Distinguishing Nucleotides Using Electrochemical Impedance Spectroscopy

All DNA sequences are composed of different combinations of the same four nucleotides, the order of which establishes the basis of our genetics. DNA methylation is one of the biological methods to control the use of DNA by providing a physical blocker to the production of relate proteins for that particular sequence. DNA base-gold affinity has also been used to evaluate genetic sequence. Initially, the affinity of nucleotides to a bare gold electrode has been established as A > C ≥ G > T (Kimura-suda et al., 2003). More recently, methylation sites were evaluated via DNA-gold affinity after a two-step preparation process: (1) bisulfate treatment to separate the strands and (2) asymmetric polymerase chain reactions to amplify the sequence of interest (Koo et al., 2014). As a point-of-care method, DNA methylation was examined using differential pulse voltammetry (Koo et al., 2014). We are attempting to improve the sensitivity of these previous results by using electrochemical impedance spectroscopy (EIS).

Electrochemical impedance spectroscopy applies an alternating potential to sense the impedance to the resulting current flow. The resistance associated with the transfer of charge can be determined through analysis, and this resistance is proportional to the amount of DNA absorbed on the gold surface rather than change of the solution resistance itself. For this work, the bare gold electrode was polished then soaked for 5 minutes in nucleic acid bases dissolved in UHP water. To investigate the reproducibility of our data, triplicates were conducted for adenine, cytosine, and guanine. We also investigated the differences in measurement of both static cell and flow cell methods. Finally, we also determined the surface resistance change when the gold electrode is exposed to individual bases and 17-base repeat oligonucleotides in a flow cell.

Currently, when statistically comparing the charge transfer resistance for the flow cell, each nucleotide base can be individually distinguished from the control. However, for longer oligonucleotide chains, adenine cannot be distinguished from the control. In both of these cases, adenine produced the smallest charge transfer resistance and guanine had the largest standard deviation between trials. Several characteristics of purine rings may contribute to these unexpected results, the most likely of which is base stacking due to the use of UHP water as the solvent. We will present our progress on reproducibility and accuracy of measurements, and our future work to expand this into a senior honors thesis.

Kimura-suda, H., Petrovykh, D.Y., Tarlov, M.J., and Whitman, L.J. (2003). Base-Dependent Competitive Adsorption of Single-Stranded DNA on Gold. 9014–9015.

Koo, K.M., Ibn Sina, A.A., Carrascosa, L.G., Shiddiky, M.J. a., and Trau, M. (2014). eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes. Analyst 139, 6178–6184.