A Robust and Convenient Fluorimetric Assay for Measuring Acetylcholinesterase Activity and Screening Acetylcholinesterase Inhibitors

by Jinfang Liao, Zhenjun Diwu

Introduction

Acetylcholinesterase (AChE) is one of the most crucial enzymes for nerve responses and neurological functions. AChE inhibitors are the key drugs for the management of Alzheimer's disease and myasthenia gravis. Although AchE is a proven drug development target, the existing technologies for measuring AChE activities and screening AChE inhibitors are a compromise between throughput, sensitivity and physiological relevance. We have developed a robust fluorimetric Thiolite™ Green-based assay for measuring AchE activities and screening AchE inhibitors.

Fig. 1

Amplite^® Fluorimetric AChE Assay Principal. The increased fluorescent signal is proportional to the increased AChE enzyme activity.

This new fluorimetric method is much more sensitive for detecting AChE activity than the existing colorimetric method that is generally based on DNTB. The assay is based on the fluorogenic reaction of Thiolite™ Green with thiocholine. This specific fluorogenic reaction can be conveniently used to quantify thiocholine produced from the AChE–induced hydrolysis of acetylthiocholine. The fluorescence intensity of Thiolite™ Green-thiocholine adduct is proportional to the formation of thiocholine, therefore the AChE activity. The assay provides a continuous and ultrasensitive fluorimetric method to detect as little as 0.01 mU/mL of AChE, which is 100 times more sensitive than the DNTB colorimetric method.

Materials and Method

All standard dilutions and AChE reactions were performed at RT with Amplite^® Fluorimetric Acetylcholinesterase Assay Kit (Cat No. 11401).
For AChE inhibitor experiment, the AChE inhibitor samples were pre-incubated with AChE (∼40 mU/mL) for 10 ∼15 min.
AChE acitivity and AChE inhibition were performed in 50 µL volumes in Costar solid black 96-well plates for 30 min.
AChE activity was measured as described in Amplite^® Fluorimetric Acetylcholinesterase Assay Kit using Gemini microplate reader at Ex/Em = 490/525 nm.

Amplite^® AChE Assay Workflow

Prepare AChE containing test samples (40 µL)
Add known AChE inhibitor or test compounds (10 µL)
Incubate at room temperature for 10 to 20 min
Add Thiolite™ Green substrate working solution (50 µL)
Incubate at room temperature for 30 to 60 min
Read fluorescence at Ex/Em = 485/525 nm

Thiolite Green™ Spectra

Fig. 2

Excitation and emission spectra of Thiolite™ Green substrate.

Results

Fig. 3

(A) AChE Activity Standard Curve: AChE dose response was measured in a 96-well black plate with Amplite^® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader. As low as 0.01 mU/well of AChE can be detected with 20 minutes incubation time (n=3). (B) AChE Inhibitor Dose Response Curve: Dose response of Tacrine inhibitory effect on AChE. AChE (40 µL/well) was incubated with 10 µL/well of various dose of 9-Amino-1,2,3,4-tetrahydroacidine hydrachloride (Tacrine) in 96-well black plate for 15 minutes. 50 µL/well of Thiolite™ Green substrate working solution (50 µL) were added, and incubated at room temperature for 30 minutes. The AChE Activity was measured at Ex/Em = 485/525 nm.

Fig. 4

AChE dose response was measured using:

(A) Amplite^® Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Cat No. 11400) - as low as 0.1 mU/well of AChE can be detected with 30 minutes incubation time (n=3).
(B) Amplite^® Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Cat No. 11402) - as low as 0.01 mU/well of acetylcholinesterase can be detected with 20 minutes incubation time (n=3).

Summary

Thiolite™ Green is a new ultrasensitive substrate characterized for quantifying AChE activities and screening AChE inhibitors as potential drug discovery targets.
Thiolite™ Green-based assay can detect as low as 0.01 mU of AChE in solution, which is much more sensitive than the DTNB–based colormetric method.
Thiolite™ Green-based assay can be performed in a convenient 384-well or 1536-well plate format. The assay is robust, and has a lower false-positive rate.

Document: 02.0162.200713r1

Last updated Mon Oct 13 2025