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El Objetivo Principal de esta Tesis Doctoral es desarrollar nuevos métodos analíticos para la determinación de vitaminas, constituyentes esenciales en la dieta humana. La Cromatografía Líquida (LC) combinada en línea con Espectrometría de Masas (MS) es una de las metodologías analíticas más sensibles y selectivas, y la aplicación de esta técnica está creciendo rápidamente en análisis de alimentos. Dependiendo de las estructuras químicas y características analíticas de los grupos de vitaminas analizados, los métodos analíticos utilizados son hifenaciones de LC con detector de diodos (LC–DAD), fluorescencia (LC–Fl) y LC–MS. La Metodología utilizada se basa en la aplicación de nuevos métodos de preparación de la muestra. Los métodos clásicos presentan inconvenientes, tales como ser tediosos y largos, y requerir volúmenes grandes de muestra y disolventes tóxicos. Teniendo como objetivo la química verde, surgen las técnicas modernas de preparación de muestra, que ofrecen la posibilidad de automatización, son limpias y selectivas, tienen mayor rapidez y eficacia, son baratas, sencillas y sin disolventes tóxicos. Así, se ha empleado la Extracción Líquida Presurizada (PLE), que es una tecnología verde emergente. Implica la extracción usando disolventes líquidos a temperatura y presión elevadas, que aumentan la solubilidad y las propiedades de transferencia de masa. También se ha utilizado la Microextracción en Fase Líquida (LPME), basada en la extracción de los analitos en una fase líquida usando cantidades muy pequeñas de disolventes orgánicos. La Microextracción Dispersiva Líquido–Líquido (DLLME) utiliza un sistema de disolventes ternario. El bajo consumo de tiempo y disolventes orgánicos son dos de las mayores ventajas de esta técnica. Finalmente, se ha empleado la Extracción en Fase Sólida (SPE), que implica una partición líquido–sólido, donde la fase extractante es un adsorbente sólido, como los nanotubos de carbono. Así pues, los Objetivos de esta Tesis Doctoral han sido estudiar los acoplamientos instrumentales de LC con detectores DAD, Fl y MS y procedimientos verdes de tratamiento de la muestra para la determinación de diferentes vitaminas hidrosolubles y liposolubles en diversos tipos de alimentos. El trabajo se ha dividido en dos partes, en función del grupo de vitaminas analizado y el tipo de detector empleado. Las conclusiones obtenidas son las siguientes: 1. Se han desarrollado métodos analíticos para la determinación de vitaminas hidrosolubles en alimentos de diferentes tipos, nutracéuticos e infantiles. Las vitaminas son cobalaminas, tiamina y sus ésteres y benfotiamina. Son compuestos polares y se analizan por LC en fase reversa con fase estacionaria RP–Amida C16 y un encapsulante de trimetilsililo. Las fases móviles son mezclas de acetonitrilo y fosfato diácido de potasio. Se utiliza DAD para cobalaminas o detección fluorescente para tiamina y sus ésteres y benfotiamina utilizando derivatización por oxidación a tiocromo. 2. Se utilizaron dos procedimientos miniaturizados para el tratamiento de la muestra. Para las cobalaminas se utiliza SPE con nanotubos de carbono multipared y un sistema de flujo para la adsorción de los analitos y su elución con dimetilsulfóxido. En el caso de la tiamina y benfotiamina se utiliza DLLME. 3. La extracción de las vitaminas se lleva a cabo mediante ultrasonidos para cobalaminas o digestión ácida y extracción enzimática para destruir las formas fosforiladas de las vitaminas B. 4. En la segunda parte de la Tesis, se desarrollan métodos para determinación de las cuatro vitaminas liposolubles (A, D, E y K) en alimentos funcionales como zumos de frutas, alimentos vegetales y lácteos, y alimentos infantiles. Se analizan por LC en fase reversa o fase normal y diferentes acoplamientos de LC con DAD o fluorescencia y MS con interfases ESI y APCI acoplados a DLLME. La extracción se realiza utilizando disolventes orgánicos, mediante PLE o saponificación alcalina. / The main objective of this Thesis, is the development of new analytical methods for the determination of vitamins, essential constituents in the human diet. Liquid chromatography (LC) combined on-line with mass spectrometry (MS) is one of the more sensitive and selective analytical methodologies, and this technique is widely applied in food analysis. Depending on the chemical structures and analytical characteristics of the groups of vitamins analyzed, the analytical methods used are hyphenations of LC with diode array detector (LC–DAD), fluorescence (LC–Fl) and LC–MS. The methodology applied is based on the application of new methods for sample preparation. Classical methods have inconveniences, as being tedious and large, requiring great volumes of sample and toxic organic solvents. With the objective of green chemistry, modern sample preparation techniques appeared, which offer the possibility of automatization, are clean and selective, have higher speed and efficiency, are cheap, simple and without toxic solvents. Thus, Pressurized liquid extraction (PLE) has been used, being an emergent green technology, based on the extraction using liquid solvents at high temperature and pressure, which increase the solubility and the properties of mass transfer. Liquid-phase microextraction (LPME) was also used and is based on the extraction of the analytes in a liquid phase using very small amounts of organic solvents. Dispersive liquid-liquid microextraction (DLLME) uses a ternary solvent system. The low time and organic solvents consume are the two main advantages of this technique. Finally, Solid phase extraction (SPE) implies a liquid-solid partition, where the extractant phase is a solid sorbent, as multiwalled carbon nanotubes. Thus, the objectives of this Thesis were to study the instrumental coupling of LC with DAD, Fl and MS detection and green sample treatment procedures for the determination of several hydrosoluble and liposoluble vitamins in different type of foods. The study is divided in two parts, depending on the group of vitamins analyzed and the type of detector used. The following conclusions have been obtained: 1. Analytical methods are developed for the determination of hydrosoluble vitamins in different type of foods, nutraceuticals and baby foods. The vitamins are cobalamins, thiamine and its esters and benfothiamine. They are polar compounds and are analyzed by LC in reversed phase with a stationary phase RP–Amide C16 and an endcapping of trimethylsilyl. The mobile phases are mixtures of acetonitrile and potassium phosphate (pH 7). DAD detection is used for cobalamins or fluorescence for thiamine and its esters and benfothiamine using derivatization by oxidation to thiochrome. 2. Two miniaturized procedures have been developed for the sample treatment. For cobalamins, SPE was used with multiwalled carbon nanotubes and a flow system for the adsorption of the analytes and its elution with dimethylsulphoxide. DLLME was used for thiamine and benfothiamine. 3. The extraction of the vitamins was carried out using ultrasounds for cobalamins or acid digestion and enzymatic extraction for destroying the phosphorilated forms of the vitamins B. 4. In the second part of the Thesis, new methods are developed for the determination of the four liposoluble vitamins (A, D, E and K) in functional foods as fruit juices, vegetables, milk foods and baby foods. They are analyzed by LC in reversed phase or normal phase and different couplings of LC with DAD or fluorescence and MS with interfaces of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) coupled to DLLME. The extraction was carried out using organic solvents, by PLE or alkaline saponification.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-05-04T10:38:55Z No. of bitstreams: 1 texto completo.pdf: 2326379 bytes, checksum: 85cf46696829782c1197895cf2033483 (MD5) / Made available in DSpace on 2016-05-04T10:38:55Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2326379 bytes, checksum: 85cf46696829782c1197895cf2033483 (MD5) Previous issue date: 2015-02-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Citrato é um ligante polidentado muito importante na complexação de metais. No solo pode também ser usado como agente quelante em fitoextração. Desse modo, sua determinação em solução e em solo é muito importante para a otimização de sistemas de liberação controlada. Assim, foi desenvolvido um método espectrofotométrico inverso baseado na relação entre a concentração analítica de citrato no meio e a diminuição da absorbância do complexo. Foi estudado incialmente o complexo de Fe(II)-ortofenantrolina. Entretanto, mesmo otimizando as condições, a sensibilidade não foi adequada. Foi necessário trabalhar em uma faixa analítica formada por concentrações mais elevadas do analito. Desse modo, julgou- se adequado a seleção de outro complexo colorido para a obtenção de um método adequado para a determinação de citrato. Para isso estudou-se o complexo cromo(III)-EDTA. As curvas analíticas apresentaram comportamento quadrático de maneira que, na otimização, foi utilizada a sensibilidade analítica mínima como resposta. Para seleção dos termos significativamente diferente de zero foram utilizadas regressões lineares múltiplas tipo stepwise. Um planejamento fatorial 23 com ponto central foi realizado para avaliar a influência do pH, concentração de cromo(III) e EDTA no meio. Apenas as duas últimas variáveis foram significativas considerando os intervalos das variáveis. Um planejamento baseado na matriz de Doehlert, para essas duas variáveis com 3 pontos centrais foi realizado. Após aplicar uma expansão da matriz o ponto ótimo foi: concentração de Cr(III) 4,2 mmol L -1 e de EDTA, 5,1 mmol L-1 em pH 4,0. O método otimizado apresentou um bom ajuste R2=0,998 e desvio padrão dos resíduos igual 0,0085, na faixa analítica de 0,29 mmol L-1 a 5,0 mmol L-1. O desvio padrão relativo (N=7) foi inferior a 1,71%. Os limites de detecção e de quantificação foram 0,087 e 0,29 mmol L-1, respectivamente. Ensaios de adição e recuperação de citrato em amostras de solo apresentaram recuperações entre 98,0% e 122,0%. Foi proposto um sistema de liberação modulada por equilíbrio de precipitação. Foi avaliada a influência do pH e da concentração de cálcio na solução receptora do sistema de liberação modulada. A concentração de citrato na solução desse sistema foi determinada pelo método espectrofotométrico baseado na competição do ligante com o complexo Cr(III)- EDTA. Para caracterizar o mecanismo de liberação, foram aplicados modelos cinéticos de pseudo 2a ordem, Noyes-Whitney e difusão intrapartícula, sendo que o modelo de Noyes foi o que apresentou o melhor ajuste, constatando que a liberação ocorre por processos de difusão. Em todas as condições testadas, as concentrações de citrato atingiram o equilíbrio após 25 horas de liberação, observando-se diferentes taxas de liberação do citrato de acordo com as condições experimentais. A influência das variáveis foi avaliada por ANOVA, com nível de confiança de 95% usando a concentração de citrato no equilíbrio como resposta. O pH e a concentração de cálcio não influenciaram significativamente a taxa de liberação, assim como a interação entre os dois fatores. / Citrate is an important polydentate ligand for complexation of metals. It can also be used as chelating agent in phytoextraction in soil. Thus, determination in solution and soil is very important for the optimization of controlled release systems. Therefore, it was developed a inverse spectrophotometric method based on the relationship between citrate concentration and decreasing absorbance of the complex. It was initially studied Fe(II)-1,10-phenantroline complex. However, even with optimization, the analytical sensitivities obtained were not high enough, it was necessary to work in an analytical range with higher concentrations of analyte. Thus, it was considered appropriate to select another colored complex to obtain an adequate method for determination of citrate. Thereby, it was studied Cr(III)-EDTA complex. In the optimization, analytical curves presented quadratic behavior, it was used the minimum analytical sensitivity. For selection of terms significantly different from zero were used linear regressions multiple stepwise. A 23 factorial design was made with a central point to evaluate the influence of pH, chromium(III) concentration and EDTA. Considering the ranges of the variables, only the last two variables were significant. A planning based on Doehlert matrix for these two variables with 3 central points was performed. After to apply a matrix expanding the optimum concentration was: concentration of Cr(III) 4.2 mmol L -1 and EDTA, 5.1 mmol L -1 at pH 4.0. The optimized method display a good adjustment R2 = 0.998 and standard deviation of residues equal to 0.0085, analitycal range between 0.29 mmol L 1. -1 to 5.0 mmol L - The relative standard deviation (N = 7) was less than 1.71%. The limits of detection and quantification were 0.087 and 0.29 mmol L -1, respectively. Recovery citrate test in soil samples presented recoveries between 98,0% and 122,0%. One modulated delivery system by precipitation equilibrium has been proposed. The influence of pH and calcium concentration in the receptor solution modulated delivery system was evaluated. The concentration of citrate in the solution of the system was determined by the spectrophotometric method based on the competition between the ligand and Cr(III)-EDTA complex. To characterize the releasing mechanism were applied kinetic models of pseudo 2nd order, Noyes-Whitney and intraparticle diffusion, and the Noyes model showed the best fitting, noting that the release occurs by diffusion processes. In all conditions tested, the citrate concentrations reached equilibrium after 25 hours of release, observing different release rates of citrate according to the experimental conditions. The influence of the variables was evaluated by ANOVA with 95% confidence level using the citrate concentration at equilibrium as a response. The pH and the calcium concentration did not influence the rate of release as well as the interaction between the two factors.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-05-05T14:41:37Z No. of bitstreams: 1 texto completo.pdf: 1441456 bytes, checksum: 0626cd90b98a0b01ba9f9e17eee8596d (MD5) / Made available in DSpace on 2016-05-05T14:41:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1441456 bytes, checksum: 0626cd90b98a0b01ba9f9e17eee8596d (MD5) Previous issue date: 2015-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho foi preparado o HDL magnético calcinado MgZnAl-Fe e caracterizado por Espectroscopia no Infravermelho (IV), Difratometria de Raios- X (DRX), Microscopia Eletrônica de Varredura (MEV), Espectrometria de Absorção Atômica e Reflectância Difusa. O desempenho fotocatalítico do MgZnAl-Fe foi avaliado utilizando o corante alaranjado de metila (corante do tipo azo) como composto modelo. Estudos fotocatalíticos com o MgZnAl-Fe foram feitos empregando-se uma concentração inicial de 100 mg L -1 do corante alaranjado de metila e uma dose de 0,2 g L -1 do fototocalisador em um fotorreator de bancada. Este fotorreator foi constituído de uma lâmpada à vapor de mercúrio de 125 W sem o bulbo protetor, envolta por um cilindro de vidro (Filtro de corte para λ > 300 nm) que foi mergulhado em um recipiente contendo 300 mL de solução de alaranjado de metila. Estudos foram realizados variando a quantidade dos constituintes do MgZnAl-Fe, onde verificou-se que uma quantidade de zinco de (4,31% m/m) e de ferro (5,23% m/m) proporcionou a maior eficiência fotocatalítica. Verificou-se que a incorporação de óxido de ferro além de proporcionar propriedades magnéticas ao compósito diminuiu a sua energia de band gap. O menor valor de band gap possibilitou que o catalisador MgZnAl-Fe deslocasse a absorção de luz para maior comprimento de onda, sendo necessário menor energia para a sua atividade fotocatalítica. Assim o menor valor de band gap obtido para o MgZnAl-Fe possibilitou empregar nos experimentos radiação com comprimento de onda superior a 300 nm. O fotocatalisador MgZnAl-Fe mostrou-se bastante eficiente na degradação do alaranjado de metila, com uma remoção de 93,4 % de cor e 91,9% do Carbono Orgânico Total (COT), após 120 minutos de reação. Nos estudos de reutilização do MgZnAl-Fe houve uma redução na porcentagem de degradação do alaranjado de metila. A atividade fotocatalítica foi reduzida de 93,4% para 78,8, 74,6, 68,9 e 63,8 %, do primeiro ao quarto ciclo de reutilização, respectivamente, mostrando um bom potencial para aplicações práticas. Desta forma, o MgZnAl-Fe apresentou um desempenho satisfatório, sendo promissor para uso em processos de fotocatálise heterogênea visando a remoção de compostos orgânicos, além de ser facilmente removido de soluções aquosas por simples processos magnéticos. / In this work the calcinated MgZnAl-Fe magnetic LDH was prepared and characterized by Infrared Spectroscopy (IR), X-ray Diffraction (XRD), scanning electron microscopy (SEM), Atomic Absorption Spectrometry and Diffuse Reflectance. The photocatalytic performance of MgZnAl Fe was evaluated using methyl orange dye (azo dye) as the model compound. Photocatalytic studies with MgZnAl-Fe were performed using an initial concentration of 100 mg L -1 of methyl orange dye and a dose of 0.2 g L -1 of the photocatalyst in a bench photoreactor. The photoreactor consisted of a 125 W mercury vapor lamp without the bulb shield, surrounded by a glass cylinder (cuttoff for λ > 300 nm) which was dipped in a recipient containing 300 mL of the methyl orange solution. Studies were performed by varying the amount of each of the constituents of the MgZnAl-Fe, where it was found that the quantities of zinc (4.31% w/w) and iron (5.23% w/w) resulted in highest photocatalytic efficiency. It was found that the incorporation of iron oxide not only provided magnetic properties to the composite but also decreased its band gap energy. The lower band gap value permitted that the catalyst MgZnAl-Fe displace the absorption of light to a greater wavelength, requiring less energy for its photocatalytic activity. Thus the lower band gap value obtained for MgZnAl-Fe allowed for using radiation with a wavelength greater than 300 nm in the experiments. The MgZnAl-Fe photocatalyst proved to be very efficient in methyl orange degradation, with a 93.4% removal of color and 91.9% removal of Total Organic Carbon (TOC) after 120 minutes of the experiment. In studies of MgZnAl-Fe reutilization, there was a small reduction in the percentage of methyl orange degradation. The photocatalytic activity was reduced from 93.4% to 78.8, 74.6, 68.9 and 63.8% in the first to fourth reuse cycle, respectively, showing good potential for practical applications. Thus, the MgZnAl-Fe showed satisfactory performance, are promising for use in heterogeneous photocatalytic processes for removing organic compounds, and is also easily removed from aqueous solutions by a simple magnetic process.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-09-08T17:11:15Z No. of bitstreams: 1 texto completo.pdf: 1835695 bytes, checksum: bf8f63b434564b64b6fde68bc988c9e5 (MD5) / Made available in DSpace on 2016-09-08T17:11:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1835695 bytes, checksum: bf8f63b434564b64b6fde68bc988c9e5 (MD5) Previous issue date: 2015-12-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização do TiO 2 como fotocatalisador apresenta algumas desvantagens como os processos de recombinação elétrons-lacuna, os quais diminuem a sua atividade fotocatalítica; somente ser fotoativado pela radiação na região do UV; e tendência a agregação das suas nanopartículas. Desta forma, este trabalho consistiu-se no preparo de compósitos contendo TiO 2 de forma a obter uma melhoria em suas propriedades funcionais. A atividade fotocatalítica foi significativamente afetada pela quantidade de TiO 2 suportada no hidróxido duplo lamelar de MgAl (hidrotalcita). Várias amostras de fotocatalisadores foram preparadas com diferentes nanopartículas metálicas (Ag, Cu ou Ni). O compósito fotocatalisador mais eficiente foi obtido na forma calcinada, com a relação molar de 2:1:1 (Mg:Al:Ti) e o metal Ag (2%, m/m) sendo denominado por HTC/TiO 2 -Ag(2). Este compósito apresentou aproximadamente 100% de degradação e 85% de mineralização do fenol, em 300 min. Compósitos contendo TiO 2 associado ao óxido de zinco obtido pela calcinação do hidróxido duplo lamelar ternário de MgZnAl também foram preparados com sucesso. Os fotocatalisadores foram avaliados em diferentes razões molares de Zn 2+ /Mg 2+ sendo que o melhor fotocatalisador foi obtido para a razão molar Zn 2+ /Mg 2+ igual a 5% e foi denominado por TiO 2 /MgZnAl-5. Este fotocatalisador removeu aproximadamente 100% de fenol e 80% de COT, em 360 minutos. Os novos compósitos fotocatalisadores preparados neste trabalho apresentaram um sinergismo de efeito ocasionado pelo suporte de hidróxido duplo lamelar, presença das nanopartículas metálicas (Ag, Cu ou Ni) ou óxido de zinco os quais, associados à fotoatividade do TiO 2, resultaram na formação de um compósito com alta capacidade fotocatalítica. Os compósitos apresentaram boa estabilidade após 5 ciclos de reuso, capacidade de recuperação e menor agregação de suas nanopartículas, mostrando assim um potencial promissor para aplicações práticas. / Use of TiO 2 as photocatalyst has some disadvantages such as electron-hole recombination processes which reduces its photocatalytic activity; only be photoactivated by radiation in UV region; and the tendency of aggregation when used as nanopowder. This study consisted in preparing composites containing TiO 2 to obtain an improvement in their functional properties. Photocatalytic activity was affected significantly by the amount of TiO 2 supported on lamellar double hydroxide of MgAl (hydrotalcite). A series of photocatalysts were prepared with different metallic nanoparticles (Ag, Cu or Ni). The most efficient photocatalyst composite was obtained calcined form, molar ratio 2:1:1 (Mg:Al:Ti) and Ag metal (2 wt.%) denominated as HTC/TiO 2 -Ag(2). This composite showed highest efficiency degradation by removing about 100% phenol by analysis via HPLC-DAD and 85% TOC after 300 minutes. Composites containing TiO 2 combined with zinc oxide obtained by the calcination of ternary (MgZnAl) layered double hydroxides were also successfully prepared in this work. The photocatalysts were evaluated at different Zn 2+ /Mg 2+ molar rations and the most efficient photocatalyst was obtained at a Zn 2+ /Mg 2+ equal to 5% (TiO 2 /MgZnAl- 5). This photocatalyst removed approximately 100% phenol and 80% COT after 360 minutes. The new composite photocatalyst prepared in this study showed a synergy effect of the layered double hydroxide support, presence of metal nanoparticles (Ag, Cu or Ni) or zinc oxide associated with photoactivity of TiO 2 , resulting in formation of a composite with high photocatalytic capacity. The composites showed good stability after 5 cycles, reduced agglomeration and ease separation by sedimentation at the end of the processes, thus showing a promising potential for practical applications.

O presente trabalho, teve como objetivo, dar continuidade ao estudo apresentado na forma de dissertação de mestrado, utilizando a complexona EDTA, totalmente \"neutralizada\". na forma de Y4-. Na titulação de íons metálicos, a hidrólise acentuada deste ânion, Y4-, causa um aumento marcante de pH após o ponto estequiométrico. Esta variação de pH pode ser acompanhada potenciometricamente, utilizando um eletrodo de vidro, ou visualmente com um indicador ácido/base como a fenolftaleina, em substituição a indicadores metalocrômicos. Uma série de parâmetros foram considerados na preparação do reagente, sua armazenagem e padronização bem como os problemas ligados a percentagem de \"neutralização\". Vários equilíbrios foram considerados no desenvolvimento de um programa em linguagem BASIC, para interpretar a curva teórica da titulação de Mg2+ com EDTA \"tetraneutralizado\", na qual participam também espécies como MgHY-, antes do ponto estequiométrico. Um método complexométrico rápido e preciso foi desenvolvido com este titulante para determinar Mg2+ e SO42- simultaneamente (este por método indireto para o excesso de íons Ba2+) com viabilidade para ser aplicado em água do mar e em concentrados salinos. O método mostrou-se adequado, também, para a determinação da mistura Ca2+ e Mg2+, em substituição ao método clássico com tampão e indicador metalocrômico, eriocromo T. / This thesis has the purpose to give continuity to a former study from the master dissertation, about the use of the complexon EDTA, totally \"neutralized\", as the species Y4-. During the titration of metallic cations the marked hydrolysis of the y4- anion causes a marked pH increase after the stoichiometric point. This change of pH can be followed potentiometrically with the glass electrode or by visual end point indication by on acid/base indicator as phenolphtalein instead of metalochromic indicators. A serie of parameters were considered in the preparation of the titrant, its storage and standardization as well as problems related with the percent neutraIization\". Equilibria were considered and used in a computer program in BASIC language in order to interpret the theoretical titration curve. The species MgHY- was found in significant contribution before the end point. A fast and precise complexometric method was developed with this titrant for Mg2+ and SO42- simultaneously (this last by titration of excess of standard Ba2+ solution). The method has been found adequate to determine mixture of Ca2+ and Mg2+ instead of the classic method which uses a buffer and eriochrome T as indicator.

Los alérgenos y contaminantes alimentarios representan uno de los problemas de salud pública más extendidos del mundo. El monitoreo de posibles contaminantes resulta importante para la protección del consumidor así como también para controlar el cumplimiento de las regulaciones internacionales de etiquetado. La seguridad alimentaria sólo puede ser asegurada a través de la implementación de sistemas de control de calidad a lo largo de toda la cadena de producción desde la materia prima hasta el consumidor final. En este contexto, la disponibilidad de métodos rápidos, confiables y altamente sensibles resulta imperativo para su utilización como alarma en la detección de contaminantes, permitiendo una respuesta inmediata. Actualmente se dirigen grandes esfuerzos al desarrollo de metodologías simples, selectivas y económicas para la detección in situ de diferentes analitos en matrices complejas. En esta tesis se han desarrollado y evaluado nuevas estrategias inmunoanalíticas para la seguridad alimentaria, basadas en la integración de micro y nanopartículas así como bionanopartículas modificadas. Dos analitos de diferentes tamaños y características fueron estudiados como modelo: por un lado la gliadina, como alérgeno proteico, y por otro, la Salmonella, como ejemplo de patógeno alimentario. Se evaluaron diferentes formatos de inmunoensayo (competitivo y sándwich, directo e indirecto), aprovechando las ventajas del uso de partículas magnéticas como soporte sólido. Los resultados obtenidos fueron evaluados tanto por detección óptica como electroquímica, utilizando magneto inmunoensayos o inmunosensores, respectivamente. En todos los casos, las señales fueron obtenidas mediante conjugados con la enzima peroxidasa como marcadores ópticos o electroquímicos, analizando tanto el desempeño analítico como el efecto matriz a través de muestras alimentarias dopadas. En el caso de la integración de partículas magnéticas para la detección de gliadina, se desarrolló una estrategia competitiva que permitiera tanto la detección de la proteína nativa como también de fragmentos hidrolizados durante la producción de alimentos. Se obtuvieron excelentes límites de detección (del orden de μg L-1), muy por debajo de los 20 mg kg-1 establecidos por las regulaciones europeas para alimentos libres de gluten. Adicionalmente se lograron excelentes valores de recuperación al analizar muestras alimentarias dopadas como leche y cerveza libre de gluten. También se evaluó por otro lado, la integración en los inmunoensayos de bionanomateriales como los bacteriófagos, utilizando el P22 como modelo para la detección de Salmonella. Se diseñaron bionanopartículas híbridas a través de i) la inmovilización de bacteriófagos sobre micro y nanopartículas magnéticas, y ii) la modificación de la cápside con marcas de biotina. Los fagos altamente biotinilados obtenidos fueron empleados tanto para la marcación de bacterias, al unirse con marcadores fluorescentes, ópticos o electroquímicos conjugados a estreptavidina, así como para la captura al ser acoplados a partículas magnéticas modificadas con estreptavidina. Las bionanopartículas fueron caracterizadas mediante numerosas técnicas, como métodos de cultivo microbiológico, electroforesis, microscopía confocal de fluorescencia, así como microscopías electrónicas de barrido y transmisión. En todos los casos, se desarrollaron estrategias no competitivas integrando los fagos como marca o elemento bioreconocimiento. Se logró una considerable reducción del tiempo de detección de 3 a 5 días necesarios en las técnicas microbiológicas clásicas, a tan sólo 2-4 hs. Adicionalmente, se alcanzaron límites de detección excepcionales, lográndose detectar por debajo de 102 CFU mL-1 de Salmonella en muestras de leche, y tan sólo 1 CFU en 25 mL luego de 6 hs de preenriquecimiento. Por último, cabe destacar que en todos los casos se obtuvo mejor sensibilidad y menor efecto matriz utilizando detección electroquímica. En consecuencia, los biosensores desarrollados resultan en una herramienta prometedora para aplicaciones dentro del campo alimentario debido a su capacidad de detección rápida e in situ, así como también la facilidad de miniaturización y compatibilidad para producción en masa. / Food allergens and food contaminants represent an important health problem worldwide. Tracking and tracing of hazard-free food production chains has become important due to consumer-safety concerns and to fulfill international labeling regulations. Food safety can only be ensured through the enforcement of quality-control systems throughout the entire food chain from the incoming raw materials until the final consumer. In this context, the availability of rapid, reliable and highly sensitive methods is mandatory for their use as an “alarm” to rapidly detect potential contaminants and take an immediate action. Therefore, great efforts are directed towards the development of simple, selective and cost-efficient methodologies for the on-site detection of different target analytes in complex food samples. This dissertation addresses a comprehensive study and assessment of novel and rapid immunoanalytical strategies in different formats by the integration of micro and nanoparticles as well as hybrid bionanoparticles for food safety. Two analytes of different sizes and characteristics (single and multivalence targets) affecting food safety, were selected as a model: the small proteic allergen gliadin and the food-borne pathogen Salmonella. Different immunoassay formats were assessed (competitive and sandwich, direct and indirect) taking advantage of the outstanding features of magnetic micro and nanoparticles as solid support. The results obtained with the novel strategies were evaluated by a dual detection through optical and electrochemical readouts, using magneto immunoassays or magneto immunosensing approaches, respectively. In all cases the signals were obtained by a horseradish peroxidase (HRP) conjugate as the enzymatic optical and electrochemical reporter and the matrix effect and analytical performance were evaluated using spiked food samples. Regarding the integration of magnetic particles in the detection of food allergens such as gliadin, a competitive approach was developed to detect not only the native protein, but also the small gliadin fragments, being thus valid for both non-treated and hydrolyzed foodstuff. Excellent detection limits (in the order of μg L-1) were achieved, much lower than the EC recommendation of 20 mg kg-1 for glutenfree food. Furthermore the matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs, such as skimmed milk and gluten-free beer, obtaining excellent recovery values. The integration of bionanomaterials as bacteriophages into the immunoassays was also explored, using P22 bacteriophage as a model to detect Salmonella. Hybrid bionanoparticles were designed and evaluated by i) immobilizing the bacteriophages on magnetic micro and nanoparticles and ii) modifying the phage capsid proteins with biotin tags. The highly biotinylated bacteriophages were applied for bacterial tagging when coupled to fluorescent, optical or electrochemical streptavidin-conjugated reporters, as well as for bacteria capturing when attached to streptavidin-modified magnetic particles. The novel hybrid bionanoparticles were extensively characterized through a wide range of techniques, such as microbiological culturing methods, electrophoresis, confocal fluorescence microscopy, scanning electron microscopy and transmission electron microscopy. In all cases, non-competitive approaches were developed by integrating the bacteriophages both for the biorecognition and tagging of Salmonella Thyphimurium. All the strategies were able to considerably reduce the time of the bacteria detection from the 3-5 days required in the conventional microbiology techniques, to as low as 2-4 h. In addition, outstanding limits of detection were achieved, being able to detect below 102 CFU mL-1 of Salmonella in milk samples, and as low as 1 CFU in 25 mL after 6 h pre-enrichment. Finally, it should be pointed out that better sensitivity and lower matrix effect were achieved with the electrochemical detection. As a result, the developed biosensing devices are promising tools for food safety applications due to their rapid and on-site testing capability as well as the compatibility with miniaturization and mass fabrication technologies.

En la actualidad, está ampliamente aceptado en Europa que la investigación y la innovación son factores clave para reforzar la capacidad industrial y las perspectivas de negocios. La tecnología es necesaria para abordar los problemas del mundo, pero también lo es la investigación, que permite la aparición de tecnologías innovadoras. Por lo tanto, invertir en investigación e innovación es esencial para el desarrollo de soluciones para los desafíos sociales, como por ejemplo la seguridad alimentaria. En este contexto, surge el término KETS (Key Enabling Technologies), que permite unificar diferentes campos de la ciencia, tales como la nanotecnología, los materiales avanzados y la biotecnología, entre otros. Esto indica una clara convergencia de la tecnología en el desarrollo de nuevas soluciones. Por ejemplo, con el fin de ofrecer soluciones en seguridad alimentaria, la investigación en química analítica no está solamente basada en el desarrollo de estrategias para obtener información cualitativa y cuantitativa sobre la composición y naturaleza de las sustancias, sino que ésta ha convergido en un campo de investigación más aplicado y multidisciplinar, formando alianzas entre diferentes áreas de conocimiento a través de la ciencia. Esta nueva concepción abre la posibilidad de crear nuevos principios analíticos, procedimientos de detección automatizados o in-situ, así como sondas de detección específica o nuevos dispositivos sensores. La presente Tesis doctoral es el resultado de dicho carácter multidisciplinar de la química analítica, y con el objetivo de proporcionar soluciones a problemas relacionados con la seguridad alimentaria, se ha desarrollado un nuevo dispositivo sensor en base a conocimientos de química analítica pero también de biotecnología, microbiología y materiales avanzados. Por ello, el primer apartado de este trabajo pretende ser una introducción general sobre la inocuidad de los alimentos y su importancia a nivel mundial, haciendo especial énfasis en los patógenos alimentarios emergentes responsables de los principales brotes y la contribución de la tecnología de los biosensores como factor conductor para el desarrollo de nuevas metodologías para la detección de bacterias transmitidas por los alimentos mediante técnicas de multiplexado. Además, se discutirá también la integración de nuevos materiales con dimensiones nano/micrométricas en biosensores electroquímicos, destacando algunas de las ventajas de las partículas magnéticas: i) su capacidad para preconcentrar las bacterias presentes en muestras complejas mediante una reacción inmunológica ii) us uso como plataforma para el bioreceptor en los dispositivos de biosensores iii) o como soporte para la inmovilización magnética en la superficie de un electrodo de trabajo por atracción magnética. Los métodos de detección para seguridad alimentaria actuales han permitido un avance significativo en relación al desarrollo de métodos rápidos y sensibles, en los que la implementación de bioensayos con capacidad de multiplexado es una de las tendencias emergentes. Sin embargo, se han descrito pocas estrategias basadas en biosensores electroquímicos para la detección simultánea de bacterias en alimentos. Por esta razón, en el presente trabajo se propone desarrollar un biosensor electroquímico que detecte simultáneamente Salmonella enterica, Listeria monocytogenes y Escherichia coli, basado en el uso de partículas magnéticas. Las estrategias presentadas en esta Tesis doctoral se basan por un lado en un reconocimiento electroquímico inmunológico, que proporciona la detección de las células bacterianas, y por otro un reconocimiento genético, que permite la detección del ADN bacteriano. Estas dos estrategias se combinan con un paso de separación inmunomagnética que da lugar a la captura y preconcentración de las bacterias a partir de muestras de alimentos. Así, se ha realizado un estudio de las partículas magnéticas de diferentes tamaños (micro y nanométrica), para después efectuarse la separación inmunomagnética de S. enterica, L. monocytogenes y E. coli. Además, se han comparado posteriormente las dos estrategias para la detección de Salmonella, usándose como modelo muestras leche. Finalmente, se describe una estrategia de PCR multiplexada combinada con un magneto-genosensor electroquímico usando partículas magnéticas de sílice para la detección simultánea de S. enterica, L. monocytogenes y E. coli. / Nowadays, it is widely recognised in Europe that research and innovation are key factors to reinforce the industrial capacities and business perspectives. We need technology to address world´s problems, but we also need research to develop innovative technologies. Therefore, investing in research and innovation is essential to develop solutions for societal challenges, as for instance Food Safety. In this context, it comes out the term KETs (Key Enabling Technologies) to unify different fields across science, such as Nanotechnologies, Advanced materials, Biotechnology, among others. This indicates a clear convergence of technologies to address new solutions. For instance, analytical chemistry research is not only based anymore on the development of strategies to obtain qualitative and quantitative information about the composition and nature of substances. In order to provide solutions in food safety, analytical chemistry research has been converging into a more applied and multidisciplinary research field, forming alliances between different fields across science. This opens the possibility for the creation of new analytical principles, automated or in-situ detection procedures, as well as specific detection probes or new sensing devices. This Dissertation is a result of the multidisciplinary character of analytical chemistry. The aim of providing solutions to problems related to food safety bonds different science fields as analytical chemistry, biotechnology and advanced materials for the development of a new sensing device. Therefore, it is intended to give a general introduction about food safety and its importance worldwide, with special focus on the emerging foodborne pathogens responsible for the main outbreaks and the contribution of biosensors technology as the driver factor for the development of new methodologies for foodborne bacteria detection with multiplexing capabilities. Furthermore, the integration of new materials with nano/micrometer dimensions on electrochemical biosensors will be also discussed, highlighting some advantages of the use of magnetic particles: i) preconcentration of the bacteria from complex samples through an immunological reaction, ii) as a platform for the biorecognition element in the biosensing devices iii) as a support for the magnetic immobilisation on the surface of a working electrode under magneto-actuation. The current state of art for detection methods for food safety shows a significant progress relative to the development rapid and sensitive methods, in which the implementation of bioassays with multiplexing capabilities is one of the emergent trends. However, few approaches based on electrochemical biosensors for the simultaneous detection of foodborne bacteria have been reported. For this reason, it is proposed to develop an electrochemical biosensor for the simultaneous detection of Salmonella enterica, Listeria monocytogenes and Escherichia coli, based on the use of magnetic particles. The strategies presented in this Dissertation are based on electrochemical magneto-immuno and genosensing, in which electrochemical magneto-immunosensing provides the detection of whole bacterial cells, whereas the electrochemical magneto-genosensing provides the detection of the bacterial DNA. These two strategies are combined with an immunomagnetic separation step to capture and preconcentrate bacteria from food samples. Hence, a study of different magnetic particles with micro and nanometer sizes will be achieved for the immunomagnetic separation of S. enterica, L. monocytogenes and E. coli. Afterwards, electrochemical magneto-immuno and genosensing will be compared for the detection of Salmonella in milk, as a model. Finally, triple-tagging multiplex PCR combined with an electrochemical magneto-genosensor using silica magnetic particles as a platform will be reported for the simultaneous detection of S. enterica, L. monocytogenes and E. coli.

En els últims anys, el camp dels sensors i bionsensors ha experimentat un desenvolupament exponencial degut a una combinació de diferents factors. Per una banda, existeixen noves tècniques per a la construcció de microdispositius, i per altra, a les bones característiques intrínseques que aquests dispositius presenten, com son la robustesa, la selectivitat, el baix cost i la rapidesa de resposta. Aquestes característiques els fan molt adequats per a la monitorització de molts paràmetres en camps tan diversos com el mediambiental, el mèdic, l’industrial, etc. Els elèctrodes selectius d’ions (ESIs) són sensors electroquímics que, degut al potencial electrostàtic de la seva membrana quan aquesta està en contacte amb una solució, permeten analitzar quantitativament l’analit al que és selectiva aquesta membrana. La selectivitat ve conferida per una espècie química (ionòfor) que interacciona de forma selectiva amb l’analit a determinar. La consistència física de la membrana, factor que defineix la viabilitat pràctica del sensor, ve determinada pel material emprat en la construcció d’aquesta, habitualment un polímer. Fins al moment, el material polimèric més emprat en la construcció d’ESIs de membrana de portador mòbil ha estat el policlorur de vinil (PVC). Una de les limitacions d’aquest polímer és que la incorporació de material biològic per a la construcció de biosensors no és fàcil degut a l’ús de dissolvents orgànics (normalment tetrahidrofurà). Les proteïnes (enzimes, anticossos) no es dissolen bé en aquests dissolvents, i se n’alteren les propietats, fet que dificulta abastament l’ús del PVC per a la construcció d’aquest tipus de dispositius. En el treball d’investigació presentat en aquesta tesi, s’estudien polímers alternatius al PVC per a la construcció de quimiosensors i biosensors potenciomètrics. Concretament, polímers amb sofre com la polisulfona i la poli(èterètercetona) sulfonada (SPEEK) que, per les seves característiques, s’han considerat que poden ser bons candidats alternatius. S’ha decidit estudiar el SPEEK perquè és un material que, a més de presentar bones característiques físiques per a la construcció de membranes potenciomètriques, té grups amb càrrega negativa que probablement afectaran la resposta del sensor. Així, s’han construït elèctrodes per a un catió (amoni) i per a un anió (nitrat). En tots dos casos la resposta ha estat raonablement correcta, però especialment positiva per a l’ió nitrat ja que la repulsió electrostàtica provocada pels grups sulfònics del polímer ha reduït l’efecte interferent dels anions estudiats. També s’ha estudiat la polisulfona que, a més de subministrar bones membranes potenciomètriques amb ionòfors convencionals per als quimiosensors, permet incorporar material d’origen biològic en l’etapa final de preparació de les membranes mitjançant el procés de precipitació del polímer per inversió de fase des d’una solució aquosa on es troben les biomolècules. D’aquesta manera no en surten malmeses i, per tant, mantenen la seva activitat. Aquesta tècnica no és viable amb el PVC i representa una gran millora en la construcció de biomembranes potenciomètriques. Com a analit model a l’hora de fabricar un quimiosensor per a anions s’ha escollit el nitrat i com a analit model per als cations, el dial.lil dimetil amoni (DADMAC). Aquest últim es va seleccionar per un requeriment d’una empresa química per a una aplicació industrial. Concretament, es volia desenvolupar un sensor per a la determinació de la concentració residual d'aquest monòmer en reaccions industrials de polímers carregats en solució. Finalment, per la major robustesa i temps de vida del ESI amb matriu de PVC, aquest és el que finalment s’ha aplicat en planta. S’han desenvolupat biosensors potenciomètrics amb membranes de polisulfona on s’han incorporat proteïnes dins la membrana polimèrica. Les proteïnes seleccionades són metal·lotioneïnes que són presents a tots els éssers vius i que presenten una elevada capacitat complexant d’ions metàl·lics. Així, per una banda s’han estudiat aquestes metal·lotioneïnes com a ionòfors, i s’ha desenvolupat un elèctrode selectiu de plata per demostrar-ne la viabilitat. Per altra banda, també s’ha construït un biosensor on aquestes proteïnes fan el paper d’agents reconeixedors de les pròpies proteïnes. Aquest darrer estudi obre una nova via pel que fa al disseny de biomembranes potenciomètriques compactes que permetrà desenvolupar sensors potenciomètrics capaços de reconèixer biomolècules carregades sense cap reacció bioquímica. / Over the last years, the field of sensors and biosensors experimented an exponential development due to the combination of several factors. On the one hand, new manufacturing technologies for microdevices have appeared and, on the other hand, the intrinsic characteristics of sensors and biosensors such as robustness, selectivity, low cost and quick response have boosted their development. These characteristics make sensors and biosensors suitable for a wide range of fields such as, inter alia, environmental, medical or industrial. Ions selective electrodes (ISEs) are electrochemical sensors, which by reason of the membrane electrostatic potential when it contacts with the solution, allow the quantitative analysis of an analyte for which the membrane is selective. This selectivity is conferred by a chemical species (ionophore) which interacts selectively with the analyte. The membrane physical consistency, which is the key factor that determines the practical viability of the sensor, is determined by the material used for its construction, usually a polymer. Up to now, polyvinyl chloride (PVC) has been the most common polymeric material used for the construction of ISE membranes of the mobile carrier type. However, one of the limitations of this polymer is the difficulty to incorporate biologic material to generate biosensors. This is due to the use of organic dissolvent - usually tetrahydrofuran. Proteins, enzymes and antibodies, do not solve properly in those organic solvents and they also suffer alterations on their characteristics. Thus, the use of PVC for such devices is not feasible. The research performed for this Ph.D. thesis is aimed at identifying alternative polymers to PVC in order to create potentiometric biosensors and quimiosensors. In particular, the thesis studies polymers containing sulfur such as polysulfone (PS) and sulfonated poly (ether ether ketone) (SPEEK) since both offer appropriate characteristics to achieve this purpose. SPEEK has been chosen – besides its good physical characteristics for potentiometric membranes- because it bears negatively charged groups, which are likely to influence on the response of the sensor. Two different paradigmatic electrodes have been built: one for a cationic analyte (amonium) and one an anionic analyte (nitrate). In both cases the results have achieved reasonably satisfactory results, especially for the anion-ISE since the electrostatic repulsion due to the sulfonic groups of the polymer reduces the interference effects. Polysulfone has also been tested as a potential candidate for making ISEs. Along with its suitability to construct potentiometric membranes with conventional ionophore for quimiosensors as carriers, this polymer also allows the incorporation of biological material in the final stage by precipitating the polymer through an inversion phase process. This process precipitates the polymer in an aqueous solution containing the desired biomolecules. By these means, biomolecules do not suffer any alteration and do maintain their biochemical activity. This option is not feasible with PVC and represents a great improvement in the construction of potentiometric membranes. Nitrate was chosen as a model analyte for making an anion quimiosensor whereas diallyldimethylammonium (DADMAC) was selected for the cation one. The latter compound was chosen following the requirement of a chemical company to conduct industrial applications, in particular for the determination of residual concentration of this monomer in industrial reactions. Following the analysis, in this case an electrode made with PVC was made on the basis of its robustness and improved life span, properties which are crucial for industrial purposes. Potentiometric biosensors have also been developed using polysulfone as matrix by studying the incorporation of proteins inside the polymeric membrane. The proteins of choice were metallothioneins, a group of proteins that can be found in every living being and which show a high affinity for metallic ions. Accordingly, a silver selective electrode was developed first to prove the feasibility of this strategy and, secondly, a biosensor was made where the metallothioneins act as detecting agent for proteins of the same family. This study provides new opportunities to design compact potentiometric biosensors since it would allow the recognition and quantification of charged biomolecules without any biochemical reaction.

En la presente disertación han sido tratados el problema de la hipersensibilidad dental y los procesos remin/demin. También han sido desarrolladas nuevas herramientas científicas para la evaluación de la eficacia de los productos dirigidos a la hipresensibilidad y a la prevención de la creación de caries dentales. Además, se han diseñado y llevado a cabo varios experimentos para investigar el estado del tejido dental mediante espectroscopia FTIR sin y con la fuente de sincrotrón. Con el objetivo de desarrollar el material oclusivo adecuado y determinar su eficacia; la porosidad de la dentina debe ser determinada. En la presente tesis doctoral ha sido desarrollada una nueva metodología para la evaluación de la eficacia del tratamiento de la hipersensibilidad dental. Este estudio se ha presentado en la primera parte del trabajo y se basa en la metodología de los discos dentinarios (dentin disc model). Los especímenes tratados con los agentes desensibilizantes han sido observados mediante la Espectroscopia Electrónica de Barrido (SEM). Las imágenes obtenidas han sido analizadas por un algoritmo automático de segmentación, desarrollado para ser usado mediante el software de MATLAB, el cual calcula el número y el área de los túbulos abiertos. Dicho método es una herramienta adecuada para que tanto científicos, como compañías privadas puedan llevar a cabo la supervisión y asistencia de los procesos remineralizantes de los tejidos dentales, o bien para el desarrollo de los nuevos productos focalizados en el problema de la hipersensibilidad dental. Las siguientes partes de la disertación se han enfocado en el problema de los procesos desmineralizantes del tejido dental. En la segunda parte de la tesis doctoral se presenta la implementación de una nueva técnica, la de Hyper Spectral Imaging (HSI) para la evaluación de la eficacia del tratamiento remineralizante. Para el análisis de los dientes humanos antes y después de la remineralización, se ha utilizado principalmente el Hyper Spectral Imaging que implementa dos tipos de cámaras de Infrarrojo Cercano (NIR) (161 y 256 canales); y como método complementario la Microscopía Confocal de Raman (CRM). Los resultados obtenidos han sido detalladamente analizados mediante una rutina de MATLAB desarrollada especialmente para analizar ese tipo de imágenes. El coeficiente de correlación y la técnica de MCR (Multivariate Curve Resolution) han sido utilizados para recabar la información necesaria de los espectros adquiridos. Gracias a su simplicidad, el método puede convertirse en una poderosa herramienta para obtener información sobre los efectos del tratamiento remineralizante aplicado sobre el tejido dental. Durante el proceso de evaluación del nuevo producto dental, ese tipo de información puede ayudar a decidir si el producto es eficaz y compararlo con otros agentes del mismo tipo. En la tercera parte de la presente tesis doctoral la Elipsometría de Infrarrojo (IRSE) y la Elipsometría con la radiación de Sincrotrón han sido escogidos para la determinación de las constantes ópticas del tejido dental humano. Los especímenes dentales han sido tratados con el producto remineralizante y analizados mediante la Elipsometría IR con la radiación de Sincrotrón. Posteriormente, una serie de experimentos y simulaciones han sido llevados a cabo para obtener una muestra de referencia adecuada para la interpretación de los datos obtenidos en el sincrotrón. En la última parte, se han estudiado los cambios de la estructura del esmalte humano y de la dentina producidos por el proceso de la remineralización. Los espectros de varios puntos de los especímenes dentales han sido comparados mediante la técnica FTIR (Fourier Transform Infrared Spectroscopy). Los datos obtenidos han sido interpretados aplicando el Análisis de Componentes Principales (PCA) y la deconvolución de los espectros (curve-fitting). Los resultados han sido comparados con las muestras de referencia de hidroxiapatita, fluorapatita y las consideradas en la bibliografía científica. / In presented dissertation a problem of hypersensitivity and remineralization/demineralization problem is treated. A novel, scientific tools for the evaluation of the efficacy of products targeting the hypersensitivity or caries creation were proposed. Moreover, experiments implementing FTIR spectroscopy as well as synchrotron techniques for the dental tissue investigation were performed. In order to design a proper sealing material and to investigate its efficacy, the porosity of the dentine has to be determined. In the present dissertation a novel methodology for the evaluation of the efficacy of the hypersensitivity treatment has been developed. It is presented in the first part of the work and is based on the dentin disc model methodology. Specimens treated with a desensitizing agent were observed under Scanning Electron Microscope (SEM) and the resulting images were analyzed by the MATLAB automatic segmentation algorithm developed to calculate the number and area of the open tubuli. The present method is a suitable scientific tool that will allow scientists and private companies the in vivo monitoring and assistance on the development of new remineralising agents. The comparison of three commercial toothpastes presented in this work showed a successful application of the developed methodology and proved it to be suitable for that kind of comparative studies. The proceeding parts of the presented dissertation were focused on the problem of the demineralization and opposite-remineralisation processes of teeth. In the second part of presented PhD thesis a novel approach implementing the Hyper Spectral Imaging (HSI) technique for the evaluation of the efficacy of the remineralising treatment is presented. Hyper Spectral Imaging (HSI) with two types of NIR cameras (161 and 256 channels) and confocal Raman microscopy (CRM) as a complementary technique were applied for analysis of teeth before and after application of different remineralizing agents. Obtained results were carefully analyzed and compared by the implementation of a MATLAB routine that used the correlation coefficient and MCR data analysis in order to obtain necessary information from the acquired spectra of dental samples. Taking into consideration the simplicity of the presented method it can be a powerful tool to obtain basic information about the performed treatment. In the process of the evaluation of a new dental product, this kind of information can lead to the decision whether to continue the development of the product or to proceed with another, more promising approach. In the third part of the presented dissertation the Infrared Spectroscopic Ellipsometry (IRSE) and synchrotron ellipsometry is proposed for the determination of the optical constants of human dental tissues. Human teeth were treated with the remineralising agent and analyzed with the synchrotron IR ellipsometer. Furtherly, a series of experiments and simulations were performed in order to obtain the most suitable reference sample for the interpretation of the obtained synchrotron data. In the last – fourth part, structural changes in the human enamel and dentin induced by the remineralization process were investigated. The spectra from various points proceeding from different locations of the tooth were compared and studied by the FTIR technique. Obtained data were interpreted applying Principal Component Analysis (PCA) and curve-fitting procedures. Obtained results were compared to the reference hydroxyl and fluorapatite powders and scientific literature.