可應用於薄膜式微流體平台之快速聚合酶連鎖反應系統開發

Abstract

COPD，慢性阻塞性肺病為受過敏原或刺激物刺激，氣管及肺泡發生慢性發炎之疾病，據統計，臺灣一年超過5000人因 COPD 死亡，且其病程為不可逆反應，目前唯一的方法為減少病人之發病機會，所以個人化之COPD檢測系統有其必要性。越來越多研究證實，miRNA之表現量與特定疾病之關係，而體內之miR-15b基因也被證實與COPD疾病有關，因此本研究選擇miR-15b作為COPD之檢測標誌，開發一種薄膜型快速聚合酶連鎖反應微流體裝置，用以作為慢性阻塞性肺病之 定點照護(Point of care)裝置。 在微流體平台的領域中，PCR技術已受到矚目，其優勢為所需樣品量較少，樣品熱質量降低，也提高了熱循環之效率，不過在許多研究中的微流體晶片使用了半導體製程，製程昂貴，商品化難度高，而塑膠微流體晶片雖然成本較低且生產容易，但塑膠材質的缺點在於塑膠熱傳導差，且常規塑膠製程下厚度不容易低於1mm，不利於PCR之熱循環。為了解決此一問題，本研究開發出了一種薄膜塑膠晶片之熱壓印技術以及薄膜熱結合技術，以吸水率極低之環烯烴聚合物薄膜，製作出厚度僅約0.288mm之薄膜晶片，突破傳統塑膠製程之厚度最低只能到1mm的限制，與之相比，本研究之薄膜晶片厚度減少了至少3.5倍，由熱傳導方程式可知，固體熱傳導速度與距離成反比，此薄膜晶片將有效改善塑膠晶片之熱傳導效率，進而加速PCR熱循環速度。另外COP低吸水性，於實驗中證明所開發的COP薄膜PCR晶片在10至40個熱循環後體積吸收率均小於1.5%。本研究中提出了兩款可應用於薄膜微流體晶片之小型化快速溫度循環系統，利用致冷晶片作為系統加熱元件，並在致冷晶片表面加入了切割後之矽晶片以及石墨片，藉此改善致冷晶片表面溫度分布均勻度，使致冷晶片表面各區最高溫度之平均值誤差小於1°C，本研究並實驗證明此裝置可在10分鐘內對原始濃度為10e11至10e7 copies/μl之miR-15b模板基因完成40個PCR循環擴增，單次循環平均為13.75秒，與擴增前之螢光值分別提高了3.73倍至2.3倍，整體工作時間比一般大型PCR儀器快7倍以上。 本研究成功整合薄膜塑膠生物晶片製程開發、薄膜晶片注射裝置、快速熱循環加熱器，成功降低了PCR 檢測之設備成本以及耗材成本。

Chronic obstructive pulmonary disease (COPD) is a chronic inflammation of the lungs that receives allergens or irritants. In Taiwan, more than 5,000 people die from COPD every year. Due to the development of COPD is an irreversible process, current treatment is to reduce the symptom. Thus, a personalized COPD detection system is necessary. Recently, studies have confirmed that the concentration of miR-15b gene in the body is related to COPD. Therefore, miR-15b was selected as the biomarker for this study, and a thin-film based rapid polymerase chain reaction device was developed for COPD point of care device. For the past decade, PCR technology has become one of major applications in Microfluidics. Its advantage is less sample consumption and high accuracy. However, many of reported semiconductor fabrication based PCR devices have disadvantages of high fabrication cost and fragile. It becomes as an obstacle for commercialization. On the other hand, plastic-based PCR devices is limited by its poor thermal conductivity and the thickness cannot be lower than 1mm under ordinary plastic. Thus, the thermal cycling process of plastic chip-based PCR cannot be shortened. To overcome this limitation, we developed a hot embossing process and bonding process to fabricate thin-film plastic PCR chip. The overall thickness is only 0.288 mm by using the Cyclo Olefin Polymer films(COP). Furthermore, COP has low water absorption rate than ordinary plastic materials. It is experimentally verified that the water loss percentage was less than 1.5% for 40 thermal cycles. In this study, two miniaturized fast temperature cyclers that can be applied to thin-film microfluidic devices are proposed. Commercial peltier module is used as a heating component, and silicon wafer and graphite sheet are added to improve temperature uniformity of peltier surface. The surface temperature variation of the peak is less than1°C. Using this device, a 40 PCR cycles can be completed to amplify 10e11 to 10e7 copies/μl miR-15b template genes in 9 minutes and 10 seconds(13.75 seconds/cycle). The fluorescent levels are 3.73 times to 2.3 times higher, respectively, and the overall process time was more than 7 times faster than that of the general PCR machine. In summary, this study successfully integrated the thin-film plastic chip fabrication processes and rapid peltier-based thermal cycling heaters, and a rapid PCR device hat can potentially apply in point-of-care diagnostics is developed.