Reagentless chemiluminescence biosensor for determination of hydrogen peroxide based on the immobilization of horseradish peroxidase on biocompatible chitosan membrane

A reagentless chemiluminescence (CL) flow sensor is developed for the determination of hydrogen peroxide. The analytical reagent luminol was immobilized on anion-exchange resins and packed in a glass tube, the horseradish peroxidase (HRP) was immobilized on biocompatible chitosan (CHIT) membrane which was formed on the glass coil to construct a transparent sensing flow cell. The two segments are integrated to fabricate a reagentless flow biosensor. The properties of CHIT, HRP and CHIT–HRP films have been carefully characterized by Fourier transform infrared (FT-IR). The results indicate that HRP retains the essential feature of its native structure in the CHIT film. The sample throughput is 120 h−1 with small sample volumes (30 μl). The two linear working regions are 1.0×10−7 to 1.0×10−5 mol l−1 and 1.0×10−5 to 2.0×10−4 mol l−1. The detection limit is 4.0×10−8 mol l−1 (S/N=3) with a relative standard deviation of 1.1% for the 4.0×10−6 mol l−1 H2O2 solution in 31 repeated measurements. The sensor could be used for 3 months.