Ultrafast Kinetic DNA Hybridization Assay Based on the Visualization of Threshold Turbidity

Abstract

We report herein the development of an ultrafast kinetic DNA hybridization assay system based on the visualization of threshold turbidity associated with the assembly of polystyrene nanospheres. Initial testing of our diagnostic protocol on a sequence associated with the anthrax lethal factor indicates that a visually identifiable, turbidity-definitive, and kinetic threshold state could be reached at a time as short as 1 min. The assay scheme allows for both target concentration quantification and differentiation of single base mismatches through registry of the threshold turbidity onset time. The positively charged environment on nanospheres not only contributes to expedited signal generation but also imparts cooperative DNA binding properties. The kinetic visual protocol complements conventionally used thermodynamic strategies and provides an entry point for the circumvention of assay issues associated with ill-defined thermodynamic end points.