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In this communication, single molecules of β-galactosidase were captured on a 1 mm femtoliter array using biotin−streptavidin binding. The femtoliter arrays, containing 24 000 individual reaction chambers, permit digital concentration readout as the percentage of reaction vessels that successfully capture a target molecule is correlated to the bulk target concentration. This capture and readout approach should prove useful for DNA and antibody assays that utilize an enzyme label to catalyze the generation of a fluorescent signal.