ABSTRACT
Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an "intrinsic disorder" mechanism to design two cooperative, three-binding-site receptors starting from a single-site-and thus noncooperative-doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors' second binding event, which serves as an allosteric "gatekeeper" defining the extent to which the system is weighted toward higher cooperativity or higher affinity.
Subject(s)
Receptors, Cell Surface/chemistry , Binding Sites , Doxorubicin/chemistry , Doxorubicin/metabolism , Kinetics , Ligands , Protein Binding , Receptors, Cell Surface/metabolismABSTRACT
Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24-48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction.
Subject(s)
Kinetics , Diffusion , Dimerization , Macromolecular Substances , Protein MultimerizationABSTRACT
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.
Subject(s)
Protein Folding , Proto-Oncogene Proteins c-fyn/chemistry , Thermodynamics , src Homology Domains , Electrochemical Techniques , Humans , Protein StabilityABSTRACT
The physics of proteins interacting with surfaces can differ significantly from those seen when the same proteins are free in bulk solution. As an example, we describe here the extent to which site-specific attachment to a chemically well-defined macroscopic surface alters the ability of several stabilizing and destabilizing cosolutes to modulate protein folding thermodynamics. We determined this via guanidinium denaturations performed in the presence of varying concentrations of cosolutes when proteins were either site-specifically attached to self-assembled monolayers on gold or free in bulk solution. Doing this we found that the extent to which guanidinium (a destabilizing Hofmeister cation), sulfate (a stabilizing Hofmeister anion), and urea (a neutral denaturant) alter the folding free energy remains indistinguishable whether proteins are surface-attached or free in bulk solution. In sharp contrast, however, neutral osmolytes sucrose and glycerol, which significantly stabilize proteins in bulk solution, do not measurably affect their stability when they are attached to a hydroxyl-terminated surface. In contrast, we recovered bulk solution-like stabilization when the attachment surface was instead carboxyl-terminated. It thus appears that chemistry-specific surface interactions can dramatically alter the way in which biomolecules interact with other components of the system.
Subject(s)
Hydroxyl Radical/chemistry , Polymers/chemistry , Proteins/chemistry , Guanidine/chemistry , Protein Folding , Sulfates/chemistry , Surface Properties , Thermodynamics , Urea/chemistryABSTRACT
Electrochemical sensors are major players in the race for improved molecular diagnostics due to their convenience, temporal resolution, manufacturing scalability, and their ability to support real-time measurements. This is evident in the ever-increasing number of health-related electrochemical sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data volumes, we describe here an open-source, easily customizable, multiplatform compatible program for the real-time control, processing, and visualization of electrochemical data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which we have called Software for the Analysis and Continuous Monitoring of Electrochemical Systems (SACMES), also includes a graphical interface allowing the user to easily change analysis parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES we use it here to analyze the real-time data output by (1) the electrochemical, aptamer-based measurement of a specific small-molecule target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the determination of the folding thermodynamics of an electrode-attached, redox-reporter-modified protein.
Subject(s)
Electrochemistry/methods , Software , Animals , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Base Sequence , Biosensing Techniques , DNA/analysis , Electronic Data Processing , Male , Protein Folding , Rats , Rats, Sprague-Dawley , Signal-To-Noise Ratio , Time FactorsABSTRACT
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 proteinâ 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 6â 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.
Subject(s)
Gold/chemistry , Proteins/chemistry , Thermodynamics , Electrochemical Techniques , Protein Folding , Protein Stability , Static Electricity , Surface PropertiesABSTRACT
Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) into new infectious PrPSc Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrPC-to-PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP.IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies.
Subject(s)
Amino Acids/chemistry , Prion Proteins/chemistry , Protein Folding , Amino Acid Substitution , Amino Acids/isolation & purification , Animals , Cattle , Disease Susceptibility , Mice , Mutation , Prion Diseases/metabolism , Prion Proteins/genetics , Prion Proteins/metabolism , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pKa values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pKa values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pKa values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pKa of 5.7 reflects the average of the pKa values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pKa of âª5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pKa of ≈10.4. We show that the pKa of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an "open" conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pKa values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pKa values of the buried groups in the different conformations.
Subject(s)
Micrococcal Nuclease/chemistry , Crystallography, X-Ray , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Micrococcal Nuclease/metabolism , Models, Molecular , Protein Conformation , ThermodynamicsABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Electrochemical Techniques/methods , Pharmaceutical Preparations/blood , Anesthetics, Local/blood , Anti-Bacterial Agents/blood , Antibiotics, Antineoplastic/blood , Calibration , Cocaine/blood , Doxorubicin/blood , Humans , Kanamycin/bloodABSTRACT
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.
Subject(s)
Electrons , Electron Transport , KineticsABSTRACT
Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Biological Transport, Active/physiology , Disulfides/chemistry , Disulfides/metabolism , Kinetics , Mitochondria/chemistry , Mitochondria/genetics , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity. In this article, we review the literature on the design of this effect, paying particular attention to the design strategies involved, the extent to which each can be rationally applied to (and optimized for) new receptors, and what each teaches us about the origins and optimization of this important phenomenon.
Subject(s)
Protein Engineering , Synthetic BiologyABSTRACT
Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding.
Subject(s)
Calcium-Binding Proteins/chemistry , Glucose/chemistry , Monosaccharide Transport Proteins/chemistry , Periplasmic Binding Proteins/chemistry , Allosteric Regulation , Amino Acid Sequence , Calcium-Binding Proteins/metabolism , Calorimetry , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Glucose/metabolism , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Monosaccharide Transport Proteins/metabolism , Periplasmic Binding Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Biosensors and bioassays, both of which employ proteins and nucleic acids to detect specific molecular targets, have seen significant applications in both biomedical research and clinical practice. This success is largely due to the extraordinary versatility, affinity, and specificity of biomolecular recognition. Nevertheless, these receptors suffer from an inherent limitation: single, saturable binding sites exhibit a hyperbolic relationship (the "Langmuir isotherm") between target concentration and receptor occupancy, which in turn limits the sensitivity of these technologies to small variations in target concentration. To overcome this and generate more responsive biosensors and bioassays, here we have used the sequestration mechanism to improve the steepness of the input/output curves of several bioanalytical methods. As our test bed for this we employed sensors and assays against neutrophil gelatinase-associated lipocalin (NGAL), a kidney biomarker for which enhanced sensitivity will improve the monitoring of kidney injury. Specifically, by introducing sequestration we have improved the responsiveness of an electrochemical aptamer based (EAB) biosensor, and two bioassays, a paper-based "dipstick" assay and an enzyme-linked immunosorbent assay (ELISA). Doing so we have narrowed the dynamic range of these sensors and assays several-fold, thus enhancing their ability to measure small changes in target concentration. Given that introducing sequestration requires only the addition of the appropriate concentration of a high-affinity "depletant," the mechanism appears simple and easily adaptable to tuning the binding properties of the receptors employed in a wide range of biosensors and bioassays.
ABSTRACT
Simplicity is one of the key feature for the spread of any successful technological product. Here, a method for rapid and low-cost fabrication of electrochemical biosensors is presented. This "plug, print & play" method involves inkjet-printing even in an office-like environment, without the need of highly specialized expertise or equipment, guaranteeing an ultra-fast idea to (scaled) prototype production time. The printed biosensors can be connected to a smartphone through its audio input for their impedance readout, demonstrating the validity of the system for point-of-care biosensing. Proper electrodes layout guarantees high sensitivity and is validated by finite element simulations. The introduction of a passivation method (wax printing) allowed to complete the devices fabrication process, increasing their sensitivity. Indeed, the wax allowed reducing the interference related to the parasitic currents flowing through the permeable coating of the employed substrates, which was used for the chemical sintering, thus avoiding the common thermal treatment after printing. As a case study, we used the devices to develop an electrochemical aptamer-based sensor for the rapid detection of neutrophil gelatinase-associated lipocalin (NGAL) in urine - a clinically important marker of acute kidney injury. The aptasensor platform is capable of detecting clinically relevant concentrations of NGAL with a simple and rapid smartphone readout. The developed technology may be extended in the future to continuous monitoring, taking advantage of its flexibility to integrate it in tubes, or to other diagnostic applications where cost/efficiency and rapidity of the research, development and implementation of point of care devices is a must.
Subject(s)
Biosensing Techniques , Electric Impedance , Electrodes , Smartphone , TechnologyABSTRACT
The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.
Subject(s)
Amino Acids/chemistry , Protein Folding , Proteins/chemistry , Amino Acid Motifs , Electrochemical Techniques , Kinetics , Protein Binding , Protein Denaturation , Static Electricity , Surface Properties , ThermodynamicsABSTRACT
Electrochemical aptamer-based (EAB) sensors, composed of an electrode-bound DNA aptamer with a redox reporter on the distal end, offer the promise of high-frequency, real-time molecular measurements in complex sample matrices and even in vivo. Here we assess the extent to which switching the aptamer terminus that is electrode-bound and the one that is redox-reporter-modified affects the performance of these sensors. Using sensors against doxorubicin, cocaine, and vancomycin as our test beds, we find that both signal gain (the relative signal change seen in the presence of a saturating target) and the frequency dependence of gain depend strongly on the attachment orientation, suggesting that this easily investigated variable is a worthwhile parameter to optimize in the design of new EAB sensors.
Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques , Electrochemical Techniques , Aptamers, Nucleotide/chemical synthesis , Cocaine/chemistry , Doxorubicin/chemistry , Electrodes , Oxidation-Reduction , Vancomycin/chemistryABSTRACT
The ability to monitor drugs, metabolites, hormones, and other biomarkers in situ in the body would greatly advance both clinical practice and biomedical research. To this end, we are developing electrochemical aptamer-based (EAB) sensors, a platform technology able to perform real-time, in vivo monitoring of specific molecules irrespective of their chemical or enzymatic reactivity. An important obstacle to the deployment of EAB sensors in the challenging environments found in the living body is signal drift, whereby the sensor signal decreases over time. To date, we have demonstrated a number of approaches by which this drift can be corrected sufficiently well to achieve good measurement precision over multihour in vivo deployments. To achieve a much longer in vivo measurement duration, however, will likely require that we understand and address the sources of this effect. In response, here, we have systematically examined the mechanisms underlying the drift seen when EAB sensors and simpler, EAB-like devices are challenged in vitro at 37 °C in whole blood as a proxy for in vivo conditions. Our results demonstrate that electrochemically driven desorption of a self-assembled monolayer and fouling by blood components are the two primary sources of signal loss under these conditions, suggesting targeted approaches to remediating this degradation and thus improving the stability of EAB sensors and other, similar electrochemical biosensor technologies when deployed in the body.
ABSTRACT
The multiplexed, point-of-care measurement of specific antibodies could improve the speed with which diseases are diagnosed and their treatment initiated. To this end, we are developing E-DNA scaffold sensors, which consist of a rigid, nucleic acid "scaffold" attached on one end to an electrode and presenting both a redox reporter and an epitope on the other. In the absence of antibody, the reporter efficiently transfers electrons when interrogated electrochemically. Binding-induced steric hindrance limits movement, reducing electron transfer in a manner that is both easily measured and quantitatively related to target concentration. Previously we have used monoclonal antibodies to explore the analytical performance of E-DNA sensors, showing that they support the rapid, single-step, quantitative detection of multiple antibodies in small volume samples. Here, in contrast, we employ authentic human samples to better explore the platform's clinical potential. Specifically, we developed E-DNA sensors targeting three HIV-specific antibodies and then compared the analytical and clinical performance of these against those of gold standard serological techniques. Doing so we find that, although the multistep amplification of an ELISA leads to a lower detection limits, the clinical sensitivity of ELISAs, E-DNA sensors and lateral-flow dipsticks are indistinguishable across our test set. It thus appears that, by merging the quantitation and multiplexing of ELISAs with the convenience and speed of dipsticks, E-DNA scaffold sensors could significantly improve on current serological practice.
ABSTRACT
The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, life-threatening conditions. In response, here we present an electrochemical aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, we have developed an electrochemical, aptamer-based (EAB) sensor against Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clinically relevant NGAL concentrations over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how we detect, monitor, and treat many important diseases.