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Microcrystals of an organic fluorescent dye, 4-octylamino-7-nitrobenz-2-oxa-1,3-diazole, were generated using the reprecipitation method, which is a solvent exchange process. In the presence of polymers, namely, poly(acrylic acid), molecular weight 5100 g mol^-1 and 15 000 g mol^-1, and poly(acrylic acid-co-maleic acid), average molecular weight about 50 000 g mol^-1, used as their sodium salts, the reprecipitation process was strongly accelerated. The reprecipitation kinetics was monitored by UV/vis absorption spectroscopy and revealed a three-step mechanism, each step being influenced by the polymer. The size and shape of the microcrystals were analyzed by fluorescence microscopy. The microcrystals obtained in the presence of polymers were smaller and more regular than those prepared in water alone and were not agglomerated. When the polymer was placed in the reprecipitation medium before introducing the dye solution, the microcrystals displayed a rectangular shape. When the polymer was introduced 20 min after the beginning of the reprecipitation process, intricately structured flowerlike microcrystals were observed. Microanalysis revealed that the microcrystals contained noticeable amounts of polymer. The measurement of the surface electric ζ potential suggested that a proportion of the polymer was present at the microcrystal surface. This work gives a thorough insight into a field where trials have until now been performed in an empirical way. It opens new perspectives to produce low-cost organic microcrystals, potentially useful in optics or pharmaceutical sciences.

Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137. The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.

Single metallic nanoparticle sensitivity in liquid solution was achieved with hyper Rayleigh scattering (HRS). The study of the HRS intensity fluctuations over time was performed for several dilutions of the nanoparticle solution. Histograms of the HRS intensity counts were then obtained. These histograms exhibit a Gaussian profile down to low concentrations before evolving towards Poisson distributions at very low concentrations, demonstrating that the single particle sensitivity is obtained. Based on these results, we were then able to achieve the three-dimensional mapping of immobile 150 nm gold metallic nanoparticles dispersed in a homogeneous transparent polyacrylamide matrix. Polarization resolved measurements were also performed allowing for a clear identification of the harmonic light generated by single gold metallic nanoparticles.