Biomedical subjects

Kuniaki Nagamine

Publications and source records attributed to Kuniaki Nagamine.

8 recordsLinked to original sources

PubMed2007

Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM).

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.

Carrier Proteins↗

PubMed2006

Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system.

Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor kappaB (NFkappaB) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NFkappaB activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrode-integrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method.

Alkaline Phosphatase↗

PubMed2005

Amperometric detection of the bacterial metabolic regulation with a microbial array chip.

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.

Electrochemistry↗

PubMed2005

On-chip transformation of bacteria.

On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs. The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.

DNA, Bacterial↗

PubMed2005

Electrochemical mutagen screening using microbial chip.

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.

Biological Assay↗

PubMed2004

Respiration activity of Escherichia coli entrapped in a cone-shaped microwell and cylindrical micropore monitored by scanning electrochemical microscopy (SECM).

The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.

Electrochemistry↗

PubMed2003

On-chip electrochemical measurement of beta-galactosidase expression using a microbial chip.

[small beta]-Galactosidase expression in a small number of Escherichia coli cells has been measured in real time with an electrochemical sensor chip. E. coli cells were embedded using collagen gel within a micropore which was microfabricated onto a chip. The activity of the expressed [small beta]-galactosidase was determined using p-aminophenyl [small beta]-d-galactopyranoside (PAPG) as a substrate.

Aminophenols↗

PubMed2003

Fabrication of microbial chip using collagen gel microstructure.

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.

Collagen↗