Label-Free Electrochemical Test of Protease Interaction with a Peptide Substrate Modified Gold Electrode

Abstract

Efficient deposition of biomolecules on the surface, maintaining their full activity and stability, is a most significant factor in biosensor construction. For this reason, more and more research is focused on the development of electrochemical biosensors that have the ability to electrically detect adsorbed molecules on electrode surface with high selectivity and sensitivity. The presented research aims to develop an efficient methodology that allows quantification of processes related to the evaluation of enzyme activity (proprotein convertase) using electrochemical methods. In this study we used impedance spectroscopy to investigate the immobilization of peptide substrate (Arg-Val-Arg-Arg) modified with 11-mercaptoundecanoic acid on the surface of gold electrode. Both the synthesis of the peptide substrate as well as the full electrochemical characteristics of the obtained electrode materials have been described. Experimental conditions, including concentration of peptide substrate immobilization, modification time, linker, and the presence of additional blocking groups have been optimized. The main advantages of the described method is that it makes it possible to observe the peptide substrate–enzyme interaction without the need to use fluorescent labels. This also allows observation of this interaction at a very low concentration. Both of these factors make this new technique competitive with the standard spectrofluorimetric method.

Keywords

• proprotein convertases
• 11-mercaptoundecanoic acid
• electrochemistry
• electrode modification
• electrochemical impedance spectroscopy
• peptide substrate