Label- and amplification-free electrochemical detection of bacterial ribosomal RNA

Grace Henihan, Holger Schulze, Damion K. Corrigan, Gerard Giraud, Jonathan G. Terry, Alison Hardie, Colin J. Campbell, Anthony J. Walton, Jason Crain, Ronald Pethig, Kate E. Templeton, Andrew R. Mount, Till T. Bachmann

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 10-40 min at room temperature without wash steps.

LanguageEnglish
Pages487-494
Number of pages8
JournalBiosensors and Bioelectronics
Volume81
Early online date18 Mar 2016
DOIs
Publication statusPublished - 15 Jul 2016
Externally publishedYes

Fingerprint

Bacterial RNA
Ribosomal RNA
Point-of-Care Systems
Amplification
Labels
Microarrays
Molecular Pathology
Pathogens
Oligonucleotide Array Sequence Analysis
Computer Simulation
Glass
Escherichia coli
Costs and Cost Analysis
Polymerase Chain Reaction
Temperature
Assays
DNA
Kinetics

Keywords

  • 16S ribosomal RNA
  • E. coli
  • electrochemical impedance spectroscopy
  • molecular diagnostics
  • pathogen detection
  • PCR-free

Cite this

Henihan, Grace ; Schulze, Holger ; Corrigan, Damion K. ; Giraud, Gerard ; Terry, Jonathan G. ; Hardie, Alison ; Campbell, Colin J. ; Walton, Anthony J. ; Crain, Jason ; Pethig, Ronald ; Templeton, Kate E. ; Mount, Andrew R. ; Bachmann, Till T. / Label- and amplification-free electrochemical detection of bacterial ribosomal RNA. In: Biosensors and Bioelectronics. 2016 ; Vol. 81. pp. 487-494.
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Henihan, G, Schulze, H, Corrigan, DK, Giraud, G, Terry, JG, Hardie, A, Campbell, CJ, Walton, AJ, Crain, J, Pethig, R, Templeton, KE, Mount, AR & Bachmann, TT 2016, 'Label- and amplification-free electrochemical detection of bacterial ribosomal RNA' Biosensors and Bioelectronics, vol. 81, pp. 487-494. https://doi.org/10.1016/j.bios.2016.03.037

Label- and amplification-free electrochemical detection of bacterial ribosomal RNA. / Henihan, Grace; Schulze, Holger; Corrigan, Damion K.; Giraud, Gerard; Terry, Jonathan G.; Hardie, Alison; Campbell, Colin J.; Walton, Anthony J.; Crain, Jason; Pethig, Ronald; Templeton, Kate E.; Mount, Andrew R.; Bachmann, Till T.

In: Biosensors and Bioelectronics, Vol. 81, 15.07.2016, p. 487-494.

Research output: Contribution to journalArticle

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T1 - Label- and amplification-free electrochemical detection of bacterial ribosomal RNA

AU - Henihan, Grace

AU - Schulze, Holger

AU - Corrigan, Damion K.

AU - Giraud, Gerard

AU - Terry, Jonathan G.

AU - Hardie, Alison

AU - Campbell, Colin J.

AU - Walton, Anthony J.

AU - Crain, Jason

AU - Pethig, Ronald

AU - Templeton, Kate E.

AU - Mount, Andrew R.

AU - Bachmann, Till T.

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N2 - 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 10-40 min at room temperature without wash steps.

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