Rapid continuous flow through polymerase chain reaction microfluidic chip

• Main Authors: Xiang Jun Liao, 廖祥竣
• Other Authors: Y. H. Lin
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/55wrqz

碩士 === 長庚大學 === 醫療機電工程研究所 === 106 === In this study, a continuous flow microfluidic chip is used for rapid polymerase chain reaction (PCR). The temperature control device consists of two PIDs, two heaters and two aluminum blocks. The reagents were run between two fixed heat sources and PCR was performed quickly. The microfluidic chip consists of PDMS combined with ultra-thin glass. We used this chip to carry out polymerase chain reaction and amplify DNA fragment, and the PCR-completed reagent is analyzed by gel electrophoresis. We explored the temperatures of denaturation and annealing, and found the most proper environment for PCR.The proper temperature of annealing is at 72.5 ℃ and it’s unable to react when temperature is higher than 74 ℃. Smear occurred in less than 70 ℃. Denaturation can react between 90 ℃ and 103 ℃, so we set the denaturation temperature at 99 ℃. The proper height of microfluidic chip flow path is between 90 μm and 56 μm. At the chip height of 90 μm, if flow rate less than 1.7 μl/min will cause the flow rate too slow. Bubbles can’t be suppressed causing evaporation and swelling, and PCR can’t be performed. At the height of 56 μm, evaporation occurs when the flow rate is less than 0.9 μl/min . The flow rate is affected by the length of the chip, longer chip length requires faster flow rate, on the contrary, shorter chip length requires slower flow rate. To summarize all the above, we found the most important factor that influencing PCR is the holding time. If the holding time too slow will cause reagent evaporate, so it is very important to find a balance for PCR. However, shortening denaturation and increasing annealing, making the ratio as 0.7 cm：1.2 cm, with the condition of keeping same temperature for 4 seconds, PCR will perform successfully with totally time of 2 minutes and 40 seconds. And doing serial dilutions, the limit value concentration of this reagent can be detected as 5 pg/λ . This rapid continuous flow PCR microfluidic chip can achieve quick response, small sample size and low cost. It is expected to shorten the testing time and reduce the chance of infection of medical staff also providing a simple and safe testing platform.