快速溶液交換之微流體元件應用於細胞電生理研究

Abstract

整合微流體系統的膜片鉗制晶片在離子通道的研究上是一強大的工具。本論文的目的在於提供ㄧ快速溶液交換之平台應用於電生理之研究，來達到高產量的離子通道訊號。在這平台上，包含微混合器和膜片鉗制晶片。利用二氧化碳雷射雕刻機來打穿硼矽酸鹽玻璃，使硼矽酸鹽玻璃因為熱回熔的效應，在晶片上產生非常光滑的孔洞。而漏斗狀的孔洞為本論文中一特殊發現。漏斗狀的孔洞剛好可以固定住細胞且使細胞膜和孔洞表面間有最大的接觸面積,來做為電阻質量測。會選擇硼矽酸鹽玻璃，是由於它的成本較為低廉且有不錯的電性質。聚二甲基矽氧烷的微混合器是利用軟微影的技術所製成的。不同的溶液可以藉著改變針筒幫浦的流速來得到，且這些溶液會經由微流道而到達已被鉗制的細胞。工作流體則選用藍色食用色素和清水。針筒幫浦的流速設定為16mm/s。 實驗結果顯示，快速溶液交換之平台可以將溶液的濃度從0% 到100% 以間隔25% 來得到不同濃度之溶液。溶液從0% 到25% 的交換時間約為25秒，而其它的50%, 75% 以及100% 等三個間段所需的交換時間，均約5秒鐘。中國倉鼠卵巢細胞與孔洞間的電阻值可以穩定的達到200~300M歐母。不管溶液以任何流速流經過細胞，已被鉗制住的細胞其電阻值不會被通過細胞表面的流體所影響。然而，孔洞的製程與達到giga等級之孔洞和細胞間電阻的成功率，還是不如預期的好。因此仍然有些關鍵性的問題需要解決，例如：雷射雕刻機的穩定度，細胞的準備，以及量測細胞和孔洞間電阻值之參數。在解決關鍵性問題後，在電生理的研究上，快速交換的微流體元件成功的達成高產量目標，是指日可待的。

The integrated patch clamp chip consisting of microfluidic system is a powerful tool for the study of ion channel. The goal of this thesis is to develop a rapid medium exchange platform for electrophysiology study to achieve high-throughput of ion channel signals. The platform consists of micro-mixer and planar patch clamp chip. Thermal reflow of glass is applied to generate very smooth surface on the chip by utilizing the CO2 laser to penetrate the borosilicate glass. A special discovery of aperture is the hourglass shaped aperture. Immobilized cells fit perfectly to the hourglass apertures offering a large contact area for sealing between the cell membrane and the surface of aperture. Borosilicate glass was chosen due to that it is inexpensive and good electrical property. Micro-mixer is made of polydimethylsiloxane (PDMS) by using soft lithography technology. Different mediums can be obtained by controlling the injected flow rate of syringe pump and then flowed through the trapped cell. Verification of the device used blue food dye and clean water as working fluid at 16mm/s. Results show that the rapid medium exchange platform can provide different concentration from 0% to 100% with increment of 25%. Exchanging times from 0~25% is almost 25seconds and other steps are almost 5seconds individually. Seal resistance of Chinese hamster ovary (CHO) achieved 200~300Mohm steadily. Flow that can pass through the cell membrane cannot affect the seal resistance of the trapped cell with any other flow rate of syringe pump. However, accuracy about fabrication of aperture and the yield of giga seal are not as good as expectation. The stability of laser engraver, cell preparation and the parameters of seal test are believed to be the key problems that need to be improved. The microfluidic device for rapid medium exchange can succeed to achieve high-throughput for electrophysiology.