Electrochemical immunosensor, and preparation method and application thereof

An immunosensor, electrochemical technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of high price, strict requirements for test operators, and inability to achieve online detection, and achieves high specificity and high sensitivity. Effect

Active Publication Date: 2014-10-08
ENSOUL TECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR, SPR, and BLI instruments and equipment require high precision and are expensive, cannot realize online detection, and have strict requirements on test operators; while fluorescent labeling technology and ELISA can only be qualitative or semi-quantitative, plus data processing and sample addition Due to the limitation of the method, it is almost impossible to realize instant, accurate quantification and online detection

Method used

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  • Electrochemical immunosensor, and preparation method and application thereof
  • Electrochemical immunosensor, and preparation method and application thereof
  • Electrochemical immunosensor, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 sensor

[0028] 1. Screening of anti-threonine phosphate lyase monoclonal antibody strains

[0029] Firstly, using conventional monoclonal antibody preparation methods, the SpvC protein antigen derived from Salmonella was injected into Balb / C mice to obtain monoclonal antibody strains, and then injected into Balb / C mice intraperitoneally, and the ascites was purified to obtain anti-threonine phosphocleavage Synthase monoclonal antibody.

[0030] Then, the anti-threonine phosphate lyase monoclonal antibody strain was screened: the biomolecular interaction instrument Octet Red96 produced by ForteBio Company of the United States was used for this experiment: five Protein A sensors A3, B3, C3, D3, and E3 were taken , five concentration gradient threonine phosphate lyase protein antigens correspond to one of them, the concentrations are: 4μM, 1.33μM, 0.44μM, 0.1481μM, 0μM. The five SpvC monoclonal antibodies used were: A2-E2 (1.129 μM); A3-E...

Embodiment 2

[0052] Embodiment 2 sensor detects Salmonella and / or Shigella

[0053] 1. Prepare standard curve:

[0054] The prepared threonine phosphate lyase double-layer nano-gold membrane electrochemical immunosensor was incubated in the Salmonella suspension diluted in 0.01M pH7.4 PBS buffer at 37°C for 15min (from low concentration to high concentration). Current-time scanning (scanning potential - 0.38V), the steady-state current value (generally the 50th s) is selected as the standard after comparison, and the change △I of the current before and after the detection and the number of Salmonella colonies (according to GB4789.2 "Food Hygiene Determination of the total number of colonies in microbiological examination "to determine the bacterial concentration) plotting, to obtain the linear relationship between △ I and the concentration of Salmonella.

[0055] 2. Sample preparation:

[0056] Choose a concentration of approximately 1×10 8 cfu / mL Salmonella suspension (obtained accordi...

Embodiment 3

[0059] Embodiment 3 sensor stability and reproducibility detection

[0060] The assembled threonine phosphate lyase double-layer gold nano-film electrochemical immunosensor was divided into 1 × 10 2 cfu / mL Salmonella and 1×10 2 The cfu / mL Shigella was continuously measured 12 times in the PBS dilution, and the relative standard deviations R.S.D of the results were 3.23% and 4.11% respectively; the sensor was stored at 4°C above the 0.01mol / L pH7.4 PBS solution, Salmonella suspension (1×10 2 cfu / mL), the current response signal on the 13th day was 86.45% of the initial current, indicating that the sensor has good stability.

[0061] Take 5 immunosensors prepared in different batches, and test the same concentration of Salmonella suspension (1×10 2 cfu / mL), Shigella suspension (1×10 2 cfu / mL) and two mixed bacterial solutions (1×10 2 cfu / mL) were measured, and the relative standard deviations R.S.D. of the response current were 4.12%, 4.66%, and 4.82%, respectively, indicat...

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Abstract

The invention discloses an electrochemical immunosensor. An antibody for resisting phosphothreonine lyase is at the center of the electrode surface of the electrochemical immunosensor, and is preferably a monoclonal antibody; a sensor preferably contains a double-layer nano-gold membrane; and an electrode of the electrochemical immunosensor is preferably a glass-carbon electrode. The phosphothreonine-lyase electrochemical immunosensor modified by the double-layer nano-gold is constructed by taking chitosan as a bridging agent, fixing a first layer of nano-gold on glass-carbon electrode and adsorbing and fixing the antibody for resisting phosphothreonine lyase, fixing a thionine-chitosan /nano-gold-horseradish peroxidase (HRP) complex to the electrode by dropping onto the electrode, and adsorbing the antibody for resisting phosphothreonine lyase. The electrochemical immunosensor is applicable to detect salmonella and/or shigella in food, is high in sensitivity and strong in specificity, and is capable of realizing rapid quantitative detection on salmonella and/or shigella.

Description

technical field [0001] The present invention relates to the technical field of biological immunodetection, in particular to an electrochemical immunosensor and its preparation method and application; sensor. Background technique [0002] With the increasingly widespread application of monoclonal antibody hybridoma technology, it is necessary to systematically analyze and identify the obtained monoclonal antibody (McAb) to determine its value in various immunological tests. In addition to analyzing the physicochemical properties of the McAb immunoglobulin chain, the specificity of the McAb binding antigen, the dissociation equilibrium constant KD, and the binding affinity K on and the dissociation rate constant K dis It is very important to select the appropriate McAb for a specific purpose. The biomolecular interaction instrument adopts Bio-Layer Interferometry (BLI) technology, does not require labeling, has the characteristics of high sensitivity and high throughput, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N33/577
Inventor 庞广昌鲁丁强耿利华
Owner ENSOUL TECH LTD
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