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Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof

An immunosensor, Mycobacterium tuberculosis technology, applied in instruments, bacteria, testing food, etc., can solve the problems of long detection methods, low accuracy, and complicated operation steps, so as to shorten the detection time, detect fast, and simplify the detection steps. Effect

Inactive Publication Date: 2011-06-08
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The task of the present invention is to provide a chitosan-nanogold enzyme immunosensor for detecting Mycobacterium tuberculosis, which has the characteristics of fast, simple and accurate detection of Mycobacterium tuberculosis, so as to overcome the time-consuming and accurate detection method of Mycobacterium tuberculosis. The shortcomings of low type and cumbersome operation steps

Method used

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  • Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof
  • Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof
  • Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof

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Embodiment 1

[0029] Rapid and accurate detection of Mycobacterium tuberculosis in milk using chitosan-nanogold-enzyme immunosensor

[0030] 1. Research on the pretreatment of milk samples

[0031] This method does not require special treatment for milk samples, and the operation is simple. Centrifuge 10ml of milk samples at 1,900g for 20min, discard the supernatant, add 5ml of PBS solution with a pH of 7.4 and a concentration of 0.01M, mix well, centrifuge at 1,900g for 10min, discard the supernatant , add 0.5ml PBS to the precipitate, mix well and set aside.

[0032] In order to improve the detection rate, the milk sample can be enriched and cultured. The specific steps are: 10ml of milk sample is centrifuged at 1,900g for 20min, the supernatant is discarded, and 10ml of Middledbrook7H9 culture solution is added, and cultured under anaerobic conditions at 37°C for 1- 2 days. Then, centrifuge at 1,900g for 20min, discard the supernatant, add 5ml of PBS solution with a pH of 7.4 and a con...

Embodiment 2

[0045] Using chitosan-nanogold-enzyme immunosensor to quickly and accurately detect Mycobacterium tuberculosis in a mixed bacterial system (containing both Escherichia coli and Mycobacterium tuberculosis):

[0046] 1. Preparation of milk samples containing mixed bacteria: take 10mL of sterilized milk samples, centrifuge at 1,900g for 20min, discard the supernatant, and then add 3 cfu / mL Mycobacterium tuberculosis and 10 7 cfu / mL Escherichia coli pH 7.4 and 0.01M PBS suspension 5mL, mix well, centrifuge at 1,900g for 10min, discard the supernatant, add 0.5ml PBS to the sediment, mix well and set aside.

[0047] 2. Preparation of chitosan-nanogold enzyme immunoelectrode

[0048] (1) Pretreatment of the glassy carbon electrode: the glassy carbon electrode (φ=3mm) is polished to a mirror surface by 0.3 μm and 0.05 μm alumina powder before modification, rinsed with three-distilled water after ultrasonic cleaning for 3 minutes, and rinsed in piranha solution (30% h 2 o 2 :H 2 S...

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Abstract

The invention relates to an enzyme immunosensor based on chitosan and nano-gold, and by using the specific combination of the monoclonal antibody of the cell wall of mycobacterium trberculosis with mycobacterium trberculosis, the enzyme immunosensor is applicable to the rapid detection of mycobacterium trberculosis in milk. The characteristic is that the enzyme immunosensor for detecting mycobacterium tuberculosis is prepared by the following steps: modifying the surface of a glassy carbon electrode through electrochemical deposition method by a mixed solution of chitosan gel, nano-gold solution, and goat anti-mouse antibody labeled by alkaline phosphatase; and fixing the mycobacterium trberculosis monoclonal mouse antibody on the surface of the glassy carbon electrode modified by the goat anti-mouse-chitosan-nano-gold film which is labeled by alkaline phosphatase through the specific antigen-antibody reaction. Quantitative analysis is performed by detecting the electric signal changegenerated before and after the incubation reaction of mycobacterium trberculosis and the modified electrode through differential pulse voltammetry. The preparation method of the sensor is simple, andthe sensor has the advantages of high sensitivity, short detection time, and simple operation, and is applicable to the method for the rapid detection of mycobacterium trberculosis.

Description

Technical field: [0001] The invention relates to a method for detecting Mycobacterium tuberculosis in a milk sample, in particular to a method for fast and accurate electrochemical detection of Mycobacterium tuberculosis by an enzyme immunosensor with a nanostructure. Background technique: [0002] Milk is an important source of protein and other nutrients for humans, but it is susceptible to contamination by pathogenic bacteria, especially Mycobacterium tuberculosis, during production, transportation and processing. Studies have found that a variety of mycobacteria such as Mycobacterium bovis and other mycobacteria were detected from commercially available milk, raw milk, bovine lung tissue, and some lymph node tissues in Nepal, Spain, and Brazil. See Vijay et al., J Vet Med Sci., 69, 819 (2007); Gutiérre et al., Vet Herit., 29, 41 (2006); Clarice et al., Mem Inst Oswaldo Cruz, Rio de Janeiro., 98, 319 (2003). There are also literatures showing that general pasteurization ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535G01N27/327G01N33/04C12N1/20
Inventor 徐顺清夏纬李媛媛许冰
Owner HUAZHONG UNIV OF SCI & TECH
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