title: Detecting the early onset of shear-induced fibril formation of insulin in situ.
abstract: A new approach is presented for detecting the early onset of amyloid fibril formation of insulin in a fluidic environment. The fibrillogenesis of insulin in a well-characterized Taylor-Couette flow cell was analyzed in situ using Raman spectroscopy in combination with principal components analysis (PCA). Raman spectra recorded using a 532.5 nm excitation laser revealed a more rapid fibrillogenesis process during the first 90 min of shearing than previously reported for samples exposed to flow. Bands corresponding to intermolecular H-bonded β-sheet structure of insulin at 1678, 1630, and 1625 cm(-1) observed in the Raman difference spectra between unsheared insulin and sheared insulin show an increase in intensity as a function of shear exposure time, which is characteristic of fibril formation, with the first changes detected after 10 min. Additional analysis of samples removed from the flow cell after specific time periods provided conformation of the flow-enhanced fibrillogenesis process, including the detection of early fibril formation after only 1 min of shearing. FT-IR spectra of the insulin solutions showed evolution of bands at 1673 and 1633 cm(-1) from an increase in H-bonded β-turn and β-sheet structures, respectively, while fluorescence emission spectra detected the presence of a new emission band at 482 nm. TEM images confirmed the early onset of fibril formation at 1 min shear exposure, before a maturation and concentration increase of fibrils with further shearing. This study highlights the ability of fluid flows to accelerate insulin fibril formation, which has important implications for biotechnology applications such as the purification process of insulin therapeutic drugs in the pharmaceutical industry, as well as the use of optical-based methods for detecting fibrillogenesis.
authors.affiliation: Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. grant.webster@manchester.ac.uk
authors.forename: Grant T
authors.initials: GT
authors.lastname: Webster
authors.forename: Jonathan
authors.initials: J
authors.lastname: Dusting
authors.forename: Stavroula
authors.initials: S
authors.lastname: Balabani
authors.forename: Ewan W
authors.initials: EW
authors.lastname: Blanch
mesh_terms: D000682:Amyloid / Q000138:chemical synthesis* / Q000737:chemistry / Q000648:ultrastructure; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D007328:Insulin / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D025341:Principal Component Analysis; D017433:Protein Structure, Secondary; D012212:Rheology; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013314:Stress, Mechanical; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T