Spatiotemporally Guided Single‐Atom Bionanozyme for Targeted Antibiofilm Treatment

Abstract The heterogeneous and dynamic microenvironment of biofilms complicates bacterial infection treatment. Nanozyme catalytic therapy has recently been promising in treating biofilm infections. However, active nanozymes designed with the required precision targeting the biofilm microenvironment are lacking. This work proposes a spatiotemporally guided single‐atom bionanozyme (BioSAzyme) for targeted antibiofilm therapy based on protein engineering of copper single‐atom nanozyme (Cu SAzyme). The Cu SAzyme, synthesized via a novel mechanochemistry‐assisted method, features highly accessible Cu–N 4 active sites exposed on 2D N‐doped carbon, exhibiting excellent triple enzyme‐like activities according to experimental results and density functional theory calculations. Inheriting biofunctionality from both glucose oxidase and concanavalin A, BioSAzyme can localize the biofilm glycocalyx and catalyze endogenous glucose into H₂O₂ and gluconic acid, thus triggering multiplex cascade reactions with pH self‐adaption to consume glucose and glutathione and generate •OH radicals. This spatiotemporally guided bionanocatalytic agent effectively inhibits E. coli O157: H7 and methicillin‐resistant S. aureus biofilms in vitro and in vivo. Taking together, this work opens up new avenues for the rational design of single‐atom nanozymes for precise antibiofilm therapy.