Principle Of The Technology

The efficient use of time resolved fluorometry is based on the use of lanthanide chelate labels with unique fluorescence properties (6-8, 13-15). The chelates can be clearly divided into those designated non-fluorescent chelates for dissociation chemistry (i.e. DELFIA chemistries) and the highly stable fluorescent chelates.

Besides DELFIA, where the labelling chelate is non-fluorescent, the fluorescent chelates can be exploited in various technologies e.g. a number of homogeneous and non-separation immuno-assays have been developed (7,19,20,21,22, 23). The fluorescence lifetime of the specific signal produced by the chelate is several orders of magnitude longer than the non-specific background. This enables the label to be measured after a delay time when the background has already declined. The large Stokes' shift, i.e. the difference between excitation and emission wavelengths, and the narrow emission peak further contributes to increasing the signal-to-noise ratio (Figure 1).

With the DELFIA chelates, the sensitivity is, furthermore, increased because of the dissociation-enhancement principle; the lanthanide ion in the chelate is dissociated and a new highly fluorescent chelate is formed inside a protective micelle (Figure 2).

1. Time-resolved Measurement of Lanthanide chelates

2. The lanthanide is dissociated from the labelling chelate into a highly fluorescent beta-diketone chelate micelle. The principle of dissociation-enhancement, the basis for assays with both reagent stability and intensive, stable fluorescence.

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