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  • Title: DNA-Functionalized Dye-Loaded Polymeric Nanoparticles: Ultrabright FRET Platform for Amplified Detection of Nucleic Acids.
    Author: Melnychuk N, Klymchenko AS.
    Journal: J Am Chem Soc; 2018 Aug 29; 140(34):10856-10865. PubMed ID: 30067022.
    Abstract:
    Going beyond the limits of optical biosensing motivates exploration of signal amplification strategies that convert a single molecular recognition event into a response equivalent to hundreds of fluorescent dyes. In this respect, Førster Resonance Energy Transfer (FRET) with bright fluorescent nanoparticles (NPs) is an attractive direction, but it is limited by poor efficiency of NPs as FRET donors, because their size is typically much larger than the Førster radius (∼5 nm). Here, we established FRET-based nanoparticle probes that overcome this fundamental limitation by exploiting a phenomenon of giant light harvesting with thousands of strongly coupled dyes in a polymer matrix. These nanoprobes are based on 40 nm dye-loaded poly(methyl methacrylate- co-methacrylic acid) (PMMA-MA) NPs, so-called light-harvesting nanoantennas, which are functionalized at their surface with oligonucleotides. To achieve this functionalization, we developed an original methodology: PMMA-MA was modified with azide/carboxylate bifunctional group that enabled assembly of small polymeric NPs and their further Cu-free click coupling with oligonucleotides. The obtained functionalized nanoantenna behaves as giant energy donor, where hybridization of target nucleic acid (encoding survivin cancer marker) with ∼23 grafted oligonucleotides/Cy5-acceptors switches on/off FRET from ∼3200 rhodamine-donors of the nanoantenna, leading to 75-fold signal amplification. In solution and on surfaces at single-particle level, the nanoprobe provides sequence-specific two-color ratiometric response to nucleic acids with limit of detection reaching 0.25 pM. It displays unprecedented brightness for a FRET biosensor: it outperforms analogous FRET-based molecular probe by >2000-fold and QDot-605 by ∼100-fold. The developed concept of amplified sensing will increase orders of magnitude sensitivity of fluorescent probes for biomolecular targets.
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