| The Plaxco laboratory has developed a new class of reagentless, electrochemical sensors that make use of electrode-bound, redox-tagged oligonucleotide probes as their sensing elements. To date, these electrochemical DNA (E-DNA) and electrochemical, aptamer-based (E-AB) sensors can detect specific DNA and RNA sequences, proteins, small molecules and inorganic ions. However, this first generation of devices had low specificity against single nucleotide polymorphisms and the inability to detect targets that do not specifically interact with oligonucleotides. This dissertation describes several improvements to this E-DNA platform via the development of new strategies that address these drawbacks.;We developed and characterized an electrode-bound DNA pseudoknot architecture that enables "signal-on" DNA sensing and increased mismatch detection capabilities. The pseudoknot consists of two stem-loop structures such that, in the absence of target, they hold a redox tag away from the electrode surface, minimizing electron transfer. Upon target binding, the pseudoknot structure is disrupted, liberating a single stranded DNA region, allowing the redox tag to collide with the electrode surface and transfer electrons, thus leading to an increase in signal. This sensor is sensitive (nanomolar detection limits), able to function in complex samples (e.g., blood serum) and, because of the improved thermodynamics of this system, can discriminate single nucleotide polymorphisms.;Many biomolecules can bind to smaller molecules with high affinity. We exploited this observation to develop a new class of sensors. Conjugation of a small molecule recognition element to a DNA scaffold allows biorecognition of a desired target. Upon target binding, the dynamics of the scaffold change, producing a measurable signal change. Detection of several protein targets has been shown with nanomolar detection limits, rapid kinetics and significant selectivity in complex media such as blood serum and environmental samples. Those sensors are also amenable to a competition assay configuration, and thus can be used for the detection of small molecules in solution. |
