Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads.
Fiche publication
Date publication
juillet 2024
Journal
Small (Weinheim an der Bergstrasse, Germany)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr PFEFFER Sébastien, Dr KLYMCHENKO Andrey
Tous les auteurs :
Cruz Da Silva E, Gaki P, Flieg F, Messmer M, Gucciardi F, Markovska Y, Reisch A, Fafi-Kremer S, Pfeffer S, Klymchenko AS
Lien Pubmed
Résumé
Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362 nt with the limit of detection down to 0.3 fm using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.
Mots clés
RNA detection, fluorescent polymeric nanoparticles, magnetic beads, molecular diagnostics, non‐enzymatic signal amplification
Référence
Small. 2024 07 16;:e2404167