摘要:細胞之離子通道為蛋白質巨分子所構成,與神經、肌肉的電訊息傳導以及許
多的新陳代謝息息相關。因此離子通道功能的缺陷往往導致許多疾病的產生,如
心律不整、囊腫性纖維化、高血壓以及神經相關的疾病等。片膜箝制技術被廣泛
地應用在研究離子通道電生理中,然而此技術需要人工精細的操作,使得產率極
低,在細胞資訊的獲得上顯得缺乏效率。
此三年的整體計畫目標在於設計以及製造一個以聚二甲基矽氧烷(PDMS)
為材料的平面片膜箝制裝置,並整合入微流體系統,以達到高效能以及高通量的
電生理晶片。以功能上而言,此裝置設計包含優良的gigaseal(即細胞膜與晶片
緊密接合,電阻超過109 歐姆)形成、快速的交換實驗溶液、細胞操控與定位、
平行化的執行、以及系統的自動化。
第一年實施內容主要專注在設計與製造原型的微流體片膜箝制裝置。此裝置
最關鍵的元件當屬與細胞相互貼合的PDMS 薄膜。此薄膜之製作將使用兩種不
同的製程,一種利用軟微影技術,另一種則利用二氧化碳雷射來進行加工。其選
擇將視何技術適合快速與方便的製作出5-15μm直徑的微孔洞而定。同步地,一
種創新的可重複使用式片膜箝制晶片亦將開始設計與製作。此兩種設計皆會透過
量測其緊密電阻值(seal resistance)來驗證其優劣與否。此外,此原形裝置將會利
用片膜箝制專用之電生理設備進行生物離子通道訊號之量測。
第二年實施內容主要專注在改善成功紀錄之產出效益。細胞表現、表面化學、
裝置之細節等等都將是影響此效益之關鍵問題。首先我們將使用電漿表面處理,
透過不同氣體的組成以及參數來進行測試。另一方面,亦將利用脂質雙層塗佈在
晶片上,使細胞與晶片的表面特性一致,達到更佳的緊閉貼合。除此之外,微流
體系統的組件將進行最佳調校,以提升產出效益。紀錄槽之設計亦將進行改良,
以達到快速實驗液體交換以及具備高品質的外界連接系統。
第三年實施內容主要專注在系統元件的整合以達到高通量分析之目標。此系
統將包含片膜箝制晶片陣列、濃度梯度產生裝置、細胞引導裝置、以及與外部世
界連接之紀錄槽。系統的自動化以及資料的擷取系統將會針對特定的實驗進行軟
體的撰寫。我們將利用哺乳動物細胞研究典型的劑量反應實驗。整體的整合系統
將進行最佳化調校以達系統之最優表現。
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Abstract: Ion channels are protein macromolecules which bear the information of electrical signal in
nerves, muscles, and plenty of synapses for metabolism.1 For this reason, ion channel defects are
responsible for disorders such as cardiac arrhythmias, cystic fibrosis, hypertension, and some
nerve-related diseases. Study of ion channel electrophysiology almost exclusively utilizes the
patch-clamp approach, which is labor-intensive for even the skillful, thus severely limiting cellular
information throughput.
The overall goal of this three-year proposal is to design and fabricate a PDMS-based planar
patch clamp device integrated with microfluidic system for high yield and high throughput operation.
Functionally, the device features quality gigaseal formation, fast solution exchange, cell
manipulation, parallel execution, and system automation.
The first year effort will focus on the design and fabrication of the prototype microfluidic patch
clamp device. Perhaps the key component of the device is the PDMS membrane which attaches the
cell. The PDMS cell-patch membrane will be fabricated either via soft lithography or engraving
with CO2 laser depending on success of rapidly fabricate the high quality aperture, with features of
about 5-15μm. Concurrently, a novel reusable patch clamp chip, which houses the PDMS
membrane, will be designed and fabricated. Both designs will be examined in seal resistance and
other functions to confirm the quality of the design. The prototype device will then be tested via
patch clamp specific software and interrogation of the data will be conducted.
The second year effort will focus on improving the yield of successful recording, which is still
a thorny issue depending cellular expressions, surface chemistry, device details etc. Surface plasma
treatment will be tested first, with different gas composition and parameters. Lipid bilayers will also
be coated on the chip surface to improve the seal between the cell and the chip surface. In addition,
parts of microfluidic system will be fine-tuned for yield enhancement. The recording chamber will
also be improved for fast solution exchange and quality chip-to-world connection.
The third year effort will integrate individual compartments to achieve high throughput
assays. The system comprises patch clamp chip array, concentration gradient generator, cell
guidance, and recording chamber with chip-to-world connection. Software will be composed
for system automation and data acquisition of specific experiment. Typically, a dose response
experiment will be studied using mammalian cells. The entire integrated system will be fine-tuned
for performance optimization.
1Bertil Hille, Ion channels of excitable membranes, 3rd ed. (Sinauer, Sunderland, Mass., 2001).
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