Robust Oxidase-Mimicking Fe/Co Single-Atom Nanozyme for Chemiluminescent Detection of Serum Acetylcholinesterase

Accurate detection of serum acetylcholinesterase (AChE) activity plays a crucial role in the clinical diagnosis and pharmacological treatment of neurological disorders. In this work, a novel method for determining serum AChE activity was developed based on the synergistically enhanced oxidase-mimicking activity of a Fe/Co single-atom nanozyme (Fe/Co-N/C). Fe/Co-N/C, featuring dual metal active sites, was successfully synthesized via high-temperature pyrolysis and demonstrated superior oxidase-like catalytic performance in the luminol-O₂ chemiluminescence (CL) reaction compared to its single-atom counterparts. In the detection system, AChE specifically catalyzes the hydrolysis of acetylthiocholine chloride (ATCh) to release thiocholine, which acts as a potent reducing agent that scavenges reactive oxygen species (ROS) generated by Fe/Co-N/C in the presence of dissolved O₂. Hence, the CL signal from the luminol-O₂-Fe/Co-N/C system decreased significantly. The inhibition rate of the CL response exhibited a good linear relationship with AChE activity over the range of 0.025 to 10.0 mU/mL. The limit of detection (LOD, 3σ/S) was determined to be 8.3 μU/mL, with a relative standard deviation (RSD) of 2.7% at an AChE concentration of 0.5 mU/mL (n = 11). The proposed method was successfully applied to the determination of AChE activity in human serum samples. This study offers a promising alternative approach to support the clinical diagnosis and management of neurological diseases.