Ikuo Satoh

Flow-injection determination of glutathione with amperometric monitoring of the enzymatic reaction

Glutathione sulfhydryl oxidase is immobilized on oxirane-acrylic beads (Eupegit-C) and packed in a small column. The simple system for glutathione comprises the immobilized enzyme column and a flow-through membrane-covered platinum/silver/silver chloride electrode pair for detection of hydrogen peroxide. The calibration graph for glutathione was linear from 0.05 to 1.0 mM for 200-μl samples. The assay took 3 min. The relative standard deviation for 0.5 mM glutathione was 2% (n=10).

High Sensitivity Flow Injection Analysis of Urea Using Composite Electropolymerized Polypyrrole‐Polyion Complex Film

A highly sensitive and rapid flow injection system for urea analysis was constructed with a composite film of electropolymerized inactive polypyrrole (PPy) and a polyion complex incorporating urease. This system shows a sensitivity of 120 mV decade 1 and a lifetime of more than 80 assays. The origin of the high sensitivity of this system is attributed to an additional potential response of inactive PPy to ammonia or ammonium ion superimposed on the response to pH change. By injecting a concentrated buffer solution immediately after the sample injection, this system is capable of assaying more than 15 samples per hour.

Flow injection analysis of potassium using an all-solid-state potassium-selective electrode as a detector

The concentration of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this laboratory demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h(-1)). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.

Biological determination of Ag(I) ion and arginine by using the composite film of electroinactive polypyrrole and polyion complex

Abstract The urea biosensor based on the combination of electropolymerized insulating polypyrrole (PPy) with urease was used for the determination of Ag(I) ion and arginine. The inhibition ratio of urease with Ag(I) ion was evaluated from the urea response of the sensor. This method enabled the detection of 1.0×10 −7 to 1.0×10 −4 mol dm −3 of Ag(I) ion. With the addition of EDTA as a masking agent for interfering heavy metal ions, the selectivity for Ag(I) ion was improved. Furthermore, arginine detection was carried out with the PPy electrode using bienzymatic combination of urease and arginase. The calibration curve for arginine was obtained in the concentration range of 1.0×10 −5 to 1.0×10 −3 mol dm −3 with a sensitivity of 50 mV decade −1 .

An apoenzyme thermistor microanalysis for zinc(II) ions with use of an immobilized alkaline phosphatase reactor in a flow system

Abstract Calorimetric microdetermination of zinc(II) ions with use of an apoenzyme thermistor in a flow stream is proposed. Alkaline phosphatase as the selective recognition element was immobilized onto oxirane-acrylic beads (Eupergit-C) and packed into a small polymer column. The flow-injection biosensing system was assembled with the immobilized enzyme reactor and a thermistor device for monitoring the enzyme activity. Zinc(II) ions were calorimetrically determined in the range 0·01–1·0 m m for 0·5 ml samples through their activation of the immobilized metal-free alkaline phosphatase (apoenzyme) reactor. The activity of the reactor was assessed by injecting 0·1 ml of 100 m m p -nitrophenyl phosphate solution. Regeneration of the reactor was performed by pumping 20 m m 2,6-pyridine dicarboxylate (pH 6·0) between successive samples. The system could be repeatedly used at least 120 times during 2 months of operation.

Flow-injection determination of inorganic pyrophosphate with use of an enzyme thermistor containing immobilized inorganic pyrophosphate

Abstract Inorganic pyrophosphate immobilized on controlled-pore glass is used in a simple flow enzyme thermistor system. The heat produced in hydrolysis of pyrophosphate is enhanced. by using Tris-HCl buffer, pH 7.2, containing 1 mM magnesium chloride, as carrier stream. The calibration graph is linear for 0.1–20 mM pyrophosphate; 500 assays are possible without loss of enzyme activity. For 0.5-ml injections of 10 mM pyrophosphate, the relative standard deviation was 2.0% (n=30). A single determination takes 6 min. Calcuim and strontium interfere.

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system.

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of lambda(ex) = 415nm and lambda(em) = 485nm. A wide, linear relationship was obtained between the concentration of urea (1.0-100muM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.

Multi-ion biosensor with use of a hybrid-enzyme membrane

Abstract A hydbrid type of metalloenzyme membrane has been applied to flow-amperometric biosensing of multi-ions based on the apoenzyme reactivation method. The hybrid-enzyme membrane prepared by co-immobilizing metal-depending enzymes, i.e., alkaline phosphatase and ascorbate oxidase, onto a partially aminated polyacrylonitrile membrane is used as the recognition element for zinc(II), cobalt(II) and copper(II) ions. This biosensing system is assembled with the enzyme membrane tightly attached onto a flow-through oxygen electrode for monitoring the enzymic activity. Biosensing of zinc(II), cobalt(II) and copper(II) ions at submillimolar levels has been amperometrically realized through the activation of each of the immobilized metal-free enzymes (apoenzymes).

Amperometric biosensing of copper(II) ions with an immobilized apoenzyme reactor

Abstract Novel amperometric biosensing is proposed for the determination of copper(II) ions by an apoenzyme sensor in flow streams. The biosensing system was assembled with an immobilized galactose oxidase reactor and flow-through electrochemical devices for monitoring the enzymatic activity. Copper(II) ions were selectively determined in the range 0.1 to 10 mM through activation of the immobilized metal-free galactose oxidase (apoenzyme) reactor. The activity was assessed by injection of d -galactose solution (0.1 ml, 40 mM) and then the consumption of dissolved oxygen by the oxidative reaction or the production of hydrogen peroxide enzymatically generated was measured independently by a polarographic oxygen electrode or a membrane-covered Pt/Ag/AgCl electrode pair. Regeneration of the reactor was carried out by pumping 10 mM N , N -di-ethyldithiocarbamate solution (pH 8.0) between successive samples.

Calorimetric Biosensing of Heavy Metal Ions with the Reactors Containing the Immobilized Apoenzymes

Trace analysis for heavy metal ions present in biofluids, foodstuffs, fermentation broth, and wastewater provides important information not only in the diagnosis of diseases, but also in quality control, process control, and environmental control. Recently, my colleagues and I have proposed novel analytical techniques using immobilized apoenzyme (cofactor-free enzyme) for heavy metal ions assay in continuous Row streams.’-’ Biosensing based on an apoenzyme reactivation method can be applied to microanalysis for cofactors, for example, nucleotides and metal ions in enzyme-catalyzed reactions. An apoenzyme thermistor described here is a unique union between an immobilized apoenzyme and a flow-calorimetric biosensing system (enzyme thermistor6). Details of the assay procedure using the apoenzyme thermistor have also been reported The enzyme thermistor possesses the advantage of unique versatility because most enzymatic reactions are accompanied by heat evolution. Thus, the calorimetric sensing method can be applied to detect any kinds of metalloenzyme activity. I n this report, a unique calorimetric biosensing method is presented for flowinjection microdetermination of heavy metal ions using the metalloenzymes (galactose oxidase. ascorbate oxidase, and carbonic anhydrase) immobilized on porous glass beads as the specific recognition elements.