Disposable, enzymatically modified printed film carbon electrodes for use in the high-performance liquid chromatographic-electrochemical detection of glucose or hydrogen peroxide from immobilized enzyme reactors.
Disposable screen-printed, film carbon electrodes (PFCE) were modified with cast-coated Osmium-polyvinylpyrridine-wired horse radish peroxidase gel polymer (Os-gel-HRP) to enable the detection of the reduction at 0 mV of hydrogen peroxide (H2O2) derived from a post-column immobilized enzyme reactor (IMER) containing acetylcholinesterase and choline oxidase. In another series of experiments PFCE were initially modified with cast-coated Os-gel-HRP and then treated with glucose oxidase in bovine serum albumin (BSA) and cross-linked with glutaraldehyde to form a bi-layer glucose-Os-gel-HRP PFCE. This bi-layer glucose-Os-gel-HRP PFCE generated a reduction current at 0 mV to H2O2 derived from the reaction of glucose oxidase and glucose in solution. These enzyme-modified PFCE were housed in a radial flow cell and coupled with cation-exchange liquid chromatographic methods to temporally separate substrates in solution for the determination of acetylcholine (ACh) and choline (Ch) in the first experimental series, or glucose in the second experimental series. These two disposable enzyme-modified PFCE exhibited linear current vs. substrate relations, were durable, being usable for approximately 40 determinations, and were sufficiently sensitive to be employed in biological sampling. Both assays utilized the same HPLC equipment. The limit of detection for ACh was 16 fmol/10 microl and that for glucose was 12 micromol/7.5 microl. ACh and Ch were measured from a microdialysate from the frontal cortex of a rat. Glucose in human urine was determined using the bi-layer glucose oxidase-Os-gel-HRP PFCE.
Osborne
P G
PG
Bioelectro Analytical Science Ltd., Department of Research and Development, Tokyo, Japan.
Yamamoto
K
K
D000109:Acetylcholine / Q000032:analysis; D000110:Acetylcholinesterase / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000737:chemistry; D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry; D002794:Choline / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005947:Glucose / Q000032:analysis*; D005949:Glucose Oxidase / Q000737:chemistry; D006735:Horseradish Peroxidase; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis*; D017551:Microdialysis; D009992:Osmium; D010084:Oxidation-Reduction; D051381:Rats; D012710:Serum Albumin, Bovine / Q000737:chemistry
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D012710:Serum Albumin, Bovine; D009992:Osmium; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D000110:Acetylcholinesterase; D005947:Glucose; D002794:Choline; D000109:Acetylcholine