Surface-Enhanced Coherent Raman Scattering (SE-CRS) with Noble Metal Nanoparticles

Abstract

Early cancer detection remains challenging due to numerous complex tempo-spatial metabolic changes in cell physiology. Based on their ability to recognise molecular structures and pathological changes at molecular levels, spectroscopic have recently emerged as promising non-invasive, non-ionising, and cost-efficient tools to help detect cancer, and other human pathologies. Raman spectroscopy is a valuable technique that provides information regarding the chemical properties of materials. Nevertheless, it has limitations due to the limited amount of Raman light scattered. Strategies for cancer diagnostics and therapies are based on the hypothesis that nanoparticles (NPs) can be precisely tailored to target cancer cells. However, the tools required to image NPs at cellular levels remain scarce in the literature. The work outlined in this thesis, for the first time, utilises noble metal NPs and Raman reporters, with the mechanisms of surface enhanced Raman scattering (SERS) and coherent anti-Stokes Raman scattering (CARS), in cancer cells and tumour spheroids to address the demerits of low spatial resolution, signal-to-noise ratio, and chemical specificity. SERS and CARS have broadly been explored in this regard. To increase the effectiveness of Raman scattering, a variety of techniques have been devised to boost its intensity. Primarily, I studied four techniques to increase Raman scattering intensity with the ultimate objective of improving sensitivity and assessing limits of various Raman methods: SERS, surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS), surface-enhanced stimulated Raman scattering (SE-SRS), and broadband coherent anti-Stokes Raman scattering (BCARS). Coherent Raman scattering (CRS) is utilised to enhance weak Raman bands. The signal is enhanced by nonlinear interaction of the excitation lasers within the sample. Despite the advantages offered over Raman, CRS has been relatively unexploited for image Raman tagged NPs. This challenge has recently been addressed using surface plasmon enhancement, which gives significantly enhanced inelastic scattering signals as well as reduced signal-to-noise ratio. Surface-enhanced coherent Raman scattering (SE-CRS) has been characterised by using a variety of techniques such as SERS, CARS, and SE-CARS. This work provides a step forward to develop plasmon enhanced SRS and CARS in addressing critical biological questions using nonlinear bio-photonics. In the first part of this thesis, I developed a reproducible substrate that mimics gold nanoparticles (AuNPs) and allows forward detection which is critical for CRS. I investigated the effects of annealing on gold films deposited on glass substrates with thicknesses from 3 nm to 15 nm as described in depth in chapter 5. In addition to this, it provides an explanation of the work that was performed to explore the interaction between Raman tags BPT (biphenyl-4-thiol), BPE trans-1,2-bis(4-pyridyl) ethylene, and IR 820 (new indocyanine green) on gold films substrates using 785 nm laser excitation. In the second part of this thesis, I investigated the interactions between Raman tags of BPT on gold films substrates using CRS and broadband CARS techniques. These experiments also offer the SE-CRS enhancement signal. The research done to examine gold thin film substrates and to offer SE-SRS and SE-CARS enhancement signals in the fingerprint region as described in chapter 6. Using CRS microscopy, the investigations in this chapter study these interactions. In the third part of this thesis, I developed a novel imaging methodology for the visualisation of AuNPs inside cellular structures and spheroids, with the intention of acquiring distinct spectroscopic fingerprints. Consequently, I undertook the task of devising protocols for visualising AuNPs and Raman reporter molecules within cancer cell models, spheroids, and animal tissues as described in chapter 7. The aim was to attain distinctive spectroscopic profiles by employing the SE-CRS technique, achieved by illuminating AuNPs along with Raman reporter molecules (BPT, BPE, IR 820) using low intensity infrared light, with both the pump and Stokes beams operating at intensities below 0.2 mW. In summary, this thesis sheds light on the development of surface plasmon resonance phenomena based on metallic nanostructures for use in nonlinear inelastic scattering systems, including surface-enhanced Raman scattering (SERS), coherent Raman scattering (CRS), and surface-enhanced coherent Raman scattering (SE- CRS). The primary focus is to use this system for disease diagnostics, rooted in SERS, reflects a commitment to advancing cancer diagnostics, based on SERS thereby enhancing the precision and discrimination of molecular signals, making a significant stride towards more effective and nuanced cancer diagnostics.