1：Experimental Design and Method Selection:

A one-pot synthesis at room temperature using MBISA as both reducing and protecting agent. Characterization includes UV-vis, fluorescence, TEM, XPS, FTIR, TGA, MALDI-TOF MS. Applications in cytotoxicity assay, cell imaging, H2S detection, and pH sensing.

2：Sample Selection and Data Sources:

SMMC-7721 cells for cytotoxicity and imaging; water and serum samples for H2S detection; various buffer solutions for pH sensing.

3：List of Experimental Equipment and Materials:

UV–vis spectrophotomter (Shimadzu UV–2450), fluorescence spectrometer (Edinburgh Instrument FLS-920), pH-meter (FE20), TEM (FEI Tecnai G220 S–TWIN), MALDI-TOF MS (Bruker Autoflex), FTIR spectrometer (Perkin–Elmer Paragon 1000), TGA analyzer (Perkin–Elmer TGA 6), XPS spectrometer (Leybold Heraeus SKL-12), ICP–AES spectrometer (Thermo iCAP 6300), confocal microscope (Zeiss LSM880 + Airyscan). Reagents include MBISA, HAuCl4, Na2S, NaOH, various ions, biothiols, MTT, DMEM, FBS, PBS, BR buffer.

4：Experimental Procedures and Operational Workflow:

Synthesis involves mixing HAuCl4 and MBISA, adding NaOH, dialyzing. Cytotoxicity assay using MTT. Cell imaging after incubation. H2S detection by adding H2S to AuNCs solution. pH response by mixing AuNCs with buffers of different pH.

5：Data Analysis Methods:

Fluorescence intensity measurements, linear regression for calibration curves, statistical analysis (RSD, recovery), comparison with ICP-AES and pH meter.