本研究開發使用磁珠於生物檢體中分離特定胞外囊泡，並萃取其所攜帶之微型核醣核酸的技術，以作為檢測疾病的生物性指標。首先使用接有CD63抗體的磁珠分離出特定胞外囊泡，將胞外囊泡膜打破後再使用接有和微型核醣核酸互補之序列的磁珠，萃取出特定微型核醣核酸，最後以即時定量聚合酶連鎖反應進行定量。由於磁珠能被磁力聚集在管壁上，可解決已往沉澱離心操作上廢液去除困難的缺點，同時磁珠的操控簡單，也易與微流道晶片結合，以開發快速簡便分離胞外囊泡所攜之微型核醣核酸技術。在此研究中選定帶有CD63胞外囊泡中的miR-21、miR-126以及miR-208a，以作為檢測心血管疾病的生物性指標。在這篇實驗中做了磁珠分步驟的效率以及效果驗證，同時也以血漿做測試得到初步結果。為了驗證CD63磁珠的性能，以奈米顆粒追蹤分析與生化分析儀的結果做比對得到差不多約70%的結果，同時也對混合時間做測試得到18小時最佳的結果；在微型核醣核酸磁珠上則將分離出的產物藉由定序來確認是否為miR-21、miR-126以及miR-208a，同時也以標準品做定量線繪製；再以血漿為樣本進行磁珠萃取，最終測量到1.3與14811 fM的miR-21和miR-126的初步結果；最後也在相關實驗晶片上做應用測試，在CD63磁珠的應用上晶片能得到和人為操作略微一致的結果。綜上所述，本篇研究已開發出能夠良好應用在微流體晶片上的胞外囊泡內微型核醣核酸分離方法並得到初步結果。

The method of isolateing plasma extracellular vesicles and extacting EV-contained microRNA has been investigated systematically, which performance is assessed by using real-time PCR quantififcation. Magnetic beads are utililzed for the development of a simple EV isolating technique that is amiable for microfluidic integration. Magnetic beads coated with CD63 antibodies are mixed with samples to isolate CD63+ EVs. EV-contained miR-21, miR-126 and miR-208a, as biomarkers for cardiovascular diseases, are extracted subsequently after mixing EV lysate with magnetic beads coated with complementary miRNA sequences. ~ 70% EVs are isolated from conditioned cell culture medium after 18 h of mixing with magnetic beads, which is assessed with a Bioanalyzer as well as nanoparticle tracking analysis. The specificity of microRNA-bead isolation is verified with sequencing results. Furthermore, calibration curves of RT-qPCR results have been established using synthesized microRNA molecules. Preliminary data indicate 1.3 and 14811 fM of miR-21 and miR-126 are present in healthy human plasma CD63+ EVs. Finally, an automated microfluidic device has shown comparable CD63+ EV isolation results. In summary, this research has demonstrated that the proposed method is capable of isolating EV-contained microRNA in human plasma samples and may potentially be implemented on microfluidic chips.

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