Nanoscale Quantitative Imaging of Single Nuclear Pore Complexes by Scanning Electrochemical Microscopy.

The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.

Transient theory for scanning electrochemical microscopy of biological membrane transport: uncovering membrane-permeant interactions.

Scanning electrochemical microscopy (SECM) has emerged as a powerful method to quantitatively investigate the transport of molecules and ions across various biological membranes as represented by living cells. Advantageously, SECM allows for the in situ and non-destructive imaging and measurement of high membrane permeability under simple steady-state conditions, thereby facilitating quantitative data analysis. The SECM method, however, has not provided any information about the interactions of a transported species, i.e., a permeant, with a membrane through its components, e.g., lipids, channels, and carriers. Herein, we propose theoretically that SECM enables the quantitative investigation of membrane-permeant interactions by employing transient conditions. Specifically, we model the membrane-permeant interactions based on a Langmuir-type isotherm to define the strength and kinetics of the interactions as well as the concentration of interaction sites. Finite element simulation predicts that each of the three parameters uniquely affects the chronoamperometric current response of an SECM tip to a permeant. Significantly, this prediction implies that all three parameters are determinable from an experimental chronoamperometric response of the SECM tip. Complimentarily, the steady-state current response of the SECM tip yields the overall membrane permeability based on the combination of the three parameters. Interestingly, our simulation also reveals the optimum strength of membrane-permeant interactions to maximize the transient flux of the permeant from the membrane to the tip.

Suppression of Resistive Coupling in Nanogap Electrochemical Cell: Resolution of Dual Pathways for Dopamine Oxidation.

A nanogap cell involves two working electrodes separated by a nanometer-wide solution to enable unprecedented electrochemical measurements. The powerful nanogap measurements, however, can be seriously interfered with by resistive coupling between the two electrodes to yield erroneous current responses. Herein, we employ the nanogap cell based on double carbon-fiber microelectrodes to suppress resistive coupling for the assessment of intrinsic current responses. Specifically, we modify a commercial bipotentiostat to compensate the Ohmic potential drop shared by the two electrodes through the common current pathway with a fixed resistance in the solution. Resistive coupling through both non-Faradaic and Faradaic processes is suppressed to eliminate erroneous current responses. Our approach is applied to investigate the mechanism of dopamine oxidation at carbon-fiber microelectrodes as important electrochemical sensors for the crucial neurotransmitter. Resistive coupling is suppressed to manifest the intrinsic current responses based on the oxidation of both adsorbed and non-adsorbed forms of dopamine to the respective forms of dopamine-o-quinone. The simultaneous dual oxidation pathways are observed for the first time and can be mediated through either non-concerted or concerted mechanisms of adsorption-coupled electron transfer. The two mechanisms are not discriminated for the two-electron oxidation of dopamine because it can not be determined whether the intermediate, dopamine semi-quinone, is adsorbed on the electrode surface. Significantly, our approach will be useful to manifest intrinsic current responses without resistive coupling for nanogaps and microgaps, which are too narrow to eliminate the common solution resistance by optimizing the position of a reference electrode.

Nanogap-Resolved Adsorption-Coupled Electron Transfer by Scanning Electrochemical Microscopy: Implications for Electrocatalysis.

Here, we demonstrate for the first time that the mechanism of adsorption-coupled electron-transfer (ACET) reactions can be identified experimentally. The electron transfer (ET) and specific adsorption of redox-active molecules are coupled in many electrode reactions with practical importance and fundamental interest. ACET reactions are often represented by a concerted mechanism. In reductive adsorption, an oxidant is simultaneously reduced and adsorbed as a reductant on the electrode surface through the ACET step. Alternatively, the non-concerted mechanism mediates outer-sphere reduction and adsorption separately when the reductant adsorption is reversible. In electrocatalysis, reversibly adsorbed reductants are ubiquitous and crucial intermediates. Moreover, electrocatalysis is complicated by the mixed mechanism based on simultaneous ACET and outer-sphere ET steps. In this work, we reveal the non-concerted mechanism for ferrocene derivatives adsorbed at highly oriented pyrolytic graphite as simple models. We enable the transient voltammetric mode of nanoscale scanning electrochemical microscopy (SECM) to kinetically control the adsorption step, which is required for the discrimination of non-concerted, concerted, and mixed mechanisms. Experimental voltammograms are compared with each mechanism by employing finite element simulation. The non-concerted mechanism is supported to indicate that the ACET step is intrinsically slower than its outer-sphere counterpart by at least four orders of magnitude. This finding implies that an ACET step is facilitated thermodynamically but may not be necessarily accelerated or catalyzed by the adsorption of the reductant. SECM-based transient voltammetry will become a powerful tool to resolve and understand electrocatalytic ACET reactions at the elementary level.

Simultaneous Intelligent Imaging of Nanoscale Reactivity and Topography by Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) enables reactivity and topography imaging of single nanostructures in the electrolyte solution. The in situ reactivity and topography, however, are convoluted in the real-time image, thus requiring another imaging method for subsequent deconvolution. Herein, we develop an intelligent mode of nanoscale SECM to simultaneously obtain separate reactivity and topography images of non-flat substrates with reactive and inert regions. Specifically, an ∼0.5 µm-diameter Pt tip approaches a substrate with an ∼0.15 µm-height active Au band adjacent to an ∼0.4 µm-wide slope of the inactive glass surface followed by a flat inactive glass region. The amperometric tip current versus tip-substrate distance is measured to observe feedback effects including redox-mediated electron tunneling from the substrate. The intelligent SECM software automatically terminates the tip approach depending on the local reactivity and topography of the substrate under the tip. The resultant short tip-substrate distances allow for non-contact and high-resolution imaging in contrast to other imaging modes based on approach curves. The numerical post-analysis of each approach curve locates the substrate under the tip for quantitative topography imaging and determines the tip current at a constant distance for topography-independent reactivity imaging. The nanoscale grooves are revealed by intelligent topography SECM imaging as compared to scanning electron microscopy and atomic force microscopy without reactivity information and as unnoticed by constant-height SECM imaging owing to the convolution of topography with reactivity. Additionally, intelligent reactivity imaging traces abrupt changes in the constant-distance tip current across the Au/glass boundary, which prevents constant-current SECM imaging.

Nanoelectrochemical Study of Molecular Transport through the Nuclear Pore Complex.

The nuclear pore complex (NPC) is the proteinaceous nanopore that solely mediates the transport of both small molecules and macromolecules between the nucleus and cytoplasm of a eukaryotic cell to regulate gene expression. In this personal account, we introduce recent progress in our nanoelectrochemical study of molecular transport through the NPC. Our work represents the importance of chemistry in understanding and controlling of NPC-mediated molecular transport to enable the efficient and safe delivery of genetic therapeutics into the nucleus, thereby fundamentally contributing to human health. Specifically, we employ nanoscale scanning electrochemical microscopy to test our hypothesis that the nanopore of the NPC is divided by transport barriers concentrically into peripheral and central routes to efficiently mediate the bimodal traffic of protein transport and RNA export, respectively, through cooperative hydrophobic and electrostatic interactions.

Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) enables high-resolution imaging by examining the amperometric response of an ultramicroelectrode tip near a substrate. Spatial resolution, however, is compromised for nonflat substrates, where distances from a tip far exceed the tip size to avoid artifacts caused by the tip-substrate contact. Herein, we propose a new imaging mode of SECM based on real-time analysis of the approach curve to actively control nanoscale tip-substrate distances without contact. The power of this software-based method is demonstrated by imaging an insulating substrate with step edges using standard instrumentation without combination of another method for distance measurement, e.g., atomic force microscopy. An ∼500 nm diameter Pt tip approaches down to ∼50 nm from upper and lower terraces of a 500 nm height step edge, which are located by real-time theoretical fitting of an experimental approach curve to ensure the lack of electrochemical reactivity. The tip approach to the step edge can be terminated at <20 nm prior to the tip-substrate contact as soon as the theory deviates from the tip current, which is analyzed numerically afterward to locate the inert edge. The advantageous local adjustment of tip height and tip current at the final point of tip approach distinguishes the proposed imaging mode from other modes based on standard instrumentation. In addition, the glass sheath of the Pt tip is thinned to ∼150 nm to rarely contact the step edge, which is unavoidable and instantaneously detected as an abrupt change in the slope of approach curve to prevent damage of the fragile nanotip.

Probing High Permeability of Nuclear Pore Complexes by Scanning Electrochemical Microscopy: Ca2+ Effects on Transport Barriers.

The nuclear pore complex (NPC) solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell to play important biological and biomedical roles. However, it is not well-understood chemically how this biological nanopore selectively and efficiently transports various substances, including small molecules, proteins, and RNAs by using transport barriers that are rich in highly disordered repeats of hydrophobic phenylalanine and glycine intermingled with charged amino acids. Herein, we employ scanning electrochemical microscopy to image and measure the high permeability of NPCs to small redox molecules. The effective medium theory demonstrates that the measured permeability is controlled by diffusional translocation of probe molecules through water-filled nanopores without steric or electrostatic hindrance from hydrophobic or charged regions of transport barriers, respectively. However, the permeability of NPCs is reduced by a low millimolar concentration of Ca2+, which can interact with anionic regions of transport barriers to alter their spatial distributions within the nanopore. We employ atomic force microscopy to confirm that transport barriers of NPCs are dominantly recessed (∼80%) or entangled (∼20%) at the high Ca2+ level in contrast to authentic populations of entangled (∼50%), recessed (∼25%), and "plugged" (∼25%) conformations at a physiological Ca2+ level of submicromolar. We propose a model for synchronized Ca2+ effects on the conformation and permeability of NPCs, where transport barriers are viscosified to lower permeability. Significantly, this result supports a hypothesis that the functional structure of transport barriers is maintained not only by their hydrophobic regions, but also by charged regions.

Nanogap-Based Electrochemical Measurements at Double-Carbon-Fiber Ultramicroelectrodes.

Electrochemical measurements with unprecedentedly high sensitivity, selectivity, and kinetic resolution have been enabled by a pair of electrodes separated by a nanometer-wide gap. The fabrication of nanogap electrodes, however, requires extensive nanolithography or nanoscale electrode positioning, thereby preventing the full exploration of this powerful method in electrode design and application. Herein, we report the simple fabrication of double-carbon-fiber ultramicroelectrodes (UMEs) with nanometer-wide gaps not only to facilitate nanogap-based electrochemical measurements but also to gain high time resolution, signal-to-background ratio, and kinetic selectivity for dopamine against ascorbic acid. Specifically, ∼7 µm-diameter carbon fibers are inserted into a double-bore glass capillary, heat-pulled, and milled by focused ion-beam technology to yield ∼50 µm-long double-cylinder UMEs. The redox cycling of the Ru(NH3)63+/2+ couple across a nanogap between voltammetric generator and amperometric collector electrodes reaches quasi-steady states at fast scan rates of 100 V/s as demonstrated experimentally and even 1000 V/s as predicted theoretically. The transient background of the amperometric collector response is suppressed ∼100 times in comparison with that of the voltammetric generator response. Nanogap voltammograms based on the collector response against the cycled generator potential are quantitatively analyzed without background subtraction to reproducibly yield nanogap widths of ∼0.18 µm and a standard electron-transfer rate constant of 0.9 cm/s. Moreover, nanogap-mediated redox cycling can be initiated by dopamine oxidation at the generator electrode to largely improve the dopamine selectivity of the collector response against ascorbic acid, which is also oxidized at the generator electrode to immediately and irreversibly produce a redox-inactive species.

Voltammetric Measurement of Adsorption Isotherm for Ferrocene Derivatives on Highly Oriented Pyrolytic Graphite.

Reversible and specific adsorption of redox-active molecules from the electrolyte solution to the electrode surface is an important process and is often diagnosed by cyclic voltammetry (CV). The entire voltammogram, however, is rarely analyzed quantitatively, thereby completely missing or incorrectly extracting inherent information about the adsorption isotherm. Herein, we report CV measurements of the adsorption isotherm for ferrocene derivatives on the basal plane of highly oriented pyrolytic graphite (HOPG) to quantitatively understand the thermodynamics of ferrocene-HOPG and ferrocene-ferrocene interactions at HOPG/water interfaces. Specifically, reversible CV of (ferrocenylmethyl)trimethylammonium, ferrocenemethanol, and 1,1'-ferrocenedimethanol is obtained at 0.05-10 V/s to confirm that only reduced forms of ferrocene derivatives are adsorbed on HOPG. Finite element analysis of the entire voltammogram yields the Frumkin isotherm to separately parametrize ferrocene-HOPG and ferrocene-ferrocene interactions. Adsorption of all ferrocene derivatives is driven by similarly weak ferrocene-HOPG interactions with free energy changes of approximately -20 kJ/mol. Adsorption of ferrocenemethanol is strengthened by intermolecular hydrogen bonding, which is quantitatively represented by a free energy change of -8 kJ/mol for surface saturation and is qualitatively characterized by a pair of sharp adsorption and desorption peaks following a pair of diffusional peaks. By contrast, adsorption of (ferrocenylmethyl)trimethylammonium and 1,1'-ferrocenedimethanol remains weak because of electrostatic repulsion and weak hydrogen bonding, respectively, which correspond to the respective free energy changes of +0.7 and -3 kJ/mol for surface saturation. The unfavorable or weakly favorable intermolecular interactions broaden or narrow a diffusional peak during the forward scan, respectively, without yielding a post peak.

Self-Inhibitory Electron Transfer of the Co(III)/Co(II)-Complex Redox Couple at Pristine Carbon Electrode.

Applications of conducting carbon materials for highly efficient electrochemical energy devices require a greater fundamental understanding of heterogeneous electron-transfer (ET) mechanisms. This task, however, is highly challenging experimentally, because an adsorbing carbon surface may easily conceal its intrinsic reactivity through adventitious contamination. Herein, we employ nanoscale scanning electrochemical microscopy (SECM) and cyclic voltammetry to gain new insights into the interplay between heterogeneous ET and adsorption of a Co(III)/Co(II)-complex redox couple at the contamination-free surface of electron-beam-deposited carbon (eC). Specifically, we investigate the redox couple of tris(1,10-phenanthroline)cobalt(II), Co(phen)32+, as a promising mediator for dye-sensitized solar cells and redox flow batteries. A pristine eC surface overlaid with KCl is prepared in vacuum, protected from contamination in air, and exposed to an ultrapure aqueous solution of Co(phen)32+ by the dissolution of the protective KCl layer. We employ SECM-based nanogap voltammetry to quantitatively demonstrate that Co(phen)32+ is adsorbed on the pristine eC surface to electrostatically self-inhibit outer-sphere ET of nonadsorbed Co(phen)33+ and Co(phen)32+. Strong electrostatic repulsion among Co(phen)32+ adsorbates is also demonstrated by SECM-based nanogap voltammetry and cyclic voltammetry. Quantitatively, self-inhibitory ET is characterized by a linear decrease in the standard rate constant of Co(phen)32+ oxidation with a higher surface concentration of Co(phen)32+ at the formal potential. This unique relationship is consistent not with the Frumkin model of double layer effects, but with the Amatore model of partially blocked electrodes as extended for self-inhibitory ET. Significantly, the complicated coupling of electron transfer and surface adsorption is resolved by combining nanoscale and macroscale voltammetric methods.

Simulation of Fast-Scan Nanogap Voltammetry at Double-Cylinder Ultramicroelectrodes.

High temporal resolution of fast-scan cyclic voltammetry (FSCV) is widely appreciated in fundamental and applied electrochemistry to quantitatively investigate rapid dynamics of electron transfer and neurotransmission using ultramicroelectrodes (UMEs). Faster potential scan, however, linearly increases the background current, which must be subtracted for quantitative FSCV. Herein, we numerically simulate fast-scan nanogap voltammetry (FSNV) for quantitative detection of diffusing redox species under quasi-steady states without the need of background subtraction while maintaining high temporal resolution of transient FSCV. These advantages of FSNV originate from the use of a parallel pair of cylindrical UMEs with nanometer-wide separation in contrast to FSCV with single UMEs. In FSNV, diffusional redox cycling across the nanogap is driven voltammetrically at the generator electrode and monitored amperometrically at the collector electrode without the transient background. We reveal that the cylindrical collector electrode can reach quasi-steady states ~104 times faster than the generator electrode with identical sizes to allow for fast scan. Double-microcylinder and nanocylinder UMEs enable quasi-steady-state FSNV at hundreds volts per second as practiced for in-vivo FSCV and megavolts per second as achieved for ultra-FSCV, respectively. Rational design and simple fabrication of double-cylinder UMEs are proposed to broaden the application of nanogap voltammetry.

In Situ Detection of the Adsorbed Fe(II) Intermediate and the Mechanism of Magnetite Electrodeposition by Scanning Electrochemical Microscopy.

Electrodeposition is an important approach that can produce functional compound materials by assembling multiple species at the electrode surface. However, a fundamental understanding of the electrodeposition mechanism has been limited by its complexity and is often gained only through ex situ studies of deposited materials. Here we report on the application of scanning electrochemical microscopy (SECM) to enable the in situ, real-time, and quantitative study of electrodeposition and electrodissolution. Specifically, we electrodeposit magnetite (Fe3O4) from an alkaline solution of Fe(III)-triethanolamine as a robust route that can prepare this magnetic and electrocatalytic compound on various conductive substrates. The powerful combination of SECM with cyclic voltammetry (CV) at a gold substrate reveals that the electrodeposition of magnetite requires the preceding adsorption of Fe(II)-triethanolamine on the substrate surface and, subsequently, is mediated through the highly complicated ECadsCmag mechanism, where both chemical steps occur at the substrate surface rather than in the homogeneous solution. SECM-based CV is obtained under high mass-transport conditions and analyzed by the finite element method to kinetically resolve all steps of the ECadsCmag mechanism and quantitatively determine relevant reaction parameters. By contrast, the adsorbed Fe(II) intermediate is unresolvable from co-deposited magnetite in situ by other electrochemical techniques and is undetectable ex situ because of the facile air oxidation of the Fe(II) intermediate. Significantly, SECM-based CV will be useful for the in situ characterization of various electrodeposited compounds to complement their ex situ characterization.

Characterization of Nanopipet-Supported ITIES Tips for Scanning Electrochemical Microscopy of Single Solid-State Nanopores.

Nanoscale scanning electrochemical microscopy (SECM) is a powerful scanning probe technique that enables high-resolution imaging of chemical processes at single nanometer-sized objects. However, it has been a challenging task to quantitatively understand nanoscale SECM images, which requires accurate characterization of the size and geometry of nanoelectrode tips. Herein, we address this challenge through transmission electron microscopy (TEM) of quartz nanopipets for SECM imaging of single solid-state nanopores by using nanopipet-supported interfaces between two immiscible electrolyte solutions (ITIES) as tips. We take advantage of the high resolution of TEM to demonstrate that laser-pulled quartz nanopipets reproducibly yield not only an extremely small tip diameter of ∼30 nm, but also a substantial tip roughness of ∼5 nm. The size and roughness of a nanopipet can be reliably determined by optimizing the intensity of the electron beam not to melt or deform the quartz nanotip without a metal coating. Electrochemically, the nanoscale ITIES supported by a rough nanotip gives higher amperometric responses to tetrabutylammonium than expected for a 30 nm diameter disc tip. The finite element simulation of sphere-cap ITIES tips accounts for the high current responses and also reveals that the SECM images of 100 nm diameter Si3N4 nanopores are enlarged along the direction of the tip scan. Nevertheless, spatial resolution is not significantly compromised by a sphere-cap tip, which can be scanned in closer proximity to the substrate. This finding augments the utility of a protruded tip, which can be fabricated and miniaturized more readily to facilitate nanoscale SECM imaging.

Ultraflat, Pristine, and Robust Carbon Electrode for Fast Electron-Transfer Kinetics.

Electron-beam (e-beam) deposition of carbon on a gold substrate yields a very flat (0.43 nm of root-mean-square roughness), amorphous carbon film consisting of a mixture of sp2- and sp3-hybridized carbon with sufficient conductivity to avoid ohmic potential error. E-beam carbon (eC) has attractive properties for conventional electrochemistry, including low background current and sufficient transparency for optical spectroscopy. A layer of KCl deposited by e-beam to the eC surface without breaking vacuum protects the surface from the environment after fabrication until dissolved by an ultrapure electrolyte solution. Nanogap voltammetry using scanning electrochemical microscopy (SECM) permits measurement of heterogeneous standard electron-transfer rate constants (k°) in a clean environment without exposure of the electrode surface to ambient air. The ultraflat eC surface permitted nanogap voltammetry with very thin electrode-to-substrate gaps, thus increasing the diffusion limit for k° measurement to >14 cm/s for a gap of 44 nm. Ferrocene trimethylammonium as the redox mediator exhibited a diffusion-limited k° for the previously KCl-protected eC surface, while k° was 1.45 cm/s for unprotected eC. The k° for Ru(NH3)63+/2+ increased from 1.7 cm/s for unprotected eC to above the measurable limit of 6.9 cm/s for a KCl-protected eC electrode.

Scanning Electrochemical Microscopy of Carbon Nanomaterials and Graphite.

Carbon materials are tremendously important as electrode materials in both fundamental and applied electrochemistry. Recently, significant attention has been given not only to traditional carbon materials, but also to carbon nanomaterials for various electrochemical applications in energy conversion and storage as well as sensing. Importantly, many of these applications require fast electron-transfer (ET) reactions between a carbon surface and a redox-active molecule in solution. It, however, has not been well understood how heterogeneous ET kinetics at a carbon/solution interface is affected by the electronic structure, defect, and contamination of the carbon surface. Problematically, it is highly challenging to measure the intrinsic electrochemical reactivity of a carbon surface, which is readily passivated by adventitious organic contaminants. This Account summarizes our recent studies of carbon nanomaterials and graphite by scanning electrochemical microscopy (SECM) not only to reveal the fast ET kinetics of simple ferrocene derivatives at their graphitic surfaces, but also to obtain mechanistic insights into their extraordinary electrochemical reactivity. Specifically, we implemented new principles and technologies to reliably and reproducibly enable nanoscale SECM measurements. We took advantage of a new SECM imaging principle to resolve the high reactivity of the sidewall of individual single walled carbon nanotubes. In addition, we developed SECM-based nanogap voltammetry to find that monolayer graphene grown by chemical vapor deposition yields an unprecedentedly high standard ET rate constant, k(0), of ≥25 cm/s, which was >1000 times higher than that reported in the literature. Remarkably, the nonideal asymmetry of paired nanogap voltammograms revealed that the high reactivity of graphitic surfaces is compromised by their contamination with airborne hydrocarbons. Most recently, we protected the clean surface of highly oriented pyrolytic graphite from the airborne contaminants during its exfoliation and handling by forming a water adlayer to obtain a reliable k(0) value of ≥12 cm/s from symmetric pairs of nanogap voltammograms. We envision that SECM of clean graphitic surfaces will enable us to reliably address not only effects of their electronic structures on their electrochemical reactivity, but also the activity of carbon-based or carbon-supported electrocatalysts for fuel cells and batteries.

Voltammetric Ion Selectivity of Thin Ionophore-Based Polymeric Membranes: Kinetic Effect of Ion Hydrophilicity.

The high ion selectivity of potentiometric and optical sensors based on ionophore-based polymeric membranes is thermodynamically limited. Here, we report that the voltammetric selectivity of thin ionophore-based polymeric membranes can be kinetically improved by several orders of magnitude in comparison with their thermodynamic selectivity. The kinetic improvement of voltammetric selectivity is evaluated quantitatively by newly introducing a voltammetric selectivity coefficient in addition to a thermodynamic selectivity coefficient. Experimentally, both voltammetric and thermodynamic selectivity coefficients are determined from cyclic voltammograms of excess amounts of analyte and interfering ions with respect to the amount of a Na(+)- or Li(+)-selective ionophore in thin polymeric membranes. We reveal the slower ionophore-facilitated transfer of a smaller alkaline earth metal cation with higher hydrophilicity across the membrane/water interface, thereby kinetically improving voltammetric Na(+) selectivity against calcium, strontium, and barium ions by 3, 2, and 1 order of magnitude, respectively, in separate solutions. Remarkably, voltammetric Na(+) and Li(+) selectivity against calcium and magnesium ions in mixed solutions is improved by 4 and >7 orders of magnitude, respectively, owing to both thermodynamic and kinetic effects in comparison with thermodynamic selectivity in separate solutions. Advantageously, the simultaneous detection of sodium and calcium ions is enabled voltammetrically in contrast to the potentiometric and optical counterparts. Mechanistically, we propose a new hypothetical model that the slower transfer of a more hydrophilic ion is controlled by its partial dehydration during the formation of the adduct with a "water finger" prior to complexation with an ionophore at the membrane/water interface.

Voltammetric Mechanism of Multiion Detection with Thin Ionophore-Based Polymeric Membrane.

The capability to detect multianalyte ions in their mixed solution is an important advantage of voltammetry with an ionophore-based polymeric membrane against the potentiometric and optical counterparts. This advanced capability is highly attractive for the analysis of physiological ions at millimolar concentrations in biological and biomedical samples. Herein, we report on the comprehensive response mechanisms based on the voltammetric exchange and transfer of millimolar multiions at a thin polymeric membrane, where an ionophore is exhaustively depleted upon the transfer of the most favorable primary ion, I(zI). With a new voltammetric ion-exchange mechanism, the primary ion is exchanged with the secondary favorable ion, J(zJ), at more extreme potentials to transfer a net charge of |zJ|/nJ - |zI|/nI for each ionophore molecule, which forms 1:nI and 1:nJ complexes with the respective ions. Alternatively, an ion-transfer mechanism utilizes the second ionophore that independently transfers the secondary ion without ion exchange. Experimentally, a membrane is doped with a Na(+)- or Li(+)-selective ionophore to detect not only the primary ion, but also the secondary alkaline earth ion, based on the ion-exchange mechanism, where both ions form 1:1 complexes with the ionophores to transfer a net charge of +1. Interestingly, the resultant peak potentials of the secondary divalent ion vary with its sample activity to yield an apparently super-Nernstian slope as predicted theoretically. By contrast, the voltammetric exchange of calcium ion (nI = 3) with lithium ion (nJ = 1) by a Ca(2+)-selective ionophore is thermodynamically unfavorable, thereby requiring a Li(+)-selective ionophore for the ion-transfer mechanism.

Origin of Asymmetry of Paired Nanogap Voltammograms Based on Scanning Electrochemical Microscopy: Contamination Not Adsorption.

Formation of a nanometer-wide gap between tip and substrate electrodes by scanning electrochemical microscopy (SECM) enables voltammetric measurement of ultrafast electron-transfer kinetics. Herein, we demonstrate the advantage of SECM-based nanogap voltammetry to assess the cleanness of the substrate surface in solution by confirming that airborne contamination of highly oriented pyrolytic graphite (HOPG) causes the nonideal asymmetry of paired nanogap voltammograms of (ferrocenylmethyl)trimethylammonium (Fc(+)). We hypothesize that the amperometric response of a 1 µm-diameter Pt tip is less enhanced in the feedback mode, where more hydrophilic Fc(2+) is generated from Fc(+) at the tip and reduced voltammetrically at the HOPG surface covered with airborne hydrophobic contaminants. The tip current is more enhanced in the substrate generation/tip collection mode, where less charged Fc(+) is oxidized at the contaminated HOPG surface. In fact, symmetric pairs of nanogap voltammograms are obtained with the cleaner HOPG surface that is exfoliated in humidified air and covered with a nanometer-thick water adlayer to suppress airborne contamination. This result disproves a misconception that the asymmetry of paired nanogap voltammograms is due to electron exchange mediated by Fc(2+) adsorbed on the glass sheath of the tip. Moreover, weak Fc(+) adsorption on the HOPG surface causes only the small hysteresis of each voltammogram upon forward and reverse sweeps of the HOPG potential. Significantly, no Fc(2+) adsorption on the HOPG surface ensures that the simple outer-sphere pathway mediates ultrafast electron transfer of the Fc(2+/+) couple with standard rate constants of ≥12 cm/s as estimated from symmetric pairs of reversible nanogap voltammograms.

Focused-Ion-Beam-Milled Carbon Nanoelectrodes for Scanning Electrochemical Microscopy.

Nanoscale scanning electrochemical microscopy (SECM) has emerged as a powerful electrochemical method that enables the study of interfacial reactions with unprecedentedly high spatial and kinetic resolution. In this work, we develop carbon nanoprobes with high electrochemical reactivity and well-controlled size and geometry based on chemical vapor deposition of carbon in quartz nanopipets. Carbon-filled nanopipets are milled by focused ion beam (FIB) technology to yield a flat disk tip with a thin quartz sheath as confirmed by transmission electron microscopy. The extremely high electroactivity of FIB-milled carbon nanotips is quantified by enormously high standard electron-transfer rate constants of ≥10 cm/s for Ru(NH3)63+. The tip size and geometry are characterized in electrolyte solutions by SECM approach curve measurements not only to determine inner and outer tip radii of down to ~27 and ~38 nm, respectively, but also to ensure the absence of a conductive carbon layer on the outer wall. In addition, FIB-milled carbon nanotips reveal the limited conductivity of ~100 nm-thick gold films under nanoscale mass-transport conditions. Importantly, carbon nanotips must be protected from electrostatic damage to enable reliable and quantitative nanoelectrochemical measurements.