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Research on the Microfluidic Chip Used in the Quick Detection of Pathogenic Microorganism

Author: GuanZuo
Tutor: ZhangHuiJing
School: Third Military Medical University
Course: Clinical Laboratory Science
Keywords: Pathogens E. coli O157: H7 Rapid detection Microfluidic Chips Immune sorting Beads ATP- bioluminescence
CLC: TN492
Type: Master's thesis
Year: 2009
Downloads: 155
Quote: 0
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Routine detection of pathogens, including non-selective and selective culture, biochemical reactions and serological identification step, the complicated operation, long life cycle (5-7d), time-consuming, can not meet the contemporary social environment, food safety supervision and infectious the need for rapid diagnosis of the disease. In recent years, with the development of biotechnology and established rapid detection techniques (such as immunoassay technology, PCR technology, etc.) in a relatively short period of time to identify the pathogen, but there are still as low sensitivity, false-positive and false-negative rate high, complex and costly defects, so the development can be accurately, sensitive, rapid detection of pathogens without delay. Microfluidic chip technology platform as the new micro analysis of its structure microfilming, functional integration, sample and reagent consumption is low, the output of the high-throughput, short analysis time, easily portable and automation advantages, in recent years, microbial detection of developing very rapidly. Existing microfluidic chips microbial detection for a unit or step in microbiological research, such as expanded enrichment / separation, pretreatment or separation and detection, the lack of a set of sample handling, the scarce sample enrichment / separation and integration testing in one technology or method, the entire study is in its infancy. And how pathogen detection all aspects of the operation of the process of integration in a microfluidic chip and form of automated pathogen detection system is a microfluidic chip for today challenges facing pathogen detection. Defects of the rapid detection of pathogens requirements and existing detection technology in view of today's society, the study constructed a microfluidic immunoassay chip the pathogens sorting / enrichment and bioluminescence detection studies carried out on the chip. Microfluidic chip immune separation technology with low antibody and reagent consumption, fast response, easy to integrate and automate the advantage; antibody with nanometer to micron-sized particle size beads as a solid phase support, not only increase the specific surface area of ??the antigen - antibody reaction, and it is easy to fill and replacement of the characteristics of the chip unit function clearer, another chip configuration is simple, inexpensive, easy to build and popularity. Quantitative bioluminescence analysis for microbial ATP-firefly luciferase - luciferin enzyme system to achieve very high sensitivity may be due to the high rate of photon production, without the light source and the complexity of the optical path, and without marked microorganism, i.e. microbial number and activity information can be obtained, can be integrated microfluidic chip ideal detection method. Therefore, the study of E.coli O157: H7 detection target, will be highly specific for the bacteria and high capture rate of immune bead enrichment chamber design chip microchannel and ATP bioluminescence system of enrichment bacteria to online quantitative analysis, the establishment of a highly specific capture / enrichment, and high-sensitivity detection of pathogens rapid detection method. First, according to the purpose of the experiment design PDMS / glass microfluidic chip configuration and size of the micro-channel chip including fluid transmission, immune capture and luminescence detection of three functional areas. The molding method to complete the production of the chip, and the production process and the manner of sealing optimized. Secondly, the silylation treatment by the particle diameter of 50 μm of the surface of the glass beads and anti-E. coli O157: H7 in the polyclonal antibodies covalently attached, the completion of the modification of the biological function of the glass beads. Filled with glass beads in the the the chip microchannel hexagonal cavity, by exploring beads transfer flow rate and the way to build a chip immunity was a single layer evenly arranged bead enrichment chamber. And under a fluorescence microscope observation by acridine orange staining of E. coli O157: H7 in the chip capturing, then verify immune enrichment chamber can effectively capture / enrichment to the bacteria. Plate culture counting the evaluation chip capture efficiency of the immune enrichment chamber, through the optimization of the chip injection and flushing parameters, filled with the actual capture rate of the the immune immune glass bead enrichment cavity bacteria samples 91.75% to 95.62%. Finally, by ATP-firefly luciferase - luciferin enzyme system and weak luminescence detector, the bioluminescent detection of bacterial chip captured, and the chips within the luminescent reaction conditions were optimized. Through the fitting of the bacterial concentration - emission intensity regression equation, to establish a method of quantitatively determining the luminous intensity of the bioluminescence reaction for bacteria. Evaluation of the linear range of the method, reliability, reproducibility, stability and specificity of the high specificity of the microfluidic chip pathogen detection system, and is able to be carried out on the the pork sample in the E.coli O157: H7 accurate quantification. In summary, this study established a good specificity, high accuracy microfluidic chip pathogens rapid detection method, and the method can be the entire pathogen detection process control in less than 20min for further development of the site rapid pathogen detection system development has laid a solid foundation.

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