封閉迴圈高輸出式微流道PCR晶片與系統之開發

Abstract

本研究以機械驅動方式同步在多組閉迴圈連續流式(closed loop flow type)晶片，實現聚合酶連鎖反應(PCR)高輸出之機構。本研究利用聚二甲基矽氧烷(PDMS)具有彈性之特性做為聚合酶連鎖反應(PCR)之晶片，利用滾輪擠壓PDMS晶片上的微流道，可精準控制微流道中流體之流動，更突破以往連續流式PCR系統只在單一微流道內表現多輸出(high-throughput)，但可能造成反應物二次汙染的問題。機械驅動系統之設計以一組滾輪同步擠壓四個晶片，有效改善同步執行連續流式PCR系統佔空間問題，節省多輸出連續流式PCR系統需多組溫控模組成本，更以自動化方式同步完成反應物注入與取出，簡化操控反應物之注入與取出繁雜之過程。 本研究利用精密雕刻機，以PMMA為材料，製作寬300μm、100μm微流道之晶片母模，以含有微粒子與氟化物之物質研磨處理後，降低母模表面粗糙度。將PDMS灌入PMMA母模所翻製之晶片，利用O2電漿 (plasma oxidizer)活化玻璃與PDMS之表面後接合以得到低成本，製作快速且耐用性高、可拋棄式晶片之優點。經實驗證明此封閉迴圈式多輸出微流道PCR系統，可同步驅動兩組容量為13.1μl之晶片，完成PCR循環時間為20分鐘，成功複製285.RI.FZ.之DNA序列。這目前的裝置容易發展成全自動注入、循環、取出裝置，和增加即時檢測(real time)功能。因此這裝置將提供高效率的聚合酶連鎖反應。

This research has developed a novel disposable closed-loop PCR chip which allows non-restricted number of thermal cycles on a small footprint. The fluid in the microchannel of the PCR chip is driven by mechanically compressing the microchannel using wheels pressing on the channel in only one direction to precisely control the flow to loop through three temperature zones. An automatic driving mechanism is also developed to achieve high throughput by driving multiple chips at the same time on a single thermal platform. The PCR chip is made by bonding a PDMS top layer to a glass substrate. The PDMS top layer is casted on a PMMA mold. The channel pattern on the mold is machined on a CNC machine then furbished by hand. The PDMS cover is then treated by O2 plasma and bond to the glass substrate. The microchannel is 300 μm wide, 100 μm high, with a total loop length of 282.24 mm to contain about 13.1 μl of fluid when full. Fluid to be processed is simply dropped onto a well of the I/O port and then taken into the channel loop automatically by the driving mechanism. When the PCR process completes, the fluid is again released through the same port. Simultaneous PCR amplification results have been successfully demonstrated on two chips. The reported PCR chip is the first flow-type PCR chip that could fully prevent cross contamination within the microchannel.