With the rapid development of micro total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micrometer-sized, spatially segregated biosensing elements on several different substrate surfaces. Both an interference pattern and a dynamic confocal patterning apparatus were used to control the dimensions and positions of immobilized regions. In both of these methods, a UV laser was used to initiate attachment of a photoactive biotin molecule to the substrate surfaces. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or nonlabeled avidin and biotinylated sensing elements such as biotinylated antibodies. Dimensions of 2-10 microm were achievable with these methods. A wide variety of materials, including glassy carbon, quartz, acrylic, polystyrene, acetonitrile-butadiene-styrene, polycarbonate, and poly(dimethylsiloxane), were used as substrates. Nitrene- and carbene-generating photolinkers were investigated to achieve the most homogeneous films. These techniques were applied to create a prototype microfluidic sensor device that was used to separate fluorescently labeled secondary antibodies.