Spatially confined multi-enzyme cascade nanoreactor for portable and real-time pesticide detection.

Multi-enzyme cascade reactions offer powerful amplification and accelerated biotransformation, yet their implementation in free-solution systems is limited by enzyme instability, diffusion losses, and low coupling efficiency. Here, we reported a spatially confined multi-enzyme cascade nanoreactor (AChE/CHO/HRP@MSN) constructed by co-immobilizing acetylcholinesterase (AChE), choline oxidase (CHO), and horseradish peroxidase (HRP) within amino-functionalized mesoporous silica nanoparticles (MSN-NH2). The ordered mesoporous architecture provides high loading capacity, efficient substrate channeling, and enhanced structural stability, enabling tightly coupled multi-step biocatalysis even in complex environments. The AChE/CHO/HRP@MSN platform supports dual-mode colorimetric and fluorescent sensing of organophosphorus pesticides (OPs) and enables real-time monitoring of pesticide degradation in plants. By coupling with a smartphone-readable hydrogel matrix, the system enables portable signal output and on-site visual monitoring of OP residues. This spatially organized, cascade-driven strategy not only addresses longstanding limitations in multi-enzyme system design but also provides a versatile framework for programmable biosensing in complex environments.