安川 智之

We have developed microdevices to handle, manipulate, culture and characterize cells and embryos. In the present study, the polarities of the human mammary epithelial cell line (S - 1) and its malignant cell line (T4 - 2) were controlled using a cellular array chip. The cells were embedded with 20 nL of reconstituted basement membrane matrix (Matrigel) on a pyramidal-shaped silicon microstructure. The proliferation behavior, the respiration activity, and the gene expression analysis have been carried out to characterize cellular functions on chip and discussed from the view point of the cellular polarity.

Technologies in tissue engineering or regenerative medicine have been applied for various organs and tissues. Some of them have already been clinically applied. However, tissue engineering of the muscle is still difficult. We clarify the difficulties of muscle tissue engineering and discuss the possibility of n-DPE (negative dielectrophresis) application for muscle tissue engineering.

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5 nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-D-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities. (c) 2005 Elsevier B.V. All rights reserved.

A quantitative analysis of the oxygen concentration profile near a poly(dimethylsiloxane) (PDMS) microfluidic device was performed using scanning electrochemical microscopy (SECM). A microchannel filled with sodium sulfite (Na2SO3) aqueous solution was imaged by SECM, showing that the oxygen diffusion layer of the PDMS microchannel was observed to be hemicylindrical. Based on a theoretical analysis of the hemicylindrical diffusion layer of the microchannel, the total oxygen mass transfer rates of oxygen to the PDMS microchannel filled with the Na2SO3 solution was calculated to be (4.01 +/- 0.30)x10(-12) mol s(-1). This is the maximum value of the oxygen transfer rate for this PDMS microchannel device. The oxygen consumption rate increased almost linearly with the logarithm of the concentration of E. coli cells (10(6)similar to 10(8) cells). The respiratory activity for a single E. coli cell was estimated to be similar to 4.31x10(-20) mol s(-1) cell(-1).

微生物の膜損傷に起因した生死状態に基づき分離が可能なチップデバイスを作製した．微生物に作用する誘電泳動力の差を利用し，生菌の捕捉と膜損傷を与えた菌の排出を行った．微生物分離チップは凹凸を有するバンドを配列させたcastellated型の透明電極（ITO）基板，直線流路パターン（幅2 mm，長さ35 mm）を有したシリコンスペーサ，入口と出口を備えたアクリル板から構成されている．無処理の大腸菌と熱処理した大腸菌をLive/Dead蛍光染色し，200 mMスクロース水溶液中に混合させた．大腸菌を流量440 μm/sでチップへ導入し，挙動を蛍光顕微鏡で観察した．ITO基板に正弦波（100 kHz，20 Vpeak-peak）を印加すると，大腸菌に正の誘電泳動が作用し電極間に捕捉された．周波数を7 MHzに切り替えると，死んでいる大腸菌への正の誘電泳動力が弱まり，電極から解放されチップから排出された．このチップを利用すると簡便で迅速な大腸菌の生死分離ができる．<br>

A miniature Clark-type oxygen sensor has been integrated with a microstructure using a novel fabrication technique. The oxygen chip consists of a glass substrate with a three-electrode configuration, which is separated and connected by a groove, and a poly(dimethylsiloxane) (PDMS) container with an immobilized PDMS oxygen-permeable membrane. The assembly of the different substrates only uses the O-2 plasma bonding technique, and the fabrication temperatures do not exceed 95 degrees C. Characteristics of the miniature sensor include the fastest response time of 6.8 s. good linearity with a correlation coefficient of 0.995, and a long lifetime of at least 60 h. The present miniature Clark oxygen sensor can be readily integrated with a microfluidic system to form a mu-TAS. (C) 2005 Elsevier B.V. All rights reserved.

Human monocytic leukemia cell line THP-1 differentiates into macrophages by phorbol myristate acetate (PMA) treatment. We report real-time monitoring of reactive oxygen species (ROS) production during the differentiation process. The production of ROS by THP-1 with several hours time scale has been detected using electrochemical and chemiluminescence methods. The increase in oxidation current derived from ROS arising from THP-1 was observed between I and 10 h after the commencement of treatment with 20 nM- PMA. The response was unusually slow compared to the quick oxidative response observed in the macrophages. Chemiluminescence methods were used to further investigate the ROS production rate by carrying out treatment with 20 nM to 100 KM PMA. The chemiluminescence responses were found to be faster for the cells treated with higher concentrations of PMA. The time scale of the oxidative response monitored using the chemiluminescence method correlated with that recorded using a microelectrode. The slow time scale of ROS production reflects the differentiation rate of THP- 1 into macrophages. The effect of inhibitors on NADPH oxidase and NO synthase were also examined to find that the superoxide anion is the main radical produced from NADPH oxidase. (c) 2005 Elsevier B.V. All rights reserved.

We describe a new method for the fabrication of robust three-dimensional (3-D) lab-on-a-chip devices comprising microfluidic channels and integrated electroanalytical sensors and show their potential application in supporting leak-free pressure driven fluid flow. The technology is based upon the lithographic patterning of a soluble negative photoresist and an insoluble, crosslinked, positive photoresist (SU-8) to produce the pattern of a large area microfluidic network, followed by coating the pattern with silicone rubber or PDMS. After curing, immersion in acetone rapidly elutes the soluble resist to yield the network of channels. As a result the channels formed are robust and it is possible to fabricate a device that supports fluids flowing at high How rates (without leakage) without the need for special pre-treatment or surface modification of the substrate or silicone covering. We also demonstrate the flexibility of the methodology by producing more complex fluidic networks that comprise bridges which enable one fluid stream to pass over (and remain physically separated from) a second fluid stream. The scaling and packaging techniques, developed for this process do not require the use of extreme conditions and are thus compatible with a broad range of lithographic processes (including, for example, the definition of and incorporation of in situ electrochemical sensor elements). Finally, measurements on a model bioelectroanalytical system applicable to analysis of a model immunoassay are shown, demonstrating the application of this new fabrication method in developing reproducible analytical methods within an encapsulated device.

On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs.The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.

Respiration, photosynthesis and peroxidase activities of living spherical samples, such as algal protoplasts, breast cancer spheroids and bovine embryos, were characterized with scanning electrochemical microscopy (SECM). The concentration profile of the metabolic product around the spherical sample was directly measured by scanning with a probe microelectrode. According to the spherical diffusion theory, the total mass transfer rate per spherical sample is linear to the multiplication of the sample radius and the concentration difference between the sample surface and the bulk of the solution. Therefore, the sample radius is a key parameter to assess the viability of the living samples. For example, we investigated the respiration and photosynthesis activities as a function of the size of the protoplast (Bryopsis plumosa). The respiration rate was linear to the cube of the sample radius. On the contrary, the photosynthesis rate was linear to the square of the sample radius, suggesting that the former is controlled by the volume of the protoplast, and the latter is controlled by the surface area of the protoplast. We will also discuss the size-dependent activity of the breast cancer spheroids and the bovine embryos. Furthermore, relations between the sample size, the concentration difference, and the oxygen consumption rate of the cryo-preserved bovine blastocysts were investigated. (c) 2004 Elsevier B.V. All rights reserved.

The oxygen consumption rate of three-dimensional cultured cells integrated in a silicon chip was non-invasively quantified by scanning electrochemical microscopy (SECM). The cells were embedded in a collagen gel matrix and entrapped in silicon microstructures. The oxygen concentration profile near the cell panel agreed well with the theoretically determined spherical diffusion. The oxygen consumption rate (F) of a single cell was calculated as (1.0 +/- 0.19) x 10(-16) mol s(-1). The oxygen consumption of a cell panel increased along with the cellular proliferation; the increase in respiration activity was in accordance with the increase in cell numbers. Our three-dimensional micro-culture system combined with SECM enables continuous quantification of the oxygen consumption rate using very small amounts of sample. (c) 2004 Elsevier B.V. All rights reserved.

The microbial chip of 3.5 nL-volume of collagen gel matrixes entrapping the bacterial cells in living state has been applied to investigate the bacterial response to high osmotic stress. Scanning electrochemical microscopy (SECM) was used to monitor the Fe(CN)(6)(4-) production that reflects the electron flow at respiratory chain located within the cytoplasmic membrane of bacteria. In the present study, we have found that the ferricyanide reduction activity in the Staphylococcus aureus, osmotolerant bacteria, is enhanced in the presence of highly concentrated salts. This result suggests that the osmotic stress causes the change in membrane structure or membrane-associated protein profile so as to enhance the cell membrane permeability to hydrophilic mediator. The low current responsibility of S. aureus can be improved not only by (1) using hydrophobic mediator, such as quinone derivatives instead of Fe(CN)(6)(3-) but also (2) exposing S. aureus cells to high osmotic stress in the presence of hydrophilic mediator. (c) 2004 Elsevier B.V. All rights reserved.

The respiratory activity of a multicellular spheroid was non-invasively monitored by scanning electrochemical microscopy (SECM) to investigate the anticancer drug sensitivity. The effects of the three anticancer drugs, cisplatin (CDDP), 5-fluorouracil (5-FU), and paclitaxel (TXL), were continuously evaluated based on respiratory activity for 5 days. The drug sensitivities obtained by SECM were higher compared to those evaluated by the spheroid volume and conformed to those evaluated by a conventional colorimetric assay. Our results show that the SECM-based assay directly correlates with the number of viable cells within the spheroid, whereas the spheroid volume does not necessarily correlate with the number of viable cells. Furthermore, the results obtained by spheroid culture (3-D) were compared to those of cells cultured in a flask (2-D) and within a collagen gel (3-D). The drug sensitivity of cells cultured in 2-D is more pronounced than that of the cells cultured in 3-D. Since the cellular proliferation status in a 3-D culture is similar to that in vivo, the drug sensitivity test performed in the spheroid culture will give meaningful results that can be extended to an in vivo application. A SECM-based assay is perfectly suitable to directly evaluate the drug sensitivity of the spheroid.

A novel three-dimensional cell culture system was constructed with an array of cell panels (4 x 5) in a silicon chip. together with multi-channel drug containers. Human breast cancer (MCF-7) cells were embedded in a collagen-gel matrix and entrapped in a pyramidal-shaped silicon hole. Each cell panel can be isolated by a channel composed of a microfluid part and a reservoir. A cell panel was exposed to 200 mm KCN for 2 days to demonstrate that each cell panel could be independently evaluated under various stimulation conditions. Based on the cellular respiration activity, the proliferation behavior was continuously monitored on the silicon-based cell array for 5 days using scanning electrochemical microscopy (SECM). The cells entrapped in the device (3-D culture) proliferated normally, and the proliferation rate was lower than that of cells grown in a monolayer cell culture (2-D culture). The effects of three anticancer drugs measured simultaneously on the cell chip were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the silicon-based device for 3D culture is appropriate for a chemosensitivity assay involving multi-chemical stimulation. (C) 2004 Elsevier Ltd. All rights reserved.

The electrochemically effective oxidation of NADH has drawn striking attention because it is widely applicable for fabricating biosensors. The mediators are normally used as electron shuttle molecules between the electrode and enzymes. Phenothiazine compounds deserve as suitable mediators due to theirs negative formal potential and large rate constant. For the practical use, both the enzyme and mediator are required to be co-immobilized on the electrode surface. In this study, gold nano-particles modified with thionine (Th+) were immobilized on the surface of the gold electrode by physical adsorption. Th+ was covalently bound to the surface of the gold nano-particles using 3,3'-dithiobis (succinimidyl propionate) (DSP) as a cross linker. The electrochemistry of Th+ modified on the gold nano-particles was characterized by voltammetry. Th+ molecules on the gold nano-particle modified electrodes showed two redox couples at −30mV and 240mV vs Ag/AgCl at pH7. A well-defined peak for the electrocatalytic oxidation of NADH was observed at 240mV. The nano-particle electrodes modified with glucose dehydrogenase (GDH) and Th+ mediators were fabricated and characterized aiming at fabricating a reagentless glucose biosensor. Upon the addition of glucose to the electrochemical cell, the oxidation current dramatically increased due to an electrocatalytic reaction.<br>

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.

A living cell-based assay is widely used for the characterization of various cellular functions, screening of drugs and environmental monitoring. Recent progress in cell culture and micro-fabrication technologies has attracted the integration of cell culture and sensors on a chip. A bioassay using a cell chip makes it possible to evaluate small-volume samples with multitudinous characterizations, and is applicable for a rapid and simple detection system. This paper reviews cell-based bioassay using living cells integrated on a chip. The cell patterning and three-dimensional cell culture facilitates the integration of cell culture technology. These in vitro culture technologies are appropriate for the recapitulation of in vivo-like cell behavior, because of the potential to control the cell, differentiation and polarity. Alternatively, the evaluation methods, such as cellular acidification, oxygen consumption and impedance measurements, are principally non-invasive, and are especially expected in the field of anticancer drug sensitivity test using a biopsy tumor.

β-Galactosidase expression in a small number of Escherichia coli cells has been measured in real time with an electrochemical sensor chip. E. coli cells were embedded using collagen gel within a micropore which was microfabricated onto a chip. The activity of the expressed β-galactosidase was determined using p-aminophenyl β-d-galactopyranoside (PAPG) as a substrate. © 2004 The Royal Society of Chemistry.

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different yTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in pTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least similar to30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.

A picoliter-volume electrochemical analytical chamber has been developed for detecting the metabolic flux resulting from the stress responses of a single plant cell. Electrochemical cells, with volumes as small as 100 pL, were fabricated by controlled electrochemical dissolution of a gold wire sealed in glass (the back-etching of the metal realizing an ultralow-volume titer chamber). In the first instance, the electrode contained within the chamber was characterized by the microinjection of standard aliquots of either ascorbic acid or hydrogen peroxide. In all cases, experimental currents obtained correlated well with theoretical calculations. Subsequently, single plant cells were micromanipulated into the chambers and were exposed to amounts of the detergent SDS (which permeabilized the cell membrane and released the intracellular contents). The flux of metabolite released from a single cell was estimated by using electrochemical-linked assays based upon the enzymes catalase, ascorbate oxidase, and horseradish peroxidase (in each case), in the presence of a mediator. In so doing, we investigated the activity of the cellular protection mechanisms through the determination of peroxides, while the individual cell was "stressed". The technique was found to provide a reliable and reproducible method for making single-cell measurements, using fabrication procedures that are both simple and do not require photolithographic methods.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was prepared by spotting a suspension of Escherichia coli on a polystyrene substrate by using a glass capillary pen. The respiration activity of the E. coli spot was imaged with SECM by mapping the oxygen concentration around the spot. The SECM images of the microbial chips clearly showed spots with lower reduction currents, indicating that E. coli in the spots uptake oxygen by respiration. The bactericidal effects of antibiotics (streptomycin and ampicillin) were measured using the E. coli-based microbial chip, and discussed in comparison with the minimum inhibitory concentration (MIC) determined by an agar plate dilution method. (C) 2001 John Wiley & Sons, Inc.

Dielectrophoretic manipulation of a single chlorella cell was performed using a dual-microdisk electrode, which consists of two Pt-Rh ultrafine wires (ca. 1-μm 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. © 2001 Elsevier Science B.V. All rights reserved.

Diaphorase was hydrophobically immobilized onto a self-assembled monolayer of alkanethiol at an interdigitated Au microarray. The diaphorase activity at the array substrates was imaged with the scanning electrochemical microscopy (SECM) in the presence of NADH and ferrocenemethanol as a redox mediator. The images demonstrate that diaphorase was immobiδ lized onto the SAM layer to form a line-and-space micropattern which reflected the pattern of the Au microarray.

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.

A novel scanning electrochemical/chemiluminescence microscopy (SECM/SCLM) was used for imaging of a spot of immobilized horse radish peroxidase (HRP) on a substrate. When the SECM tip scanned over the spot in an aqueous luminol solution, the immobilized HRP catalyzed the oxidation of luminol by the electrogenerated H2O2 at the tip to emit chemiluminescence which was detected by a photon-counter. The photon-counting intensity was plotted against the position of the tip to give the image indicating the activity of immobilized HRP. The topography image was also obtained by mapping the oxygen reduction current, since the current was sensitive to the topography of substrate.

Two types (A and B; see Figure 1 in the text) of dual-Pt microdisk electrodes (disk radius, 0.5-4.0 mu m; whole tip radius, 5.0-15 mu m) were fabricated to simultaneously detect two electroactive species near a single algal protoplast (radius, 25 mu m). At the tip of type A, the two disks were on the same plane. Two disk planes of electrode type B formed an angle of similar to 135 degrees at the tip. Cyclic voltammetric investigation indicated that, compared with type At one disk in type B only slightly collected the species electrogenerated at the other disk. These dual-microdisk electrodes were applied to electrochemical measurements of a single protoplast in artificial seawater. The topographic profiles Of the single protoplast were obtained from the variation of oxidation current for 1.0 mM Fe(CN)(6)(4-) at one disk. The photosynthetic oxygen generation from the protoplast was also monitored by detecting the reduction current for oxygen at another disk. The electrode of type B was used as a probe for scanning electrochemical microscopy (SECM) to obtain dual-SECM images of topography and activity of a single protoplast.

The effects of p-benzoquinone (BQ) on photosynthetic and respiratory electron transport in a single algal protoplast (radius, 100 μm) 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 electrontransport 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 ~(2.6 Π 1.0) x 10-13 mol/s, which was ~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.

Scanning electrochemical microscopy (SECM) based on the reduction current for oxygen was used for imaging of respiratory and photosynthetic activities of single, living protoplasts. In the dark, the image of the protoplast appeared as a spot with lower oxygen concentration due to consumption of oxygen by respiration. Under light irradiation, the protoplast appeared as the opposite image because it generated oxygen by photosynthesis. SECM images clearly displayed a decrease in photosynthetic activity of the protoplast injected with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), a photosynthetic inhibitor.

Permeation of several redox species through a cell membrane of a single algal protoplast (radius 100 mu m) was investigated by amperometry with a Pt microdisk electrode (disk radius, 6.5 mu m) 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 (P-m) 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 P-m values for Fe(CN)(6)(4-), Fe(CN)(6)(3-), Co(phen)(3)(2+), ferrocenyl methanol(FMA) and p-hydroquinone(QH(2)) were less than or equal to 1.0 X 10(-4), less than or equal to 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 P-m values, the concentration changes inside a model cell and chloroplast were theoretically calculated. (C) 1998 Elsevier Science B.V.

Scanning electrochemical microscopy (SECM) based on oxygen reduction current was used for imaging of respiration activity of living animal cells (SW-480). The addition of KCN into the culture medium paled the image of the cells. The semiquantitative analysis of the SECM responses suggested that the membrane permeability of SW-480 cells to CN is on the order of 10-7 cm/s.