An Ag-MOF–hydrogel hybrid enhances Cd2 + aptamer recognition by balancing conformational stability and flexibility

Excessive multi-site confinement impairs induced-fit Cd 2+ aptamer binding. • Ag-MOF/Pen co-regulation balances structural rigidity and binding flexibility. • Interfacial anchoring and H-bond regulation enhance apparent affinity 16.1-fold. • Label-free fluorescence enables accurate Cd 2+ analysis in pea seedling tissues. Aptamer immobilization can improve structural stability, but excessive confinement may restrict the conformational rearrangements required for induced-fit target recognition. A key challenge is therefore to stabilize recognition-competent conformations while preserving the structural adaptability required for binding. Using a Cd 2+ aptamer as a model, we first demonstrated that conventional multi-site immobilization increased the apparent dissociation constant by approximately eightfold relative to the free aptamer. To address this issue, we developed a surface-imprinting strategy that confines the aptamer within hydrogel through single-point tethering of the aptamer while modulating its local microenvironment. The Cd 2+ -bound aptamer was first assembled on Ag-MOF, followed by hydrogel polymerization in the presence of penicillamine. During imprinting, Ag-MOF promoted localized aptamer anchoring, while penicillamine regulated the hydrogen-bonding network surrounding the Cd 2+ -aptamer complex. Subsequent Cd 2+ removal generated an imprinted microenvironment that partially preserved the preorganized recognition conformation without over-constraining flexible regions required for rebinding. The optimized hybrid exhibited an apparent dissociation constant of 0.19 μM, corresponding to a 16.1-fold decrease relative to the free aptamer, and reached binding equilibrium within 10 min. Integration with label-free Tb 3+ -sensitized fluorescence enabled Cd 2+ detection with a limit of detection of 0.7 nM, approximately 1.1–1114-fold lower than nine representative reported methods, while maintaining satisfactory selectivity and reproducibility. The method was further validated by quantifying Cd²⁺ in different tissues of hydroponically grown pea seedlings and food matrices, with results consistent with ICP-MS. These results establish moderate, microenvironment-assisted confinement as a general design principle for balancing conformational stabilization and induced-fit adaptability at aptamer recognition interfaces. Download: Download high-res image (212KB) Download: Download full-size image