Scanning Probe Microscopy for Nanoscale Electrochemical Imaging

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Scanning Probe Microscopy for Nanoscale Electrochemical Imaging Yasufumi Takahashi, Akichika Kumatani, Hitoshi Shiku, and Tomokazu Matsue Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04355 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Analytical Chemistry

Scanning Probe Microscopy for Nanoscale Electrochemical Imaging

Yasufumi Takahashi a,b, Akichika Kumatani c,d, Hitoshi Shiku e, Tomokazu Matsue c,d,* a

Division of Electrical Engineering and Computer Science, Kanazawa University, 920-1192, Japan;

b

Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and

Technology Agency (JST), Saitama 332-0012, Japan c

WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan;

d e

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan;

Department of Applied Chemistry, Graduate School of Engineering, Tohoku University, Sendai

980-8579, Japan

*Corresponding authors: Yasufumi Takahashi Akichika Kumatani Hitoshi Shiku

Tomokazu Matsue, e-mail: [email protected]

ACS Paragon Plus Environment


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This article presents an overview of scanning probe microscopy (SPM) methods to study nanoscale electrochemical functionalities. Because the dynamics of chemical and biological processes at interfaces underpin a wide range of phenomena, it is important to gain information about the phenomena occurring at these interfaces by measuring chemical and biochemical fluxes on the nanoscale to understand the origins of their functions. SPM employs fine probe scanning over a surface, exploiting the local interactions between the probe and the surface to maintain a constant probe-sample distance. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to monitor key chemical and biological processes at cell surfaces. The distance of the probe is precisely controlled to attain high resolution topographical and/or functional imaging of samples. The greatest advantage of SPM over conventional microscopy is that various microprobes can be equipped to sense localized physical or chemical conditions on a variety of surfaces in situ under wet conditions. Electrode miniaturization enables localized space measurement; its small size offers several advantages, such as low double-layer charging currents, low ohmic drops, fast mass transport, and diffusion rate-limiting steady-state current, which are particularly useful to elucidate phenomena occurring on the nanometer scale. In this review, we introduce three electrochemical imaging methods: scanning electrochemical microscopy (SECM), scanning ion conductance microscopy (SICM), and scanning electrochemical cell microscopy (SECCM). Several recent reviews are also helpful to understand the research field of SECM.1-14 Here, we aim to provide an overview of mainly the last three years of electrochemical imaging-based techniques applicable for the characterization of live cells, biomolecules, catalysts, corrosion processes, energy storage, and energy conversion materials; we also discuss the advantages of SPM-based approaches.

Scanning electrochemical microscopy (SECM) SECM is a technique that is used to characterize the local electrochemical environment of various materials by scanning with a probe microelectrode. The probe current reflects the electrochemical processes occurring in the small space surrounding the probe and the substrate. SECM has been used to characterize and image the local electrochemical environment of various materials by scanning sample surfaces with an ultramicroelectrode (UME) tip. SECM has been widely applied for analyzing numerous electrochemical systems, such as electrode surfaces, nanomaterials, polymers, biomaterials, and liquid/liquid interfaces. Recent progress in the fabrication of nanometer-scale electrodes and the development of electrode-sample distance control systems has greatly enhanced the applicability of SECM systems. Here we present an overview of various measurement modes for high-resolution SECM and the wide-range applications of electrochemical imaging systems.


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Measurement modes and recent progress Feedback mode (FB-mode) is used to image the redox activity of a sample surface. In a solution containing a suitable redox species (mediator), the amperometric response at the UME tip depends on the tip–sample distance. The response change (approach curves) is used to determine the distance and quantify the rate constant at the surface of the substrate electrode. Chronoamperograms and cyclic voltammograms (CVs) at a fixed distance provide useful electrochemical information in a limited area. A mediator is oxidized or reduced at the UME tip and diffuses to the substrate electrode, where it is regenerated; this establishes a redox cycle and enhances the current through the UME. This situation is called positive feedback. If the sample surface is an inert electrical insulator, the redox cycle does not occur; thus, the UME current decreases. This situation is called negative feedback. In both positive and negative FB-modes, the tip current largely depends on the tip– substrate distance. Therefore, FB-mode has high spatial resolution compared to the generation/collection (G/C) mode. FB-mode SECM operation is allowed in the region where the tip-substrate distance is less ten times the UME tip radius. The region corresponds to the length where the tip current evidently deviates from the tip current in the bulk; it is critically dependent on the size of the UME tip radius. In G/C mode, the UME tip collects redox species generated from a substrate electrode. For biological applications, electrochemically active species can be generated from the samples (enzymes and cells) located on an insulator without applying a potential. This is also referred to as G/C mode. In the same manner, liquid/liquid interfaces, solid/liquid interfaces, air/liquid interfaces, and membranes can supply redox species to be collected at the UME tip during G/C mode SECM experiments. Therefore, G/C mode generally denotes substrate generation-tip collection (SG-TC) mode. SG-TC mode SECM provides high sensitivity measurements because the background current is usually very low. SG/TC mode is used to image the concentration profiles of redox species and evaluate the flux of redox species generated from a sample. Conversely, tip generation-substrate collection (TG-SC) mode is an experimental setup, in which the substrate electrode collects redox species generated from the UME tip. The current signal can be observed only when the tip-substrate distance is sufficiently short; namely, only when the diffusion layer of the tip (which is hemispherical in shape) reaches the surface of the substrate electrode. In this situation, positive or negative feedback occurs when the UME tip approaches a conductive or nonconducting substrate electrode, respectively. This is the same situation as FB-SECM; however, the redox species is not limited to “reversible” redox species in TG-SC SECM. The surface interrogation mode of scanning electrochemical microscopy (SI-SECM) has been recently introduced as an in situ technique for the detection and quantification of adsorbed intermediates in electrochemical systems.15 In this technique, the UME tip is used to monitor transient current in the FB-SECM configuration. SI-SECM enables quantification of an adsorbed



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species that is generated at the substrate electrode at a controlled potential or under open circuit conditions. Time-dependent response is observed at the UME tip because the mass of the reactant is remarkably limited at the surface area of the substrate electrode as the adsorbed species. Ahn et al. investigated the density of an active cobalt phosphate (CoPi) catalyst by SI-SECM.16 The same group successfully used rapid switching to improve the time resolution of the SI-SECM system.17 Similarly, the hydrogen evolution reaction (HER) activities of molybdenum disulfide (MoS2) catalysts deposited on an Au substrate electrode were evaluated. The HER active sites on a MoS2 surface are known to be molybdenum-terminated edge sites. Mo-H is generated by sending a reducing potential pulse to the substrate electrode. The reaction between FcDM+ and Mo-H will deliver a feedback current until the titrand that is monitored at the UME tip electrode is fully consumed (Figure 1).18 Arroyo-Curras et al. quantified adsorbed OH and H species generated at the surface of an Ir electrode in alkaline solution.19 Nickel-iron mixed metal oxyhydroxides were evaluated by SI-SECM as oxygen evolution reaction catalysts for solar fuel renewable energy applications.20 SI-SECM has been also applied to study photoprocesses at semiconducting materials.21-24 Simpson et al. reported SECM imaging of photoelectrocatalysts in situ and performed nanoscale redox titration of adsorbed reactive oxygen species (ROS) using SI-SECM.25 K+ permeation through poly(para-nitrostyrene) (PNS) films on a substrate electrode was evaluated by SI-SECM with a Hg-capped SECM tip to supply K+; the performance of K+ was compared to other cations, TBA+ and Li+.26

SECM with distance regulation Control of the distance between the SECM tip electrode and the sample is critical to achieve electrochemical measurements with higher spatial and temporal resolutions. However, nanoscale electrochemical imaging by SECM remains a challenge because of the difficulty of miniaturizing the UME. For constant-distance SECM operation, the distance between the tip and the sample can be regulated by shear force (SF), AFM, ion conductance, and Faraday current. Conversely, SECM imaging captured without feedback regulation, called “constant height” mode, requires consideration of the convolution of topographic and electrochemical information regarding the current responses observed at the UME tip. AFM-SECM probes with high reproducibility show future potential for processing at the wafer level.27,

28

Various insulation materials (Parylene C, silicon nitride, and

intrinsic diamond) have been tested, and the influences of the material composition and the layer sequence on the overall shapes and properties of the combined probes have been evaluated. The fabricated bifunctional AFM-SECM probes were characterized using CV and applied for imaging.29, 30

Topographical and EC images of carbon nanotube (CNT) networks in 20 mM K3Fe(CN)6 and 0.5

M KCl electrolyte were simultaneously collected by AFM-SECM (Figure 2). Owing to the small


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effective distance, CNT networks were successfully visualized without short-circuiting problems, even in contact mode.30 AFM-SECM with peak force tapping (PFT) enables the determination of nanomechanical properties, including adhesion, deformation, and Young’s modulus. The versatility of this combined scanning probe approach has been demonstrated by mapping topographical, electrochemical, and nanomechanical properties of gold microelectrodes and of gold electrodes patterned onto polydimethylsiloxane.31 Scanning Li-nanopipette and probe microscopy (SLi-NPM) has been developed; this technique utilizes an open-ended MWCNT as the AFM-SECM tip. Li can be controllably pipetted to or from the MWCNT tip to deposit a Li thin film on a Si(111) substrate.32 Izquierdo et al. applied AFM-SECM to visualize the pit nucleation and propagation.33 AFM-SECM has been explored to monitor copper ions generated by the dissolution of copper samples while recording the induced changes in topography.34 Nault et al. reported AFM-SECM for in situ mapping of the distribution of proteins on individual virus particles.35 Shear force-based tip–sample distance control is a successful feedback mechanism for high-resolution imaging. In particular, the feedback mechanism using a tuning fork, a relatively simple and low-cost design, has become the standard system for controlling the probe position. Schuhmann and coworkers reported shear-force feedback SECM (SF-SECM).36 A reduced graphene oxide-field emission transistor (rGO-FET) device was characterized by SF-SECM.37 We reported living cell topography and electrochemical imaging by SF-SECM.38, 39 The topographies and the electrochemical activities of a blank SiO2/Si substrate, a GO substrate, and a rGO substrate were also investigated. SF-SECM has been used in association with Raman microspectrometry to perform combined electrochemical and spectrochemical analysis of reactive interfaces.40 The effectiveness of this method was evaluated by analyzing local corrosion phenomena in damaged Zn(Mg, Al) self-healing coatings deposited on steel. Plettenberg et al. applied SF-based distance control for electrochemical reactivity mapping to correlate the topographical data of sample electrodes.41 SECM-SICM has been applied to visualize the reactive chemistry of electroactive features at the 100 to 150 nm scale and to image of biomolecules and electrocatalytic activity. The applications of SECM-SICM are described in the SICM section. The constant-current mode has been shown to be capable of high-resolution imaging; however, it has only been used for topography to date.42, 43 Voltage-switching mode (VSM)-SECM was developed to achieve constant-distance mode measurements with simultaneous electrochemical flux measurements at the same location.44 The UME tip is moved toward the sample surface at the substrate to detect the distance-dependent current for the hindered diffusion of a redox-active species to the tip. By conducting this process at numerous points over the surface, the topography can be mapped. When the electrode reaches the desired position at each point, the applied voltage can be switched to a positive value for electrochemical (flux) imaging of the sample surface. Intermittent contact (IC)-SECM was introduced as a new tip positioning method that



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allows the surface topography and activity to be resolved simultaneously and independently. Changes in the oscillation amplitude, caused by IC of the UME tip with the substrate surface, are used as a feedback signal to control the tip height. Imaging of gold bands on a glass substrate demonstrates the capabilities of this method for simultaneous topography and activity mapping.45 IC-SECM was applied to quantify the rates of electron transfer across single crystal boron-doped diamond (BDD) electrodes.46 Scanning electrochemical impedance microscopy (SEIM) is a type of alternating current (AC) SECM. SEIM allows the extraction of many parameters, including capacitance and charge transfer resistance. However, those parameters are dependent on the tip–sample distance. Local capacitance maps can be obtained and converted into local thickness distribution maps with the aid of finite element simulations.47 AC-SECM and X-ray dispersive techniques have been used to study corrosion processes on a-brass (Cu65-Zn35).48

Probe fabrication and miniaturization Advances in tip fabrication have contributed to expanding the research applications of SECM. Miniaturization, multi-probes, and integration of microfluidic channels have been performed. Reviews on the fabrication of nanoelectrodes and their applications for electrocatalysis, single-cell analysis,

and

high-resolution

electrochemical

imaging

have

been

published

recently.2

Electrochemical push-pull probes have been applied to locally alter the microenvironment of small numbers of adherent living cells. Localized fluorescence labeling and pH changes were introduced to validate the probe as a tool for studying adherent cancer cells.49 A novel finite conical nanoelectrode was fabricated for probing single synapses and monitoring individual vesicular exocytotic events in real time.50 Michalak et al. fabricated a voltammetric nanosensor for reliable pH assessment between pH 2 and 12 with high spatial resolution.51 Prussian Blue (PB) deposited on a nanoelectrode has been applied as an amperometric hydrogen peroxide sensor.52 A single Ni(OH)2 (nickel hydroxide) nanoparticle was deposited on a carbon nanoelectrode and characterized in terms of its energy storage properties and electrocatalysis for the oxygen evolution reaction (OER).53 BLMs were formed at the tip openings of pulled theta pipettes, and the rates of permeation of different carboxylic acids across a bilayer from within the pipette into bulk solution were determined.54 Solid-state ion-selective electrodes have been used as SECM probes. Three-dimensional pH and calcium ion distribution mapping were also successfully performed using these solid-state probes.55 Kiss and Nagy improved potentiometric SECM by using a model system with a high scan rate; the image was then deconvoluted using the inverse of the potentiometric response function to calculate the equilibrium potential.56, 57


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Characterization of electrocatalysis and corrosion SECM has been applied in the study of carbon-supported Pd and Au catalyst arrays on glassy carbon electrode (GCE). Significant enhancements in overall activity for the oxygen reduction reaction (ORR) were observed on both the C/Pd and C/Au catalysts under conditions involving metal oxide reduction.58 Electrocatalytic ORR on Pt nanoelectrode (NE) ensembles with ultramicroelectrode dimensions (UME-NEEs) and nanoparticle electrodes (UME-NPEs) in 0.1 M H2SO4 was evaluated.59 SG-TC SECM was applied for characterization of nanosheet-structured ZnCo2O4/CNTs composite as an ORR catalyst.60 Dobrzeniecka et al. reported ORR on multiwalled carbon nanotubes (MWCNTs) and a composite of MWCNTs and cobalt (IX) protoporphyrin.61 The screening of an electrocatalyst array with SG-TC SECM was reported by Minguzzi et al. using pulsed potential with short times to induce the oxygen evolution reaction (OER) on a substrate electrode where electrocatalyst spots were arrayed.62 SECM has been applied for detecting charged reactive intermediates and has also been applied to the mechanistic analysis of ORR. A nanopipette filled with an organic phase (benzotrifluoride (BTF)) was used as a SECM tip to detect superoxide anion.63 Programmable fabrication of sample microarrays by microprinting followed by comparative SECM was investigated for quick optimization and characterization for specific purposes.64 SG-TC SECM was employed for studying HER at the surface of AZ31 as a model Mg alloy.65 Jamali et al. characterized the active and passive domains on AZ31 using different modes of SECM.65, 66 The oxidation current of FcMeOH mediator recorded at conventional Pt microelectrodes is systematically perturbed by parasitic faradaic contributions from the oxidation of hydrogen.67, 68 Carbon microelectrodes were found to be effective to remove unwanted faradaic contributions, enabling in situ time lapse monitoring of the growth of corrosion product layers.69, 70 Asmussen et al. applied SECM for the characterization of two Al-Mn materials.71 Schaller et al. reported the detection of pre-dissolved hydrogen across an ultra-high strength stainless steel surface.72 Yanagisawa et al. characterized dual-phase carbon steel with ferrite and martensite phases.73 SECM was applied to study the effects of hydrogen sulfide ions (HS−) on the passivity of stainless steel.74 The corrosion behaviors and inhibition effects of aryl-triazino-benzimidazole-2-thiones on carbon steel were investigated by SECM SG-TC using the Fe2+/Fe3+ redox couple as a redox mediator.75 The effects of hydrogen on the surface reactivity of X80 pipeline steel and the interaction of thiosulfate with Alloy 800 in aqueous chloride solutions were studied.76, 77

Characterization of nanomaterials An extremely small nanoelectrode was used for SECM mapping of Au nanoparticles (NPs) on a carbon surface78 and Pt NPs on highly oriented pyrolytic graphite (HOPG)

79

. It is noteworthy

that high-resolution SECM images were captured without the feedback regulation of tip-sample distance. A constant-height SECM image with a ferrocene derivative (Figure 3 (a)) showed



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enhanced currents above the five PtNPs. The same area was scanned by monitoring the hydrogen oxidation reaction (HOR) (Figure 3 (b)). The Pt NPs were better resolved than those shown in Figure 3 (a) because of the closer tip distance.79 SECM was applied to screen Ag NPs for the electrocatalytic degradation of CHCl3.80 The electrochemical activities of AuPd alloys for H2O2 and FcMeOH+ reduction reactions have been investigated in both redox-competition and FB modes.80 Scanning droplet cell microscopy (SDCM) was used to characterize various materials, including organic semiconductors,81 Ce-rich OER catalysts,82 and solar fuel photoanodes in CuO-V2O583. A multiple-type detection system called multi-SDCM has been developed.84 Impedance spectroscopy of localized individual droplets has been performed.85 MoS2, with its active edge sites, is expected to be an alternative to platinum for catalyzing HER. SECM investigation indicated that MoS2 with a strained S vacancy showed an electron transfer rate 4 times higher than that of MoS2 with an unstrained S vacancy.86 Characterization of CNTs embedded in alginate hydrogel beads demonstrated an apparent increase in the number of conductive spots with increasing CNT concentration.87 Blanchard et al. reported SECM characterization of a thiolated aryl multilayer film obtained by electrografting thiophenol diazonium on HOPG.88 SECM was used to introduce carboxylate groups at the graphene substrate.89 Dual Cu-Au UMEs were applied to study the patterning of ferrocene derivatives on their surfaces.90 A nanopipette filled with aryl diazonium cation in DCE was applied for local substrate patterning to induce submicrometer-size grafted spots on a HOPG surface.91

The Belousov-Zhabotinsky (BZ)

oscillating reaction was characterized by indirectly monitoring the short-lived highly reactive radical intermediate BrO2 •.92 Localized reduction of thin (2 to 3 nm) GO films based on the electrogeneration of naphthalene radical anions and electrografting of reduced GO with a diazonium salt was performed.

93

Rapino et al. reported FB-SECM imaging of GO flakes on insulating and

conducting substrates. They evaluated the rate constant, the conductivity of the substrate, and the electrostatic properties of the GO and rGO sheets.94 The oxidation of FcTMA+ at HOPG was used as a model system to demonstrate the effects of reversible reactant adsorption on SECM response.95 Numerical simulations were conducted using a model including a potential-dependent adsorption reaction of the mediator (H+/H2 mediator) at the substrate electrode.96 The impact of the adsorption of ferrocene derivatives in aqueous solutions on graphite HOPG for voltammetric studies has been discussed.97 Chen et al. demonstrated that the asymmetry of paired nanogap voltammograms of FcTMA+ is affected by the cleanliness of the substrate surface in solution by confirming the airborne contamination of HOPG.98

Characterization of battery-related materials SECM has been recognized as a powerful analytical tool for the characterization of lithium-ion batteries (LIBs), including visualization of the electroactive sites of positive and negative


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electrode materials, electronic and ion transport processes, and the stabilities of electrolytes, to evaluate their nanoscale electrochemical functionalities.1,

4, 5, 99-101

The formation of a solid

electrolyte interphase (SEI) on a lithiated graphite composite electrode in inert atmosphere was characterized. FB-SECM imaging revealed heterogeneous active sites on the substrate electrode. Prolonged imaging revealed fluctuation of the passivating properties on time scales between 2 min and 20 h, with inhomogeneous distribution over the sample.99-101 Zampardi et al. demonstrated in operando SECM to evaluate the SEI formation process on GCE using ferrocene as a mediator.102, 103 The electrochemical insertion of Li ions into TiO2 nanoparticles was investigated by FB-SECM at different substrate potentials.103 In operando SECM was also employed to monitor the evolution of the electrically insulating character of the surface of a Si electrode during lithiation and delithiation. The SEI formed on Si electrodes was shown to be intrinsically electrically insulating. However, volume changes upon (de-)lithiation lead to the loss of the protecting character of the initially formed SEI.104 SECM was applied to detect the influx of oxygen in electrolyte close to the outer surface of a gas diffusion electrode (GDE)105. The oxygen reduction current at the SECM tip was monitored to evaluate the oxygen permeation of GDEs with passivation films of different thicknesses.100, 105 AFM and SECM were combined for in situ investigation of surface reactions in LIBs. SEI formation on GCE has been investigated.106 AFM revealed changes in topography during SEI formation. FB-SECM visualized the local activity of the sample where the AFM tip was used to partially remove the SEI by scratching Li2O2 and solvent degradation and oxidation products on a gold electrode in 0.1 M LiPF6 DMSO electrolyte with soluble tetrathiofulvalene (TTF) using a variety of techniques: rotating ring disk electrode (RRDE), SECM, electrochemical quartz crystal microbalance (EQCM), and AFM.106 Hg-capped Pt ultramicroelectrodes were tested as probes for Li ions in propylene carbonate (PC) in an oxygen- and water-free environment. We reported on the quantitative, spatially resolved study of ionic processes for energy materials in nonaqueous environments by in situ electrochemical methods on the micro and nanoscale.107 Schwager et al. characterized the generation of superoxides in Li-oxygen battery systems. Superoxides formed during the charge and discharge reactions in lithium ion-containing DMSO electrolytes and were released into the solution.108

Characterization of biomaterials The advantage of applying SECM technology to the evaluation of biological samples is the ability to screen a targeted sample on a single solid substrate chip arrayed with biomaterials of many different types. In addition, SECM has been applied for the characterization of various cellular functions of living cells and cellular aggregates under physiological conditions. Gdor et al. reported SECM imaging of printed patterns of multiple enzymes.109 Various



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arrays of enzymes were printed with an inkjet printer. The operation of the enzyme-based logic gates was studied. A SECM-based DNA biosensing platform has been introduced by Chen et al.110 This platform exhibited a low detection limit of 0.18 aM of target DNA by immobilization of a thiol-tethered DNA capture probe on an Au substrate. SECM was applied for the sensitive detection of single base mismatches (SBMs) in sandwiched dsDNA.111 A combination of SECM and a continuous nanoflow (CNF)

system was

used to characterize horseradish peroxidase

(HRP)-amplified imaging of protein microarrays.112 Kinase-catalyzed phosphorylation has been detected using a ferrocene-labeled substrate.113 For the characterization of living cells, a non-invasive analytical tool with high sensitivity and capability is demanded to correlate the morphology and activity of live cells. Vasanthan et al. reported SECM investigation of live HepG2 cells cultured on galactose-incorporated scaffolds.

114

Sridhar et al. used a combination of SECM and large-scale microtissue arrays formed in a stamped petri dish to conduct non-invasive cellular assays on 3D cellular models.115 The respiratory activity of live HeLa microtissues has been assessed by monitoring the oxygen reduction current in constant height mode SECM. We reported SECM investigation of the respiration activity of mouse embryonic stem cells,

116, 117

rat primary hepatocyte spheroids,118 muscle cells embedded in a

hydrogel-metallic glass wire complex,119 the β-galactosidase activity of cellular aggregates as a biomarker for senescence,120 the alkaline phosphatase activity of single cells entrapped in microwells with ring-electrode arrays121 and intracellular enzymatic activities.122 Membrane protein expression levels have also been evaluated using SECM.123, 124 Lin et al. mapped the prognostic indicator tyrosinase (TyR) in non-metastatic and metastatic melanoma tissues using soft-stylus microelectrodes.125 TyR activity was also mapped as a prognostic indicator for non-metastatic and metastatic melanoma tissues by adapting an immunochemical method.126 Single contractile myotubes have been investigated by SECM.127 The oxygen reduction current at the SECM tip was observed to immediately decrease after application of 1.0 Hz electric pulses because of oxygen consumption due to the contraction of myotubes accompanied by consumption of energy. SECM was used to determine the membrane permeability of single live human bladder cancer (T24) cells affected by Cd2+. SECM affords a microscopic view of the heterogeneous flux distribution across porous membranes.128 Dongmo et al. reported electropolymerization onto a grafted monolayer of plumbagin (PLG) on glassy carbon (PLG/GCE) to yield continuous growth of a polymer film (polyPLG/PLG/GCE).129 Kuss and Polcari et al. prepared co-cultured cells of nonresistant human cancer cells and their multidrug-resistant variants using a stencil-based patterning method. The constant-height mode SECM measurements of the patterned cells enabled mapping of the activity of ATP-binding cassette transporters.130 SECM was applied for real-time characterization of the dynamic formation process of a biofilm of Shewanella oneidensis MR-1 on an electrode by monitoring the redox mediator current of Ru(NH3)6Cl3.131


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Scanning Scanning ion conductance microscopy (SICM) Scanning ion conductance microscopy (SICM), which was invented by Paul Hansma in 1989, is a unique and promising technique for noncontact topographical imaging under physiological conditions.132 SICM uses a nanopipette as a probe to detect the ionic current between an electrode inside the pipette and an electrode located in a bath. The pipette–sample distance is regulated by the ionic currents used as feedback signals. When the pipette approaches a sample surface, the resistance between the two electrodes increases, and the ionic current decreases. The magnitude of the ionic current depends on the pipette–sample distance. SICM has been used for the visualization of live cell dynamic structure changes and functional imaging with hybrid systems of SICM and other analytical tools.133, 134 Here, we show the recent progress and applications of SICM.

Recent progress in imaging modes The scanning method is a key element for measuring convoluted live cell surface topographies because the feedback distance control system cannot predict height information in advance. In conventional scanning methods, the tip–sample distance is controlled by maintaining the set point of a feedback signal at a constant value. This deforms the live cell surface and generates artifacts during the lateral motion of the tip. Imaging high-aspect-ratio structures, such as the narrow axon and large cell body of a neuron, is especially difficult. Recently, Pavel and coworkers developed hopping mode SICM. Hopping mode, in which the probe repeatedly approaches the sample surface and retracts at each point, is a promising method for imaging the convoluted surface topographies of living cells.135 However, this image acquisition is very time consuming compared with conventional lateral scanning methods. This limitation was resolved by changing the step size during measurement and prescanning the area of interest. When visualizing the topography of nanoscale cavities or protrusions, such as clathrin pits and stereocilia, overshoot due to a delay of the Z axis piezo actuator during falling causes fatal deformation of the structure of the sample. To compensate for this overshoot, Andrew and coworkers installed another small high resonance frequency piezo actuator on top of the Z piezo actuator (Figure 4 (a)).136 This short travel piezo actuator was used only at the most critical moments. This system, called a “brake booster,” was used for time-lapse imaging of clathrin-dependent endocytosis136 and nanoparticle internalization137. The patch-clamp technique is used to study the characteristics of ion channels in living cells. Ion channels are frequently associated with specific subcellular structures and are not uniformly distributed on the cell surface. However, it is difficult to identify the position of an ion channel at the cell surface because a patch-clamp pipette is typically positioned using manual



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adjustments while focusing between the pipette and sample under a light microscope. Gorelik and coworkers developed the smart patch-clamp method.138 In this method, a pipette is used for SICM topography imaging and patch-clamp recording. They used it to characterize channel L-type calcium channel currents in the T-tubules of adult rat cardiomyocytes. In the conventional smart-patch technique, topography imaging and patch-clamp recording are performed using the same micropipette. The size of the micropipette limits the resolution. Especially, resolving individual synaptic boutons and axons on topographical images requires high-resistance pipettes with small inner tip diameters (∼100 nm). However, it is difficult to break the presynaptic cell membrane with the high-resistance pipettes used for whole-cell patch-clamp recordings. To overcome this limitation, Pavel and coworkers optimized a method to widen the ultra-fine pipette tip after high-resolution topography imaging.139 They broke the ultra-fine pipette tip on the glass coverslip using the programmable feedback control of the SICM scanner controller (Figure 4(b)). Using this technique, they acquired whole-cell recordings of small presynaptic boutons with a characteristic size of ∼1 µm. Determining the distribution of ion channels on the bulk of tissue has also become essential for understanding cellular mechanisms. However, currently available SICM tips are placed directly above the specimen; it is difficult to combine the SICM setup with phase contrast or high magnification upright optics. Andrew and coworkers developed angle scan SICM, which enables mounting of the SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle (Figure 4 (c)).140 This angle can be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, they obtained topographical images of islets of Langerhans, hippocampal neurons, and cardiomyocytes. They also found NaV1.5 clusters at the intercalated disk of a cardiomyocyte (Figure 5).141

SECM-SICM hybrid system A high-spatial-resolution chemical detection tool in physiological conditions is needed to evaluate the relationship between the localized topography and the function of a substrate. For this purpose, SICM has been combined with SECM to obtain topography and ionic/electrochemical properties under physiological conditions without physical contact. Two types of SECM-SICM probe, ring142,

143

and double barrel,144 were developed for SECM-SICM hybrid systems for

simultaneous topography and electrochemical imaging. A ring-type probe was applied to reveal the distribution of the activity of enzyme spots on uneven surfaces with submicrometer resolution. The topographic information of differentiated PC12 and neuro-transmitter release events were obtained by SECM-SICM. An advantage of SECM-SICM is that the probe filled with electrolyte solution can be used to inject different reagents for localized stimulation of the cell. Therefore, voltage-driven


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application of K+ ions was realized by SECM-SICM to achieve both local depolarization of the cell membrane and detection of a neurotransmitter (Figure 6(a)). The high spatial resolution of SECM-SICM is effective to evaluate the catalytic activity of nanoparticles. Au nanoparticles (AuNPs) were chemically imaged by detecting localized H2O2 generation145 or positive FB current amplification with FcMeOH as a mediator.146 SECM-SICM was also applied to monitor the morphological changes and appearance of surface defects on degraded Nafion® 212 (N212) membranes treated with Fenton's reagent under a series of degradation conditions.147 Potentiometric SICM is also effective to evaluate local pH. Unwin and coworkers developed double-barrel pH probes. The probes incorporate an iridium oxide-coated carbon electrode for pH measurement and an SICM barrel for distance control, enabling simultaneous pH and topography mapping. They measured both pH and topographical changes during the dissolution of a calcite microcrystal in aqueous solution.148 Baker and coworkers fabricated a polyaniline (PANi) thin film on the surface of a gold electrode for detecting pH.149 They also developed potentiometric scanning ion conductance microscopy (P-SICM) with a dual-barrel probe; in this method, the probe position is controlled by the current measured in one barrel, and the potential is measured in a second barrel.150 They used P-SICM to quantify paracellular conductance heterogeneity in epithelial cells. Zhang and coworkers developed nanometric field-effect-transistor (FET) sensors on the tips of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes.151 A channel of electrodeposited polypyrrole (PPy) exhibits high sensitivity toward pH changes and can be applied to real-time monitoring of ATP (Figure 6 (b)). 151

Hybrid system of SICM and fluorescence microscope Fluorescence techniques are important to identify the relationship between the dynamic phenomena of a cell surface and interacting biomolecules. Gorelik and coworkers developed scanning surface confocal microscopy (SSCM), a combination of SICM and scanning confocal microscopy, to simultaneously record topographic and quantitative fluorescence images of the cell surface.138 They visualized single-nanoparticle interactions with live membrane processes. Endocytic pits are suitable targets for SSCM because their nanoscale cavities and dynamic structural changes are difficult to visualize by conventional optical microscopy. Andrew and coworkers identified the precise positions and sizes of pits by SICM and determined the GFP-tagged molecular nature of the pits, e.g., clathrin-coated or caveolae, by SSCM.152 A fluorescence resonance energy transfer (FRET) and SICM combination system has also been reported to determine both the position and function of endogenous β-adrenergic receptors (βARs).153



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Local chemical injection and collection SICM nanopipettes are also effective tools for local chemical injection/ejection. The nanopipette can be easily navigated to a specific position by the support of topography images. Klenerman and coworkers used SICM to pattern biomolecules on a substrate in solution.154-162 The amount of biomolecule ejection can be controlled by the applied voltage. Local cell stimulation has also been performed using this technique. 163 It is very difficult to inject ultrasmall volumes (fL to aL level) of solution or neutral micro-molecules using conventional voltage- or pressure-driven systems. Mirkin and coworkers developed an electrochemistry-based solution manipulation system called an electrochemical attosyringe.164 The application of voltage across the liquid/liquid interface changes the surface tension, providing a driving force to move the position of the liquid/liquid interface in the nanopipette; this enables control of the injection/ejection and collection of a solution. This technique can be used to control attoliter-to-picoliter (10−18 to 10−12 liter) volumes of solution. Paolo and coworkers used this technique to collect the cytosol of a single cell for mRNA analysis.165 A hybrid system of SICM and an attosyringe was fabricated to evaluate the localization of mRNA in single cells.166 Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions that were used for SICM and as an electrochemical attosyringe, respectively. SICM imaging can be utilized to analyze the localization of mRNA in single cells; the small aperture of the nanopipette is also effective to ionize chemicals for electrospray. Baker and coworkers used a nanopipette with

Scanning Probe Microscopy for Nanoscale Electrochemical Imaging

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