Owe Orwar, Chalmers University of Technology, Gothenburg, Sweden, and colleagues in France and the USA have used a steady-state microfluidic delivery device to expose individual cells to a controlled amount of a substance that opens a pore in the cell membrane, digitonin. They were then able to administer fluorescein diphosphate (FDP) to the cells. This compound is a fluorogenic alkaline phosphatase substrate. By also exposing the cell to levamisole, an inhibitor of this enzyme, the team could obtain dose-response and dose-inhibition curves based on fluorescence for the enzyme functioning in a single cell. Monitoring is possible within a couple of minutes of setup.
The team suggests that their approach could open up new avenues for research into enzyme kinetics that have never before been tested in situ.
- Probing Enzymatic Activity Inside Single Cells,
Jessica Olofsson, Shijun Xu, Gavin D. M. Jeffries, Aldo Jesorka, Helen Bridle, Ida Isaksson, Stephen G. Weber, Owe Orwar,
Anal. Chem. 2013.
DOI: 10.1021/ac4013122
