安川 智之

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.

The permeability for oxygen and hydrogen peroxide through a poly(dimethylsiloxane) (PDMS) layer with various thicknesses on a platinum electrode was investigated in order to develop a glucose sensor based on the detection of a reduction current of oxygen, which was consumed by a catalytic reaction of glucose oxidase (GO<sub>x</sub>) in the presence of glucose. PDMS layers with various thicknesses were prepared on platinum electrodes by casting various concentrations of the PDMS emulsion. Oxygen and hydrogen peroxide permeated through the PDMS layers on a Pt electrode were detected by cyclic voltammetry in buffer solutions. The PDMS layer allowed us to permeate the oxygen, but also acted as a barrier for the permeation of hydrogen peroxide. The concentration of oxygen in the PDMS layers was roughly estimated by quantitative analysis using an amperometric technique, and was found to be >2.5 mM, which is >10-times higher than that in an air-saturated buffer solution with 0.22 mM oxygen. Finally, PDMS-modified Pt electrodes combined with a poly(vinyl alcohol) stilbazole quaternized (PVA-SbQ) membrane containing GO<sub>x</sub> were fabricated, which were applied to glucose sensing. The incorporation of PDMS layers with oxygen permselectivity to glucose sensing enhanced the current responses for oxygen reduction because of a restriction of the permeation for hydrogen peroxide and the high concentration level in PDMS. However, the response time increased with increasing the thickness of the PDMS layer. Therefore, a PDMS layer with 50 μm − 100 μm is suitable to fabricate glucose sensors based on the detection of oxygen.

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.

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.

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.

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.

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.

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 glucose-sensing electrode based on the cathodic detection of oxygen, which was consumed with the glucose oxidase-catalyzed reaction in the presence of glucose, was prepared by immobilizing the enzyme on a surface-modified poly(dimethylsiloxane) (PDMS) layer. A PDMS layer was prepared on a platinum electrode by casting an emulsion of the polymer, the polymer surface was treated with oxygen plasma to replace silane groups with silanol groups, and then a GOx layer was prepared on the PDMS layer by applying silanization and cross-linking chemistries. The oxygen plasma-treatment of PDMS did not cause significant changes in the permselectivity with the polymer layer so as to discriminate hydrophilic solutes, such as hydrogen peroxide, from oxygen. Thus, the resulting electrode could be used for the measurement for glucose (0.02 - 1.8 mM) without the error caused by L-ascorbic acid and uric acid.

A novel electrochemical technique for DNA detection has been developed by a simple combination cisplatin and a glassy carbon electrode. The complexation of cisplatin with DNA caused suppression of the electroreduction of cisplatin owing to the steric hindrance and, subsequently, the electrolytic current for reducing proton that is catalyzed by the platinum deposited on the glassy carbon electrode by the electroreduction of cisplatin. Thus the DNA content was determined from the decrease in the catalytic activity of the electrode: an enhanced current response for measuring the current for reducing proton as compared with that for reducing cisplatin itself could provide the highly-sensitive measurement of cisplatin. The detection limit for herring DNA was as low as 0.1 ng/mL.

A novel electrochemical enzyme immunoassay system with a detection limit of 2 pM (10 ng L-1) was developed for the determination of insulin, an important hormone for clinical diagnosis of diabetes. An insulin-containing sample solution was added to a solution containing a certain concentration of glucose oxidase (GO(x))-labeled anti-insulin antibody to undergo an immunological reaction, and unbound GO.-antibody conjugate was trapped on the insulin-immobilized glass plate. Then hydrogen peroxide generated by the enzymatic reaction with the trapped conjugate was coulometrically determined as follows. The hydrogen peroxide generated oxidized [Os(bpy)(2)Cl](+) to [Os(bpy)(2)Cl](2+) in a horseradish peroxidase (HRP)-containing polymer layer on an electrode under the open-circuit condition, and then the [Os(bpy)(2)Cl](2+) accumulated was reduced by applying a negative potential. The accumulation brought about a large current response and a low detection limit for hydrogen peroxide (0.5 nM): the detection limit was lowered by more two orders of magnitude than that obtained for conventional amperometric method using the same polymer-coated electrode. The enhanced sensitivity for hydrogen peroxide enabled the determination of a trace concentration of insulin with the immunoassay using the antibody modified with a hydrogen peroxide-producing enzyme. The assay time was ca. 30 min. The results suggested the possible application of the proposed method to an on-site diabetic sensing system. (c) 2008 Elsevier B.V. All rights reserved.

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.

We report here a rapid and separation-free immunoassay using a dielectrophoresis(DEP) device consisting of an interdigitated microarray (IDA) electrode and a polydimethylsiloxane (PDMS) substrate. On applying an AC voltage to the IDA in a negative-DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microbeads moved to the surface of the PDMS substrate placed above the IDA. The microbeads accumulated at designated areas of the PDMS surface that had been precoated with anti-mouse IgG. When the fluorescence microbeads bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microbeads trapped the analyte to form immunocomplexes on microbeads. The microbeads reacted with mouse IgG accumulated and were captured at the designated areas of the PDMS surface via an antibody-antigen-antibody (sandwich) reaction. The captured microbeads were detected selectively by fluorescence measurements at the focused designated areas, regardless of the presence of uncaptured microbeads suspended in 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, a period as short as 30 s was sufficient to detect the immunoreaction at the surface. The fluorescence intensity of the captured microbeads at the designated areas increased with analyte concentration in the range 0.01 -10 ng/mL The present procedure therefore yields a rapid, sensitive, and separation-free immunoassay in a simple device. (C) 2008 Elsevier B.V. All rights reserved.

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 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.

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.

A coulometric signal accumulation system to detect a trace concentration of hydrogen peroxide (H2O2) has been applied to develop a novel electrochemical enzyme immunosensing system for insulin, an important hormone for clinical diagnosis of diabetes. Glucose oxidase (GOx)-labeled anti-insulin IgG antibody was trapped on the substrate immobilized with insulin by an indirect competitive immunoassay. H2O2 generated by the enzymatic reaction with the trapped conjugate was detected by the coulometric signal accumulation system. The H2O2 generated oxidized [Os(bpy)2Cl]+ to [Os(bpy)2Cl] 2+ in a horseradish peroxidase (HRP)-containing polymer layer on an electrode under the open-circuit condition, and then the [Os(bpy) 2Cl]2+ accumulated was reduced by applying a negative potential. The accumulation brought about a large current response and a low detection limit for H2O2 (0.5 nM). The enhanced sensitivity for H2O2 enabled the determination of a trace concentration of insulin with the immunoassay using the antibody modified with GOx, a H2O2-producing enzyme. © The Electrochemical Society.

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.

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.

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.

Scanning electrochemical microscopy (SECM) was applied to a dual enzyme immunoassay for the detection of pepsinogen 1 (PG1) 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 PG1 and PG2 spots. Relationships between the reduction current in the SECM images and the concentrations of PG1 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 μm physical barrier on the substrate. Pyramidal hole arrays with 100 μm × 100 μm 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. © 2007 Elsevier B.V. All rights reserved.

ニトロセルロース膜を抗体の固定化担体として用いた酵素免疫測定法（enzyme-linked immunosorbent assay）により膜に捕捉された標識酵素の活性を電気化学的に計測しテストステロンの定量検出を行った．膜に抗テストステロン抗体を固定化し，テストステロン及び西洋わさびペルオキシダーゼ（horseradish peroxidase，HRP）標識テストステロンを競争的に反応させた．電子伝達メディエーターであるフェロセンメタノール及びHRPの基質である過酸化水素を添加すると，捕捉された標識HRPの酵素反応により酸化型フェロセンメタノール（Fc<sup>＋</sup>OH）が生成される．金ディスク電極を用いて生成されるFc<sup>＋</sup>OHをアンペロメトリックに計測した．電極先端及び膜表面間の距離を常に一定に保持することが可能な電気化学測定用チャンバーを自作し，抗体を固定化したニトロセルロース膜及び金ディスク電極を設置した．ニトロセルロース膜の種類，抗体固定化の際に介在させるウシ血清アルブミン濃度，競合物質であるHRP標識テストステロン濃度及び電気化学計測における緩衝液の種類について検討し，条件の最適化を行った．得られた最適条件を用いてテストステロンを競合的に捕捉し，HRPの酵素活性を電気化学的に検出することによりテストステロンの定量を行った．テストステロン濃度の増加に伴い，還元電流応答の減少が観測され0.5～20 ng/mLの濃度範囲において相関が得られた．この手法を用いると，通常のウェルプレートを用いた発色法と比較して1けた高い感度が得られた．<br>

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.

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.

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.

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5 alpha was applied for the screening of recombinant protein solubilities. The a-fragment of beta-galactosidase (beta Gal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular Peal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response' obtained using SECM increased with an increase in the beta Gal activity and therefore; with the soluble fraction of MBP in the host cells.

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 degrees 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-microwefls 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.

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 ∼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. © 2006 Elsevier Ltd. All rights reserved.

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.

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<sup>+</sup>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<sup>+</sup>OH was detected with a microelectrode at 0.05 V <i>vs</i>. 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 mm 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.<br>

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.

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.

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.

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.