Escola Superior de Biotecnologia
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Browsing Escola Superior de Biotecnologia by Field of Science and Technology (FOS) "Ciências Naturais::Ciências Químicas"
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- Development of sequential injection enzymatic and bead injection assays in lab on valve formatPublication . Vidigal, Susana Maria Socorro de Matos Peixoto; Rangel, António O. S. S.; Tóth, Ildikó V.The development of bioengineering and biotechnology has promoted the interest for faster and more reliable methods for monitoring biochemical processes. Flowbased systems stand out as the most effective in the automation and miniaturization of these methods. A significantly reduction on the analysis time and dispensing skilled labour is achieved, thus becoming an important tool for implementation and enforcement of these assays. The main objective was the development of new methodologies for the miniaturization of biochemical analysis system using a sequential injection lab-onvalve (SI-LOV) system. In this context, analytical methodologies were developed based on enzymatic assays for the detection of enzyme substrates, for measurements of enzyme activity, or for enzymatic inhibition studies. Under this program, some enzymatic methodologies were developed: determination of ethanol in alcoholic beverages; determination of hydrogen peroxide in cleaning solutions for contact lenses, with and without dilution in line; and determination of the activity of peroxidase in vegetable extracts. The developed method for the enzymatic assay of ethanol in beverages (Chap.3) was based on the conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH), using spectrophotometric detection. The quantification of hydrogen peroxide (H2O2) (Chap.4) and peroxidase activity (Chap.5) was based on the reaction of oxidation of 2,2’-Azino-bis(3-Ethilbenzothiazoline 6-sulfonic acid) (ABTS) with hydrogen peroxide in the presence of peroxidase (HRP). Another major goal was to explore the potential of the SI-LOV systems to execute solid phase spectrophotometry, demonstrating its suitability for handling complex samples. This process, in the form of bead injection, was implemented inside the flow system. For this purpose, we used the resin "NitriloaceticAcid (NTA) Superflow", capable of retaining metal ions, which can be derivatized and measured by spectrophotometry (solid-phase spectrophotometry). These methods were used for the quantification of iron and protein content in wine samples. The developed method for the quantification of iron in wine samples (Chap. 6) was based on the reaction of Fe3+ with SCN-, where the Fe3+ ions were retained at the surface of the NTA beads. The total iron content was achieved by performing the inline oxidation of the Fe2+ to Fe3+. The methodology for total protein content in white wine samples (Chap.7) was based on the Lowry method. The NTA beads were initially charged with Cu2+ to complex with the proteins present in the sample. The quantification was achieved by the colour reaction of the proteins with the Folin Ciocalteau reagent. As for detection system, UV/Vis spectrophotometry was used in all of the developed methodologies.
- Development of solid phase extraction flow-based tools for environmental monitoringPublication . Pedro, Tânia Cristina Ferreira Ribas Vaz; Rangel, António Osmaro; Tóth, Ildikó VargánéThe development of new analytical tools can be considered a non-stop challenge due to the constant search for new improved features and also to the emerging environmental contaminants. Flow-based methodologies stand out in contributing for this analytical challenge, providing the automation and miniaturization of the analysis including sample pre-treatment. This thesis was developed based on two major objectives, one of them was to develop new miniaturized and automated analytical tools based on flow analysis for environmental monitoring. When designing new methodologies, another essential objective was to simplify sample preparation by coupling these techniques, based on solid phase extraction (SPE), within the developed flow-based system. The developed methodologies were optimized based on the same principles: minimize the use of reagent, make greener choices of the reagents, minimize the effluent production, lower the limits of detection and quantification, simplify and minimize sample/reagent handling. The use of the in-line SPE strategy showed to bring advantageous features to the analytical method (lowering limits of detection and quantification). The in-line SPE was achieved by using commercial resins (NTA and Chelex 100) and also a lab-made polymer inclusion membrane (Chapter 3). A biparametric sequential injection system for the determination of copper and zinc in water and soil leachates was developed (Chapter 3). The strategy was to use a non-specific coulour reagent (4-(2-Pyridylazo)resorcinol – PAR) and explore the use of two different sorbent materials to selectively separate the two different metal ions in the same manifold. A polymer inclusion membrane (PIM) and the commercial resin Chelex 100 were the chosen materials to selectively retain zinc and copper, respectively. It was the first time that a PIM was used with this purpose in a flow system. A spectrophotometric method for iron quantification using a newly designed chromogenic chelator was developed (Chapter 4). This low toxicity iron chelator was a specially designed 3-hydroxy-4-pyrydinone functionalized with ethers. Furthermore, this reagent demonstrated to display high affinity and specificity for iron ions. With the main objective of quantifying iron in a variety of water samples (fresh and marine water) a strategy including SPE was added to the manifold. By using an in-line SPE process, resorting to a NTA resin column coupled to the flow system, sample matrix clean-up and also the enrichment of the analyte was achieved. A method for the screening of biogenic amines in waters was developed (Chapter 5). The system was divided in two analytical parts. The first one was devoted to the pre-concentration of the analyte using a column packed with Chelex 100; the second was the derivatization of the biogenic amines using fluorescamine for the fluorescent detection of the analyte. This method intended to be a suitable and ease to operate system to obtain real-time information about biogenic amines content in water. A flow injection system for the spectrophotometric determination of the total zinc content in plant digests was developed (Chapter 6). By using a NTA resin column, zinc pre-concentration and the removal of possible interferences was accomplished. A specially designed multi-reflection flow cell coupled with a light emitting diode was the chosen detection system for the spectrophotometric determination of zinc using Zincon as colour reagent. The physical configuration of the flow cell contributed to improve the limit of detection and minimize refractive index gradients produced by the mixture of the reagents.