l-Glutamate enzyme electrode involving amplification by substrate recycling

Yao, Takeshi; Yamamoto, Hironori; Wasa, Tamotsu

doi:10.1016/s0003-2670(00)83345-0

Cited by 32 publications

(15 citation statements)

References 7 publications

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“…Although these sensors exhibit a dynamic range of 10-6 -10-3 M comparable to that of the present GLOD/HRP/PPy electrode, the output current density is 10-to 102-fold lower. Yao et al 4 and Schubert et a1.13 constructed GLOD/glutamate-pyruvate transaminase bienzyme-immobilized electrodes with amplified response by substrate recycling. These sensors provide a much lower detection limit of 10-' M13 and 10-9 M4 L-glutamic acid.…”

Section: Resultsmentioning

confidence: 99%

Glutamate Sensors Carrying Glutamate Oxidase/Peroxidase Bienzyme System on Tin Oxide Electrode

1995

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Amperometric L-glutamic acid sensors were fabricated by depositing L-glutamate oxidase (GLOD) and horseradish peroxidase (HRP) on the tin oxide surface by means of either encapsulation into electropolymerized pyrrole or sequential chemical modification via surface hydroxyl groups. After optimization for the mixing ratio of the two enzymes during polypyrrole (PPy) formation and for the thickness of the PPy film, the GLOD/HRP/PPy electrode gave a nearly linear response to L-glutamic acid in the concentration range of 10-' to 10.4 M, with a response time of about 1 min. The GLOD/HRP bilayer-modified electrode showed a sensitivity comparable to that of the PPy-encapsulated bienzyme electrode. Both type of electrodes exhibited excellent substrate specificity. The pH dependence and lifetime of the sensor response are also given.

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Section: Resultsmentioning

confidence: 99%

Glutamate Sensors Carrying Glutamate Oxidase/Peroxidase Bienzyme System on Tin Oxide Electrode

1995

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Section: Preparation Of Fmn Sensormentioning

confidence: 99%

“…In contrast, biocatalytic approach can be performed by choosing a combination of coupled enzymes, such that the product of the first enzyme is the substrate of the second; such an amplification technique has been applied to the highly sensitive detection of various analytes by using an enzyme electrode [11][12][13] or an enzyme reactor 14,15 involving amplification based on substrate recycling.…”

Section: 10mentioning

confidence: 99%

“…The bioelectrocatalytic approach is based on substrate recycling between an electrode and an enzyme, and is therefore limited to reversible redox species, such as catechol, 5,6 hydroquinone 7,8 and phenol. 9,10 In contrast, biocatalytic approach can be performed by choosing a combination of coupled enzymes, such that the product of the first enzyme is the substrate of the second; such an amplification technique has been applied to the highly sensitive detection of various analytes by using an enzyme electrode [11][12][13] or an enzyme reactor 14,15 involving amplification based on substrate recycling.We are particularly interested in preparing an "amplified" electrode of flavin mononucleotide (FMN), which is one of the physiologically important coenzymes. However, an amplified electrode that functions for FMN has not yet been developed.…”

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confidence: 99%

“…The enzyme electrode was then constructed by cross-linking NADH-FMN oxidoreductase and gelatin with glutaraldehyde on the same end of the GC electrode. The method was similar to that described previously 11 and was as follows.The NADH-FMN oxidoreductase (5 mg; 0.22 U) and 6 µl of 10% (w/v) aqueous gelatin were added to 16 µl of 0.05 M sodium phosphate buffer (pH 6.5). A 2.5 µl portion of a 1% (v/v) solution of glutaraldehyde was added and mixed well.…”

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Flavin Mononucleotide Enzyme Electrode Amplified by a Bioelectrocatalytic Cycle

Yao

Nishimura

2002

ANAL. SCI.

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Substrate recycling is an interesting approach 1 for producing a highly sensitive enzyme electrode that can selectively measure substrates in the nanomolar range. Various sensors involving amplification by substrate recycling have been developed using either a bioelectrocatalytic 2,3 or a biocatalytic 4 approach. The bioelectrocatalytic approach is based on substrate recycling between an electrode and an enzyme, and is therefore limited to reversible redox species, such as catechol, 5,6 hydroquinone 7,8 and phenol. 9,10 In contrast, biocatalytic approach can be performed by choosing a combination of coupled enzymes, such that the product of the first enzyme is the substrate of the second; such an amplification technique has been applied to the highly sensitive detection of various analytes by using an enzyme electrode [11][12][13] or an enzyme reactor 14,15 involving amplification based on substrate recycling.We are particularly interested in preparing an "amplified" electrode of flavin mononucleotide (FMN), which is one of the physiologically important coenzymes. However, an amplified electrode that functions for FMN has not yet been developed. This paper describes a bioelectrocatalytic approach to prepare an FMN electrode involving amplification based on substrate recycling between NADH-FMN oxidoreductase and the electrode, because FMN is an electrochemically reversible redox species. ExperimentalReagents NADH-FMN oxidoreductase (EC 1.6.99.3, 0.043 U mg -1 of solid from Vibrio harveyi) was obtained from Sigma and NADH (reduced nicotinamide adenine dinucleotide) was from Oriental Yeast. FMN, bovine serum albumin (BSA, 96 -99% albumin), gelatin, and glutaraldehyde (20% solution) were obtained from Wako. They were used as received. All other chemicals were of analytical reagent grade. Phosphate buffers were prepared from sodium dihydrogenphosphate. Distilled water purified using a Millipore Milli-Q system (Nippon Millipore) was used throughout. Preparation of FMN sensorPrior to an enzyme coating, a glassy carbon (GC) disk electrode (3 mm in diameter) was first polished with 1 µm diamond particles (BAS, code no. 11-2054) and then 0.05 µm alumina particles, then washed with distilled water in an ultrasonic bath and allowed to dry in air. The enzyme electrode was then constructed by cross-linking NADH-FMN oxidoreductase and gelatin with glutaraldehyde on the same end of the GC electrode. The method was similar to that described previously 11 and was as follows.The NADH-FMN oxidoreductase (5 mg; 0.22 U) and 6 µl of 10% (w/v) aqueous gelatin were added to 16 µl of 0.05 M sodium phosphate buffer (pH 6.5). A 2.5 µl portion of a 1% (v/v) solution of glutaraldehyde was added and mixed well. A 4 µl aliquot of the resulting solution was spin-coated on the surface of a GC electrode. The membrane was allowed to form for half a day at room temperature, open to the air. The completed enzyme electrode was stored in a sodium phosphate buffer (pH 6.7, 0.1 M) at 4 -5˚C when not in use. Apparatus and proceduresCyclic voltammetric an...

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