Su_2025_Adv.Sci.(Weinh)__e17919

Enzyme-based biosensors with rapid and on-site detection capabilities possess great potential for practical application. However, conventional enzyme immobilization strategies often enhance stability at the expense of substantial enzyme activity loss, thereby limiting the detection performance of enzyme-based biosensors. Herein, a highly sensitive and robust biosensor is constructed based on defect-engineered amorphous metal-organic frameworks, enabling on-site detection of organophosphate pesticides in complex food matrices. Defective acetylcholinesterase@amorphous metal-organic frameworks (denoted as AChE@AMOF-74) can be in situ tailored via a defect-engineered strategy to provide a suitable microenvironment and obtain high porosity for enzyme encapsulation, while its porous architecture enhances the catalytic activity of immobilized enzyme. Impressively, the catalysis activity and target recognition ability of AChE@AMOF-74 are 3.4-fold and 5.6-fold higher than those of nanoarchitectures with a regular crystalline structure. Benefiting from the structural advantages, a robust AChE@AMOF-74-based biosensor is constructed for the sensitive detection of pesticides, enabling quantitative analysis of paraoxon with a detection limit of 0.05 ng.mL(-1) using an image processing algorithm. This work demonstrates the significant potential of AMOFs in constructing high-performance enzyme-based biosensors and has been successfully applied to detect pesticide in complex food matrices, providing a new protocol for on-site application.