Marta Ribeiro

Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions.

Staphylococcus comprises up to two-thirds of all pathogens in orthopedic implant infections and they are the principal causative agents of two major types of infection affecting bone: septic arthritis and osteomyelitis, which involve the inflammatory destruction of joint and bone. Bacterial adhesion is the first and most important step in implant infection. It is a complex process influenced by environmental factors, bacterial properties, material surface properties and by the presence of serum or tissue proteins. Properties of the substrate, such as chemical composition of the material, surface charge, hydrophobicity, surface roughness and the presence of specific proteins at the surface, are all thought to be important in the initial cell attachment process. The biofilm mode of growth of infecting bacteria on an implant surface protects the organisms from the host immune system and antibiotic therapy. The research for novel therapeutic strategies is incited by the emergence of antibiotic-resistant bacteria. This work will provide an overview of the mechanisms and factors involved in bacterial adhesion, the techniques that are currently being used studying bacterial-material interactions as well as provide insight into future directions in the field.

Multi-pumping flow systems: an automation tool

Multi-pumping flow systems (MPFS) are one of the most recent developments in terms of the design, conception and implementation of continuous flow methodologies, for sample and reagent handling and for the automation of analytical procedures. Based on the utilisation of multiple solenoid micro-pumps they enable the configuring of fully automated and easily controlled and operated analytical systems since all the fundamental operations involved in carrying out a sample analysis, including sample insertion, reagent addition and signal measurement could be carried out by the same manifold component, reducing the number of system parts and minimising its control or the occurrence of mal-functions. On the other hand, micro-pumps actuation produce a pulsed flow characterised by a chaotic movement of the solutions, which contributes to a fast sample/reagent homogenisation with low axial dispersion yielding improved analytical signals. The combination of such advantageous features resulted in simple, compact, versatile, fast, low-cost analytical procedures, exhibiting low reagent and low sample consumption, reducing the production of undesirable wastes and minimising operator intervention.

Development and validation of a GC-MS method for determination of 39 common pesticides in estuarine water – targeting hazardous amounts in the Douro River estuary

An analytical method based on solid-phase extraction followed by gas chromatography mass spectrometry (GC-MS) was developed and validated for the quantification of 39 pesticides with distinct physico-chemical characteristics (including some degradates), in estuarine water samples. The method includes pre-concentration of samples (2500-fold factor), in OASIS HLB cartridges, by solid phase extraction. The analysed pesticides included organochlorines, organophosphorous, triazines, pyrethroids and other miscellaneous compounds, that are usually analysed separately. Method detection limits were between 3.6 and 61.2 ng L−1. The obtained sensitivity and accuracy, associated with the inherent confirmatory potential of GC-MS, validate the method as a toll in environmental monitoring. Analyses of water samples (n = 84) taken from the Douro River estuary, from March to May 2009, showed the presence of all assayed pesticides, among which 71.6% where above the limits of quantification. Besides, the feasibility of the method for chemical monitoring was highlighted by establishing a first pattern of spatial-temporal fluctuation for all classes of pesticides in the estuary, showing a significant increase of their concentrations in April and mainly at the margin bordering the city of Porto. Moreover, the finding of environmental hazardous amounts of hexachlorbenzene, endrin, endosulfan (all forms), DDTs, chlorfenvinfos Z and simazine pose a clear risk for local aquatic wildlife, and eventually to humans, by direct exposure or by consumption of contaminated fish.

Endocrine disruptors in the Leça River and nearby Porto Coast (NW Portugal): presence of estrogenic compounds and hypoxic conditions

The Leça River and two beaches close to the north and south margins of the estuary, nowadays an important seaport harbor, show signs of contamination. However, the chemical nature of that contamination is uncertain. Therefore, this study checked for the presence of 11 endocrine disrupting compounds (EDCs) of animal (17β-estradiol and estrone), pharmaceutical (17α-ethynylestradiol), and industrial (alkylphenols, alkylphenol ethoxylates, and bisphenol A) origins in water samples collected from six points at the river gradient and at two sites in the coastline (Atlantic Ocean). Along with the EDCs, evaluated by gas chromatography – mass spectroscopy (GC-MS) during spring, summer, and autumn, physico-chemical parameters were also assessed and particular attention was given to dissolved oxygen (DO) levels. Data showed the presence of high amounts of estrogens (up to 10 ng L−1 for estrone) and industrial compounds (up to 2 µg L−1 for the nonylphenol ethoxylates) at both river and seacoast. Along with this, clear signs of hypoxia were found in the river (DO < 3 mg L−1 at several sampling sites). Taking into account these issues, it was concluded that there are local conditions for the occurrence of endocrine disruption in aquatic animals.

Development and optimisation of a GC-MS method for the evaluation of oestrogens and persistent pollutants in river and seawater samples

This paper describes the development and validation of a GC-MS method which allows the simultaneous quantification of 11 endocrine disrupting compounds (EDCs) in surface water samples from both estuary and sea. The analysed EDCs are oestrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), 4-tert-octylphenol, 4-n-octylphenol, 4-nonylphenol, bisphenol A and finally, mono and diethoxylates of 4-nonylphenol and 4-octylphenol. The method includes the pre-concentration of water samples, 1000-fold factor, in OASIS HLB cartridges by solid phase extraction, the derivatisation of all EDCs by N,O-bis(trimethylsilyl)trifluoroacetamide added with 1% trimethylchlorosilane and pyridine (at 65°C for 30 min) and, finally the stabilisation of the EDCs-silylated derivatives, in hexane, for 72 h. The validation parameters revealed that this method was highly specific for all target compounds using real samples. The linearity of the calibration curves (r 2) showed correlation factors higher than 0.990. The detection limits ranged from 0.10 to 1.45 ng L−1, depending on each analysed compound, and recoveries were satisfactory for most of the assayed EDCs (>60%). Analysis of samples from four polluted areas of Douro River estuary and from two points of the Atlantic Ocean (Portugal) showed high amounts of E1 (up to 1.96 ng L−1), E2 (up to 14.36 ng L−1) and EE2 (up to 2.76 ng L−1).

The role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. The aim of this work was to study the role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels. Hydrogels were prepared after 3 and 7 days of dialysis and the effect of freezing was analyzed. For that purpose, a part of the fibroin hydrogels underwent freezing at -20 °C for 24 h, followed by lyophilization and the rest of the hydrogels were kept at 8 °C for 24 h, with further lyophilization. The fibroin hydrogels were characterized by X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Measurements by XRD and FTIR indicated that silk I and silk II structures were present in the fibroin hydrogels and that the secondary structure of fibroin is transformed mostly to β-sheet during the gelation process. Thermal analysis indicated that fibroin hydrogels are thermally stable with the degradation peak at around 330–340 °C. SEM micrographs showed porous structures and the fibroin hydrogels subjected to freezing presented a much larger pore size. Results indicate that the dialysis time and freezing did not alter the material crystallinity, conformation or thermal behavior; however, hydrogel microstructure was strongly affected by dialysis time and freezing, showing controlled pores size. This study provides fundamental knowledge on silk fibroin hydrogels preparation and properties and the studied hydrogels are promising to be used in the biomaterial field.

Single reaction interface in flow analysis

The dual or multiple reaction interface concept, commonly associated to the distinct flow techniques, was replaced by a single interface concept, which do not no rely on the utilisation of a well-defined and compelling sample volume but only on mutual penetration of sample and reagent zones at a single reaction interface where both sample and reagent met together prior to detection. In the proposed approach basic principles of flow analysis, such as controlled dispersion and reaction zone formation, are not influenced by sample and reagent volumes but determined exclusively by the extension of the overlap of two adjoining quasi-infinite zones enhanced by multiple flow reversals and the pulsed nature of the flowing streams. The detector is positioned at the core of the flow manifold (not in the conventional terminal position), and repetitive flow reversals enable interface manipulations, including multi-detection of the entire reaction interface or the monitoring of the evolution of a pre-selected interface zone by using suitable reversal cycle times. The implementation of the developed approach was facilitated due to the configuration simplicity and operational versatility of multi-pumping flow systems. Its performance was evaluated by monitoring processes involving two or four-solution reaction interfaces.

Rapid chemiluminometric determination of gabapentin in pharmaceutical formulations exploiting pulsed‐flow analysis

In this study, a straightforward and automated pulsed flow-based procedure was developed for the chemiluminometric determination of gabapentin [1-(aminomethyl)cyclo-hexaneacetic acid], a new generation antiepileptic drug, in different formulated dosage forms. The software-controlled time-based injection method capitalizes on the decrease of the background chemiluminescence (CL) readout of the luminol-hypochlorite reaction in the presence of gabapentin. In short, gabapentin works as a hypochlorite scavenger. The analytical procedure was implemented in a multi-pumping flow network furnished with a suite of microdispensing solenoid-actuated pumps. The diaphragm-type micropumps might be configured to operate as fluid propellers, commutation units and metering injectors. A dynamic linear working range for gabapentin concentrations in the range 60-350 micromol/L was obtained, with an estimated detection limit of 40 micromol/L. The flow analyser handles about 41 injections/h and yields precise results (RSD < 2%). The miniaturized flow analyser thus has potential to be exploited for in-line monitoring of drug manufacturing within the quality assurance framework of modern pharmaceutical companies.

Fatty Acid Profile of Human Milk of Portuguese Lactating Women: Prospective Study from the 1st to the 16th Week of Lactation

Background/Aims: Fatty acid (FA) composition varies over the course of the day and during lactation. The aim of this study was to evaluate FA composition and its compositional stability in human milk, from day 7 to week 16 of lactation. Methods: Human milk was collected from all feedings over 24 h at day 7 and weeks 4, 8, 12 and 16 of lactation in 31 lactating women. FAs were analyzed through gas chromatography. Comparisons were made with analysis of variance. Results: Total monounsaturated FAs decreased from 33.04 ± 2.58% wt/wt at day 7 to 31.48 ± 3.32% wt/wt at week 16 of lactation, much at the expenses of the decrease in the major monounsaturated FA found in human milk, oleic acid. Main polyunsaturated FAs n-6 and n-3 showed fluctuations from day 7 up to week 16 of lactation, but with no statistical significance. Arachidonic acid significantly decreased from transitional to mature milk. Conclusions: The FA profile obtained throughout the study time points presented very low levels of oleic acid and very high linoleic acid/α-linoleic acid ratios which reflect recent changes in Portuguese women’s food patterns. Despite this, the ascorbate/dehydroascorbate ratio remained constant during the study, suggesting a protective metabolic mechanism.

Multipumping Flow Systems: An Alternative Approach to Sample Handling in Spectroscopy Measurements

Abstract Multipumping flow systems (MPFSs) are one of the most recent developments in terms of flow‐based analytical methodologies. They differ from conventional flow‐based modalities such as flow injection analysis (FIA) and sequential injection analysis (SIA) in several aspects, including manifold components and configuration, operational mode, and flow hydrodynamics. This paper presents an overview of MPFSs and discusses their basic features, including methodological implementation, operational characteristics, analytical performance, potential, and limitations. Applications to different types of samples involving different detection techniques are discussed.