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Zeptomole detection sensitivity of prostate‐specific antigen in a rapid microtitre plate assay using time‐resolved fluorescence
Author(s) -
Härmä Harri,
Soukka Tero,
Lönnberg Stefan,
Paukkunen Janika,
Tarkkinen Piia,
Lövgren Timo
Publication year - 2000
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/1522-7243(200011/12)15:6<351::aid-bio624>3.0.co;2-3
Subject(s) - streptavidin , biotinylation , chemistry , analyte , immunoassay , chromatography , biotin , fluorescence , antibody , biochemistry , biology , physics , quantum mechanics , immunology
Prostate‐specific antigen (PSA) was detected in microtitre wells coated with a PSA‐specific antibody using biotinylated antibody and streptavidin‐coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by β‐diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 × 10 −21 mol/L) of PSA. The high specific activity and low non‐specific binding of the streptavidin‐coated nanoparticles improved the sensitivity of the PSA assay 100‐fold compared to the conventional europium‐labelled streptavidin tracer in the same assay format. Additionally, the streptavidin‐coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin–biotin complex and a high number of binding 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 nanoparticles reacting with the surface‐captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto‐ and histochemistry, multianalyte DNA‐chip assays and single‐particle assays. Copyright © 2000 John Wiley & Sons, Ltd.