A portable paper sensor for clothianidin based on triple-helix aptamer-grafted nanozymes with steric hindrance switch

A steric-hindrance-mediated “on-off” switchable probe is developed. • Triple-helix aptamer blocks nanozyme sites, inhibiting activity until target binding. • Solution assay detects clothianidin at 5.345 ng/mL with high sensitivity. • The probe is integrated into a portable paper-based device (PAD). • The PAD enables on-site screening with an LOD of 68.221 ng/mL. While the integration of triple-helix aptamer (THA) recognition with nanozyme catalytic activity offers great potential for on-site detection, the commonly used label-free THA-regulated nanozyme strategy suffers from an inherently high background signal, which significantly limits its applicability in environmental and food samples. In this study, a THA-grafted nanozymes was developed based on a steric-hindrance-mediated “on-off” switching mechanism for clothianidin (CLO) detection. THA was immobilized on Fe 3 O 4 @Cu-MOF nanozymes as a steric blocker, which physically masked the active sites via their spatial conformation, effectively suppressing the nanozyme's catalytic activity (“off” state). In the presence of CLO, specific binding induced a conformational change of THA. This conformational change exposes the previously masked active sites, “turning on” the nanozyme's activity and generating a quantitative colorimetric signal. Leveraging this precise switching mechanism, we established a highly sensitive solution-phase assay with a limit of detection (LOD) of 5.35 ng/mL and recoveries ranging from 94.05% to 102.14%, which was comparable to those obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To translate this system into a field-deployable platform, the probe was immobilized on paper to fabricate a paper-based analytical device (PAD). The resulting PAD achieved CLO detection with an LOD of 68.22 ng/mL. Overall, the developed method provides a portable, user-friendly, and practical platform for on-site screening of CLO in food samples. Download: Download high-res image (154KB) Download: Download full-size image