High-density capture surfaces, where probe molecules are immobilized at densities beyond the limit of diffraction (>109 molecules cm-2), can expand the capabilities of assays based on single-molecule fluorescence detection. Assays performed at high-density capture surfaces provide several advantages, including less nonspecific adsorption, since a larger fraction of the surface is composed of probe molecules, and expanded dynamic range of measurable equilibrium constants, since single-molecule events can be detected at concentrations far below the Kd. These surfaces have similar densities of probe molecules as SPR and ELISA capture assays, making the results relevant to these commonly used clinical techniques.  In this work, we describe surface immobilization and super-resolution imaging techniques to measure reversible single-molecule DNA hybridization events at high densities of short single-stranded probe DNA (ssDNA).  Probe ssDNA is immobilized through covalent attachment using temperature or exposure time to control surface density.  Then the surface is passivated through attachment of sulfonate groups, resulting in low nonspecific adsorption and high specificity for complementary versus non-complementary target DNA.  With such low nonspecific adsorption, the dissociation time of hybridized probe-target DNA can be determined from the residence times of target molecules at the interface � no additional molecular registration (fluorescence resonance energy transfer, co-localization, etc.) is needed to discern specific from nonspecific interactions.  To determine the density of probe sites needed to report association rates, we have recently adapted a spatial autocorrelation technique that uses a super-resolution spatial map of target DNA hybridization to probe DNA locations on the surface. With both the measured dissociation and association rates, we can characterize the equilibria and kinetics of the hybridization process.  Detecting single-molecules captured by a high-density of probe molecules on the surface is a sensitive method to determine a few hundred DNA target molecules in a sample without chemical amplification.

Fluorescence microscopy of single-molecule DNA hybridization at high density interfaces

248th National Meeting (2014)

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