(503e) Kinetic Modeling of Surface-Based Hybridization Through Electrostatics, Secondary Structures, and Probe-Probe Interactions and the Comparison with Langmuir and 2nd Order Langmuir Kinetic Models

DNA-DNA biosensor devices use single-stranded DNA as surface-tethered "probes" that interact, through base-sequence specific recognition, with DNA or RNA "targets" from solution. These types of DNA layers can also be used to study DNA-drug or DNA-protein interactions. A central limiting factor in DNA-DNA hybridization is the poor fundamental understanding of how target sequences compete for probes and of how the complex interfacial environment biases the hybridization kinetics and thermodynamics.  In this work a "map" of hybridization kinetics is measured to better understand how electrostatic and contact interactions influence surface hybridization, and to elucidate how competition between different sequence targets influences approach to equilibrium.

In this work a mixed SAM composed of a 20 bp probe sequence (P1) and Mercaptohexanol passivator, is end tethered to a gold electrode, to prepare a probe layer of known coverage.  To study the initial kinetic rates of binding, a model system consisting of a fully complementary, 18 bp DNA target sequence (T1) is used.  DNA are conjugated with Ferrocene derivative redox couples, and hybridization kinetics are tracked with time using cyclic voltammetry; hydrodynamic conditions are controlled using a rotating disk electrode, that allows for decoupling of mass-transport effects and surface hybridization kinetics.  Kinetics are studied over a wide parameter space of counterion concentration (C_K+) and probe coverages for P1 and T1, showing significant deviation from the Langmuir model, even in the pseudo-Langmuir regime.  These results motivated a study of four additional target sequences (T2, T3, T4, T5) of varying length and complementarity, under pseudo-Langmuir (PL) and moderate probe (MP) coverages and a counterion concentration C_K+=1.0mol L^-1 chosen to effectively screen electrostatic interactions.  The results suggest a kinetic mechanism where long-lived imperfect duplex structures relax to perfect duplexes as equilibrium is approached.  Nearest neighbor calculations are used as a benchmark for investigating the possibility of probe-probe interactions, showing two such possible secondary structures for P1.  The qualitative contribution of these effects are investigated under PL and MP for C_K+=1.0mol L^-1.  The results suggest that hybridization is described by: (1) electrostatic effects from membrane potentials in the probe layer before and during hybridization (2) long-lived P-T secondary structures in the layer (3) probe-probe interactions in the probe layer.