Development of micro-analytical and diagnostic methods using microfluidic paper based analytical devices and nanomaterials (Doctoral thesis)

Κάππη, Φωτεινή

The aim of the PhD thesis is the development of novel analytical methods and signal readout approaches for the quantitative determination of biothiols in biological fluids which are easy to use, do not require instrumental analytical techniques and use stable and commercially available reagents which do not require specialized handling and treatment (such as those used in immunoassays or enzymatic assays). Firstly, the thesis describes a new approach for the determination of free biothiols in biological fluids that exploits some of the basic principles of early photographic chemistry — that was based on silver-halide recording materials — and uses broadly-available imaging devices (i.e. flatbed scanners) as detectors. Specifically, the proposed approach relies on the ability of biothiols to bind to silver ions and dissociate the silver halide crystals thus changing the photosensitivity of silver halide crystal suspension. The changes induced by biothiols on the light intensity transmitted through the silver halide suspension, after photochemical reduction, were measured with a simplified photometric approach that employs a flatbed scanner operating in transmittance mode. The overall analytical procedure for the determination of biothiols was easily executable, fast and could be applied with inexpensive and commercially available materials and reagents. What is more, physiologically relevant biothiol levels could be inspected even by the unattended eye. The developed assay was successfully applied to the determination of biothiols in urine and blood plasma samples with detection limits as low as 10 μM, satisfactory recoveries (92-97%), good reproducibility (6.7-8.8%) and high selectivity against other major components of biological fluids. The utility of the method to the determination of reduced/oxidized thiol ratio’s as well as its application under natural light illumination, without external energy sources, was also demonstrated and is discussed with regard to point-of need applications in facility-limited settings.In the next chapter the thesis describes the development of paper-based devices for the determination of biothiols. The devices are inexpensive (composed of paper and silver halide particles), and the analytical protocol is easily executable with minimum technical expertise and without the need of specialized equipment; the user has to add a test sample, illuminate the device with a UV lamp, and read the color change of the sensing area using a simple imaging device (i.e., cell-phone camera) or a bare eye. The detection mechanism of the assay is based on the biothiols-mediated photoreduction of nanometer-sized silver chloride particles deposited on the surface of paper; photoreduced silver chloride particles have a grayish coloration that depends on the concentration of biothiols in the tested solution. This is the first time that the UV-mediated photoreduction of solid silver halides particles is used for analytical purposes. The performance of the devices has been tested on the detection of total biothiols content of artificial body fluids and protein–free human blood plasma samples, and the results were satisfactory in terms of sensitivity, selectivity, recoveries and reproducibility. Finally, a new approach for performing analytical assays on paper-based analytical devices that relies on the principles of multiple standard additions calibration, and uses calibrant-loaded paper devices, is described. Calibrant-loaded devices have multiple sensing areas pre-loaded with an excess amount of the necessary colorimetric reagents and known amounts (standard solutions) of the tested analyte; thus, a colored product is developed before analysis in each sensing area. After sample addition, the analytical signal from each sensing area corresponds to the total concentration of the analyte, which is the sum of the concentration of the analyte in the tested sample plus the known amount of the analyte pre-stored in the device. The combined signal retrieved from each sensing zone is plotted to form a standard addition calibration curve that is used to calculate the concentration of the analyte in the sample using linear or non-linear models (i.e., first or higher order polynomials). This new approach could reduce the influence of matrix effects, paper inhomogeneity and environmental conditions on the results, and it could simplify the analysis as it may eliminate the steps associated with on-spot calibration that should ideally be performed using standard solutions that are prepared or transported in-situ. The utility and applicability of this approach is demonstrated as proof-of-concept on five assays (i.e., for the determination of iron, protein, hydrogen peroxide, acetic acid and carbonate ions) that use different reactions (i.e. complexation, oxidation and neutralization). This work provides a demonstration of a multiple standard addition methodology applied on calibrant-loaded paper-based analytical devices and illustrate its use as a sample-in result-out tool for point-of-use applications.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας
Subject classification: Φωτοαγωγιμότητα
Keywords: Αλογονίδια του αργύρου,βιοθειόλες,χαμηλού κόστους αναλύσεις,κοινές ηλεκτρονικές συσκευές,φωτοαναγωγή,βιολογικά ρευστά,αναλυτικές διατάξεις χάρτου,σταθερή προσθήκη,προ-βαθμονομημένες διατάξεις,μη γραμμική βαθμονόμηση,silver halides,biothiols,low-cost assays,consumer devices,photoreduction,biological fluids,paperbased devices,standard addition,Calibrant-loaded devices,nonlinear calibration
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