Centro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.
Chemphyschem. 2010 Jun 7;11(8):1749-56. doi: 10.1002/cphc.201000015.
We achieve very high hybridization efficiencies by using a new method to immobilize DNA strands on the surface of thermoresponsive polymer nanoparticles. Hybridization efficiencies of about 70 % are obtained between the DNA immobilized in the particles and a complementary strand in solution, even at very low ionic strengths (1 mM). The polymer nanoparticles have a glassy poly(methylmethacrylate) (PMMA) core and a thermoresponsive shell of poly(N-isopropylacrylamide) (PNIPAM) containing positive charges. After a DNA strand labeled with a fluorescence probe is loaded onto the particles at room temperature, the temperature is increased above the volume phase transition temperature of the PNIPAM shell, TVPT approximately 28 degrees C. The collapse of the particle shell immobilizes the DNA while maintaining its availability for hybridization with a complementary strand. Förster resonance energy transfer (FRET) is used to detect the hybridization with a complementary DNA strand labeled with a FRET acceptor probe.
我们通过一种新的方法将 DNA 链固定在热敏聚合物纳米粒子的表面上，从而实现了非常高的杂交效率。即使在非常低的离子强度（1 mM）下，固定在粒子中的 DNA 与溶液中的互补链之间的杂交效率也约为 70%。聚合物纳米粒子具有玻璃状的聚（甲基丙烯酸甲酯）（PMMA）核和含有正电荷的热敏聚（N-异丙基丙烯酰胺）（PNIPAM）壳。在室温下将带有荧光探针标记的 DNA 链加载到粒子上后，将温度升高到 PNIPAM 壳的体积相转变温度（TVPT）以上，约 28 摄氏度。粒子壳的坍塌固定了 DNA，同时保持其与互补链杂交的可用性。利用Förster 共振能量转移（FRET）检测与带有 FRET 受体探针标记的互补 DNA 链的杂交。
