安川 智之

The rapid manipulation technique with positive dielectrophoresis (p-DEP) was applied to the formation of single-cell pairs in a microwells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower microwell array electrode fabricated on an ITO substrate. Myeloma cells were trapped within 1 s in the microwells restricted the size to two vertically aligned cells by p-DEP. Different types of cells were paired within 1 min and a pairing efficiency of approximately 50% was achieved. The use of the present device allows large numbers of cell pairs (over 5,000 pairs) with the vertical alignment.

We present single-cell pairing of different types of cells with a rapid manipulation based on positive dielectrophoresis (p-DEP). The DEP device for the manipulation of cells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO electrode with 10,000 (100 100) microwells. The width (14 mu m) and depth (25 mu m) of the individual microwells restricted the size to two vertically aligned cells. Cells stained in blue and stained in green were continuously trapped in the microwells. Cells were paired within only 1 min and a pairing efficiency of 53% was achieved.

Rapid conversion of the line formation with the cells based on dielectrophoresis (DEP) was applied to simple and rapid distinction of cells with specific surface antigens from a cell population. The gap area of the interdigitated band array (IDA) electrode was modified with anti-CD33 antibody. Human promyelocytic leukemia cells (HL-60) which express CD33 surface antigen were accumulated on the surface in the gap area between both bands of the IDA electrode by negative DEP (n-DEP). Switching of the applied voltage of the band electrode brought about the removal of accumulated cells to form another pattern due to the formation of the different pattern of the electric field in the device. The modification with the antibody inhibits the removal of the cells with specific surface antigen due to the irreversible capture with immunoreactions during the first pattern formation. Therefore, we should discriminate the cells with specific antigen from the cell remained on the gap area. The time required for the assay was substantially short, 60 s for forcing and 60 s for the separation of unbounded cells. Furthermore, the present method does not require pretreatment such as target labeling or washing of unbound cells.

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.

Based on the substitutional stripping voltammetry (SSV) that is a highly sensitive electrochemical measurement, we have developed a new measurement system employing a gel made from hydrophobic ionic liquid and polymer. Since a salt bridge and an auxiliary electrochemical solution were necessary to conduct SSV, the conventional measurement system was somewhat complicated. The problem has hindered the application of SSV in compact chemical sensors. A gel coating on the silver electrode could replace both the salt bridge and the auxiliary electrochemical solution. As a result, all electrodes for SSV could be prepared on a planar substrate and the amount of the sample solution was enormously reduced to only 30 mu l. The conventional SSV consists of two electrochemical steps: pre-electrolysis and stripping. In order to shorten the duration of measurement, the potential shifts of the auxiliary electrode were monitored before and after the pre-electrolysis to skip stripping, maintaining the sensitivity equivalent to the conventional SSV. (C) 2013 Elsevier Ltd. All rights reserved.

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.

We report on control of line pattern positioning with particles fabricated by negative dielectrophoresis (n- DEP) using the applied intensity and phase of an AC electric field. Line patterns were fabricated in a microfluidic device consisting of upper conductive indium-tin-oxide (ITO) substrates and lower ITOinterdigitated microband array (IDA) electrodes used as the template. A 6-μm-diameter polystyrene particles suspension was introduced into the device between upper ITO and the bottom ITO-IDA substrate. An AC electric signal of a typically 20 peak-to-peak voltage and 1.0 MHz was then applied to upper ITO and bands on lower IDA, resulting in the formation of line patterns with low electric-field gradient regions. AC voltage was applied to bands A and B on lower IDA with the opposite phase and the same frequency and intensity. When the signal identical to band A was applied to upper ITO, particles were aligned above band A because relatively lower electric fields were produced in these regions. In contrast, the application of a signal identical to band B formed line patterns with particles aligned above band B due to the generation of a strong electric field between band A and upper ITO and the disappearance of the strong electric field between band B and upper ITO. The decrease in applied intensity to upper ITO shifted the accumulated position of particles to the center between bands A and B because of the balance of electric fields generated between band A or B and upper ITO. We thus fabricated line patterns with particles at desired positions in the fluidic device.

We investigated oxidation and reduction responses of hydrogen peroxide using a carbon electrode with platinum particles deposited by cathodic reduction of cis-diamminedichloroplatinum（II）（cisplatin）. Platinum deposition led to a marked increase of oxidation and reduction currents for hydrogen peroxide compared to currents observed using the carbon electrode without platinum. The reduction response is particularly useful for determination of cisplatin because the electrochemical reaction forms no bubbles.

We applied a rapid and simple fabrication method of the island pattern of particles and cells to discriminating cells with specific surface antigen. The Upper interdigitated microband array (IDA) electrode was mounted on the lower substrate with the same design to fabricate a microfluidic-channel device for dielectrophoretic manipulation. Grid formation of electrodes was fabricated by rotating the upper template IDA by 90 degrees relative to the lower IDA. A suspension of particles modified with anti-CD33 antibody or/and HL60 cells was introduced into the channel. AC electric signal (typically 20 V peak-to-peak, 100 kHz) 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. The accumulated particles and cells were fixed to produce the complexes through the immunoreactions between the antibody immobilized on the particle and CD33 on the cell surface. The complexes were only produced by the corresponded pair of antigen-antibody. It is noted that the time required for single sensing is as short as 6 min in the presented procedure. Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells.

Antibody spots were used to demonstrate that cells with a specific antigen on the membrane can be captured selectively and simply. By dropping a few microliters of solution containing antibody, antibody spots were prepared on three substrates: glass modified with amino group, glass modified with amino group with high density, and polystyrene substrate. Cells with specific surface antigen were captured on the antibody spots after the substrates were washed, but almost no cell without antigen was captured. The densities of cells captured on spots were estimated. Results show that the highest density occurred on the polystyrene substrate. Almost equal densities of captured cells were obtained for spots prepared using various volumes of dropped solutions（0.5-5.0 μL）. Mixtures of cells with and without surface antigen were dropped on the antibody spot to investigate the selective capture capability. When cells with antigen were treated with fluorescent molecules, fluorescent signals were observed from almost all captured cells. In contrast, no captured cell displayed fluorescence when cells without antigen were treated. The results indicate that the cells with antigen were captured on the antibody spots by an immunorecognition event between the antibody on the substrate and the antigen expressed on the cell membrane. The method does not require target labeling. Moreover, a single assay was completed in as little as 10 min. The present simple and rapid method might prove useful in widely various characterization applications for surface antigens on cell membranes. Results show that human acute monocytic leukemia cell line with the surface antigen C3b receptor can be captured with anti-C3b antibody.

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 report the fabrication of two different cell patterns based on negative dielectrophoresis (n-DEP) and apply it to simple and rapid distinction of cells with specific surface antigens from a cell population. The DEP device for cell manipulation comprised a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO-interdigitated band array (ITO-IDA) electrode modified with an antibody. Cells immediately accumulated on the surface in the gap area between both bands of the ITO-IDA electrode by n-DEP upon AC voltage between the upper ITO and both lower bands. Switching of the applied band electrode voltage resulted in the removal of accumulated cells to form another pattern because of the formation of a different electric field pattern in the device. Modifying the ITO-IDA surface with the antibody inhibited the removal of the cells with a specific surface antigen for irreversible capture by immunoreactions during the first accumulation. In this study, we targeted the CD33 surface antigen expressed on human promyelocytic leukemia cells (HL-60). The time required for the assay was substantially short: 60 s for forcing and 60 s for separating the unbound cells. Furthermore, the present method does not require pretreatment such as target labeling or washing of unbound cells. Moreover, the use of the swing technique considerably improved cell binding to the antibody-modified surface for cells with a specific surface antigen. The distinct integration of cells with n-DEP in the high conductivity medium provided higher cell binding efficiency compared to that obtained in our previous study (Hatanaka, H.; Yasukawa, T.; Mizutani, F. Anal. Chem., 2011, 83, 7207-7212) without loss of rapidity and simplicity.

We have demonstrated the electrochemistry of a redox active species, Fe(CN)(6)(4-), flowing in a nitrocellulose membrane incorporated into two plates with a pole array and electrodes, and applied it by measuring the activity of an enzyme, glucose oxidase (GOx), captured by immunoreaction to develop an electrochemical immunochromatographic format. The flow in the membrane between the flat surfaces with electrodes is unstable because the solution seeps into the surfaces by capillary force and the substrate support and electrode materials have different wettabilities. Thus, electrochemical responses obtained were also unstable. we prepared the small pole array and incorporated a microelectrode in a pole to diminish the total contact area used to support the membrane. The solution flowed uniformly and stably in the membrane supported on the plates with the pole array, and a steady-state oxidation current of Fe(CN)(6)(4-) was observed by amperometry. The mouse IgG used as a model target was captured in the antibody immobilization area prepared in the membrane. Subsequent flowing of GOx to conjugate the antibody allowed the labeling of the captured target by the formation of a sandwich-type immunocomplex. Fe(CN)(6)(4-) produced through the enzyme reaction in the presence of glucose flowed downstream to give an electrochemical response with the electrode arranged 2 mm from the antibody immobilization area. Electrochemical detection of redox species flowing in the membrane indicated the possibility for developing a single-step immunochromatographic assay format with electrochemical quantitation. (c) 2012 Elsevier Ltd. All rights reserved.

In this work, a novel immunosensing method has been developed on the basis of the sensitive determination of a product generated by an enzyme reaction with dual amplification system combining an electrochemical-redox cycling and coulometric signal transduction using a galvanic cell. Analytes were captured on microparticles to form sandwich-type immunocomplexes and then labeled with beta-galactosidase (beta-gal). 4-Aminophenol (PAP) produced by enzyme reaction of beta-gal was introduced into the anode compartment consisting of a comb type of an interdigitated array (IDA) electrode. PAP was oxidized at the IDA electrode by the coupled reduction of silver ions at the glassy carbon (GC) electrode of the cathode, resulting in the deposition of silver metal on the GC electrode. The other comb of the IDA electrode was used to reduce quinoneimine generated by the oxidation of PAP, regenerating PAP. The deposited silver was collectively converted to a signal by anodic stripping voltammetry. The amount of silver deposited corresponded to the degree of PAP oxidation by redox cycling, which leads to an enhancement of the stripping signal due to the conversion of the product (PAP) and accumulation of the insoluble silver metal. Using carcinoembryonic antigen as a model analyte, the present immunosensing method showed linear behavior over two orders of magnitude with detection limits down to 0.01 ng/mL. Dual signal amplification with redox cycling and coulometric signal transduction provides a promising, sensitive, and simple method for the determination of marker proteins. (C) 2012 Elsevier B.V. All rights reserved.

In this paper, we describe a development of the rapid and simple discrimination system of cells with specific surface antigen by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). The time required for the determination of the surface antigen is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells.

The rapid and simple discrimination system of cells with specific surface antigen was developed based on the pattern switching of cellular array formed by the positive and negative dielectrophoresis (p-DEP and n-DEP). p-DEP was used to accumulate the cells and capture by the immunoreactions between the specific surface antigen and antibody immobilized on the surface, while n-DEP was to remove of unbound cells. The time required for the determination of the surface antigen is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells.

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.

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.

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 mu m- and 20 mu m-diameter particles was introduced into the channel with 30 mu m height at 700 mu m/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 forming the flow paths with different electric fields. The microparticles separately flow in line along the paths formed between the band electrodes, the 10 mu m-diameter particles mainly flow through the narrow path and 20 mu m-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.

We describe an improvement of the detectable range of glucose oxidase (GOx)-based microsensors to higher concentrations. Oxygen consumed by the enzyme reaction of GOx was detected cathodically by the microelectrode modified with poly(dimethylsiloxane) (PDMS) and GOx layers. PDMS was used to prevent the permeation of hydrogen peroxide that was produced by the enzyme reaction. The prepared microsensors were positioned above microband electrodes which were used to produce oxygen by the electrolysis of water, allowing a high oxygen concentration (0.8 mM) to be maintained around the microsensors. By applying this system, the detectable concentration range for glucose was extended up to 20 mM. (C) The Electrochemical Society of Japan, All rights reserved.

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. © 2012 The Japan Society for Analytical Chemistry.

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.

In this study, a rapid immunosensing system has been developed for simultaneous analysis of two tumor markers, alpha-fetoprotein (AFP) and prostate-specific antigen (PSA). The strategy for rapid multisensing is based on rapid immunoreactions occurring on the surface of microparticles and the spatial separation of different particles that exhibit distinct dielectrophoretic (DEP) properties. Recognition events for immunoreactions have been performed on the surfaces of two different microparticles conjugated with two different antibodies: polystyrene (PS) microparticles with an anti-AFP antibody and gold-coated (50 nm) PS microparticles with an anti-PSA antibody. The DEP devices consisted of an upper indium tin oxide (ITO) glass and a lower ITO electrode with a castellated structure. Sandwich structured immunocomplexes of AFP and PSA were created on the microparticles and then labeled with fluorescent molecules via a secondary antibody. After introducing the particles into the DEP devices, an alternating current (AC) voltage (20 V peak-to-peak voltage and 30 kHz) was applied between the upper ITO and lower electrodes to manipulate the particles with negative dielectrophoresis (n-DEP). The uncoated PS particles and the gold-coated PS particles rapidly moved and separated to form wave-like line and triangular aggregates, respectively. The measurements of the fluorescence signals from the uncoated and gold-coated PS particles directed to different regions of the DEP device permit the determination of the concentrations of AFP and PSA simultaneously. No cross-reactivity was observed for either of the immunorecognition events. Limits of detection achieved for the AFP and PSA assays were 0.18 and 1.1 ng mL(-1), respectively, which satisfy medical requirements for both antigens in human serum. The total assay time required for the simultaneous detection of the two different analytes in this study (25 min) was shortened compared to the conventional enzyme-linked immunosorbent assay. (C) 2011 Elsevier B.V. All rights reserved.

Rapid determination of surface antigens on cells is possible by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). The DEP device for cell manipulation comprises a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO microband array electrode (band electrode) modified with an antibody. Cells with the surface antigen introduced into the channel immediately accumulated on the surface of the band electrode during p-DEP generated by the application of ac voltage between the ITO electrode and the band electrode to immobilize by the specific antibody. The removal of accumulated cells to the gap region during n-DEP was used for rapid estimation of the residual cells with a specific surface antigen. We demonstrate here that human promyelocytic leukemia cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody. The time required for the determination of the surface antigen using this compelled accumulation of cells by p-DEP and the separation of unbound cells by n-DEP is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells and thereby enhancing throughput in the clinic and in cytobiology studies.

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.

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.

In this work, we designed a new immunodevice that combines competitive immunoreactions on the microparticles, accumulation of these particles' by negative dielectrophoresis (n-DEP), and their subsequent capture through hybridization among single-stranded DNAs (ssDNAs). Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to test the resulting simultaneous detection system. For sensing, we prepared two different sets of microparticles: one modified with atrazine-conjugated bovine serum albumin (BSA-2d) and ssDNA-J1(up) and the other with bromopropylate-conjugated aminodextran (AD-155) and ssDNA-J2(up). The microparticles were incubated in a mixture of analyte-specific antibody and analyte at different concentrations to trap the unreacted antibodies prior to being labeled with antibodies conjugated with a fluorescence molecule. A suspension containing both types of microparticles was introduced into an n-DEP device consisting of an interdigitated microarray (IDA) electrode and channel modified with ssDNA-J1(down) and ssDNA-J2(down) which are complementary to ssDNA-J1(up) and ssDNA-J2(up), respectively. The n-DEP force generated by applying AC voltage to the IDA electrode displaced the microparticles toward the encoded areas, causing them to rapidly accumulate on the upper surfaces. Hybridization allowed us to distinguish the microparticles and sense multiple analytes by spatial recognition in the DNA encoded areas. The fluorescence intensity of the captured particles, which depends on analyte concentrations, was measured selectively by focusing on specific areas. The strategy is advantageous for sensitivity dui: to the equivalent trapping efficiency by DNA hybridization and large surface area of the microparticle for immunoreactions. The rapidity and simplicity were still supported by particle manipulation. Using this concept, we detect atrazine and bromopropylate simultaneously with limits of detection (LODs) of 0.2 mu g . L-1, Which covered the maximum residue level (MRL) in food samples established the European Union (EU) and Japan Ministry of Health, Labor and Welfare (MHLW).

Simple and rapid detection of surface antigens on cells is possible by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). Cells with the surface antigen accumulated on the band electrode modified with specific antibody during p-DEP generated by the application of AC voltage between the indium tin oxide (ITO) electrode and the band electrode. The removal of accumulated cells to the gap region during n-DEP was used for rapid estimation of the residual cells with a specific surface antigen. We demonstrate here that human promyelocytic leukemia cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody. The time required for the determination of the surface antigen is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). The present method does not require pretreatment such as target labeling or washing of unbound cells.

An anti-fouling ability of diamond-like carbon (DLC) electrodes to biological macromolecules has been investigated from a decrease in the electrochemical redox current of Fe(CN)(6)(4-/3-), used as a redox marker. A DLC electrode and a glassy carbon (GC) electrode were immersed in a solution containing bovine serum albumin (BSA) or DNA. The GCs treated with biological macromolecules gave rise to a significant decrease in the currents, while there was no signal decreases from the treated DLCs. The signals from the DLCs remain essentially unchanged for at least 24 h at a 10 mg/mL concentration level of BSA.

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.

In this work, we have developed glucose biosensors based on chemical amplification with glucose cycling between glucose oxidase (GO<sub>x</sub>) and glucose dehydrogenase (GDH) immobilized on a poly(dimethylsiloxane) (PDMS) layer. PDMS-coated platinum electrodes modified with GO<sub>x</sub> and GDH have been prepared from poly(L-lysine) (polymer backbone) and glutaraldehyde (cross-linking agent). The reduction currents of oxygen were amperometrically monitored by adding glucose. In the presence of NADH, D-glucono-<i>δ</i>-lactone generated by glucose oxidation with an enzyme reaction of GO<sub>x</sub> is reduced back to glucose by the enzyme reaction of GDH. Since the reproduced glucose acts as a substrate of GO<sub>x</sub> again, the current response for oxygen is amplified by oxygen consumption with GO<sub>x</sub>. The PDMS layer with selective permeability for oxygen was used to prevent interference from hydrogen peroxide produced by the GO<sub>x</sub> reaction and the others in biological samples. The current response was amplified around 10 times and appeared at a concentration as low as 1.0 μM by using the present system. The glucose concentration could be determined in 500-fold diluted human serum in the presence of 5.0 mM NADH.

We report determination of the apparent Michaelis constant of glucose oxidase (GOx) immobilized on a microelectrode with respect to oxygen. We used a GOx-modified microelectrode as a probe for scanning electrochemical microscopy. We detected hydrogen peroxide generated by the enzyme reaction at the microelectrode under controlling the oxygen concentration using water electrolysis at an interdigitated array (IDA) electrode. The response depends on the oxygen concentration, which is regulated by the microelectrode position and the potential applied to the IDA electrode. We estimated the apparent Michaelis constant with respect to oxygen in this experimental condition to be about 0.28 mM.

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 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.

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.

A novel immunosensor is presented based oil a charge accumulation system using all electrode modified with a polymer containing [osmium(4,4'-dimethyl-2,2'-bipyridine)(2)-chloride](+/2+) and horseradish peroxidase (Os/HRP polymer) to detect N-1,N-12-diacetylspertnine (DiAcSprn). Glucose oxidase (GOx)-labeled antibody captured with immunoreaction on Os/HRP polymer catalyzed the production of hydrogen peroxide, which oxidizes [Os(bpy)(2)Cl](+) in polymer with HRP. [Os(bpy)(2)Cl](2+) was gradually accumulated in the polymer and reduced back to [Os(bpy)(2)Cl](+) by applying -0.1 V. This accumulation system of redox species allows enhancement of the response.

We prepared a microfluidic device with an interdigitated array electrode modified with antibodies to develop immunosensors. A comb-type array (W1) 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 W1. 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.

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.

Microcontact printing (μCP) with various proteins has been widely applied to biosensors and cell biology research. However, the mechanism of protein patterning in the μCP process is not clear in detail We have electrochemically estimated enzyme concentration on glass slide patterned with μCP 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 μCP. Our result suggested that it was possible to transfer enzyme monolayer from PDMS to glass surfaces with 100% efficiency with μCP. Immunoglobulin G (IgG) patterned with μCP 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.

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.

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.

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.

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.

Poly(dimethylsiloxane) (PDMS)-coated platinum electrodes modified with glucose oxidase (GOx) have been prepared from Poly(L-lysine) (polymer backbone), glutaraldehyde (cross-linking agent) and poly(ethylene glycol) units. To fabricate a GOx layer by applying cross-linking chemistries, the PDMS layer was treated with oxygen plasma to replace silane groups with silanol groups. Optimization for the chemical fabrication of a GOx layer resulted in a simple preparation of sensors with a wide detectable range (0.1 - 6.0 mM) and without interference from hydrogen peroxide produced by a GOx reaction and the other in biological samples.