The Design、Fabrication And Experimental Research Of Time-domain PCR Chip

Global burdens from existing or emerging infectious diseasesemphasize the need for point-of-care (POC) diagnostics to enhance timelyrecognition and intervention. Molecular approaches based on PCRmethods have made significant inroads by improving detection time andaccuracy but are still largely hampered by resource-intensive processing incentralized laboratories, thereby precluding their routine bedside-or field-use. Microfluidic technologies have enabled miniaturization of PCRprocesses onto a chip device with potential benefits including speed, cost,portability, throughput, and automation. In addition, develop uTAS on onebiochip with real-time fluorescence detection technology is also the currenttrend in related field.The purpose of this study is to improve the fabrication technology toproduce an efficient, cheap and easily operated practical PCR chip throughoptimizing the structure design, and to realize the integration of theportable PCR diagnosis platform through combining with the integratedexternal temperature manage system and real-time fluorescence detectionequipment.In this paper, the main research contents and results are as follows:1. Optimizing the existing PCR chip design, including materialselection, general layout and structural parameters of the parts setting up;Improving the PCR chip fabrication method, including mask, PDMS coverplates, glass substrates and sealing molding; Making12×12mm squarethree layers structure of time-domain PCR chip, compact structured andconvenient sampling. 2. Designing temperature measurement and control system, combinedwith the PCR reaction platform constructed by preparative PCR chip.Temperature measurement and control system including hardware controlcircuit and software acquisition program of temperature control, thehardware control circuit is composed by the power supply, signalamplification module, temperature acquisition module and the chiptemperature cooling modules, then, make it PCB; The software part iswritten into the project with LabVIEW, including temperature conversion,temperature presetting, and PID control procedures; Connecting the chipand test temperature control effect after water injection, to meet the PCR’srequirements to temperature.3. Based on the experimental observation, the reason of PCR’s failureand mechanism of bubble is studied, then, on this basis, a set of simple andpractical PCR pretreatment process is figured out and proved to be a goodinhibition to the generation of air bubbles. Choosing the better way ofsyringe continuous sampling mode, and then successfully amplified thejellyfish GFP gene in the time-domain PCR chip, analyzing electrophoresisresults after compared with traditional PCR analysis.4. Analyzing the current separate ends of amplification product flawsand the demand of real-time fluorescence detection, concrete plans ofreal-time fluorescence detection to match the time-domain PCR chip is putforward, contains specific need to purchase the device and thecorresponding advantages and disadvantages.