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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Main Authors: Jennifer A. Martin, Yaroslav Chushak, Jorge L. Chávez, Joshua A. Hagen, Nancy Kelley-Loughnane
Format: Article
Language:English
Published: Hindawi Limited 2016-01-01
Series:Journal of Nucleic Acids
Online Access:http://dx.doi.org/10.1155/2016/9718612
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spelling 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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