Solution-based biophysical characterization of conformation change in structure-switching aptamers.

Structure-switching aptamers have become ubiquitous in several applications, notably in analytical devices such as biosensors, due to their ease of supporting strong signaling. Aside from their ability to bind specifically with their respective target, this class of aptamers also undergoes a conformational rearrangement upon target recognition. While several well-studied and early-developed aptamers (e.g., cocaine, ATP, and thrombin) have been found to have this structure-switching property, the vast majority do not. As a result, it is common to try to engineer aptamers into switches. This proves challenging in part because of the difficulty in obtaining structural and functional information about aptamers. In response, we review various readily available biophysical characterization tools that are capable of assessing structure switching of aptamers. In doing so, we delve into the fundamentals of these different techniques and detail how they have been utilized in characterizing structure-switching aptamers. While each of these biophysical techniques alone has utility, their real power to demonstrate the occurrence of structural change with ligand binding is when multiple techniques are used. We hope that through a deeper understanding of these techniques, researchers will be better able to acquire biophysical information about their aptamer-ligand systems and accelerate the translation of aptamers into biosensors.

Truncations and in silico docking to enhance the analytical response of aptamer-based biosensors.

Aptamers are short oligonucleotides capable of binding specifically to various targets (i.e., small molecules, proteins, and whole cells) which have been introduced in biosensors such as in the electrochemical aptamer-based (E-AB) sensing platform. E-AB sensors are comprised of a redox-reporter-modified aptamer attached to an electrode that undergoes, upon target addition, a binding-induced change in electron transfer rates. To date, E-AB sensors have faced a limitation in the translatability of aptamers into the sensing platform presumably because sequences obtained from Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are typically long (>80 nucleotides) and that obtaining structural information remains time and resource consuming. In response, we explore the utility of aptamer base truncations and in silico docking to improve their translatability into E-AB sensors. Here, we first apply this to the glucose aptamer, which we characterize in solution using NMR methods to guide design and translate truncated variants in E-AB biosensors. We further investigated the applicability of the truncation and computational approaches to four other aptamer systems (vancomycin, cocaine, methotrexate and theophylline) from which we derived functional E-AB sensors. We foresee that our strategy will increase the success rate of translating aptamers into sensing platforms to afford low-cost measurements of molecules directly in undiluted complex matrices.

Whole blood multiplex measurements using electrochemical aptamer-based biosensors.

Simultaneous measurements of various molecules ("multiplex") using electrochemical biosensors typically require multiple electrode implementations, which for neonates, hemophiliacs, etc. is problematic. Here, we introduce the oxazine ATTO 700 into electrochemical aptamer-based biosensors to achieve "true" multiplex, continuous and real-time measurements of two different molecules in undiluted whole blood using a single electrode.

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.

Electrochemical Aptamer-Based Biosensors for Measurements in Undiluted Human Saliva.

Although blood remains a gold standard diagnostic fluid for most health exams, it involves an unpleasant and relatively invasive sampling procedure (finger pricking or venous draw). Saliva contains many relevant and useful biomarkers for diagnostic purposes, and its collection, in contrast, is noninvasive and can be obtained with minimal effort. Current saliva analyses are, however, achieved using chromatography or lateral flow assays, which, despite their high accuracy and sensitivity, can demand expensive laboratory-based instruments operated by trained personnel or offer only semiquantitative results. In response, we investigated electrochemical aptamer-based (E-AB) biosensors, a reagentless sensing platform, to allow for continuous and real-time measurements directly in undiluted, unstimulated human whole saliva. As a proof-of-concept study, we developed E-AB biosensors capable of detecting low-molecular-weight analytes (glucose and adenosine monophosphate (AMP)). To our knowledge, we report the first E-AB sensor for glucose, an approach that is inherently independent of its chemical reactivity in contrast to home glucometers. For these three sensors, we evaluated their figures of merits, stability, and reusability over short- and long-term exposure directly in saliva. In doing so, we found that E-AB sensors allow rapid and convenient molecular measurements in whole saliva with unprecedented sensitivities in the pico- to nanomolar regime and could be regenerated and reused up to 7 days when washed and stored in phosphate-buffered saline at room temperature. We envision that salivary molecular measurements using E-AB sensors are a promising alternative to invasive techniques and can be used for improved point-of-care clinical diagnosis and at-home measurements.

DNA Chimeras as Electrochemical Biosensors for Host-Guest Measurements in Blood.

Few sensing platforms have become ubiquitous to enable rapid and convenient measurements at the point-of-care. Those, however, are "one-off" technologies, meaning that they can only detect a single target and are hardly adaptable. In response, we plan to develop a sensing platform that can be extended to detect other classes of molecules and that affords rapid, convenient, continuous measurements directly in undiluted complex matrices. For this, we decided to rely on a host molecule that presents reversible interactions toward specific guest molecules to develop a new class of sensors that we coined "Electrochemical DNA-host chimeras". As a proof-of-concept for our sensor, we decided to use cyclobis(paraquat-p-phenylene) ("blue box") that we attached on an electrode-bound DNA to allow measurements of electron-rich guests such as dopamine and aspirin. Doing so allows to promote host-guest complex that could be quantified using blue box's electrochemistry. Because of this unique sensor architecture, we achieve, to our knowledge, the first reagentless, continuous and rapid (<5 min) host-guest measurements in undiluted whole blood. We envision that given the library of electroactive host molecules that this will allow the development of a sensing platform for measurements of several classes of molecules in complex matrices at the point-of-care.

Pulcherriminic acid modulates iron availability and protects against oxidative stress during microbial interactions.

Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.

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.

Redox Reporter - Ligand Competition to Support Signaling in the Cocaine-Binding Electrochemical Aptamer-Based Biosensor.

Electrochemical aptamer-based (E-AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode-bound, redox-reporter-modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding-induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance-dependent change in electron transfer rates in E-AB sensors. In response, we investigate here the signal transduction of the well-studied cocaine-binding aptamer with different analytical methods and found that this sensor relies on a redox-reporter - ligand competition mechanism rather than a ligand-induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine-binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine-binding aptamer, given the prevalence of methylene blue in E-AB sensors, a similar competition-based may occur in other systems.

Finding the Lost Dissociation Constant of Electrochemical Aptamer-Based Biosensors.

Electrochemical aptamer-based (E-AB) biosensors afford real-time measurements of the concentrations of molecules directly in complex matrices and in the body, offering alternative strategies to develop innovative personalized medicine tools. While different electroanalytical techniques have been used to interrogate E-AB sensors (i.e., cyclic voltammetry, electrochemical impedance spectroscopy, and chronoamperometry) to resolve the change in electron transfer of the aptamer's covalently attached redox reporter, square-wave voltammetry remains a widely used technique due to its ability to maximize the redox reporter's faradic contribution to the measured current. Several E-AB sensors interrogated with this technique, however, show lower aptamer affinity (i.e., µM-mM) even in the face of employing aptamers that have high affinities (i.e., nM-µM) when characterized using solution techniques such as isothermal titration calorimetry (ITC) or fluorescence spectroscopy. Given past reports showing that E-AB sensor's response is dependent on square-wave interrogation parameters (i.e., frequency and amplitude), we hypothesized that the difference in dissociation constants measured with solution techniques stemmed from the electrochemical interrogation technique itself. In response, we decided to compare six dissociation constants of aptamers when characterized in solution with ITC and when interrogated on electrodes with electrochemical impedance spectroscopy, a technique able to, in contrast to square-wave voltammetry, deconvolute and quantify E-AB sensors' contributions to the measured current. In doing so, we found that we were able to measure dissociation constants that were either separated by 2-3-fold or within experimental errors. These results are in contrast with square-wave voltammetry-measured dissociation constants that are at the most separated by 2-3 orders of magnitude from ones measured by ITC. We thus envision that the versatility and time scales covered by electrochemical impedance spectroscopy offer the highest sensitivity to measure target binding in electrochemical biosensors relying on changes in electron-transfer rates.

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.

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

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

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

The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clinical practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary molecules in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, we describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a critical part of the standard of care, and its adaptation into an electrochemical aptamer-based (E-AB) sensor. Using this sensor, we then demonstrate: (i) rapid (seconds) and convenient (single-step and calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (ii) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (iii) high-precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience) but also actively control plasma drug levels provides an unprecedented route toward improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacological interventions.

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.

Surface Attachment Enhances the Thermodynamic Stability of Protein†L.

Despite the importance of protein-surface interactions in both biology and biotechnology, our understanding of their origins is limited due to a paucity of experimental studies of the thermodynamics behind such interactions. In response, we have characterized the extent to which interaction with a chemically well-defined macroscopic surface alters the stability of protein L. To do so, we site-specifically attached a redox-reporter-modified protein variant to a hydroxy-terminated monolayer on a gold surface and then used electrochemistry to monitor its guanidine denaturation and determine its folding free energy. Comparison with the free energy seen in solution indicates that interaction with this surface stabilizes the protein by 6 kJ mol-1 , a value that is in good agreement with theoretical estimates of the entropic consequences of surface-induced excluded volume effects, thus suggesting that chemically specific interactions with this surface (e.g., electrostatic interactions) are limited in magnitude.

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.

Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors.

Electrochemical, aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small molecules directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously we have used voltammetry (cyclic, alternating current, and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, we demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concentration is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the number of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resolution of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach we demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resolution and unprecedented, few-percent precision in the determination of its pharmacokinetic phases.

Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood.

The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clinically relevant (±20% for this application) accuracy, for example, commercial continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, we demonstrate a "dual-frequency" approach for achieving the calibration-free operation of electrochemical biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, we use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochemical aptamer-based (E-AB) biosensors as our test bed, we demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor.

Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry.

The efficiency with which square-wave voltammetry differentiates faradic and charging currents makes it a particularly sensitive electroanalytical approach, as evidenced by its ability to measure nanomolar or even picomolar concentrations of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extraction of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, we demonstrate here a numerical approach by which square-wave data can be used to determine electron transfer rates. Specifically, we have developed a numerical approach in which we model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate constant and the electron transfer coefficient. As validation of the approach, we have used it to determine electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods.