Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode

• A graphene/ionic liquid-modified glassy carbon electrode was prepared.
• Cholesterol oxidase and catalase were co-immobilized on the electrode surface.
• Both enzymes showed improved electrochemical behaviors at the modified electrode.
• Highly sensitive amperometric response for cholesterol was achieved.
• The biosensor could be used within neutral pH values.

Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR–IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163 mA mM−1 cm−2, a response time less than 6 s, and a linear range of 0.25–215 μM (R2>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis–Menten constant (KMapp) was calculated to be 2.32 mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDs<5%) with minimal interference from the coexisting electroactive compounds such as ascorbic acid and uric acid. The CAT/ChOx/GR–IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples.