Plasmonic metal nanoparticles for studying complex interactions and dynamics in biological systems using surface-enhanced raman spectroscopy: insights from biofilms

Abstract

This PhD thesis addresses the challenges posed by complex biological systems on molecular detection and analysis using biofilms as a model system. Biofilms, with their sticky and heterogeneous extracellular matrix of polysaccharides, proteins and nucleic acids, exhibit strong matrix interference. The presence of multiple interacting components complicates the identification of specific interactions and alters the behavior of probing molecules or nanoparticles, affecting detection sensitivity and reproducibility. Surface-enhanced Raman spectroscopy (SERS) was used in this thesis as a robust, label-free analytical tool to investigate these challenges and advance its application for analyzing complex biological systems. In Chapter 3, interactions between SERS-active nanoparticles (Au and Agnanospheres and Au nanostars) and ex-situ biofilms were systematically studied. The ex-situ biofilm reduced SERS signals through impaired nanoparticle aggregation and surface site blocking. Aunanostars (NS), however, were less affected, providing reproducible signals, making them suitable for more complex in-situ biofilms. Building on this, SERS with NS was used to probe how interactions within ex-situ biofilms affect the detection of the antibiotics, levofloxacin and ampicillin. This study demonstrated, for the first time, label-free SERS detection of antibiotics in ex-situ biofilms at clinically relevant concentrations, although with reduced sensitivity. The decrease in sensitivity was attributed to interactions between biofilm components and the NS, which are governed by coupled equilibria rather than simple additive effects. Finally, SERS with NS was used to monitor levofloxacin diffusion within in-situ biofilms. By embedding NS at specific depths, Levo penetration was quantified using a label-free approach that allows direct detection of unmodified drug molecules while avoiding artefacts associated with fluorescence-based techniques. The method proved to be reproducible across multiple biofilm samples, demonstrating the reliability of SERS with NS for studying antibiotic transport and behavior in biofilms. Thesis is embargoed until 31 July 2027.