Visual screening of butyrylcholinesterase inhibitors by a cellulose membrane biosensor with amide-bonded immobilization of butyrylcholinesterase in a solid-phase enzyme-catalyzed reaction

The rapid discovery of highly active butyrylcholinesterase (BChE) inhibitors is key to the treatment of the late stages of Alzheimer's disease. Herein, a colorimetric cellulose membrane (CM)-based biosensor was developed. The CM was employed as a carrier, which can be immobilized with the BChE and 5,5′-dithio-(2-nitrobenzoic acid) (DTNB) to prepare the biosensor for the solid-phase enzyme-catalyzed reaction. Specifically, the CM was oxidized to the oxidized cellulose membrane (OCM), which can further covalently bind the BChE to prepare the BChE-immobilized cellulose membrane (BCM). Then, the DTNB was adsorbed to the BCM to prepare the BDCM biosensor. The BDCM biosensor can visually detect S-butyrylthiocholine iodide at the yellow light signal with several characters, including sensitivity (LOD 1.56 μM), affinity (Km = 140.48 ± 5.42 μM), stability (over 1 month), via the BChE-catalyzed solid-phase reaction. Furthermore, the BDCM biosensor was applied to screen and determine the specific commercial BChE inhibitors tacrine and rivastigmine, with the IC50 values of 30.99 ± 12.20 nM and 2.02 ± 0.13 μM, respectively, from 13 different commercial inhibitors. This work provided a new method of visual BChE inhibitor screening and a useful strategy for the design of enzyme-based immobilization technology cellulose membrane biosensors. •BChE was immobilized on the cellulose membranes via covalent amide bonds.•DTNB was loaded on the cellulose membranes functionalized as a signal converter.•The BDCM biosensor can visually catalyze BTC substrate via a solid-phase reaction.•The BDCM biosensor was successfully used to screen the highly active BChEIs.•The BDCM biosensor can determine the IC50 value of tacrine and rivastigmine.