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 −0.210 V (vs. SCE) in 0.1 mol/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 (ks) was estimated to be 0.78 s−1. The maximal surface coverage (Г) of the immobilized Hb was 8.3 × 10−12 mol cm−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.

Graphical abstractBased on the click and carbodiimide reaction, hemoglobin-terminated self assembled monolayer was formed on the Au surface and the direct electron transfer and biocatalysis was successfully achieved.Figure optionsDownload full-size imageDownload high-quality image (101 K)Download as PowerPoint slideResearch highlights► Hemoglobin was covalent immobilized on the azide-terminated SAMs via click reaction and carbodiimide reaction. ► Hemoglobin maintains its bioactivity and the direct electrical communication to electrode surface is achieved. ► Mole fraction of azide-terminated alkanethiol effects on the immobilization and electrochemical characteristics of hemoglobin.