Alerjen Spesifik İmmunoglobulin E Tayini İçin Yüzey Plazmon Biyosensörlerin Geliştirilmesi

Abstract

Allergy is a mysterious chronic disease that causes the digestive system to be exposed to certain foods, the respiratory system to dust or pollen, or the skin to chemicals. Allergy is a very common disease affecting 10-20% of the world's population. Allergy affects 2% of the adult population and 8% of children. Immunoglobulin E (IgE) level rises in allergic conditions. The main allergenic sources are pollen, foods, mites, molds and poisons. Therefore, the increase in IgE level is important for the diagnosis of allergic diseases such as asthma, eczema and hay fever. Food allergy is defined by the adverse reactions of the immune system when triggered by the consumption of certain foods. Food allergy reactions occur as a result of the recognition of allergenic proteins by allergen-specific IgE antibodies. For some allergic individuals, selective food contact can develop into life-threatening reactions (such as anaphylactic shock). Traditional methods for allergen detection are Enzyme-linked immunosorbent analysis (ELISA), which usually requires enzymatic labeling. This labeling process is time consuming and can alter the immunochemical activity of target biomolecules. Surface plasmon resonance (SPR) is a well-known technology for the measurement of interactions between biomolecules. In this thesis, optical-based surface plasmon resonance biosensors were prepared for allergen-specific serum IgE determination. The SPR biosensor gold chip surface is primarily functionalized with 3-mercaptopropionic acid. The covalent coupling of N-ethyl-N'-(3-diethylaminopropyl) carbodiimide (EDC)/sulfo-N-hydroxysulfosuccinimide (NHS) has been performed to activate carboxylic acid functions. Anti-immunoglobulin E (anti-IgE) with terminal amine groups on the antibody were then immobilized to the surface as recognition sites. The prepared SPR biosensor chip surface was characterized by the contact angle, atomic force microscope (AFM) and ellipsometry. Surface roughness values were found as 0.28 nm and 8.36 nm for the bare chip surface and the modified chip surface, respectively. The detection of IgE has been carried out in the broad range of 1.0 ng/mL-1000 ng/mL. The detection of IgE was performed with the SPR biosensor in plasma and aqueous solutions very selectively with an analysis time of approximately 10 minutes. The limit of detection (LOD) value for IgE was found to be 0.22 ng/mL. Selectivity studies were performed with albumin, immunoglobulin G, and myoglobin as similar proteins. In reusability studies, IgE samples were given to the SPR biosensor system five times in a sequence and were examined the stability of the signal response. Scatchard, Langmuir, Freundlich and Langmuir-Freundlich adsorption isotherm models were calculated to examine the surface homogeneity of the designed SPR biosensor gold chips. The adsorption behavior of both chips was found following the Langmuir isotherm model.