Viscosity monitoring with a piezoelectric quartz crystal and its application to determination of endotoxin by gelation of limulus amebocyte lysate

A piezo-electric quartz crystal is used to monitor viscosity changes in a liquid. A resistance, included in the electrical equivalent circuit of the crystal, and the resonance frequency change of the crystal, measured by an impedance analyzer, are used for the viscosity measurement. Endotoxin concentrations are determined with this system by the gelation of Limulus amebocyte lysate. The gelation time is computed by using an approximation to a polynomial equation from the resistance or the resonance frequency change. The gelation time measured was in good agreement with that obtained conventionally. The detection limit is 1 pg ml^?1. The maximum rate of change of the resistance was confirmed as a good index of endotoxin concentration in order to shorten the measurement time to ? 40 min. The changes in the resistance or resonance frequency were also considered as indices of endotoxin concentration.     

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10^?7-10^?11 g ml^?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10^?7-10^?11 g ml^?1.     

Integrated biosensor for glucose and galactose

A glucose microsensor based on quinoprotein glucose dehydrogenase (GDH) and electron mediators is described. It is unaffected by the concentration of dissolved oxygen. Its calibration graph is linear below 10 mg dl ^?1. The insensitivity to oxygen arises because the concentration of the oxidized forms of the mediators is insufficient to oxidize the reduced form of GDH. An integrated microsensor for glucose and galactose based on GHD and galactose oxidase was constructed. Glucose and galactose concentrations were determined from the current increase due to oxidation of the mediators or the current decrease due to reduction of oxygen.     

Microbial electrode sensor for vitamin B12

A microbial sensor consisting of immobilized Escherichia coli 215 and an oxygen electrode is described for the determination of vitamin B₁₂. When the sample solution is injected into the microbial electrode system, the increased consumption of oxygen by the micro-organisms causes a decrease in the dissolved oxygen around the porous membrane of the oxygen electrode and the current decreases gradually with time until a steady state is reached. The response time for a rate measurement is 2 h. When 0.5 mg of Escherichia coli 215 is immobilized, a linear relationship is obtained between the current decrease and the vitamin B₁₂ concentration between 5 × 10^?9 and 25 × 10^?9 g ml^?1.     

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.     

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.     

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.     

Nanomaterial-based electrochemical biosensors for medical applications

With recent advances in nanotechnology, great progress has been made in biosensors based on nanomaterials, but there are still numerous challenges to overcome. We describe nanomaterial-based biosensors for researchers new to the field, paying particular attention to metal nanoparticles and carbon nanotube (CNT)-based label-free approaches. Label-free monitoring of biorecognition events provides a promising platform, which is simple, cost-effective, and requires no external modification to biomolecules. Using examples from recent reports, we illustrate the diversity of biological recognition events and the range of experimental techniques employed for metal-nanoparticle-based and label-free characterization.     

Anomalous behavior of radioiodine: Solvent extraction from acidic solution

Anomalous behavior of radioiodine was found in solvent extraction. Iodine was extracted into CS₂ from acidic solution over a wide range of iodine concentration. The distribution ratio was obtained by measuring the -rays from iodine-131. The ratio drastically changed at about 10^–5 mol l^–1. The change is readily explained by the kinetic behavior of radioiodine.