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

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Vargas Orgaz, Eva and Povedano Muñumel, Eloy and Ruiz Valdepeñas Montiel, Víctor and Torrente Rodríguez, Rebeca Magnolia and Zouari, Mohamed and Montoya Miñano, Juan José and Raouafi, Noureddine and Campuzano Ruiz, Susana and Pingarrón Carrazón, José Manuel (2018) Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads. Sensors, 18 (3). p. 863. ISSN 1424-8220

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Official URL: https://doi.org/10.3390/s18030863




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.


Item Type:Article
Uncontrolled Keywords:miRNA; screen-printed electrode; amperometry; competitive assay; magnetic beads; cancer cells
Subjects:Sciences > Chemistry > Analytic chemistry
ID Code:66794
Deposited On:07 Jul 2021 13:57
Last Modified:07 Jul 2021 15:34

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