Silica colloidal crystals for enhanced fluorescence detection in microarrays.

Silica colloidal crystals were investigated for their potential as high surface area materials to enhance sensitivity over planar surfaces for microarrays using fluorescence detection. A relation was derived showing how crystal thickness and transmission, as well as colloid size, combine to determine the optically accessible surface area for enhancing sensitivity. Experimentally, crystals of 250-nm colloids were prepared with thicknesses determined by SEM to be 1.6, 4.2, and 11.0 microm. The material was sintered at 1000 degrees C to make it durable without affecting the crystalline structure, as confirmed by SEM. UV/visible spectrometry showed the depth of penetration (1/e) to be 8.4 microm at 488 nm for these materials. Fluorescein-labeled streptavidin and biotin were used as a model ligand-receptor pair. For the fluorescence measurements, biotin was covalently bonded to the silica surfaces, and the fluorescence was detected from the captured streptavidin-fluorescein. The observed fluorescence enhancement agreed well with the theory developed here. Compared to a planar surface, the colloidal crystal of 11.0 microm in thickness enhanced the fluorescence by nearly a factor of 80, with only a 0.3% increase in fluorescence background.