DNA Electrochemistry: Charge-Transport Pathways through DNA Films on Gold

Abstract

Over the past 25 years, collective evidence has demonstrated that the DNA base-pair stack serves as a medium for charge transport chemistry in solution and on DNA-modified gold surfaces. Since this charge transport depends sensitively upon the integrity of the DNA base pair stack, perturbations in base stacking, as may occur with DNA base mismatches, lesions, and protein binding, interrupt DNA charge transport (DNA CT). This sensitivity has led to the development of powerful DNA electrochemical sensors. Given the utility of DNA electrochemistry for sensing and in response to recent literature, we describe critical protocols and characterizations necessary for performing DNA-mediated electrochemistry. We demonstrate DNA electrochemistry with a fully AT DNA sequence using a thiolated preformed DNA duplex and distinguish this DNA-mediated chemistry from that of electrochemistry of largely single-stranded DNA adsorbed to the surface. We also demonstrate the dependence of DNA CT on a fully stacked duplex. An increase in the percentage of mismatches within the DNA monolayer leads to a linear decrease in current flow for a DNA-bound intercalator, where the reaction is DNA-mediated; in contrast, for ruthenium hexammine, which binds electrostatically to DNA and the redox chemistry is not DNA-mediated, there is no effect on current flow with mismatches. We find that, with DNA as a well hybridized duplex, upon assembly, a DNA-mediated pathway facilitates the electron transfer between a well coupled redox probe and the gold surface. Overall, this report highlights critical points to be emphasized when utilizing DNA electrochemistry and offers explanations and controls for analyzing confounding results.

Figure 1

Electrochemistry at all-AT 40-mer DNA sequences. Left: cyclic voltammetry of MB-dsDNA well-matched sequences (in green), and MB-dsDNA sequences containing a single AC mismatch (red). Right: cyclic voltammetry of MB-ssDNA deposited on the gold surface, 5′-MB-AA TAA AAA ATA AAA TAA AAT AAA AAT AAA TAA AAA ATA AT-3′ (blue), and cyclic voltammetry after addition of its complementary strand thiol (PG) protected, 5′-PG-AT TAT TTT TTA TTT ATT TTT ATT TTA TTT TAT TTT TTA TT-3′ (orange). Methylene blue is depicted as a blue sphere connected to the DNA strand. Voltammograms were collected in buffered solutions (5 mM NaH₂PO₃, 50 mM NaCl, pH 7.0), at 100 mV/s scan rate.

Figure 2

Schematic representation of self-assembled DNA monolayers on the gold surface. Left: densely packed monolayers self-assembled in the presence of MgCl₂. Right: loosely packed monolayers self-assembled without MgCl₂ and passivated with 6-mercaptohexanol.

Figure 3

AFM measurement of DNA film height. Upper level: schematic representation showing the height measurement of DNA films. Lower level, left: AFM images (750 nm × 750 nm) of DNA-modified gold (sequence: 5′-SH-AGT ACA GTC ATC GCG-3′) after removal of a small patch (∼100 nm²) of DNA by mechanically scrapping the probe tip against the surface as indicated by the cartoon representation. The images were recorded under fluid solution (0.1 M potassium phosphate buffer, pH 7), and the height of the DNA was calculated by measuring the depth of the square patch. Moving from left to right are images recorded under electrochemical control as the applied potential was poised negative of the Au/thiol reduction potential; as the thiolated DNA is electrochemically stripped off, the underlying surface features of the gold substrate are revealed. Bottom right: plot of the maximum film height (measured at ∼100 mV vs Ag) measured for MB-DNA duplexes possessing 15 bases (5′-SH-AGT ACA GTC ATC GCG-3′), 18 bases (5′-SH-AGT ACA GTC GTA GTC GCG-3′), and 20 bases (5′-SH-AGT ACA GAT CGT AGC TCG CG-3′). These data show a slope of 3.2 Å/bp, close to the predicted value of 3.4 Å/bp. The intercept, ∼7 Å, is somewhat smaller than the ∼16 Å expected for a fully extended alkylthiol linker, likely due to compression of the film owing to the vertical tip force.

Figure 4

Ru(NH₃)₆³⁺ vs daunomycin interaction with duplex DNA. Ru(NH₃)₆³⁺ (RuHex) complexes bind to DNA electrostatically and are generally not sequence-specific. Since the binding is to the phosphates, the electrochemistry is not expected to be DNA-mediated, and the electrochemical readout from RuHex does not change in the presence of a mismatch site in the duplex DNA. As seen in the plot, the surface concentration of bound Ru(NH₃)₆³⁺ (Γ_Ru, mol/cm²) in the DNA film with varying % of well-matched (WM) duplexes remains constant. In contrast, a mismatch perturbation switches off the signal from the covalently bound DNA intercalator daunomycin. Consequently, the readout of surface concentration of bound daunomycin (Γ_daunomycin, mol/cm²) decreases linearly with decreasing % of well-matched dsDNA in the monolayer. The DNA sequence is 5′-SH-ATC CTC AAT CAT GGA C-3′, where GG represents the daunomycin cross-linking site and C represents the site of an AC mismatch for mismatched duplexes.