Wataru Tonomura

Electrophysiological Biosensor with Micro Channel Array for Sensing of Signals from Single Cells

This paper describes a novel electrophysiological biosensor with micro channel array (hereafter MCA) for sensing of signals from individual single cells. By using microelectromechanical systems (MEMS) technologies, we have developed the batch-measurement biosensor with MCA for sensing of electrophysiological signals gathered from single cells. We design the multipoint-measurement biosensor with MCA for observation of individual single cells. Detecting electrodes of the multipoint-measurement biosensor with MCA are independent from each other in order to analyze individual single cells. Biosensors with MCA employ the measurement principles of the patch-clamp technique. In this study, we evaluate electrophysiological signals of the mPerl-luc slice culture in response to chemical stimulus by the multipoint-measurement biosensor with MCA.

Spatially Arranged Microelectrodes using Wire Bonding Technology for Spatially Distributed Chemical Information Acquision

This paper presents spatially arranged microelectrodes to allow real-time monitoring of behavior of spatially distributed chemical. Out-of-plane microelectrodes standing on a substrate with gradation in height are developed for the purpose. Wire-bonding-based probe technology [1] makes it possible to provide spatially arranged microelectrodes. The wire-bonding-based probe technology combines wire bonding and laser machining. Bonded metal wires are converted to probe arrays by cutting the bridge. Spatially arranged microelectrodes can detect spatially distributed chemical in real time would understand various phenomena caused by spatially distributed chemical. This paper demonstrates spatially arranged microelectrodes could be used as the working electrodes of spatially electrochemical sensors in chronoamperometric measurements using K3Fe (CN)6.

Parallel multipoint recording of aligned and cultured neurons on micro channel array toward cellular network analysis

This paper describes an advanced Micro Channel Array (MCA) for recording electrophysiological signals of neuronal networks at multiple points simultaneously. The developed MCA is designed for neuronal network analysis which has been studied by the co-authors using the Micro Electrode Arrays (MEA) system, and employs the principles of extracellular recordings. A prerequisite for extracellular recordings with good signal-to-noise ratio is a tight contact between cells and electrodes. The MCA described herein has the following advantages. The electrodes integrated around individual micro channels are electrically isolated to enable parallel multipoint recording. Reliable clamping of a targeted cell through micro channels is expected to improve the cellular selectivity and the attachment between the cell and the electrode toward steady electrophysiological recordings. We cultured hippocampal neurons on the developed MCA. As a result, the spontaneous and evoked spike potentials could be recorded by sucking and clamping the cells at multiple points. In this paper, we describe the design and fabrication of the MCA and the successful electrophysiological recordings leading to the development of an effective cellular network analysis device.

Parallel multipoint recording of aligned and cultured neurons on corresponding Micro Channel Array toward on-chip cell analysis

This paper describes an advanced Micro Channel Array (MCA) so as to record neuronal network at multiple points simultaneously. Developed MCA is designed for neuronal network analysis which has been studied by co-authors using MEA (Micro Electrode Arrays) system. The MCA employs the principle of the extracellular recording. Presented MCA has the following advantages. First of all, the electrodes integrated around individual micro channels are electrically isolated for parallel multipoint recording. Sucking and clamping of cells through micro channels is expected to improve the cellular selectivity and S/N ratio. In this study, hippocampal neurons were cultured on the developed MCA. As a result, the spontaneous and evoked spike potential could be recorded by sucking and clamping the cells at multiple points. Herein, we describe the successful experimental results together with the design and fabrication of the advanced MCA toward on-chip analysis of neuronal network.

Electrophysiological biosensor with micro channel array for multipoint measurement of signals from distributed cells

We have studied and developed electrophysiological biosensors by MEMS technology. Micro channel arrays (hereafter MCA) integrated with suction holes and electrodes were previously presented for electrophysiological biosensors for the drug screening. This paper proposes novel electrophysiological biosensors with MCA for both batch measurement and multipoint measurement of cell signals based on our previous results. Report on experimental results of feasible study with using developed biosensors will follow the description of design and fabrication of devices.

Lift-off process with bi-layer photoresist patterns for conformal-coated superhydrophilic pulsed plasma chemical vapor deposition-SiOx on SiCx for lab-on-a-chip applications

In this work, a lift-off process with bi-layer photoresist patterns was applied to the formation of hydrophobic/hydrophilic micropatterns on practical polymer substrates used in healthcare diagnostic commercial products. The bi-layer photoresist patterns with undercut structures made it possible to peel the conformal-coated silicon oxide (SiOx) films from substrates. SiOx and silicon carbide (SiCx) layers were deposited by pulsed plasma chemical vapor deposition (PPCVD) method which can form roughened surfaces to enhance hydrophilicity of SiOx and hydrophobicity of SiCx. Microfluidic applications using hydrophobic/hydrophilic patterns were also demonstrated on low-cost substrates such as poly(ethylene terephthalate) (PET) and paper films.

Cellular aggregate capture by fluidic manipulation device highly compatible with micro-well-plates

This paper proposes a capture device to manipulate and transport a cellular aggregate in a micro-well. A cellular aggregate (a few hundreds μm in diameter) is currently manipulated by a pipette. The manual manipulation by a pipette has problems; low reliability, low throughput, and difficulty in confirmation of task completion. We took into account of compatibility with existing methods such as a micro-well-plate and designed for the capture device of a cellular aggregate in a micro-well. A newly developed capture device flows and carries a cellular aggregate from a bottom of a well to a trap of the capture device. We designed a curved surface at the bottom of the capture device to form a space to act as a channel between the inner wall of the micro-well. This paper presents concept, design, fabrication, and of the proposed cellular aggregate capture, followed by successful experimental results.

A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology

Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with “patterned” or “randomly‐mixed” co‐culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co‐cultured with neonatal rat ventricular myocytes (NRVMs) on multi‐electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca2+ imaging using a gene‐encoded Ca2+ indicator, G‐CaMP2. In the monoculture of NRVMs (control), conduction was well‐organized. In the randomly‐mixed co‐culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the “patterned” co‐culture where an en bloc SKMB‐layer was inserted into the NRVM‐layer, excitation homogenously propagated although conduction was distorted by the SkMB‐area. The 4‐mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM‐area did not change (25 ± 3 ms), although CT through the SkMB‐area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat‐to‐beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca2+ transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249–2256, 2016.

High-sensitivity erectrochemical sensor using pyrolyzed polymer-gold 3D probe arrays for spatial chemical sensing

This paper reports spatially arranged pyrolyzed polymer-gold probes to allow high-sensitivity monitoring of spatially distributed chemicals. Pyrolyzed polymer is a promising carbon material for applications of electrochemical sensors. Out-of-plane gold microelectrodes coated by polymer film (parylene-C) are transformed to conductive carbon-gold materials by annealing at 1000°C for 2h in a vacuum chamber. 3D probe technology using wire bonding makes it possible to provide the spatially arranged microelectrodes. This paper demonstrates pyrolyzed polymer-gold 3D probes have high sensitivity and wider electrochemical potential window than typical electrochemical electrode materials such as gold to realize spatial chemical sensing.

Hetero multilayer structures by rapid prototyping for simultaneous encapsulation and interconnection of microchips

This paper presents a novel use of photopolymerization-based rapid prototyping for simultaneous encapsulation and interconnection of MEMS and μTAS chips. Recent photopolymerization-based rapid prototyping achieves high machining resolution so as to be combined with micromachining. This paper demonstrates combination of rapid prototyped structure and MEMS and μTAS chips such as pressure sensor and fluidic structures of PDMS (polydimethylsiloxane). Supporting material for photopolymerization-based rapid prototyping can be used as sacrificial structures. Sacrificial etching of supporting material allows complicated three dimensional structures by rapid prototyping. Hetero connected channels will be also demonstrated through combination of RP and micromachining.