Yasukazu Asano

A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptide

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen-antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001 degrees was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol-BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10-250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.

Evaluation of beef aging by determination of hypoxanthine and xanthine contents: application of a xanthine sensor

Abstract The changes of ATP-related compounds were measured during the aging of sirloin meat of eight bullocks, together with changes in myofibrillar fragmentation index (FI) and inosine-5-monophosphate (IMP) content. The increase in hypoxanthine (Hx) content, (Hx + 1 2 Xanthine (X)) content and K value correlated well with the observed increase in FI (R2 = 0.804, 0.819 and 0.801, respectively) and decrease of IMP content (R2 = 0.809, −0.839 and −0.965, respectively). An immobilized xanthine oxidase biosensor was tested for its usefulness as a convenient method for measurement of (Hx + 1 2 X ) content. Also, organoleptic evaluation and measurement of FI, free amino acids and ATP-related compounds were carried out. The findings suggested that the xanthine sensor could provide accurate measurements of the (Hx + 1 2 X ) content, and was useful for assessing the progress of aging by the estimation of the changes in tenderness and IMP content.

Development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine

A simple immunosensor based on a conductivity method was developed for determination of methamphetamine (MA, a stimulant drug) in urine. Anti-MA antibody was immobilized onto the surface of a pair of platinum electrodes. The reaction of MA with the antibody causes a decrease in the conductivity of the anti-MA immobilized layer between the electrodes. A linear relationship was obtained between the conductivity and MA concentration in the range of 1-10 micrograms/ml. The method requires the sample to be rinsed with water on the electrodes after the immunoreaction. This detection system was applied to the determination of MA in urine and proved to be a useful and a simple detection technique of MA in forensic science in comparison with a gas chromatography-mass spectrometry method.

Application of semiconductor gas sensor to quality control of meat freshness in food industry

Abstract We have investigated the possibility of utilizng a semiconductor gas sensor for quality control of meat freshness in the food industry. A semiconductor gas sensor based on In 2 O 3 is exploited for detecting ethyl acetate, a typical flavour volatile component produced in the initial bacterial putrefaction of meat. Of the sensor elements examined, Rh-La 2 O 3 -In 2 O 3 is found to show excellent sensitivity and selectivity to ethyl acetate at 300 °C. The sensor response signal increases with increasing meat-storage time and reflects rather well the increase of bacteria determined by a conventional colony-counting method. The multiple correlation factor between both methods is 0.8.

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2-100ngmL(-1)) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2ngmL(-1).

Changes in the concentration of biogenic amines and application of tyramine sensor during storage of beef

Abstract The vacuum-packaged beef was stored at 10, 5 and 0 °C, and biogenic amines, viable counts and tenderness with the passage of time were measured. Of the biogenic amines analyzed, only tyramine was detected in viable cell counts in the order of 105~ 106 before the appearance of a faint putrid smell (initial stage of putrefaction) at all three storage temperatures. Cadaverine was detected before the initial stage of putrefaction only at 5 °C. The changes in tenderness ceased in 5 days (10 °C), 10 days (5 °C), 28 days (0 °C), and the meat retained freshness judging from the viable counts and organoleptic evaluation. To estimate bacterial spoilage conventionally, a tyramine sensor which was composed of a tyramine oxidase-immobilized column and an oxygen electrode was applied. The sensor first detected tyramine at 5 days (10 °C), 13 days (5 °C) and 32 days (0 °C). It was confirmed that the tyramine sensor was useful for estimating the bacterial spoilage in aging beef.

Continuous monitoring for cyanide in waste water with a galvanic hydrogen cyanide sensor using a purge system.

A continuous monitoring system for cyanide with a galvanic hydrogen cyanide sensor and an aeration pump for purging was developed. Hydrogen cyanide evolved from cyanide solution using a purging pump was measured with the hydrogen cyanide sensor. The system showed good performance in terms of stability and selectivity. A linear calibration curve was obtained in the concentrating range from 0 to 15 mg dm(3) of cyanide ion with a slope of -0.24 microA mg(-1) dm(-3). The lower detection limit was 0.1 mg dm(-3). The 90% response time of the sensor system was within 3.5 min for a 0.5 mg dm(-3) cyanide solution, when the flow rate of the purging air was 1 dm(3) min(-1). The system maintained the initial performance for 6 months in the field test. The developed galvanic sensor system was not subject to interference from sulfide and residual chlorine, compared with a potentiometric sensor system developed previously. The analytical results obtained by the present system were in good agreement with those obtained by the pyridine pyrazolone method. The correlation factor and regression line between both methods were 0.979 and Y=2.30 x 10(-4)+1.12X, respectively. This system was successfully applied for a continuous monitoring of cyanide ion in waste water.

Measurement of pork freshness using potentiometric sensor

This study evaluated pork freshness using potentiometric solid-state electrodes in order to detect chemical indices such as reduced compounds, organic compounds and sulfides, which are produced during the initial stage of putrefaction in meat. Pt, CuS and Ag(2)S electrodes selected as solid-state electrodes have, respectively, been used to detect the organic compounds (regarded as chemical indices of deterioration in meat freshness). The outputs of these electrodes have been analyzed by principal component analysis (PCA) and multiple regression analysis (MRA) in order to find the correlation with the results of viable bacterial counts. By using the potentiometric sensor, the pork freshness was evaluated and the PCA and MRA corresponded to the degree of bacterial increases more simply and rapidly than other methods such as viable bacterial counts or a biosensor.

Development of a chemiluminescence detector with photodiode detection for flow-injection analysis and its application to L-lactate analysis

A low cost chemiluminescence detector with a photodiode for flow-injection analysis (FIA) of l-lactate in food was developed for quality control in food industry. In this system, l-lactate is oxidized by the enzymatic reaction with l-lactate oxidase immobilized on the column to produce hydrogen peroxide. Then chemiluminescence caused by mixing hydrogen peroxide with the chemiluminescence reagent was detected by the photodiode. This light intensity was proportional to l-lactate concentration. For detecting weak chemiluminescence efficiently a flow-through cell of the detector was specially designed. A photodiode was used for the purpose of reducing the costs of FIA systems instead of a photomultiplier tube. As a result, a linear working curve was obtained from 10−7to 10−3 mol l−1l-lactate concentration. We applied the present system with a photodiode detector for food samples and compared the results with those obtained by the conventional HPLC method. The data obtained by the present FIA method were fairly in good agreement with those obtained by the conventional HPLC method. The correlation factor and regression line between both methods were 0.992 and Y = 1.07X-0.15, respectively. The present FIA system with the photodiode detector is available as a simple, easy-handling instrument for quality control in food industry.

Improvement of the silver/silver chloride reference electrode and its application to pH measurement

When a silver/silver chloride (Ag AgCl ) reference electrode was used continuously in a low conductivity solution or reductive solution, it was often observed that stability of the liquid junction potential was lost. This phenomenon was remarkable with a Ag AgCl reference electrode compared to a calomel reference electrode. We found that 340 mg l(-1) of silver was dissolved in 3 M potassium chloride (KCl) internal solution as silver complex ions (AgCl(-(x-1))(x)) for x = 2 or 3. However, only 1.93 mg l(-1) of silver chloride (AgCl) can theoretically be dissolved in water. The complex ion that effused into the sample solution through the liquid junction clogged the liquid junction (e.g. porous ceramic) as AgCl, or as metallic silver (Ag) in reducing solution. Therefore, the constant effusion of KCl internal solution was inhibited, and the liquid junction potential became unstable or fluctuating. A new reference electrode was developed, which can eliminate AgCl(-(x-1))(x) in 3 M KCl internal solution by the use of chelating resins. A combination of this reference electrode with a pH electrode made long-term stable pH measurements possible.