利用石英晶體微天秤發展細胞檢測平台

USING QUARTZ CRYSTAL MICROBALANCE TO DEVELOP THE CELL DETECTION PLATFORM

周鴻哲(Hung-che Chou)

指導教授 ： 顏聰榮

大同大學/工程學院/生物工程學系所/博士(2010年)

摘要

石英晶體微天秤(Quartz crystal microbalance，簡稱QCM)經簡易處理後，結合可供細胞生長之細胞培養槽，加上震盪電路的整合，可以建構成為細胞分析系統。細胞在 QCM 晶片表面上生長時，隨著細胞數目在晶片上增加， QCM 震盪頻率會逐漸下降，此情形顯示出由於細胞貼附於晶片上時，細胞膜貼附在電極表面使得QCM晶片表面的環境產生較高的密度與黏滯度，導致QCM 震盪頻率下降，當細胞數量在晶片表面生長達到飽和時，加入 trypsin-EDTA 後使細胞從晶片表面分離，便使得震盪頻率快速上升。在經由細胞存活度實驗確認細胞相容性，利用氨水溶液與過氧化氫溶液使晶片來增加 QCM 表面上親水度，幫助細胞的貼附在晶片表面上。
之後利用已建立之細胞檢測系統評估細胞緊密接合的完整度。利用 QCM 對於溶液不同時會產生對應感應之特性檢測細胞緊密接合的密合度。以 Caco-2 細胞為測試細胞株，培養於 QCM 晶片上生長並偵測狀態。根據實驗結果顯示，隨著細胞緊密接合的密合度越高 QCM 晶片所產生對應頻率變化量越小，當頻率變化量小於100 Hz時細胞的密合度將達90%以上，證實量測 Caco-2 細胞緊密接合的可行性。
最後部分則利用電漿沉積 Hexamethyldisilazane (HMDSZ) 所產生電漿薄膜與光接枝聚合法建立出一保護膜應用於保護銀電極QCM。確立了 HMDSZ 電漿膜的製成條件後，針對三種可以增加晶片親水性方式選擇出最適於應用。綜合水接觸角試驗、細胞存活率實驗與電極保護試驗顯示光接枝聚合 Acrylamide (AAm) 的效果可使水接觸角下降60度，可使晶片脫落時間延長7天，故為最佳條件。最後將修飾後銀電極 QCM 應用於細胞檢測系統證實可以將銀電極 QCM 應用於細胞偵測，在未來將可應用於其他細胞檢測上。

關鍵字

石英晶體微天秤 ； 細胞檢測

並列摘要

A cell detecting system was fabricated in this study. The system includes three major parts. One is the cell incubator chamber, another is the QCM sensor chip, and the other is the cell circuit system. The frequency is decreased with more cells growing on QCM chip. The result indicates that when cell grow on the chip the cell membrane adhered on the surface of electrode and produced higher density and viscosity, and it made the decrease of frequency. When cells were full of chip surface, we added trypsin-EDTA solution inducing the cells leaved chip surface. Thus, the frequency change was increased. After we confirmed the biocompatibility by cell survival experiment, using NH4OH and H2O2 solution to increase the surface hydrophilic can help cell adhere on chip surface.
We used the cell detecting system to estimate the completeness of cell tight junction. The completeness of cell tight junction would be detected by using different solution in QCM system. We choose Caco-2 cell as test cell line, and detected its growth condition when Caco-2 cells grew on the QCM chip. The result showed that the QCM resonance frequency changes less when a better tight cell junction is formed. The cell tight junction integrality is completed up to 90% when the change of frequency was less than 100Hz. We proved the possibility of detecting Caco-2 cell tight junction by using QCM system.
We established a membrane process by plasma treatment and graft polymerization to form a protection film to protect QCM sliver electrode. After establishing the process condition of HMDSZ plasma film, we choose one of three ways to increase the chip hydrophilic ability. For overall concerned with water contact angle test, cell survival experiment and electrode protective analysis, it suggested graft polymerization AAm be the better effect. We proved that using improved silver electrode QCM with a protection film can detect cell growth of Caco-2, and believed that this system can be used for detection of other kind cell growth.

並列關鍵字

QUARTZ CRYSTAL MICROBALANCE ； CELL DETECTION

參考文獻

田育彰。2002。固定生醫單體之生物活性與結構穩定性之探討，中原大學醫工所博士論文。

李宗星。1993。聚丙烯不織布經乙烯單體皆知與膠原蛋白及幾丁聚醣交聯固定其生物相容性及物化性之影響，中國文化大學材料科學與製造研究所碩士論文。

施証耀。1996。以乙炔/氮氣電漿改質高分子氣體分離膜之研究，中原大學化學工程學系碩士學位論文。

陳俊光。2008。同時解析分子質量與液相阻尼之相位補償式石英微質量天平，臺灣大學應用力學研究所博士論文。

楊士賢。2005。以脈衝式電漿輔助化學氣相沉積法製備氟化非晶碳膜之研究，中原大學化工所碩士論文。