Toshio Yao

A chemically-modified enzyme membrane electrode as an amperometric glucose sensor

Abstract A chemically-modified enzyme membrane electrode for glucose was constructed by cross-linking glucose oxidase with bovine serum albumin using glutaraldehyde onto a platinum electrode silanized with 3-aminopropyltriethoxysilane. The response time is only 10 s, the calibration graph is linear for 10−7–2 × 10−3 M glucose, and the sensor can be used repeately at room temperature for at least 30 days with little deterioration in response.

Amperometric biosensor with a composite membrane of sol-gel derived enzyme film and electrochemically generated poly(1,2-diaminobenzene) film

A sol-gel silicate-based biosensor for glucose was made by utilizing a composite membrane of sol-gel enzyme film and electrochemically generated poly(1,2-diaminobenzene) film to improve the selectivity of the sol-gel enzyme sensors. The stability of the sensor was improved by exposing the enzyme layer to glutaraldehyde vapor. The glucose sensor responded rapidly (ca. 15 s) to glucose at 0.6 V (versus Ag/AgCl), without any interferences from electroactive species such as L-ascorbate and urate below 0.2 mM. Reliable results were obtained in the assays of glucose in controlled human sera, with both the steady-state and flow-injection measurements. Subsequently, the same sol-gel procedure was applied to the preparation of the sensors for galactose and cholesterol. The galactose and cholesterol sensors responded rapidly to galactose and cholesterol, respectively, although the sensitivity of these sensors was inferior to that of glucose sensor.

Electrochemical enzymatic determinations of ethanol and l-lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD+) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD+ is converted to NADH by oxidation of ethanol and l-lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD+, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or l-lactic acid in the range 0.05–2 × 10-9 mol.

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.

Amperometric assays of total and free cholesterols in serum by the combined use of immobilized cholesterol esterase and cholesterol oxidase reactors and peroxidase electrode in a flow injection system.

A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2-microliter sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2-160 mg/dl and to total cholesterol in the range 3-300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively.

High-performance liquid chromatographic detection of L- and D-amino acids by use of immobilized enzyme electrodes as detectors

Abstract Two enzyme electrodes based on immobilized L - and D -amino acid oxidases give specific responses to L - and D -amino acids, respectively. They are used as amperometric detectors for high-performance liquid chromatography, by splitting the flow after elution from the column and detecting D -isomers in one line, L -isomers in the other. The detection limit is about 2 pmol for some amino acids (methionine, tyrosine, leucine, and phenylalanine). The procedure is useful for the specific detection of L - and D -amino acids without complicated pretreatment. The electrodes retain most of their original activities after repetitive use for one month.

Amperometric detection of acetylcholine and choline in a liquid chromatographic system with an immobilized enzyme reactor

Abstract Acetylcholinesterase and choline oxidase were co-immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, which was incorporated as the enzyme reactor in an h.p.l.c. system for the determination of acetylcholine and choline. The hydrogen peroxide produced enzymatically in the enzyme reactor, after the separation of acetylcholine and choline by the reverse-phase column, was monitored amperometrically. The detection limits were 1.2 pmol for choline and 1.8 pmol for acetylcholine.

Flow injection analysis for cholinesterase in blood serum by use of a choline-sensitive electrode as an amperometric detector

Abstract A platinum electrode chmically modified with choline oxidase is used as an electrochemical detector for cholinesterase. The flow injection system for the determination of cholinesterase in blood serum comprises a copper(II) dithiocarbamate-modified silica gel column for removing interfering substances, a mixing coil to allow hydrolysis of acetylcholine chloride in phosphate buffer at pH 7.5 in the carrier stream, and the choline-sensitive electrode in a flow cell. The peak current caused by the hydrogen peroxide produced is linearly related to the cholinesterase activity in the range 2.5 × 10−4–0.1 IU; 40 samples per hour can be processed with a r.s.d. below 2% without interference. The electrode is stable for about two months.

Simultaneous determination of l(+)- and d(−)-lactic acid by use of immobilized enzymes in a flow-injection system

Abstract The system comprises l (+)- and d (−)-lactate dehydrogenase reactors in parallel and a diaphorase electrode. Separate peaks are obtained for l (+)- and d (−)-lactic acid. The peak current is linearly related to the concentration of both isomers in the range 1 × 10−5−2 × 10−3 M.

Amperometric determination of glucose in blood serum with a chemically modified enzyme membrane electrode in a continuous flow system

Abstract Glucose oxidase cross-linked with bovine serum albumin and chemically immobilized on silanized platinum is used as the electrochemical detector for glucose in a continuous flow system. The peak current is linearly related to the glucose concentration in the range 1–800 mg dl−1, for injections of 5-μl samples. Glucose in blood serum can be determined at 100 samples per hour with satisfactory precision (1% r.s.d.); interferences are removed with a precolumn containing copper(II) diethyldithiocarbamate-modified silica gel. The flow sensor retains most of its original activity after repetitive use for two months.