Thioaromatic DNA monolayers for target-amplification-free electrochemical sensing of environmental pathogenic bacteria

• Published in: Biosensors & bioelectronics 2017-06, Vol.92, p.162-170
• Main Authors: Miranda-Castro, Rebeca, Sánchez-Salcedo, Raquel, Suárez-Álvarez, Beatriz, de-los-Santos-Álvarez, Noemí, Miranda-Ordieres, Arturo J., Jesús Lobo-Castañón, María
• Format: Article
• Language: English
• Subjects: Aniline Compounds - chemistry; Backfilling method; Bacterial RNA detection; Benzoates - chemistry; Biosensing Techniques - methods; DNA sensing surfaces; Electrochemical Techniques - methods; Gold - chemistry; Humans; Immobilized Nucleic Acids - chemistry; Inserting method; Legionella pneumophila; Legionella pneumophila - isolation & purification; Legionnaires' Disease - microbiology; Limit of Detection; Nucleic Acid Hybridization - methods; RNA, Bacterial - analysis; RNA, Ribosomal - analysis; Sulfhydryl Compounds - chemistry; Surface Properties; Thioaromatic self-assembled monolayers; Water Microbiology
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2017.02.017

Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p-mercaptobenzoic acid (p-MBA) and p-aminothiophenol (p-ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p-ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p-MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria. [Display omitted] •New methods for preparing finely tuned DNA sensing layers on gold using aromatic thiols.•A sensing platform for the direct detection of bacterial rRNA.•Improved sensitivity and storage stability versus classical mercaptohexanol monolayers.