Screening of photocatalysts by scanning electrochemical microscopy

A method for rapid screening of photocatalysts employing a form of scanning electrochemical microscopy (SECM) is described. A piezoelectric dispenser was used to deposit arrays composed of approximately 300-microm-size photocatalyst spots with different compositions onto conducting glass, fluorine-doped tin oxide substrate. The scanning tip of the SECM was replaced by a fiber optic connected to a xenon lamp and was rapidly scanned over the array. In this arrangement, the photocatalytic performance of the spots was evaluated by measuring the photocurrent at the substrate of the array. A fiber optic with a ring electrode can also be used to electrochemically detect products of the photoreaction. Several iron oxide-based bimetallic oxide combinations were found to exhibit enhanced photocatalytic activity, when compared to pure alpha-Fe2O3. These combinations included iron-palladium, iron-europium, and iron-rubidium in specific ratios. A trimetallic bismuth-vanadium-zinc oxide combination was also found to show a higher photocurrent, by approximately 40%, compared to BiVO3.     

Microimaging of algal bioconvection by scanning electrochemical microscopy

Local bioconvection generated by algal flagellar movement was imaged by scanning electrochemical microscopy. As a microelectrode probe vertically approached an individual multicellular flagellate alga, Volvox carteri, an oxidation current of a coexisting redox marker ([Fe(CN)6]4-) increased gradually, due to bioconvective enhancement of mass transport, and eventually decreased because the algal body blocked the diffusion of the marker. Two-dimensional imaging of the bioconvection of an individual alga was also possible. The bioconvective enhancement of the current was hindered by a toxic compound that inhibits the flagellar movement.     

Scanning electrochemical microscopy: approach curves for sphere-cap scanning electrochemical microscopy tips

The parameters of functions used to predict diffusion-controlled scanning electrochemical microscopy approach curves under positive and negative (hindered diffusion) feedback for sphere-cap tips are reported. These functions were obtained by fitting approach curves simulated with an error-bounded adaptive finite element algorithm. Several geometries corresponding to different sphere-cap dimensions were considered including the effect of the tip insulating sheath. The simulated approach curves were successfully compared with experimental ones obtained with mercury sphere caps electrodeposited onto platinum microdisk electrodes.     

Microfluidic push-pull probe for scanning electrochemical microscopy

This paper presents a microfluidic push-pull probe for scanning electrochemical microscopy (SECM) consisting of a working microelectrode, an integrated counter/reference electrode and two microchannels for pushing and pulling an electrolyte solution to and away from a substrate. With such a configuration, a droplet of a permanently renewed redox mediator solution is maintained just at the probe tip to carry out SECM measurements on initially dry substrates or in microenvironments. For SECM imaging purposes, the probe fabricated in a soft polymer material is used in a contact regime. SECM images of various gold-on-glass samples demonstrate the proof-of-concept of a push-pull probe for local surface activity characterization with high spatial resolution even on vertically oriented substrates. Finite element computations were performed to guide the improvement of the probe sensitivity.     

Noncontact electrochemical imaging with combined scanning electrochemical atomic force microscopy

Combined scanning electrochemical atomic force microscopy (SECM-AFM) is a recently introduced scanned probe microscopy technique where the probe, which consists of a tip electrode and integrated cantilever, is capable of functioning as both a force sensor, for topographical imaging, and an ultramicroelectrode for electrochemical imaging. To extend the capabilities of the technique, two strategies for noncontact amperometric imaging-in conjunction with contact mode topographical imaging-have been developed for the investigation of solid-liquid interfaces. First, SECM-AFM can be used to image an area of the surface of interest, in contact mode, to deduce the topography. The feedback loop of the AFM is then disengaged and the stepper motor employed to retract the tip a specified distance from the sample, to record a current image over the same area, but with the tip held in a fixed x-y plane above the surface. Second, Lift Mode can be employed, where a line scan of topographical AFM data is first acquired in contact mode, and the line is then rescanned to record SECM current data, with the tip maintained at a constant distance from the target interface, effectively following the contours of the surface. Both approaches are exemplified with SECM feedback and substrate generation-tip collection measurements, with a 10-microm-diameter Pt disk UME serving as a model substrate. The approaches described allow electrochemical images, acquired with the tip above the surface, to be closely correlated with the underlying topography, recorded with the tip in intimate contact with the surface.     

Oxygen consumption of single bovine embryos probed by scanning electrochemical microscopy

Oxygen consumption of individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). A probe microelectrode was used to scan near a single embryo surface in a culture medium to monitor the oxygen reduction current at 37 degrees C, under a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profiles near the embryos were in good agreement with the theoretical spherical diffusion. When an embryo reached the stage of a morula with a 74-microm radius on day 6 after in vitro fertilization, the oxygen concentration difference (deltaC) between the bulk solution and the morula surface was 6.90 +/- 1.35 microM. The oxygen consumption rate (F) of the single morula was estimated to be (1.40 +/- 0.27) x 10(-14) mol s(-1). After the SECM measurement, the embryo was continuously cultured for another 2 days and grew to the stage of a blastocyst with a 100-microm radius. For the blastocyst, the deltaC values for the inner cell mass side and the trophoblast side were 16.40 +/- 1.83 and 9.14 +/- 1.68 microM, respectively. The oxygen consumption rate of the blastocyst was found to be in the range of (2.50 +/- 0.46) x 10(-14) mol s(-1) < F < (4.49 +/- 0.50) x 10(-14) mol s(-1). We have carried out SECM measurements for 19 embryos, and the results were compared in detail with these from an optical microscopic observation. The deltaC values for the morulae on day 6 after in vitro fertilization were strongly related to the morphological embryo quality. The morulae showing a larger deltaC value developed into blastocysts of a larger size, and the deltaC value after the subsequent 2 days of cultivation was found to be increased.     

Soft microelectrode linear array for scanning electrochemical microscopy

A linear array of eight individual addressable microelectrodes has been developed in order to perform high-throughput scanning electrochemical microscopy (SECM) imaging of large sample areas in contact regime. Similar to previous reports, the soft microelectrode array was fabricated by ablating microchannels on a polyethylene terephthalate (PET) film and filling them with carbon ink. Improvements have been achieved by using a 5 μm thick Parylene coating that allows for smaller working distances, as the probe was mounted with the Parylene coating facing the sample surface. Additionally, the application of a SECM holder allows scanning in contact regime with a tilted probe, reducing the topographic effects and assuring the probe bending direction. The main advantage of the soft microelectrode array is the considerable decrease in the experimental time needed for imaging large sample areas. Additionally, soft microelectrode arrays are very stable and can be used several times, since the electrode surface can be regenerated by blade cutting. Cyclic voltammograms and approach curves were recorded in order to assess the electrochemical properties of the device. An SECM image of a gold on glass chip was obtained with high resolution and sensitivity, proving the feasibility of soft microelectrode arrays to detect localized surface activity. Finite element method (FEM) simulations were performed in order to establish the effect of diffusion layer overlapping between neighboring electrodes on the respective approach curves.     

Impedance feedback control for scanning electrochemical microscopy.

A new constant-distance imaging method based on the relationship between tip impedance and tip-substrate separation has been developed for the scanning electrochemical microscope. The tip impedance is monitored by application of a high-frequency ac voltage bias between the tip and auxiliary electrode. The high-frequency ac current is easily separated from the dc-level faradaic electrochemistry with a simple RC filter, which allows impedance measurements during feedback or generation/collection experiments. By employing a piezo-based feedback controller, we are able to maintain the impedance at a constant value and, thus, maintain a constant tip-substrate separation. Application of the method to feedback and generation/collection experiments with tip electrodes as small as 2 microm is presented.     

Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy

A new technique combining scanning electrochemical microscopy (SECM) and single-molecule fluorescence spectroscopy was developed to accomplish locally and temporally defined pH adjustments in buffer solutions and on surfaces monitored by fluorescence alteration of pH-sensitive fluorophores in real time. Local pH gradients were created by electrochemical generation of H(+) or OH(-) during redox reactions at ultramicro- or nanoelectrodes with radii from 5 microm to 35 nm. Ratiometric fluorescence measurements were performed with a confocal laser microscope using two detectors for different spectral regions. Time-resolved pH measurements were carried out with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCD camera. The fluorophore SNAFL-succinimidyl ester was bound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplished with an accuracy of 0.2 unit. The measured pH profiles showed a strong dependence on the tip diameter, the buffer/mediator concentration ratio, and the tip-surface distance. As an application for bionanotechnology using SECM-induced pH changes on the molecular level, the proton-driven ATP synthesis by single membrane-bound F(0)F(1)-ATP synthases was investigated. ATP synthesis resulted in stepwise subunit rotation within the enzyme that was monitored by single-molecule fluorescence resonance energy transfer.     

Detection of hydrogen peroxide produced during electrochemical oxygen reduction using scanning electrochemical microscopy

The substrate-generation/tip-collection mode of scanning electrochemical microscopy was used to detect hydrogen peroxide formed as an intermediate during oxygen reduction at various electrodes. The experiment is conceptually similar to rotating ring-disk experiments but does not require the production of a ring-disk assembly for the specific electrode material in question. In order to limit the extension of the diffusion layer above the sample, the sample electrode potential is pulsed while the Pt ultramicroelectrode probe (UME) is held at a constant potential for oxidative amperometric detection of hydrogen peroxide. The signal at UME is influenced by the sample region within the diffusion length of hydrogen peroxide during the pulse of 2.5 s. The method is tested with three model electrodes showing different behavior with respect to the oxygen reduction reaction (ORR) in acidic solution. Simple analytical models were used to extract effective rate constants for the most important reaction paths of ORR at gold and palladium-cobalt samples from the chronoamperometric response of the UME to a reduction pulse at the sample electrode.     

Detection of microspotted carcinoembryonic antigen on a glass substrate by scanning electrochemical microscopy

Microspots of carbinoembryonic antigen (CEA) on glass substrates were characterized by scanning electrochemical microscopy (SECM). CEA was immobilized via a sandiwch method using horseradish peroxidase (HRP)-labeled anti-CEA. The reduction current of the oxidized form of ferrocenylmethanol generated by the HRP reaction was monitored to view SECM images. This method detects as low as ～10(4) CEA molecules in a single 20-μm-radius spot.     

Cathodic electropaint insulated tips for electrochemical scanning tunneling microscopy

A method of preparing tungsten tips insulated for in situ scanning tunneling microscopy (STM) work is presented. Tips were electrocoated at low applied voltages using an organic solution of the cathodic electropaint rather than the more frequently utilized electropaint emulsions. The insulated tips were then characterized using cyclic voltammetry and electrochemical STM (EC-STM). They displayed low Faradaic leakage currents under electrochemical conditions and provided for high-resolution STM imaging in electrolyte solution. Flat terraces on a monocrystalline Au surface and adlayers of 5,10,15,20-tetraphenyl-21H,23H-porphine cobalt(II) were imaged under potential control in dilute HClO4 using the fabricated tips, and atomic or molecular resolution images were obtained in both cases. The developed procedure allows for the fast, reproducible generation of large numbers of electrically insulated tungsten tips suitable for EC-STM imaging experiments.     

Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.     

Study of electron transfer across the liquid/ice-like matrix interface by scanning electrochemical microscopy

In this work, we report the findings of a study on scanning electrochemical microscopy (SECM) to investigate the interfacial electron-transfer (ET) reaction between the 7,7,8,8-tetracyanoquinodimethane radical anion (TCNQ*-) in 1,2-dichloroethane and ferricyanide in an ice-like matrix (a mixture of insulting ice and conductive liquid) under low temperatures. Experimental results indicate that the formed liquid/ice-like matrix interface is superficially similar in electrochemical characteristics to a liquid/liquid interface at temperatures above -20 degrees C. Furthermore, imaging data show that the surface of the ice-like matrix is microscopically flat and physically stable and can be applied as either a conductive or an insulting substrate for SECM studies. Perchlorate ion was selected as the common ion in both phases, the concentrations of which controlled the interfacial potential difference. The effect of perchlorate concentration in the DCE phase on interfacial reactions has been studied in detail. The apparent heterogeneous rate constants for TCNQ*- oxidation by Fe(CN)6(3-) in another phase under different temperatures have been calculated by a best-fit analysis, where the experimental approach curves are compared with the theoretically derived relationships. Reaction rate data obey Butler-Volmer formulation before and after the freezing point, which is similar to most other known cases of ET reactions at liquid/liquid interfaces. However, there is a sharp change observed for heterogeneous rate constants around the freezing point of the aqueous phase, which reflects the phase transition. At temperatures below -20 degrees C, surface-confined voltammograms for the reduction of ferricyanide were obtained, and the ice-like matrix became an insulating one, which indicates that the aqueous phase is really a frozen phase.     

Nanowire probes for high resolution combined scanning electrochemical microscopy - atomic force microscopy

We describe a method for the production of nanoelectrodes at the apex of atomic force microscopy (AFM) probes. The nanoelectrodes are formed from single-walled carbon nanotube AFM tips which act as the template for the formation of nanowire tips through sputter coating with metal. Subsequent deposition of a conformal insulating coating, and cutting of the probe end, yields a disk-shaped nanoelectrode at the AFM tip apex whose diameter is defined by the amount of metal deposited. We demonstrate that these probes are capable of high-resolution combined electrochemical and topographical imaging. The flexibility of this approach will allow the fabrication of nanoelectrodes of controllable size and composition, enabling the study of electrochemical activity at the nanoscale.     

Visualization of chlorine evolution at dimensionally stable anodes by means of scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) has been used to detect and visualize the local electrocatalytic activity of dimensionally stable anodes (DSA) for Cl(2) evolution from brine. The sample generation-tip collection (SG-TC) mode of SECM shows limitations arising from complications connected with the reduction of Cl(2) at the SECM tip due to the presence of a significant amount of nondissolved Cl(2) gas. Because only dissolved Cl(2) can be electrochemically reduced at the tip, a large amount of the Cl(2) gas which is produced at active spots of the DSA is not detected. Additionally, a decrease of the cathodic current at the tip may occur owing to the adhesion of gas bubbles and blocking of the electrode surface. To overcome this limitation, the redox competition mode of SECM was extended and applied to the local visualization of Cl(2) evolution from highly concentrated brine solutions. High concentrations of Cl(2) produced at the sample can cause inhibition of the same reaction at the tip by accumulation of Cl(2) in the proximity of the SECM tip. In this way the tip current is decreased, which can be used as a measure for the catalytic activity of the sample underneath the tip.     

Spatially addressed deposition and imaging of biochemically active bead microstructures by scanning electrochemical microscopy

A new procedure is described to deposit paramagnetic beads on surfaces to form microscopic agglomerates. By using surface-modified beads, microscopic structures with defined biochemical activity are formed. The shape and size of agglomerates were characterized by scanning electron microscopy (SEM), and the biochemical activity was mapped with scanning electrochemical microscopy (SECM). This approach is demonstrated using beads modified with anti-mouse antibodies (Ab). After allowing them to react with a conjugate of mouse IgG and alkaline phosphatase (ALP), the beads were deposited as agglomerates of well-defined size and shape. The biochemical activity was recorded in the generation-collection SECM mode by oxidizing 4-aminophenol formed in the ALP-catalyzed hydrolysis of 4-aminophenyl phosphate at the surface of the beads. The signal height correlated with both the amount of beads present in one agglomerate and the proportion of Ab binding sites saturated with the ALP mouse IgG conjugate. The feedback mode of the SECM was used to image streptavidin-coated beads after reaction with biotinylated glucose oxidase.     

Examining ultramicroelectrodes for scanning electrochemical microscopy by white light vertical scanning interferometry and filling recessed tips by electrodeposition of gold

In this paper, we present a technique to rapidly and directly examine ultramicroelectrodes (UMEs) by white light vertical scanning interferometry (VSI). This technique is especially useful in obtaining topographic information with nanometer resolution without destruction or modification of the UME and in recognizing tips where the metal is recessed below the insulating sheath. Two gold UMEs, one with a metal radius a = 25 μm and relative insulating sheath radius RG = 2 and the other with a = 5 μm and RG = ～1.5, were examined, and the average depth of the gold recessions was determined to be 1.15 μm and 910 nm, respectively. Electrodeposition of gold was performed to fill the recessed hole, and the depth was reduced to ～200 nm. With the electrodeposited gold electrode and a conventional microelectrode (a = 25 μm) as a tip and substrate, respectively, a tip/substrate distance, d, of 600 nm was achieved allowing scanning electrochemical microscopy (SECM) in positive feedback mode at a close distance, which is useful for measuring fast kinetics.     

In situ spatial and time-resolved studies of electrochemical reactions by scanning transmission X-ray microscopy

The first in situ measurements with scanning transmission X-ray microscopy (STXM) of an active electrochemical cell are reported. An electrochemical wet cell, consisting of an electrodeposited polyaniline thin film on a thin Au film covered by an overlayer of 1 M HCl solution sitting between two X-ray transparent silicon nitride windows, was assembled. X-ray absorption images and spatial and time-resolved spectra of this system under potential control were examined using the beamline 5.3.2 STXM at the Advanced Light Source. The chemical state of the polyaniline film was reversibly converted between reduced (leucoemeraldine) and oxidized (emeraldine chloride) states by changing the applied potential. The electrochemical changes were monitored by spatially resolved C 1s and N 1s X-ray absorption spectroscopy and chemical-state selective imaging. Comparison of differences between images at two energies at different potentials provided electrochemical contrast with a resolution better than 50 nm, thereby monitoring that component of the polyaniline film that was electrochemically active. Kinematic studies in the subsecond regime are demonstrated.     

Screening of oxygen evolution electrocatalysts by scanning electrochemical microscopy using a shielded tip approach

Oxygen evolution electrocatalysts in acidic media were studied by scanning electrochemical microscopy (SECM) in the substrate generation-tip collection (SG-TC) imaging mode with a 100 microm diam tip. Pure IrO2 and Sn(1-x)Ir(x)O2 combinatorial mixtures were prepared by a sol-gel route to form arrays of electrocatalyst spots. The experimental setup has been developed to optimize screening of electrocatalyst libraries under conditions where the entire array is capable of the oxygen evolution reaction (OER). The activity of individual spots was determined by reducing the interference from the reaction products of neighboring spots diffusing to the tip over the spot of interest. A gold layer deposited on the external wall of the SECM tip was used as a tip shield. In this study the shield was kept at a constant potential to reduce oxygen under mass transfer controlled conditions. The tip shield consumes oxygen coming from the neighbor spots in the array and enables the tip to correctly detect the activity of the spot below the tip. Simulations and experimental results are shown, demonstrating the effectiveness of the tip shield with the SG-TC setup in determining the properties of the composite materials and imaging arrays.