RT Journal Article
T1 Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs
A1 Vargas Orgaz, Eva
A1 Torrente Rodríguez, Rebeca Magnolia
A1 Ruiz Valdepeñas Montiel, Víctor
A1 Povedano Muñumel, Eloy
A1 Pedrero Muñoz, María
A1 Montoya Miñano, Juan José
A1 Campuzano Ruiz, Susana
A1 Pingarrón Carrazón, José Manuel
AB 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.
PB MDPI
SN 1422-0067
YR 2017
FD 2017-11-09
LK https://hdl.handle.net/20.500.14352/19185
UL https://hdl.handle.net/20.500.14352/19185
LA eng
NO Ministerio de Economía y Competitividad (MINECO)
NO Comunidad de Madrid
DS Docta Complutense
RD 7 abr 2025