Smart microsystems for cell manipulations

This thesis documents the design, manufacture and testing of a passive and non-invasive micro-scale planar particle-from-fluid filter for segregating cell types from a homogeneous suspension. The microfluidics system can be used to separate spermatogenic cells from testis biopsy samples, providing a mechanism for filtrate retrieval for assisted reproduction therapy. The system can also be used for point-of-service diagnostics applications for hospitals, lab-on-a-chip pre-processing and field applications such as clinical testing in the third world. Various design concepts are developed and manufactured, and are assessed based on etched structure morphology, robustness to variations in the manufacturing process, and design impacts on fluid flow and particle separation characteristics. Segregation was measured using image processing algorithms that demonstrate efficiency is more than 55% for 1 µl volumes at populations exceeding 1 x 107. the technique supports a significant reduction in time over conventional processing, in the separation and identification of particle groups, offering a potential reduction in the associated cost of the targeted procedure. The thesis has developed a model of quasi-steady wetting flow within the micro channel and identifies the forces across the system during post-wetting equalisation. The model and its underlying assumptions are validated empirically in microfabricated test structures through a novel Micro-Particle Image Velocimetry technique. The prototype devices do not require ancillary equipment nor additional filtration media, and therefore offer fewer opportunities for sample contamination over conventional processing methods. The devices are disposable with minimal reagent volumes and process waste. Optimal processing parameters and production methods are identified with any improvements that could be made to enhance their performance in a number of identified potential applications.

Aspects of the prevention and repair of chloride-induced corrosion of steel in concrete

Sodium formate, potassium acetate and a mixture of calcium and magnesium acetate (CMA) have all been identified as effective de-icing agents. In this project an attempt has been made to elucidate potentially deleterious effects of these substances on the durability of reinforced concrete. Aspects involving the corrosion behaviour of embedded steel along with the chemical and physical degradation of the cementitious matrix were studied. Ionic diffusion characteristics of deicer/pore solution systems in hardened cement paste were also studied since rates of ingress of deleterious agents into cement paste are commonly diffusion-controlled. It was found that all the compounds tested were generally non-corrosive to embedded steel, however, in a small number of cases potassium acetate did cause corrosion. Potassium acetate was also found to cause cracking in concrete and cement paste samples. CMA appeared to degrade hydrated cement paste although this was apparently less of a problem when commercial grade CMA was used in place of the reagent grade chemical. This was thought to be due to the insoluble material present in the commercial formulation forming a physical barrier between the concrete and the de-icing solution. With the test regimes used sodium formate was not seen to have any deleterious effect on the integrity of reinforced concrete. As a means of restoring the corrosion protective character of chloride-contaminated concrete the process of electrochemical chloride removal has been previously developed. Potential side-effects of this method and the effect of external electrolyte composition on chloride removal efficiency were investigated. It was seen that the composition of the external electrolyte has a significant effect on the amount of chloride removed. It was also found that, due to alterations to the composition of the C3A hydration reaction products, it was possible to remove bound chloride as well as that in the pore solution. The use of an external electrolyte containing lithium ions was also tried as a means of preventing cathodically-induced alkali-silica reaction in concretes containing potentially reactive aggregates. The results obtained were inconclusive and further practical development of this approach is needed.

Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2

Lipopolysaccharide (LPS), which generally activates Toll-like receptor 4 (TLR4), is expressed on commensal colonic bacteria. In a number of tissues, LPS can act directly on epithelial cells to increase paracellular permeability. Such an effect in the colon would have an important impact on the understanding of normal homeostasis and of pathology. Our aim was to use a novel primary culture of colonic epithelial cells grown on Transwells to investigate whether LPS, or Pam(3)CSK( 4), an activator of TLR2, affected paracellular permeability. Consequently, [(14)C]-mannitol transfer and transepithelial electrical resistance (TEER) were measured. The preparation consisted primarily of cytokeratin-18 positive epithelial cells that produced superoxide, stained for mucus with periodic acid-Schiff reagent, exhibited alkaline phosphatase activity and expressed TLR2 and TLR4. Tight junctions and desmosomes were visible by transmission electron microscopy. Basally, but not apically, applied LPS from Escherichia coli increased the permeability to mannitol and to a 10-kDa dextran, and reduced TEER. The LPS from Helicobacter pylori increased paracellular permeability of gastric cells when applied either apically or basally, in contrast to colon cells, where this LPS was active only from the basal aspect. A pan-caspase inhibitor prevented the increase in caspase activity caused by basal E. coli LPS, and reduced the effects of LPS on paracellular permeability. Synthetic Pam(3)CSK(4) in the basal compartment prevented all effects of basal E. coli LPS. In conclusion, LPS applied to the base of the colonic epithelial cells increased paracellular permeability by a mechanism involving caspase activation, suggesting a process by which perturbation of the gut barrier could be exacerbated. Moreover, activation of TLR2 ameliorated such effects.

Liposome-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Luminescent techniques for studying free radical production and cell viability in relation to cardiovascular disease and HRT

Epidemiological studies have suggested that hormone replacement therapy (HRT) offers protection from atherosclerosis, a precursor of cardiovascular disease (CVD), in postmenopausal women. There is good evidence that oxidation of low-density lipoprotein (LDL) by leucocyte-derived reactive oxygen species plays a key role in development of an atherosclerotic plaque. Therefore we have investigated whether the possible protection against CVD by HRT could be due to immunomodulation, specifically of free radical production. The study involves 2 approaches: I) analysing the production of free radicals by leucocytes from women on HRT, 2) investigating the effect of I7p-oestradiol and progesterone on cultured myeloid cells (HL60 and U937). Free radical production by leucocytes was determined using a recently developed bioluminescent assay. In the assay, Pholasin® emits light in the presence of free radicals produced by the NADPH oxidase system of leucocytes stimulated with PMA or fMLP. Cell viability was also investigated using a bioluminescent assay (Cell Titer-Glo®) in which cytosolic ATP levels were measured by the production of luminescence in the presence of Luciferin/Luciferase reagent. Studies of leucocytes from HRT patients showed considerable variation in free radical production, which appeared to be dependent on HRT regime. Studies on the cultured cells showed that there was no cell proliferation at low hormone concentrations, while high concentrations caused cytotoxicity. The effect of hormones on free radical production in this in vitro model system is currently being investigated. The results show that the effects of the hormones on cells of the immune system are very dose dependent, and that both beneficial and adverse effects may occur. In conclusion, luminescent techniques offer a valuable and sensitive approach to studying inflammatory and oxidative processes both in vivo and in vitro.

CD4+ T cell surface alpha enolase is lower in older adults

To identify novel cell ageing markers in order to gain insight into ageing mechanisms, we adopted membrane enrichment and comparison of the CD4+ T cell membrane proteome (purified by cell surface labelling using Sulfo-NHS-SS-Biotin reagent) between healthy young (n=9, 20-25y) and older (n=10; 50-70y) male adults. Following two-dimensional gel electrophoresis (2DE) to separate pooled membrane proteins in triplicates, the identity of protein spots with age-dependent differences (p<0.05 and >1.4 fold difference) was determined using liquid chromatography-mass spectrometry (LC-MS/MS). Seventeen protein spot density differences (ten increased and seven decreased in the older adult group) were observed between young and older adults. From spot intensity analysis, CD4+ T cell surface α-enolase was decreased in expression by 1.5 fold in the older age group; this was verified by flow cytometry (n=22) and qPCR with significantly lower expression of cellular α-enolase mRNA and protein compared to young adult CD4+ T cells (p<0.05). In an independent age-matched case-control study, lower CD4+ T cell surface α-enolase expression was observed in age-matched patients with cardiovascular disease (p<0.05). An immune-modulatory role has been proposed for surface α-enolase and our findings of decreased expression suggest that deficits in surface α-enolase merit investigation in the context of immune dysfunction during ageing and vascular disease.

Facile synthesis of polymer-peptide conjugates via direct amino acid coupling chemistry

Polymer-peptide conjugates (also known as biohy-brids) are attracting considerable attention as injectable materials owing to the self-assembling behavior of the peptide and the ability to control the material properties using the polymer component. To this end, a simple method for preparing poly(ethylene oxide)-oligophenylalanine polymer-peptide conjugates (mPEOm-F n-OEt) using isobutylchloroformate as the activating reagent has been identified and developed. The synthetic approach reported employs an industrially viable route to produce conjugates with high yield and purity. Moreover, the approach allows judicious selection of the precursor building blocks to produce libraries of polymer-peptide conjugates with complete control over the molecular composition. Control over the molecular make-up of the conjugates allows fine control of the physicochemical properties, which will be exploited in future studies into the prominent self-assembling behavior of such materials. © 2013 Wiley Periodicals, Inc.

Syntheses of aza analogs of kainoids

The kainoids are a class of non-proteinogenic pyrrolidine dicarboxylates that exhibit both excitatory and excitotoxic activities. These activities are a result of the ability of the kainoids to act as glutamate receptor agonists by activating ionotropic glutamate receptors. The parent of this group of compounds is α-kainic acid. Kainic acid is isolated from the seaweed Diginea simplex and has been used in Asian countries as a treatment for intestinal worms in children. In addition it is used extensively by neuropharmacologists for the study of glutamate receptors. Several years ago, the world's sole supplier of kainic acid discontinued this product. Since that time, other sources have appeared, however, the price of kainic acid remains significantly higher than it once was. We have thus been working on synthesizing aza analogs of kainoids which would be less costly but potentially potent alternatives to kainic acid via the dipolar cycloadditions of diazoalkanes with trans diethyl glutaconate. These 1, 3-dipolar cycloadditions yielded 2-pyrazolines or pyrazoles. The 2-pyrazolines may be precursors to aza analogs of kainoids. The regioselectivity of these 1, 3-dipolar cycloadditions and isomerization of the 1-pyrazolines to 2-pyrazolines was evaluated. Reductions of the 2-pyrazolines yielded aza analogs of kainoids.^ TMS diazomethane, due to the commercial availability, has been frequently used as a synthetic reagent in 1, 3-dipolar cycloadditions, particularly in the preparation of novel amino acid analogs. A survey of the recent literature indicates that the regioselectivity of the double bond isomerization of TMS substituted 1-pyrazolines is variable and at first glance, unpredictable. In an effort to develop a mechanistic rational for the isomerization which could account for the products obtained, a systematic survey of dipolar cycloadditions between TMS diazomethane and α, β-unsaturated dipolarophiles was undertaken. It was suggested that the steric demand of the dipolarophiles had a profound effect on both the relative stereochemistry of dipolar cycloaddition reactions of TMSCHN2 and the preferred direction of isomerization of the resulting 1-pyrazoline.^

Comparison of Common Field/Clinical Measures to Standard Laboratory Measures of Hydration Status

Context: Accurately determining hydration status is a preventative measure for exertional heat illnesses (EHI). Objective: To determine the validity of various field measures of urine specific gravity (Usg) compared to laboratory instruments. Design: Observational research design to compare measures of hydration status: urine reagent strips (URS) and a urine color (Ucol) chart to a refractometer. Setting: We utilized the athletic training room of a Division I-A collegiate American football team. Participants: Trial 1 involved urine samples of 69 veteran football players (age=20.1+1.2yr; body mass=229.7+44.4lb; height=72.2+2.1in). Trial 2 involved samples from 5 football players (age=20.4+0.5yr; body mass=261.4+39.2lb; height=72.3+2.3in). Interventions: We administered the Heat Illness Index Score (HIIS) Risk Assessment, to identify athletes at-risk for EHI (Trial 1). For individuals “at-risk” (Trial 2), we collected urine samples before and after 15 days of pre-season “two-a-day” practices in a hot, humid environment(mean on-field WBGT=28.84+2.36oC). Main Outcome Measures: Urine samples were immediately analyzed for Usg using a refractometer, Diascreen 7® (URS1), Multistix® (URS2), and Chemstrip10® (URS3). Ucol was measured using Ucol chart. We calculated descriptive statistics for all main measures; Pearson correlations to assess relationships between the refractometer, each URS, and Ucol, and transformed Ucol data to Z-scores for comparison to the refractometer. Results: In Trial 1, we found a moderate relationship (r=0.491, p<.01) between URS1 (1.020+0.006μg) and the refractometer (1.026+0.010μg). In Trial 2, we found marked relationships for Ucol (5.6+1.6shades, r=0.619, p<0.01), URS2 (1.019+0.008μg, r=0.712, p<0.01), and URS3 (1.022+0.007μg, r=0.689, p<0.01) compared to the refractometer (1.028+0.008μg). Conclusions: Our findings suggest that URS were inconsistent between manufacturers, suggesting practitioners use the clinical refractometer to accurately determine Usg and monitor hydration status.

Estudo sobre tratamento térmico e mudança superficial da diatomita de Punaú/RN para o desenvolvimento de um auxiliar de filtração

The diatomite is a natural material that has numerous applications due to changes in their physical and chemical properties after processing. It is currently used in the industry as a sound insulator , filter aid and industrial load . The filter material shall be inert chemical composition , which will diatomite confers a high commercial value and performance not found in other particulate materials , for this application. The diatomite surface undergoes changes after thermal treatment at high temperatures , from 800ºC , with properties that enable its application in the food , beverage , pharmaceutical , cosmetic and textiles . In this work , we developed a study on thermal treatment on natural diatomite to adapt their properties to the application as a filter aid . The heat treatments were performed in an open oven at temperatures of 800ºC , 1000ºC and 1200ºC for a time of 24 hours. Reagents were added in the constitution of the samples analyzed. The reagents used were sodium carbonate (Na2CO3 ) and sodium chloride (NaCl) . The samples were characterized by x - ray diffraction , x -ray fluorescence , scanning electron microscopy , analysis and particle size distribution , specific surface area by the BET method , and pore volume by BJH method. The results showed a reduction in porosity of the material as well as a significant increase in specific surface area after heat treatment and the reactants in the ratio of 3 wt%. The diatomaceous earth , after heat treatment , undergone changes in its coloration , varying in white, cream and beige , which directly interferes with the speed of filtration materials process. All results obtained before and after heat treatment of the material with the values obtained for samples already used industrially , Brazilian and American industry , which were characterized using the same test methods performed with the samples in the study were compared and showed promising efficiency when material studied in the region of Punaú - RN , after processing , reagent addition and heat treatment, as an element in the composition of filter .

Degradação fotocatalítica oxidativa do fenol utilizando carvão obtido da pirólise de diferentes biomassas

The modern industrial progress has been contaminating water with phenolic compounds. These are toxic and carcinogenic substances and it is essential to reduce its concentration in water to a tolerable one, determined by CONAMA, in order to protect the living organisms. In this context, this work focuses on the treatment and characterization of catalysts derived from the bio-coal, by-product of biomass pyrolysis (avelós and wood dust) as well as its evaluation in the phenol photocatalytic degradation reaction. Assays were carried out in a slurry bed reactor, which enables instantaneous measurements of temperature, pH and dissolved oxygen. The experiments were performed in the following operating conditions: temperature of 50 °C, oxygen flow equals to 410 mL min-1 , volume of reagent solution equals to 3.2 L, 400 W UV lamp, at 1 atm pressure, with a 2 hours run. The parameters evaluated were the pH (3.0, 6.9 and 10.7), initial concentration of commercial phenol (250, 500 and 1000 ppm), catalyst concentration (0, 1, 2, and 3 g L-1 ), nature of the catalyst (activated avelós carbon washed with dichloromethane, CAADCM, and CMADCM, activated dust wood carbon washed with dichloromethane). The results of XRF, XRD and BET confirmed the presence of iron and potassium in satisfactory amounts to the CAADCM catalyst and on a reduced amount to CMADCM catalyst, and also the surface area increase of the materials after a chemical and physical activation. The phenol degradation curves indicate that pH has a significant effect on the phenol conversion, showing better results for lowers pH. The optimum concentration of catalyst is observed equals to 1 g L-1 , and the increase of the initial phenol concentration exerts a negative influence in the reaction execution. It was also observed positive effect of the presence of iron and potassium in the catalyst structure: betters conversions were observed for tests conducted with the catalyst CAADCM compared to CMADCM catalyst under the same conditions. The higher conversion was achieved for the test carried out at acid pH (3.0) with an initial concentration of phenol at 250 ppm catalyst in the presence of CAADCM at 1 g L-1 . The liquid samples taken every 15 minutes were analyzed by liquid chromatography identifying and quantifying hydroquinone, p-benzoquinone, catechol and maleic acid. Finally, a reaction mechanism is proposed, cogitating the phenol is transformed into the homogeneous phase and the others react on the catalyst surface. Applying the model of Langmuir-Hinshelwood along with a mass balance it was obtained a system of differential equations that were solved using the Runge-Kutta 4th order method associated with a optimization routine called SWARM (particle swarm) aiming to minimize the least square objective function for obtaining the kinetic and adsorption parameters. Related to the kinetic rate constant, it was obtained a magnitude of 10-3 for the phenol degradation, 10-4 to 10-2 for forming the acids, 10-6 to 10-9 for the mineralization of quinones (hydroquinone, p-benzoquinone and catechol), 10-3 to 10-2 for the mineralization of acids.

Desenvolvimento de métodos para quantificação de benzilpenicilina em medicamentos veterinários e seus resíduos em leite bovino e caprino por UFLC-DAD

The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.

Desenvolvimento de métodos para quantificação de benzilpenicilina em medicamentos veterinários e seus resíduos em leite bovino e caprino por UFLC-DAD

The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.

Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.