Specific enzymatic synthesis of 2,3-diaminophenazine and copper nanoclusters used for dual-emission ratiometric and naked-eye visual fluorescence sensing of choline

This work demonstrates a novel, facile and efficient biosensor for dual-emission ratiometric and visual fluorescence detection of choline. Copper nanoclusters (CuNCs) were prepared by using N2H4H2O as the reducing agent and lysozyme as the stabilizer, based on an ambient hydrothermal method. The prepared CuNCs were blue-emitting and showed an emission peak centered at 440 nm. In the mixture system consisting of CuNCs, choline oxidase, horseradish peroxidase (HRP) and o-phenylenediamine (OPD), the added choline was catalyzed by choline oxidase to generate H2O2. OPD was oxidized by H2O2 and HRP to generate 2,3-diaminophenazine (DAP). As the oxidation product of OPD, DAP emitted a fluorescence peaked at 550 nm and the fluorescence exhibited a regular enhancement with the increase of the choline concentration. Meanwhile, the fluorescence of the CuNCs was reduced by DAP due to an internal filtration effect-induced fluorescence quenching. There is an optimal linear relationship (r = 0.9992) between the ratiometric fluorescence intensity (I-DAP/I-CuNCs, I-550/I-440) and the choline concentration in the range of 0.1-80 M, with a low limit of detection of 25 nM. The response of I-DAP/I-CuNCs to choline was sensitive and selective over potential interferents existing in real samples. This developed sensor enabled the naked-eye visual detection of choline in aqueous solutions and on immersed filter papers. In human serum and liquid milk samples, this sensor showed a superior performance for choline detection.