Nanopipettes for the Electrochemical Study of Enhanced Enzymatic Activity in a Femtoliter Space

The chemical reaction in a confined space is known to be accelerated due to a high collision probability; however, the study of this confinement effect in a supersmall space down to femtoliter (fL) is seldom reported. Here, an adjustable volume [from picoliter (pL) to fL] of the aqueous phase is retrained at the tip of a nanopipette by an organic solvent so that the confinement effect on the specific activity of glucose oxidase is investigated. The activity is determined by the amount of hydrogen peroxide generated from the reaction between the oxidase and glucose using a nanoelectrode inside the nanopipette. As compared with the activity in bulk solution (82 U/mg), the activity increases up to 7500 U/mg in a 105 fL space. The 2 orders of magnitude increase in the enzymatic activity is the highest amplification in the volume-confined enzyme reaction as reported. A near-exponential drop in the activity is observed with the increase in the space volume, revealing the dominant enhancement in the confined space at the fL level for the first time. The established electrochemical nanopipettes should not only provide a strategy for the study of the enzymatic activity in supersmall confined space but also help understand the confinement effect of enzyme-catalyzed reactions.