An “Off−On” Room-Temperature Phosphorescence Aptasensor for Direct Acetylcholinesterase Detection

Abstract Most acetylcholinesterase (AChE) assays quantify catalytic activity, which does not necessarily reflect protein abundance. Herein, an “off−on” room-temperature phosphorescence (RTP) aptasensor (CDs@SiO2/PEG-Apt-Q1) based on Förster resonance energy transfer (FRET) was developed for direct AChE protein detection. An AChE-binding DNA aptamer (Apt) was immobilized on CDs@SiO2 and hybridized with a Black Hole Quencher 1 (BHQ1)-labeled complementary strand (Q1), resulting in RTP quenching. Upon AChE recognition, the Apt/Q1 assembly underwent target-induced reorganization, weakening donor−acceptor coupling and restoring the RTP signal. The aptasensor exhibited a linear response from 0.01 to 25 mg/mL with a limit of detection of 0.009 mg/mL for AChE. RTP readout reduced short-lived fluorescence interference from fetal bovine serum (FBS) relative to fluorescence detection, and AChE-spiked FBS samples gave recoveries of 86.32−103.12% with relative standard deviations of ≤4.32%. This work integrates direct protein recognition, target-responsive DNA reorganization, and long-lived RTP transduction for AChE sensing in complex biological matrices.