title: Enzymatic amplification-free nucleic acid hybridisation sensing on nanostructured thick-film electrodes by using covalently attached methylene blue.
abstract: Amplification-free (referring to enzymatic amplification step) detection methodologies are increasing in biosensor development due to the need of faster and simpler protocols. However, for maintaining sensitivity without this step, highly detectable molecules or very sensitive detection techniques are required. The nanostructuration of transducer surfaces with carbon nanotubes (CNTs), gold nanoparticles (AuNPs) or both in nanohybrid configurations has been employed in this work for DNA hybridisation sensing purposes. Methylene blue (MB), covalently attached to single stranded DNA, (ssDNA) was incubated with a complementary sequence immobilized on nanostructured screen-printed electrodes (AuSPEs). Although CNTs can increase notoriously the signal of the marker, adsorptive properties should also be considered when bioassays are performed because non-specific adsorption (NSA) phenomena are magnified. In this work, strategies for decreasing NSA were thoroughly evaluated for the detection of Mycoplasma pneumoniae (MP) on CNTs-nanostructured screen-printed electrodes. Among them, the employ of UV-radiation or long incubation times (72h) allowed obtaining higher signals for the complementary strand with respect to the non-complementary one. The use of CNTs/AuNPs nanohybrids, together with the use of streptavidin-biotin (ST-B) interaction allows the higher differentiation (with a 3.5 ratio) in the genosensing of M. pneumoniae.
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: García-González
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain.
authors.forename: Agustín
authors.initials: A
authors.lastname: Costa-García
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain. Electronic address: mtfernandeza@uniovi.es.
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Fernández-Abedul
mesh_terms: D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry; D008751:Methylene Blue / Q000737:chemistry; D009177:Mycoplasma pneumoniae / Q000235:genetics*; D037742:Nanotubes, Carbon; D009693:Nucleic Acid Hybridization; D019809:Streptavidin / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D037742:Nanotubes, Carbon; D001710:Biotin; D006046:Gold; D019809:Streptavidin; D008751:Methylene Blue
keywords: Amplification-free biosensors; Methylene blue; Nanohybrids; Nanostructured gold screen-printed electrodes; Nucleic acid strands detection