Development of in situ electrochemical scanning electron microscopy with ionic liquids as electrolytes.

We report that ionic liquids (ILs) can be observed by electron microscopy without any charging of the liquid. Based on this, we present an in situ electrochemical scanning electron microscopy (in situ ECSEM) system. The key technology that enables in situ ECSEM is that charges can be removed from an IL by grounding it with a Pt wire, even if the IL is in an insulating glass cell. As a first demonstration, we describe the redox reaction of a polypyrrole (PPy) film accompanied by changes in its thickness when it is polarized by the film-deposited Pt electrode in the IL. Furthermore, energy-dispersive X-ray fluorescence (EDX) analysis can be employed for the electrode polarized in the IL. The component analysis by EDX of PPy in an IL containing K+ as a marker, reveals doping of electrolyte cations into the PPy film upon the latter's reduction and dedoping of cations from the film upon oxidation.

Dielectrophoretic micropatterning with microparticle monolayers covalently linked to glass surfaces.

Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 mum) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (DSS) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity.

Effects of omega-functional groups on pH-dependent reductive desorption of alkanethiol self-assembled monolayers.

Self-assembled monolayers (SAMs) of alkanethiols having various terminal groups on their omega-positions were formed on a Au111 electrode, and their reductive desorption was studied by linear sweep voltammetry, focusing on effects of solution pH on the desorption behavior. The peak potentials (Ep) of cathodic waves representing reductive desorption were found to be reflected by the pKa value of the thiol group and were negatively shifted with an increase in pH of the electrolyte solution. The magnitude of the pH dependency of Ep was greatly influenced by the hydrophobicity of the terminal groups. In the cases of alkanethiol SAMs having pH-sensitive terminal groups such as carboxyl and amino groups, their basicity was estimated from bending points appearing in the pH titration profile of Ep. This method allows direct determination of not only the pKa value of the arrayed groups but also that of the groups dissolved in solution simultaneously. The pKa values of the arrayed carboxyl groups in SAMs were larger by ca. 3 pH units than their original ones, while those for amino groups were smaller by ca. 2 pH units.

Microelectrochemical approach to induce local cell adhesion and growth on substrates.

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring.

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance.

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.

Scanning electrochemical microscopy-based drug sensitivity test for a cell culture integrated in silicon microstructures.

The respiratory activity of collagen-embedded living cells was imaged by scanning electrochemical microscopy (SECM) with the objective to study anticancer drug sensitivity. Two kinds of cancer cells, the human erythroleukemia cell line (K562) and its adriamycin-resistant subline (K562/ADM), were immobilized at the array of microholes micromachined on a silicon wafer for comparative characterization of their sensitivity to the anticancer drug, ADM. The results obtained by the SECM method showed correspondence to a conventional colorimetric assay (SDI assay). Furthermore, since the SECM assay is based on the noninvasive measurement of the respiration activity, continuous monitoring of a dose response was possible.

Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection.

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.

Fabrication of microbial chip using collagen gel microstructure.

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.