Masato Suzuki

Many sexually mature women experience premenstrual syndrome (PMS) or premenstrual dysphoric mood disorder (PMDD). Current approaches for managing PMS and PMDD rely on daily mental condition recording, which many discontinue due to its impracticality. Hence, there's a critical need for a simple, objective method to monitor mental symptoms. One of the principal symptoms of PMDD is a dysfunction in emotional regulation, which has been demonstrated through brain-function imaging measurements to involve hyperactivity in the amygdala and a decrease in functionality in the prefrontal cortex (PFC). However, most research has been focused on PMDD, leaving a gap in understanding of PMS. Near-infrared spectroscopy (NIRS) measures brain activity by spectroscopically determining the amount of hemoglobin in the blood vessels. This study aimed to characterize the emotional regulation function in PMS. We measured brain activity in the PFC region using NIRS when participants were presented with emotion-inducing pictures. Furthermore, moods highly associated with emotions were assessed through questionnaires. Forty-six participants were categorized into non-PMS, PMS, and PMDD groups based on the gynecologist's diagnosis. POMS2 scores revealed higher negative mood and lower positive mood in the follicular phase for the PMS group, while the PMDD group exhibited heightened negative mood during the luteal phase. NIRS results showed reduced emotional expression in the PMS group during both phases, while no significant differences were observed in the PMDD group compared to non-PMS. It was found that there are differences in the distribution of mood during the luteal and follicular phase and in cerebral blood flow responses to emotional stimuli between PMS and PMDD. These findings suggest the potential for providing individuals with awareness of PMS or PMDD through scores on the POMS2 and NIRS measurements.

Many sexually mature females suffer from premenstrual syndrome (PMS), but effective coping methods for PMS are limited due to the complexity of symptoms and unclear pathogenesis. Awareness has shown promise in alleviating PMS symptoms but faces challenges in long-term recording and consistency. Our research goal is to establish a convenient and simple method to make individual female aware of their own psychological, and autonomic conditions. In previous research, we demonstrated that participants could be classified into non-PMS and PMS groups based on mood scores obtained during the follicular phase. However, the properties of neurophysiological activity in the participants classified by mood scores have not been elucidated. This study aimed to classify participants based on their scores on a mood questionnaire during the follicular phase and to evaluate their autonomic nervous system (ANS) activity using a simple device that measures pulse waves from the earlobe. Participants were grouped into Cluster I (high positive mood) and Cluster II (low mood). Cluster II participants showed reduced parasympathetic nervous system activity from the follicular to the menstrual phase, indicating potential PMS symptoms. The study demonstrates the feasibility of using mood scores to classify individuals into PMS and non-PMS groups and monitor ANS changes across menstrual phases. Despite limitations such as sample size and device variability, the findings highlight a promising avenue for convenient PMS self-monitoring.

Surface plasmon resonance is an optical phenomenon that can be applied for label-free, real-time sensing to directly measure biomolecular interactions and detect biomarkers in solutions. Previous studies using plasmonic nanohole arrays have monitored and detected various biomolecules owing to the propagating surface plasmon polaritons (SPPs). Extraordinary optical transmission (EOT) that occurs in the near-infrared (NIR) and infrared (IR) regions is usually used for detection. Although these plasmonic nanohole arrays improve the sensitivity and throughput for biomolecular detection, these arrays have the following disadvantages: (1) molecular diffusion in the solution (making the detection of biomolecules difficult), (2) the device fabrication's complexities, and (3) expensive equipments for detection in the NIR or IR regions. Therefore, there is a need to fabricate plasmonic nanohole arrays as biomolecular detection platforms using a simple and highly reproducible procedure based on other SPP modes in the visible region instead of the EOT in the NIR or IR regions while suppressing molecular diffusion in the solution. In this paper, we propose the combination of a polymer-based gold nanohole array (Au NHA) obtained through an easy process as a simple platform and dielectrophoresis (DEP) as a biomolecule manipulation method. This approach was experimentally demonstrated using SPP and LSPR modes (not EOT) in the visible region and simple, label-free, rapid, cost-effective trapping and enrichment of nanoparticles (trapping time: <50 s) and bovine serum albumin (trapping time: <1000 s) was realized. These results prove that the Au NHA-based DEP devices have great potential for real-time digital and Raman bioimaging, in addition to biomarker detection.

Simultaneous electrorotation in microwells during chemical stimulation label-free monitoring effect of chemicals in single-cell manner.

A cascade of the formation of cell arrays, the discrimination of cells secreting specific molecules, and the selective retrieval of cells has been developed to harvest antibody-secreting hybridomas in heterogeneous cell populations simply and rapidly. The microwell array device consisted of three-dimensional microband electrodes by assembling both upper and lower substrates perpendicularly. Arrays of hybridomas secreting specific antibodies were prepared by aligning hybridomas in each microwell based on the attractive force of positive dielectrophoresis (p-DEP). Antibody secreted by the hybridomas in the microwells was recognized by the antigen immobilized on the microwells or the membrane surfaces of hybridomas to discriminate hybridomas with the secretion ability. Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was applied to release the target hybridomas from the microwell array. To harvest the target hybridoma, AC signals could be modulated in the n-DEP frequency region and applied to a pair of microband electrodes located above and below each microwell containing target hybridoma. Thus, the cell-based array system described in this study allowed selective retrieval of single target hybridomas by merely switching from p-DEP to n-DEP after selecting the antibody-secreting hybridomas trapped in each microwell. The development of this high-affinity device could be useful to recover hybridomas producing antibodies in large populations of cells rapidly and effectively.

多くの成人女性は,月経前症候群(PMS)や月経前不快気分障害(PMDD)を経験する.PMSやPMDDの症状は多岐にわたり,正確な診断はむずかしい.本研究では,Profile of Mood States 2nd Edition短縮版(POMS2)を用いて,健常女性とPMSもしくはPMDDの判別を目的とした.対象は,20～22歳の女性(PMDD群3名,PMS群5名,non-PMS群11名)である.POMS2の得点に対してWard法によるCluster分析を行った.卵胞期では2つのClusterがみられた.Cluster1に分類された被験者は9人のNon-PMSで構成され,「友好」と「活気-活力」において高得点であった.一方,Cluster2はPMSまたはPMDDを有するすべての被験者と,軽度のPMSの可能性がある2人のNon-PMSで構成された.Cluster1は9名のnon-PMS群,Cluster2はすべてのPMS群とPMDD群から構成された.黄体期でも2つのClusterがみられたが,両Clusterにもnon-PMS群が存在した.以上より卵胞期のPOMS2の得点から健常女性とPMSもしくはPMDDの判別の可能性が示唆された.(著者抄録)

This paper reports a superiority of the asymmetric electric field formed in the rectangle microwell array for the electrofusion of splenocytes and myeloma cells with different diameters. The upper substrate with microband electrodes was mounted on the lower substrate with the microwell array. Two electrodes were arranged at the both sides of the microwells on the bottom surface. An attractive force of positive dielectrophoresis was employed to capture splenocytes with smaller diameter and myeloma cells with larger diameter at the right and left of microwells by applying AC electric field. The splenocytes and myeloma cells were fused by the asymmetric electric field that was generated in the microwells by applying DC electric pulse to the bottom electrode at the right side. The asymmetric field could allow to the formation of small openings on the membrane for the fusion of smaller splenocytes by experiencing higher field and the suppression for the disruption of larger myeloma cells by experiencing lower field.

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

We proposed selective manipulation techniques for retrieving and retaining target cells arrayed in microwells based on dielectrophoresis (DEP). The upper substrate with microband electrodes was mounted on the lower substrate with microwells based on the same design of microband electrodes by 90 degree relative to the lower substrate. A repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target cells from the microwell array selectively. Furthermore, the target cells were retained in the microwells after other cells were removed by n-DEP. Thus, the system described in this study could make it possible to retrieve and recover single target cells from a microwell array after determining the function of cells trapped in each microwell.

Introduction: We have developed manipulation techniques to form cell-based arrays by positive dielectrophoresis (p-DEP) and to retrieve target cells from cell−based arrays selectively by negative dielectrophoresis (n-DEP). The novel devices with microwell arrays on microband electrodes were employed to manipulate cells. Hybridomas with the secretion ability of antibodies were trapped to form cell-based arrays. Then, the ability was discriminated by trapping the antibodies to the antigen immobilized on the bottom of wells. Finally, individual cells trapped in microwells were selectively retrieved by regulating the generation of electric fields in individual microwells. The development of series of these techniques could be useful to recover hybridomas producing antibodies with high affinity in large populations of cells without repeated steps of a culture and a limiting serial-dilution. Experimental: The device comprises the upper substrate with indium-tin-oxide (ITO) microband electrodes and the lower substrate with microwell arrays on ITO microband electrodes. Hybridomas producing an anti-rabbit serum albumin (RSA) antibody suspended in the DEP medium were introduced in the channel. AC signal (3 MHz, 2 Vpp) was then applied to the upper and lower microband electrodes with opposite phase to form a cell-based array. Anti-RSA antibodies secreted from hybridomas trapped in the microwells were captured by RSA immobilized on the electrodes. Cell arrays were then treated with anti-mouse IgG antibody conjugated with Alexa 488 (a secondary antibody). Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target hybridomas from the microwell array. To retrieve the target hybridoma, an AC signal in the n-DEP frequency region was applied to a pair of microband electrodes above and below the microwell with the target hybridoma. Results and discussion: Cell−based arrays were formed with the occupancy efficiency of over 90% in a few seconds by p-DEP. Antibodies secreted in microwell arrays were captured to discriminate target hybridomas in a few hours without repeated steps of a culture and a limiting serial-dilution. Furthermore, hybridomas trapped in microwells were retrieved from the cell−based array by applying an AC signal to band electrodes. The sequential system for forming cell−based arrays, discriminating hybridomas secreting specific antibodies, and retrieving target hybridomas was developed by using the novel microwell array device comprising 3-D microband array electrodes with an orthogonal arrangement.

Introduction Electrical properties of cells (i.e. membrane capacitance and cytoplasm conductivity) have been studied to understand the complex physiological states of cells and used as markers for determining cell types. Electrorotation (ROT) that is one of the alternating current electrokinetic phenomena has been utilized to characterize the electrical properties of single cells. In ROT techniques, quadrupole electrodes were conventionally employed to induce a rotating electric field at the center of them by applying sine waves with 90° differences of each phase. However, a relatively long experimental period was required because several single cells must be repeatedly arranged to a center of quadrupole electrodes. In this presentation, we demonstrated the simultaneous ROT measurement of K562 cells by using the 3D-interdigitated array (3D-IDA) device to determine the membrane capacitances and cytoplasm conductivities of the different types of cells [1]. The rotating electric fields in over 1,500 grids were simultaneously generated by this device to obtain the distribution of rotation rates of over 50 cells with single operation [2]. The grid is defined as regions surrounded by four microband electrodes. We have also developed a new 3D-IDA device with microwells to maintain the cells in them during a chemical stimulation by a fluid flow. We investigated ROT rates accompanied with the changes of the dielectric properties of cells during the stimulation with an activator regent for Jurkat cells. This is the first report to monitor the changes of ROT rates upon chemical stimuli to investigate the effect of chemical agents to cellular function. Experimental Methods Construction of 3D-IDA device The device consisted of two IDA electrodes (20 μm in width, 30 μm gaps) made from indium tin-oxide (ITO). The IDA electrode was overlaid on another IDA electrode by an orthogonal arrangement via a double-sided adhesive film (30 μm in thickness) (Fig. A). A constant rotational electric field was generated in the grids by applying AC voltages with 90° differences of each phase to four microband electrodes. Construction of 3D-IDA device with microwells Microwells with rectangle shape (26 mm in long side and 20 mm in short side) were fabricated with an insulative photoresist (20 mm in thickness) on the gaps of ITO-IDA electrodes. Two ITO-electrodes (3 µm × 20 µm) were arranged at both short sides on the bottoms of microwells. Two gold electrodes (3 µm × 20 µm) were also arranged at both long sides on the insulative photoresist (Fig. B). Results and Conclusions Simultaneous ROT measurement of K562 cells using the 3D-IDA device K562 cells were resuspended in a ROT solution with the conductivity of 360 mS m⁻¹ (the mixture of 75% (v/v) of 300 mM mannitol and 25% (v/v) of RPMI 1640 medium) and subjected to ROT measurement by 3D-IDA devices. When AC signals (700 kHz, 5 Vpp) were applied, K562 cells dispersed randomly started to rotate and move to the center of each grid. The ratio of the grids occupied with single cells was 45%, when the cell suspension with the concentration of 4.0 × 10⁷ cells mL-1 was injected (Fig, A). Thus, we could obtain the rotation rates of 450–680 cells (40–60 cells in single images) simultaneously and estimated the ROT rate as 8.53 radian s^-1(Fig. C). Monitoring of ROT behavior of single cells stimulated with calcium ionophore The ROT device with the microwells were filled with Jurkat cells resuspended in the ROT solution adjusted to 5 × 10⁵ cells mL^-1 and incubated for 2.5 min to arrange cells in the microwells. The ratio of microwells occupied with single cells were 27% (Fig. B). When AC voltages (2 Vpp, 300 kHz) with different phases were applied to four electrodes, the cells began to rotate (Fig. D). The average ROT rate of Jurkat cells was estimated and found to be 1.01 radian s^-1. Then, the solution containing 1 mM ionomycin which is well known as an activator for Jurkat cells to lead to the decrease of the membrane capacitance [3] was injected in the device. Although the solution above the microwells was disturbed by the fluidic flow (40 mm s^-1), the cells rotated with the constant rate in the microwells. The ROT rate gradually decreased and reached at 0.73 radian s^-1 40 s after the injection. The result could reflect the decrease of the membrane capacitance due to the activation of Jurkat cells induced by the stimulus of ionomycin. The 3D-IDA device with microwells would be applied to the high through-put screening of drugs and discrimination of highly responsible cells to drugs. References (1) Ino, K.; Ishida, A.; Inoue, K. Y.; Suzuki, M.; Koide, M.; Yasukawa, T.; Shiku, H.; Matsue, T. Electrorotation Chip Consisting of Three-Dimensional Interdigitated Array Electrodes. Sensors Actuators, B Chem.2011, 153 (2), 468–473. https://doi.org/10.1016/j.snb.2010.11.012. (2) Kawai, S.; Suzuki, M.; Arimoto, S.; Korenaga, T.; Yasukawa, T. Determination of Membrane Capacitance and Cytoplasm Conductivity by Simultaneous Electrorotation. Analyst2020, 145 (12), 4188–4195. https://doi.org/10.1039/d0an00100g. (3) Pethig, R.; Bressler, V.; Carswell‐Crumpton, C.; Chen, Y.; Foster‐Haje, L.; García‐Ojeda, M. E.; Lee, R. S.; Lock, G. M.; Talary, M. S.; Tate, K. M. Dielectrophoretic Studies of the Activation of Human T Lymphocytes Using a Newly Developed Cell Profiling System. Electrophoresis2002, 23 (13), 2057–2063. https://doi.org/10.1002/1522-2683(200207)23:13&lt;2057::AID-ELPS2057&gt;3.0.CO;2-X. Figure 1 <p></p>

We developed a simple, rapid, and label-free method to obtain the ratio of cells with a specific surface protein from heterogeneous cell populations, and applied it to estimate the cell differentiation states. The repulsive force of negative dielectrophoresis was used to form the first pattern of HL60 cells on a substrate immobilized with anti-CD13 or anti-CD11b antibody. Next, the patterned cells were converted to form the second pattern by switching the pattern of the electric field. The cells exhibiting a specific protein remained in the original position due to the immunorecognition event, while the unwanted cells that were not bound to the antibody on the substrates could be simply removed. The cell-binding efficiencies of substrates modified with anti-CD13 and anti-CD11b decreased and increased, respectively, with increasing duration of cell culture in medium containing differentiation-inducing agents, including all-trans retinoic acid. This is explained by the downregulation of CD13 and upregulation of CD11b throughout the differentiation process of HL60 cells. Furthermore, the assay was applied to investigate the effects of various differentiation-inducing agents. The total assay time required for discriminating the proteins expressed on the cell surface in each differentiation state was as short as 120 s. No fluorescence label is required for the proposed assay. The method could be useful to estimate the cell differentiation and factors that influence the differentiation trajectory for numerous cell types.

The electrorotation (ROT) rates of K562 cells accompanied by erythroid differentiation were estimated to identify the differentiation status by using a novel electrorotation device with a microwell arranged on polynomial electrodes. Successive estimations of individual cells were achieved by sequential manipulations which involve trapping of the cell by positive dielectrophoresis (DEP), rotating by ROT, and removing by negative DEP. The ROT rate increased with the differentiation of K562 cells, because the cytoplasm conductivity would increase with an increase of the concentration of iron ions to produce hemoglobin. The ROT rate could be utilized to estimate the stage of cell differentiation without labeling.

We have developed the effective electrofusion of cells with different sizes in microwells with a rectangle shape. B-cells were captured by positive dielectrophoresis on the right side of the microwell and myeloma cells on the left side to form pairs. When DC electric pulses were applied to the right electrodes against the left electrode and the upper electrode, B-cells and myeloma cells were fused. On the other hand, no electrofusion was observed, when B-cells and myeloma cells were oppositely arranged. This is due to the steep gradient of the electric field at the right side in the microwells.

Introduction Cell microarrays which sectionalized cells into microwells are powerful tool for elucidating exhaustively functions of cells at single cell level. However, there are some difficulties in taking advantages of the cell array. At first, the “single-cell occupancy”, which is the ratio of microwells containing one cell to total microwell, is low. Typically, the sedimentation of cells with their own weight was used to form cell array, resulting in a low “single-cell occupancy” as low as about 50%. The second difficulty is that it takes time and effort to retrieve the target cell due to the precise and careful manipulation of the micropippet at resolution of micrometer. The broad generalization of cell microarrays requires the development of techniques to efficiently fabricate cell arrays and to easily pick up cells from cell populations. Dielectrophoresis (DEP) has become attractive because it allows for easy, rapid, and mass manipulation of cells. We have previously demonstrated the dielectrophoretic trapping cells in microwells within a few seconds with an occupation efficiency over 95% using a microwell array device. The device was fabricated by placing the upper indium-tin oxide (ITO) substrate on top of the bottom ITO electrode covered with an insulating layer with a microwell pattern. Although this device has the advantage of producing cell arrays rapidly at high density, it did not selectively manipulate the cells on the microwells. In this study, we propose a simple device for flexible dielectrophoretic manipulation of cells based on the combination of positive DEP (p-DEP) and negative DEP (n-DEP). The use of the present device allows for accurate retrieval of the target cells from a microwell array with retaining the undesired on microwells, not to mention the forming of cell arrays. This device was comprised an upper substrate with microband electrodes mounted on a lower substrate with microwells on the same design of microband electrodes by 90 degree relative to the bottom substrate (Fig. A). The layout of electrodes enables to retrieve the target cell by the repulsive force of n-DEP induced by applying an AC voltage to two microband electrodes arranged above and below the microwell containing the target cell. Naturally, cell arrays can also be fabricated by the attractive force to bottom of microwell induced with p-DEP by applying an AC voltage to all microband electrodes. Experiments and Results The upper and lower ITO substrates with patterns of microband array were fabricated by a conventional photolithographic method. Width of the microband electrode and gap between the electrodes were 40 µm and 80 µm, respectively. The pattern of microwell (16 µm in diameter and 10 µm in height) on the microband electrodes on the lower substate was made of a negative photoresist SU-8. Microband array electrodes on the upper substrate were located 30 µm above the microwells on the lower substrate to form 144 intersections comprising microband electrodes containing microwells. Cell mixtures (3.0 × 10⁶ cells/mL) of hybridoma stained in green and red were prepared in a ratio of 10:1 to demonstrate the retrieval of red cells designated as the target cell. The application of the AC signal (1 MHz, 3 Vpp) to both the upper and the lower microband electrodes with opposite phasing resulted in the formation of the cell array. A single red cell was trapped in the 1–G well and eight green cells were trapped in the others (Fig. B). Subsequently, the frequency applied to band electrode 1 on the upper substrate and the band electrode G on the lower substrate was switched from 1 MHz for p-DEP to 300 kHz for n-DEP, while the frequency applied to the other band electrodes was maintained at 1 MHz for p-DEP (Fig. C). The target red cell in 1–G well was gradually removed over a few seconds after switching the frequency, and it was then transferred downward in the image by slight fluidic flow. In contrast, the other green cells remained in the original position. The results indicated that the repulsive force of n-DEP from the strong electric field region acts on the cell in the 1–G well that comprises both band electrodes switched in the n-DEP frequency region. It is noted that p-DEP still acted on cells in wells comprising band electrodes that applied an AC signal in the p-DEP and n-DEP frequency regions, respectively. Thus, this system would make it possible to retrieve target cells selectively from the array of cells and recover them in an outlet without the microdispensers. Figure 1 <p></p>

We provide a novel method to discriminate the cells binding with molecules immobilized on a glass substrate by using the repulsive force of negative dielectrophoresis. The device consists of a lower substrate with interdigitated (IDA) patterns (12 μm in width and 50 μm in gap) of indium-tin oxide (ITO) and an upper ITO substrate disposed on the lower substrate. When an AC voltage is applied to a pair of microband electrodes comprising IDA to the upper ITO substrate connected to the ground, the cells are pressed onto gaps between the microband electrodes, and adhesion of the cells to the substrate is promoted (1st patterning in Fig. A). Subsequently, when an AC voltage is microband electrode “a” against the microband “b” and the upper ITO connected to the ground, the weakest region of the electric field is formed just above the microband “b”, and the cells move there by the repulsive force of negative dielectrophoresis (2nd patterning in Fig. B). At this time, if the cells adhered to the substrate via binding to molecules immobilized on the lower substrate, the cells remain on the gaps. Cells that do not interact with the molecules are expelled on the microband electrode. These procedures provide a rapid method for evaluating the expression of surface proteins that interact with molecules on the lower substrate in as little as 2 min; 60 sec in an adhesion and 60 sec in an expelling. The proposed method was applied to identify the differentiation status of HL60. Undifferentiated HL60 is known to express CD13 but not CD11b, while HL60 differentiated into neutrophils and monocytic lineages is known to up-regulate CD11b expression and down-regulate CD13 expression. In the device pretreated with anti-CD11b antibody, almost all HL60 formed well-defined linear patterns on the gaps between the microband electrodes at the 1st patterning by applying the ac voltage (100 kHz in frequency and 20 Vpp in voltage) for 60 seconds and aligned on the microband electrode “b” after 2nd patterning (day 0 in Fig. B). This result suggests that CD11b rarely expressed in HL60 at the state of undifferentiation. It was also confirmed that the expression levels of CD11b were extremely low by immunobiological fluorescent imaging. Conversely, HL60s differentiated with 1 μM all-trans retinoic acid (ATRA), which is well known as a inducer of HL60 to neutrophil, were randomly adhered on the gaps, and only a few of the adhered HL60s moved onto the microband electrodes after 2nd patterning (day 4 inf Fig. B). The increase of number of cells remaining on the gaps in differentiated HL60 represented the upregulation of CD11b expression. The expression of CD11b in differentiated cells cultured in ATRA-containing medium for 4 days was confirmed by immunostaining. There was a good correlation between the expression levels of CD11b obtained by the present method and those obtained by conventional fluorescent method. In addition, the expression of CD13 was investigated using the device pretreated with anti-CD13. In this case, the majority of undifferentiated cells adhered on the gap between the microband electrode and the differentiated cells were expelled from the gap onto the microband electrode “b”. Thus, using the repulsive force of n-DEP to expel cells unbound to the substrate, the differentiation status can be elucidated in as little as 120 seconds. Figure 1 <p></p>

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

Membrane capacitances and cytoplasm conductivities of hematopoietic cells were investigated by simultaneous electrorotation (ROT) systems of multiple cells. Simultaneous ROT was achieved by the rotation of electric fields in grid arrays formed with three-dimensional interdigitated array (3D-IDA) electrodes that can be easily fabricated using two substrates with IDA electrodes. When AC signals were applied to four microband electrodes with a 90° phase difference to each electrode, cells dispersed randomly in the 3D-IDA device started to rotate and moved to the center of each grid. Multiple cells were simultaneously rotated at the center of grids without friction from contact with other cells and substrates. The averages and variance of ROT rates of cells at each frequency can be measured during a single operation of the device within 5 min, resulting in the acquisition of ROT spectra. Membrane capacitances and cytoplasm conductivities of hematopoietic cells (K562 cells, Jurkat cells, and THP-1 cells) were determined by fitting ROT spectra obtained experimentally to the curves calculated theoretically. The values determined by using the simultaneous ROT systems well coincided with the values reported previously. The membrane capacitances and cytoplasm conductivities of WEHI-231 cells were firstly determined to be 8.89 ± 0.25 mF m-2 and 0.28 ± 0.03 S m-1, respectively. Furthermore, the difference of the ROT rates based on the difference of the electric properties of cells was applied to discriminate the types of cells. The acquisition of rotation rates of multiple cells within a single operation makes the statistical analysis extremely profitable for determining the electrical properties of cells.

Introduction: We have developed manipulation techniques to form cell-based arrays by positive dielectrophoresis (p-DEP) and to retrieve target cells from cell−based arrays selectively by negative dielectrophoresis (n-DEP). The novel devices with microwell arrays on microband electrodes were employed to manipulate cells. Hybridomas with the secretion ability of antibodies were trapped to form cell-based arrays. Then, the ability was discriminated by trapping the antibodies to the antigen immobilized on the bottom of wells. Finally, individual cells trapped in microwells were selectively retrieved by regulating the generation of electric fields in individual microwells. The development of series of these techniques could be useful to recover hybridomas producing antibodies with high affinity in large populations of cells without repeated steps of a culture and a limiting serial-dilution. Experimental: The device comprises the upper substrate with indium-tin-oxide (ITO) microband electrodes and the lower substrate with microwell arrays on ITO microband electrodes. Hybridomas producing an anti-rabbit serum albumin (RSA) antibody suspended in the DEP medium were introduced in the channel. AC signal (3 MHz, 2 Vpp) was then applied to the upper and lower microband electrodes with opposite phase to form a cell-based array. Anti-RSA antibodies secreted from hybridomas trapped in the microwells were captured by RSA immobilized on the electrodes. Cell arrays were then treated with anti-mouse IgG antibody conjugated with Alexa 488 (a secondary antibody). Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target hybridomas from the microwell array. To retrieve the target hybridoma, an AC signal in the n-DEP frequency region was applied to a pair of microband electrodes above and below the microwell with the target hybridoma. Results and discussion: Cell−based arrays were formed with the occupancy efficiency of over 90% in a few seconds by p-DEP. Antibodies secreted in microwell arrays were captured to discriminate target hybridomas in a few hours without repeated steps of a culture and a limiting serial-dilution. Furthermore, hybridomas trapped in microwells were retrieved from the cell−based array by applying an AC signal to band electrodes. The sequential system for forming cell−based arrays, discriminating hybridomas secreting specific antibodies, and retrieving target hybridomas was developed by using the novel microwell array device comprising 3-D microband array electrodes with an orthogonal arrangement.

Point-of-care testing (POCT) is a test that’s been designed to obtain valuable information for diagnostics, therapeutics, nursing care, disease prevention, and promotion of healthy lifestyles at a patient’s bedside. The analytical apparatuses for POCT need to be portable enough to bring to the patient’s location, simple to use, and quick to obtain diagnostic results. Although using an antibody would be an excellent way to recognize and trap target molecules selectively, currently there is no conversion ability of signal molecules to antibodies. However, an immunochromatography assay (ICA) can achieve a signal conversion by accumulating gold nanoparticles, along with an automatic separation of unreacted signal source. In this chapter, we describe the principles behind ICA and the present state of its use in POCT apparatuses that apply an antigen-antibody reaction. We also describe the development of a quantitative ICA device that incorporates an electrochemical detection system and a quick, simple, and quantitative immunosensing method using particle manipulation techniques based on dielectrophoresis.

A revolution in functional brain imaging techniques is in progress in the field of neurosciences. Optical imaging techniques, such as high-density diffuse optical tomography (HD-DOT), in which source-detector pairs of probes are placed on subjects’ heads, provide better portability than conventional functional magnetic resonance imaging (fMRI) equipment. However, these techniques remain costly and can only acquire images at up to a few measurements per square centimetre, even when multiple detector probes are employed. In this study, we demonstrate functional brain imaging using a compact and affordable setup that employs nanosecond-order pulsed ordinary laser diodes and a time-extracted image sensor with superimposition capture of scattered components. Our technique can simply and easily attain a high density of measurement points without requiring probes to be attached, and can directly capture two-dimensional functional brain images. We have demonstrated brain activity imaging using a phantom that mimics the optical properties of an adult human head, and with a human subject, have measured cognitive brain activation while the subject is solving simple arithmetical tasks.

We applied a fabrication method for the formation of island organization of cells based on a three-dimensional (3D) device for negative dielectrophoresis (n-DEP) to produce cell aggregates with uniform numbers of cells rapidly and simply. The intersections formed by rotating the interdigitated array (IDA) with two combs of band electrodes on the upper substrate by 90° relative to the IDA with two combs on the lower substrate were prepared in the device. The AC voltage was applied to a comb on the upper substrate and a comb on the lower substrate, while AC voltage with opposite phase was applied to another comb on the upper substrate and another comb on the lower substrate. Cells dispersed randomly were directed toward the intersections with relatively lower electric fields due to n-DEP, which formed by AC voltage applied bands with the identical phase, resulting in the formation of island patterns of cells. The cells accumulated at intersections were promoted to form the cell aggregates due to the close contact together. The production of cell aggregations adhered together was easily found by the dispersion behavior after switching the applied frequency to convert the cellular pattern. When cells were accumulated at the intersections by n-DEP for 45 min, almost accumulations of cells were adhered together, and hence a formations of cell aggregations. By using the present method, we can rapidly and simply fabricate cell aggregations with a uniform number of cells.

Positioning and patterning of polystyrene particles on a silicon nitride (SiN) membrane with a microhole array has been demonstrated by positive dielectrophoresis (p-DEP). A chamber with an SiN membrane with the well-aligned microholes as the bottom substrate was positioned on an indium-tin-oxide (ITO) electrode with a 1 mm space between the bottom substrate and the ITO electrode. The chamber and the space were filled with water and a suspension of particles (10 µm diameter) in water, respectively. An AC electric signal was then applied to a microelectrode positioned at 10 µm above the SiN membrane, while the ITO electrode was connected to the ground. Particles present in the space between the SiN membrane and ITO electrode gradually moved toward the lower surface of the SiN membrane directly under the microelectrode owing to the strong electric field generated on and in localized microholes and accumulated at this position to form aggregates. For particles of 3 µm diameter, one particle was deposited in each hole (2 µm diameter) in the region directly under the microelectrode. The horizontal movement of the microelectrode gave rise to the formation of a line pattern of particles along the trail of the microelectrode because of the shift of the region with the strong electric field. These demonstrations could be applicable to arranging the particles at desired positions and in desired holes, and form particle patterns with highly flexible designs.

We have developed a simple and rapid formation of a cell-based array on microwell array electrodes by an attractive force of positive dielectrophoresis (p-DEP), even after removing an upper disk electrode stick that was used as a counter electrode to the microwell array electrodes. The attractive force of p-DEP generated by the scanning of the disk electrode allows the formation of a cell-based array on all microwell arrays. We demonstrated an exploration of target cells spiked with a low ratio after removing the disk electrode.

Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology.

Microelectrode arrays (MEAs) are very powerful sensor devices for the non-invasive measurement of extracellular field potentials from excited cells. In this study, new types of interfaces were developed on MEAs by exploiting the electrochemical polymerization of conducting polymers with biomolecules. The composite films were composed of poly(3,4-ethylenedioxythiophen) (PEDOT) and anionic polysaccharide (gellan gum). The PEDOT/biomolecule composite films had no cytotoxic effect, and reduced the electrode impedance at 1 kHz to 8.84 ± 0.50 kΩ. This biointerface enabled us to record extracellular field potentials derived from the spontaneous beating of cardiomyocytes.

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays.

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 π/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. © 2010 Elsevier B.V. All rights reserved.

Arrays of gold nanoparticles and gold nanorods have been fabricated in the spatially localized pores of a porous alumina membrane using positive dielectrophoresis (p-DEP). The porous alumina membrane with a pore size of 200 nm and a thickness of 60μm was sandwiched between the lower microarray electrode and the upper bare ITO electrode. Then, a 100-nm-diameter gold nanoparticle suspension (0.6-1.0×1012 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 30Vpp, 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. © 2008 Elsevier B.V. All rights reserved.

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 μm- and 10 μm-diameter particles were introduced into the channel with 300 μm height at 30 μl/min. An AC electric field (20 V 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 μm-diameter particles mainly flow through the narrow path and 10 μm-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. © 2009 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 μm 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 1 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 nm diameter pores, the particles penetrated the membrane with 400 nm 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. © 2007 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 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 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. © 2008 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 μm 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 Vpeak 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 μg/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. © 2006 Elsevier B.V. All rights reserved.

Microparticle patterns have been fabricated on a nonconductive glass substrate and a conductive indium tin oxide (ITO) substrate using negative dielectrophoresis (n-DEP). The patterned microparticles on the substrate were immobilized by covalent bonding or embedded into polymer sheets or strings. The patterning device consisted of an ITO interdigitated microband array (IDA) electrode as the template, a glass or ITO substrate, and a polyester film (10-μm thickness) as the spacer. A suspension of 2-μm-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. © 2007 American Chemical Society.

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.

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.

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

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

Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 μm) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (D3S) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity.