Nanokits for the electrochemical quantification of enzyme activity in single living cells.

Abstract

The development of more intricate devices for the analysis of enzyme activity in single cells, and even in individual intracellular compartments, would advance the knowledge of cellular heterogeneity and protein function at subcellular locations. This chapter describes construction and implementation of nano-capillary electrodes kits, named as "Nanokits," for unprecedented cost-effective analysis of enzyme activity within single living cells and even at single lysosomes. The nanokit that is specific for the target enzyme is assembled in a nanometer-sized capillary with a working electrode, and electrochemically loaded into the cell allowing the electrochemical quantification of the enzyme activity. Furthermore, the enzyme activity in individual intracellular compartments can be characterized by reversed electrochemical pumping to confine the targeted organelle in the nano-capillary tip with the nanokit. The use of commercially available reagent kits marketed for cell population studies permits direct application of the nano-capillary electrode for targeting a variety of enzymes in single cells. This protocol will likely spark expansion in the number of groups embarking on enzyme activity investigations by clearly providing a cookbook description of new cost-effective technology for single cell analysis.