Here we demonstrate a new class of reagentless, single-step sensors for the detection of proteins and peptides that is the electrochemical analog of fluorescence polarization (fluorescence anisotropy), a versatile optical approach widely employed to this same end. Our electrochemical sensors consist of a redox-reporter-modified protein (the “receptor”) site-specifically anchored to an electrode via a short, flexible polypeptide linker. Interaction of the receptor with its binding partner alters the efficiency with which the reporter approaches the electrode surface, thus causing a change in redox current upon voltammetric interrogation. As our first proof-of-principle we employed the bacterial chemotaxis protein CheY as our receptor. Interaction with either of CheY’s two binding partners, the P2 domain of the chemotaxis kinase, CheA, or the 16-residue “target region” of the flagellar switch protein, FliM, leads to easily measurable changes in output current that trace Langmuir isotherms within error of those seen in solution. Phosphorylation of the electrode-bound CheY decreases its affinity for CheA-P2 and enhances its affinity for FliM in a manner likewise consistent with its behavior in solution. As expected given the proposed sensor signaling mechanism, the magnitude of the binding-induced signal change depends on the placement of the redox reporter on the receptor. Following these preliminary studies with CheY, we also developed and characterized additional sensors aimed at the detection of specific antibodies using the relevant protein antigens as the receptor. These exhibit excellent detection limits for their targets without the use of reagents or wash steps. This novel, protein-based electrochemical sensing architecture provides a new and potentially promising approach to sensors for the single-step measurement of specific proteins and peptides.

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.

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.

Recent years have seen increasing study of stimulus-responsive hydrogels constructed from aptamer-connected DNA building blocks. Presumably due to a lack of simple, quantitative tools with which to measure gel responsiveness, however, the literature describing these materials is largely qualitative. In response, we demonstrate here simple, time-resolved, multiscale methods for measuring the response kinetics of these materials. Specifically, by employing trace amounts of fluorophore-quencher labeled cross-linkers and the rheology of entrapped fluorescent particles, we simultaneously measure dissolution at molecular, hundred-nanometer, and hundred-micron length-scales. For our test-bed system, an adenine-responsive hydrogel, we find biphasic response kinetics dependent on both effector concentration and depth within the gel and a dissolution pattern uniform at scales longer than a few times the monomer–monomer distance. Likewise, we find that, in agreement with theoretical predictions, dissolution kinetics over the hundred nanometer length scale exhibit a power-law-like dependence on the fraction of disrupted cross-links before a distinct crossover from solid-like to liquid-like behavior.Keywords: aptamers; gels; macromolecular assemblies; rheology; smart materials; soft matter;

AbstractThe 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.

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.

The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell. This forces evolution to focus at least as much effort on achieving optimal sensitivity and specificity as on achieving low detection limits, leading to the “invention” of a number of mechanisms, such as allostery and cooperativity, by which the useful dynamic range of receptors can be tuned, extended, narrowed, or otherwise optimized by design, rather than by sample manipulation. As the use of biomolecular receptors in artificial technologies matures (i.e., moves away from multistep, laboratory-bound processes and toward, for example, systems supporting continuous in vivo measurement) and these technologies begin to mimic the reagentless single-step convenience of naturally occurring chemoperception systems, the ability to artificially design receptors of enhanced sensitivity and specificity will likely also grow in importance. Thus motivated, we have begun to explore the adaptation of nature’s solutions to these problems to the biomolecular receptors often employed in artificial biotechnologies. Using the population-shift mechanism, for example, we have generated nested sets of receptors and allosteric inhibitors that greatly expanded the normally limited (less than 100-fold) useful dynamic range of unmodified molecular and aptamer beacons, enabling the single-step (e.g., dilution-free) measurement of target concentrations across up to 6 orders of magnitude. Using this same approach to rationally introduce sequestration or cooperativity into these receptors, we have likewise narrowed their dynamic range to as little as 1.5-fold, vastly improving the sensitivity with which they respond to small changes in the concentration of their target ligands. Given the ease with which we have been able to introduce these mechanisms into a wide range of DNA-based receptors and the rapidity with which the field of biomolecular design is maturing, we are optimistic that the use of these and similar naturally occurring regulatory mechanisms will provide viable solutions to a range of increasingly important analytical problems.

The continuous, real-time monitoring of specific molecular targets in unprocessed clinical samples would enable many transformative medical applications. Electrochemical aptamer-based (E-AB) sensors appear to be a promising approach to this end because of their selectivity (performance in complex samples, such as serum) and reversible, single-step operation. E-AB sensors suffer, however, from often-severe baseline drift when challenged in undiluted whole blood. In response we report here a dual-reporter approach to performing E-AB baseline drift correction. The approach incorporates two redox reporters on the aptamer, one of which serves as the target-responsive sensor and the other, which reports at a distinct, nonoverlapping redox potential, serving as a drift-correcting reference. Taking the difference in their relative signals largely eliminates the drift observed for these sensors in flowing, undiluted whole blood, reducing drift of up to 50% to less than 2% over many hours of continuous operation under these challenging conditions.

Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless biosensor architectures employing biomolecules modified via the attachment of a redox-active “reporter.” Here we survey a large set of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter “sets” signaling at distinct, nonoverlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-stranded DNA “probe” that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an “E-DNA” sensor responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scanning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale–we wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have investigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox reporters.

Electrochemical DNA (E-DNA) sensors have emerged as a promising class of biosensors capable of detecting a wide range of molecular analytes (nucleic acids, proteins, small molecules, inorganic ions) without the need for exogenous reagents or wash steps. In these sensors, a binding-induced conformational change in an electrode-bound “probe” (a target-binding nucleic acid or nucleic-acid-peptide chimera) alters the location of an attached redox reporter, leading to a change in electron transfer that is typically monitored using square-wave voltammetry. Because signaling in this class of sensors relies on binding-induced changes in electron transfer rate, the signal gain of such sensors (change in signal upon the addition of saturating target) is dependent on the frequency of the square-wave potential pulse used to interrogate them, with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor. Here, we show that, because it alters the driving force of the redox reaction and thus electron transfer kinetics, signal gain in this class of sensors is also strongly dependent on the amplitude of the square-wave potential pulse. Specifically, we show here that the simultaneous optimization of square-wave frequency and amplitude produces large (often more than 2-fold) increases in the signal gain of a wide range of E-DNA-type sensors.

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

The high packing densities and fixed geometries with which biomolecules can be attached to macroscopic surfaces suggest that crowding effects may be particularly significant under these often densely packed conditions. Exploring this question experimentally, we report here the effects of crowding on the stability of a simple, surface-attached DNA stem-loop. We find that crowding by densely packed, folded biomolecules destabilizes our test-bed biomolecule by ∼2 kJ/mol relative to the dilute (noninteracting) regime, an effect that presumably occurs due to steric and electrostatic repulsion arising from compact neighbors. Crowding by a dense brush of unfolded biomolecules, in contrast, enhances its stability by ∼6 kJ/mol, presumably due to excluded volume and electrostatic effects that reduce the entropy of the unfolded state. Finally, crowding by like copies of the same biomolecule produces a significantly broader unfolding transition, likely because, under these circumstances, the stabilizing effects of crowding by unfolded molecules increase (and the destabilizing effects of neighboring folded molecules decrease) as more and more neighbors unfold. The crowding of surface-attached biomolecules may thus be a richer, more complex phenomenon than that seen in homogeneous solution.

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and “smart” materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using “Hill-type” cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder–based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.

We describe an electrochemical analog of fluorescence polarization that supports the quantitative measurement of a specific protein, the chemokine IP-10, directly in undiluted blood serum. The sensor is label-free, wash-free, and electronic, suggesting it could support point-of-care detection of diagnostic proteins in largely unprocessed clinical samples.

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here, we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 μg/mL crude nuclear extracts) in a convenient, low-reagent process requiring only 10 μL of sample. Our approach thus appears a promising means of monitoring transcription factor levels.

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged—but otherwise apparently inert—surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ∼130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.

The development of rapid, low-cost point-of-care approaches for the quantitative detection of antibodies would drastically impact global health by shortening the delay between sample collection and diagnosis and by improving the penetration of modern diagnostics into the developing world. Unfortunately, however, current methods for the quantitative detection of antibodies, including ELISAs, Western blots, and fluorescence polarization assays, are complex, multiple-step processes that rely on well-trained technicians working in well-equipped laboratories. In response, we describe here a versatile, DNA-based electrochemical “switch” for the rapid, single-step measurement of specific antibodies directly in undiluted whole blood at clinically relevant low-nanomolar concentrations.

The population-shift mechanism can be used for rational re-engineering of structure-switching biosensors to enable their allosteric inhibition and activation. As a proof-of-principle example of this, we have introduced distal allosteric sites into molecular beacons, which are optical sensors for the detection of specific nucleic acid sequences. The binding of inhibitors and activators to these sites enabled the rational modulation of the sensor’s target affinity—and thus its useful dynamic range—over 3 orders of magnitude. The convenience with which this was done suggests that the population-shift mechanism may prove to be a useful method by which allosteric regulation can be introduced into biosensors, “smart” biomaterials, and other artificial biotechnologies.

The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid “scaffold” modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.

Biomolecular recognition has long been an important theme in artificial sensing technologies. A current limitation of protein- and nucleic acid-based recognition, however, is that the useful dynamic range of single-site binding typically spans an 81-fold change in target concentration, an effect that limits the utility of biosensors in applications calling for either great sensitivity (a steeper relationship between target concentration and output signal) or the quantification of more wide-ranging concentrations. In response, we have adapted strategies employed by nature to modulate the input–output response of its biorecognition systems to rationally edit the useful dynamic range of an artificial biosensor. By engineering a structure-switching mechanism to tune the affinity of a receptor molecule, we first generated a set of receptor variants displaying similar specificities but different target affinities. Using combinations of these receptor variants (signaling and nonsignaling), we then rationally extended (to 900000-fold), narrowed (to 5-fold), and edited (three-state) the normally 81-fold dynamic range of a representative biosensor. We believe that these strategies may be widely applicable to technologies reliant on biorecognition.

The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here “transcription factor beacons,” binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-κB), and measure binding activity directly in crude nuclear extracts.

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ∼800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.

Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomolecules into a general platform for molecular sensing. Despite significant effort, however, so-called “biosensors” have almost entirely failed to achieve their potential as reagentless, real-time analytical devices; the only quantitative, reagentless biosensor to achieve commercial success so far is the home glucose monitor, employed by millions of diabetics. The fundamental stumbling block that has precluded more widespread success of biosensors is the failure of most biomolecules to produce an easily measured signal upon target binding. Antibodies, for example, do not change their shape or dynamics when they bind their recognition partners, nor do they emit light or electrons upon binding. It has thus proven difficult to transduce biomolecular binding events into a measurable output signal, particularly one that is not readily spoofed by the binding of any of the many potentially interfering species in typical biological samples. Analytical approaches based on biomolecular recognition are therefore mostly cumbersome, multistep processes relying on analyte separation and isolation (such as Western blots, ELISA, and other immunochemical methods); these techniques have proven enormously useful, but are limited almost exclusively to laboratory settings. In this Account, we describe how we have refined a potentially general solution to the problem of signal detection in biosensors, one that is based on the binding-induced “folding” of electrode-bound DNA probes. That is, we have developed a broad new class of biosensors that employ electrochemistry to monitor binding-induced changes in the rigidity of a redox-tagged probe DNA that has been site-specifically attached to an interrogating electrode. These folding-based sensors, which have been generalized to a wide range of specific protein, nucleic acid, and small-molecule targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concentrations), and reagentless. They are also greater than 99% reusable, are supported on micrometer-scale electrodes, and are readily fabricated into densely packed sensor arrays. Finally, and critically, their signaling is linked to a binding-specific change in the physics of the probe DNA, and not simply to adsorption of the target onto the sensor head. Accordingly, they are selective enough to be employed directly in blood, crude soil extracts, cell lysates, and other grossly contaminated clinical and environmental samples. Indeed, we have recently demonstrated the ability to quantitatively monitor a specific small molecule in real-time directly in microliters of flowing, unmodified blood serum. Because of their sensitivity, substantial background suppression, and operational convenience, these folding-based biosensors appear potentially well suited for electronic, on-chip applications in pathogen detection, proteomics, metabolomics, and drug discovery.

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to “recycle” target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 °C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 °C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.

Electrode-bound, redox-reporter-modified oligonucleotides play roles in the functioning of a number of electrochemical biosensors, and thus the question of electron transfer through or from such molecules has proven of significant interest. In response, we have experimentally characterized the rate with which electrons are transferred between a methylene blue moiety on the distal end of a short, single-stranded polythymine DNA to a monolayer-coated gold electrode to which the other end of the DNA is site-specifically attached. We find that this rate scales with oligonucleotide length to the −1.16 ± 0.09 power. This weak, approximately inverse length dependence differs dramatically from the much stronger dependencies observed for the rates of end-to-end collisions in single-stranded DNA and through-oligonucleotide electron hopping. It instead coincides with the expected length dependence of a reaction-limited process in which the overall rate is proportional to the equilibrium probability that the end of the oligonucleotide chain approaches the surface. Studies of the ionic strength and viscosity dependencies of electron transfer further support this “chain-flexibility” mechanism, and studies of the electron transfer rate of methylene blue attached to the hexanethiol monolayer suggest that heterogeneous electron transfer through the monolayer is rate limiting. Thus, under the circumstances we have employed, the flexibility (i.e., the equilibrium statistical properties) of the oligonucleotide chain defines the rate with which an attached redox reporter transfers electrons to an underlying electrode, an observation that may be of utility in the design of new biosensor architectures.

In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a “sticky end.” When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry.

An “XOR” gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.

Water-soluble, cationic conjugated polymer binds single-stranded DNA with higher affinity than it binds double-stranded or otherwise “folded” DNA. This stronger binding results from the greater hydrophobicity of single-stranded DNA. Upon reducing the strength of the hydrophobic interactions, the electrostatic attraction becomes the important interaction that regulates the binding between the water-soluble conjugated polymer and DNA. The different affinities between the cationic conjugated polymer and various forms of DNA (molecular beacons and its open state; single-stranded DNA and double-stranded DNA and single-stranded DNA and complex DNA folds) can be used to design a variety of biosensors.

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.

We report the preparation of 20 and 65 nm radii glass nanopores whose surface is modified with DNA aptamers controlling the molecular transport through the nanopores in response to small molecule binding.

Electrochemical sensors employing redox-tagged, electrode-bound oligonucleotides have emerged as a promising new platform for the reagentless detection of molecular analytes. Signal generation in these sensors is linked to specific, binding-induced changes in the efficiency with which an attached redox tag approaches and exchanges electrons with the interrogating electrode. We present here a straightforward means of optimizing the signal gain of these sensors that exploits this mechanism. Specifically, using square-wave voltammetry, which is exquisitely sensitive to electrode reaction rates, we can tune the frequency of the voltammetric measurements to preferentially enhance the signal associated with either the unbound or target-bound conformations of the probe. This allows us to control not only the magnitude of the signal gain associated with target binding but also the sign of the signal change, generating “signal-on” or “signal-off” sensors. This optimization parameter appears to be quite general: we show here that tuning the square-wave frequency can significantly enhance the gain of the sensors directed against specific oligonucleotide sequences, small molecules, proteins, and protein−small molecule interactions.

Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2′-hydroxyl groups outside of the aptamer’s antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor.

Electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform. Combining the generality and specificity of aptamer–ligand interactions with the selectivity and convenience of electrochemical readouts, this approach affords the detection of a wide variety of targets directly in complex, contaminant-ridden samples, such as whole blood, foodstuffs and crude soil extracts, without the need for exogenous reagents or washing steps. Signaling in this class of sensors is predicated on target-induced changes in the conformation of an electrode-bound probe aptamer that, in turn, changes the efficiency with which a covalently attached redox tag exchanges electrons with the interrogating electrode. Aptamer selection strategies, however, typically do not select for the conformation-switching architectures, and as such several approaches have been reported to date by which aptamers can be re-engineered such that they undergo the binding-induced switching required to support efficient E-AB signaling. Here, we systematically compare the merits of these re-engineering approaches using representative aptamers specific to the small molecule adenosine triphosphate and the protein human immunoglobulin E. We find that, while many aptamer architectures support E-AB signaling, the observed signal gain (relative change in signal upon target binding) varies by more than two orders of magnitude across the various constructs we have investigated (e.g., ranging from −10% to 200% for our ATP sensors). Optimization of the switching architecture is thus an important element in achieving maximum E-AB signal gain and we find that this optimal geometry is specific to the aptamer sequence upon which the sensor is built.

We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly “universal” biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise “folded” DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient—picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.

We herein demonstrate a sandwich assay based on single aptamer sequences is suitable for the direct detection of small molecule targets in blood serum and other complex matrices. By splitting an aptamer into two pieces, we convert a single affinity reagent into a two-component system in which the presence of the target drives formation of a complex comprised of the target and the two halves of the aptamer. To demonstrate the utility of this approach we have used single anticocaine and anti-ATP aptamers to fabricate electrochemical sensors directed against the representative small molecules coaine and ATP. Both targets are detected at low micromolar concentrations, in seconds, and in a convenient, general, readily reusable, electrochemical format. Moreover, both sensors are selective enough to deploy directly in blood, crude cellular lysates and other complex sample matrices.

Here we have demonstrated a general, sensitive, and selective approach for the detection of macromolecules that bind to specific small molecule recognition elements. Our electrochemical approach utilizes a redox-tagged DNA signaling scaffold that is conjugated to a small molecule recognition element and is covalently attached to an interrogating electrode. The binding of a protein to the small molecule recognition element alters the dynamics of the scaffold, increasing or decreasing the efficiency with which the redox tag collides with the electrode and thus altering the observed faradaic current. We optimized the scaffold using a biotin recognition element and streptavidin as a target to determine the variables that define sensor performance before then applying the approach to detection of anti-digoxigenin antibodies using the steroid as the recognition element. We generated streptavidin sensors exhibiting both signal-on (target binding increases the faradaic current) and signal-off behavior, of which only the signal-off approach was generalizable to the detection of antibodies. Sensors for both targets are sensitive (detection limits in the low nanomolar range), rapid (minutes), reusable, and selective enough to function directly in complex matrices including blood serum, soil, and foodstuffs.

Here we report a versatile method by which the interaction between a protein and a small molecule, and the disruption of that interaction by competition with other small molecules, can be monitored electrochemically directly in complex sample matrices.

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.

Alkane thiol self-assembled monolayers (SAMs) have seen widespread utility in the fabrication of electrochemical biosensors. Their utility, however, reflects a potentially significant compromise. While shorter SAMs support efficient electron transfer, they pack poorly and are thus relatively unstable. Longer SAMs are more stable but suffer from less efficient electron transfer, thus degrading sensor performance. Here we use the electrochemical DNA (E-DNA) sensor platform to compare the signaling and stability of biosensors fabricated using a short, six-carbon monothiol with those employing either of two commercially available trihexylthiol anchors (a flexible Letsinger type and a rigid adamantane type). We find that all three anchors support efficient electron transfer and E-DNA signaling, with the gain, specificity, and selectivity of all three being effectively indistinguishable. The stabilities of the three anchors, however, vary significantly. Sensors anchored with the flexible trithiol exhibit enhanced stability, retaining 75% of their original signal and maintaining excellent signaling properties after 50 days storage in buffer. Likewise these sensors exhibit excellent temperature stability and robustness to electrochemical interrogation. The stability of sensors fabricated using the rigid trithiol anchor, by comparison, are similar to those of the monothiol, with both exhibiting significant (>60%) loss of signal upon wet storage or thermocycling. Employing a flexible trithiol anchor in the fabrication of SAM-based electrochemical biosensors may provide a means of improving sensor robustness without sacrificing electron transfer efficiency or otherwise impeding sensor performance.

Binding-induced biomolecular switches are used throughout nature and, increasingly, throughout biotechnology for the detection of chemical moieties and the subsequent transduction of this detection into useful outputs. Here we show that the thermodynamics of these switches are quantitatively described by a simple 3-state population-shift model, in which the equilibrium between a nonbinding, nonsignaling state and the binding-competent, signaling state is shifted toward the latter upon target binding. Because of this, their performance is determined by the tradeoff inherent to their switching thermodynamics; while a switching equilibrium constant favoring the nonbinding, nonsignaling, conformation ensures a larger signal change (more molecules are poised to respond), it also reduces affinity (binding must overcome a more unfavorable conformational free energy). We then derive and employ the relationship between switching thermodynamics and switch signaling to rationally tune the dynamic range and detection limit of a representative structure-switching biosensor, a molecular beacon, over 4 orders of magnitude. These findings demonstrate that the performance of biomolecular switches can be rationally tuned via mutations that alter their switching thermodynamics and suggest a mechanism by which the performance of naturally occurring switches may have evolved.

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.

We review the development of reagentless, electrochemical sensors for the sequence-specific detection of nucleic acids that are based on the target-induced folding or unfolding of electrode-bound oligonucleotides. These devices, which are sometimes termed E-DNA sensors, are comprised of an oligonucleotide probe modified on one terminus with a redox reporter and attached to an electrode at the other. Hybridization of this probe DNA to a target oligonucleotide influences the rate at which the redox reporter collides with the electrode, leading to a detectable change in redox current. Because all sensing elements of this method are strongly linked to the interrogating electrode, E-DNA sensors are label-free, operationally convenient and readily reusable. As E-DNA signaling is predicated on a binding-specific change in the dynamics of the probe DNA (rather than simply monitoring the adsorption of a target to the sensor surface) and because electroactive contaminants (interferents) are relatively rare, this class of sensors is notably resistant to false positives arising from the non-specific adsorption of interferents, and performs well even when challenged directly with blood serum, soil and other complex sample matrices. We review the history of and recent advances in this promising DNA and RNA detection approach.

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.

We have developed a new biosensor architecture, which is comprised of a polypeptide–peptide nucleic acid tri-block co-polymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.

We report an electrochemical method for the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium. Using an asymmetric PCR and the electrochemical E-DNA detection scheme, single-stranded amplicons were produced from as few as 90 gene copies and, without subsequent purification, rapidly identified. The detection is specific; the sensor does not respond when challenged with control oligonucleotides based on the gyrB genes of either Escherichia coli or various Shigella species. In contrast to existing sequence-specific optical- and capillary electrophoresis-based detection methods, the E-DNA sensor is fully electronic and requires neither cumbersome, expensive optics nor high voltage power supplies. Given these advantages, E-DNA sensors appear well suited for implementation in portable PCR microdevices directed at, for example, the rapid detection of pathogens.

Hybridization-induced conformational changes have been successfully used in biosensors for the transduction of DNA-binding events into readily observable optical or electronic signals. Similar signal transduction has not, however, proven of equal utility in proteinbased biosensors. The discrepancy arises because, unlike ssDNA, most proteins do not undergo significant conformational changes upon ligand binding. Here, we describe a solution to this problem. We show that an arbitrarily selected, normally well folded protein can be rationally engineered such that it undergoes ligand-induced folding. The engineered protein responds rapidly (milliseconds) and selectively to its target, and it couples recognition with the largest possible conformational change: folding. These traits suggest that ligand-induced folding could serve as an ideal signal-transduction mechanism. Consistent with this claim, we demonstrate a label-free optical biosensor based on the effect that is sufficiently selective to detect its target even in complex, contaminant-ridden samples such as blood serum.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R G) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R G of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r 2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

We report a strategy for the reagentless transduction of DNA hybridization into a readily detectable electrochemical signal by means of a conformational change analogous to the optical molecular beacon approach. The strategy involves an electroactive, ferrocene-tagged DNA stem–loop structure that self-assembles onto a gold electrode by means of facile gold-thiol chemistry. Hybridization induces a large conformational change in this surface-confined DNA structure, which in turn significantly alters the electron-transfer tunneling distance between the electrode and the redoxable label. The resulting change in electron transfer efficiency is readily measured by cyclic voltammetry at target DNA concentrations as low as 10 pM. In contrast to existing optical approaches, an electrochemical DNA (E-DNA) sensor built on this strategy can detect femtomoles of target DNA without employing cumbersome and expensive optics, light sources, or photodetectors. In contrast to previously reported electrochemical approaches, the E-DNA sensor achieves this impressive sensitivity without the use of exogenous reagents and without sacrificing selectivity or reusability. The E-DNA sensor thus offers the promise of convenient, reusable detection of picomolar DNA.

NMR experiments with partially aligned protein molecules in strongly denaturing conditions suggest that the unfolded state is less chaotic than is widely believed. Hence protein folding is probably less paradoxical than Levinthal originally thought.

Theory suggests that the otherwise rapid folding of simple heteropolymer models becomes “glassy”—dominated by multiple kinetically trapped misfolded states—at low temperatures or when the overall bias toward the native state is reduced relative to the depth of local minima. Experimental observations of nonsingle-exponential protein-folding kinetics have been taken as evidence that the protein-folding free energy landscape is similarly rough. No equivalent analysis, however, has been reported for a simple single-domain protein lacking prolines, disulfide bonds, prosthetic groups, or other gross structural features that might complicate folding. In an effort to characterize the glassiness of a folding free energy landscape in the absence of these potentially complicating factors, we have monitored the folding of a kinetically simple protein, peptostreptococcal protein L (protein L). We observe no statistically significant deviation from homogeneous single-exponential relaxation kinetics across temperatures ranging from near the protein's melting temperature to as low as −15°C. On the basis of these observations, we estimate that, if there is a glass transition in the folding of protein L, it occurs at least 45°C and possibly more than 145°C below the freezing point of water. Apparently the folding free energy landscape of protein L is extremely smooth, which may be indicative of a rate-limiting step in folding that is, effectively, a nonglassy process.

•We review current methods for electrochemical real-time nucleic acid amplification.•Electrochemical detection rivals optical methods due to low cost and complexity.•Initial methods were solid phase and suffered from low amplification efficiency.•New solution phase methods achieve clinically relevant sensitivity and specificity.•Solution phase methods can pave the road towards use in point-of-care applications.Real-time nucleic acid amplification, whereby the amplification rate is used to quantify the initial copy number of target DNA or RNA, has proven highly effective for monitoring pathogen loads. Unfortunately, however, current optical methods are limited to centralized laboratories due to complexity, bulk and cost. In response, recent efforts aim to develop lower-cost, electrochemical real-time amplification platforms for point-of-care applications, with researchers already having developed platforms that not only perform in situ and concurrent electrochemical detection during amplification, but also deliver sensitivity and specificity potentially rivaling bench-top optical systems. This report chronicles the evolution of the different strategies, describes the current state of the art, and identifies challenges of bringing the power of real-time detection to the point-of-care.

Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of −3.49 ± 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length—an observation that sheds new light on the origins of the “internal friction” observed in the dynamics of many polymer systems.

The results of more than a dozen single-molecule Förster resonance energy transfer (smFRET) experiments suggest that chemically unfolded polypeptides invariably collapse from an expanded random coil to more compact dimensions as the denaturant concentration is reduced. In sharp contrast, small-angle X-ray scattering (SAXS) studies suggest that, at least for single-domain proteins at non-zero denaturant concentrations, such compaction may be rare. Here, we explore this discrepancy by studying protein L, a protein previously studied by SAXS (at 5 °C), which suggested fixed unfolded-state dimensions from 1.4 to 5 M guanidine hydrochloride (GuHCl), and by smFRET (at 25 °C), which suggested that, in contrast, the chain contracts by 15–30% over this same denaturant range. Repeating the earlier SAXS study under the same conditions employed in the smFRET studies, we observe little, if any, evidence that the unfolded state of protein L contracts as the concentration of GuHCl is reduced. For example, scattering profiles (and thus the shape and dimensions) collected within ∼ 4 ms after dilution to as low as 0.67 M GuHCl are effectively indistinguishable from those observed at equilibrium at higher denaturant. Our results thus argue that the disagreement between SAXS and smFRET is statistically significant and that the experimental evidence in favor of obligate polypeptide collapse at low denaturant cannot be considered conclusive yet.Download high-res image (72KB)Download full-size imageHighlights► smFRET results suggest that the unfolded state is collapsed at low denaturant. ► SAXS suggests that the low- and high-denaturant unfolded states are indistinguishable. ► Poor overlap between the data sets has historically obscured the significance of this discrepancy. ► Parallel studies on one protein under one set of conditions confirm the discrepancy.

Intramolecular collision dynamics play an essential role in biomolecular folding and function and, increasingly, in the performance of biomimetic technologies. To date, however, the quantitative studies of dynamics of single-stranded nucleic acids have been limited. Thus motivated, here we investigate the sequence composition, chain-length, viscosity, and temperature dependencies of the end-to-end collision dynamics of single-stranded DNAs. We find that both the absolute collision rate and the temperature dependencies of these dynamics are base-composition dependent, suggesting that base stacking interactions are a significant contributor. For example, whereas the end-to-end collision dynamics of poly-thymine exhibit simple, linear Arrhenius behavior, the behavior of longer poly-adenine constructs is more complicated. Specifically, 20- and 25-adenine constructs exhibit biphasic temperature dependencies, with their temperature dependences becoming effectively indistinguishable from that of poly-thymine above 335 K for 20-adenines and 328 K for 25-adenines. The differing Arrhenius behaviors of poly-thymine and poly-adenine and the chain-length dependence of the temperature at which poly-adenine crosses over to behave like poly-thymine can be explained by a barrier friction mechanism in which, at low temperatures, the energy barrier for the local rearrangement of poly-adenine becomes the dominant contributor to its end-to-end collision dynamics.

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.

Proteins unfolded by high concentrations of chemical denaturants adopt expanded, largely structure-free ensembles of conformations that are well approximated as random coils. In contrast, globular proteins unfolded under less denaturing conditions (via mutations, or transiently unfolded after a rapid jump to native conditions) and molten globules (arising due to mutations or cosolvents) are often compact. Here we explore the origins of this compaction using a truncated equilibrium-unfolded variant of the 57-residue FynSH3 domain. As monitored by far-UV circular dichroism, NMR spectroscopy, and hydrogen-exchange kinetics, CΔ4 (a 4-residue carboxy-terminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, CΔ4 is quite compact under these conditions, with a hydrodynamic radius only slightly larger than that of the native protein. In order to understand the origins of this molten-globule-like compaction, we have characterized a random sequence polypeptide of identical amino acid composition to CΔ4. Notably, we find that the hydrodynamic radius of this random sequence polypeptide also approaches that of the native protein. Thus, while native-like interactions may contribute to the formation of compact “unfolded” states, it appears that non-sequence-specific monomer–monomer interactions can also account for the dramatic compaction observed for molten globules and the “physiological” unfolded state.

Here we report a versatile method by which the interaction between a protein and a small molecule, and the disruption of that interaction by competition with other small molecules, can be monitored electrochemically directly in complex sample matrices.

We report the preparation of 20 and 65 nm radii glass nanopores whose surface is modified with DNA aptamers controlling the molecular transport through the nanopores in response to small molecule binding.

We have developed a new biosensor architecture, which is comprised of a polypeptide–peptide nucleic acid tri-block co-polymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.