Effects of Physiological-Scale Variation in Cations, pH, and Temperature on the Calibration of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors are the first technology supporting high-frequency, real-time, in vivo molecular measurements that is independent of the chemical reactivity of its targets, rendering it easily generalizable. As is true for all biosensors, however, EAB sensor performance is affected by the measurement environment, potentially reducing accuracy when this environment deviates from the conditions under which the sensor was calibrated. Here, we address this question by measuring the extent to which physiological-scale environmental fluctuations reduce the accuracy of a representative set of EAB sensors and explore the means of correcting these effects.

Dual-Purpose Aptamer-Based Sensors for Real-Time, Multiplexable Monitoring of Metabolites in Cell Culture Media.

The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in blood and tissues.

Effects of storage conditions on the performance of an electrochemical aptamer-based sensor.

The electrochemical aptamer-based (EAB) sensor platform is the only molecular monitoring approach yet reported that is (1) real time and effectively continuous, (2) selective enough to deploy in the living body, and (3) independent of the chemical or enzymatic reactivity of its target, rendering it adaptable to a wide range of analytes. These attributes suggest the EAB platform will prove to be an important tool in both biomedical research and clinical practice. To advance this possibility, here we have explored the stability of EAB sensors upon storage, using retention of the target recognizing aptamer, the sensor's signal gain, and the affinity of the aptamer as our performance metrics.

A high-precision view of intercompartmental drug transport via simultaneous, seconds-resolved, in situ measurements in the vein and brain.

Background And Purpose: The ability to measure specific molecules at multiple sites within the body simultaneously, and with a time resolution of seconds, could greatly advance our understanding of drug transport and elimination.

Experimental Approach: As a proof-of-principle demonstration, here we describe the use of electrochemical aptamer-based (EAB) sensors to measure transport of the antibiotic vancomycin from the plasma (measured in the jugular vein) to the cerebrospinal fluid (measured in the lateral ventricle) of live rats with temporal resolution of a few seconds.

Key Results: In our first efforts, we made measurements solely in the ventricle.

Dual-Frequency, Ratiometric Approaches to EAB Sensor Interrogation Support the Calibration-Free Measurement of Specific Molecules In Vivo.

Electrochemical aptamer-based (EAB) sensors represent the first molecular measurement technology that is both (1) independent of the chemical reactivity of the target, and thus generalizable to many targets and (2) able to function in an accurate, drift-corrected manner in situ in the living body. Signaling in EAB sensors is generated when an electrode-bound aptamer binds to its target ligand, altering the rate of electron transfer from an attached redox reporter and producing an easily detectable change in peak current when the sensor is interrogated using square wave voltammetry. Due to differences in the microscopic surface area of the interrogating electrodes, the baseline peak currents obtained from EAB sensors, however, can be highly variable.

Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors, a minimally invasive means of performing high-frequency, real-time measurement of drugs and biomarkers in situ in the body, have traditionally been fabricated by depositing their target-recognizing aptamer onto an interrogating gold electrode using a "sequential" two-step method involving deposition of the thiol-modified oligonucleotide (typically for 1 h) followed by incubation in mercaptohexanol solution (typically overnight) to complete the formation of a stable, self-assembled monolayer. Here we use EAB sensors targeting vancomycin, tryptophan, and phenylalanine to show that "codeposition", a less commonly employed EAB fabrication method in which the thiol-modified aptamer and the mercaptohexanol diluent are deposited on the electrode simultaneously and for as little as 1 h, improves the signal gain (relative change in signal upon the addition of high concentrations of the target) of the vancomycin and tryptophan sensors without significantly reducing their stability. In contrast, the gain of the phenylalanine sensor is effectively identical irrespective of the fabrication approach employed.

The Use of Xenonucleic Acids Significantly Reduces the In Vivo Drift of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision.

Real-Time Monitoring of Antibiotics in the Critically Ill Using Biosensors.

By ensuring optimal dosing, therapeutic drug monitoring (TDM) improves outcomes in critically ill patients by maximizing effectiveness while minimizing toxicity. Current methods for measuring plasma drug concentrations, however, can be challenging, time-consuming, and slow to return an answer, limiting the extent to which TDM is used to optimize drug exposure. A potentially promising solution to this dilemma is provided by biosensors, molecular sensing devices that employ biorecognition elements to recognize and quantify their target molecules rapidly and in a single step.

Implantable Hydrogel-Protective DNA Aptamer-Based Sensor Supports Accurate, Continuous Electrochemical Analysis of Drugs at Multiple Sites in Living Rats.

The ability to track the levels of specific molecules, such as drugs, metabolites, and biomarkers, in the living body, in real time and for long durations, would improve our understanding of health and our ability to diagnose, treat, and monitor disease. To this end, we are developing electrochemical aptamer-based (EAB) biosensors, a general platform supporting high-frequency, real-time molecular measurements in the living body. Here we report that the use of an agarose hydrogel protective layer for EAB sensors significantly improves their signaling stability when deployed in the complex, highly time-varying environments found in vivo.

Precise Electrochemical Sizing of Individual Electro-Inactive Particles.

Nanoimpact electrochemistry enables the time-resolved in situ characterization (e.g., size, catalytic activity) of single nanomaterial units, providing a means of elucidating heterogeneities that would be masked in ensemble studies.

Calibration-Free, Seconds-Resolved In Vivo Molecular Measurements using Fourier-Transform Impedance Spectroscopy Interrogation of Electrochemical Aptamer Sensors.

Electrochemical aptamer-based (EAB) sensors are capable of measuring the concentrations of specific molecules in vivo, in real time, and with a few-second time resolution. For their signal transduction mechanism, these sensors utilize a binding-induced conformational change in their target-recognizing, redox-reporter-modified aptamer to alter the rate of electron transfer between the reporter and the supporting electrode. While a variety of voltammetric techniques have been used to monitor this change in kinetics, they suffer from various drawbacks, including time resolution limited to several seconds and sensor-to-sensor variation that requires calibration to remove.

Continuous molecular monitoring of human dermal interstitial fluid with microneedle-enabled electrochemical aptamer sensors.

The ability to continually collect diagnostic information from the body during daily activity has revolutionized the monitoring of health and disease. Much of this monitoring, however, has been of physical "vital signs", with the monitoring of molecular markers having been limited to glucose, primarily due to the lack of other medically relevant molecules for which continuous measurements are possible in bodily fluids. Electrochemical aptamer sensors, however, have a recent history of successful demonstrations in rat animal models.

Survey of oligoethylene glycol-based self-assembled monolayers on electrochemical aptamer-based sensor in biological fluids.

The ability to monitor levels of endogenous markers and clearance profiles of drugs and their metabolites can improve the quality of biomedical research and precision with which therapies are individualized. Towards this end, electrochemical aptamer-based (EAB) sensors have been developed that support the real-time monitoring of specific analytes in vivo with clinically relevant specificity and sensitivity. A challenge associated with the in vivo deployment of EAB sensors, however, is how to manage the signal drift which, although correctable, ultimately leads to unacceptably low signal-to-noise ratios, limiting the measurement duration.

Using seconds-resolved pharmacokinetic datasets to assess pharmacokinetic models encompassing time-varying physiology.

Aim: Pharmacokinetics have historically been assessed using drug concentration data obtained via blood draws and bench-top analysis. The cumbersome nature of these typically constrains studies to at most a dozen concentration measurements per dosing event. This, in turn, limits our statistical power in the detection of hours-scale, time-varying physiological processes.

A tight squeeze: geometric effects on the performance of three-electrode electrochemical-aptamer based sensors in constrained, placements.

Electrochemical, aptamer-based (EAB) sensors are the first molecular monitoring technology that is (1) based on receptor binding and not the reactivity of the target, rendering it fairly general, and (2) able to support high-frequency, real-time measurements in the living body. To date, EAB-derived measurements have largely been performed using three electrodes (working, reference, counter) bundled together within a catheter for insertion into the rat jugular. Exploring this architecture, here we show that the placement of these electrodes inside or outside of the lumen of the catheter significantly impacts sensor performance.

Efforts toward the continuous monitoring of molecular markers of performance.

Objectives: Technologies supporting the continuous, real-time measurement of blood oxygen saturation and plasma glucose levels have improved our ability to monitor performance status. Our ability to monitor other molecular markers of performance, however, including the hormones known to indicate overtraining and general health, has lagged. That is, although a number of other molecular markers of performance status have been identified, we have struggled to develop viable technologies supporting their real-time monitoring in the body.

On the Rational Design of Cooperative Receptors.

Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity.

Conformational-switch biosensors as novel tools to support continuous, real-time molecular monitoring in lab-on-a-chip devices.

Recent years have seen continued expansion of the functionality of lab on a chip (LOC) devices. Indeed LOCs now provide scientists and developers with useful and versatile platforms across a myriad of chemical and biological applications. The field still fails, however, to integrate an often important element of bench-top analytics: real-time molecular measurements that can be used to "guide" a chemical response.

Real-Time, Seconds-Resolved Measurements of Plasma Methotrexate In Situ in the Living Body.

Dose-limiting toxicity and significant patient-to-patient pharmacokinetic variability often render it difficult to achieve the safe and effective dosing of drugs. This is further compounded by the slow, cumbersome nature of the analytical methods used to monitor patient-specific pharmacokinetics, which inevitably rely on blood draws followed by post-facto laboratory analysis. Motivated by the pressing need for improved "therapeutic drug monitoring", we are developing electrochemical aptamer-based (EAB) sensors, a minimally invasive biosensor architecture that can provide real-time, seconds-resolved measurements of drug levels in situ in the living body.

On the Disinfection of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors encompass the only biosensor approach yet reported that is simultaneously: (1) independent of the chemical or enzymatic reactivity of its target, rendering it general; (2) continuous and real-time; and (3) selective enough to deploy in the living body. Consistent with this, EAB sensors supporting the seconds-resolved, real-time measurement of multiple drugs and metabolites have been reported, suggesting the approach may prove of value in biomedical research and the diagnosis, treatment, and monitoring of disease. However, to apply these devices in long-duration animal models, much less in human patients, requires that they be free of any significant pathogen load.

Pulsed transistor operation enables miniaturization of electrochemical aptamer-based sensors.

By simultaneously transducing and amplifying, transistors offer advantages over simpler, electrode-based transducers in electrochemical biosensors. However, transistor-based biosensors typically use static (i.e.

The sequestration mechanism as a generalizable approach to improve the sensitivity of biosensors and bioassays.

Biosensors and bioassays, both of which employ proteins and nucleic acids to detect specific molecular targets, have seen significant applications in both biomedical research and clinical practice. This success is largely due to the extraordinary versatility, affinity, and specificity of biomolecular recognition. Nevertheless, these receptors suffer from an inherent limitation: single, saturable binding sites exhibit a hyperbolic relationship (the "Langmuir isotherm") between target concentration and receptor occupancy, which in turn limits the sensitivity of these technologies to small variations in target concentration.

Real-Time, Molecular Monitoring Using Electrochemical Aptamer Based Sensors: Opportunities and Challenges.

The continuous, real-time measurement of specific molecules in the body would greatly improve our ability to understand, diagnose, and treat disease. The vast majority of continuous molecular sensing technologies, however, either (1) rely on the chemical or enzymatic reactivity of their targets, sharply limiting their scope, or (2) have never been shown (and likely will never be shown) to operate in the complex environments found . Against this background, here we review electrochemical aptamer-based (EAB) sensors, an electrochemical approach to real-time molecular monitoring that has now seen 15 years of academic development.