Highly sensitive amperometric detection of bilirubin using enzyme and gold nanoparticles on sol–gel film modified electrode

We describe the development of a simple and highly sensitive electrochemical (amperometric) sensing of bilirubin based on bilirubin oxidase (BOx) incorporated into the gold nanoparticles (AuNPs). This nanoelectrode platform with self-assembled enzyme is highly sensitive toward the electrochemical oxidation of bilirubin and increased the bilirubin concentration linearly from 1 to 5000 μM with a correlation coefficient of 0.9960, and an apparent Michaelis constant (KM,app) of 44 ± 0.4 μM. Using an amperometric method, the detection limit for bilirubin at the enzyme-modified electrode was 1.4 nM (signal-to-noise ratio = 3). The modified electrode retained a stable response for 2 days while losing only ca. 3.4% of its initial sensitivity during a 10 days storage period in 0.2 M phosphate buffer solution (pH = 8.4) at ≤4 °C. The practical application of the modified electrode was demonstrated by measuring the concentration of bilirubin in blood serum sample.