Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor

Pyruvate oxidase (E.C. 1.2.3.3.) is immobilized by adsorption on a porous acetylcellulose membrane, and combined with an oxygen electrode to provide a sensor for pyruvate (0.1–0.8 mM). The response time is 2 min. Glutamate pyruvate transaminase (0.5–180 × 10^-3 I.U. ml^-1) is determined by its effect on pyruvate production by the alanine—α-ketoglutarate reaction. The sensor is stable for more than 10 days and 100 assays.     

Amplification immunoassay for the determination of Hepatitis B surface antigen

A sensitive sandwich immunoassay for the determination of Hepatitis B surface antigen (HBs) was developed, using a cascade system of Limulus amebocyte lysate as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to anti-HBs antibody. Anti-HBs antibody was adsorbed to polystyrene beads. First, HBs were reacted to solid phase anti-HBs antibody (a-HBs). After the reaction, the beads were rinsed, and were then reacted with a-HBs-LPS. Then, LPS activity specifically bound to the beads was measured. HBs could be measured in the range of 10(-10)-10(-12) g/mL.     

Enzymatic hydrolysis of starch in water-immiscible organic solvent,two-phase systems

Enzyme-catalyzed hydrolyzations of starch by α-amylase have been studied in various two-phase systems, consisting of water and a water-immiscible organic solvent. The hydrolytic conversion of soluble starch to malto-oligosaccharides by α-amylase was greatly accelerated in 10% (v/v) water content of water-dodecane two-phase systems. However, a rapid inactivation of the enzyme has been observed in these systems. Addition of surfactant to these systems, such as polyoxyethylene (20) sorbitan monopalmitate (Tween 60) or bis(2-ethylhexyl) sodium sulfosuccinate (AOT), was effective for the enzyme stability. Effects of enzyme immobilization on the stability of α-amylase, using Ca-alginate and chitosan beads, also have been studied. The stability of immobilized enzyme was clearly enhanced in a 5–10% (v/v) water content two-phase system, whereas the free enzyme was inactivated within 41 h, remaining at a relative activity of 47–76% after 41 h of treatment. Furthermore, scanning electron micrographs (SEM) were taken to observe the effect of the two-phase system on the hydrolysis of starch. Potato starch granules have been extremely swelled and burst out in the stirred 10% (v/v) water content system, which did not contain enzymes.     

Amperometric determination of total cholesterol in serum with use of immobilized cholesterol esterase and cholesterol oxidase

Cholesterol esterase and cholesterol oxidase were immobilized on octyl-agarose gel, activated with cyanogen bromide and placed in a reactor. The sensor system for total cholesterol was assembled with the immobilized enzyme reactor, a hydrogen peroxide electrode and a peristaltic pump. Characteristics of the sensor system were investigated by using cholesterol palmitate as a standard substrate. A linear relationship was obtained between peak current and cholesterol palmitate concentration below 1000 mg dl^-1 (10.3 mM). A 10-μl sample could be assayed in 5 min. Total cholesterol in human serum was determined in the range 100–400 mg dl^-1. The standard deviation for the determination of 50 samples of 300 mg dl^-1 was 6 mg dl^-1 (2%). The system was used for 300 assays without loss of enzymatic activity. The correlation coefficient was 0.94 for 27 samples of human sera analyzed by the system proposed and by the conventional chemical method.     

Specific liquid DNA hybridization kinetics measured by fluorescence polarization

The kinetics of specific DNA hybrid formation were monitored directly in solution. Detection was based on fluorescence polarization measurements of labelled oligonucleotides at different stages during the hybridization. The effect of mismatched base pairs on the kinetics was measured. Significant differences could be observed in the kinetics when as few as three mismatches were introduced by the polymerase chain reaction technique in the target DNA sequences.     

Rapid hybridization at high salt concentration and detection of bacterial DNA using fluorescence polarization

The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46 degrees C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl(2) were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl(2). Moreover, in the presence of 0.2 M MgCl(2), there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

A surface plasmon resonance probe without optical fibers as a portable sensing device

A surface plasmon resonance (SPR) sensor integrating a small sensor probe, a laser emission diode, a photo detector, and a polarizer was developed as a portable sensing device. The sensor probe was made with a glass cylinder, 50 mm long and 1.5 mm in diameter, that was connected directly to a beam splitter without optical fibers. The SPR spectrum obtained with this probe system showed a 10% reflectivity minimum at 690 nm. Shifts of the SPR spectrum induced by refractive index (RI) changes in the sample were measured by detecting the reflection light intensity at 670 nm. When the sensitivity was compared using a BIAcore™ SPR instrument, the lowest sensor response of 1 mV observed with the SPR probe system coincided with 1.4 × 10⁻⁶ of the RI changes. The RI resolution of the SPR probe was estimated with experimentally evaluated noise on the signal, and, consequently, it was concluded that the RI resolution was 1.2 × 10⁻⁵. Moreover, immunoreaction was demonstrated with adsorbed bovine serum albumin (BSA) and anti-BSA antibody as an analyte. As a result, 50 ng mL⁻¹ of the lower detection limit was estimated.     

Preparation, structural characterization of 1,2-digermacyclobut-3-enes, and their palladium-catalyzed insertion of alkynes

1,2-Digermacyclobut-3-enes were prepared by the treatment of Z-α,β-bis(chlorodialkylgermyl)ethenes with Na metal in boiling toluene and their structures were fully established by spectroscopic methods coupled with X-ray crystallography. In the presence of an appropriate catalyst, 1,2-digermacyclobut-3-enes reacted smoothly with alkynes to give the corresponding insertion products, 1,4-digermacyclohexa-2,5-dienes in moderate to good yields. Conventional complexes, such as [Pd(PPh₃)₄] and [Pt(PPh₃)₄], serve as efficient catalysts. The mechanism of the insertion reaction of alkynes into the germanium-germanium bond of 1,2-digermacyclobut-3-enes is discussed in terms of a key intermediate, a 1,4-digerma-2-buten-1,4-diylpalladium.