Construction of Bio-Nano Interfaces on Nanozymes for Bioanalysis

Nanomaterials with enzyme-like activity (nanozymes) have been of great interest in broad applications ranging from biosensing to biomedical applications. Despite that much effort has been devoted to the development of the synthesis and applications of nanozymes, it is essential to understand the interactions between nanozymes and most commonly used biomolecules, i.e., avidin, streptavidin (SA), bovine serum albumin (BSA), immunoglobulin G (IgG), and glutathione (GSH), yet they have been rarely explored. Here, a series of bio-nano interfaces were constructed through direct immobilization of proteins on a variety of iron oxide and carbon-based nanozymes with different dimensions, including Fe₃O₄ nanoparticles (NPs, 0D), Fe₃O₄@C NPs (0D), Fe₃O₄@C nanowires (NWs, 1D), and graphene oxide nanosheets (GO NSs, 2D). Such interfaces enabled the modulation of the catalytic activities of the nanozymes with varying degrees, which allowed a good identification of multiplex proteins with high accuracy. Given the maximum inhibition on Fe₃O₄@C NP by BSA, we established molecular switches based on aptamer and toehold DNA, as well as Boolean logic gates (AND and NOR) in response to both DNA and proteins. Also importantly, we developed an on-particle reaction strategy for colorimetric detection of GSH with ultrahigh sensitivity and good specificity. The proposed sensor achieved a broad dynamic range spanning 7 orders of magnitude with a detection limit down to 200 pg mL^-1, which was better than that of an in-solution reaction-based biosensor by 2 orders of magnitude. Furthermore, we explored the mechanisms of the interactions at bio-nano interfaces by studying the interfacial factors, including surface coverage, salt concentration, and the curvature of the nanozyme. This study offered new opportunities in the elaborate design and better utilization of nanozymes for bioanalysis in clinical diagnosis and in vivo detection.