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.

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.

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.

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.

ERK-Directed Phosphorylation of mGlu5 Gates Methamphetamine Reward and Reinforcement in Mouse.

Methamphetamine (MA) is a highly addictive psychomotor stimulant drug. In recent years, MA use has increased exponentially on a global scale, with the number of MA-involved deaths reaching epidemic proportions. There is no approved pharmacotherapy for treating MA use disorder, and we know relatively little regarding the neurobiological determinants of vulnerability to this disease. Extracellular signal-regulated kinase (ERK) is an important signaling molecule implicated in the long-lasting neuroadaptations purported to underlie the development of substance use disorders, but the role for this kinase in the propensity to develop addiction, particularly MA use disorder, is uncharacterized. In a previous MA-induced place-conditioning study of C57BL/6J mice, we characterized mice as MA-preferring, -neutral, or -avoiding and collected tissue from the medial prefrontal cortex (mPFC). Using immunoblotting, we determined that elevated phosphorylated ERK expression within the medial prefrontal cortex (mPFC) is a biochemical correlate of the affective valence of MA in a population of C57BL/6J mice. We confirmed the functional relevance for mPFC ERK activation for MA-induced place-preference via site-directed infusion of the MEK inhibitor U0126. By contrast, ERK inhibition did not have any effect upon MA-induced locomotion or its sensitization upon repeated MA treatment. Through studies of transgenic mice with alanine point mutations on T1123/S1126 of mGlu5 that disrupt ERK-dependent phosphorylation of the receptor, we discovered that ERK-dependent mGlu5 phosphorylation normally suppresses MA-induced conditioned place-preference (MA-CPP), but is necessary for this drug's reinforcing properties. If relevant to humans, the present results implicate individual differences in the capacity of MA-associated cues/contexts to hyper-activate ERK signaling within mPFC in MA Use Disorder vulnerability and pose mGlu5 as one ERK-directed target contributing to the propensity to seek out and take MA.

Increased Alcohol-Drinking Induced by Manipulations of mGlu5 Phosphorylation within the Bed Nucleus of the Stria Terminalis.

The bed nucleus of the stria terminalis (BNST) is part of the limbic-hypothalamic system important for behavioral responses to stress, and glutamate transmission within this region has been implicated in the neurobiology of alcoholism. Herein, we used a combination of immunoblotting, neuropharmacological and transgenic procedures to investigate the role for metabotropic glutamate receptor 5 (mGlu5) signaling within the BNST in excessive drinking. We discovered that mGlu5 signaling in the BNST is linked to excessive alcohol consumption in a manner distinct from behavioral or neuropharmacological endophenotypes that have been previously implicated as triggers for heavy drinking. Our studies demonstrate that, in male mice, a history of chronic binge alcohol-drinking elevates BNST levels of the mGlu5-scaffolding protein Homer2 and activated extracellular signal-regulated kinase (ERK) in an adaptive response to limit alcohol consumption. Male and female transgenic mice expressing a point mutation of mGlu5 that cannot be phosphorylated by ERK exhibit excessive alcohol-drinking, despite greater behavioral signs of alcohol intoxication and reduced anxiety, and are insensitive to local manipulations of signaling in the BNST. These transgenic mice also show selective insensitivity to alcohol-aversion and increased novelty-seeking, which may be relevant to excessive drinking. Further, the insensitivity to alcohol-aversion exhibited by male mice can be mimicked by the local inhibition of ERK signaling within the BNST. Our findings elucidate a novel mGluR5-linked signaling state within BNST that plays a central and unanticipated role in excessive alcohol consumption.SIGNIFICANCE STATEMENT The bed nucleus of the stria terminalis (BNST) is part of the limbic-hypothalamic system important for behavioral responses to stress and alcohol, and glutamate transmission within BNST is implicated in the neurobiology of alcoholism. The present study provides evidence that a history of excessive alcohol drinking increases signaling through the metabotropic glutamate receptor 5 (mGlu5) receptor within the BNST in an adaptive response to limit alcohol consumption. In particular, disruption of mGlu5 phosphorylation by extracellular signal-regulated kinase within this brain region induces excessive alcohol-drinking, which reflects a selective insensitivity to the aversive properties of alcohol intoxication. These data indicate that a specific signaling state of mGlu5 within BNST plays a central and unanticipated role in excessive alcohol consumption.

Real-time measurement of small molecules directly in awake, ambulatory animals.

The development of a technology capable of tracking the levels of drugs, metabolites, and biomarkers in the body continuously and in real time would advance our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by high-resolution, patient-specific pharmacokinetics (including feedback-controlled drug delivery), opening new dimensions in personalized medicine. In response, we demonstrate here the ability of electrochemical aptamer-based (E-AB) sensors to support continuous, real-time, multihour measurements when emplaced directly in the circulatory systems of living animals. Specifically, we have used E-AB sensors to perform the multihour, real-time measurement of four drugs in the bloodstream of even awake, ambulatory rats, achieving precise molecular measurements at clinically relevant detection limits and high (3 s) temporal resolution, attributes suggesting that the approach could provide an important window into the study of physiology and pharmacokinetics.

Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data.

Electrochemical sensors are major players in the race for improved molecular diagnostics due to their convenience, temporal resolution, manufacturing scalability, and their ability to support real-time measurements. This is evident in the ever-increasing number of health-related electrochemical sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data volumes, we describe here an open-source, easily customizable, multiplatform compatible program for the real-time control, processing, and visualization of electrochemical data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which we have called Software for the Analysis and Continuous Monitoring of Electrochemical Systems (SACMES), also includes a graphical interface allowing the user to easily change analysis parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES we use it here to analyze the real-time data output by (1) the electrochemical, aptamer-based measurement of a specific small-molecule target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the determination of the folding thermodynamics of an electrode-attached, redox-reporter-modified protein.

PI3K activation within ventromedial prefrontal cortex regulates the expression of drug-seeking in two rodent species.

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50µM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction.

High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors.

The electrochemical, aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific molecular targets (irrespective of their chemical reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model we employ, entails devices less than 200 µm in diameter. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response we have developed an electrochemical roughening approach that enhances the signaling of small electrochemical sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, our method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufacturing. Using these high surface area electrodes, we demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the determination of drug disposition parameters.

Cocaine craving during protracted withdrawal requires PKCε priming within vmPFC.

In individuals with a history of drug taking, the capacity of drug-associated cues to elicit indices of drug craving intensifies or incubates with the passage of time during drug abstinence. This incubation of cocaine craving, as well as difficulties with learning to suppress drug-seeking behavior during protracted withdrawal, are associated with a time-dependent deregulation of ventromedial prefrontal cortex (vmPFC) function. As the molecular bases for cocaine-related vmPFC deregulation remain elusive, the present study assayed the consequences of extended access to intravenous cocaine (6 hours/day; 0.25 mg/infusion for 10 day) on the activational state of protein kinase C epsilon (PKCε), an enzyme highly implicated in drug-induced neuroplasticity. The opportunity to engage in cocaine seeking during cocaine abstinence time-dependently altered PKCε phosphorylation within vmPFC, with reduced and increased p-PKCε expression observed in early (3 days) and protracted (30 days) withdrawal, respectively. This effect was more robust within the ventromedial versus dorsomedial PFC, was not observed in comparable cocaine-experienced rats not tested for drug-seeking behavior and was distinct from the rise in phosphorylated extracellular signal-regulated kinase observed in cocaine-seeking rats. Further, the impact of inhibiting PKCε translocation within the vmPFC using TAT infusion proteins upon cue-elicited responding was determined and inhibition coinciding with the period of testing attenuated cocaine-seeking behavior, with an effect also apparent the next day. In contrast, inhibitor pretreatment prior to testing during early withdrawal was without effect. Thus, a history of excessive cocaine taking influences the cue reactivity of important intracellular signaling molecules within the vmPFC, with PKCε playing a critical role in the manifestation of cue-elicited cocaine seeking during protracted drug withdrawal.

A Biomimetic Phosphatidylcholine-Terminated Monolayer Greatly Improves the In Vivo Performance of Electrochemical Aptamer-Based Sensors.

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.

Prefrontal glutamate correlates of methamphetamine sensitization and preference.

Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA-drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated MA sensitized drug-induced glutamate release and lowered indices of N-methyl-d-aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low-dose MA is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency.

Deficits in ventromedial prefrontal cortex group 1 metabotropic glutamate receptor function mediate resistance to extinction during protracted withdrawal from an extensive history of cocaine self-administration.

Anomalies in prefrontal cortex (PFC) function are posited to underpin difficulties in learning to suppress drug-seeking behavior during abstinence. Because group 1 metabotropic glutamate receptors (mGluRs) regulate drug-related learning, we assayed the consequences of extended access to intravenous cocaine (6 h/d; 0.25 mg/infusion for 10 d) on the PFC expression of group 1 mGluRs and the relevance of observed changes for cocaine seeking. After protracted withdrawal, cocaine-experienced animals exhibited a time-dependent intensification of cue-induced cocaine-seeking behavior and an impaired extinction of this behavior. These behavioral phenomena were associated with a time-dependent reduction in mGluR1/5 expression within ventromedial PFC (vmPFC) of cocaine-experienced animals exposed to extinction testing but not in untested ones. Interestingly, pharmacological manipulations of vmPFC mGluR1/5 produced no immediate effects on cue-induced cocaine-seeking behavior but produced residual effects on a subsequent test for cocaine seeking. At 3 d withdrawal, cocaine-experienced rats infused intra-vmPFC with mGluR1/5 antagonists, either before or after an initial test for cocaine seeking, persisted in their cocaine seeking akin to cocaine-experienced rats in protracted withdrawal. Conversely, cocaine-experienced rats infused with an mGluR1/5 agonist before the initial test for cocaine-seeking at 30 d withdrawal exhibited a facilitation of extinction learning. These data indicate that cue-elicited deficits in vmPFC group 1 mGluR function mediate resistance to extinction during protracted withdrawal from a history of extensive cocaine self-administration and pose pharmacological stimulation of these receptors as a potential approach to facilitate learned suppression of drug-seeking behavior that may aid drug abstinence.

Adaptations underlying the development of excessive alcohol intake in selectively bred mice.

BACKGROUND: This commentary discusses the important contributions of the article in this issue by Matson and colleagues entitled "Selectively bred crossed high-alcohol-preferring mice drink to intoxication and develop functional tolerance, but not locomotor sensitization during free-choice ethanol access" as well as providing comparison to studies on other drugs of abuse. METHODS: The findings of the target article are evaluated and compared to the larger literature of intake escalation and vulnerability to addiction observed with other drugs of abuse. RESULTS: In their study, Matson and colleagues report that mice derived by crossing different lines selectively bred for high alcohol intake exhibit initial alcohol intakes associated with motor impairment followed by marked escalation of consumption and tolerance to the effects of alcohol on motor coordination. In contrast, no evidence of pharmacokinetic tolerance or sensitization of alcohol-induced locomotion was observed. These results demonstrate that the cHAP mice constitute an appropriate model for the study of excessive drinking, which is produced by escalated alcohol intake and functional changes, leading to excessive intoxication. CONCLUSIONS: Future work should assess adaptations in motivational processes and subjective effects of alcohol as well as the potential genetic and epigenetic bases of escalated alcohol intake.

Alcohol exposure inhibits adult neural stem cell proliferation.

Alcohol exposure can reduce adult proliferation and/or neurogenesis, but its impact on the ultimate neurogenic precursors, neural stem cells (NSCs), has been poorly addressed. Accordingly, the impact of voluntary consumption of alcohol on NSCs in the subventricular zone (SVZ) of the lateral ventricle was examined in this study. The NSC population in adult male C57BL/6J mice was measured after voluntary alcohol exposure in a two-bottle choice task using the neurosphere assay, while the number of NSCs that had proliferated 2 weeks prior to tissue collection was indexed using bromodeoxyuridine (BrdU) retention. There was a significant decrease in the number of BrdU-retaining cells in alcohol-consuming mice compared with controls, but no difference in the number of neurosphere-forming cells that could be derived from the SVZ of alcohol-consuming mice compared with controls. Additionally, PCNA-labeled cells in the SVZ tended to be lower, but there was no difference in BrdU labeling in the dentate gyrus following alcohol exposure. To determine alcohol's direct impact on NSCs and their progeny, neurospheres derived from naïve mice were treated with alcohol in vitro. Neurosphere formation was reduced by 100 mM alcohol without reducing cell viability. These findings are the first to assess the impact of moderate voluntary alcohol consumption on selective measures of adult NSCs and indicate that such exposure alters NSC proliferation dynamics in vivo and alcohol has direct but dissociable effects on the expansion and viability on NSCs and their progeny in vitro.

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.

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.

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.