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

► The enzyme incorporated AuNPs on sol–gel film electrode is highly sensitive toward the electro-oxidation of bilirubin. ► The electrocatalytic oxidation current of bilirubin increased linearly from 1 to 5000μM at enzyme-modified electrode with a correlation coefficient of 0.9960. ► Using an amperometric method, the detection limit of bilirubin was found to be 1.4nM (S/N=3). ► The practical application of the modified electrode was demonstrated by measuring the concentration of bilirubin in blood serum. 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 5000μM with a correlation coefficient of 0.9960, and an apparent Michaelis constant (KM,app) of 44±0.4μM. Using an amperometric method, the detection limit for bilirubin at the enzyme-modified electrode was 1.4nM (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 0.2M 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.