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Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics

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Abstract

A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8 %); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

Pictorial representation of the working principle of the proposed structurally responsive oligonucleotide-based single-probe lateral-flow test and example of the test response as a function of increasing concentration of target analyte.

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Acknowledgments

Kamalodin Kor is grateful to the Ministry of Science, Research and Technology of Iran for financial support (project ref. 214140) to carry out this project at Linköping University, Sweden.

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Authors and Affiliations

  1. Biosensors and Bioelectronics Centre, Department of Physics, Chemistry and Biology (IFM), Linköping University, 58183, Linköping, Sweden

    Kamalodin Kor, Anthony P. F. Turner, Valerio Beni & Wing Cheung Mak

  2. School of Chemistry, Damghan University, Damghan, 36716-41167, Iran

    Kamalodin Kor, Kobra Zarei & Morteza Atabati

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  1. Kamalodin Kor
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  2. Anthony P. F. Turner
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Correspondence to Valerio Beni or Wing Cheung Mak.

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Kor, K., Turner, A.P.F., Zarei, K. et al. Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics. Anal Bioanal Chem 408, 1475–1485 (2016). https://doi.org/10.1007/s00216-015-9250-9

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  • DOI: https://doi.org/10.1007/s00216-015-9250-9

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