Spectroscopical properties of organic/metal nanohybrids.

The aim of our work was to prepare stable nanohybrids of controlled size and shape consisting of a noble metal core decorated with polydiacetylenes (PDAs). Due to the combination of the outstanding linear and nonlinear optical properties of the polydiacetylenic chains with the electromagnetic field-enhancing capability of metal nanostructures, these novel composites can find potential application in different fields. In particular, the different colours exhibited by PDAs in relation to the chemical nature of the monomer and the polymerization procedure, as well as in response to environmental perturbations, make them excellent materials for the fabrication of sensing devices. On the basis of our previous work on PDA self-assembled monolayers on flat metal surfaces, the results of which are briefly reported, we prepared differently-shaped gold and silver nanocores (spheres, cages) coated with various diacetylenic monomers having end-groups able to firmly anchor to the metal surface. These nanohybrids exhibit in aqueous colloidal solution an enhanced photochemical polymerization monitored step by step with UV-Vis and SERS techniques. It is shown that in these stable assemblies an intra-particle polymerization takes place and that the dominant PDA form is conditioned by the core size and geometry. While the nanoparticles are SERS active in the visible, the nanocages should be excellent SERS substrates from the visible to the near infrared regions.

Photopolymerization of diacetylene-capped gold nanoparticles.

Gold nanoparticles (AuNPs) coated with the diacetylene henicosa-10,12-diyn-1-yl (DS9) disulfide were successfully prepared by direct synthesis in toluene solutions. The average size of the nanohybrid metal core was finely adjusted by manipulation of the preparative conditions in the diameter range from 1.6 to 7.5 nm, as determined by TEM characterization. The topochemical polymerization of DS9 chemisorbed onto the gold nanoclusters of different size was carried out in colloidal suspensions by exposure to UV radiation and the process was monitored by UV-Vis and Raman spectroscopies. The results showed that in these assemblies the monomer undergoes an intra-particle polymerization and that the dominant polydiacetylene phase present is ruled by the core size. The deposition of the photoirradiated colloids onto different substrates was found to leave the polydiacetylene conjugation unaltered.

UV polymerization of self-assembled monolayers of a novel diacetylene on silver: a spectroscopic analysis by surface plasmon resonance and surface enhanced Raman scattering.

UV polymerization of self-assembled monolayers of a novel carbazolyl-diacetylene (CDS9) chemisorbed on silver films was demonstrated by surface plasmon resonance (SPR) and surface enhanced Raman scattering (SERS) experiments. SPR tests performed during UV exposure permitted one to observe the growth of the absorption coefficient, associated with the formation of the polymeric backbone. The Raman spectra of polymerized monolayers exhibited the bands associated with the C=C stretching modes of the conjugated backbone, typical of the blue and red polymeric phases usually present in polydiacetylenes, with a clear predominance of the red form. Moreover, the strong surface enhancement of the Raman band corresponding to the aromatic C=C stretching modes suggested that carbazolyl groups arrange nearly perpendicularly to the metal surface. In contrast, the absence of a SERS signal in the region of conjugated C[triple bond]C bond stretchings confirmed a polymerization scheme with conjugated triple bonds nearly parallel to the plane of the metal.

Separation of human fibrinopeptides by capillary zone electrophoresis.

We describe a method for the simultaneous determination of the five fibrinopeptide forms derived from the thrombin-promoted activation of human fibrinogen by capillary zone electrophoresis (CZE). The fibrinopeptide mixture was first desalted by a solid-phase extraction (SPE) step. The analysis was performed in reversed polarity in a highly cross-linked polyethylene glycol (PEG)-coated capillary with UV-light absorption detection at 200 nm. Several parameters including buffer concentration and pH, presence of an organic modifier, temperature, and applied voltage, have been tested. The best separations were obtained within 20 min, utilizing a 20 mM sodium phosphate buffer without organic modifier, in the narrow 6.1-6.2 pH range, at 25 degrees C, with an applied voltage of 20 kV. Quantitative analysis is made possible by the use of sheep fibrinopeptide A as an internal standard to correct for both extraction and injection errors.

The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module.

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using (15)N NMR relaxation collected at two (15)N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.

Kinetics of fibrinopeptide release by thrombin as a function of CaCl2 concentration: different susceptibility of FPA and FPB and evidence for a fibrinogen isoform-specific effect at physiological Ca2+ concentration.

The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigated on unfractionated fibrinogen samples as a function of CaCl(2) concentration. A 50 mM Tris, 104 mM NaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na(2) (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mM CaCl(2) (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter beta likely reflects substrate polydispersity (beta = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found beta approximately 1, with corresponding normalized rate constants (K(a)) of 3.8, 4.2, 2.7, and 1.9 x 10(-5) [(NIHu/L)s](-1). Surprisingly, in TBC2.5 we found beta = 0.69, with an "average" K(a) of 3.5 x 10(-5) [(NIHu/L)s](-1). This effect disappeared [beta = 0.97, K(a) = 2.7 x 10(-5) [(NIHu/L)s](-1)] with an increase in the ionic strength I to that of TBC30 with 186 mM NaCl (TBCaNa buffer). FPB releases were instead consistent with a nonstretched consecutive exponential growth function, except in TBC30 where some FPB appeared to be cleaved independently. Log-log plots of K(a) versus Ca(2+) concentration, Cl(-) concentration, or I showed a strong linear correlation with only the latter two except in TBCaNa, again suggesting specific effects of the physiological Ca(2+) concentration and I on FPA release. The corresponding K(b) plots showed instead that both total depletion and high Ca(2+) hampered FPB release. To further investigate the TBC2.5 beta = 0.69 effect, FG polydispersity was assessed by Western blot analyses. The thrombin-binding gamma'-chain isoform was approximately 4%, resulting in a bound:free thrombin ratio of approximately 25:75. With regard to the C-terminal ends of the Aalpha-chains, approximately 45% were either intact or lightly degraded, while the remaining approximately 55% were more degraded. Fitting the % FPA release data in TBC2.5 with a sum of two exponentials resulted in a faster component and a slower component (K(a1)/K(a2) approximately 6), with a ratio of approximately 48:52. While a role for the gamma'-chain isoform cannot be excluded, this good correlation with the C-terminal degradation of the Aalpha-chains suggests their calcium-dependent involvement in FPA release.