Next Article in Journal
A Comprehensive Study on Pyrolysis Mechanism of Substituted β-O-4 Type Lignin Dimers
Next Article in Special Issue
The Dark Side of the Epitranscriptome: Chemical Modifications in Long Non-Coding RNAs
Previous Article in Journal
Physical, Structural, Barrier, and Antifungal Characterization of Chitosan–Zein Edible Films with Added Essential Oils
Previous Article in Special Issue
miRNAs, Melanoma and Microenvironment: An Intricate Network
Article Menu
Issue 11 (November) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2017, 18(11), 2151; https://doi.org/10.3390/ijms18112151

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs

1
Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
2
Cannan Research and Investment & Faculty of Medicine, University Complutense of Madrid, 28040 Madrid, Spain
*
Authors to whom correspondence should be addressed.
Received: 17 September 2017 / Revised: 8 October 2017 / Accepted: 9 October 2017 / Published: 9 November 2017
(This article belongs to the Collection Regulation by Non-Coding RNAs)
View Full-Text   |   Download PDF [1798 KB, uploaded 9 November 2017]   |  

Abstract

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA–RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at −0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA–RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNAt) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues. View Full-Text
Keywords: miRNA-21; electrochemical sensor; anti DNA–RNA hybrid antibody; ProtA-PolyHRP40; cancer cells; breast human tissues miRNA-21; electrochemical sensor; anti DNA–RNA hybrid antibody; ProtA-PolyHRP40; cancer cells; breast human tissues
Figures

Graphical abstract

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).

Share & Cite This Article

MDPI and ACS Style

Vargas, E.; Torrente-Rodríguez, R.M.; Ruiz-Valdepeñas Montiel, V.; Povedano, E.; Pedrero, M.; Montoya, J.J.; Campuzano, S.; Pingarrón, J.M. Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs. Int. J. Mol. Sci. 2017, 18, 2151.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top