Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence
✍ Scribed by Harri Härmä; Tero Soukka; Stefan Lönnberg; Janika Paukkunen; Piia Tarkkinen; Timo Lövgren
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 64 KB
- Volume
- 15
- Category
- Article
- ISSN
- 1522-7235
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✦ Synopsis
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 b-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 Â 10 À21 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 singleparticle assays.