Colorimetric assay for acetylcholinesterase and inhibitor screening based on the Ag [I] ion–3,3′,5,5′-tetramethylbenzidine (TMB)

Abstract A novel colorimetric method for the detection of acetylcholinesterase (AChE) and its inhibitor by taking utilization of Ag [I] ion–3,3′,5,5′-tetramethylbenzidine (TMB) detection system has been proposed. Ag [I] ion could oxidize TMB to induce a blue color and an absorption peak centered at 652 nm. In the presence of AChE, acetylthiocholine (ACh) was hydrolyzed to thiocholine. Ag [I] ion could interact with thiocholine and thiocholine can reduce the oxidized TMB (oxTMB) to TMB, both of which suppressed the oxidation capacity of Ag [I] ion, resulting in a blue color fading and a decrease of the absorbance at 652 nm. The developed assay is highly sensitive with a low detection limit of 4.3 μU/mL. Moreover, the method was also applied in AChE inhibitor sensing. The obtained assay is fairly simple, inexpensive and sensitive without the utilization of nanomaterials, which may be not only used for the sensing of other hydrolytic enzyme activities with properly selected substrates, but also used for the screening of potential inhibitor drugs.