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While high density MicroWell plates, including 384 well and 1536 well plates, offer advantages of low volume reaction or growth vessels in a standardized footprint allowing robotic manipulation, the greatest benefits lie in the increased well surface area to reaction volume ratios. This surface area to volume benefit can be utilized to optimize assay development by utilizing the molecular surface as a reactant. The well geometry and optical properties are also enhanced in high-density plates, allowing microscopic imaging and increased signal detection for fluorescence and luminescence.    The molecular characteristics of high density MicroWell plates were examined in regard to base materials and induced surface modification. Various modification methods including injection-molding parameters, exposure to high and low energy sources and exposure to specific physical conditions were studied. The surfaces were molecularly characterized and then utilized as reactants in high throughput and low volume in vitro assays. Polystyrene plates exhibited a natural relatively hydrophobic surface due to the hydrocarbon backbone of the polystyrene along with the repeating benzene ring. Exposure to direct electrical discharge, and several forms of irradiation in the presence of air and oxygenated atmosphere resulted in surfaces exhibiting various levels of oxidation. These levels were measured and the molecular species were calculated.    These specific surfaces were utilized as reactants in solid phase, cell based and cell culture assays. Specific surfaces demonstrated specific efficacy in regard to particular applications. For example, a highly charged surface exhibiting hydroxyl, carbonyl, and some carboxyl moieties was best for adherent cell culture. Utilizing passive adsorption procedures, a variant surface exhibiting more hydroxyl and carbonyl groups did not bind IgG well but did bind phospholipids, providing a surface “high-binding” for lipids while maintaining low background due to non-specific adsorption. A surface, commonly referred to as “high binding,” having slightly lower surface energy than the other two studied and characterized by aromatic hydrocarbons, hydroxyl and some few carbonyl groups did not bind hydrophobic antigens, but did bind glycoproteins such as IgG very well under slightly basic, passive conditions.    The geometries of wells were also examined. Well geometries contributed significantly by eliminating reagent wicking or creeping, enhancing detection of signals, and increasing the active surface area to reagent volume, allowing the utilization of decreased substrate concentrations along with low reagent volumes without loss of sensitivity.

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Assay Development in High Density MicroWell® Plates: Use of Well Geometries, Format, Surface Modification and Optical Properties to Achieve Optimal Assay Performance