Detection of Ligand-Induced Stabilization of DNA Based on Thermal Profiles from an Electronic Microscale Platform

S.M. Robinson, J.R. Askim, H.O. Sintim, S. Semancik
University of Maryland,
United States

Keywords: electrochemistry, temperature, electrodes, biosensor, DNA, ligand-based stabilization


Rapid monitoring of biomolecular conformational changes and binding-induced stability variation is being investigated with a microscale platform to enable technology for point-of-care diagnostics and high-throughput screening. Electrochemical detection with redox-active labeling has been employed to realize fast, cost-effective signal acquisition with high signal-to-noise ratios. Our electrochemical platform includes an embedded microheater for localized thermal control that allows temperature-dependent probing of conformational changes of DNA nanostructures tethered to gold working electrodes. Thermal profiles and the associated melting temperatures (Tm) offer insight into the inherent stability of a wide range of bioanalytes as well as the effects of ligand-based stabilization for identifying potential drug molecules. Measurement capabilities have been demonstrated with individual 3-electrode elements that incorporate an embedded platinum microheater and a ~10 µL microfluidic cell. The wafer-based fabrication design used to produce the platform prototypes is fully compatible with the assembly of arrays that can have potential utility in applications from disease diagnosis to drug discovery. As an illustration of the capabilities of the platform for examining biomolecular interactions, the thermal stability of duplex DNA was analyzed for the binding of two known small-molecule therapeutics. Ligand-based stabilization would result in a Tm increase, ΔTm. Stabilization effects were observed for a ligand that intercalates between the base pairs, Proflavine, and for a minor-groove binding ligand, diminazene aceturate (DMZ or Berenil). These ligands were chosen to show proof-of-concept with known duplex binders, but the method could be easily employed to examine effects for a variety of other ligands.