A novel homogeneous assay format utilising proximity dependent fluorescence energy transfer between particulate labels

Particle labels feature considerably higher specific activity than small molecule labels due to high number of label molecules incorporated in one unit. We developed a novel homogeneous assay relying on time-resolved detection of fluorescence energy transfer between particulate labels. The long-lifetime fluorescent Eu(III)-chelate-doped donor nanoparticles and the prompt near-infrared fluorescent TransFluoSphere™ acceptor particles possess high specific activity, resulting in great sensitivity potential. The proximity required in fluorescence energy transfer was achieved via antibody-antigen interactions, where the model analyte was prostate specific antigen (PSA), and donor and acceptor particles were coated with monoclonal anti-PSA-antibodies specific for discrete PSA epitopes. Due to long-lived donor emission and near-infrared acceptors, the sensitized acceptor emission was detectable in delayed measurement window free of autofluorescence. Due to these properties and low non-specific binding of the particles, the detection limit of below 0.1 ng/mL PSA and low variations, typically 0.1-7%, were achieved. The developed homogeneous assay setup is applicable to sensitive indirect competitive or non-competitive high-throughput screening.