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Related Experiment Videos

Homogeneous assay technology based on upconverting phosphors.

Katri Kuningas1, Terhi Rantanen, Telle Ukonaho

  • 1Department of Biotechnology, University of Turku, Finland. katri.kuningas@utu.fi

Analytical Chemistry
|November 16, 2005
PubMed
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This study presents a novel luminescence resonance energy-transfer assay using upconverting lanthanide phosphors. This method offers sensitive detection with reduced background, simplifying instrumentation for various applications.

Area of Science:

  • Materials Science
  • Analytical Chemistry
  • Biochemistry

Background:

  • Autofluorescence and scattered excitation light pose challenges in homogeneous luminescence assays.
  • Upconversion photoluminescence (UPL) offers a solution by utilizing near-infrared excitation and visible emission.
  • Lanthanide-based upconverting phosphors (UPC) provide narrow-banded anti-Stokes emission, minimizing background noise.

Purpose of the Study:

  • To develop a luminescence resonance energy-transfer (LRET) based assay using UPC phosphors as donors.
  • To demonstrate the feasibility of this assay for detecting small molecules like biotin.
  • To overcome limitations of existing luminescence-based assay technologies.

Main Methods:

  • Utilized inorganic upconverting (UPC) lanthanide phosphor (Er3+,Yb3+-doped) as a donor and fluorescent protein as an acceptor.

Related Experiment Videos

  • Constructed a competitive model assay for biotin using streptavidin-conjugated UPC phosphor and biotinylated phycobiliprotein.
  • Excited UPC phosphor at near-infrared (980 nm) and measured sensitized acceptor emission at red wavelengths (600 nm) using a microtitration plate fluorometer.

    • Main Results:

    • Successfully demonstrated a proximity-dependent LRET assay with minimal background.
    • Achieved a lower limit of detection in the subnanomolar concentration range for biotin.
    • The assay showed simplified instrumentation compared to time-resolved fluorometry.

    Conclusions:

    • The developed UPC phosphor-based LRET assay is a sensitive and robust method.
    • This technology is suitable for analyzing complex samples, including those that are strongly colored or fluorescent.
    • The simplified instrumentation and reduced background make it advantageous for clinical immunoassays and high-throughput screening.