Author, as appears in the article.: Yenice, CP; Chahin, N; Jauset-Rubio, M; Hall, M; Biggs, P; Dimai, HP; Obermayer-Pietsch, B; Ortiz, M; O'Sullivan, CK
Department: Enginyeria Química
URV's Author/s: Chahin, Nassif / Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Yenice, Cansu Pinar
Keywords: Primer extension product pcr oligonucleotides echinomycin dna biosensor
Abstract: 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.
Thematic Areas: Química Medicina ii Medicina i Materiais Interdisciplinar Geociências General medicine Farmacia Ensino Engenharias iv Engenharias iii Engenharias ii Enfermagem Ciências biológicas iii Ciências biológicas ii Ciências biológicas i Ciências ambientais Ciências agrárias i Ciência de alimentos Ciência da computação Chemistry, analytical Biotecnología Biodiversidade Astronomia / física Analytical chemistry
licence for use: https://creativecommons.org/licenses/by/3.0/es/
Author's mail: mayreli.ortiz@urv.cat cansupinar.yenice@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat cansupinar.yenice@urv.cat
Author identifier: 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055
Record's date: 2024-08-03
Papper version: info:eu-repo/semantics/publishedVersion
Link to the original source: https://pubs.acs.org/doi/10.1021/acs.analchem.3c01668
Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
Papper original source: Analytical Chemistry. 95 (38): 14192-14202
APA: Yenice, CP; Chahin, N; Jauset-Rubio, M; Hall, M; Biggs, P; Dimai, HP; Obermayer-Pietsch, B; Ortiz, M; O'Sullivan, CK (2023). Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood. Analytical Chemistry, 95(38), 14192-14202. DOI: 10.1021/acs.analchem.3c01668
Article's DOI: 10.1021/acs.analchem.3c01668
Entity: Universitat Rovira i Virgili
Journal publication year: 2023
Publication Type: Journal Publications
Repositori URV