Ultrasmall CuFeMn nanozymes with multienzyme activity under neutral conditions: A smartphone-based colorimetric visualization of multiple biomarkers

• A trimetallic CuFeMn nanozyme was creatively synthesized. • CF4M exhibits dual enzyme activity of POD-like and OXD-like at neutral pH. • The colorimetric platforms for glutathione, glucose and glucose-6-phosphate dehydrogenase were constructed effectively. • The smartphone detection platform for GSH, Glu and G6PD was established successfully. This study presents a novel nanozyme design with the development of ultrasmall trimetallic CuFeMn nanozymes (CF4M), which uniquely demonstrate dual peroxidase (POD)-like and oxidase (OXD)-like enzymatic activities under neutral pH conditions. By circumventing the limitation of conventional nanozymes that require acidic environments for optimal function, CF4M enables biosensing workflows without the need for buffer adjustments, significantly enhancing practical applicability. The CF4M utilizes its multivalent properties of Cu, Fe and Mn to ensure POD-like and OXD-like enzyme activity in neutral environments, addressing a major challenge in the field of biosensing. A versatile colorimetric platform utilizing CF4M achieves detection of multiple biomarkers, including glutathione (GSH), glucose (Glu), and glucose-6-phosphate dehydrogenase (G6PD) through cascading enzymatic reactions and TMB chromogenic responses. Integrated with smartphone-based imaging technology mediated by gold nanorods, the system facilitates user-friendly and visual quantification of multiple biomarkers. The CF4M-based biosensing platform establish a new paradigm for neutral-pH-compatible biosensors, offering broad utility in detect clinical biomarkers.