Probing redox reaction of azurin protein immobilized on hydroxyl-terminated self-assembled monolayers with different lengths

摘要

Immobilization of proteins with controlled electron transfer properties is an important task for future biomedical applications. In this work, immobilization of azurin on gold electrodes modified with 6-mercaptohexanol (MCH), and 11-mercaptoundecanol (MUD) self-assembled monolayers (SAMs) was studied by electrochemical techniques. The immobilized azurin showed a quasi-reversible redox response on MCH/Au and MUD/Au electrodes, with E-1/2 = 0.11 V vs. Ag/AgCl and Delta E-p = 10 mV and 40 my, respectively. Cyclic voltammograms (CVs) of azurin at different pHs revealed that proton-coupled redox reaction of azurin is a one-electron, three-proton process. The tunneling electron transfer rate constant (k(0)) of immobilized azurin on MCH/Au electrode was estimated 135 (+/- 0.05) x 10(3) s(-1) using a fast scan CV method, and that on MUD/Au electrode was measured as 120 +/- 10 s(-1) by scanning electrochemical microscopy (SECM). In SECM studies, the approach curves were recorded at different substrate overpotentials with different surface coverages of azurin. For extraction of kinetic parameters, azurin was reduced on MUD/Au electrode via tunneling electron transfer across the SAM, and oxidized back via solution-phase tip-generated ferrocyanide. The bimolecular electron transfer rate constant (k(BI)) between immobilized azurin and [Fe(CN)(6)](4-) mediator was estimated as 3 x 10(8) mol(-1) cm(3) s(-1). Based on distribution of hydrophilic and hydrophobic amino acids on azurin structure, and the distinct voltammetric behavior of azurin on MCH and MUD SAMs as hydroxyl-terminated SAMs vs. methyl- and thiol-terminated SAMs, possible orientations of azurin on different SAMs were also assessed. (C) 2015 Elsevier B.V. All rights reserved.