Micro- and nanochamber array system for single enzyme assays

Arrays of small reaction containers, ranging from 624 femtoliters (10-15 L) to 270 attoliters (10-18 L), for capturing a single enzyme molecule and measuring the activity were developed together with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be carried out over a long period of time. The hydrolysis rates of β-D-galactosidase (β-gal), kcat, were decreased according to the chamber size decrease, and the overall tendency seems to be symmetrical relationship to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localisation of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environments and molecular crowding conditions.