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An electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus and its detection method

A technology of nuclear polyhedron and immune sensor, which is applied in the field of virus epidemic diagnosis, can solve the problem of early detection, early prevention and timely cutting off the secondary infection route of pathogen transmission, high requirements for detection equipment and operators, difficult virus Infection diagnosis and other problems, to achieve the effect of improving sensitivity and biocompatibility, convenient electrochemical properties, and short time consumption

Active Publication Date: 2021-07-20
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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  • An electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus and its detection method
  • An electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus and its detection method
  • An electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of electrochemical immunosensor for detection of Bombyx mori 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 is 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 and 30% (mass ratio) hydrogen peroxide in a volume ratio of 3:1) for 10 min, take out the electrode and rinse it with ultrapure water for 3 times, wash the ear balls and blow dry, on the polishing cloth, successively use the diameter of 0.3 and 0.05 μm. After polishing the electrode to a mirror surface with alumina powder, take an appropriate amount of double-distilled water to clean the dirt on the surface of the electr...

Embodiment 2

[0060] Example 2: Detection of BmNPV using electrochemical immunosensor

[0061] Specific steps are as follows:

[0062] (1) Using an electrochemical workstation at 10 mmol / 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 in the frequency range of 100mHz to 100kHz.

[0063] (2) A series of BmNPV polyhedrin antigen solutions of different concentrations were added dropwise to the surface of the working electrode prepared in Example 1, incubated at 37°C for 30 min, washed with PBS and air-dried, and the electrochemical impedance spectroscopy was measured respectively, 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 on the surface of the working electrode prepared in Example 1, incubate at 37°C for 30 min, measure the electro...

Embodiment 3

[0065] Example 3: Sensitivity detection of electrochemical immunosensors

[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. The polyhedrin solution was incubated at 37°C for 30 min, washed with PBS and then air-dried. The electrodes before and after antigen binding were placed at 10 mmol / L [Fe(CN) 6 ] 4 / 3- In +0.1mol / L KCl PBS solution (pH 7.4), the impedance value of the prepared working electrode was measured with a disturbance amplitude of 10mV in the frequency range of 100mHz to 100kHz. According to the difference between the obtained impedance values ​​and the logarithm of the BmNPV antigen concentration Linear relationship, draw the working curve; the determination results show that the BmNPV antigen concentration is in the linear range of 0.0001-100ng / mL, the detection limit is: 14.54fg / m...

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Abstract

The invention discloses an electrochemical immunosensor for detecting silkworm nuclear polyhedrosis virus and a detection method thereof. The present invention uses undecaptoundecanoic acid / β-mercaptoethanol mixed self-assembled membrane to modify the gold electrode, which is used to immobilize BmNPV polyhedrin antibody, and utilizes the specific combination of antigen and antibody to complete the capture of BmNPV polyhedrin protein , Ultrasensitive detection of silkworm nuclear polyhedrosis virus based on hybrid self-assembled membrane electrocatalysis. The detection method has the characteristics of simplicity, rapidity, good specificity and high sensitivity, with a linear range of 0.0001-100ng / mL and a detection limit of 14.54fg / mL, which is suitable for the early and rapid diagnosis and prevention of silkworm karyotype 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 Patents(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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