Probing Enzymatic Activity Inside Single Cells

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• 作者：Jessica Olofsson ; Shijun Xu ; Gavin D. M. Jeffries ; Aldo Jesorka ; Helen Bridle ; Ida Isaksson ; Stephen G. Weber ; Owe Orwar
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：November 5, 2013
• 年：2013
• 卷：85
• 期：21
• 页码：10126-10133
• 全文大小：349K
• 年卷期：v.85,no.21(November 5, 2013)
• ISSN：1520-6882

文摘

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant K_m of 15.3 渭M 卤 1.02 (mean 卤 standard error of the mean (SEM), n = 16) and an inhibition constant K_i of 0.59 mM 卤 0.07 (mean 卤 SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.