A label-free fluorescence biosensor based on a bifunctional MIL-101(Fe) nanozyme for sensitive detection of choline and acetylcholine at nanomolar level

Abstract A label-free and high sensitive fluorescence biosensor for the detection of choline and acetylcholine (ACh) was developed based on the MIL-101(Fe) nanozyme with dual functions of mimetic peroxidase and fluorescent emission. This fluorescence sensing strategy involves the reaction of acetylcholinesterase (AChE) catalyzing the hydrolysis of ACh into choline that is in turn oxidized by choline oxidase (ChOx) to generate H2O2, and the as-produced H2O2 is decomposed into highly reactive hydroxyl radicals under the catalysis of MIL-101(Fe) nanozyme, thereby the non-fluorescent organic ligand terephthalic acid of MIL-101(Fe) is oxidized by hydroxyl radicals to form a highly fluorescent 2-hydroxy terephthalic acid. By virtue of this finding, the label-free detection of choline and ACh via multienzyme cascade reaction was realized with low detection limits of 20.0 nM and 8.9 nM, respectively. Moreover, the practicality of the developed bifunctional MIL-101(Fe)-based sensing strategy was successfully validated through the detection of choline in milk and ACh in human plasma. This proposed assay not only exhibited high sensitivity and selectivity towards choline and ACh, but also simplified the sensing systems, reduced the detection cost, and avoided the potential risks caused by the fluorescent reagents, which opened a green avenue for the analysis of complex biological samples.