In the present work, a fluorescent H₂O₂ biosensor was constructed by encapsulating fluorescent probe Rhodamine B (RhmB) in the hydrophobic cavity of the cyclodextrin (β-CD) and immobilizing catalase (CAT) on the 2-NH₂ of chitosan (CTS) in a chitosan 6-OH immobilized β-cyclodextrin derivative (CTS-6-CD). The inclusion complex of CTS-6-CD to RhmB (CTS-6-CD-RhmB) was prepared by a solution method. Its structure and inclusion efficiency were determined by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and fluorescence spectroscopy (FL). CAT was immobilized on CTS-6-CD-RhmB to eventually form the functional membrane, CTS-6-CD-RhmB-CAT, via glutaraldehyde crosslinking, which was further characterized by FTIR and FL, and used as a H₂O₂ biosensor. The functional membrane was used to simultaneously oxidize and detect H₂O₂. The detection condition was optimized as pH 8, a reaction temperature of 25 °C, and an immobilized enzyme concentration of 2 × 10-4 mol/L. The fluorescence response of the biosensor exhibited a good linear relationship with the concentration of H₂O₂ in the range of 20 mΜ-300 μM and the detection limit of 10-8 mol/L.
Keywords: Rhodamine B; catalase; chitosan; fluorescent biosensor; β-cyclodextrin.