  The clinical need for ultra-sensitive molecular analysis has motivated the
development of several endpoint assay technologies capable of single molecule
readout. These endpoint assays are now primarily limited by the affinity and
specificity of the molecular recognition agents for the analyte of interest. In
contrast, a kinetic assay with single molecule readout could distinguish
between low abundance, high affinity (specific analyte) and high abundance, low
affinity (nonspecific background) binding by measuring the duration of
individual binding events at equilibrium. Here we describe such a kinetic
assay, in which individual binding events are detected and monitored during
sample incubation. This method uses plasmonic gold nanorods and interferometric
reflectance imaging to detect thousands of individual binding events across a
multiplex solid phase sensor with a large area approaching that of leading
bead-based endpoint assay technologies. A dynamic tracking procedure is used to
measure the duration of each event. From this, the total rates of binding and
de-binding as well as the distribution of binding event durations are
determined. We observe a limit of detection of 15 femtomolar for a
proof-of-concept synthetic DNA analyte in a 12-plex assay format.
