Colloidal silica films for high-capacity DNA probe arrays

The use of arrays of immobilized DNA "probes" for high-throughput analysis of genomic samples is expanding rapidly. The detection sensitivity of these arrays depends on the quantity and density of immobilized probe molecules as well as on the thermodynamics and kinetics of nucleic acid hybridization. We have prepared and investigated substrates with a porous, "three-dimensional" surface layer as a means of increasing the surface area available for the synthesis or immobilization of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target per unit area. Surfaces with pores 5 nm and larger were created by spin-coating colloidal suspensions of silica particles, followed by thermal curing. DNA arrays were synthesized on the resulting surfaces by photolithographic patterning, and the performance on the high-capacity substrates was compared to that on standard flat glass surfaces. The colloidal silica films created via this route show equivalent performance to flat glass substrates in terms of the efficiency of chemical synthesis and resolution of photolithographic patterning. DNA targets are able to penetrate the porous layers, and under saturating conditions, the quantity of bound target is proportional to the layer thickness. The result is an enhanced hybridization signal that is 20 times higher than flat glass for a colloidal particle layer that is 0.5 mum thick. The thermodynamic stability of probe/target duplexes in the matrix is the same as that for their counterparts on flat surfaces, although the colloidal silica films reach saturation more slowly than flat surfaces.