A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria

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