Amyloid fibril based bionanotechnologies.

Abstract

Amyloid protein nanofibrils (PNFs) are self-assembling, misfolded structures formed by many proteins when exposed to denaturing conditions. PNFs offer several attractive features for utilisation in the creation of new bionanomaterials, such as nanometre dimensions, strength and stability, and ease of functionalisation through amino acid residues. However, if amyloid PNFs are to be used in bionanotechnology, then methods need to be developed to utilise inexpensive, crude proteins instead of pure protein preparations. This research focuses on utilising PNFs obtained from a low cost source, including fish eye lens crystallins, and whey protein isolate to develop novel PNF-based nanoscaffolds, with potential applications in bionanotechnology. To ensure that crystallin PNFs can be effectively used as a versatile bionanoscaffold, biocompatibility and stability studies were done using cell viability and Thioflavin T dye binding assays, Transmission electron microscopy, and Infrared microspectroscopy. Crystallin PNFs were shown to be stable over all the conditions studied (pH and temperature extremes, presence of proteases and solvents), and showed no evidence of cytotoxicity. The results obtained from IR microspectroscopy illustrated the long-term (up to 3 years) structural integrity of crystallin PNFs. To obtain PNF-based functional nanoscaffolds, several enzymes of industrial relevance were successfully immobilised onto the crystallin PNFs via a versatile glutaraldehyde-based crosslinking approach. Dual-functionalised PNFs were also obtained by co-immobilising enzymes onto the whey PNF scaffold. The functional PNF-based nanoscaffolds provided a significant increase in thermostability and reusability of industrial enzymes relative to the free enzyme in solution under the same conditions. To demonstrate that PNF-based scaffolds can be potentially used for creating active bionanomaterials, electrochemistry, surface-assembly, and cell attachment and proliferation experiments were done. Electrochemistry studies demonstrated that functionalised PNFs can be successfully used to develop single enzyme-, or dual enzyme-based biosensing elements, for glucose and lactose analysis. Surface-assembly studies established that PNF scaffold provides more surface area for biomolecule immobilisation, and can be used to create PNF-based active surfaces. The use of PNF-based scaffold for cellular growth resulted in improved attachment and proliferation of fibroblasts, suggesting a role for PNFs obtained from crude protein sources in the biomedical field. This work demonstrated the potential of PNFs obtained from crude proteins to develop functional nanoscaffolds with broad applications. The described work contributes to the existing knowledge of PNF-based bionanomaterials, and is very significant for the technological development and future applications of PNF-based materials in bionanotechnology.