利用磁珠與數位微流體進行DNA萃取

Abstract

｢個人化醫療｣ (personalized medicine)是針對患者個體的差異，進行正確疾病檢驗並做出合適的醫療決策，以有效縮短疾病治療的時間。其主要的關鍵技術便是針對患者個體進行的｢基因診斷｣ (genetic testing)。而所謂的生物實驗室晶片 (Lab-on-chip)，則是將一連串繁複的生化試驗處理的功能，縮小並整合在一片為小的晶片上。例如：在病患的血液樣本取得後到進行基因定序或診斷需進行生化處理程序。 本文是利用數位微流體(Digital Microfluidics)技術當作生物實驗室的平台，並且和磁珠結合使用，來進行DNA萃取。數位微流體以電場精準的控制微小單位體積液體，移動試驗所需反應之試劑，減少試劑用量和加熱孵化的過程，從90 nl的血液中取出約1..85×〖10〗^(-2) ng/μl的DNA。 試劑於晶片上均有獨立的路徑進入反應區，避免彼此間的交叉污染。同時，在晶片上利用磁珠進行DNA的萃取，免除了在傳統DNA萃取過程中繁複的離心分離程序。於磁珠的分離及清洗的過程中，透過電極的設計及操作，移去絕大部分的懸浮液體，確實的控制殘存的液體量，改善洗滌時的效率，降低操作過程的時間以及大幅減少試劑及樣本的消耗。

To develop and realize the personalized medicine and point-of-care applications in genetic testing, the full sequences of lab processes should be integrated. DNA extraction is essential and often required for a sensitive nucleic acid testing. This thesis introduces the implementation of magnetic beads (MB) based DNA extractions on electrowetting- based digital microfluidics (DMF). The reagents from two different extraction kits, ITRI and Agencourt Genfind v2, are characterized as a droplet on DMF. These droplets can be precisely manipulated by using electric signals, which simplifies the whole extraction procedures, and promises the possibility of process automation The results from the on-chip DNA extraction protocols are validated and quantified. DNA at 1.85 〖×10〗^(-2) ng/μl is successfully extracted from a droplet of 90 nl whole blood. Finally, our DMF microchip has been optimized in the following three aspects: (1) it has independent paths of the electrodes for different reagents to avoid the cross-contamination problem. (2) It utilizes MBs to replace the complex centrifugation in tradition DNA exrtaction procedures. (3) Ratio separation electrodes are designed to re-suspend the MBs and to improve the efficiency of the wasing process. Therefore, our DMF microchip not only can successfully extract the DNA from whole blood, but also demonstrate the possibility to use less sample/reagent and shorter process time to purify DNA on chip for point-of-care genetic testing.