Upconversion fluorescence enables homogeneous immunoassay in whole blood.
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Blood Chemical Analysis↗
Biomedical subjects
Telle Ukonaho
Publications and source records attributed to Telle Ukonaho.
Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol.
We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.
Estradiol↗
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.
Biosensing Techniques↗