Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence

Prostate-specific antigen (PSA) was detected in microtitre wells coated wit h a PSA-specific antibody using biotinylated antibody and streptavidin-coat ed, highly fluorescent 107 nm nanoparticles, which contained more than 3000 0 europium ions entrapped by beta -diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensiti vity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomole s (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-spec ific binding of the streptavidin-coated nanoparticles improved the sensitiv ity of the PSA assay 100-fold compared to the conventional europium-labelle d streptavidin tracer in the same assay format. Additionally, the streptavi din-coated nanoparticle label made very rapid assays possible, due to the h igh affinity of the streptavidin-biotin complex and a high number of bindin g sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated na noparticles reacting with the surface-captured analyte and biotinylated ant ibody was favoured and factors influencing this are discussed. This univers al labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sens itivities in many areas of biochemical analysis, such as cyto- and histoche mistry, multianalyte DNA-chip assays and single-particle assays. Copyright (C) 2000 John Wiley & Sons, Ltd.