A Tetrazine–Metal–Polyphenol Nanoprobe for Single-Tube Multimodal Sulfide Analysis

Reliable sulfide analysis in complex matrices remains challenging because single-signal probes are susceptible to interference and ambiguous readouts. Here, we report a tetrazine-metal-polyphenol nanoprobe, RhB@Tz-MPN, for a single-tube sequential multimodal workflow for sulfide analysis. Sulfide produces three role-differentiated responses: a tetrazine-associated UV-vis blue shift for rapid screening, fluorescence recovery accompanying framework reorganization for quantitative readout, and suppression of peroxidase-like activity as a substrate-dependent cross-checking signal. All three readouts are obtained sequentially from the same sample tube, reducing sample transfer and intertube variation. Exploratory residual analysis showed a low observed residual correlation between the two direct optical channels (r = 0.06), while the nanozyme response was more appropriately treated as a sequential cross-checking readout. Under the acidic workflow condition, the fluorescence channel showed a linear range of 5-50 μM with an LOD of 0.79 μM. Spike-and-recovery experiments in tap water and lake water gave recoveries of 102.50-118.16% with RSDs below 4%. The platform also responded to stimulation-induced, cell-associated H2S generation in HepG2 cells and differentiated accumulated spoilage-associated headspace exposure from pork. This work establishes a role-differentiated single-tube strategy for integrating screening, quantification, and cross-checking in sulfide analysis.