Some fluorescent dyes, like Atto635 or Cy5, exhibit alterations in fluorescence emission in the presence of various metal
ions and metal ion complexes. We used this effect to design dye-ligand conjugates that can be immobilized on glass
surfaces and allow studying metal-ion binding using time-resolved single-molecule fluorescence spectroscopy (SMFS).
Double-stranded DNA served as a rigid scaffold carrying 2,2'-bipyridene-4,4'-dicarboxylic acid as chelating ligand and
a fluorescent dye as reporter, placed in close vicinity to the ligand. In the absence of metal ions, the probes showed high
fluorescence quantum yield, whereas strong fluorescence quenching upon binding of Cu2+-ions was observed. Time-resolved
single-molecule measurements revealed stochastic switching between a highly fluorescent ("on") and a low
fluorescent ("off") state. The coordination of the metal ion to the ligand is thus indicated by intramolecular fluorescence
quenching of the dye. We screened various fluorescent dyes for their sensitivity to Cu2+-coordination, and found that
both Atto620 and MR121 are well-suited for this application. Ensemble studies of the fluorescence lifetimes of metalsensors
with Atto620 showed only small dependence on the metal-ion concentration, while single-molecule studies
reported strong changes in the fluorescence lifetimes which were correlated with the observed on- and off-states. Our
results further indicate that the fluorescence of Atto620 is not completely quenched upon association of the metal-ion
complex; either because a less fluorescent complex is formed or because of intramolecular collisional quenching due to
conformational changes of the C6-linker used for covalent coupling of the fluorescent dye.