biostudies-literatureChen YNatural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)National Natural Science Foundation of China (National Science Foundation of China)1863https://www.ebi.ac.uk/biostudies/studies/S-EPMC11845792biostudies-literatureUnknown16(1)Synthetic drugs fundamentally reshape the illicit drug market due to their low cost, ease of production, and rapid manufacturing processes. However, current drug detection methods, which rely on complex instruments, have limited applicability and often neglect the influence of pH fluctuations, leading to potential bias and unreliable results. Herein, we propose co-calibration DNA probes on a nanoconfined biosensor (N_CB_S), covering the range of sweat pH 3-8 to achieve significantly enhanced target signal recognition. The N_CB_S exhibits a linear response range of 10³-10⁸ fM with a low limit of detection (LOD) of 3.58 fM in artificial sweat. Compared to the single-aptamer N_CB_S, the dual-aptamer N<sub>C</subNature communicationsSub-femtomolar drug monitoring via co-calibration mechanism with nanoconfined DNA probes.PMC11845792LD21B0500022090050, 2217618022474049, 52003103Li XHuang YHe ZLi FYu WChen YWang YYue XShi YXia FfalseSub-femtomolar drug monitoring via co-calibration mechanism with nanoconfined DNA probes.Synthetic drugs fundamentally reshape the illicit drug market due to their low cost, ease of production, and rapid manufacturing processes. However, current drug detection methods, which rely on complex instruments, have limited applicability and often neglect the influence of pH fluctuations, leading to potential bias and unreliable results. Herein, we propose co-calibration DNA probes on a nanoconfined biosensor (N_CB_S), covering the range of sweat pH 3-8 to achieve significantly enhanced target signal recognition. The N_CB_S exhibits a linear response range of 10³-10⁸ fM with a low limit of detection (LOD) of 3.58 fM in artificial sweat. Compared to the single-aptamer N_CB_S, the dual-aptamer N<sub>C</sub2025-01-01T00:00:00Z2025 Feb2025-04-04T07:32:44.458Z2025-04-04T07:32:44.458ZS-EPMC118457923998444110.1038/s41467-025-57112-1