電化學方法偵測細胞活性最適化條件探討

Feasibility study of electrochemical detection for assessing cell viability

蔡尚恆(Shang-Heng Tsai)

指導教授 ： 蔡惠燕

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00124

摘要

細胞活性評估被用來作為藥物活性及有效成分篩選。在生化領域中常用MTT或顯微鏡計數方式檢測，但其操作繁複、耗時，不利於即時偵測。 本研究希望以電化學建立一即時監測細胞活性，且可避免生化染劑的使用減少環境污染之方法。利用細胞進行代謝作用時與赤血鹽產生還原物質(黃血鹽)，進而透過計時安培法( Chronoamperometry )間接測定細胞活性；另外以電化學阻抗( Electrochemical impedance spectroscopy )觀察細胞貼附結合網版印刷電極(Screening printed electrode)阻抗值的直接變化，藉此觀察細胞的活動情形。 此研究探討細胞所需之雙介質及電化學偵測之最適化條件，以及細胞阻抗量測之最佳頻率與合理的阻抗等效電路，並以已知有毒性之藥物試驗。同時以電化學分析法、MTT及DAPI染色相互驗證，探討其可行性。結果顯示計時安培方法具有即時監測細胞活性之優點，且可避免生化染劑的使用，利用拋棄式網版印刷三合一電極可節省試劑及減少細胞樣品交叉污染。

關鍵字

計時安培法 ； 阻抗 ； 網版印刷電極 ； 細胞活性

並列摘要

A cell viability evaluation can be used to screen the toxicity of a medicine and effective components. The common approach for cell viability detection uses MTT assay and a microscope to perform a direct count, but that operation is complicated and time consuming, rendering it unsuited to real-time detection. The present study aims to establish real-time monitoring of cell viability while avoiding the use of biochemical dyes to lower environmental pollution. Electrochemical measurements of cell viability are based on electrochemical mediators which act as electrons shuttle between intracellular reducing center and an external electrode. For example, ferricyanide was converted to ferrocyanide by cell respiration. The concentration of ferrocyanide, was in proportion to the cell viability. The impedance change in the screen-printed electrodes was measured during cell adhesion through an impedance sensing method in order to record cellular activities through the impedance changes. This research explored double-mediator system and the optimal conditions for electrochemical detection, in addition to exploring the optimal frequency for electric cell-substrate impedance sensing and equivalent circuit models of impedance. We also applied the developed method for in-vitro assessment of drug effects on cell growth. At the same time, MTT assay and imaging through an optical microscope were to confirm that impedance changes were due to cell viability and attachment status. The results show that the method developed in this study has the advantage of real-time monitoring of cell activity. The disposable screen- printed three-electrode system reduced the amount of reagents used and sample cross-contamination.

並列關鍵字

Chronoamperometry ； impedance ； screen- printed electrode ； cell viability

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