Construction of an amperometric bilirubin biosensor based on covalent immobilization of bilirubin oxidase onto zirconia coated silica nanoparticles/chitosan hybrid film.

A method is described for the construction of a highly sensitive electrochemical biosensor for the detection of bilirubin. The sensor is based on covalent immobilization of bilirubin oxidase (BOx) onto zirconia coated silica nanoparticles (SiO2@ZrONPs)/chitosan (CHIT) composite electrodeposited onto Au electrode. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infra-red spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 2s at pH 8.5 (0.1M Tris-HCl) and 35°C, when operated at 20 mV s(-1). The biosensor exhibited excellent sensitivity (detection limit as 0.1 nM), fast response time and wider linear range (from 0.02 to 250 μM). Analytical recovery of added bilirubin was 95.56-97.0%. Within batch and between batch coefficients of variation were 3.2% and 3.35% respectively. The enzyme electrode was used 150 times over a period of 120 days, when stored at 4°C. The biosensor measured bilirubin levels in sera of apparently healthy and persons suffering from jaundice, which correlated well with a standard colorimetric method (r=0.99).