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.

Development of sub-micron patterned carbon electrodes for immunoassays.

Sub-micron sized domains of a carbon surface are derivatized with antibodies using biotin/avidin technology. These sites are spatially-segregated from, and directly adjacent to, electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains are kept to a minimum (e.g. less than a micron) to maintain the high sensitivity required for the measurement of enzyme-linked cofactors in an enzyme-linked immunoassay (ELISA). This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This allows the construction of microscopic arrays of active ELISA sites on a carbon substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators; thus maximizing sensitivity and detection of the enzyme mediator. The carbon electrode surface is characterized with respect to its chemical structure and electron transfer properties following each step of the antibody immobilization process. The characterization of specific modifications of micron regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas-Red labeled antibody) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product.

Localized avidin/biotin derivatization of glassy carbon electrodes using SECM.

Different forms of the microreagent mode of SECM were used to attach biotin or make "clean" spots on micron-sized regions on the surface of a carbon electrode. In the direct-write mode, the SECM probe tip is used as an electrochemical "pen" depositing biotin in micron-sized lines on the carbon substrate as it is scanned across its surface. In the negative microreagent mode, the SECM probe tip is used as an electrochemical "eraser" cleaning of the surface attached molecules and leaving clean spots on the surface of a globally derivatized carbon surface. This type of simple micromodification of the surface of a carbon electrode will allow the fabrication of biosensors that can potentially be tailor-made for a variety of applications.

Electron transfer kinetics at a biotin/avidin patterned glassy carbon electrode.

Photolithographic techniques using a laser interference pattern were used to attach photobiotin to micron-sized stripes on the surface of a carbon electrode. Fluorophore-tagged avidin was attached to this spatially-patterned biotin with essentially no loss in spatial resolution. The kinetics of the glassy carbon surface were examined to see if electron transfer sites could indeed be segregated from the attachment sites of photobiotin-immobilized avidin. The ECL of luminol and SECM were used to verify the segregation between underivatized sites (which exhibit normal electron transfer kinetics) and extensively derivatized biotin/avidin surfaces (which presumably exhibit slow electron transfer kinetics). Both techniques were found to be capable of differentiating the protein-covered surface from bare carbon with sufficient resolution to tell whether a significant portion of the carbon surface is still active and available to detect the product of an enzyme generated analyte. These results indicate that extensive biotin/avidin derivatization of the surface does decrease the electron transfer rate of a carbon electrode, and that the photolithographic approach was able to modify specific sections of the electrode surface, while leaving other regions untouched and available for facile electron transfer. This leads to a more general protocol for the construction of enzyme-based biosensors which utilize diffusable mediators.

Generation of biotin/avidin/enzyme nanostructures with maskless photolithography.

Micrometer-sized domains of a carbon surface are modified to allow derivatization to attach redox enzymes with biotin/avidin technology. These sites are spatially segregated from and directly adjacent to electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains must be kept to a minimum (e.g., less than 5 microns) to maintain the fast response time and high sensitivity required for the measurement of neurotransmitter dynamics. This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This will allow the construction of microscopic arrays of active enzyme sites on a carbon fiber substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators, thus maximizing enzyme activity and detection of the enzyme mediator. The ultimate sensitivity of these sensors will be realized only through careful characterization of the carbon electrode surface with respect to its chemical structure and electron transfer properties following each step of the enzyme immobilization process. The characterization of specific modifications of micrometer regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas Red-labeled avidin) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product. An atomic force microscope was used to obtain high-resolution images that probe the heterogeneity of the enzyme sites.

Electrocatalytic surface for the oxidation of NADH and other anionic molecules of biological significance.

A simple electrochemical treatment of a carbon fiber electrode surface has been found to dramatically improve the voltammetry of NADH and several other anionic molecules under steady-state and fast scan (100 V/s) conditions. The electrocatalytic surface is generated through the electrochemical oxidation of NADH on a carbon fiber electrode that exhibits product adsorption. The oxidative product is reacted with ascorbic acid at elevated temperatures to create a surface which has very little overpotential for the oxidation of dopamine and many metabolites such as NADH, DOPAC, uric acid, and ascorbate. The electrochemical properties of the modified surface were examined voltammetrically at both slow and fast scan rates. The surface shown in this paper shifts the oxidation overpotentials different magnitudes for each analyte tested, thus allowing discrimination between analytes of interest and their major interferences. Another benefit of this new electrocatalytic wave is that it decreases the limit of detection for NADH by approximately 1 order of magnitude. Therefore, this new carbon surface not only gives better discrimination between two analytes but also gives better detection limits for certain analytes of interest.