Robert P. Johnson

A Label-Free, Electrochemical SERS-Based Assay for Detection of DNA Hybridization and Discrimination of Mutations

A label-free, surface-enhanced Raman spectroscopy-based assay for detecting DNA hybridization at an electrode surface and for distinguishing between mutations in DNA is demonstrated. Surface-immobilized DNA is exposed to a binding agent that is selective for dsDNA and acts as a reporter molecule. Upon application of a negative potential, the dsDNA denatures into its constituent strands, and the changes in the spectra of the reporter molecule are monitored. This method has been used to distinguish between a wild-type, 1653C/T single-point mutation and ΔF508 triplet deletion in the CFTR gene. The use of dsDNA-selective binding agents as reporter molecules in a discrimination assay removes the burden of synthetically modifying the target to be detected, while retaining flexibility in the choice of the reporter molecule.

SERS from molecules bridging the gap of particle-in-cavity structures

We demonstrate that by combining silver nanoparticles and structured gold SSV surfaces the SERS for those molecules that bridge the nanoparticle-cavity gap is preferentially enhanced using 4-mercaptoaniline and 4-mercaptobenzoic acid as examples.

Denaturation of dsDNA immobilised at a negatively charged gold electrode is not caused by electrostatic repulsion

Double-stranded DNA immobilised through a thiol anchor at a gold electrode surface can be unwound and denatured by applying a negative potential. One proposed mechanism for this electrochemical denaturation is that electrostatic field effects are responsible for the destabilisation of the dsDNA through repulsion of the DNA sugar-phosphate backbone away from the electrode surface. Herein, we demonstrate conclusively that electrochemical melting at gold electrodes cannot be explained solely as a simple repulsion mechanism by showing that immobilised DNA denatures at high ionic strengths, where the DNA base-pairs are situated outside of the electrochemical double-layer (and outside the influence of the electric field), and further, that oligomers comprised of the mimic peptide nucleic acid (PNA) can also be denatured at negative potentials, despite the absence of a negatively charged backbone.

The effect of base-pair sequence on electrochemically driven denaturation

Application of a voltage ramp can result in denaturation of dsDNA and strand separation. We show that the potential at which half of the surface immobilised duplexes denature (the melting potential, E(m)) directly correlates with the calculated nearest neighbour and experimental melting temperatures; T(m), for a duplex is solution. The results demonstrate that the electrochemical melting potential measures the stability of the dsDNA, and therefore existing nearest neighbour melting models can be utilized to design DNA probes with predictable electrochemical melting potentials for future assay applications.

Real-time surface-enhanced Raman spectroscopy monitoring of surface pH during electrochemical melting of double-stranded DNA

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.

The effect of temperature on electrochemically driven denaturation monitored by SERS.

Scanning the electrochemical potential negative results in the gradual denaturation of dsDNA immobilised at a nanostructure gold electrode, the DNA melting is monitored by SERS. We demonstrate the effect of the experimental temperature on the electrochemically driven melting (E-melting) by carrying out experiments between 10 and 28 °C using two DNA duplexes (20 and 21 base pairs). Significant temperature dependence for both the melting potentials, Em, and the steepness of the melting curves was found over the range 10 to 18 °C. Above 18 °C the results were found to be independent of temperature. The relative temperature insensitivity of the melting potentials above 18 °C is advantageous for the application of the electrochemically driven melting technique because precise temperature control is not necessary for measurements that are carried out around room temperature. Conversely temperature dependence below 18 °C offers a way to improve discrimination for highly similar DNA sequences.