一種新型迴路式毛細管熱對流聚合酶連鎖反應載具之開發─超音波加工試劑載具

Abstract

迴路式熱對流聚合酶連鎖反應（Capillary Loop Convective Polymerase Chain Reaction，CLCPCR）為熱對流聚合酶連鎖反應的新式應用，該方法係在一迴路式載具底部提供單一加熱源使載具內部的流體間存在溫度差，產生熱對流帶動試劑沿著迴路無間斷循環流動完成PCR所需的溫度循環，省卻傳統機台耗時的熱傳導升降溫過程，亦解決CCPCR內部試劑流向不穩定的問題。本論文針對CLCPCR機台的試劑載具進行改良，開發全新製程製作載具，探討實驗相關參數以驗證此載具的可行性。 本研究利用超音波加工法在玻璃基板上銑削加工，依照設計的流道形狀銑出立體凹槽，再以耐高溫抗油的silicone膠封裝玻璃基板與蓋玻片，最後使用清潔劑清洗內部中空區域並使用高壓滅菌爐去除生物性物質，完成試劑載具的製作。為了確認載具內部溫度場與流場符合PCR需求，在載具內部注入含有聚醯胺微粒的溶液後放至加熱油槽從底部單一加熱，檢驗封裝的牢固性且觀察流場運動情形，待內部流體穩定循環流動再測量內部溫度分佈，在底部加熱溫度設定130 ℃及環境溫度為25 ℃的條件下，載具內部流體的最高溫及最低溫皆處於PCR溫度循環的合理範圍。同時，探討載具底部及側壁的表面粗糙度對加熱過程氣泡生成的影響，並針對載具製程中接觸的化學物質進行生物干擾性測試，確保載具內部並無影響DNA擴增的因素。 實驗結果證明，此超音波加工玻璃載具能在反應時間25分鐘內擴增HBV檢體，相比過去CLCPCR機台的260 μL玻璃迴路式毛細管，整體試劑用量減少至180 μL，目前擴增成功的初始模板濃度最低為107 copies/ ml，試劑載具已能規格化生產，解決過去載具間尺寸相異結果缺乏重複性的隱憂。

Capillary Loop Convective Polymerase Chain Reaction (CLCPCR) is a new application of convective PCR. It can complete the temperature cycle of PCR with a single heater. By heating the bottom of the loop container, the fluid would form a Rayleigh–Bénard convection cell and generate a circulating flow continuously. The purpose of this thesis is developing a new manufacturing procedure for the reagent container and analyzing relevant factors to prove the possibility of the new container. A glass substrate is fabricated with the groove of loop-shaped with an ultrasonic machining approach. The fabricated glass substrate is bonded with a top glass cover with the silicone glue to form the test piece. Last, we remove toxic materials to DNA in the container by using the cleaner and the autoclave. To ensure the fluid field and the temperature distribution in the container suitable for PCR, we inject a solution of PSP into the container to make flow visualization and put thermocouples into the container to do the temperature measurement. In the condition of the heater temperature at 130 ℃ and the surrounding temperature at 25 ℃, the highest and the lowest temperature measured in the container are in the temperature range of PCR thermocycling. Meanwhile, we discuss the effect of the roughness to bubble formation during the heating process and check which material may affect DNA amplification by biocompatible tests. The experimental results show that the HBV template with initial concentration 107 copies/ ml can be amplified in the container made by ultrasonic machining at present. It costs 180 μL of reagent each time and takes 25 minutes to amplify DNA. Now the reagent container can be produced in specification and we have eliminated the doubt about reproducibility of the old containers.