A fluorometric biosensor based on H2O2-sensitive nanoclusters for the detection of acetylcholine

We have constructed a simple, cost-effective, label-free, selective and sensitive biosensor based on the fluorescence quenching of protein-stabilized nanoclusters (NCs) for the detection of acetylcholine (ACh). In this assay, acetylcholineaterase (AChE) catalyzes the hydrolysis of ACh to form choline, which in turn is oxidized by choline oxidase (ChOx) to generate H2O2 which induces the fluorescence quenching of NCs. By virtue of this specific response, the detection limit of a fluorescent biosensor for determination of ACh was 5 pM (signal/noise=3), and the linear range was 0.1-20 nM. In addition, we use the proposed method to detect ACh in human blood, in which the concentration of ACh is ca. 6.83 nM, and the recoveries of the spiked sample range from 93% to 102%. The result agree with a commercial assay kit, suggesting that the excellent biosensor can be used in the practice detection of ACh.