Reports on the use multi electrode arrays (MEA) systems for screening the electrophysiological effects of drug candidates on cardiac tissues as a response to the need of the pharmaceutical industry for a high-throughput screening method have increased in the past decades. Though the field potential (FP) measured in MEA have been reported coherent to the progression of the well-established progression of action potential (AP), certain pharmacological effects do not accurately reflect in the observed FP waveform alteration. Here, we revisit the imaging analysis algorithm developed in our lab based on the pixel intensity variation that can be utilized to improve the FP measurement and interpretation. Imaging analysis was done on videos recorded with a microscope camera at 30 Hz frame rate and 680 × 510 pixel resolution resulting to a relative lower system requirement compared to other methods. It is expected that combining our method with MEA systems can greatly aid in developing a screening platform for cardiovascular effect profiling of candidate drugs.
Single-stranded deoxyribonucleic acid (ssDNA) with guanine-rich sequences can form four-stranded structures so called G-quadruplex (Gq), which binds hemin, an iron-containing porphyrin. The hemin/Gq is an electroactive complex and exhibits peroxidase activity, and thus the hemin/Gq complexes have attracted great attention as a signal generator. This short review surveys the use of the hemin/Gq complex as an electrochemical signal generator. Electrochemical methods for detecting the hemin/Gq complex, approaches to signal amplification, and targeting strategies are herein discussed. Compared with protein-based signal generators, hemin/Gq complexes have some advantages: a wide variety of ssDNA sequences with various chemical modifications are easy to obtain, they can be immobilized on an electrode easily, and they can be designed as desired to connect functional domains. Also there are many molecular-biological tools to handle them. Hemin/Gq complexes have been successfully used to detect bioanalytes ranging from low molecular weight compound to macromolecules such as proteins, specific nucleic acids, or living cancer cells. Such bioanalytes are critical to the investigation of cellular function. Thus, DNA-based probes that contain hemin/Gq as a signal generator are a promising tool for the electrochemical analysis of cellular function, offering a competent alternative to a conventional protein-based signal generator.
Neuronal patterning is useful for understanding signal propagation between neurons as well as for biosensors and cell-based assays. The patterning of living cells has been made possible by employing surface physicochemical and topographic features. This study investigated neuronal growth on patterned nanopillars. Rat cortical neurons were cultivated on quartz substrates with amorphous silicon (a-Si) and Au pillars 100 and 500 nm in diameter. The neurites grew better with the larger diameter pillars, and the partly-selective neurite growth was observed for a-Si pillars but not for Au pillars. These results reveal the possibility of controlling neuronal growth by using a-Si nanopillars.
Cell-cell communication in animals is predominantly conducted via gap junction (GJ) plaque comprising 20 connexin (Cx) isoforms. However the GJ plaques are formed at only limited part of cell-cell interface and such an irregular localization is a current problem. To solve this problem, we developed an experimental platform to analyze the role of each Cx isoform in the regulation of GJ plaque localization and in the intercellular molecular movement. A tetracysteine-tagged Cx expressed in HeLa cell deficient in Cx was found feasible for viable imaging of GJ plaques. Femtoinjection enabled the quantitative analysis of intercellular dye diffusion.
Oxygen consumption rate (OCR) of mouse embryoid body (EB) was measured repeatedly in glucose-depleted medium or PBS solution. Non-invasive nature of the scanning electrochemical microscopy allowed sequential OCR measurement of the identical EB sample for more than 24 h. We found that the OCR value for the undifferentiated EB (cultured for 2 days) reached almost zero after 12 h exposure in PBS. On the contrary, the OCR value for the differentiated EB (cultured for 8 days) indicated 40 to 28% normal to the OCR at 0 h-incubation after 24 h exposure in PBS-based solution. It is suggested that differentiated cells indicated a stronger adaptation to glucose deprivation comparing to the undifferentiated cells.
We describe a new method for delivering a zinc finger protein directly into cells using a nanoneedle array and an ATP aptamer. The zinc finger protein can be used for site-specific DNA modification by combining with an enzyme that can cut or modify DNA. This controlled release method utilizes aptamer conformational changes to deliver various biomolecules specifically into cells.
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.
The present study investigated extracellular electron transfer (EET) of Pseudomonas stutzeri, a model organism for bacterial denitrification. Electrochemical cultivation of P. stutzeri in a lithotrophic medium with Fe2+ ions generated the clear cathodic current associated with denitrification reactions. The EET ability of P. stutzeri was greatly strengthened by the addition of acetate, which correlated with the enhanced rate of the enzymatic oxidation of Fe2+. Since the addition of acetate induced the change of cellular metabolisms from lithotrophic denitrification to mixotrophic one, the present finding suggests the effectiveness of EET monitoring as a descriptor of bacterial denitrification activity and changing metabolisms according to the environmental conditions.
The rate of heterogeneous direct electron transfer of laccase immobilized on single-walled carbon nanotube (SWCNT) and carbon paper electrodes was evaluated by cyclic voltammetry and background-current-corrected steady-state linear voltammetry. These rates indicated that the molecular orientation of laccase immobilized on the SWCNT electrode was more favorable for direct electron transfer, than that of laccase immobilized on the carbon paper electrode. The inhibition of the bioelectrocatalytic O2 reduction current of the two electrodes by chloride and fluoride were tested. The results indicated differing inhibition mechanisms by these two halides. Laccase immobilized on the SWCNT electrode exhibited high stability and high resistance to chloride inhibition.
Positive dielectrophoresis was used to increase cell leading effect into microwells on the single cell based microwell array system. Cell suspension prepared with low conductivity buffer was applied to PDMS microwell array sheet which was put on an ITO electrode. Then it was covered with another ITO electrode across a silicone spacer and high frequency sinusoidal voltage was applied between the two ITO electrodes. The optimal condition for positive dielectrophoresis was 10 MHz and 10 V. Implanted ratio (ratio of cells in wells comparing with total cells in the measured area) and occupied ratio (ratio of wells filled with a cell in the measured area) were increased up to ca. 90%. By using positive dielectrophoresis, ca. 80% cells in a sample can be utilized for cell analysis. Comparing with negative dielectrophoresis (26%), efficiency of cell analysis could be extremely improved. Positive dielectrophoresis also contributes to release prevention of cells in microwells. The present method was successfully applied to two kinds of microwell-arrayed chemical sensors for single cell analysis.
In this study, a method involving polyamidoamine dendron-modified magnetic nanoparticles (PAMAM-MNPs) along with application of an alternating magnetic field (AMF) was developed for inactivation of bacteria in water samples. The PAMAM-MNPs efficiently bound to Escherichia coli cells, resulting in magnetic recovery of cells from aqueous solutions. By applying the AMF (5 kW, 250 kHz) to the cell suspension, E. coli cells were successfully inactivated within 10 min in the presence of the MNPs, while no effect was observed in the absence of the MNPs. The use of PAMAM-MNPs could increase the inactivation rate of E. coli under the applied AMF. E. coli cells with PAMAM-MNPs stained by propidium iodide (PI) exhibited apparent fluorescence after exposure to the AMF, suggesting the occurrence of membrane damage in the cells because of direct heat transfer from the PAMAM-MNPs. Our technique can be used to address bacterial contamination with wide varieties of microorganisms in water samples.
Proton transport from an aqueous phase (W1) to another one (W2) across a planar bilayer lipid membrane (BLM) was driven by the electron transport system. The electron transport system was composed of the oxidation of D-fructose by an oxidized form of D-fructose dehydrogenase (FDH) at the W1|BLM interface, the oxidation of a reduced form of FDH by an oxidized form of 7,7,8,8-tetracyanoquinodimethane (TCNQ) at the W1|BLM interface and the oxidation of a reduced form of TCNQ by [Fe(CN)6]3− at the W2|BLM interface. Then the negative current due to the electron transfer from W1 to W2 was clearly observed around 0 V. The zero-current potential varied to hold the electroneutrality in all phases by balancing the proton transport with the electron transport.
Fluorescent dye is a useful tool in qualitative analysis. Acridine orange (AO) is one such dye, which fluoresces when bound to biomolecules. In this study, we investigated whether the electrochemical activity of AO can be used for bacterial quantification. We observed that the oxidation peak current of AO depended on the amount of AO-labeled bacteria present on the electrode. The electrochemical response was estimated as 8.4 × 10−15 A for a single cell. In addition, it was found that the response obtained in a dead bacterium was 3 times larger than that obtained in a living cell. We concluded that the difference in the electrochemical response is dependent on the biological functions such as metabolism, respiration, and viability of bacteria.
This paper describes a method of hairpin DNA (hpDNA) unzipping analysis using a biological nanopore array. Various lengths of hpDNAs were captured and translocated through the nanopore and the individual duration times were monitored. The correlation between the unzipping time and the simulated hybridization energy of the duplexes was able to be modeled as first-ordered reaction. This method is one of the approach for understanding of basic biological reactions in DNA replication and RNA transcription.
We newly identified FAD-dependent glucose dehydrogenase (FADGDH) gene homologs from thermophilic filamentous fungus Thermoascus aurantiacus. The gene homologs were cloned from two strains of Th. aurantiacus, NBRC 6766 and NBRC 9748. Recombinant FADGDHs of the two strains were prepared by using Escherichia coli and Pichia pastoris as hosts. Absorption spectra and enzymatic characterization clearly showed that these enzymes contained oxidized FAD as a coenzyme and exhibited glucose dehydrogenase activity. Analysis of thermal stability revealed that the denaturation midpoints (Tm) of these FADGDHs were at least above 71.4°C. The results showed that these FADGDHs exhibited remarkably high thermal stability. We also performed bioelectrochemical experiments using unglycosylated and glycosylated FADGDHs of Th. aurantiacus NBRC 6766, which exhibited higher affinities for glucose than those of Th. aurantiacus NBRC 9748 FADGDHs. Th. aurantiacus NBRC 6766 FADGDH-immobilized electrodes effectively showed current responses to glucose. Therefore, these thermostable FADGDHs could be applied to a bioelectro-catalyst for glucose oxidation with long-term storage and continuous use.
Sensitive electrochemical detection of nereistoxin (NEX), a widely used insecticide, by reductive desorption is studied. NEX is adsorbed onto a gold single-crystal electrode via its disulfide group to form a self-assembled monolayer (SAM), which shows a reductive desorption peak in alkaline solution. The detection limit depended on the modification time and the crystallographic orientation of Au. When a Au(111) electrode was used as a substrate, the limit of detection was 10 nM. This limit was decreased to 1 nM when a Au(100) electrode was used. The NEX-SAM on a Au(111) and Au(100) electrode also showed a characteristic reductive wave, which allowed NEX to be distinguished from other similar compounds such as aminoethanthiol, N-dimethylaminoethanthiol, and aminohexanethiol.
Microelectrode arrays (MEAs) are very powerful sensor devices for the non-invasive measurement of extracellular field potentials from excited cells. In this study, new types of interfaces were developed on MEAs by exploiting the electrochemical polymerization of conducting polymers with biomolecules. The composite films were composed of poly(3,4-ethylenedioxythiophen) (PEDOT) and anionic polysaccharide (gellan gum). The PEDOT/biomolecule composite films had no cytotoxic effect, and reduced the electrode impedance at 1 kHz to 8.84 ± 0.50 kΩ. This biointerface enabled us to record extracellular field potentials derived from the spontaneous beating of cardiomyocytes.
We electrically collected membrane-intact and dehydrogenase-positive chemosynthetic symbiotic bacteria from fresh gill tissues of the deep-sea bivalve Calyptogena okutanii. The symbiotic bacteria in the homogenized gill tissues were attached to an indium tin oxide/glass electrode by a −0.3-V vs. Ag/AgCl constant potential application for 2 h at 4°C. The attached symbiotic bacteria were detached by applying a ±10-mV vs. Ag/AgCl, 9-MHz triangular wave potential. This electrical collection method holds potential for molecular and cellular analyses of both host–symbiont interactions and symbiont metabolism in chemosynthetic symbioses.
We describe here the dielectrophoretic manipulation using a microdisk electrode with microcavity for picking up, positioning and relocating single target cells. The attractive force of positive dielectrophoresis (p-DEP) was used to trap target cells at the electrode tip, while trapped cells were released by the repulsive force of negative DEP (n-DEP). The capturing of cells in the cavity allows them to transfer at the desired position even after turning off the attractive force by p-DEP. We demonstrate an application of the present technique in the patterning with living cells.
We here report the electrochemical imaging of cell adhesion using a large-scale integration (LSI)-based amperometric device, called a Bio-LSI device. The device consists of 400 sensor electrodes arranged with a pitch of 250 µm. The device surface was modified with collagen to assist in the culture of MCF-7 cells and promote their adhesion. The cells disturb the electrochemical reaction of redox mediators, allowing the electrochemical signal to be used to evaluate cell adhesion at the single-cell level. This approach was applied to a cell detachment test. The results show that the Bio-LSI device is a promising tool for single-cell analysis.
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