Upconverting phosphors in a dual-parameter LRET-based hybridization assay.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Materials Science

Background:

• Upconverting phosphors (UCPs) are lanthanide-doped particles emitting visible light under infrared excitation.
• Conventional fluorescence assays face limitations like autofluorescence and spectral overlap.

Purpose of the Study:

• To develop a dual-parameter, homogeneous sandwich hybridization assay using UCPs and lanthanide resonance energy transfer (LRET).
• To demonstrate simultaneous and quantitative detection of two different oligonucleotide sequences.

Main Methods:

• Utilized UCPs with dual emission bands (540 nm and 653 nm) as donors.
• Employed two distinct fluorophores as acceptors for LRET.
• Performed homogeneous sandwich hybridization assay with measurements at 600 nm and 740 nm.

Main Results:

• Achieved simultaneous and quantitative detection of two target oligonucleotides.
• Demonstrated a dynamic detection range from 0.03 to 0.4 pmol (0.35-5.4 nM).
• Avoided autofluorescence limitations by measuring at shorter wavelengths than excitation.

Conclusions:

• UCPs facilitate multiplexed homogeneous LRET assays with a single excitation wavelength.
• This approach simplifies detection and broadens the application of upconversion in bioanalysis.