Gaitada Gizli Kan Tanısında Kullanılacak Moleküler Baskılanmış Polimer Tabanlı Hasta Başı Test Kiti İçin Altın Nanopartikül İşaretli Sentetik Hemoglobin Molekülünün Geliştirilmesi ve Karakterizasyonu
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Immunochromatographic tests are the systems that enable to detect the existence of the target analyte in the liquid sample without needing a special device. This test is based on a porous paper or capillary beds in series as sintered polymers. The components of this system have the liquid bearing capacity instinctively. Once the capillary bed in the first part is interacted with the liquid sample, it transports the liquid to the second component of the system. In this part, the antibody (ligand), the diagnosis component of the test, is found as immobilized to the solid support material. The analyte (antigen) is found in biological matrix, where ligand-analyte complex occurs after their interaction. Next, the modified conjugate enzyme that has affinity to the analyte, attaches to this complex. Finally, the test finishes by the color changer substrate is given to the medium. The biological based molecules used in the conventional fecal occult blood (FOB) test kits, such as antibody and enzyme, may be affected by the heat and humidity of the environment. This situation causes a significant reduction of the test kits shelf life. In this thesis, the developed test system is based on a competitive affinity chromatography method. Apart from the test systems mentioned above, the second component of the test consists of the MIP (Molecular Imprinted Polymer), specific to hemoglobin molecule that is going to detect in FOB tests and the synthetic hemoglobin molecules labeled with colloidal gold nanoparticles which is attached to the cavities on these MIP by weak bonds. Due to the liquid sample and the transported hemoglobin molecules to this area have higher affinities, they are going to attach to the cavities in the second part by removing the labeled hemoglobin molecules. Removed synthetic hemoglobins are going to pass to the third part by means of the liquid sample and these molecules are going to settle in the relevant cavities of the MIP which are specific to the labeling materials, colloidal gold nanoparticles. In the third part of the test system (the diagnosis part), the colloidal gold nanoparticles, settled in the specific cavities, are going to lead a color change in the test strip. The length of the area that shows the color change correlates with the amount of hemoglobin in the sample and this enables the quantitative analysis. For this purpose, the molecules that are going to immobilize to the gold nanoparticles were determined by docking simulation. According to the results obtained from docking simulation, 11-MUA and Ethanolamine hydrochloride modified Aldehyde-PEG-Ester were immobilized to the synthesized equidimensional, uniform and spheric gold nanoparticles. For the size and shape analysis of the synthesized gold nanoparticles; Transmission electron microscopy (TEM) and for the characterization of the modified gold nanoparticles; UV Visible Spectroscopy, X-ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FT-IR) and Dynamic Light Scattering (DLS-Zetasizer) were used. The interaction between the synthetic hemoglobin molecules prepared during the thesis and the surface of the hemoglobin specific MIP was investigated by Surface Plasmon Resonance (SPR). In addition, the balance and kinetic analysis were carried out. The isotherm graphs were design as a result of the SPR analysis and by this way the interaction of the molecules was calculated. Thus, in this thesis, it was laid a foundation of a novel test kit, with the properties of competing with the conventional FOB test kits, having a longer shelf life and enabling the quantitative measurement.