Blue ‘turn-on’ fluorescent probes for the direct detection of free radicals and nitric oxide in Pseudomonas aeruginosa biofilms

Abstract

Two novel cell-permeable and bacteria-compatible 'turn-on' fluorescent probes, designed to be compatible in a multi-dye system and to fluoresce in the blue region exhibiting emission maxima of 440-490 nm, were synthesized. The profluorescent nitroxide probe (11) was developed to visualize and quantify free radical and redox processes, and the Cu(II)-complexed coumarin-based probe (16a) was developed for NO detection. Confocal laser-scanning microscopy and subsequent digital analysis of Pseudomonas aeruginosa biofilms stained with 11 or 16a determined that free radical and redox processes and NO generation occur predominantly in live cells during normal biofilm growth.

Photophysical insights and guidelines for blue “turn‐on” fluorescent probes for the direct detection of nitric oxide (NO•) in biological systems

“…Lippard et al developed a strategy to transform fluorophores into sensors for NO • , which utilises a non‐fluorescent copper complex that turns on fluorescence upon replacement of the metal by NO • . If coumarin and its derivatives, which are known blue‐fluorescent dyes, would be used as chromophore, some of us recently suggested that Lippard's replacement approach could provide access to blue “turn‐on” fluorescent probes and hereupon synthesised the probe CB5 to test its potential to detect NO • (Scheme ) …”

“…Mechanism of “turn‐on” fluorescence through replacement of Cu(II) by NO • in the recently developed coumarin based probe CB5 …”

Heteroorganic molecules and bacterial biofilms: Controlling biodeterioration of cultural heritage

“…(45) to be used to quantify and visualize changes in redox status and associated stress conditions within the biofilm during dispersal. 349 …”

“…105,106 In an attempt to discover the function of free radicals and the role of oxidative stress in biofilm formation and dispersal, Barzegar Amiri Olia et al, developed a novel profluorescent nitroxide (45) that detects free radical and redox processes associated with oxidative stress during P. aeruginosa biofilm growth (Figure 18). 349 Confocal laser-scanning microscopy and subsequent co-localization studies using digital image analysis revealed that conditions of oxidative stress occur predominantly in the EPS and in live cells during normal biofilm growth. 349 Profluorescent nitroxides (such as 45) therefore provide a sensitive technique to detect, quantify and visualize oxidative stress conditions and alterations in the redox status of the cellular environment.…”

“…349 Confocal laser-scanning microscopy and subsequent co-localization studies using digital image analysis revealed that conditions of oxidative stress occur predominantly in the EPS and in live cells during normal biofilm growth. 349 Profluorescent nitroxides (such as 45) therefore provide a sensitive technique to detect, quantify and visualize oxidative stress conditions and alterations in the redox status of the cellular environment. (45) to be used to quantify and visualize changes in redox status and associated stress conditions within the biofilm during dispersal.…”