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The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase