Developing a sensor based on functionalised gold nanoparticles for bacterial detection by surface-enhanced Raman spectroscopy

Details

Supervisors

Gillis, Annika ; Hermans, Sophie

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enIn 2019, 7.7 million deaths were associated with 33 pathogenic bacterial species, representing 13.6% of all global fatalities. In parallel, antimicrobial resistance is increasing worldwide and is projected to cause more annual deaths than cancer by 2050. Rapid and reliable bacterial detection methods are therefore essential. However, current detection techniques, such as culture-based methods, enzyme-linked immunosorbent assays (ELISA), and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS), are limited by long analysis times and/or insufficient sensitivity. Consequently, new detection technologies are required. One promising approach is based on Surface-Enhanced Raman Spectroscopy (SERS), which exploits the optical properties of gold nanoparticles to amplify Raman signals. In this work, gold nanostars (AuNS) were synthesised using a seed-mediated growth protocol. A bioorthogonal Raman reporter was synthesised and grafted onto the AuNS surface. The nanoparticles were then coated with either silica or polyethylene glycol (PEG), followed by chemical modification to introduce aldehyde or carboxylic acid functional groups. Anti-green fluorescent protein (GFP) antibodies were grafted onto the modified coating through EDC/NHS coupling. SERS measurements were performed either in liquid phase or on porous silicon (PSi) free standing membranes (FSM). In parallel, Escherichia coli harbouring a plasmid encoding GFP was cultured and lysed using lysozyme. Successful synthesis of the Raman reporter was confirmed by a characteristic band at 2232 cm−1, located within the biological silent region where biological materials do not generate Raman signals. Silica was selected over PEG as the coating material due to its stronger Raman reporter signal, despite lower reproducibility. The most effective surface modification strategy was achieved using succinic anhydride with triethylamine, introducing carboxylic acid groups and yielding a ζ potential of -34.2 mV at pH 7.4. SERS measurements in liquid phase did not allow the detection of Raman bands originating from the bacterial lysate, and only the Raman reporter signal was observed. In contrast, measurements performed on PSi FSM yielded Raman bands attributable to bacterial lysate components, although no specific GFP peaks could be unequivocally identified. Importantly, and for the first time, as far as we know, the Raman reporter remained detectable simultaneously with the lysate. In conclusion, simultaneous detection of a bacterial lysate and the Raman reporter was more effective on the PSi FSM platform than in liquid phase, likely due to SERS hot-spot formation. Although further optimisation is required, these results demonstrate the potential of this approach for future bacterial detection applications.