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The clinical need for ultra-sensitive molecular analysis has motivated thedevelopment of several endpoint assay technologies capable of single moleculereadout. These endpoint assays are now primarily limited by the affinity andspecificity of the molecular recognition agents for the analyte of interest. Incontrast, a kinetic assay with single molecule readout could distinguishbetween low abundance, high affinity (specific analyte) and high abundance, lowaffinity (nonspecific background) binding by measuring the duration ofindividual binding events at equilibrium. Here we describe such a kineticassay, in which individual binding events are detected and monitored duringsample incubation. This method uses plasmonic gold nanorods and interferometricreflectance imaging to detect thousands of individual binding events across amultiplex solid phase sensor with a large area approaching that of leadingbead-based endpoint assay technologies. A dynamic tracking procedure is used tomeasure the duration of each event. From this, the total rates of binding andde-binding as well as the distribution of binding event durations aredetermined. We observe a limit of detection of 15 femtomolar for aproof-of-concept synthetic DNA analyte in a 12-plex assay format.

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events