prepared as follows:

[0049] 1. Gold electrode pretreatment:

[0050] Put the gold electrode (d=2mm) into the freshly prepared piranha solution (concentrated H 2 SO 4 Soak in 30% (mass ratio) hydrogen peroxide (volume ratio: 3:1) for 10 minutes, take out the electrode and rinse it with ultrapure water for 3 times, wash the ear ball and dry it, and use a polishing cloth with a diameter of 0.3 and 0.05 μm successively After polishing the electrode to the mirror surface with alumina powder, take an appropriate amount of double distilled water to clean the dirt on the surface of the electro...

Embodiment 2

[0060] Embodiment 2: Utilize electrochemical immunosensor to detect BmNPV

[0061] Specific steps are as follows:

[0062] (1) Use an electrochemical workstation at 10mmol / L [Fe(CN) 6 ] 4 / 3- In the PBS (pH 7.4) solution of +0.1mol / L KCl, the impedance value of the working electrode prepared in Example 1 was measured with a disturbance amplitude of 10mV within the frequency range of 100mHz to 100kHz.

[0063] (2) Add a series of different concentrations of BmNPV polyhedrin antigen solution dropwise on the surface of the working electrode prepared in Example 1, incubate at 37° C. for 30 min, wash with PBS and dry, respectively measure the electrochemical impedance spectrum, according to step (1) Draw a standard working curve with the change of the electrochemical impedance value measured in step (2).

[0064] (3) Drop the sample to be tested onto the surface of the working electrode prepared in Example 1, incubate at 37° C. for 30 min, measure the electrochemical impedance sp...

Embodiment 3

[0065] Embodiment 3: Sensitivity detection of electrochemical immunosensor

[0066] 20 μL of BmNPV with concentrations of 0.0001ng / mL, 0.001ng / mL, 0.01ng / mL, 0.1ng / mL, 1ng / mL, and 100ng / mL were added dropwise to the working electrode of the electrochemical immunosensor successfully constructed in Example 1. Polyhedrin solution, incubated at 37°C for 30min, washed with PBS and dried. Place the electrodes before and after binding the antigen at 10mmol / L [Fe(CN) 6 ] 4 / 3- In the PBS solution (pH 7.4) of +0.1mol / L KCl, measure the impedance value of the prepared working electrode with the perturbation amplitude of 10mV in the frequency range of 100mHz to 100kHz, according to the logarithm of the obtained impedance value difference and BmNPV antigen concentration Linear relationship, draw working curve; Determination result shows that BmNPV antigen concentration is in the linear range of 0.0001-100ng / mL, detection limit is: 14.54fg / ml (S / N=3), linear correlation coefficient square...

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Abstract

The invention discloses an electrochemical immunosensor for detecting a bombyx mori nuclear polyhedrosis virus and a detection method thereof. The preparation method comprises the following steps: modifying a gold electrode by using an undecylmercaptoundecanoic acid/beta-mercaptoethanol mixed self-assembled membrane, wherein the hybrid self-assembly membrane is used for immobilizing a BmNPV polyhedrosis protein antibody; completing the capturing of BmNPV polyhedrosis protein through specific binding of an antigen and the antibody, and achieving the ultra-sensitive detection of the bombyx morinuclear polyhedrosis virus based on the electro-catalysis effect of the hybrid self-assembly membrane. The detection method has the characteristics of simplicity, convenience, quickness, good specificity and high sensitivity, the linear range is 0.0001-100ng/mL, the detection limit is 14.54 fg/mL, and the detection method is suitable for early-stage quick diagnosis and prevention of the bombyx mori nuclear polyhedrosis.

Description

technical field [0001] The invention relates to an electrochemical immunosensor, in particular to an electrochemical immunosensor based on a mixed self-assembled membrane, and belongs to the technical field of virus epidemic diagnosis. Background technique [0002] Bombyx mori Nuclear Polyhedrosis Virus belongs to Baculoviridae (Baculoviridae), Baculovirus (Baculovirus), silkworm nuclear polyhedrosis virus (Bombyx moriNuclear Polyhedrosis Virus), referred to as BmNPV. The nuclei of the trachea and other cells form the virus polyhedron and get its name. Its polyhedron is a protein crystal coated with long rod-shaped virus particles inside. The size is generally between 2-6 μm, with an average of 3.2 μm, and can be observed under an optical microscope. Polyhedrin protein Polh, as the main protein of polyhedron crystallization matrix, is a highly expressed protein of baculovirus, and its accumulation in cells at the late stage of infection can be as high as 30% to 50%. The si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/02G01N33/543G01N33/569G01N27/38
CPCG01N27/02G01N27/3275G01N27/38G01N33/54366G01N33/56983
Inventor 高绘菊宋贵珍任春久王彦文
Owner SHANDONG AGRICULTURAL UNIVERSITY
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