A Long Fluorescence\nLifetime Probe for Labeling of\nGram-Negative Bacteria

Bacterial resistance, primarily stemming from misdiagnosis,\nmisuse,\nand overuse of antibacterial medications in humans and animals, is\na pressing issue. To address this, we focused on developing a fluorescent\nprobe for the detection of bacteria, with a unique featurean\nexceptionally long fluorescence lifetime, to overcome autofluorescence\nlimitations in biological samples. The polymyxin-based probe (ADOTA-PMX)\nselectively targets Gram-negative bacteria and used the red-emitting\nfluorophore azadioxatriangulenium (with a reported fluorescence lifetime\nof 19.5 ns). Evaluation of ADOTA-PMX’s bacterial labeling efficacy\nrevealed strong specificity for Gram-negative bacteria, and full spectral\nfluorescence lifetime imaging microscopy demonstrated the potential\nof ADOTA-PMX for bacterial imaging applications. The probe exhibited\na lifetime of 4.5 ns when bound to bacteria, possibly indicating interactions\nwith the bacterial outer membrane. Furthermore, the fluorescence lifetime\nmeasurements of ADOTA-PMX labeled bacteria could be performed using\na benchtop fluorimeter without the need of sophisticated microscopes.\nThis study represents the first targeted probe for fluorescence lifetime\nimaging, offering sensitivity for detecting Gram-negative bacteria\nand enabling multiplexing via fluorescence lifetime imaging.