Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Spectroscopy

Background:

• Fluorescence correlation spectroscopy (FCS) is widely used for studying reversible chemical reactions.
• Traditional FCS is limited for reactions with high binding constants (>10^4 M^-1) due to insufficient association/dissociation events.
• Metal-ligand complexation often involves high binding constants, posing challenges for conventional kinetic measurements.

Purpose of the Study:

• To explore the applicability of FCS for measuring reaction rates of metal-ligand complexation reactions.
• To investigate the binding kinetics of iron, europium, uranyl, and americium ions with the fluorescent chelator calcein.
• To demonstrate FCS's utility for studying reactions with high equilibrium constants and radioactive samples.

Main Methods:

• Utilized FCS to monitor changes in fluorescence intensity fluctuations upon metal-ligand binding.
• Exploited fluorescence quenching of calcein upon metal ion coordination to generate measurable signals.
• Applied FCS to measure binding kinetics of various metal ions, including radioactive 241Am3+.

Main Results:

• Demonstrated that FCS can accurately measure reaction rates for metal-ligand complexes with high binding constants.
• Observed strong intensity fluctuations due to fluorescence quenching, leading to partial correlation decay in FCS.
• Achieved sensitive measurements for radioactive americium ions at negligible radioactivity levels.

Conclusions:

• FCS is a versatile technique applicable to metal-ligand binding reactions with high equilibrium constants.
• The method allows for kinetic studies of complexation reactions, even with challenging samples like radioactive ions.
• FCS offers high sensitivity, enabling measurements at low concentrations and volumes, crucial for radioactive materials.