While breakthroughs in peroxidase-like nanozymes for bioanalysis have been made, most of current nanozyme biosensing systems are based on a single signal output. Such sensing systems could be easily influenced by environmental and personal factors. We envision that nanozyme sensing systems with ratiometric signal outputs would provide more reliable and robust sensing performance. Herein, to construct such ratiometric sensing systems, three fluorescent graphitic carbon nitride (C₃N₄)-based nanozymes (i.e., C₃N₄-Ru, C₃N₄-Cu, and C₃N₄-hemin) with excellent peroxidase-like activities were prepared. These fluorescent nanozymes emitted a fluorescence at 438 nm when excited at 385 nm. Interestingly, when o-phenylenediamine (OPD) was catalytically oxidized to oxidized OPD (OPDox) in the presence of H₂O₂ and nanozymes, the OPDox not only emitted an emerging fluorescence at 564 nm but also quenched the fluorescence at 438 nm of the nanozymes. We therefore employed the ratio of the fluorescent intensity at 564 and 438 nm (i.e., F₅₆₄/F₄₃₈) as the signal output to construct the ratiometric biosensing systems. First, we used the C₃N₄-Ru nanozyme to construct the ratiometric H₂O₂ sensing system, which showed not only the enhanced robustness but also wider linear range and better sensitivity than most reported H₂O₂ sensors based on nanozymes. Second, with the assistance of glucose oxidase, glucose can be detected by such ratiometric sensing systems. Third, we used three different C₃N₄-based nanozymes to construct ratiometric sensor arrays for the detection and discrimination of five phosphates. This study provides new insights for constructing robust nanozyme biosensing systems.

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