Electrochemical sensors based on DNA-mediated charge transport chemistry

The base pair stack within double helical DNA provides an effective medium for charge transport. The π-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to minor perturbations in DNA structure and base stacking makes DNA-mediated charge transport chemistry an ideal platform for DNA sensing. Electrochemical methods through DNA-modified electrode surfaces that exploit this sensitivity for efficient biosensing are described. Gold electrodes are modified with DNA double helices and used to monitor the electrochemistry of bound redox-active intercalators. The efficiency of electrochemical reduction of the intercalated redox-probe, in a DNA-mediated reaction, provides an indicator of base stacking within the surface-bound duplexes. Perfectly stacked DNA is capable of mediating the electrochemical reduction, while duplexes containing π-stacking perturbations, such as single base mismatches, do not support current flow to the intercalator. All single base mismatches, including thermodynamically stable GT and GA mismatches, as well as many common base damage products can be detected within DNA and DNA/RNA hybrid duplexes using this assay. Moreover, mismatches can be detected as a small percentage of a perfectly matched film, making it possible to detect mutations associated with genetic disorders in only a small fraction of cells. This assay is also compatible with DNA based chip technology. Furthermore, electrochemistry at DNA films is found to provide a novel and sensitive method for probing protein dependent changes in DNA structure and enzymatic reactions. The efficient transport of charge through self-assembled monolayers of thiol-terminated duplexes on gold therefore offers an extremely sensitive probe for the integrity of DNA sequences. Completely new approaches to single base mismatch detection as well as assaying protein-DNA interactions and reactions on surfaces are now available. This technology is generally applicable as a tool for directly measuring base pair stacking in nucleic acid duplexes.