1：Experimental Design and Method Selection:

The study focuses on synthesizing surfactant-free copper nanoclusters (sf-CuNCs) and investigating their fluorescence quenching properties in the presence of Fe3+ and Fe(III)-biomolecules (hemin, cytochrome C, ferritin). Mechanistic insights are gained through various spectroscopic and microscopic techniques.

2：Sample Selection and Data Sources:

Aqueous dispersions of sf-CuNCs are prepared. Metal ions (e.g., Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+, Pb2+, Cd2+, Al3+, Li+, Na+, K+) and anions (F?, Cl?, Br?, I?, NO3?, OAc?, SO42?, P4O72?) are screened. Fe(III)-biomolecules (hemin, cytochrome C, ferritin) and vitamin C are used for interaction studies.

3：List of Experimental Equipment and Materials:

Instruments include Perkin-Elmer Lamda-750 spectrophotometer for UV-Vis spectra, Malvern Nano ZS 90 for zeta potential measurements, Horiba TCSPC setup for fluorescence decays, JEOL JEM 2100 electron microscope for TEM, and a hand-held UV lamp for fluorescence imaging. Materials include copper(II) nitrate trihydrate, DMF, various salts, proteins, and vitamin C from Sigma-Aldrich, Fisher Scientific, and Merck.

4：Experimental Procedures and Operational Workflow:

sf-CuNCs are synthesized by heating DMF, adding Cu(NO3)2 solution, refluxing, cooling, centrifuging, evaporating, and redispersing in water. Fluorescence quenching is measured by adding analytes to CuNCs dispersions and recording emission spectra. Mechanistic studies involve time-resolved fluorescence, zeta potential, TEM, and effects of vitamin C addition.

5：Data Analysis Methods:

Stern-Volmer plots are used to calculate KSV and LOD. Image-J software analyzes TEM images for size distribution. Statistical analysis of quenching percentages and potential changes is performed.