A copper-mediated Fenton-like dual-mode optical sensor for sensitive determination of acetylcholinesterase activity in biological fluids

Abstract

Acetylcholinesterase (AChE) is a clinically important enzyme, and its accurate and sensitive determination in serum and erythrocytes is of considerable significance for biomedical and toxicological applications. Herein, a novel dual-mode optical sensing platform is reported for the quantitative determination of AChE activity, based on coupling enzymatic thiocholine generation with a copper-mediated Fenton-like redox system. AChE catalyzes the hydrolysis of acetylthiocholine to produce thiocholine, which strongly coordinates Cu(I) and suppresses copper redox cycling. In the absence of AChE, the Cu(I)/H₂O₂ system proceeds efficiently, generating hydroxyl radicals that oxidize N,N-dimethyl-p-phenylenediamine (DMPD) to a colored product, while concurrently formed Cu(II) quenches the fluorescence of nitrogen- and sulfur-doped carbon dots (N,S-CDs). In the presence of AChE, thiocholine production progressively suppresses radical generation and limits free Cu(II) availability, resulting in decreased absorbance and simultaneous fluorescence recovery. The fluorometric mode exhibited linearity over 0.02–0.6 mU mL⁻¹ with a limit of detection of 0.0068 mU mL⁻¹, while the colorimetric mode showed linearity over a broader range of 0.1–10.0 mU mL⁻¹ with a limit of detection of 0.028 mU mL⁻¹. Applied to spiked serum and erythrocyte samples, the fluorometric and colorimetric modes yielded mean recoveries of 96.50–100.50% and 96.37–102.30%, respectively. The proposed platform offers complementary sensitivity ranges, cross-validated readouts, and practical applicability in complex biological matrices, representing a reliable tool for AChE activity monitoring in clinical and environmental contexts.