Probing Enzymatic Activity Inside Single Cells.

Analytical chemistry

PubMedID: 24003961

Olofsson J, Xu S, Jeffries GD, Jesorka A, Bridle H, Isaksson I, Weber SG, Orwar O. Probing Enzymatic Activity Inside Single Cells. Anal Chem. 2013;.
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 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 Km of 15.3 µM ± 1.02 (mean ± SEM, n=16), and Ki of 0.59 mM ± 0.07 (mean ± SEM, n=14). Enzymatic activity could be monitored just 40 s after permeabilization, and 5 point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.