Analytical Sciences

Flow-through Micro Sensor Using Immobilized Peroxidase with Chemiluminometric FIA System for Determining Hydrogen Peroxide

Nobutoshi KIBA,*  Toyonari TOKIZAWA,* Sayaka KATO,* Masaki TACHIBANA,* Kazue TANI,* Hitoshi KOIZUMI,* Masaharu EDO,** and Eiichi YONEZAWA**

*Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu 400-8511, Japan
**Functional Divice Laboratory, Fuji Electric Corporate Research and Development, Yokosuka 240-0194, Japan

A micromachined flow cell (overall size; 25 x 25 x 1 mm³) was designed for the fast determination of hydrogen peroxide, based on a luminol-H₂O₂ chemiluminescence reaction catalyzed by immobilized peroxidase (POD). The flow cell consisted of a sandwich of anisotropically etched silicon and glass chips and contained a spiral channel (20 turns, 50 cm long, 150 µm wide, 20 µm depth, channel volume 1.4 µl) and two holes (1 mm diameter). POD was covalently immobilized with 3-(trimethoxysilyl)propyldietylenetriamine and glutaraldehyde on the inner surface of the channel. The chip was placed in front of a window of a photomultiplier tube and used as a flow cell in a single-line flow-injection analysis system using a luminol solution as a carrier solution. The sample volume for one measurement was 0.2 µl. The maximal sampling rate was 315 h^-1 at a carrier solution flow rate of 10 µl min^-1. A calibration graph for H₂O₂ was linear for 5 nM - 5 µM; the detection limit (signal-to-noise = 3) was 1 nM (7 fg in 0.2 µl injection). The H₂O₂ concentration in rainwater was determined using this sensor system.

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