Quantitative Measurements of Electrocatalytic Reaction Rates with NanoSECM.

Scanning electrochemical microscopy (SECM) has been extensively used for mapping electrocatalytic surface reactivity; however, most of the studies were carried out using micrometer-sized tips, and no quantitative kinetic experiments on the nanoscale have yet been reported to date. As the diffusion-limited current density at a nanometer-sized electrode is very high, an inner-sphere electron-transfer process occurring at a nanotip typically produces a kinetic current at any attainable overpotential. Here, we develop a theory for substrate generation/tip collection (SG/TC) and feedback modes of SECM with a kinetic tip current and use it to evaluate the rates of hydrogen and oxygen evolution reactions in a neutral aqueous solution from the current-distance curves. The possibility of using chemically modified nanotips for kinetic measurements is also demonstrated. The effect of the substrate size on the shape of the current-distance curves in SG/TC mode SECM experiments is discussed.

Efficient Voltage-Driven Oxidation of Water and Alcohols by an Organic Molecular Catalyst Directly Attached to a Carbon Electrode.

The integration of heterogeneous electrocatalysis and molecular catalysis is a promising approach to designing new catalysts for the oxygen evolution reaction (OER) and other processes. We recently showed that the electrostatic potential drop across the double layer contributes to the driving force for electron transfer between a dissolved reactant and a molecular catalyst immobilized directly on the electrode surface. Here, we report high current densities and low onset potentials for water oxidation attained using a metal-free voltage-assisted molecular catalyst (TEMPO). Scanning electrochemical microscopy (SECM) was used to analyze the products and determine faradic efficiencies for the generation of H2O2 and O2. The same catalyst was employed for efficient oxidations of butanol, ethanol, glycerol, and H2O2. DFT calculations show that the applied voltage alters the electrostatic potential drop between TEMPO and the reactant as well as chemical bonding between them, thereby increasing the reaction rate. These results suggest a new route for designing next-generation hybrid molecular/electrocatalysts for OER and alcohol oxidations.

Photo-scanning Electrochemical Microscopy Observation of Overall Water Splitting at a Single Aluminum-Doped Strontium Titanium Oxide Microcrystal.

Particulate photocatalysts for the overall water splitting (OWS) reaction offer promise as devices for hydrogen fuel generation. Even though such photocatalysts have been studied for nearly 5 decades, much of the understanding of their function is derived from observations of catalyst ensembles and macroscopic photoelectrodes. This is because the sub-micrometer size of most OWS photocatalysts makes spatially resolved measurements of their local reactivity very difficult. Here, we employ photo-scanning electrochemical microscopy (photo-SECM) to quantitatively measure hydrogen and oxygen evolution at individual OWS photocatalyst particles for the first time. Micrometer-sized Al-doped SrTiO3/Rh2-yCryO3 photocatalyst particles were immobilized on a glass substrate and interrogated with a chemically modified SECM nanotip. The tip simultaneously served as a light guide to illuminate the photocatalyst and as an electrochemical nanoprobe to observe oxygen and hydrogen fluxes from the OWS. Local O2 and H2 fluxes obtained from chopped light experiments and photo-SECM approach curves using a COMSOL Multiphysics finite-element model confirmed stoichiometric H2/O2 evolution of 9.3/4.6 µmol cm-2 h-1 with no observable lag during chopped illumination cycles. Additionally, photoelectrochemical experiments on a single microcrystal attached to a nanoelectrode tip revealed a strong light intensity dependence of the OWS reaction. These results provide the first confirmation of OWS at single micrometer-sized photocatalyst particles. The developed experimental approach is an important step toward assessing the activity of photocatalyst particles at the nanometer scale.

Elucidating the Shape of Current Transients in Electrochemical Resistive-Pulse Sensing of Single Liposomes.

Electrochemical resistive-pulse (ERP) sensing with conductive carbon nanopipettes (CNPs) has recently been developed and employed for the detection of single liposomes and biological vesicles, and for the analysis of redox molecules contained in such vesicles. However, the origins of different shapes of current transients produced by the translocation of single vesicles through the CNP remain poorly understood. Herein, we report extensive finite-element simulations of both portions of an ERP transient, the current blockage by a vesicle approaching and passing through the pipet orifice and the faradaic current spike due to oxidation/reduction of redox species released from a vesicle on the carbon surface, for different values of parameters defining the geometry and dynamics of the vesicle/CNP system. The effects of the pipet geometry, surface charge, transport, vesicle trajectory, and collision location on the shape of current transients are investigated. The possibility of quantitative analysis of experimental ERP transients produced by translocations of liposomes and extracellular vesicles by fitting them to simulated curves is demonstrated. The developed theory can enable a more reliable interpretation of complicated ERP signals and characterization of the size and contents of single biological and artificial vesicles.

Electrochemical Resistive-Pulse Sensing of Extracellular Vesicles.

Extracellular vesicles (EVs) released from biological cells have attracted considerable interest due to their potential for cancer diagnostics and important role in cell signaling. Most previously reported studies have been concerned with the detection of EVs in biofluids and analysis of proteins and nucleic acids they contain. Electrochemical resistive-pulse (ERP) sensing enables direct detection of single EVs released from a specific cell and analysis of reactive oxygen and nitrogen species in such vesicles. Here, we demonstrate the applicability of ERP sensing to distinguish between nontransformed and cancerous breast cell lines as well as between breast cancer cell lines with different metastatic potential. Another application of ERP sensing is in real-time monitoring of changes in a single cell induced by a chemical agent. This approach is potentially useful for evaluating the efficacy of therapeutic agents, including those that trigger breast cancer cell death by inducing intense oxidative stress.

Decoupling Through-Tip Illumination from Scanning in Nanoscale Photo-SECM.

The use of scanning electrochemical microscopy (SECM) for nanoscale imaging of photoelectrochemical processes at semiconductor surfaces has recently been demonstrated. To illuminate a microscopic portion of the substrate surface facing the SECM probe, a glass-sealed, polished tip simultaneously served as a nanoelectrode and a light guide. One issue affecting nanoscale photo-SECM experiments is mechanical interactions of the rigid optical fiber with the tip motion controlled by the piezo-positioner. Here we report an improved experimental setup in which the tip is mechanically decoupled from the fiber and light is delivered to the back of the tip capillary using a complex lens system. The advantages of this approach are evident from the improved quality of the approach curves and photo-SECM images. The light intensity delivered from the optical fiber to the tip is not changed significantly by their decoupling.

Direct high-resolution mapping of electrocatalytic activity of semi-two-dimensional catalysts with single-edge sensitivity.

The catalytic activity of low-dimensional electrocatalysts is highly dependent on their local atomic structures, particularly those less-coordinated sites found at edges and corners; therefore, a direct probe of the electrocatalytic current at specified local sites with true nanoscopic resolution has become critically important. Despite the growing availability of operando imaging tools, to date it has not been possible to measure the electrocatalytic activities from individual material edges and directly correlate those with the local structural defects. Herein, we show the possibility of using feedback and generation/collection modes of operation of the scanning electrochemical microscope (SECM) to independently image the topography and local electrocatalytic activity with 15-nm spatial resolution. We employed this operando microscopy technique to map out the oxygen evolution activity of a semi-2D nickel oxide nanosheet. The improved resolution and sensitivity enables us to distinguish the higher activities of the materials' edges from that of the fully coordinated surfaces in operando The combination of spatially resolved electrochemical information with state-of-the-art electron tomography, that unravels the 3D complexity of the edges, and ab initio calculations allows us to reveal the intricate coordination dependent activity along individual edges of the semi-2D material that is not achievable by other methods. The comparison of the simulated line scans to the experimental data suggests that the catalytic current density at the nanosheet edge is ∼200 times higher than that at the NiO basal plane.

Voltage-Driven Molecular Catalysis of Electrochemical Reactions.

Heterogeneous electrocatalysis and molecular redox catalysis have developed over several decades as two distinct ways to facilitate charge-transfer processes essential for energy conversion and storage. Whereas electrocatalytic reactions are driven by the applied voltage, molecular catalytic processes are driven by the difference between standard potentials of the catalyst and the reactant. Here, we demonstrate that the rate of electron transfer between a dissolved reactant and a molecular catalyst immobilized directly on the surface of a carbon nanoelectrode is governed by combination of chemical driving force and electrostatic potential drop across the double layer. DFT calculations show that varying the applied voltage alters the potential drop between the surface-bound and dissolved redox species. These results suggest a new route for designing next-generation hybrid molecular/electrocatalysts.

Mediated Charge Transfer at Nanoelectrodes: A New Approach to Electrochemical Reactivity Mapping and Nanosensing.

Scanning electrochemical microscopy (SECM) is a powerful tool for mapping surface reactivity. Electrochemical mapping of electrocatalytic processes at the nanoscale is, however, challenging because the surface of a nanoelectrode tip is easily fouled by impurities and/or deactivated by products and intermediates of innersphere surface reactions. To overcome this difficulty, we introduce new types of SECM nanotips based on bimolecular electron transfer between the dissolved electroactive species and a redox mediator attached to the surface of a carbon nanoelectrode. A tris(2,2'-bipyridine)ruthenium complex, Ru(bpy)3, that undergoes reversible oxidation/reduction reactions at both positive and negative potentials was used to prepare the SECM nanoprobes for mapping a wide range of electrocatalytic processes through oxidation of H2, reduction of O2, and both oxidation and reduction of H2O2 at the tip. In addition to high-resolution reactivity mapping and localized kinetic measurements, chemically modified nanoelectrodes can serve as nanosensors for a number of important analytes such as reactive oxygen and nitrogen species and neurotransmitters.

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.

Correlating Molecule Count and Release Kinetics with Vesicular Size Using Open Carbon Nanopipettes.

In this work, open carbon nanopipettes (CNPs) with radius between 50 and 600 nm were used to control translocation of different-sized vesicles through the pipette orifice followed by nanoelectrochemical analysis. Vesicle impact electrochemical cytometry (VIEC) was used to determine the number of catecholamine molecules expelled from single vesicles onto an inner-wall carbon surface, where the duration of transmitter release was quantified and correlated to the vesicle size all in the same nanotip. This in turn allowed us to both size and count molecules for vesicles in a living cell. Here, small and sharp open CNPs were employed to carry out intracellular VIEC with minimal invasion and high sensitivity. Our findings with VIEC reveal that the vesicular content increases with vesicle size. The release kinetics of vesicular transmitters and dense core size have the same relation with the vesicle size, implying that the vesicular dense core size determines the speed of each release event. This direct correlation unravels one of the complexities of exocytosis.

Resistive-Pulse Sensing Inside Single Living Cells.

Resistive-pulse sensing is a technique widely used to detect single nanoscopic entities such as nanoparticles and large molecules that can block the ion current flow through a nanopore or a nanopipette. Although the species of interest, e.g., antibodies, DNA, and biological vesicles, are typically produced by living cells, so far, they have only been detected in the bulk solution since no localized resistive-pulse sensing in biological systems has yet been reported. In this report, we used a nanopipette as a scanning ion conductance microscopy (SICM) tip to carry out resistive-pulse experiments both inside immobilized living cells and near their surfaces. The characteristic changes in the ion current that occur when the pipet punctures the cell membrane are used to monitor its insertion into the cell cytoplasm. Following the penetration, cellular vesicles (phagosomes, lysosomes, and/or phagolysosomes) were detected inside a RAW 264.7 macrophage. Much smaller pipettes were used to selectively detect 10 nm Au nanoparticles in the macrophage cytoplasm. The in situ resistive-pulse detection of extracellular vesicles released by metastatic human breast cells (MDA-MB-231) is also demonstrated. Electrochemical resistive-pulse experiments were carried out by inserting a conductive carbon nanopipette into a macrophage cell to sample single vesicles and measure reactive oxygen and nitrogen species (ROS/RNS) contained inside them.

Thin layer cell behavior of CNT yarn and cavity carbon nanopipette electrodes: Effect on catecholamine detection.

Carbon nanotube yarn microelectrodes (CNTYMEs) are an alternative to carbon-fiber microelectrodes (CFMEs) with interesting electrochemical properties because analyte is momentarily trapped in cavities between the CNTs. Here, we compare fast-scan cyclic voltammetry (FSCV) detection of catecholamines, including dopamine, norepinephrine, and epinephrine, at CNTYMEs, CFMEs, as well as cavity carbon nanopipette electrodes (CNPEs). At CFMEs, current decreases dramatically at high FSCV repetition frequencies. At CNTYMEs, current is almost independent of FSCV repetition frequency because the analytes are trapped in the crevices between CNTs, and thus the electrode acts like a thin-layer cell. At CFMEs, small cyclization product peaks are observed due to an intramolecular cyclization reaction to form leucocatecholamine, which is electroactive, and these peaks are largest for the secondary amine epinephrine. At CNTYMEs, more of the leucocatecholamine cyclization product is detected for all catecholamines because of the enhanced trapping effects, particularly at higher repetition rates where the reaction occurs more frequently and more product is accumulated. For epinephrine, the secondary peaks have larger currents than the primary oxidation peaks at 100 Hz, and similar trends are observed with faster scan rates and 500 Hz repetition frequencies. Finally, we examined CNPEs, which also momentarily trap neurotransmitters. Similar to CNTYMEs, at CNPEs, catecholamines have robust cyclization peaks, particularly at high repetition rates. Thus, CNTYMEs and CNPEs have thin layer cell behavior that facilitates high temporal resolution measurements, but catecholamines CVs are complicated by cyclization reactions. However, those additional peaks could be useful in discriminating the analytes, particularly epinephrine and norepinephrine.

Electrochemical Resistive-Pulse Sensing.

Resistive-pulse sensing with biological or solid-state nanopores and nanopipettes has been widely employed in detecting single molecules and nanoparticles. The analytical signal in such experiments is the change in ionic current caused by the molecule/particle translocation through the pipet orifice. This paper describes a new version of the resistive-pulse technique based on the use of carbon nanopipettes (CNP). The measured current is produced by electrochemical oxidation/reduction of redox molecules at the carbon surface and responds to the particle translocation. In addition to counting single entities, this technique enables qualitative and quantitative analysis of the electroactive material they contain. Using liposomes as a model system, we demonstrate the capacity of CNPs for (1) conventional resistive-pulse sensing of single liposomes, (2) electrochemical resistive-pulse sensing, and (3) electrochemical identification and quantitation of redox species (e.g., ferrocyanide, dopamine, and nitrite) contained in a single liposome. The small physical size of a CNP suggests the possibility of single-entity measurements in biological systems.

Electrochemical Measurements of Reactive Oxygen and Nitrogen Species inside Single Phagolysosomes of Living Macrophages.

The release of reactive oxygen and nitrogen species (ROS/RNS) by macrophages undergoing phagocytosis is crucial for the efficiency of the immune system. In this work, platinized carbon nanoelectrodes were used to detect, characterize, and quantify for the first time the intracellular production rates of the four primary ROS/RNS (i.e., H2O2, ONOO-, NO•, and NO2-) inside single phagolysosomes of living RAW 264.7 murine macrophages stimulated by interferon-γ and lipopolysaccharide (IFN-γ/LPS) to mimic an in vivo inflammatory activation. The time-dependent concentrations of the four primary ROS/RNS in individual phagolysosomes monitored using a four-step chronoamperometric method evidenced a high variability of their production rates. This intrinsic variability unravels the complexity of phagocytosis.

TEM-Assisted Fabrication of Sub-10 nm Scanning Electrochemical Microscopy Tips.

High-resolution scanning electrochemical microscopy (SECM) is a powerful technique for mapping surface topography and reactivity on the nanoscale and investigating heterogeneous processes at the level of single nanoparticles. The ability to fabricate ultrasmall nanoelectrode tips is critical for the progress in nano-SECM. Despite long-term efforts to improve previously developed procedures, the preparation and characterization of disk-type polished tips with the radius <∼25 nm remains challenging and unpredictable. One of the problems is that the geometry of such tips is hard to characterize by either SEM or atomic force microscopy (AFM) that has been employed for examination of somewhat larger nanoelectrodes. Herein, we report a new approach to more predictable and reproducible two-step fabrication of ultrasmall (≤10 nm radius) polished Pt electrodes assisted by transmission electron microscopy (TEM) imaging. Both voltammetric and SECM responses of the prepared nanoelectrodes are consistent with the size and geometry extracted from TEM images. These tips can be used to attain sub-10 nm spatial resolution of SECM imaging and kinetic studies.

Surface-Charge Effects on Voltammetry in Carbon Nanocavities.

Ion transport controlled by electrostatic interactions is an important phenomenon in biological and artificial membranes, channels, and nanopores. Here, we employ carbon-coated nanopipets (CNPs) for studying permselective electrochemistry in a conductive nanopore. A significant accumulation (up to 2000-fold) of cationic redox species and anion depletion inside a CNP by diffuse-layer and surface-charge effects in a solution of low ionic strength were observed as well as the shift of the voltammetric midpeak potential. Finite-element simulations of electrostatic effects on CNP voltammograms show permselective ion transport in a single conducting nanopore and semiquantitatively explain our experimental data. The reported results are potentially useful for improving sensitivity and selectivity of CNP sensors for ionic analytes.

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.

Ultrasensitive Detection of Dopamine with Carbon Nanopipets.

Carbon fiber micro- and nanoelectrodes have been extensively used to measure dopamine and other neurotransmitters in biological systems. Although the radii of some reported probes were ≪1 µm, the lengths of the exposed carbon were typically on the micrometer scale, thus limiting the spatial resolution of electroanalytical measurements. Recent attempts to determine neurotransmitters in single cells and vesicles have provided additional impetus for decreasing the probe dimensions. Here, we report two types of dopamine sensors based on carbon nanopipets (CNP) prepared by chemical vapor deposition of carbon into prepulled quartz capillaries. These include 10-200 nm radius CNPs with a cavity near the orifice and CNPs with an open path in the middle, in which the volume of sampled solution can be controlled by the applied pressure. Because of the relatively large surface area of carbon exposed to solution inside the pipet, both types of sensors yielded well-shaped voltammograms of dopamine down to ca. 1 nM concentrations, and the unprecedented voltammetric response to 100 pM dopamine was obtained with open CNPs. TEM tomography and numerical simulations were used to model CNP responses. The effect of dopamine adsorption on the CNP detection limit is discussed along with the possibilities of measuring other physiologically important analytes (e.g., serotonin) and eliminating anionic and electrochemically irreversible interferences (e.g., ascorbic acid).

Cavity Carbon-Nanopipette Electrodes for Dopamine Detection.

Microelectrodes are typically used for neurotransmitter detection, but nanoelectrodes are not because there is a trade-off between spatial resolution and sensitivity that is dependent on surface area. Cavity carbon-nanopipette electrodes (CNPEs), with tip diameters of a few hundred nanometers, have been developed for nanoscale electrochemistry. Here, we characterize the electrochemical performance of CNPEs with fast-scan cyclic voltammetry (FSCV) for the first time. Dopamine detection using cavity CNPEs, with a depth equivalent to a few radii, is compared with that using open-tube CNPEs, an essentially infinite geometry. Open-tube CNPEs have very slow temporal responses that change over time as the liquid rises in the CNPE. However, a cavity CNPE has a fast temporal response to a bolus of dopamine that is not different from that of a traditional carbon-fiber microelectrode. Cavity CNPEs, with tip diameters of 200-400 nm, have high currents because the small cavity traps and increases the local dopamine concentration. The trapping also leads to an FSCV frequency-independent response and the appearance of cyclization peaks that are normally observed only with large concentrations of dopamine. CNPEs have high dopamine selectivity over ascorbic acid (AA) because of the repulsion of AA by the negative electric field at the holding potential and the irreversible redox reaction. In mouse-brain slices, cavity CNPEs detected exogenously applied dopamine, showing they do not clog in tissue. Thus, cavity CNPEs are promising neurochemical sensors that provide spatial resolution on the scale of hundreds of nanometers, which is useful for small model organisms or for locations near specific cells.