Detection and structural characterization of insulin prefibrilar oligomers using surface enhanced Raman spectroscopy

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface‐Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single‐molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time‐dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:488–495, 2014