整合細胞內外微流交換及光學檢測於平面式片膜鉗制晶片之電生理量測系統

Abstract

離子通道在人體中扮演重要的生理訊號傳遞的角色。傳統操作玻璃針尖的片膜鉗制技術(patch-clamp technique)為目前最為可靠用來了解離子通道的經典技術，能從細胞膜上獲得豐富的電生理資訊。但長久的訓練與複雜的操作過程使此技術進入門檻高且效能低。因此近年來許多平面式片膜鉗制晶片的研發，希望能夠減少傳統研究之入門門檻。本研究之前身，為團隊於2007年之發現：當二氧化碳雷射聚焦於150微米的蓋玻片上時，因玻璃回融之現象，而產生出1~30微米尺度的獨特沙漏構型微孔洞，可利用替代傳統玻璃針管，進行離子通道之量測。 本篇論文之主軸藉由調控雷射的頻率、時間、個數，將先前發現的孔洞做定性定量的分析，並搭配特殊二階段雷射製程方法，使孔洞能穩定縮小至1-3微米成為適合電生理量測之平面孔洞晶片，並進一步將晶片整合微流體交換與光學檢測之功能。結果顯示，在不同頻率、時間、個數之雷射製成下，孔洞形式會有四種迥異之結果，而其中能獲得較平滑的第一階段孔洞。配合二階段雷射方法，穩定的改變與縮小孔洞的最終尺寸，使1-3微米孔洞製程良率大幅提升。數種細胞株whole-cell的電流，皆可於此平台上確實量測。細胞內溶液也藉由交換perforated patch量得Jurkat 之whole-cell電流。HEK 293T之細胞外溶液從原本正常生理鹽水交換成以鉀離子為主的溶液，whole-cell電流改變也確實量測得到。當細胞外溶液交換成calcein AM溶液時，細胞螢光強度的變化也被確實檢測。我們也成功嘗試同時將九顆同時抓取與觀測。本團隊相信此研究經濟、方便、微流體整合、螢光檢測的特性，是目前首見的。其也具有與市場商品相抗衡的競爭優勢。

Ion-channel plays an important role in signaling and functioning throughout human body. The most pervasive technique to interrogate ion-channel is patch-clamp owing to its potent and reliable information extracted from cell, but traditional patch-clamp technique requires high skill-dependent and laborious manipulation that impedes its throughput. Our lab have previously proposed a simple laser drilling technique on a plain cover glass in fabrication of planar patch-clamp chip in order to lessen traditional exquisite operation. This paper presents planar patch-clamp chip integrated with microfluidic system and optical detection for ion-channel recording. Results show that whole-cell recording is feasible for various cell lines, such as pancreatic beta cell of HIT-T15 and RIN-m5F, suspension lymphoma Jurkat cell, and HEK 293T cells that are generally used as transfection model for channel expression,. Intracellular solution exchange was performed via internal perfusion of perforated patch, and whole-cell current is consistent with typical patch recording via voltage zap. Extracellular solution was successfully exchanged from standard extracellular saline to symmetrical K+-containing solution. Fluorescence was measured via optical detection while extracellular solution exchanged with calcein AM stain. Nine cells were concurrently captured and aligned in 3x3 array configuration. It is believed to be the first proposition of economical fabrication, microfluidics integration, and optical detection, which is compatible with existing commercial products, and has great potential for drug-related applications.