Shinya Nakamoto

A lift-off method for patterning enzyme-immobilized membranes in multi-biosensors

Abstract A new method for preparing enzyme-immobilized membranes of multi- biosensors has been developed and demonstrated by fabricating a multi- biosensor for glucose and urea, based on the pH-selective ion-sensitive field effect transitor. The enzyme solution, containing glutaraldehyde, is spin coated onto a water covered with a patterned photoresist. The resulting enzyme-immobilized membrane is lifted off by ultrasonic vibration in acetone, except for the membrane on the transitor gates. The deposited membrane is precisely patterned and difficult to peel off. The method is compatible with IC processes and applicable to multi-biosensor mass production

A Micro Planar Ampehometric Glucose Sensor Using an Isfet as a Reference Electrode

Abstract A very small glucose sensor has been realized, which consists of a gold working electrode with a glucose oxidase immobilized membrane on it, and a gold counter electrode, all made on a sapphire substrate. By using the pH sensitive ISFET as a reference electrode, the potential for a solution, whose pH is constant, can be measured and irreversible metal electrodes, such as gold or platinum, can be used as working electrode and counter electrode. The sensor is very suitable for miniaturizing and mass production, because the Integrated Circuit (IC) fabrication process can be applied. The glucose oxidase immobilized membrane was also deposited by a lift off method, one of the IC processes. A glucose concentration, from 1 to 100 mg/dl, was measured with good linear current output.

A newly isolatedBacillus stearotheromophilus K1041 and its transformation by electroporation

Optimal conditions for the plasmid transformation of a newly isolatedBacillus stearothermophilus K1041 by electroporation were investigated. The optimal conditions allowed a transformation efficiency of 5.8×105 transformants per μg plasmid pUB110.

Evaluation of an albumin-based, spin-coated, enzyme-immobilized membrane for an ISFET glucose sensor by computer simulation

Abstract An ISFET biosensor response was analyzed and evaluated using computer simulation, based on a ping-pong mechanism model [1], one of the two commonly used substrate models. Diffusion coefficients and Km values for glucose and oxygen were determined by comparing calculated responses with glucose sensor responses measured with an albumin-based membrane formed by the lift-off method. Diffusion coefficients for glucose and oxygen were estimated to be 2.5 x 10-9 cm2 sec and 7.5 x 10-9 cm2/sec, respectively. These low diffusion coefficients values mean that the membrane has a relatively dense structure. Km values for glucose and oxygen were estimated to be 8 mM and 0.05 raM, respectively. These values suggest that the enzyme activity in the membrane is influenced by the membrane electric charge or mechanical condition. Responses for a biosensor with a membrane less than 1 μm thick were well described by the model.

Thermostabilization of lactate oxidase by random mutagenesis

SummaryA lactate oxidase (LOD) gene from Aerococcus viridans was cloned and sequenced to generate thermostable LOD. One mutant LOD selected from a set of variants created by random mutagenesis had a half life of 6.2 min at 65 °C, approximately three times longer than that of the wild type LOD. This mutant exhibited an Asn to Asp point mutation at position 212 in the amino acid sequence.

Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, bacillus subtilis, and Escherichia coli

SummaryA shuttle vector that could replicate in B. stearothermophilus, B. subtilis, and E. coli was constructed from B. stearothermophilus cryptic plasmid pSTK1, E. coli vector pUC19, and a thermostable kanamycin-resistance marker. This new vector was stably maintained in B. stearothermophilus at 67°C without selective pressure.

Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

SummaryThe complete nucleotide sequence of pSTK1, a cryptic plasmid isolated from B. stearothermophilus TK015, has been determined. pSTK1 has been shown to be 1883 bp in length and contain three open reading frames (ORFs), one of which has a helix-turn-helix motif typical of DNA-binding proteins. Also identified was a region that can form an extensive secondary structure, which would show a high degree of similarity to palA, an origin for minus strand elongation in rolling circle replication.

A new shuttle vector forBacillus stearothermophilus andEscherichia coli

SummaryA new shuttle vector was constructed by inserting a 3.1 kbp-DNA fragment from thermophilicBacillus sp. plasmid pIH41 intoEscherichia coli plasmid pUC18. The resultant hybrid replicates in bothE. coli andB. stearothermophilus. This vector has ten unique restriction sites within a part oflacZ gene. Insertion of foreign DNA into these sites can be readily detected by a coloration method.

A novel oligonucleotide cassette for the overproduction ofEscherichia coli aspartate transcarbamylase inBacillus stearothermophilus

SummaryA novel double-stranded oligonucleotide cassette was devised in order to express heterologous genes inBacillus stearothermophilus. By linking the cassette to the upstream ofEscherichia coli aspartate transcarbamylase gene, this enzyme was overexpressed inB. stearothermophilus cells.

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

SummaryA kanamycin-resistant plasmid possessing a thermostable replicon derived from Bacillus stearothermophilus cryptic plasmid pSTK1 was constructed. The plasmid could transform not only B. stearothermophilus and Bacillus subtilis, but also Gram-negative Escherichia coli. The behavior of the plasmid in the hosts was examined. The plasmid was stably maintained even at 67°C in B. stearothermophilus without selective pressure. During the plasmid replication, single-stranded DNA (ssDNA) intermediates were found in E. coli, while these were not found in B. subtilis.