Browsing by Author "Puolakka, Aura"

The aim of this thesis was to present some non-antibody-based analytical techniques and methods for protein analysis and separation. The first part of the thesis focused on the possibilities of aptamers, chemically synthesized oligonucleotide affinity tools, in protein analysis. Aptamer-based affinity chromatography, affinity capillary electrophoresis, and biosensor applications in protein analysis published in the past ten years (2007−2017) were studied. Also, microfluidic applications are discussed shortly. The literature review revealed the outstanding prospects of aptamer-based techniques in clinical diagnostics and biomedical research. Especially aptamer-based biosensors are expected to become an important technique in diagnostics assays of disease-related biomarkers. The detection limits achieved with aptasensors were in the picomolar to attomolar range in most of the reviewed studies, thereby, being comparable (or even superior) to many of the detection limits achieved with traditional antibody-based methods. However, challenges, such as the special expertise needed when working with aptamers and the low availability of appropriate aptamers mean that a commercial breakthrough of these applications is still being waited to happen. The second, the experimental, part of the thesis studied the possibility to separate lipoproteins (HDL2, LDL, VLDL, and IDL) by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) using UV-VIS detection. Different parameters, such as separation voltage, injection style, and capillary dimensions were investigated. The capillaries were coated with poly(N-methyl-2-vinylpyridinium iodide-block-ethylene oxide (P2QVP-b-PEO) in order to prevent the adsorption of lipoproteins onto the capillary wall. It was found that CZE under physiological conditions (phosphate buffer at pH 7.4) resulted in broad low-intensity lipoprotein peaks. Furthermore, a lipoprotein mixture of all three fractions (HDL2, LDL, and VLDL/IDL) migrated as two peaks. CGE with linear polyacrylamide, on the other hand, resulted in sharp high-intensity peaks for the lipoproteins. The separation of lipoprotein mixture, however, did not succeed. The gel used in CGE studies consisted of 4% m/v acrylamide and 0.1% sodium dodecyl sulfate in Tris-aspartic acid buffer at pH 8.0. For further studies, a more systematic research plan needs to be constructed.