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Microchimica Acta

, 186:426 | Cite as

Nucleic acid lateral flow assays using a conjugate of a DNA binding protein and carbon nanoparticles

  • Gülsen Betül Aktas
  • Jan H. Wichers
  • Vasso Skouridou
  • Aart van AmerongenEmail author
  • Lluis MasipEmail author
Original Paper
  • 32 Downloads

Abstract

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 103 CFU·mL−1. The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence.

Graphical abstract

Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles.

Keywords

Nucleic acid lateral flow assay Immunochromatographic test Single-chain Cro Enzyme conjugate Signal enhancement Escherichia coli 

Notes

Acknowledgements

This work was supported financially by the FP7-PEOPLE-2011-CIG DeCoDeB project grant awarded to LM. GBA acknowledges the Universitat Rovira i Virgili for the doctoral fellowship and EMBO for the short-term fellowship. The authors thank Prof. M. C. Mossing (University of Mississippi, USA) for the gift of the pscCro16 construct.

Compliance with ethical standards

The authors declare that they have no competing interests.

Supplementary material

604_2019_3544_MOESM1_ESM.pdf (254 kb)
ESM 1 (PDF 254 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Interfibio, Bioengineering and Bioelectrochemistry Group, Departament d’Enginyeria QuímicaUniversitat Rovira i VirgiliTarragonaSpain
  2. 2.BioSensing & Diagnostics, Wageningen Food & Biobased ResearchWageningen University & ResearchWageningenThe Netherlands

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