1：Experimental Design and Method Selection:

The study involved designing and synthesizing a pyrene-based Schiff base chemosensor (chemosensor 1) through condensation of 1-pyrenecarboxaldehyde and nicotinic hydrazide in ethanol. Spectroscopic methods (UV-Vis, fluorescence) were used to study metal ion binding, with selectivity and sensitivity assessed in DMSO-H2O buffer.

2：Sample Selection and Data Sources:

Metal ions (e.g., Bi3+, Al3+) were prepared from nitrate salts. Biological samples included RAW

3：7 cell lines for cytotoxicity and imaging studies. List of Experimental Equipment and Materials:

2 Instruments included a Bruker 400 MHz NMR spectrometer, Perkin Elmer FT-IR spectrophotometer, Shimadzu UV-240 spectrophotometer, Jasco FP-8200 spectrofluorometer, Bruker D8 QUEST diffractometer for X-ray crystallography, and Synergy H1 multi-plate reader for MTT assay. Materials included nicotinic hydrazide, 1-pyrenecarboxaldehyde, DMSO, HEPES buffer, and cell culture reagents.

4：Experimental Procedures and Operational Workflow:

Synthesis involved refluxing reactants in ethanol, followed by purification. Spectroscopic titrations were performed by adding metal ions to chemosensor solutions and measuring changes. Biological assays included MTT for cytotoxicity and fluorescence microscopy for cell imaging.

5：Data Analysis Methods:

Data were analyzed using Job's plot for stoichiometry, Benesi-Hildebrand method for association constants, and statistical methods for detection limits and cell viability.