DOI: 10.1002/cphc.201000015 DNA Hybridization in Thermoresponsive Polymer Nanoparticles Leila M. Moura, Jose M. G. Martinho, and Jose Paulo S. Farinha* [a] Dedicated to Prof. Dr. M. A. Winnik 1. Introduction The detection of specific single-stranded DNA sequences by hybridization with the corresponding complementary DNA probe is of fundamental importance in medical sciences, envi- ronmental sciences, etc. Hybridization can be detected using either surface sensors, for example, DNA chips, or solution- based sensors. The later can be directly used in biological sys- tems with very high efficiencies, especially when supported in particles of nanometer dimension. Fluorescence has been widely used in DNA hybridization detection due to its sensitivi- ty and selectivity. In this work, we use “smart” polymer nano- particles to support and immobilize fluorescently labeled DNA test sequences for hybridization with the complementary DNA probe labeled with an appropriate Fçrster resonance energy transfer (FRET) dye. “Smart” polymer materials show a sharp re- sponse to slight changes in their environment, such as pH, ionic strength, electromagnetic radiation, electric field, specific ions or solvents, etc. The study and application of these mate- rials at the nanoscale have been increasing in areas such as drug delivery, biosensors, biocatalysts, thermoresponsive surfa- ces, chemical valves, biomimetic actuators, immunoassays, bio- separation. [1–6] One of the most representative examples of stimuli-responsive polymers is poly(N-isopropylacrylamide) (PNIPAM), a thermoresponsive polymer with a volume phase transition temperature in water (T VPT ) of about 30 8C. Below the T VPT , PNIPAM is hydrophilic and its chains are expanded due to hydrogen bonding with water. At temperatures above T VPT , the enthalpic contribution due to hydrogen bonding between water and PNIPAM is lost and the increase in entropy associat- ed with water expulsion prevails. The polymer becomes hydro- phobic and collapses to a globular conformation. [1, 3, 5, 7, 8] We have prepared core–shell polymer nanoparticles with a glassy poly(methyl methacrylate) (PMMA) core and a cross- linked thermoresponsive shell of PNIPAM and aminoethyl methacrylate hydrochloride (AEMH). [9] The positively charged co-monomer is used to increase the absorption of the nega- tively charged DNA strands. Our objective is to use these parti- cles as a support for the detection of DNA hybridization using FRET between complementary DNA sequences, each labeled with either rhodamine X at the 3’ end (ROX, energy donor) or malachite green at the 5’ end (MG, energy acceptor) (Figure 1). [10–13] As a test DNA strand we use a sequence chosen from the F5 gene, specifically containing the 1691G-A mutation, associated with the presence of the Leiden V factor, which leads to a fivefold increase in the risk of developing blood clots (thrombosis). [14–16] The efficiency of FRET, F ET , depends strongly on the distance r between the donor and acceptor dyes [Eq. (1)]: [10] F ET ¼ 1 1 þ r = R 0   6 ð1Þ where R 0 is the Fçrster critical radius, corresponding to the dis- tance at which the transfer probability is 50 %. The value of R 0 for the ROX/MG pair has been determined as R 0 = 6.8 nm. [17] The hybridization between two fluorescently labeled comple- mentary DNA strands approximates the dyes, increasing the ef- ficiency of energy transfer and therefore decreasing the emis- sion of the donor dye. If the dyes are in very close proximity [a] L. M. Moura, Prof. J. M. G. Martinho, Prof. J. P. S. Farinha Centro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology Instituto Superior TØcnico, 1049-001 Lisboa (Portugal) Fax: (+ 351) 218464455 E-mail : farinha@ist.utl.pt Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.200000015. We achieve very high hybridization efficiencies by using a new method to immobilize DNA strands on the surface of thermo- responsive 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 thermo- responsive shell of poly(N-isopropylacrylamide) (PNIPAM) con- taining positive charges. After a DNA strand labeled with a fluorescence probe is loaded onto the particles at room tem- perature, the temperature is increased above the volume phase transition temperature of the PNIPAM shell, TVPT  28 8C. The collapse of the particle shell immobilizes the DNA while maintaining its availability for hybridization with a complementary strand. Fçrster resonance energy transfer (FRET) is used to detect the hybridization with a complementa- ry DNA strand labeled with a FRET acceptor probe. ChemPhysChem 2010, 11, 1749 – 1756  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1749