Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform

Abstract

Development of technologies for rapid screening of DNA secondary structure thermal stability and the effects on stability for binding of small molecule drugs is important to the drug discovery process. In this report, we describe the capabilities of an electrochemical, microdevice-based approach for determining the melting temperatures (T_m) of electrode-bound duplex DNA structures. We also highlight new features of the technology that are compatible with array development and adaptation for high-throughput screening. As a foundational study to exhibit device performance and capabilities, melting-curve analyses were performed on 12-mer DNA duplexes in the presence/absence of two binding ligands: diminazene aceturate (DMZ) and proflavine. By measuring electrochemical current as a function of temperature, our measurement platform has the ability to determine the effect of binding ligands on T_m values with high signal-to-noise ratios and good reproducibility. We also demonstrate that heating our three-electrode cell with either an embedded microheater or a thermoelectric module produces similar results. The ΔT_m values we report show the stabilizing ability of DMZ and proflavine when bound to duplex DNA structures. These initial proof-of-concept studies highlight the operating characteristics of the microdevice platform and the potential for future application toward other immobilized samples.

Keywords: DNA; electrochemical sensor; ligand-based stabilization; melting profiles; microfabrication; microheater; rapid temperature control; square wave voltammetry.

Figure 1

Structures of ligands used in this study.

Figure 2

The electrochemical microscale platform architecture: (a) a top-view schematic of the platform (without the PDMS sample chamber); and (b) a side-view schematic (not to scale), including a sample well. The Pt heater/PRT fabricated on a glass wafer was isolated by a SiO₂ insulating layer. Au and Pt electrodes were fabricated by two lift-off e-beam evaporated depositions. The PDMS chamber and lid were centered above the electrode system.

Figure 3

Scanning electron micrograph (SEM) of the embedded microheater: before electrodes were patterned on top (a); and after the fabrication was complete (b).

Figure 4

Melting analysis protocol for duplex DNA to probe for ligand-induced stabilization and to minimize measurement variability.

Figure 5

Melting-curve analysis of duplex DNA in the presence/absence of binding ligands performed with an embedded heater. (a) Square wave voltammograms of 2 µmol/L MB-labeled duplex DNA in 10 mmol/L PBS with 100 mmol/L NaCl, pH 7.4. Melting curves (normalized) of duplex in the absence (b) and presence of 13 µmol/L ligands: proflavine (c), and diminazene aceturate (DMZ) (d). (b–d) The first baseline curve is shown in black, and the second melting analysis with or without ligand is shown in its respective color.

Figure 6

Melting-curve analysis of duplex DNA in the presence/absence of binding ligands performed with a thermoelectric module. (a) Square wave voltammograms of 2 µmol/L MB-labeled DNA duplex in 10 mmol/L PBS with 100 mmol/L NaCl, pH 7.4. Melting curves (normalized) of duplex in the absence (b) and presence of 13 µmol/L ligands: proflavine (c), and diminazene aceturate (DMZ) (d). (b–d) The first baseline curve is shown in black, and the second melting analysis with or without ligand is shown in its respective color.