| Objective:E. coli O157: H7is a common pathogenic bacteria, which can cause humanhemorrhagic diarrhea and others serious illness, so the rapid detection of E. coli O157:H7has important clinical significance. In recent years, the microfluidic chiptechnology gain great interesting in rapid detection of bacteria as it has a lot ofadvantages, including short analysis time, small reagent consumption, and easy tominiaturization, etc. However, the most recent pathogen rapid detection microfluidicchip are still suffer from low detection throughput, and the cost of a single sampletesting is still relatively high. In order to solve this problem, this study proposedintroduce microvalve and microchannel large-scale integration technology inpathogen detection microfluidic chip to achieve high throughput, automaticallycontrol of E. coli O157: H7rapid detection by using integrated microfluidic chipoperation parallelism.Methods:1. Developed automatic integrated microfluidic chip reaction system: using autoCADsoftware to design the CAD image of integrated microfluidic chip, which havemulti-channels and multiple sets of pneumatic valve. Then making the integratedPDMS microfluidic chip by molding and multilayer soft lithography. Assembling adedicated micro valve controller with single chip microcomputer, electromagneticvalve. After that, build an automated integrated microfluidic chip reaction system byconnecting the microfluidic chip, valve controller, injection pump, syringes andinverted fluorescent microscope, and then test this system by liquid dye.2. Investigated and sifted out the best method of surface modification and antibodyimmobilization method in integrated microfluidic chip: used glass as carrier,comparing the protein fixed ability of three surface modification method through asystematic research. Then fixing the protein G and Streptavidin on the glass surfacebased on the best method of surface modification, and comparing the ability ofimmobilizing active antibody of these two affinity antibody immobilization method, to sift the best antibody immobilization method.3. Build a system for rapid detection of E.coli O157：H7based on the integratedmicrofluidic chip, and investigated its application value: by using the integratedmicrofluidic chips automated response system as reaction platform,MPTS-GMBS-SA method as antibody immobilization strategy, the air valvecontrolled chip channel and reaction order,10detection units were built at the chipchannel junction. On this basis, bulid integrated microfluidic chip-based E. coli O157:H7rapid detection system by using immunofluorescence technique as detectionmethod, streptavidin-antibody-target bacteria-detected antibody as detection signalamplification system, and optimize the reaction conditions of detection. Then, appliedthis rapid detection system in test of mineral water, milk and feces suspension sample,and comparing with culture method (the gold standard), verify its practicalapplication value.Results：1. A integrated microfluidic chip was successfully developed, which have40pneumatic valves and11microchannels. In addition, a pneumatic microvalvecontroller was developed based on single chip microcomputer and theelectromagnetic valve. On this basis, a integrated microfluidic chip automatedreaction system was successfully set up, which used the integrated microfluidic chipas reaction unit, the microvalve controller as the control center, the ten channelinjection pump as the power source, the CCD and conventional inverted fluorescentmicroscope as detection unit. And it was also proved that by operating the microvalvecontroller, the multi–channels of the chip can be simply, quickly and convenientlycontrolled at the same time, and realized high-throughput parallel operation and fluidautomated control.2. Through the experimental study, it was found that the final antibodyimmobilization efficiency can be impacted by the reagent type, reagent concentration,reaction temperature and time. Through comparative experiments, MPTS-GMBSmethod was proved is the best surface modification method, which have the highestprotein fixed efficiency, and the streptavidin signal amplification system is the bestantibody immobilization method, which can maintain the highest activity of antibody. Hence, MPTS-GMBS-SA was chose as the antibody immobilization strategy in thisstudy.3.10detection units were built at the chip channel junction. On this basis, rapiddetection of E. coli O157: H7without cultivation was achieved by utilizingimmunofluorescence technique as detection method, theSA-antibody-bacteria-fluorescent system as signal amplification. Under the optimumdetection conditions, the testing throughput of this system is10samples per time, andthe total testing time and reagent consumption is only1h and5ul respectively with alow detection limit of1.6x103CFU/mL and good specificity and repeatability. Inaddition, this integrated microfluidic chip detection system was applied in rapiddetection of E. coli O157: H7in mineral water, milk and feces suspension sample,proved that this system has good consistency with conventional detection method,and has practical valve of application.Conclusion：1. Highly integrated microfluidic chip can be developed through large-scale integratedtechnology and multi-layer soft lithography; special chip controller can be assembledusing single-chip microcomputer and electromagnetic valve; integrated microfluidicchip-based automatic response system can realize high-throughput parallel operationand fluid automation of operation, has very good feasibility and flexible operation.2. MPTS-GMBS method have higher proteins fixed ability than physical adsorption,APTES-GA method and GPTS method; streptavidin have higher ability ofimmobilizing reactive antibody affinity than protein G affinity method;MPTS-GMBS-SA method is more suitable for antibody immobilization inmicrofluidic chip.3. Integrated microfluidic chip reaction system can be applied in E.coli O157: H7rapid detection. Compared with the conventional bacteria detection method, thissystem features high detection throughput, low cost, fast speed, high degree ofautomation advantages, which has certain research and practical application value... |
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