以微流道整合表面電漿子共振晶片開發微型聚合酶鏈鎖反應高通量快速檢驗裝置
Development of microscale PCR high-throughput rapid test device by integrating microfluidic and surface plasma resonance chip
計畫主持人：臺大應力所-沈弘俊、中研院應科中心-魏培坤
本研究成功開發一整合裝置，透過往復式循環之微流道，使樣本成功進行PCR，並與SPR晶片進行結合，達到提高檢測極限及即時檢測之目的。因應疫情，本研究選擇COVID-19作為檢測樣本，且因近期有文獻指出，體內有EB病毒之COVID-19患者，其症狀會加劇，因此本研究最終應用於COVID-19及EB Virus之同時檢測。本研究所採用之COVID-19 N-gene DNA序列參照臺灣衛生福利部疾病管制署所公布之文件去進行合成，而LMP1 DNA序列則參照文獻去進行合成。
此整合裝置主要由三部分所組成，加熱系統、光學檢測系統以及反應晶片。加熱系統為雙區恆溫加熱，改善傳統PCR儀器反覆升降溫的能量及時間損耗；光學系統則為直線光路收光系統，透過一般光源垂直入射SPR晶片表面，便能直接激發表面電漿並產生訊號，此系統之靈敏度高、檢測極限也較佳，改善傳統膠體電泳極限不足之缺點；最後，反應晶片由壓克力所製成，並以CNC雕刻機於表面進行微流道之雕刻，透過壓克力此材質，不但能夠大量製造，也可以重複進行使用，大幅降低裝置成本。
本研究成功找到整合裝置之最佳流速，並能夠應用於多重PCR之裝置；並且透過紅移量之檢測，分別建立COVID-19 N-gene及LMP1之檢量線，另外還針對兩者樣本進行交叉實驗，以確立其專一性，結果證明，COVID-19 N-gene與LMP1並不會有顯著之交叉影響。最終使用多重PCR整合裝置進行實驗，成功同時複製兩樣本，並且能夠同時檢測到兩樣本。
Results:
This research has successfully developed an integrated device, through the reciprocating microfluidic channel, the specimen can be successfully replicated and the device combined with the SPR chip to achieve the purpose of increasing the detection limit and real-time detection. In response to the epidemic, this study chose COVID-19 as the specimen, and because recent literature pointed out that the symptoms of COVID-19 patients with EB virus in their body will be exacerbated, so this study was finally applied to the simultaneous detection of COVID-19 and EB virus. The COVID-19 N-gene DNA sequence used in this study was synthesized by referring to the documents published by the Taiwan Center of Disease Control, while the LMP1 DNA sequence was synthesized by referring to the literature.
This integrated device is mainly composed of three parts, heating system, optical inspection system and SPR chips. The heating system is dual-zone constant temperature heating, which improves the energy and time loss of the traditional PCR instrument’s repeated temperature rise and fall. The optical system is a linear light-receiving system, which can directly excite the surface plasma and generate a signal through a normal light source perpendicular to the surface of the SPR chip. This system has high sensitivity and better detection limit, which can improve the shortcomings of electrophoresis. Finally, the reaction chip is made of acrylic, and the microfluidic channels are engraved on the surface with the CNC engraving machine. Through the acrylic material, not only can it be manufactured in large quantities, it can also be reused, which greatly reduces the cost of the device.
This study successfully found the optimal flow rate of the integrated device, which can be applied to multiple PCR devices. Through the measurement of redshift, the calibration lines of COVID-19 N-gene and LMP1 were established respectively. In addition, cross-experiments were performed on the two samples to establish their specificity. The results proved that COVID-19 N-gene and LMP1 There will be no significant cross-effects. In the end, a multiplex PCR integration device was used for experiments, and two samples were successfully copied at the same time, and the two samples could be detected simultaneously.
