Simultaneous Use of Time-Resolved Fluorescence and Anti-Stokes Photoluminescence in a Bioaffinity Assay

• Published in: Analytical chemistry (Washington) Vol. 77; no. 9; pp. 2826 - 2834
• Main Authors: Kuningas, Katri, Rantanen, Terhi, Karhunen, Ulla, Lövgren, Timo, Soukka, Tero
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.05.2005
• Subjects: Analytical chemistry; Biological Assay - methods; Biomedical research; Biotechnology; Chemistry; Exact sciences and technology; Fluorescence; Fluorometry - methods; Immunoassay; Lanthanoid Series Elements - chemistry; Light; Luminescent Measurements - methods; Serum Albumin, Bovine - analysis; Spectrometric and optical methods; Time resolution; Lifetime; Lanthanide; Chemical analysis; Detection limit; Linearity; Photoluminescence; Anti Stokes line; Solid phase; Optical frequency conversion; Europium; Fluorescence spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac048186y

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 × 103 particles in solution and 1 × 104 particles in solid phase, and for europium label same values were 9 × 106 and 9 × 107 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.