(324e) Selectivity Enhancements in Gel-Based DNA-Nanoparticle Assays By Membrane-Induced Isotachophoresis: Thermodynamics Versus Kinetics

Selectivity improvements in microfluidic chips for nucleic acid sensing remain challenging because of both thermodynamic and kinetic limitations. The thermodynamically based dissociation constants of DNA largely determine an assayâ??s ability to discriminate between matched (target) and unmatched (nontarget) sequences. Furthermore, techniques which improve limits of detection and shorten assay times by altering thermodynamic equilibrium, such as isotachophoretic preconcentration, do little to enhance the selectivity because these improvements affect targets and nontargets equally. Introducing external forces to facilitate hydrodynamic or electrical shear may increase selectivity for assays with surface-bound probes. However, elimination of nontarget signals are also met with substantial losses in target signals thereby leading to poor limits of detection. To overcome both the thermodynamic and kinetic limitations of current DNA sensors, we introduce a platform based on depletion isotachophoresis in agarose gel generated by an ion-selective membrane. The ion-selective membrane permits us to capture target molecules with probe-functionalized nanoparticles, concentrate them, and lower the effective dissociation by more than four orders of magnitude. The membrane also generates a depletion zone to create high electrical shear forces which enable irreversible dehybridization of the nontargets. By varying the applied voltage during the depletion step, we enhance the selectivity beyond the thermodynamic limit by five-fold for nontargets with only two base mismatches while minimizing any loss in the targetsâ?? signal. We also explore the effect of mismatch location on selectivity and demonstrate the importance the dehybridization rate constant in determining the magnitude of the selectivity.