Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes

Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels, and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB Molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA) dominated by the on off' signal change mode at nanomolar levels' of the analyzed DNA. When MB bears an additional carboxylic group; the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami, E.; Clima, L.; Gothelf, K.; Ferapontova, E. E. Anal. Chem 2011, 83, 1594) providing the robust off on nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of -electrochemical hybridization sensors exploiting DNA:nanoswitchable architectures.