| Description |
In this work, single molecule fluorescence microscopy techniques are used to investigate the role of electrostatics in deoxyribonucleic acid (DNA) hybridization, and in the interactions of DNA and colloidal particles with charged surfaces. Single molecule total internal reflection fluorescence (TIRF) microscopy provides the sensitivity and interfacial specificity needed to probe electrostatic interactions in the microscopic electrical double-layer region between charged molecules and surfaces. Image analysis has been developed to quantitatively detect single molecule spots in TIRF images by sampling their diffraction-limited point-spread function by multiple pixels on the imaging sensor. By detecting spots with multiple pixels above an intensity threshold, single molecules can be located with signal-to-noise ratios as low as 2.5, minimizing false positive and false negative probabilities. Single molecule imaging was used to monitor the time-course of individual complementary strand DNA hybridization events. Target single stranded DNA (ssDNA) was immobilized at an interface, and its absolute surface density and association constant were determined from the binding isotherm of fluorescently labeled complimentary strand probe ssDNA. Dissociation rate constants of the DNA duplex were determined from the dissociation times, and association rates were calculated from the association constant and the dissociation rate assuming a two-state binding model. From the dependence of association constants, association rates, and dissociation rates on ionic strength, an Eyring model was used to determine the electrostatic contribution to the free energy of the transition state and the fully hybridized double-helix. The electrostatic interactions between large DNA plasmids and a potential-controlled indium tin oxide (ITO) semiconductor surface were investigated by measuring DNA populations and diffusion near the semiconductor surface as a function of applied potential. DNA populations increased exponentially with positive applied potentials, while maintaining free-solution-like diffusion coefficients and no surface adsorption. A Boltzmann model indicates that interfacial DNA has a net charge less than one electron equivalent, suggesting that much of its charge is screened by counterions. Similar accumulation with increasing positive applied potential was observed with 100 nm carboxylate-polystyrene colloidal particles. These colloidal particles were used to investigate shifts in surface charge of the ITO-aqueous interface induced by photoexcitation of charge carriers in the semiconductor. |
