Single Voltammetric Sweep Calibration-Free Interrogation of Electrochemical Aptamer-Based Sensors Employing Continuous Square Wave Voltammetry.

Continuous square wave voltammetry (cSWV) is a technique that enables the continuous collection of current data (at 100 kHz) to maximize the information content obtainable from a single voltammetric sweep. This data collection procedure results in the generation of multiple voltammograms corresponding to different effective square wave frequencies. The application of cSWV brings significant benefits to electrochemical aptamer-based (E-AB) sensors. The E-AB sensor platform permits continuous real-time monitoring of small biological molecules. Traditionally, E-AB sensors report only on changes in analyte concentration rather than absolute quantification in matrices when basal concentrations are not known a priori. This is because they exhibit a voltammetric peak current even in the absence of a target. However, using a dual-frequency approach, calibration-free sensing can be performed effectively, eliminating the sensor-to-sensor variation by taking ratiometric current responses obtained at two different frequencies from two different voltammetric sweeps. In employing our approach, cSWV provides a great advantage over the conventionally used square wave voltammetry since the required voltammograms are collected with a single sweep, which improves the temporal resolution of the measurement when considering the current at multiple frequencies for improved accuracy and reduced surface interrogation. Moreover, we show here that using cSWV provides significantly improved concentration predictions. E-AB sensors sensitive to ATP and tobramycin were interrogated across a wide range of concentrations. With this approach, cSWV allowed us to estimate the target concentration, retaining up to an ±5% error of the expected concentration when tested in buffer and complex media.

Controlling the Collision Type and Frequency of Single Pt Nanoparticles at Chemically Modified Gold Electrodes.

Studying the electrochemical response of single nanoparticles at an electrode surface gives insight into the dynamic and stochastic processes that occur at the electrode interface. Herein, we investigated single platinum nanoparticle collision dynamics and type (elastic vs inelastic) at gold electrode surfaces modified with self-assembled monolayers (SAMs) of varying terminal chemistries. Collision events are measured via the faradaic current from catalytic reactions at the Pt surface. By changing the terminal, solution-facing group of a thiolate monolayer, we observed the effect of hydrophobicity at the solution-electrode interface on single-particle collisions by employing either a hydrophobic -CH3 terminal group (1-hexanethiol), a hydrophilic -OH terminal group (6-mercaptohexanol), or an equimolar mixture of the two. Changes in the terminal group lead to alterations in collision-induced current magnitude, collisional frequency, and the distinct shape of the collision event current transient. The effects of the terminal group of the SAM were probed by measuring quantitative differences in the events monitored through both the hydrogen evolution reaction (HER) and hydrazine oxidation. In both cases, a platinum nanoparticle (PtNP) favors adsorption to bare and hydrophilic surfaces but demonstrates elastic collision behavior when it collides with a hydrophobic surface. In the case of a mixed monolayer, distinct characteristics of hydrophobic and hydrophilic surfaces are observed. We report how single nanoparticle collisions can reveal nanoscale surface heterogeneity and can be used to manipulate the nature of single-particle interactions on an electrode surface by functionalized self-assembled monolayers.

Utilization of Spontaneous Alkyne-Gold Self-Assembly Chemistry as an Alternative Method for Fabricating Electrochemical Aptamer-Based Sensors.

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.

Tuning the Probe-Bilayer Architecture of Silver Nanoneedle-based Ion Channel Probes.

Ion channel probes, as one of the ion channel platforms, provide an appealing opportunity to perform localized detection with a high precision level. These probes come basically in two classes: glass and metal. While the glass-based probes showed the potential to be employed for molecular sensing and chemical imaging, these probes still suffer from limited resolution and lack of control over protein insertion. On the other hand, metal-based nanoneedle probes (gold and silver) have been recently developed to allow reducing probe dimensions to the nanoscale geometry. More specifically, silver probes are preferable owing to their ability to mitigate the channel current decay observed with gold probes and provide a stable DC channel current. However, there are still some challenges related to the probe design and bilayer curvature that render such probes insensitive to small changes in the tip-substrate distance. Herein, we introduce two main pathways to control the probe-bilayer architecture; the first is by altering the probe shape and geometry during the fabrication process of silver probes. The second pathway is by altering the surface characteristics of the silver probe via an electrophoretic deposition process. Our findings reveal that varying the electrochemical etching parameters results in different probe geometries and producing sharper tips with a 2-fold diameter reduction. In addition, the electrophoretic deposition of a cathodic paint on the silver nanoneedle surface led to a miniaturized exposed silver tip that enables the formation of a confined bilayer. We further investigated the characteristics of bilayers supported on both the sharper nanoneedles and the HSR-coated silver probes produced by controlling the etching conditions and electrodeposition process, respectively. We believe this work paves the way to rationally design silver nanoneedle ion channel probes, which are well suited for localized molecular sensing and chemical imaging.

Effects of Nucleic Acid Structural Heterogeneity on the Electrochemistry of Tethered Redox Molecules.

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.

Observing Real-Time Formation of Self-Assembled Monolayers on Polycrystalline Gold Surfaces with Scanning Electrochemical Cell Microscopy.

Self-assembled monolayers (SAMs) of alkanethiols on gold have become a central focus of controllable surface chemistry because they can be easily formed from the solution phase and characterized using various techniques. Understanding the formation processes occurring at a nanoscale level is crucial to form defect-free SAMs for tailored applications in bio- and nanotechnology. Although many reports study and characterize SAMs after they are formed on gold surfaces, typical methods have not extensively studied the SAM formation process at the nanoscale. This paper focuses on the formation of defect-free SAMs and elucidates the formation mechanism occurring at the nanoscale level during the formation process. Exploiting the strength of scanning electrochemical cell microscopy, we monitored SAM formation via a soluble redox reporter on a polycrystalline gold foil using voltammetric and amperometric techniques. We formed SAMs by varying the concentration of 3-mercapto-1-propanol [HS(CH2)3OH], 6-mercapto-1-hexanol [HS(CH2)6OH], and 9-mercapto-1-nonanol [HS(CH2)9OH] to determine the effects of the thiol chain length, concentration, and location on the substrate (grain boundaries) on monolayer formation. We observed real-time changes in the quasisteady-state current of our redox reporter during the self-assembly process. Importantly, we formed defect-free SAMs at the nanoscale level using different concentrations of HS(CH2)6OH and HS(CH2)9OH and found that SAM formation at the nanoscale is concentration-dependent and varies when at a boundary between two crystal grains.

Silver Nanoneedle Probes Enable Sustained DC Current, Single-Channel Resistive Pulse Nanopore Sensing.

Resistive pulse sensing using ion channel proteins (biological nanopores) has been evolving as a single-molecule approach to detect small biomolecules owing to atomically precise pore size reproducibility, high signal-to-noise ratio, and molecular selectivity. The incorporation of biological nanopores in sensing platforms requires a stable lipid membrane that can be formed by a variety of methods such as the painting method and droplet-based techniques. However, these methods are limited by the fragility of the unsupported bilayer or the need for specific microdevices. Electrode-supported bilayers, in which a metal electrode is used as a support structure, have been recently developed using a fine gold nanoneedle. We previously described the utility of the gold nanoneedle-supported ion channel probe to detect small molecules with high spatial resolution; however, it exhibited a channel current decay over time, which affected the binding frequency of the target molecule to the protein pore as well. Here, we introduce a silver nanoneedle probe to support the lipid bilayer formation and ion channel measurements. The silver nanoneedle mitigates the current decay observed on gold electrodes and produces stable DC channel currents. Our findings propose the formation of a AgCl layer creating a nonpolarizable electrode. The new nanoneedle is successfully applied for single-molecule detection of sulfonated ß-cyclodextrin (S7ßCD) using αHL as a test bed protein. We believe that this new silver nanoneedle platform has great potential given the relative ease of lipid bilayer formation and stable open channel currents.

Effects of Experimental Conditions on the Signaling Fidelity of Impedance-Based Nucleic Acid Sensors.

Electrochemical impedance spectroscopy (EIS), an extremely sensitive analytical technique, is a widely used signal transduction method for the electrochemical detection of target analytes in a broad range of applications. The use of nucleic acids (aptamers) for sequence-specific or molecular detection in electrochemical biosensor development has been extensive, and the field continues to grow. Although nucleic acid-based sensors using EIS offer exceptional sensitivity, signal fidelity is often linked to the physical and chemical properties of the electrode-solution interface. Little emphasis has been placed on the stability of nucleic acid self-assembled monolayers (SAMs) over repeated voltammetric and impedimetric analyses. We have studied the stability and performance of electrochemical biosensors with mixed SAMs of varying length thiolated nucleic acids and short mercapto alcohols on gold surfaces under repeated electrochemical interrogation. This systematic study demonstrates that signal fidelity is linked to the stability of the SAM layer and nucleic acid structure and the packing density of the nucleic acid on the surface. A decrease in packing density and structural changes of nucleic acids significantly influence the signal change observed with EIS after routine voltammetric analysis. The goal of this article is to improve our understanding of the effect of multiple factors on EIS signal response and to optimize the experimental conditions for development of sensitive and reproducible sensors. Our data demonstrate a need for rigorous control experiments to ensure that the measured change in impedance is unequivocally a result of a specific interaction between the target analyte and nucleic recognition element.

Nucleic Acid Identity, Structure, and Flexibility Affect the Electrochemical Signal of Tethered Redox Molecules upon Biopolymer Collapse.

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.

Repetitive drug releases from light-activatable micron-sized liposomes.

In this work, a novel light activatable micron-sized liposomal drug carrier that has a unique capability to release drug repetitively in proportion to the cycle number of short irradiation (5 s) of near-infrared (NIR) pulsed laser is reported. We synthesized methotrexate (MTX)-loaded liposomes based on a modified reverse-phase evaporation method. Gold nanorods (AuNR) were attached to the liposomal surfaces, enabling the liposomes to release drug under short NIR irradiation via the photothermal effect. The concentrations of methotrexate (MTX) released from the liposomes were 10.6, 29.8, 43.7 and 65.9 µg/mL after one, two, three or four NIR laser cycles (1.1 W at 1064 nm, 5 s per cycle), respectively. The current finding will provide possible solution to the previously reported inconsistency in drug release from light activatable liposomal drug carriers at each activation cycle. The repeatability of drug release described in this work is believed to be due to reversible nature of the liposomes. The liposomes release drug via lipid bilayer melting when irradiated by laser due to gold nanorods' plasmonic heat on the lipid bilayer surface and quickly regain their original structure once the laser source is removed. We provided evidence of the reversible liposomal structures by monitoring the change of number densities of liposomes using a microelectrode sensor with different laser irradiation durations and powers. We also assessed the micron-sized liposome with respect to long-term stability, drug encapsulation efficiency, and drug-releasing efficiency, demonstrating the possibility of utilizing these liposomes as long-term drug delivery vehicles for various drugs.

Recessed Ag/AgCl Microelectrode-Supported Lipid Bilayer for Nanopore Sensing.

Biological nanopores reconstituted into supported lipid bilayer membranes are widely used as a platform for stochastic nanopore sensing with the ability to detect single molecules including, for example, single-stranded DNA (ssDNA) and miRNA. A main thrust in this area of research has been to improve overall bilayer stability and ease of measurements. These improvements are achieved through a variety of clever strategies including droplet-based techniques; however, they typically require specific microfabrication techniques to prepare devices or special manipulation techniques for microdroplets. Here, we describe a new method to prepare lipid bilayers using a recessed-in-glass Ag/AgCl microelectrode as a support structure. The lipid bilayer is formed at the tip of the microelectrode by immersing the microelectrode into a layered bath solution consisting of an oil/lipid mixture and an aqueous electrolyte solution. In this paper, we demonstrate this stable, supported lipid bilayer structure for channel current measurements of pore-forming toxins and single-molecule detection of ssDNA. This Ag/AgCl-supported lipid bilayer can potentially be widely adopted as a lipid membrane platform for nanopore sensing because of its simple and easy procedure needed to prepare lipid bilayers.

Analysis of Membrane Protein Deinsertion-Associated Currents with Nanoneedle-Supported Bilayers to Discover Pore Formation Mechanisms.

Analysis of the pore formation mechanisms of biological nanopores can provide insight into pore-forming peptide-induced diseases and into the characterization of nanopores employed in sensing methods. Evaluation of pore formation mechanisms is typically performed using microscopy including atomic force microscopy, transmission electron microscopy, as well as electrically via channel current measurements using a patch-clamp amplifier. However, due to the relatively low temporal resolution of the above-mentioned microscopy techniques and the low analysis accuracy of the channel current measurements, new analytical methods are required. Here, we describe a new analytical strategy to measure and analyze both ionic currents associated with biological nanopore insertion and deinsertion into and out of lipid bilayers to determine pore formation mechanisms for several representative proteins. The current changes associated with protein deinsertion are monitored as the lipid membrane leaflets are pulled apart-a unique phenomenon enabled by our gold nanoneedle measurement probe. This deinsertion current analysis (DiCA) is performed using a gold nanoneedle-supported lipid bilayer at which a bilayer membrane is formed by bringing together two lipid monolayers on the surface of the nanoneedle and at the interface of an aqueous solution and a lipid/oil mixture. The lipid bilayer can be pulled apart by removing the nanoneedle from this interface. In this study, we demonstrate the determination of pore formation mechanisms for four different pore-forming proteins and peptides-α-hemolysin, streptolysin O, alamethicin, and amyloid ß 1-42 using DiCA. As a result, we successfully discern the pore formation mechanism, either addition or expansion, for each protein/peptide by analyzing the ratio and magnitude of insertion and deinsertion current events.

Effect of Laser Irradiation on Reversibility and Drug Release of Light-Activatable Drug-Encapsulated Liposomes.

Although several studies have demonstrated repetitive drug release using light-activatable liposomes, inconsistent drug release at each activation limits widespread usage. Here, we report reversible plasmonic material-coated encapsulated liposomes for proportional controlled delivery of methotrexate (MTX), which is a common drug for cancer and autoimmune diseases, using repetitive laser irradiation. Our results suggest a proportional increase in total drug release after repetitive laser irradiation. We hypothesize that the drug is released via "melted" lipid bilayers when the plasmonic materials on the liposome surface are heated by laser irradiation followed by reversible formation of the liposome. To evaluate our hypothesis, the number density of liposomes after laser irradiation was measured using single-particle (liposome) collision experiments at an ultramicroelectrode. Collisional frequency data suggest that the number density of liposomes remains unaltered even after 60 s of laser irradiation at 1.1 and 1.8 W, indicating that the liposome structure is reversible. The results were further compared with gold nanorod-coated nanodroplets where drug is released via irreversible phase transition. In contrast to what was observed with the liposome particles, the number density of the nanodroplets decreased with increasing laser irradiation duration. The structure reversibility of our liposome particles may be responsible for repetitive drug release with laser heating. We also studied the temperature rise in the lipid bilayer by incorporating polymerized 10,12-pentacosadiynoic acid (PCDA) in the lipid composition. The red shift in the UV-vis spectrum due to the structural change in PCDA lipids after laser irradiation indicates a rise in temperature above 75 °C, which is also above the chain-melting temperature of the main lipid used in the liposomes. All these results indicate that drug is released from the light-activatable liposomes due to reversible nanostructural alteration in the lipid bilayer by plasmonic resonance heating. The liposomes have potential to be a drug carrier for dose-controlled repetitive drug delivery.

Electrochromic, Closed-Bipolar Electrodes Employing Aptamer-Based Recognition for Direct Colorimetric Sensing Visualization.

In this paper, we adapt the electrochemical, aptamer-based (E-AB) sensor platform to develop colorimetric aptamer-based sensors using a closed-bipolar electrode (C-BPE) system. The C-BPE E-AB sensors provide quantitative detection of target molecules based on the rate of color change of an electrochromic Prussian blue (PB) thin-film indicator electrode. The C-BPE cathode, or sensing electrode, is modified with a redox-labeled aptamer that binds to a specific target. More specifically, we employed sequences specific for adenosine triphosphate (ATP) and tobramycin as test-bed targets because these sequences are well vetted. The C-BPE anode, or indicator electrode, was coated with an electrochromic thin film comprising Prussian white (PW) that, when reduced to PB, is accompanied by a corresponding color change used for analytical detection. The rate of color change from PW to PB is facilitated by a potassium ferricyanide-catalyzed oxidation of leucomethylene blue (LB) to methylene blue (MB), the redox label conjugated to the aptamer on the sensing electrode. We demonstrate that the rate of color change is quantitatively related to the concentration of target analyte, which provides a means for naked eye determination. When combined with smartphone-based colorimetric detection, these C-BPE E-AB sensors present a user-friendly alternative to traditional E-AB sensors that rely on voltammetric analysis and a potentiostat, opening up the possibility of point-of-use applications.

Perspective on the Future Role of Aptamers in Analytical Chemistry.

It has been almost 30 years since the invention of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology and the description of the first aptamers. In retrospect over the past 30 years, advances in aptamer development and application have demonstrated that aptamers are potentially useful reagents that can be employed in diverse areas within analytical chemistry, biotechnology, biomedicine, and molecular biology. While often touted as artificial antibodies with an ability to be selected for any target, aptamer development, unfortunately, lags behind development of analytical methodologies that employ aptamers, hindering deeper integration into the application of analytical tool development. This perspective covers recent advances in SELEX methodology for improving efficiency of the SELEX procedure and enhancing affinity and specificity of the selected aptamers, what we view as a critical barrier in the future role of aptamers in analytical chemistry. We discuss postselection modifications that can be used for enhancing performance of the selected aptamers in an analytical device by including understanding intermolecular interaction forces in the binding domain. While highlighting promising properties of aptamers that enable several analytical advances, we provide discussion on the challenges of penetration of aptamers in the analytical field.

Electrochemical Studies of Cation Condensation-Induced Collapse of Surface-Bound DNA.

In this paper, we demonstrate the ability to control and electrochemically monitor nucleic acid conformation by inducing collapse of short, surface-bound nucleotides (7-28 nucleotides). More specifically, we monitored changes in a 5'-electrode-bound DNA structure via changes in the faradaic current related to the reduction/oxidation of a 3'-terminal-appended redox molecule. Reversible DNA collapse was induced by cation condensation achieved by either reducing the dielectric permittivity of the interrogation solution or by the addition of multivalent cations such as the polyamine spermidine (3+). Additionally, we find that while the change in electrochemical signal associated with surface bound DNA collapse is dependent on nucleic acid length and surface packing density, the solution conditions (e.g., dielectric permittivity) required for collapse remain constant. As such, we find that collapse of the short DNA strands occurs when the effective charge of the DNA backbone is ∼73-89% neutralized by cations in solution/buffer, according to Manning's theory on cation condensation. This work provides new insight into the structure function relationship of surface-bound nucleic acids and how this is manifested in electrochemical signaling.

Quantitative Framework for Stochastic Nanopore Sensors Using Multiple Channels.

Membrane protein channels employed as stochastic sensors offer large signal-to-noise ratios and high specificity in single molecule binding measurements. Stochastic events in a single ion channel system can be measured using current-time traces, which are straightforward to analyze. Signals arising from measurement using multiple ion channels are more complicated to interpret. We show that multiple independent ion channels offer improved detection sensitivity compared to single channel measurements and that increased signal complexity can be accounted for using binding event frequency. More specifically, the leading edge of binding events follows a Poisson point process, which means signals from multiple channels can be superimposed and the association times (between each binding event leading edge), allow for sensitive and quantitative measurements. We expand our calibration to high ligand concentrations and high numbers of ion channels to demonstrate that there is an upper limit of quantification, defined by the time resolution of the measurement. The upper limit is a combination of the instrumental time resolution and the dissociation time of a ligand and protein which limits the number of detectable events. This upper limit also allows us to predict, in general, the measurement requirements needed to observe any process as a Poisson point process. The nanopore-based sensing analysis has wide implications for stochastic sensing platforms that operate using multiple simultaneous superimposable signals.

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA.

Electrochemical aptamer-based (E-AB) sensors offer advantageous analytical detection abilities due to their rapid response time (seconds to minutes), specificity to a target, and selectivity to function in complex media. Ribonucleic acid (RNA) aptamers employed in this class of sensor offer favorable binding characteristics resulting from the ability of RNA to form stable tertiary folds aided by long-range intermolecular interactions. As a result, RNA aptamers can fold into three-dimensional structures more complex than those of their DNA counterparts and consequently exhibit better binding ability to target analytes. Unfortunately, RNA aptamers are susceptible to degradation by nucleases, and for this reason, RNA-based sensors are scarce or require significant sample pretreatment before use in clinically relevant media. Here, we combine the usefulness of a collagen I hydrogel membrane with entrapped ribonuclease inhibitors (RI) to protect small molecule RNA E-AB sensors from endogenous nucleases in complex media. More specifically, the biocompatibility of the naturally polymerized hydrogel with encapsulated RI promotes the protection of an aminoglycoside-binding RNA E-AB sensor up to 6 h, enabling full sensor function in nuclease-rich environments (undiluted serum) without the need for prior sample preparation or oligonucleotide modification. The use of collagen as a biocompatible membrane represents a general approach to compatibly interface E-AB sensors with complex biological samples.

Bioinspired Protein Channel-Based Scanning Ion Conductance Microscopy (Bio-SICM) for Simultaneous Conductance and Specific Molecular Imaging.

The utility of stochastic single-molecule detection using protein nanopores has found widespread application in bioanalytical sensing as a result of the inherent signal amplification of the resistive pulse method. Integration of protein nanopores with high-resolution scanning ion conductance microscopy (SICM) extends the utility of SICM by enabling selective chemical imaging of specific target molecules, while simultaneously providing topographical information about the net ion flux through a pore under a concentration gradient. In this study, we describe the development of a bioinspired scanning ion conductance microscopy (bio-SICM) approach that couples the imaging ability of SICM with the sensitivity and chemical selectivity of protein channels to perform simultaneous pore imaging and specific molecule mapping. To establish the framework of the bio-SICM platform, we utilize the well-studied protein channel α-hemolysin (αHL) to map the presence of ß-cyclodextrin (ßCD) at a substrate pore opening. We demonstrate concurrent pore and specific molecule imaging by raster scanning an αHL-based probe over a glass membrane containing a single 25-µm-diameter glass pore while recording the lateral positions of the probe and channel activity via ionic current. We use the average channel current to create a conductance image and the raw current-time traces to determine spatial localization of ßCD. With further optimization, we believe that the bio-SICM platform will provide a powerful analytical methodology that is generalizable, and thus offers significant utility in a myriad of bioanalytical applications.

Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors.

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.