Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of Living Cells Using H₂O₂‑Sensitive Conjugated Polymers

A new water-soluble conjugated poly­(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor-1) via PEG linker has been designed and synthesized. In the presence of H₂O₂, the peroxyfluor-1 group can transform into green fluorescent fluorescein by deprotecting the boronate protecting groups. In this case, upon selective excitation of PFP-FB backbone at 380 nm, efficient fluorescence resonance energy transfer (FRET) from PFP-FB backbone to fluorescein occurs, and accordingly, the fluorescence color of PFP-FB changes from blue to green. Furthermore, the emission color of PFP-FB and the FRET ratio change in a concentration-dependent manner. By taking advantage of PFP-FB, ratiometric detection of choline and acetylcholine (ACh) through cascade enzymatic reactions and further dynamic monitoring of the choline consumption process of cancer cells have been successfully realized. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting the chemical transmitter of living cells.