Herein, we successfully synthesized two-dimensional iron-doped carbon-based nanosheets (Fe-N800 CS) with catalase-like activity through doping Fe into Zn MOF and introducing graphitic C3N4 (g-C3N4). The interaction of the Fe-N800 CS with hydrogen peroxide could generated abundant reactive oxygen species (ROS) and further oxidize o-Phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) which has constant fluorescence at 560 nm. Ascorbic acid (AA) could be generated via the hydrolysis reaction between alkaline phosphatase (ALP) and ascorbic acid 2-phosphate (AAP). AA can be oxidized to dehy-droascorbic acid (DHA) by ROS, and then combined with OPD to generate 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) with fluorescence at 440 nm, which could increase as the concentration of AA enhanced. DHA could also be generated through oxidation of AA by ascorbate oxidase (AAO). Thus, by monitoring the fluorescence ratio (I560/I440), a ratiometric fluorescence biosensing platform for ALP and AAO was established with the linear ranges in 0.2-10 U/L and 1-60 U/L, respectively. The limit of detection for ALP and AAO were 0.12 U/L and 0.59 U/L. Furthermore, the biosensing platform was successfully applied for the detection of ALP and AAO activity in human serum samples. This work provides a potential tool for future biomedical diagnostics.
Keywords: Alkaline phosphatase; Ascorbate oxidase; Metal-organic framework; Nanozyme; Ratiometric fluorescence.
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