Immobilization Protocols of Nanozyme with Different Morphologies in Microfluidic Chips for Biosensing

Owing to their capabilities for product separation and controlled substance flow, microfluidic chips (MFC) offer a highly efficient and integrated reaction platform for nanozyme-based cascade catalysis and continuous detection. A critical factor in the design of immobilized cascade nanozyme chips with enhanced catalytic performance is the optimization of the balance between immobilization and active site exposure. Herein, cobalt-doped carbon nanozymes with diverse morphologies (zero-dimensional, one-dimensional, and two-dimensional) were employed as model systems to propose tailored immobilization strategies that balance simplicity, stability, and catalytic efficiency. Mechanism studies showed that the immobilization of nanozymes primarily relies on their close contact with the inner channel walls of the MFC, which results in the physical blockage of active sites on the nanozyme surface. Compared with the compact and spherical morphology of zero-dimensional (0D) nanozymes, the elongated and extended architectures of one-dimensional (1D) and two-dimensional (2D) nanozymes exhibit cross-linked and rich wrinkle structures, which facilitate effective immobilization while minimizing the loss of active sites. Consequently, incorporation of a binder─particularly for 0D nanozymes─was necessary, as it prevents deep embedding into the PDMS substrate and allows greater exposure of catalytically active surfaces. As a proof of concept, a microfluidic biosensor for dopamine detection was developed, demonstrating markedly enhanced stability and sensitivity following optimization of the immobilization interfaces. This work provides guidance for the selection of immobilization methods of different nanozymes in MFCs, thereby improving the practicability of nanozyme-modified MFC systems.