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

Abstract

Upconverting phosphors (UCPs) are lanthanide-doped sub-micrometer-sized particles, which produce multiple narrow and well-separated anti-Stokes emission bands at visible wavelengths under infrared excitation (980 nm). The advantageous features of UCPs were utilized to construct a dual-parameter, homogeneous sandwich hybridization assay based on a UCP donor and lanthanide resonance energy transfer (LRET). UCPs with two emission bands (540 nm and 653 nm) were exploited together with two appropriate fluorophores as acceptors. The energy transfer excited emissions of the acceptors were measured at 600 nm and 740 nm without any significant interference from each other. The autofluorescence limitation associated with conventional fluorescence was totally avoided as the measurements were carried out at shorter wavelength relative to the excitation. In the sandwich hybridization assay two different single-stranded target-oligonucleotide sequences were detected simultaneously and quantitatively with a dynamic range from 0.03 to 0.4 pmol (corresponding 0.35-5.4 nM). The UCPs enable multiplexed homogeneous LRET-based assay requiring only a single excitation wavelength, which simplifies the detection and extends the applicability of upconversion in bioanalytical measurements.

Upconverting Phosphor Labels for Bioanalytical Assays

“…The fluorescence quantum yield of the intercalating dye increases significantly when bound to a double-stranded DNA compared to a single-stranded one, and an increase in sensitized acceptor emission intensity was observed. The LOD (0.1 nM) of this simple sensor was close to that our group achieved with a traditional sandwich-type hybridization assay (LOD <0.35 nM) [180]. The SYBR Green I absorbs at blue wavelengths and, therefore, Yb 3+ -Tm 3+ -doped UCPs were used as donors.…”

“…The analyte types vary from proteins and hormones to carbohydrates, oligonucleotides, toxins, enzymes, and even metal ions. Also multiparametric detection has been demonstrated to quantitatively identify two 186 T. Riuttamäki (née Rantanen) and T. Soukka analytes at the same time [180,183]. Green-, red-, and blue-emitting Er 3+ and Tm…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…In most cases the UCNPs donors are coupled with the traditional organic fluorophore acceptors, although there have been reports of UCNPs coupling with QD [393] or Au NP [394] acceptor NMs. LRET-based in vitro sensors using UCNPs have been successfully developed for small molecules, such as ammonia [395] and glucose [394], nucleic acid hybridization [396], and protein detection, such as caspase-3 [397] and avidin [392]. UCNPs combined with energy transfer assays have also been used in various imaging studies, for example, to look at the intracellular fate of small interference RNA (siRNA) upon uptake into cells [398].…”

Photonen aufkonvertierende Nanopartikel zur optischen Codierung und zum Multiplexing von Zellen, Biomolekülen und Mikrosphären

“…Dank der Unterscheidbarkeit einzelner Fehlpaarungen in der Nucleotidsequenz wurde es mçglich, Punktmutationen nachzuweisen, die mit der Sichelzellenanämie in Verbindung gebracht werden. [227,228] Die Gruppe von Soukka [229] nutzte die mehrfachen Emissionslinien der UCNPs zum parallelen Nachweis von DNA. Auf aminfunktionalisierten UCNPs des Typs NaYF 4 :Yb,Er wurden zwei unterschiedliche Sequenzen einzelsträngiger Fänger-DNA angebracht.…”