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Sensors 2018, 18(3), 863; https://doi.org/10.3390/s18030863

Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads

1
Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
2
Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Department of Chemistry, Faculty of Science, University of Tunis El Manar, Rue Béchir Salem Belkheria, Tunis El-Manar, 2092 Tunis, Tunisia
3
Cannan Research and Investment & Faculty of Medicine, University Complutense of Madrid, 28040 Madrid, Spain
*
Authors to whom correspondence should be addressed.
Received: 6 February 2018 / Revised: 7 March 2018 / Accepted: 12 March 2018 / Published: 15 March 2018
(This article belongs to the Special Issue I3S 2017 Selected Papers)
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Abstract

This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs) and amperometric transduction at screen-printed carbon electrodes (SPCEs). The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC)-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp) immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs). Upon labeling, the FITC-modified miRNA attached to the MBs with horseradish peroxidase (HRP)-conjugated anti-FITC Fab fragments and magnetic capturing of the MBs onto the working electrode surface of SPCEs. The cathodic current measured at −0.20 V (versus the Ag pseudo-reference electrode) was demonstrated to be inversely proportional to the concentration of the target miRNA. This convenient biosensing method provided a linear range between 0.7 and 10.0 nM and a limit of detection (LOD) of 0.2 nM (5 fmol in 25 μL of sample) for the synthetic target miRNA without any amplification step. An acceptable selectivity towards single-base mismatched oligonucleotides, a high storage stability of the b-Cp-MBs, and usefulness for the accurate determination of miRNA-21 in raw total RNA (RNAt) extracted from breast cancer cells (MCF-7) were demonstrated. View Full-Text
Keywords: miRNA; screen-printed electrode; amperometry; competitive assay; magnetic beads; cancer cells miRNA; screen-printed electrode; amperometry; competitive assay; magnetic beads; cancer cells
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Vargas, E.; Povedano, E.; Montiel, V.R.-V.; Torrente-Rodríguez, R.M.; Zouari, M.; Montoya, J.J.; Raouafi, N.; Campuzano, S.; Pingarrón, J.M. Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads. Sensors 2018, 18, 863.

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