Analytical and Bioanalytical Chemistry

, Volume 409, Issue 12, pp 3261–3269 | Cite as

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection

  • Jonathan Sabaté del Río
  • Ivan Magriñà Lobato
  • Olena Mayboroda
  • Ioanis Katakis
  • Ciara K. O’Sullivan
Research Paper

Abstract

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10−15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples.

Graphical abstract

Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Keywords

Electrochemical genosensor Solid-phase recombinase polymerase amplification Surface chemistry Real samples from hares 

Notes

Acknowledgements

This work has been carried out with partial financial support from Spanish Ministerio de Economía y Competitividad (SEASENSING BIO2014-56024-C2-1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_269_MOESM1_ESM.pdf (471 kb)
ESM 1(PDF 470 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Jonathan Sabaté del Río
    • 1
  • Ivan Magriñà Lobato
    • 1
  • Olena Mayboroda
    • 1
  • Ioanis Katakis
    • 1
  • Ciara K. O’Sullivan
    • 1
    • 2
  1. 1.Departament d’Enginyeria QuímicaUniversitat Rovira i VirgiliTarragonaSpain
  2. 2.Institució Catalana de Recerca i Estudis AvançatsBarcelonaSpain

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