The level of free bilirubin is a considerable index for the characterization of jaundice-related diseases. Herein, a biosensor was fabricated via the immobilization of bilirubin oxidase (BOx) on graphene oxide (GO) and polyaniline (PANI) that were electrochemically co-precipitated on indium tin oxide (ITO) conductive glass. The structural enzyme electrode was characterized by FTIR, XRD, and Raman spectroscopy, while the spectral and thermal properties were investigated by UV-vis and thermogravimetric analysis (TGA). Owing to the activity of the fabricated BOx/GO@PANI/ITO biosensor, it could detect free bilirubin with good selectivity and sensitivity in a low response time. The electrochemical response was studied using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). At polarization potential 0.2 V vs. Ag/AgCl, the fabricated sensor illustrated a response in only 2 s at 30 °C and pH 7.5. The LOD and LOQ for the BOx/GO@PANI/ITO biosensor were calculated and found to be 0.15 nM and 2.8 nM, respectively. The electrochemical signal showed a linear response in the concentration range 0.01-250 μM. At 5 °C, the biosensor demonstrated a half-time of 120 days, through which it could be utilized 100 times at this temperature conditions. By using a common colorimetric method, the data on bilirubin levels in serum showed a determination coefficient (R2) of 0.97.
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