On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9 cm of the capillary inlet (total length of capillary 60.2 cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0 min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K i and IC 50 were 0.551 μmol L −1 and 1.52 μmol L −1 , respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan. Figure ( a ) Schematic representation of the immobilized enzymatic microreactor. ( b ) Typical electropherograms for AChE enzymatic hydrolysis reaction. Peaks: S, substrate (AThCh); P, product (ThCh). CE conditions: fused-silica capillary, dimensions, 75 μm × 60.2 cm (50 cm to detection window); running buffer, 20 mM Tris-HCl (pH 8.0); cartridge temperature, 37 ℃; sample injection, 0.5 psi, 10 s; incubation time, 2 min; separation potential, 25 kV; detection wavelength, 230 nm; concentrations of AChE and substrate: 0.37 mg mL −1 (containing 20 mM MgSO 4 and 1.0 g L −1 sodium alginate) and 10 mM, respectively.