Autor segons l'article: Yenice, CP; Chahin, N; Jauset-Rubio, M; Hall, M; Biggs, P; Dimai, HP; Obermayer-Pietsch, B; Ortiz, M; O'Sullivan, CK
Departament: Enginyeria Química
Autor/s de la URV: Chahin, Nassif / Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Yenice, Cansu Pinar
Paraules clau: Primer extension product pcr oligonucleotides echinomycin dna biosensor
Resum: 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.
Àrees temàtiques: 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
Accès a la llicència d'ús: https://creativecommons.org/licenses/by/3.0/es/
Adreça de correu electrònic de l'autor: mayreli.ortiz@urv.cat cansupinar.yenice@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat cansupinar.yenice@urv.cat
Identificador de l'autor: 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055
Data d'alta del registre: 2024-08-03
Versió de l'article dipositat: info:eu-repo/semantics/publishedVersion
URL Document de llicència: https://repositori.urv.cat/ca/proteccio-de-dades/
Referència a l'article segons font original: Analytical Chemistry. 95 (38): 14192-14202
Referència de l'ítem segons les normes 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
Entitat: Universitat Rovira i Virgili
Any de publicació de la revista: 2023
Tipus de publicació: Journal Publications
Repositori URV