Influence of Temperature on Alkaline Phosphatase Activity in Single Cells
We report a new approach for the determination of influence of temperature on enzymatic activity within a single fixed cell. We investigate enzyme activity at different temperature (22, 27, 32,37 and 42°C) by means of directed infrared laser heating. Our method generates individual cells at selected location, through the use of a flexible microfluidic delivery device and IR fiber heating system. Using timed exposure to the pore-forming agent digitonin, we successfully controlled the extent of poration within the plasma membrane of individual NG-108 cells. Mildly permeabilized cells (∼100 pores) were exposed to substrate concentrations for alkaline phosphotase and exposure to a series of temperature. We generated quantitative estimates for intracellular enzyme activity at different temperature at the single-cell level. This rapid approach offers a new methodology for characterizing influence of temperature on enzyme activity within single cells.View Large Image | View Hi-Res Image | Download PowerPoint Slide