New pathogen biosensor published
Label- and amplification-free electrochemical detection of bacterial ribosomal RNA
Grace Henihana*,Holger Schulzea*, Damion K. Corrigana,b, Gerard Giraudc, Jonathan G. Terrye, Alison Hardief, Colin J. Campbellb, Anthony J. Waltone, Jason Crainc, d, Ronald Pethige, Kate E. Templetona, f, Andrew R. Mountb, Till T. Bachmanna
a Division of Infection and Pathway Medicine, College of Medicine and Veterinary Medicine , The University of Edinburgh
b School of Chemistry, The University of Edinburgh
c School of Physics and Astronomy, The University of Edinburgh
d National Physical Laboratory
e Institute for Integrated Micro and Nano Systems, School of Engineering, The University of Edinburgh
f Department of Laboratory Medicine, Royal Infirmary of Edinburgh
Corresponding author: phone: +44 131 242 9438, fax: +44 131 242 6244; email: Till.Bachmann@ed.ac.uk
* These authors contributed equally to this study
Biosensors & Bioelectronics (2016) doi:10.1016/j.bios.2016.03.037
| Abstract |
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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 E. 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 labelled 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. |