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. Doing so we find that, although the collection of hundreds of concentration values over a single drug lifetime enables high-precision estimates of the parameters describing intracranial transport, due to a mathematical equivalence, the data produce two divergent descriptions of the drug's plasma pharmacokinetics that fit the in-brain observations equally well. The simultaneous collection of intravenous measurements, however, resolves this ambiguity, enabling high-precision (typically of ±5 to ±20% at 95% confidence levels) estimates of the key pharmacokinetic parameters describing transport from the blood to the cerebrospinal fluid in individual animals. CONCLUSIONS AND IMPLICATIONS: The availability of simultaneous, high-density 'in-vein' (plasma) and 'in-brain' (cerebrospinal fluid) measurements provides unique opportunities to explore the assumptions almost universally employed in earlier compartmental models of drug transport, allowing the quantitative assessment of, for example, the pharmacokinetic effects of physiological processes such as the bulk transport of the drug out of the CNS via the dural venous sinuses.

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 situ 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. Doing so we find that low-temperature (-20 °C) storage is sufficient to preserve sensor functionality for at least six months without the need for exogenous preservatives.

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.

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. This sensor, however, appears to employ binding-induced displacement of the redox reporter rather than binding-induced folding as its signal transduction mechanism, suggesting in turn a mechanism for the improvement observed for the other two sensors. Codeposition thus not only provides a more convenient means of fabricating EAB sensors but also can improve their performance.

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.

High-Affinity Aptamers for In Vitro and In Vivo Cocaine Sensing.

The ability to quantify cocaine in biological fluids is crucial for both the diagnosis of intoxication and overdose in the clinic as well as investigation of the drug's pharmacological and toxicological effects in the laboratory. To this end, we have performed high-stringency in vitro selection to generate DNA aptamers that bind cocaine with nanomolar affinity and clinically relevant specificity, thus representing a dramatic improvement over the current-generation, micromolar-affinity, low-specificity cocaine aptamers. Using these novel aptamers, we then developed two sensors for cocaine detection. The first, an in vitro fluorescent sensor, successfully detects cocaine at clinically relevant levels in 50% human serum without responding significantly to other drugs of abuse, endogenous substances, or a diverse range of therapeutic agents. The second, an electrochemical aptamer-based sensor, supports the real-time, seconds-resolved measurement of cocaine concentrations in vivo in the circulation of live animals. We believe the aptamers and sensors developed here could prove valuable for both point-of-care and on-site clinical cocaine detection as well as fundamental studies of cocaine neuropharmacology.

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. The improved stability is sufficient that these hydrogel-protected sensors achieved good baseline stability and precision when deployed in situ in the veins, muscles, bladder, or tumors of living rats without the use of the drift correction approaches traditionally required in such placements. Finally, our implantable gel-protective EAB sensors achieved good biocompatibility when deployed in vivo in the living rats without causing any severe inflammation.

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.

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. Given the recent advent of in vivo electrochemical aptamer-based (EAB) sensors, however, we can now obtain hundreds of concentration measurements per administration. Our aim in this paper was to assess the ability of these time-dense datasets to describe time-varying pharmacokinetic models with good statistical significance. METHODS: We used seconds-resolved measurements of plasma tobramycin concentrations in rats to statistically compare traditional one- and two-compartmental pharmacokinetic models to new models in which the proportional relationship between a drug's plasma concentration and its elimination rate varies in response to changing kidney function. RESULTS: We found that a modified one-compartment model in which the proportionality between the plasma concentration of tobramycin and its elimination rate falls reciprocally with time either meets or is preferred over the standard two-compartment pharmacokinetic model for half of the datasets characterized. When we reduced the impact of the drug's rapid distribution phase on the model, this one-compartment, time-varying model was statistically preferred over the standard one-compartment model for 80% of our datasets. CONCLUSIONS: Our results highlight both the impact that simple physiological changes (such as varying kidney function) can have on drug pharmacokinetics and the ability of high-time resolution EAB sensor measurements to identify such impacts.

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. Motivated by the correction of signal drift, in this paper, we have explored the use of oligoethylene glycol (OEG), a widely employed antifouling coating, to reduce the signal drift in EAB sensors. Counter to expectations, however, when challenged in 37 °C whole blood in vitro, EAB sensors employing OEG-modified self-assembled monolayers exhibit both greater drift and reduced signal gain, compared with those employ a simple, hydroxyl-terminated monolayer. On the other hand, when EAB sensor was prepared with a mix monolayer using MCH and lipoamido OEG 2 alcohol, reduced signal noise was observed compared to the same sensor prepared with MCH presumably due to improved SAM construction. These results suggest broader exploration of antifouling materials will be required to improve the signal drift of EAB sensors.

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.

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. Here we review biosensor approaches that may support such measurements, as well as the molecules potentially of greatest interest to monitor. DESIGN: Narrative literature review. METHOD: Literature review. RESULTS: Significant effort has been made to harness the specificity, affinity, and generalizability of biomolecular recognition in a platform technology supporting continuous in vivo molecular measurements. Most biosensor approaches, however, are either not generalizable to most targets, or fail when challenged in the complex environments found in vivo. Electrochemical aptamer-based sensors, in contrast, are the first technology to simultaneously achieve both of these critical attributes. In an effort to illustrate the potential of this platform technology, we both critically review the literature describing it and briefly survey some of the molecular performance markers we believe will prove advantageous to monitor using it. CONCLUSIONS: Electrochemical aptamer-based sensors may be the first truly generalizable technology for monitoring specific molecules in situ in the body and how adaptation of the platform to subcutaneous microneedles will enable the real-time monitoring of performance markers via a wearable, minimally invasive device.

Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

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.

Electrochemical Aptamer-Based Sensors: A Platform Approach to High-Frequency Molecular Monitoring In Situ in the Living Body.

The monitoring of specific molecules in the living body has historically required sample removal (e.g., blood draws, microdialysis) followed by analysis via cumbersome, laboratory-bound processes. Those few exceptions to this rule (e.g., glucose, pyruvate, the monoamines) are monitored using "one-off" technologies reliant on the specific enzymatic or redox reactivity of their targets, and thus not generalizable to the measurement of other targets. In response we have developed in vivo electrochemical aptamer-based (E-AB) sensors, a modular, receptor-based measurement technology that is independent of the chemical reactivity of its targets, and thus has the potential to be generalizable to a wide range of analytes. To further the adoption of this in vivo molecular measurement approach by other researchers and to accelerate its ultimate translation to the clinic, we present here our standard protocols for the fabrication and use of intravenous E-AB sensors.

Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures.

Introduction: Incubation of drug-craving refers to a time-dependent increase in drug cue-elicited craving that occurs during protracted withdrawal. Historically, rat models of incubated cocaine craving employed extended-access (typically 6 h/day) intravenous drug self-administration (IV-SA) procedures, although incubated cocaine craving is reported to occur following shorter-access IV-SA paradigms. The notoriously low-throughput of extended-access IV-SA prompted us to determine whether two different short-access IV-SA procedures akin to those in the literature result in qualitatively similar changes in glutamate receptor expression and the activation of downstream signaling molecules within prefrontal cortex (PFC) subregions as those reported previously by our group under 6h-access conditions. Methods: For this, adult, male Sprague-Dawley rats were trained to intravenously self-administer cocaine for 2 h/day for 10 consecutive days (2-h model) or for 6 h on day 1 and 2 h/day for the remaining 9 days of training (Mixed model). A sham control group was also included that did not self-administer cocaine. Results: On withdrawal day 3 or 30, rats were subjected to a 2-h test of cue-reinforced responding in the absence of cocaine and a time-dependent increase in drug-seeking was observed under both IV-SA procedures. Immunoblotting of brain tissue collected immediately following the cue test session indicated elevated phospho-Akt1, phospho-CaMKII and Homer2a/b expression within the prelimbic subregion of the PFC of cocaine-incubated rats. However, we failed to detect incubation-related changes in Group 1 metabotropic glutamate receptor or ionotropic glutamate receptor subunit expression in either subregion. Discussion: These results highlight further a role for Akt1-related signaling within the prelimbic cortex in driving incubated cocaine craving, and provide novel evidence supporting a potential role also for CaMKII-dependent signaling through glutamate receptors in this behavioral phenomenon.

A Protein-Based Biosensor for Detecting Calcium by Magnetic Resonance Imaging.

Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer a promising approach for noninvasive brain-wide monitoring of neural activity at any arbitrary depth. Current examples of MRI-based calcium probes involve synthetic molecules and nanoparticles, which cannot be used to examine calcium signaling in a genetically encoded form. Here, we describe a new MRI sensor for calcium, based entirely on a naturally occurring calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese ions, thereby limiting Mn2+ enhanced paramagnetic relaxation of nearby water molecules. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 based MRI signals in response to biologically relevant calcium concentrations. The resulting response amplitude, i.e., change in relaxation time, is comparable to existing MRI-based calcium sensors as well as other reported protein-based MRI sensors. As a preliminary demonstration of its biological applicability, we used calprotectin to detect calcium in a lysed hippocampal cell preparation as well as in intact Chinese hamster ovary cells treated with a calcium ionophore. Calprotectin thus represents a promising path toward noninvasive imaging of calcium signaling by combining the molecular and cellular specificity of genetically encodable tools with the ability of MRI to image through scattering tissue of any size and depth.