This project addressed the quest for a new nanoparticle-based biosensing platform to study the chemical signaling within bacterial microbiome, bridging the gap from nanotechnology to biodiagnostic application. Healing-impaired and chronic wounds are a snowballing threat to public health and the economy. Their efficient treatment requires insight into the local wound microbiome, namely the composition and temporal evolution of bacterial communities. However, such bacterial communities are still poorly understood.
To improve our understanding, we need access to the chemical cell-to-cell communication (quorum sensing, QS), to enable monitoring of changes in biofilms, specifically the competition of bacterial communities for resources and space, and the population density-dependent change from non-virulent to virulent. To realize this goal, NANOBIOME developed a plasmonic 2D screening platform for sensitive, rapid, and robust detection of bacterial QS signaling by surface-enhanced Raman scattering (SERS) spectroscopy. Contrary to other approaches, NANOBIOME followed the concept of (1) well-defined “sensing pixels” in the form of self-assembled plasmonic nanoclusters and anisotropic nanostructures; and (2) their ordered assembly on solid supports with defined interparticle spacing - to avoid plasmonic coupling (crosstalk) between pixels. Consequently, the decoupling of optical properties and assembly structure allowed for optical tailoring and by localized post-modification. (3) The controlled growth of the “sensing pixels” transformed the preassembled array into a “plasmonic particle library” with a gradient in sizes, morphology, and optical properties – ideally suited for rapid screening for highest SERS activity. This SERS screening platform has been used for the detection of different bacterial QS molecules (pyocyanin, violacein). Such hydrophobic biomarkers pose a major challenge for SERS detection because of their low adsorption tendency on gold surfaces. For this purpose, the polymer coating around the “sensing pixels” was used as a molecular trap to chemically harvest and accumulate hydrophobic analyte molecules near the particle surface.
We achieved a proof-of-principle for the quantitative detection of pyocyanin (PYO), a QS signaling molecule of P. aeruginosa, with a lower limit of detection between 10^-7-10^-6 M. This is 1-2 orders of magnitude below concentrations of PYO commonly found in clinical samples (10^-5-10^-4 M PYO, HPLC after 24 h). In clinical samples, the concentration of PYO is traditionally determined by extraction with chloroform and subsequently high-performance liquid chromatography (HPLC), which is a time-consuming, cost-intensive, and laborious analysis. Since P. aeruginosa is the only known micro-organism producing PYO, it could serve as a biomarker for infections.
We are confident that studies of the intra- and interspecies signaling in bacterial communities will improve our understanding of bacterial competition in wound microbiomes. Our sensing platform may provide guidelines for alternative designs and future studies of bacterial model systems (e.g. pathogens versus commensal bacteria).
