Prussian blue nanocubes peroxidase mimetic-based colorimetric assay for screening acetylcholinesterase activity and its inhibitor

In this paper, a novel, convenient and sensitive colorimetric assay for acetylcholinesterase (AChE) activity and its inhibitor screening is successfully proposed based on the peroxidase-like activity of Prussian blue nanocubes (PB NCs). PB NCs can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to develop a blue color and an absorption peak centered at 652 nm. AChE mediates the hydrolysis of acetylthiocholine (ATCh) to yield a reducing agent thiocholine (TCh) that cause the reduction of oxidized TMB and it can also chelate with Fe3+, both of which result in a blue color fading and a decrease of the absorbance. The generation of TCh is inhibited and the absorbance intensity is recovered after the introduction of AChE inhibitor. Consequently, the assay is also utilized in AChE inhibitor screening. The PB NCs-H2O2-TMB based platform is highly sensitive for AChE activity sensing with a low detection limit of 0.04 mU/mL. In addition, this assay has great potential in discriminatively determining AChE over other enzymes. The proposed method is fairly novel, simple and sensitive, which may pave the way for the detection of other hydrolytic enzyme activities with properly selected substrates.