ABSTRACT
Glycosaminoglycans (GAGs) serve as a biomarker for mucopolysaccharidoses disease. In this study, a novel fluorometric method was developed to measure total GAGs in urine. Graphene oxide (GO) and rhodamine B (RhB), a cationic fluorescent dye, were employed in the development of the method. RhB attaches to the GO surface via electrostatic attraction, leading to the quenching of its fluorescence upon the establishment of the RhB-GO complex. However, the presence of GAGs prompts a resurgence of intense fluorescence. The linear range of the method is between 5.00 and 70.00 mg/L. The total GAG levels of urine samples analyzed using the method agree with the results of the biochemistry analysis laboratory (65.85 and 79.18 mg/L; 73.30 ± 1.76 and 72.21 ± 2.21). The method is simple, accurate, and sensitive and may be used for both first-step diagnosis of the mucopolysaccharidoses and detection of individual GAGs for studies of GAG-related research and other biological applications.
Subject(s)
Glycosaminoglycans , Graphite , Graphite/chemistry , Glycosaminoglycans/urine , Glycosaminoglycans/chemistry , Humans , Spectrometry, Fluorescence , Rhodamines/chemistry , Fluorescent Dyes/chemistry , Fluorescence , Mucopolysaccharidoses/urine , Mucopolysaccharidoses/diagnosisABSTRACT
The measurement of phenylalanine in biological fluids for the diagnosis of phenylketonuria (PKU) in newborns and the monitoring/therapeutic drug monitoring of individuals with PKU are especially important. Owing to the importance of PKU monitoring in clinical medicine, a new fluorometric method was developed for the determination of L-phenylalanine in serum samples. This method is based on the relationship between phenylalanine ammonia-lyase (PAL) and o-phthalaldehyde (OPA). PAL catalyzes the conversion of phenylalanine to ammonia and trans-cinnamic acid. The formed ammonia reacts with OPA in the presence of sodium sulfite, giving a fluorescent product. The presence of sulfide in an alkaline environment prevents OPA from reacting with other amino acids while allowing it to react only with ammonia. Method characterization and optimization studies, such as the effects of pH, temperature, and interferents, were carried out. The amount of L-phenylalanine in a human serum sample was successfully determined by using the fluorescence emission intensity of the fluorescent product formed as a result of the reaction between OPA and ammonia. The linear range of the method is between 10 µM and 10 mM. The obtained result showed good agreement with the results of the biochemistry analysis laboratory. Recoveries of 95.41% and 73.39% were obtained for phenylalanine and ammonia, respectively. This PAL-OPA-based fluorometric method for phenylalanine is practical, easy to operate, low cost, highly sensitive, and selective and can also be used for the simultaneous determination of ammonia in human serum samples.
Subject(s)
Phenylalanine Ammonia-LyaseABSTRACT
A new fluorometric method was developed for the determination of α-amylase activity in human serum samples. Firstly, a saturated starch-iodine complex (SI) was prepared. The SI complex was combined with sodium fluorescein to form a starch-iodine-sodium fluorescein complex (SIF). As the SIF complex decomposes with the α-amylase enzymatic hydrolysis of starch, the intensity of its fluorescence emission increases. The α-amylase activity is determined using the increased fluorescence emission intensity following hydrolysis of the SIF complex by α-amylase. The optimum pH, optimum buffer concentration, optimum temperature, and interference effect were identified for the developed fluorometric measurement method. Under the optimum conditions, a linear calibration curve was obtained between 0.18 and 9.00 U/L for α-amylase. The α-amylase activity in the human serum sample was also determined by our prepared measurement system and compared with the result from a medical center. Both methods are in good agreement with each other. Because this newly developed fluorometric method for α-amylase activity in serum samples is inexpensive, easy to use, and carried out to detect a very low amount of human serum α-amylase with sensitivity, it can be proposed this method for alpha-amylase activity assay in all other biological samples.
