Made available in DSpace on 2016-08-19T12:56:35Z (GMT). No. of bitstreams: 1 DARLAN FERREIRA DA SILVA.pdf: 808482 bytes, checksum: cec93c6177a364a96efd7df7673d26a0 (MD5) Previous issue date: 2010-10-11 / Organophosphate insecticides, especially those based on the active ingredient methyl parathion, have been extensively used in crop protection of rice in the State of Maranhão. In this work, an optimized chromatographic method for determination of the methyl parathion insecticide by using solid phase microextraction in a confined environment (headspace) followed by analysis employing gas chromatography with mass spectrometric detection (HS-SPME-GC/MS). Electroanalytical methods using amperometric biosensors were also developed and used to analyze the composite samples of fresh rice. The optimized method using SPME resulted in satisfactory results for sensitivity (limit of detection: 0.026 mg/L-1), precision (coefficients of variation between 6.1 and 22.4%, 8.8% and 32.5 and 19.7 and 24.6%, for milled rice samples, rice with shells and full rice, respectively) and accuracy (recoveries ranging from 73.2 to 90% for milled rice samples, from 88.7 to 89.5%, for paddy rice samples and from 59.5 to 60.6% for full rice samples). In the case of biosensors, these have been built based on acetylcholinesterase enzyme (AChE), commercially obtained or genetically modified, having the sensors prepared with carbon paste modified with TCNQ (tetracyanoquinodimethane) mediator. Better sensitivities were observed with sensors based on AChE enzymes extracted from Drosophila melanogaster. Detection limits of 10.0, 0.05 and 0.001 μg.L-1 were found for the sensors based on AChE (ee), AChE (eb) and mutant enzymes (AChE (Dros) B08 e B12), respectively. The efficiency of the detection method of the methyl parathion insecticide in rice samples without any previous treatment was evidenced by an average recovery of 103.9%, 101%, 102.3% and 105.6%, for the sensors prepared with AChE (ee), AChE (eb), AChE (Dros) B08 and B12 enzymes, respectively. In general, the chromatographic technique used was practical and efficient for direct detection of insecticide residues in rice samples in a wider range of concentration (0.1 a 1 mg.Kg-1). Since the biosensor was more sensitive and effective in smaller amounts (0.0005 a 0.01 mg.Kg-1) of such insecticide in rice, considering the additional advantage of speed and decreasing cost of the analytical technology. / Os inseticidas organofosforados, em especial aqueles à base do princípio ativo paration metílico, têm sido extensivamente usados na proteção de cultivos de arroz no Estado do Maranhão. Neste trabalho, foi otimizado um método cromatográfico para determinação do inseticida paration metílico usando microextração em fase sólida em ambiente confinado (headspace), seguido de análise empregando cromatografia a gás com detecção por espectrometira de massas (HS-SPME-GC/MS). Métodos eletroanalíticos empregando biossensores amperométricos também foram desenvolvidos, tendo sido empregados para análise do composto em amostras de arroz in natura. O método otimizado empregando SPME resultou em resultados satisfatórios quanto à sensibilidade (limite de detecção: 0,026 mg/L-1), precisão (coeficientes de variação entre 6,1 e 22,4%; 8,8 e 32,5% e 19,7 a 24,6%, para as amostras de arroz polido, arroz com casca e arroz integral respectivamente) e exatidão (recuperações na faixa de 73,2 a 90%, para as amostras de arroz polido; de 88,7 a 89,5 %, para as amostras de arroz com casca e de 59,5 a 60,6%, para as amostras de arroz integral). No caso dos biossensores, estes foram construídos à base de enzimas acetilcolinesterase (AChE), comerciais e geneticamente modificadas, tendo sido os sensores preparados com pasta de carbono modificada com o mediador TCNQ (tetracianoquinodimetano). Melhores sensibilidades foram verificadas com sensores preparados à base de enzimas AChE extraídas da Drosophila Melanogaster. Limites de detecção de 10; 0,05 e 0,001μg.L-1 foram encontrados para os sensores preparados com as enzimas AChE (ee), AChE (eb) e mutantes (AChE (Dros) B08 e B12), respectivamente. A eficiência do método de detecção do inseticida paration metílico em amostras de arroz sem nenhum prévio tratamento foi comprovada pelas recuperações médias de 103,9%; 101 %; 102,3% e 105,6 % para os sensores preparados com as enzimas AChE (ee), AChE (eb), AChE (Dros) B08 e B12 respectivamente. De modo geral, a técnica cromatográfica empregada foi prática e eficiente para detecção direta de resíduos do inseticida em amostras de arroz, em uma faixa maior de concentração (0,1 a 1 mg.Kg-1). Já os biossensores foram mais sensíveis e eficientes em teores menores (0,0005 a 0,01 mg.Kg-1) do inseticida no arroz, considerando ainda a vantagem adicional da rapidez e menor custo da técnica analítica.

Made available in DSpace on 2016-08-19T12:56:39Z (GMT). No. of bitstreams: 1 Dissertacao Marconiel.pdf: 1507450 bytes, checksum: 7ab99051017cb605155d971d0a8bc70b (MD5) Previous issue date: 2009-01-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Honey is a product of proven therapeutic and nutritional properties, and which may act as an excellent bioindicator, given the way the bees gather and process the raw material used in its preparation. The monitoring of pesticide residues in honey is important both to control product quality in domestic and international markets, and in assessing the potential for contamination in areas near agricultural crops. Considering these facts, we developed a method for analyzing carbamate insecticides (carbaryl and carbofuran), organochlorine (aldrin and dieldrin), organophosphates (dimethoate and parathion methyl) and pyrethroid (permethrin), and the group of anilides herbicide (propanil) and fenoxiácidos (2,4-D) in honey. Hence, in this work the analytical conditions were established for the proper determination of residues of pesticides cited using the technique of solid phase extraction and liquid chromatography with UV detection with a diode array. The study aimed to compare different analytical conditions for the determination of pesticides, looking for better separation, sensitivity, precision and accuracy in the determination of pesticide residues in samples of honey bees. Standard solutions at concentrations ranging from 1.0 to 20.0 μ g mL-1 were evaluated in different mobile phases, using a C18 column (250 x 4.6 mm id, 5 μ m). The mobile phase composition more suitable was acetonitrile: water at pH 3.0 (acidified with acetic acid) in gradient mode, for monitoring the compounds at a wavelength of 220 nm. In the established conditions, except for aldrin and dieldrin and dimeotate, it was observed recoveries between 75.26% and 113.58%, with variation Coefficients Between 2.16% and 11.31% for concentration levels raging from 0:10 to 1.00 μ gg-1 (correlation coefficients between 0.9978 and 0.9998). Recoveries lower than 50% to the cited insecticides were observed when using ethyl acetate solvent extractor and the solvent elution by ethyl acetate, in such a way new studies can be done to obtain better recoveries for these compounds. / O mel é um produto de reconhecidas propriedades nutritivas e terapêuticas e, o qual pode atuar como um excelente bioindicador, dada à forma como as abelhas recolhem e processam a matéria-prima utilizada na sua elaboração. O monitoramento de resíduos de pesticidas no mel é importante, tanto para o controle de qualidade do produto nos mercados nacional e internacional, quanto na avaliação do potencial de contaminação em áreas próximas a cultivos agrícolas. Considerando tais fatos, foi desenvolvido um método para análise dos inseticidas carbamatos (carbaril e carbofurano), organoclorados (aldrin e dieldrin), organofosforados (dimetoato e parationa metílica) e piretróide (permetrina), além do herbicida do grupo das anilidas (propanil) e fenoxiácidos (2,4-D) em mel. Para tanto, neste trabalho foram estabelecidas condições analíticas para a adequada determinação dos resíduos dos citados pesticidas utilizando a técnica de extração em fase solida e cromatografia líquida com detecção espectrofotométrica com arranjo de diodos. O trabalho teve como objetivo comparar diferentes condições analíticas para a determinação dos pesticidas, buscando-se melhor separação, sensibilidade, precisão e exatidão na determinação de resíduos dos pesticidas em amostras de mel de abelhas. Soluções-padrão em concentrações variando de 1,0 a 20,0 μg mL-1 foram avaliadas em diferentes fases móveis, utilizando-se coluna C18 (250 x 4.6 mm d.i., 5 μm). A composição da fase móvel mais adequada foi acetonitrila:água em pH 3,0 (acidificada com ácido acético), em modo gradiente, monitorando-se os compostos em comprimento de onda de 220 nm. Nas condições estabelecidas, excetuando-se aldrin, dieldrin e dimetoato, foram observadas recuperações entre 75,26% a 113,58%, com coeficientes de variação entre 2,16% a 11,31%, para níveis de concentração entre 0,10 a 1,00 μg.g-1 (coeficientes de correlação entre 0,9978 e 0,9998). Recuperações inferiores a 50 % foram, entretanto, observadas para os citados inseticidas, utilizando-se solvente extrator acetato de etila e eluição com acetato de etila, sendo necessários novos estudos para obtenção de melhores recuperações para tais compostos.

No presente estudo, duas espécies de fungos isoladas do ambiente marinho tiveram seus extratos brutos ativos contra Staphylococcus aureus e Candida albicans para uma espécie de P. citrinum e, atividade citotóxica e contra Mycobacterium tuberculosis para uma espécie de P. oxalicum. Estas foram estudadas com a finalidade de otimizar suas condições de crescimento para produção de metabólitos secundários. Foram realizadas análises multivariadas utilizando planejamento fatorial fracionário. Foram obtidas duas condições ótimas de crescimento para as duas linhagens de Penicillium. O crescimento em maior volume em condições de cultura otimizadas para P. oxalicum permitiu observar a presença de três metabólitos secundários no extrato do meio de cultura. Dois puderam ser isolados e identificados: a meleagrina 52 e a oxalina 26. A metodologia utilizada para se obter uma maior quantidade de metabólitos secundários proporcionou, ainda, o incremento em cerca de 150% na área do pico da oxalina. A partir do crescimento em maior volume em condições de cultura otimizadas para P. citrinum foi possível observar presença de pelo menos onze diferentes metabólitos na análise dos extratos obtidos do meio de cultura. Foi possível identificar e isolar quatro compostos já conhecidos: a (8E)-1-(2,3-diidropirrol-1-il)-2-metildec-8-eno-1,3-diona 56; a 1-(2,3-diidropirrol-1-il)-2-metildecano-1,3-diona 58; a 2-((E)-hept-5-enil)-6,7,8,8a-tetraidro-3-metilpirrolo[2,1-b][1,3]oxazin-4-ona 59 e a citrinina 31. Também foram isolados dois novos alcalóides indólicos contendo um grupo nitro na molécula que foram nomeadas de citrinalinas A 60 e B 62. Foi realizado, também, um estudo com P. citrinum visando a maior produção das citrinalinas, que possibilitou o incremento na produção da citrinalina B. / In this study, two species of fungi isolated from the marine environment had their active extracts against Staphylococcus aureus and Candida albicans to a strain of P. citrinum, and activity cytotoxic and against Mycobacterium tuberculosis to a strain of P. oxalicum. This studied aims the optimization their growth conditions for the production of secondary metabolites. Multivariate analysis using a fractional factorial design were used to establish optimal growth conditions for both Penicillium strains. Two optimal growth conditions were obtained for both Penicillium strains. A largest growth volume of P. oxalicum using optimized conditions enabled the detection of three secondary metabolites in the culture media crude extract. Two of such compounds were isolated and identified: meleagrin 52 and oxaline 26. The methodology used to increase the production of secondary metabolites by P. citrinum enabled an increase of 150% in the peak area of oxaline. A largest growth volume of P. citrinum led to the detection of eleven different metabolites in the culture media. Four of these compounds were isolated and identified as the known (8E)-1-(2,3-dihydropyrrol-1-yl)-2-methyldec-8-ene-1,3-dione 56; the 1-(2,3-dihydropyrrol-1-yl)-2-methyldecane-1,3-dione 58; the 2-((E)-hept-5-enyl)-6,7,8,8a-tetrahydro-3-methylpyrrolo[2,1-b][1,3]oxazin-4-one 59 and citrinin 31. Two new indole alkaloids containing a nitro group were also isolated and identified, named citrinalins A 60 and B 62. A further study with P. citrinum aiming an enhanced production of citrinalins allowed a significant increase in the production of citrinalin B.

Orientadores: Helena Teixeira Godoy, Fabio Augusto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T13:02:51Z (GMT). No. of bitstreams: 1 Kowalski_ClaudiaHoffmann_D.pdf: 1521730 bytes, checksum: 5077a83f5292bcd2dd1b651ce2fc229c (MD5) Previous issue date: 2008 / Resumo: Um estudo sobre a contaminação do leite materno com bifenilas policloradas (PCB) foi desenvolvido objetivando um conhecimento mais amplo dessa problemática no nosso país. Para isso, amostras de leite foram coletadas em nove estados brasileiros e questionários contendo perguntas sobre hábitos alimentares, condições sócio-econômicas, locais de habitação entre outras foram aplicados às mães doadoras. A determinação dos PCB foi feita por Microextração em Fase Sólida no modo headspace (HS-SPME) combinada com Cromatografia Gasosa e Detector de Captura de Elétrons (GC-ECD). As etapas da SPME foram otimizadas univariadamente e através de planejamento experimental Doehlert. A análise de variância mostrou que a temperatura de extração e a força iônica do meio (estudada através da adição de NaCl) foram as variáveis que mais influenciaram nas quantidades de PCB extraídas. Em seguida, refinaram-se as condições experimentais através de uma abordagem neuro-genética (rede neural Bayesiana aliada a algoritmos genéticos) que identificou como o ponto experimental ótimo a seguinte condição: concentração de NaCl de 0,36 g mL-1, temperatura de extração de 95,0 °C, tempo de extração de 60 minutos e adição de 210 µL de metanol. Após a otimização, o método foi validado através do estudo das figuras de mérito e apresentou-se linear na faixa estudada (1 a 16 µg L-1) com r > 0,9884, além de boa precisão (RSD < 12 %, n = 5), recuperação aceitável (71 a 127 %) e limites de quantificação entre 0,45 µg L-1 e 2,42 µg L-1. A fim de estabelecer correlações entre os resultados das amostras (expressos na forma de áreas cromatográficas) e os resultados obtidos nos questionários, fez-se uso da rede neural de Kohonen. Assim, foi possível verificar que nas cidades metropolitanas o acúmulo de PCB no leite é mais expressivo que em outras regiões. Por exemplo, em São Paulo 58 % das amostras estavam contaminadas com algum PCB, principalmente os congêneres 180 e 153. Outro resultado gerado pela rede foi que, em geral, as amostras de leite maduro (maior porcentagem de gordura) estavam mais contaminadas com PCB. O número de gestações também foi um fator importante a ser considerado, pois o estudo mostrou que o primeiro filho normalmente recebe uma dose maior de PCB do que os demais. Ainda, maior contaminação foi encontrada no leite de mães que moram nas proximidades de indústrias e/ou rios poluídos, corroborando com o fato de que esses compostos chegam facilmente ao meio ambiente e em seguida aos seres humanos / Abstract: A study about the contamination of the breast milk with polychlorinated biphenyls (PCB) was developed aiming a wider knowledge of this problematic in our country. For that, breast milk samples were collected in nine Brazilian states and questionnaires with questions regarding to food habits, social and economic conditions, places of dwelling between others were applied to the donors. The determination of the PCB was done by headspace Solid-Phase Microextraction (HS-SPME) combined with Gas Chromatography and Electron Capture Detector (GC-ECD). The SPME steps were optimized through univariated procedures and Doehlert experimental design. The analysis of variance showed that the temperature of extraction and the ionic strength of the media (studied by the addition of NaCl) were the most significant variables in the quantities of extracted PCB. After that, the experimental conditions were refined considering a neurogenetic approach (Bayesian neural network and genetic algorithms) and it was identified as the optimum experimental point the following condition: concentration of NaCl of 0.36 g mL-1, temperature of extraction of 95.0 °C, time of extraction of 60 minutes and addition of 210 µL of methanol. After the optimization, the method was validated through the study of the figures of merit. It showed linear in the studied range (1 to 16 µg L-1) with r > 0.9884, besides good precision (RSD < 12 %, n = 5), acceptable recuperation (71 to 127 %) and limits of quantization between 0.45 µg L-1 and 2.42 µg L-1. In order to establish correlations between the results of the samples (expressed as chromatographic areas) and the results obtained in the questionnaires, Kohonen neural network had been used. Thus, it was possible to check that in the metropolitan cities the accumulation of PCB in the milk is more expressive than in other regions. For example, in Sao Paulo state, 58 % of the samples were contaminated by some PCB, mainly the congener 180 and 153. Another result produced by the net was that the samples of hindmilk (higher fat percentage) were the most contaminated. The number of gestations also was an important factor to be considered, since the study showed that the first son normally receives a higher dosage of PCB compared to the others. Also higher levels of contamination was found in the milk of mothers who lived in the proximities of industries and/or polluted rivers, corroborating with the fact that these compounds are easily spread in the environment and consequently in the human beings / Doutorado / Doutor em Ciência de Alimentos

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-23T19:00:13Z No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Made available in DSpace on 2017-03-23T19:00:13Z (GMT). No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polímeros de impressão molecular (MIPs) foram sintetizados e aplicados como adsorventes na técnica de extração em fase sólida (EFS). O método de polimerização por precipitação foi utilizado para a síntese dos polímeros, devido à simplicidade de preparo, altos rendimentos e obtenção de partículas mais uniformes, devido a não trituração do polímero. O MIP foi sintetizado com ácido metacrílico (MAA) como monômero funcional, trimetacrilato de trimetilolpropano (TRIM) e dimetacrilato de etilenoglicol (EDMA) como agentes de reticulação e o cloridrato de amilorida (AMI) foi escolhido como molécula-molde. Diferentes proporções de MAA, TRIM, EDMA, volume e tipo de solvente foram utilizadas para ajuste das condições ideais de síntese. Os MIP foram avaliados quanto à capacidade de adsorção comparando-se a polímeros sintetizados na ausência da molécula-molde (NIP, polímeros não impressos). O solvente de elevada polaridade empregado na síntese (THF:MeOH:H2O) permitiu o emprego da técnica para moléculas polares como AMI. O controle no volume de solvente permitiu a obtenção de partículas maiores, de modo que a EFS foi realizada em condições usuais, o que confere um potencial para aplicação dessa técnica de polimerização na preparação de adsorventes para EFS. O polímero que apresentou maior capacidade adsortiva no ensaio realizado em tampão citrato-acetato pH 6,5 foi o MIP/NIP 12 (AMI:MAA:TRIM 1:8:10), com uma taxa média de adsorção de 83 e 88% para NIP e MIP, respectivamente. A adsorção foi elevada devido a interação iônica entre MAA e AMI promovida pelo controle de pH, porém foi não específica. O polímero MIP/NIP 12 foi aplicado como adsorvente na EFS, onde a recuperação de AMI foi avaliada nos resíduos de carregamento e eluição com solventes. O carregamento com tampão citrato-acetato pH 6,5 foi o ideal, favorecendo a interação iônica do polímero com o analito. A eluição total de AMI do cartucho somente ocorre após lavagem com o solvente na presença de ácido, que protona os grupos carboxila do polímero, rompendo assim a interação iônica com o analito / Molecularly imprinted polymers (MIPs) were synthesized and applied as adsorbents in solid-phase extraction technique (SPE). The polymers have been synthesized by precipitation polymerization method because of its simplicity, high yields and good control of final size and shape of particles. MIP was synthesized using methacrylic acid (MAA) as functional monomer, trimethylolpropane trimethacrylate (TRIM) and ethyleneglycol dimethacrylate (EDMA) as cross-linker and amiloride hydrochloride (AMI) was chosen as template. Different ratios of MAA, TRIM and EDMA, volume and type of solvent were used to adjust the optimal synthesis conditions. The MIP were tested for adsorption capacity compared to the polymers synthesized in the absence of template molecule (NIP, non-imprinted polymers). The polar solvent mixture used (THF:MeOH:H2O) allowed the synthesis of MIP of polar molecules as AMI. The solvent volume control afforded the larger particles so the SPE was performed in the usual conditions, giving a potential application for this polymerization technique in the preparation of adsorbents for SPE. The polymers with higher adsorption capacity at the test performed in citrateacetate buffer pH 6,5 was MIP/NIP 12 (AMI:MAA:TRIM 1:8:10) with adsorption rate of 83 and 88% for NIP and MIP, respectively. The recognition of MIP was due to ionic interaction between MAA and AMI promoted by pH control, but was not specific. The polymer MIP/NIP 12 was used as a solid-phase extraction sorbent and the recoveries of AMI was evaluated using different loading and elution conditions. The loading with buffer citrate-acetate pH 6,5 was optimal, due to ionic interaction of the polymer with the analyte. Total elution of AMI bound to the polymers only occurs after washing with a acid-containing solvent, because of protonation of the carboxyl groups of the polymer and disrupting the ionic interaction with the analyte

This work is focused on the occurrence of surface-active substances in the environment. It deals with the development and optimization of methods for the determination of selected surfactants in samples from rivers and from influent and effluent of wastewater treatment plants. Representatives of anionic, nonionic and zwitterionic surfactants were selected as target compounds. Based on optimization of analytical methods and their pilot testing abroad on samples of surface water, the selection of analytes for the conditions in the Czech Republic were corrected and operatively expanded. For the final analysis liquid chromatography with mass spectrometry was used. Analysis of cationic surfactants was realized using liquid chromatograph with UV-VIS detector. This optimized method for analysis of anionic surfactants was applied to real samples. Samples of surface running water were from the River Thurso in Scotland and then from watercourses in the catchment area of Moravia river were collected. In addition to the grab sampling also continuous weekly sampling of water from Tvaroženský potok and Litava was realized. Waste water was collected at inflow and outflow of three wastewater treatment plants (WWTP) in South Moravia (Brno – Modřice, Břeclav and Hodonín).

Les nanomatériaux combinant des fonctions de ciblage, d'imagerie, de thérapie et de détection font l'objet de nombreuses recherches dans le domaine de la santé. Les travaux présentés dans cette thèse concernent la multi‐fonctionnalisation de nanoparticules (NPs) par un procédé de synthèse supportée. Le support solide développé dans cette étude est constitué d'un matériau poreux en verre sur lequel sont greffées de manière temporaire des nanoparticules de silice. La fonctionnalisation de la surface des nanoparticules a été réalisée de façon automatisée par une chimie de synthèse dite aux phosphoramidites. Dans un premier temps, cette technique a permis d'obtenir des densités de greffage de l'ordre de 5000 à 7000 oligonucléotides par nanoparticule, ce qui représente une fonctionnalisation 10 à 20 fois supérieure à celles obtenues par des méthodes de greffage en solution. Les brins d'ADN synthétisés sur les NPs ont montré une bonne accessibilité pour l'hybridation avec un brin d'ADN complémentaire, ouvrant la voie à des applications thérapeutiques ou à l'intégration de ces objets dans des systèmes de détection. La deuxième partie de ces travaux est consacrée à la vectorisation d'une protéine thérapeutique, le G‐CSF (facteur de croissance de colonies de granulocytes), par des nanoparticules présentant également des propriétés d'imagerie. Ces nanovecteurs thérapeutiques ont montré des propriétés de stimulation cellulaire in vitro et de ciblage de la rate, organe réservoir de neutrophiles, in vivo. Enfin il a été démontré que la modification de NPs sur support ouvre des perspectives intéressantes pour la préparation d'assemblages complexes de nanoparticules (dimères et NPs dissymétriques) / Nanomaterials combining targeting, imaging, therapy and sensing properties are of growing interest for biomedical applications. The work reported in this thesis concerns nanoparticle (NP) multifunctionalization by solid phase synthesis. The solid support developed in this study is composed of a porous glass material on which silica NPs are temporarily grafted. Nanoparticle surface functionalization was performed by automated synthesis using phosphoramidite chemistry. Firstly, high surface loadings from 5000 to 7000 oligonucleotides per NP were achieved, representing a functionalization 10 to 20‐fold greater than those obtained by coupling methods in solution. DNA strands synthesized on NPs showed a good accessibility for hybridization with a complementary DNA strand, paving the way for therapeutic applications or integration of these objects in detection systems. The second part of this work was devoted to the vectorization of a therapeutic protein, GCSF (Granulocyte‐Colony Stimulating Factor) by nanoparticles that also exhibited imaging properties. These therapeutic nanocarriers showed cell stimulating properties in vitro and spleen targeting, which is a reservoir of neutrophils, in vivo. Finally, it was demonstrated that the solid phase modification of NPs opens interesting perspectives for the production of complex nanoparticle assemblies (dimers and asymmetric NPs)

Cette thèse décrit la synthèse de nanoparticules de polymères à empreintes moléculaires (MIP, de l’anglais molecularly imprinted polymer) pour la reconnaissance de protéines, par une approche de synthèse en phase solide. Les polymères à empreintes moléculaires sont des récepteurs biomimétiques synthétisés sur mesure par un processus de nanomoulage du polymère autour de la molécule unique. Ils possèdent ainsi des cavités de reconnaissance spécifiques pour leur molécule cible. La technique de l'impression moléculaire pour les petites molécules cibles est bien établie, alors que l'impression de protéines reste encore un défi en raison de la flexibilité et complexité de leur structure native et de leurs nombreux sites fonctionnels, mais aussi en raison de leur faible stabilité dans des conditions inhabituelles. Par conséquent, une approche de synthèse en phase solide a été développée ici où la protéine est immobilisée sur un support avant la synthèse de nanoparticules hydrosolubles de MIP par polymérisation radicalaire. Les MIPs obtenus ont des affinités comparables à celles des anticorps, et des réactivités croisées faibles. Ils possèdent des avantages tels qu'une stabilité meilleure, un coût plus faible et peuvent potentiellement être régénérés et réutilisés, devenant ainsi des alternatives prometteuses aux anticorps naturels. Nous avons fabriqué des MIPs contre des protéases à sérine, telles la trypsine et la kallikréine, mais aussi contre un épitope peptidique de la protéine gp41 du VIH. Des nanogels de MIP thermosensibles ont été synthétisés dans un réacteur sous la forme d’une colonne thermostatée ou une boîte de Pétri, par polymérisation radicalaire initiée par voie thermique ou photochimique. Un simple changement de la température permet de libérer les MIPs de la protéine immobilisée. Ces MIPs sont hydrosolubles en fonction de la température et ont un diamètre inférieur à 100 nm. Leur affinité pour leur cible est élevée, avec un Kd du nano ou picomolaire. Ces 'anticorps synthétiques' ont été appliqués dans des tests d'adsorption sur microbalance à cristal de quartz, mais également comme 'chaperons synthétiques'. Des études préliminaires de la protection des protéines d'une dénaturation thermique ou par un pH défavorable ont été effectuées. L'utilisation d'un iniferter pour initier la photopolymérisation vivante du MIP a permis de synthétiser des nanogels de type core-shell. En introduisant des marqueurs fluorescents dans les MIPs, les tests d’immunoessai dans des fluides biologiques ont été démontrés, ce qui indique le grand potentiel de ces MIPs dans le diagnostic clinique. En conclusion, nous avons développé une nouvelle approche de synthèse de nanoparticules de MIP hydrosoluble ayant une haute affinité pour une protéine, utilisables à la place des anticorps dans des applications dans le monde réel tel que la détection de protéines biomarqueurs dans des échantillons complexes, et potentiellement comme principe actif in vivo. / This thesis describes the synthesis, by a solid-phase synthesis approach, of nanoparticles of molecularly imprinted polymers (MIPs) for the recognition of proteins. Molecularly imprinted polymers are biomimetic receptors synthesized by a nanomolding process of the polymer around single molecules. They therefore possess specific recognition cavities for their target molecule. The technique of molecular imprinting for small target molecules is well established, while protein imprinting remains a challenge due to the flexibility and complexity of their native structure and functional sites, but also because of their low stability under unusual conditions. Therefore, a solid-phase synthesis approach has been developed where the protein is immobilized on a support before the synthesis of water-soluble MIP nanogel particles by radical polymerization. The MIPs obtained have affinities comparable to those of antibodies, and low cross-reactivities. They have advantages such as better stability, lower cost, and can potentially be regenerated and reused, thus becoming promising alternatives to real antibodies. We have synthesized MIPs against serine proteases such as trypsin, and kallikrein, but also against a peptide epitope of the HIV gp41 protein. Thermosensitive MIP nanogels were synthesized in a thermostated column-type reactor or a petri dish, by thermally or photo-initiated radical polymerization. Their thermosensitivity allows the MIPs to be released from the immobilized protein by a simple temperature change. They are water-soluble as a function of temperature and have a diameter of less than 100 nm. Their affinity for their target is strong, with a Kd in the nano or picomolar range. These 'synthetic antibodies' have been applied in binding assays with quartz crystal microbalance, but also as 'synthetic chaperones'. Preliminary studies of the protection of proteins from thermal denaturation or from denaturation by an unfavorable pH have been carried out. The use of an iniferter to initiate the living photopolymerization of MIP made it possible to synthesize nanogels of core-shell type. By introducing fluorescent markers into MIPs, immunoassay applications in biological fluids have been demonstrated, indicating the great potential of these MIPs in clinical diagnostics. In conclusion, we have developed a novel approach to the synthesis of soluble MIP nanoparticles having high affinity for a protein, usable in place of antibodies in real world applications such as the detection of biomarker proteins in complex samples, and potentially as an active principle in vivo.

Dans cette thèse, nous avons démontré la faisabilité de la préparation de polymères à empreinte moléculaires (MIP) hautement sélectifs pour la reconnaissance dans des matrices aqueuses complexes avec des applications dans les cosmétiques et en technologie alimentaire. Les MIP (de l'anglais molecularly imprinted polymers) sont des récepteurs synthétiques comparables aux anticorps, qui sont synthétisés par co-polymérisation de monomères fonctionnels et réticulants en présence d'un gabarit moléculaire. Leurs propriétés de reconnaissance moléculaire, associées à leur grande stabilité, robustesse mécanique, faible coût et leur synthèse facile les rendent extrêmement intéressants comme matériaux de capture sélective, avec des applications dans les séparations analytiques, la détection et la vectorisation des médicaments. Cependant, leur reconnaissance sélective dans des milieux aqueux reste toujours problématique et c'est l'une des raisons de leur expansion commerciale restreinte. Dans une première partie, nous avons développé un MIP fonctionnant en milieu aqueux pour son application comme ingrédient actif dans un déodorant. Les odeurs corporelles sont principalement dues à des acides gras volatils générés à partir de leurs précurseurs, des conjugués de glutamine par des enzymes hydrolytiques produites à partir de bactéries présentes sur la peau. La plupart des anti-transpirants et des déodorants actuellement commercialisés contiennent des sels d'aluminium et des agents antibactériens non spécifiques, respectivement. Cependant, l'utilisation extrêmement étendue de ces produits nécessite des solutions alternatives en ce qui concerne divers problèmes (environnement, respect de l'écosystème de la peau, toxicité, etc.). Pour cette raison, un MIP a été synthétisé pour capturer les précurseurs conjugués de glutamine afin qu'ils ne soient plus disponibles aux bactéries, empêchant ainsi leur transformation en composés malodorants. Afin de générer des liaisons sélectifs dans des environnements aqueux, un monomère à base d'amidinium qui peut former une interaction électrostatique stoechiométrique forte avec les groupes carboxyle sur le gabarit moléculaire a été synthétisé. Le MIP, mélangé dans une formulation dermo-cosmétique, pourrait capter sélectivement les précurseurs conjugués de glutamine, au milieu d'une multitude d'autres molécules présentes dans la sueur humaine. En outre, le MIP n’affecte pas les bactéries de la peau, ouvrant la voie à des déodorants innovateurs de nouvelle génération, moins problématiques pour la santé. Dans une deuxième partie, nous avons développé une procédure rapide et efficace basée sur l'extraction en phase solide à empreinte moléculaire (MISPE) pour la purification sélective de la bétanine et de son stéréoisomère l’isobétanine à partir d'extraits de betterave. La bétanine est un pigment naturel ayant un fort pouvoir antioxydant et dont les propriétés pharmacologiques sont de plus en plus étudiées. Ce pigment est actuellement utilisé comme simple colorant alimentaire. Dans notre étude, l'acide dipicolinique a été utilisé comme gabarit moléculaire pour la synthèse de MIP, en raison de sa similarité structurelle avec le groupe chromophore de la bétanine. Les procédures MISPE ont été optimisées permettant l'élimination presque complète des glucides issus de la matrice végétale ainsi que la majorité des protéines, ce qui permet d'obtenir un rendement élevé d'extraction de la bétanine / isobétanine en une seule étape. De plus, toute la procédure d'extraction a été réalisée dans des solvants respectueux de l'environnement, tels que l'éthanol ou l'eau. Pour conclure, nous sommes convaincus que ce travail pave le chemin au développement d'une nouvelle génération des MIP fonctionnant en milieu aqueux avec des propriétés de reconnaissance améliorées dans des environnements complexes, qui pourra s'appliquer également à d'autres domaines biotechnologiques et biomédicaux. / In this thesis, we have demonstrated the feasibility of preparing highly selective molecularly imprinted polymers (MIPs) for recognition in complex aqueous matrices with applications in cosmetics and food technology. MIPs are synthetic tailor-made receptors, with binding affinities and specificities comparable to those of natural antibodies. Their molecular recognition properties, combined with their high stability, mechanical robustness, low cost and easy synthesis make them extremely attractive as selective capture materials with applications in analytical and preparative separations, sensing and drug delivery, among others. However, their selective recognition in aqueous samples still remains problematic and is one of the reasons for their so far lilited commercial expansion. In the first part, we developed a water compatible MIP for its application as an active ingredient in a deodorant. Body odors are mainly due to volatile fatty acids generated from their glutamine conjugate precursors by hydrolytic enzymes from bacteria present on the skin. Most currently marketed anti-perspirants and deodorants contain, respectively aluminum salts and unspecific antibacterials. However, the extremely wide use of these products requires alternative solutions with regard to various problems (environmental, respect of skin ecosystem, toxicity, etc.). For this reason, a MIP was developed to capture the glutamine conjugate precursors so that they are no longer available to the bacteria, thus preventing their transformation to malodorous compounds. In order to generate binding selectivity in aqueous environments, an amidinium-based monomer which can form a strong stoichiometric electrostatic interaction with the carboxyl groups on the template, was synthesized. The MIP, blended in a dermo-cosmetic formulation, could capture selectively the glutamine precursors, amidst a multitude of other molecules present in human sweat. Furthermore, the MIP did not affect the skin bacteria, paving the way to an innovative and 'safer ' future-generation deodorant. In the second part, we developed a fast and efficient procedure based on molecularly imprinted solid­ phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from red beetroot extracts. Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. Dipicolinic acid was used as template for the MIP synthesis, because of its structural similarity to the chromophore group of betanin The MISPE procedures were optimized allowing the almost complete removal of carbohydrates and the majority of proteins, resulting in high extraction recovery of betanin / isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. To conclude, we believe that this study paves the way towards the development of a new generation of water compatible MIPs with improved recognition properties in highly complex aqueous environments, and should be applicable to other biotechnological and biomedical areas as well.

As a result of increased terrorist activity around the world, the development of a canine training aid suitable for daily military operations is necessary to provide effective canine explosive detection. Since the use of sniffer dogs has proven to be a reliable resource for the rapid detection of explosive volatiles organic compounds, the present study evaluated the ability of the Human Scent Collection System (HSCS) device for the creation of training aids for plasticized / tagged explosives, nitroglycerin and TNT containing explosives, and smokeless powders for canine training purposes. Through canine field testing, it was demonstrated that volatiles dynamically collected from real explosive material provided a positive canine response showing the effectiveness of the HSCS in creating canine training aids that can be used immediately or up to several weeks (3) after collection under proper storage conditions. These reliable non-hazardous training aids allow its use in areas where real explosive material aids are not practical and/or available.