Generation of Biotin/Avidin/Enzyme Nanostructures with Maskless Photolithography

Abstract

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.

Covalent modification of graphitic carbon substrates by non-electrochemical methods

“…Kuhr and co-workers have developed this approach for covalently grafting biotin to graphitic carbon surfaces [79,80,81,82].…”

“…Using this chemistry, Kuhr's group demonstrated two methods for generating micron-scale patterns of biotin on GC surfaces. In their first approach to photopatterning, a GC surface coated with a film of photobiotin was exposed to a laser diffraction pattern (325 nm) for 60 s, resulting in 5 µm lines of biotin, with a spacing of 10 µm [79,80]. In their second method, UV laser light (325 nm) was focused on the photobiotin-coated GC surface and the GC was moved in the controlled manner to 'write' covalently-attached biotin features on the surface, with line widths of 5 -10 µm [82].…”

Development of a versatile biotinylated material based on SU-8

“…The specic binding of biotin to streptavidin presents a very high affinity (K a ¼ 10 15 M À1 ), 34 and its tetravalency allows for a simultaneous binding to several biotins emerging from the surface via a biotin-streptavidin-biotin bridge. 35,36 Upon UV irradiation (l ¼ 350 nm), the photoactivatable group of Phb gives rise to a nitrene from an azide, which can be inserted readily into both C-H as well as other functional groups, such as OH, NH, NO 2 , C]C, etc.…”

Photopolymerizable Materials in Biosensorics

“…Typically photo-linking prepared polymers are used (Jae Ho Shin et al, 1998;Jobst et al, 1993;Nakako at al., 1986;Barie et al, 1998;Dobrikov & Shishkin, 1983a, 1983bDontha et al, 1997;Leca et al, 1995;Nakayama & Matsuda, 1992;Nakayama et al, 1995;Navera et al, 1991). Polyvinyl derivatives such as polyvinyl chloride were widely used at the creation of biosensors (Jae Ho Shin et al, 1998).…”