Via functional groups following Via ligand chemistry; few established protocols Often protocols available Several several dyes bind to a single Binding to biomolecules bind to a single biomolecule Labeling-induced biomolecules QD Very little information effects on spectroscopic available on labeling-induced properties of reporter studied for effects many common dyes 6–60 nm (hydrodynamic Size 0.5 nm; molecule diameter); colloid Dependent on dye class; can be High; depends on shell or Thermal stability critical for NIR-wavelength dyes ligands Sufficient for many applications High (visible and NIR (visible wavelength), but can be wavelengths); orders of Photochemical insufficient for high-light flux magnitude higher than that of stability applications; often problematic for organic dyes; can reveal NIR-wavelength dyes photobrightening Little known yet (heavy metal From very low to high; dependent Toxicity leakage must be prevented, on dye potential nanotoxicity) Reproducibility of Good, owing to defined molecular Limited by complex structure labels (optical, structure and established methods and surface chemistry; limited chemical of characterization; available from data available; few properties) commercial sources commercial systems available Applicability to Moderate; limited by single-molecule Good; limited by blinking photobleaching analysis Few examples; Well-described FRET pairs; mostly single-donor– configurations possible; FRET configurations; enables limitation of FRET efficiency optimization of reporter properties due to nanometer size of QD coating
Binding to biomolecules Via functional groups following established protocols Often several dyes bind to a single biomolecule Labeling-induced effects on spectroscopic properties of reporter studied for many common dyes Via ligand chemistry; few protocols available Several biomolecules bind to a single QD Very little information available on labeling-induced effects Size 0.5 nm; molecule 6–60 nm (hydrodynamic diameter); colloid Thermal stability Dependent on dye class; can be critical for NIR-wavelength dyes High; depends on shell or ligands Photochemical stability Sufficient for many applications (visible wavelength), but can be insufficient for high-light flux applications; often problematic for NIR-wavelength dyes High (visible and NIR wavelengths); orders of magnitude higher than that of organic dyes; can reveal photobrightening Toxicity From very low to high; dependent on dye Little known yet (heavy metal leakage must be prevented, potential nanotoxicity) Reproducibility of labels (optical, chemical properties) Good, owing to defined molecular structure and established methods of characterization; available from commercial sources Limited by complex structure and surface chemistry; limited data available; few commercial systems available Applicability to single-molecule analysis Moderate; limited by photobleaching Good; limited by blinking FRET Well-described FRET pairs; mostly single-donor– configurations; enables optimization of reporter properties Few examples; configurations possible; limitation of FRET efficiency due to nanometer size of QD coating