Publications

Abstract An in-situ approach based in multiple spectroscopic techniques and benchmarked with DSC was used to characterise beta-Lg thermally-induced transitions. The methodology applied overcomes previously reported limitations by ensuring similar experimental conditions in different determinations, non-aggregation conditions and allowing differentiation between fluorescent variations due to collisional quenching and structural modifications. These experimental improvements along with the correlation of complementary data from the assessment of several unfolding-related events, allowed a real time, precise and detailed description of the unfolding/refolding pathways of beta-Lg. The existence of a complex multi-step unfolding mechanism was confirmed, with a focus on the reversible conformational changes. The elusive unfolding intermediates were characterised in terms of structural swelling, hydrophobic sites accessibility and tryptophan exposure. This approach allowed establishing a clear order of events during thermally-induced structural changes, representing a step forward in the understanding of protein stability and interactions, useful, e.g., when establishing heat treatments of dairy products.

Abstract The effects of light-absorbing atmospheric particles on climate forcing have been integrated into climate models, but the absence of brown carbon (BrC) in these models has led to differences between model predictions and measured data. Herein, we have used density functional theory (DFT) to generate models for the atmospheric absorption of polycyclic aromatic hydrocarbons (PAHs), major contributors to BrC light absorption, found over Seoul (South Korea), considering their seasonal and yearly variation. Winter was found to be the most problematic season, with significant absorption from the PAHs, while the absorption was more moderate in the autumn and in the spring. In the summer, the absorption is relatively quite weak. This is in line with the higher concentration of PAHs during winter, followed by autumn and spring, while being lower during summer. Moreover, these models showed that PAHs absorb strongly in the UVA and UVB regions of the UV spectrum, followed by moderate absorption in the UVC region and weak absorption in the visible region. Nevertheless, only absorption at the UVA and the visible region should be relevant for climate forcing. Finally, fluoranthene and benzo[a]anthracene are the most relevant contributors for UVB absorption, while benzo[a]pyrene, benzo[g,h,i]perylene, indeno[1,2,3-cd]pyrene and benzo[k]fluoranthene are the main responsible for absorption in the visible region. Thus, our modelling approach allowed us to identify which should be the most relevant PAHs for climate forcing on this region of the globe.

Abstract alpha-Synuclein (alpha syn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an alpha-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on alpha-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on alpha syn secondary-structure formation, using human-, elephant- and whale -alpha syn. Our results confirm that negatively charged lipids induce alpha syn folding in h-alpha syn and e-alpha syn but not in w-alpha syn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 degrees C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying alpha syn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as alpha syn into nontoxic alpha-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and alpha-helix formation by alpha syn, paramount to the proposal of new methods to avoid protein aggregation and disease.

Abstract The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very important public health issue, due to the worldwide increase of resistant pathogens. Within this effort, much research has been done on antimicrobial peptides, because having the membrane as a target, they represent a new antibiotic paradigm. Among these, cyclic peptides (CPs) made of sequences of D- and L-amino acids have emerged as a new class of potential antimicrobial peptides, due to their expected higher resistance to protease degradation. These CPs are planar structures that can form Self-assembled Cyclic Peptide Nanotubes (SCPNs), in particular in the presence of lipid membranes. Aiming at understanding their mechanism of action, we used biophysical experimental techniques (DSC and ATR-FTIR) together with Coarse-grained molecular dynamics (CG-MD) simulations, to characterize the interaction of these CPs with model membranes of different electrostatic charges' contents. DSC results revealed that the CPs show a strong interaction with negatively charged membranes, with differences in the strength of interactions depending on peptide and on membrane charge content, at odds with no or mild interactions with zwitterionic membranes. ATR-FTIR suggested that the peptides self-assemble at the membrane surface, adopting mainly a beta-structure. The experiments with polarized light showed that in most cases they lie parallel to the membrane surface, but other forms and orientations are also apparent, depending on peptide structure and lipid:peptide ratio. The nanotube formation and orientation, as well as the dependence on membrane charge were also confirmed by the CG-MD simulations. These provide detail on the position and interactions, in agreement with the experimental results. Based on the findings reported here, we could proceed to the design and synthesis of a second-generation CPs, based on CP2 (soluble peptide), with increased activity and reduced toxicity.

Abstract While TiO2 nanoparticles have shown potential as photocatalysts in the degradation of organic contaminants, their inability to absorb efficiently visible light has limited their industrial application. One strategy for solving this problem is monodoping TiO2 photocatalysts with transition metals, which has worked in the degradation of several pollutants. However, it is not clear if this improvement is enough to offset the potential environmental impacts of adding metal ions to the synthesis of TiO2. Herein, we have used Life Cycle Assessment (LCA) to determine the sustainability of monodoping TiO2 with transition metals (Fe, Co, Mn and Ni, with a 1% weight ratio) to enhance the photocatalytic properties of the photocatalyst toward the degradation of Carbamazepine and Methyl Orange, under UV-A and visible light irradiation. We found that the addition of transition-metals has no significant effect on the environmental impacts associated with the synthesis of TiO2, when a weight-based functional unit was considered. However, when photocatalytic activity was considered, major differences were found. Thus, our results demonstrate that the sustainability of monodoping with different transition metals is solely determined by their ability to enhance (or not) the photocatalytic activity of TiO2. Our data also demonstrated that isopropyl alcohol constitutes a critical point in the synthesis of TiO2 photocatalysts, with ethanol being a potential substitute.

Abstract Carbon dots (CDs) are carbon-based nanoparticles with remarkable luminescent properties, which have made them exciting and suitable alternatives to more traditional fluorophores and even to more recent luminescent nanomaterials (such as metal-based quantum dots). However, despite this high interest on CDs, there has been no focus on their sustainable development and fabrication, and so, there is lacking concrete data on their environmental impacts. A life cycle assessment (LCA) approach was used here to compare and understand the environmental impacts of carbon dots (CDs) obtained via six representative bottom-up synthetic strategies (cradle-to-gate). These routes consist on hydrothermal and microwave-assisted synthesis of CDs derived of citric acid (with the occasional addition of urea), which represent current trends in the synthesis of CDs. Results show that for hydrothermal synthesis the use of electricity is dominant for almost all environmental categories, while citric acid produces most impacts for microwave-assisted synthesis. A performance-based comparison was also made by rescaling results with the fluorescence quantum yield of the CDs. This approach changed the rank order of preference in all categories by a significant margin. While previous analysis indicated microwave-assisted synthesis of citric acid-derived CDs to be the most benign in environmental terms, now the option is the synthesis (either by hydrothermal or microwave-assisted treatment) of urea and citric acid-derived CDs.

Abstract This paper proposes for the first time: (a) a qualitative analytical method based on portable and benchtop backscattering Raman spectrometers coupled to hierarchical cluster analysis (HCA) and multivariate curve resolution - alternating least-squares (MCR-ALS) to identify two polymorphs of antimalarial quinine sulfate in commercial pharmaceutical tablets in their intact forms and (b) a quantitative analytical method based on gold nanoparticles (AuNPs) as active substrates for surface-enhanced Raman scattering (SERS) in combination with MCR-ALS to quantify quinine sulfate in commercial pharmaceutical tablets in solution. The pure concentration and spectral profiles recovered by MCR-ALS proved that both formulations present different polymorphs. These results were also confirmed by two clusters observed in the HCA model, according to their similarities within and among the samples that provided useful information about the homogeneity of different pharmaceutical manufacturing processes. AuNPs-SERS coupled to MCR-ALS was able to quantify quinine sulfate in the calibration range from 150.00 to 200.00 ng mL(-1) even with the strong overlapping spectral profile of the background SERS signal, proving that it is a powerful ultrahigh sensitivity analytical method. This reduced linearity was validated throughout a large calibration range from 25.00 to 175.00 mu g mL(-1) used in a reference analytical method based on high performance liquid chromatography with a diode array detector (HPLC-DAD) coupled to MCR-ALS for analytical validation purposes, even in the presence of a coeluted compound. The analytical methods developed herein are fast, because second-order chromatographic data and first-order SERS spectroscopic data were obtained in less than 6 and 2 min, respectively. Concentrations of quinine sulfate were estimated with low root mean square error of prediction (RMSEP) values and a low relative error of prediction (REP%) in the range 1.8-4.5%.

Abstract The impact of structural differentiation between phenylcarbazoles (PhC, mCP, CBP, TCB) and phenylamines (TPA, BDB, TPB, TDAB) on the phase equilibria, optical spectrum, band gap, and thin-film morphology is evaluated and discussed. The carbazolyl units lead to a lower electronic conjugation contributing to a wide band gap when compared with the diphenylamine analogs. The fusion and sublimation equilibria indicate that entropic contribution is the key factor for the distinguished melting behavior and solid-phase volatility between phenylcarbazole derivatives and phenylamine analogs. The molecular differentiation between the two classes of compounds is not reflected in the crystal packing and intermolecular interactions. However, compared with the diphenylamino groups, the incorporation of carbazolyl moieties contributes to a less flexible molecule. Moreover, the results evidence that intermolecular bonding disruption along the fusion transition is more extensive for phenylamine derivatives. Due to the asymmetric nonplanar structure, mCP is characterized by a ratio of {T-g/T-m approximate to 3/4} while the more symmetric CBP and TCB molecules display ratios closer to {T-g/T-m approximate to 2/3}. Vapor-deposited thin films of mCP, CBP, and TCB are amorphous and their morphology is highly dependent on the substrate roughness. The lower flexibility of nonplanar phenylcarbazoles induces the formation of a glassy state due to the harder packing mechanism leading to the lower ability of the crystallization process.

Abstract Carbon dots (CDs) are carbon-based nanoparticles with very attractive luminescence features. Furthermore, their synthesis by bottom-up strategies is quite flexible, as tuning the reaction precursors and synthesis procedures can lead to an endless number of CDs with distinct properties and applications. However, this complex variability has made the characterization of the structural and optical properties of the nanomaterials difficult. Herein, we performed a systematic evaluation of the effect of three representative bottom-up strategies (hydrothermal, microwave-assisted, and calcination) on the properties of CDs prepared from the same precursors (citric acid and urea). Our results revealed that these synthesis routes led to nanoparticles with similar sizes, identical excitation-dependent blue-to-green emission, and similar surface-functionalization. However, we have also found that microwave and calcination strategies are more efficient towards nitrogen-doping than hydrothermal synthesis, and thus, the former routes are able to generate CDs with significantly higher fluorescence quantum yields than the latter. Furthermore, the different synthesis strategies appear to have a role in the origin of the photoluminescence of the CDs, as hydrothermal-based nanoparticles present an emission more dependent on surface states, while microwave- and calcination-based CDs present an emission with more contributions from core states. Furthermore, calcination and microwave routes are more suitable for high-yield synthesis (similar to 27-29%), while hydrothermal synthesis present almost negligible synthesis yields (similar to 2%). Finally, life cycle assessment (LCA) was performed to investigate the sustainability of these processes and indicated microwave synthesis as the best choice for future studies.