Recovery of silver (I) using a thiourea-modified chitosan resin

This work describes the preparation of a chelating resin from chemically modified chitosan. The resin was synthesized by using O-carboxymethylated chitosan to cross-link a polymeric Schiffs base of thiourea/glutaraldehyde and characterized by IR. Batch method was applied for testing the resin's adsorption behavior. Adsorption experiments showed the resin had good adsorption capacity and high selectivity for Ag(I) in aqueous solution. The maximum uptake of Ag(I) exhibited was 3.77 mmol/g, at pH 4.0. The results also indicated that the adsorption process was exothermic and fit well with the pseudosecond-order kinetic model. Ag(I) desorption could reach 99.23% using 0.5 M thiourea-2.0 M HCl solution. (C) 2010 Elsevier B.V. All rights reserved.

Synthesis and evaluation of a thiourea-modified chitosan derivative applied for adsorption of Hg(II) from synthetic wastewater

In this work, a thiourea-modified chitosan derivative (TMCD) was synthesized through two steps, O-carboxymethylated first and then modified by a polymeric Schiff's base of thiourea/glutaraldehyde. The adsorption behavior of mercury (II) ions onto TMCD was investigated through batch method. The maximum adsorption capacity for Hg(II) was found to be 6.29 mmol/g at pH 5.0 and both kinetic and thermodynamic parameters of the adsorption process were obtained. The results indicated that adsorption process was spontaneous exothermic reaction and kinetically followed pseudo-second-order model. The adsorption experiments also demonstrated TMCD had high adsorption selectivity towards Hg(II) ions when coexisted with Cu(II), Zn(II), Cd(II) and Ca(II) in solution and it could be easily regenerated and efficiently reused. (C) 2010 Elsevier B.V. All rights reserved.

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase

We examined the effects of cofactors and DNA on the stability, oligomeric state and conformation of the human mitochondrial DNA helicase. We demonstrate that low salt conditions result in protein aggregation that may cause dissociation of oligomeric structure. The low salt sensitivity of the mitochondrial DNA helicase is mitigated by the presence of magnesium, nucleotide, and increased temperature. Electron microscopic and glutaraldehyde cross-linking analyses provide the first evidence of a heptameric oligomer and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleoside diphosphate. We find that single-stranded DNA binding occurs in the absence of cofactors and renders the mitochondrial DNA helicase more susceptible to proteolytic digestion. Our studies indicate that the human mitochondrial DNA helicase shares basic properties with the SF4 replicative helicases, but also identify common features with helicases outside the superfamily, including dynamic conformations similar to other AAA⁺ ATPases.

Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

Previous studies have shown that glycation of insulin occurs in pancreatic beta -cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limper haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I-125-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED50) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Retinal and choroidal responses to panretinal photocoagulation: an ultrastructural perspective

BACKGROUND: There have been few histological or ultrastructural studies of the outer retina and choriocapillaris following panretinal photocoagulation therapy. This investigation examines the long-term morphological effects of panretinal photocoagulation in two patients with type II diabetes who had received laser treatment more than 6 months prior to death.

METHODS: Regions of retina and choroid from each patient were fixed in 2.5% glutaraldehyde, dissected out and examined using light microscopy and scanning and transmission electron microscopy.

RESULTS: After removing the neural retina, scanning electron microscopy of non-photocoagulated areas of the eye cups revealed normal cobblestone-like retinal pigment epithelial (RPE) cells. Regions with laser scars showed little RPE infiltration into the scar area, although large rounded cells often appeared in isolation within these areas. Sections of the retina and choroid in burn regions showed a complete absence of the outer nuclear layer and photoreceptor cells, with the inner retinal layers lying in close apposition to Bruch's membrane. Non-photocoagulated regions of the retina and choroid appeared normal in terms of both cell number and cell distribution. The RPE layer was absent within burn scars but many RPE-like cells appeared markedly hypertrophic at the edges of these regions. Bruch's membrane always remained intact, although the underlying choriocapillaris was clearly disrupted at the point of photocoagulation burns, appearing largely fibrosed and non-perfused. Occasional choroidal capillaries occurring in this region were typically small in profile and had plump non-fenestrated endothelium.

CONCLUSIONS: This study outlines retinal and choroidal cell responses to panretinal photocoagulation in diabetic patients and demonstrates an apparent reduction in the capacity of these tissues to repair laser damage.

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.

Electroanalysis of urinary L-dopa using tyrosinase immobilized on gold nanoelectrode ensembles

The performance of an amperometric biosensor constructed by associating tyrosinase (Tyr) enzyme with the advantages of a 3D gold nanoelectrode ensemble (GNEE) is evaluated in a flow-injection analysis (FIA) system for the analysis of l-dopa. GNEEs were fabricated by electroless deposition of the metal within the pores of polycarbonate track-etched membranes. A simple solvent etching procedure based on the solubility of polycarbonate membranes is adopted for the fabrication of the 3D GNEE. Afterward, enzyme was immobilized onto preformed self-assembled monolayers of cysteamine on the 3D GNEEs (GNEE-Tyr) via cross-linking with glutaraldehyde. The experimental conditions of the FIA system, such as the detection potential (−0.200 V vs. Ag/AgCl) and flow rates (1.0 mL min−1) were optimized. Analytical responses for l-dopa were obtained in a wide concentration range between 1 × 10−8 mol L−1 and 1 × 10−2 mol L−1. The limit of quantification was found to be 1 × 10−8 mol L−1 with a resultant % RSD of 7.23% (n = 5). The limit of detection was found to be 1 × 10−9 mol L−1 (S/N = 3). The common interfering compounds, namely glucose (10 mmol L−1), ascorbic acid (10 mmol L−1), and urea (10 mmol L−1), were studied. The recovery of l-dopa (1 × 10−7 mol L−1) from spiked urine samples was found to be 96%. Therefore, the developed method is adequate to be applied in the clinical analysis.

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-Screen Printed Electrodes

A gold screen printed electrode (Au-SPE) was modified by merging Molecular Imprinting and Self-Assembly Monolayer techniques for fast screening cardiac biomarkers in point-of-care (POC). For this purpose, Myoglobin (Myo) was selected as target analyte and its plastic antibody imprinted over a glutaraldehyde (Glu)/cysteamine (Cys) layer on the gold-surface. The imprinting effect was produced by growing a reticulated polymer of acrylamide (AAM) and N,N′-methylenebisacrylamide (NNMBA) around the Myo template, covalently attached to the biosensing surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were carried out in all chemical modification steps to confirm the surface changes in the Au-SPE. The analytical features of the resulting biosensor were studied by different electrochemical techniques, including EIS, square wave voltammetry (SWV) and potentiometry. The limits of detection ranged from 0.13 to 8 μg/mL. Only potentiometry assays showed limits of detection including the cut-off Myo levels. Quantitative information was also produced for Myo concentrations ≥0.2 μg/mL. The linear response of the biosensing device showed an anionic slope of ~70 mV per decade molar concentration up to 0.3 μg/mL. The interference of coexisting species was tested and good selectivity was observed. The biosensor was successfully applied to biological fluids.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

The effect of age on the structure and compostition of the cell walls of Choanephora cucurbitarum

The effect of age on the structure and composition of isolated and purified cell walls from cultures of Choanephora cucurbitarum was investigated by microchemical analyses, visible and infrared spectrophotometry, x-ray diffractometry and electron microscopy. Qualitative evaluation revealed the presence of lipids, proteins, neutral sugars, strong alkali soluble sugars, chitin, chitosan and uronic acids in the cell walls of both the 1 and 7 day old cultures. As the mycelium aged, there was a slight but statistically significant increase in the protein content, and a pronounced rise in the chitin and neutral sugar constituents of the cell walls. Conversely, the decrease in the chitosan content during this period had the net effect of altering the chitin: chitosan ratio from near unity in the younger cultures, to a 2:1 ratio in the 7 day old cell wall samples. Glutaraldehyde-osmium fixed thin sections of the 1 day old vegetative hyphae of £. curbitarum revealed the presence of a monolayered cell wall, which upon aging became bilayered. Replicas of acid hydrolysed cell walls demonstrated that both the 1 and 7 day old samples possessed an outer layer which was composed of finely granular amorphous material and randomly distributed microfibrils. The deposition of an inner secondary layer composed of parallel oriented microfibrils in the older hypha was correlated with an increase in the chitin content in the cell wall. The significance of these results with respect to the intimate relationship between composition and structure is discussed.

X-ray diffraction studies of muscle : observations on the anterior byssus retractor muscle of Mytilus edulis and the insect flight muscle from Sarcophaga bullata

The interfilament spacing of the anterior byssus retractor muscle from Mytilus edulis was studied as the muscle was extended. It was found that variations in this spacing were very small and consistent with the hypothesis that the interfilament spacing was independent of the extension of the muscle. It was observed that the interfilament spacing was dependent on the osmolarity of the bathing medium. In concentrated solutions of the artificial seawater, the interfilament spacing decreased; while in dilute solutions of artificial seawater, it was observed that the interfilament spacing was increasing. X-ray diffraction patterns were obtained from fresh, and glutaraldehyde fixed, specimens of insect flight muscle from Sarcophaga bullata. There patterns were in general agreement with previous X-ray diffraction studies of insect flight muscle. A reflexion G at 93A was observed and interpreted as arising from diffraction in the mitochondria. Specimens of dried insect flight muscle produced a diffraction pattern consisting of arc and ring reflexions. This was interpreted as suggesting an ordered arrangement of cristae, in the mitochondria from these muscles.

Pigment orientation changes detected by low temperature linear dichroism spectroscopy: cold-hardening transition in phycobilisome-containing organisms

Low temperature (77K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. pee 6301, and cold-hardening in winter rye (Secale cereale L. cv. Puma). Samples were oriented for spectroscopy using the gel squeezing method (Abdourakhmanov et aI., 1979) and brought to 77K in liquid nitrogen. The linear dichroism (LD) spectra of Synechococcus 6301 phycobilisome/thylakoid membrane fragments cross-linked in light state 1 and light state 2 with glutaraldehyde showed differences in both chlorophyll a and phycobilin orientation. A decrease in the relative amplitude of the 681nm chlorophyll a positive LD peak was observed in membrane fragments in state 2. Reorientation of the phycobilisome (PBS) during the transition to state 2 resulted in an increase in core allophycocyanin absorption parallel to the membrane, and a decrease in rod phycocyanin parallel absorption. This result supports the "spillover" and "PBS detachment" models of the light state transition in PBS-containing organisms, but not the "mobile PBS" model. A model was proposed for PBS reorientation upon transition to state 2, consisting of a tilt in the antenna complex with respect to the membrane plane. Linear dichroism spectra of PBS/thylakoid fragments from the red alga, Porphyridium cruentum, grown in green light (containing relatively more PSI) and red light (containing relatively more PSll) were compared to identify chlorophyll a absorption bands associated with each photosystem. Spectra from red light - grown samples had a larger positive LD signal on the short wavelength side of the 686nm chlorophyll a peak than those from green light - grown fragments. These results support the identification of the difference in linear dichroism seen at 681nm in Synechococcus spectra as a reorientation of PSll chromophores. Linear dichroism spectra were taken of thylakoid membranes isolated from winter rye grown at 20°C (non-hardened) and 5°C (cold-hardened). Differences were seen in the orientation of chlorophyll b relative to chlorophyll a. An increase in parallel absorption was identified at the long-wavelength chlorophyll a absorption peak, along with a decrease in parallel absorption from chlorophyll b chromophores. The same changes in relative pigment orientation were seen in the LD of isolated hardened and non-hardened light-harvesting antenna complexes (LHCII). It was concluded that orientational differences in LHCII pigments were responsible for thylakoid LD differences. Changes in pigment orientation, along with differences observed in long-wavelength absorption and in the overall magnitude of LD in hardened and non-hardened complexes, could be explained by the higher LHCII monomer:oligomer ratio in hardened rye (Huner et ai., 1987) if differences in this ratio affect differential light scattering properties, or fluctuation of chromophore orientation in the isolated LHCII sample.

Marquage fluorescent des protéines pour étudier les enzymes protéolytiques solubles et immobilisées par la cartographie peptidique électrophorétique

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991.