İdrarda Total Glikozaminoglikan SemiKantitatif Ölçümü İçin Hızlı Test Yöntemi Geliştirilmesi
Project no. 23133 · Status: Kapanmış (Yayın bekleniyor) · 2021
Summary
<p>Mucopolysaccharidoses (MPS) is a genetic disease because of defects in lysosomal enzymes that catalyze the breakdown of glycosaminoglycans (GAGs) and are a subset of lysosomal storage diseases (LSD).</p><p>MPS results in the accumulation of GAG in tissues leading to organ dysfunction. Therefore, GAGs are important biomarkers for MPS. Without any treatment, patients with severe forms of MPS die within the first twenty years of life. For this reason, accurate, fast, sensitive, and specific measurements of GAGs are required. Accumulated GAGs are detected using various analysis techniques. One of the most common methods available is based on the quantification of a 1.9 dimethyl methylene blue (DMB) cationic dye by binding to negatively charged GAGs.</p><p>A fast and easy paper-based semi-quantitative diagnostic control integrated with the existing DMB dye-binding method was developed for the analysis of total GAG in urine for use in MPS preliminary analysis.</p><p>A purple color was observed when urine with chondroitin sulfate (CS) was dripped onto the pretreated and then DMB-impregnated papers. Then the test system; Paper type, type of alcohol solution used for pretreatment, DMB concentration, stabilizer addition, time and temperature, chondroitin sulfate concentration, and interference effect were evaluated. A standard graph was created by choosing the optimal test strip.</p><p>According to the results obtained from the optimization studies, the paper was selected and a rapid test system was created. The paper was incubated in ethanol and stained with DMB10x and no stabilizer was added. Drying at room temperature overnight was found suitable. Bilirubin did not cause interference in test strips up to 0.03 mg/ml, ascorbic acid 5 mg/ml, and glucose 20 mg/ml.</p><p>The lowest CS concentration in the urine where the difference between CS-containing and CS-free urine was observed was 0,04 mg/ml.</p><p>After the optimization studies, the calibration curve of the CS concentration in the urine was drawn against the mean color differences calculated according to the software, and a linear graph (R2=0.9722 ) was obtained.</p><p>A simple, rapid, and cost-effective first paper-based test based on color changes of DMB dye was developed for the semi-quantitative determination of total GAG. The designed test has shown that normal urines can be distinguished from urines containing CS. This study demonstrated its potential utility as a tool for pre-diagnosing patients at risk for MPS.</p>