Micropatterning of biomolecules on glass surfaces modified with various functional groups using photoactivatable biotin

Biomolecule patterning by photolithographic methods has considerable advantages because a large number of different biomolecules can be assembled on a spatial area by a combinatorial method and complex biomolecule patterning can be created in situ in closed environments such as microfluidic channels. Here, a photobiotin was used as the photoactivatable reagent to create patterned arrays of biomolecules. The variability of photobiotin deposition on glass substrates modified with a variety of materials having carboxyl, lysine, aldehyde, amine groups, and BSA (bovine serum albumin) was characterized by subsequent derivatization with Cy3-labeled streptavidin. The fluorescence images of the photobiotin patterned glass surfaces showed that the BSA/aldehyde-coated glass could be considered as the most appropriate substrate to immobilize photobiotin, in view of the homogeneous immobilization of biomolecules with high density in defined regions and the reduction of nonspecific binding to the surface. In streptavidin equilibrium adsorption assays, the maximum amount of streptavidin-Cy3 bound to the BSA/aldehyde-coated glass surface continued to rise with increasing streptavidin-Cy3 concentration until 12.0 microg/mL was reached and the surface then became saturated. Also, a line array of biotin-labeled single-strand probe DNAs was created on the BSA/aldehyde-coated glass by photolysis of photobiotin through a slit-type mask and biotin/streptavidin/biotin chemistry, extended to a quantitative measurement of the concentrations of target DNA. The results of target DNA analysis showed linearity over a wide range from 0.5 ng/mL to 5 microg/mL and were reproducible.