Laser interference pattern ablation of a carbon fiber microelectrode: biosensor signal enhancement after enzyme attachment.

Fluorescence microscopy was used to visualize the accumulated fluorescent product of the enzyme alkaline phosphatase to indicate where active covalently bound enzyme remained on the surface after application of a Nd: YAG laser interference pattern to a surface that was first globally derivatized with the covalently bound enzyme. The electrochemical kinetics of the same carbon fiber surface were examined through the electrogenerated chemiluminescence of Ru(bpy)(3)2+ to determine that electron-transfer sites were indeed segregated from the enzyme-binding sites. The enzyme-derivatized areas are determined to be separate and distinct from the areas of enhanced electron transfer. Two other enzymes, glucose oxidase and malic dehydrogenase, were then covalently bound to carbon fiber microelectrode surfaces in order to verify the change in detection limit of their respective cofactors, NADH or H2O2, under a variety of surface conditions. The S/N of an enzyme-modified electrode after laser interference pattern photoablation and electrocatalytic treatment is improved by more than 1 order of magnitude over that observed at an electrode that is globally enzyme modified.

A laser ablation method for the spatial segregation of enzyme and redox sites on carbon fiber microelectrodes.

A laser-generated interference pattern was used to remove enzyme from micrometer-wide stripes on an enzyme-covered carbon fiber microelectrode surface to create regions of facile electron transfer. Fluorescence microscopy was used to visualize fluorophore-tagged enzyme to indicate where the adsorbed enzyme remained on the surface. The electrochemical kinetics of the carbon fiber surface were examined to see if electron-transfer sites could indeed be segregated from enzyme adsorbed across the entire surface. CCD imaging of the electrochemical luminescence of Ru(bpy)3(2+) was used to verify the segregation between photoablated sites (with facile electron-transfer kinetics) and surfaces with adsorbed enzyme (which exhibit slow electron-transfer kinetics). The laser-ablated surface could also be distinguished from the enzyme-covered carbon surface with atomic force microscopy. Thus, photoablation of the surface of a protein-covered carbon fiber microelectrode with an interference pattern generated by a Nd:YAG laser allows the activation of 1.7-micron-wide bands of the electrode surface (available for facile electron transfer) while leaving 2.6-micron-wide enzyme-modified areas intact, thereby producing electroactive regions directly adjacent to enzyme modified regions of the same surface.