Ken-ichi Inatomi

Electrophoresis of DNA in micro-pillars fabricated in polydimethylsiloxane

We describe the fabrication of a device containing micro-pillars and separation of DNA. It was made of polydimethylsiloxane (PDMS) and made by a replica molding method using a patterned silicon wafer as a mold. The device has a micro-channel in which 15 µm pillars are arranged in a hexagonal lattice with 1 µm spacing. Electrophoresis of DNA in the pillars showed the average speed of DNA migration was dependent on its lengths. The PDMS device not only demonstrates the possibility of rapid DNA separation, but also has an advantage in mass production cost by using the replication method.

Glucose-sensitive field-effect transistor with a membrane containing co-immobilized gluconolactonase and glucose oxidase

Abstract A glucose-sensitive field-effect transistor (FET) with a two-enzyme membrane containing gluconolactonase and glucose oxidase is investigated. The two-enzyme membrane (ca. 1 μm thick) is formed on the ion-sensitive gate of the FET by photopolymerization. The gluconolactonase used was a partially purified product prepared from crude glucose oxidase by gel filtration. A glucose sensor with only purified glucose oxidase has little response for glucose, but the co-immobilization of gluconolactonase and glucose oxidase considerably enhanced the response amplitude of the glucose sensor. The composition of the two-enzyme/photopolymer solution is optimized; gluconolactonase with an activity at least twice that of glucose oxidase is necessary. The linear calibration graph extends from 0.2 to 2 mM glucose.

Application of a microfluidic device for counting of bacteria.

Aims:  To develop a miniaturized analytical system for counting of bacteria.

F- and V-type ATPases in the hyperthermophilic bacterium Thermotoga neapolitana.

Abstract. Two gene clusters encoding F- or V-type ATPases were found in genomic DNA of the hyperthermophilic bacterium Thermotoga neapolitana. The subunit genes of each ATPase formed an operon. While the gene arrangement in the operon of the F-type ATPase resembled those in eukaryotic organelles and bacteria, that of the V-type ATPase was different from those reported for archaea, bacteria, or eukaryotes. Both ATPases were found to be expressed in the cells of T. neapolitana by Western blot analysis. Although V-type ATPase could not be rendered soluble, F-type ATPase was solubilized with 1% Triton X-100 and characterized. This is the first report of the coexistence of both F- and V-type ATPases in hyperthermophilic bacteria. It has recently been shown by a genome analysis that Thermotoga maritima has no V-type ATPase gene cluster but does have an F-type ATPase gene cluster; however, part of a gene for the D-subunit of the V-type ATPase gene has been reported in the T. maritima genome. Evolution of the two types of ATPases in Thermotoga is discussed.

Application of Amido black staining to enumerating bacteria grown on membrane filters.

A method to detect and enumerate bacterial colonies grown on membrane filters (MF) was described. The colonies were stained with an ethanolic solution of 0.1% Amido black 10B. The procedure yielded the rapid detection of colonies as compared to a conventional plate counting method.

A CAD study on micro flow cytometer and its application to bacteria detection

We have designed and fabricated a micro flow cytometer for application to bacteria detection. In this paper, we present a CAD study which has been done to obtain optimal working conditions of the device. In the simulation, we have investigated flow rate ratios of sample and sheath flow to confirm hydrodynamic focusing at a desired position and proper sample stream width at the detection position. As a result, we could obtain adequate flow rate ratios of sample and sheath flow, which is in the vicinity of 1:5 for our device. A bacteria enumeration experiment has also been carried out, based on the simulation results, and we have achieved hydrodynamic focusing at the expected position and proper sample flow rate.

Molecular Orientation And Photoelectric Properties Of Molecular Heterojunction Using Flavin-cytochrome c Multilayers

In order to realize electronic devices based on molecular electronic function, it is of great importance to control the structure and the electron transport function of molecular organization. From the structural standpoint, cytochrome c (cyt c) adsorbed on flavin monolayers was found to show specific in-plane molecular orientation by the polarized fluorescence method. As for the electron transport function, the rectifying property was observed in the MIM (Metal-InsulatorMetal) cell using FMN / cyt c / EDTA solid film under illumination.

Alpha- and beta-subunits of a V-type membrane ATPase in a hyperthermophilic sulfur-dependent archaeum, Thermococcus sp. KI

The genes encoding alpha- and beta-subunits of a V-type ATPase in a sulfur-dependent hyperthermophilic archaeum, Thermococcus sp. KI, were cloned and sequenced. The deduced amino acid sequences were approximately 60, 50 and 25% identical to those of other archaeal, eukaryotic V-type and E. coli F-type ATPase, respectively. Phylogenetic analysis revealed that Thermococcus ATPase was closely related to that of Thermus, and those of Methanosarcina and Halobacterium.