Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers
Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking bi...
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Series: | Journal of Nucleic Acids |
Online Access: | http://dx.doi.org/10.1155/2016/9718612 |
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doaj-fa6ace6106c748769201eedaed7e1cb72020-11-24T20:49:45ZengHindawi LimitedJournal of Nucleic Acids2090-02012090-021X2016-01-01201610.1155/2016/97186129718612Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of AptamersJennifer A. Martin0Yaroslav Chushak1Jorge L. Chávez2Joshua A. Hagen3Nancy Kelley-Loughnane4Human Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433, USAHuman Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433, USAHuman Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433, USAHuman Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433, USAHuman Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433, USAImmobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface.http://dx.doi.org/10.1155/2016/9718612 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jennifer A. Martin Yaroslav Chushak Jorge L. Chávez Joshua A. Hagen Nancy Kelley-Loughnane |
spellingShingle |
Jennifer A. Martin Yaroslav Chushak Jorge L. Chávez Joshua A. Hagen Nancy Kelley-Loughnane Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers Journal of Nucleic Acids |
author_facet |
Jennifer A. Martin Yaroslav Chushak Jorge L. Chávez Joshua A. Hagen Nancy Kelley-Loughnane |
author_sort |
Jennifer A. Martin |
title |
Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers |
title_short |
Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers |
title_full |
Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers |
title_fullStr |
Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers |
title_full_unstemmed |
Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers |
title_sort |
microarrays as model biosensor platforms to investigate the structure and affinity of aptamers |
publisher |
Hindawi Limited |
series |
Journal of Nucleic Acids |
issn |
2090-0201 2090-021X |
publishDate |
2016-01-01 |
description |
Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface. |
url |
http://dx.doi.org/10.1155/2016/9718612 |
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