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Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia
Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplifi...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663036/ https://www.ncbi.nlm.nih.gov/pubmed/33142935 http://dx.doi.org/10.3390/s20216240 |
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author | Sierpe, Rodrigo Kogan, Marcelo J. Bollo, Soledad |
author_facet | Sierpe, Rodrigo Kogan, Marcelo J. Bollo, Soledad |
author_sort | Sierpe, Rodrigo |
collection | PubMed |
description | Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method. |
format | Online Article Text |
id | pubmed-7663036 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-76630362020-11-14 Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia Sierpe, Rodrigo Kogan, Marcelo J. Bollo, Soledad Sensors (Basel) Article Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method. MDPI 2020-10-31 /pmc/articles/PMC7663036/ /pubmed/33142935 http://dx.doi.org/10.3390/s20216240 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Sierpe, Rodrigo Kogan, Marcelo J. Bollo, Soledad Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title | Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title_full | Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title_fullStr | Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title_full_unstemmed | Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title_short | Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia |
title_sort | label-free oligonucleotide-based spr biosensor for the detection of the gene mutation causing prothrombin-related thrombophilia |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663036/ https://www.ncbi.nlm.nih.gov/pubmed/33142935 http://dx.doi.org/10.3390/s20216240 |
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