1：Experimental Design and Method Selection:

The study involved synthesizing a DAHK-TMBB probe by coupling a tetramethyl BODIPY dye to the DAHK tetrapeptide. Fluorescence lifetime correlation spectroscopy (FLCS) was used to measure species-specific autocorrelation functions based on fluorescence lifetime differences. Titration experiments with copper(II) ions were conducted with and without metal ion buffers (NTA3?/CuNTA?) to control free copper concentrations.

2：Sample Selection and Data Sources:

The probe DAHK-TMBB was synthesized and purified (>95% purity). Measurements were performed in PBS buffer (9 mM, ionic strength 154 mM with NaCl, pH

3：54) or suitable Good's buffers for pH-dependent studies. Metal salts and buffer substances were commercially sourced. List of Experimental Equipment and Materials:

UV/Vis and fluorescence spectrometers (Jasco V650, Jasco FP-6500), quantum yield measurement system (Hamamatsu Quantaurus-QY), home-built TCSPC setup with pulsed diode laser (LDH-P-C-470B), laser driver (PDL-808 Sepia), SPAD detectors (PDM series, SPCM-AQR-14), TCSPC electronics (PicoHarp 300), software (SymPhoTime 64), inverted microscope (Axiovert 200), water immersion objective (C-Apochromat 63x/

4：2 NA), bandpass filters (HQ525/50), dichroic mirror (z405/470rpc XT), and chemicals from Sigma-Aldrich and Carl Roth. Experimental Procedures and Operational Workflow:

Synthesis involved reacting Fmoc-DAHK with TMBB-Su in THF, followed by Fmoc deprotection and purification via HPLC. Spectral properties were characterized using absorption and fluorescence spectroscopy. Fluorescence titrations were performed by adding CuSO4 or using metal ion buffers, with measurements of intensity, lifetime, and FLCS. FLCS data were processed to generate species-specific autocorrelation curves.

5：Data Analysis Methods:

Fluorescence decays were fitted with biexponential models using SymPhoTime 64 software. Titration data were fitted to derived equations (e.g., Eq. 5 and Eq. 9) to determine dissociation constants. FLCS filters were applied to extract species-specific autocorrelation functions, fitted with a 3D diffusion model.