Enzyme Mimics of Au/Ag Nanoparticles for Fluorescent Detection of Acetylcholine

We have developed a highly sensitive and selective fluorescent assay for the detection of acetylcholine (ACh) based on enzyme mimics of Au/Ag nanoparticles (NPs). These NPs were prepared via a one-step solution phase reaction between 13 nm Au NPs and Ag+ ions in the presence of stabilizing agents such as adenosine triphosphate (ATP) and polyethylene glycol (PEG). Our sensing strategy involves reacting ACh with acetylcholinesterase (AChE) to form choline that is in turn oxidized by choline oxidase (ChOx) to produce betaine and H2O2, which reacts with Amplex UltraRed (AUR) in the presence of bimetallic NPs catalyst to form a fluorescent product. The fluorescence intensity (excitation/emission wavelengths of 540/592 rim) is proportional to the concentration of ACh over a range of 1-100 nM (R-2 = 0.998), with a limit of detection of 0.21 nM (signal/noise = 3). When compared with Au NPs and horseradish peroxidase, the Au/Ag NPs provide 150- and 115-fold higher catalytic activity toward the H2O2-mediated AUR reaction. The practicality of the assay has been validated by determining the concentrations of ACh in plasma and blood samples, with results of 2.69 +/- 0.84 nM (n = 5) and 6.75 +/- 1.42 nM (n = 5), respectively. Thus, the present assay holds great potential for the analysis of ACh in biological samples.