We have estimated the effect of a chemical reagent based on the oxygen consumption and the motion of zebrafish embryos by using an LSI-based amperometric sensor array device (BioLSI). Bio-LSI with 400 gold microelectrodes in a 6 mmx6 mm region enabled us to visualize the distribution of the concentration of redox species as images. We applied -0.2 V vs. Ag/AgCl to all electrodes of a Bio-LSI chip to obtain the distribution of the oxygen concentrations in the presence of zebrafish embryos. The motions of the embryos were observed and traced by a CMOS camera. The reduction currents, obtained at the electrodes immediately under the embryos, are smaller than that obtained at the electrodes without the embryos. When the embryos moved to the position above other electrodes, the positions of electrodes with the low reduction current synchronized to the position of the embryos. This was due to the consumption of oxygen by the respiration of embryos and the inhibition of the diffusion of oxygen to the electrodes. When a solution containing 2,4-dinitrophenol (DNP), that is an uncoupler of oxidative phosphorylation, was added, the reduction current drastically decreased and then gradually returned to the original level. The active motions of the embryos coupled with the decrease of the reduction current were observed immediately after adding DNP, while no motion was observed after turning toward original level. Embryos would transiently enhance the consumption of oxygen by respiration due to the compensation of its depression of ATP synthesis, and then be led to death. The present system based on the reduction currents of oxygen and the motions of embryos would provide simple and high-throughput toxicity tests for various chemical agents.

© 2020 by the authors. A microfluidic device is presented for the continuous separation of red blood cells (RBCs) and white blood cells (WBCs) in a label-free manner based on negative dielectrophoresis (n-DEP). An alteration of the electric field, generated by pairs of slanted electrodes (separators) that is fabricated by covering parts of single slanted electrodes with an insulating layer is used to separate cells by their sizes. The repulsive force of n-DEP formed by slanted electrodes prepared on both the top and bottom substrates led to the deflection of the cell flow in lateral directions. The presence of gaps covered with an insulating layer for the electric field on the electrodes allows the passing of RBCs through gaps, while relatively large WBCs (cultured cultured human acute monocytic leukemia cell line (THP-1 cells) flowed along the slanted separator without passing through the gaps and arrived at an edge in the channel. The passage efficiency for RBCs through the gaps and the arrival efficiency for THP-1 cells to the upper edge in the channel were estimated and found to be 91% and 93%, respectively.

© 2019 Elsevier B.V. In this study, we have developed a novel electrochemical device (IDEA-Bio-LSI) incorporating interdigitated electrodes array (IDEA) and a LSI-based amperometric device (Bio-LSI) for high speed (4–200 ms) and selective imaging of an analyte diffusion and cellar activities such as dopamine release. The amplification factor (ηamp) and capture efficiency (CE) of IDEA of the device were 2.17 and 0.767, respectively. Compared with previously reported IDE based imaging sensor, the acquisition speed of the present device to acquire one image was improved up to 50–250 times. In addition, the dopamine release from PC12 spheroids in the presence of ascorbic acid was successfully obtained by using the IDEA-Bio-LSI. Therefore, IDEA-Bio-LSI can apply to rapid analyte diffusion biological events such as release of dopamine release.

<p>In this study, we developed a bipolar electrochemical microscopy (BEM) using a closed bipolar electrode (cBPE) array with an electrochemiluminescence (ECL) detecting system. Because cBPEs are not directly connected to...</p>

<jats:p> Lithium-ion battery is one of the most popular secondary batteries for practical applications such as mobile phones and electrical cars because of their high energy density and good cyclability. For further improvement of their total performance with highly efficient cyclability, it is necessary to be in precise control of lithium-ion transport at the interface between electrolyte and electrodes. There is a major issue for efficient ion transport due to solid electrolyte interface (SEI) on negative composite electrodes such as graphite and/or silicon with some binders and electrical additives. The SEI is in general formed by decomposition of electrolyte. However, the SEI formation process or SEI itself is not still clear how the decomposition process proceeds due to the surface structure, binders or dispersion of active materials. Here, in this work we have used a scanning electrochemical microscopy (SECCM) with a single barrel nanopipette inside glove-box for analyzing local electrochemical reaction for SEI formation. A 50 nm radius nanopipette was filled with organic electrolyte (1.0 M LiPF<jats:sub>6</jats:sub> in ethylene carbonate:diethyl carbonate = 1:1 in vol. %) with a Li reference electrode. When the pipette was approached to the sample surface, a meniscus was created. Then, through the meniscus as a nanoscale electrochemical simulator, cyclic voltammogram was measured to introduce the SEI in localized area. The location SEI formed and their created potential were mapped by scanning the pipette on the sample. Further, on the particular structure such as basal and edge of graphite, the decomposition process was also analyzed for investigation of material structures for SEI formation. Those information would be particularly important to understand their mechanism and to control SEI formation. </jats:p>

<jats:p> To understand the metal oxide coating effect on battery performance, the following two techniques are required: 1) constructing a flat thin-film electrode surface to realize a well-defined interface and 2) analyzing the electrode/electrolyte interface reaction with nanoscale resolution. We previously studied flat LiCoO<jats:sub>2</jats:sub> thin-film electrodes using<jats:italic> in situ</jats:italic> surface-sensitive X-ray absorption spectroscopy (XAS) and reported that Co reduction at the LiCoO<jats:sub>2</jats:sub> surface resulting from electrolyte contact caused the initial degradation. We also showed that the ZrO<jats:sub>2</jats:sub> layer successfully prevented physical contact between LiCoO<jats:sub>2</jats:sub> and the electrolyte. And it confirmed that a thicker ZrO<jats:sub>2</jats:sub> layer (above 2 nm) increased the diffusion resistance of the lithium ions in the ZrO<jats:sub>2</jats:sub> layer. However, since XAS lacks in-plane resolution and provides only averaged information, it is impossible to analyze the ZrO<jats:sub>2</jats:sub> morphology in detail. Recently, Taguchi et al. investigated a thin Li-Zr-layer (ca. 2 nm) on a LiCoO<jats:sub>2</jats:sub> composite electrode by transmission electron microscopy (TEM). They suggested that this thin layer could improve the durability.<jats:sup /> However, it is difficult to analyze the electrochemical properties using TEM. To evaluate the intrinsic mechanism of the metal oxide coating effect, it is necessary to develop a novel <jats:italic>in-situ</jats:italic> method that can analyze the surface morphology with high spatial resolution and simultaneously determine the local electrochemical properties. </jats:p> <jats:p>Scanning electrochemical microscopy (SECM) is a powerful technique for linking the surface morphology of a sample to its electrochemical properties. For the battery materials, the SECM feedback mode is effective in monitoring solid electrolyte interphase formation. To directly and quantitatively investigate spatially resolved ionic processes, mercury-capped platinum ultramicroelectrodes were developed and employed for Li<jats:sup>+</jats:sup> imaging based on Li stripping. However, it is difficult to visualize the Li<jats:sup>+</jats:sup> flux in battery materials at the sub-micrometer scale by SECM. Scanning electrochemical cell microscopy (SECCM), which uses a nanopipette as a probe and forms a local electrochemical cell, is effective in characterizing surface reactivity. We recently applied SECCM for visualization of electrochemical activities on a lithium-ion battery cathode material at sub-micrometer resolution. The SECCM was applied to collect or provide Li in specified area confined by the nanopipette. Further, it collection visualized the electrochemical properties by scanning the nanopipette as an image. There are some strong advantages in SECCM for battery material research such as its high spatial resolution, small capacitive current, and isolated electrochemical cell. </jats:p> <jats:p>In this report, we applied SECCM to characterize a ZrO<jats:sub>2</jats:sub>-coated LiCoO<jats:sub>2</jats:sub> thin-film electrode prepared by pulsed laser deposition. Local cyclic voltammetry (CV) and galvanostatic charge/discharge were performed to characterize the cycle durability and rate performance of ZrO<jats:sub>2</jats:sub>-coated LiCoO<jats:sub>2</jats:sub> thin-film electrodes and to reveal the relationship between the ZrO<jats:sub>2</jats:sub> morphology and thickness. </jats:p>

This paper reports a portable aptamer based allergen sensing system for food safety. A complete device with illumination and luminescence observation optical system, signal processor, power management system and data transmitter was fabricated. The luminescence signal from the sensing sheet could be successfully distinguished by the developed system.

In this study, we developed a novel electrochemical imaging system using a closed bipolar electrode (cBPE) with electrochemiluminescence (ECL) detecting system to realize the high spatio-temporal resolution imaging. This novel imaging system can obtain high spatial resolution images because each cBPEs need no connecting lines, which severely limited the spatial resolution of the conventional electrochemical images obtained by the micro-electrode array method. We successfully applied the system to monitor the spreading activity of potassium ferricyanide solution in high spatio-temporal resolution.

© 2019 The Chemical Society of Japan Here we report the synthesis and electrocatalytic properties of polymer films containing nitrogen and iron. The films were prepared by calcination of polydopamine, polyethyleneimine and iron. The composite films were self-assembled at the air/ water interface, and exhibited higher activity for the oxygen reduction reaction than glassy carbon or calcined polydopamine films. These results suggest that such calcined composite films have potential applications as cathodes in polymer electrolyte fuel cells.

© 2018 John Wiley &amp; Sons, Ltd. Scanning electrochemical cell microscopy with a single barrel micro-/nano-pipette (SECCM) was applied to lithium iron phosphate (LiFePO4) composite positive electrodes and an isolated LiFePO4 secondary particle for lithium-ion batteries. To analyze lithium-ion (Li+) charge or discharge process on the electrodes using local probe, a pipette filled with LiCl electrolyte solution and Ag/AgCl quasi-reference counter electrode (QRCE) was used. Both the local electrochemical activities of LiFePO4 on the composite electrodes and a single particle were revealed by SECCM as in-situ direct measurement of current response-related Li+ transport from mapping and cyclic voltammogram at confined area by the pipette. The mapping has visualized Li+ deintercalation process from LiFePO4 at +0.65 V (applied between the sample-QRCE). We show that the SECCM system is a strong analytical tool for a characterization of local Li+ behavior of active electrode materials in lithium-ion batteries.

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Herein, we present the fabrication of micropatterned Nafion membranes on an electrode array using a combination of photolithographic and lift-off techniques. The Nafion-modified electrodes were applied for selective assays based on the charges of redox compounds. In addition, redox compounds with positive charges were successfully accumulated on the Nafion layers, so that redox signals were improved with the anodic stripping. To the best of our knowledge, this is the first report on micropatterning of Nafion membranes using photolithographic and lift-off techniques.

In this study, composite gelatin-polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced myotube formation on composite gelatin-PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced myotube maturation. Thus, in myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation-contraction (E-C) coupling apparatus, calcium transients, and myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

The O-2 consumption rate of embryos has been attracting much attention as a key indicator of cell metabolisms and development. In this study, we propose an on-chip device that enables the accurate, easy, and noninvasive measurement of O-2 consumption rates of single embryos. Pt electrodes and micropits for embryo settlement were fabricated on Si chips via microfabrication techniques. The configuration of the device enables the detection of O-2 concentration profiles surrounding the embryos by settling embryos into the pits with a mouth pipet. Moreover, as the detection is based on an electrochemical method, the influence of O-2 consumption on the electrodes was also considered. By using a simulator (COMSOL Multiphysics), we estimated the O2 concentration profiles in the device with and without the effects of the electrodes. Based on the simulation results, we developed a normalization process to calculate the precise O-2 consumption rate of the sample. Finally, using both the measurement system and the algorithm for the analysis, the respiratory activities of mouse embryos were successfully measured.

We incorporated bovine serum albumin (BSA)-functionalized graphene in poly(ethylene glycol) diacrylate (PEG) hydrogels. The graphene alignment in PEG hydrogels was changed using dielectrophoresis (DEP) forces. Effect of graphene concentration (2.55 and 5.1 mg/mL) and alignment (vertical and horizontal) on electrical and mechanical properties of hybrid graphene-PEG gels was evaluated. In addition, the molecular interaction of BSA peptides with graphene in the presence and absence of an electric field was assessed. The incorporation of graphene in PEG improved mechanical properties of hybrid gels. Dielectrophoretically aligned graphene-PEG hydrogels showed an anisotropic electrical conductivity. Young's modulus of 5.1 mg/mL graphene-PEG was almost three times higher than that of pristine PEG. Viability and proliferation of mouse embryonic stem cells on graphene-PEG gels were comparable with control PEG hydrogels. Ab initio calculations showed longer peptides had higher binding affinity towards graphene and caused a change in dipole moment due to the presence of electric field. The electric field also expanded graphene and peptide chains, which confirms the graphene alignment using DEP forces. Hybrid aligned graphene-PEG hydrogels may serve as functional biomaterials to engineer biological tissues and fabricate biomedical devices. (C) 2017 Elsevier Ltd. All rights reserved.

We embedded carbon nanotubes (CNTs) in mouse embryoid bodies (EBs) for modulating mechanical and electrical cues of the stem cell niche. The CNTs increased the mechanical integrity and electrical conductivity of the EBs. Measured currents for the unmodified EBs (hereafter, EBs) and the EBs-0.25 mg/mL CNTs were 0.79 and 26.3 mA, respectively, at voltage of 5 V. The EBs had a Young's modulus of 20.9 +/- 6.5 kPa, whereas the Young's modulus of the EB-0.1 mg/mL CNTs was 35.2 +/- 5.6 kPa. The EB-CNTs also showed lower proliferation and greater differentiation rates compared with the EBs as determined by the expression of pluripotency genes and the analysis of EB sizes. Interestingly, the cardiac differentiation of the EB-CNTs was significantly greater than that of the EBs, as confirmed by high-throughput gene analysis at day 5 of culture. Applying electrical stimulation to the EB-CNTs specifically enhanced the cardiac differentiation and beating activity of the EBs.

Here we report on the flattening of water droplets using an ultrathin membrane of autopolymerized polydopamine at the air/water interface. This has only been previously reported with the use of synthetic or extracted peptides, two-dimensional designed synthetic peptide thin films with thicknesses of several tens of nanometers. However, in the previous study, the shape of the water droplet was changed irreversibly and the phenomenon was observed only at the air/water interface. In the present study, an ultrathin polydopamine membrane-stabilized droplet induced the flattening of a water droplet at the air/liquid and liquid/liquid interfaces because a polydopamine membrane was spontaneously formed at these interfaces. Furthermore, a reversible transformation of the droplet to flat and dome shape droplets were discovered at the liquid/liquid interface. These are a completely new system because the polydopamine membrane is dynamically synthesized at the interface and the formation speed of the polydopamine membrane overcomes the flattening time scale. These results will provide new insight into physical control of the interfacial shapes of droplets.

Observation of nanoscale structure dynamics on cell surfaces is essential-to understanding cell functions. Hopping Mode, scanning ion conductance microscopy (SICM) was used to visualize the topography, of fragile convoluted nanoscale structures on cell surfaces under noninvasive conditions. However, conventional hopping mode SICM does not have sufficient temporal resolution to observe cell-surface dynamics in situ because of the additional time required for performing vertical probe movements of the nanopipette. Here, we introduce a new scanning algorithm for high speed SICM measurements using low capacitance and high-resonance-frequency piezo stages. As a result, a topographic image is taken within 18 s with a 64 x 64 pixel resolution at 10 X 10 mu m. The high speed SICM is applied to the characterization of microvilli dynamics on surfaces, which shows clear structural changes after the epidermal growth factor stimulation.

Motion tracking of microorganisms is useful to investigate the effects of chemical or physical stimulation on their biological functions. Herein, we describe a novel electrochemical imaging method for motion tracking of microorganisms using a large-scale integration (LSI)-based amperometric device. The device consists of 400 electrochemical sensors with a pitch of 250 mm. A convection flow caused by the motion of microorganisms supplies redox species to the sensors and increases their electrochemical responses. Thus, the flow is converted to electrochemical signals, enabling the electrochemical motion tracking of the microorganisms. As a proof of concept, capillary vibration was monitored. Finally, the method was applied to monitoring the motion of Daphnia magna. The motions of these microorganisms were clearly tracked based on the electrochemical oxidation of [Fe(CN)(6)](4-) and reduction of O-2.

While digital detection improves the sensitivity of electrochemical analysis, adapting it for array-based high throughput assays has been challenging. The reason is that when integrating many electrochemical sensors in a small chip device, there may not be sufficient space for multiple lead connections to the external equipment. Here, we propose a new approach to prepare multiple sensors on one working electrode, and use the device for simultaneous digital electrochemical detection of droplet arrays. Based on the binary number system, sensor electrodes 4,2, and 1 mm2 in size are prepared on a single working electrode, and droplets with or without a target redox compound are placed on each sensor. The total signal from the working electrodes is converted to a three-digit binary number that specifies the presence or absence of the analyte at each position. Therefore, the droplets can be monitored simultaneously via a single line. This new concept allows us to prepare multiple electrochemical sensors on a small device for digital detection and digital imaging. (C) 2017 Elsevier B.V. All rights reserved.

This manuscript reports on a novel substrate for the amperometric detection of cellular apoptosis, Asp-Glu-Val-Asp-p-methoxyaniline (DEVD-pMA). This substrate showed electrochemical inertia from -0.2 to 0.7V versus Ag/AgCl in phosphate-buffered saline (PBS), but was cleaved by caspase-3, releasing pMA, which was detected with cyclic voltammetry and chronoamperometry. Compared with p-aminophenol (pAP), pMA showed lower toxicity to the human hepatocellular carcinoma cell line (HepG2) and higher stability in PBS. By using this substrate, we successfully detected caspase-3 in the range of 0.31-2.5x10(-2) units/mL and cellular apoptosis in HepG2 cells by using simple amperometry without cell lysis. This work will help to develop a simple method for the electrochemical monitoring of apoptosis, which can be applied in biological research and drug development.

Investigation of the positional heterogeneity of messenger RNA (mRNA) expression in tissues requires a technology that facilitates analysis of mRNA expression in the selected single cells. We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control. The MP was used for continuous collection of different nucleic acid samples and sequential evaluation of gene expression with mRNA barcoding tags. First, we found that the aqueous phases could be laminated into five individual layers and separated by the plugs of the organic phases in a nanopipette when the salt (THATPBCl) concentration in the organic phase was 100 mM. Second, the aspiration rate of the MP was stabilized and the velocity of the aqueous phase in the MP was lowered at higher THATPBCl concentrations in the organic phase. This was because the force during ingression of the aqueous phase into the organic - phase-filled nanopipette induced an electro-osmotic flow between the inside wall of the nanopipette and THATPBCl in the organic phase. Third, inclusion of mRNA barcoding tags in the MP facilitated complementary DNA construction and sequential analysis of gene expression. This technique has potential to be applicable to RNA sequencing from different cell samples across the life sciences.

Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co-cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf-5, myogenin, Mefc2, MLP), muscle maturation markers (MHC-IId/x, MHC-IIa, MHC-IIb, MHC-pn, alpha-actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR-epsilon) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright (C) 2014 John Wiley & Sons, Ltd.

© 2017 The Electrochemical Society of Japan, All rights reserved. We developed three modes of high resolution scanning probe microscope system based on electrochemical principles, scanning ion conductance microscopy (SICM), scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM), and scanning electrochemical cell microscopy (SECCM). Firstly, a developed SICM system was constructed with a nanopipette filled with electrolyte solution as a probe. High resolution topographic images of NanoCulture®Plate with hexagonal chambers with 2 μm-width banks, electrodeposited PEDOT (poly(3,4-ethylenedioxythiophane)) film, and fixed human squamous cell carcinoma were captured by the SICM. Second, SECM-SICM was performed with a double-barrel carbon nanoprobe. The one barrel of the nanoprobe was filled with carbon and used for SECM apparatus, the other barrel was filled with electrolyte for SICM configurations. Using the SECM-SICM system, simultaneous topographic and electrochemical images of micro-band electrodes with 10 μm-width line and space, and a cathode site of corrosion in the aluminum die gusto with submicron spatial resolution were obtained. Thirdly, the SECCM featuring a nanopipette probe filled with LiCl electrolyte was applied for obtaining topographies and images of current activity of a LiFePO4 electrode.

<p>A novel and simple method for constructing cell sheets is described in which culture surfaces were modified with RGD peptide-coupled alginate hydrogels using an electrodeposition method. Cells were cultured on the hydrogels to form a contiguous cell sheet-like structure. Then, the hydrogels were dissolved by addition of an EDTA solution and the cell sheets rapidly detached from the surface. This is the first report of the fabrication of cell sheets on RGD peptide-coupled alginate hydrogels using an electrodeposition method. We believe the technique is useful for cell sheet engineering.</p>

Nitrogen-doped graphene is favored as a catalyst for oxygen reduction reaction (ORR) over rare metals. However, the effects of bonding state, nitrogen doped site and defects on catalytic conversion are still unclear. Here, we investigate oxygen reduction reaction using nitrogen-doped graphene with selective bonding state through pyridinic and graphitic nitrogen selective approaches. Both types show ORR activity and the catalytic reaction is clarified to be a four electron reaction path. Graphitic nitrogen with a low level of defects is found superior from the viewpoint of using single graphene sheet for the ORR application. Our investigation provides useful information for various applications using doped graphene. (C) 2016 Elsevier B.V. All rights reserved.

Electrochemical techniques have been widely utilized for evaluation of oxygen consumption of cells. For high throughput cell analysis and imaging, several electrode-array devices have been developed. However, it is difficult to incorporate many sensors into a small area using a simple arrangement. In the present study, we developed a novel molecular electrochemical switching element for the incorporation of many sensors for detection of oxygen consumption of cells. The switching element is based on the competition of molecular consumption at an electrode pair. The switching elements are incorporated into an electrochemical imaging device, so that n(2) electrochemical sensors are prepared with only 2n connector pads. The device was then applied to evaluate the respiratory activity of cell aggregates. The detection system is a useful tool for the electrochemical imaging of cell respiratory activity without any damage to samples.(C) 2016 Elsevier B.V. All rights reserved.

Stem cells are one of the key components in tissue engineering (TE) for tissue repair and regeneration. However, further studies are necessary in order to provide a suitable microenvironment for stem cells to differentiate and thereby regenerate tissues. Carbon-based nanomaterials (i.e., carbon nanotubes (CNTs) and graphene) have recently attracted much significant attention as tools for investigating and controlling stem cell biology and fate due to their remarkable characteristics, including unique mechanical properties, tunable surface chemistry, and high electrical conductivity. In this review paper, we describe applications of CNTs and graphene in stem cell differentiation and consequently tissue formation in both in vitro and in vivo conditions. Cytotoxicity of CNTs and graphene is also addressed. Finally, we discuss potential challenges and future directions for applications of CNTs and graphene in the stem cell culture and differentiation.

Here we propose a novel electrochemical lithography methodology for fabricating calcium-alginate hydrogels having controlled shapes. We separated the chambers for Ca2+ production and gel formation with alginate with a semipermeable membrane. Ca2+ formed in the production chamber permeated through the membrane to fabricate a gel structure on the membrane in the gel formation chamber. When the calcium-alginate hydrogels were modified with collagen, HepG2 cells proliferated on the hydrogels. These results show that electrochemical hydrogel lithography is useful for cell culture.

Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

Electrochemical imaging is an excellent technique to characterize an activity of biomaterials, such as enzymes and cells. Large scale integration-based amperometric sensor (Bio-LSI) has been developed for the simultaneous and continuous detection of the concentration distribution of redox species generated by reactions of biomolecules. In this study, the Bio-LSI system was demonstrated to be applicable for simultaneous detection of different analytes in multiple specimens. The multiple specimens containing human immunoglobulin G (hIgG) and mouse IgG (mIgG) were introduced into each channel of the upper substrate across the antibody lines for hIgG and mIgG on the lower substrate. Hydrogen peroxide generated by the enzyme reaction of glucose oxidase captured at intersections was simultaneously detected by 400 microelectrodes of a Bio-LSI chip. The oxidation current increased with increasing the concentrations of hIgG, which can be detected in the range of 0.01-1.0 mu g mL(-1). Simultaneous detection of hIgG and mIgG in multiple specimens was achieved by using line patterns of both antibodies. Therefore, the presence of different target molecules in the multiple samples would be quantitatively and simultaneously visualized as a current image by the Bio-LSI system.

Oxygen consumption rate (OCR) of mouse embryoid body (EB) was measured repeatedly in glucose-depleted medium or PBS solution. Non-invasive nature of the scanning electrochemical microscopy allowed sequential OCR measurement of the identical EB sample for more than 24 h. We found that the OCR value for the undifferentiated EB (cultured for 2 days) reached almost zero after 12 h exposure in PBS. On the contrary, the OCR value for the differentiated EB (cultured for 8 days) indicated 40 to 28% normal to the OCR at 0 h-incubation after 24 h exposure in PBS-based solution. It is suggested that differentiated cells indicated a stronger adaptation to glucose deprivation comparing to the undifferentiated cells. (C) The Electrochemical Society of Japan, All rights reserved.

Single-cell analyses are important for providing new insights into cellular biology. Here we report an electrochemical reporter gene assay for single cells using a scanning electrochemical microscope (SECM)-microwell system. Each microwell trapped a single cell that synthesized a reporter protein, secreted alkaline phosphatase (SEAP). The SEAP catalyzed the hydrolysis of p-aminophenyl phosphate to p-aminophenol (PAP). A disk electrode in the SECM was positioned above the microwell and monitored the oxidation currents of PAP derived from SEAP. In addition, ring electrodes were prepared on the microwell device to induce redox cycling between the ring and disk electrodes, thus amplifying the electrochemical signals from the reporter protein. The redox cycling-based electrochemical reporter gene assay is useful for single-cell analyses. (C) The Electrochemical Society of Japan, All rights reserved.

We here report the electrochemical imaging of cell adhesion using a large-scale integration (LSI)-based amperometric device, called a Bio-LSI device. The device consists of 400 sensor electrodes arranged with a pitch of 250 pm. The device surface was modified with collagen to assist in the culture of MCF-7 cells and promote their adhesion. The cells disturb the electrochemical reaction of redox mediators, allowing the electrochemical signal to be used to evaluate cell adhesion at the single-cell level. This approach was applied to a cell detachment test. The results show that the Bio-LSI device is a promising tool for single-cell analysis. (C) The Electrochemical Society of Japan, All rights reserved.

A liquid-junction-free electrochemical system has been developed for substitutional stripping voltammetry (SSV). SSV is a type of stripping analysis that changes the redox current to metal deposition. Generally, SSV requires a liquid junction to maintain electrical conductivity between separated cells. In this report, a closed bipolar electrode system(cBPES) was used to perform SSV without a liquid junction. In this system, electrical conductivity between separated cells was maintained using driving electrodes inserted into each cell. We demonstrated quantification of p-aminophenol (pAP) using a liquid-junction-free system for SSV. We investigated the effect of electrode area ratio on the BPE and the concentration ratio of redox species present in each cell. Then, we calibrated the pAP. A linear relationship between pAP concentration and SSV electrical charge was successfully obtained using our liquid-junction-free system, based on cBPES. This novel analytical system is a promising method for the fabrication of a compact SSV sensor for highly sensitive detections. (C) 2016 Elsevier B.V. All rights reserved.

All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques.

This paper describes potentiometric bioimaging for enzyme activity using a large-scale integration (LSI)-based electrochemical device with 400 sensors. Potentiometric detection is useful for bioimaging because redox species are not consumed or produced during the detection process; therefore, there is no effect on cell activity and the detectable signal is sustained. In this study, the potentiometer mode of the LSI-based device was applied for the detection of glucose oxidase (GOx) and alkaline phosphatase (ALP) activity. The enzyme activities were quantitatively detected within the concentration ranges of 25-250 mu g/mL and 0.10-5.0 ng/mL. In addition, GOx activity in hydrogels and the ALP activity of embryoid bodies (EBs) from embryonic stem (ES) cells were successfully imaged based on detection of the open circuit potentials of individual sensors in real time. To the best of our knowledge, this is the first report of potentiometric imaging using LSI-based electrochemical arrays to detect enzyme activity in ES cells. The LSI-based device is thus demonstrated to be a promising tool for bioimaging of enzyme activity. (C) 2015 Elsevier B.V. All rights reserved.

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

Carbon nanotubes (CNTs) were aligned in gelatin methacryloyl (GelMA) hydrogels using dielectrophoresis approach. Mouse embryoid bodies (EBs) were cultured in the microwells fabricated on the aligned CNT-hydrogel scaffolds. The GelMA-dielectrophoretically aligned CNT hydrogels enhanced the cardiac differentiation of the EBs compared with the pure GelMA and GelMA-random CNT hydrogels. This result was confirmed by Troponin-T immunostaining, the expression of cardiac genes (i.e., Tnnt2, Nkx2-5, and Actc1), and beating analysis of the EBs. The effect on EB properties was significantly enhanced by applying an electrical pulse stimulation (frequency, 1 Hz; voltage, 3 V; duration, 10 ms) to the EBs for two continuous days. Taken together, the fabricated hybrid hydrogel-aligned CNT scaffolds with tunable mechanical and electrical characteristics offer an efficient and controllable platform for electrically induced differentiation and stimulation of stem cells for potential tissue regeneration and cell therapy applications. Statement of significance Dielectrophoresis approach was used to rapidly align carbon nanotubes (CNTs) in gelatin methacryloyl (GelMA) hydrogels resulting in hybrid GelMA-CNT hydrogels with tunable and anisotropic electrical and mechanical properties. The GelMA-aligned CNT hydrogels may be used to apply accurate and controllable electrical pulses to cell and tissue constructs and thereby regulating their behavior and function. In this work, it was demonstrated that the GelMA hydrogels containing the aligned CNTs had superior performance in cardiac differentiation of stem cells upon applying electrical stimulation in contrast with control gels. Due to broad use of electrical stimulation in tissue engineering and stem cell differentiation, it is envisioned that the GelMA-aligned CNT hydrogels would find wide applications in tissue regeneration and stem cell therapy. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials.

<br><br>NanoSECCMは電解液を充填したナノピペットをプローブとして用い，各種材料表面のナノメートルスケールの電気化学反応を捉え，可視化できる新しいプローブ顕微鏡である．本講演では，NanoSECCMの動作原理，特徴，装置構成を紹介し，電池材料の局所機能解析に適用した例を紹介する．

Proper surface characteristics for a titanium implant are crucial for the formation of different cellular protrusions known as filopodia and lamellipodia, both of which have a significant impact on cell attachment, spreading, migration, and proliferation. Microstructural features such as grain boundaries and defects of implant surface can modulate the cellular components and structure at the leading edge of cells. Here, a nano-grained Ti-29Nb-13Ta-4.6Zr (NG TNTZ) substrate was produced by high-pressure torsion (HPT) for improved biofunctionality. Cellular response of human osteoblast cells on nano-grained TNTZ substrates is evaluated and compared with the cellular response of those on coarse-grained TNTZ. High wettability, which depends on high internal energy due to the nano-sized grains that are full of boundaries, interfaces, and high dislocation density, influenced the hOBs cells on NG TNTZ to form highly developed cellular protrusions. Large number of filopodia protrusions resulted in excellent cell attachment as consistent with high level of vinculin and superior cell proliferation. This study demonstrates the advantages of nanocrystalline surface modification using HPT for processingmetallic biomaterials that are proper for orthopedic implants.

The mouse embryonic stem (ES) cell-derived angiogenesis model is widely used as a 3D model, reproducing cell cell interactions in the living body. Previously, many methods to analyze localized cellular function, including in situ hybridization and laser capture microdissection, have been reported. In this study, we achieved a collection of localized cells from the angiogenesis model in hydrogel. The gene expression profiles of the endothelial cells derived from mouse ES cells were evaluated. First, we collected localized cells from the live tissue model embedded in hydrogel using the double barrel carbon probe (DBCP) and quantified mRNA expression. Second, we found that vascular marker genes were expressed at a much higher level in sprouting vessels than in the central core of the embryoid body because the cells in sprouting vessels might significantly differentiate into endothelial linages, including tip/stalk cells. Third, the gene expression levels tended to be different between the top and middle regions in the sprouting vessel due to the difference in the degree of differentiation in these regions. At the top region of the vessel, both the tip and stalk cells were present. The cells in the middle region became more mature. Collectively, these results show that DBCP is very useful for analyzing localized gene expression in cells collected from 3D live tissues embedded in hydrogel. This technique can be applied to comprehensive gene expression analyses in the medical field.

Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.

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Electrochemical imaging has been dramatically developed. Electrochemical imaging can provide images of surface chemical kinetics, and its technique has been used for several applications, such as bioanalysis. Although several electrode array devices have been proposed for electrochemical imaging, it is difficult to incorporate many electrochemical sensors into a simple electrode array device due to the lack of spaces for electrodes. To solve this problem, we developed a novel electrochemical imaging system. In this system, redox cycling is based so as to incorporate many sensors in a chip device. In this review, our strategy of the detection system and device construction are described. Finally, electrochemical bioimaging using the device is described.

In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.

In the present study, we monitored the alkaline phosphatase (ALP) activity of embryoid bodies (EBs) of mouse embryonic stem (ES) cells using a large-scale integration (LSI)-based amperometric device with 400 sensors and a pitch of 250 μm. In addition, a simulation analysis was performed to reveal the positional relationship between the EBs and the sensor electrodes toward more precise measurements. The study shows that simulation analysis can be applied for precise electrochemical imaging of three-dimensionally cultured cells by normalization of the current signals.

© Springer Japan 2015. This article presents an overview of the recent progress in scanning electrochemical microscopy (SECM) for imaging single cells and biomolecules. SECM is a technique for characterizing the local electrochemical nature of various materials by scanning a probe microelectrode. The probe current reflects the electrochemical processes occurring in the small space surrounded by the probe and the substrate. The spatial resolution of SECM is inferior to conventional scanning probe microscopes such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM), since the fabrication of the probe microelectrodes with nanometer sizes is quite difficult. However, recent progress in the fabrication of nanometer-scale electrodes and the development of electrode-sample distance control systems has greatly enhanced the capacity of SECM systems to solve problems in cell biology. The topics reviewed include the following: enzyme activity evaluation, electrochemical enzyme-linked immunosorbent assays, membrane permeability evaluation, respiratory activity measurements, reporter gene assays, membrane protein imaging, and neurotransmitter detection.

Gradient biomaterials have been developed and employed as an important tool in tissue engineering and biology research since the discovery that tissues and organs are non-homogeneous, exhibiting natural functional gradients in their structure or composition. Gradient biomaterials consist of relatively gradual continuous transitions in either compositional or mechanical properties. They have been used to study cellular responses such as cell adhesion, migration, proliferation, and differentiation, and may also be useful tools in drug discovery and development. Gradients made of hydrogels and nanofibers are widely used scaffolds in tissue engineering, which have aroused great interest owing to their tunable properties and analogy to the microenvironment of native tissues. In this chapter, we classify gradient biomaterials into two main cohorts, physical and chemical/biological gradients, and describe their features and applications, particularly as tooth or bone tissue scaffolds.

Dielectrophoresis (DEP) approach was employed to achieve highly aligned multi-walled carbon nanotubes (MWCNTs) within the gelatin methacrylate (GelMA) hydrogels in a facile, rapid, inexpensive, and reproducible manner. This approach enabled us to make different CNTs alignments (e.g., vertical or horizontal alignments) within the GelMA hydrogel using different electrode designs or configurations. Anisotropically aligned GelMA-CNTs hydrogels showed considerably higher conductivity compared to randomly distributed CNTs dispersed in the GelMA hydrogel and the pristine and non-conductive GelMA hydrogel. Adding 0.3 mg/mL CNTs to the GelMA hydrogel led to a slight increase in the mechanical properties of the GelMA and made it to behave as a viscoelastic material. Therefore, it can be used as a suitable scaffold for soft tissues, such as skeletal muscle tissue. 3D microarrays of skeletal muscle myofibers were then fabricated based on the GelMA and GelMA-CNTs hydrogels and they were characterized in terms of gene expressions related to the muscle cell differentiation and contraction. Owing to high electrical conductivity of aligned GelMA-CNTs hydrogels, the engineered muscle tissues cultivated on these materials demonstrated superior maturation and functionality particularly after applying the electrical stimulation (voltage 8 V, frequency 1 Hz, and duration 10 ms for 2 days) compared to the corresponding tissues obtained on the pristine GelMA and randomly distributed CNTs within the GelMA hydrogel.

Stem cells are a key element in tissue engineering and regenerative medicine. However, they require a suitable microenvironment to grow and regenerate. Carbon nanotubes (CNTs) have attracted much attention as promising materials for stem cell research due to their extraordinary properties, such as their extracellular matrix-like structure, high mechanical strength, optical properties, and high electrical conductivity. Of particular interest is the use of CNTs as biomimetic substrates to control the differentiation of stem cells. CNTs have also been combined with commonly used scaffolds to fabricate functional scaffolds to direct stem cell fate. CNTs can also be used for stem cell labeling due to their high optical absorbance in the near-infrared regime. In this paper, we review and discuss the applications of CNTs in stem cell research along with CNT toxicity issues.

We propose a novel application of dielectrophoresis (DEP) to make three-dimensional (3D) methacrylated gelatin (GelMA) hydrogels with gradients of micro- and nanoparticles. DEP forces were able to manipulate micro- and nanoparticles of different sizes and materials (i.e., C2C12 myoblasts, polystyrene beads, gold microparticles, and carbon nanotubes) within GelMA hydrogels in a rapid and facile way and create 3D gradients of these particles in a microchamber. Immobilization of drugs, such as fluorescein isothiocyanate-dextran (FITC-dextran) and 6-hydroxydopamine (6-OHDA), on gold microparticles allowed us to investigate the high-throughput release of these drugs from GelMA-gold microparticle gradient systems. The latter gradient constructs were incubated with C2C12 myoblasts for 24 h to examine the cell viability through the release of 6-OHDA. The drug was released from the microparticles in a gradient manner, inducing a cell viability gradient. This novel approach to create 3D chemical gradients within hydrogels is scalable to any arbitrary length scale. It is useful for making anisotropic biomimetic materials and high-throughput platforms to investigate cell-microenvironment interactions in a rapid, simple, cost-effective, and reproducible manner. (C) 2014 Elsevier B.V. All rights reserved.

We evaluated the intracellular NAD(P) H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione-ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 mu M) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 mu M), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system. (C) 2014 Elsevier B.V. All rights reserved.

The activities of glucose oxidase (GOx) and lactate oxidase (LOx) immobilized on a substrate were obtained as images of oxidation currents of H2O2 using a Bio-LSI system. In the presence of appropriate substrates, the image of the enzyme appeared as lines with a high oxidation current. The time required to obtain the current image was 10s, a significant decrease from the time required by the scanning method with a miniaturized probe.

Nanoscale cell surface topography was visualized using a scanning ion conductance microscope (SICM), where a nanopipette was used as the scanning probe to detect ionic current as feedback signal. SICM was an effective tool for noncontact topographical imaging of live cells, because measurements were performed under physiological conditions. This breakthrough technique opens up a wealth of possible new experiments in membrane and cell biology. (C) The Electrochemical Society of Japan, All rights reserved.

This paper reports a novel approach for the simple detection of cell apoptosis using an electrochemical technique. This method uses caspase-3 activity as an indicator of apoptosis. Caspase-3 activity was detected with differential plus voltammetry (DPV) as an alternative to conventional spectrometry. In this method, p-nitroaniline (pNA) released from Asp-Glu-Val-Asp-pNA by caspase-3 enzyme reaction was measured with DPV by using a glassy carbon electrode. Using this method, we successfully detected cell apoptosis occurring inside living HepG2 cells without the need for a cell lysis step. This method provides an easy assay procedure and, more importantly, allows a live cell apoptosis detection format. This novel electrochemical apoptosis assay using living cells instead of typically used cell lysates will expand the applicable range of the apoptosis assay to include cell activity assays for drug discovery and cell transplantation medicine.

A new local redox cycling-based electrochemical (LRC-EC) device integrated with many electrochemical sensors has been developed into a small chip device. The LRC-EC chip device was successfully applied for detection of alkaline phosphatase and horseradish peroxidase activity in substrate generation/chip collection (SG/CC) and extended feedback modes, respectively. The new imaging approach with extended feedback mode was particularly effective for sharpening of the image, because this mode uses feedback signals and minimizes the undesired influence of diffusion. The LRC-EC chip device is considered to be a useful tool for bioanalysis.

Biological scaffolds with tunable electrical and mechanical properties are of great interest in many different fields, such as regenerative medicine, biorobotics, and biosensing. In this study, dielectrophoresis (DEP) was used to vertically align carbon nanotubes (CNTs) within methacrylated gelatin (GelMA) hydrogels in a robust, simple, and rapid manner. GelMA-aligned CNT hydrogels showed anisotropic electrical conductivity and superior mechanical properties compared with pristine GelMA hydrogels and GelMA hydrogels containing randomly distributed CNTs. Skeletal muscle cells grown on vertically aligned CNTs in GelMA hydrogels yielded a higher number of functional myofibers than cells that were cultured on hydrogels with randomly distributed CNTs and horizontally aligned CNTs, as confirmed by the expression of myogenic genes and proteins. In addition, the myogenic gene and protein expression increased more profoundly after applying electrical stimulation along the direction of the aligned CNTs due to the anisotropic conductivity of the hybrid GelMA-vertically aligned CNT hydrogels. We believe that platform could attract great attention in other biomedical applications, such as biosensing, bioelectronics, and creating functional biomedical devices.

A rapid, ultrasensitive, and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First, we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated, and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 mu g/L) and a low detection limit (LOD) of 0.001 mu g/L, which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning, an LOD of 0.052 mg/kg-oil was achieved, which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally, the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs, and the results agreed well with the Japanese official method (HRGC/HRMS).

Amorphous hydrocarbon (aCH) material is receiving plenty of attention due to its possible wide application. However, one hurdle facing this application is that high temperature is required to express conductivity of aCH, e.g., post annealing or deposition at high temperature. To form a conductive aCH on a substrate controlled below room temperature, we have developed a neutral beam enhanced chemical vapor deposition (NBECVD) method to control a hydrocarbon molecular structure that has a large conjugated system with delocalized pi electrons in film. For material gas, we prepared toluene. As a result, we obtained a highly conductive carbon on a Si substrate with -50 degrees C using only toluene by optimizing the state of disassociated material gas. From an evaluation of film structure, a polycyclic aromatic hydrocarbon molecular structure was grown and contained in film because NBECVD could avoid irradiating UV to the Si substrate during deposition. Thus, an excited large conjugated chain structure generated by toluene in plasma could be maintained and polymerized on the Si substrate. Furthermore, the conductive aCH film could work as electrode in solution by electrochemical examination. Additionally, we found that nitrogen doped into conductive aCH could increase the working current of an electrode. (C) 2013 Elsevier Ltd. All rights reserved.

As a complementary tool to nanofluidics, bio-molecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.

A flexible sensor based on SU-8 photoresist was fabricated and its electrochemical performance was investigated using cyclic voltammetry. The device consisted of interdigitated array (IDA) electrodes on an SU-8 layer. It exhibited a clear electrochemical response during redox cycling of ferrocenemethanol at the IDA electrodes. Since the device was flexible, it could be inserted into a narrow bent space to monitor electrochemical responses. The observed electrochemical behavior was found to be consistent with that predicted by simulations based on redox compound diffusion.

【<b>Objective】</b>Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.<br>【<b>Methods】</b>Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and divided into two groups, including a control group (n=9) and tacrolimus-treated group (n=7). Changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system.<br>【<b>Results】</b>The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p＜0.05). Although the expression of <i>Vegfa</i> (p＜0.05) and <i>Ccnd1</i> (p＜0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.<br>【<b>Conclusion】</b>The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.<br>*This article is a secondary publication in Japanese of "Tacrolimus inhibits the revascularization of isolated pancreatic islets" published by Nishimura R, Nishioka S, Fujisawa I, <i>et al</i>. PLoS One.2013 Apr 17; 8. DOI: 10.1371/journal.pone.56799

Dielectrophoresis (DEP) approach was employed to achieve highly aligned multi-walled carbon nanotubes (MWCNTs) within the gelatin methacrylate (GelMA) hydrogels in a facile, rapid, inexpensive, and reproducible manner. This approach enabled us to make different CNTs alignments (e.g., vertical or horizontal alignments) within the GelMA hydrogel using different electrode designs or configurations. Anisotropically aligned GelMA-CNTs hydrogels showed considerably higher conductivity compared to randomly distributed CNTs dispersed in the GelMA hydrogel and the pristine and non-conductive GelMA hydrogel. Adding 0.3 mg/mL CNTs to the GelMA hydrogel led to a slight increase in the mechanical properties of the GelMA and made it to behave as a viscoelastic material. Therefore, it can be used as a suitable scaffold for soft tissues, such as skeletal muscle tissue. 3D microarrays of skeletal muscle myofibers were then fabricated based on the GelMA and GelMA-CNTs hydrogels and they were characterized in terms of gene expressions related to the muscle cell differentiation and contraction. Owing to high electrical conductivity of aligned GelMA-CNTs hydrogels, the engineered muscle tissues cultivated on these materials demonstrated superior maturation and functionality particularly after applying the electrical stimulation (voltage 8 V, frequency 1 Hz, and duration 10 ms for 2 days) compared to the corresponding tissues obtained on the pristine GelMA and randomly distributed CNTs within the GelMA hydrogel. Copyright © Materials Research Society 2014.

Electrically conductive reinforced hydrogels offer a wide range of applications as three-dimensional scaffolds in tissue engineering. We report electrical and mechanical characterization of methacrylated gelatin (GelMA) hydrogel, containing palladium-based metallic glass nanofibers (MGNF). Also we show that the fibers are biocompatible and C2C12 myoblasts in particular, planted into the hybrid hydrogel, tend to attach to and elongate along the fibers. The MGNFs in this work were created by gas atomization. Ravel of fibers were embedded in the GelMA prepolymer in two different concentrations (0.5 and 1.0 mg/ml), and then the ensemble was cured under UV light, forming the hybrid hydrogel. The conductivity of the hybrid hydrogel was proportional to the fiber concentration.

© 14CBMS. In the present study, we present a method of eletrodeposittion of hydrogels to fabricate cell sheets. In the method, calcium-alginate hydrogels are electrodeposited by water electrolysis. 3T3 cells were cultured on the hydrogels to form cell sheets, and the cell sheets were collected by dissolving the hydrogels. Intact cell sheets can be collected, indicating that the method is useful to create cell sheets.

Construction of an in vitro drug screening method for evaluating drug metabolism and toxicity by using cells is required instead of the conventional in vivo one that uses animals. In order to realize the in vitro study, analyzing the cellular activity or viability noninvasively in advance of the screening is essential. The aim of the current study is to establish a method that can evaluate the cellular activity depending on spheroid sizes by means of oxygen consumption and to determine the valid diameter of hepatocyte spheroids. To measure the respiratory activity of the spheroids, which were formed on a nanopillar sheet, we applied scanning electrochemical microscopy (SECM). From the viewpoint of high respiratory activity and its small variation, we determined that spheroids with 70 mu m in diameter were adequate. We then performed a gene expression analysis by using a real-time PCR to evaluate the correlation with respiratory activity. As a result, a higher expression level of Hnf4 alpha, which is essential for hepatocytes to fulfill many liver functions and is the indicator of well-differentiated hepatocytes, showed relatively higher respiratory activity. We concluded that the noninvasive SECM technique could evaluate the cellular activity of a single spheroid. Noninvasively measuring cellular activity by SECM makes it possible to evaluate the cellular activity prior to a nonclinical test and enables the continued monitoring of the drug response by using single spheroid. SECM becomes a powerful tool to satisfying the increasing demand for an in vitro system in the course of new drug development. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

A novel measurement system to determine oxygen consumption rates via respiration in migrating Zebrafish (Danio rerio) has been developed. A signal equalization system was adapted to detect oxygen in a chamber with one fish, because typical electrochemical techniques cannot measure respiration activities for migrating organisms. A closed chamber was fabricated using a pipet tip attached to a Pt electrode, and a columnar Vycor glass tip was used as the salt bridge. Pt electrode, which was attached to the chamber with one zebrafish, and Ag electrode were immersed in 10 mM potassium iodide (KI), and both the electrodes were connected externally to form a galvanic cell. Pt and Ag electrodes act as the cathode and anode to reduce oxygen and oxidize silver, respectively, allowing the deposition of insoluble silver iodide (AgI). The AgI acts as the signal source accumulated on the Ag electrode by conversion of oxygen. The amount of AgI deposited on the Ag electrode was determined by cathodic stripping voltammetry. The presence of zebrafish or its embryo led to a decrease in the stripping currents generated by a 10 min conversion of oxygen to AgI. The conversion of oxygen to AgI is disturbed by the migration of the zebrafish and allows the detection of different equalized signals corresponding to respiration activity. The oxygen consumption rates of the zebrafish and its embryo were estimated and determined to be similar to 4.1 and 2.4 pmol.s(-1), respectively. The deposited AgI almost completely disappeared with a single stripping process. The signal equalization system provides a method to determine the respiration activities for migrating zebrafish and could be used to estimate environmental risk and for effective drug screening.

We have previously reported a local redox cycling-based electrochemical (LRC-EC) system for the incorporation of many electrochemical sensors into a small chip device. In the present study, a new type of LRC-EC chip device was fabricated for the detection of a droplet array. To detect electrochemically redox compounds in droplets, Pt pseudo-reference/counter electrodes were incorporated into the individual sensors of the LRC-EC chip device. Cyclic voltammetry for the LRC-EC chip device with internal Pt pseudo-reference electrodes indicated well-defined voltammograms based on redox cycling for the individual sensor points. The device was successfully applied to the addressable detection of alkaline phosphatase (ALP) activity of HeLa cells in single droplets on the sensor points. Therefore, the LRC-EC chip device is considered to be a useful device for the bioanalysis of droplet systems.

In this study, we introduce the double-barrel carbon probe (DBCP)-a simple, affordable microring electrode-which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

The electrical conductivity of graphene provides a unique opportunity to modify the behavior of electrically sensitive cells. Here, we demonstrate that C2C12 myoblasts that were cultured on ultrathin thermally reduced graphene (TR-Graphene) films had more favorable cell adhesion and spreading compared to those on graphene oxide (GO) and glass slide substrates, comparable with conventional Petri dish. More importantly, we demonstrate that electrical stimulation significantly enhanced myoblast cell differentiation on a TR-Graphene substrate compared to GO and glass slide surfaces as confirmed by the expression of myogenic genes and proteins. These results highlight the potential applications of graphene-based materials for cell-based studies, bioelectronics, and biorobotics.

We have developed a novel method for detection of endotoxin with extra-high sensitivity by using substitutional stripping voltammetry (SSV). In this method, a p-aminophenol (pAP) conjugated peptide (Boc-Leu-Gly-Arg-pAP; LGR-pAP) was used as a substrate for a protease, which is activated at the last step of the endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. Extra-highly sensitive detection of pAP liberated by the endotoxin-induced LAL reaction was successfully realized with SSV, based on the accumulation of an amperometric signal owing to exchange of the oxidation current of pAP generated at an electrode in a reaction cell with silver deposition on another electrode in a deposition cell. This reaction is driven by the difference in the redox potential between pAP/quinoneimine and silver/silver ion. The amount of the deposited silver is quantified by anodic stripping voltammetry (ASV). This SSV-based endotoxin assay was performed with a chip device comprising two cells, each of which was connected via a liquid junction made of Vycor (R) glass. The reaction cell and the deposition cell contained a standard endotoxin sample with LAL regents containing LGR-pAP and AgNO3 solution, respectively. After the cells were electrically connected for 60 min, ASV was conducted in the deposition cell to quantify the total electrical charge derived by the oxidation of free pAP in the reaction cell. The ASV signal increased with the increase of the endotoxin concentration in the sample solution in the range of 0.5-1000 EU L-1.

Skeletal muscle tissues play a significant role to maintain the glucose level of whole body and any dysfunction of this tissue leads to the diabetes disease. A culture medium was created in which the muscle cells could survive for a long time and meanwhile it did not interfere with the glucose sensing. We fabricated a model of skeletal muscle tissues in vitro to monitor its glucose uptake. A nanoporous gold as a high sensitive nanobiosensor was then successfully developed and employed to detect the glucose uptake of the tissue models in this medium upon applying the electrical stimulation in a rapid, and non-invasive approach. The response of the glucose sensor was linear in a wide concentration range of 1-50 mM, with a detection limit of 3 mu M at a signal-to-noise ratio of 3.0. The skeletal muscle tissue was electrically stimulated during 24 h and glucose uptake was monitored during this period. During the first 3 h of stimulation, electrically stimulated muscle tissue consumed almost twice the amount of glucose than counterpart non-stimulated sample. In total, the glucose consumption of muscle tissues was higher for the electrically stimulated tissues compared to those without applying the electrical field. (C) 2013 Elsevier B.V. All rights reserved.

A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5 h, and their ALP activity in a glucose-free buffer decreased after 2 h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3 h. The chip is expected to be useful for the evaluation of cell activities. (C) 2013 Elsevier B.V. All rights reserved.

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

Control of the flow position of cells in a microchannel is useful for developing cell separation systems. We demonstrated that cells with different sizes were transported through different gaps by a repulsive force generated by negative dielectrophoresis (n-DEP). A device was fabricated by sandwiching a polyester film with a fluidic channel between upper and lower substrates with design same as that of navigator and separator electrodes which were used to concentrate the flowing cells in the center of the channel and to guide them to gaps of different sizes, respectively. The performance of the system was assessed using a human acute monocytic leukemia cell line (THP-1) and red blood cells (RBCs) from preserved equine blood as model cells. The cells flowed along the edges of navigator electrodes to concentrate in the center of the channel because of a strong repulsive force between the upper and lower substrates induced by the application of an AC electric field. THP-1 and RBCs passed through gaps of different sizes in a separator consisting of a microelectrode array. Passage efficiencies for THP-1 and RBCs through the desired gaps were found to be 88% and 44%, respectively. The results indicate the possibility of the continuous separation of cells with different sizes in the fluidic device based on n-DEP. (C) 2013 Elsevier B.V. All rights reserved.

A local redox cycling-based electrochemical (LRC-EC) chip device was used to investigate the relationship between cardiomyocyte differentiation from embryonic stem (ES) cells and alkaline phosphatase (ALP) activity. In the LRC-EC chip device, ring-type interdigitated array electrodes were incorporated at n × n measurement points with only 2n bonding pads for external connection. Microwells were also fabricated at each measurement point to trap cell aggregates. To differentiate ES cells into cardiomyocytes, ES cells were three-dimensionally cultured to form simple and cystic embryoid bodies (EBs). ALP activity of these EBs was then detected using the LRC-EC chip device. The electrochemical responses for ALP activity decreased concurrently with the differentiation of ES cells into cardiomyocytes, indicating that an LRC-EC chip device is useful for evaluating cell differentiation. © 2013 The Electrochemical Society of Japan, All rights reserved.

Dual carbon electrodes (DCEs) are quickly, easily, and cheaply fabricated by depositing pyrolytic carbon into a quartz theta nanopipet. The size of DCEs can be controlled by adjusting the pulling parameters used to make the nanopipet. When operated in generation/collection (G/C) mode, the small separation between the electrodes leads to reasonable collection efficiencies of ca. 30%. A three-dimensional finite element method (FEM) simulation is developed to predict the current response of these electrodes as a means of estimating the probe geometry. Voltammetric measurements at individual electrodes combined with generation/collection measurements provide a reasonable guide to the electrode size. DCEs are employed in a scanning electrochemical microscopy (SECM) configuration, and their use for both approach curves and imaging is considered. G/C approach curve measurements are shown to be particularly sensitive to the nature of the substrate, with insulating surfaces leading to enhanced collection efficiencies, whereas conducting surfaces lead to a decrease of collection efficiency. As a proof-of-concept, DCEs are further used to locally generate an artificial electron acceptor and to follow the flux of this species and its reduced form during photosynthesis at isolated thylakoid membranes. In addition, 2-dimensional images of a single thylakoid membrane are reported and analyzed to demonstrate the high sensitivity of G/C measurements to localized surface processes. It is finally shown that individual nanometer-size electrodes can be functionalized through the selective deposition of platinum on one of the two electrodes in a DCE while leaving the other one unmodified. This provides an indication of the future versatility of this type of probe for nanoscale measurements and imaging.

In this study, we fabricated a probe consisting of a carbon nanoelectrode and an Ag/AgCl reference electrode for detecting the activity of cells in single droplets. HeLa cells were confined into a single droplet, and the alkaline phosphatase (ALP) activity of the cells was electrochemically measured using the probe inserted into the droplet. The ALP of the confined cells catalyzed the hydrolysis of p-aminophenyl phosphate (PAPP) to yield p-aminophenol (PAP) that gave electrochemical responses. Since the tip of the carbon-Ag/AgCl probe is very small, it is useful for electrochemical analysis of cells using droplets. © 2013 American Chemical Society.

In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering. © 2012 The Society for Biotechnology, Japan.

Aims: Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.Methods: Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system.Results: The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of Vegfa (p<0.05) and Ccnd1 (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.Conclusions: The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.

Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields. © 2012 Springer Science+Business Media, LLC.

The micro/nanoelectrode has unique characteristics, which are not expected for conventional electrodes, such as the capability to perform localized measurements, low double layer charging currents, low ohmic drops, and fast mass transport. Scanning electrochemical microscopy (SECM) with a micro/nanoelectrode for detecting electroactive chemical species and is an effective tool for the investigation of the localized chemical properties of sample surfaces and interfaces. Because SECM has high temporal resolution under physiological conditions, it is particularly well suited for quantitative measurements of chemicals like neurotransmitters, nitric oxide, reactive oxygen species, and oxygen, which are released/consumed by living cells. This article presents an overview of the recent progress of SECM.

Mercury is considered the most important heavy-metal pollutant, because of the likelihood of bioaccumulation and toxicity. Monitoring widespread ionic mercury (Hg2+) contamination requires high-throughput and cost-effective methods to screen large numbers of environmental samples. In this study, we developed a simple and sensitive analysis for Hg2+ in environmental aqueous samples by combining a microfluidic immunoassay and solid-phase extraction (SPE). Using a rnicrofluidic platform, an ultrasensitive Hg2+ He immunoassay, which yields results within only 10 min and with a lower detection limit (LOD) of 0.13 mu g/L, was developed. To allow application of the developed immunoassay to actual environmental aqueous samples, we developed an ion-exchange resin (IER)-based SPE for selective Hg2+ extraction from an ion mixture. When using optimized SPE conditions, followed by the microfluidic immunoassay, the LOD of the assay was 0.83 mu g/L, which satisfied the guideline values for drinking water suggested by the United States Environmental Protection Agency (USEPA) (2 mu g/L; total mercury), and the World Health Organisation (WHO) (6 mu g/L; inorganic mercury). Actual water samples, including tap water, mineral water, and river water, which had been spiked with trace levels of Hg2+, were well-analyzed by SPE, followed by microfluidic Hg2+ immunoassay, and the results agreed with those obtained from reduction vaporizing atomic adsorption spectroscopy.

The differentiation status of single live embryonic stem (ES) cells was quantitatively evaluated by monitoring the activity of alkaline phosphatase (ALP), an undifferentiation marker of ES cells, using scanning electrochemical microscopy (SECM).

Multicellular spheroids of human breast cancer cells (MCF-7) formed with two different three-dimensional (3D) culture methods were evaluated in detail on the basis of respiratory activity and highthroughput gene expression analysis. The spheroids formed with poly(dimethylsiloxane) (PDMS) microwell arrays indicated significant restriction of the spheroid size, whereas their respiratory activity was 2-fold greater than that formed with the hanging drop culture method. Fluidigm BioMark dynamic array was used for comprehensive and quantitative real-time polymerase chain reaction (qRT-PCR) analysis on the samples whose respiratory activity had been measured. Genes involved in cellular senescence and glucose metabolism indicated significantly higher values for the PDMS microwell culture method than for the hanging drop culture method (P &lt 0.05). Interestingly, samples formed with the PDMS microwell culture method showed stronger responses for glycolysis than those formed with the hanging drop method. These results illustrate the power of multiparameter analysis to characterize multicellular spheroids cultured in different microenvironments even if they have the same morphology. © 2013 Elsevier Inc.

In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

We developed a novel protease detection method based on amperometry using a p-aminophenol (pAP) conjugated substrate. We prepared Boc-Leu-Gly-Arg-pAP (LGR-pAP) as a novel substrate for a clotting enzyme, which is a protease activated by an endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. The basic study using cyclic voltammetry revealed that the oxidation peak potentials of LGR-pAP and pAP were sufficiently separated from each other (0.25 V) to conduct amperometric detection of protease activity. We combined simple amperometric detection with a screen-printed electrode chip to produce a practical protease sensor. As an application of the sensor, we demonstrated quantitative endotoxin sensing. The endotoxin activated zymogens contained in the LAL to generate pAP, which was then electrochemically detected by potential step chronoamperometry (PSCA). The observed oxidation current increased with the concentration of endotoxin in the LAL assay solution. This PSCA detection was performed with a disposable chip sensor consisting of a screen-printed electrode and a fluidic channel with a hydrophilic cover. This chip sensor successfully detected 10-1000 EU L-1 endotoxin within 60 min. This novel amperometric measurement with a screen-printed electrode not only provides compact, low-cost, and easy-to-use sensors for on-site monitoring of endotoxin, but also shows promise for use in other in vitro protease assays for biochemical research, diagnosis, and drug development.

Mouse embryoid bodies (mEBs) were evaluated in detail on the basis of respiratory activity and high-throughput quantitative reverse transcription-PCR (RT-qPCR) analysis. The hanging drop culture method was applied to prepare various sizes of mEBs ranging from 100 to 250 mm in radius by causing the aggregation of embryonic cells. The respiratory activity of individual mEBs was noninvasively measured using scanning electrochemical microscopy in a cone-shaped microwell. For gene expression analysis, we used 48.48 Dynamic Array chips (Fluidigm) integrating microfluidic circuits, which allowed high-throughput qPCR analysis in parallel. The respiratory activity of the mEBs that were cultured for 1 to 6 days could predict the mRNA levels of undifferentiation and differentiation markers. However, the sizes of the mEBs could also predict the gene expression of the undifferentiation/differentiation markers because the radii of the mEBs increased by more than 2-fold after incubation in hanging drop culture for 6 days. Next, mEBs with identical sample sizes were evaluated for respiratory activity and gene expression. For mEBs cultured at 1500 cells per droplet for 3 days, the respiratory activity was negatively correlated with the mRNA levels of pluripotent markers such as Nanog and Sox2. Many differentiation markers were positively correlated with the respiratory activity. However, there was no significant difference in respiration activity between the beating and nonbeating samples on day 3. Finally, principal component analysis (PCA) confirmed the relationship between respiratory activity and the mRNA levels of undifferentiation/differentiation markers.

Island organization of particles have been fabricated in a microfluidic device consisted of upper and lower conductive indium-tin-oxide (ITO) substrates with interdigitated microband array (IDA) electrode used as the template based on a dielectrophoresis (DEP). Grid formation of electrodes was fabricated by rotating the upper template ITO-IDA by 90 degrees relative to the lower ITO-IDA. A suspension of polystyrene particles with a 3-mu m diameter was introduced into the device. AC electric signal (typically 20V peak-to-peak, 1.0 MHz) was then applied to the bands on the upper and lower IDA, resulting in the formation of island patterns at the intersections with low electric fields. When the AC voltage with same frequency and same phase was applied to the bands on upper and lower IDA, particles were accumulated at the intersections consisted of the bands applied voltage and bands connected to the ground because the relatively lower electric fields were produced at those intersections; on contrast, the application of the AC voltage with different frequencies to the bands allowed to the formation of second pattern due to the generation of the strong electric field at the intersections applied the AC voltage with different frequencies. Moreover, it is possible to convert the second patterns reversibly by choosing the band applying the different frequencies. The particles forming the patterns were immobilized in a photoreactive hydrogel polymer. The well-ordered particles embedded in the flexible hydrogel sheet firmly were obtained by ultraviolet irradiation to the entire device with patterns. The accumulated particles were fixed through the immunoreactions between the antibody immobilized on the particle surface and analytes in the solution. The presence of the specific antigens allowed to the fixed complexes of particles. It is noted that the time required for single sensing is as short as 5 min and separation steps are eliminated in the presented procedure, while we decide only the presence of target analytes above the limit of detection. We demonstrated the rapid and simple immunosensing using the aggregation of particles accumulated with DEP. (C) 2012 Elsevier Ltd. All rights reserved.

We propose a novel electrochemical detection system for alkaline phosphatase (ALP) activity using the difference in water and oil solubilities between the substrate, ferrocene ethyl phosphate ester (FcEtOPO(3)(2-)), and the enzymatic product, ferroceneethanol (FcEtOH). In this system, water droplets containing ALP and FcEtOPO(3)(2-) were placed on a Pt disk microelectrode and surrounded by a mineral oil. By the ALP-catalyzed reaction, FcEtOPO(3)(2-) was converted to FcEtOH, which was then transferred to the mineral oil from the water droplets with FcEtOPO(3)(2-) remaining in the water droplets. After partitioning FcEtOH from the water droplets, FcEtOPO(3)(2-) was detected at the Pt disk microelectrode to estimate the ALP activity. Using this novel system, the ALP activity of embryoid bodies was successfully detected. We believe that the present system will be widely applicable to ALP-based bioassays.

A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4?x?4 array of microwells having a size of 100?mu m?x?100?mu m (diameter?x?depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay. Biotechnol. Bioeng. 2012; 109:21632167. (c) 2012 Wiley Periodicals, Inc.

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5. the half-wave potential (E-1/2) for IFe(CN)(6)](4-) oxidation and 02 reduction measured versus the internal Pt pseudo reference was shifted by about -0.2 V. compared with the E-1/2 measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O-2)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R-2 = 0.90). The slope of the least squares method was 1. In a 1.0 mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E-1/2 for PAP oxidation measured versus the Pt layer was not shifted, compared with the E-1/2 versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier. (C) 2012 Elsevier B.V. All rights reserved.

Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction.

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K-d-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act. (C) 2011 Elsevier B.V. All rights reserved.

我々は，微細加工技術を利用して種々のマイクロ／ナノ電極を開発し，生体物質の局所領域の機能解析を行うとともに，新しいバイオセンシングデバイス・システムへと展開してきたが，本講演では，マイクロ／ナノ電極をプローブとした走査型電気化学顕微鏡(SECM)システム，基板に多数のマイクロ／ナノ電極が配列したバイオセンサアレイを利用したバイオイメージングに焦点を絞り，最近の研究を紹介する．

Rapid accumulations of particles and living cells with negative dielectrophoresis (n-DEP) have been applied to develop the rapid and simple immunosensing method. Grid formation of electrodes was fabricated by rotating the upper template interdigitated microband array (IDA) electrode by 90 degrees relative to the lower IDA. When AC electric signal was applied to the bands on the upper and lower IDA, island organization was rapidly formed at the intersections with low electric fields. The accumulated particles were fixed through the immunoreactions between the antibody immobilized on the particle surface and analytes in the solution. The presence of the specific antigens allowed the formation of fixed complexes of particles. It is noted that the time required for single sensing is as short as 5 min and separation steps are eliminated in the presented procedure. We demonstrated the rapid and simple immunosensing using the aggregation of particles accumulated with DEP.

In this study, we have developed a novel electrochemical detection system containing many electrochemical sensors. The detection system is based on local redox cycling to incorporate many electrochemical sensors into small chip device. The density of the electrochemical sensors is the highest in the field of the electrochemical lab-on-a-chip devices. By using the chip device, comprehensive electrochemical imaging can be achieved. In this study, the chip device was applied for cell analysis of three-dimensional culture cells.

We have previously proposed an excellent method to incorporate many electrochemical sensors into a single chip device using two sets of microelectrode array to induce local redox cycling, and we have designated the novel methodology as local redox cyclingbased electrochemical (LR-EC) system. In this study, we developed a LRC-EC chip device consisting of 256 electrochemical sensors. At the electrochemical sensors, ring-type interdigitated array (IDA) electrodes were placed to induce local redox cycling. The LRC-EC chip device was applied to evaluate three-dimensional (3D) culture cells, such as embryoid bodies (EBs).

In this work, we report the control of a microparticle position within fluid flow based on its size by using a repulsive force generated with negative dielectrophoresis (n-DEP). The n-DEP based fluidic channel, which was consisted of navigator and separator electrodes, was used to manipulate the particle flow in the center of channel and to control the particle position in the fluidic flow. The mixture of 10 μm-and 20 μm-diameter particles was introduced into the channel with 30 μm height at 700 μm/s. On applying an AC voltage (23 V peak-peak and 7 MHz) to the navigator electrodes on the upper and lower substrates in a n-DEP frequency region, the suspended microparticles were guided to the center of the fluidic channel and then channelled through the passage gate positioned at the center of the channel. The AC electric field was also applied to separator electrodes, resulting in a formation of flow paths with low electric fields. The separator was consisted of the five band electrodes with the different gap spaces with the adjacent band, which allow to fomvng the flow paths with different electric fields. The microparticles separately flow in line along the paths formed between the band electrodes, the 10 μm-diameter particles mainly flow through the narrow path and 20 μm-diameter particles through the wide path arranged at the outside from the center. These results indicated that positions of two types of microparticles in the fluidic channel were easily separated and controlled using the n-DEP. The present procedure therefore yields a procedure for the DEP based simple and miniaturized separators. © 2012 TSI® Press.

A microfluidic device with analytical chambers for electrochemical measurements has been employed to detect photosynthetic activity at single cell level. The flowing cells (Microcystis viridis) in a main channel are individually guided to the chamber with microelectrodes by an electrophoretic manipulation. The reduction current of oxygen was continuously monitored to determine the photosynthetic activity upon light irradiation. The average rates for oxygen generation were estimated and found to be 10(-18) mol/s level.

Detectable sensitivity for immobilized enzymes was improved using a constant-distance mode of scanning electrochemical microscopy (SECM). Redox species generated by an enzyme reaction captured via immuno-recognition were detected with a microelectrode used as a probe. The probe maintained considerably close to the target surface (50 nm) allowed the enhancement of the current response because of cycling of redox species between the probe and the captured enzymes. The sensitivity of di(n-butyl)phthalate detection with a competitive assay by using constant-distance mode SECM was at least one order of magnitude higher than that of the probe scanned in a constant z-position. (C) The Electrochemical Society of Japan, All rights reserved.

Establishing the 3D microscale organization of cells has numerous practical applications, such as in determining cell fate (e. g., proliferation, migration, differentiation, and apoptosis) and in making functional tissue constructs. One approach to spatially pattern cells is by dielectrophoresis (DEP). DEP has characteristics that are important for cell manipulation, such as high accuracy, speed, scalability, and the ability to handle both adherent and non-adherent cells. However, widespread application of this method is largely restricted because there is a limited number of suitable hydrogels for cell encapsulation. To date, polyethylene glycol-diacrylate (PEG-DA) and agarose have been used extensively for dielectric patterning of cells. In this study, we propose gelatin methacrylate (GelMA) as a promising hydrogel for use in cell dielectropatterning because of its biocompatibility and low viscosity. Compared to PEG hydrogels, GelMA hydrogels showed superior performance when making cell patterns for myoblast (C2C12) and endothelial (HUVEC) cells as well as in maintaining cell viability and growth. We also developed a simple and robust protocol for co-culture of these cells. Combined application of the GelMA hydrogels and the DEP technique is suitable for creating highly complex microscale tissues with important applications in fundamental cell biology and regenerative medicine in a rapid, accurate, and scalable manner.

This report describes the electrochemical detection of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device consisting of 256 sensors. The time-course analyses showed that the redox compound in the droplet was dynamically changed during droplet evaporation or mass transfer through a water/oil interface.

We have developed an LSI-based amperometric sensor called "Bio-LSI" with 400 measurement points as a platform for electrochemical bio-imaging and multi-point biosensing. The system is comprised of a 10.4 mm x 10.4 mm CMOS sensor chip with 20 x 20 unit cells, an external circuit box, a control unit for data acquisition, and a DC power box. Each unit cell of the chip contains an operational amplifier with a switched-capacitor type I-V converter for in-pixel signal amplification. We successfully realized a wide dynamic range from +/- 1 pA to +/- 100 nA with a well-organized circuit design and operating software. In particular, in-pixel signal amplification and an original program to control the signal read-out contribute to the lower detection limit and wide detection range of Bio-LSI. The spacial resolution is 250 mu m and the temporal resolution is 18-125 ms/400 points, which depends on the desired current detection range. The coefficient of variance of the current for 400 points is within 5%. We also demonstrated the real-time imaging of a biological molecule using Bio-LSI. The LSI coated with an Os-HRP film was successfully applied to the monitoring of the changes of hydrogen peroxide concentration in a flow. The Os-HRP-coated LSI was spotted with glucose oxidase and used for bioelectrochemical imaging of the glucose oxidase (GOx)-catalyzed oxidation of glucose. Bio-LSI is a promising platform for a wide range of analytical fields, including diagnostics, environmental measurements and basic biochemistry.

Engineered skeletal muscle tissues could be useful for applications in tissue engineering, drug screening, and bio-robotics. It is well-known that skeletal muscle cells are able to differentiate under electrical stimulation (ES), with an increase in myosin production, along with the formation of myofibers and contractile proteins. In this study, we describe the use of an interdigitated array of electrodes as a novel platform to electrically stimulate engineered muscle tissues. The resulting muscle myofibers were analyzed and quantified in terms of their myotube characteristics and gene expression. The engineered muscle tissues stimulated through the interdigitated array of electrodes demonstrated superior performance and maturation compared to the corresponding tissues stimulated through a conventional setup (i.e., through Pt wires in close proximity to the muscle tissue). In particular, the ES of muscle tissue (voltage 6 V, frequency 1 Hz and duration 10 ms for 1 day) through the interdigitated array of electrodes resulted in a higher degree of C2C12 myotube alignment (similar to 80%) as compared to ES using Pt wires (similar to 65%). In addition, higher amounts of C2C12 myotube coverage area, myotube length, muscle transcription factors and protein biomarkers were found for myotubes stimulated through the interdigitated array of electrodes compared to those stimulated using the Pt wires. Due to the wide array of potential applications of ES for two-and three-dimensional (2D and 3D) engineered tissues, the suggested platform could be employed for a variety of cell and tissue structures to more efficiently investigate their response to electrical fields.

A novel surface plasmon resonance (SPR) biosensor which is capable of monitoring proteomic biomarker secretion from living cells is reported here. Vascular endothelial growth factor (VEGF) secretion from living SKOV-3 ovarian cancer cells was measured for concept demonstration.

A lab-on-a-chip device is described for the electrochemical detection of alkaline phosphatase (ALP) secreted by transformed single HeLa cells. Detection on the chip device is based on local redox cycling at 256 individually addressable sensor points. Ring-disk electrodes (generator/collector) are arranged at individual sensor points to amplify the signal due to redox-cycling with only 32 connector pads. The surface of each sensor point is modified with antibodies for secreted alkaline phosphatase (SEAP) immobilization, which facilitates separation and detection of SEAP. Separation of SEAP from HeLa cells enables elimination of endogenous ALP and prevents HeLa cells from damage due to exposure to high level pH used during electrochemical detection. The large number of sensor points enables the simultaneous analysis of a large amount of single cells using the chip. The system is useful for gene reporter assays and for the detection of several types of secreted proteins.

A novel device was designed for the multipoint addressable detection of DNA hybridization. Row and column electrodes array were orthogonally arranged, and the microwells were assembled on the crossing points of the row/column electrodes to form a 4 x 4 microwell array. Amperometric signals at the individual microwells could be detected separately on the basis of redox cycling of localized electroactive species occurring between the electrodes. Immobilization and hybridization of DNA could block the redox cycling of Fe(CN)(6)(4-)/Fe(CN)(6)(3-) at the designated microwells, resulting in the reduction of current response. This device had been used to detect DNA hybridization with excellent sensitivity (0.03 mu M) and selectivity. The device can be applied to comprehensive and high-throughput detection and imaging of biochemical species. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 +/- 0.33 X 10(-15) mol s(-1), 1.38 +/- 0.58 x 10(-15) mol s(-1), and 3.44 +/- 2.07 x 10(-15) mol s(-1), respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM). (C) 2011 Elsevier B.V. All rights reserved.

The influence of the tip reaction on the substrate generation/tip collection mode SECM imaging of a single yeast cell was investigated by using various sizes of probe electrodes in mu m-nm scales because the tip reaction would disturb the concentration profile of intracellular enzyme reactant formed around the cell. GC mode measurements of a single yeast cell were performed using a dual mediator system with lipophilic menadione and hydrophilic ferricyanide. We found downscaling of the tip size enabled quantitative SECM imaging of a single cell in GC mode without disturbing the concentration profile of the mediator formed around the cell by the tip reaction.

We have developed an electrorotation (ER) chip that has a sandwich structure in which interdigitated array (IDA) electrodes are arranged face-to-face. These IDA electrodes on the top and bottom of the chip were orthogonally arranged to form over 2000 square regions having rotating electric fields between the IDA electrodes. Since rotating electric fields can be generated by arranging the electrical connections to produce a pi/2 phase difference between adjacent electrodes, a large number of measurement areas for ER were incorporated within a single ER chip. The ER properties of glass microrods at the individual measurement areas were investigated using this ER chip. The present ER chip was found to be a useful tool for performing high-throughput assays to analyze the dielectric properties of microparticles. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.

Electrochemical devices have been fabricated with 2 platinum (Pt) microelectrodes located at the bottom of microwells to characterize the photosynthetic activity of a single Microcystis viridis (cyanobacterium) cell. The cell was manipulated into the microwells by performing electrophoresis by applying dc voltage (typically 1.30V peak-to-peak) to the Pt microelectrode. After removing the excess cells around the microwells, the microwells were covered using a urethane cover-plate, which blocked the diffusion of oxygen generated from the trapped cells thus leading to oxygen accumulation in the chamber. The cells trapped in the microwells were irradiated with a light-emitting diode light for 10 min to stimulate oxygen evolution by photosynthesis. The amount of oxygen accumulated in the chamber was determined by amperometry. The average rate of oxygen generation from a single cyanobacterium was calculated from the charge that was calculated from the responses and found to be at the level of 10(-17) molts. Our system will be applicable to fundamental studies of cell biology and on-site monitoring of environmental toxicity. (C) 2010 Elsevier B.V. All rights reserved.

Here, we introduce several applications for single-cell gene-expression analysis utilizing electrical cell lysis technique with a Pt-ring electrode probe. Glass capillary probes featuring a Pt-ring electrode were utilized to collect single cell with two or three dimensional culture systems. An electric pulse with 100 V height and 10 mu s width was applied to lyse the cellular membrane of the single cell in 0. 2 M sucrose solution. Multicellular spheroid of human breast cancer cell line MCF-7 was formed according to hanging drop method or on-top culture on a matrigel sheet. It was shown that evaluation of mRNA expression level is possible at single-cell level collected at the surface of the multicellular spheroid.

An electrochemical device is proposed for high-throughput electrochemical detection that consists of 32 row and 32 column electrodes on a single glass substrate. The row and column electrodes are connected to interdigitated array (IDA) electrodes to form 1024 (32 x 32) addressable sensor points in the device. Electrochemical responses from each of the 1024 sensors were successfully acquired on the device within 1 min using redox cycling at individual IDA electrodes, which ensures application of the device to comprehensive, high-throughput electrochemical detection for enzyme-linked immunosorbent assay (ELISA), reporter gene assay for monitoring gene expressions, and DNA analysis.

We report a scanning electrochemical microscopy (SECM)-based receptor-mediated endocytosis detection method. Epidermal growth factor receptor (EGFR), which is one of the key membrane proteins associated with cancer, was used as a model for receptor-mediated endocytosis. EGFR molecules on the outer cell membrane were detected by SECM by using alkaline phosphatase (ALP) as a labeling enzyme. Since SECM detected the ALP activity on the outer membrane, the procedure helped discriminate the EGFR on the outer membrane from the intracellular EGFR involved in endocytosis. SECM showed a marked decrease in the current responses generated due to ALP activity by 93% on addition of the epidermal growth factor, indicating clearly that EGF triggered the endocytosis, which led to the withdrawal of most EGFRs from the outer membrane.

An electrochemical platform for parallel monitoring of secreted alkaline phosphatase (SEAP) has been microfabricated on a device with a mammalian-cell array chip. A 4 x 4 ring-ring electrode array was designed at the rim of the round cellular pattern with a diameter of 270 mu m. Electrochemical characterization was carried out, and it was found that the collection efficiency was about 50% in dual mode when the inner-ring and the outer-ring electrodes were selected as the collector and generator electrodes, respectively. The current amplification ratio for the dual mode normal to single mode was 2.84. SEAP expressing from the cells was parallelly monitored by using a multiplexer switching system at the 16 round cellular spots. The reduction current for HeLa cells transfected with plasmid encoding SEAP observed at the collector outer ring electrode was found to be significantly higher than that for wild-type HeLa. Finally, the top of the microwell with the round cellular pattern was covered with a poly(dimethylsiloxane) block for 5 min to accumulate the secreted enzyme and the product of the enzyme reaction so that further signal enhancement could be observed.

Single-cell analysis has become a powerful method to construct a new type of whole cell sensor integrating with individual cellular responses all together However sensitivity of electrochemical detection system in the present stage is insufficient to discuss in detail about the results obtained from a large number of data set because the individual single-cell responses are largely fluctuated To improve sensitivity chronoamperometry (CA) and electric charge analysis were performed for a single-cell entrapped within a poly(dimethylsiloxisane) (PDMS) microwell HeLa cells transfected with vectors encoding SEAP (secreted alkaline phosphatase) have been seeded in the cylindrical PDMS microwell arrays with and without groove A microelectrode tip located the top of the PDMS microwell to isolate the volume of the measuring solution containing p-aminophosphate (PAPP) as an enzymatic substrate After a certain time period (t(accu)) to promote the SEAP reaction the tip potential was stepped from 00 to 0 3 V and the CA response for oxidation of p-aminophenol (PAP) was converted to the electric charge by time integration of the current The electrochemical response from HeLa cells transfected with SEAP (HeLa-SEAP) was significantly larger than that from wild-type HeLa for both the PDMS microwell with and without groove For the PDMS microwell without groove accumulation of PAP was evidently observed with increasing t(accu) For the PDMS microwell with a groove of 5 mu m width and 5 mu m depth the PAP oxidation current was large but the accumulation of PAP was not evident with increasing t(accu) due to the promoted mass transfer of PAP and electric connection according to the groove (C) 2010 Elsevier Ltd All rights reserved

mRNA from single cells was quantified using real-time RT-PCR after recording the address and reporter protein activity with chemiluminescence, fluorescence, and electrochemical techniques, using luciferase, green fluorescent protein, and secreted alkaline phosphatase. mRNA copy number ranging from below 103 to 107 in single cells showed a lognormal distribution for both externally introduced reporter genes and internally expressed genes. The fluctuation in the gene expression decreased with the increase of the number of cells picked but did not decrease with the increase of mRNA copy number per cell. We found that the correlation coefficients for mRNA and protein expression in logarithmic plot at single-cell level were much lower than 1.00. (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

We have developed a zymogen-based electrochemical sensor. Zymogen is an inactive enzyme precursor (proenzyme) and it is necessary to transform it biochemically (e.g., by hydrolysis and conformational change) to make it an active enzyme. In this study, we demonstrated the detection of endotoxin by using recombinant Factor C (rFC), which is a protease zymogen activated by endotoxin binding. The activated rFC hydrolyzes a synthetic substrate of Boc-Val-Pro-Arg-p-nitroanilnide to generate an electrochemical active compound, p-nitroaniline (pNA). The liberated pNA was detected by differential pulse voltammetry at -0.75 V. By using this electrochemical process, 5000 endotoxin units (EU) L(-1) and 1000 EU L(-1) were detected in a Tris-Ac buffer with a pH of 7.5 at 37 degrees C for reaction times of 1 h and 3 h, respectively. The concept of zymogen-based electrochemical sensors is expected to lead to the development of new biosensors. (C) 2010 Elsevier By. All rights reserved.

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/ SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

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. (C) 2010 Elsevier B.V. All rights reserved.

In this work, we have applied particle manipulation based on negative dielectrophoresis (n-DEP) to develop rapid and separation-free immunosensing systems. Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to demonstrate competitive immunosensing based on the rapid manipulation of microparticles. A suspension of the fluorescence microparticles modified with a specific antibody was injected into the n-DEP device consisting of the interdigitated microarray (IDA) electrode and indium-tin-oxide (ITO) substrate immobilized by protein conjugation with antigen. The application of 2 MHz AC voltage (16V peak-to-peak) to the IDA forced most of the particles to form a line pattern on the upper ITO over the gaps of IDA within 60 s. In the absence of analytes, patterned microparticles were irreversibly captured on the ITO by the construction of immuno-complexes. When the microparticles bearing anti-atrazine IgG antibody were suspended in an analyte (atrazine) solution, irreversible capturing of microparticles on the ITO was inhibited because of the occupation of the binding sites of the antibodies with free-atrazine. As a result, the analyte molecules were re-dispersed from the ITO to disintegrate the line formation after turning off the voltage. We could discriminatively detect the fluorescence intensity of the captured microparticles at the designated areas from that of the uncaptured microparticles suspended in the solution. Thus, the separation steps usually required for conventional immunoassay are eliminated in the present procedure. A pre-incubation of microparticles for 3 min in an orange juice solution containing analyte allowed for the determination of the atrazine and bromopropylate concentrations with a limit of detection of 4 and 1.5 mu g L(-1), respectively, providing sufficient detectability to achieve international regulations regarding pesticide residues in food samples. The assay was significantly accelerated by the rapid particle manipulation with n-DEP and totally accomplished within 5 min. We also demonstrated the possibility of the simultaneous determination of two pesticide residues by using the DEP devices with two channels modified with specific competitors for atrazine and bromopropylate. (c) 2010 Elsevier B.V. All rights reserved.

We developed an electrochemical-sensing device for continuous monitoring extracellular hydrogen peroxide (H(2)O(2)). The device consists of an indium-tin-oxide electrode coated with osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) and a poly-dimethyl siloxane well to house the cells on the chip. Granulocyte-like differentiated HL-60 cells were accommodated in the well and stimulated with phorbol 12-myristate 13-acetate (PMA), which triggered the generation of H(2)O(2). The extracellular H(2)O(2) released from the cells was enzymatically reduced at the Os-HRP-modified electrode chip using Os(II) as an electron donor, resulting in reduction current responses by the device. The reduction current increased immediately upon PMA stimulation and this current transient was similar to that obtained by conventional chemiluminescence assays using sodium luminol. Apocynin, an inhibitor of NADPH oxidase activation, eliminated both the electrochemical and chemiluminescence signals. On the other hand, superoxide dismutase (SOD) increased the amperometric signals and catalase (CAT) decreased, whereas SOD decreased luminescence emission and CAT did not. These results were in accordance with the expected reaction mechanism, and strongly indicate that this new electrochemical-sensing device successfully detects extracellular H(2)O(2) production. (C) 2009 Elsevier B.V. All rights reserved.

We prepared a microfluidic device with an interdigitated array electrode modified with antibodies to develop immunosensors. A comb-type array (WI) was used for forming immunocomplexes and another (W2) for detecting mediators generated by the enzyme reaction. We used electropolymerization and avidin-biotin complexes for an addressable immobilization of antibodies on WI. Since the microfluidics significantly accelerated the formation of the immunocomplexes, a period as short as 10 min was sufficient to detect the responses. The current observed at bare array (W2) increased with increasing analyte (mouse IgG) concentration in the range of 1.0-100 ng/mL. Therefore, present procedure is suitable for rapid immunosensing in a simple device.

Microcontact printing (mu CP) with various proteins has been widely applied to biosensors and cell biology research. However, the mechanism of protein patterning in the mu CP process is not clear in detail. We have electrochemically estimated enzyme concentration on glass slide patterned with mu CP technique. We also estimated the enzyme concentration at the surface of poly(dimethylsiloxane) (PDMS), which corresponded to the enzyme activity at the PDMS stamp just before mu CP. Our result suggested that it was possible to transfer enzyme monolayer from PDMS to glass surfaces with 100% efficiency with mu CP. Immunoglobulin G (IgG) patterned with mu CP was also characterized by tagging with enzyme labeled anti-IgG. Scanning electrochemical microscopy (SECM) image was obtained to visualize line and space pattern of IgG monolayer.

This is the first report on addressable electrochemiluminescence (ECL) based on redox-cycling of tris(2,2&apos;-bipyridine)-ruthenium(II) (Ru(bpy)(3)(2+)). By changing the column or row electrodes addressed, the ECL at each address point can be detected separately.

The present study describes a new electrochemical imaging method for monitoring the population and growth of adherent cells with a novel addressable microelectrode array.

Scanning ion conductance microscopy (SICM) using a nanopipette as a probe and ionic current as a feedback signal was introduced as a novel technique to study live cells in a physiological environment. To avoid contact between the pipette tip and cells during the conventional lateral scanning mode, we adopted a standing approach (STA) mode in which the probe was moved vertically to first approach and then retracted from the cell surface at each measurement point on an XY plane. The STA mode ensured non-contact imaging of the topography of live cells and for a wide range of uneven substrates (500 x 300 mu m to 5 x 5 mu m). We also used a field-programmable gate array (FPGA) board to enhance feedback distance regulation. FPGA dramatically increased the feedback speed and decreased the imaging time (450 s per image) with enhanced accuracy and quality of live cell images. To evaluate the potential of the STA mode for SICM, we carried out imaging of a convoluted surface of live cell in various scan ranges and estimated the spatial resolutions of these images.

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 30 mu m and a depth of 25 mu m. 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 3.0 Vpp and 1 MHz 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 (NF kappa B) that was used as the model for on-chip monitoring of cellular signal transduction. (C) 2009 Elsevier B.V. All rights reserved.

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 3 7 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.

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative-dielectrophoresis (n-DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non-uniform electric fields. The MLAG electrode was mounted to a target substrate in a face-to-face configuration with a spacer. When an AC voltage (4.60 V(rms) and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 mu m diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009;104: 709-718. (C) 2009 Wiley Periodicals, Inc.

Individual ZnO wires were deposited on two closely spaced electrodes in a microfluidic channel by using the positive dielectrophoretic (p-DEP) alignment technique. By using the p-DEP technique in the presence of a microfluidic force, efficient and rapid alignment of single wires was achieved; this was a result of spatial confinement, uniform wire orientation in the channel, and the competition between microfluidic and DEP forces. The technique helped achieve a capturing efficiency of up to 86% within a few tens of seconds. In addition, we fabricated an electrochemically gated field-effect transistor (EC-FET) using single ZnO wires. The conductivity of the as-aligned wire was enhanced by carrying out post-treatments such as the deposition of metal layers at both ends of the wire, annealing, and functionalization with dodecanedioic acid. By applying a voltage across the electrolyte, it was confirmed that the fabricated EC-FET had stable n-channel FET characteristics.

We report here the control of the microparticles position within fluid flow based on its size by using dielectrophoresis (DEP) with a microelectrode array consisted of rectangular features with the different size of width and gap. 3 mu m- and 10 mu m-diameter particles were introduced into the channel with 300 mu m height at 30 mu l/min. An AC electric field (20V peak-peak and 2 MHz) was then applied to microelectrode arrays to form dielectrophoretic fluid cage, resulting in a formation of flow paths with low electric fields on the arrays. The microparticles separately flow in line streams along the paths formed between the rectangular features of the arrays, the 3 mu m-diameter particles mainly flow through the narrow path and 10 mu m-diameter particles through the wide path. These results indicated that positions of two types of microparticles in the fluidic channel were easily separated and controlled using the n-DEP. (C) 2009 Elsevier B.V. All rights reserved.

Endocrine disruptors that act like hormones in the endocrine system might have toxic effects. Therefore, it is important to develop a portable device that can detect hormone active chemicals in samples rapidly and easily. in this study, a microfluidic device was developed for the detection of hormone active chemicals using genetically engineered yeast cells. The yeast cells were used as biosensors since they were genetically engineered to respond to the presence of hormone active chemicals by synthesizing beta-galactosidase (beta-gal). For achieving further sensitivity, we incorporated interdigitated array (IDA) electrodes (width, 1.2 mu m; gap, 0.8 mu m) with 40 electrode fingers into the analytical chamber of the microfluidic device. The yeast cells precultured with a hormone active chemical, 17 beta-estradiol (E2), were trapped from the main channel of the device to the analytical camber by electrophoresis. After trapping in the analytical chamber, we performed electrochemical detection of beta-gal induced in the yeast cells with the IDA electrodes. Actually, electrochemical detection was performed on p-aminophenol that was converted from p-aminophenyl-beta-D-galactopyranoside with beta-gal. The electrochemical signals from the yeast cells precultured with 17 beta-estradiol were successfully detected with the device. Furthermore, the inhibitory effects of antagonists such as tamoxifen were also detected electrochemically by using the device. Thus, the present microfluidic device can be used for highly sensitive detection of hormone active chemicals.

We microfabricated a novel device consisting of a 4 x 4 array microchamber sandwiched with the two microband electrode array. This device allows dielectrophoretic (DEP) manipulation of microbeads to introduce into and release out a certain address of the V-shaped microchamber, by applying AC voltage (10 V(pp), 10 kHz) on the basis of DEP forces. The design and the position of the two electrodes (row and column electrodes) at each microchamber were optimized by simulation based on a finite element method. More importantly, electrochemical generation-collection measurement was possible to evaluate enzymatic activity. After microbeads immobilized with glucose oxidase (GOD) was entrapped in the V-shaped microchamber with DEP, a measuring solution containing 3 mM ferrocenemethanol (FcCH(2)OH) and 0.1 M glucose was introduced. The medium in the V-shaped microwell was immediately exchanged into the measuring solution whereas microbeads stayed within the microwell without applying DEP voltage, because the flow within the microchamber was isolated from that of the main channel. Then the potential of the row and column electrodes were set at 0.5 and 0.1 V vs Ag/AgCl. The GOD activity can be monitored as the decrease in the [FcCh(2)OH](+) reduction current. (C) 2009 Elsevier B.V. All rights reserved.

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.

Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), Or kappa B (binding site for NF kappa B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 mu m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL(-1) dexamethasone, 10 ng mL(-1) forskolin,or 100 ng mL(-1) TNF-alpha (tumor necrosis factor alpha) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring Solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAR, pCRE-SEAP, and pNF kappa B-SEAP patterned oil the same solid Support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 101 cells per well. (c) 2009 Elsevier B.V. All rights reserved.

Arrays of gold nanoparticles and gold nanorods have been fabricated in the spatially localized pores of a porous alumina membrane using positive dielectrophoresis (p-DEP). The porous alumina membrane with a pore size of 200 nm and a thickness of 60 mu m was sandwiched between the lower microarray electrode and the upper bare ITO electrode. Then, a 100-nm-diameter gold nanoparticle suspension (0.6-1.0 x 10(12) particles ml(-1)) in pure water was introduced in the space between the alumina membrane and the upper ITO substrate. The application of an AC voltage (typically 30V(pp), 50-100 kHz) for p-DEP to the lower and upper electrodes forced the nanoparticle to move into the straight pores of the membrane above the ITO microarray electrode, forming arrays of nanoparticles inside the membrane. When the distances between the adjacent gold nanoparticles were close, the particles fused together to form rod-like structures with a well-defined diameter. The present procedure for fabrication of ordered nanocomposites in nanopores can be applied to electrical and optical devices and sensors. (c) 2008 Elsevier B.V. All rights reserved.

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 mu m diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgC) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 mu g L(-1) and in pre-treated wine samples of 6.8 mu g L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 mu g L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples. (C) 2008 Elsevier B.V. All rights reserved.

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer Solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles. (C) 2008 Elsevier Inc. All rights reserved.

Endocrine disrupting chemicals that act like hormones is a potential hazard for human health. Therefore, it is necessary to develop a device that can detect easily hormone active chemicals. In this study, a microfludic device with some analytical chambers was fabricated for manipulating the yeast cells that were genetically engineered to respond to the presence of hormone active chemicals by synthesizing β-galactosidase (β-gal). Interdigitated array (IDA) electrodes with 40 electrode fingers were incorporated into the device for sensitive detection of the β-gal activity electrochemically, and hormone active chemicals were successfully detected by using the systems. © 2009 CBMS.

In the work, microfluidic device consisting of an interdigitated microarray (IDA) electrode was developed for a rapid, and separation-free immuno-sensors based on a manipulation technique of microparticles by dielectrophoresis (DEP). A poly-dimethylsiloxane (PDMS) substrate with microfluidic channel was placed on the IDA plate to allow to fabricating the device. On applying AC voltage to the IDA in a negative DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microparticles moved to the surface of PDMS substrate placed above the IDA by n-DEP force to accumulate at the designated areas of the PDMS surface, where anti-mouse IgG was precoated. When the fluorescence microparticles bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microparticles trapped the analyte to form microparticle-conjugates. The conjugates were accumulated and captured at the designated areas of the PDMS surface via antibody-antigen-antibody (sandwich) reaction. The captured microparticles were detected selectively by fluorescence measurements at the focused, designated areas regardless of the presence of uncaptured microparticles in the suspended solution. Thus, the separation and washing-out steps, usually required for conventional immunoassay, are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, as short as 30 sec was enough to detect the immunoreaction at the surface. The fluorescence intensity of the captured microparticles at the designated area increased with the analyte in the range, 0.01 ∼ 10 ng/mL. The present procedure realizes a rapid, sensitive and separation-free immunoassay in a simple device. ©2009; IEEE.

In this work, we manipulated recombinant yeast cells onto a sensing electrode by using hydraulic and electrophoretic force and thus the current response of trace enzyme activity was increased in p-aminophenyl- β -D - galactopyranoside (PAPG) solution. The recombinant yeast which can generate β-D -galactosidase (β- Gal) in response to steroidal hormone 17 -estradiol were employed as the sensing element of the developed cell-based biosensor. Cells was trapped and stabilized within a microwell structure when they flowed through the sensing electrode. A vertical trapping force was generated by applying +2.0 V across the sensing electrode and an Indium Tin Oxide (ITO). The geometrical electrode area ( 100 × 30 μm2) was defined by a photoresister layer. Since the treated cells were collected on the electrode surface, PAP concentration can be significantly changed around the sensing electrode. Consequently, the detection limit was extend to 0.5 ppb, and the response time was shorten as fast as 15 min by using this device. © 2009 IEEE.

Single-cell mRNA analysis has been performed by electrical cell lysis technique with Pt-ring electrode-capillary probe and RT real-time PCR. Four different hose-keeping genes were detected from single cell and it was found that our multi-functional probe technique allowed quantitative single-cell mRNA analysis at 10 copies/cell. Single-cell mRNA analysis in reporter system has been also performed to validate our mRNA analysis technique by using GFP reporter system as a model. Messenger RNA and protein expressed from identical individual cells were analyzed with real-time PCR and fluorescence microscope, respectively. Basically, the fluorescence intensity was linear to the GFP mRNA copy number of the individual transfected HeLa cell.

Introduction. The current methods for evaluating islet potency are not useful in clinical transplantation. Therefore, we need reliable, rapid methods enabling accurate prediction of islet quality. Materials and Methods. We evaluated respiratory activity using scanning electrochemical microscopy (SECM), glucose-stimulated respiratory activity, glucose-stimulated insulin release, ADP/ATP assays, insulin/DNA levels, and Trypan blue exclusion tests as predictive methods for the ability of isolated rat islets to cure syngeneic diabetic rats. Results. Although glucose-stimulated respiratory activity, basal respiratory activity, ADP/ATP ratio, and glucose-stimulated insulin release were significantly correlated with the outcome of transplantation into diabetic rats, there was no correlation between outcomes, insulin/DNA ratios, and Trypan blue exclusion tests. The glucose-stimulated respiratory activity in islet preparations that could cure diabetic rats was significantly greater than those unable to cure diabetes. Rat islets with &gt;1.5-fold glucose-stimulated respiratory activity consistently cured diabetic rats, whereas those with a value &lt;1.5 hardly cured any rats. Conclusion. Measurement of the glucose-stimulated respiratory activity using SECM technique is a novel method that may be useful as a rapid, potent predictor of the outcome of clinical islet transplantation.

A highly sensitive and quantitative analysis was performed using a poly(dimethylsiloxane) (PDMS) microwell array in a scanning electrochemical microscopy setup. A microelectrode with a relatively large seal radius was used to cover the top of the cylindrical PDMS microwell (96 pL). The voltammogram for 4 mM ferrocyanide resulted in a charge value of 38 nC, suggesting that almost 100% of the reductant in the microwell was converted to the oxidation current. When genetically modified yeast cells were entrapped in the microwell, the accumulation of p-aminophenol (PAP) produced by expressing beta-galactosidase (beta GAL) was successfully observed.

A chip with integrated electrophoretic and electrochemical systems was developed to manipulate either an individual microbead or a cell inside a microwell electrode (MWE) for electrochemical measurement. The optimal MWE geometry (30 mm diameter and 25 mm depth) was designed to accommodate the micro particles according to the simulated results. A chip device was sequentially built from a slide patterned with Pt electrodes, an adhesive tape defined with a flow channel (200 mm in width and 25 mm in height), and an indium tin oxide (ITO) cover. The MWE not only generated an active electrophoretic force to trap the particle but also provided a low flow velocity area (LFVA) to stabilize the trapped bead or cell in a continuous flow. Scanning electrochemical microscopy (SECM) theory was employed to explain the electrochemical behaviors of the MWE. An enhanced current was confirmed as the redox recycling effect on the conductive ITO cover. The catalytic reaction of an individual alkaline phosphatase coated microbead (ALP-bead) was electrochemically detected with the MWE after being trapped. The ALP on the trapped ALP-bead catalyzed the hydrolysis of p-aminophenylphosphate (PAPP) to p-aminophenol ( PAP), and then a decaying amperogram (+0.3 V vs. Ag/AgCl) due to a tiny PAP quantity around the MWE was observed.

Well aligned ZnO nanowall arrays with submicron pitch were grown on a periodically polarity-inverted ZnO template using a carbothermal reduction process. Under the conditions of a highly dense Au catalyst for increasing nucleation sites, ZnO nanowalls with a thickness of 126 10nm, an average height of 3.4νm, and a length of about 10mm were formed on the template. The nanowalls were only grown on a Zn-polar surface due to a different growth mode with an O-polar surface. The results of x-ray diffraction and photoluminescence (PL) measurements revealed a single crystalline, vertical alignment on the template, and a large surface to volume ratio of the ZnO nanowalls. © 2009 IOP Publishing Ltd.

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 12 V(pp), 1 MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1 min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5 min 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 15 min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types. (C) 2008 Elsevier B.V. All rights reserved.

Column and row electrodes on two different glass substrates were orthogonally arranged in order to assemble an addressable microelectrode device for the purpose of comprehensive electrochemical detection. Amperometric signal at the individual crossing point of the column and row electrodes was detected separately on the basis of redox cycling of localized electroactive species occurring between the electrodes. The addressable microelectrode device was simple and could be easily assembled; however, it comprised as many as 10 x 10 addressable detection points on a single chip. The basic electrochemical performance of the device was investigated by using the ferricyanide/ferrocyanide redox couple. Electrochemical responses at 100 individual points could be collected within 22 s. The present device was successfully used for imaging the spots of alkaline phosphatase on the array substrate. The results indicate that the device can be applied to comprehensive and higb-throughput detection and imaging of biochemical species.

We developed a cell-based assay device for the detection of endotoxin, the potentially toxic compound that induces septic shock. Genetically-engineered cells that secrete alkaline phosphatase (SEAP) on exposure to endotoxin were cultured in an electrochemical cell device in medium containing p-aminophenyl phosphate and various concentrations of endotoxin. After 24 hr incubation, p-aminophenol (pAP), generated by SEAP-catalyzed hydrolysis, was detected by amperometry at +0.35 V. The amperometric response increased with the concentration of endotoxin in the range of 0.01-1 ng/ml.

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 mu m in width, 10 pm in height) and an analytical chamber (100 x 20 x 10 mu m(3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain (Saccharomyces cereisiae Y190) carrying the P-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17 beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta-D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.

We report here a rapid formation of island arrays with nanoparticles on and within polycarbonate (PC) membrane based on positive dielectrophoresis (p-DEP). For the fabrication of the patterning device, PC membranes with 10 mu m thickness and 100, 200 or 400 nm pore size were sandwiched by an upper bare ITO substrate and a lower disk array ITO electrode which was defined by insulation layer of negative photoresist. A suspension of 190 nm diameter polymethylmethacrylate (PMMA) particles containing rhodamine 6G (R6G) fluorescent molecules was introduced into the device between the upper ITO and the PC membrane. AC electric signal (typically 20 Vpp, 70 kHz) was then applied to the ITO, resulting in the formation of island patterns with high electric fields gradient regions on and in the PC membrane. Particles patterns with island shape were assembled on membrane within I s after applying AC electric field. The electrodes can be used repeatedly as the template of subsequent patterning. Although, particles islands were only formed on the PC membrane with 100 and 200 ran diameter pores, the particles penetrated the membrane with 400 nut pores to form patterns on the back surface. Since the strong electric fields were formed at the edges of disks, particles on the back surface were the projection of the disk array of ITO to form ring shapes. The unique structure with particles was explained based on the simulation of electric field distribution. The present proposal offers a procedure to fabricate particle arrays with extremely simple, rapid and highly reproducible manner. (C) 2007 Elsevier B.V. All rights reserved.

A shear force-based ion-selective probe for scanning electrochemical microscopy was fabricated for the imaging of localized potassium ions. A solution of dibenzo-18-crown-6/1,2-dichloroethane was added to a pulled capillary which was contact with a tuning fork attached to a piezoelectric buzzer. 10 mu m of a Nation island on glass was imaged, and the topography and current image originating from localized potassium ion concentration were obtained.

A shear force-based scanning capillary microscope was fabricated by contacting a glass capillary with a quartz crystal tuning fork and was used for the patterning of gold surfaces with hexadecanethiol. A micrometer-sized pattern was successfully obtained by moving the capillary, through which the hexadecanethiol solution was introduced, along the gold surface. Dotted and line patterns were observed by high-resolution scanning electrochemical microscopy using a standing approach mode in (ferrocenylmethyl)trimethylammonium (FA(+)) solution without destroying the morphology of the surface.

Negative dielectrophoresis (n-DEP) have been used to manipulate microparticles with immunoreagents (antigens or antibodies) in a microfluidic channel, and applied to develop a rapid immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 μm diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area by using n-DEP. A sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing FITC-labeled anti-mouse IgG antibody, into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 μg/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. This immunosensing system using the n-DEP technique is faster and simpler than conventional enzyme-linked immunosorbent assay (ELISA) using microtiter plates, and has the significant advantage that sensing requires simple and easy handling since unreacted immunomolecules are flushed from the signal detection area by the fluidic stream. The device can be reused by removing the microparticles. The automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP. © 2008 IEEE.

Respiration is useful parameter for evaluating embryo quality as it provides important information about metabolic activity. In the present study, we employed scanning electrochemical microscopy (SECM) to accurately determine die oxygen consumption of single, identical human embryos at different developmental stages. The oxygen consumption rates of single embryos were low at 2-8-cell stages (0.51 +/- 0.05 X 10(14)/mol . s(-1), n = 18) but increased by the morula (0.61 +/- 0.11 X 10(14)/mol . s(-1), n = 5) and early blastocyst (0.72 +/- 0.06 X 10(14)/mol . s(-1), n = 14) stages. Later blastocysts exhibited even higher oxygen consumption rates (1.00 +/- 0.19 X 10(14)/mol . s(-1), n = 4). Ultrastructural studies revealed that most mitochondria in embryos Lip to the 8-cell stage were immature and had a spherical or ovoid shape. However, by the morula stage, die mitochondria had elongated cristae, with the elongated morphology even more pronounced in mitochondria present in blastocysts. The maturation of mitochondria con-elated with the increase of oxygen consumption rate during the development of embryos. The SECM technique may be a valuable tool for accurately assessing the mitochondrial function and quality of human embryos.

Scanning electrochemical microscopy (SECM) is a technique in which the tip of a microelectrode is used to scan and monitor die local distribution of electro-active species near die sample surface. In this study, we have studies on the SECM technique, to establish the accurate method for measurement of respiratory activity of single pig oocytes. The oxygen consumption rates of pig oocytes cultured in modified TCM199 medium were evaluated by the SECM system. After the measuring, distribution of active mitochondria and ATP content was investigated in die identical oocytes. The oocytes were classified in three types (Type-I, Type-II or Type-III) according to the pattern of active mitochondria distribution. There was no difference in die oxygen consumption rate (F X 10(14) /mol s(-1)) between Type-II and Type-III (0.59 and 0.60, respectively). However, the ATP content (pmol/oocyte) was significantly higher in Type-III (2.38) compared with that of Type-II (1.53). Meanwhile, the oxygen consumption rate and ATP content of Type-I were very low (0.02 and 0.06, respectively). These results suggest that the oxygen consumption rate and ATP content of oocytes was significantly affected by category of mitochondrial distribution. In the present study, we succeeded in the multiple analysis of respiratory activity in die identical oocytes. This novel system may be a valuable tool for accurately assessing the mitochondrial functions.

Micropatterns of diaphorase (Dp) were fabricated on glass substrates by the microcontact printing (mu CP) method and characterized with atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM). AFM images of the printed samples revealed that the mean height of the Dp patterns was 3-5 nm, indicating the formation of a monolayer pattern. The Dp molecules on the surface organized themselves into two-dimensional arrays. We used two kinds of inking solutions: Dp-phosphate buffer solution (PBS) (pH 7.0) and Dp-PBS (pH 7.0) with glutaraldehyde (GA, 1% v/v) as a cross-linking reagent. Although the AFM imaging showed high-quality Dp monolayer patterns in both cases, SECM measurements indicated that the enzymatic activity of Dp was almost lost when Dp-PBS with GA was used as the inking solution, whereas clear enzymatic activity was found when Dp-PBS was used. (C) 2007 Elsevier B.V. All rights reserved.

Inclusion behavior of negatively charged host molecules, tiliacalix[4]arene-p-tetrasulfonate (TC4AS) and [6]arene-p-hexasulfonate (TC6AS), toward (ferrocenylmethyl)trimethylammonium (FcCH(2)NMe(3)(+)), hydroxymethylferroceiie (FcCH(2)OH), ferrocenecarboxylic acid (FcCOOH), and 1,1'-ferrocenedicarboxylic acid (Fc(COOH)(2)) was studied in aqueous solutions (pH 7.0) with cyclic voltammetry. Upon increasing the concentration of TC4AS to 4-fold of each guest, the anodic peak current density (j(p,a)) decreased, suggesting inclusion of the ferrocenyl guests in TC4AS. Also oxidation half-wave potential (E-1/2) of FcCH(2)N Me-3(+), FcCH(2)OH, and FcCOOH was shifted to cathodic direction, showing preferential inclusion of the oxidative state. Inclusion of neutral guests such as FcCH(2)OH and Fc(+)COO(-) implies that hydrophobic interaction between TC4AS and the guests is the chief driving force for formation of host-guest assembly. The decrease of E-1/2 for each guest was in the order: FcCH(2)NMe(3)(+) &gt; FcCH(2)OH &gt; FcCOOH, suggesting that electrostatic interaction con trols the preference toward oxidative form of the guest. Dicarboxylic Fc(COOH)(2) showed decrease of j(p.a) but increase of E-1/2 upon inclusion, suggesting TC4AS preferred reduced form Fc(COOH)(2) to oxidized form Fc(+)(COO-)(2). TC6AS behaved similarly to TC4AS but with larger decrease in of E-1/2 and j(p,a). The larger shift of E-1/2 for inclusion of FcCOOH, the oxidative form of which is also neutral (Fc(+)COO(-)), than that attained with TC4AS endorses main role of hydrophobic interaction between TCnAS (n = 4, 6) and ferrocenyl guest molecules. Having the most preferential electrostatic interaction, kinetically stable complex was formed between TC6AS and FcCH(2)NMe(3)(+). (C) 2007 Elsevier B.V. All rights reserved.

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 3.2 mm x 3.2 mm 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 H2O2 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 1-625 ng/ml. (c) 2007 Elsevier B.V. All rights reserved.

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 pin above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10,us between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 mu L of the lysed solution was collected using a microinjector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the singlecell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-die aggregated cellular masses formed during the three-dimensional culture-as a model system to isolate small cellular clusters from tissues and organs.

Micro-batteries were fabricated by using BAB block copolymer as dry polymer electrolyte, which consisted of polyethylene oxide and polystyrene and had relatively high ionic conductivity at room temperature. The micro-batteries were fabricated by a sol-gel method combined with micro-injection system. Two types of micro-battery were fabricated. One consists of a single cell and another of 3-cells connected in series. LiMn2O4 and Li4/3Ti5/3O4 were used as active materials in positive and negative electrode, respectively. The microarray batteries were operated at room temperature without any plasticizer in the polymer electrolyte. The operation voltages were 2.45 V and 7.40 V for a single cell and 3-cell array, respectively. The discharge capacities estimated from cyclic voltammetry measurements were 245 nA It for a single cell and 12.1 nA h for a 3-cell array, which corresponded to the energy densities of 8.48 mu W h cm(-2) and 4.54 mu W h cm(-2), respectively. (c) 2007 Elsevier B.V. All rights reserved.

The detection of oxygen consumption as an indicator of the bovine embryo activity has attracted Much attention. A microfluidic chip with the built-in amperometric detector array was successfully constructed to transport, immobilize, and in situ measure a single bovine embryo for the evaluation of oxygen consumption characteristics. The rnicrofluidic chip consisted of a poly(dimethylsiloxane) slab containing a size-limited transportation microchannel and a glass substrate with a 4-working-electrode amperometric detector array located on the bottom of a groove. A single embryo could be transported along the groove and be safely immobilized at the gate position of the microchannel due to the size limitation by using the flow rate of 10 mu 1/min. Subsequently, the oxygen consumption characteristics of the embryo were in situ measured with the amperometric detector array. The average value of oxygen concentration difference (Delta C) between the bulk solution and the Day-6 embryo surface based on the analysis of spherical diffusion theory was 2.80 0.72 mu M at room temperature. Moreover, the correlation coefficient of more than 0.92 indicates that the spherical diffusion theory could be suitable for depicting the oxygen consumption layer of an embryo at the morula stage in the microchannel. The success of the presently used microfluidic chip not only greatly simplifies the expensive instrument requirements such as scanning electrochemical microscope and stereo-positioning manipulator but also makes the transportation, immobilization, and detection of a single embryo feasible within a microchip. (c) 2007 Elsevier B.V. All rights reserved.

Three cell lines, that is, the human breast cancer cell line (MCF-7) and the human mammary epithelial cell line (S-1) and its malignant form (T4-2) were embedded in a reconstituted basement membrane (Matrigel) that had 20-nL pyramid-shaped silicon microstructures. The proliferative behavior of the MCF-7 cells was dependent on the surrounding conditions (2-D, collagen gel, or Matrigel), whereas the respiratory activity of a single cell (Fc) was almost identical under different culture conditions. The Fc value changed with cellular polarity. The Fc value for the S-1 cells was observed to decrease slightly, whereas that of the T4-2 cells increased 2 days after cultivation in the microstructures within the Matrigel. However, when the T4-2 cells were cultured in the presence of tyrphostin AG 1478 (T4-2 tyr) to inhibit epidermal growth factor (EGF) signaling, the Fc value decreased slightly and remained almost constant for an additional 1 week this was similar to the behavior of the S-1 cells. Further, fluorescence images showed that the T4-2 tyr cells formed polar structures that were similar to those formed by the S-1 cells whereas the T4-2 cells did not form such structures. These results indicate that cellular polarity can be assessed by measuring cellular respiratory activity. © 2006 Wiley Periodicals, Inc.

A quantitative electrochemical detection of testosterone was performed using a nitrocellulose membrane as a support for antibodies. Testosterone competitively reacted with horseradish peroxidase (HRP)-labeled testosterone, and was captured with an anti-testosterone antibody immobilized on the membrane. The activity of the labeled HRP was electrochemically estimated with a gold-disk electrode (diameter, 300 gm) by detecting the oxidized form of ferrocenemethanol (FcOH), which was produced by the HRP-catalyzed oxidation of FcOH with H2O2. The electrochemical detection chamber was fabricated in order to maintain a constant distance between the electrode and the membrane surface. We optimized some factors, especially nitrocellulose membranes and the concentration of bovine serum albumin (BSA) and HRP-labeled testosterone for the electrochemical analysis of testosterone with a high reliability. Testosterone was detected by a competitive amperometric measurement under the optimized conditions. As a result, the current response decreased with increasing testosterone concentration, and a linear current response was observed in the range of 0.5 similar to 20 ng/mL on a semi-log graph. This method provides high sensitivity by one order of magnitude, compared with the conventional well plate assay.

Scanning electrochemical microscopy (SECM) was applied to a dual enzyme immunoassay for the detection of pepsinogen 1 (PG I) and pepsinogen 2 (PG2). Sandwich-type immunocomplexes labeled with horseradish peroxidase (HRP) were constructed on microspots consisting of anti-PG1 IgG antibody and anti-PG2 IgG antibody. These microspots were fabricated on a hydrophobic glass substrate using a capillary microspotting technique. In the presence of H2O2 and ferrocenemethanol (FcOH; used as an electron mediator), the labeled HRP catalyzed the oxidation of FcOH by H2O2 to generate the oxidized form of FcOH (Fc(+)OH) at localized areas corresponding to microspots containing both immunocomplexes. The enzymatically generated Fc(+)OH was reduced and detected with a SECM probe (0.05 V versus Ag/AgCl), and the substrate surface was mapped to generate SECM images of the PG I and PG2 spots. Relationships between the reduction current in the SECM images and the concentrations of PG I and PG2 were obtained in the range 1.6-60.3 ng/ml protein. Dual imaging of PG1 and PG2 was achieved using microspots containing PG1 and PG2 immunocomplexes separated by a 200 mu m physical barrier on the substrate. Pyramidal hole arrays with 100 mu m x 100 mu m openings on the silicon wafer were utilized to fabricate spots using antibodies on poly(dimethylsiloxane) (PDMS) membranes. Current responses obtained from microspots fabricated with pyramidal holes are significantly sharper compared to the responses obtained from spots fabricated using the capillary method. (c) 2007 Elsevier B.V. All rights reserved.

Three cell lines, that is, the human breast cancer cell line (MCF-7) and the human mammary epithelial cell line (S-1) and its malignant form (T4-2) were embedded in a reconstituted basement membrane (Matrigal) that had 20-nL pyramid-shaped silicon microstructures. The proliferative behaviour of the MCF-7 cells was dependent on the surrounding conditions (2-D, collagen gel, or Matrigel), whereas the respiratory activity of a single cell (F-c) was almost identical under different culture conditions. The F-c value changed with cellular polarity. The F-c value for the S-1 cells was observed to decrease slightly, whereas that of the T4-2 cells increased 2 days after cultivation in the microstructures within the Matrigel. However, when the T4-2 cells were cultured in the presence of tyrphostin AG 1478 (T4-2 tyr) to inhibit epidermal growth factor (EGF) signaling, the F-c value decreased slightly and remained almost constant for an additional 1 week; this was similar to the behaviour of the S-1 cells. Further, fluorescence images showed that the T4-2 tyr cells formed polar structures that were similar to those formed by the S-1 cells whereas the T4-2 cells did not form such structures. These results indicate that cellular polarity can be assessed by measuring cellular respiratory activity.

Microparticles have been manipulated in a microfluidic channel by means of negative dielectrophoresis (n-DEP), and the approach applied to a heterogeneous immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 mu m diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area when surrounded by intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 6-15 V-peak and frequencies over 500 kHz were applied to the two facing microelectrodes. A heterogeneous sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing a secondary antibody with a signal source (FITC-labeled anti-mouse IgG antibody), into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 mu g/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. Thus, the automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP. (c) 2006 Elsevier B.V. All rights reserved.

Microarray electrodes of LiMn2O4 and Li4/3Ti5/3O4 were prepared on a glass substrate using a sol-gel method. The prepared LiMn2O4 and Li4/3Ti5/3O4 microarray electrodes were characterized with scanning electron microscopy, Raman spectroscopy, and cyclic voltammetry. Using a polymer-gel electrolyte, lithium ion microbattery of Li4/3Ti5/3O4/polymer-gel/LiMn2O4 (cell area: 6.6 x 10(-2) cm(2)) was successfully constructed. The microbattery operated reversibly at 2.5 V, and the discharge capacity was 300 nA h, which corresponded to an energy density of 11 mu W h cm(-2). (c) 2006 Elsevier B.V. All rights reserved.

Microparticle patterns have been fabricated on a nonconductive glass substrate and a conductive indium tin oxide (ITO) substrate using negative dielectrophoresis (n-DEP). The patterned microparticles on the substrate were immobilized by covalent bonding or embedded into polymer sheets or strings. The patterning device consisted of an ITO interdigitated microband array (IDA) electrode as the template, a glass or ITO substrate, and a polyester film (10-mu m thickness) as the spacer. A suspension of 2-mu m-diameter polystyrene particles was introduced into the device between the upper IDA and the bottom glass or ITO support. An ac electrical signal (typically 20 Vpp, 3 MHz) was then applied to the IDA, resulting in the formation of line patterns with low electric field gradient regions on the bottom support. When the glass substrate was used as the bottom support, the particles aligned under the microband electrodes of the IDA within 5 s because the aligned areas on the support were regions with the weakest electric field; however, for the ITO support, the particles were directed to the regions under the electrode gap and aligned on the support because these regions had the weakest electric field. The width of the particle lines could be roughly controlled by regulating the initial concentration of the suspended particles. The particles forming the line and grid patterns with single-particle widths were immobilized by using a cross-linking reaction between the amino groups on the aligned particles and N-hydroxysuccinimide-activated ester on the glass substrate activated by succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB). The patterned particles were also embedded in a photoreactive hydrogel polymer. A prepolymer solution of poly(ethylene glycol) diacrylate (PEG-DA) was used as the suspension medium to maintain the particle patterns in the polymerized hydrogel sheet and string following photopolymerization. The hydrogel sheets with particle patterns were fabricated by ultraviolet (UV) irradiation through the ITO-IDA template for 120 s. Hydrogel strings with the aligned particles were fabricated by using a conductive ITO support and a Pt-IDA template. Pt-IDA was used as a template as well as a photomask to block UV transmission. The present procedure affords extremely simple, rapid, and highly reproducible fabrication of particle arrays. The reusability of the template IDA electrode is also a substantial advantage over previous methods.

Scanning electrochemical Microscopy (SECM) has been used to noninvasively characterize oxygen consumption rate of single mammalian embryos and oocytes under physiological condition in culure medium at 37 °C. Local oxygen concentration profile near the embryo sample was monitored by scanning with a Pt microelectrode probe, and then mass transfer rate for oxygen has been estimated based on spherical diffusion theory. A bovine embryo at two-cell stage was located in either a conventional culture dish or a cone-shaped microwell and compared the differences in concentration profile and diffusion behavior. We found that the cone-shaped microwell functions to amplify the oxygen concentration difference between the sample surface and the bulk. Further more, a measuring plate equipped with the cone-shaped six-microwells was developed to easily handle many embryos in a short time. The respiration activities significantly increased with the embryo development for both bovine and mouse. © 2007 IEEE.

Respiration is useful parameter for evaluating embryo quality as it provides important information about metabolic activity. In the present study, we employed scanning electrochemical microscopy (SECM) to accurately determine the oxygen consumption of single, identical human embryos at different developmental stages. The oxygen consumption rates of single embryos were low at 2-8-cell stages (0. 51 +/- 0.05 x 10(14)/mol. s(-1), n = 18) but increased by the morula (0. 61 +/- 0. 11 x 10(14)/mol. s(-1), n = 5) and early blastocyst (0. 72 +/- 0.06 x 10(14)/mol. s(-1) n = 14) stages. Later blastocysts exhibited even higher oxygen consumption rates. (1.00 +/- 0.19 x 10(14)/mol. s(-1), n = 4). Ultrastructural studies revealed that most mitochondria in embryos up to the 8-cell stage were immature and had a spherical or ovoid shape. However, by the morula stage, the mitochondria had elongated, with the elongated morphology even more pronounced in mitochondria present in blastocysts. The maturation of mitochondria correlated with the increase of oxygen consumption rate during the development of embryos. The SECM technique may be a valuable tool for accurately assessing the mitochondrial function and quality of human embryos.

Respiration is useful parameter for evaluating embryo quality as it provides important information about metabolic activity. In the present study, we employed scanning electrochemical microscopy (SECM) to accurately determine the oxygen consumption of single, identical human embryos at different developmental stages. The oxygen consumption rates of single embryos were low at 2-8-cell stages but increased by the morula and early blastocyst stages. Later blastocysts exhibited even higher oxygen consumption rates. Ultrastructural studies revealed that the maturation of mitochondria correlated with the increase of oxygen consumption rate during the development of embryos. The SECM technique may be a valuable tool for accurately assessing the mitochondrial function and quality of human embryos.

We have applied the microfluidic probe (MFP) to collect mRNA from single adherent cells. The probe prepared from a theta (theta-shaped capillary can be positioned at any target single adherent cell. The cell lysis buffer was introduced from the injection aperture, and the cell-lysed solution was recovered from the aspiration aperture for further mRNA analysis based on reverse transcription (RT) real-time PCR. This method achieves not only sensitivity at the single cell level but identifies the differences in cell types based on the relative gene expression profiles. The collection efficiency for mRNA was quantitatively evaluated using real-time PCR.

In this paper, a versatile, rapid and reproducible 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 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 12 Vpp, 1 MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1 min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5 min allows the cells to adsorb onto the cell culture slide. After disassembling the device and removing excess cells, the culture slide was assembled again with the IDA electrode, and was rotated 90 degrees relative to the previous setup. The second cell type was patterned in lines using the same method as with the first set of cells, forming a grid pattern on the slide.

We describe a novel multicellular spheroid culture system that facilitates the easy preparation and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of similar to 2 weeks) is available on the chip. Measurement of albumin production in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 weeks. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochemical microscopy. Responses to four different chemical stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells. (c) 2006 Elsevier Ltd. All rights reserved.

Scanning electrochemical microscopy (SECM) has been applied to enzyme immunoassay for the detection of C-reactive protein (CRP). The immunocomplexes of the sandwich type with horseradish peroxidase (HRP) labeling were constructed at the microspots of an anti-CRP IgG antibody fabricated on a hydrophobic glass substrate by capillary microspoting. In the presence of ferrocenemethanol (FcOH) as an electron mediator and hydrogen peroxide as a substrate of HRP, the oxidized form of FcOH (Fc(+)OH) was generated at localized areas corresponding to the microspot of immunocomplexes by an enzymatic reaction of captured antibodies with HRP label. The reduction current of Fc+OH was detected with a microelectrode at 0.05 V vs. Ag/AgCl and mapped by scanning the microelectrode to view SECM images of the spots for CRP. An amperometric determination of CRP was also performed using the microelectrode positioned at 10 mu m above the microspots. Relationships between the reduction current in SECM images and the concentration of CRP, were obtained in the range of 0.1 ng/ml to 100 ng/ml. A system for multi-samples measurements has been developed using amperometric determination and antibody array chips.

We developed a high-resolution scanning electrochemical microscope (SECM) for the characterization of various biological materials. Electrode probes were fabricated by Ti/Pt sputtering followed by parylene C-vapor deposition polymerization on the pulled optical fiber or glass capillary. The effective electrode radius estimated from the cyclic voltammogram of ferrocyanide was found to be 35 nm. The optical aperture size was less than 170 nm, which was confirmed from the cross section of the near-field scanning optical microscope (NSOM) image of the quantum dot (QD) particles with diameters in the range of 10-15 nm. The feedback mechanism controlling the probe-sample distance was improved by vertically moving the probe by 0.1-3 mu m to reduce the damage to the samples. This feedback mode, defined as "standing approach (STA) mode" (Yamada, H.; Fukumoto, H.; Yokoyama, T.;Koike, T. Anal. Chem. 2005, 77, 1785-1790), has allowed the simultaneous electrochemical and topographic imaging of the axons and cell body of a single PC12 cell under physiological conditions for the first time. STA-mode feedback imaging functions better than tip-sample regulation by the conventionally available AFM. For example, polystyrene beads ( diameter similar to 6 mu m) was imaged using the STA-mode SECM, whereas imaging was not possible using a conventional AFM instrument.

The quality of in vitro-produced bovine embryos was investigated by a scanning electrochemical microscopy (SECM)-based instrument for measuring the respiratory activity of individual bovine embryos. The measurement was performed by using a measuring cell equipped with a six cone-shaped microwell, a temperature-controlling unit, an auto-approach program to noninvasively locate the probe-microelectrode very close to the sample bovine embryo, a program for semi-automatic probe-scanning, and analyzing software to estimate the respiration activity of a single bovine embryo based on a spherical diffusion theory.

Vertically-aligned zinc oxide (ZnO) nano-needles have been selectively grown on the Si (100) substrates using chemical vapor transport and condensation method without metal catalyst. The selective nucleation of nano-needles was achieved by the controlled treatment of substrate surface using zinc acetate aqueous solution. The nano-needles were selectively grown on the zinc acetate treated area, while the nano-tetrapod structures were formed on the non-treated area. The nano-needles have uniform tip-diameter and length, about 10 nm and 2-3 mu m, respectively. The angle of the ZnO nano-needles from the substrate was 90 +/- 0.2 degrees. The structural and optical properties of nanoneedles and nanotetrapod structures were investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and photoluminescence (PL). The results showed that ZnO nano-needles grow along the c-axis of the crystal plane due to the c-oriented ZnO nanoseeds formed by zinc acetate treatment. The nano-needles have strong ultraviolet emission peak of 3.29 eV with green emission of 2.3 eV at room temperature. This selective growth technique of vertical nano-needles using aqueous solution method has potential applications in the field emission devices or optoelectronic devices hybridized with silicon based electronic devices.

Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor kappa B (NF kappa B) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NF kappa B activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrodeintegrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method.

A novel cytokine assay has been designed using a cellular chip by combining a collagen gel embedded cell culture technique with scanning electrochemical microscopy-enzyme linked immunosorbent assay (SECM-ELISA). An array of cell-collagen gel mixture (2 mu L) was spotted on an antibody-coated chip and incubated for 0.5-24 h. The very small trace amounts of cytokines produced by the activated leukocytes on the chip were effectively entrapped within the collagen gel matrix, and these were collected with the immobilized antibodies on the chip. The chip was further treated with horseradish peroxidase (HR-P)-labeled antibodies via the sandwich method after removing the cell-collagen gel spots from the chip. Scanning electrochemical microscopy (SECM) was used to quantitatively evaluate the cytokines from the activated leukocytes produced on the chip, and the SECM images were obtained to visualize the position and concentration of IL-1 beta secreted from THP-1 and HL-60 cell lines at concentration levels of 10-350pg mL(-1). Based on the chemiluminescence method, the sensitivity of the cytokine assay system in combination with SECM-ELISA is comparable to that of the marketed cytokine assay kit; further, the sample volume required for a single assay is drastically reduced. (c) 2006 Elsevier B.V. All rights reserved.

An electrochemical microdevice with separable electrode and antibody chips has been developed and applied to detect atrophic gastritis-related proteins, pepsinogen 1 (PG1) and pepsinogen 2 (PG2), based on sandwich-type enzyme-linked immunosorbent assays (ELISAs) with horseradish peroxidase (HRP)-labeled antibody. To fabricate the electrochemical device for simultaneous analysis of several proteins, the electrode chip with eight electrode elements was assembled along with an antibody chip with eight cavities containing immobilized anti-PG1 or anti-PG2. The immunoreactions occurring in the cavities of the device were detected simultaneously by amperometry. The labeled HRP in the cavity in the presence of hydrogen peroxide catalyzed the oxidation of ferrocenemethanol (FMA) to FMA(+), which was detected electrochemically by the electrode chip. The amperometric responses of respective cavities in the device increased with increasing concentration of PG1 or PG2 of 0-50 ng/ml, ensuring the simultaneous detection of PG1 and PG2. The detection limits for both PG I and PG2 were 0.6 ng/ml (S/N = 2). The electrode chip was recovered easily by disassembling the electrochemical device; thereby, it was used repeatedly, whereas the antibody chip was discarded. No marked decrease in electrochemical responses was detected after repeated use. Reuse of the electrode chip is beneficial to reduce costs of protein analysis. (c) 2005 Elsevier B.V. All rights reserved.

We report on the growth of uniquely shaped ZnO nanowires with high surface area and patterned over large areas by using a poly(dimethylsiloxane) (PDMS) microfluidic channel technique. The synthesis uses first a patterned seed template fabricated by zinc acetate solution flowing though a microfluidic channel and then growth of ZnO nanowire at the seed using thermal chemical vapor deposition on a silicon substrate. Variations the ZnO nanowire by seed pattern formed within the microfluidic channel were also observed for different substrates and concentrations of the zinc acetate solution. The photocurrent properties of the patterned ZnO nanowires with high surface area, due to their unique shape, were also investigated. These specialized shapes and patterning technique increase the possibility of realizing one-dimensional nanostructure devices such as sensors and optoelectric devices.

Scanning electrochemical microscopy (SECM) was employed to quantitatively characterize the oxygen permeation behaviors of poly(dimethylsiloxane) (PDMS) and surface-modified PDMS. The mass-transfer process of oxygen from the PDMS substrate to the tip electrode is diffusion limited, whereas the oxygen permeability of PDMS subjected to oxygen plasma treatment or albumin adsorption is critically restricted. Our results suggest that the oxygen permeability of PDMS is possibly affected by O-2 plasma irradiation and albumin adsorption at the PDMS surfaces.

Biological cells have been manipulated in a microdevice by means of negative dielectrophoresis (n-DEP) to form a micropatterns with two different cell types. The device with interdigitated array (IDA) electrodes was used to manipulate cells and to create a periodical line patterns with cells on a cell culture slide that was placed on 30 μm above IDA electrodes. Mouse myoblast cells (C2C12) were used as model cells to regulate the cultivation in weaker electric field regions in the micropatterning device filled with a cell culture medium. Specifically, cells formed the line pattern within 30 sec on the cell culture slide when a 1 MHz ac voltage (12 Vpeak-to-peak) was applied to the IDA electrodes. Most of patterned cells adsorbed on the slide after several minutes incubation under the application of ac voltage. The micropatterning with alternate lines of two different cell types was achieved by subsequently injecting another cell suspension in the device, and applying ac voltage to a different set of IDA electrodes. The method based on n-DEP permits the quick and easy fabrication of micropatterns composed from two different cell types without chemical modification of substrates. © 2006 Society for Chemistry and Micro-Nano Systems.

We have fabricated a microfluidic device comprising a 100 pL scale analytical chamber for electrochemical measurement of cellular activities. The microfluidic main channel and the analytical chamber were fabricated using poly(dimethylsiloxane) (PDMS). The chamber contained two Pt microelectrodes served as an electrochemical working electrode and an electrophoretic electrode to manipulate cells into the chamber. Yeast cells were introduced to the chamber by the electrophoresis, and its β-galactosidase (βGal) activities were characterized by amperometry. p-Aminophenol (PAP), generated by the enzyme-catalyzed hydrolysis of p-aminophenyl-β-D-galactopyranoside (PAPG), was detected electrochemically. © 2006 Society for Chemistry and Micro-Nano Systems.

Scanning electrochemical microscopy (SECM) is a technique in which the tip of a microelectrode is used to scan and monitor the local distribution of electro-active species near the sample surface. In this study, we have studies on the SECM technique, to establish the accurate method for measurement of respiratory activity of single bovine oocytes. With SECM procedure, oxygen consumption of single, identical bovine cumulus-oocyte complexes (COCs) and denuded oocytes has been monitored. Oxygen consumption rates (X 10(-14) moL center dot s(-1)) of the immature COC and oocytes (immediately upon recovery from ovary) were 5.48 and 0.67, respectively. Although respiration rate of denuded oocytes was lower than that of cumulus cells, oxygen consumption rate by a single bovine oocyte was quantitatively measured by SECM. Furthermore, oxygen consumption has been monitored in COCs and oocytes cultured in serum-free medium for oocyte maturation. An increase in oxygen consumption rate was found in oocytes (1.10), whereas the oxygen consumption by COCs (3.15) decreased during in vitro maturation (IVM). To analyze the metabolic activity by mitochondrial respiration, ATP content and mitochondrial distribution in oocytes have been examined. ATP content of oocytes after the maturation culture was significantly higher than that of immature oocytes. In immature oocytes, staining with MitoTracker Orange revealed mitochondrial clumps with a strong signal in the periphery of the cytoplasm. After IVM, mitochondrial clumps were allocated more toward the center of the cytoplasm. Electron microscopic study confirmed the mitochondrial reorganization in the bovine oocytes during the oocyte maturation. These results showed that the respiratory activity of bovine oocytes increased during IVM and mitochondrial reorganization may be partly to the respiratory activity. The SECM procedure is a useful technique to evaluate the metabolic activity and quality of single oocyte.

Scanning electrochemical microscopy (SECM) is a technique in which the tip of a microelectrode is used to scan and monitor the local distribution of electro-active species near the sample surface. In this study, we have studies on the SECM technique, to establish the accurate method for measurement of respiratory activity of single bovine oocytes. With SECM procedure, oxygen consumption of single, identical bovine cumulus-oocyte complexes (COCs) and denuded oocytes has been monitored. Oxygen consumption rates ( X 10(-14) mol center dot s(-1)) of the immature COC and oocytes (immediately upon recovery from ovary) were 5.48 and 0.67, respectively. Although respiration rate of denuded oocytes was lower than that of cumulus cells, oxygen consumption rate by a single bovine oocyte was quantitatively measured by SECM. Furthermore, oxygen consumption has been monitored in COCs and oocytes cultured in serum-free medium for oocyte maturation. An increase in oxygen consumption rate was found in oocytes (1.10), whereas the oxygen consumption by COCs (3.15) decreased during in vitro maturation (IVM). To analyze the metabolic activity by mitochondrial respiration, ATP content and mitochondrial distribution in oocytes have been examined. ATP content of oocytes after the maturation culture was significantly higher than that of immature oocytes. In immature oocytes, staining with MitoTracker Orange revealed mitochondrial clumps with a strong signal in the periphery of the cytoplasm. After IVM, mitochondrial clumps were allocated more toward the center of the cytoplasm. Electron microscopic study confirmed the mitochondrial reorganization in the bovine oocytes during the oocyte maturation. These results showed that the respiratory activity of bovine oocytes increased during IVM and mitochondrial reorganization may be partly to the respiratory activity. The SECM procedure is a useful technique to evaluate the metabolic activity and quality of single oocyte.

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.

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).

The dielectrophorefic separation of micro-organisms, based on cellular membrane damage, was carried out using a microfabricated fluidic device. The fluidic device was composed of an indium-tin oxide electrode with castellated electrode patterns, an acrylic board with inlet and outlet holes for micro-organisms suspension, and a silicone separator with a fluidic channel (width, 2 mm; length, 35 mm) between the electrode substrate and acrylic board. Dielectrophoretic separation was demonstrated for a mixture of live and heat-treated Escherichia coli bacteria labeled by fluorescent stains. The mixture was injected into the fluidic device at a flow rate of 440 mu m/sec. Both live and dead bacteria were collected around castellated electrode when an alternative (sinusoidal) electric field (frequency 100 kHz, voltage 20 Vpeak-to-peak) was applied to the castellated electrode. The dielectrophoretic separation was found by changing the electric field frequency from 100 kHz to 7 MHz. Only the heat-treated E. coli. cells were flown out from the fluidic device, while the live E. coli cells remained being captured between the electrodes. The results demonstrated that the fluidic device equipped with a microelectrode array provides a convenient way for the dielectrophoretic concentration and separation of targeted bio-particles in biomedical applications.

Two-dimensional cross-linked polysiloxane Langmuir-Blodgett (LB) films were prepared and applied to nitric oxide (NO)-permselective membranes in order to block other electroactive interfering species. The cross-linked siloxane LB films deposited on platinum micro-disc electrodes (10 mu m in diameter) offered revealing high performances as a permselective membrane for NO sensor such as high sensitivity to NO (detection limit, 40 nM) and high selectivity (e.g., the ratio of current response for acetaminophen or uric acid on the modified electrode to that on the bare electrode, less than 10(-3)). Furthermore, the permselective membrane could be easily deposited irrespective of the size and shape of electrode. (C) 2005 Elsevier Ltd. All rights reserved.

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.

An amperometric nitric oxide (NO)-sensing microelectrode was constructed using two-dimensional (213) cross-linked Langmuir-Blodgett (LB) films of siloxane copolymer as permselective membranes in order to block other electroactive interfering species. The 2D cross-linked siloxane LB films deposited on platinum micro-disc electrodes (10 mu m in diameter) were highly effective in the elimination of the interfering responses (e.g., the ratio of current response for dopamine on the modified electrode to that on the bare electrode, less than 10(-4)). Furthermore, high sensitivity to NO (detection limit, 40 nM) was also remained with modified LB films consisting of monolayers. (c) 2005 Elsevier B.V. All rights reserved.

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 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.

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose. (c) 2004 Elsevier B.V. All rights reserved.

To create an enzyme-based biological fuel cell generating electricity from glucose and O-2, we modified a glassy carbon electrode with a bi-layer polymer membrane, the inner layer containing diaphorase (Dp) and the outer, glucose dehydrogenase (GDH, an NAD(+)-dependent enzyme). The Dp membrane was formed from a newly synthesized 2-methyl-1,4-naphthoquinone (Vitamin K-3; VK3)-based polymer. This polymer showed reversible redox activity at a potential close to that of free VK3 (-0.25 V vs. Ag/AgCl sat. KCl), and served as an electron mediator of Dp for the electrocatalytic oxidation of NADH to NAD(+). The addition of Ketjenblack into the Dp/VK3 film enhanced the generation of NAD(+). The outer GDH membrane oxidized glucose continuously using NAD(+) generated at the inner Dp film. To construct the glucose/O-2 biological fuel cell, we coupled the enzyme-modified anode with a polydimethylsiloxane-coated Pt cathode. The cell's open circuit voltage was 0.62 V and its maximum power density was 14.5 mu W/cm(2) at 0.36 V in an air-saturated phosphate buffered saline solution (pH 7.0) at 37 degrees C containing 0.5 mM NADH and 10 mM glucose. Although its performance deteriorated to ca. 4 mu W/cm(2) over 4 days, the cell subsequently maintained this power density for more than 2 weeks. (C) 2005 Elsevier B.V. All rights reserved.

We have investigated a technique for the local delivery of chemicals to part of a cellular network. The device for the local stimulation was prepared by binding a permeable substrate made of a porous polycarbonate membrane and a micromask made of an elastomeric poly(dimethylsiloxane) (PDMS) film having a patterned hole (diameter, 50-200 mu m). As the model of the cellular network, primary cardiac myocytes were cultured on the membrane as a monolayer sheet and a line pattern, and then octanol (a gap junctional inhibitor) was locally delivered to the cells from the opposite phase of the porous substrate through a hole in the micromask. Intercellular communications of these cells were evaluated by observing the cytosolic Ca2+ transients. The synchronous Ca2+ transition on a myocyte sheet was stopped at only the area defined by the micromask. Based on the experiments using the line-patterned myocytes, it was proved that the electrically conjugating cellular network was divided into independent parts by the octanol stimulation across the cellular network. This research was carried out with the objective of developing a biosensing system based on a cellular network. (c) 2005 Elsevier B.V. All rights reserved.

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.

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.

Two-dimensional (2D) cross-linked polysiloxane Langmuir-Blodgett (LB) films, which were prepared by polymer-polymer cross-linking on an airwater interface, were applied to selective permeable membranes for nitric oxide (NO) sensor. The 2D cross-linked siloxane LB films deposited on platinum electrodes were remarkably effective in the elimination of the interfering responses (e.g., acetaminophen, L-cysteine, and L-ascorbic acid) and a rapid response to NO remained with modified LB films consisting of monolayers.

The microcontact printing (μCP) is a well-established method to pattern a material of interest using an elastomeric stamp. We have developed two techniques which assist the μCP-based cell-patterning for the controlled growth guidance of cultured neuronal cells on substrates. (i) Contact-transfer of extracellular matrix (ECM) protein on a microelectrode array substrate was achieved by spatially designing the microstamp to allow printing proteins even on the surface having uneven structures, and the differentiated PC12 cells showed selective adhesion and growth in the pre-determined locations on the electrode array. (ii) Cell alignment onto the pre-patterned ECM protein was also succeeded by using the microstructured silicon wafer having a band array of microholes, and the placed PC12 cells extended their axons along the protein pattern. These researches were carried out with the objective to developing a cell-based device based on a cellular network.

The respiration activity of single bovine embryos entrapped in a cone-shaped microwell was monitored by scanning electrochemical microscopy (SECM). A measuring cell composed of six cone-shaped microwells was employed. The height and radius of the cone-shaped microwell were both 2 mm. Individual embryos were located in either a culture dish or the rnicrowell to compare the oxygen concentration gradients of the surrounding medium solution at 37 degreesC in a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profile of the embryo located on the flat bottom of the culture dish is well described by the spherical diffusion theory. When the embryo was transferred into the cone-shaped microwell, the concentration profile changed. To analyze the diffusion behavior of the embryo in the microwell, a sphere whose center is located at the top of the cone was appropriate rather than one whose center is located at the sample center. The oxygen consumption rate of the embryo in the microwell was found to be 0.7 fold of that in the culture dish. (C) 2004 Elsevier B.V. All rights reserved.

We describe a novel anticancer drug sensitivity assay on a silicon chip applicable for tumors extirpated from in vivo mammalians. Human promyelocytic leukemia (HL-60) cells were subcutaneously (s.c.) inoculated in SCID mice, then removed 31 days after the inoculation. The cells were embedded in a small volume (18 nL) of a collagen-gel matrix on a pyramid-shaped silicon microstructure for further cultivation. The respiration activity of the cells on the chip was measured by scanning electrochemical microscopy (SECM). The proliferation behavior was continuously monitored for 6 days. It seemed that the proliferation rate of the cells removed from the mice was lower than that cultured in a flask and conformed to that in mice. The effects of cisplatin (CDDP) and etoposide (VP-16) on the HL-60 cultured in vivo were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the SECM-based assay is appropriate for biopsy specimens in a relatively short-time evaluation. (C) 2003 Wiley-Liss, Inc.

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.

The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.

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. (C) 2003 Elsevier Science B.V. All rights reserved.

The cardiac myocytes patterned on a single cell level were prepared by microcontact printing, and the response to chemical stimuli was studied using confocal fluorescent Ca2+ imaging. The patterned iriyocytes were found to conjugate by forming gap junction. It was confirmed for the patterned myocytes that gap junction communication was reversibly inhibited by 1-octanol, but activated by caffeine. Localized stimulation with chemicals was also attempted using a microinjection system for the myocyte patterns formed by single cell alignment. This research was carried out with the objective of developing a bioassay system based on a cellular network. (C) 2003 Elsevier Science Ltd. All rights reserved.

Scanning electrochemical microscopy (SECM) was applied to the Study of the spatial distribution of the respiratory activity in a PC12 neuronal cell. The SECM imaging of the oxygen concentration around the cells indicated that the axon of PC12 as well as its cell body consumed oxygen as the result of mitochondrial respiration. The variation in respiratory activity during NGF-induced axonal growth was monitored at the growth cone of the axon. The respiratory activity at the growth cone Was found to be particularly activated by the NGF treatment. (C) 2003 Elsevier Ltd. All rights reserved.

The peroxidase activity in a single protoplast of alga Bryopsis plumosa is quantitatively characterized by scanning electrochemical microscopy. The generation of ferriceniummethanol (FMA(+)) at the protoplast surface is directly detected by the microelectrode tip scanned close to the sample surface in seawater containing ferrocenemethanol (FMA) and hydrogen peroxide, an electron mediator and an enzyme-substrate, respectively. The oxidation reaction requires hydrogen peroxide, which clearly shows FMA(+) generation due to the peroxidase (POD) catalytic reaction Occurring in the protoplast. The FMA+ generation and the FMA accumulation rates at a single alga protoplast were equivalent. A plot of the FMA(+) generation rates according to the hydrogen peroxide concentrations was well allowed with a Michaelis-Menten-type reaction. An estimation of the mass-transfer rate and a determination of the K are quite important advantages of the SECM technique that cannot be realized using other techniques. The POD activity has been further investigated from the viewpoint of the size of the protoplast. The POD activity of the alga in the adult stage is also Visualized by SECM. The noninvasive nature of the SECM technique has been confirmed by observing the developmental process after measurements.

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. (C) 2003 Elsevier B.V. All rights reserved.

A hybrid system consisting of a scanning electrochemical microscope (SECM) and scanning confocal microscope (SCFM) was fabricated and used for micropatterning and imaging of diaphorase immobilized on a glass substrate. Simultaneous imaging of the diaphorase spots demonstrated that the SECM can provide information on a localized enzyme reaction, while the SCFM affords information on the location of the active enzyme. By using this SECM/SCFM system, spatially selective deactivation of diaphorase was performed by inducing a local electrochemical reaction or by irradiating with the focused laser. The resulting patterns of diaphorase were simultaneously imaged with the SECM/SCFM system. 2003 Elsevier Science B.V. All rights reserved.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was fabricated by filling the micropores on a glass substrate with collagen gel embedding Escherichia coli (E.coli) cells. For measuring the activity of E.coli cells, the SECM measurement using ferricyanide as an electron mediator showed that the respiration activity of the immobilized E.coli cells increased with the concentration of glucose in the solution. The results suggested that the microbial chip can be used as a chemical sensor for nutritive species.

The immobilization of two enzymes on a pair of Au microband electrodes was performed by using electrochemical desorption of self-assembled monolayer (SAM) of alkanethiol. Both of the An electrodes were coated with the SAM of n-octadecanethiol (ODT-SAM) at first. The ODT-SAM on one of the Au electrodes was electrochemically removed by applying reductive potential. The resulting naked Au surface was re-coated with the SAM of 2-aminoethanethiol (AET). These treatments resulted in a couple of Au electrodes coated with the ODT-SAM and AET-SAM, respectively. Horseradish peroxidase. (HRP) was selectively immobilized on the AET-SAM by using crosslinking agent, glutaraldehyde. On the other hand, diaphorase (Dp) was immobilized on the surface of ODT-SAM by hydrophobic interaction. The imaging of the resulting substrate with scanning electrochemical microscope (SECM) demonstrated the enzymatic activities of HRP and Dp at the AET- and ODT- treated An electrodes, respectively.

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.

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. (C) 2003 Elsevier Science B.V. All rights reserved.

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 erythro-leukemia 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.

The patterning of cardiac myocytes on a micron scale (similar to5 mum) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca2+ transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca2+ concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope. (C) 2003 Wiley Periodicals, Inc.

A simply designed valveless switch for microparticle sorting was fabricated on a glass chip. A successful sorting of 10 mum diameter polystyrene latex beads was performed by the microfluidic system consisted of a unique electrophoretic switch and pair of parallel laminar flow streams. In applying the voltage to the electrodes placed on the banks of the flow through channel, microparticles were electrophoretically diverted across the boundary between two distinct laminar flows. (C) 2003 Elsevier Science B.V. All rights reserved.

Micropatterns of the electrically conjugating cardiomyocytes were prepared on a single cell level by microcontact printing (mCP), and the localized chemical stimulations were applied to the cellular pattern using multiple laminar flows. The locally delivered 1-octanol inactivated part of the myocyte patterns, while the other areas retained the activity showing spontaneous and synchronous pulsatility. Since both the cells and flows were well defined as micropatterns and each integrated on a chip, the obtained results simply demonstrate the cellular responses in a single-cell network.

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.

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.

The micropatterns (similar to5 mum) of HeLa cells were maintained for more than 4 days on a glass surface modified by the corresponding pattern of a covalently attached polyethylene glycol (PEG) monolayer, even in the presence of serum proteins.

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1 similar to 2 mum) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s(-1) while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable. (C) 2002 Elsevier Science B.V. All rights reserved.

The micropatterns of mammalian cells (HeLa cells) were prepared on glass substrates, and the respiration of the patterned cells was studied by microelectrode techniques, mainly by scanning electrochemical microscopy (SECM). The cellular patterns on a micrometer scale were prepared by microcontact printing of an extracellular matrix protein, fibronectin, onto a hydrophobic glass plate. The oxygen concentration in the vicinity of the patterned cells was mapped by scanning a Pt microelectrode, and the obtained SECM images proved that the cells in patterns were living with the uptake of oxygen. HeLa cells in the band patterns were well spread, while the cells in the small island patterns were restricted in their shape. The respiratory activities of these cells were evaluated by measuring the difference in the oxygen concentration between the bulk solution and the cell surface, and it was shown that a spreading cell consumed a significantly larger amount of oxygen than a round cell.

Scanning chemiluminescence microscopy (SCLM) was applied to visualize bienzyme reaction at a microspot (50 mum in radius) composed of glucose oxidase (GOD) and horseradish peroxidase (HRP). In an aqueous solution Containing glucose, a glass capillary tip was scanned over the bienzyme spot with luminol injected continuously from the tip to induce local chemiluminescence. The GOD catalyzes the oxidation of glucose by oxygen to form H2O2 which oxidizes luminol in the presence of HRP to generate chemiluminescence. Since the enzymatic luminescence reaction proceeds only within a bienzyme spot, even an array of spots was clearly imaged by SCLM without any significant interference between neighbor spots.

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.

Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H2O2 to increase FMA(+) concentration. Two-dimensional reduction current profiles for FMA(+) produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles. (C) 2001 Elsevier Science B.V. All rights reserved.

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 degreesC, 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-mum radius on day 6 after in Nitro fertilization, the oxygen concentration difference (DeltaC) between the bulk solution and the morula surface was 6.90 +/- 1.35 muM. 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-mum 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 muM, 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 &lt; F &lt; (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.

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

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a γ-aminobutyric acidA (GABAA) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABAA receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABAA receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABAA receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis. © 2001 Elsevier Science Ireland Ltd.

Scanning electrochemical/chemiluminescence microscopy (SECM/SCLM) was developed for simultaneous imaging of chemiluminescence and topography of immobilized horseradish peroxidase (HRP) on a glass substrate. When the SECM tip for electrochemical generation of H2O2 scanned over the immobilized spot in an aqueous luminol solution, the immobilized enzymes catalyzed the oxidation of luminol to emit chemiluminescence, which was detected by a photon counter. The photon-counting intensity was plotted against the position of the tip to give a two-dimensional image, indicating the activity of immobilized enzymes. Since the current was sensitive to the topography of substrate, the topography image was also obtained by mapping the oxygen reduction current. (C) 2001 Academic Press.

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.

Scanning electrochemical microscopy (SECM) was applied to the immunoassay of leukocidin, which is a toxic protein produced by methicillin-resistant Staphylococcus aureus (MRSA), with the intention of developing and early diagnostic for MRSA infection. An antibody-chip for leukocidin was prepared by self-assembling of anti-leukocidin on a protein A-coated glass substrate. A sample solution containing leukocidin was spotted onto the antibody-chip, followed by labeling with horseradish peroxidase (HRP) via a sandwich method. The reduction current of the oxidized form of ferrocenylmethanol generated by the HRP reaction was monitored to view SECM images of the spot of captured leukocidin, The amplitude of reduction current depended on the concentrations of sample solutions used for making spots. This SECM-based immunoassay detects as low as 5.25 pg mL(-1) leukocidin.

We developed a novel method for the fabrication of a multichannel glutamate sensor based on a planar electrode array. By scanning and holding the electrode potential in a solution containing glutamate oxidase and a polymer to which horseradish peroxidase and electron-transfer mediator were bound, we successfully immobilized these enzymes and a mediator on the electrodes of the electrode array through electrochemical deposition of the polymer. We confirmed the resulting sensor possessed high sensitivity and selectivity to glutamate. This multichannel sensor may be useful for the non-invasive, real-time monitoring of the glutamate distribution in biological samples.

A novel approach for micropatterning of surfaces with organic and biological microstructures using the scanning electrochemical microscope (SECM) is described. The approach is based on the introduction of the spatial resolution by local deposition of gold particles followed by monolayer formation and functionalization. Specifically, gold patterns were deposited locally on silicon wafers with the SECM as a result of the controlled anodic dissolution of a gold microelectrode. The gold patterns were further used as microsubstrates for assembling cystamine monolayers to which either fluoresceine isothiocyanate (FIT) or glucose oxidase (GOD) were covalently attached. Characterization of the organic monolayers, as well as the biological activity of the enzyme patterns, was carried out by fluorescence microscopy and the SECM, respectively.

Communication: Microstructured TiO2 films with hemispheric pores have been used here to control the size of protoplasts of the marine alga Bryopsis plumosa. The Figure shows protoplasts prepared on a 20 mu m pitch TiO2 template, illustrating the small range of diameters that are obtained. This technique serves as a convenient way to provide cell models for in vitro physiological studies.

A novel scanning chemiluminescence microscopy (SCLM) was used for imaging localized horse radish peroxidase (HRP) or glucose oxidase (GOD) immobilized at a solid substrate. SCLM equips a scanning capillary tip (diameter, 1 μm) for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The chemiluminescense induced by the enzyme-catalyzed reaction was detected with a photon-counter. Two-demensional mapping of the photon-counting intensity against the tip position gave images indicating the activity of immobilized HRP and GOD.

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.

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.

A technique was developed for simultaneous monitoring of temporal changes in hydrogen peroxide (H2O2) and dopamine (DA) levels by using Pt-disk microelectrodes ( phi 30 mu m). H2O2 and DA were determined by differential double-pulse amperometry (for H2O2; 1st step, 750 mV, 1000 ms; 2nd step, 1100 mV, 1000 ms: far DA; 1st step, 150 mV, 600 ms; 2nd step, 250 mV, 50 ms). The electrode for H2O2 or DA is capable of determining each target substance and these substances do not interfere with each other. For an in vivo application, the electrodes were implanted into the striatum of rats and used successfully to monitor intrastriatal changes in H2O2 and DA simultaneously after intraperitoneal injection of methamphetamine while the animals were moving freely.

The effects of p-benzoquinone (BQ) on photosynthetic and respiratory electron transport in a single algal protoplast (radius, 100 mu m) 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 (QH(2)) by (2.7 +/- 0.3) x 10(-13) mol/s, suggesting that BQ was quantitatively reduced to QH(2) via the intracellular photosynthetic electron-transport chain. The generation of QH(2) increased with light intensity and with concentration of BQ added to the outside solution but became saturated when the tight 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 QH(2) 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 similar to(2.6 +/- 1.0) x 10(-13) mol/s, which was similar to 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.

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.

An uniform, dense film of spinel LiMn2O4 (thickness, about 0.5 mu m) was prepared by the electrostatic spray deposition method onto a Pt film electrode on Si substrate kept at 400 degrees C. Electrochemical properties of the LiMn2O4 film in a solution of 1 M LiClO4/propylene carbonate were studied by cyclic voltammetry and potential-step chronoamperometry. The oxidation and reduction of the film, which are accompanied by the extraction/insertion of lithium ions, proceeded almost quantitatively around 4 V vs. Li/Li+ with excellent coulombic reversibility. The apparent chemical diffusion coefficient of Li+ in Li1-xMn2O4 spinel phase varied on the order between 10(-10) and 10(-12) cm(2) s(-1) as a function of electrode potentials with minima at the potentials corresponding to the voltammetric current peaks.

The solid-state diffusion of lithium ion in carbon was directly investigated in single spherical particles of graphitized mesocarbon microbeads (MCMB, Osaka Gas Co.). In order to achieve electrochemical measurements focusing on a single particle, a molybdenum-filament microelectrode was maneuvered to make electrical contact with a single MCMB placed in an organic solvent mixture containing lithium ions as an electrolyte. This novel microelectrode technique gives electrochemical information about a MCMB particle, itself, without any dilution with additives such as organic binders. The chemical diffusion coefficient (D) of lithium ion was determined from potential-step chronoamperograms assuming spherical diffusion of lithium ions in the MCMB particle. The values of D were found to change the order between 10(-9) and 10(-11) cm(2)/s depending on the electrode potential. Plots of D showed pronounced minima at the potentials where the transformation of staging level of lithium intercalation compounds occurred. (C) 1998 The Electrochemical Society, Inc. S1099-0062(98)01-019-0.

Thin films of LiMn2O4 have been prepared by RF magnetron sputtering on interdigitated microarray electrodes. In situ conductivity-potential profiles and cyclic voltammograms during extraction/insertion processes of Li ions were obtained simultaneously in nonaqueous and aqueous electrolyte solutions (1 M LiClO4/propylene carbonate and 1 M LiCl/water). The electronic conductivity of Li1-xMn2O4 was found not to show metallic transition and maintain its semiconducting state during the extraction/insertion of Li ion. A slight decrease in conductivity was observed with increasing the anodic potential, i.e., with increasing x (lithium extraction) and recovered reversibly when the potential returned to the cathodic side (re-insertion of Li ions). Similar results were obtained in both aqueous and nonaqueous electrolyte solutions.

Photoelectrochemical properties of spinel Li1-xMn2O4 (0 &lt; x &lt; 1) thin film on ITO electrodes were studied in propylenecarbonate (PC) containing 1 M LiClO4. Cathodic photocurrents were clearly observed, suggesting the occurrence of photo-assisted lithium insertion reaction at the Li1-xMn2O4 film electrode.

It has been hypothesized that free radicals play a causative role in tardive dyskinesia, which is an inveterate movement disorder caused by chronic administration of neuroleptics. To verify this hypothesis, rats were reared while being regularly treated with water containing a neuroleptic, haloperidol (HPD), for 1 year (HPD group). The changes in the striatal hydrogen peroxide content of the rats in the HPD and control groups were measured by using a Pt-disk microelectrode while the animals were in a freely moving state following intraperitoneal administration of HPD (HPD challenge). We also performed electron spin resonance (ESR) detection of lipid radicals in the striatum before the HPD challenge. HPD challenge led to significant elevation of the intrastriatal hydrogen peroxide in all animals, but the elevation in the HPD group was smaller than that in the control group. However, in the KPD group, marked ESR signals of intrastriatal lipid radicals were observed. We think that these results support the hypothesis on the role of free radicals in tardive dyskinesia. (C) 1998 Elsevier Science Inc.

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.

Transport of ferrocenylmethanol and Co(phen)(3)(2+) through a nitrobenzene/water interface was examined by an electrochemical method using a microelectrode. The transfer rate of Co(phen)(3)(2+) through nitrobenzene / water interface was determined to be 0.05 cm/s by digital simulation analysis. Transfer ferrocenylmethanol was very fast, with the transfer estimated to be larger than 0.1 cm/s.

Scanning electrochemical microscopy (SECM) was used for localized electrogeneration of hydroxyl radical to create patterns with diaphorase on the substrates immobilized with self-assembled monolayers (SAMs) of four different alkylsilane derivatives. Hydroxyl radical was generated at an SECM tip (microelectrode) in a solution containing H2O2 and Fe3+ by applying a reduction potential pulse of 0.00 V vs Ag/AgCl. The electrogenerated hydroxyl radical degraded the SAMs and removed them from the glass surfaces. Diaphorase patterns were formed on the substrates by physical adsorption onto the hydrophobic area or by chemical linkage to the hydroxyl radical-attacked area. Local diaphorase activity was visualized by SECM by detecting the diaphorase-catalyzed current of ferrocenylmethanol coupled with oxidation of reduced nicotinamide adenine dinucleotide.

A dual imtnunoassay of human chorionic gonadotropin (HCG) and human placental lactogen (HPL) at a microfabricated substrate was studied using scanning electrochemical microscopy (SECM). Anti-HCG and anti-HPL were microspotted on 50 × 50 μm2 raised flat squares separated from each other by 150 μm at a glass substrate. A 10 μ1 sample solution containing HCG and/or HPL was then dropped onto the substrate followed by treatment with a solution of horseradish peroxidase (HRP)-labeled anti-HCG and HRP-labeled anti-HPL. The SECM measurements were carried out at 0.05V vs. Ag|AgCl in the presence of ferrocenylmethanol (FMA) and H2O2 to detect FMA+ generated by the HRP reaction. The SECM images of the substrates showed clearly the large response areas which corresponded to the squares with HCG and/or HPL. The detection limits were 0.1 IU ml-1 for HCG and 3 ng ml-1 for HPL. © 1997 Elsevier Science S.A.

We fabricated a Pt-disk microelectrode (diameter 30 μm) to conduct differential double-pulse amperometry (first step: 750 mV, 1 s second step: 1100 mV, 1 s) to detect hydrogen peroxide in the brain of a freely moving animal. This measurement determined hydrogen peroxide (detection limit, 0.03 μM) without any observable influence from other oxidizable species, such as dopamine (DA), ascorbic acid, or uric acid. The electrode was implanted into the right striatum of a rat. After intraperitoneal injection of methamphetamine (MAP), hydrogen peroxide concentrations were directly detected while the behavioral changes were monitored. MAP injection led to significant augmentation of hydrogen peroxide, the elevation of which depended on the dose of MAP. This is consistent with a previous report on the increase of DA-release caused by amphetamines and indirect evidence of the production of hydrogen peroxide via DA-metabolism.

A dual immunoassay of human chorionic gonadotropin (HCG) and human placental lactogen (HPL) at a microfabricated substrate was studied using scanning electrochemical microscopy (SECM). Anti-HCG and anti-HPL were microspotted on 50 X 50 mu m(2) raised flat squares separated from each other by 150 mu m at a glass substrate. A 10 mu l sample solution containing HCG and/or HPL was then dropped onto the substrate followed by treatment with a solution of horseradish peroxidase (HRP)-labeled anti-HCG and HRP-labeled anti-HPL The SECM measurements were carried out at 0.05 V vs. Ag/AgCl in the presence of ferrocenylmethanol (FMA) and H2O2 to detect FMA(+) generated by the HRP reaction. The SECM images of the substrates showed clearly the large response areas which corresponded to the squares with HCG and/or HPL. The detection limits were 0.1 IU ml(-1) for HCG and 3 ng ml(-1) for HPL. (C) 1997 Elsevier Science S.A.

Simultaneous measurements of in situ Raman spectroscopy and cyclic voltammetry have been carried out for thin film electrodes of LixCoO2 in propylene carbonate containing 1 M LiClO4. The Raman lines of 485 and 587 cm(-1) observed at LixCoO2 electrodes were attributed to the A(1g) and E-g modes of the LiCoO2, respectively. The Raman intensity of two lines changed drastically during the insertion/extraction of lithium ions. This effect can be explained by the reduction of the optical skin depth due to the conductivity change of LixCoO2. Phase transition from Raman-active phase to Raman-inactive phase is also conceivable. (C) 1997 Elsevier Science S.A.

Micropatterns with living cells were rapidly formed on glass substrates using negative dielectrophoretic force induced at a template microarray electrode consisting of assembly of microband electrodes. The template electrode was placed above the substrate with a 50 mu m-thick spacer and cell suspension was sucked into the space between the substrate and electrode. When an ac voltage (typically, 7 V rms, 10 kHz) was applied to the template electrode, the induced dielectrophoretic force repelled the cells from the electrode and pushed at the glass substrate to form the cell pattern which reflected the electrode shape. The magnitude and frequency of the applied ac voltage affected the resolution and accuracy of the cell patterns. The width of the pattern was controlled by changing the band width of the template. Cell patterns with a single cell alignment appeared when the band width of the template electrode was 20 mu m. (C) 1997 Elsevier Science Ltd.

In situ conductivity-potential profiles and cyclic voltammograms of an LiCoO2 thin film were obtained simultaneously by using an interdigitated microarray electrode with a constant potential difference. The electronic conductivity of Li1-xCoO2 increased exponentially with potential during lithium extraction from 2.7 to 4.0 V. The conductivity changes drastically when very small amounts of Li (0 &lt; x &lt; 0.1) are extracted.

Horseradish peroxidase (HRP) and xanthine oxidase (XOD) reactions were investigated using a multichannel electrochemical detector with 16 independent band electrodes held at different potentials. Although the enzyme reaction media gave complicated electrochemical responses in flow injection analysis, responses for a redox species can be extracted easily from the multichannel responses. The course analysis over time of the extracted responses enabled the Michaelis constants and molecular activities of HRP for various electron donors to be established. The electrochemical responses of O-2(-) on the mu M order in aqueous solution were also washed out from the responses in the XOD reaction media.

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 similar to 10(4) CEA molecules in a single 20-mu m-radius spot.

Two types of microelectroporation capillaries (two-electrode and three-electrode poration capillaries) for injection of genes or drugs into single cells were fabricated and their performance was investigated using Fe(CN)₆^3- as a model substance. The two-electrode poration capillary consisted of a glass capillary with a sputtered Pt film at the outside and a Pt microwire at the inside. The three-electrode poration capillary possesses another Ag/AgCl microelectrode in the capillary. When an electric pulse was applied between the Pt film and Pt wire of the two-electrode capillary (Pt film positive, Pt wire negative), Fe(CN)₆^3- dissolved in the solution inside the capillary was released by electromigration. The amount of the released Fe(CN)₆^3- can be controlled by adjusting the magnitude and period of the pulse. We applied the two-electrode poration capillary to inject Fe(CN)₆^3- into a single protoplast. The application of a electric pulse brought about a reversible membrane breakdown to form small pores in the membrane and simultaneously repelled Fe(CN)₆^3- from the capillary by electromigration. The injection of Fe(CN)₆^3- into the protoplast was confirmed by detecting the reduction current of Fe(CN)₆^3- at an ultramicrodisk electrode inserted into the cell. In the three-electrode poration system, we applied two consecutive pulses for reversible membrane breakdown and for electromigration. The three-poration capillary was found to be effective for injection of charged substances.

We directly and continuously monitored the endogenous nitric oxide (NO) in the rat hippocampus after intraperitoneal (i.p.) administration of N-methyl-D-aspartic acid (NMDA) in freely moving state. The sensor used here was Malinski-type porphyrinic probe modified so as to measure in the freely moving animals. When the rats received the subconvulsive dose of NMDA (20 mg/kg), a significant increase of the NO concentration in the hippocampus was continuously observed, The increase was up to 1 mu M at 25-30 min past administration. This elevation in NO concentration was totally blocked by pretreatment with an NO synthase inhibitor, N-G-monomethyl-L-arginine (30 mg/kg, i.p.). These findings indicate that our sensor successfully measured the NMDA-induced NO in the hippocampus catalyzed by NO synthase.

The electrochemical behaviour of V-groove microelectrodes (10-100 mu m wide) was investigated and compared with that of planar band microelectrodes with the same sizes. The V-groove electrodes were fabricated by anisotropic etching on a [100] silicon wafer in KOH solutions. The cyclic voltammetric investigation at the V-groove electrodes revealed a different scan rate dependence of the peak current from that at the planar band microelectrodes. When the V-groove electrode was covered with a platinum plate lid electrode, the redox cycling between the two electrodes proceeds efficiently, exhibiting higher collection efficiencies than at the ordinary interdigitated microarray electrode with the same size. The high collection efficiency originated from the small effective diffusion length.

The ESR spectra of stable free radical in solution have first been detected through potential change between two electrodes inserted in the solution. The working electrode (phi 50 mu m) was biased by a constant current source under microwave irradiation and modulated magnetic field. The AC component of the potential on resonance was preamplified and detected by a lockin amplifier. The sensitivity of this method, 5 X 10(6) spins/G, is 10(3) higher than those of conventional ESR spectrometers.

The conductivity change of a vacuum deposited V2O5 thin film during lithium intercalation/deintercalation was investigated using an interdigitated microarray electrode. The conductivity-potential profile showed two peaks (ca. 1.7x10(-3) S cm(-1)) at 3.23 V and 3.41 V vs. Li/Li+, corresponding to the peaks in the cyclic voltammogram for Li intercalation/deintercalation. The conductivity decreased below 10(-5) S cm(-1) when the potential was scanned to the region more negative than 3.0 V vs. Li/Li+ and an irreversible conductivity change was observed when the potential was scanned back.

We fabricated an electrochemical photo memory device using a Pt-TiO₂-Pt microarray electrode coated with a polypyrrole film. The TiO₂ microband electrode, positioned between two comb-shaped Pt electrodes, was an n-type semiconductor to oxidize the polypyrrole film under light irradiation. Because oxidation enhances the conductivity of polypyrrole, light irradiation is detected by the increase in the conductivity of polypyrrole between the two Pt comb electrodes. Enhanced conductivity remained even after light irradiation was terminated. This photo memory behavior was observed when the potential of the titanium oxide electrode was set to -0.3V vs. SCE.

Scanning electrochemical microscopy (SECM) was used to microfabricate and quantify diaphorase-patterned glass surfaces. Deactivated circular and linear micropatterns were produced at diaphorase-immobilized substrates by a localized surface reaction. The oxidation of Br- and Cl- at a microelectrode generated a reactive species which deactivated the localized enzyme molecules at the substrate. The diaphorase-patterned surfaces were characterized by SECM on the basis of detection of catalytic current of ferrocenylmethanol coupled with oxidation of NADH. The concentration of the immobilized diaphorase was mapped from the quantitative analysis of the catalytic current.

The electrochemical behavior and electrical conductivity of C-60 thin films were studied in acetonitrile containing M(bpy)(3)(2+) (M = Fe, Ni, Ru or Os; bpy = 2,2'-bipyridine). Cyclic voltammetry of C,, films showed stable reduction-reoxidation waves. In situ conductivity measurements indicated that an enhanced conductivity (on the order of 10(-2) S cm(-1)) appeared reversibly on electrochemical reduction accompanied by doping of M(bpy)(3)(2+) ions into the C-60 films. The electrochemical reduction also induced some structural change.

A conductometric glucose sensor was fabricated by coating a twin-microband electrode with a bilayer of a membrane composed of polyaniline and glucose oxidase/gluconolactonase films. The enzyme-catalyzed hydrolysis of glucose induces a change in the conductivity of the pH-sensitive polyaniline film. The conductivity change was detected by the current flowing between the two-band electrode. The sensor demonstrates clear responses to glucose up to 1 mM.

The permeability of Ru(NH3)(6)(3+), Fe(CN)(6)(3-), and I- through voltage-gated alamethicin ion channels in a planar lipid membrane was investigated by amperometric measurements using a microelectrode located near the membrane. The redox current at the microelectrode initiated by application of a potential pulse to the BLM was quantitatively analyzed to obtain permselectivity of the channel. The permeability of Fe(CN)(6)(3-) was ca. 10 times smaller than that of Ru(NH3)(6)(3+) due to electrostatic repulsion, indicating that the channel gives a high barrier to the permeation of multicharged negative ions.

Paired cell alignment of mouse myeloma by dielectrophoretic phenomena was carried out using a jagged microarray electrode. A.c. voltage application (0.1-1 V rms, 0.1-10 MHz) forced the cells to move into projection gaps where non-uniform, locally-intense electric fields were created. A highly selective one-to-one cell alignment (ca. 50%) was observed at the array electrode with 10 mu m projection gaps. Since the dielectrophoretic force acted effectively on living cells, the present array electrode selected living cells to form one-to-one cell alignment from the suspension mixture of living and dead cells.

Crystallographic changes of C-60 thin film during electrochemical reduction and reoxidation in acetonitrile containing of tetra-n-butylammonium (TBA(+)) were studied by X-ray diffraction (XRD) and scanning electron microscope (SEM). XRD spectra showed a reversible structure change due to the electrochemical doping of TBA(+).

The heterogeneous electron-transfer rate constants for Co(phen)3(2+/3+) and Fe(CN)6(4-/3-) at the bilayer lipid membrane (BLM) saturated with tetracyanoquinodimethane (TCNQ) were respectively (3 +/- 1) X 10(-4) and &lt;2 X 10(-8) cm/s, determined by ac impedance spectroscopy. The large difference in the rate constants was explained by hydrophobic interaction between the interior of the BLM and the redox species, by the electrostatic interaction between the negatively-charged surface and the redox species in solution, and by the difference in formal potential between TCNQ and the redox species.

Dielectrophoretic manipulation of a single myeloma cell was carried out using microring-ring electrodes (tip radius, 3-5 mum). When a.c. voltages were applied, the cell nearby the electrode tip was forced to move and trapped at the tip due to dielectrophoretic force induced by locally intense electric field.

Polypyrrole grows effectively along the glass substrates pretreated with n-alkylsilane reagents in electropolymerization from aqueous solutions, resulting in the formation of an ultrathin polypyrrole film at twin-microband electrodes. However, no effective promotion of lateral growth of polypyrrole was observed when the substrate was pretreated with 3-aminopropyltriethoxysilane or when the electrolysis was conducted in acetonitrile, suggesting that the hydrophobicity of the substrate is essential for promotion of the lateral growth of polypyrrole. The promotion effect is accounted for by adsorption of pyrrole monomers and selective deposition of intermediate oligomers at the hydrophobic surfaces. The polymerization anisotropy (the ratio of lateral growth rate to vertical growth rate) was ca. 25. The partial pretreatment provided a way of micropatterning with polypyrrole on an insulating substrate. Such a promotion effect at hydrophobic surfaces is amplified in the presence of a small amount of an anionic surfactant. Polyaniline also grows effectively along the hydrophobic surface with the polymerization anisotropy of &gt;100.

The activity of diaphorase (from Bacillus stearothermophilus) immobilized on glassy carbon (GC) electrodes was determined by analyzing the catalytic currents for oxidation of the immobilized enzyme using digital simulation techniques, which gives the concentration of the active enzyme at the electrode surface. Results show that the immobilization by the cross-linking reaction with glutaraldehyde deactivates the enzyme and only about 10% of the total enzyme remains active at the electrode surface.

Electrochemical stability of the adsorbed monolayers of n-alkanethiols and aromatic thiols on Au and Ag electrodes was investigated by AC impedance and in-situ surface-enhanced Raman scattering(SERS). The differential capacity measurements were carried out at the monolayer-coated-electrode/0.1M Na 2SO4 solution interfaces to examine a stable potential region of the thiol adsorption. Under the same conditions as the capacity measurement, SERS spectra were obtained to confirm the presence of thiol molecules adsorbed on the electrode surface in comparison with the the differential capacity-potential curves. The capacity value, which was unchanged in the stable adsorption potential range, increased rapidly at cathodic potentials more negative than ca.-0.6 Vvs.SCE, indicating the desorption or some structure changes in the adsorbed thiol monolayer. According to SERS spectra, the intensity of nu(C-S) peaks of n-alkanethiols remained unchanged even at -1.2 V, indicating high stability of n-alkanethiol monolayer. However, the peaks in SERS spectra completely disappeared at -1.2 V for aromatic thiols which are more hydrophilic than n-alkanethiols.

An oxygen ultramicroelectrode was fabricated by pyrolysis of butane inside a glass capillary, followed by electrodeposition of Pt. The intracellular determination of oxygen in an algal protoplast (diameter, approx. 80-mu-m) with the ultramicroelectrode indicated that at a light intensity of 25 klx, the oxygen efflux from a single protoplast caused by the light reactions is approx. 5 . 10(-13) mol/s and that corresponding to the dark reactions approx. 1 . 10(-13) mol/s.

The electrochemical reduction of self-assembled monolayers formed from p-nitrothiophenol has been studied in situ by surface-enhanced infrared (IR) absorption and Raman scattering (SEIRA and SERS, respectively) spectroscopies. The sensitivity of SEIRA spectroscopy is 20 times higher than that of IR reflection-absorption (IR-RAS) spectroscopy. Combined use of SEIRA and SERS spectroscopies is useful for detailed analyses of electrode reactions and vibrational properties of adsorbed molecules.

A multichannel electrochemical detection system for flow injection analysis (FIA) and high-performance liquid chromatography (HPLC) has been fabricated using a 16-channel microelectrode array. The 16-channel detection was performed by collecting current responses at 16 microband electrodes held at potentials differed stepwise by means of a multipotentiostat to show three-dimensional results, i.e., time, current, and potential. The detection limit for the ferrocene derivative is 10(-8) M, and the response shows good linearity over the concentration range 10(-3)-10(-8) M at 16-channel detection. The 80-channel detection was also carried out by applying a 5-step potential staircase of 10-mV step height to the 16 individual electrodes. The faradaic current in each potential step was sampled in the time region where the charging current decreased negligibly. A hydrodynamic voltammogram comprising 80 data points can be obtained in 0.26 s. This rapid detection system was applied to FIA and HPLC of a mixture of several electroactive chemicals, such as ferrocene derivatives, ascorbic acid, uric acid, and catecholamines. These hydrodynamic voltammograms provide useful electrochemical information, i.e., concentration, half-wave potential, and reversibility. The utility of this system is demonstrated by the detection of uric acid and ascorbic acid in blood and urine.