利用光誘導細胞裂解法於微流體平台進行連續式細胞核萃取之應用

Nucleus Extraction from Cells by Performing Optically-Induced Cell Lysis on a Continuous-flow Platform

黃士軒(Huang, Shih Hsuan)

指導教授 ： 李國賓

國立清華大學/工學院/動力機械工程學系/碩士(2015年)

摘要

摘要
本研究提供一種創新的微流體技術應用到連續式平台進行細胞核萃取。先前研究所提出的微流體細胞裂解法主要可分為幾類:加熱、電、化學、聲波、光學以及機械微結構法，這些方法可以被應用到後端的化學處理與細胞內物質之分析。相較於其他裂解方式，創新的光誘導細胞裂解平台提供更具精確性、選擇性、甚至更有效率。更重要的是，只要可以精確地控制光誘導法的條件與參數，達到僅裂解細胞膜但可留下完整細胞核之成果。為了可以將光誘導裂解法應用到連續式細胞萃取，我們更進一步整合光介電泳到我們的微流體系統。此創新整合型微流體系統解決許多傳統細胞核萃取常會遇到的問題，包含:無法快速萃取完整的細胞核以及需要專業人員的技術操作。因此，我們的新的微流控系統提供了有效的方法，自動化四個步驟包含:集中並傳輸人類胚胎腎臟細胞（human embryonic kidney 293T）、進行細胞膜的光誘導裂解並釋放出細胞核，緊接著利用光介電泳分離細胞核，並萃取細胞核於最後端的收集槽。而此系統平均每秒可以同時對1顆細胞進行細胞核萃取。為了證明整合式微流體系統的所有功能，我們利用綠色螢光(SYTO Green) 標定細胞核，紅色螢光(CellMask Deep Red Plasma Membrane) 標定細胞膜以利於觀察細胞。本研究藉由觀察螢光訊號來證明整合式系統的可行性，結果顯示紅色螢光不見而綠色螢光維持存在，表示細胞膜裂解但細胞核沒被破壞，而量化後的光誘導細胞膜裂解效率以及細胞核分離效率分別為78.04 ± 5.70 % 和80.90 ± 5.98 %。因此，可以得到細胞核萃取效率為58.21 ± 2.21 % 。不僅如此，我們也成功將流體操控模組整合到整個系統，希望本系統可以控制精確的定量的細胞進行細胞核萃取，以利於化學處理和疾病檢測以及分析之應用。

關鍵字

細胞裂解 ； 光誘導電細胞裂解 ； 細胞核萃取 ； 微流體

並列摘要

Abstract
This study presents an innovative microfluidics-based approach for nucleus extraction and collection in a continuous format. Previously proposed micro-devices for cell lysis can be classified into several types, including thermal, electrical, chemical, acoustic, optical, and mechanical approaches, and can be combined with subsequent chemical treatments and analysis of extracted intracellular components. Comparing with previous micro-devices, a novel optically-induced cell lysis (OICL) device proposed in this study shows the advantages with significant flexibility, selectivity and efficiency. More importantly, once can precisely control the operating conditions of this new device to lyse cell membranes without disrupting nucleus. To facilitate this OICL module for continuous nucleus extraction, we further integrate an optically-induced dielectrophoresis (ODEP) module with OICL device into a microfluidic system. This novel approach avoids critical issues such as difficulty to extract intact nucleus rapidly and the requirement of professional skills to operate the traditional equipments, which are posed as common disadvantages to the traditional nucleus extraction approaches. Our new microfluidic system therefore provides a powerful method for automating four steps, including automatically focusing and transporting human embryonic kidney 293T cells, releasing the nucleus on the OICL module, nucleus isolation on the ODEP module and finally nucleus collection in the outlet chamber. The average throughput of nucleus extraction and collection was measured to be 1 cells/s. In order to prove the full function of this integrated microfluidic system, the nucleus was stained with green fluorescence dye (SYTO Green) and plasma membrane was stained with red fluorescence dye (CellMask Deep Red Plasma Membrane) for cell observation. The feasibility of this integrated system was demonstrated by observing fluorescent results showing no red fluorescence and maintenance of green fluorescence, indicating cell membrane lysis without disruption of the nucleus. The cell membrane lysis rate and ODEP nucleus separation rate were measured to be 78.04 ± 5.70 % and 80.90 ± 5.98 %, respectively. Therefore, the overall efficiency of nucleus extraction rate can be quantifiably up to 58.21 ± 2.21%. Furthermore, a flow control module was successfully integrated with whole micro-system, suggesting that the developed system is capable of transportation metered cells, releasing nucleus, and isolation of extracted nucleus for subsequent chemical treatments, applications and specific analysis of diseases.

並列關鍵字

Cell Lysis ； Optically-Induced Cell Lysis ； Nucleus Extraction ； Microfluidic

參考文獻

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