Imaging biocatalytic activity of enzyme-polymer spots by means of combined scanning electrochemical microscopy/electrogenerated chemiluminescence.

The purpose of this study was to develop a scanning electrochemical microscopy (SECM) and scanning electrogenerated chemiluminescence (SECL) setup to visualize the localized enzymatic activity using glucose oxidase as a model. Combination of SECM and electrogenerated chemiluminescence (ECL) was made possible by integrating a photomultiplier tube (PMT) within a SECM setup which is mounted on top of an inverted microscope. An enzyme-polymer spot formed on a glass slide and placed on top of the entrance window of the PMT was used as a model sample to evaluate the potential of the combined SECM/ECL setup. Hydrogen peroxide, which was locally generated by the glucose oxidase (GOx)-catalyzed reaction, reacted with oxidized luminol which was simultaneously electrochemically generated at the positioned SECM electrode tip. By using the phase-sensitive lock-in amplifier, the potential applied to the SECM tip was sinusoidally swept to invoke an associated oscillation of the ECL. Thus, sensitivity of SECL could be substantially enhanced. Images of the local immobilized enzyme activity obtained both by ECL and generator/collector (GC) mode of SECM were compared to elucidate the pathway in which the SECM and SECL signals are generated.