A ratiometric fluorescence probe based on graphene quantum dots and o-phenylenediamine for highly sensitive detection...

By using graphene quantum dots (GQDs) and o-phenylenediamine (OPD), a ratiometric fluorescence probe was designed for the highly sensitive and selective detection of AChE. GQDs with strong fluorescence were synthesized by the one-step hydrothermal method. The optimal emission wavelength of GQDs was 450 nm at the excitation wavelength of 375 nm. MnO₂ nanosheets with a wide absorption band of 300-600 nm were prepared at room temperature. Because of the extensive overlap between the absorption spectrum of MnO₂ nanosheets and the excitation and emission spectra of GQDs, the fluorescence of GQDs at 450 nm was efficiently quenched by the inner-filter effect. Meanwhile, due to the peroxidase-like activity of MnO₂ nanosheets, OPD was catalytically oxidized to 2,3-diaminophenazine (oxOPD), a yellow fluorescent substance with a new emission peak at 572 nm. When AChE was present, the substrate acetylthiocholine (ATCh) was hydrolyzed to thiocholine (TCh) that is capable of decomposing MnO₂ nanosheets. Therefore, the quench of GQDs and the oxidation of OPD by MnO₂ nanosheets were suppressed, resulting in the fluorescence recovery of GQDs at 450 nm, while the fluorescence decrease of oxOPD at 572 nm. Utilizing the fluorescence intensity ratio F₄₅₀/F₅₇₂ as the signal readout, the ratiometric fluorescence method was established to detect AChE activity. The ratio F₄₅₀/F₅₇₂ against the AChE concentration demonstrated two linear relationships in the range 0.1-2.0 and 2.0-4.5 mU mL^-1 with a detection limit of 0.09 mU mL^-1. The method was applied to the detection of positive human serum samples and the analysis of the inhibitor neostigmine. Due to the advantages of high sensitivity, favorable selectivity, and strong anti-interference, the method possesses an application prospect in clinical diagnosis of AChE and the screening of inhibitors. Graphical abstract Schematic presentation of a ratiometric fluorescence method for the detection of acetylcholinesterase (AChE). The fluorescence of graphene quantum dots (GQDs) is quenched and o-phenylenediamine (OPD) is oxidized to generate fluorescent product 2,3-diaminophenazine (oxOPD) by MnO₂ nanosheets. When AChE is present, acetylthiocholine iodide (ATCh) is hydrolyzed to thiocholine (TCh) with reducibility for decomposing MnO₂ nanosheets. Due to the decomposition of MnO₂ nanosheets, the quenching of GQDs and oxidation of OPD are suppressed. The fluorescence of GQDs at 450 nm is enhanced, while the fluorescence of oxOPD at 572 nm is reduced. The fluorescence intensity ratio F₄₅₀/F₅₇₂ is used to establish the ratiometric fluorescence method for AChE activity.