Synthesis and application of SERS active gold nanostar probes to monitor viral evolution
Creators: name: Dardir, Kholud
Publisher: name: No Publisher Supplied
Year: 2019

description: Gold nanoparticles have become increasingly popular due to their unique physical and optical properties. Their nanoscale dimensions place them in the ideal size range to study and manipulate biological systems. The shape and plasmonic properties of these particles contribute to significant signal enhancement in spectroscopic techniques, which is extremely relevant toward the analysis of biological samples. Furthermore, the gold surface allows for accurate surface conjugation, which holds tremendous potential for various biomedical applications, including drug delivery and biosensing. Researchers have made significant progress in the advancement of gold nanoparticles for applications in medicine; however, with respect to the eradication of viral diseases, the study of viral replication and mutation has not been approached with methods that leverage gold nanoparticle-based tools. Nonetheless, gold nanoparticles hold tremendous potential in this respect, especially for tracking viral mutations and understanding complex mechanisms of viral evolution.In this thesis, gold nanostars were utilized to monitor intracellularly viral mutations at the single cell level. The synthesis of gold nanostars and their functionalization with various coatings and ligands for optimized cellular uptake was the first step to creating the proper tool for intracellular applications, in particular for the purpose of tracking viral RNA mutations intracellularly and provide mechanistic insight into viral evolution.We demonstrated that gold nanostars, functionalized with a surface enhanced Raman spectroscopy (SERS) beacon, can create a sensitive tool for high resolution single nanoparticle-based influenza A virus (IAV) detection without the need of PCR amplification and sequencing. The initial step was to create a probe that could detect viral RNA intracellularly with sensitivity to single base RNA. We have demonstrated for the first time, both in buffer and at the single cell level, that the SERS signal intensity of a fluorophore covalently bonded to the beacon is an ideal reporter of mutations, as it has been shown to be dependent on the number of mutations present on the target viral RNA. We demonstrated the selectivity of the nanoparticle probe assay to its target by introducing other viral sequences and observing a measurable SERS signal only in the presence of the targeted strand. To overcome obstacles with cellular uptake, and in particular to ensure that the nanostars would circulate in the cytosol (where the viral RNA is located) rather than being trapped in endosomes, we combined gold nanoparticles with trisaminocyclopropenium (TAC) ion-functionalized macro-molecules to provide an efficient route to nanoparticle endosomal escape. TAC polymers can be readily wrapped around nanoparticles through electrostatic interactions and have been shown to be less toxic than their counterpart poly(ethyleneimine) (PEI). We electrostatically layered TAC polymers onto gold nanostars for cellular uptake. These particles were able to escape the endosomal membrane and diffuse within the cytoplasm without affecting cellular viability. In addition to being less toxic, the TAC coated nanostars diffused within the cytoplasm more rapidly than PEI-coated nanostars, making them an ideal tool to match the four-hour replication kinetics of IAV in cells. This platform will not replace current assays but rather become an invaluable tool to add to the virologist toolbox.