Micrometer dimension derivatization of biosensor surfaces using confocal dynamic patterning

Using laser scanning confocal optics in conjunction with avidin/biotin tech nology, micrometer-sized patterns of biomolecules were fabricated on glassy -carbon and fused-silica surfaces. Photoactive biotin was immobilized using the 325-nm line of a Helium-Cadmium laser, which was focused through a 25x or 100x quartz microscope objective. A three-dimensional piezoelectric mic romanipulator was used to position the sample surface in the focal plane of the microscope objective and to create patterns on the focused surface. Bi otin patterns with line widths of 5-20 mu m were produced by varying the sc an speed of the micromanipulator while exposing the surface to the laser. T he integrity of the immobilized biotin was confirmed by subsequent derivati zation with fluorescently labeled avidin. Fluorescence microscopy with a co oled charge coupled device (CCD) imaging system was used to visualize the d istribution of biotin and fluorescent avidin within the patterns created by the laser.