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.

Electrochemical, aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small molecules directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously we have used voltammetry (cyclic, alternating current, and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, we demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concentration is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the number of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resolution of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach we demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resolution and unprecedented, few-percent precision in the determination of its pharmacokinetic phases.

The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, lifethreatening conditions. In response, here we present an electrochemical aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, we have developed an electrochemical, aptamer-based (EAB) sensor against Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clinically relevant NGAL concentrations over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how we detect, monitor, and treat many important diseases.

The ability to monitor drugs, metabolites, hormones, and other biomarkers in situ in the body would greatly advance both clinical practice and biomedical research. To this end, we are developing electrochemical aptamer-based (EAB) sensors, a platform technology able to perform real-time, in vivo monitoring of specific molecules irrespective of their chemical or enzymatic reactivity. An important obstacle to the deployment of EAB sensors in the challenging environments found in the living body is signal drift, whereby the sensor signal decreases over time. To date, we have demonstrated a number of approaches by which this drift can be corrected sufficiently well to achieve good measurement precision over multihour in vivo deployments. To achieve a much longer in vivo measurement duration, however, will likely require that we understand and address the sources of this effect. In response, here, we have systematically examined the mechanisms underlying the drift seen when EAB sensors and simpler, EAB-like devices are challenged in vitro at 37 °C in whole blood as a proxy for in vivo conditions. Our results demonstrate that electrochemically driven desorption of a self-assembled monolayer and fouling by blood components are the two primary sources of signal loss under these conditions, suggesting targeted approaches to remediating this degradation and thus improving the stability of EAB sensors and other, similar electrochemical biosensor technologies when deployed in the body.

By, in effect, rendering pharmacokinetics an experimentally adjustable parameter, the ability to perform feedbackcontrolled dosing informed by high-frequency in vivo drug measurements would prove a powerful tool for both pharmacological research and clinical practice. Efforts to this end, however, have historically been thwarted by an inability to measure in vivo drug levels in real time and with sufficient convenience and temporal resolution. In response, we describe a closed-loop, feedbackcontrolled delivery system that uses drug level measurements provided by an in vivo electrochemical aptamer-based (E-AB) sensor to adjust dosing rates every 7 s. The resulting system supports the maintenance of either constant or predefined time-varying plasma drug concentration profiles in live rats over many hours. For researchers, the resultant high-precision control over drug plasma concentrations provides an unprecedented opportunity to (1) map the relationships between pharmacokinetics and clinical outcomes, (2) eliminate inter-and intrasubject metabolic variation as a confounding experimental variable, (3) accurately simulate human pharmacokinetics in animal models, and (4) measure minute-to-minute changes in a drug's pharmacokinetic behavior in response to changing health status, diet, drug−drug interactions, or other intrinsic and external factors. In the clinic, feedback-controlled drug delivery would improve our ability to accurately maintain therapeutic drug levels in the face of large, often unpredictable intra-and interpatient metabolic variation. This, in turn, would improve the efficacy and safety of therapeutic intervention, particularly for the most gravely ill patients, for whom metabolic variability is highest and the margin for therapeutic error is smallest.

By analogy to the revolution the "home glucose monitor" created in the treatment of diabetes, the availability of a modular, "platform" technology able to measure nearly any metabolite, biomarker, or drug "at-home" in unprocessed, finger-prick volumes of whole blood could revolutionize the monitoring and treatment of disease. Thus motivated, we have adapted here the electrochemical aptamer-based sensing platform to the problem of rapidly and conveniently measuring the level of phenylalanine in the blood, an ability that would aid the monitoring and management of phenylketonuria (PKU). To achieve this, we exploited a previously reported DNA aptamer that recognizes phenylalanine in complex with a rhodium-based "receptor" that improves affinity. We reengineered this to undergo a large-scale, binding-induced conformational change before modifying it with a methylene blue redox reporter and attaching it to a gold electrode that supports the appropriate electrochemical interrogation. The resultant sensor achieves a useful dynamic range of 90 nM to 7 μM. When challenged with finger-prick-scale sample volumes of the whole blood (diluted 1000-fold to match the sensor's dynamic range), the device achieves the accurate (±20%), calibration-free measurement of blood phenylalanine levels in minutes.

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.

A wide range of new devices aimed at in vivo molecular detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics associated with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approximately −2.2. Such behavior is consistent with a diffusioncontrolled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the observed rates are, at 5−400 s −1 , orders of magnitude slower than the intramolecular dynamics of single-stranded oligonucleotides when free in solution. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equivalent terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is negatively charged at the redox potential of methylene blue. Consistent with this, changing monolayer composition so as to increase the negative charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theoretical models and computer simulations performed in support of our experimental studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.