Sequence Detection of Unlabeled DNA Using the Sandwich Assay: Strand-Displacement, Hybridization Efficiency, and Probe- Conformation Considerations for the Tethered Surface

We report the development of a “sandwich-hybridization” electrochemical DNA-detection assay on gold electrodes using osmium tetroxide-labeled reporter strands, while avoiding chemical modification of analyte strands. Successful design of the oligonucleotides used in the assay required a significant adjustment to solution-based estimates of melting temperature for the immobilized-probe/target duplex, to achieve optimal displacement kinetics. This allowed for reduced hybridization times and temperatures, and a higher sensitivity at low concentrations of DNA-analyte than oligos designed using solution-based melting temperatures as a guide. In addition we found that for the highest ranges of probe surface-density, signal response was sluggish until the DNA self-assembled monolayer (SAM) was exposed to several hybridization procedures, suggesting a modification of the SAM in response to a hybridization experiment. Use of a less-dense SAM eliminated this effect, suggesting increased influence of molecular crowding on the sandwich assay versus a simple hybridization. The high sensitivity, quick response time, relative simplicity and low cost demonstrated here helps pave the way for a field-level electrochemical genetic sensor using our method, as the unmodified target from samples in question can be tested directly without a chemical labeling step.