Thesis

Detection of galectin proteins using advanced raman rechniques

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2024
Thesis identifier
  • T16999
Person Identifier (Local)
  • 201771953
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Galectin-3 protein (Gal-3) has emerged as a potential indicator suitable for the prognosis of many diseases, such as heart failure, viral infection, kidney disease, tumour formation, etc, thus monitoring its expression could assist in clinical decision-making. This study focuses on the development of new diagnostic and imaging approaches for the detection of serum and cellular Gal-3. Here, a label-free, rapid and cost-effective platform for Gal-3 detection is proposed based on specifically designed 3-mercaptopropyl β-D-lactoside-coated nanoparticles (AuML3) that present changes of surface plasmon resonance (SPR) band after interactions with Gal-3 protein. Surface modification of citrate gold nanoparticles with a lactose-containing ligand such as 3-mercaptopropyl β-D-lactoside (ML3) provides multiple binding areas for Gal-3 protein that enhance the protein-carbohydrate-binding affinities and increase the detection sensitivity. More specifically, the increase of Gal-3 concentration resulted in the dampening of the 530 nm extinction peak and the appearance of a second extinction band present at 700 nm, leading to 0.1 nM limit of detection (LoD). Additionally, the interactions of AuML3 nanosensors with a control protein such as bovine serum albumin (BSA) or with 10 nM of Gal-3 previously treated with lactose solution, that has high affinity towards Gal-3 did not display any SPR band shift verifying the selectivity of the AuML3 glycoconjugates against Gal-3 and the lack of significance of the non-specific interactions. Previous studies have shown that the interactions between multivalent proteins and SE(R)RS nanotags generate aggregates that lead to the formation of hotspots and enhancement of the SE(R)RS signal. Therefore, SE(R)RS advantages were exploited in designing a more sensitive approach based on AuCit nanoparticles functionalised with a Raman reporter such as MGITC and an ML3 ligand that interacts specifically with Gal- 3. More specifically, the interaction of the SERRS nanosensors with Gal-3 led to similar SPR changes as the label-free approach. Furthermore, the recognition of Gal-3 by the SERRS nanosensors resulted in colour changes of the solution from pink-red to purple and increased hydrodynamic diameter with increasing protein concentrations, indicating the formation of lactose-Gal-3 aggregates. We additionally demonstrated that the interaction of SERRS nanotags with Gal-3 enhanced the SERRS signal. However, a clear connection between the detected SERRS signal and Gal-3 concentration was not possible due to the fluctuations caused by uncontrolled aggregation. We believe that the SERRS signal could potentially be quantitative with further optimisation of SERRS nanosensors. A variety of imaging techniques have been widely used in imaging biological samples to understand the underlying biochemistry of cells and tissues. Advanced bioorthogonal Raman labels have been employed in developing stimulated Raman spectroscopy (SRS) and Raman immunostaining to overcome some limitations of the current fluorescence tags used in immunocytochemistry such as photobleaching and limited multiplexing capability. Here, a novel approach to immunofluorescence, called immuno-SRS microscopy, was designed to selectively detect Gal-3, through the reaction of biotinylated antibodies with alkyne-modified streptavidin-HRP. During the immuno-SRS staining the cells were first incubated with AntiGal-3 biotinylated antibodies and then stained with alkyne-modified streptavidin. The successful synthesis of alkyne-modified streptavidin was confirmed by UV-vis and Raman spectroscopy. This approach allows the specific detection of intercellular Gal-3 proteins in various permeabilised cell lines, such as Michigan cancer foundation-7 (MCF-7), Henrietta Lacks (HeLa) and the human lung cancer cell line H1975, by demonstrating the breakthrough potential of alkyne tags for intracellular immune-labelling with high spatial resolution using rapid SRS imaging. These results displayed negligible background signal in off-resonance frequency. Additionally, increasing the number of triple bonds in the polyyne chain of the alkyne tag led to alterations of the detected Raman shifts, which could lead to the simultaneous visualisation of different molecular species. The designed alkyne-modified streptavidin tags were also implemented in alkyne-tag Raman imaging (ATRI) immunolabelling for the quantification of Raman intensity of the Gal-3 and α-tubulin population in the cellular, nuclei and cytoplasmatic areas. To our knowledge, this is the first example of ATRI immunolabelling based on particle-free alkyne-conjugates and more conventional Raman techniques. The signal amplification achieved in this new method is based on the combination of streptavidin-HRP-conjugates, which increase the binding sites of strconjugates per antibody and produce bright Raman alkyne tags. Therefore, this study highlighted that ATRI immunolabelling offers a powerful tool for screening and diagnostic accuracy involving intracellular imaging and understanding of protein mechanisms and metabolism in different cell types.
Advisor / supervisor
  • Graham, Duncan (Professor of Chemistry)
  • Faulds, Karen
  • Dhaliwal, Kev
Resource Type
DOI
Date Created
  • 2023
Funder
Embargo Note
  • The digital version of this thesis is restricted to Strathclyde users only until 13th June 2029.

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