Alasiri_2026_RSC.Adv_16_20448

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)/H(2)O(2) 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.