Phosphonic Acid Fluorescent Organic Nanoparticles for High-Contrast and Selective Staining of Gram-Positive Bacteria

Fast and selective detection of pathogens represents high challenges given the considerably high proliferation rate and mutation potential of bacteria against antibiotics. With this aim, anionic red-orange-emitting fluorescent organic nanoparticles (FONs), characterized by high brightness and photostability, are developed to selectively stain Staphylococcus aureus after only 5 min of exposure. No cytotoxicity effects are observed as a result of the negatively charge surface of the employed FONs. By contrast, no staining can be observed with Pseudomonas aeruginosa strains. The exclusive labeling of Gram-positive bacteria is ascribed to originate from the phosphonic acid moieties incorporated in the FON-constituting fluorophores because model FONs, devoid of phosphonic acids, show no adhesion under the same experimental conditions. Tight hydrogen bonding between the FON acidic units and the peptidoglycan (PG) layers comprising the outer wall of S. aureus is suspected to be the prevailing factor for the encountered selective interactions. PG layers from S. aureus are employed to apprehend the interactions developed between FONs and the bacteria membrane. Correlative light electron microscopy using confocal fluorescence microscopy and SEM reveal FONs mainly located at extensively reorganized or "dented" PG areas. Such privileged localizations tend to suggest multivalent physicochemical interactions to aggregate a multifold of nanoparticles. Finally, spectral follow-up of the FON-stained bacteria membrane shows significant hypsochromic shift of the fluorescence emission, signaling progressive disassembly of FONs and a change of the surrounding polarity. This feature offers promising perspectives to use doped FONs as theranostic agents to liberate encapsulated antibiotics upon FON disintegration inside the bacteria membrane.