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theses

The need for early detection of cancer has resulted in the development of a number of molecular identification techniques that can screen for cancer biomarkers or cellular components indicative of a cancerous state. Biomarkers play a crucial role in cancer diagnosis and their identification and quantification can help monitor disease progression and therefore significantly contribute to clinical prognosis and to individualization and optimization of systemic therapy. Nanomaterial-based imaging systems provide the sensitivity, selectivity, and high multiplexing capability needed for molecular detection when leveraging the optical phenomenon of surface-enhanced Raman scattering (SERS). SERS is a vibrational spectroscopic technique that can be used to detect molecules present on or near the surface of plasmonic nanomaterials such as gold nanoparticles. The objective of this dissertation was to develop a SERS-based imaging tool for phenotype assessment of cancer cells and tissues by utilizing the optical properties of gold nanostars (i.e. gold nanoparticles (AuNPs) with a spherical core and sharp protruding spikes) together with the effective targeting ability of aptamers. It was hypothesized that gold nanostars would be able to provide excellent SERS enhancement factors that would enable the quantification of biomarker expression at the single cell level. However, toxicity of the nanoparticles is a critical issue that needs to be taken into consideration before their implementation in vitro. For this purpose, detailed multi-parametric assessment of gold nanoparticle toxicity was carried out in cancerous (glioblastoma cells, U-87) and healthy cells (human fibroblasts) where the effects of nanoparticle shape, surface chemistry, and size on cell toxicity and cellular uptake were assessed. The study demonstrated that gold nanostars can be effectively taken up by both cell types without inducing significant toxicity when capped by a suitable ligand. They were therefore considered to be less toxic when compared to other nanoparticle shapes with the same surface coating and at the same dosage. Gold nanostars were then implemented in the development of a SERS based sensing platform for the recognition of soluble and cell membrane embedded epithelial cell adhesion molecule (EpCAM) protein with high sensitivity and selectivity. EpCAM is a key epithelial biomarker that is overexpressed in several types of cancer and changes in its expression have been associated with the onset of metastasis. The developed biosensor enabled the detection and quantification of EpCAM at the single cell level in two cancer cells with varying expression levels, MCF-7 and PC-3. The highly sensitive cellular targeting with a detection limit of 10 pM was achieved by using EpCAM aptamers. The results demonstrated potential in using this approach for detecting cells that undergo phenotype variations during cancer metastasis. Finally, the gold nanostar aptamer-based SERS tags were implemented to distinguish between early and late stage prostate cancer based on the expression of the prostate specific membrane antigen (PSMA) biomarker. The SERS tags enabled sensitive detection of the PSMA biomarker in the prostate cancer cell LNCaP and in a tissue microarray containing samples from prostate cancer patients. Results showed elevated PSMA expression with progression of the disease and was found to outperform immmunofluorescence. Overall, the dissertation established the importance of cancer diagnosis and staging based on biomarker expression and demonstrated the potential of SERS based diagnostic tools for molecular detection.

ETD_9137

doi:10.7282/T3VH5SGQ

Ph.D.

Includes bibliographical references

by Manjari Bhamidipati

ETD doctoral

The author owns the copyright to this work.