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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...

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Autores principales: Yenice, Cansu Pinar, Chahin, Nassif, Jauset-Rubio, Miriam, Hall, Matthew, Biggs, Phil, Dimai, Hans-Peter, Obermayer-Pietsch, Barbara, Ortiz, Mayreli, O’Sullivan, Ciara K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
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.
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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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