▎ 摘　　要

A novel label-free fluorescence 'turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti4+. The as-prepared rGO@PDA-T-i4+-FMNs (nanosensor), fluoresce only weakly due to the ineffective Forster resonance energy transfer between the FMNs and rGO@PDA-Ti4+. The experimental findings revealed that the microwave-assisted interaction of the nanosensor with alpha-, beta-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti4+), leading to an excellent fluorescence 'turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of alpha-, beta-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.