Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-free Staining of Bacteria

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.