Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay.
A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.
Kuningas
Katri
K
Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. katri.kuningas@utu.fi
Rantanen
Terhi
T
Karhunen
Ulla
U
Lövgren
Timo
T
Soukka
Tero
T
D001681:Biological Assay / Q000379:methods*; D005470:Fluorometry / Q000379:methods; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods; D012710:Serum Albumin, Bovine / Q000032:analysis*
D028581:Lanthanoid Series Elements; D012710:Serum Albumin, Bovine