利用超快雷射實現PCR微流體元件於新冠肺炎核酸檢測

Abstract

本研究利用超快雷射製程技術(Ultrafast laser technique)之超短脈衝(Ultrashort pulses)與低熱影響區(Low heat-affected zone)機制，在玻璃基材製作陣列微柱(Micro-array structures)之微流體元件(Microfluidic devices)。本研究使用之陣列微柱為毛細流的微泵，應用於流感病毒(Influenza virus)的電性檢測(Electrical detection)和SARS-CoV-2的核酸(Deoxyribonucleic acid, DNA)擴增。本研究在元件設計概念上以毛細力(Capillary force)驅動作為其特點，於微流道底部設計6種不同間距之陣列微柱(10 µm; 20 µm; 40 µm; 80 µm; 160 µm；無微柱)，根據不同的生物分子檢測應用，本研究所設計之陣列微柱可提供多功能應用。在不同生物分子(101至106 PFU/µl(cells/µl))的電性檢測上，以奈米銀顆粒(Silver nanoparticle, AgNPs)覆蓋在元件結構，測試在陣列微柱於直徑30 µm分子的攔截效力，作為電性檢測依據。在聚合酶連鎖反應(Polymerase chain reaction, PCR)中，藉由有限元素法(Finite element method, FEM)進行設計的陣列微柱間距，最佳化流體行為與熱傳於微柱PCR反應，再透過程式控制與電路設計實現PCR所需的溫度控制。本研究成功在30次的溫度循環中成功擴增72 bp的SARS-CoV-2核酸片段，檢測限度可達到2.8 pg/µl。In this study, the advantages of ultrashort pulses and low heat-affected zone in ultrafast laser technique were used to fabricate the micro-array pillar within the glass microfluidic device. The micro-array pillar in this study can be a capillary-flow pump for the applications in electrical detection for influenza virus and DNA amplification for SARS-CoV-2. Six types of micro-array pillar can be designed at different pitches with the microchannel devices, offering the multiple functions in this study. In electrical detection of biomolecules with different concentrations, the micro-array pillar can not only capture biomolecules in diameter of 30 μm, but also provide a large conductive area based on silver nanoparticles (AgNPs) electrodes by titration approach for electrical detection. In the polymerase chain reaction (PCR), the design of micro-array pillar at the different piches can used finite element method (FEM) to optimize the fluid behavior and heat transfer in PCR device. Through the program control and circuit design, the required temperature in PCR device can be doned. By using the microfluidic system, it can perform the stationary temperature in the PCR device to amplify 72 bp of N fragment of SARS-CoV-2 DNA for 30 times of thermal cycle, the detection limit can reach the concertration of 2.8 pg/μl.