安川 智之

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.

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 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>

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>

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

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.

幹細胞移植治療や再生医療には、細胞集団の中から特定の細胞の選択的な回収が必要不可欠である。最も汎用的な方法は、標的細胞に特異的に発現する表面抗原を利用する。表面抗原の発現状態は培養状況によって変化し、また非標的細胞と明確に区別が可能な表面抗原の種類は限定的である。表面抗原を用いず細胞の大きさ、密度、硬さ、電気特性、付着力など、細胞の物理特性を利用して細胞を回収する方法が注目されている。誘電泳動は、"細胞の動きの観察"という簡単な方法で、非侵襲的に細胞の電気特性を同定することができる。本稿では、誘電泳動現象を利用したノンラベルな細胞の識別方法について概説する。(著者抄録)

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.

© The Japan Society for Analytical Chemistry. 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.

© MYU K.K. 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.

© The Japan Society for Analytical Chemistry. 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.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.

© The Japan Society for Analytical Chemistry. Arrays with cell aggregations and single-cell arrays embedded in hydrogel sheets were fabricated by negative dielectrophoretic (n-DEP) cell-manipulation techniques and hydrogel gelation. Cells suspended randomly in a prepolymer solution were rapidly manipulated to form an island-like organization of cells through the repulsive force of n-DEP by using a DEP device consisting of grid electrodes. The cell patterns were retained by irradiating ultraviolet (UV) light so as to urge gelation. Moreover, control of the optical transparency of the grid electrode allows for the fabrication of cubes with single cells and cell aggregation.

A single-step electrochemical immunochromatography has been developed: the device was based on two pieces of nitrocellulose membrane, a sample pad with anti-mouse IgG antibody labeled with glucose oxidase (GOx-labeled antibody), a conjugate pad with glucose, and a Pt working electrode. Either antibody or antigen was immobilized on the membrane. The addition of a solution containing mouse IgG, a model target, allows for the dissolution of GOx-labeled antibody in the sample pad to form an immunocomplex. The produced immunocomplex was automatically separated by capturing to the antibody immobilized on the membrane with the sandwich structure or by passing through the membrane modified with an antigen for the competitive reaction. The separated GOx label arrived at the conjugate pad with glucose to undergo the enzyme reaction. Hydrogen peroxide generated by this reaction was detected at the Pt electrode prepared on the second nitrocellulose membrane downstream from the conjugate pad. The results demonstrated that the designed immunochromatography can be applied to quantitative detection with a single-step procedure, because both the GOx-labeled antibody for revealing the immunoreactions and the substrate for the enzyme reaction were prepared in the device. Moreover, the initial concentration of the GOx-labeled antibody permitted control of the detectable concentration for mouse IgG.

In this study, we investigated the effect of positive dielectrophoresis (DEP) on gene expression in mesenchymal stem cells. When applying an alternating current voltage, human bone marrow derived mesenchymal stem cells (UE7T-13) exhibited a positive DEP, and were compressed onto the electrode surface. The constructed device can easily control the DEP force to the cells by changing the frequency. Interestingly, gene expressions of the cell differentiation marker in UE7T-13 cells and the mechanical stimulation-susceptible one were changed by applying a positive DEP. These results suggested that the gene expression in mesenchymal stem cells can be regulated by applying mechanical stimulation derived from DEP.

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

We describe here the dielectrophoretic manipulation using a microdisk electrode with microcavity for picking up, positioning and relocating single target cells. The attractive force of positive dielectrophoresis (p-DEP) was used to trap target cells at the electrode tip, while trapped cells were released by the repulsive force of negative DEP (n-DEP). The capturing of cells in the cavity allows them to transfer at the desired position even after turning off the attractive force by p-DEP. We demonstrate an application of the present technique in the patterning with living cells. (C) The Electrochemical Society of Japan, All rights reserved.

We report on a biosensor for cocaine based on the conformation change of DNA aptamer by capturing the cocaine molecules. The oxidation current of ferrocene conjugated on the terminal end of aptamer immobilized on an Au electrode increased with increasing cocaine concentration. The sensor response has been improved by a simple heat treatment after immobilization, since the aggregates of DNA aptamer generated during the immobilization step could be dissociated and rearranged on the electrode.

Scanning electrochemical microscopy (SECM) has been applied to determining the oxygen consumption of individual myotubes. Reduction currents of oxygen monitored by Pt microelectrode positioned above the myotube decreased after the continuous contraction of myotubes was induced by the application of electric pulses.

The attractive force of positive dielectrophoresis has been used to form the pairs of different types of cells rapidly and simply. Two different types of cells were successively directed into the individual wells fabricated on the microwell array electrodes. The width (16 mu m) and depth (25 mu m) of the microwells restricted the size to two vertically aligned cells. Mouse myeloma cells stained in blue were trapped within 1 s in the microwells by p-DEP by applying an alternating current voltage. The cells were retained inside the wells even after switching off the voltage and washing with a fluidic flow. K562 cells stained in green were then trapped in the microwells occupied by the myeloma cells to form the vertical cell pairing in the microwells. Two types of cells were paired within only 1 min and a pairing efficiency of 53% was achieved.

We present the development of the method for picking up, positioning and relocating single cells based on the dielectrophoresis (DEP). The target cell was captured in the microcavity fabricated on the top of the microdisk electrode by positive DEP (p- DEP). After the trapped cell was transferred to the desired position, negative DEP (n- DEP) was used to release the trapped cell at the desired position. The cell patterns can be formed by the capture and relocation of individual cells with p- and n-DEP.

The oxygen consumption of single contractile myotubes was investigated by amperometric monitoring of the reduction current for oxygen using scanning electrochemical microscope (SECM) equipped with Pt microelectrode. The contraction of myotubes was induced by the application of electric pulse stimulation. The reduction current for oxygen immediately decreased after application of the electric pluses with 1.0 Hz and almost reached steady state because the activity of the oxygen consumption by respiration increased due to the contraction of myotubes accompanied with a consumption of energy.

Formation of line pattern with cells based on dielectrophoresis (DEP) was applied to simple and rapid distinction of cells with specific surface antigens from a cell population. Dispersed cells were accumulated to gap areas of the interdigitated band array (IDA) electrode modified with antibody within 5 s by negative DEP (n-DEP) and captured by immunoreactions. Unbounded cells without the specific antigen on the membrane were removed to form another pattern by switching the applied voltage of the band electrode. 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.

An electrochemical detection system for DNA has been investigated based on the current increase due to the catalytic oxidation of hydrogen peroxide with platinum deposited by the electrochemical reduction of chloro-2,2':6',2 ''-terpyridine platinum (II) chloride dihydrate (Pt complex) on a screen-printed carbon electrode. The platinum deposited on the screen-printed carbon electrode that has shown no catalytic activity for oxidation of hydrogen peroxide gives rise to catalytic activity. Cyclic voltammetry was used to reduce the Pt complex to deposit platinum metal on the carbon electrode. The oxidation current of hydrogen peroxide increased with increasing concentration of the Pt complex in the electrolytic deposition. The intercalation of the Pt complex into double-stranded DNA (dsDNA) decreased the concentration of free Pt complex and caused a decrease in the diffusion coefficient of the intercalated Pt complex. Moreover, the reduction of the Pt complex was inhibited due to steric hindrance. Thus, the oxidation current for hydrogen peroxide by platinum deposited on the carbon electrode decreased with an increase in the concentration of dsDNA. This procedure is absolutely simple without the need to immobilize DNA. Furthermore, the use of inexpensive screen-printed carbon electrodes will allow for the development of disposable sensing systems.

A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6 3- to Fe(CN)6 4-, which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL-1, respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

We fabricated an electrochemical detection system involving redox species flowing in a nitrocellulose membrane incorporated into two plates with both poles and electrodes. We prepared an upper substrate with poles inserted a Pt microelectrode and a Ag/AgCl electrode as well as a bottom substrate with a small pole array and poles that were used as supports of nitrocellulose membranes. The presence of poles allowed a uniform flow of a solution in the membrane and a steady-state oxidation current of hydrogen peroxide. A solution containing different concentrations of glucose was introduced into the device with membranes to be immobilized by glucose oxidase (GOx). Hydrogen peroxide produced through the enzyme reaction in the presence of glucose flowed downstream to give an electrochemical response with the electrode arranged 6 mm away from the GOx immobilization area. Oxidation currents of hydrogen peroxide increased with increasing the concentration of glucose. Thus, an electrochemical detection system of redox species flowing in the membrane indicated the possibility for developing an enzymatic and immunological assay format with electrochemical quantitation.

We report a novel electrochemical sensing system for single-stranded DNA (ssDNA) with a specific sequence based on the catalytic reduction of protons with platinum deposited by the electrochemical reduction of chloro-2,2':6',2''-terpyridine platinum(II) chloride dihydrate (Pt complex) on a glassy carbon (GC) electrode. There was no catalytic property observed for proton reduction at the GC electrode, while the platinum deposited by the reduction of the Pt complex shows the catalytic activity of proton reduction. The intercalation of the Pt complex with double-stranded DNA (dsDNA) decreased the concentration of the free Pt complex with a concomitant diminution in the electrochemical catalytic current due to steric hindrance and a decrease in the diffusion coefficient of the intercalated Pt complex. Thus, the catalytic current of proton reduction by platinum deposited on a GC electrode decreased with an increase in the concentration of target ssDNA, when capture DNA with a complementary sequence was present in the solution to form the hybrid dsDNA. A detectable concentration range was estimated and found to be 0.1-1.0 mu M. The catalytic current was significantly larger than the reduction current of the Pt complex, resulting in the sensitive detection of ssDNA. Furthermore, the present method is simply due to the immobilization of capture DNA being unnecessary.

A rapid and simple method for the fabrication of the island patterns with particles and cells was applied to detect the presence of specific antigens on the cell surface. An upper interdigitated microband array (IDA) electrode was mounted on a lower substrate with the same design to fabricate a microfluidic-channel device for dielectrophoretic manipulation. The electrode grid structure was fabricated by rotating the upper template IDA by 90 relative to the lower IDA. A suspension of anti-CD33 modified particles and HL-60 cells was introduced into the channel. An AC electrical signal (typically 20 V peak-to-peak, 100 kHz) was then applied to the bands of the upper and lower IDAs, resulting in the formation of island patterns at the intersections with low electric fields. Immunoreactions between the antibodies immobilized on the accumulated particles and the CD33 present on the surface of the cells led to the formation of complexes comprising corresponding antigen-antibody pairs. Non-specific pairs accumulated at the intersection, which did not form complexes, were then dispersed after removal of the applied field. The time required for the detection of the formation/dispersion of the complexes is as short as 6 min in the present procedure. Furthermore, this novel cell binding assay does not require pretreatment such as target labeling or washing of the unbound cells. (C) 2014 Elsevier B.V. All rights reserved.

We developed a simple device with 400 (20 x 20) microwells that integrated the cell positioning and cell pairing of two different types of cells based on positive dielectrophoretic manipulation. We fabricated a star-shaped micropole array on a conductive substrate and used the areas encircled with micropoles as microwells for positioning the cells. The different types of cells were successively trapped to form vertical cell pairs in the microwells. The time required for the formation of the array of cell pairs was as short as 1 min

We report a simple device with an array of 10 000 (100 x 100) microwells for producing vertical pairs of cells in individual microwells with a rapid manipulation based on positive dielectrophoresis (p-DEP). The areas encircled with micropoles which fabricated from an electrical insulating photosensitive polymer were used as microwells. The width (14 mu m) and depth (25 mu m) of the individual microwells restricted the size to two vertically aligned cells. The DEP device for the manipulation of cells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower microwell array electrode fabricated on an ITO substrate. Mouse myeloma cells stained in green were trapped within 1 s in the microwells by p-DEP by applying an alternating current voltage between the upper ITO and the lower microwell array electrode. The cells were retained inside the wells even after switching off the voltage and washing with a fluidic flow. Other myeloma cells stained in blue were then trapped in the microwells occupied by the cells stained in green to form the vertical cell pairing in the microwells. Cells stained in different colors were paired within only 1 min and a pairing efficiency of over 50% was achieved.

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