A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunochemistry

Background:

• Current dissociative fluorescence enhancement techniques struggle with unstable lanthanide chelate probes in one-step assays.
• High concentrations of complexones or metal ions interfere with lanthanide chelates conjugated to detector molecules.

Purpose of the Study:

• To develop and test stable lanthanide chelates for one-step immunoassays.
• To improve fluorescence enhancement techniques for complex biological samples.

Main Methods:

• Conjugated EuDTPA derivatives to antibodies as tracers.
• Developed and tested enhancement solutions based on β-diketones.
• Evaluated fluorescence intensity, analytical sensitivity, kinetics, and signal stability.

Main Results:

• EuDTPA probes demonstrated stability in the presence of high concentrations of ethylenediaminetetraacetate (EDTA) and metal ions.
• Fast complex formation (5 min) and stable, intensive signals (4 h).
• Achieved high analytical sensitivity: Eu (40 fmol/L), Tb (130 fmol/L), Sm (2.1 pmol/L), Dy (8.5 pmol/L).

Conclusions:

• The improved fluorescence enhancement technique enables analysis of complex samples (EDTA, citrate plasma) in one-step immunoassays.
• The method is suitable for elevated temperatures and facilitates four-plexing.
• Offers a wide dynamic range and high analytical sensitivity for immunoassays.