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.

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.

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.

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.

Rational design to control the trade-off between receptor affinity and cooperativity.

Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an "intrinsic disorder" mechanism to design two cooperative, three-binding-site receptors starting from a single-site-and thus noncooperative-doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors' second binding event, which serves as an allosteric "gatekeeper" defining the extent to which the system is weighted toward higher cooperativity or higher affinity.

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein.

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

Protein-Protein Communication Mediated by an Antibody-Responsive DNA Nanodevice.

We report here the rational design and optimization of an antibody-responsive, DNA-based device that enables communication between pairs of otherwise non-interacting proteins. The device is designed to recognize and bind a specific antibody and, in response, undergo a conformational change that leads to the release of a DNA strand, termed the "translator," that regulates the activity of a downstream target protein. As proof of principle, we demonstrate antibody-induced control of the proteins thrombin and Taq DNA polymerase. The resulting strategy is versatile and, in principle, can be easily adapted to control protein-protein communication in artificial regulatory networks.

Catalytic Interruption Mitigates Edge Effects in the Characterization of Heterogeneous, Insulating Nanoparticles.

Blocking electrochemistry, a subfield of nanochemistry, enables nondestructive, in situ measurement of the concentration, size, and size heterogeneity of highly dilute, nanometer-scale materials. This approach, in which the adsorptive impact of individual particles on a microelectrode prevents charge exchange with a freely diffusing electroactive redox mediator, has expanded the scope of electrochemistry to the study of redox-inert materials. A limitation, however, remains: inhomogeneous current fluxes associated with enhanced mass transfer occurring at the edges of planar microelectrodes confound the relationship between the size of the impacting particle and the signal it generates. These "edge effects" lead to the overestimation of size heterogeneity and, thus, poor sample characterization. In response, we demonstrate here the ability of catalytic current amplification (EC') to reduce this problem, an effect we term "electrocatalytic interruption". Specifically, we show that the increase in mass transport produced by a coupled chemical reaction significantly mitigates edge effects, returning estimated particle size distributions much closer to those observed using ex situ electron microscopy. In parallel, electrocatalytic interruption enhances the signal observed from individual particles, enabling the detection of particles significantly smaller than is possible via conventional blocking electrochemistry. Finite element simulations indicate that the rapid chemical kinetics created by this approach contributes to the amplification of the electronic signal to restore analytical precision and reliably detect and characterize the heterogeneity of nanoscale electro-inactive materials.

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.

Nanometer-Scale Force Profiles of Short Single- and Double-Stranded DNA Molecules on a Gold Surface Measured Using a Surface Forces Apparatus.

Using a surface forces apparatus (SFA), we have studied the nanomechanical behavior of short single-stranded and partially and fully double-stranded DNA molecules attached via one end to a self-assembled monolayer on a gold surface. Our results confirm the previously proposed "mushroom-like" polymer structure for surface-attached, single-stranded DNA at low packing density and a "brush-like" structure for the same construct at higher density. At low density we observe a transition to "rigid rod" behavior upon addition of DNA complementary to the surface-attached single strand as the fraction of molecules that are double-stranded increases, with a concomitant increase in the SFA-observed thickness of the monolayer and the characteristic length of the observed repulsive forces. At higher densities, in contrast, this transition is effectively eliminated, presumably because the single-stranded state is already extended in its "brush" state. Taken together, these studies offer insights into the structure and physics of surface-attached short DNAs, providing new guidance for the rational design of DNA-modified functional surfaces.

Quantitative measurements of protein-surface interaction thermodynamics.

Whereas proteins generally remain stable upon interaction with biological surfaces, they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date, however, only a small number of such artificial surfaces have been reported, and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated, we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chemically well-defined, macroscopic surfaces. Comparison with the folding free energies seen in bulk solution then provides a quantitative measure of the extent to which surface interactions alter protein stability. As proof-of-principle, we have characterized the FynSH3 domain site-specifically attached to a hydroxyl-coated surface. Upon guanidinium chloride denaturation, the protein unfolds in a reversible, two-state manner with a free energy within 2 kJ/mol of the value seen in bulk solution. Assuming that excluded volume effects stabilize surface-attached proteins, this observation suggests there are countervening destabilizing interactions with the surface that, under these conditions, are similar in magnitude. Our technique constitutes an unprecedented experimental tool with which to answer long-standing questions regarding the molecular-scale origins of protein-surface interactions and to facilitate rational optimization of surface biocompatibility.

Attachment of Proteins to a Hydroxyl-Terminated Surface Eliminates the Stabilizing Effects of Polyols.

The physics of proteins interacting with surfaces can differ significantly from those seen when the same proteins are free in bulk solution. As an example, we describe here the extent to which site-specific attachment to a chemically well-defined macroscopic surface alters the ability of several stabilizing and destabilizing cosolutes to modulate protein folding thermodynamics. We determined this via guanidinium denaturations performed in the presence of varying concentrations of cosolutes when proteins were either site-specifically attached to self-assembled monolayers on gold or free in bulk solution. Doing this we found that the extent to which guanidinium (a destabilizing Hofmeister cation), sulfate (a stabilizing Hofmeister anion), and urea (a neutral denaturant) alter the folding free energy remains indistinguishable whether proteins are surface-attached or free in bulk solution. In sharp contrast, however, neutral osmolytes sucrose and glycerol, which significantly stabilize proteins in bulk solution, do not measurably affect their stability when they are attached to a hydroxyl-terminated surface. In contrast, we recovered bulk solution-like stabilization when the attachment surface was instead carboxyl-terminated. It thus appears that chemistry-specific surface interactions can dramatically alter the way in which biomolecules interact with other components of the system.

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.

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.

An Electrochemical Biosensor Architecture Based on Protein Folding Supports Direct Real-Time Measurements in Whole Blood.

The ability to monitor drug and biomarker concentrations in the body with high frequency and in real time would revolutionize our understanding of biology and our capacity to personalize medicine. The few in vivo molecular sensors that currently exist, however, all rely on the specific chemical or enzymatic reactivity of their targets and thus are not generalizable. In response, we demonstrate here an electrochemical sensing architecture based on binding-induced protein folding that is 1) independent of the reactivity of its targets, 2) reagentless, real-time, and with a resolution of seconds, and 3) selective enough to deploy in undiluted bodily fluids. As a proof of principle, we use the SH3 domain from human Fyn kinase to build a sensor that discriminates between the protein's peptide targets and responds rapidly and quantitatively even when challenged in whole blood. The resulting sensor architecture could drastically expand the chemical space accessible to continuous, real-time biosensors.

High-Precision Electrochemical Measurements of the Guanine-, Mismatch-, and Length-Dependence of Electron Transfer from Electrode-Bound DNA Are Consistent with a Contact-Mediated Mechanism.

Despite 25 years' effort, serious questions remain regarding the mechanism(s) underlying electron transfer through (or from) electrode-bound double-stranded DNA. In part this is because a control experiment regarding the putatively critical role of guanine bases in the most widely proposed transport mechanism (hopping from guanine to guanine through the π-stack) appears to be lacking from the prior literature. In response, we have employed chronoamperometry, which allows for high-precision determination of electron transfer rates, to characterize transfer to a redox reporter appended onto electrode-bound DNA duplexes. Specifically, we have measured the effects of guanines and base mismatches on the electron transfer rate associated with such constructs. Upon doing so, we find that, counter to prior reports, the transfer rate is, to within relatively tight experimental confidence intervals, unaffected by either. Parallel studies of the dependence of the electron transfer rate on the length of the DNA suggest that transfer from this system obeys a "collision" mechanism in which the redox reporter physically contacts the electrode surface prior to the exchange of electrons.

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.