Single-Molecule Fluorescence Imaging of DNA at a Potential-Controlled
Interface
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Abstract
Many interfacial
chemical phenomena are governed in part by electrostatic
interactions between polyelectrolytes and charged surfaces; these
phenomena can influence the performance of biosensors, adsorption
of natural polyelectrolytes (humic substances) on soils, and production
of polyelectrolyte multilayer films. In order to understand electrostatic
interactions that govern these phenomena, we have investigated the
behavior of a model polyelectrolyte, 15 kbp fluorescently labeled
plasmid DNA, near a polarized indium tin oxide (ITO) electrode surface.
The interfacial population of DNA was monitored in situ by imaging
individual molecules through the transparent electrode using total-internal-reflection
fluorescence microscopy. At applied potentials of +0.8 V versus Ag/AgCl,
the DNA interfacial population near the ITO surface can be increased
by 2 orders of magnitude relative to bulk solution. The DNA molecules
attracted to the interface do not adsorb to ITO, but rather they remain
mobile with a diffusion coefficient comparable to free solution. Ionic
strength strongly influences the sensitivity of the interfacial population
to applied potential, where the increase in the interfacial population
over a +300 mV change in potential varies from 20% in 30 mM ionic
strength to over 25-fold in 300 μM electrolyte. The DNA accumulation
with applied potential was interpreted using a simple Boltzmann model
to predict average ion concentrations in the electrical double layer
and the fraction of interfacial detection volume that is influenced
by applied potential. A Gouy–Chapman model was also applied
to the data to account for the dependence of the ion population on
distance from the electrode surface, which indicates that the net
charge on DNA responsible for interactions with the polarized surface
is low, on the order of one excess electron. The results are consistent
with a small fraction of the DNA plasmid being resident in the double-layer
and with counterions screening much of the DNA excess charge