Analysis of layered assays and volume microarrays in stratified media

Changing traditional microarray methods by using both sides of a substrate or stacking microarrays combined with optical sectioning enables the detection of more than one assay along the z-axis. Here we demonstrate two sided substrates, multilayer arrays with up to 5 substrates, and 2- and 3-dimensional antigen microarrays. By replacing standard substrates with multiple 30 μm layers of glass or mica, high density multilayer and 3-dimensional volume arrays were created within a stratified medium. Although a decrease in fluorescence intensity with increasing number of substrate layers was observed together with a concomitant broadening of the axial resolution, quantitative results were obtained from this stratified system using calibrated intensities. Two- and three-dimensional antigen microarrays were generated via microcontact printing and detected as indirect immunoassays with quantum dot conjugated antibodies. Volume arrays were analysed by confocal laser scanning microscopy producing clear patterns, even when the assays were overlapped spatially.