Nanomaterials from waste

Abstract

Amyloid fibrils are a class of insoluble nanofibers, which are self-assembled from a wide range of peptides and proteins under denaturing conditions. They have been increasingly exploited for a broad range of applications in bionanotechnology such as biosensors, nanowires, hydrogels, thin films and drug delivery. One of the persistent challenges faced in the field of amyloid fibrils is the high costs associated with purified proteins and peptides used for fibril formation. Hence this thesis has investigated hemoglobin extracted from slaughterhouse waste blood as a cheap source of amyloid fibril synthesis at large scale. The conditions for amyloid fibril formation from commercially available hemoglobin were first screened due to limited literature on hemoglobin amyloid fibrils. The incubation of 10 mg/mL apo-hemoglobin aqueous solutions at pH 2.0 at 80 oC for 24 h resulted in β-sheet rich, nanofibrils with a diameter of 7.6 ± 1 nm as confirmed by the ThT assay and TEM imaging. To isolate hemoglobin from bovine waste blood, the red blood cell lysis with 0.1% NaCl was found to be efficient, yielding 30.56 g of hemoglobin from 1 L of blood. The fibrillation of extracted hemoglobin was shown to be possible in its apo-form under the same conditions used with pure hemoglobin. AHB nanofibrils demonstrated a high stability over a range of temperatures, pHs, organic solvents and proteases, thereby suggesting their potential as nanomaterials for versatile applications. The optimization of AHB fibril formation revealed a pH dependent shift in the morphology from long, straight to short, worm-like. Moreover, a significant increase in fibril yield was achieved with the addition of moderate NaCl concentrations. Optimized conditions for AHB fibril formation was determined to be incubation of 10 mg/mL solutions at pH 2.8 at 80 oC for 24 h in the presence of 125 mM NaCl. The amyloid cross-β quaternary structure and unbranched morphology were confirmed by X-ray fiber diffraction and AFM, thereby providing conclusive evidence for amyloid fibril formation from hemoglobin for the first time. Finally, AHB fibril formation was successfully scaled up from 1 mL to 250 mL with a yield of 25.6 g from a liter of blood.