Microfabrication of electrode surfaces for biosensors.

摘要

This work describes the strategies used to develop an amperometric biosensor for the detection of glutamate in vivo on a physiologically relevant time scale. Glutamate is not electrochemically active at conditions amenable to normal brain chemistry, but amperometric detection is made possible by immobilization of an enzyme that has the appropriate characteristics needed for chemical selectivity (i.e. substrate consumption and product formation) on a carbon fiber microelectrode surface. Unfortunately, effective modification of electrode surfaces leads to slow electrode response times due to inhibition of diffusion to the electrocatalytic surface and/or inhibition of electron transfer by the immobilized enzyme. A Nd:YAG laser-generated interference pattern, used following derivatization of the entire microelectrode surface, removes 2-micron wide stripes of enzyme, restores facile electron transfer in a spatially segregated manner, and allows sub-second response time for the modified electrode.; Two methods were then developed to enhance enzyme coverage of the electrode surface prior to Nd:YAG laser pattern ablation. Electrochemical deposition of an active form of biotin was characterized and verified by attachment of fluorescently labeled avidin. This electrodeposition is faster and avidin coverage is more extensive than an previous strategies. Biotinylated Jeffamines were synthesized and used to enhance stability and further increase enzyme loading by allowing formation of dendrimeric enzyme structures on the electrode surface. This enzyme attachment scheme resulted in stable active glucose oxidase or glutamate oxidase bound to the electrode surface. Time resolution for flow injection fast scan cyclic voltammetry of glutamate at these electrodes, following treatment with the Nd:YAG laser-generated interference pattern, is on the order of 200 milliseconds. Sinusoidal voltammetry and subsequent signal processing is demonstrated to further enhance sensitivity to low micromolar levels while simultaneously allowing electrochemical signal discrimination.