Enhanced hemoglobin electroactivity on carbon in electrolytes or binders containing water-miscible primary alcohols

Abstract

Herein we demonstrate for the first time, the commonly overlooked impact of water-miscible primary alcohols on hemoglobin (Hb) electroactivity. When Hb is cast into biosensor films, the binder solvent is often an alcohol; however, the impact of alcohols on Hb electroactivity is unknown. It is well-established that Hb electroactivity is inhibited by the protein structure and that alcohols disturb this structure; thus, probing the alcohol-Hb relationship is vital to evaluate electrochemical results using binder-immobilized Hb. Using differential pulse voltammetry on glassy carbon in neutral pH phosphate buffer, we show a twofold higher Hb response when the Nafion binder contains alcohol, indicating the alcohol significantly changes Hb electroactivity. Significant changes are seen for alcohol concentrations as low as 10%. When binders are necessary for a Hb-containing film, a low methanol content is preferred; ethanol and propanol should be avoided.Conversely, Hb quantification is 53× higher in alcohol-containing electrolytes, due to changes to both the Hb and carbon. Methanol-containing electrolyte exhibits an ultra-low 0.78 nM Hb detection limit. This detection limit is twofold lower than any previously reported for solution-based Hb detection and on par with surface-immobilized Hb, while having the advantages of simplicity, ease of application and low cost.