An EIS study of the heterogeneity of redox labeled DNA SAMs on gold before and after hybridization

Redox active labels on DNA SAMs are used for transducing hybridization via the change in the rate of contact-mediated redox with the electrode surface. This change in the fraction hybridized can be measured using CV or SWV. Here, we detail the use of EIS to quantify the change in the redox rate and thereby assess the hybridization characteristics for DNA SAMs. EIS of methylene blue (MB) labeled DNA SAMs on smooth single crystal bead electrodes were measured and the results fit to a series of equivalent circuits representing one redox rate, two redox rates or a distribution of redox rates. The rate of MB redox decreased with increasing complementary strand (cDNA) concentration, interpreted as an increase in the amount of hybridized dsDNA in the SAM. The EIS method was able to accurately measure the average rate of MB redox, but EIS was unable to independently measure the ssDNA and dsDNA populations as the redox rates were not sufficiently distinct. A majority of the redox active MB was one average time constant, but a consistent 10% of the MB had much faster redox rates which were interpreted as due to non-specifically adsorbed MB-labeled DNA. The EIS fits were not improved by using a CPE in place of C d l . However, the EIS fits were improved by replacing the RC circuit element with a distributed circuit element based on the Cole-Cole model, replacing a R with a CPE in one RC branch of the equivalent circuit. This distributed model better explained the EIS results giving the most probable redox rate and a distribution of rates. The EIS measurement approach was verified with a higher coverage DNA SAM which showed slower redox rates as expected. Interestingly, in all cases the amount of redox active MB ( Γ M B ) decreased with an increase in the [cDNA], suggesting that some MB was in an environment that prevented it from reaching the surface. This decrease in Γ M B with [cDNA] was reflected in the SWV results since it was sensitive to both the change in redox rate and the amount of redox active MB. In contrast, the EIS results were found to measure the k E T independent of the amount of redox active MB. Hybridization isotherms produced using either SWV or EIS results showed a significant difference between SWV (50 Hz) and k E T when fit to a Langmuir-Hill isotherm. These results suggested the SWV results must be interpreted with caution as the K A and Hill co-operativity coefficient ( n ) were different than the EIS measurements, but in a way that strongly depended on the coverage and the SWV frequency chosen. Overall, EIS of the redox labeled DNA SAM was successfully interpreted using a distributed redox rate expression based on the Cole-Cole model. EIS measured k E T provided a hybridization isotherm that was not convoluted with the amount of redox active MB, enabling an accurate assessment of the DNA SAM interface.