High surface density immobilization of oligonucleotide on silicon.

Oligonucleotide (11-mer) molecules are immobilized on silicon in high surface population using either a permanent thioether bond or a chemo-selectively reversible disulfide bond to the surface thiol functionality. Substrate hydroxy groups are first silanized with an 11 carbon trichlorosilane containing a terminal, protected thiol moiety. Oligonucleotide modified with a tether possessing a terminal thiol group is further derivatized with a water-soluble, halobenzylic bifunctional reagent, which allows the complete conjugate to be attached to the surface through a permanent thioether bond. Alternatively, the oligonucleotide-tether complex can be combined with a pyridyldisulfide compound, which, in turn, facilitates the formation of a reversible disulfide bond with surface thiol. The amount of immobilized oligonucleotide was determined by radiochemical labeling with 32P. Additional verification of surface amounts was obtained from X-ray photoelectron spectroscopic analysis of substrates. The results of the immobilization protocols are compared with the oligonucleotide surface population achieved through the conventional silanizing agent, mercaptopropyltrimethoxysilane. Finally, a preliminary confirmation of duplex formation of a TTU-attached 25-mer with its complementary strand is outlined.