The objective of this research is to utilize laser scanning confocal microscope (LSCM) optics in conjunction with avidin/biotin technology to fabricate biopolymer sensor arrays of micron to submicron dimensions. Many areas of biochemistry and biomedical research would benefit from ultratrace analyses ( few thousand molecules) of very small volumes (less then a microliter). One example the analysis of complex and very small biological samples such as pediatric blood sample. Here an array of antibodies with different affinities would allow a complete blood analysis (involving 256 analyses) to be run on a sensor surface less then 10 micrometers squared with a total sampling volume of about 1 picoliter (10 to the -15th L). Alternatively, one cold read an array of submicron sensor elements with the same selectivity to gain extremely high spatial resolution of the distribution of a single component such as the secretion of a neurotransmitter from a single nerve cell. In the work propped here, a LSCM will be used as a light source to generate these sensors via the use of photoliable groups, primarily photobiotin, that can be incorporate into the surface derivatization chemistry. These groups can be photolytically cleaved from or attach to specific regions of the substrate surface via the application of appropriate wavelengths of light. This will allow the production of enzyme-based and/or affinity-based sensors such as hybridized DNA or antibodies. The sensitively of these sensors will be realized through characterization of carbo, glass or plastic substrate surfaces after patterning with respect to chemical structure and reactive properties following each step of the immobilization process. characterization of specific modifications of micron regions of the surfaces will be accomplished with fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from FITC-labeled avidin) where products can be monitored with florescence microscopy.
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