Nanoconfinement effects: glucose oxidase reaction kinetics in nanofluidics

Size-tunable nanofluidic devices coupled to an electrochemical detector have been designed and then used to study glucose oxidase (GOx) reaction kinetics confined in nanospaces. The devices are fabricated via a photochemical decomposition reaction, which forms nanochannels covered with carboxyl groups. The generated carboxyl groups enable us to chemically pattern biological molecules on the polymer surfaces via covalent bonding. With this approach, the activity of the immobilized biological molecules confined in nanospaces with different sizes has been investigated. GOx species are chemically immobilized on the surface of the nanochannels, catalyzing the oxidation of substrate glucose as it flows through the channels. The enzyme reaction product, hydrogen peroxide, passing through the nanochannels, reaches an electrochemical detector, giving rise to an increase in anodic current. This steady-state electrochemical current, which responds to various glucose concentrations, can be used to evaluate the GOx activity under confinement conditions. The results show significant nanoconfinement effects that are dependent on the channel size where the reaction occurs, demonstrating the importance of spatial confinement on the GOx reaction kinetics. The present approach provides an effective method for the study of enzyme activity and other bioassay systems, such as cell assays, drug discovery, and clinical diagnosis.