Chimeric Interaction of Nitrogenase-Like Reductases with the MoFe Protein of Nitrogenase.

The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.

Patterning Conjugated Polymers by Laser: Synergy of Nanostructure Formation in the All-Polymer Heterojunction P3HT/PCDTBT.

In this work we report a broad scenario for the patterning of semiconducting polymers by laser-induced periodic surface structures (LIPSS). Based on the LIPSS formation in the semicrystalline poly(3-hexylthiophene) (P3HT), we have extended the LIPSS fabrication to an essentially amorphous semiconducting polymer like poly[N-90-heptadecanyl-2,7-carbazole-alt-5,5-(40,70-di-2-thienyl-20,10,30-benzothiadiazole)] (PCDTBT). This polymer shows a good quality and well-ordered nanostructures not only at the 532 nm laser wavelength, as in the case of P3HT, but also at 266 nm providing gratings with smaller pitch. In addition, we have proven the feasibility of fabricating LIPSS in the P3HT/PCDTBT (1:1) blend, which can be considered as a model bulk-heterojunction for all-polymer solar cells. In spite of the heterogeneous roughness, due to phase separation in the blend, both P3HT and PCDTBT domains present well-defined LIPSS as well as a synergy for both components in the blend when irradiating at wavelengths of 532 and 266 nm. Both, P3HT and PCDTBT in the blend require lower fluence and less pulses in order to optimize LIPSS morphology than in the case of irradiating the homopolymers separately. Near edge X-ray absorption fine structure and Raman spectroscopy reveal a good chemical stability of both components in the blend thin films during LIPSS formation. In addition, scanning transmission X-ray spectro-microscopy shows that the mechanisms of LIPSS formation do not induce a further phase segregation neither a mixture of the components. Conducting atomic force microscopy reveals a heterogeneous electrical conductivity for the irradiated homopolymer and for the blend thin films, showing higher electrical conduction in the trenches than in the ridge regions of the LIPSS.

Concentration-controlled formation of myoglobin/gold nanosphere aggregates.

Gold nanoparticles are being increasingly proposed as biotechnological tools for medical diagnosis and therapy purposes. Their safety for human beings and the environment is therefore becoming an emerging issue, which calls for basic research on the interactions between nanostructured gold particles and biological materials, including physicochemical studies of model systems. In this Article, we focus on the "reaction products" of a widely known nanoparticle type, citrate-capped 30 nm gold nanospheres, with a model protein, horse myoglobin. Protein adsorption and partial denaturation were accompanied by the formation of nanoparticle aggregates with strongly distinct optical spectroscopy properties and shapes, as observed by transmission electron microscopy. We singled out the concentration of myoglobin as the determinant of these differences, and verified on this basis that surface-enhanced Raman scattering (SERS) spectra can only be obtained by aggregates with strong interparticle optical coupling, which are obtained at low protein concentration. The results can be useful both in improving the spectroscopy of biomolecules and in understanding the formation of the protein corona in biomedical applications.

Adsorption study and detection of the high performance organic pigments quinacridone and 2,9-dimethylquinacridone on Ag nanoparticles by surface-enhanced optical spectroscopy.

In this work, surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) were employed in the study of the adsorption and detection of the pigments quinacridone (QA) and 2,9-dimethylquinacridone (2,9-DMQA). These pigments are of great relevance in artwork and textile, plastic, and photochemical industries due to their condition as high performance pigments since they possess a high tinting strength. Due to this fact, they have been employed at relatively low concentrations. Therefore, the analysis and detection of these pigments requires the application of a highly sensitivity technique, such as SERS and SEF. The adsorption of QA and 2,9-DMQA on silver nanoparticles was extensively studied by means of SERS at different surface coverages. This study was completed by carrying out an in depth vibrational (Raman and IR) analysis of these pigments in solid state by ab initio density functional theory (DFT) calculations. In addition, UV-vis spectroscopy was employed to investigate the aggregation undergone by both pigments in solid state and in solution. 2,9-DMQA was demonstrated to have a lower tendency toward aggregation due to the presence of methyl groups. Even so, this molecule follows a BET adsorption mechanism on the metal nanoparticles due to its high tendency toward intermolecular interaction. From the analysis of the adsorption mechanism of this molecule, the limit of detection was deduced to be ca. 55 ppb.

Electrochemical SERS study on a copper electrode of the insoluble organic pigment quinacridone quinone using ionic liquids (BMIMCl and TBAN) as dispersing agents.

SERS detection of quinacridone quinone (QAQ), an insoluble synthetic organic pigment relevant to modern artworks, is reported here. The use of ionic liquids (BMIMCl and TBAN) as dispersing agents has allowed us to carry out electrochemical SERS experiments of QAQ in aqueous solution using a Cu electrode. No SERS spectra were obtained either from the ionic liquids (ILs) or from QAQ when silver/gold colloids were employed. The spectra of the Cu electrode in 0.1 M KCl aqueous solutions containing either BMIMCl or TBAN indicate that the corresponding cations approach the metal surface through formation of an ion pair with specifically adsorbed Cl⁻, but the corresponding cyclic voltammograms only show reduction and oxidation of the Cu surface. In the case of QAQ dispersed in BMIMCl and TBAN, two different SERS spectra due to QAQ species are observed between -0.4 and -1.0 V. One of the spectra agrees with the SERS of QAQ previously reported by us, using calixarenes as dispersing agents and silver colloids as SERS substrates. The other spectrum reveals a reallocation of the charge of the QAQ molecule, concomitant with a change in its relative orientation to the Cu surface. The SERS detection of QAQ is free from interferences of bands corresponding to the ILs.

Surface enhanced fluorescence of anti-tumoral drug emodin adsorbed on silver nanoparticles and loaded on porous silicon.

Fluorescence spectra of anti-tumoral drug emodin loaded on nanostructured porous silicon have been recorded. The use of colloidal nanoparticles allowed embedding of the drug without previous porous silicon functionalization and leads to the observation of an enhancement of fluorescence of the drug. Mean pore size of porous silicon matrices was 60 nm, while silver nanoparticles mean diameter was 50 nm. Atmospheric and vacuum conditions at room temperature were used to infiltrate emodin-silver nanoparticles complexes into porous silicon matrices. The drug was loaded after adsorption on metal surface, alone, and bound to bovine serum albumin. Methanol and water were used as solvents. Spectra with 1 µm spatial resolution of cross-section of porous silicon layers were recorded to observe the penetration of the drug. A maximum fluorescence enhancement factor of 24 was obtained when protein was loaded bound to albumin, and atmospheric conditions of inclusion were used. A better penetration was obtained using methanol as solvent when comparing with water. Complexes of emodin remain loaded for 30 days after preparation without an apparent degradation of the drug, although a decrease in the enhancement factor is observed. The study reported here constitutes the basis for designing a new drug delivery system with future applications in medicine and pharmacy.

New insights on the Au(core)/Pt(shell) nanoparticle structure in the sub-monolayer range: SERS as a surface analyzing tool.

Surface-enhanced Raman spectroscopy (SERS) was used as a powerful surface analyzing tool to investigate the core-shell structural evolution of Au@Pt nanoparticles, revealing the templating role of the underlying Au atoms on the nanoscale Pt-phase structure in the sub-monolayer range.

Solution SERS of an insoluble synthetic organic pigment-quinacridone quinone-employing calixarenes as dispersive cavitands.

A possibility of getting SERS spectra of insoluble aromatic compounds in colloidal silver solutions is described. The method tested for the organic pigment quinacridone quinone consists of dispersing it in calix[n]arenes. The potentials of such cavitands, both as dispersing and as silver functionalization agents, is reported as a function of the substitution in their lower rim and their cavity size.

Aggregation of antitumoral drug emodin on Ag nanoparticles: SEF, SERS and fluorescence lifetime experiments.

We have studied the fluorescence and Raman emission of the anthraquinone drug emodin immobilized molecules on nanostructured silver surfaces, prepared through two different methods. Two different pHs (pH = 10 and pH = 6) have been used. The dye aggregation favors SEF at pH = 6, whereas quenching of fluorescence is observed at pH = 10, due to the short distance between emodin and Ag particles. Along with these results, SERS spectra have given us information about the different species present in the solution. We have used two different reducing agents to obtain the Ag nanoparticles: hydroxylamine hydrochloride and sodium citrate. In all the cases analysed, the enhancement of both SEF and SERS spectra, is larger for hydroxylamine hydrochloride than that for sodium citrate. We have also measured fluorescence lifetime, observing a shorter lifetime for emodin molecules near Ag nanoparticles than that for emodin solved in pure water, thus corroborating the results obtained in fluorescence emission spectra.

Self-assembly of alpha,omega-aliphatic diamines on Ag nanoparticles as an effective localized surface plasmon nanosensor based in interparticle hot spots.

The adsorption and self-assembly of alpha,omega-aliphatic diamines on silver nanoparticles is studied in this work by surface-enhanced Raman scattering (SERS) spectroscopy and plasmon resonance. These bifunctional diamines can act as linkers of metal nanoparticles (NPs) inducing the formation of hot spots (HS), i.e. interparticle junctions or gaps between metal NPs, which are points where a huge intensification of the electromagnetic field occurs. In addition, the dicationic nature of these diamines leads to the formation of cavities just at the induced hot spots which can be applied to molecular recognition of analytes. The influence of the surface coverage and the aliphatic chain length in diamines on their self-assembly was tested by the vibrational spectra and correlated to the different plasmon resonances of the dimers detected in the extinction spectra. These factors can be used for tuning the plasmon resonance of dimers formed by two metal nanoparticles where interparticle hot spots are formed. Finally, the analytical potential of these functionalized Ag nanoparticles is demonstrated for the trace detection of the pesticide aldrin.

Self-assembly of a dithiocarbamate calix[4]arene on Ag nanoparticles and its application in the fabrication of surface-enhanced Raman scattering based nanosensors.

The absorption and self-assembly of a dithiocarbamate calix[4]arene derivative (DTCX) on Ag nanoparticles (NPs) was characterized in this work by surface-enhanced Raman scattering (SERS). This study was carried out on Ag NPs prepared by chemical reduction of silver nitrate with two different reducing agents: sodium citrate or hydroxylamine hydrochloride. SERS was able to discriminate between the different conformations and interaction geometries adopted by DTCX when adsorbed and self-assembled on Ag NPs at different surface coverings. The identification of structural marker bands was crucial in this study. Furthermore, the structure adopted by DTCX is important to determine the activity of this calixarene as a molecular host to detect pollutants, such as pyrene (PYR), since an excessively open or closed cavity is not efficient regarding the analyte detection.

Nanosensors based on viologen functionalized silver nanoparticles: few molecules surface-enhanced Raman spectroscopy detection of polycyclic aromatic hydrocarbons in interparticle hot spots.

The functionalization of silver nanoparticles (Ag NPs) by viologen dications (VGDs) is reported in this work as well as their applications in the surface-enhanced Raman scattering (SERS) detection of polycyclic aromatic hydrocarbons (PAHs). VGDs are able to form intermolecular cavities at interparticle junctions (SERS hot spots) where the analyte can be allocated. This leads to a giant intensification of the Raman emission of the target molecule. This effect was applied in the detection of PAHs, one of the most widespread and dangerous group of pollutants existing in the atmosphere and waters. A comparison between sensing-systems based on different VGDs (lucigenin, diquat, and paraquat) was done for the detection of two PAHs (pyrene and benzo[c]phenanthrene). The functionalization with lucigenin (LG) provided the most powerful and stable VGD-NPs sensor, which allowed the SERS detection of pyrene (PYR) down to 10(-9) M in the macro setup and in the zeptomole range for spectra obtained by single NPs aggregates (micro setup). Besides, SERS spectra afforded important structural information about the interaction mechanism of VGD and PAHs, revealing the formation of a CT complex between the VGD and PYR and changes in the host conformation. The position of the nu(Ag-Cl) band and the plasmon resonance contribution assigned to Ag dimers were also used as spectral markers to monitor the host-guest interaction.

Sensing polycyclic aromatic hydrocarbons with dithiocarbamate-functionalized ag nanoparticles by surface-enhanced Raman scattering.

Trace detection of polycyclic aromatic hydrocarbons is reported in this work on dithiocarbamate calix[4]arene functionalized Ag nanoparticles by using surface-enhanced Raman scattering (SERS). SERS spectra informed about the existence of the pollutant by measuring its characteristic fingerprint vibrational features. In addition, SERS revealed important structural information from both the host and the analyte which was crucial to understand and deduce the host-guest interaction mechanism. The effectiveness of this system was checked for a group of PAHs: pyrene, benzo[c]phenanthrene, triphenylene, and coronene. From the analyzed results, the affinity constants and the limit of detection were deduced for each pollutant.

Identification of the antitumoral drug emodin binding sites in bovine serum albumin by spectroscopic methods.

Using SERS, fluorescence, circular dichroism and stopped-flow, we have unequivocally characterized the binding sites of emodin in bovine serum albumin. Emodin interacts with protein through two different binding sites corresponding to Sudlow's sites 1 and 2. Site 2, where the binding drug presents, in the cavity, a form between neutral and mono-anionic species slightly displaced to the neutral one, is the primary interaction site, with higher association binding constant, and hence, higher affinity than the other binding site. This interaction changes considerably the alpha-helical content of the protein and it occurs mainly within the interval [emodin]/[protein] < or = 2.0. The process involves a fast reaction and the observed rate constant increases when increasing the [emodin]/[protein] ratio. The secondary emodin interaction site corresponds to the Sudlow's site 1, where the drug shows a similar form to that deduced for site 2, but in this case, it is more displaced to mono-anionic species. This interaction does not change the alpha-helical content of bovine serum albumin, and it occurs mainly for [emodin]/[protein] > 2.0 ratios, the process implies a slower reaction than the union process to the site 2, with an observed rate constant that is invariable within the studied interval.

Functionalization of Ag nanoparticles with dithiocarbamate calix[4]arene as an effective supramolecular host for the surface-enhanced Raman scattering detection of polycyclic aromatic hydrocarbons.

We report the use of 25,27-diethyl-dithiocarbamic-26,28-dihydroxy-p-tert-butylcalix[4]arene in the functionalization of Ag nanoparticles for pyrene detection by surface-enhanced Raman scattering (SERS). SERS spectra provided information about the calixarene orientation on the metal surface and the interaction mechanism with pyrene. Thus, in this work, we have combined a powerful spectroscopy technique such as SERS, the electronic plasmon-based properties of nanostructured metals, the molecular size-selective recognition of calixarene, and the strong chelating properties of the dithiocarbamate group toward the metal surface in the detection of polycyclic aromatic hydrocarbons.

Interaction of antitumoral 9-aminoacridine drug with DNA and dextran sulfate studied by fluorescence and surface-enhanced Raman spectroscopy.

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy are applied to study the interaction of the drug 9-aminoacridine (9AA) with DNA and dextran sulfate. The effect of the electrostatic interaction between the positively charged 9AA and negatively charged groups in relation to the excimer or exciplex emission is investigated. The exciplex emission of 9AA is connected to the intercalation of this drug between nucleic base residues. The importance of negative groups in this interaction is evaluated by using dextran and dextran sulfate as model polymers. The existence of negative charges seems to induce an increase of the drug concentration in the vicinity of the polymers. The role of electrostatic attraction in the 9AA dimerization is confirmed by the excimer emission of 9AA in the presence of dextran sulfate. In the case of DNA, the phosphate groups may induce the drug approach to the DNA chain, but the exciplex fluorescence emission could be due to a charge transfer between the drug and adenine-rich sequences of DNA.

Light scattering from self-affine fractal silver surfaces with nanoscale cutoff: far-field and near-field calculations.

We study the light scattered from randomly rough, one-dimensional, self-affine fractal silver surfaces with nanoscale lower cutoff illuminated by s- or p-polarized Gaussian beams a few micrometers wide. By means of rigorous numerical calculations based on the Green's theorem integral equation formulation (GTIEF), we obtain both the far- and near-field scattered intensities. The influence of diminishing the size of the fractal lower-scale irregularities (from approximately 50 nm to a few nanometers) is analyzed in the case of both single realization and ensemble-average magnitudes. For s polarization, variations are small in the far field, being significant only in the higher-spatial-frequency components of evanescent character in the near field. In the case of p polarization, however, the nanoscale cutoff has remarkable effects stemming from the roughness-induced excitation of surface-plasmon polaritons. In the far field, the effect is noticed both in the speckle pattern variation and in the decrease of the total reflected energy upon ensemble averaging, as a result of increased absorption. In the near field, more efficient excitation of localized optical modes is achieved with smaller cutoff, which in turn leads to huge surface electric field enhancements.