Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733793
Title: Protein-nanoparticles interaction and assembly
Author: Ma, Wenwei
ISNI:       0000 0004 6495 3839
Awarding Body: University of Lincoln
Current Institution: University of Lincoln
Date of Award: 2017
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Abstract:
Nanoparticles are increasingly important in biotechnology as they are extensively used as drug delivery carriers and in biosensors. In both these two contexts, protein-nanoparticle interactions are often involved. Proteins that are present in body fluids inevitably interact with nanoparticle based drug carriers and typically surround them forming the so called “protein corona”. Biosensors that are based on nanoparticles often have proteins deliberately attached to their surface, for example antibodies that bind specific analytes. The understanding of the assembly mechanisms at the protein-nanoparticle interface and the ability to engineer proteins that interact with nanoparticles in the desired way, are therefore two essential requisites for the future development of nano-medicines and nano-biosensors. In this work, we focused on the interaction of proteins with gold nanoparticles (GNPs). GNPs are available with a broad range of surface chemistries, suitable for the conjugation of many biomolecules. Although there are at least three decades of studies on gold colloids with different surface chemistries, there is still quite little known about what are the exact features of a protein that determine its adsorption onto gold. We developed methods to study this and applied them to characterise the adsorption on GNPs of Glutathione-S-Transerase (GST), which was reported previously as a protein that strongly binds gold. We determined its affinity and kinetics of binding and unravelled the mechanism of its thiol-mediated chemisorption. We found that GST binds to GNPs even more efficiently than other known gold-binding proteins, such as Bovine Serum Albumin (BSA). We concluded that GST could be considered a very useful gold-protein interface, especially considering that GST fusion is routinely used for affinity purification of recombinant proteins and therefore well established. We also fused self-assembling proteins to GST or chemically cross-linked them to BSA. The scope was to explore the feasibility of hierarchical and ordered assembly of designer proteins onto GNPs, with the ultimate goal of providing a convenient tool for modular assembly of proteins onto nanomaterials. It is known that proteins tend to denature and lose their function when in contact with GNPs, which is not optimal for biosensors or in nanomedicine. We found that it is possible to use GST or BSA to form a sacrificial layer on gold, which exposes linked, self-assembling proteins that are able to bind their counterpart, unaffected by the GNP surface. We reported two proof-of-concepts: the first based on mimics of the self-assembling neuronal SNARE proteins and the second based on the pair SpyCatcher/SpyTag, derived from Streptococcus pyogenes proteins and used in bio-conjugation for their ability to self-catalyse the formation of isopeptidic bonds. We believe that the novel methods and original results presented in this thesis apply to both the understanding and the engineering of the protein-nanoparticle interface and will be beneficial for the broad nanobiotechnology community. In fact, our findings have potential applications in a broad range of fields, spanning from the improvement of the circulation life-time of nanomedicines by preventing the binding of serum protein and opsonisation, to the improvement of the manufacturing of GNPs-based immune-biosensors such as those used in lateral flow devices.
Supervisor: Ferrari, Enrico Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.733793  DOI: Not available
Keywords: F110 Applied Chemistry ; F200 Materials Science
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