Electrochemical DNA (E-DNA) sensors have emerged as a promising class of biosensors capable of detecting a wide range of molecular analytes (nucleic acids, proteins, small molecules, inorganic ions) without the need for exogenous reagents or wash steps. In these sensors, a binding-induced conformational change in an electrode-bound "probe" (a target-binding nucleic acid or nucleic-acid-peptide chimera) alters the location of an attached redox reporter, leading to a change in electron transfer that is typically monitored using square-wave voltammetry. Because signaling in this class of sensors relies on binding-induced changes in electron transfer rate, the signal gain of such sensors (change in signal upon the addition of saturating target) is dependent on the frequency of the square-wave potential pulse used to interrogate them, with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor. Here, we show that, because it alters the driving force of the redox reaction and thus electron transfer kinetics, signal gain in this class of sensors is also strongly dependent on the amplitude of the square-wave potential pulse. Specifically, we show here that the simultaneous optimization of square-wave frequency and amplitude produces large (often more than 2-fold) increases in the signal gain of a wide range of E-DNA-type sensors.

Despite 25 years' effort, serious questions remain regarding the mechanism(s) underlying electron transfer through (or from) electrode-bound double-stranded DNA. In part this is because a control experiment regarding the putatively critical role of guanine bases in the most widely proposed transport mechanism (hopping from guanine to guanine through the π-stack) appears to be lacking from the prior literature. In response, we have employed chronoamperometry, which allows for high-precision determination of electron transfer rates, to characterize transfer to a redox reporter appended onto electrode-bound DNA duplexes. Specifically, we have measured the effects of guanines and base mismatches on the electron transfer rate associated with such constructs. Upon doing so, we find that, counter to prior reports, the transfer rate is, to within relatively tight experimental confidence intervals, unaffected by either. Parallel studies of the dependence of the electron transfer rate on the length of the DNA suggest that transfer from this system obeys a "collision" mechanism in which the redox reporter physically contacts the electrode surface prior to the exchange of electrons.

The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clinically relevant (±20% for this application) accuracy, for example, commercial continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, we demonstrate a "dual-frequency" approach for achieving the calibration-free operation of electrochemical biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, we use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochemical aptamer-based (E-AB) biosensors as our test bed, we demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor.

Whereas proteins generally remain stable upon interaction with biological surfaces, they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date, however, only a small number of such artificial surfaces have been reported, and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated, we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chemically well-defined, macroscopic surfaces. Comparison with the folding free energies seen in bulk solution then provides a quantitative measure of the extent to which surface interactions alter protein stability. As proof-of-principle, we have characterized the FynSH3 domain site-specifically attached to a hydroxyl-coated surface. Upon guanidinium chloride denaturation, the protein unfolds in a reversible, two-state manner with a free energy within 2 kJ/mol of the value seen in bulk solution. Assuming that excluded volume effects stabilize surface-attached proteins, this observation suggests there are countervening destabilizing interactions with the surface that, under these conditions, are similar in magnitude. Our technique constitutes an unprecedented experimental tool with which to answer long-standing questions regarding the molecular-scale origins of protein-surface interactions and to facilitate rational optimization of surface biocompatibility.

The efficiency with which square-wave voltammetry differentiates faradic and charging currents makes it a particularly sensitive electroanalytical approach, as evidenced by its ability to measure nanomolar or even picomolar concentrations of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extraction of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, we demonstrate here a numerical approach by which square-wave data can be used to determine electron transfer rates. Specifically, we have developed a numerical approach in which we model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate constant and the electron transfer coefficient. As validation of the approach, we have used it to determine electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods.

Despite the importance of protein-surface interactions in both biology and biotechnology, our understanding of their origins is limited due to a paucity of experimental studies of the thermodynamics behind such interactions. In response, we have characterized the extent to which interaction with a chemically well-defined macroscopic surface alters the stability of protein L. To do so, we site-specifically attached a redox-reporter-modified protein variant to a hydroxy-terminated monolayer on a gold surface and then used electrochemistry to monitor its guanidine denaturation and determine its folding free energy. Comparison with the free energy seen in solution indicates that interaction with this surface stabilizes the protein by 6 kJ mol-1 , a value that is in good agreement with theoretical estimates of the entropic consequences of surface-induced excluded volume effects, thus suggesting that chemically specific interactions with this surface (e.g., electrostatic interactions) are limited in magnitude.

The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clinical practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary molecules in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, we describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a critical part of the standard of care, and its adaptation into an electrochemical aptamer-based (E-AB) sensor. Using this sensor, we then demonstrate: (i) rapid (seconds) and convenient (single-step and calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (ii) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (iii) high-precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience) but also actively control plasma drug levels provides an unprecedented route toward improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacological interventions.

The real-time monitoring of specific analytes in situ in the living body would greatly advance our understanding of physiology and the development of personalized medicine. Because they are continuous (wash-free and reagentless) and are able to work in complex media (e.g., undiluted serum), electrochemical aptamer-based (E-AB) sensors are promising candidates to fill this role. E-AB sensors suffer, however, from often-severe baseline drift when deployed in undiluted whole blood either in vitro or in vivo. We demonstrate that cell-membrane-mimicking phosphatidylcholine (PC)-terminated monolayers improve the performance of E-AB sensors, reducing the baseline drift from around 70 % to just a few percent after several hours in flowing whole blood in vitro. With this improvement comes the ability to deploy E-AB sensors directly in situ in the veins of live animals, achieving micromolar precision over many hours without the use of physical barriers or active drift-correction algorithms.

Knowledge of drug concentrations in the brains of behaving subjects remains constrained on a number of dimensions, including poor temporal resolution and lack of real-time data. Here, however, we demonstrate the ability of electrochemical aptamer-based sensors to support seconds-resolved, real-time measurements of drug concentrations in the brains of freely moving rats. Specifically, using such sensors, we achieve <4 μM limits of detection and 10-s resolution in the measurement of procaine in the brains of freely moving rats, permitting the determination of the pharmacokinetics and concentration-behavior relations of the drug with high precision for individual subjects. In parallel, we have used closed-loop feedback-controlled drug delivery to hold intracranial procaine levels constant (±10%) for >1.5 hours. These results demonstrate the utility of such sensors in (i) the determination of the site-specific, seconds-resolved neuropharmacokinetics, (ii) enabling the study of individual subject neuropharmacokinetics and concentration-response relations, and (iii) performing high-precision control over intracranial drug levels.