Chemical sensing at the nanoscale: Local field enhanced coherent Raman scattering micro-spectroscopy near a plasmonic nano-antenna

Recchia, Martina 2024. Chemical sensing at the nanoscale: Local field enhanced coherent Raman scattering micro-spectroscopy near a plasmonic nano-antenna. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy is a promising technology for label-free chemical sensing and imaging at high-speed. It has been extensively developed over the last two decades, allowing for mapping of endogenous biomolecules in living systems, from single cells to large area tissues, without the drawback of staining artifacts and photobleaching associated with fluorescence microscopy, at speeds up to video rate. To date, we are still missing a label-free non-invasive detection method able to achieve single-molecule detection and measure nanoscale regions of interest such as lipid nanodomains in living cells with high spatio-temporal resolution. Despite all the advantages, with a CARS-based technique is still not possible to achieve single-molecule detection. The strong enhancement of the light field occurring at the nanoscale region near the surface of a metallic nanostructure has helped to overcome this limitation. In plasmon-enhanced CARS, the generated anti-Stokes signal is the result of the locally enhanced pump and Stokes laser fields with an additional amplification of the generated anti-Stokes field by the plasmonic nanostructure. In this work, we first show proof-of-principle experiments to chemically detect lipid molecules label-free using an epi heterodyne detected CARS (eH-CARS) technique and exploiting the local field enhancement (LFE) occurring in the nanoscale region near a single plasmonic silica-coated gold nanorod (SiAuNR). Such technique is thus named LFE eH-CARS. For optimum LFE effect, the localized surface plasmon resonance (LSPR) of the selected SiAuNRs was chosen to coincide with the CARS wavelength of the CH stretch vibration in lipid (∼2900cm−1), i.e. 660nm in our set-up. For this purpose, the extinction cross-section spectrum of each individual SiAuNR was measured. We additionally developed an optical sizing tool able to estimate parameters describing the geometry of SiAuNRs via comparison of quantitative experimental and numerical results. Furthermore, we developed an elaborate simulation model, reproducing the experimental setup, both from the point of view of the excitation and detection, to gain a better understanding of the LFE provided by a gold nanorod (NR) in CARS. Such a model has been a significant new development, to date not shown in the literature. The established technique was then used to perform correlative fluorescence and LFE eH-CARS sensing measurements at single gold nanobowtie (AuNB) antennas, by exploiting fluorescently labeled PS beads moving in and out of the antenna LFE volume. Moving forward, we interrogated the plasma membrane of HEK293 living cells over-expressing a GFP-tagged P2X7, which is a membrane receptor thought to partition in lipid nanodomains, rich in cholesterol and saturated lipids. The AuNBs were entirely designed and fabricated within the project and protocols to attach the HEK293 cells on top of the nanoantennas and keep them alive sufficiently long to perform the sensing measurements were successfully established. While eH-CARS from the PS beads in the absence of the nano-antenna was not sufficiently strong to be detected and correlated with two photon fluorescence (TPF) simultaneously detected in our set-up, we found evidence of LFE eH-CARS correlatively with TPF fluorescence when measuring onto the antennas. Notably, we detected LFE eH-CARS correlatively with TPF fluorescence also when measuring on living cells, suggesting the presence of transient lipid nanodomains/raft, exhibiting CARS signals, close to a GFP-tagged P2X7. Further studies are needed to draw robust conclusions. The proposed LFE eH-CARS correlatively with TPF technique is biocompatible and serves as the foundation to investigate the dynamics of individual proteins within living cell membranes and their association with lipid nanodomains, with chemical specificity and sensitivity at the nanoscale.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	23 July 2024
Last Modified:	24 Jul 2024 11:39
URI:	https://orca.cardiff.ac.uk/id/eprint/170849

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