Step-and-repeat photopatterning of protein features using caged/biotin-BSA: Characterization and resolution

This paper characterizes the caged-biotin-BSA system developed for selectively patterning biotinylated proteins into patterns on glass slides. Methyl alpha-nitropiperonyloxycarbonyl biotin, caged biotin, is coupled to a bovine serum albumin (BSA) carrier molecule using succinimide chemistry and.then employed in a four-step patterning process: (1) caged-biotin BSA compound is adsorbed onto a glass slide; (2) the slide is irradiated with 353-nm light through a chrome-on-glass photomask; (3) streptavidin is selectively bound to the irradiated regions; and (4) biotinylated protein is bound to the selectively immobilized streptavidin. A step-and-repeat scheme was used to demonstrate that streptavidin and then biotinylated BSA can be sequentially immobilized with reproducible feature density and little interfeature binding. Eight-minute irradiation of a mixed monolayer of 25% native BSA and 75% caged-biotin-BSA yielded the highest feature contrast, required the minimum use of reagent, and produced the least nonspecific binding. FITC-labeled streptavidin and biotinylated BSA served as the patterned protein films. Pattern resolution decreased with both decreasing feature size and increasing substrate thickness, primarily due to pattern spreading effects. The molecular density of the patterned protein, determined via fluorescence microscopy, is 3.9 x 10(10) molecules/mm(2) for caged-biotin - BSA, 9 x 10(9) molecules/mm(2) for patterned streptavidin, and 1.5 x 10(9) molecules/mm(2) for biotinylated BSA. These results indicate that the immobilization of streptavidin and of biotinylated BSA (steps 3 and 4) are both on the order of 20% efficient, for an overall efficiency of approximately 4%. A series of three step-and-repeat procedures are used to produce a pattern of three different biotinylated and fluorescently labeled proteins.