Two-potential electrochemical probe for study of DNA immobilization.
A two-potential electrochemical method is applied to study DNA immobilization, by the simultaneous characterization of capture probe DNA self-assembled monolayers and hybridized target DNA molecules on a given gold electrode surface. Capture probe and target DNA strands are labeled with ferrocenes composed of differing chemical environments, to permit their simultaneous, yet independent signaling at different formal potentials, so that their respective signals may be de-convoluted and assessed for relative surface concentration. Some special attributes of the nondestructive two-potential electrochemical probe described herein include surface sensitivity, chemical and orientation specificity, and the ability to provide a real-time, in situ probe that does not need any wash steps for stringency. This electrochemical probe is applied to study the kinetics, surface architecture, coverage, and orientation of DNA during its immobilization on gold. On the basis of our results primarily from this electrochemical probe, and validated by N(1s) core-level X-ray photoelectron spectra, we judge significant DNA deposition within 5 min of incubation in the deposition solutions, with the capture probe DNA anchored predominantly via the thiol end, even at low coverages. Surface coverage for DNA immobilization plateaus within 30 min of incubation time to approximately 2 x 10(13) molecules/cm(2) and the immobilization kinetics as determined from this electrochemical method are consistent with surface re-organization as the rate-determining step.