Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay

authored by

Marc Prante, Torsten Schuling, Bernhard Roth, Kort Bremer, Johanna Walter

Abstract

Detection of the small molecule 25-hydroxyvitamin D (25(OH)D) as the most relevant marker for vitamin D supply suffers from a high variability of results using the current detection methods, such as high-performance liquid chromatography (HPLC) and immunoassays. A new detection approach using a highly specific aptamer directed against 25(OH)D was established in this study based on the target-induced dissociation (TID) sensing approach. In this work, the aptamer was investigated regarding its structural properties as well as its binding affinity by using microscale thermophoresis (MST). Moreover, complementary oligonucleotides were designed based on the aptamer structure and were evaluated in MST experiments. Binding experiments of immobilized aptamers were conducted in microarray experiments. It could be shown that the aptamer exhibited the usual B-DNA structure and did not form any G-quadruplexes. The design of complementary oligonucleotides for the TID assay identified a putative 25(OH)D binding site within the aptamer. The limit of detection of the established competitive assay was determined to be 5.4 nM, which sets the stage for the development of a biosensor system.

Organisation(s)

Institute of Technical Chemistry
Hannover Centre for Optical Technologies (HOT)
PhoenixD: Photonics, Optics, and Engineering - Innovation Across Disciplines

Type

Article

Journal

Biosensors

Volume

9

Publication date

12.2019

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Clinical Biochemistry

Electronic version(s)

https://doi.org/10.3390/bios9040134 (Access: Open)
https://doi.org/10.15488/8795 (Access: Open)