title: Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.
abstract: Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.
authors.affiliation: Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK.
authors.forename: Grace
authors.initials: G
authors.lastname: Henihan
authors.affiliation: Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK.
authors.forename: Holger
authors.initials: H
authors.lastname: Schulze
authors.affiliation: Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK; School of Chemistry, The University of Edinburgh, Joseph Black Building, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK.
authors.forename: Damion K
authors.initials: DK
authors.lastname: Corrigan
authors.affiliation: School of Physics and Astronomy, The University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JZ, Scotland, UK.
authors.forename: Gerard
authors.initials: G
authors.lastname: Giraud
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, The University of Edinburgh, Alexander Crum Brown Road, Edinburgh EH9 3FF, Scotland, UK.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Terry
authors.affiliation: Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, Scotland, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Hardie
authors.affiliation: School of Chemistry, The University of Edinburgh, Joseph Black Building, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Campbell
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, The University of Edinburgh, Alexander Crum Brown Road, Edinburgh EH9 3FF, Scotland, UK.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Walton
authors.affiliation: School of Physics and Astronomy, The University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JZ, Scotland, UK; National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, UK.
authors.forename: Jason
authors.initials: J
authors.lastname: Crain
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, The University of Edinburgh, Alexander Crum Brown Road, Edinburgh EH9 3FF, Scotland, UK.
authors.forename: Ronald
authors.initials: R
authors.lastname: Pethig
authors.affiliation: Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK; Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, Scotland, UK.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Templeton
authors.affiliation: School of Chemistry, The University of Edinburgh, Joseph Black Building, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Mount
authors.affiliation: Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK. Electronic address: Till.Bachmann@ed.ac.uk.
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D058266:Dielectric Spectroscopy / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics*; D004927:Escherichia coli Infections / Q000382:microbiology*; D006801:Humans; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S ribosomal RNA; E. coli; Electrochemical impedance spectroscopy; Molecular diagnostics; PCR-free; Pathogen detection