Particulate and soluble Eu(III)-chelates as donor labels in homogeneous fluorescence resonance energy transfer based immunoassay

Many well-established homogeneous separation free immunoassays rely on particulate label technologies. Particles generally contain a high concentration of the embedded label and they have a large surface area, which enables conjugation of a large amount of protein per particle. Eu(III)-chelate dyed nanoparticles have been successfully used as labels in heterogeneous and homogeneous immunoassays. In this study, we compared the characteristics of two homogeneous competitive immunoassays using either soluble Eu(III)-chelates or polystyrene particles containing Eu(III)-chelates as donors in a fluorescence resonance energy transfer based assay. The use of the particulate label significantly increased the obtained sensitized emission, which was generated by a single binding event. This was due to the extremely high specific activity of the nanoparticle label and also in some extent the longer Förster radius between the donor and the acceptor. The amount of the binder protein used in the assay could be decreased by 10-fold without impairing the obtainable sensitized emission, which subsequently led to improved assay sensitivity. The optimized assay using particulate donor had the lowest limit of detection (calculated using 3 × S.D. of the 0 nM standard) 50 pM of estradiol in the assay well, which was approximately 20-fold more sensitive than assays using soluble Eu(III)-chelates.