Development of QTF-based mass sensitive immunosensor for phenylketonuria diagnosis

Phenylketonuria, an autosomal recessive disorder, is a serious disease that causes accumulation of L-phenylalanine in the body. Phenylketonuria is caused by the mutation that occurs in chromosome 12, which mainly synthesizes phenylalanine hydrogenase enzyme. If not treated, the amount of L-phenylalanine in the body fluids increases; epilepsy, microcephaly, eczema and scleroderma-like skin lesions, development and intelligence. It is of great importance that this disease, which has serious consequences at this level, should be diagnosed early and started to be treated without losing time. Today, the diagnosis of this disease is being made with complex and expensive devices, which require a tough preliminary process within the neonatal screening program. For this reason, it is important that the economic diagnosis methods which can be used in the diagnosis of the diseased, easy to apply, fast / correct answer and clinical diagnosis are given. For this reason, a QTF-based immunosensor has been developed for use in the diagnosis of phenylketonuria in a doctoral dissertation study, which has been modified with plasma to provide a high performance, rapid response, and economical properties and to measure the level of L-phenylalanine in the ex-vivo environment. To this end, QTF's surfaces have been functionalized in two steps by plasma polymerization process. At the beginning of the functionalization process, with the aim of overcoming the stability problem of amine-rich thin films, the QTF surfaces were modified by plasma polymerization of n-hexane which is a hydrocarbon-based precursor, with different discharge power and exposure times. As a result of the stability tests and chemical characterization, the optimum conditions for stable thin film for 30 days were obtained with 75 W- 10 minutes plasma parameters. In the second step; QTF's surfaces were modified with ethylenediamine (EDA), which is amine-rich monomer, and functionalized for antibody immobilization. As a result of the characterization process, optimal plasma parameters for the production of functional thin films are obtained with 75 W-1, -5 and -10 minutes plasma parameters. Then, functionalized QTF's surfaces were treated with glutaraldehyde at different times and media in order to activate surfaces for antibody immobilization and optimum conditions were obtained as a result of a 2 hour incubation period in a 25% (v: v) glutaraldehyde solution. During the design of immunosensor, the production biological recognition layer on substrate was performed by decorating the activated transducer surfaces with anti-phenylalanine at concentration intervals of 1-20 μg / μl. The developed immunosensor was followed up against L-phenylalanine and the antibody amount was optimized to 10 μg / μl and the measurement performance was examined. As a consequence, QTF-based mass-sensitive immunosensor were designed for the first time in the literature within this study. It is thought that the produced immunosensor platform will be an infrastructure for future immunosensor studies.