title: Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.
abstract: Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.
authors.affiliation: Department of Physical Chemistry, University of Sevilla , Profesor García González 1, 41012, Sevilla, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Olloqui-Sariego
authors.affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences , Leninsky Prospect 33/2, Moscow, 119071, Russia.
authors.forename: Galina S
authors.initials: GS
authors.lastname: Zakharova
authors.affiliation: Department of Chemistry, Lomonosov Moscow State University , Vorob'evy Gory 1, Moscow, 119991, Russia.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Poloznikov
authors.affiliation: Department of Physical Chemistry, University of Sevilla , Profesor García González 1, 41012, Sevilla, Spain.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Calvente
authors.affiliation: Department of Chemistry, Lomonosov Moscow State University , Vorob'evy Gory 1, Moscow, 119991, Russia.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Hushpulian
authors.affiliation: Department of Biochemistry and Structural Biology, University of Lund, Kemicentrum , Box 118, 221 00, Lund, Sweden.
authors.forename: Lo
authors.initials: L
authors.lastname: Gorton
authors.affiliation: Department of Physical Chemistry, University of Sevilla , Profesor García González 1, 41012, Sevilla, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Andreu
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D055664:Electrochemical Techniques; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry*; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D004800:Enzymes, Immobilized; D006108:Graphite; D009195:Peroxidase