ADVANTAGES OF LANTHANIDES
The lanthanide complexes are very bright. In this family of lanthanide complexes, the energy is transferred with very high efficiency from the ligand to the luminescent lanthanide.
The complexes have long emission lifetimes and their emission can be time-resolved. Time-resolved measurements use a time delay between an excitation pulse and the time at which the emission is recorded. This temporal discrimination allows the lanthanide probe to be seen clearly–shorter-lived background signals due to scattering, Raman, and fluorescent impurities decays more rapidly than most lanthanide luminescence. Gated detection and a pulsed excitation source technologies are available to enhance this superior signal to noise sensitivity.
- The complexes transfer (intermolecularly) energy very efficiently to acceptors. Lanthanides have exceptionally long Förster distances for fluorescence resonance energy transfer (FRET).
- The wavelengths of excitation (310 – 400 nm) and emission (ca 500 – 750 nm) are far apart. This reduces inner-filter effects (when dyes reabsorb their own emitted light).
- The complexes can be made with a range of lanthanides emitting in the visible and in the near infra red, including terbium (Tb), europium (Eu), samarium (Sm) and dysprosium (Dy), thus enabling multiplexed measurements (photograph at right).
- The complexes have very narrow emission lines further facilitating multiplexing by limiting the overlap between the emission spectra. Also, using a narrower filter than with traditional dyes, more of the fluorescent signals can be observed.
- Lumiphore’s lanthanide complexes are very stable. They are not easily oxidized or photobleached. They can be stored at room temperature for years.
- The complexes are designed with linkers, and can easily be conjugated to a variety of substrates.
- The complexes can be easily and inexpensively synthesized, thus enabling a wide range of commercial applications.
The Limit of Detection
A major advantage of small organic dyes is their small size, which means they will probably not alter natural interactions. Small organic fluorophores that absorb and emit over a range of wavelengths are available, but these molecules have broad absorption and emission lines (which limit the use of multiple dye probes in a single experiment) and are susceptible to photobleaching and self-quenching when their local concentrations are high. In addition, their short fluorescent lifetimes (1-100 ns) correlate with natural fluorescent background found in biological samples, making signal/noise levels unsatisfactory for many applications.
Luminescent lanthanide metal complexes, on the other hand, have many properties making them desirable for homogeneous applications in biological assays. Their long-lived (us-ms) luminescence lifetimes, of the lanthanides emitting in the visible region, lead to especially high sensitivity and high signal-noise ratios because short-lived auto fluorescence of samples and containers is removed by time-gated detection. The advantage of using time-resolved measurements are clearly demonstrated.