Mediator-free total cholesterol estimation using a bi-enzyme functionalized nanostructured gold electrode

We report the fabrication of a bi-enzyme functionalized nanostructured Au electrode for the mediator-free determination of total cholesterol. A one-step electrochemical route for the synthesis, functionalization and deposition of Au nanostructures via the electroreduction of gold chloride onto indium tin oxide (ITO) coated glass plates has been proposed. The covalent biofunctionalization of the optimized Au electrode was done with cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) to investigate the kinetic parameters and the sensing characteristics. The ChEt–ChOx/Glu–NanoAu/ITO bioelectrode has a surface-controlled electrode reaction with an electron transfer coefficient and a charge transfer rate constant of 0.68 and 7.09 s−1, respectively. Under the optimal conditions, the bioelectrode undergoes a direct electron transfer reaction and exhibits a high sensitivity of 0.53 mA mM−1 cm−2 and a low detection limit of 1.57 μM for cholesterol ester without the use of any redox mediator. In addition, the kinetic analysis reveals that the bioelectrode exhibits a surface concentration of 8.82 × 10−12 mol cm−2. The sensor has also been validated with clinical samples. The proposed biosensor shows good sensitivity, stability and selectivity towards total cholesterol and may thus find implications in the fabrication of biosensing devices.