Design of phage-templated gold assemblies as contrast agent.

Sokullu, Esen (2017). Design of phage-templated gold assemblies as contrast agent. Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, Doctorat en sciences de l'énergie et des matériaux, 206 p.

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Résumé

“Plasmonics”, the study of the interaction between an electromagnetic field and free electrons in a metal, is a growing field due to the recent developments in nanotechnology and nanofabrication that enable the design of new materials with properties suitable for locally concentrating light. One strategy to concentrate light is to create small gaps between metallic nanoparticles by organizing them into assemblies that display new, collective properties. The resulting enhancement of optical properties is mainly due to the coupling of the localized electric fields of the assembled nanoparticles, which is a phenomenon that depends on the inter-particle distance. Amongst the different metallic nanoparticles that have been explored for the production of plasmonic nanostructures, gold nanoparticles (AuNPs) have been the most popular owing to their general chemical inertness, biocompatibility, ease of synthesis, and simple surface modification. Given these important features, ‘isolated’ AuNPs have been investigated for several applications, including imaging biomaterials, via their ability to generate contrast for surface-enhanced and non-linear spectroscopies. Moving forward, the generation of assemblies of gold nanostructures could yield greater contrast than their isolated nanoparticle building blocks, because of the coupling of their plasmonic properties. As a consequence, this could enable the use of smaller AuNPs that do not intrinsically exhibit plasmonic features. As such, it is exciting and timely to investigate new plasmonic structures that exploit the collective properties of ensembles of AuNPs. Generally, plasmonic nanostructures are fabricated by ‘top-down’ techniques such as electron beam lithography, focused beam lithography, and nanoimprint lithography, which are limited by high cost, low-speed, and limited ability to generate three-dimensional structures. On the other hand, the self-assembly of AuNPs onto a template material is emerging as an alternative ‘bottom-up’ approach for producing colloidal plasmonic structures that could potentially have the ability to label biological structures. In this context, the use of very small AuNPs could be particularly advantageous because of their lesser expected detrimental effect on binding target biological molecules/structures, and better image resolution (i.e., smaller contrast agents). For the ‘bottom-up’ approach, nanoparticles are immobilized onto pre-determined binding sites on a template material. Viruses are one of the most studied biological template materials because of their great structural diversity and the ability to precisely position binding motifs in a controlled manner on their surface by genetic manipulation. Moreover, viruses naturally target and bind to cells, which make them suitable for producing colloidal contrast agents for specific types of cell. Notwithstanding, developing procedures for producing virus–AuNP assemblies can be technically complex (depends on AuNP size), and little information is available on the plasmonic properties of such assemblies prepared with very small AuNPs. In the present work, two bacteriophage templates (virus that selectively infect bacteria), M13 and T4, were examined as biological templates to create well-defined assemblies of AuNPs. These phage were selected because of their considerably different geometries (filamentous vs icosahedral for M13 and T4, respectively), which will allow us to investigate both the effect of short-range and longer-range ordering of the AuNPs on plasmonic properties. Because of the paucity of data on the assembly of very small (<13 nm) AuNPs on bacteriophage, several gold-binding motifs were explored and, for T4, the relative disposition of these motifs on the phage surface was also varied. In Chapters 2 and 3, the genetic engineering of the phage is presented, and protocols for the assembly of 3, 9, and 13-nm AuNPs were established. The resulting assemblies were characterized by several complementary analytical techniques. Furthermore, the ability of the assemblies to bind to bacteria, despite the presence of AuNPs covering the surface of the phage, was verified. This finding supports the investigation of very small AuNPs in this work, because target binding is an important property for developing imaging contrast agents. In Chapters 4 and 5, the plasmonic properties of the phage–AuNP assemblies were examined by surface-enhanced Raman scattering (SERS) and two-photon excitation fluorescence (2PEF) microscopy. These Chapters evaluate the influence of the structural parameters of the assemblies on their stability (i.e., suitability to typical sample handling procedures such drop casting and drying on microscope slides), their ability to enhance SERS and 2PEF, and the influence of binding to bacteria on the observed SERS/2PEF signals. For SERS, only the assemblies prepared with 13 nm AuNPs were able to generate strong local electric field enhancements yielding signals which were clearly distinguishable from the background. The recorded SERS spectra corresponded to peaks associated with the stabilizing ligand on the surface of AuNPs, which was used as SERS reporter, indicating that signal enhancement originated from within the gaps formed between adjacent AuNPs. Binding of the assemblies to bacteria did not significantly affect the SERS spectrum or signal enhancement. Higher SERS enhancement was observed for M13-templated assemblies compared to T4-templated assemblies, and the ensemble of data suggests that short-range ordering is the dominant factor affecting enhancement. It was further demonstrated that covalent attachment of AuNPs to phage via a gold–thiolate bond was indispensable for stability of the assemblies, given that assemblies designed with non-covalent gold-binding motifs disassembled during sample handling, resulting in little or no SERS enhancement compared to AuNPs alone. For 2PEF, several of the phage–AuNP assemblies demonstrated signal enhancement. However, in contrast to SERS, the absolute intensity of 2PEF signals obtained for the clusters was closer to that of the background noise. Therefore, an image processing techniques was employed to objectively discriminate 2PEF signals from background, which enabled straightforward quantitative analysis. Reproducible 2PEF signal enhancement was observed for M13-templated assemblies prepared with 9 and 13 nm AuNPs, but not with 3 nm. Only a small effect of AuNP size was observed between the 9 and 13 nm AuNPs. In contrast, assemblies prepared with T4 typically did not exhibit 2PEF and signals and were only rarely distinguishable from the background. Only one T4-templated assembly, prepared with 13 nm AuNPs, produced reliable 2PEF signal, and the magnitude of the intensity was comparable in magnitude albeit slightly lower than that observed for equivalent assemblies prepared with M13. This difference could be explained with different spatial geometries of AuNP assemblies templated on M13 and T4 phages and their corresponding stabilities. It should be noted that isolated AuNPs (3–13 nm) never enhanced SERS of 2PEF in any of the experiment conducted. Indeed, these AuNPs are much smaller than those typically examined for their ability to enhance SERS/2PEF. Overall, this thesis presents the design, production, and characterization of new colloidal plasmonic structures combining AuNPs and bacteriophage. It should be emphasized that this work explores very small AuNPs, for which a great paucity of data exists in the literature, both in terms of chemistry (i.e., conditions for assembly onto biological templates) and plasmonic properties. Drop casting and drying these assemblies, followed by their analysis by SERS and 2PEF provided essential information on how the design of the assemblies affected their performance as targeted imaging contrast agents, in terms of signal enhancement, stability, and effect of binding to cells. Screening a wide range of design parameters susceptible to influence these properties above has led to the identification of phage–AuNP assemblies that are suitable for use as biological contrast agents for SERS and 2PEF, and the relevant opportunities and limitations are discussed in each appropriate chapter. This body of work sets the stage for future experiments, which can now be performed because of the refinement achieved from within the large library of phage–AuNP assemblies above. For instance, analysis of the plasmonic properties of the assemblies in solution, while technically challenging, should provide additional data on some of the less-stable or meta-stable assemblies, which did not survive sample handling. This could enable a comparison of optical properties with, for instance, plasmonic characteristics determined by simulation. Furthermore, an additional level of matrix complexity can now be examined, such as the use of the assemblies as contrast agents within complex aqueous matrices (e.g., bacteria detection in wastewater) or for mixtures of cell populations. Indeed, the ability of the assemblies to be re-engineered to bind to different targets (beyond their native bacterial targets), may make them suitable for several imaging- and detection-related applications.

Type de document:	Thèse Thèse
Directeur de mémoire/thèse:	Ozaki, Tsuneyuki
Co-directeurs de mémoire/thèse:	Gauthier, Marc-André
Mots-clés libres:	plasmonics; nanotechnology; nanofabrication; optical properties; gold nanoparticles (AuNPs)
Centre:	Centre Énergie Matériaux Télécommunications
Date de dépôt:	03 juill. 2018 13:53
Dernière modification:	30 sept. 2021 19:41
URI:	https://espace.inrs.ca/id/eprint/6932

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