Fluorescent Graphitic Carbon Nitride-Based Nanozymes with Peroxidase-Like Activities for Ratiometric Biosensing.

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.