Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

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.

Bioimaging using bipolar electrochemical microscopy with improved spatial resolution.

In this study, we developed bipolar electrochemical microscopy (BEM) using a closed bipolar electrode (cBPE) array with an electrochemiluminescence (ECL) detecting system. Because cBPEs are not directly connected to a detector, high spatio-temporal resolution imaging can be achieved by fabricating a microelectrode array in which each electrode point is arranged in a short interval. A cBPE array with individual cBPEs arranged in 41 µm intervals was successfully fabricated by depositing gold in the pores of a track-etched membrane using electroless plating. Using BEM with the cBPE array, which has a higher density of electrode points than the conventional multi-electrode array, we effectively demonstrated the imaging of [Fe(CN)6]3- diffusion and the respiratory activity of MCF-7 spheroids with high spatio-temporal resolution.

High-Resolution Electrochemical Mapping of the Hydrogen Evolution Reaction on Transition-Metal Dichalcogenide Nanosheets.

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.

A highly sensitive endotoxin sensor based on redox cycling in a nanocavity.

We report a highly sensitive and rapid electrochemical method for the detection of endotoxin, based on a Limulus amebocyte lysate (LAL) assay using redox cycling at a pair of electrodes in a nanocavity for electrochemical signal amplification. We have previously developed Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as a substrate for the amperometric LAL assay, and in this work, Z-Leu-Gly-Arg-aminomethylferrocene (LGR-AMF) was newly prepared. They were examined as substrates for a LAL-based endotoxin assay using a nanocavity device. During the last step of the endotoxin-induced LAL cascade reaction, pAP or AMF is generated from the substrate, which can be detected electrochemically with efficient signal amplification by redox cycling between the two electrodes in the nanocavity. A device with a 190 nm-high nanocavity was fabricated by photolithography. With the fabricated device in model assay solutions prepared by mixing LGR-pAP and pAP, we demonstrated that pAP could be quantitatively detected from the difference in oxidation potentials between LGR-pAP and pAP. For LGR-AMF and AMF, a difference in the formal potential of 0.1 V was obtained which was considered to be insufficient to distinguish AMF from LGR-AMF. However, we showed for the first time that analytes such as AMF can be detected by differences in diffusion coefficients between the analyte and coexisting molecules (such as LGR-AMF) using a device with high redox-cycling efficiency. Next, the endotoxin assay was performed using the fabricated nanocavity device. Using this method, endotoxin was detected at concentrations as low as 0.2 and 0.5 EU L-1 after LAL reaction times of 1 h and 30 min, respectively, using the LGR-pAP substrate. However, the endotoxin assay using LGR-AMF was not successful because the clotting enzyme did not react with LGR-AMF. This problem might be solved by further design of the substrate. Our nanocavity device represents an effective platform for the simple and rapid detection of endotoxin with high sensitivity.

High Speed Scanning Ion Conductance Microscopy for Quantitative Analysis of Nanoscale Dynamics of Microvilli.

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 × 64 pixel resolution at 10 × 10 µm. 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.

Development of Oxygen Consumption Analysis with an on-Chip Electrochemical Device and Simulation.

The O2 consumption rate of embryos has been attracting much attention as a key indicator of cell metabolisms and development. In this study, we propose an on-chip device that enables the accurate, easy, and noninvasive measurement of O2 consumption rates of single embryos. Pt electrodes and micropits for embryo settlement were fabricated on Si chips via microfabrication techniques. The configuration of the device enables the detection of O2 concentration profiles surrounding the embryos by settling embryos into the pits with a mouth pipet. Moreover, as the detection is based on an electrochemical method, the influence of O2 consumption on the electrodes was also considered. By using a simulator (COMSOL Multiphysics), we estimated the O2 concentration profiles in the device with and without the effects of the electrodes. Based on the simulation results, we developed a normalization process to calculate the precise O2 consumption rate of the sample. Finally, using both the measurement system and the algorithm for the analysis, the respiratory activities of mouse embryos were successfully measured.

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

Carbon nanotubes embedded in embryoid bodies direct cardiac differentiation.

We embedded carbon nanotubes (CNTs) in mouse embryoid bodies (EBs) for modulating mechanical and electrical cues of the stem cell niche. The CNTs increased the mechanical integrity and electrical conductivity of the EBs. Measured currents for the unmodified EBs (hereafter, EBs) and the EBs-0.25 mg/mL CNTs were 0.79 and 26.3 mA, respectively, at voltage of 5 V. The EBs had a Young's modulus of 20.9 ± 6.5 kPa, whereas the Young's modulus of the EB-0.1 mg/mL CNTs was 35.2 ± 5.6 kPa. The EB-CNTs also showed lower proliferation and greater differentiation rates compared with the EBs as determined by the expression of pluripotency genes and the analysis of EB sizes. Interestingly, the cardiac differentiation of the EB-CNTs was significantly greater than that of the EBs, as confirmed by high-throughput gene analysis at day 5 of culture. Applying electrical stimulation to the EB-CNTs specifically enhanced the cardiac differentiation and beating activity of the EBs.

Reversible Shape Transformation of Ultrathin Polydopamine-Stabilized Droplet.

Here we report on the flattening of water droplets using an ultrathin membrane of autopolymerized polydopamine at the air/water interface. This has only been previously reported with the use of synthetic or extracted peptides, two-dimensional designed synthetic peptide thin films with thicknesses of several tens of nanometers. However, in the previous study, the shape of the water droplet was changed irreversibly and the phenomenon was observed only at the air/water interface. In the present study, an ultrathin polydopamine membrane-stabilized droplet induced the flattening of a water droplet at the air/liquid and liquid/liquid interfaces because a polydopamine membrane was spontaneously formed at these interfaces. Furthermore, a reversible transformation of the droplet to flat and dome shape droplets were discovered at the liquid/liquid interface. These are a completely new system because the polydopamine membrane is dynamically synthesized at the interface and the formation speed of the polydopamine membrane overcomes the flattening time scale. These results will provide new insight into physical control of the interfacial shapes of droplets.

Micro/nanoelectrochemical probe and chip devices for evaluation of three-dimensional cultured cells.

Herein, we present an overview of recent research progress in the development of micro/nanoelectrochemical probe and chip devices for the evaluation of three-dimensional (3D) cultured cells. First, we discuss probe devices: a general outline, evaluation of O2 consumption, enzyme-modified electrodes, evaluation of endogenous enzyme activity, and the collection of cell components from cell aggregates are discussed. The next section is focused on integrated chip devices: a general outline, electrode array devices, smart electrode array devices, droplet detection of 3D cultured cells, cell manipulation using dielectrophoresis (DEP), and electrodeposited hydrogels used for fabrication of 3D cultured cells on chip devices are discussed. Finally, we provide a summary and discussion of future directions of research in this field.

Continuous collection and simultaneous detection of picoliter volume of nucleic acid samples using a mille-feuille probe.

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. Graphical abstract We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control.

3D electrochemical and ion current imaging using scanning electrochemical-scanning ion conductance microscopy.

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.

Electrochemical Motion Tracking of Microorganisms Using a Large-Scale-Integration-Based Amperometric Device.

Motion tracking of microorganisms is useful to investigate the effects of chemical or physical stimulation on their biological functions. Herein, we describe a novel electrochemical imaging method for motion tracking of microorganisms using a large-scale integration (LSI)-based amperometric device. The device consists of 400 electrochemical sensors with a pitch of 250 µm. A convection flow caused by the motion of microorganisms supplies redox species to the sensors and increases their electrochemical responses. Thus, the flow is converted to electrochemical signals, enabling the electrochemical motion tracking of the microorganisms. As a proof of concept, capillary vibration was monitored. Finally, the method was applied to monitoring the motion of Daphnia magna. The motions of these microorganisms were clearly tracked based on the electrochemical oxidation of [Fe(CN)6 ]4- and reduction of O2 .

Localized Gene Expression Analysis during Sprouting Angiogenesis in Mouse Embryoid Bodies Using a Double Barrel Carbon Probe.

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.

Nanoscale imaging of an unlabeled secretory protein in living cells using scanning ion conductance microscopy.

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 (∼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.

Improving the electrochemical imaging sensitivity of scanning electrochemical microscopy-scanning ion conductance microscopy by using electrochemical Pt deposition.

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.

Electrochemical Imaging of Dopamine Release from Three-Dimensional-Cultured PC12 Cells Using Large-Scale Integration-Based Amperometric Sensors.

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 µm for rapid electrochemical imaging of large areas. PC12 spheroids were stimulated with K(+) to release dopamine. Poststimulation dopamine release from the PC12 spheroids was electrochemically imaged using the Bio-LSI device. Bio-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. Our 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.

Microtubule guiding in a multi-walled carbon nanotube circuit.

In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy.

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.

Molecular motor-powered shuttles along multi-walled carbon nanotube tracks.

As a complementary tool to nanofluidics, biomolecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.