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

TL;DR

This paper introduces a kinetic biosensor assay that uses dynamic tracking of single binding events with plasmonic nanorods, achieving ultra-sensitive detection limits surpassing traditional endpoint assays.

Contribution

The study presents a novel kinetic assay method utilizing plasmonic nanorods and interferometric imaging to monitor individual binding events, enhancing sensitivity beyond existing biosensor technologies.

Findings

Achieved a detection limit of 15 femtomolar for DNA analyte.

Monitored thousands of binding events across a multiplex sensor.

Demonstrated differentiation of specific and nonspecific binding through event duration analysis.

Abstract

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.