The ability to track the concentrations of specific molecules in the body in real time would vastly improve our ability to diagnose, understand, and treat disease. Most real-time molecular sensing technologies, however, rely on the adsorption of target to a receptor-modified surface. Because of this, they fail when challenged directly in complex biological media, such as whole blood. In such media, proteins, cells, and other interferents bind to the sensing interface, which leads to false positives or poor quantification. Electrochemical aptamer-based (EAB) sensors, in contrast, report on the concentration of their target via a binding-induced change in electron transfer from a redox reporter attached to a target-recognizing aptamer. They thus enable real-time molecular sensing directly in undiluted blood and even in situ in the living body. And because aptamers can be artificially selected for many target species, EAB sensors are a platform technology generalizable to a range of small molecules, metabolites, and proteins. Consistent with this, to date, EAB sensors have enabled real-time, in vivo measurements of numerous pharmaceuticals, drugs of abuse, and metabolites.

In this thesis, I described my work focused on the improvement of EAB sensor time resolution, spatial resolution, and accuracy. Specifically, I first detailed the application of the phase component of electrochemical impedance spectroscopy to push EAB time resolution to sub-second measurements. Then, with the aim of rendering them less invasive and improving their spatial resolution, I miniaturized the sensors by six-fold using nanoporous gold substrates. Finally, I improved the calibration used for in vivo quantification by examining how media selection, temperature, and age impact the accuracy of EAB sensor measurements.

Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or other target-induced conformational changes in electrode-bound oligonucleotides, with examples reported to date including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These of sensors, termed Electrochemical DNA-based (E-DNA) sensors, are comprised of an electrode modified with surface immobilized, redox-reporter-tagged DNA probes. This technique has emerged as a promising new biosensor platform due to its sensitive and selective measurement of specific molecular targets without the need for additional reagents, wash steps or complex and costly equipment. My thesis work has focused on expanding and improving this increasingly important sensing platform. We have expanded the number of signaling redox reporter for multiplxing and ratiometric auto-calibration sensing. As well as we developed a simple strategy to rationally edit the useful dynamic range of our electrochemical DNA sensors. To make our platform more generic for sensing, we designed a novel protein-based electrochemical sensing architecture achieves good specificity and sensitivity, providing a new approach for the quantitative, single-step measurement of protein-macromolecule interactions. At the end, we have used surface forces apparatus (SFA) to study the behavior of surface grafted single-stranded and double-stranded DNAs.

By coupling the specific and selective binding of biomolecules with conformation-linked signaling and electrochemical outputs, electrochemical aptamer-based sensors (E-AB sensors) provide a unique ability to measure molecules in real-time directly in complex media. Because E-AB sensors do not require washing steps or reagent additions, they open the door for measurements directly where they are needed – in complex sample streams such as foodstuffs, clinical samples, and even directly within the living body. To further advance this promising technology, my work has addressed both improvements in the physical sensor and improvements in how we use the electron transfer kinetics underlying the signaling in these sensors to circumvent the drift associated with performing measurements directly in complex media. That is, using new electrode formats and new analytical signal-processing techniques, my thesis work created fully realized devices capable of measuring specific molecular targets in real time in relevant, highly complex sample streams. As examples, in my thesis I describe sensors supporting the measurement of a mycotoxin directly within the flowing sample stream of a foodstuff affected by that mycotoxin, the measurement of a malaria diagnostic protein in clinically-relevant ranges directly in a fingerprick-sized sample of human serum, a new technique to measure a protein of interest directly in unprocessed whole blood – without pre-calibration, and new deployments of thin, surgically-implanted wire electrodes with sensors that measure, for the first time, in vivo blood concentrations in real time of a variety of small-molecule drugs as well as an arbitrary circulating protein of interest.

Current methodologies for patient diagnosis require the use of skilled workers, expensive reagents, and instrumentation, and most importantly, time. As these methodologies encourage inconsequential follow-up between patients and their doctors, new methods must be developed. These methods should follow the WHO guidelines of ASSURED (Accessible to those in need, Sensitive to the clinical range of the target of interest, Specific to the target of interest with no false positives or negatives, User-friendly, Rapid, under one hour for results, Equipment free, and Delivered to those in need) to create point of care technologies available for all. To this end, my studies focus on the development of convenient calibration methods for use with fluorescent, aptamer-based biosensors (aptamer beacons). Specifically, I describe here the creation, characterization, and application of a one-tube method for the calibration of aptamer beacons using sensors for the detection of DNA, silver ions, and thrombin as my test bed.

A significant hurdle in the point of care testing of biomarkers in complex solutions is that there is sample variation, requiring large volumes of the target of interest and complex and cumbersome calibration. In response, I have created a simple, three-step, one-tube method for the calibration of aptamer beacons supporting the direct quantification of specific target molecules within complex sample matrices. Specifically, I have shown that the sequential addition of two inexpensive and stable oligonucleotide-based reagents allows for the determination of the absolute minimum and maximum fluorescence produced by the sensor in each solution, providing the information to calibrate in the face of sample-to-sample variations in a simple, single-tube format. Using the three associated measurements together with the pre-determined disassociation constant of the aptamer I demonstrate the single-tube calibrated quantification of multiple targets directly in complex samples.

Electrochemistry serves as a sensitive and rapid signal transduction platform for sensing applications. In contrast to commonly used diagnostic platforms which rely on polymerase-chain reaction for target amplification or immunological recognition, electrochemical-based systems are generalizable to many targets at various length-scales. At the ensemble scale, electrochemical aptamer-based (EAB) sensors convert target-recognition into an electrochemical signal via the binding-induced change in electron transfer from a redox reporter. Because aptamers are artificially selected for the detection of a wide range of small molecule, metabolite, and protein targets, EAB sensors are not restricted to chemically specific ligand-binding events and represent a generalizable platform technology that enable real-time, in vivo measurements. On a single particle scale, single-entity electrochemistry (SEE) offers unparalleled insights into the fundamental electron-transfer events that enable life. With this technique, we can parse individual differences, typically masked by some average thermodynamic response, by studying biological molecules one at a time. Doing so, we can size molecules, determine individual catalytic activity, and more broadly, characterize heterogeneous behaviors.

In this dissertation, I describe my work focused on utilizing electrochemistry to poke, prod, and improve bioanalytical sensing systems. I first address the practical needs of transitioning EAB sensors from the laboratory to the clinic; namely, I use electrochemistry to characterize disinfected or stored EAB sensors to ensure optimal device performance. Second, I tackle a longstanding issue within the electrochemistry literature that has resulted in imprecise SEE measurements. I discovered that by introducing a homogeneous chemical reaction in solution, I was able to shift the rate limiting step in current generation at an electrode from mass transport of a redox mediator to its local regeneration by a substrate. Doing so, I can achieve a ten-fold gain in measurement precision when sizing insulating particles using nanoimpact electrochemistry. Finally, with the imprecision associated with SEE greatly reduced, I lay out a foundation to explore biological interactions at a nano-scale electrode. In closing, I hope this work adds to the current body of literature establishing electrochemistry as a powerful tool for nanoscale bioanalytical systems.