Search

The fluorescence of a rhodamine X dye covalently linked to the 5′ terminus of a 25-mers thymine oligodeoxynucleotide (dT25-ROX), adsorbed on the shell of thermo-responsive core-shell polymer particles, was used to probe the polarity, mobility, and distribution of the oligodeoxynucleotides (ODNs) in the shell. The particles have a glassy core of poly(methyl methacrylate) (PMMA) with a 67-nm radius, and a thermoresponsive shell of poly(N-isopropyl acrylamide) (PNIPAM) whose thickness changes from 42 nm at 11 °C to 5 nm at 45 °C. The variation in polarity of the shell with temperature was obtained both from the lifetimes and from the solvatochromic shifts of the dye and shows a sharp transition at the volume phase transition temperature (TVPT) of the PNIPAM shell. Förster resonance energy transfer (FRET) between dT25-ROX and a malachite green (MG)-labeled ODN (dT25-MG) was used to obtain the distribution of the ODNs in the thermoresponsive shell. Our results show that at 23 °C (below TVPT) the ODNs are distributed inside the shell, sensing an environment similar to water. At this temperature, the PNIPAM shell is composed of hydrated chains with high mobility, as probed by the fluorescence anisotropy of dT25-ROX. By increasing the temperature above TVPT, the shell collapses and the chain mobility drastically slows down owing to the anchoring of the ODN to the dense shell of PNIPAM. Furthermore, FRET shows that the ODNs are absorbed on the 5-nm-thick collapsed shell but extend into the water. The polarity probed by the ROX averages the dyes distributed in the interior of the particle shell and in water, with 60 % of the dyes outside the particle shell (i.e., sensing pure water). Another indication that above the TVPT most of the ODNs are oriented with the dye toward the water phase is that the mobility of the dye covalently bound to the ODNs is identical in water and in the collapsed particle shell. The hybridization efficiency between an ODN supported in the particle shell (by adsorbing the ODN below TVPT and subsequently increasing the temperature above TVPT) and the complementary ODN in solution is identical to that of hybridization in water. This result opens good perspectives toward the use of the core-shell thermoresponsive nanoparticles as supports in DNA bioassays. [ABSTRACT FROM AUTHOR]

Chargedmolecules based on the quinolizinum cation have potentialapplications as labels in fluorescence imaging in biological mediaunder nonlinear excitation. A systematic study of the linear and nonlinearphotophysics of derivatives of the quinolizinum cation substitutedby either dimethylaniline or methoxyphenyl electron donors is performed.The effects of donor strength, conjugation length, and symmetry inthe two-photon emission efficiency are analyzed in detail. The bestperforming nonlinear fluorophore, with two-photon absorption crosssections of 1140 GM and an emission quantum yield of 0.22, is characterizedby a symmetric D-π-A+-π-D architecture basedon the methoxyphenyl substituent. Application of this molecule asa fluorescent marker in optical microscopy of living cells revealedthat, under favorable conditions, the fluorophore can be localizedin the cytoplasmatic compartment of the cell, staining vesicular shapeorganelles. At higher dye concentrations and longer staining times,the fluorophore can also penetrate into the nucleus. The nonlinearlyexcited fluorescence lifetime imaging shows that the fluorophore lifetimeis sensitive to its location in the different cell compartments. Usingfluorescence lifetime microscopy, a multicolor map of the cell isdrafted with a single dye. [ABSTRACT FROM AUTHOR]