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Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood
[Image: see text] The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase elect...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10534999/ https://www.ncbi.nlm.nih.gov/pubmed/37713191 http://dx.doi.org/10.1021/acs.analchem.3c01668 |
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author | Yenice, Cansu Pinar Chahin, Nassif Jauset-Rubio, Miriam Hall, Matthew Biggs, Phil Dimai, Hans-Peter Obermayer-Pietsch, Barbara Ortiz, Mayreli O’Sullivan, Ciara K. |
author_facet | Yenice, Cansu Pinar Chahin, Nassif Jauset-Rubio, Miriam Hall, Matthew Biggs, Phil Dimai, Hans-Peter Obermayer-Pietsch, Barbara Ortiz, Mayreli O’Sullivan, Ciara K. |
author_sort | Yenice, Cansu Pinar |
collection | PubMed |
description | [Image: see text] The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing. |
format | Online Article Text |
id | pubmed-10534999 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-105349992023-09-29 Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood Yenice, Cansu Pinar Chahin, Nassif Jauset-Rubio, Miriam Hall, Matthew Biggs, Phil Dimai, Hans-Peter Obermayer-Pietsch, Barbara Ortiz, Mayreli O’Sullivan, Ciara K. Anal Chem [Image: see text] The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing. American Chemical Society 2023-09-15 /pmc/articles/PMC10534999/ /pubmed/37713191 http://dx.doi.org/10.1021/acs.analchem.3c01668 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Yenice, Cansu Pinar Chahin, Nassif Jauset-Rubio, Miriam Hall, Matthew Biggs, Phil Dimai, Hans-Peter Obermayer-Pietsch, Barbara Ortiz, Mayreli O’Sullivan, Ciara K. Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood |
title | Semiautomated
Electrochemical Melting Curve Analysis
Device for the Detection of an Osteoporosis Associated Single Nucleotide
Polymorphism in Blood |
title_full | Semiautomated
Electrochemical Melting Curve Analysis
Device for the Detection of an Osteoporosis Associated Single Nucleotide
Polymorphism in Blood |
title_fullStr | Semiautomated
Electrochemical Melting Curve Analysis
Device for the Detection of an Osteoporosis Associated Single Nucleotide
Polymorphism in Blood |
title_full_unstemmed | Semiautomated
Electrochemical Melting Curve Analysis
Device for the Detection of an Osteoporosis Associated Single Nucleotide
Polymorphism in Blood |
title_short | Semiautomated
Electrochemical Melting Curve Analysis
Device for the Detection of an Osteoporosis Associated Single Nucleotide
Polymorphism in Blood |
title_sort | semiautomated
electrochemical melting curve analysis
device for the detection of an osteoporosis associated single nucleotide
polymorphism in blood |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10534999/ https://www.ncbi.nlm.nih.gov/pubmed/37713191 http://dx.doi.org/10.1021/acs.analchem.3c01668 |
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