Layer by layer assembly of glucose oxidase and thiourea onto glassy carbon electrode: Fabrication of glucose biosensor

For the first time a novel, simple and facile approach is described to construct highly stable glucose oxidase (GOx) multilayer onto glassy carbon (GC) electrode using thiourea (TU) as a covalent attachment cross-linker. The layer by layer (LBL) attachment process was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and Fourier transform infrared reflection spectroscopy (FT-IR-RS) techniques. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation using ferrocenemethanol as artificial electron transfer mediator and biosensor response was directly correlated to the number of bilayers. The surface coverage of active GOx per bilayer, heterogeneous electron transfer rate constant (ks) and Michaelis–Menten constant (KM), of immobilized GOx were 1.50 × 10−12 mol cm−2, 9.2 ± 0.5 s−1 and 3.42(±0.2) mM, respectively. The biosensor constructed with four-bilayers of TU/GOx showed good stability, high reproducibility, long life-time, fast amperometric response (5 s) with the high sensitivity of 5.73 μA mM−1 cm−2 and low detection limit of 6 μM at concentration range up to 5.5 mM.

► Although various enzymes immobilization have been approve for the construction of glucose biosensor, a layer by layer (LBL) technique has attracted more attention due to simplicity of the procedure, wide choice of materials that can be used, controllability of film thickness and unique mechanical properties.
► In this paper, we described a novel and simple strategy for developing an amperometric glucose biosensor based on layer-by-layer self assembly of glucose oxidase on the glassy carbon electrode modified by thiourea.
► Thiourea has two amino groups that the one can be immobilized on the activated glassy carbon electrode and the other can be used for the coupling of glucose oxidase enzyme.
► The biosensor exhibited good performance for electrocatalytic oxidation of glucose, such as high sensitivity, low detection limit, short response time and wide concentration range.
► Finally, the new method is strongly recommended for immobilization of many other enzymes or proteins containing carbaldehyde or carboxylic groups for fabricating third generation biosensors and bioelectronics devices.