Homogeneous assay technology based on upconverting phosphors.
Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.
Kuningas
Katri
K
Department of Biotechnology, University of Turku, Finland. katri.kuningas@utu.fi
Rantanen
Terhi
T
Ukonaho
Telle
T
Lövgren
Timo
T
Soukka
Tero
T
D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D010758:Phosphorus / Q000737:chemistry*; D052979:Phycobiliproteins / Q000737:chemistry; D017186:Titrimetry
D052979:Phycobiliproteins; D010758:Phosphorus; D001710:Biotin