Upconverting phosphors in a dual-parameter LRET-based hybridization assay.
Upconverting phosphors (UCPs) are lanthanide-doped sub-micrometer-sized particles, which produce multiple narrow and well-separated anti-Stokes emission bands at visible wavelengths under infrared excitation (980 nm). The advantageous features of UCPs were utilized to construct a dual-parameter, homogeneous sandwich hybridization assay based on a UCP donor and lanthanide resonance energy transfer (LRET). UCPs with two emission bands (540 nm and 653 nm) were exploited together with two appropriate fluorophores as acceptors. The energy transfer excited emissions of the acceptors were measured at 600 nm and 740 nm without any significant interference from each other. The autofluorescence limitation associated with conventional fluorescence was totally avoided as the measurements were carried out at shorter wavelength relative to the excitation. In the sandwich hybridization assay two different single-stranded target-oligonucleotide sequences were detected simultaneously and quantitatively with a dynamic range from 0.03 to 0.4 pmol (corresponding 0.35-5.4 nM). The UCPs enable multiplexed homogeneous LRET-based assay requiring only a single excitation wavelength, which simplifies the detection and extends the applicability of upconversion in bioanalytical measurements.
Rantanen
Terhi
T
Department of Biotechnology, University of Turku, Tykistökatu 6A 6th floor, 20520 Turku, Finland. tetrant@utu.fi
Järvenpää
Marja-Leena
ML
Vuojola
Johanna
J
Arppe
Riikka
R
Kuningas
Katri
K
Soukka
Tero
T
D004735:Energy Transfer; D005456:Fluorescent Dyes; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010758:Phosphorus / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D028581:Lanthanoid Series Elements; D010758:Phosphorus