ABSTRACT
Electrical impedance spectroscopy (EIS) appears a promising label-free methodology for the investigation of processes related to the aggregation of macromolecules in solution. Here, we explore the EIS technique as a convenient tool for studying the irreversible aggregation of human insulin and describing its corresponding fibrillation kinetics. The in situ measurement of the electrical response of pure insulin solutions at 60°C allows for the real-time monitoring of the protein fibrillation as a function of the incubation time. The fitting of the EIS data through an equivalent circuit based on a constant phase element provides a simple set of electric parameters whose abrupt changes can be associated to transitions occurring in the organization of the macromolecules. For establishing the reliability of the method proposed, we have compared the protein aggregation profile collected from the EIS data to that obtained from a conventional fluorescence methodology where Thioflavin T (ThT) is used as a dye probe. The description of the fibrillation process is quite similar in both cases, since characteristic times of the same order were found for the consecutive processes associated to the initial lag phase of insulin fibrillation, to the rapid growth of amyloidal aggregates and to the final saturation step. Our results suggest that in situ EIS can be considered as a promising approach for the real-time label-free monitoring of protein fibril formation.
