We report the use of gold nanoband electrodes ranging from 60 to 180 nm in width to study collision and oxidation of single Ag nanoparticles (NPs). The use of nanoscale electrodes has enabled the observation of unique single-NP collision responses indicating a strong electrode size effect when the critical dimension of the electrode (the bandwidth) is reduced to that of NPs. In addition to multipeak events, NP collision on a nanoband electrode displays reduced collision frequency, significantly higher probability of single-peak events, and fewer subpeaks. More importantly, the average charge transferred in a single-peak event is about 50% less than that of the first subpeak of a multipeak event. The reduced charge of single-peak collisions and the more frequent appearance on nanoelectrodes are strong evidence that NPs start to behave differently at the electrode/solution interface when the size of the electrode is reduced to be comparable to that of the NPs. The reduced charge is likely due to a weaker particle–electrode interaction when the particle collides on the edge of the nanoband electrode. Random walk numerical simulation was used to further understand the electrode size effect in single-particle collision and oxidation. The simulated results are in good agreement with the experiments. A detailed analysis of the collision signal reveals that a Ag NP is more likely to diffuse away after making its initial contact with a nanoband electrode, due to the electrode’s smaller critical dimension and a possible strong edge effect from the negatively charged silicon nitride/oxide. This study offers a deeper insight into the dynamic collision behavior of metal NPs on the electrode surface.

Recent high-bandwidth recordings of the oxidation and dissolution of 35 nm radius Ag nanoparticles at a Au microelectrode show that these nanoparticles undergo multiple collisions with the electrode, generating multiple electrochemical current peaks. In the time interval between observed current peaks, the nanoparticles diffuse in the solution near the electrolyte/electrode interface. Here, we demonstrate that simulations of random nanoparticle motion, coupled with electrochemical kinetic parameters, quantitatively reproduce the experimentally observed multicurrent peak behavior. Simulations of particle diffusion are based on the nanoparticle-mass-based thermal nanoparticle velocity and the Einstein diffusion relations, while the electron-transfer rate is informed by the literature exchange current density for the Ag/Ag+ redox system. Simulations indicate that tens to thousands of particle–electrode collisions, each lasting ∼6 ns or less (currently unobservable on accessible experimental time scales), contribute to each experimentally observed current peak. The simulation provides a means to estimate the instantaneous current density during a collision (∼500–1000 A/cm2), from which we estimate a rate constant between ∼5 and 10 cm/s for the electron transfer between Ag nanoparticles and the Au electrode. This extracted rate constant is approximately equal to the thermal collisional velocity of the Ag nanoparticle (4.6 cm/s), the latter defining the theoretical upper limit of the electron-transfer rate constant. Our results suggest that only ∼1% of the surface atoms on the Ag nanoparticles are oxidized per instantaneous collision. The combined simulated and experimental results underscore the roles of Brownian motion and collision frequency in the interpretation of heterogeneous electron-transfer reactions involving nanoparticles.

Spiroiminodihydantoin (Sp) is a hyperoxidized form of guanine (G) resulting from oxidation by reactive oxygen species. The lesion is highly mutagenic, and the stereocenter renders the two isomers with distinct behaviors in chemical, spectroscopic, enzymatic, and computational studies. In this work, the α-hemolysin (αHL) latch sensing zone was employed to investigate the base pairing properties of the Sp diastereomers embedded in a double-stranded DNA. Duplexes containing (S)-Sp consistently gave deeper current blockage, and a baseline resolution of ∼0.8 pA was achieved between (S)-Sp:G and (R)-Sp:G base pairs. Ion fluxes were generally more hindered when Sp was placed opposite pyrimidines. Analysis of the current noise of blockade events further provided dynamics information about the Sp-containing base pairs. In general, base pairs comprised of (S)-Sp generated current fluctuations larger than those of their (R)-Sp counterparts, suggesting enhanced base pairing dynamics. The current noise was also substantially affected by the identity of the base opposite Sp, increasing in the following order: A < G < T < C. This report provides information about the dynamic structure of Sp in the DNA duplex and therefore has implications for the enzymatic repair of the Sp diastereomers.

Herein, we use Pt nanodisk electrodes (apparent radii from 4 to 80 nm) to investigate the nucleation of individual O2 nanobubbles generated by electrooxidation of hydrogen peroxide (H2O2). A single bubble reproducibly nucleates when the dissolved O2 concentration reaches ∼0.17 M at the Pt electrode surface. This nucleation concentration is ∼130 times higher than the equilibrium saturation concentration of O2 and is independent of electrode size. Moreover, in acidic H2O2 solutions (1 M HClO4), in addition to producing an O2 nanobubble through H2O2 oxidation at positive potentials, individual H2 nanobubbles can also be generated at negative potentials. Alternating generation of single O2 and H2 bubbles within the same experiment allows direct comparison of the critical concentrations for nucleation of each nanobubble without knowing the precise size/geometry of the electrode or the exact viscosity/temperature of the solution.

The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pretreatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine–cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base-flipping at the mismatch site. Base-flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically relevant, but poorly understood, modifications to cytosine affect the local conformational dynamics of a DNA duplex in a confined environment.

This article describes the dynamic steady state of individual H2 nanobubbles generated by H+ reduction at inlaid and recessed Pt nanodisk electrodes. Electrochemical measurements coupled with finite element simulations allow analysis of the nanobubble geometry at dynamic equilibrium. We demonstrate that a bubble is sustainable at Pt nanodisks due to the balance of nanobubble shrinkage due to H2 dissolution and growth due to H2 electrogeneration. Specifically, simulations are used to predict stable geometries of the H2/Pt/solution three-phase interface and the width of exposed Pt at the disk circumference required to sustain the nanobubble via steady-state H2 electrogeneration. Experimentally measured currents, iss, corresponding to the electrogeneration of H2, at or near the three-phase interface, needed to sustain the nanobubble are between 0.2 and 2.4 nA for Pt nanodisk electrodes with radii between 2.5 and 40 nm. However, simple theoretical analysis shows that the diffusion-limited currents required to sustain such a single nanobubble at an inlaid Pt nanodisk are 1–2 orders larger than the observed values. Finite element simulation of the dynamic steady state of a nanobubble at an inlaid disk also demonstrates that the expected steady-state currents are much larger than the experimental currents. Better agreement between the simulated and experimental values of iss is obtained by considering recession of the Pt disk nanoelectrode below the plane of the insulating surface, which reduces the outward flux of H2 from the nanobubble and results in smaller values of iss.

Ion current rectification (ICR) refers to the asymmetric potential-dependent rate of the passage of solution ions through a nanopore, giving rise to electrical current–voltage characteristics that mimic those of a solid-state electrical diode. Since the discovery of ICR in quartz nanopipettes two decades ago, synthetic nanopores and nanochannels of various geometries, fabricated in membranes and on wafers, have been extensively investigated to understand fundamental aspects of ion transport in highly confined geometries. It is now generally accepted that ICR requires an asymmetric electrical double layer within the nanopore, producing an accumulation or depletion of charge-carrying ions at opposite voltage polarities.Our research groups have recently explored how the voltage-dependent ion distributions and ICR within nanopores can induce novel nanoscale flow phenomena that have applications in understanding ionics in porous materials used in energy storage devices, chemical sensing, and low-cost electrical pumping of fluids. In this Account, we review our most recent investigations on this topic, based on experiments using conical nanopores (10–300 nm tip opening) fabricated in thin glass, mica, and polymer membranes. Measurable fluid flow in nanopores can be induced either using external pressure forces, electrically via electroosmotic forces, or by a combination of these two forces. We demonstrate that pressure-driven flow can greatly alter the electrical properties of nanopores and, vice versa, that the nonlinear electrical properties of conical nanopores can impart novel and useful flow phenomena.Electroosmotic flow (EOF), which depends on the magnitude of the ion fluxes within the double layer of the nanopore, is strongly coupled to the accumulation/depletion of ions. Thus, the same underlying cause of ICR also leads to EOF rectification, i.e., unequal flows occurring for the same voltage but opposite polarities. EOF rectification can be used to electrically pump fluids with very precise control across membranes containing conical pores via the application of a symmetric sinusoidal voltage.The combination of pressure and asymmetric EOF can also provide a means to generate new nanopore electrical behaviors, including negative differential resistance (NDR), in which the current through a conical pore decreases with increasing driving force (applied voltage), similar to solid-state tunnel diodes. NDR results from a positive feedback mechanism between the ion distributions and EOF, yielding a true bistability in both fluid flow and electrical current at a critical applied voltage. Nanopore-based NDR is extremely sensitive to the surface charge near the nanopore opening, suggesting possible applications in chemical sensing.

The Young–Laplace equation is central to the thermodynamic description of liquids with highly curved interfaces, e.g., nanoscale droplets and their inverse, nanoscale bubbles. The equation relates the pressure difference across an interface to its surface tension and radius of curvature, but the validity in using the macroscopic surface tension for describing curved interfaces with radii smaller than tens of nanometers has been questioned. Here we present electrochemical measurement of Laplace pressures within single H2 bubbles between 7 and 200 nm radius (corresponding, respectively, to between 200 and 7 atm). Our results demonstrate a linear relationship between a bubble’s Laplace pressure and its reciprocal radius, verifying the classical thermodynamic description of H2 nanobubbles as small as ∼10 nm.

The dynamic collision behavior of the electro-oxidation of single Ag nanoparticles is observed at Au microelectrodes using stochastic single-nanoparticle collision amperometry. Results show that an Ag nanoparticle collision/oxidation event typically consists of a series of 1 to ∼10 discrete “sub-events” over an ∼20 ms interval. Results also show that the Ag nanoparticles typically undergo only partial oxidation prior to diffusing away from the Au electrode into the bulk solution. Both behaviors are characterized and shown to exist under a variety of experimental conditions. These previously unreported behaviors suggest that nanoparticle collision and electro-dissolution is a highly dynamic process driven by fast particle–electrode interactions and nanoparticle diffusion.

Ion transport near interfaces is a fundamental phenomenon of importance in electrochemical, biological, and colloidal systems. In particular, electric double layers in highly confined spaces have implications for ion transport in nanoporous energy storage materials. By exploiting redox cycling amplification in lithographically fabricated thin-layer electrochemical cells comprising two platinum electrodes separated by a distance of 150–450 nm, we observed current enhancement during cyclic voltammetry of the hexaamineruthenium(III) chloride redox couple (Ru(NH3)63/2+) at low supporting electrolyte concentrations, resulting from ion enrichment of Ru(NH3)63/2+ in the electrical double layers and an enhanced ion migration contribution to mass transport. The steady-state redox cycling was shown to decrease to predominately diffusion controlled level with increasing supporting electrolyte concentration. Through independent biasing of the potential on the individual Pt electrodes, the voltammetric transport limited current can be controlled without changing the electrochemical nature at the system. Using finite-element simulations based on numerical solutions to the Poisson and Nernst–Planck equations with Butler–Volmer type boundary conditions, we are able to semiquantitatively predict the voltammetric behavior of the nanogap cell that results from coupling of surface electrostatics and ion transport.

Rotational tumbling of nanorods as they translocate through a glass nanopipet modulates the flux of charge carrying ions, generating a resistive pulse with multiple peaks. The measured times between maxima and minima in the resistive pulse correspond to an average rotation of approximately 90° and can be used to compute the rotational diffusion coefficient, Dr. Analytical expressions for the rotational diffusion coefficient (Dr) in terms of the nanorod length (L) allow the calculation of the rod length. We report experiments in which an individual Au nanorod (nominal length of 77–122 nm) is driven repeatedly through the nanopipet orifice by voltage switching at up to 30 Hz, allowing rapid measurement of Dr and L of individual nanorods with ∼15% error. Measured values of Dr between 2000 and 4000 rad2 s–1 for Au nanorods of 77–122 nm length are in good agreement with theoretical predictions.

The latch region of the wild-type α-hemolysin (α-HL) protein channel can be used to distinguish single base modifications in double-stranded DNA (dsDNA) via ion channel measurements upon electrophoretic capture of dsDNA in the vestibule of α-HL. Herein, we investigated the use of the latch region to detect a nick in the phosphodiester DNA backbone. The presence of a nick in the phosphodiester backbone of one strand of the duplex results in a significant increase in both the blockade current and noise level relative to the intact duplex. Differentiation between the nicked and intact duplexes based on blockade current or noise, with near baseline resolution, allows real-time monitoring of the rate of T3-DNA ligase-catalyzed phosphodiester bond formation. Under low ionic strength conditions containing divalent cations and a molecular crowding agent (75 mg mL–1 PEG), the rate of enzyme-catalyzed reaction in the bulk solution was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the α-HL protein channel vestibule. Enzyme kinetic results obtained from the nanopore experiments match those from gel electrophoresis under the same reaction conditions, indicating the α-HL nanopore measurement provides a viable approach for monitoring enzymatic DNA repair activity.Keywords: DNA ligase; enzyme kinetics; latch zone; nicked duplex; α-hemolysin;

A method for identifying and differentiating DNA duplexes containing the mismatched base pairs CC and CA at single molecule resolution with the protein pore α-hemolysin (αHL) is presented. Unique modulating current signatures are observed for duplexes containing the CC and CA mismatches when the mismatch site in the duplex is situated in proximity to the latch constriction of αHL during DNA residence inside the pore. The frequency and current amplitude of the modulation states are dependent on the mismatch type (CC or CA) permitting easy discrimination of these mismatches from one another, and from a fully complementary duplex that exhibits no modulation. We attribute the modulating current signatures to base flipping and subsequent interaction with positively charged lysine residues at the latch constriction of αHL. Our hypothesis is supported by the extended residence times of DNA duplexes within the pore when a mismatch is in proximity to the latch constriction, and by the loss of the two-state current signature in low pH buffers (<6.3), where the protonation of one of the cytosine bases increases the stability of the intrahelical state.

Exploring the nucleation of gas bubbles at interfaces is of fundamental interest. Herein, we report the nucleation of individual N2 nanobubbles at Pt nanodisk electrodes (6–90 nm) via the irreversible electrooxidation of hydrazine (N2H4 → N2 + 4H+ + 4e–). The nucleation and growth of a stable N2 nanobubble at the Pt electrode is indicated by a sudden drop in voltammetric current, a consequence of restricted mass transport of N2H4 to the electrode surface following the liquid-to-gas phase transition. The critical surface concentration of dissolved N2 required for nanobubble nucleation, CN2,criticals, obtained from the faradaic current at the moment just prior to bubble formation, is measured to be ∼0.11 M and is independent of the electrode radius and the bulk N2H4 concentration. Our results suggest that the size of stable gas bubble nuclei depends only on the local concentration of N2 near the electrode surface, consistent with previously reported studies of the electrogeneration of H2 nanobubbles. CN2,criticals is ∼160 times larger than the N2 saturation concentration at room temperature and atmospheric pressure. The residual current for N2H4 oxidation after formation of a stable N2 nanobubble at the electrode surface is proportional to the N2H4 concentration as well as the nanoelectrode radius, indicating that the dynamic equilibrium required for the existence of a stable N2 nanobubble is determined by N2H4 electrooxidation at the three phase contact line.

Measured apparent activation energies, EA, of ion transport (K+ and Cl–) in conical glass nanopores are reported as a function of applied voltage (−0.5 to 0.5 V), pore size (20–2000 nm), and electrolyte concentration (0.1–50 mM). EA values for transport within an electrically charged conical glass nanopore differ from the bulk values due to the voltage and temperature-dependent distribution of the ions within the double layer. Remarkably, nanopores that display ion current rectification also display a large decrease in EA under accumulation mode conditions (at applied negative voltages versus an external ground) and a large increase in EA under depletion mode conditions (at positive voltages). Finite element simulations based on the Poisson–Nernst–Planck model semiquantitatively predict the measured temperature-dependent conductivity and dependence of EA on applied voltage. The results highlight the relationships between the distribution of ions with the nanopore, ionic current, and EA and their dependencies on pore size, temperature, ion concentration, and applied voltage.

Coulter counters measure the size of particles in solution by passing them through an orifice and measuring a resistive pulse, i.e., a drop in the ionic current flowing between two electrodes placed on either side of the orifice. The magnitude of the pulse gives information on the size of the particle; however, resolution is limited by variability in the path of the translocation, due to the Brownian motion of the particle. We present a simple yet powerful modified Coulter counter that uses programmable data acquisition hardware to switch the voltage after sensing the resistive pulse of a nanoparticle passing through the orifice of a nanopipet. Switching the voltage reverses the direction of the driving force on the particle and, when this detect–switch cycle is repeated, allows us to pass an individual nanoparticle through the orifice thousands of times. By measuring individual particles more than 100 times per second we rapidly determine the distribution of the resistive pulses for each particle, which allows us to accurately determine the mean pulse amplitude and deliver considerably improved size resolution over a conventional Coulter counter. We show that single polystyrene nanoparticles can be shuttled back and forth and monitored for minutes, leading to a precisely determined mean blocking current equating to sub-angstrom size resolution.Keywords: Coulter method; nanoparticles; nanopipet; nanopore; particle sizing; resistive-pulse analysis;

Ion transport near an electrically charged electrolyte/electrode interface is a fundamental electrochemical phenomenon that is important in many electrochemical energy systems. We investigated this phenomenon using lithographically fabricated thin-layer electrochemical cells comprising two Pt planar electrodes separated by an electrolyte of nanometer thickness (50–200 nm). By exploiting redox cycling amplification, we observed the influence of the electric double layer on transport of a charged redox couple within the confined electrolyte. Nonclassical steady-state peak shaped voltammograms for redox cycling of the ferrocenylmethyltrimethylammonium redox couple (FcTMA+/2+) at low concentrations of supporting electrolyte (≤10 mM) results from electrostatic interactions between the redox ions and the charged Pt electrodes. This behavior contrasts to sigmoidal voltammograms with a diffusion-limited plateau observed in the same electrochemical cells in the presence of sufficient electrolyte to screen the electrode surface charge (200 mM). Moreover, steady-state redox cycling was depressed significantly within the confined electrolyte as the supporting electrolyte concentration was decreased or as the cell thickness was reduced. The experimental results are in excellent agreement with predictions from finite-element simulations coupling the governing equations for ion transport, electric fields, and the redox reactions. Double layer effects on ion transport are generally anticipated in highly confined electrolyte and may have implications for ion transport in thin layer and nanoporous energy storage materials.Keywords: electric double layer; electrostatic depletion; finite-element simulations; redox cycling; thin-layer electrochemical cells;

Resistive-pulse sensing has generated considerable interest as a technique for characterizing nanoparticle suspensions. The size, charge, and shape of individual particles can be estimated from features of the resistive pulse, but the technique suffers from an inherent variability due to the stochastic nature of particles translocating through a small orifice or channel. Here, we report a method, and associated automated instrumentation, that allows repeated pressure-driven translocation of individual particles back and forth across the orifice of a conical nanopore, greatly reducing uncertainty in particle size that results from streamline path distributions, particle diffusion, particle asphericity, and electronic noise. We demonstrate ∼0.3 nm resolution in measuring the size of nominally 30 and 60 nm radius Au nanoparticles of spherical geometry; Au nanoparticles in solution that differ by ∼1 nm in radius are readily distinguished. The repetitive translocation method also allows differentiating particles based on surface charge density, and provides insights into factors that determine the distribution of measured particle sizes.Keywords: Coulter method; nanoparticles; nanopore; particle sizing; resistive-pulse analysis;

The α-hemolysin (α-HL) nanopore can detect DNA strands under an electrophoretic force via many regions of the channel. Our laboratories previously demonstrated that trapping duplex DNA in the vestibule of wild-type α-HL under force could distinguish the presence of an abasic site compared to a G:C base pair positioned in the latch zone at the top of the vestibule. Herein, a series of duplexes were probed in the latch zone to establish if this region can detect more subtle features of base pairs beyond the complete absence of a base. The results of these studies demonstrate that the most sensitive region of the latch can readily discriminate duplexes in which one G:C base pair is replaced by an A:T. Additional experiments determined that while neither 8-oxo-7,8-dihydroguanine nor 7-deazaguanine opposite C could be differentiated from a G:C base pair, in contrast, the epigenetic marker 5-methylcytosine, when present in both strands of the duplex, yielded new blocking currents when compared to strands with unmodified cytosine. The results are discussed with respect to experimental design for utilization of the latch zone of α-HL to probe specific regions of genomic samples.Keywords: base pair detection; duplex DNA; epigenetic marker detection; latch zone; α-hemolysin;

We report the electrochemical generation of a single hydrogen bubble within the cavity of a recessed Pt nanopore electrode. The recessed Pt electrode is a conical pore in glass that contains a micrometer-scale Pt disk (1–10 μm radius) at the nanopore base and a nanometer-scale orifice (10–100 nm radius) that restricts diffusion of electroactive molecules and dissolved gas between the nanopore cavity and bulk solution. The formation of a H2 bubble at the Pt disk electrode in voltammetric experiments results from the reduction of H+ in a 0.25 M H2SO4 solution; the liquid-to-gas phase transformation is indicated in the voltammetric response by a precipitous decrease in the cathodic current due to rapid bubble nucleation and growth within the nanopore cavity. Finite element simulations of the concentration distribution of dissolved H2 within the nanopore cavity, as a function of the H+ reduction current, indicate that H2 bubble nucleation at the recessed Pt electrode surface occurs at a critical supersaturation concentration of ∼0.22 M, in agreement with the value previously obtained at (nonrecessed) Pt disk electrodes (∼0.25 M). Because the nanopore orifice limits the diffusion of H2 out of the nanopore cavity, an anodic peak corresponding to the oxidation of gaseous and dissolved H2 trapped in the recessed cavity is readily observed on the reverse voltammetric scan. Integration of the charge associated with the H2 oxidation peak is found to approach that of the H+ reduction peak at high scan rates, confirming the assignment of the anodic peak to H2 oxidation. Preliminary results for the electrochemical generation of O2 bubbles from water oxidation at a recessed nanopore electrode are consistent with the electrogeneration of H2 bubbles.

This paper describes a fundamental study of the effect of electrostatic interactions on the resistive pulse waveshape associated with translocation of charged nanoparticles through a conical-shaped, charged glass nanopore. In contrast to single-peak resistive pulses normally associated with resistive-pulse methods, biphasic pulses, in which the normal current decrease is preceded by a current increase, were observed in the current–time recordings when a high negative potential (lower than −0.4 V) is applied between the pore interior and the external solution. The biphasic pulse is a consequence of the offsetting effects of an increased ion conductivity induced by the surface charge of the translocating particle and the current decrease due to the volume exclusion of electrolyte solution by the particle. Finite-element simulations based on the coupled Poisson–Nernst–Planck equations and a particle trajectory calculation successfully capture the evolution of the waveshape from a single resistive pulse to a biphasic response as the applied voltage is varied. The simulation results demonstrate that the surface charges of the nanopore and the particle are responsible for the voltage-dependent shape evolution. Additionally, the use of high ionic strength solution or high pressures to drive particle translocation was found to eliminate the biphasic response. The former is due to the screening of the electrical double layer, while the latter results from the solution flow preventing formation of an equilibrium double layer ion distribution within the nanopore, similar to the previously reported elimination of ion current rectification when solution flows through a nanopore.

Studies on the interaction of hairpin DNA with the α-hemolysin (α-HL) nanopore have determined hairpin unzipping kinetics, thermodynamics, and sequence-dependent DNA/protein interactions. Missing from these results is a systematic study comparing the unzipping process for fishhook (one-tail) vs internal (two-tail) hairpins when they are electrophoretically driven from the cis to the trans side of α-HL via a 30-mer single-stranded tail. In the current studies, fishhook hairpins showed long unzipping times with one deep blockage current level. In contrast, the internal hairpins demonstrated relatively fast unzipping and a characteristic pulse-like current pattern. These differences were further explored with respect to stem length and sequence context. Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins. Interestingly, these studies were able to resolve a current difference of ∼6% between hairpin DNA immobilized in the nanopore waiting to unzip vs the translocating unzipped DNA, with the latter showing a deeper current blockage level. This demonstration of different currents for immobilized and translocating DNA has not been described previously. These results were interpreted as fishhook hairpins unzipping inside the vestibule, while the internal hairpins unzip outside the vestibule of α-HL. Lastly, we used this knowledge to study the unzipping of a long double-stranded DNA (>50 base pairs) outside the vestibule of α-HL. The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids.

Redox cycling in nanometer-wide thin-layer cells holds great promise in ultrasensitive voltammetric detection and in probing fast heterogeneous electron-transfer kinetics. Quantitative understanding of the influence of the nanometer gap distance on the redox processes in the thin-layer cells is of crucial importance for reliable data analysis. We present theoretical consideration on the voltammetric behaviors associated with redox cycling of electroactive molecules between two electrodes separated by nanometer widths. Emphasis is placed on the weakness of the commonly used Butler–Volmer theory and the classic Marcus–Hush theory in describing the electrochemical heterogeneous electron-transfer kinetics at potentials significantly removed from the formal potential of redox moieties and, in addition, the effect of the electric-double-layer on the electron-transfer kinetics and mass transport dynamics of charged redox species. The steady-state voltammetric responses, obtained by using the Butler–Volmer and Marcus–Hush models and that predicted by the more realistic electron-transfer kinetics formulism, which is based on the alignments of the density of states between the electrode continuum and the Gaussian distribution of redox agents, and by inclusion of the electric-double-layer effect, are compared through systematic finite element simulations. The effect of the gap width between the electrodes, the standard rate constant and reorganization energy for the electron-transfer reactions, and the charges of the redox moieties are considered. On the basis of the simulation results, the reliability of the conventional voltammetric analysis based on the Butler–Volmer kinetic model and diffusion transport equations is discussed for nanometer-wide thin-layer cells.Keywords: electric double layer; finite element simulation; heterogeneous electron transfer; nanogap effects; thin-layer cells; voltammetric responses;

The effect of an electrolyte cation on the unzipping of furan-containing double-stranded DNA in an α-hemolysin (αHL) nanopore is described. The current through an open αHL channel increases in proportion to the ion mobility. However, the ionic current measured during residence of a DNA duplex inside of the protein pore shows a more complex dependence on the choice of cation, indicating that the current measured during DNA residence in the pore is modulated by the specific interactions of the cations with the DNA and/or αHL. The residence time (stability) of the DNA duplex inside of the pore prior to unzipping is also highly dependent on the cation, in striking contrast to the small variation in duplex stability (as measured by the melting temperature) in bulk electrolyte solution. A missing base in DNA can be detected in the latch region of αHL with optimal current resolution in RbCl, while optimal time resolution is possible in LiCl.Keywords: cations; DNA damage; latch zone; nanopore; α-hemolysin;

Single H2 nanobubble nucleation is studied at Pt nanodisk electrodes of radii less than 50 nm, where H2 is produced through electrochemical reduction of protons in a strong acid solution. The critical concentration of dissolved H2 required for nanobubble nucleation is measured to be ∼0.25 M. This value is ∼310 times larger than the saturation concentration at room temperature and pressure and was found to be independent of acid type (e.g., H2SO4, HCl, and H3PO4) and nanoelectrode size. The effects of different surfactants on H2 nanobubble nucleation are consistent with the classic nucleation theory. As the surfactant concentration in H2SO4 solution increases, the solution surface tension decreases, resulting in a lower nucleation energy barrier and consequently a lower supersaturation concentration required for H2 nanobubble nucleation. Furthermore, amphiphilic surfactant molecules accumulate at the H2/solution interface, hindering interfacial H2 transfer from the nanobubble into the solution; consequently, the residual current decreases with increasing surfactant concentration.Keywords: nanobubble; nanoelectrodes; nucleation; supersaturation; surface tension;

A solid-state nanopore separating two aqueous solutions containing different concentrations of KCl is demonstrated to exhibit negative differential resistance (NDR) when a constant pressure is applied across the nanopore. NDR refers to a decrease in electrical current when the voltage applied across the nanopore is increased. NDR results from the interdependence of solution flow (electroosmotic and pressure-engendered) with the distributions of K+ and Cl– within the nanopore. A switch from a high-conductivity state to a low-conductivity state occurs over a very narrow voltage window (<2 mV) that depends on the nanopore geometry, electrolyte concentration, and nanopore surface charge density. Finite element simulations based on a simultaneous solution of the Navier–Stokes, Poisson, and Nernst–Planck equations demonstrate that NDR results from a positive feedback mechanism between the ion distributions and electroosmotic flow, yielding a true bistability in fluid flow and electrical current at a critical applied voltage, i.e., the NDR “switching potential”. Solution pH and Ca2+ were separately employed as chemical stimuli to investigate the dependence of the NDR on the surface charge density. The NDR switching potential is remarkably sensitive to the surface charge density, and thus to pH and the presence of Ca2+, suggesting possible applications in chemical sensing.Keywords: electrical feedback and bistability; finite element simulation; nanopore; negative differential resistance; sensor

The steady-state i–V behavior of an electrochemical cell comprising two polarizable Au microelectrodes in a mixed redox solution containing reversible and soluble redox species (FcCH2OH and Ru(NH)63+) is reported. Analytical solutions describing the cell i–V response in the absence of a well-poised reference electrode are presented, and these expressions are shown to agree with the experimental i–V results. Depending on the relative areas of the electrodes, the i–V response in the absence of a reference electrode is particularly sensitive to the presence of redox impurities. Specifically, we demonstrate that FcCH2OH+ impurity in the solution yields diffusion limited current plateaus that are equal to the currents for the primary redox species present in nearly 100-fold excess.Highlights► We describe the voltammetric response of a cell comprising two polarizable microelectrodes. ► The computed i–V response is in excellent agreement with experimental data. ► Impurity species dominate the behavior for cells with electrodes of unequal areas.

The influence of DNA duplex structural destabilization introduced by a single base-pair modification was investigated by nanopore measurements. A series of 11 modified base pairs were introduced into the context of an otherwise complementary DNA duplex formed by a 17-mer and a 65-mer such that the overhanging ends comprised poly(dT)23 tails, generating a representative set of duplexes that display a range of unzipping mechanistic behaviors and kinetic stabilities. The guanine oxidation products 8-oxo-7,8-dihydroguanine (OG), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) were paired with either cytosine (C), adenine (A), or 2,6-diaminopurine (D) to form modified base pairs. The mechanism and kinetic rate constants of duplex dissociation were determined by threading either the 3′ or 5′ overhangs into an α-hemolysin (α-HL) channel under an electrical field and measuring the distributions of unzipping times at constant force. In order of decreasing thermodynamic stability (as measured by duplex melting points), the rate of duplex dissociation increases, and the mechanism evolves from a first-order reaction to two sequential first-order reactions. These measurements allow us to rank the kinetic stability of lesion-containing duplexes relative to the canonical G:C base pair in which the OG:C, Gh:C, and Sp:C base pairs are, respectively, 3–200 times less stable. The rate constants also depend on whether unzipping was initiated from the 3′ versus 5′ side of the duplex. The kinetic stability of these duplexes was interpreted in terms of the structural destabilization introduced by the single base-pair modification. Specifically, a large distortion of the duplex backbone introduced by the presence of the highly oxidized guanine products Sp and Gh leads to a rapid two-step unzipping. The number of hydrogen bonds in the modified base pair plays a lesser role in determining the kinetics of duplex dissociation.

The electrochemical generation of individual H2 nanobubbles at Pt nanodisk electrodes immersed in a 0.5 M H2SO4 solution is reported. A sudden drop in current associated with the transport-limited reduction of protons is observed in the i–V response at Pt nanodisk electrodes with radii of less than 50 nm. This decrease in current (∼95% blockage) corresponds to the formation of a single H2 nanobubble attached to the nanoelectrode that blocks proton transport to the surface. The current at which nanobubble formation occurs, inbp, is independent of scan rate and H2SO4 concentration (for [H2SO4] > 0.1 M), indicating a critical concentration profile of electrogenerated H2 required to nucleate a nanobubble. Finite element simulation based on Fick’s first law, combined with the Young–Laplace equation and Henry’s law, indicates that the concentration of H2 near the nanoelectrode surface at inbp exceeds the saturation concentration necessary to generate a nanobubble with a size comparable to the electrode size. The rapid dissolution of the nanobubble due to the high inner Laplace pressure is precisely balanced by the electrogeneration of H2 at the partially exposed Pt surface, resulting in a dynamically stabilized nanobubble. Preliminary measurements of the i–t response during nanobubble formation indicate a two-step nucleation and growth mechanism with time scales on the order of 100 μs (or less) and ∼1 ms, respectively.

The unzipping kinetics for lesion-containing DNA duplexes was studied in an α-hemolysin (α-HL) nanopore. The lesion of focus was the guanine two-electron oxidation product, 8-oxo-7,8-dihydroguanine (OG), and its further oxidation products, the hydantoins guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). The voltage-driven unzipping of individual duplex DNA molecules with symmetrical overhangs was carried out by pulling one strand of the duplex through the α-HL channel using an electrical field. Entry from the 3′ or 5′ end produced distinct current blockages, allowing directional effects on unzipping kinetics to be investigated. We find that the strand dissociation of complementary duplexes or duplexes containing the slightly destabilizing lesion OG follows a first-order kinetic model, while opening of duplexes that contain the highly destabilizing lesions Gh or Sp is described by two sequential first-order reactions, in which the intermediate state is proposed to correspond to the duplex unzipped to the lesion site within the channel. The rate constants for strand separation of the duplexes containing single lesions were obtained from kinetic model fits to histograms of unzipping duration. For all duplexes, the rate constants for strand separation displayed a significant dependence on the direction of entry into the nanopore. For duplexes containing Gh, truncated duplexes were used to assign the measured rate constants for the first and second unzipping steps of symmetrically designed duplexes.

Electrical current signatures of DNA adducts were investigated during immobilization of strands inside the membrane-bound α-hemolysin ion channel. The current blockages produced by these adducts were found to depend on both size and shape, providing insights into the DNA–protein interactions and the size limitation of bulky adducts to be translocated.

DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na+ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li+, Na+ or K+) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement.

The influence of diffusional motion on the capture and release of individual nanoparticles as they are driven through a conical-shaped glass nanopore membrane (GNM) by pressure-induced flow is reported. In these experiments, one to several hundred particles are driven through the orifice of the nanopore. Following the initial translocation, the pressure is reversed and the particles are driven through the GNM orifice in the reverse direction. The resistive-pulse technique is used to monitor the temporal sequence of particle capture and release translocations. The size of the particles (120–160 nm) and the direction of translocation can be determined from the pulse amplitude and shape. The stochastic influence of diffusion on particle trajectories has been investigated, including instantaneous transfer rate, release probability, and cumulative release success rate. We demonstrate that the sequence of particle translocations in the capture step (a, b, c... where the letters represent different particles) is largely preserved and can be read out by resistive-pulse signature during the release translocations (...c, b, a). The observed stochastic events are in good agreement with a convective diffusion model of particle trajectory within the confined geometry of the nanopore. The pressure-reversal technique opens new avenues for chemical analysis of particles using resistive-pulse methods.Keywords: capture and release; Coulter counter; diffusion; nanopore; resistive pulse

Liquid-phase negative differential resistance (NDR) is observed in the i–V behavior of a conical nanopore (∼300 nm orifice radius) in a glass membrane that separates an external low-conductivity 5 mM KCl solution of dimethylsulfoxide (DMSO)/water (v/v 3:1) from an internal high-conductivity 5 mM KCl aqueous solution. NDR appears in the i–V curve of the negatively charged nanopore as the voltage-dependent electro-osmotic force opposes an externally applied pressure force, continuously moving the location of the interfacial zone between the two miscible solutions to a position just inside the nanopore orifice. An ∼80% decrease in the ionic current occurs over less that a ∼10 mV increase in applied voltage. The NDR turn-on voltage was found to be tunable over a ∼1 V window by adjusting the applied external pressure from 0 to 50 mmHg. Finite-element simulations based on solution of Navier–Stokes, Poisson, and convective Nernst–Planck equations for mixed solvent electrolytes within a negatively charged nanopore yield predictions of the NDR behavior that are in qualitative agreement with the experimental observations. Applications in chemical sensing of a tunable, solution-based electrical switch based on the NDR effect are discussed.Keywords: electro-osmosis; ion current; nanopore; negative differential resistance; pressure

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, Tc. All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the Tc were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above Tc. The results provide insights into the deformation and translocation of individual liposomes through a porous material.

Translocation measurements of intact DNA strands with the ion channel α-hemolysin (α-HL) are limited to single-stranded DNA (ssDNA) experiments as the dimensions of the channel prevent double-stranded DNA (dsDNA) translocation; however, if a short oligodeoxynucleotide is used to interrogate a longer ssDNA strand, it is possible to unzip the duplex region when it is captured in the α-HL vestibule, allowing the longer strand to translocate through the α-HL channel. This unzipping process has a characteristic duration based on the stability of the duplex. Here, ion channel recordings are used to detect the presence and relative location of the oxidized damage site 8-oxo-7,8-dihydroguanine (OG) in a sequence-specific manner. OG engages in base pairing to C or A with unique stabilities relative to native base Watson–Crick pairings, and this phenomenon is used here to engineer probe sequences (10–15mers) that, when base-paired with a 65mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (Tm). Unzipping times also depend on the direction from which the duplex enters the vestibule if the stabilities of leading base pairs at the ends of the duplex are significantly different. It is shown here that the presence of a single DNA lesion can be distinguished from an undamaged sequence and that the relative location of the damage site can be determined based on the duration of duplex unzipping.

Ion current rectification that occurs in conical-shaped glass nanopores in low ionic strength solutions is shown to be dependent on the rate of pressure-driven electrolyte flow through the nanopore, decreasing with increasing flow rate. The dependence of the i–V response on pressure is due to the disruption of cation and anion distributions at equilibrium within the nanopore. Because the flow rate is proportional to the third power of the nanopore orifice radius, the pressure-driven flow can eliminate rectification in nanopores with radii of ∼200 nm but has a negligible influence on rectification in a smaller nanopore with a radius of ∼30 nm. The experimental results are in qualitative agreement with predictions based on finite-element simulations used to solve simultaneously the Nernst–Planck, Poisson, and Navier–Stokes equations for ion fluxes in a moving electrolyte within a conical nanopore.

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.

This report presents a fundamental study of nanoparticle transport phenomena in conical-shaped pores contained within glass membranes. The electrophoretic translocation of charged polystyrene (PS) nanoparticles (80- and 160-nm-radius) was investigated using the Coulter counter principle (or “resistive-pulse” method) in which the time-dependent nanopore current is recorded as the nanoparticle is driven across the membrane. Particle translocation through the conical-shaped nanopore results in a direction-dependent and asymmetric triangular-shaped resistive pulse. Because the sensing zone of conical-shaped nanopores is localized at the orifice, the translocation of nanoparticles through this zone is very rapid, resulting in pulse widths of ∼200 μs for the nanopores used in this study. A linear dependence between translocation rate and nanoparticle concentration was observed from 107 to 1011 particles/mL for both 80- and 160-nm-radius particles, and the magnitude of the resistive pulse scaled approximately in proportion to the particle volume. A finite-element simulation based on continuum theory to compute ion fluxes was combined with a dynamic electric force-based nanoparticle trajectory calculation to compute the position- and time-dependent nanoparticle velocity as the nanoparticle translocates through the conical-shaped nanopore. The computational results were used to compute the resistive pulse current–time response for conical-shaped pores, allowing comparison between experimental and simulated pulse heights and translocation times. The simulation and experimental results indicate that nanoparticle size can be differentiated based on pulse height, and to a lesser extent based on translocation time.

The resistive-pulse sensing technique was used to investigate the deformation and dehydration of individual 570 nm radius poly(N-isopropylacrylamide-co-acrylic acid) microgels during their translocation through a glass membrane containing a single conical nanopore with orfice radii ranging from 200 to 700 nm. Microgel translocation rates were found to be dependent on both the applied pressure and the pore radius, and a translocation threshold pressure was found to be a function of the pore size. Importantly, current–time traces resulting from translocation events demonstrate changes in the conductivity of the microgel, due to compression and partial dehydration during translocation. A minimum nanopore-to-microgel radius ratio of ∼0.4 is observed for translocation, suggesting a theoretical limit imposed by the compressibility of the microgel and Columbic repulsion with the pore walls. Our results provide fundamental insight into microgels including their compressibility and conductivity, as well as the ability of soft particles to permeate porous membranes and pass through voids of dimensions smaller than the particles themselves.

Deformation of 570 nm radius poly(N-isopropylacrylamide-co-acrylic acid) microgels passing through individual 375−915 nm radius nanopores in glass has been investigated by the resistive-pulse method. Particle translocation through nanopores of dimensions smaller than the microgel yields electrical signatures reflecting the dynamics of microgel deformation. Translocation rates, and event duration and peak shape, are functions of the conductivities of microgel and electrolyte. Our results demonstrate that nanopore resistive-pulse methods provide new fundamental insights into microgel permeation through porous membranes.

Experimental, theoretical, and finite-element simulation investigations of the pressure-driven translocation of nanoparticles across a conical-shaped glass nanopore membrane (GNM) are presented. The translocation of the particles is experimentally analyzed by measuring the shape of transient pulses when current flowing between two Ag/AgCl electrodes, located on opposite sides of the GNM, is momentarily interrupted as a particle passes through the nanopore. Asymmetric triangular-shaped resistive pulses are observed for the translocation of 120 nm radius particles through a 210 nm radius GNM at a transmembrane pressure between −2 and −160 mmHg. A linear dependence is observed between the particle translocation frequency and the applied pressure. Analytical theory and finite-element simulation for pressure-driven flow through a conical-shaped pore were developed to compute the volumetric flow rate, the position-dependent particle velocity, and the particle translocation frequency. The translocation frequencies computed from theory and simulation as a function of pressure were found to be in agreement with experimental observations. The particle translocation pulse shape was also computed by a combination of finite-element simulation with a dynamic nanoparticle trajectory calculation. Surprisingly, the simulations demonstrate that pulse widths are nearly independent of the nanopore radius. The independence of pulse width on nanopore size is a consequence of both the solution velocity and the width of the electrical sensing zone increasing in proportion to the orifice radius for conical-shaped pores.

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.

A method is described for fabricating 25−75 μm thick fused quartz membranes containing a single conical shaped nanopore (orifice radius ranging from 10 to 1000 nm). The quartz nanopore membrane (QNM) provides an excellent solid support structure for lipid bilayers in ion channel recordings due to the large electrical resistivity of fused quartz. Electrical measurements demonstrate that the leakage current through 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers suspended across a 500−1000 nm radius QNM orifice is immeasurably small, corresponding to a bilayer resistance greater than 1012 ohms. Translocation of single-stranded DNA oligomers (poly dA 50-mer and poly dA 20-mer) through a protein ion channel (α-hemolysin) reconstituted in a DPhPC bilayer suspended across the QNM orifice is demonstrated.

Alternating current, phase-sensitive stochastic detection using between 1 and 26 α-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 αHL channels are noninteracting and that significant gains in sensitivity are possible without sacrificing the simplicity of single-molecule detection strategies.

Translocation of single-stranded DNA through α-hemolysin (α-HL) channels is investigated in glycerol/water mixtures containing 1 M KCl. Experiments using glass nanopore membranes as the lipid bilayer support demonstrate that the translocation velocities of poly(deoxyadenylic acid), poly(deoxycytidylic acid), and poly(deoxythymidylic acid) 50-mers are decreased by a factor of ∼20 in a 63/37 (vol %) glycerol/water mixture, relative to aqueous solutions. The ion conductance of α-HL and the entry rate of the polynucleotides into the protein channel also decrease with increasing viscosity. Precise control of translocation parameters by adjusting viscosity provides a potential means to improve sequencing methods based on ion channel recordings.

The mechanism of molecule-based electrostatic gating of redox fluxes at conical glass nanopore (GNP) electrodes has been investigated using finite-element simulations. The results demonstrate that the fluxes of cationic redox molecules through the nanopore orifice can be reduced to negligibly small values when the surface charge of the nanopore is switched from a negative to a positive value. Electrostatic charge reversal can be affected by ionization of surface-bound moieties in response to environmental stimuli (e.g., photoionization or acid protonation), but only if the negative charge of the glass is included in the analysis. Numerical simulations of the responses of GNP electrodes are based on a simultaneous solution of the Poisson and Nernst−Planck equations and are in excellent agreement with our previously reported experimental results for electrostatic gating of the fluxes of Ru(NH3)63+ and Fe(bpy)32+ at GNP electrodes with orifice radii between 15 and 100 nm. The gating mechanism is discussed in terms of three components: (1) migration of ionic redox species in the depletion layer adjacent to the electrode surface; (2) migrational transport along the charged pore walls; (3) electrostatic rejection of charged molecules at the pore orifice. The numerical results indicate that all three components are operative, but that ion migration along the pore walls is dominant.

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.

The steady-state i–V behavior of an electrochemical cell comprising two polarizable Au microelectrodes in a mixed redox solution containing reversible and soluble redox species (FcCH2OH and Ru(NH)63+) is reported. Analytical solutions describing the cell i–V response in the absence of a well-poised reference electrode are presented, and these expressions are shown to agree with the experimental i–V results. Depending on the relative areas of the electrodes, the i–V response in the absence of a reference electrode is particularly sensitive to the presence of redox impurities. Specifically, we demonstrate that FcCH2OH+ impurity in the solution yields diffusion limited current plateaus that are equal to the currents for the primary redox species present in nearly 100-fold excess.Highlights► We describe the voltammetric response of a cell comprising two polarizable microelectrodes. ► The computed i–V response is in excellent agreement with experimental data. ► Impurity species dominate the behavior for cells with electrodes of unequal areas.

Nanopores have been investigated as a simple and label-free tool to characterize DNA nucleotides when a ssDNA strand translocates through the constriction of the pore. Here, a wild-type α-hemolysin protein nanopore was used to monitor DNA repair enzyme activity based on base-specific interactions of dsDNA with the vestibule constriction “latch”, a previously unrecognized sensing zone in α-hemolysin specific for dsDNA structure. The presence of a single abasic site within dsDNA that is in proximity to the latch zone (±2 nucleotides) results in a large increase in ion channel current, allowing accurate quantitation of the kinetics of base repair reactions involving an abasic site product. Taking advantage of the high resolution for abasic site recognition, the rate of uracil-DNA glycosylase hydrolysis of the N-glycosidic bond, converting 2′-deoxyuridine in DNA to an abasic site, was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the ion channel vestibule. Our work suggests use of the nanopore as an enzymology tool and provides a means to identify single base structural changes in dsDNA.

Electrical current signatures of DNA adducts were investigated during immobilization of strands inside the membrane-bound α-hemolysin ion channel. The current blockages produced by these adducts were found to depend on both size and shape, providing insights into the DNA–protein interactions and the size limitation of bulky adducts to be translocated.