| Combining the versatility, specificity and affinity of biological recognition with the convenience of modern electronics to create user-friendly, inexpensive, and miniaturizable detection platforms is the drive underlying my thesis research. My studies have focused on the development of a new class of reagentless, electrochemical DNA sensors for the detection of oligonucleotide, protein and small molecule targets termed, E-DNA (electrochemical DNA) and E-AB (electrochemical aptamer-based) sensors. Here I describe the characterization and application of the first of these E-DNA platforms, as well as, the development of several new nucleic acid detection schemes.;The first E-DNA construct comprised a redox-tagged stem-loop DNA molecule chemi-adsorbed onto a gold electrode via thiol modification that in the absence of target, produces a large faradaic current. Hybridization to a complementary oligonucleotide forces the redox tag away from the electrode, impeding electron transfer and producing a readily detectable decrease in the faradaic current. Here we describe the first detailed characterization of this platform, including its target specificity, reusability and performance characteristics when deployed directly in complex sample matrices such as clinically relevant fluids, soil extracts, and foodstuffs. Given E-DNA's traits, we demonstrate a real-world application in detecting molecular markers (information-encoding oligonucleotide "tags") used for a variety of identification and authentication purposes.;In an effort to determine important design parameters and how they affect E-DNA performance, we studied the effects of probe geometry, length, and redox-tag positioning on the platform. We found that linear geometries are more efficient at signaling than their stem-loop counterparts, longer probes outperform shorter probes if the redox-tag is positioned in similar proximity to the electrode surface, but shorter probes produce improved target specificity.;Finally, we explored alternative E-DNA architectures aimed at improving signaling and sensitivity. We describe a new "signal-on" oligonucleotide sensing construct comprising a "recognition arm" and complementary redox-tagged "signaling arm" that form a rigid helix restricting probe dynamics and redox-tag electron transfer. Target hybridization liberates the signaling arm allowing for facile electron transfer from the redox-tag and an increase in the observed faradaic current. This bifurcated E-DNA construct demonstrates target specificity, nanomolar sensitivity and reusability even after detection in blood serum. |
