University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Osamu Nozaki

Abbrev:
Nozaki, O.
Other Names:
Address:
Department of Clinical Pathology, Kinki University, 377-2 Ohno-Higashi, Osaka 5898511, Japan
Phone:
+81-72-366-0221
Fax:
+81-72-366-0206

Citations 7

"Reactivation Of Horseradish Peroxidase With Imidazole For Continuous Determination Of Hydrogen Peroxide Using A Microflow Injection-chemiluminescence Detection System"
Luminescence 2003 Volume 18, Issue 4 Pages 203-206
Osamu Nozaki*, Hiroko Kawamoto

Abstract: A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H2O 2). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H2O2) possible by microflow injection based on horseradish-catalyzed luminol chemiluminescence. For reproducible determination of H2O2 with HRP. the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H2O2 must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the microflow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within-run reproducibility, CV = 4.0%) and the detection limit for H 2O2 was 5 pmol.

"Determination Of Hydrogen Peroxide By Micro-flow Injection-chemiluminescence Using A Coupled Flow Cell Reactor Chemiluminometer"
Luminescence 2000 Volume 15, Issue 3 Pages 137-142
Osamu Nozaki, Hiroko Kawamoto

Abstract: A novel flow cell reactor was developed for micro-flow injection determination of hydrogen peroxide (H2O2) using horseradish peroxide (HRP)-catalyzed luminol chemiluminescence. The newly developed how cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1-0.01 mL/min, resulting in increased sensitivity of detection of light. The how cell reactor was made by packing HRP-immobilized gels into a flow cell (Teflon tube; 6 cm x 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow-through type). The HRP-immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H2O2 specimens (50 µL) were injected into a stream of water delivered at a how rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within-run reproducibility of the assay of H2O2 was 2.4% (4.85 µmol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H2O2 assay was influenced by the flow rates and the injection intervals of the H2O2 specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3-8 mmol/L, but 0.56 mmol/L was sufficient for assay of H2O2 in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H2O2 (0-9.7 µmol/L) was Y = 27.5 X-2 + 394 X + 58.9 (Y = light intensity; X = concentration of H2O2) and the detection limit of H2O2 was 0.2 µmol/L. This method was used to assay glucose (2.7-16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X-2 - 351 X + 1484 (Y = light intensity; X = glucose). The within-run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% (n = 5) and 5.70% (n = 9), respectively. Copyright
Enzyme Chitosan

"Total Free Catecholamines Assay By Identification Of Its Two Functional Groups And Micro-flow Injection Chemiluminescence"
Luminescence 1999 Volume 14, Issue 6 Pages 369-374
Osamu Nozaki, Hiroko Kawamoto, Hiroyuki Moriyama

Abstract: We have developed a novel method of assaying total free catecholamines using sulfuric acid-derivatized beads for extracting and identifying catecholamine (CA) on the surface, and assaying the peroxide produced from CA by chemiluminescence (CL). Current assay methods for CA by electrochemical determination, fluorescence and chemiluminescence need a time-consuming separation by high-performance liquid chromatography. We eliminated this separation step by identifying the two functional groups of CA using a derivatized bead and this resulted in a highly specific CA assay. The principle is as follows: the amino group of CA was trapped by ion binding with a sulfuric acid derivative immobilized on a bead, and the diol of the CA bound to the bead was converted to peroxide with imidazole under alkaline conditions. The peroxide produced was assayed by microflow injection-horseradish peroxidase-catalyzed luminol chemiluminescence. We synthesized three types of sulfuric acid-derivative immobilized beads (6.5 mm i.d.). The types of immobilized sulfuric acid derivative used were straight-chain, branched chain and benzenesulfonic, respectively. The order of the three types of beads for extracting CA was: bezenesulfonic type > branched type > straight-chain type. The optimal incubation time for generating peroxide was 30 min. The peroxide generated in the reaction solution was stable with within-run reproducibility of CV 5.7% after incubation for 80 min. The regression equation of a standard curve for dopamine was Y = 12.8X(2) + 476X -373 (where Y = light intensity (RLU), X = concentration of dopamine (µmol/L)). The minimum detection limit of dopamine was 0.1 µmol/L, and the within-run reproducibility of dopamine (10.5 µmol/L) was CV 4.7% (n = 5). This method is applicable to assay of total free CA without use of HPLC. Copyright

"Chemiluminescent Detection Of Catecholamines By Generation Of Hydrogen Peroxide With Imidazole"
Luminescence 1999 Volume 14, Issue 3 Pages 123-127
Osamu Nozaki, Toshinao Iwaeda, Hiroyuki Moriyama, Yoshio Kato

Abstract: A novel detection method for catecholamines using imidazole was investigated using a chemiluminescence coupled flow injection system. Imidazole catalyzed decomposition of catecholamines to generate hydrogen peroxide, then the hydrogen peroxide was detected by chemiluminescence. The optimal condition for generation of hydrogen peroxide from a catecholamine was to incubate the catecholamines (53 pmol) in an imidazole solution (50 mmol/L, pH 9.0, 1.0 mt) at 60°C for 30 min. Peroxide was detected by peroxyoxalate chemiluminescence, and the rank order of the light emission intensities was as follows; dopamine (100%) >epinephrine (78%) >L-DOPA (62%) >norepinephrine (58%) >deoxyepinephrine (51%) >isoproterenol (43%) >dihydroxybenzylamine (25%). The light intensities of the reaction mixtures (corresponding to 1.06 pmol catecholamines) varied depending on the chemiluminescence (CL) detection reaction, and the rank order of the light intensity was as follows; luminol CL catalyzed with horseradish peroxidase (HRP) (371%) >peroxyoxalate chemiluminescence (100%) >luminol CL catalyzed with ferrycianide (62%) >lucigenin CL (15%) >pyrogallol CL (0.8%) >purpurogallin CL (0.4%) >luminol CL (0.3%). The luminol CL reaction catalyzed by HRP is recommended for the detection of peroxide in this method for catecholamines. Copyright
Temperature

"Amines For Detection Of Dopamine By Generation Of Hydrogen Peroxide And Peroxyoxalate Chemiluminescence"
J. Biolumin. Chemilumin. 1996 Volume 11, Issue 6 Pages 309-313
Osamu Nozaki*, Toshinao Iwaeda, Yoshio Kato

Abstract: Of various N compounds tested for the generation of H2O2 from dopamine for determination by peroxyoxalate chemiluminescence, the best was imidazole; after incubation with 50 mM imidazole (pH 9.5-10.5) at 60°C for 30 min in the dark, followed FIA of a 20 µL portion with chemiluminescence detection, the detection limit was 10.5 nmol of dopamine. Imidazolidin-2-one and allantoin performed in the same way as imidazole.
Dopamine Chemiluminescence

"Detection Of Substances With Alcoholic Or Phenolic Hydroxyl Groups By Generation Of Hydrogen Peroxide With Imidazole And Peroxyoxalate Chemiluminescence"
J. Biolumin. Chemilumin. 1995 Volume 10, Issue 6 Pages 339-344
Osamu Nozaki, Toshinao Iwaeda, Yoshio Kato

Abstract: Online detection of substances with an alcoholic or phenolic hydroxyl group using imidazole and peroxyoxalate chemiluminescence was investigated qualitatively using a flow injection method. The substances tested included six polyphenols, five monophenols and six sugars. After incubation at 80°C with an imidazole buffer (pH 9.5) the substances were detected by peroxyoxalate chemiluminescence. The polyphenols tested (e.g., pyrogallol, purpurogallin, and dopamine) showed the strongest light emission. The sugars with hydroxyl groups (e.g., fructose and lactose) and the monophenols (e.g., phenol, serotonin, and β-estradiol) produced only a weak light emission. Imidazole served two roles, it catalyzed the reaction with the hydroxyl compound and initiated peroxyoxalate chemiluminescence online. A novel reactor formed by packing glass beads into a flow cell (Teflon) of a chemiluminometer improved the sensitivity of light detection.
Pyrogallol Purpurogallin Dopamine Fructose Lactose Phenol Serotonin β-Estradiol Chemiluminescence Glass beads Solid phase detection

"Reactivation Of Inactivated Horseradish Peroxidase With Ethyleneurea And Allantoin For Determination Of Hydrogen Peroxide By Micro-flow Injection Horseradish Peroxidase-catalyzed Chemiluminescence"
Anal. Chim. Acta 2003 Volume 495, Issue 1-2 Pages 233-238
Osamu Nozaki and Hiroko Kawamoto

Abstract: A method for reactivation of inactivated horseradish peroxidase (HRP) with ethyleneurea and allantoin was studied and exploited in a continuous assay of hydrogen peroxide by micro-flow injection HRP-catalyzed luminol chemiluminescence. It is necessary to maintain the activity of immobilized HRP constant during the assay of hydrogen peroxide because the HRP is used repeatedly. If no reactivating reagents are used (control), light emission from H2O2 (9.7 µmol l-1) decreased in the course of the assay resulting in poor reproducibility (CV=22.9%, n=3). However, if ethyleneurea (100 mmol l-1) is added to the luminol solution (0.56 mmol L-1 in Tricine buffer, pH 9.2) the reproducibility of the assay improved remarkably (CV=2.9%, n=5). Allantoin (10 mmol l-1) also improved the reproducibility of the assay (CV=4.33%, n=10). However, a side effect was the suppression of light emission from H2O2 in a dose-dependent manner with both ethyleneurea and allantoin. The 3s detection limit of H2O2 using ethyleneurea (100 mmol l-1) was 0.6 pmol per injection. The mechanism of the inactivation of HRP after reaction with H2O2 and reactivation of the inactivated HRP was postulated as follows: the active site of HRP attracts a proton from H2O2 resulting in protonated HRP (inactive form). Exogenous ethyleneurea or allantoin removes the proton attached at the active site of the protonated HRP to return the enzyme to the active form.