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. To overcome this, we have historically performed single-point calibration using measurements performed in a single sample of known target concentration. Here, however, we explore approaches to EAB sensor operation that negate the need to perform even single-point calibration of individual sensors. These are a ratiometric approach employing the ratio of the peak currents observed at two distinct square wave frequencies, and a kinetic differential measurement approach that employs the difference between peak currents seen at the two frequencies. Using in vivo measurements of vancomycin and phenylalanine as our test bed, we compared the output of these methods with that of the same sensor when single-point calibration was employed. Doing so we find that both methods support accurately drift-corrected measurements in vivo in live rats, even when employing rather crudely handmade devices. By removing the need to calibrate each individual sensor in a sample of known target concentration, these interrogation methods should significantly simplify the use of EAB sensors for in vivo applications.

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. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant "xenonucleic acid" 2'O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2'O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2'O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2'O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.

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. Here, however, we show that the use of fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate EAB sensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. To showcase these benefits, we demonstrate the approach's ability to perform real-time molecular measurements in the veins of living rats.

High-precision monitoring of and feedback control over drug concentrations in the brains of freely moving rats.

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.

A tight squeeze: geometric effects on the performance of three-electrode electrochemical-aptamer based sensors in constrained, in vivo 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 situ in the living body. To date, EAB-derived in vivo 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. Specifically, we find that retaining the counter electrode within the catheter increases the resistance between it and the working electrode, increasing the capacitive background. In contrast, extending the counter electrode outside the lumen of the catheter reduces this effect, significantly enhancing the signal-to-noise of intravenous molecular measurements. Exploring counter electrode geometries further, we find that they need not be larger than the working electrode. Putting these observations together, we have developed a new intravenous EAB architecture that achieves improved performance while remaining short enough to safely emplace in the rat jugular. These findings, though explored here with EAB sensors may prove important for the design of many electrochemical biosensors.

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. A key advantage of EAB sensors is that they are generalizable to the detection of a wide range of therapeutic agents because they are independent of the chemical or enzymatic reactivity of their targets. Three of the four therapeutic drug classes that have, to date, been shown measurable using in vivo EAB sensors, however, bind to nucleic acids as part of their mode of action, leaving open questions regarding the extent to which the approach can be generalized to therapeutics that do not. Here, we demonstrate real-time, in vivo measurements of plasma methotrexate, an antimetabolite (a mode of action not reliant on DNA binding) chemotherapeutic, following human-relevant dosing in a live rat animal model. By providing hundreds of drug concentration values, the resulting seconds-resolved measurements succeed in defining key pharmacokinetic parameters, including the drug's elimination rate, peak plasma concentration, and exposure (area under the curve), with unprecedented 5 to 10% precision. With this level of precision, we easily identify significant (>2-fold) differences in drug exposure occurring between even healthy rats given the same mass-adjusted methotrexate dose. By providing a real-time, seconds-resolved window into methotrexate pharmacokinetics, such measurements can be used to precisely "individualize" the dosing of this significantly toxic yet vitally important chemotherapeutic.

Improved calibration of electrochemical aptamer-based sensors.

Electrochemical aptamer-based (EAB) sensors support the real-time, high frequency measurement of pharmaceuticals and metabolites in-situ in the living body, rendering them a potentially powerful technology for both research and clinical applications. Here we explore quantification using EAB sensors, examining the impact of media selection and temperature on measurement performance. Using freshly-collected, undiluted whole blood at body temperature as both our calibration and measurement conditions, we demonstrate accuracy of better than ± 10% for the measurement of our test bed drug, vancomycin. Comparing titrations collected at room and body temperature, we find that matching the temperature of calibration curve collection to the temperature used during measurements improves quantification by reducing differences in sensor gain and binding curve midpoint. We likewise find that, because blood age impacts the sensor response, calibrating in freshly collected blood can improve quantification. Finally, we demonstrate the use of non-blood proxy media to achieve calibration without the need to collect fresh whole blood.

Nanoporous Gold for the Miniaturization of In Vivo Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based sensors enable real-time molecular measurements in the living body. The spatial resolution of these measurements and ability to perform measurements in targeted locations, however, is limited by the length and width of the device's working electrode. Historically, achieving good signal to noise in the complex, noisy in vivo environment has required working electrode lengths of 3-6 mm. To enable sensor miniaturization, here we have enhanced the signaling current obtained for a sensor of given macroscopic dimensions by increasing its surface area. Specifically, we produced nanoporous gold via an electrochemical alloying/dealloying technique to increase the microscopic surface area of our working electrodes by up to 100-fold. Using this approach, we have miniaturized in vivo electrochemical aptamer-based (EAB) sensors (here using sensors against the antibiotic, vancomycin) by a factor of 6 while retaining sensor signal and response times. Conveniently, the fabrication of nanoporous gold is simple, parallelizable, and compatible with both two- and three-dimensional electrode architectures, suggesting that it may be of value to a range of electrochemical biosensor applications.

Seconds-Resolved, In Situ Measurements of Plasma Phenylalanine Disposition Kinetics in Living Rats.

Current knowledge of the disposition kinetics of endogenous metabolites is founded almost entirely on poorly time-resolved experiments in which samples are removed from the body for later, benchtop analysis. Here, in contrast, we describe real-time, seconds-resolved measurements of plasma phenylalanine collected in situ in the body via electrochemical aptamer-based (EAB) sensors, a platform technology that is independent of the reactivity of its targets and thus is generalizable to many. Specifically, using indwelling EAB sensors, we have monitored plasma phenylalanine in live rats with a few micromolar precision and a 12 s temporal resolution, identifying a large-amplitude, few-seconds phase in the animals' metabolic response that had not previously been reported. Using the hundreds of individual measurements that the approach provides from each animal, we also identify inter-subject variability, including statistically significant differences associated with the feeding status. These results highlight the power of in vivo EAB measurements, an advancement that could dramatically impact our understanding of physiology and provide a valuable new tool for the monitoring and treatment of metabolic disorders.

Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors.

Recent years have seen the development of a number of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resolution to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochemical phase interrogation, as a means of collecting high time resolution measurements with sensors in this class. Specifically, using changes in the electrochemical phase to monitor target binding in an electrochemical-aptamer based (EAB) sensor, we achieve subsecond temporal resolution and multihour stability in measurements performed directly in undiluted whole blood. Electrochemical phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equivalent circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochemical biosensor architectures rely on this signaling mechanism, we believe that electrochemical phase interrogation may prove generalizable toward subsecond measurements of molecular targets.

Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery.

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.

An electrochemical aptamer-based sensor for the rapid and convenient measurement of L-tryptophan.

The field of precision medicine-the possibility to accurately tailor pharmacological treatments to each specific patient-would be significantly advanced by the ability to rapidly, conveniently, and cost-effectively measure biomarkers directly at the point of care. Electrochemical aptamer-based (E-AB) sensors appear a promising approach to this end due to their low cost, ease of use, and good analytical performance in complex clinical samples. Thus motivated, we present here the development of an E-AB sensor for the measurement of the amino acid L-tryptophan, a diagnostic marker indicative of a number of metabolic and mental health disorders, in urine. The sensor employs a previously reported DNA aptamer able to recognize the complex formed between tryptophan and a rhodium-based receptor. We adopted the aptamer to the E-AB sensing platform by truncating it, causing it to undergo a binding-induced conformational change, modifying it with a redox-reporting methylene blue, and attaching it to an interrogating electrode. The resulting sensor is able to measure tryptophan concentrations in the micromolar range in minutes and readily discriminates between its target and other aromatic and non-aromatic amino acids. Using it, we demonstrate the measurement of clinically relevant tryptophan levels in synthetic urine in a process requiring only a single dilution step. The speed and convenience with which this is achieved suggest that the E-AB platform could significantly improve the ease and frequency with which metabolic diseases are monitored. Graphical Abstract.

Persistent effect of withdrawal from intravenous methamphetamine self-administration on brain activation and behavioral economic indices involving an effort cost.

Exposure to drugs of abuse produces maladaptive changes in cost-benefit decision-making, including the evaluation of time and risk. Studies probing the effects of drug exposure on such evaluations have primarily used experimenter-administered drug regimens. Similarly, while much is known about the neural bases of effort, there have been relatively fewer investigations of the effects of drug experience on effort-based choices. We recently reported that experimenter-administered methamphetamine (meth) resulted in steeper discounting of effort for food rewards in rats, when assessed in protracted withdrawal. Here, we studied rats that underwent withdrawal from weeks of meth intravenous self-administration that later could freely select between a high effort, preferred option (progressive ratio lever pressing for sucrose pellets) versus a low effort, less preferred option (freely-available lab chow). We found decreased effort for the preferred reward and changes in a behavioral economic index demonstrating an increased sensitivity to effort in meth-experienced rats. Critically, the decreased effort for the preferred option was only present in the context of a competing option, not when it was the only option. We also confirmed rats preferred sucrose pellets over chow when both were freely available. These long-lasting changes were accompanied by decreased c-Fos activation in ventral striatum and basolateral amygdala, regions known to be important in effort-based choices. Taken together with our previous observations, these results suggest a robust and enduring effect of meth on value-based decision-making, and point to the underlying neural mechanisms that support the evaluation of an effort cost.

Steep effort discounting of a preferred reward over a freely-available option in prolonged methamphetamine withdrawal in male rats.

RATIONALE: Drug addiction can be described as aberrant allocation of effort toward acquiring drug, despite associated costs. It is unclear if this behavioral pattern results from an overvaluation of reward or to an altered sensitivity to costs. OBJECTIVE: Present experiments assessed reward sensitivity and effortful choice in rats following 1 week of withdrawal from methamphetamine (mAMPH). METHODS: Rats were treated with either saline or an escalating dose mAMPH regimen, then tested after a week without the drug. In experiment 1, rats were given a free choice between water and various concentrations of sucrose solution to assess general reward sensitivity. In experiment 2, rats were presented with a choice between lever-pressing for sucrose pellets on a progressive ratio schedule or consuming freely-available chow. RESULTS: In experiment 1, we found no differences in sucrose preference between mAMPH- and saline-pretreated rats. In experiment 2, when selecting between two options, mAMPH-pretreated rats engaged in less lever-pressing for sucrose pellets (p < 0.01) and switched from this preferred reward to the chow sooner than saline-pretreated rats (p < 0.05). This effect was not consistent with general reward devaluation or loss of motivation. CONCLUSIONS: These findings demonstrate that mAMPH exposure and withdrawal lead to steeper discounting of reward value by effort, an effect that is consistent with the effect of mAMPH on discounting by delay, and which may reflect an underlying shared mechanism.

Anterior cingulate cortex supports effort allocation towards a qualitatively preferred option.

The anterior cingulate cortex (ACC) is known to be involved in effortful choice, yet its role in cost-benefit evaluation of qualitatively different rewards (more/less preferred), beyond magnitude differences (larger/smaller), is poorly understood. Selecting between qualitatively different options is a decision type commonly faced by humans. Here, we assessed the role of ACC on a task that has primarily been used to probe striatal function in motivation. Rats were trained to stable performance on a progressive ratio schedule for sucrose pellets and were then given sham surgeries (control) or excitotoxic NMDA lesions of ACC. Subsequently, a choice was introduced: chow was concurrently available while animals could work for the preferred sucrose pellets. ACC lesions produced a significant decrease in lever presses for sucrose pellets compared to control, whereas chow consumption was unaffected. Lesions had no effect on sucrose pellet preference when both options were freely available. When laboratory chow was not concurrently available, ACC-lesioned rats exhibited similar lever pressing as controls. During a test under specific satiety for sucrose pellets, ACC-lesioned rats also showed intact devaluation effects. The effects of ACC lesions in our task are not mediated by decreased appetite, a change in food preference, a failure to update value or a learning deficit. Taken together, we found that ACC lesions decreased effort for a qualitatively preferred option. These results are discussed with reference to effects of striatal manipulations and our recent report of a role for basolateral amygdala in effortful choice.