Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/elsa.202300015

Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcc.3c02690

Language：English   Publishing type：Research paper (scientific journal)  

Understanding the catalytic mechanism of highly active two-dimensional electrocatalysts is crucial to their rational design. Herein, we reveal the element dependence of the reactivity of two-dimensional metal dichalcogenide sheets for electrocatalytic CO2 reduction. We found that tin(IV) disulfide (SnS2) and molybdenum(IV) disulfide (MoS2) sheets exhibited Faradaic efficiencies of 63.3% and ∼0%, respectively, for formic acid. Scanning electrochemical cell microscopy and theoretical calculations were used to identify the catalytically active sites of SnS2 as terraces and edges. Owing to the effective utilization of the entire surface area, SnS2 can effectively accelerate catalytic reactions. This finding provides a direction for material research in two-dimensional electrocatalysts for energy-efficient chemical production from electrochemical CO2 reduction, as well as for other energy devices.

DOI： 10.1021/acsnano.2c12627

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.electacta.2023.142192

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.electacta.2022.141783

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Although various spectroscopic methods have been developed to capture ion-concentration profile changes, it is still difficult to visualize the ion-concentration profile and surface topographical changes simultaneously during the charging/ discharging of lithium-ion batteries (LIBs). To tackle this issue, we have developed an operando scanning ion conductance microscopy (SICM) method that can directly visualize an ion concentration profile and surface topography using a SICM nanopipette while controlling the sample potential or current with a potentiostat for characterizing the polarization state during charging/discharging. Using operando SICM on the negative electrode (anode) of LIBs, we have characterized ion-concentration profile changes and the reversible volume changes related to the phase transition during cyclic voltammetry (CV) and charge/ discharge of the graphite anode. Operando SICM is a versatile technique that is likely to be of major value for evaluating the correlation between the electrolyte concentration profile and nanoscale surface topography changes.

DOI： 10.1021/jacsau.2c00677

Web of Science

Scopus

PubMed

Publishing type：Research paper (scientific journal)  

Nanopore sensing measures the changes in charge and physical state around the nanopores as changes in ionic current. In this study, we performed vancomycin (VCM) sensing with nanopores modified via Au−S bonds with dithiobis(4-butyrylamino-m-phenylboronic acid) (DTBA-PBA), a derivative of phenylboronic acid (PBA) bearing a thiol. First, we modified a 3-mm-diameter Au electrode with a DTBA-PBA self-assembled monolayer (SAM) to confirm the VCM response of the PBA interface by cyclic voltammetry. DTBA-PBA was then immobilized in the same manner on a nanopore pipette coated with an Au layer. We measured the VCM concentration from the change in ion current using the nanopore pipette. A VCM concentration-dependent ionic current response was observed in the range of 0.01–1 mM. Many kinds of pharmaceuticals can bind to PBA; therefore, this method could be used for quick, easy, in situ quantification of not only VCM but also other pharmaceuticals with serious side effects.

DOI： 10.1002/elan.202300173

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Extracellular fine particles of various sizes and origins can be taken up by cells, affecting their function. Understanding the cellular uptake processes is crucial for understanding the cellular effects of these particles and the development of means to control their internalization. Although macropinocytosis is a possible pathway for the cellular uptake of particles larger than 0.2 μm, its contribution to cellular uptake in non-phagocytic cells is controversial. Using 3 μm polystyrene beads as a model particle, we aimed to assess the detailed modes of their cellular uptake by non-phagocytic HeLa cells. Cellular uptake was assessed using confocal, scanning electron, and scanning ion conductance microscopy analyses, together with inhibitor studies. Our results revealed that 3 μm beads were taken up by HeLa cells by an actin-, cholesterol-, and membrane protrusions-dependent noncanonical endocytic pathway, different from the canonical macropinocytic and phagocytic pathways. Our work provides a framework for studying the cellular uptake of extracellular fine particles.

DOI： 10.1039/d2bm01353c

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Site-selective chemistry opens new paths for the synthesis of technologically important molecules. When a reactant is placed inside a Fabry-Perot (FP) cavity, energy exchange between molecular vibrations and resonant cavity photons results in vibrational strong coupling (VSC). VSC has recently been implicated in modified chemical reactivity at specific reactive sites. However, as a reaction proceeds inside an FP cavity, the refractive index of the reaction solution changes, detuning the cavity mode away from the vibrational mode and weakening the VSC effect. Here we overcome this issue, developing actuatable FP cavities to allow automated tuning of cavity mode energy to maintain maximized VSC during a reaction. As an example, the site-selective reaction of the aldehyde over the ketone in 4-acetylbenzaldehyde is achieved by automated cavity tuning to maintain optimal VSC of the ketone carbonyl stretch during the reaction. A nearly 50% improvement in site-selective reactivity is observed compared to an FP cavity with static mirrors, demonstrating the utility of actuatable FP cavities as microreactors for organic chemistry.

DOI： 10.1002/chem.202201260

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The biodistribution of chemotherapy compounds within tumor tissue is one of the main challenges in the development of antineoplastic drugs, and techniques for simple, inexpensive, sensitive, and selective detection of various analytes in tumors are of great importance. In this paper we propose the use of platinized carbon nanoelectrodes (PtNEs) for the electrochemical detection of platinum-based drugs in various biological models, including single cells and tumor spheroids in vitro and inside solid tumors in vivo. We have demonstrated the quantitative direct detection of Pt(II) in breast adenocarcinoma MCF-7 cells treated with cisplatin and a cisplatin-based DNP prodrug. To realize the potential of this technique in advanced tumor models, we measured Pt(II) in 3D tumor spheroids in vitro and in tumor-bearing mice in vivo. The concentration gradient of Pt(II) species correlated with the distance from the sample surface in MCF-7 tumor spheroids. We then performed the detection of Pt(II) species in tumor-bearing mice treated intravenously with cisplatin and DNP. We found that there was deeper penetration of DNP in comparison to cisplatin. This research demonstrates a minimally invasive, real-time electrochemical technique for the study of platinum-based drugs.

DOI： 10.1021/acs.analchem.2c00136

Web of Science

Scopus

PubMed

Language：Japanese  

Point-of-care testing (POCT) devices have evolved to provide beneficial information about an individual's health whenever needed. Enzyme-based analytical devices have facilitated the highly selective detection of numerous biological molecules and ions. Enzymes are commonly used as the tags of recognition components, such as antibodies, to generate and amplify detection signals. Particularly, alkaline phosphatase (ALP) is one of the most widely used enzymes because of its high turnover number and low cost. Rapid response time and the incorporation of many sensors fabricated by micro/nano processing technologies are the advantages in using electrochemical devices as analytical tools. Therefore, ALP-based electrochemical devices have potential applications for more practical POCT platforms. This review summarizes recent research progress of ALP-based electrochemical devices for POCT. In addition to ALP substrates, the application of ALP-based immunosensors, aptasensors, and DNAzyme sensors are discussed.

DOI： 10.1002/elan.202100294

Scopus

Language：Japanese   Publishing type：Research paper (scientific journal)  

Photoelectrochemical (PEC) water splitting is an important and rapidly developing technology that produces H2 as a renewable resource, but local surface investigations remain a major challenge. Using scanning electrochemical cell microscopy (SECCM), the PEC catalytic effects of anatase TiO2-nanotube arrays grown on Ti felt are explored. The SECCM imaging is performed both perpendicular and parallel to the nanotube growth direction. In contrast to bulk cyclic voltammetry measurements, SECCM measures only the upper region of the nanotubes that remain in contact with the electrolyte, which provides a better understanding of the phenomena connected to the longitudinal charge transport. Despite the presence of regions with higher and lower photocurrent, the PEC reactivities of the nanotube tops and walls are roughly comparable with each other. The data support the model of orthogonal electron-hole separation. This model facilitates the photogenerated hole diffusion over the short distance to the electrolyte interface due to the sufficient transport of photoexcited electrons along the long axial direction of TiO2 nanotubes and is often applied to one-dimensional systems. Observed results were additionally supported by the nanotube decoration with photoelectrochemically deposited PbO2 particles.

DOI： 10.1021/acscatal.1c04910

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

The discharged state affects the charge transfer resistance of lithium-ion secondary batteries (LIBs), which is referred to as the depth of discharge (DOD). To understand the intrinsic charge/discharge property of LIBs, the DOD-dependent charge transfer resistance at the solid-liquid interface is required. However, in a general composite electrode, the conductive additive and organic polymeric binder are unevenly distributed, resulting in a complicated electron conduction/ion conduction path. As a result, estimating the DOD-dependent rate-determining factor of LIBs is difficult. In contrast, in micro/nanoscale electrochemical measurements, the primary or secondary particle is evaluated without using a conductive additive and providing an ideal mass transport condition. To control the DOD state of a single LiFePO4 active material and evaluate the DOD-dependent charge transfer kinetic parameters, we use scanning electrochemical cell microscopy (SECCM), which uses a micropipette to form an electrochemical cell on a sample surface. The difference in charge transfer resistance at the solid-liquid interface depending on the DOD state and electrolyte solution could be confirmed using SECCM.

DOI： 10.1021/acs.analchem.1c02851

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.coelec.2021.100739

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

DOI： 10.1002/adhm.202101186

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.carbon.2021.06.004

Web of Science

Scopus

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/elsa.202100053

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/elsa.202100053

Language：English   Publishing type：Research paper (scientific journal)  

Boron-doped diamond (BDD) is most often grown by chemical vapor deposition (CVD) in polycrystalline form, where the electrochemical response is averaged over the whole surface. Deconvoluting the impact of crystal orientation, surface termination, and boron-doped concentration on the electrochemical response is extremely challenging. To tackle this problem, we use CVD to grow isolated single-crystal microparticles of BDD with the crystal facets (100, square-shaped) and (111, triangle-shaped) exposed and combine with hopping mode scanning electrochemical cell microscopy (HM-SECCM) for electrochemical interrogation of the individual crystal faces (planar and nonplanar). Measurements are made on both hydrogen- (H-) and oxygen (O-)-terminated single-crystal facets with two different redox mediators, [Ru(NH3)6]3+/2+ and Fe(CN)64-/3-. Extraction of the half-wave potential from linear sweep and cyclic voltammetric experiments at all measurement (pixel) points shows unequivocally that electron transfer is faster at the H-terminated (111) surface than at the H-terminated (100) face, attributed to boron dopant differences. The most dramatic differences were seen for [Ru(NH3)6]3+/2+ when comparing the O-terminated (100) surface to the H-terminated (100) face. Removal of the H-surface conductivity layer and a potential-dependent density of states were thought to be responsible for the behavior observed. Finally, a bimodal distribution in the electrochemical activity on the as-grown H-terminated polycrystalline BDD electrode is attributed to the dominance of differently doped (100) and (111) facets in the material.

DOI： 10.1021/acs.analchem.1c00053

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.

DOI： 10.1039/d0nr08349f

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

DOI： 10.1021/acs.analchem.0c04097

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.

DOI： 10.1021/acs.analchem.0c05174

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：The Japan Society of Applied Physics  

DOI： 10.11470/jsapmeeting.2021.1.0_1370

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

Electrochemical CO2 reduction is a key technology to recycle CO2 as a renewable resource, but adsorbing CO2 on the catalyst surface is challenging. We explored the effects of reduced graphene oxide (rGO) in Sn/rGO composites and found that the CO2 adsorption ability of Sn/rGO was almost 4-times higher than that of bare Sn catalysts. Density functional theory calculations revealed that the oxidized functional groups of rGO offered adsorption sites for CO2 toward the adjacent Sn surface and that CO2-rich conditions near the surface facilitated the production of formate via COOH∗ formation while suppressing CO∗ formation. Scanning electrochemical cell microscopy directly indicated that CO2 reduction was accelerated at the interface, together with the kinetic suppression of undesirable and competitive hydrogen evolution at the interface. Thus, the synergism of Sn/rGO ensures a substantial/rapid supply of CO2 from the functional groups to the Sn surface, thereby enhancing the Faradaic efficiency 1.8-times compared with that obtained with bare Sn catalysts.

DOI： 10.1021/acscatal.0c04887

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Transient amperometry with a microelectrode was applied to a SrTiO₃ photocatalyst for water splitting. <italic>Operando</italic> O₂ detection at intervals of 0.1 s indicated light-triggered O₂ adsorption and desorption in addition to photocatalytic O₂ evolution.

DOI： 10.1039/d1cp03264j

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Nanopore pH sensing is based on the interaction between the surface charge of the nanopore and ions passing through the nanopore. The nanopore surface charge can be derived from the acid-base dissociation equilibrium of the modified polyelectrolyte. Various polyelectrolytes have been selected based on the acid dissociation constant of the monomer units, and various techniques have been applied to modify nanopores. However, they have been developed without clear guidelines for characterizing the surface modification status or surface charge. One reason has been the difficulty in accurately estimating the surface charge of nanopores in solution. Thus, in this study, the dissociation constant (pKa ) of the surface charge of a modified polyelectrolyte nanopore was quantitatively estimated via electrochemical measurements. Previously, the modification status of nanopores has been evaluated using the ion current response. In addition, we monitored in real-time the polyelectrolyte modification status using a quartz crystal microbalance (QCM). Some polyelectrolytes were difficult to immobilize directly on the nanopore surface, and those polymers could be effectively modified by the layer-by-layer (LbL) technique. Therefore, we produced a guideline for the fabrication of a nanopore sensor for pH measurements under physiological conditions by quantitative evaluation of the pKa via electrochemical methods, the monitoring of the modification status by QCM, and the development of an effective modification method via the LbL technique. app app

DOI： 10.1002/elan.202100041

Web of Science

Scopus

Language：Japanese   Publisher：The Japan Society of Vacuum and Surface Science  

<p>To improve the performance of catalysts and batteries, it is very important to visualize the surface reactivity with the structure on a micro/nanoscale to understand the crystal orientation and grain boundary-dependent reactivity. Therefore, we have developed scanning electrochemical cell microscopy that uses a nanopipette as a probe to locally form an electrochemical cell and scan the nanopipette on the sample surface to visualize the reactivity of the sample surface. Using SECCM, we characterized the hydrogen evolution reaction of nanosheet material and the charge/discharge property of the lithium-ion secondary battery.</p>

DOI： 10.14886/jvss.2021.0_1da05

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

© Water splitting to produce H2 and O2 is a fundamental reaction for artificial photosynthesis on semiconductor photocatalysts. The mechanism of the multistepped reaction, especially four-electron oxidation to O2, has not yet been understood. Although some intermediate states have been detected in transient spectroscopy, O2 evolution kinetics remain unknown at the end of consecutive reaction steps. We apply operando O2 detection with a microelectrode to determine the absolute evolution rate on a highly efficient SrTiO3 photocatalyst film casted on a glass plate. The evolution rate was determined with a time resolution of 0.1 s, which was improved by 1000 times compared with that in widely used gas-chromatographic detection. The observed rate did not respond instantaneously to excitation light irradiation. When light was turned on, the photocatalyst film was inactive for evolution and light-activated in seconds. It was proposed that the first absorbed photons were consumed to fill trap states on SrTiO3 surface and then the latter photons drove steady O2 evolution. When excitation light stopped, the O2 evolution rate exponentially decayed in seconds. The microelectrode method demonstrated herein will be useful for understanding many other reaction kinetics at liquid-solid interfaces.

DOI： 10.1021/acscatal.0c04115

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

To visualize the electrochemical reactivity and obtain the diffusion coefficient of the anode of lithium-ion batteries, we used scanning electrochemical cell microscopy (SECCM) in a glovebox. SECCM provided the facet-dependent diffusion coefficient on a Li4Ti5O12 (LTO) thin-film electrode and detected the metastable crystal phase of LixFePO4.

DOI： 10.1039/d0cc02865g

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

DOI： 10.1021/acs.analchem.0c01256

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Copyright © 2020 American Chemical Society. Confinement of hydrogen molecules at graphene-substrate interface has presented significant importance from the viewpoints of development of fundamental understanding of two-dimensional material interface and energy storage system. In this study, we investigate H2 confinement at a graphene-Au interface by combining selective proton permeability of graphene and the electrochemical hydrogen evolution reaction (electrochemical HER) method. After HER on a graphene/Au electrode in protonic acidic solution, scanning tunneling microscopy finds that H2 nanobubble structures can be produced between graphene and the Au surface. Defect dependence of the bubble formation suggests that intrinsic defects in graphene, which have high hydrogen permeation barrier but are permeable for protons, are involved in the fundamental mechanism of bubble formation. Strain analysis by Raman spectroscopy also shows that atomic size roughness on the graphene/Au surface originating from the HER-induced strain relaxation of graphene plays significant role in formation of the nucleation site and H2 storage capacity. The result presented herein would provide further understanding of molecular confinement at graphene-based interface and development of novel energy material.

DOI： 10.1021/acs.jpcc.0c00995

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim High-resolution scanning electrochemical cell microscopy (SECCM) is used to image and quantitatively analyze the hydrogen evolution reaction (HER) catalytically active sites of 1H-MoS2 nanosheets, MoS2, and WS2 heteronanosheets. Using a 20 nm radius nanopipette and hopping mode scanning, the resolution of SECCM was beyond the optical microscopy limit and visualized a small triangular MoS2 nanosheet with a side length of ca. 130 nm. The electrochemical cell provides local cyclic voltammograms with a nanoscale spatial resolution for visualizing HER active sites as electrochemical images. The HER activity difference of edge, terrace, and heterojunction of MoS2 and WS2 were revealed. The SECCM imaging directly visualized the relationship of HER activity and number of MoS2 nanosheet layers and unveiled the heterogeneous aging state of MoS2 nanosheets. SECCM can be used for improving local HER activities by producing sulfur vacancies using electrochemical reaction at the selected region.

DOI： 10.1002/anie.201912863

Web of Science

Scopus

PubMed

Language：English  

Dynamic reassembly of the cytoskeleton and structural changes represented by dendritic spines, cargo transport, and synapse formation are closely related to memory. However, the visualization of the nanoscale topography is challenging because of the diffraction limit of optical microscopy. Scanning ion conductance microscopy (SICM) is an effective tool for visualizing the nanoscale topography changes of the cell surface without labeling. The temporal resolution of SICM is a critical issue of live-cell time-lapse imaging. Here, we developed a new scanning method, automation region of interest (AR)-mode SICM, to select the next imaging region by predicting the location of a cell, thus improving the scanning speed of time-lapse imaging. The newly developed algorithm reduced the scanning time by half. The time-lapse images provided not only novel information about nanoscale structural changes but also quantitative information on the dendritic spine and synaptic bouton volume changes and formation process of the neural network that are closely related to memory. Furthermore, translocation of plasmalemmal precursor vesicles (ppvs), for which fluorescent labeling has not been established, were also visualized along with the rearrangement of the cytoskeleton at the growth cone.

DOI： 10.1021/acs.analchem.9b04775

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：The Electrochemical Society of Japan  

<p></p>

DOI： 10.5796/denkikagaku.20-FE0022

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

DOI： 10.1038/s41467-019-13535-1

Web of Science

Scopus

PubMed

Publishing type：Research paper (scientific journal)   Publisher：The Electrochemical Society  

<jats:p> Lithium-ion battery is one of the most popular secondary batteries for practical applications such as mobile phones and electrical cars because of their high energy density and good cyclability. For further improvement of their total performance with highly efficient cyclability, it is necessary to be in precise control of lithium-ion transport at the interface between electrolyte and electrodes. There is a major issue for efficient ion transport due to solid electrolyte interface (SEI) on negative composite electrodes such as graphite and/or silicon with some binders and electrical additives. The SEI is in general formed by decomposition of electrolyte. However, the SEI formation process or SEI itself is not still clear how the decomposition process proceeds due to the surface structure, binders or dispersion of active materials. Here, in this work we have used a scanning electrochemical microscopy (SECCM) with a single barrel nanopipette inside glove-box for analyzing local electrochemical reaction for SEI formation. A 50 nm radius nanopipette was filled with organic electrolyte (1.0 M LiPF<jats:sub>6</jats:sub> in ethylene carbonate:diethyl carbonate = 1:1 in vol. %) with a Li reference electrode. When the pipette was approached to the sample surface, a meniscus was created. Then, through the meniscus as a nanoscale electrochemical simulator, cyclic voltammogram was measured to introduce the SEI in localized area. The location SEI formed and their created potential were mapped by scanning the pipette on the sample. Further, on the particular structure such as basal and edge of graphite, the decomposition process was also analyzed for investigation of material structures for SEI formation. Those information would be particularly important to understand their mechanism and to control SEI formation. </jats:p>

DOI： 10.1149/ma2019-01/47/2267

Publishing type：Research paper (scientific journal)   Publisher：The Electrochemical Society  

<jats:p> To understand the metal oxide coating effect on battery performance, the following two techniques are required: 1) constructing a flat thin-film electrode surface to realize a well-defined interface and 2) analyzing the electrode/electrolyte interface reaction with nanoscale resolution. We previously studied flat LiCoO<jats:sub>2</jats:sub> thin-film electrodes using<jats:italic> in situ</jats:italic> surface-sensitive X-ray absorption spectroscopy (XAS) and reported that Co reduction at the LiCoO<jats:sub>2</jats:sub> surface resulting from electrolyte contact caused the initial degradation. We also showed that the ZrO<jats:sub>2</jats:sub> layer successfully prevented physical contact between LiCoO<jats:sub>2</jats:sub> and the electrolyte. And it confirmed that a thicker ZrO<jats:sub>2</jats:sub> layer (above 2 nm) increased the diffusion resistance of the lithium ions in the ZrO<jats:sub>2</jats:sub> layer. However, since XAS lacks in-plane resolution and provides only averaged information, it is impossible to analyze the ZrO<jats:sub>2</jats:sub> morphology in detail. Recently, Taguchi et al. investigated a thin Li-Zr-layer (ca. 2 nm) on a LiCoO<jats:sub>2</jats:sub> composite electrode by transmission electron microscopy (TEM). They suggested that this thin layer could improve the durability.<jats:sup /> However, it is difficult to analyze the electrochemical properties using TEM. To evaluate the intrinsic mechanism of the metal oxide coating effect, it is necessary to develop a novel <jats:italic>in-situ</jats:italic> method that can analyze the surface morphology with high spatial resolution and simultaneously determine the local electrochemical properties. </jats:p>
<jats:p>Scanning electrochemical microscopy (SECM) is a powerful technique for linking the surface morphology of a sample to its electrochemical properties. For the battery materials, the SECM feedback mode is effective in monitoring solid electrolyte interphase formation. To directly and quantitatively investigate spatially resolved ionic processes, mercury-capped platinum ultramicroelectrodes were developed and employed for Li<jats:sup>+</jats:sup> imaging based on Li stripping. However, it is difficult to visualize the Li<jats:sup>+</jats:sup> flux in battery materials at the sub-micrometer scale by SECM. Scanning electrochemical cell microscopy (SECCM), which uses a nanopipette as a probe and forms a local electrochemical cell, is effective in characterizing surface reactivity. We recently applied SECCM for visualization of electrochemical activities on a lithium-ion battery cathode material at sub-micrometer resolution. The SECCM was applied to collect or provide Li in specified area confined by the nanopipette. Further, it collection visualized the electrochemical properties by scanning the nanopipette as an image. There are some strong advantages in SECCM for battery material research such as its high spatial resolution, small capacitive current, and isolated electrochemical cell. </jats:p>
<jats:p>In this report, we applied SECCM to characterize a ZrO<jats:sub>2</jats:sub>-coated LiCoO<jats:sub>2</jats:sub> thin-film electrode prepared by pulsed laser deposition. Local cyclic voltammetry (CV) and galvanostatic charge/discharge were performed to characterize the cycle durability and rate performance of ZrO<jats:sub>2</jats:sub>-coated LiCoO<jats:sub>2</jats:sub> thin-film electrodes and to reveal the relationship between the ZrO<jats:sub>2</jats:sub> morphology and thickness. </jats:p>

DOI： 10.1149/ma2019-01/47/2259

Language：Japanese   Publisher：The Japan Society of Applied Physics  

DOI： 10.11470/jsapmeeting.2019.1.0_1521

CiNii Research

Language：Japanese   Publisher：The Japan Society of Applied Physics  

DOI： 10.11470/jsapmeeting.2019.1.0_3620

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

© 2018 John Wiley &amp; Sons, Ltd. Scanning electrochemical cell microscopy with a single barrel micro-/nano-pipette (SECCM) was applied to lithium iron phosphate (LiFePO4) composite positive electrodes and an isolated LiFePO4 secondary particle for lithium-ion batteries. To analyze lithium-ion (Li+) charge or discharge process on the electrodes using local probe, a pipette filled with LiCl electrolyte solution and Ag/AgCl quasi-reference counter electrode (QRCE) was used. Both the local electrochemical activities of LiFePO4 on the composite electrodes and a single particle were revealed by SECCM as in-situ direct measurement of current response-related Li+ transport from mapping and cyclic voltammogram at confined area by the pipette. The mapping has visualized Li+ deintercalation process from LiFePO4 at +0.65 V (applied between the sample-QRCE). We show that the SECCM system is a strong analytical tool for a characterization of local Li+ behavior of active electrode materials in lithium-ion batteries.

DOI： 10.1002/sia.6538

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

© 2019 The Authors. Published by WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim Carbon-based metal-free catalysts for the hydrogen evolution reaction (HER) are essential for the development of a sustainable hydrogen society. Identification of the active sites in heterogeneous catalysis is key for the rational design of low-cost and efficient catalysts. Here, by fabricating holey graphene with chemically dopants, the atomic-level mechanism for accelerating HER by chemical dopants is unveiled, through elemental mapping with atomistic characterizations, scanning electrochemical cell microscopy (SECCM), and density functional theory (DFT) calculations. It is found that the synergetic effects of two important factors—edge structure of graphene and nitrogen/phosphorous codoping—enhance HER activity. SECCM evidences that graphene edges with chemical dopants are electrochemically very active. Indeed, DFT calculation suggests that the pyridinic nitrogen atom could be the catalytically active sites. The HER activity is enhanced due to phosphorus dopants, because phosphorus dopants promote the charge accumulations on the catalytically active nitrogen atoms. These findings pave a path for engineering the edge structure of graphene in graphene-based catalysts.

DOI： 10.1002/advs.201900119

Web of Science

Scopus

PubMed

Language：Japanese  

Web of Science

Language：Japanese   Publisher：The Japan Society for Analytical Chemistry  

<p>Scanning probe microscopy is an effective tool for nanoscale imaging and characterization at a solid-liquid interface. Scanning electrochemical microscopy (SECM) and the scanning ion conductance microscopy (SICM) using microelectrodes and nanopipettes as probes are applied to cell surface chemical and topographical imaging, respectively. In this review, we introduce our recent study concerning the development of nanoscale electrodes and a system for high-resolution of SECM, electrochemical imaging of membrane proteins, electrochemical evaluation of differentiation state of embryonic stem cells, hybrid system of SECM and SICM, neurotransmitters measurement, high-speed SICM, and chemical field effect transistor.</p>

DOI： 10.2116/bunsekikagaku.68.33

Scopus

CiNii Research

J-GLOBAL

Language：Japanese   Publisher：公益社団法人 日本薬理学会  

&lt;p&gt;Primary cilia are non-motile cilia consisting of a centriole-derived basal body and a microtubule-based axoneme. In recent years, the structure and function of primary cilia have been attracting attention due to the relation with the onset of ciliary disease. Scanning ion conductance microscopy (SICM) is a probe microscopy used to measure the topography and functions of living cells at nanoscale. Furthermore, the labelling procedure is not necessary for SICM measurement compare to fluorescence imaging. We compared the structures of primary cilia of human retinal pigment epithelial cell line (RPE-1 cells) and Madin-Darby canine kidney cell line (MDCK cells) at nanoscale by using SICM. In addition, high resolution SICM images have also succeeded in visualizing ciliary pockets that difficult to be fluorescently labeled.&lt;/p&gt;

DOI： 10.1254/fpj.154.192

Scopus

PubMed

CiNii Research

Language：Japanese   Publisher：公益社団法人 日本薬理学会  

&lt;p&gt;Scanning electrochemical microscopy (SECM), which utilizes microelectrodes as a probe to measure the chemicals released and consumed by cells as current signal, is a promising tool for measuring the metabolites of cells. We have improved SECM resolution for single cell imaging by miniaturizing the size of the electrode and developing hybrid system of SECM and scanning ion conductance microscopy (SICM), which utilizes nanopipette as a probe to measure live cell topography. SECM-SICM provides simultaneous imaging of concentration profiles of chemical substances and cell surface topography. Using this system, we successfully measured the release of neurotransmitters from PC12 cells. In addition, the nanoscale electrodes are useful for intracellular chemical detection by inserting the electrodes into cells and measured reactive oxygen species (ROS).&lt;/p&gt;

DOI： 10.1254/fpj.153.267

Scopus

PubMed

CiNii Research

Language：Japanese   Publisher：Japanese Pharmacological Society  

<p>Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.</p>

DOI： 10.1254/jpssuppl.92.0_1-s09-3

CiNii Research

Language：Japanese   Publisher：Japanese Pharmacological Society  

<p>Local metabolite and ionic strength are important factors for maintaining the functions of living cells. We have developed micro-nanoscale electrode and electrochemical sensor based scanning probe microscopy to measure the spatial electrochemical metabolite and ion concentration profile near the sample surface with nanoscale resolution. Scanning electrochemical microscopy (SECM) uses an ultramicroelectrode as a probe for detecting electroactive chemical species (oxygen, ATP, and reactive oxygen species (ROS), and neurotransmitter). SECM has been recognized as an effective tool for investigating micrometer-scale local chemical flux. Miniaturization of the electrode is an important factor for improving SECM resolution.Electrode–sample distance control is also an important factor for measuring fast chemical flux and improving SECM resolution. Distance control by ion current feedback is a promising way for the non-contact investigation of soft materials, SECM–scanning ion-conductance microscopy (SICM) has been used in a hybrid system to improve SECM resolution by controlling the electrode probe and sample distance in solutions without direct contact. SICM is also useful for detecting the ion concentration profile and charge measurement in a solution. We measured the 3D chemical and ion current using SECM–SICM. In this presentation, we report the SICM topography images of gastric surface mucous cell lines (GSM06), which produce periodic acid-schiff and concanavalin A positive glycoproteins. To visualize the damage process of the mucosal layer, we added ethanol to GSM06 cells and imaged the topography change using SICM. We also performed topography and electrochemical simultaneous imaging using SICM-SECM to identify the mucosal layer without labelling.</p>

DOI： 10.1254/jpssuppl.92.0_2-s14-3

CiNii Research

Language：Japanese  

J-GLOBAL

Language：Japanese   Publishing type：Research paper (scientific journal)  

Cathode surface coating with metal-oxide thin layers has been intensively studied to improve the cycle durability of lithium-ion batteries. A comprehensive understanding of the metal-oxide morphology and the local electrochemical properties is essential for figuring out the metal-oxide coating effect. In this study, scanning electrochemical cell microscopy (SECCM) is used to analyze the surface morphology with high spatial resolution, together with the local electrochemical properties.

DOI： 10.1039/c8cc08916g

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

© 2018 Wiley-VCH Verlag GmbH &amp; Co. KGaA, Weinheim An antibody-induced internalization of epidermal growth factor receptor (EGFR) was quantitatively monitored using scanning electrochemical microscopy (SECM) at the single-cell level. The antibody Cetuximab is used for cancer treatment because of its capability to bind and internalize EGFR, which is one of the key membrane proteins associated with many cancers. To monitor antibody-induced EGFR internalization, Cetuximab was simply labelled with enzymes and the resulting enzymatic products were then electrochemically detected by using a microelectrode. By optimizing experimental parameters, such as immunostaining condition, enzyme type, and buffer solution, we were able to successfully monitor the EGFR internalization by using SECM, from which we found a decrease in current responses from a single living cell by 20 % in the first 60 min, suggesting different internalization pathways from inherent ligand-induced internalization.

DOI： 10.1002/celc.201800563

Web of Science

Scopus

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.

DOI： 10.1021/acs.analchem.7b05112

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The article An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution, written by Hiroshi Hibino, Madoka Takai, Hidenori Noguchi, Seishiro Sawamura, Yasufumi Takahashi, Hideki Sakai and Hitoshi Shiku, was originally published Online First without open access.

DOI： 10.1007/s12576-017-0572-6

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：The Physical Society of Japan  

DOI： 10.11316/jpsgaiyo.73.1.0_3049

CiNii Research

Language：Japanese  

DOI： 10.11477/mf.2425200655

CiNii Research

J-GLOBAL

Language：English   Publisher：Elsevier B.V.  

Carbon nanoelectrodes are emerging as a powerful tool for local electrochemical measurements. In addition to the advantages of carbon electrodes, i.e., their electrochemical catalytic property, high current density, low ohmic drop, low capacitive current, wide potential window, and minimal electrode fouling, nanoscale spatial resolution opens up new possibilities for single synapse and intracellular measurements as well as for high sensitive chemical measurements. In this review, we present the recent progress made in the applications of carbon nanoelectrodes, mainly in electrochemical sensing and electrochemical imaging.

DOI： 10.1016/j.coelec.2017.07.014

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Local cell-membrane permeability and ionic strength are important factors for maintaining the functions of cells. Here, we measured the spatial electrochemical and ion concentration profile near the sample surface with nanoscale resolution using scanning electrochemical microscopy (SECM) combined with scanning ion-conductance microscopy (SICM). The ion current feedback system is an effective way to control probe-sample distance without contact and monitor the kinetic effect of mediator regeneration and the chemical concentration profile. For demonstrating 3D electrochemical and ion concentration mapping, we evaluated the reaction rate of electrochemical mediator regeneration on an unbiased conductor and visualized inhomogeneous permeability and the ion concentration 3D profile on a single fixed adipocyte cell surface.

DOI： 10.1039/c7cp05157c

Web of Science

Scopus

PubMed

Language：English   Publisher：SPRINGER JAPAN KK  

In vivo, cells are immersed in an extracellular solution that contains a variety of bioactive substances including ions and water. Classical electrophysiological analyses of epithelial cells in the stomach and small intestine have revealed that within a distance of several hundred micrometers above their apical plasma membrane, lies an extracellular layer that shows ion concentration gradients undetectable in the bulk phase. This "unstirred layer", which contains stagnant solutes, may also exist between the bulk extracellular solution and membranes of other cells in an organism and may show different properties. On the other hand, an earlier study using a bacterial planar membrane indicated that H+ released from a transporter migrates in the horizontal direction along the membrane surface much faster than it diffuses vertically toward the extracellular space. This result implies that between the membrane surface and unstirred layer, there is a "nanointerface" that has unique ionic dynamics. Advanced technologies have revealed that the nanointerface on artificial membranes possibly harbors a highly ordered assembly of water molecules. In general, hydrogen bonds are involved in formation of the ordered water structure and can mediate rapid transfer of H+ between neighboring molecules. This description may match the phenomenon on the bacterial membrane. A recent study has suggested that water molecules in the nanointerface regulate the gating of K+ channels. Here, the region comprising the unstirred layer and nanointerface is defined as the interphase between the plasma membrane and bulk extracellular solution (iMES). This article briefly describes the physicochemical properties of ions and water in the iMES and their physiological significance. We also describe the methodologies that are currently used or will be applicable to the interphase research.

DOI： 10.1007/s12576-017-0530-3

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Observation of nanoscale structure dynamics on cell surfaces is essential-to understanding cell functions. Hopping Mode, scanning ion conductance microscopy (SICM) was used to visualize the topography, of fragile convoluted nanoscale structures on cell surfaces under noninvasive conditions. However, conventional hopping mode SICM does not have sufficient temporal resolution to observe cell-surface dynamics in situ because of the additional time required for performing vertical probe movements of the nanopipette. Here, we introduce a new scanning algorithm for high speed SICM measurements using low capacitance and high-resonance-frequency piezo stages. As a result, a topographic image is taken within 18 s with a 64 x 64 pixel resolution at 10 X 10 mu m. The high speed SICM is applied to the characterization of microvilli dynamics on surfaces, which shows clear structural changes after the epidermal growth factor stimulation.

DOI： 10.1021/acs.analchem.7b00584

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

DOI： 10.1002/anie.201703578

Web of Science

Scopus

PubMed

Language：Chinese   Publishing type：Research paper (scientific journal)   Publisher：ELECTROCHEMICAL SOC JAPAN  

We developed three modes of high resolution scanning probe microscope system based on electrochemical principles, scanning ion conductance microscopy (SICM), scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM), and scanning electrochemical cell microscopy (SECCM). Firstly, a developed SICM system was constructed with a nanopipette filled with electrolyte solution as a probe. High resolution topographic images of NanoCulture (R) Plate with hexagonal chambers with 2 mu m-width banks, electro-deposited PEDOT (poly(3,4-ethylenedioxythiophane)) film, and fixed human squamous cell carcinoma were captured by the SICM. Second, SECM-SICM was performed with a double-barrel carbon nanoprobe. The one barrel of the nanoprobe was filled with carbon and used for SECM apparatus, the other barrel was filled with electrolyte for SICM configurations. Using the SECM-SICM system, simultaneous topographic and electrochemical images of micro-band electrodes with 10 pm-width line and space, and a cathode site of corrosion in the aluminum die gusto with submicron spatial resolution were obtained. Thirdly, the SECCM featuring a nanopipette probe filled with LiCI electrolyte was applied for obtaining topographies and images of current activity of a LiFePO4 electrode. (C) The Electrochemical Society of Japan, All rights reserved.

DOI： 10.5796/electrochemistry.85.319

Web of Science

Scopus

CiNii Research

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Investigation of the positional heterogeneity of messenger RNA (mRNA) expression in tissues requires a technology that facilitates analysis of mRNA expression in the selected single cells. We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control. The MP was used for continuous collection of different nucleic acid samples and sequential evaluation of gene expression with mRNA barcoding tags. First, we found that the aqueous phases could be laminated into five individual layers and separated by the plugs of the organic phases in a nanopipette when the salt (THATPBCl) concentration in the organic phase was 100 mM. Second, the aspiration rate of the MP was stabilized and the velocity of the aqueous phase in the MP was lowered at higher THATPBCl concentrations in the organic phase. This was because the force during ingression of the aqueous phase into the organic - phase-filled nanopipette induced an electro-osmotic flow between the inside wall of the nanopipette and THATPBCl in the organic phase. Third, inclusion of mRNA barcoding tags in the MP facilitated complementary DNA construction and sequential analysis of gene expression. This technique has potential to be applicable to RNA sequencing from different cell samples across the life sciences.

DOI： 10.1007/s00216-016-0006-y

Web of Science

Scopus

PubMed

Language：English   Publisher：AMER CHEMICAL SOC  

DOI： 10.1021/acs.analchem.6b04355

Web of Science

Scopus

PubMed

Language：Japanese  

CiNii Books

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical Society of Japan  

A novel and simple method for constructing cell sheets is described in which culture surfaces were modified with RGD peptide-coupled alginate hydrogels using an electrodeposition method. Cells were cultured on the hydrogels to form a contiguous cell sheet-like structure. Then, the hydrogels were dissolved by addition of an EDTA solution and the cell sheets rapidly detached from the surface. This is the first report of the fabrication of cell sheets on RGD peptide-coupled alginate hydrogels using an electrodeposition method. We believe the technique is useful for cell sheet engineering.

DOI： 10.1246/cl.170003

Scopus

Language：Japanese   Publishing type：Research paper (scientific journal)  

CiNii Books

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELECTROCHEMICAL SOC JAPAN  

This article reviews our recent progress in the development of high-resolution scanning electrochemical microscopy (SECM) and its application to biological samples. SECM uses an ultramicroelectrode (UME) as a probe and a scanning mechanical stage for controlling the probe position. To improve the resolution of SECM, we have developed a fabrication method for pyrolytic carbon nanoelectrodes and a current feedback system for probe sample distance control. The current feedback system effectively provides high-quality electrochemical and non contact topography images because the current signal depends on the probe-sample distance. High-resolution SECM has overcome the limit of microscale imaging resolution and enabled the imaging of local regions within cells. In this study, we address four topics: nanoelectrode fabrication, current feedback probe-sample distance control systems, membrane protein imaging, and neurotransmitter detection. (C) The Electrochemical Society of Japan, All rights reserved.

DOI： 10.5796/electrochemistry.84.662

Web of Science

Scopus

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

DOI： 10.1021/acsnano.6b02753

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

A liquid-junction-free electrochemical system has been developed for substitutional stripping voltammetry (SSV). SSV is a type of stripping analysis that changes the redox current to metal deposition. Generally, SSV requires a liquid junction to maintain electrical conductivity between separated cells. In this report, a closed bipolar electrode system(cBPES) was used to perform SSV without a liquid junction. In this system, electrical conductivity between separated cells was maintained using driving electrodes inserted into each cell. We demonstrated quantification of p-aminophenol (pAP) using a liquid-junction-free system for SSV. We investigated the effect of electrode area ratio on the BPE and the concentration ratio of redox species present in each cell. Then, we calibrated the pAP. A linear relationship between pAP concentration and SSV electrical charge was successfully obtained using our liquid-junction-free system, based on cBPES. This novel analytical system is a promising method for the fabrication of a compact SSV sensor for highly sensitive detections. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.elecom.2016.02.014

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELECTROCHEMICAL SOC JAPAN  

Single-cell analyses are important for providing new insights into cellular biology. Here we report an electrochemical reporter gene assay for single cells using a scanning electrochemical microscope (SECM)-microwell system. Each microwell trapped a single cell that synthesized a reporter protein, secreted alkaline phosphatase (SEAP). The SEAP catalyzed the hydrolysis of p-aminophenyl phosphate to p-aminophenol (PAP). A disk electrode in the SECM was positioned above the microwell and monitored the oxidation currents of PAP derived from SEAP. In addition, ring electrodes were prepared on the microwell device to induce redox cycling between the ring and disk electrodes, thus amplifying the electrochemical signals from the reporter protein. The redox cycling-based electrochemical reporter gene assay is useful for single-cell analyses. (C) The Electrochemical Society of Japan, All rights reserved.

DOI： 10.5796/electrochemistry.84.308

Web of Science

Scopus

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

DOI： 10.1021/acsnano.5b05211

Web of Science

Scopus

PubMed

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The mouse embryonic stem (ES) cell-derived angiogenesis model is widely used as a 3D model, reproducing cell cell interactions in the living body. Previously, many methods to analyze localized cellular function, including in situ hybridization and laser capture microdissection, have been reported. In this study, we achieved a collection of localized cells from the angiogenesis model in hydrogel. The gene expression profiles of the endothelial cells derived from mouse ES cells were evaluated. First, we collected localized cells from the live tissue model embedded in hydrogel using the double barrel carbon probe (DBCP) and quantified mRNA expression. Second, we found that vascular marker genes were expressed at a much higher level in sprouting vessels than in the central core of the embryoid body because the cells in sprouting vessels might significantly differentiate into endothelial linages, including tip/stalk cells. Third, the gene expression levels tended to be different between the top and middle regions in the sprouting vessel due to the difference in the degree of differentiation in these regions. At the top region of the vessel, both the tip and stalk cells were present. The cells in the middle region became more mature. Collectively, these results show that DBCP is very useful for analyzing localized gene expression in cells collected from 3D live tissues embedded in hydrogel. This technique can be applied to comprehensive gene expression analyses in the medical field.

DOI： 10.1021/acs.analchem.5b04338

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：日本生物物理学会 = The Biophysical Society on Japan  

DOI： 10.2142/biophys.56.179

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本表面科学会  

&lt;p&gt;Scanning electrochemical cell microscopy with a single barrel nanopipette (nanoSECCM), which is one of scanning electrochemical microscope (SECM) families, is a powerful tool for analyzing electrochemical activities (e.g. redox reaction and lithium-ion (de) intercalation processes) at localized area. A glass nano-pipette is used as a probe of nanoSECCM filled with electrolyte and a reference electrode. As the pipette is in proximity of sample surface, a meniscus is created. Through the meniscus, electrochemical activities can be obtained at localized area on the sample. Further, as the pipette scans the sample surface, electrochemical activities can be also visualized. Our experiments demonstrate that nanoSECCM is applicable to investigation of current response related to lithium-ion transport, redox cycling on various types of electrodes.&lt;/p&gt;

DOI： 10.1380/jsssj.37.494

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In the present study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to image dopamine release from three-dimensional (3D)-cultured PC12 cells (PC12 spheroids). The Bio-LSI device consists of 400 sensor electrodes with a pitch of 250 mu m for rapid electroehemical imaging of large, areas. PC12 spheroids were stimulated with K+ to release dopamine. Poststimulation dopai-nine release fibril the PC12 spheroids was electrochemically imaged using the Bio-LSI device. Bia-LSI clearly showed the effects of the dopaminergic drugs L-3,4-dihydroxyphenylalanine (L-DOPA) and reserpine on K+-stimulated dopamine release from PC12 spheroids. Out results demonstrate that dopamine release from PC12 spheroids can be monitored using the device, suggesting that the Bio-LSI is a promising tool for use in evaluating 3D-cultured dopaminergic cells and the effects of dopaminergic drugs. To the best of our knowledge, this report is the first to describe electrochemical imaging of dopamine release by PC12 spheroids using LSI-based amperometric sensors.

DOI： 10.1021/acs.analchem.5b01307

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We fabricated a platinum-based double barrel probe for scanning electrochemical microscopy scanning ion conductance microscopy (SECM-SICM) by electrodepositing platinum onto the carbon nanoelectrode of the double barrel probe. The deposition conditions were optimized to attain highly sensitive electrochemical measurements and imaging. Simultaneous SECM-SICM imaging of electrochemical features and noncontact topography by using the optimized probe afforded high-resolution images of epidermal growth factor receptors (EGFR) on the membrane surface of the A431 cells.

DOI： 10.1021/acs.analchem.5b00027

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (similar to 500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.

DOI： 10.1021/ac5046388

Web of Science

Scopus

PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

This article presents an overview of the recent progress in scanning electrochemical microscopy (SECM) for imaging single cells and biomolecules. SECM is a technique for characterizing the local electrochemical nature of various materials by scanning a probe microelectrode. The probe current reflects the electrochemical processes occurring in the small space surrounded by the probe and the substrate. The spatial resolution of SECM is inferior to conventional scanning probe microscopes such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM), since the fabrication of the probe microelectrodes with nanometer sizes is quite difficult. However, recent progress in the fabrication of nanometer-scale electrodes and the development of electrode-sample distance control systems has greatly enhanced the capacity of SECM systems to solve problems in cell biology. The topics reviewed include the following: enzyme activity evaluation, electrochemical enzyme-linked immunosorbent assays, membrane permeability evaluation, respiratory activity measurements, reporter gene assays, membrane protein imaging, and neurotransmitter detection.

DOI： 10.1007/978-4-431-55190-4_16

Scopus

Language：Japanese   Publishing type：Research paper (international conference proceedings)  

© 15CBMS-0001. We report a large-scale integration (LSI) based amperometric chip device containing 400 sensors with a pitch of 250 μm and its applications for biological analysis of dopamine release from three-dimensional (3D) cultured cells using rat pheochromocytoma (PC12) cells as a neural cell model. The 3D cultured PC12 cells (PC12 spheroids) were stimulated with a high K+ solution to induce dopamine release. We showed clearly drug effects of dopaminergic drugs L-3,4-dihydroxyphenylalanine, (L-DOPA) and reserpine on dopamine release from PC12 spheroids. Our results indicate that the device is a useful tool for evaluating the effects of dopaminergic drugs.

Scopus

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1380/jsssj.36.313

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Intercalation and deintercalation of lithium ions at electrode surfaces are central to the operation of lithium-ion batteries. Yet, on the most important composite cathode surfaces, this is a rather complex process involving spatially heterogeneous reactions that have proved difficult to resolve with existing techniques. Here we report a scanning electrochemical cell microscope based approach to define a mobile electrochemical cell that is used to quantitatively visualize electrochemical phenomena at the battery cathode material LiFePO4, with resolution of similar to 100 nm. The technique measures electrode topography and different electrochemical properties simultaneously, and the information can be combined with complementary microscopic techniques to reveal new perspectives on structure and activity. These electrodes exhibit highly spatially heterogeneous electrochemistry at the nanoscale, both within secondary particles and at individual primary nanoparticles, which is highly dependent on the local structure and composition.

DOI： 10.1038/ncomms6450

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We evaluated the intracellular NAD(P) H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione-ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 mu M) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 mu M), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aca.2014.06.047

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：Angewandte Chemie - International Edition  

DOI： 10.1002/anie.201404979

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELECTROCHEMICAL SOC JAPAN  

Nanoscale cell surface topography was visualized using a scanning ion conductance microscope (SICM), where a nanopipette was used as the scanning probe to detect ionic current as feedback signal. SICM was an effective tool for noncontact topographical imaging of live cells, because measurements were performed under physiological conditions. This breakthrough technique opens up a wealth of possible new experiments in membrane and cell biology. (C) The Electrochemical Society of Japan, All rights reserved.

DOI： 10.5796/electrochemistry.82.331

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A new local redox cycling-based electrochemical (LRC-EC) device integrated with many electrochemical sensors has been developed into a small chip device. The LRC-EC chip device was successfully applied for detection of alkaline phosphatase and horseradish peroxidase activity in substrate generation/chip collection (SG/CC) and extended feedback modes, respectively. The new imaging approach with extended feedback mode was particularly effective for sharpening of the image, because this mode uses feedback signals and minimizes the undesired influence of diffusion. The LRC-EC chip device is considered to be a useful tool for bioanalysis.

DOI： 10.1021/ac500435d

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

A flexible sensor based on SU-8 photoresist was fabricated and its electrochemical performance was investigated using cyclic voltammetry. The device consisted of interdigitated array (IDA) electrodes on an SU-8 layer. It exhibited a clear electrochemical response during redox cycling of ferrocenemethanol at the IDA electrodes. Since the device was flexible, it could be inserted into a narrow bent space to monitor electrochemical responses. The observed electrochemical behavior was found to be consistent with that predicted by simulations based on redox compound diffusion.

DOI： 10.2116/analsci.30.305

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

In this study, we introduce the double-barrel carbon probe (DBCP)-a simple, affordable microring electrode-which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

DOI： 10.1007/s00216-013-7430-z

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5-200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.

DOI： 10.1021/nn405612q

Web of Science

Scopus

PubMed

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S189_4

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S245_3

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have previously reported a local redox cycling-based electrochemical (LRC-EC) system for the incorporation of many electrochemical sensors into a small chip device. In the present study, a new type of LRC-EC chip device was fabricated for the detection of a droplet array. To detect electrochemically redox compounds in droplets, Pt pseudo-reference/counter electrodes were incorporated into the individual sensors of the LRC-EC chip device. Cyclic voltammetry for the LRC-EC chip device with internal Pt pseudo-reference electrodes indicated well-defined voltammograms based on redox cycling for the individual sensor points. The device was successfully applied to the addressable detection of alkaline phosphatase (ALP) activity of HeLa cells in single droplets on the sensor points. Therefore, the LRC-EC chip device is considered to be a useful device for the bioanalysis of droplet systems.

DOI： 10.1039/c3lc51156a

Web of Science

Scopus

PubMed

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S266_3

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level or single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.

DOI： 10.1021/ac4021769

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

A local redox cycling-based electrochemical (LRC-EC) chip device was used to investigate the relationship between cardiomyocyte differentiation from embryonic stem (ES) cells and alkaline phosphatase (ALP) activity. In the LRC-EC chip device, ring-type interdigitated array electrodes were incorporated at n × n measurement points with only 2n bonding pads for external connection. Microwells were also fabricated at each measurement point to trap cell aggregates. To differentiate ES cells into cardiomyocytes, ES cells were three-dimensionally cultured to form simple and cystic embryoid bodies (EBs). ALP activity of these EBs was then detected using the LRC-EC chip device. The electrochemical responses for ALP activity decreased concurrently with the differentiation of ES cells into cardiomyocytes, indicating that an LRC-EC chip device is useful for evaluating cell differentiation. © 2013 The Electrochemical Society of Japan, All rights reserved.

DOI： 10.5796/electrochemistry.81.682

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Dual carbon electrodes (DCEs) are quickly, easily, and cheaply fabricated by depositing pyrolytic carbon into a quartz theta nanopipet. The size of DCEs can be controlled by adjusting the pulling parameters used to make the nanopipet. When operated in generation/collection (G/C) mode, the small separation between the electrodes leads to reasonable collection efficiencies of ca. 30%. A three-dimensional finite element method (FEM) simulation is developed to predict the current response of these electrodes as a means of estimating the probe geometry. Voltammetric measurements at individual electrodes combined with generation/collection measurements provide a reasonable guide to the electrode size. DCEs are employed in a scanning electrochemical microscopy (SECM) configuration, and their use for both approach curves and imaging is considered. G/C approach curve measurements are shown to be particularly sensitive to the nature of the substrate, with insulating surfaces leading to enhanced collection efficiencies, whereas conducting surfaces lead to a decrease of collection efficiency. As a proof-of-concept, DCEs are further used to locally generate an artificial electron acceptor and to follow the flux of this species and its reduced form during photosynthesis at isolated thylakoid membranes. In addition, 2-dimensional images of a single thylakoid membrane are reported and analyzed to demonstrate the high sensitivity of G/C measurements to localized surface processes. It is finally shown that individual nanometer-size electrodes can be functionalized through the selective deposition of platinum on one of the two electrodes in a DCE while leaving the other one unmodified. This provides an indication of the future versatility of this type of probe for nanoscale measurements and imaging.

DOI： 10.1021/ac401476z

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we fabricated a probe consisting of a carbon nanoelectrode and an Ag/AgCl reference electrode for detecting the activity of cells in single droplets. HeLa cells were confined into a single droplet, and the alkaline phosphatase (ALP) activity of the cells was electrochemically measured using the probe inserted into the droplet. The ALP of the confined cells catalyzed the hydrolysis of p-aminophenyl phosphate (PAPP) to yield p-aminophenol (PAP) that gave electrochemical responses. Since the tip of the carbon-Ag/AgCl probe is very small, it is useful for electrochemical analysis of cells using droplets. © 2013 American Chemical Society.

DOI： 10.1021/ac303569t

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering. © 2012 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2012.10.014

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

DOI： 10.1039/c3lc50455g

Web of Science

Scopus

PubMed

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S155_3

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S149_6

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The differentiation status of single live embryonic stem (ES) cells was quantitatively evaluated by monitoring the activity of alkaline phosphatase (ALP), an undifferentiation marker of ES cells, using scanning electrochemical microscopy (SECM).

DOI： 10.1039/c3cc43126f

Web of Science

Scopus

PubMed

Language：English   Publisher：The Surface Science Society of Japan  

The micro/nanoelectrode has unique characteristics, which are not expected for conventional electrodes, such as the capability to perform localized measurements, low double layer charging currents, low ohmic drops, and fast mass transport. Scanning electrochemical microscopy (SECM) with a micro/nanoelectrode for detecting electroactive chemical species and is an effective tool for the investigation of the localized chemical properties of sample surfaces and interfaces. Because SECM has high temporal resolution under physiological conditions, it is particularly well suited for quantitative measurements of chemicals like neurotransmitters, nitric oxide, reactive oxygen species, and oxygen, which are released/consumed by living cells. This article presents an overview of the recent progress of SECM.

DOI： 10.1380/jsssj.34.488

CiNii Books

J-GLOBAL

Language：Japanese   Publisher：Materials Research Society Symposium Proceedings  

By incorporating a localized heating system within a scanning ion-conductance microscopy (SICM) system, we have performed stable 'hopping-mode' (HPICM) imaging for live cells maintained at temperatures ranging up to human body temperature. This allows the accurate study of cell volume and morphology variation versus temperature over extended periods of time. The integration of SICM with scanning electrochemical microscopy (SECM) provides the simultaneous mapping of electrochemical and topographic information for soft samples, such as live cells. This combined technique overcomes the limitations of resolution and topographical artifacts typically associated with SECM. However, previously reported SECM-SICM probe production required expensive and time-consuming focused ion beam (FIB) methods and produced pipettes that are typically hundreds of nanometers in diameter. We report a simple and rapid production method for SECM-SICM double-barrel probes with apertures down to 20 nm in diameter. The characterization of these SECM-SICM probes using scanning electron microscopy (SEM) imaging, cyclic voltammetry (CV) and Raman spectroscopy is also detailed. These SECM-SICM probes were subsequently used to study the morphology and electrochemical activity of several samples, ranging from hard metallic/insulating samples to live cells. © 2012 Materials Research Society.

DOI： 10.1557/opl.2012.489

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We propose a novel electrochemical detection system for alkaline phosphatase (ALP) activity using the difference in water and oil solubilities between the substrate, ferrocene ethyl phosphate ester (FcEtOPO(3)(2-)), and the enzymatic product, ferroceneethanol (FcEtOH). In this system, water droplets containing ALP and FcEtOPO(3)(2-) were placed on a Pt disk microelectrode and surrounded by a mineral oil. By the ALP-catalyzed reaction, FcEtOPO(3)(2-) was converted to FcEtOH, which was then transferred to the mineral oil from the water droplets with FcEtOPO(3)(2-) remaining in the water droplets. After partitioning FcEtOH from the water droplets, FcEtOPO(3)(2-) was detected at the Pt disk microelectrode to estimate the ALP activity. Using this novel system, the ALP activity of embryoid bodies was successfully detected. We believe that the present system will be widely applicable to ALP-based bioassays.

DOI： 10.1021/ac301429n

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

DOI： 10.1073/pnas.1203570109

Web of Science

Scopus

PubMed

Language：Japanese  

DOI： 10.5796/electrochemistry.80.271

CiNii Books

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.201201602

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

This report describes the electrochemical detection of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device consisting of 256 sensors. The time-course analyses showed that the redox compound in the droplet was dynamically changed during droplet evaporation or mass transfer through a water/oil interface.

DOI： 10.1039/c2cc34264b

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The influence of the tip reaction on the substrate generation/tip collection mode SECM imaging of a single yeast cell was investigated by using various sizes of probe electrodes in mu m-nm scales because the tip reaction would disturb the concentration profile of intracellular enzyme reactant formed around the cell. GC mode measurements of a single yeast cell were performed using a dual mediator system with lipophilic menadione and hydrophilic ferricyanide. We found downscaling of the tip size enabled quantitative SECM imaging of a single cell in GC mode without disturbing the concentration profile of the mediator formed around the cell by the tip reaction.

DOI： 10.1002/elan.201000595

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：FUTURE MEDICINE LTD  

Cells naturally operate on the nanoscale level, with molecules combining together to form complex molecular machines, which can work together to enable normal cell function or go wrong as in the case of many diseases. Visualizing these key processes on the nanoscale has been difficult and two main approaches have been used to date; nanometer resolution imaging of fixed cells using electron microscopy, or imaging live cells using optical or fluorescence microscopy, with a resolution of a few hundred nanometers. Scanning probe microscopy has the potential to allow live cells to be imaged at nanoscale resolution and a noncontact method based on the use of a nanopipette probe has been developed over the last 10 years that allows both topographic and functional imaging. The rapid progress in this area of research over the last 4 years is reviewed in this article, which shows that imaging of complex cellular structures and tissues is now possible and that these methods are now sufficiently mature to provide new insights into important diseases.

DOI： 10.2217/NNM.10.154

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.201102796

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

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.

DOI： 10.1039/c1cp21886g

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

mRNA from single cells was quantified using real-time RT-PCR after recording the address and reporter protein activity with chemiluminescence, fluorescence, and electrochemical techniques, using luciferase, green fluorescent protein, and secreted alkaline phosphatase. mRNA copy number ranging from below 103 to 107 in single cells showed a lognormal distribution for both externally introduced reporter genes and internally expressed genes. The fluctuation in the gene expression decreased with the increase of the number of cells picked but did not decrease with the increase of mRNA copy number per cell. We found that the correlation coefficients for mRNA and protein expression in logarithmic plot at single-cell level were much lower than 1.00. (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2010.08.008

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/ SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

DOI： 10.1021/ja1029478

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

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

DOI： 10.1039/c002607g

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Electrochemical Society of Japan  

Microcontact printing (μCP) with various proteins has been widely applied to biosensors and cell biology research. However, the mechanism of protein patterning in the μCP process is not clear in detail We have electrochemically estimated enzyme concentration on glass slide patterned with μCP technique. We also estimated the enzyme concentration at the surface of poly(dimethylsiloxane) (PDMS), which corresponded to the enzyme activity at the PDMS stamp just before μCP. Our result suggested that it was possible to transfer enzyme monolayer from PDMS to glass surfaces with 100% efficiency with μCP. Immunoglobulin G (IgG) patterned with μCP was also characterized by tagging with enzyme labeled anti-IgG. Scanning electrochemical microscopy (SECM) image was obtained to visualize line and space pattern of IgG monolayer.

DOI： 10.5796/electrochemistry.78.122

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Gene-transfected single HeLa cells were characterized using a scanning electrochemical/optical microscope (SECM/OM) system with shear-force-based probe-sample distance regulation to simultaneously capture electrochemical, fluorescent, and topographic images. The outer and inner states of single living cells were obtained as electrochemical and fluorescent signals, respectively, by using an optical fiber-nanoelectrode probe. A focused ion beam (FIB) was used to mill the optical aperture and the ring electrode at the probe apex (the inner and outer radii of the ring electrode were 3 7 and 112 nm, respectively). To apply an appropriate shear force between the probe tip and the living cell surface, we optimized the amplitude of oscillation of the tuning fork to which the probe was attached. Field-programmable gate arrays (FPGA) were adopted to drastically increase the feedback speed of the tip-sample distance regulation, shorten the scanning time for imaging, and enhance the accuracy and quality of the living cell images. In employing these improvements, we simultaneously measured the cellular expression activity of both secreted alkaline phosphatase outside and GFP inside by using the SECM/OM with shear force distance regulation.

DOI： 10.1021/ac901796r

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

We microfabricated a novel device consisting of a 4 x 4 array microchamber sandwiched with the two microband electrode array. This device allows dielectrophoretic (DEP) manipulation of microbeads to introduce into and release out a certain address of the V-shaped microchamber, by applying AC voltage (10 V(pp), 10 kHz) on the basis of DEP forces. The design and the position of the two electrodes (row and column electrodes) at each microchamber were optimized by simulation based on a finite element method. More importantly, electrochemical generation-collection measurement was possible to evaluate enzymatic activity. After microbeads immobilized with glucose oxidase (GOD) was entrapped in the V-shaped microchamber with DEP, a measuring solution containing 3 mM ferrocenemethanol (FcCH(2)OH) and 0.1 M glucose was introduced. The medium in the V-shaped microwell was immediately exchanged into the measuring solution whereas microbeads stayed within the microwell without applying DEP voltage, because the flow within the microchamber was isolated from that of the main channel. Then the potential of the row and column electrodes were set at 0.5 and 0.1 V vs Ag/AgCl. The GOD activity can be monitored as the decrease in the [FcCh(2)OH](+) reduction current. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2009.05.028

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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.

DOI： 10.1021/ac900195m

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer Solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2008.10.039

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.200805743

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

A chip with integrated electrophoretic and electrochemical systems was developed to manipulate either an individual microbead or a cell inside a microwell electrode (MWE) for electrochemical measurement. The optimal MWE geometry (30 mm diameter and 25 mm depth) was designed to accommodate the micro particles according to the simulated results. A chip device was sequentially built from a slide patterned with Pt electrodes, an adhesive tape defined with a flow channel (200 mm in width and 25 mm in height), and an indium tin oxide (ITO) cover. The MWE not only generated an active electrophoretic force to trap the particle but also provided a low flow velocity area (LFVA) to stabilize the trapped bead or cell in a continuous flow. Scanning electrochemical microscopy (SECM) theory was employed to explain the electrochemical behaviors of the MWE. An enhanced current was confirmed as the redox recycling effect on the conductive ITO cover. The catalytic reaction of an individual alkaline phosphatase coated microbead (ALP-bead) was electrochemically detected with the MWE after being trapped. The ALP on the trapped ALP-bead catalyzed the hydrolysis of p-aminophenylphosphate (PAPP) to p-aminophenol ( PAP), and then a decaying amperogram (+0.3 V vs. Ag/AgCl) due to a tiny PAP quantity around the MWE was observed.

DOI： 10.1039/b817705h

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Column and row electrodes on two different glass substrates were orthogonally arranged in order to assemble an addressable microelectrode device for the purpose of comprehensive electrochemical detection. Amperometric signal at the individual crossing point of the column and row electrodes was detected separately on the basis of redox cycling of localized electroactive species occurring between the electrodes. The addressable microelectrode device was simple and could be easily assembled; however, it comprised as many as 10 x 10 addressable detection points on a single chip. The basic electrochemical performance of the device was investigated by using the ferricyanide/ferrocyanide redox couple. Electrochemical responses at 100 individual points could be collected within 22 s. The present device was successfully used for imaging the spots of alkaline phosphatase on the array substrate. The results indicate that the device can be applied to comprehensive and higb-throughput detection and imaging of biochemical species.

DOI： 10.1021/ac801389d

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008f.0.950.0

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Micropatterns of diaphorase (Dp) were fabricated on glass substrates by the microcontact printing (mu CP) method and characterized with atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM). AFM images of the printed samples revealed that the mean height of the Dp patterns was 3-5 nm, indicating the formation of a monolayer pattern. The Dp molecules on the surface organized themselves into two-dimensional arrays. We used two kinds of inking solutions: Dp-phosphate buffer solution (PBS) (pH 7.0) and Dp-PBS (pH 7.0) with glutaraldehyde (GA, 1% v/v) as a cross-linking reagent. Although the AFM imaging showed high-quality Dp monolayer patterns in both cases, SECM measurements indicated that the enzymatic activity of Dp was almost lost when Dp-PBS with GA was used as the inking solution, whereas clear enzymatic activity was found when Dp-PBS was used. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.elecom.2007.08.024

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 pin above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10,us between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 mu L of the lysed solution was collected using a microinjector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the singlecell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-die aggregated cellular masses formed during the three-dimensional culture-as a model system to isolate small cellular clusters from tissues and organs.

DOI： 10.1021/ac071050q

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：PHOENIX PUBL & MEDIA NETWORK  

We have applied the microfluidic probe (MFP) to collect mRNA from single adherent cells. The probe prepared from a theta (theta-shaped capillary can be positioned at any target single adherent cell. The cell lysis buffer was introduced from the injection aperture, and the cell-lysed solution was recovered from the aspiration aperture for further mRNA analysis based on reverse transcription (RT) real-time PCR. This method achieves not only sensitivity at the single cell level but identifies the differences in cell types based on the relative gene expression profiles. The collection efficiency for mRNA was quantitatively evaluated using real-time PCR.

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/la0611763

Scopus

PubMed

Language：Japanese  

DOI： 10.1380/jsssj.27.613

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

Web of Science

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

Language：English   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2013.03.006

Web of Science

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

Web of Science

Language：Japanese  

CiNii Books

Language：Japanese  

Language：Japanese  

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

File： takahashi 受賞講演3.pdf

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：English   Presentation type：Symposium, workshop panel (public)  

Language：Japanese  

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

File： 学会4.pdf

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：English   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Language：English   Presentation type：Oral presentation (invited, special)  

File： sample_Word_english4.pdf

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (invited, special)  

File： 抄録登録 [10044].eml

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (general)  

File： takahashi1.pdf

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

File： 日本語4.pdf

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Oral presentation (keynote)  

File： SECM8_abstract-template3.doc

Language：English   Presentation type：Oral presentation (invited, special)  

Language：Japanese  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

File： ABSTRACTS_TEMPLATE.doc

Language：English   Presentation type：Poster presentation  

File： abstract.pdf

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Oral presentation (general)  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Applicant：国立大学法人東北大学

Application no：特願2008-175120  Date applied：2008.7

Announcement no：特開2010-014558  Date announced：2010.1

Patent/Registration no：特許第5176235号  Date issued：2013.1

J-GLOBAL

Applicant：国立大学法人東北大学

Application no：特願2008-175120  Date applied：2008.7

Announcement no：特開2010-014558  Date announced：2010.1

J-GLOBAL