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Open AccessArticle

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

1
Institute of Technical Chemistry, Leibniz University of Hannover, 30167 Hannover, Germany
2
Hannover Centre for Optical Technologies, Leibniz University of Hannover, 30167 Hannover, Germany
3
Cluster of Excellence PhoenixD, (Photonics, Optics, and Engineering—Innovation Across Disciplines), 30167 Hannover, Germany
*
Author to whom correspondence should be addressed.
Biosensors 2019, 9(4), 134; https://doi.org/10.3390/bios9040134
Received: 20 September 2019 / Revised: 7 November 2019 / Accepted: 8 November 2019 / Published: 13 November 2019
(This article belongs to the Special Issue Microarray for Biomarker Detection and Discovery)
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. View Full-Text
Keywords: 25(OH)D; aptamer; biosensing; small molecules; microarray 25(OH)D; aptamer; biosensing; small molecules; microarray
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MDPI and ACS Style

Prante, M.; Schüling, T.; Roth, B.; Bremer, K.; Walter, J. Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay. Biosensors 2019, 9, 134.

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