1：Experimental Design and Method Selection:

The study employed a one-step hydrothermal carbonization method to synthesize N,S-CDs using citric acid and reduced glutathione as precursors. The experimental design focused on optimizing the synthesis conditions (200°C for 4 h) and characterizing the optical, structural, and sensing properties of the N,S-CDs. Theoretical models included fluorescence quenching mechanisms (e.g., static quenching for MTX detection) and temperature-dependent fluorescence behavior.

2：Sample Selection and Data Sources:

Samples included synthesized N,S-CDs, various metal ions (e.g., Fe3+, Cu2+, Zn2+), biomolecules (e.g., L-arginine, glucose, urea, VPA, PB, folic acid), and human blood plasma from healthy volunteers. Data were acquired through fluorescence spectroscopy, UV-vis absorption, TEM, XPS, FT-IR, and element analysis.

3：List of Experimental Equipment and Materials:

Equipment included a Teflon-lined stainless steel autoclave, centrifuge, dialysis membrane bag (MWCO = 1000 Da), UV1800 UV-vis spectrophotometer (Shimadzu), RF-6000 spectro-fluorophotometer (Shimadzu), IRAffinity-1S FT-IR spectrophotometer (Shimadzu), X-ray photoelectron spectrometer (K-Alpha, Thermo Fisher), JEM-2100 (HR) TEM (JEOL), Edinburgh FLS920 spectrometer (Edinburgh Instruments), and Elementar Vario EL III for element analysis. Materials included citric acid, reduced glutathione, various metal salts, biomolecules, and quinine sulfate as a reference for quantum yield measurements.

4：Experimental Procedures and Operational Workflow:

The synthesis involved mixing citric acid and reduced glutathione in deionized water, hydrothermal treatment at 200°C for 4 h, centrifugation at 4000 rpm for 20 min, and dialysis. For sensing applications, N,S-CDs were mixed with analytes (Fe3+ or MTX) in phosphate buffer solution (PBS, pH 7.0), incubated for specific times (5 min for Fe3+, 12 min for MTX), and fluorescence spectra were recorded at excitation wavelength 340 nm. Human plasma samples were diluted and spiked with MTX for recovery tests.

5：0), incubated for specific times (5 min for Fe3+, 12 min for MTX), and fluorescence spectra were recorded at excitation wavelength 340 nm. Human plasma samples were diluted and spiked with MTX for recovery tests. Data Analysis Methods:

5. Data Analysis Methods: Data analysis included calculating quantum yield using quinine sulfate as a reference, plotting calibration curves for Fe3+ and MTX detection, determining detection limits (LOD = 3σ/κ), and assessing selectivity and interference. Statistical techniques involved linear regression (R2 values) and standard deviation calculations for repeated experiments.