Browsing by Author "Vidigal, Susana Maria Socorro de Matos Peixoto"
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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.