Developing a gold nanoparticle-based colorimetric assay for Bacillus cereus detection using betatectiviral cell wall-binding domains

Details

Supervisors

Gillis, Annika

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

Foodborne contamination by members of the Bacillus cereus group represents a persistent public health challenge, owing to their widespread environmental prevalence, capacity to form highly resistant spores, and ability to produce toxins responsible for emetic and diarrhoeal syndromes. Existing detection methods lack the combination of speed, selectivity, and field accessibility required for effective on-site monitoring, motivating the development of alternative detection systems. This work therefore aimed to develop a colorimetric biosensing platform for the rapid and selective detection of B. cereus group members, combining phage-derived cell wall-binding domains (CBDs) as biorecognition elements with the visible colorimetric shift produced by gold nanoparticle (AuNP) aggregation, mediated through multivalent thiolated dendrimer scaffolds. Bioinformatic analysis of betatectivirus GIL16 identified two endolysins, Gp27 and Gp31, whose C-terminal regions were selected as CBD candidates for recombinant expression. GFP-fused constructs of both CBDs were successfully expressed in soluble form and purified. Fluorescence-based cell wall decoration assays revealed that Gp27_CBD showed no detectable binding, whereas Gp31_CBD selectively recognised a defined subset of B. cereus group strains spanning multiple species, while showing no interaction with non-target organisms, establishing it as a strain-selective biorecognition element. Citrate-capped AuNPs of 15–20 nm were synthesised and confirmed to remain colloidally stable in the presence of bacterial cultures. Bis-MPA-COOH dendrimers were successfully functionalised with cysteamine and reliably induced AuNP aggregation, generating a readily observable colorimetric shift at dendrimer concentrations as low as 1 µM. However, subsequent conjugation of Gp31_CBD to the thiolated dendrimer scaffold was unsuccessful across multiple attempts, preventing assembly of the complete detection system. This work identified and characterised a selective phage-derived biorecognition element targeting specific B. cereus group strains, and established a dendrimer-mediated AuNP aggregation system producing a clear colorimetric readout. Together, these results lay the groundwork for a CBD-directed colorimetric biosensing platform, and highlight the potential of phage-derived recognition elements combined with plasmonic nanoparticles as a promising strategy for the development of rapid, selective, and equipment-free bacterial detection tools.