Highly sensitive detection of 25-HydroxyvitaminD(3) by using a target-induced displacement of aptamer

초록

For the prevention of 25-HydroxyvitaminD3 deficiency, in this study, aptamers which can bind to 25HydroxyvitaminD(3) with high specificity and affinity, were successfully developed by using immobilization -free, graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX) method. The 9 sequences including VDBA14 aptamer were obtained out of 16 aptamer candidates, based on the specificity and affinity of the aptamers confirmed by both the gold nanoparticles (AuNPs)-based calorimetric assay and the isothermal titration calorimetry (ITC) method. Among them, the aptamer, VDBA14, developed in this study was found to show a great affinity to 25-HydroxyvitaminD(3), with 11 nM of its Kd value. Moreover, the circular dichroism (CD) analysis data indicated the target-induced displacement of the aptamer VDBA14clearly. In addition, this target-induced change of the aptamer was also confirmed again by conducting two different experimental formats, the use of streptavidin-coated 96 well plates and the use of magnetic beads. The results clearly indicated that the structure of VDBA14 aptamer was changed upon the binding of the target, 25-HydroxyvitaminD3, and so the indicator sequences (partially complementary to the aptamer sequence) tagged with an enzyme as a signaling molecule could be de-hybridized from the aptamer. Finally, the limit of detection for vitamin D based on AuNPs-based calorimetric assay using VDBA14 aptamer was found to be 1 M. All these results were taken together, the aptamer which was developed could play an exquisite role in the fields of early medical diagnosis of vitamin D deficiency with accurate, rapid and simple analytical method. (C) 2016 Elsevier B.V. All rights reserved.

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