新型皮升級濃縮微孔用於偵測胞外囊泡中微核糖核酸與分子信標雜合之螢光訊號增強

Novel Picoliter Condensation in Microwell for Amplifying the Fluorescent Signal Detection of Extracellular Vesicle MicroRNA Hybridized with Molecular Beacon

洪瑞鴻(RUI-HONG HONG)

指導教授 ： 林政鞍

中原大學/工學院/生物醫學工程研究所/碩士(2020年)

https://doi.org/10.6840/cycu202000073

摘要

癌症是國人十大死因之首，早期檢測為臨床癌症醫學之重要課題，現今癌症的檢測常以內視鏡及電腦斷層掃描為主要依據，然近年癌症基因檢測的技術日益精進，有機會利用癌症病患血清中胞外囊泡進行基因訊息的篩檢，針對特定種類基因片段進行數量計數，癌症的病患囊泡內之特定基因會有異常的表現。最新臨床研究中可利用不破壞囊泡本身檢測囊泡中特定基因序列之方式，改以利用自製脂質體奈米粒子包覆反序列分子信標，和人體血清囊泡進行雜合，使用螢光強弱的方式判斷囊泡中miRNA之數量，但由於弱螢光物質難以觀測且影像訊雜比過低容易導致誤判，故本研究建構新型微量濃縮的概念，得以增強低螢光訊號之待測樣品，並製作以親疏水性差異為導向的微米級孔洞，使得訊號集中並侷限於設計之微孔中，讓樣品液體體積從微升（μL）等級濃縮至皮升（pL）等級，使得濃縮倍率高達萬倍以上，再透過影像分析軟體（Image J）搭配濃縮結果利用灰階數值計算兩種奈米粒子接合前後之螢光強度變化，以數據化的方式得出影像訊雜比，結果與高感度螢光光譜儀檢測數據成高度正相關，可做為未來微量檢測之新標準。

關鍵字

胞外囊泡 ； miRNA ； 脂質體奈米粒子 ； 微孔 ； 親疏水差異 ； 皮升級濃縮 ； 訊雜比

並列摘要

Recently, cancer is still the top 10 cause of death in the world, and its early detection is important for current cancer medicine. Endoscopy and computed tomography are basic tools for cancer detection, and the genetic diagnostics also become the advanced tools in the last two decades. Researchers analyzed the extracellular vesicles (EVs) from the serum of cancer patients to screen for genetic information, to count the number of specific gene fragments, and to compare with those with health people. At the latest progress, clinical researcher has created a way to detect specific gene sequences in vesicles without destroying themselves. The self-assembled lipoplex nanoparticles were embedded with molecular beacons to sense the miRNA of human EVs in a fluorescent way. Due to the cost and its weak fluorescent signals during detection, this study intended to use the concept of condensation to enhance the original source of samples with low fluorescent signals, and making micron-level guided wells to enhance the signals. The sample liquid volume can vary from the microliter (μL) level to the picoliter (pL) level. The image analysis was also used to quantify the concentration by calculating the change in fluorescence intensity upon the lipoplex-EVs hybridization. This techniques bring the alternative way of micro-detection in the future

並列關鍵字

extracellular vesicles ； miRNA ； lipoplex nanoparticles ； microwells ； hydrophilic ； hydrophobic ； pico-liter condensation ； signal-to-noise ratio

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