Nanoscale Scanning Electrochemical Microscopy Research Articles

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.

Mechanistic Assessment of Metabolic Interaction between Single Oral Commensal Cells by Scanning Electrochemical Microscopy.

The human oral microbiome heavily influences the status of oral and systemic diseases through different microbial compositions and complex signaling between microbes. Recent evidence suggests that investigation of interactions between oral microbes can be utilized to understand how stable communities are maintained and how they may preserve health. Herein, we investigate two highly abundant species in the human supragingival plaque, Streptococcus mitis and Corynebacterium matruchotii, to elucidate their real-time chemical communication in commensal harmony. Specifically, we apply nanoscale scanning electrochemical microscopy (SECM) using a submicropipet-supported interface between two immiscible electrolyte solutions as an SECM probe not only to image the permeability of S. mitis and C. matruchotii membranes to tetraethylammonium (TEA+) probe ions but also to real-time visualize the metabolic interaction between two microbes via lactate production/consumption at a single-cell level. The metabolic relationship between two strains is quantitatively assessed by determining (1) the passive permeability of both bacterial membranes of 2.4 × 10-4 cm/s to the free diffusion of TEA+, (2) 0.5 mM of the lactate concentration produced by a single S. mitis strain at a rate of 2.7 × 10-4 cm/s, and (3) a lactate oxidation rate ≥5.0 × 106 s-1 by an individual C. matruchotii strain. Significantly, this study, for the first time, describes a mechanism of in situ metabolic interaction between oral commensals at the single-cell level through quantitative analysis, which supports the observed in vivo spatial arrangements of these microbes.

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.

(Invited) Photo-SECM Measurements of Water Splitting at Single Semiconductor Particles

Understanding and improving the properties of photocatalysts can have important implications for energy conversion and storage. Because the activity of heterogeneous catalysts depends strongly on local atomic structures, probing (photo)catalytic reactions at specific local sites is critically important. We have recently reported the use of scanning electrochemical microscopy (SECM) for nanoscale imaging of photoelectrochemical processes at semiconductor surfaces. To illuminate a microscopic portion of the substrate surface facing the SECM probe, a glass-sealed, polished Pt tip simultaneously serves as a nanoelectrode and a light guide. In this paper, we present the results of first nanoscale SECM and photo-SECM experiments with single BiVO4 and SrTiO3 particles. Local mapping of oxygen and hydrogen fluxes produced by water splitting at single semiconductor particles was carried out using chemically modified SECM nanotips.

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.

Scanning Electrochemical and Photoelectrochemical Microscopy on Finder Grids: Toward Correlative Multitechnique Imaging of Surfaces.

Scanning electrochemical microscopy (SECM) is a powerful technique for mapping surface reactivity and investigating heterogeneous processes on the nanoscale. Despite significant advances in high-resolution SECM and photo-SECM imaging, they cannot provide atomic scale structural information about surfaces. By correlating the SECM images with atomic scale structural and bonding information obtained by transmission electron microscopy (TEM) techniques with one-to-one correspondence, one can elucidate the nature of the active sites and understand the origins of heterogeneous surface reactivity. To enable multitechnique imaging of the same nanoscale portion of the electrode surface, we develop a methodology for using a TEM finder grid as a conductive support in SECM and photo-SECM experiments. In this paper, we present the results of our first nanoscale SECM and photo-SECM experiments on carbon TEM grids, including imaging of semiconductor nanorods.

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.

(Invited) SECM and Photo-SECM on TEM Grids: Toward Correlated Electrochemical/TEM Imaging of Catalysts

Understanding and improving the properties of photocatalysts and electrocatalysts can have important implications for energy conversion and storage. Studying heterogeneous catalysts is challenging because their activity depends strongly on local atomic structures. Therefore, probing electrocatalytic reactions at specific local sites is critically important. By correlating the scanning electrochemical microscopy (SECM) data with atomic scale structural and bonding information obtained by transmission electron microscopy (TEM) techniques with one-to-one correspondence, one can elucidate the nature of the active sites. To enable multi-technique imaging of the same nanoscale portion of the catalytic surface, we are developing methodology for using a TEM finder grid as a substrate in SECM experiments. In this paper, we present the results of first nanoscale SECM and photo-SECM experiments on carbon TEM grids, including imaging of semiconductor nanorods.

Photo-Scanning Electrochemical Microscopy on the Nanoscale with Through-Tip Illumination.

Scanning electrochemical microscopy (SECM) has previously been employed in probing photoelectrochemical processes at semiconductor surfaces. However, the spatial resolution of these studies has not yet matched the nanoscale SECM resolution attained without substrate illumination. Herein, we introduce nanoscale photo-SECM with a glass-sealed, polished tip simultaneously serving as a nanoelectrode and a light guide to produce a microscopic light spot on the substrate surface. The advantages of this approach are demonstrated by comparing current transients obtained using through-tip and global illumination of the sample. The spot of light on the substrate surface facing the nanotip was sufficiently bright to measure the diffusion-controlled positive feedback current in good agreement with the theory. We employed this approach for high-resolution photoelectrochemical mapping of ferrocenemethanol oxidation and oxygen evolution reactions at the Nb:TiO2 rutile (110) single crystal surface. The images obtained using 40-50 nm radius tips showed only minor and random variations in photoelectrochemical reactivity for both processes, pointing to essentially uniform distribution of the Nb dopant over the TiO2 surface and no measurable segregation on the ∼50 nm scale.

Direct Observation of C2O4•- and CO2•- by Oxidation of Oxalate within Nanogap of Scanning Electrochemical Microscope.

Oxalate oxidation in the presence of different oxidized luminophores leads to the emission of light and has been studied extensively in electrogenerated chemiluminescence (ECL). The proposed mechanism involves the initial formation of the oxalate radical anion, C2O4•-. The ensuing decomposition of C2O4•- produces a very strong reductant, CO2•-, which reacts with the oxidized luminophores to generate excited states that emit light. Although the mechanism has been proposed for decades, the experimental demonstration is still lacking, because of the complexity of the system and the short lifetimes of both radical anions. To address these issues, we studied oxalate oxidation at platinum ultramicroelectrodes (UMEs) in anhydrous N, N-dimethylformamide (DMF) solution by nanoscale scanning electrochemical microscopy (SECM) with the tip generation/substrate collection (TG/SC) mode. A Pt nanoelectrode was utilized as the SECM generator for oxalate oxidation, while another Pt UME served as the SECM collector and was used to capture the generated intermediates. We studied the influence of the gap distance, d, on the substrate current ( is). The results indicate that, when 73 nm < d < 500 nm, the species captured by the substrate were primarily CO2•-, while C2O4•- was the predominant intermediate measured when d was below 73 nm. A half-life of 1.3 μs for C2O4•- was obtained, which indicates a stepwise mechanism for oxalate oxidation. The relevance of these observations to the use of oxalate as the coreactant in ECL systems is also discussed.

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.

Hopping mode SECM imaging of redox activity in ionic liquid with glass-coated inlaid platinum nanoelectrodes prepared using a heating coil puller

Major difficulties in nanoscale scanning electrochemical microscopy (SECM), resulting from the short tip-to-sample distance, are tilt misalignment and vertical thermal drift. This renders constant height mode nanoscale electrochemical imaging experimentally problematic. Nanoscale SECM also requires application of reliable nanoprobes. Although SECM nanotips are commercially available, they are rather disposable, fragile and their shelf-life is limited. Many SECM laboratories employ self-prepared nanoprobes, fabricated using advanced apparatus. In this paper, we employ a hopping mode approach to nanoscale electrochemical imaging, which resolves the mentioned common difficulties. The applied platinum nanoelectrodes, insulated with borosilicate glass, were prepared using affordable equipment and materials. They were applied for nanoscale mapping of redox mediator regeneration at a model interdigitated microsensor, composed of Pt microband electrodes, in a solution of ferrocene in viscous hydrophobic room temperature ionic liquid (RTIL) – 1-butyl-3-methylimidazoliumbis(trifluoro-methylsulfonyl)imide (C4mim N(Tf)2). The presented nanosensors were also characterized with cyclic voltammetry in aqueous electrolyte containing hexamineruthenium (III) chloride as redox mediator.

Imaging resolution of biocatalytic activity using nanoscale scanning electrochemical microscopy

Scanning electrochemical microscopy represents a powerful tool for electro(chemical) characterization of surfaces, but its applicability has been limited in most cases at microscale spatial resolution, and the greatest challenge has been the scaling down to the nanoscale for fabrication and the use of nanometer-sized tips. Here, Pt nanoelectrodes with nanometer electroactive area were fabricated and employed for imaging a distribution of gold nanoparticles (AuNPs) and bioelectrocatalytic activity of a redox-active enzyme immobilized on gold surfaces.

Bipolar Electrochemistry on a Nanopore-Supported Platinum Nanoparticle Electrode.

In this Technical Note, we describe a method to fabricate nanopore-supported Pt nanoparticle electrodes and their use in bipolar electrochemistry. A Pt nanoparticle is deposited on the orifice of a solid-state nanopore inside a focused-ion beam (FIB) system. Complete blockage of the nanopore with Pt metal forms a closed bipolar nanoparticle electrode whose size and shape can be tunable in one simple step. Nanoparticle electrodes and their arrays can be prepared on different substrates such as the tip of a glass pipet, a double-barrel pipet, and a freestanding silicon nitride membrane. Steady-state voltammetry can be performed on such nanoparticle electrodes via bipolar electrochemistry. Moreover, an array of Pt nanoparticles can be used for fluorescence-enabled electrochemical microscopy. Future use of highly advanced FIB systems may allow nanoparticles of <10 nm to be fabricated which may enable coupled electrochemical reactions of single redox molecules. Pipette-supported single particle electrodes may also find useful applications in high resolution imaging with nanoscale scanning electrochemical microscopy (SECM) and neurochemical analysis inside single cells.

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.

(Invited) High-Resolution Scanning Electrochemical Microscopy of Biological and Artificial Nanopores

In this presentation, we discuss applications of scanning electrochemical microscopy (SECM) to high-resolution imaging of biological and artificial nanopores. SECM imaging enables us to obtain unprecedented information about the kinetics, pathways, and mechanism of molecular transport through single nanopores. We characterize nanopipet electrodes as nanoscale SECM probes by transmission electron microscopy to verify their small diameters of ~30 nm. The resultant high spatial resolution of our SECM instrument is quantitatively demonstrated by imaging ion transport through the single nanopores of a thin solid silicon membrane. The nanoscale SECM approach thus established is applied to image a biological nanopore, the nuclear pore complex (NPC), in aqueous solution, which eliminates artifacts that are caused when the NPC is imaged by electron microscopy in vacuum and atomic force microscopy in air. Our SECM study of single NPCs is significant, because it will led to a greater mechanistic understanding of molecular transport through the NPC, which plays crucial roles in the gene expression regulation and gene delivery and is linked to many human diseases and therapeutics of genetic disorders.

Nanoscale scanning electrochemical microscopy: Emerging advances in applications and theory

Nanoscale scanning electrochemical microscopy is emerging as a powerful electrochemical technique due to recent pioneering advances in tip fabrication and handling, instrument and cell design, and awareness of airborne and solution organic contamination which impact tip and substrate handling. Applications, accompanied by theory usually as finite-element simulations, are beginning to appear as discussed in this review.

Visualizing and Calculating Tip-Substrate Distance in Nanoscale Scanning Electrochemical Microscopy Using 3-Dimensional Super-Resolution Optical Imaging.

We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.

Advanced Electrochemistry of Individual Metal Clusters Electrodeposited Atom by Atom to Nanometer by Nanometer.

Metal clusters are very important as building blocks for nanoparticles (NPs) for electrocatalysis and electroanalysis in both fundamental and applied electrochemistry. Attention has been given to understanding of traditional nucleation and growth of metal clusters and to their catalytic activities for various electrochemical applications in energy harvesting as well as analytical sensing. Importantly, understanding the properties of these clusters, primarily the relationship between catalysis and morphology, is required to optimize catalytic function. This has been difficult due to the heterogeneities in the size, shape, and surface properties. Thus, methods that address these issues are necessary to begin understanding the reactivity of individual catalytic centers as opposed to ensemble measurements, where the effect of size and morphology on the catalysis is averaged out in the measurement. This Account introduces our advanced electrochemical approaches to focus on each isolated metal cluster, where we electrochemically fabricated clusters or NPs atom by atom to nanometer by nanometer and explored their electrochemistry for their kinetic and catalytic behavior. Such approaches expand the dimensions of analysis, to include the electrochemistry of (1) a discrete atomic cluster, (2) solely a single NP, or (3) individual NPs in the ensemble sample. Specifically, we studied the electrocatalysis of atomic metal clusters as a nascent electrocatalyst via direct electrodeposition on carbon ultramicroelectrode (C UME) in a femtomolar metal ion precursor. In addition, we developed tunneling ultramicroelectrodes (TUMEs) to study electron transfer (ET) kinetics of a redox probe at a single metal NP electrodeposited on this TUME. Owing to the small dimension of a NP as an active area of a TUME, extremely high mass transfer conditions yielded a remarkably high standard ET rate constant, k0, of 36 cm/s for outer-sphere ET reaction. Most recently, we advanced nanoscale scanning electrochemical microscopy (SECM) imaging to resolve the electrocatalytic activity of individual electrodeposited NPs within an ensemble sample yielding consistent high k0 values of ≥2 cm/s for the hydrogen oxidation reaction (HOR) at different NPs. We envision that our advanced electrochemical approaches will enable us to systematically address structure effects on the catalytic activity, thus providing a quantitative guideline for electrocatalysts in energy-related applications.

Nanometer Scale Scanning Electrochemical Microscopy Instrumentation.

We report the crucial components required to perform scanning electrochemical microscopy (SECM) with nanometer-scale resolution. The construction and modification of the software and hardware instrumentation for nanoscale SECM are explicitly explained including (1) the LabVIEW code that synchronizes the SECM tip movement with the electrochemical response, (2) the construction of an isothermal chamber to stabilize the nanometer scale gap between the tip and substrate, (3) the modification of a commercial bipotentiostat to avoid electrochemical tip damage during SECM experiments, and (4) the construction of an SECM stage to avoid artifacts in SECM images. These findings enabled us to successfully build a nanoscale SECM, which can be utilized to map the electrocatalytic activity of individual nanoparticles in a typical ensemble sample and study the structure/reactivity relationship of single nanostructures.