Fabrication and characterization of probes for combined scanning electrochemical/optical microscopy experiments

A technique that combines scanning electrochemical microscopy (SECM) and optical microscopy (OM) was implemented with a new probe tip. The tip for scanning electrochemicaVoptical microscopy (SECM/OM) was constructed by insulating a typical gold-coated near-field scanning optical microscopy tip using electrophoretic anodic paint. Once fabricated, the tip was characterized by steady-state cyclic voltammetry, as well as optical and electrochemical approach experiments. This tip generated a stable steady-state current and well-defined SECM approach curves for both conductive and insulating substrates. Durable tips whose geometry was a ring with < 1 microm as outer ring radius could be consistently fabricated. Simultaneous electrochemical and optical images of an interdigitated array electrode were obtained with a resolution on the micrometer scale, demonstrating good performance of the tip as both an optical and an electrochemical probe for imaging microstructures. The SECM feedback current measurements were successfully employed to determine tip-substrate distances for imaging.     

Scanning electrochemical microscopy. 55. Fabrication and characterization of micropipet probes

The fabrication and characterization of novel micropipet probes for use in scanning electrochemical microscopy (SECM) are described. These can be used to dispense small (pL) amounts of a solution while monitoring the electrochemical response at a substrate and at a ring electrode tip on the micropipet probe. The probes were constructed by insulating gold-coated borosilicate micropipets with electrophoretic paint and exposing a ring electrode at the tip by heat treatment. Characterization of the probes was performed using scanning electron microscopy, cyclic voltammetry, and SECM approach curve experiments. Routine construction of tips with diameters of the order of 3 microm was possible using this technique. The probes exhibited stable steady-state currents and positive and negative feedback approach curves that agreed with those predicted by theory. Demonstrative SECM imaging experiments were performed using a picodispenser to continuously dispense an electroactive solution (ferrocenemethanol) to the SECM cell while the probe was located within a few micrometers of a Pt substrate surface. Oxidation of the dispensed electroactive solution was performed at the substrate, and feedback currents were measured at the probe tip by holding the gold ring at a reducing potential. This mode of tip-dispensing SECM was used to obtain images of a platinum substrate electrode while monitoring both the substrate current and the feedback current at the probe.     

Probing heterogeneous electron transfer at an unbiased conductor by scanning electrochemical microscopy in the feedback mode

The theory of the feedback mode of scanning electrochemical microscopy is extended for probing heterogeneous electron transfer at an unbiased conductor. A steady-state SECM diffusion problem with a pair of disk ultramicroelectrodes as a tip and a substrate is solved numerically. The potential of the unbiased substrate is such that the net current flow across the substrate/solution interface is zero. For a reversible substrate reaction, the potential and the corresponding tip current depend on SECM geometries with respective to the tip radius including not only the tip-substrate distance and the substrate radius but also the thickness of the insulating sheath surrounding the tip. A larger feedback current is obtained using a probe with a thinner insulating sheath, enabling identification of a smaller unbiased substrate with a radius that is approximately as small as the tip radius. An intrinsically slow reaction at an unbiased substrate as driven by a SECM probe can be quasi-reversible. The standard rate constant of the substrate reaction can be determined from the feedback tip current when the SECM geometries are known. The numerical simulations are extended to an SECM line scan above an unbiased substrate to demonstrate a "dip" in the steady-state tip current above the substrate center. The theoretical predictions are confirmed experimentally for reversible and quasi-reversible reactions at an unbiased disk substrate using disk probes with different tip radii and outer radii.     

Scanning electrochemical microscopy. 38. Application of SECM to the study of charge transfer through bilayer lipid membranes

The use of the scanning electrochemical microscope (SECM) to probe the kinetics of charge-transfer processes at bilayer lipid membranes (BLM) is presented. Analysis of the SECM tip response demonstrates that an unmodified BLM behaves as an insulator, whereas a BLM doped with iodine shows some positive feedback. The SECM technique thus allows one to probe processes at a BLM and determine the kinetics of the charge-transfer process. The SECM can also be used to determine the shape of the BLM.     

Scanning electrochemical microscopy, 52. Bipolar conductance technique at ultramicroelectrodes for resistance measurements

The bipolar conductance, BICON, technique for the measurement of solution resistance, based on the application of microsecond current pulses, as originally described by Enke and co-workers for measurements with conventional electrodes, was extended for use with ultramicroelectrodes, with a focus on its application in scanning electrochemical microscopy (SECM). When the plateau time used to make the measurement lies within the BICON conditions, the solution conductance can be obtained directly from the output without the need for calibration curves. However, decreasing the size of the ultramicroelectrode decreases the range of values that satisfy these conditions, and one must resort to calibration curves to obtain solution conductance from the measured current, which was nevertheless found to be proportional to electrolyte concentration with electrodes as small as 5 mum in diameter. BICON/SECM approach curves over insulating substrates followed SECM negative feedback theory and approach curves in the presence of low (micromolar) or no added electrolyte are possible once the background conductivity is taken into account. Approach curves to a conducting substrate at open circuit potential are influenced by the solution time constant (solution resistance at the electrode tip x electrode double layer capacitance), which is a function of the tip/substrate distance, as well as the substrate size.     

Application of scanning electrochemical microscope in the study of corrosion of metals

Scanning electrochemical microscope (SECM) has become a very useful and powerful technique for probing a variety of electrochemical reactions in corrosion process due to its high spatial resolution and electrochemical sensitivity to characterize the topography and redox activities of the metal/electrolyte solution interface. Its capability for the direct identification of chemical species in localized corrosion processes with high spatial resolution would be more advantageous compared to other local probe techniques with only morphological characterization. In this review, the applications of the SECM in the study of early stages of localized corrosion, electroactive defect sites in passive films, local initiation of pits, degradation of coating properties on steels, and some combined methods through SECM integrated with other techniques have been summarized and commented. Finally, the optimization for SECM’s experiment design and operation as well as foreseeable application range has been proposed.     

Alternating current impedance imaging of high-resistance membrane pores using a scanning electrochemical microscope. Application of membrane electrical shunts to increase measurement sensitivity and image contrast

Whether an individual pore in a porous membrane can be imaged using scanning electrochemical microscopy (SECM), operated in ac impedance mode, is determined by the magnitude of the change in the total impedance of the imaging system as the SECM tip is scanned over the pore. In instances when the SECM tip resistance is small relative to the internal pore resistance, the total impedance changes by a negligible amount, rendering the pore invisible during impedance imaging. A simple solution to this problem is to introduce a low-impedance electrical shunt (i.e., a salt bridge) across the membrane. This principle is demonstrated by imaging polycarbonate membranes (6-12-microm thickness) containing between 1 and 2000 conical-shaped pores (60-nm- and 2.5-microm-diameter openings) using an approximately 1-microm-radius Pt tip. Theory and experiments show that image contrast (the change in ac current measured as the probe is scanned over the pore) is inversely proportional to the total resistance of the membrane and can be increased by a factor of approximately 50x by introducing a low-resistance electrical shunt across the membrane. Remarkably, SECM images of membranes containing a single high-resistance (approximately 1 G Omega) pore can only be imaged by short-circuiting the membrane. Image contrast also becomes independent of membrane resistance when an electrical shunt is used, allowing for more quantitative comparisons of the features in ac impedance images of different membranes.     

Scanning electrochemical microscopy: theory and characterization of electrodes of finite conical geometry

Finite conical electrodes, which are of particular interest as probes for imaging of surfaces using scanning electrochemical microscopy (SECM), in kinetic studies and in probing thin films were investigated. Theoretical SECM tip current-distance feedback (approach) curves for a finite conical electrode were calculated by numerical (finite element) analysis and compared to an earlier approximate model. The SECM curves obtained depended on the ratio of the base radius of the cone to the height of the cone and on the thickness of the insulating sheath. A new approach to fabricating conical tips of Pt in glass is described. These were used to obtain approach curves over both electrically conducting and insulating substrates. Comparison of experimental and simulated SECM approach curves provided a sensitive method of evaluating the size and shape of finite conical electrodes.     

Use of catechol as selective redox mediator in scanning electrochemical microscopy investigations

The use of catechols, and more specifically of dopamine, as a specific redox mediator for scanning electrochemical microscopy (SECM) investigations was evaluated in the challenging situation of an ultrathin layer deposited on a conductive substrate (carbon materials). Experiments show that dopamine is a well-adapted redox system for SECM in feedback mode and in unbiased conditions. Used as a redox mediator, catechol permits the investigations of modified surfaces without an electrical connection of the sample thanks to fast charge transfer kinetics but with a surface selectivity that does not exist in classical outer-sphere redox mediators. The interest of catechol in SECM as a sensitive redox mediator is exemplified by monitoring several modification steps of an ultrathin (<1 nm) hierarchically porous organic monolayer deposited on carbon substrates. For quantitative analysis, the SECM approach curves using dopamine could simply be characterized with an irreversible electron transfer kinetics model in a large range of pH.     

Local feedback mode of scanning electrochemical microscopy for electrochemical characterization of one-dimensional nanostructure: theory and experiment with nanoband electrode as model substrate

Local feedback mode is introduced as a novel operation mode of scanning electrochemical microscopy (SECM) for electrochemical characterization of a single one-dimensional (1D) nanostructure, for example, a wire, rod, band, and tube with 1-100-nm width and micrometer to centimeter length. To demonstrate the principle, SECM feedback effects under diffusion limitation were studied theoretically and experimentally with a disk probe brought near a semi-infinitely long band electrode as a geometrical model for a conductive 1D nanostructure. As the band becomes narrower than the disk diameter, the feedback mechanism for tip current enhancement is predicted to change from standard positive feedback mode, to positive local feedback mode, and then to negative local feedback mode. The negative local feedback effect is the only feedback effect that allows observation of a 1D nanostructure without serious limitations due to small lateral dimension, available tip size, or finite electron-transfer rate. In line-scan and approach-curve experiments, an unbiased Pt band electrode with 100-nm width and 2.6-cm length was detectable in negative local feedback mode, even using a 25-microm-diameter disk Pt electrode. Using a 2-microm-diameter probe, both well-defined and defected sites were observed in SECM imaging on the basis of local electrochemical activity of the nanoband electrode. Noncontact and spatially resolved measurement is an advantage of this novel SECM approach over standard electrochemical approaches using electrodes based on 1D nanostructure.     

Scanning electrochemical microscopy of living cells. 3. Rhodobacter sphaeroides.

The scanning electrochemical microscope (SECM) was used to probe the redox activity of individual purple bacteria (Rhodobacter sphaeroides). The approaches developed in our previous studies of mammalian cells were expanded to measure the rates and investigate the pathway of transmembrane charge transfer in bacteria. The two groups of redox mediators (i.e., hydrophilic and hydrophobic redox species) were used to shuttle the electrons between the SECM tip electrode in solution and the redox centers inside the cell. The analysis of the dependencies of the measured rate constant on formal potential and concentration of mediator species in solution yielded information about the permeability of the outer cell membrane to different ionic species and intracellular redox properties. The maps of redox reactivity of the cell surface were obtained with a micrometer or submicrometer spatial resolution.     

Distinction of the two phases of CuTCNQ by scanning electrochemical microscopy

The two known phases of CuTCNQ and TCNQ (TCNQ = 7,7',8,8'-tetracyanoquinodimethane) have been probed by scanning electrochemical microscopy (SECM) in the feedback mode. The first use of this technique for distinguishing differences in the electronic properties of semiconductor phases exploits the large differences in conductivity that exist between CuTCNQ and the parent TCNQ material and also between the CuTCNQ phases I and II. However, the packing density of the individual CuTCNQ crystals in a film structure also is shown to influence the SECM feedback response. Finally, it is shown that films of pure phase II material or mixtures of the phases can be mapped using feedback mode SECM. The SECM method provides valuable insights for elucidating properties of semiconducting solids that are mounted on insulating substrates.     

Polished nanopipets: new probes for high-resolution scanning electrochemical microscopy

Nanometer-sized pipets pulled from glass or quartz capillaries have been extensively used as probes for scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM). A small separation distance between such a probe and the sample, which is required for high-resolution SECM measurements, may be hard to attain because of considerable roughness of the pipet tip. In this Letter, we report the preparation and characterization of polished nanopipet SECM probes with a much smoother tip edge. Using polished pipets, quantitative SECM measurements were performed at extremely short tip/substrate distances (e.g., d ≈ 1 nm).     

Integrated scanning Kelvin probe-scanning electrochemical microscope system: development and first applications

The integration of a scanning Kelvin probe (SKP) and a scanning electrochemical microscope (SECM) into a single SKP-SECM setup, the concept of the proposed system, its technical realization, and first applications are presented and discussed in detail. A preloaded piezo actuator placed in a grounded stainless steel case was used as the driving mechanism for oscillation of a Pt disk electrode as conventionally used in SECM when the system was operated in the SKP mode. Thus, the same tip is recording the contact potential difference (CPD) during SKP scanning and is used as a working electrode for SECM imaging in the redox-competition mode (RC-SECM). The detection of the local CPD is established by amplification of the displacement current at an ultralow noise operational amplifier and its compensation by application of a variable backing potential (V(b)) in the external circuit. The control of the tip-to-sample distance is performed by applying an additional alternating voltage with a much lower frequency than the oscillation frequency of the Kelvin probe. The main advantage of the SKP-SECM system is that it allows constant distance measurements of the CPD in air under ambient conditions and in the redox-competition mode of the SECM in the electrolyte of choice over the same sample area without replacement of the sample or exchange of the working electrode. The performance of the system was evaluated using a test sample made by sputtering thin Pt and W films on an oxidized silicon wafer. The obtained values of the CPD correlate well with known data, and the electrochemical activity for oxygen reduction is as expected higher over Pt than W.     

Scanning electrochemical microscopy. 44. Imaging of horseradish peroxidase immobilized on insulating substrates

Scanning electrochemical microscopy (SECM) was used to study horseradish peroxidase (HRP) immobilized with copolymer on insulating substrates (glass slide or polycarbonate membrane filter). Two methods were used to immobilize HRP: In the first, HRP was coimmobilized by cross-linking on a glass slide with a copolymer swelled in water to form a hydrogel; in the second, the same copolymer and avidin were coimmobilized on the glass slide and biotin-labeled HRP was conjugated to the avidin of the film. SECM was then used to detect the presence of the bound enzyme by observing the feedback current in a solution of benzoquinone and hydrogen peroxide, when hydroquinone was generated at the tip. A detection limit less than 7 x 10(5) HRP molecules within a approximately 7-microm-diameter area was demonstrated.     

Stabilizing nanometer scale tip-to-substrate gaps in scanning electrochemical microscopy using an isothermal chamber for thermal drift suppression

The control of a nanometer-wide gap between tip and substrate is critical for nanoscale applications of scanning electrochemical microscopy (SECM). Here, we demonstrate that the stability of the nanogap in ambient conditions is significantly compromised by the thermal expansion and contraction of components of an SECM stage upon a temperature change and can be dramatically improved by suppressing the thermal drift in a newly developed isothermal chamber. Air temperature in the chamber changes only at ~.2 mK/min to remarkably and reproducibly slow down the drift of tip-substrate distance to ~0.4 nm/min in contrast to 5-150 nm/min without the chamber. Eventually, the stability of the nanogap in the chamber is limited by its fluctuation with a standard deviation of ±0.9 nm, which is mainly ascribed to the instability of a piezoelectric positioner. The subnanometer scale drift and fluctuation are measured by forming a ~20 nm-wide gap under the 12 nm-radius nanopipet tip based on ion transfer at the liquid/liquid interface. The isothermal chamber is useful for SECM and, potentially, for other scanning probe microscopes, where thermal-drift errors in vertical and lateral probe positioning are unavoidable by the feedback-control of the probe-substrate distance.     

Feedback-independent Pt nanoelectrodes for shear force-based constant-distance mode scanning electrochemical microscopy

A new generation of platinum nanoelectrodes for constant-distance mode scanning electrochemical microscopy (CD-SECM) has been prepared, characterized, and used for high spatial resolution electrochemical measurements and visualization of electrochemically induced concentration gradients in microcavities. The probes have long (1-2 cm), narrow quartz tips that were conically polished and have a Pt nanoelectrode that is slightly offset from center. Because of the size and location of the electrode on the probe, it does not exhibit SECM feedback while approaching the analyzed sample surfaces even to distances within a few hundred nanometers. The probe was positioned near the surface while scanning and performing electrochemical measurements through use of nonoptical shear force control of the tip-to-sample distance. Test structures consisted of cylindrically shaped microcavities that are 50 microm in diameter with three individually addressable electrodes: a gold disk at 8-microm depth, a crescent-shaped gold ring at 4-microm depth along the wall, and a top gold electrode at the rim. Different electrodes within the microcavity were used to reduce and oxidize redox species in 250 microL of a solution of 5 mM hexaamineruthenium(III) chloride and 0.1 M potassium chloride, protected from evaporation by mineral oil, while the SECM tip followed the topography of the structures and monitored the current from the oxidation of [Ru(NH3)6]2+. Electrochemically generated concentration profiles were obtained from these complex test structures that are not possible with any other SECM technology at this time.     

Combined scanning electrochemical/optical microscopy with shear force and current feedback

A technique that combines scanning electrochemical microscopy (SECM) and scanning optical microscopy (OM) was developed. Simultaneous scanning electrochemical/optical microscopy (SECM/OM) was performed by a special probe tip, which consists of an optical fiber core for light passage, surrounded by a gold ring electrode, and an outermost electrophoretic insulating sheath, with the tip attached to a tuning fork. To regulate the tip-substrate distance, either the shear force or the SECM tip current was employed as the feedback signal. The application of a quartz crystal tuning fork (32.768 kHz) for sensing shear force allowed simultaneous topographic, along with SECM and optical imaging in a constant-force mode. The capability of this technique was confirmed by obtaining simultaneously, for the first time, topographic, electrochemical, and optical images of an interdigitated array electrode. Current feedback from SECM also provided simultaneous electrochemical and optical images of relatively soft samples, such as a polycarbonate membrane filter and living diatoms in a constant-current mode. This mode should be useful in mapping the biochemical activity of a living cell.     

Quantitative determination of enzyme activity in single cells by scanning microelectrode coupled with a nitrocellulose film-covered microreactor by means of a scanning electrochemical microscope

An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose film-covered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-microm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference.