微流碟盤系統應用於血液中外吐小體之免疫親合抓取之研究

Abstract

癌症轉移是目前最主要的癌症死亡原因，對於其轉移的機制至今仍待釐清。透過分析現今所認定之癌症轉移因子，盼望對於其轉移的預防能有更進一步的貢獻。近年來，外吐小體已被許多研究證實在癌症轉移中扮演著相當重要的角色，而用其當作早期癌症轉移的標的也漸趨明朗。如何精準且快速的抓取與分析高純度的外吐小體也就成為現今很重要的一個課題。本研究提出一套能運用在自動化機台的微流碟盤系統。此系統由微結構碟盤與特殊設計的微量離心管承載器所構成，碟盤可直接從全血開始進行自動化血漿分離以及外吐小體的磁珠免疫親合抓取，其免疫磁珠與血漿比例和外吐小體抓取效率則是經由100到600微升的全血進行驗證，而結果是由西方墨點法進行蛋白檢測。此外，透過與超高速離心法和高分子試劑抓取的比較，實驗結果證實，碟盤系統可從三位乳癌患者成功的分離出血漿中的外吐小體。且可從全血分離98.3%的紅血球以達到血漿分離。此系統透過自動化機台和免疫親合法的專一抗體結合抗原特性，減少了人為的操作實驗的誤差，同時省去了超高速離心繁瑣步驟和常用試劑的非專一抓取的問題。盼望此系統能對於未來臨床研究者提供妥善的協助。

Cancer management can be better served by suitable biomarkers ranging from diagnosis and monitoring of therapeutic progress. Over the past few years, clinical relevance of exosomes as a marker during tumor progression and early disease detection has been validated. However, the enrichment of exosomes remains technically challenging, where purity, reproducibility and automation are highly desirable. In this thesis, a centrifugal microfluidic platform was presented to enrich exosomes directly from blood. The platform contains a microfluidic disk and a mechanism to collect plasma into an Eppendorf tube. A range of parameters of immunomagnetic beads to plasma ratio and system performance could be obtained from 100 to 600 μl of human whole blood. Western blotting was used for protein quantification. Besides, the performance of exosome enrichment was compared with that from ultracentrifugation and a commercial exosome isolation kit. Results showed that the microfluidic device successfully enriches exosomes from three breast cancer patients directly from whole blood. Averaged 98.3% red blood cells from whole blood was depleted in the plasma separation process. Taken together, our microfluidic platform provides a simple-to-use and robust approach to enrich specific exosomes by recognizing the exosomal surface markers. Moreover, the automated system reduces variation in operator biases and may serve as a standard device for clinical uses.