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Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays

The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilize...

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Autores principales: Hallerbach, Kira, Khederlou, Khadijeh, Wentland, Lael, Senten, Lana, Brentano, Steven, Keefe, Brian, Fu, Elain
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608873/
https://www.ncbi.nlm.nih.gov/pubmed/37893373
http://dx.doi.org/10.3390/mi14101936
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author Hallerbach, Kira
Khederlou, Khadijeh
Wentland, Lael
Senten, Lana
Brentano, Steven
Keefe, Brian
Fu, Elain
author_facet Hallerbach, Kira
Khederlou, Khadijeh
Wentland, Lael
Senten, Lana
Brentano, Steven
Keefe, Brian
Fu, Elain
author_sort Hallerbach, Kira
collection PubMed
description The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin–biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM.
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spelling pubmed-106088732023-10-28 Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays Hallerbach, Kira Khederlou, Khadijeh Wentland, Lael Senten, Lana Brentano, Steven Keefe, Brian Fu, Elain Micromachines (Basel) Article The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin–biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM. MDPI 2023-10-16 /pmc/articles/PMC10608873/ /pubmed/37893373 http://dx.doi.org/10.3390/mi14101936 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hallerbach, Kira
Khederlou, Khadijeh
Wentland, Lael
Senten, Lana
Brentano, Steven
Keefe, Brian
Fu, Elain
Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title_full Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title_fullStr Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title_full_unstemmed Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title_short Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays
title_sort protein-based anchoring methods for nucleic acid detection in lateral flow format assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608873/
https://www.ncbi.nlm.nih.gov/pubmed/37893373
http://dx.doi.org/10.3390/mi14101936
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