Research On The Microfluidic Chip Used In The Quick Detection Of Pathogenic Microorganism

Detection of pathogenic microorganism has played an important role in maintaining the public health and researches on the detection protocol of pathogenic microorganism has sustained continuous development. Detection methods at present have some disadvantages, such as long time of inspection, complexity of operation, low sensitivity, easy trend of error, needs of highly expensive equipment or well-trained technicians. Moreover, all the analysis must be finished in labs, which means the impossibility of field inspection on samples.Microfluidic chip, a novel analytical technology, has been developed quickly during these years due to its advantages of micro structure, integration of function, low consumption of samples and reagents, high-flux output, short time of analyzing, automation and portability. It is an ideal platform for pathogenic microorganism detection because the characterization and counting of microorganism can be finished simultaneously, the whole detection time can be shortened to several minutes and the automation and portability can be realized. At present the researches on the detection of pathogenic microorganism by microfluidic chip mainly focuse on some certain units or procedure, such as enriching/separation, fore treatment or separation. Methods or techniques for the integration of sample processing and enriching/separation, fore treatment or separation are few, and related researches are in the initial stage. It still remains a great challenge that how to establish automatic micro-fluidic integrating sophisticated fore treatment of samples and detection methods and integrate the inspection equipment into microfluidic chip in order to form the portable system for spot operation.Considering the shortcomings of the present techniques, a new kind of highly sensitive detection system based on microfluidic chip for pathogenic microorganism and integrating immunocapture/enriching and bioluminescent assay has been established in this study. Bioluminescent assay based on micro-fluidic chip has the advantages of low consumption of antibody and reagent, fast speed of reaction, easy realization of automation and integration. Using microbead of nanometer to micrometer as solid phase bearer possesses the merits of not only increasing specific surface of antigen antibody reaction, but also being easy to fill and supersede. Because of the merit of high sensitivity without luminaire, complicated optical paths or marking the microorganism, quantitating microorganism by bioluminescent assay of ATP- firefly luciferin- luciferase system can obtain the information of the number and activity of microorganism, which is an ideal method for inspection based on microfluidic chip.Herein, taking E.coli O157∶H7 as the detection target, high-specificity and high-capture-rate immune-microbead chamber has been integrated into chip's microchannel, on-line quantitative analysis for microorganism enriched has been carried out by ATP bioluminescence, and the quick inspection system integrating high-specificity capture/enriching and highly sensitive detection has been set accordingly.Firstly, enriching chamber has been designed for the filling of immune-microbead, and numerical simulations of flow fields in different microchannels have been done by ANASYS software. According to the result, hexagon has been chosen as the structure for immune enriching chamber.Then, by surface silanization of microbead of 50μm and covalent linkage with anti-E.coli O157∶H7 polyclonal antibody, bio-functionalization of microbead has been completed. Filling the microbeads into hexagon chamber and letting them in the arrangement of monolayer and uniformity, the enriching chamber has been formed. By observing the capture of E.coli O157∶H7 stained by AO in fluorescence microscope, it has been proved that immune enriching chamber can capture/enrich bacteria effectively. Capture rate has been evaluated by plate culture method. Through the optimization of parameters of sample injection and washing, the real bacteria capture rate of immune enriching chamber formed by the way of filling immune microbead has reached 91.75%～95.62%.Finally, bioluminescence inspection of the bacteria captured in the chip has been finished through ATP-firefly luciferin-luciferase system and ultra weak chemiluminescence analyzer, and conditions of lumination reaction in the chip have been optimized. The method of quantitating bacteria by the luminous intensity of reaction is set up through the drawing of standard curve of bacteria concentration-lumination intensity. It has been proved that the accuracy rating, sensitivity and degree of precision are high and stability good according to the evaluation of the method's linear range, detectability, confidence, reproducibility and stability. The study of the chip's performance proves the high specificity of this microbiological detection system and that the system can be used to quantitate E.coli O157∶H7 in the pork samples accurately.From the above-mentioned, the establishment of microorganism quick inspection system with high specificity and high sensitivity has provided a platform for the development of the system for the highly-sensitive spot quick inspection for pathogenic microorganism.