微流道聚合酶連鎖反應裝置結合奈米狹縫表面電漿共振感測器用於LMP1 DNA 之檢測

Abstract

EB病毒是一種與鼻咽癌及淋巴癌高度相關的病毒，而 LMP1是EB病毒中常見的表現蛋白之一，因此本研究便以LMP1 DNA作為檢測標的，利用微流道聚合酶連鎖反應裝置結合奈米狹縫表面電漿共振感測器，開發出一套結合DNA複製及檢測的裝置化整合平台，能夠針對LMP1 DNA及其真實檢體進行免標定、即時、高靈敏度的檢測。 本研究微流道PCR採用分段加溫的方式，可以省去傳統PCR反覆升降溫的多餘能源及時間消耗。感測器使用奈米狹縫SPR晶片，是一種免標定、即時檢測、高靈敏度的生物感測器，由於其體積小、光路設計簡單，因此相當適合與微流道PCR整合。微流道製作方式採用CNC雕刻機雕刻壓克力搭配有機溶液黏合法，成本低且製造快速。奈米狹縫SPR晶片採用自動化奈米壓印技術，於PC板上壓印奈米級光柵結構，製作方式簡單，適合大量製造。 微流道PCR裝置在流速5 g/mL之下，能達到儀器PCR 30 cycles的複製效果，且反應時間僅需要36分鐘，比傳統儀器節省了將近70分鐘。針對低濃度LMP1 DNA的檢測，最低檢測極限濃度達到10 pg/mL，比傳統凝膠電泳檢測方式擁有更低的檢測範圍。真實檢體檢測方面，亦能成功從癌細胞萃取出的DNA中檢測出LMP1基因，顯示本研究裝置應用於真實檢體檢測的可行性。

EB virus is highly associated with Nasopharyngeal carcinoma and Lymphoma, and LMP1 is one of the main performance proteins of the EB virus. A combined device of microfluidic Polymerase Chain Reaction with nanoslit Surface Plasmon Resonance sensor for LMP1 DNA detection has been developed, which enables amplification and detection of LMP1 gene on a combined platform. The combined device performed a label-free, real-time, highly sensitive, low cost, and rapid detection versus LMP1 DNA and its relative real samples. The continued-flow PCR has been used, which can avoid unnecessary energy loss and time consumption, and nanoslit SPR chips have been used as a label-free, real-time, high sensitivity sensor. Considering the small volume and simple optical setup, the nanoslit SPR chip is quite feasible to integrate with microfluidic PCR. Microchannel was fabricated by CNC engraving and solvent bonding of PMMA. Furthermore, automatic Nanoimprinting Lithography has been used to fabricate SPR chips by printing nanoscale grating structure, owning the advantage of low cost and mass production. An optimized flow rate has been found out. For the 30 cycles reaction of PCR, the new method only took 36 minutes, almost 70 minutes less than the conventional method with the same DNA amplification effect. Besides, the different initial concentration of LMP1 DNA has been detected by the combined device. The detection limit of LMP1 DNA is 10 pg/mL. Moreover, the combined device is capable of detecting real samples of extracted DNA from EBV-positive cells, showing the potential of clinical diagnosis.