1：Experimental Design and Method Selection:

The study designed a fluorescent sensing strategy based on inner filter effect (IFE) using polymer dots (PDs) synthesized from 1,4-benzoquinone and ethylenediamine. The mechanism involves the hydrolysis of PNPF by AFu to produce PNP, whose absorption overlaps with the excitation spectrum of PDs, causing fluorescence quenching.

2：Sample Selection and Data Sources:

AFu solutions with various concentrations (0 to 1000 U L-1) were prepared in buffer. Human serum samples were diluted and spiked with AFu for analysis.

3：List of Experimental Equipment and Materials:

Materials included AFu from Sigma-Aldrich, PNPF from TCI, quinine sulfate, BSA, enzymes, amino acids, metal ions, ethylenediamine, 1,4-benzoquinone, PNP, citric acid, sodium hydrogen phosphate, and other salts from Sinopharm Chemical Reagent Co., Ltd. Equipment included RF-6000 fluorescence spectrometer (Shimadzu), UV-2450 spectrophotometer (Shimadzu), Avatar 330 FT-IR spectrometer (Nicolet), Tecnai F20 TEM (FEI), AXIS-ULTRA DLD-600W XPS (Kratos), Brucker D8 Advance X Diffractometer, FL920 fluorescence spectrometer (Edinburgh), and Millipore Milli-Q water purification system.

4：Experimental Procedures and Operational Workflow:

PDs were synthesized by heating a mixture of ethylenediamine and 1,4-benzoquinone at 70°C for 3 hours, followed by purification. AFu activity was determined by incubating AFu with PNPF at 37°C for 20 minutes, then adding to PDs solution and measuring fluorescence at 410 nm excitation. Selectivity was tested with interfering substances. Serum samples were analyzed similarly after dilution.

5：Data Analysis Methods:

Fluorescence intensity was measured, and a calibration curve was established. Detection limit was calculated based on S/N=3. Statistical analysis included recovery and RSD calculations for serum samples.