Fe₃C@NC nanozyme-based platform for highly sensitive dual-readout sequential detection of cysteine and Hg²

Simultaneous detection of biologically relevant thiols and toxic heavy metal ions is important for biomedical analysis, food safety, and environmental monitoring. Cysteine (Cys) is a key sulfur-containing amino acid involved in redox regulation, whereas mercury(II) (Hg²⁺) is a highly toxic pollutant that binds strongly to thiol groups. This Hg²⁺-thiol interaction provides a chemical basis for sequential Cys/Hg²⁺ sensing. However, many nanozyme-based optical assays rely on H₂O₂-dependent peroxidase-like activity, and the instability of H₂O₂ can compromise assay reproducibility and practical use. Here, we synthesized a core-shell Fe₃C@NC nanozyme through a two-step hydrothermal-calcination strategy. Fe₃C nanoparticles were encapsulated within a nitrogen-doped carbon shell to support stable oxidase-like catalysis. Fe₃C@NC catalyzed the H₂O₂-independent oxidation of o-phenylenediamine to fluorescent 2,3-diaminophenazine using dissolved oxygen. This reaction was coupled with Cys-mediated signal inhibition and Hg²⁺-induced signal recovery to construct a sequential ON-OFF-ON dual-readout platform. Cys suppressed fluorescence by scavenging reactive oxygen species, whereas Hg²⁺ restored the signal through coordination with the thiol group of Cys. The fluorescence detection limits were 28 nM for Cys and 12 nM for Hg²⁺. Smartphone-assisted RGB analysis enabled portable visual quantification and showed good consistency with fluorescence measurements. The method was validated in human serum, milk, and environmental water samples, giving satisfactory recoveries and acceptable precision. This work presents an H₂O₂-free Fe₃C@NC nanozyme platform that links oxidase-like OPD oxidation with thiol-mediated signal regulation, providing a simple strategy for sensitive laboratory analysis and portable visual detection in complex samples.