A high- power ultrasonic method was used to prepare bilirubin oxidase nanoparticles (BOxNPs) which were immobilized on polyethylene (PE) film. The characterization of PE film bound to BOxNPs and BOxNPs was carried out using "Dynamic Light Scattering (DLS)," "Transmission Electron Microscopy (TEM)," and "Scanning Electron Microscopy (SEM)." The PE film was treated with nitric acid (HNO3) for its activation. BOxNPs bound to PE film exhibited optimal activity (pH-8), incubation time (11 s) with temperature 35 °C. A linear relationship was observed between the bilirubin concentrations (0.02-250 μM), with an apparent Km value and Vmax for PE- bound BOxNPs, at 0.015 μM and 2.56 μmol/mL/min. The mean recoveries of added serum bilirubin were 94.5 % at a level of 5 mM whereas 98.5 % were observed at 10 mM which showed the satisfactory reliability of BOxNPs immobilized on PE film. The coefficient of variation for serum bilirubin ranged between 4.52%-5.25%, measured on the first day (within batch) and after seven days of storage (between batch).This current method has showed a good correlation for bilirubin values when compared to the standard enzymatic colorimetric method using free enzyme. BOxNPs bound to PE film were reutilized 150 times with storage at 4 °C for 120 days.
Keywords: Bilirubin; Bilirubin oxidase; Bilirubin oxidase nanoparticles; Polyethylene film; Serum; Ultrasonic method.
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