An amperometric bienzymatic cholesterol biosensor based on functionalized graphene modified electrode and its electrocatalytic activity towards total cholesterol determination

• Published in: Talanta (Oxford) Vol. 99; pp. 302 - 309
• Main Authors: Manjunatha, Revanasiddappa, Shivappa Suresh, Gurukar, Savio Melo, Jose, D'Souza, Stanislaus F., Venkatarangaiah Venkatesha, Thimmappa
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.09.2012; Elsevier
• Subjects: Analytical chemistry; Biological and medical sciences; Biosensing Techniques - methods; Biosensors; Biotechnology; Catalysis; Chemistry; Cholesterol - analysis; Cholesterol - blood; Cholesterol esterase; Cholesterol oxidase; Cholesterol Oxidase - chemistry; Cholesterol Oxidase - metabolism; Dielectric Spectroscopy; Direct electrochemistry; Electrochemical methods; Electrodes; Electron Transport; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; General, instrumentation; Graphite - chemistry; Humans; Hydrogen peroxide; Hydrogen Peroxide - chemistry; Methods. Procedures. Technologies; Sterol Esterase - chemistry; Sterol Esterase - metabolism; Total cholesterol; Various methods and equipments; Cholesterol oxidase; Hydrogen peroxide; Direct electrochemistry; Cholesterol esterase; Total cholesterol; Ascorbic acid; Immobilization; Sterol esterase; Electron transfer; Esterases; Glucose; Cyclic voltammetry; Electrocatalysis; Detection limit; Amperometry; Peak; Rate constant; Enzyme; Use; Interference; Uric acid; Chemical sensor; Enzyme electrode; Cholesterol; Carboxylic ester hydrolases; Sensitivity; Biosensor; Graphite; Hydrolases; Electrochemical impedance spectroscopy
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2012.05.056

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto functionalized graphene (FG) modified graphite electrode. Enzymes modified electrodes were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). FG accelerates the electron transfer from electrode surface to the immobilized ChOx, achieving the direct electrochemistry of ChOx. A well defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH2 of ChOx. The electron transfer coefficient (α) and electron transfer rate constant (Ks) were calculated and their values are found to be 0.31 and 0.78s−1, respectively. For the free cholesterol determination, ChOx-FG/Gr electrode exhibits a sensitive response from 50 to 350μM (R=−0.9972) with a detection limit of 5μM. For total cholesterol determination, co-immobilization of ChEt and ChOx on modified electrode, i.e. (ChEt/ChOx)-FG/Gr electrode showed linear range from 50 to 300μM (R=−0.9982) with a detection limit of 15μM. Some common interferents like glucose, ascorbic acid and uric acid did not cause any interference, due to the use of a low operating potential. The FG/Gr electrode exhibits good electrocatalytic activity towards hydrogen peroxide (H2O2). A wide linear response to H2O2 ranging from 0.5 to 7mM (R=−0.9967) with a sensitivity of 443.25μAmM−1cm−2 has been obtained. ► ChOx and ChEt have been covalently immobilized onto functionalized graphene modified electrode. ► A well defined redox peak was observed, corresponding to the DET of the FAD/FADH2 of ChOx. ► For the free cholesterol determination, ChOx-FG/Gr electrode exhibits a sensitive response from 50 to 350μM. ► For total cholesterol determination, (ChEt/ChOx)-FG/Gr electrode showed a linear range from 50 to 300μM. ► Some common interferents like glucose, AA and UA did not cause any interference.