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Accueil du site > Animations Scientifiques > Séminaires 2012 > José Paulo Farinha - DNA Hybridization in Smart Polymer Nanoparticles

José Paulo Farinha - DNA Hybridization in Smart Polymer Nanoparticles

Speaker : José Paulo Farinha Centro de Química-Física Molecular, IN-Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, 1049-001 Lisboa, Portugal

When : Thursday 29 november, 11:00

Where : Salle CO23 (grande salle de réunions du CBP rez-de-chaussée LR6)

Title : DNA Hybridization in Smart Polymer Nanoparticles

The detection of specific single-stranded DNA sequences by hybridization with the corresponding complementary DNA probe can be detected using either surface sensors, for example, DNA chips, or solution based sensors. The later can be directly used in biological systems with very high efficiencies, especially when supported in particles of nanometer dimension. We use “smart” polymer nanoparticles to support and immobilize fluorescently labeled DNA test sequences for hybridization with the complementary DNA probe labeled with an appropriate Förster resonance energy transfer (FRET) dye. Smart polymer materials show a sharp response to changes in their environment, such as pH, ionic strength, electromagnetic radiation, etc. We prepare smart polymer nanoparticles with a glassy core of poly(methylmethacrylate) and an anionic shell with thermoresponsive poly(N isopropylacrylamide). Above the volume phase transition temperature of the shell, TVPT 30ºC, the shell collapses to ca. 10% of its low temperature volume, with a sharp decrease in its polarity. The variation in shell properties strongly influences the distribution and dynamics of DNA adsorbed onto the nanoparticles. Fluorescence anisotropy shows that the adsorbed DNA suffers a strong decrease in mobility above the TVPT, while FRET reveals that the distribution of DNA in the shell becomes much broader than the shell thickness, indicating that the DNA strands are anchored in the collapsed shell but protruding into the water. When a test DNA sequence is adsorbed onto the particles at 20ºC and fixed at the surface by increasing the temperature to 40ºC, the hybridization with a complementary sequence in solution can be detected by FRET with hybridization efficiencies ca. one order of magnitude higher than in surface sensors, even at very low ionic strength.

Figure : A ROX-labeled DNA sequence (cGene-ROX, chosen from the F5 gene, containing the 1691G-A mutation, associated with the Leiden V factor which leads to a fivefold increase in the risk of thrombosis) are adsorbed onto the nanoparticles at 20ºC : The temperature is increased to 40ºC and the complementary Gene MG is added. Hybridization leads to a strong decrease in ROX fluorescence.

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