Photopatterning of antibodies on biosensors

The immobilization of biomolecules on surfaces in defined micropatterns has become increasingly important for the development of new diagnostic device s and high-throughput genetic and drug screening protocols. We describe the synthesis and testing of thiol-reactive, photoactivatable linkers that wil l permit laser micropatterning or photolithographic patterning of surfaces. In these linkers, a benzophenone photophore is tethered through a variable -length poly(ethylene glycol) hydrophilic spacer to a maleimide group. Spac ers containing one to five ethylene glycol units were examined. Antibodies were photoimmobilized on polystyrene waveguides and the resulting biosensor s were used for fluorescence immunoassays. The spacer with five ethylene gl ycol units optimally decreased the steric interactions among large molecule s (antibodies and antigens) and increased binding capacity and response rat e of the biosensor. Two different sandwich assay protocols were examined. I n the first, the antigen and fluorescently labeled second antibody were add ed sequentially to the biosensor ("stepwise"). In the second, the antigen a nd antibody were premixed before injection into the biosensor ("premixed"). The stepwise protocol gave a significantly higher response than that of th e premixed protocol. Although the premixed protocol is more convenient, the stepwise protocol provides enhanced sensitivity.