Addressable nanowell arrays formed using reversibly sealable hybrid elastomer-metal stencils

There are two major array formats used in life science research and biomedical analysis. The first is the microwell plate format with millimeter-sized wells each with microliter capacity addressed individually and repeatedly during experiments. The second is the microarray format with micrometer-sized spots that are patterned initially but not addressable individually thereafter. Here, we present an addressable nanoliter-well plate with micrometer sized wells that combines the advantages of the two array formats. The nanowells are formed by reversibly sealing a steel stencil featuring an array of micrometer-scale openings to an optically transparent substrate. The nanowells have a capacity of approximately 1 nL, are approximately 140 microm in diameter, and are arrayed at a density of 1600 wells cm(-2). A soft polymer is patterned photolithographically around each opening so as to form a microgasket for pressure sensitive, liquid tight, and reversible sealing to any type of smooth substrate, either hydrophilic or hydrophobic. The rigidity of the steel prevents the distortion that occurs in soft, all-polymeric stencils and permits accurate registration across the entire array, which in turn allows for repeated, individual addressing of wells using an inkjet spotter. The stencils are used to pattern cells, make protein microarrays, and create nanowells on surfaces to study reverse transfection by first spotting plasmids encoding fluorescent proteins into the wells, seeding cells, and monitoring the transfection of the cells in real time using time-lapse imaging. The hybrid elastomer-metal stencils (HEMSs) are versatile and useful for multiplexed analysis of drugs, biomolecules, and cells with microarray density.