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Electrochemical aptamer-based (E-AB) sensors are a promising class of biosensors which use structure-switching redox-labeled oligonucleotides (aptamers) codeposited with passivating alkanethiol monolayers on electrode surfaces to specifically bind and detect target analytes. Signaling in E-AB sensors is an outcome of aptamer conformational changes upon target binding, with the sequence of the aptamer imparting specificity toward the analyte of interest. The change in conformation translates to a change in electron transfer between the redox label attached to the aptamer and the underlying electrode and is related to analyte concentration, allowing specific electrochemical detection of nonelectroactive analytes. E-AB sensor measurements are reagentless with time resolutions of seconds or less and may be miniaturized into the submicron range. Traditionally these sensors are fabricated using thiol-on-gold chemistry. Here we present an alternate immobilization chemistry, gold-alkyne binding, which results in an increase in sensor lifetimes under ideal conditions by up to ∼100%. We find that gold-alkyne binding is spontaneous and supports efficient E-AB sensor signaling with analytical performance characteristics similar to those of thiol generated monolayers. The surface modification differs from gold-thiol binding only in the time and aptamer concentration required to achieve similar aptamer surface coverages. In addition, alkynated aptamers differ from their thiolated analogues only by their chemical handle for surface attachment, so any existing aptamers can be easily adapted to utilize this attachment strategy.

Microscale electrodes offer the advantages of increased mass transport rates, high sensitivity, and rapid measurement capabilities. Fabricating electrochemical aptamer-based (E-AB) sensors on these electrode platforms opens new applications to chemical and biological sensing but has remained challenging due to low signal-to-noise ratios and monolayer instability. In this article, we report the development and characterization of E-AB sensors on a gold microelectrode platform (∼500 nm radius). To overcome the small current response, we modified the electrodes by growing nanostructures via electrodeposition. We interrogated the sensors with two different electroanalytical techniques, square wave voltammetry (SWV) and intermittent pulse voltammetry (IPA), to measure the representative response of an ATP sensor and determine aptamer-target binding and dissociation time scales. We find robust and stable sensor performance with an increased response rate over sensors fabricated on macroscale electrodes. These results demonstrate that sensors developed on this microelectrode platform can be employed for enhanced spatiotemporal resolution measurements in chemical and biological environments.

The ability to monitor dynamic changes in neuropeptide Y (NPY) levels in complex environments can have an impact on many fields, including neuroscience and immunology. Here, we describe the development of an electrochemical, aptamer-based (E-AB) sensor for the dynamic (reversible) measurement of physiologically relevant (nanomolar) concentrations of neuropeptide Y. The E-AB sensors are fabricated using a previously described 80 nucleotide aptamer 1 reported to specifically bind NPY with a binding affinity K d = 0.3 ± 0.2 uM. We investigated two redox tag placement locations on the aptamer sequence (terminal vs internal) and various sensor fabrication and interrogation parameters to tune the performance of the resulting sensor. The best-performing sensor architecture displayed a physiologically relevant dynamic range (nM) and low limit of detection and is selective among competitors and similar molecules. The development of this sensor accomplishes two breakthroughs: first, the development of a nonmicrofluidic aptamer-based electrochemical sensor that can detect NPY on a physiologically relevant (seconds to minutes) time scale and across a relevant concentration range; second, the expansion of the range of molecules for which an electrochemical, aptamer-based sensor can be used.

Electrochemical aptamer-based sensors (EABs) using self-assembled monolayers on gold working-electrodes have now been in-vivo demonstrated for multiple-analytes, demonstrating their sensitivity and specificity even in a continuous sensing format. However, longevity has been demonstrated for only 24 hours and sensitivity has been challenging for highly dilute analytes (nM regime). A novel approach is reported here using electrochemical aptamer-based sensing that is not covalently-bound to a gold-working electrode but where aptamers are freely mobile in solution. This alternative approach has the potential to improve longevity by reducing electrode surface degradation and improving sensitivity using aptamer binding constructs that are not available for aptamers when covalently bound to the electrode. Specifically, a molecular-beacon (fluorescent) cortisol aptamer was adapted into an amperometry solution-phase cortisol EAB sensor, demonstrating ∼5% signal gain starting at only 10 nM and a saturated signal gain of ∼70% at several μM. A robust signal was achieved due to use of methylene-blue redox-tagged aptamer that was measured through amperometry with interdigitated electrodes. While this result demonstrates the basic feasibility of solution-phase EAB sensors, the result also required a self-assembled monolayer alkylthiolate blocking-layer on the gold working electrode which restricts potential device longevity. These results cumulatively suggest that initial significance of solution-phase EAB sensors may be strongest for point-of-care type testing applications and further development would be required for long-lasting continuous sensing applications.

The cation condensation-induced collapse of electrode-bound nucleic acids and the resulting change in the electrochemical signal is a useful tool to predict the structure and redox probe location of heterogeneous structures of surface-tethered DNA probes─a common architecture employed in the development of electrochemical sensors. In this paper, we measure the faradaic current of an appended redox molecule at the 3' position of the nucleic acid using cyclic voltammetry before and after nucleic acid collapse for various nucleic acid architectures and heterogeneous mixtures on the same electrode surface. The voltammetric peak current change with collapse correlates with the proximity of the redox molecules from the surface. For stem-loop probes, the terminal methylene blue is initially held closer to the surface, such that inducing collapse, by reducing the dielectric permittivity of the interrogation solution, results in a ∼30% increase in current. However, when incorporating pseudoknot probes that hold methylene blue further away from the electrode surface, the current change is much larger (∼120%), indicating a larger conformation change. Upon a 50:50 ratio of the two, we observe a change in current that relates to the ratiometric distribution of the probe used to make the surfaces. Additionally, using cyclic voltammetry, we find that the change between diffusion-limited and diffusion-independent peak currents is dependent upon the distinct structural characteristics of DNA probes on the surface (stem-loop or pseudoknot), as well as the ratios of different DNA probes on the surface. Thus, we demonstrate that the heterogeneous nature of DNA probes governs the corresponding electrochemical signals, which can lead to a better understanding on how to predict the structures of functional nucleic acids on electrode surfaces and how this affects surface-to-surface variability and electrochemical response.

We demonstrate the ability to rapidly prototype and fabricate an epoxy-embedded electrode platform and microfluidic device suitable for using electrochemical biosensors under flow conditions. We utilize three-dimensional (3-D) printing to rapidly prototype molds to fabricate epoxy-embedded electrodes in addition to molds for rapid prototyping of PDMS microfluidic components. We characterize the bare gold epoxy-embedded electrodes using ferricyanide as a redox indicator and then characterize the performance of an adenosine triphosphate (ATP) specific electrochemical, aptamer-based (E-AB) sensor. We then incorporate the ATP specific E-AB sensors into the microfluidic device to study and take advantage of the dynamic response this class of sensor offers. We were able to flow varying concentrations of target analyte and monitor the dynamic response of the sensors to the changing concentration. This work demonstrates the ability to rapidly prototype E-AB sensors under flow conditions using 3-D printing which can lead to rapid and affordable point-of-care or fieldable applications where dynamic measurements of concentration, specificity and sensitivity and multiplex detection are necessary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We demonstrate that cation condensation can induce the collapse of surface-bound nucleic acids and that the electrochemical signal from a tethered redox molecule (methylene blue) upon collapse reports on nucleic acid identity, structure, and flexibility. Furthermore, the correlation of the electrochemical signal and structure is consistent with theoretical considerations of nucleic acid collapse. Changes in solution dielectric permittivity or the concentration of trivalent cations cause the structure of nucleic acids to become more compact due to an increase in attractive electrostatic interactions between the charged biopolymer backbone and multivalent ions in the solution. Consequently, the compaction of nucleic acids results in a change in the dynamics and location of the terminally appended redox marker, which is reflected in the faradaic current measured using cyclic voltammetry. In comparison to ssDNA, nucleic acid duplexes (dsDNA, DNA/peptide nucleic acid, and dsRNA) require nucleic-acid-composition-specific solution conditions for the collapse to occur. Moreover, the magnitude of current increase observed after the collapse is different for each nucleic structure, and we find here that these changes are dictated by physical parameters of the nucleic acids including the axial charge spacing and the periodicity of the helix. The work here aims to provide quantitative and predicative measures of the effects of the nucleic acid structure on the electrochemical signal produced from distal-end appended redox markers. This architecture is commonly employed in functional nucleic acid sensors and a better understanding of structure-to-signal correlations will enable the rational design of sensitive sensing architectures.

The advent of electrochemical affinity assays and sensors evolved from pioneering efforts in the 1970s to broaden the field of analytes accessible to the selective and sensitive performance of electrochemical detection. The foundation of electrochemical affinity assays/sensors is the specific capture of an analyte by an affinity element and the subsequent transduction of this event into a measurable signal. This review briefly covers the early development of affinity assays and then focuses on advances in the past decade. During this time, progress on electroactive labels, including the use of nanoparticles, quantum dots, organic and organometallic redox compounds, and enzymes with amplification schemes, has led to significant improvements in sensitivity. The emergence of nanomaterials along with microfabrication and microfluidics technology enabled research pathways that couple the ease of use of electrochemical detection for the development of devices that are more user friendly, disposable, and employable, such as lab-on-a-chip, paper, and wearable sensors.

Single-nucleotide polymorphisms (SNPs), insertion/deletion (indel) polymorphisms, and DNA methylation are the most frequent types of genetic variations. As such, DNA polymorphisms play significant roles in genetic mapping and diagnostics. Thus, analytical methods enabling DNA polymorphism detection will provide an invaluable means for early disease diagnosis. However, no single electrochemical nucleic acid-based sensor has achieved the detection of the three major polymorphisms (SNPs, indel polymorphisms, and DNA methylation) with sufficient specificity and sensitivity. In response, we explore the utilization of a catalytic reaction between methylene blue (MB) covalently linked to surface-bound nucleic acid and freely diffusing ferricyanide (Fe(CN) 6 3- ) to improve specificity and sensitivity of DNA polymorphism detection. We find that the dynamics of the nucleic acid tether is an additional rate-limiting factor for the electrocatalytic reaction, in addition to the more traditional kinetic and excess factors. Our proof-of-concept experiments demonstrate that the use of electrocatalysis enables differentiation of the three polymorphisms when target sequences are present at 10 nM. We hypothesize that this ability is a result of the distinct dynamics of the DNA probe with each respective polymorphism. In addition to the specificity the sensor displays, the sensor achieves a 20 pM limit of detection. We believe that the electrocatalysis between nucleic acid-tethered MB and Fe(CN) 6 3- is highly promising for electrochemical nucleic acid-based sensors to achieve better specificity and sensitivity.

The design and development of advanced electrocatalysis have been extensively explored for efficient energy conversion and electrochemical biosensing. Both ferricyanide (Fe(CN) 6 3- ) and methylene blue (MB) have been widely used in the development of electrochemical biosensing strategies. However, the electrocatalytic mechanism between nucleic acid-tethered MB and Fe(CN) 6 3- remains unexplored. In this manuscript, we aim to provide readers in our community molecular insights into the electrocatalytic mechanism. The exploration of the electrocatalytic mechanism starts with a kinetic zone diagram for a one-electron homogeneous electrocatalytic reaction. Two factors-the excess factor γ and the kinetic parameter λ-are important for a homogeneous electrocatalytic reaction; as such, we studied both. The excess factor parameter was controlled by applying Fe(CN) 6 3- with various concentrations (50, 100, and 200 μM), and the kinetic parameter effect on the electrocatalytic process was examined by varying scan rates of cyclic voltammetry (CV) or frequencies of square-wave voltammetry (SWV). Moreover, we discovered that the probe dynamics of the nucleic acid tether is the third rate-limiting factor for the electrocatalytic reaction. As the probe dynamics switch of electrode-bound nucleic acid is often utilized as a mechanism in electrochemical nucleic acid-based sensors, we believe the electrocatalysis between nucleic acid-tethered MB and Fe(CN) 6 3- is capable of enhancing sensitivity and specificity of electrochemical nucleic acid-based sensors with covalent redox tags.

In this manuscript, we describe the development and application of electrochemical aptamer-based (E-AB) sensors directly interfaced with astrocytes in three-dimensional (3D) cell culture to monitor stimulated release of adenosine triphosphate (ATP). The aptamer-based sensor couples specific detection of ATP, selective performance directly in cell culture media, and seconds time resolution using squarewave voltammetry for quantitative ATP-release measurements. More specifically, we demonstrate the ability to quantitatively monitor ATP release into the extracellular environment after stimulation by the addition of calcium (Ca 2+ ), ionomycin, and glutamate. The sensor response is confirmed to be specific to ATP and requires the presence of astrocytes in culture. For example, PC12 cells do not elicit a sensor response after stimulation with the same stimulants. In addition, we confirmed cell viability in the collagen matrix for all conditions tested. Our hydrogel-sensor interface offers the potential to study the release of small molecule messengers in 3D environments. Given the generality of electrochemical aptamer-based sensors and the demonstrated successful interfacing of sensors with tissue scaffold material, in the long term, we anticipate our sensors will be able to translate from in vitro to in vivo small molecule recordings.

In this paper, we developed an electrochemical, aptamer-based (E-AB) for the real-time monitoring of insulin. The sensor utilizes a redox label-modified guanine-rich aptamer which folds into a G-quadruplex for specific recognition of insulin. To develop a reproducible E-AB sensor employing insulin aptamer probes for the detection of insulin, 10% sodium dodecyl sulfate (SDS) pretreatment is crucial as it disrupts interstrand G-quartets. After sensor pretreatment with 10% SDS, a more uniform sensor response is obtained. Upon introduction of the insulin target, binding-induced steric hindrance quantitatively reduces the efficiency of electron transfer of a distal-end redox label leading to the rapid signal change within ∼60 s. Testing demonstrates that the E-AB insulin exhibits a limit of detection of 20 nM and can be used to discriminate against both glucagon and somatostatin in Krebs-Ringer bicarbonate buffer, typically used in perfusion experiments. These results demonstrate that this assay has potential for rapid, specific, and quantitative analysis of insulin.

In this manuscript, we employ the technique intermittent pulse amperometry (IPA) to interrogate equilibrium and kinetic target binding to the surface of electrochemical, aptamer-based (E-AB) sensors, achieving as fast as 2 ms time resolution. E-AB sensors comprise an electrode surface modified with a flexible nucleic acid aptamer tethered at the 3'-terminus with a redox-active molecule. The introduction of a target changes the conformation and flexibility of the nucleic acid, which alters the charge transfer rate of the appended redox molecule. Typically, changes in charge transfer rate within this class of sensor are monitored via voltammetric methods. Here, we demonstrate that the use of IPA enables the detection of changes in charge transfer rates (i.e., current) at times <100 μs after the application of a potential pulse. Changes in sensor current are quantitatively related to target analyte concentration and can be used to create binding isotherms. Furthermore, the application of IPA enables rapid probing of the electrochemical surface with a time resolution equivalent to as low as twice the applied potential pulse width, not previously demonstrated with traditional voltammetric techniques employed with E-AB sensors (alternating current, square wave, cyclic). To visualize binding, we developed false-color plots analogous to those used in the field of fast-scan cyclic voltammetry. The use of IPA is universal, as demonstrated with two representative small molecule E-AB sensors directed against the aminoglycoside antibiotic tobramycin and adenosine triphosphate (ATP). Intermittent pulse amperometry exhibits an unprecedented sub-microsecond temporal response and is a general method for measuring rapid sensor performance.

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.

Structure-switching sensors utilize recognition elements that undergo a conformation change upon target binding that is converted into a quantitative signal. Electrochemical, aptamer-based sensors achieve detection of analytes through a conformation change in an electrode-bound, oligonucleotide aptamer by measuring changes in electron transfer efficiencies. The analytical performance of these sensors is related to the magnitude of the conformation change of the aptamer. The goal of the present work is to develop a general method to predictably tune the analytical performance (sensitivity and linear range) of electrochemical, aptamer-based sensors by utilizing a mixture of rationally designed aptamer sequences that are specific for the same target but with different affinities on the same electrode surface. To demonstrate control over sensor performance, we developed heterogeneous sensors for two representative small molecule targets (adenosine triphosphate and tobramycin). We demonstrate that mixtures of modified sequences can be used to tune the affinity, dynamic range, and sensitivity of the resulting sensors predicted by a bi-Langmuir-type isotherm.

Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3' terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM-4 μM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein-DNA binding mechanisms.

The utility of biological nanopores for the development of sensors has become a growing area of interest in analytical chemistry. Their emerging use in chemical analysis is a result of several ideal characteristics. First, they provide reproducible control over nanoscale pore sizes with an atomic level of precision. Second, they are amenable to resistive-pulse type measurement systems when embedded into an artificial lipid bilayer. A single binding event causes a change in the flow of millions of ions across the membrane per second that is readily measured as a change in current with excellent signal-to-noise ratio. To date, ion channel-based biosensors have been limited to well-behaved proteins. Most demonstrations of using ion channels as sensors have been limited to proteins that remain in the open, conducting state, unless occupied by an analyte of interest. Furthermore, these proteins are nonspecific, requiring chemical, biochemical, or genetic manipulations to impart chemical specificity. Here, we report on the use of the pore-forming abilities of heat shock cognate 70 (Hsc70) to quantify a specific analyte. Hsc70 reconstitutes into phospholipid membranes and opens to form multiple conductance states specifically in the presence of ATP. We introduce the measurement of "charge flux" to characterize the ATP-regulated multiconductance nature of Hsc70, which enables sensitive quantification of ATP (100 μM-4 mM). We believe that monitoring protein-induced charge flux across a bilayer membrane represents a universal method for quantitatively monitoring ion-channel activity. This measurement has the potential to broaden the library of usable proteins in the development of nanopore-based biosensors.

Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.

A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients' health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system's versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at subminute temporal resolution. We show that MEDIC can also obtain pharmacokinetic parameters for individual animals in real time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe that MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.

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.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this "E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

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.

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.

Alternating current, phase-sensitive stochastic detection using between 1 and 26 alpha-hemolysin ion channels reconstituted in a lipid bilayer, suspended over a 160-nm-radius orifice glass nanopore, is reported. As predicted by the binomial distribution, simultaneous analyte detection at large numbers of channels is effectively zero, independent of the number of ion channels. The results indicate that alphaHL channels are noninteracting and that significant gains in sensitivity are possible without sacrificing the simplicity of single-molecule detection strategies.

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.

Protein ion-channel recordings using a glass nanopore (GNP) membrane as the support structure for lipid bilayer membranes are presented. The GNP membrane is composed of a single conical-shaped nanopore embedded in a approximately 50 microm-thick glass membrane chemically modified with a 3-cyanopropyldimethylchlorosilane monolayer to produce a surface of intermediate hydrophobicity. This surface modification results in lipid monolayer formation on the glass surface and a lipid bilayer suspended across the small orifice (100-400 nm-radius) of the GNP membrane, while allowing aqueous solutions to fully wet the glass nanopore. The GNP membrane/bilayer structures, which exhibit ohmic seal resistances of approximately 70 GOmega and electrical breakdown voltages of approximately 0.8 V, are exceptionally stable to mechanical disturbances and have lifetimes of at least 2 weeks. These favorable characteristics result from the very small area of bilayer (10(-10)-10(-8) cm(2)) that is suspended across the GNP membrane orifice. Fluorescence microscopy and vibrational sum frequency spectroscopy demonstrate that a lipid monolayer forms on the 3-cyanopropyl-dimethylchlorosilane modified glass surface with the lipid tails oriented toward the glass. The GNP membrane/bilayer structure is well suited for single ion-channel recordings. Reproducible insertion of the protein ion channel, wild-type alpha-hemolysin (WTalphaHL), and stochastic detection of a small molecule, heptakis(6-O-sulfo)-beta-cyclodextrin, are demonstrated. In addition, the insertion and removal of WTalphaHL channels are reproducibly controlled by applying small pressures (-100 to 350 mmHg) across the lipid bilayer. The electrical and mechanical stability of the bilayer, the ease of which bilayer formation is achieved, and the ability to control ion-channel insertion, coupled with the small bilayer capacitance of the GNP membrane-based system, provide a new and nearly optimal system for single ion-channel recordings.

A simple benchtop method of fabricating glass-sealed nanometer-sized Au and Pt disk electrodes, glass nanopore electrodes, and glass nanopore membranes is reported. The synthesis of all three structures is initiated by sealing the tips of electrochemically sharpened Au and Pt microwires into glass membranes at the end of a soda lime or lead glass capillary. Pt and Au nanodisk electrodes are obtained by hand polishing using a high-input impedance metal oxide semiconductor field effect transistor (MOSFET)-based circuit to monitor the radius of the metal disk. Proper biasing of the MOSFET circuit, based on a numerical analysis of the polishing circuit impedance, allows for the reproducible fabrication of Pt disk electrodes of radii as small as 10 nm. Significantly smaller background currents in voltammetric measurements are obtained using lead glass capillaries, a consequence of the lower mobility of Pb(2+) (relative to Na(+)) in the glass matrix. Glass nanopore electrodes and glass nanopore membranes are fabricated, respectively, by removal of part or all of the metal sealed in the glass membranes. The nanostructures are characterized by atomic force microscopy, steady-state voltammetry, and ion conductivity measurements.