Härmä H, Soukka T, Lönnberg S, Paukkunen J, Tarkkinen P, Lövgren T
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland.
Luminescence. 2000 Nov-Dec;15(6):351-5. doi: 10.1002/1522-7243(200011/12)15:6<351::AID-BIO624>3.0.CO;2-3.
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 beta-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 x 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.
使用生物素化抗体和包被链霉亲和素的、高荧光的107纳米纳米颗粒，在包被有前列腺特异性抗原（PSA）特异性抗体的微量滴定孔中检测PSA。这些纳米颗粒含有30000多个被β-二酮捕获的铕离子。无需任何额外增强步骤，可直接在孔表面监测PSA。该检测方法的灵敏度为1.6纳克/升，相当于50飞摩尔/升或250zeptomoles（250×10⁻²¹摩尔/升）的PSA。与相同检测形式下的传统铕标记链霉亲和素示踪剂相比，包被链霉亲和素的纳米颗粒的高比活性和低非特异性结合将PSA检测的灵敏度提高了100倍。此外，由于链霉亲和素 - 生物素复合物的高亲和力以及表面上可用于追踪生物素化抗体的大量结合位点，包被链霉亲和素的纳米颗粒标记使得非常快速的检测成为可能。由于用生物素化抗体和包被链霉亲和素的纳米颗粒复合物追踪分析物存在固有问题，因此更倾向于包被链霉亲和素的纳米颗粒与表面捕获的分析物和生物素化抗体发生反应，并对影响此反应的因素进行了讨论。这种通用标记技术可应用于检测溶液或固相中的任何生物素化分子，以提高生物化学分析许多领域的检测灵敏度，如细胞和组织化学、多分析物DNA芯片检测和单颗粒检测。
