Development of new strategies for the design of in situanalysis devices: nano and biomaterials

La Tesis describe el concepto de análisis in situ y biomateriales que han dado lugar al desarrollo de varios dispositivos de análisis in situ. Estos dispositivos se basan principalmente en la inmovilización de reactivos en soportes sólidos. Se han empleado nano y (bio) materiales en el desarrollo de varios (bio) sensores o kits. Para el desarrollo de los dispositivos se consideraron los siguientes dos puntos críticos: i) la selección de los materiales de soporte donde tiene lugar la inmovilización de los reactivos, ii) la reacción involucrada en el procedimiento. Es importante estudiar la reacción entre el material y los reactivos para comprender la liberación del reactivo a la disolución o la entrada de los analitos al soporte sólido. El material de soporte no debe reaccionar con el reactivo ni interferir con la medida. Algunos de los materiales propuestos en esta Tesis como soportes sólidos para el diseño de sensores in situ han sido zeína, nailon, PDMS y nanocelulosa. Asimismo, se ha realizado la caracterización de los dispositivos desarrollados. Se han propuesto nuevas metodologías de análisis in situ basadas en el empleo de (bio) sensores para la determinación de compuestos relevantes como H2O2, fosfato o fármacos en matrices reales complejas como muestras de suero, orina o agua ambiental. Estos análisis han empleado diferentes respuestas analíticas en función de la sensibilidad requerida.There is great need, in general, to develop sustainable analytical methodologies based on the miniaturization, simplification and analytical process automation, with the aim of reducing the environmental impact without compromising the selectivity and sensitivity. Specifically, biosensors, are emerging as a fast and simple method for the in situ detection of compounds in several fields such as healthcare and food and drink industry including environmental and security monitoring among others. The advantages of in situ analysis are (1) in most cases the determination is carried out without isolation of the analyte from its environment, so the sample is not altered from its original conditions and (2) the analytical process including sampling is physically carried out in space and in time reducing the time of the analysis. Also, in situ analyses generally do not require sample treatment which, according to Green Analytical Chemistry principles, minimizes waste generation. On the other hand, Green Chemistry includes the use of safe and clean material and methods to decrease the adverse effects of pollution on the environment. The use of polymer-based biomaterials such as polysaccharides and proteins appears as a responsible option since it allows microorganisms to degrade these materials and directly reduce waste generation. Moreover, biomaterials have generated great interest due to their biocompatibility in food and medical area. The Thesis describes the concept of in situ analysis and biomaterials which have been resulting in the development of several situ devices. These devices are mainly based on the reagent immobilization in solid supports. Nano and (bio)materials have been employed in the development of several (bio)sensors or kits. In order to develop the devices, the two critical points were considered: i) the selection of the support materials where the immobilization of the reagents takes place, ii) the reaction involved in the procedure. It is important to study the reaction between the material and the reagents in order to understand the reagent release to dissolution or the entry of analytes to the solid support. The support material must not react with the reagent or interfere with the measurement. Some of the materials proposed in this Thesis as solid supports for the design of in-situ sensors have been zein, nylon, PDMS and nanocellulose. The characterization of the devices has been performed. New methodologies for analysis in situ based on the employment of (bio)sensors have been proposed for the determination of relevant compounds such as H2O2, phosphate or drugs in complex real matrices like serum, urine or environmental water samples. These analyses have employed different analytical responses depending on the sensibility required. These approaches have been validated and its analytical properties have been compared with other already existing

Development of thermochemiluminescence-based sensitive probes: synthesis, optimization, and characterization of c2- and c7-substituted acridine-containing 1,2-dioxetanes

After initial efforts in the late 1980s, the interest in thermochemiluminescence (TCL) as an effective detection technique has gradually faded due to some drawbacks, such as the high temperatures required to trigger the light emission and the relatively low intensities, which determined a poor sensitivity. Recent advances made with the adoption of variably functionalized 1,2-dioxetanes as innovative luminophores, have proved to be a promising approach for the development of reagentless and ultrasensitive detection methods exploitable in biosensors by using TCL compounds as labels, as either single molecules or included in modified nanoparticles. In this PhD Thesis, a novel class of N-substituted acridine-containing 1,2-dioxetanes was designed, synthesized, and characterized as universal TCL probes endowed with optimal emission-triggering temperatures and higher detectability particularly useful in bioanalytical assays. The different decorations introduced by the insertion of both electron donating (EDGs) and electron withdrawing groups (EWGs) at the 2- and 7-positions of acridine fluorophore was found to profoundly affect the photophysical properties and the activation parameters of the final 1,2-dioxetane products. Challenges in the synthesis of 1,2-dioxetanes were tackled with the recourse to continuous flow photochemistry to achieve the target parent compound in high yields, short reaction time, and easy scalability. Computational studies were also carried out to predict the olefins reactivity in the crucial photooxygenation reaction as well as the final products stability. The preliminary application of TCL prototype molecule has been performed in HaCaT cell lines showing the ability of these molecules to be detected in real biological samples and cell-based assays. Finally, attempts on the characterization of 1,2-dioxetanes in different environments (solid state, optical glue and nanosystems) and the development of bioconjugated TCL probes will be also presented and discussed