Electrochemical monitoring of hydrogen peroxide released from leucocytes on horseradish peroxidase redox polymer coated electrode chip.
We developed an electrochemical-sensing device for continuous monitoring extracellular hydrogen peroxide (H(2)O(2)). The device consists of an indium-tin-oxide electrode coated with osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) and a poly-dimethyl siloxane well to house the cells on the chip. Granulocyte-like differentiated HL-60 cells were accommodated in the well and stimulated with phorbol 12-myristate 13-acetate (PMA), which triggered the generation of H(2)O(2). The extracellular H(2)O(2) released from the cells was enzymatically reduced at the Os-HRP-modified electrode chip using Os(II) as an electron donor, resulting in reduction current responses by the device. The reduction current increased immediately upon PMA stimulation and this current transient was similar to that obtained by conventional chemiluminescence assays using sodium luminol. Apocynin, an inhibitor of NADPH oxidase activation, eliminated both the electrochemical and chemiluminescence signals. On the other hand, superoxide dismutase (SOD) increased the amperometric signals and catalase (CAT) decreased, whereas SOD decreased luminescence emission and CAT did not. These results were in accordance with the expected reaction mechanism, and strongly indicate that this new electrochemical-sensing device successfully detects extracellular H(2)O(2) production.
Inoue
Kumi Y
KY
Graduate School of Environmental Studies, Tohoku University, 6-6-11 Aoba, Aramaki, Aoba, Sendai 980-8579, Japan. inoue@bioinfo.che.tohoku.ac.jp
Ino
Kosuke
K
Shiku
Hitoshi
H
Kasai
Shigenobu
S
Yasukawa
Tomoyuki
T
Mizutani
Fumio
F
Matsue
Tomokazu
T
D001681:Biological Assay / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002460:Cell Line; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003217:Conductometry / Q000295:instrumentation* / Q000379:methods; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018922:HL-60 Cells; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D011108:Polymers; D006735:Horseradish Peroxidase