Flow-through micro sensor using immobilized peroxidase with chemiluminometric FIA system for determining hydrogen peroxide

A micromachined flow cell (overall size; 25 x 25 x 1 mm(3)) 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 mum wide, 20 mum depth, channel volume 1.4 mul) and two holes (1 mm diameter). POD was covalently immobilized with 3-(trimethoxysiiyl)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 mul. The maximal sampling rate was 315 h(-1) at a carrier solution flow rate of 10 mul min(-1). A calibration graph for H2O2 was linear for 5 nM - 5 muM: the detection limit (signal-to-noise = 3) was 1 nM (7 fg in 0.2 mul injection). The H2O2 concentration in rainwater was determined using this sensor system.