亮場型與暗場型LED螢光定量PCR感測模組開發及微小化

Abstract

定量聚合酶鏈鎖反應(quantitative polymerase chain reaction, qPCR)是一種結合DNA複製與螢光感測技術以進行快速致病原檢測的分析方法。隨著防疫觀念的興起，防疫人員期望能發展在採樣現場即時作業的可攜式定量PCR檢測器。然而檢測器的微小化往往會造成螢光訊號微弱的問題。為了增進微小檢測器的效能，我們期望能運用暗場(dark-field)光學機構的概念，以光遮片(light stop)與聚光器(condenser)設計來抑制背景光源的干擾並提高訊號對比度。本研究研發以高發光效率的LED作為光源之可攜式定量PCR核酸檢測器，並提出一種新型的暗場光學設計藉以增進核酸偵測極限。檢測器之光學機構使用壓克力為基材，以CNC铣床進行加工，再將LED、光電二極體、濾光鏡、透鏡等元件安置其中。同時設計亮場(傳統直線式)與暗場(新型同側式)的光學機構進行性能比較，亮場與暗場的激發光強度分別為37.4 klx與15.2 klx，進行檢測時各有50.2與28.6的訊噪比。暗場型的光遮片設計為160度的5 mm縫隙時，具有最佳的感測效能。檢測器進行核酸定量時，可具有0.5 ng/ml(亮場)與0.16 ng/ml(暗場)的偵測極限。在定量PCR系統的驗證中，我們採用沙門氏菌(Salmonella spp.)的特徵序列InvA gene作為標的，以市售PCR機器進行核酸擴增，再加入SYBR Green I螢光分子後染(post-stain)，並使用螢光檢測器分析。PCR反應的擴增曲線以sigmoid model進行曲線配適，可得其擴增效率為0.56(亮場)與1.02(暗場)。而在30 cycles定量PCR分析中，檢測器的檢測極限分別為103 copies(亮場)與102 copies(暗場)。本研究發展的暗場型螢光檢測器可成功的應用於定量PCR檢測，並獲得較亮場型光學機構更佳的偵測極限。未來若能整合微型化的溫度控制模組，成為可攜式的即時定量PCR儀器(real-time qPCR)，將能有效提升檢疫人員於現場分析的機動性，對防疫工作有很大的助益。

Quantitative polymerase chain reaction (qPCR) is a rapid pathogen detection method. It combines DNA amplification and fluorescence detection technology. Emerging concept of point-of-care demands that the quarantine officers expect to develop portable qPCR systems, which can be used for on-site detection. However, the miniaturization of system may hamper the fluorescence signal. In order to enhance the system’s detection limit, we adapted and elaborated the dark-field optics that was developed for the microscope application. This design combines light stop and condenser in order to improve the contrast of fluorescent image by inhibiting the background light interference. In this study, we have developed and tested a portable qPCR device using the high luminous efficiency LED and a novel dark-field optical design against the detection limit. The device is constructed out by the CNC machining, and it is modularized compactly in order to accommodate self-sufficiently the LED, the photodiode, the filters and the lens. Our design features, two kinds of optical structure – bright field and dark-field are designed in order to facilitate system performance comparison as well as application flexibility. The results show that the excitation intensity were 37.4 klx(bright-field) and 15.2 klx(dark-field) and the signal to noise ratio were 50.2(bright-field) and 28.6(dark-field), respectively. Optimum sensing performance was identified with a 160 degree at 5 mm slit. When the light stop in dark-field optics has 160 degress and 5 mm width slit, the system has better sensing performance. For the quantification of SYBR Green binding to the DNA, the detection limit were 0.5 ng/ml(bright-field) and 0.16 ng/ml(dark-field). Finally, the system verification was done by detecting the InvA gene sequence which is the biomarker of Salmonella spp. and amplified them by commercial PCR machine. The PCR products were post-stained with SYBR Green I and detected by optical detector. The record signal were analyzed by sigmoid model fitting to calculate the amplified efficiency, and the efficiency were found to be 0.78(bright-field) and 1.02(dark-field). In the quantitative PCR analysis, the bright-field and dark-field device can detect 103 and 102 copies of templates, respectively, in 30 cycles of operation. In summary, the dark-field fluorometer developed in this research can be successfully applied to quantitative PCR and gets better sensing performance than bright-field optical apparatus. If a thermal control module can be integrated with this optical system to develop a real-time qPCR device, we conclude that our development will facilitate the mobility of quarantine officers in the detection site a lot. It will also advance the point-of-care medicine service in near future.