Covalent immobilization of redox protein via click chemistry and carbodiimide reaction: Direct electron transfer and biocatalysis

In this paper, a simple, facile and general strategy was proposed for the covalent biofunctionalization of Au surface with hemoglobin (Hb). Structurally well-defined azide-terminated organic self-assembled monolayers (SAMs) were formed on Au surface from a mixture solution of azidoundecanethiol and dilute thiol. 4-Pentynoic acid was used as a bifunctional linker to immobilized Hb via a selective, reliable, robust click reaction and a widely used carbodiimide reaction. The surface modification was characterized by reflectance absorption infrared (RAIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The experimental data indicate that the proposed methodology is a highly versatile strategy for quantitative, stable and covalent attachment of biomacromolecules onto solid interface. The Hb functionalized Au electrode shows a pair of well-defined and quasi-reversible peaks at about -0.210V (vs. SCE) in 0.1mol/L pH 5.0 buffers, which stands for the reduction and oxidation of Hb Fe(III)/Fe(II) redox couple. The electron transfer rate constant (k_s) was estimated to be 0.78s^-1. The maximal surface coverage (G{cyrillic}) of the immobilized Hb was 8.3×10^-12molcm^-2, indicating the formation of a monolayer. Moreover, the biocatalytic activity of the Hb biofunctonalized electrode was investigated and an excellent electrocatalytic reduction toward O₂ and H₂O₂ was observed.