Abstract
Single-signal fluorescent sensing mode for sequential detection of Ag⁺ and L-cysteine (L-Cys) in complex biological matrices are susceptible to environmental interference and false-positive responses. Herein, we develop a ratiometric fluorescent sensing mode based on mixed quantum dots (R-QDs) by combining blue-emitting N-CQDs (450 nm) and red-emitting CdTe QDs (630 nm). Although both QDs are quenched by Ag⁺, their differential quenching provides a concentration-dependent F630/F455 ratio and an internal referencing effect. The cascade mechanism enables dual-target detection: Ag⁺ quenches both QDs (LOD = 0.04 µM), while L-Cys restores fluorescence via competitive binding (LOD = 8.7 µM). The advantage of the ratiometric design becomes particularly evident in complex matrices. In human serum, the R-QDs sensor exhibited a minimal increase in detection limit (from 8.7 µM to 15.1 µM), whereas the single-signal sensors showed substantially higher detection limits (reaching 38.9-50.1 µM). This differential performance correlated with superior recovery rates for the ratiometric sensor (95.2-110.1%, RSD < 5.1%). This work provides a reliable ratiometric platform where self-calibration advantages become critical for accurate detection in real-world biological samples.