Investigating lysozyme amyloid fibrillization by electrochemical impedance spectroscopy for application in lysozyme sensor

Abstract

• GO/SPE was used for investigation of lysozyme fibrillization. • Fibril and amorphous structures of lysozyme were investigated by the non-Faradaic EIS. • Lysozyme protofibril-modified GO/SPE could detect lysozyme in phosphate buffered saline and in wine. • The binding specificity of lysozyme onto its protofibril structure was advantaged for lysozyme biosensor. Amyloid fibrils have garnered increasing attention as viable building blocks for designing and synthesizing of biomedical material. The structure of amyloid fibril of most of the proteins are well known, but the electrical property of their conformational changes during synthesis is not fully understood. Herein, we investigated this relationship by using non-Faradaic electrochemical impedance spectroscopy (EIS). Lysozyme (Lyz) from hen egg white was used as a model protein and incubated under heat (60 °C) at pH 2 which led to the amyloid fibrillization. Morphological changes during fibrillization were confirmed by AFM. Raman spectroscopy was used to distinguish amyloid fibrils from their native and amorphous structures. For EIS measurement, graphene oxide coated screen-printed electrode (GO/SPE) was immobilized with Lyz aggregates obtained from different incubation times. The charge transfer resistance (R ct ) of incubated Lyz was measured to determine the fibrillization pathway of amyloid fibril i.e., unfolding and nucleation, protofibril (PF, a short fibril) and mature fibril. Next, PF-modified GO/SPE (PF/GO/SPE) was demonstrated to determine Lyz molecules dissolved in PBS and in wine using EIS. The dependence of R ct of PF/GO/SPE on Lyz concentration was reported. Linear range and limit of detection (LOD) of detecting Lyz molecules in PBS and in wine were the same. The linear range and LOD were 0.005–0.08 μM and 0.001 μM, respectively. PF/GO/SPE sensor was found to have good selectivity to recognize Lyz. These findings show that PF/GO/SPE sensor provides an attractive component for future electrochemical sensing devices.