Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen

Background: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional bioc hemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecule s in a microtiter plate format. Methods: Streptavidin was covalently coated on 107-nm nanoparticles contain ing >30 000 europium molecules entrapped with P-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-speci fic antigen (PSA) in a microtiter plate format. Results: The detection limit (mean + 3 SD of the zero calibrator) of biotin ylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 x 10(-21) moles) of PSA. Moreover, single nanoparticles, representing indivi dual PSA molecules, were visualized in the same microtiter wells with a tim e-resolved fluorescence microscope using a x10 objective. Single nanopartic les, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer. Conclusions: The universal streptavidin-coated europium(III) nanoparticle l abel is suitable for detection of any biotinylated molecule either in solut ion or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immuno chemical and multianalyte DNA-chip assays as well as histo- and cytochemist ry to improve detection sensitivities. (C) 2001 American Association for Cl inical Chemistry.