Cascade catalysis-based signal amplification for colorimetric detection of acetylcholinesterase and its inhibitors

Cascade catalysis represents a fundamental physiological process and serves as a highly effective strategy for signal amplification in biosensing. Acetylcholinesterase (AChE) is a pivotal enzyme in neurological function, acting not only as a critical biomarker for neurodegenerative diseases but also as a primary target for pharmaceuticals and pesticides. Consequently, the detection of AChE activity and the screening of its inhibitors are essential for clinical diagnostics, drug development, and environmental monitoring. Reliable methods for trace-level AChE analysis remain a critical challenge that needs to be addressed promptly.

We reported a novel AChE-urease cascade catalysis amplification strategy for the colorimetric detection of AChE activity and its inhibitors. In this system, AChE catalyzes the hydrolysis of thioacetylcholine chloride (ATCh) to produce thiocholine (TCh). TCh then binds to Ag⁺ via its thiol group, thereby alleviating the inhibitory effect of Ag⁺ on urease activity. Urease with high activity catalyzes urea hydrolysis, leading to a rise in pH, which is monitored using the pH indicator phenol red. Leveraging cascade catalysis, this method achieves highly sensitive detection of AChE with a limit of detection (LOD) as low as 0.0116 mU/mL. The feasibility for inhibitor screening was validated using dipterex and berberine as model inhibitors, yielding IC₅₀ values of 28 ng/mL and 14.4 μM, and LODs of 0.394 ng/mL and 0.23 μM, respectively.

These results present a novel signal amplification strategy relying on enzyme cascade catalysis. This approach boasts advantages like simplicity, high sensitivity, and low cost. It not only holds promising applications in detecting AChE and its inhibitors for agriculture, medicine, and biosensing but also may extend to other enzymes that mediate thiol transformations.