Details
| Original language | English |
|---|---|
| Article number | 134 |
| Journal | Biosensors |
| Volume | 9 |
| Issue number | 4 |
| Early online date | 13 Nov 2019 |
| Publication status | Published - Dec 2019 |
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.
Keywords
- 25(OH)D, Aptamer, Biosensing, Microarray, Small molecules
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Clinical Biochemistry
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In: Biosensors, Vol. 9, No. 4, 134, 12.2019.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay
AU - Prante, Marc
AU - Schuling, Torsten
AU - Roth, Bernhard
AU - Bremer, Kort
AU - Walter, Johanna
N1 - Funding Information: Acknowledgments: We gladly thank Patrick Stolle from the Institute of Microbiology, Leibniz University of AckHnaonwnolevdegr,mfoerntthse: aWbielitgylatodrlyectohrdanthkePCaDtriscpkecSttroalulesefdroimnththiseaIrntisctliet.uTtheeopfuMbliiccraotiboinoloofgtyh,isLaeritbicnleizwUansifvuenrdseitdy of HanbnyotvheerO, fpoernthAecacebsilsitfyuntodroefcLoerdibtnhize UCDnivseprescittärat Husaendnoinvetrh.is article. The publication of this article was funded by the Open Access fund of Leibniz Universität Hannover. ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest. Conflicts of Interest: The authors declare no conflicts of interest. Funding Information: Funding: B.R. acknowledges funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy within the Cluster of Excellence PhoenixD (EXC 2122, Funding: B.R. acknowledges funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Project ID 390833453). J.W. and K.B. acknowledge support from the Bundesministerium für Wirtschaft und ID 390833453). J.W. and K.B. acknowledge support from the Bundesministerium für Wirtschaft und Energie
PY - 2019/12
Y1 - 2019/12
N2 - 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.
AB - 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.
KW - 25(OH)D
KW - Aptamer
KW - Biosensing
KW - Microarray
KW - Small molecules
UR - http://www.scopus.com/inward/record.url?scp=85075536385&partnerID=8YFLogxK
U2 - 10.3390/bios9040134
DO - 10.3390/bios9040134
M3 - Article
C2 - 31766203
AN - SCOPUS:85075536385
VL - 9
JO - Biosensors
JF - Biosensors
IS - 4
M1 - 134
ER -