Determination of membrane capacitance and cytoplasm conductivity by simultaneous electrorotation.

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.

Label-Free Rapid Separation and Enrichment of Bone Marrow-Derived Mesenchymal Stem Cells from a Heterogeneous Cell Mixture Using a Dielectrophoresis Device.

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.

A DNA hybridization sensor based on catalytic response by platinum deposition.

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 µM. 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.

Cell pairing using microwell array electrodes based on dielectrophoresis.

We report a simple device with an array of 10,000 (100 × 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 µm) and depth (25 µm) 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.

Detection of the oxygen consumption rate of migrating zebrafish by electrochemical equalization systems.

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

Development of a simultaneous electrorotation device with microwells for monitoring the rotation rates of multiple single cells upon chemical stimulation.

Here, we described a unique simultaneous electrorotation (ROT) device for monitoring the rotation rate of Jurkat cells via chemical stimulation without fluorescent labeling and an algorithm for estimating cell rotation rates. The device comprised two pairs of interdigitated array electrodes that were stacked orthogonally through a 20 µm-thick insulating layer with rectangular microwells. Four microelectrodes (two were patterned on the bottom of the microwells and the other two on the insulating layer) were arranged on each side of the rectangular microwells. The cells, which were trapped in the microwells, underwent ROT when AC voltages were applied to the four microelectrodes to generate a rotating electric field. These microwells maintained the cells even in fluid flows. Thereafter, the ROT rates of the trapped cells were estimated and monitored during the stimulation. We demonstrated the feasibility of estimating the chemical efficiency of cells by monitoring the ROT rates of the cells. After introducing a Jurkat cell suspension into the device, the cells were subjected to ROT by applying an AC signal. Further, the rotating cells were chemically stimulated by adding an ionomycin (a calcium ionophore)-containing aliquot. The ROT rate of the ionomycin-stimulated cells decreased gradually to 90% of the initial rate after 30 s. The ROT rate was reduced by an increase in membrane capacitance. Thus, our device enabled the simultaneous chemical stimulation-induced monitoring of the alterations in the membrane capacitances of many cells without fluorescent labeling.

Dielectrophoretic trapping of nanosized biomolecules on plasmonic nanohole arrays for biosensor applications: simple fabrication and visible-region detection.

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.

Simple detection of surface antigens on living cells by applying distinct cell positioning with negative dielectrophoresis.

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

Selective retrieval of antibody-secreting hybridomas in cell arrays based on the dielectrophoresis.

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.

Electrofusion of cells with different diameters by generating asymmetrical electric field in the microwell array.

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.

Sensitive and spatially multiplexed detection system based on dielectrophoretic manipulation of DNA-encoded particles used as immunoreactions platform.

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

Detection of surface antigens on living cells through incorporation of immunorecognition into the distinct positioning of cells with positive and negative dielectrophoresis.

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

Electrochemical detection of receptor-mediated endocytosis by scanning electrochemical microscopy.

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

Electrorotation Rates of K562 Cells Accompanied by Erythroid Differentiation Induced by Sodium Butyrate.

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.

Selective Trapping and Retrieval of Single Cells Using Microwell Array Devices Combined with Dielectrophoresis.

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.

Discrimination of cell-differentiation using a cell-binding assay based on the conversion of cell-patterns with dielectrophoresis.

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.

Topographic imaging of convoluted surface of live cells by scanning ion conductance microscopy in a standing approach mode.

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

Microfluidic Separation of Blood Cells Based on the Negative Dielectrophoresis Operated by Three Dimensional Microband Electrodes.

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.

Transfected single-cell imaging by scanning electrochemical optical microscopy with shear force feedback regulation.

Gene-transfected single HeLa cells were characterized using a scanning electrochemical/optical microscope (SECM/OM) system with shear-force-based probe-sample distance regulation to simultaneously capture electrochemical, fluorescent, and topographic images. The outer and inner states of single living cells were obtained as electrochemical and fluorescent signals, respectively, by using an optical fiber-nanoelectrode probe. A focused ion beam (FIB) was used to mill the optical aperture and the ring electrode at the probe apex (the inner and outer radii of the ring electrode were 37 and 112 nm, respectively). To apply an appropriate shear force between the probe tip and the living cell surface, we optimized the amplitude of oscillation of the tuning fork to which the probe was attached. Field-programmable gate arrays (FPGA) were adopted to drastically increase the feedback speed of the tip-sample distance regulation, shorten the scanning time for imaging, and enhance the accuracy and quality of the living cell images. In employing these improvements, we simultaneously measured the cellular expression activity of both secreted alkaline phosphatase outside and GFP inside by using the SECM/OM with shear force distance regulation.

Electrochemical detection of epidermal growth factor receptors on a single living cell surface by scanning electrochemical microscopy.

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.