Hücre Ölüm Yolaklarının Tespiti İçin Kağıt Tabanlı Biyosensör Tasarımı

Abstract

The aim of the presented thesis, a paper-based biosensor design has been designed to be used in the detection of early (extrinsic pathway) and late (instrinsic pathway) stages of apoptosis. In the study, in order to eliminate the disadvantages of traditional apoptosis determination methods such as high cost, large sampling requirement, appropriate laboratory and equipment conditions, a paper-based system was developed that is cheaper, easier, and provides more precise quantitative results. In the first phase of the study, firstly, the synthesis of magnetic nanoparticles to be interacted with the sample and immobilization of primary caspase antibodies on the surface of these nanoparticles were performed and characterized with the scanning electron microscopy (SEM), Zeta potential and size measurement, Fourier transformed infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimeter (DSC). Optimization and selectivity studies of the complex formed by primary antibody immobilized magnetic nanoparticles with standard caspase proteins were examined by HPLC system. Within the scope of optimization studies, protein concentrations (11.2-0.7 mg / mL), optimal duration (5-90 min), temperature (4-42 ° C) parameters were examined at pH: 7.4 environment. Optimal conditions was determined as pH: 7.4, concentration; 5.6 μg / mL, time: 45 min and temperature: room temperature. Within the scope of selectivity studies, the adsorption of competitive proteins (Caspase-3, Caspase-8, iv Caspase-9 and lysozyme) was investigated. The same competing proteins were also interacted with bare magnetic nanoparticles as a control group. Synthesis of upconversion silica nanoparticles and immobilization of the secondary antibody to these particles was performed and characterized with scanning electron microscopy (SEM), Zeta potential and size measurement, Fourier transformed infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC). Subsequently, zeta potential and size measurement and fluorescence emission spectrometry analysis of the supracomplex formed in dispersion medium by standard caspase proteins treated with primary antibody immobilized magnetic nanoparticles under optimal conditions, secondary antibody immobilized upconversion silica nanoparticles were performed. As a result of these analyzes, it was observed that the sandwich ELISA model formed in the dispersion medium maintained its upconversion feature and that the amount of protein added to the structure decreased due to this feature. This enabled a quantitative result to be obtained. In the second stage of the study, cell culture experiments were carried out. In this context, MCF-7 breast cancer cell line treated with 100 μM Cisplatin and not treated was used and the proteins obtained from the lysate of these cells were analyzed in dispersion medium with the designed system and the caspase protein amounts were determined. The results obtained were compared with traditional apoptosis determination methods (Double staining, Annexin-V, MTT). In the third stage of the study, a paper-based system was designed. For this purpose, the size of the paper to be used, the secondary antibody immobilized upconversion silica nanoparticle volume to be immobilized on the collection area (2.5-12.5 μL) and its distance from the sample area, the volume of primary antibody immobilized magnetic nanoparticles (10-100 μL) to be dropped into the sample area were determined. Subsequently, studies performed with standard caspase proteins and cell lysates in dispersion medium were examined on paper. Validation has been made by comparing the results obtained from the dispersion medium with the results obtained on paper. As a result of all these studies, it has been shown that more precise and reliable results can be obtained than traditional methods with the paper-based system.