Tomoyuki Yasukawa

Characterization and Imaging of Single Cells with Scanning Electrochemical Microscopy

This article reviews the applications of scanning electrochemical microscopy (SECM) with ultramicroelectrode (UME) as a probe for characterization and imaging of single living cells. The permeation of cell membranes of several redox species was quantitatively estimated from the SECM measurements of the species in the vicinity region of the cells. The rates for the oxygen generation by photosynthesis and the oxygen consumption by respiration were determined for the detailed analysis of localized oxygen concentration around single cells. The images for photosynthetic and respiration activities were obtained with SECM based on the oxygen reduction current. A dual-microdisk electrode was used for simultaneous imaging based on electroactive species to characterize single cells.

Electrochemical Detection of Epidermal Growth Factor Receptors on a Single Living Cell Surface by Scanning Electrochemical Microscopy

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.

Negative dielectrophoretic patterning with different cell types

In this paper, a novel method for patterning different cell types based on negative dielectrophoresis (n-DEP), without any special pretreatment of a culture slide, has been described. An interdigitated array (IDA) electrode with four independent microelectrode subunits was fabricated with indium-tin-oxide (ITO) and used as a template to form cellular micropatterns. A suspension of C2C12 cells was introduced into the patterning device between the upper slide and the bottom IDA. In the present system, the n-DEP force is induced by applying an ac voltage (typically 12V(pp), 1MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5min allows the cells to adsorb onto the cell culture slide. After removing excess cells, the second cell type was patterned in lines using the same method as with the first set of cells. Periodic and alternate cell lines incorporating two cell types were also fabricated by changing the ac voltage mode. A second cell type was introduced into the device and guided to other areas to form a different pattern. The described system enables two cell types to be patterned in 15min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types.

Transfected Single-Cell Imaging by Scanning Electrochemical Optical Microscopy with Shear Force Feedback Regulation

Gene-transfected single HeLa cells were characterized using a scanning electrochemical/optical microscope (SECM/OM) system with shear-force-based probe-sample distance regulation to simultaneously capture electrochemical, fluorescent, and topographic images. The outer and inner states of single living cells were obtained as electrochemical and fluorescent signals, respectively, by using an optical fiber-nanoelectrode probe. A focused ion beam (FIB) was used to mill the optical aperture and the ring electrode at the probe apex (the inner and outer radii of the ring electrode were 37 and 112 nm, respectively). To apply an appropriate shear force between the probe tip and the living cell surface, we optimized the amplitude of oscillation of the tuning fork to which the probe was attached. Field-programmable gate arrays (FPGA) were adopted to drastically increase the feedback speed of the tip-sample distance regulation, shorten the scanning time for imaging, and enhance the accuracy and quality of the living cell images. In employing these improvements, we simultaneously measured the cellular expression activity of both secreted alkaline phosphatase outside and GFP inside by using the SECM/OM with shear force distance regulation.

Electrochemical single-cell gene-expression assay combining dielectrophoretic manipulation with secreted alkaline phosphatase reporter system

Scanning electrochemical microscopy (SECM) was used for the analysis of single-cell gene-expression signals on the basis of a reporter system. We microfabricated a single-cell array on an Indium tin oxide (ITO) electrode comprising 4 x 4 SU-8 microwells with a diameter of 30microm and a depth of 25microm. HeLa cells transfected with plasmid vectors encoding the secreted alkaline phosphatase (SEAP) were seeded in the microwell at a concentration of 1 cell per well by positive-dielectrophoresis (pDEP). A pDEP pulse of 3.0Vpp and 1MHz was applied between the microwell array/ITO electrode and an ITO counter electrode located on the top of the flow-cell assembly of the microdevice. The electrochemical responses of the individual HeLa cells transfected with SEAP were significantly larger than those of the wild-type HeLa cells. The electrochemical response of the transfected single cells was statistically distinguishable from that of wild-type HeLa cells. The size of the wells and the material of the single-cell array were optimized in order to evaluate the tumor necrosis factor alpha (TNF-alpha)-induced activation process of nuclear factor kappa B (NFkappaB) that was used as the model for on-chip monitoring of cellular signal transduction.

Microamperometric Measurements of Photosynthetic Activity in a Single Algal Protoplast

The effects of p-benzoquinone (BQ) on photosynthetic and respiratory electron transport in a single algal protoplast (radius, 100 microm) was investigated quantitatively by amperometric measurements using microelectrodes. Under light irradiation (25 kLx) in the presence of 1.00 mM BQ, a single protoplast consumed BQ by (2.9 +/- 0.2) x 10(-13) mol/s and generated p-hydroquinone (QH2) by (2.7 +/- 0.3) x 10(-13) mol/s, suggesting that BQ was quantitatively reduced to QH2 via the intracellular photosynthetic electron-transport chain. The generation of QH2 increased with light intensity and with concentration of BQ added to the outside solution but became saturated when the light intensity was above 15 kLx or the BQ concentration was higher than 0.75 mM. The addition of 3-(3, 4-dichlorophenyl)-1,1-dimethylurea, a photosynthetic electron-transport inhibitor, decreased the generation of QH2 upon light irradiation, suggesting that BQ accepts electrons from a site in the photosynthetic electron-transport chain after the photosystem II site. The presence of 1.00 mM BQ increased the generation of photosynthetic oxygen by approximately (2.6 +/- 1.0) x 10(-13) mol/s, which was approximately 1.5-2 times larger than that expected from the consumption of BQ. The electrons produced by the additional generation of oxygen is used to reduce intracellular species as well as to reduce BQ.

PERMEATION OF REDOX SPECIES THROUGH A CELL MEMBRANE OF A SINGLE, LIVING ALGAL PROTOPLAST STUDIED BY MICROAMPEROMETRY

Permeation of several redox species through a cell membrane of a single algal protoplast (radius 100 microns) was investigated by amperometry with a Pt microdisk electrode (disk radius, 6.5 microns) located near the membrane. The redox current observed at the microelectrode decreased as the microelectrode approached the cell membrane since the membrane acted as a barrier for diffusion of redox species from bulk to the microelectrode. Permeability coefficient (Pm) of the protoplast membrane was determined by the quantitative analysis of the variation of the redox current with microelectrode-membrane distance using digital simulation. The Pm values for Fe(CN)6(4-), Fe(CN)6(3-), Co(phen)3(2+), ferrocenyl methanol(FMA) and p-hydroquinone(QH2) were < or = 1.0 x 10(-4), < or = 1.0 x 10(-4), 1.0 x 10(-3), 5.0 x 10(-3) and 2.0 x 10(-2) cm/s, respectively. Using these Pm values, the concentration changes inside a model cell and chloroplast were theoretically calculated.

A competitive immunochromatographic assay for testosterone based on electrochemical detection

An immunochromatographic assay using nitrocellulose membrane was combined with electrochemical detection using an electrode chip in order to quantitatively detect testosterone as a model analyte. The electrode chip consisted of a gold working electrode, a counter electrode and a pseudo-reference electrode, all fabricated on the bottom of a 3.2mmx3.2mm well. Competitive immunoreactions on the membrane were initiated by flowing a solution containing testosterone and horseradish peroxidase (HRP)-labeled testosterone (a competitor) over the membrane. Prepared membrane was placed in a solution containing ferrocenemethanol (FcOH) and H(2)O(2) in the well of the electrode chip, and the enzyme reaction was detected by amperometry. Labeled HRP captured on the membrane catalyzed the oxidation of FcOH to the oxidized form FcOH(+), which was reduced electrochemically by the electrode chip. The electrochemical response of the reduction current decreased with increasing concentration of testosterone over the range 1-625ng/ml.

Dielectrophoretic manipulation of a single chlorella cell with dual-microdisk electrode

Dielectrophoretic manipulation of a single chlorella cell was performed using a dual-microdisk electrode, which consists of two Pt-Rh ultrafine wires (ca. 1-microm radius) sealed in a glass capillary. An attractive or repulsive force was induced on the chlorella depending on the frequency of the ac voltage applied between the two disk electrodes. To avoid the direct contact of a chlorella with the metal, a dual electrode with retracted disks was fabricated and used for forming a micropattern of chlorellas at a solid substrate. The effect of both the frequency and ion concentration of the solutions on the dielectrophoretic force exerted on a chlorella cell was investigated in detail based on the theories of dielectrophoresis.

Rapid and simple immunosensing system for simultaneous detection of tumor markers based on negative-dielectrophoretic manipulation of microparticles

We report here a rapid, simple, and simultaneous immunosensing method for two tumor markers, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), by applying the negative-dielectrophoretic (n-DEP) manipulation of microparticles. Microparticles modified with different antibodies rapidly accumulated to designated areas of poly(dimethylsiloxane) (PDMS) fluidic channels modified with different antibodies within 1 min by n-DEP upon the application of AC voltage. The presence of specific antigens, AFP or CEA, permitted the irreversible capture of microparticles via the formation of immuno-complexes between the PDMS surface and the microparticles. Uncaptured microparticles redispersed after switching off the AC voltage. The fluorescent intensity from the irreversibly captured microparticles allowed us to determine the concentration of AFP and CEA in the sample. Neither the unreacted analytes nor the microparticles required separation steps, since we detected the fluorescent signals only from the microparticles captured on the PDMS surface. The detectable concentration range shifted to lower values when the amount of the antibody on the PDMS surface increased. The range for both AFP and CEA assays was 0.1-100 ng/mL, which was sufficient to cover the concentration required for the medical diagnoses. We simultaneously detected the concentrations of AFP and CEA by using a device, with two channels modified for different antibodies. Since n-DEP was used for the rapid manipulation of the microparticles toward the PDMS surface, the time required for the assay was substantially short; 1 min for forcing and 5 min for redispersion of the microparticles and sensing.