Layer-by-layer self-assembly film of PEI-reduced graphene oxide composites and cholesterol oxidase for ultrasensitive cholesterol biosensing

A novel electrochemical platform composed of water soluble polyethylene imine-reduced graphene oxide (PEI-rGO) composites and cholesterol oxidase (ChOx) based on layer-by-layer (LBL) technique has been triumphantly developed. The PEI-rGO composites could effectively improve the electron transfer ability between the assembled enzyme and the surface of electrode. Furthermore, LBL technique could immobilize more enzyme under mild conditions, which was benefit to keep the bioactivity of redox enzyme. TEM, UV–vis and FT-IR showed that the PEI-rGO composites behaved as individual nanosheet structure and were successfully prepared. The self-assembly process of (ChOx/PEI-rGO) n film was monitored by UV–vis spectroscopy, demonstrating that the ChOx had been immobilized orderly without denaturation. Owing to the good conductivity of the PEI-rGO composites, the electron transfer ability of the immobilized ChOx was greatly improved on glassy carbon electrode (GCE). The PEI/(ChOx/PEI-rGO) 3 /GCE showed excellent electrochemical performances toward the detection of cholesterol. Under optimized conditions, the fabricated electrode exhibited a wide linear range in the cholesterol concentration from 0.10 × 10 −6 to 9.331 × 10 −3 mol L −1 with a detection limit of 0.021 μmol L −1 estimated at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant ( K m ) was 0.0431 mmol L −1 . This result indicated that a good affinity between the immobilized ChOx and the cholesterol. Meanwhile, the biosensor presented good stability, reproducibility and anti-interference ability with good recoveries for real sample detection.