Detection and imaging of bacterial biofilms with glutathione-stabilized gold nanoclusters

Bacterial biofilms colonize chronic wounds and surfaces of medical devices, thus making the development of reliable methods for imaging and detection of biofilms crucial. Although fluorescent identification of bacteria is sensitive and non-destructive, the lack of biofilm-specific fluorescent dyes limits the application of this technique to biofilm detection. Here, we demonstrate, for the first time, that fluorescent glutathione-stabilized gold nanoclusters (GSH-AuNCs) without targeting ligands can specifically interact with extracellular matrix components of Gram-negative and Gram-positive bacterial biofilms resulting in fluorescent staining of bacterial biofilms. By contrast, fluorescent bovine serum albumin-stabilized gold nanoclusters and 11-mercaptoundecanoic acid - stabilized gold nanoclusters do not stain the extracellular matrix of biofilms. According to molecular docking studies, GSH-AuNCs show affinity to several targets in extracellular matrix, including amyloid-anchoring proteins, matrix proteins and polysaccharides. Some experimental evidence was obtained for the interaction of GSH-AuNCs with the lipopolysaccharide (LPS) that was isolated from the matrix of Azospirillum baldaniorum biofilms. Based on GSH-AuNCs properties, we propose a new fluorescent method for the measurement of biofilm biomass with a limit of detection 1.7 × 10⁵ CFU/mL. The sensitivity of the method is 10-fold higher than the standard biofilm quantification with the crystal violet assay. There is a good linear relationship between the fluorescence intensity from the biofilms and the number of CFU from the biofilms in the range from 2.6 × 10⁵ to 6.7 × 10⁷ CFU/mL. The developed nanocluster-mediated method of biofilm staining was successfully applied for quantitative detection of biofilm formation on urinary catheter surface. The presented data suggest that fluorescent GSH-AuNCs can be used to diagnose medical device-associated infections.