Reduced fluoresceinamine as a fluorescent sensor for nitric oxide

A new fluorescent sensor for nitric oxide (NO) is presented that is based on its reaction with a non fluorescent substance, reduced fluoresceinamine, producing the highly fluorescent fluoresceinamine. Using a portable homemade stabilized light source consisting of 450 nm LED and fiber optics to guide the light, the sensor responds linearly within seconds in the NO concentration range between about 10–750 µM with a limit of detection (LOD) of about 1 µM. The system generated precise intensity readings, with a relative standard deviation of less than 1%. The suitability of the sensor was assessed by monitoring the NO generated by either the nitrous acid decomposition reaction or from a NO-releasing compound. Using relatively high incubation times, the sensor also responds quantitatively to hydrogen peroxide and potassium superoxide, however, using transient signal measurements results in no interfering species.

Síntese e caracterização de redes metalo-orgânicas de fármacos

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Processos de purificação/ valorização do glicerol proveniente da produção de biodiesel

Mestrado em Engenharia Química - Ramo optimização energética na indústria química

Square-wave adsorptive-stripping voltammetric detection in the quality control of fluoxetine

Electroanalytical methods based on square-wave adsorptive-stripping voltammetry (SWAdSV) and flow-injection analysis with square-wave adsorptive-stripping voltammetric detection (FIA-SWAdSV) were developed for the determination of fluoxetine (FXT). The methods were based on the reduction of FXT at a mercury drop electrode at -1.2 V versus Ag/AgCl, in a phosphate buffer of pH 12.0, and on the possibility of accumulating the compound at the electrode surface. The SWAdSV method was successfully applied in the quantification of FXT in pharmaceutical products, human serum samples, and in drug dissolution studies. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was possible to quantify FXT in several pharmaceutical products using FIA-SWAdSV. This method enables analysis of up to 120 samples per hour at reduced costs.

Electroanalytical study of fluvoxamine

Fluvoxamine (FVX) can be reduced at a mercury- drop electrode, with a maximum peak current intensity being obtained at a potential of -0.7 V vs. Ag/ AgCl, in an aqueous electrolyte solution of pH 2. The compound was determined in a pharmaceutical product and in spiked human serum by square-wave adsorptivestripping voltammetry (SWAdSV) after accumulation at the electrode surface, under batch conditions. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was also possible to determine FVX in the pharmaceutical product by use of a flow-injection analysis (FIA) system with SWAdSV detection. The methods developed were validated and successfully applied to the quantification of FVX in a pharmaceutical product. Recoveries between 76 and 89% were obtained in serum analysis. The FIA– SWAdSV method enabled analysis of up to 120 samples per hour at reduced cost, implying the possibility of competing with the chromatographic methods usually used for this analysis.

Electroanalytical determination of paroxetine in pharmaceuticals

Electroanalytical methods based on square-wave adsorptive-stripping voltammetry (SWAdSV) and flow-injection analysis with SWAdSV detection (FIA-SWAdSV) were developed for the determination of paroxetine (PRX). The methods were based on the reduction of PRX at a mercury drop electrode at −1.55V versus Ag/AgCl, in a borate buffer of pH 8.8, and the possibility of accumulating the compound at the electrode surface. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was also possible to determine PRX using FIASWAdSV. This method enables analysis of up to 120 samples per hour at reduced costs. Both methods developed were validated and successfully applied to the quantification of PRX in pharmaceutical products.

Chromatographic techniques for the determination of free phenol in foundry resins

Phenol is a toxic compound present in a wide variety of foundry resins. Its quantification is important for the characterization of the resins as well as for the evaluation of free contaminants present in foundry wastes. Two chromatographic methods, liquid chromatography with ultraviolet detection (LC-UV) and gas chromatography with flame ionization detection (GC-FID), for the analysis of free phenol in several foundry resins, after a simple extraction procedure (30 min), were developed. Both chromatographic methods were suitable for the determination of phenol in the studied furanic and phenolic resins, showing good selectivity, accuracy (recovery 99–100%; relative deviations <5%), and precision (coefficients of variation <6%). The used ASTM reference method was only found to be useful in the analysis of phenolic resins, while the LC and GC methods were applicable for all the studied resins. The developed methods reduce the time of analysis from 3.5 hours to about 30 min and can readily be used in routine quality control laboratories.

Study of the voltammetric behaviour of metam and its application to an amperometric flow system

The electrochemical behaviour of the pesticide metam (MT) at a glassy carbon working electrode (GCE) and at a hanging mercury drop electrode (HMDE) was investigated. Different voltammetric techniques, including cyclic voltammetry (CV) and square wave voltammetry (SWV), were used. An anodic peak (independent of pH) at +1.46 V vs AgCl/Ag was observed in MTaqueous solution using the GCE. SWV calibration curves were plotted under optimized conditions (pH 2.5 and frequency 50 Hz), which showed a linear response for 17–29 mg L−1. Electrochemical reduction was also explored, using the HMDE. A well defined cathodic peak was recorded at −0.72 V vs AgCl/ Ag, dependent on pH. After optimizing the operating conditions (pH 10.1, frequency 150 Hz, potential deposition −0.20 V for 10 s), calibration curves was measured in the concentration range 2.5×10−1 to 1.0 mg L−1 using SWV. The electrochemical behaviour of this compound facilitated the development of a flow injection analysis (FIA) system with amperometric detection for the quantification of MT in commercial formulations and spiked water samples. An assessment of the optimal FIA conditions indicated that the best analytical results were obtained at a potential of +1.30 V, an injection volume of 207 μL and an overall flow rate of 2.4 ml min−1. Real samples were analysed via calibration curves over the concentration range 1.3×10−2 to 1.3 mg L−1. Recoveries from the real samples (spiked waters and commercial formulations) were between 97.4 and 105.5%. The precision of the proposed method was evaluated by assessing the relative standard deviation (RSD %) of ten consecutive determinations of one sample (1.0 mg L−1), and the value obtained was 1.5%.

Assessment of workers’ exposure to Aflatoxin B1 in a Portuguese waste industry

Aflatoxin B1 (AFB1) is considered by different International Agencies as a genotoxic and potent hepatocarcinogen. However, despite the fact that the fungi producing this compound are detected in some work environments, AFB1 is rarely monitored in occupational settings. The aim of the present investigation was to assess exposure to AFB1 of workers from one Portuguese waste company located in the outskirt of Lisbon. Occupational exposure assessment to AFB1 was done with a biomarker of internal dose that measures AFB1 in the serum by enzyme-linked immunosorbent assay. Forty-one workers from the waste company were enrolled in this study (26 from sorting; 9 from composting; 6 from incineration). A control group (n = 30) was also considered in order to know the AFB1 background levels for the Portuguese population. All the workers showed detectable levels of AFB1 with values ranging from 2.5ng ml−1 to 25.9ng ml−1 with a median value of 9.9±5.4ng ml−1. All of the controls showed values below the method’s detection limit. Results obtained showed much higher (8-fold higher) values when compared with other Portuguese settings already studied, such as poultry and swine production. Besides this mycotoxin, other mycotoxins are probably present in this occupational setting and this aspect should be taken into consideration for the risk assessment process due to possible synergistic reactions. The data obtained suggests that exposure to AFB1 occurs in a waste management setting and claims attention for the need of appliance of preventive and protective safety measures.

Electroanalytical study of the pesticide ethiofencarb

A detailed study of voltammetric behavior of ethiofencarb (ETF) is reported using glassy carbon electrode (GCE) and hanging mercury drop electrode (HMDE). With GCE, it is possible to verify that the oxidative mechanism is irreversible, independent of pH, and the maximum intensity current was observed at +1.20 V vs. AgCl/Ag at pH 1.9. A linear calibration line was obtained from 1.0x10-4 to 8.0x10-4 mol L-1 with SWV method. To complete the electrochemical knowledge of ETF pesticide, the reduction was also explored with HMDE. A well-defined peak was observed at –1.00V vs. AgCl/Ag in a large range of pH with higher signal at pH 7.0. Linearity was obtained in 4.2x10-6 and 9.4x10-6 mol L-1 ETF concentration range. An immediate alkaline hydrolysis of ETF was executed, producing a phenolic compound (2-ethylthiomethylphenol) (EMP), and the electrochemical activity of the product was examined. It was deduced that it is oxidized on GCE at +0.75V vs. AgCl/Ag with a maximum peak intensity current at pH 3.2, but the compound had no reduction activity on HMDE. Using the decrease of potential peak, a flow injection analysis (FIA) system was developed connected to an amperometric detector, enabling the determination of EMP over concentration range of 1.0x10-7 and 1.0x10-5 mol L-1 at a sampling rate of 60 h-1. The results provided by FIA methodology were performed by comparison with results from high-performance liquid chromatography (HPLC) technique and demonstrated good agreement with relative deviations lower than 4%. Recovery trials were performed and the obtained values were between 98 and 104%.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

CuxSnSx+1 (x = 2, 3) thin films grown by sulfurization of metallic precursors deposited by dc magnetron sputtering

We report the results of the growth of Cu-Sn-S ternary chalcogenide compounds by sulfurization of dc magnetron sputtered metallic precursors. Tetragonal Cu2SnS3 forms for a maximum sulfurization temperature of 350 ºC. Cubic Cu2SnS3 is obtained at sulfurization temperatures above 400 ºC. These results are supported by XRD analysis and Raman spectroscopy measurements. The latter analysis shows peaks at 336 cm-1, 351 cm-1 for tetragonal Cu2SnS3, and 303 cm-1, 355 cm-1 for cubic Cu2SnS3. Optical analysis shows that this phase change lowers the band gap from 1.35 eV to 0.98 eV. At higher sulfurization temperatures increased loss of Sn is expected in the sulphide form. As a consequence, higher Cu content ternary compounds like Cu3SnS4 grow. In these conditions, XRD and Raman analysis only detected orthorhombic (Pmn21) phase (petrukite). This compound has Raman peaks at 318 cm-1, 348 cm-1 and 295 cm-1. For a sulfurization temperature of 450 ºC the samples present a multi-phase structure mainly composed by cubic Cu2SnS3 and orthorhombic (Pmn21) Cu3SnS4. For higher temperatures, the samples are single phase and constituted by orthorhombic (Pmn21) Cu3SnS4. Transmittance and reflectance measurements were used to estimate a band gap of 1.60 eV. For comparison we also include the results for Cu2ZnSnS4 obtained using similar growth conditions.

Study of polycrystalline Cu2ZnSnS4 films by Raman scattering

Cu2ZnSnS4 (CZTS) is a p-type semiconductor that has been seen as a possible low-cost replacement for Cu(In,Ga)Se2 in thin film solar cells. So far compound has presented difficulties in its growth, mainly, because of the formation of secondary phases like ZnS, CuxSnSx+1, SnxSy, Cu2−xS and MoS2. X-ray diffraction analysis (XRD), which is mostly used for phase identification cannot resolve some of these phases from the kesterite/stannite CZTS and thus the use of a complementary technique is needed. Raman scattering analysis can help distinguishing these phases not only laterally but also in depth. Knowing the absorption coefficient and using different excitation wavelengths in Raman scattering analysis, one is capable of profiling the different phases present in multi-phase CZTS thin films. This work describes in a concise form the methods used to grow chalcogenide compounds, such as, CZTS, CuxSnSx+1, SnxSy and cubic ZnS based on the sulphurization of stacked metallic precursors. The results of the films’ characterization by XRD, electron backscatter diffraction and scanning electron microscopy/energy dispersive spectroscopy techniques are presented for the CZTS phase. The limitation of XRD to identify some of the possible phases that can remain after the sulphurization process are investigated. The results of the Raman analysis of the phases formed in this growth method and the advantage of using this technique in identifying them are presented. Using different excitation wavelengths it is also analysed the CZTS film in depth showing that this technique can be used as non destructive methods to detect secondary phases.

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.