[Show abstract][Hide abstract] ABSTRACT: Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials.
Full-text · Article · Feb 2016 · Frontiers in Physiology
[Show abstract][Hide abstract] ABSTRACT: 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 towards pH changes. This property is exploited by immobilising 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.
[Show abstract][Hide abstract] ABSTRACT: Proper surface characteristic for a titanium implant is crucial for the formation of different cellular protrusions known as filopodia and lammellipodia, both of which have a significant impact on cell attachment, spreading, migration, and proliferation. Microstructural features such as grain boundaries and defects of implant surface can modulate the cellular components and structure at a leading edge cells. Here, a nano-grained Ti-29Nb-13Ta-4.6Zr (NG TNTZ) substrate was produced by high-pressure torsion (HPT) for improved biofunctionality. Cellular response of human osteoblast cells on nano-grained TNTZ substrates is evaluated and compared with the cellular response of those on coarse-grained TNTZ. High wettability, which depends on high internal energy due to the nano-sized grains that are full of boundaries, interfaces, and high dislocation density, influenced the hOBs cells on NG TNTZ to form highly developed cellular protrusions. Large number of filopodia protrusions resulted in excellent cell attachment as consistent with high level of vinculin and superior cell proliferation. This study demonstrates the advantages of nanocrystalline surface modification using HPT for processing metallic biomaterials that are proper for orthopedic implants.
[Show abstract][Hide abstract] ABSTRACT: We report on the effect of molecular structure and substrate material on amorphous carbon nitride (a-CN:H) electrode properties including film adhesion to the substrate and electrochemical properties. Films were prepared by neutral beam enhanced chemical vapor deposition on different substrate materials (p-type Si, Cu, Ti, and Pt) below room temperature. When depositing on Si, doping nitrogen into carbon improved the electrochemical properties despite weak adhesion to the substrate. Nitrogen in a-CN:H formed two different bonding configurations: incorporation into aromatic carbon rings and hydrogen nitride by infrared (IR) spectroscopy. Therefore, delocalization of π bonds by incorporation of nitrogen affected the electrochemical improvement of the a-CN:H electrode. For samples deposited on a different metal substrate, the adhesion to substrate increased as a function of decreasing oxygen concentration on the metal substrate surface; the Pt substrate performed well with no delamination in our evaluation. The electrochemical properties were improved only in the case of deposition on Pt. Moreover, Pt surface modification by hydrogen beam was also effective; consequently, the electrochemical property of the a-CN:H electrode was superior to the graphite electrode with high temperature annealing. The observed increases in IR spectra of aromatic clusters were in line with the electrochemical improvements of a-CN:H.
[Show abstract][Hide abstract] ABSTRACT: 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 cap-ture 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.
No preview · Article · Nov 2015 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Cellular senescence is a physiological phenomenon by which normal cells irreversibly lose their proliferative potential. Understanding the mechanisms of senescence may be helpful to elucidate the causes of ageing and for the development of anti-tumor drugs. As an indicator of senescent cells, the senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6.0 has been used widely for fundamental senescence research. However, the conventional SA-β-Gal assay is not optimal for evaluating complex three-dimensional multicellular samples. We established a highly sensitive method for the direct quantification of the SA-β-Gal activity of individual multicellular spheroids within 6 min, based on scanning electrochemical microscopy (SECM). Using SECM, we successfully detected the differences in SA-β-Gal activity between MCF-7 spheroids undergoing all-trans retinoic acid (ATRA)-induced senescence and control spheroids. The SECM-based method demonstrated in this study may be widely applicable to characterize senescence processes involved in physiology and pathology.
No preview · Article · Nov 2015 · Electrochimica Acta
[Show abstract][Hide abstract] ABSTRACT: Statement of significance:
Dielectrophoresis approach was used to rapidly align carbon nanotubes (CNTs) in gelatin methacryloyl (GelMA) hydrogels resulting in hybrid GelMA-CNT hydrogels with tunable and anisotropic electrical and mechanical properties. The GelMA-aligned CNT hydrogels may be used to apply accurate and controllable electrical pulses to cell and tissue constructs and thereby regulating their behavior and function. In this work, it was demonstrated that the GelMA hydrogels containing the aligned CNTs had superior performance in cardiac differentiation of stem cells upon applying electrical stimulation in contrast with control gels. Due to broad use of electrical stimulation in tissue engineering and stem cell differentiation, it is envisioned that the GelMA-aligned CNT hydrogels would find wide applications in tissue regeneration and stem cell therapy.
Full-text · Article · Nov 2015 · Acta biomaterialia
[Show abstract][Hide abstract] ABSTRACT: This paper describes potentiometric bioimaging for enzyme activity using a large-scale integration (LSI)-based electrochemical device with 400 sensors. Potentiometric detection is useful for bioimaging because redox species are not consumed or produced during the detection process; therefore, there is no effect on cell activity and the detectable signal is sustained. In this study, the potentiometer mode of the LSI-based device was applied for the detection of glucose oxidase (GOx) and alkaline phosphatase (ALP) activity. The enzyme activities were quantitatively detected within the concentration ranges of 25-250μg/mL and 0.10-5.0ng/mL. In addition, GOx activity in hydrogels and the ALP activity of embryoid bodies (EBs) from embryonic stem (ES) cells were successfully imaged based on detection of the open circuit potentials of individual sensors in real time. To the best of our knowledge, this is the first report of potentiometric imaging using LSI-based electrochemical arrays to detect enzyme activity in ES cells. The LSI-based device is thus demonstrated to be a promising tool for bioimaging of enzyme activity.
No preview · Article · Oct 2015 · Biosensors & Bioelectronics
[Show abstract][Hide abstract] ABSTRACT: An electrochemical device, which consists of electrode arrays, nanocavities, and microwells, was developed for multi-electrochemical detection with high sensitivity. A local redox cycling-based electrochemical (LRC-EC) system was used for multi-electrochemical detection and signal amplification. The LRC-EC system consists of n(2) sensors with only 2n bonding pads for external connection. The nanocavities fabricated in the sensor microwells enable significant improvement of the signal amplification compared with the previous devices we have developed. The present device was successfully applied for evaluation of embryoid bodies (EBs) from embryonic stem (ES) cells via electrochemical measurements of alkaline phosphatase (ALP) activity in the EBs. In addition, the EBs were successfully trapped in the sensor microwells of the device using dielectrophoresis (DEP) manipulation, which led to high-throughput cell analysis. This device is considered to be useful for multi-electrochemical detection and imaging for bioassays including cell analysis.
[Show abstract][Hide abstract] ABSTRACT: Electrochemical imaging has been dramatically developed. Electrochemical imaging can provide images of surface chemical kinetics, and its technique has been used for several applications, such as bioanalysis. Although several electrode array devices have been proposed for electrochemical imaging, it is difficult to incorporate many electrochemical sensors into a simple electrode array device due to the lack of spaces for electrodes. To solve this problem, we developed a novel electrochemical imaging system. In this system, redox cycling is based so as to incorporate many sensors in a chip device. In this review, our strategy of the detection system and device construction are described. Finally, electrochemical bioimaging using the device is described.
[Show abstract][Hide abstract] ABSTRACT: Hydrogels with tunable electrical and mechanical properties have a wide range of biological applications in tissue engineering, biosensing, and biorobotics. In this work, palladium-based metallic glass sub-micron wires (PdMGSMWs) were employed to enhance the conductivity and mechanical strength of gelatin methacryloyl (GelMA) gels. The values of electrical resistivity and stiffness of hybrid GelMA-PdMGSMW hydrogels were varied by the concentration of the sub-micron wires in the gels. Compared with pristine GelMA gels, hybrid GelMA-PdMGSMW gels were more efficient in regulating adhesion and spreading of C2C12 myoblasts. Formation, contractility, and metabolic activity of C2C12 myotubes in GelMA hydrogels also increased upon inclusion of the PdMGSMWs and applying electrical stimulation. The latter phenomenon is likely because of the electrical conductivity of hybrid GelMA gels.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Aug 2015 · Biomedical Microdevices
[Show abstract][Hide abstract] ABSTRACT: In the present study, we monitored the alkaline phosphatase (ALP) activity of embryoid bodies (EBs) of mouse embryonic stem (ES) cells using a large-scale integration (LSI)-based amperometric device with 400 sensors and a pitch of 250 μm. In addition, a simulation analysis was performed to reveal the positional relationship between the EBs and the sensor electrodes toward more precise measurements. The study shows that simulation analysis can be applied for precise electrochemical imaging of three-dimensionally cultured cells by normalization of the current signals.
[Show abstract][Hide abstract] ABSTRACT: Flk-1 (VEGF receptor 2) is a well-defined mesodermal progenitor marker and the Flk-1-positive (Flk-1(+)) cells derived from embryonic stem cells (ESCs) have been known to generate hemangioblasts and cardiovascular progenitor cells, which are formed in the early and late stages of differentiation, respectively. In this study, we separated Flk-1(+) and Flk-1(-) cells from spontaneously differentiating embryoid bodies (EBs) of mouse ESCs. We found cell aggregates derived from late stage Flk-1(+) cells had a relatively small size and low oxygen consumption rate (OCR) compared with those derived from Flk-1(-) cells. Furthermore, using single-cell comprehensive gene expression analysis, we found that both Flk-1(+) and Flk-1(-) cells could be categorized into subgroups with either low or high glucose metabolic activity. We observed that metabolic suppression occurs in cells expressing an intermediate level of both Nanog and Pou5f1. Taken together, our data suggested the temporary metabolic suppression is an intrinsic feature of mesodermal differentiation.
No preview · Article · Jul 2015 · Molecular BioSystems
[Show abstract][Hide abstract] ABSTRACT: 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 um for rapid electrochemical imaging of large areas. PC12 spheroids were stimulated with K+ to release dopamine. Post-stimulation dopamine release from the PC12 spheroids was electrochemically imaged using the Bio-LSI device. The 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.
No preview · Article · May 2015 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 A431 cells.
No preview · Article · Feb 2015 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2015 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: We proposed a facile, low cost, and green approach to produce stable aqueous graphene dispersions from graphite through sonication in aqueous bovine serum albumin (BSA) solution for biomedical applications. The production of high quality graphene was confirmed using microscopy images, Raman spectroscopy, UV-vis spectroscopy, and XPS. In addition, ab initio calculations revealed molecular interactions between graphene and BSA. The processability of aqueous graphene dispersions was demonstrated by fabricating conductive and mechanically robust hydrogel-graphene materials.
[Show abstract][Hide abstract] ABSTRACT: Feedback mode-based electrochemical imaging of conductivity and topography for large substrate surfaces is presented using a large-scale integration (LSI)-based amperometric chip device with 400 sensors at a pitch of 250 μm. The LSI-based chip device has enabled rapid electrochemical imaging of large substrate surfaces, compared to scanning electrochemical microscope (SECM). Substrates modified with conductive and insulating materials were placed onto the device to acquire electrochemical signals from the substrate surface using positive and negative feedback signals. The conductivity and topography of the substrate were successfully imaged, indicating that the feedback mode-based electrochemical imaging with such a device is useful to characterize large-area substrate surfaces.
No preview · Article · Jan 2015 · Journal of Electroanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Live cell imaging is important to understand the cell function such as membrane dynamics. Scanning probe microscopy (SPM) is used for evaluation of cell surface topography with nano-scale, but in most cases the measurement induced cell damage when probe contact with the cell surface. Scanning Ion Conductance Microscopy (SICM) uses ion current as a feedback signal for nanopipette probe-sample distance control. SICM allows non-contact live cell imaging and high-resolution characterization of dynamic changes of cell surface. Furthermore, SICM can combine with other analytical tool as distance control technique.