Fenilketonüri Hastalığının Erken Teşhisi ve Takibi için Disk Tabanlı Hasta Başı Tanı Sisteminin Geliştirilmesi

Abstract

Phenylketonuria (PKU) is an autosomal recessive metabolic disease due to dysfunction of phenylalanine hydroxylase (PAH) enzyme, which converts phenylalanine (Phe) amino acid to tyrosine amino acid. Phe amino acid and byproduct metabolites (phenylpyruvic acid, phenyllactic acid, etc.) that accumulate in blood, urine and other body fluids disrupt the myelination in the Central Nervous Systems (CNS) and cause mental - motor retardation. Turkey ranks first in terms of the PKU prevalence and the patient population in the world. Since mental retardation can be prevented by Phe poor diet in patients diagnosed during the neonatal period, early diagnosis, treatment and follow-up of the disease hold great importance. In routine tests, commonly used methods are Guthrie test, fluorometric methods, enzymatic colorimetric test, HPLC (for direct amino acid analysis) and tandem mass spectrometry (MS/MS). To conduct such tests, it is necessary to have fully equipped health centers with technical infrastructure, advanced tools and well-trained staff. In Turkey, there are only two health centers which are located in Istanbul and Ankara that collect and analyse the blood samples of PKU patients. Since the screen test cannot be done routinely in the provinces where some of the PKU patients are located, they do not give the blood samples at regular intervals. Furthermore, storage and delivery of the blood samples to these cities increase the cost. Therefore, it is quite important to develop an easy, low-cost and point of care applicable blood Phe monitoring system. For all these reasons, the purpose of the completed thesis was to develop a Lab-on-a-CD based bedside test system that allows the early diagnosis and the follow-up of the treatment process of PKU. To achieve that CD shaped disposable cartridges, and electrodes to be used in the chambers of these cartridges were developed. Prepared Lab-on-a-CD system is based on the electrochemical measurements for the diagnosis and the treatment of PKU. Developed test system proved to be a portable device that provides quantitative results. The test kit consists of disposable cartridges designed with siphon system geometry, and a biocompatible polymethyl methacrylate (PMMA, plexi) material with a diameter of 10 cm and a thickness of 3,5 mm, which can be customized according to the desired test. Screen-printed gold electrodes were used for the electrochemical measurements. Electrode modification was conducted by electrochemical graphene oxide reduction and deposition on the electrode surface. The sensitivity of the measurement was 1,5 fold increased as a result of the modification. In the electrochemical measurement of Phe, amperometric detection was applied during the oxidation of NAD+ to NADH in the presence of phenylalanine dehydrogenase (PDH) enzyme. Between 0-20 mg/dL measuring range (R2 = 0,9955), calculated limit of detection (LOD), limit of quantification (LOQ), and sensitivity were 0,0524, 0,1587 mg/dL and 0,3338 (mg/dL)/µA, respectively. The system's specificity to Phe was determined by conducting selectivity studies. In the final stage of the thesis, the developed system was compared with the validation method (HPLC). Calculated percent success in proximity to Phe concentrations in samples for HPLC and developed test kits were 83,1 and 84,1, respectively