Enzyme Biosensor for Rapid Detection of 17- Alpha-OHP; A New Perspective for Screening Congenital Adrenal Hyperplasia in Newborn Ebru Dündar Yenilmez, Umut Kökbaş, Abdullah Tuli Çukurova University Faculty of Medicine, Department of Medical Biochemistry, Adana-Turkey.
[email protected] Objective: Congenital Adrenal Hyperplasia (CAH) is a family of autosomal recessive disorders characterized by deficiency in one or another of the enzymes of cortisol biosynthesis. The most prevalent form of the disorder is 21-hydroxylase (21-OH) deficiency which is the most frequent inborn metabolism error and 17-Alpha-OHP is secreted in abundant excess. Measurement of 17-Alpha-OHP is therefore valuable in the initial diagnosis of CAH. Newborn screening procedures for CAH are still suboptimal because of low specificity, particularly in premature infants. In this study, a biosensor designed to detect 17-Alpha-OHP for a new alternative method for newborn screening of CAH.
Figure 1. Detection methods for 17-alpha-OHP ; detection in dried blood spot , mass measurement of metabolits and detection with biosensors
Methods: The electrochemical measurements were performed using a gold electrode coated with Au-Poly HemaMac, combined with the reference Ag/AgCl electrode and the auxiliary Au/Pd (98/2%) electrode. UV immobilization performed with 17-Alpha-OHP-horseradish peroxidase on the modified gold electrode surface with anilin (20µL enzyme and 20µL anilin).
Figure 2. General principle of biosensors
Results: Optimization studies determine the most suitable working conditions for using the biosensor. Polymerization time was 2h, the enzyme concentration used 0.5mg/mL, temperature was 35°C, pH was 6.5 with phosphate buffer. After the characterization studies of the biosensor the detection limit was 0.015ng/mL-7.5ng/mL, repeatability was 2.98±0.04.
Figure 3. Cyclic voltammogram of biosensor without sample
Figure 4. Change of biosensor response at different concentrations of 17-Alpha-OHP standarts.
Figure 5. Optimum temperature of designed biosensor
Conclusion: The demonstrated method for 17-Alpha-OHP detection in newborn is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to the traditional tests in use. (**This study is supported by the scientific research unit of Çukurova University-TSA-2017-7293) References 1. Kopacek C, Martins de Castro S, Prado MJ., Neonatal screening for congenital adrenal hyperplasia in Southern Brazil: a population based study with 108,409 infants. BMC Pediatrics 2017; 17 :22. 2. Olgemoller B, Roscher AA, Liebl B, Fingerhut R. Screening for Congenital Adrenal Hyperplasia: Adjustment of 17-Hydroxyprogesterone Cut-Off Values to Both Age and Birth Weight Markedly Improves the 3. Predictive Value. The Journal of Clinical Endocrinology & Metabolism 2003; 88 (12): 5790–5794. 3. N Linder N, Davidovitch N, Kogan A, Barzilai A, Kuint J, Mazkeret R, Sack J. Longitudinal measurements of 17á-hydroxyprogesterone in premature infants during the first three months of life Arch Dis Child Fetal Neonatal Ed 1999; 81: F175–F178.
