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

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.

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

“…The achieved LOD is similar (0.2 versus 0.4 nM) [ 27 ] or higher than those reported for other MBs-based approaches for the determination of miRNAs (6 aM [ 28 ], 0.4 pM [ 31 ], 2.4 pM [ 29 ], 40 pM [ 26 ], and 60 pM [ 32 ]). However, the methodology reported here is carried out in a single incubation step (once the MBs were modified with the b-Cp) with a very low concentration of synthetic nucleic acid probe (2.5 nM) and without any amplification strategy.…”

“…These advantages have been coupled in many cases with those provided by planar disposable electrodes (versatility of fabrication and mass production at low cost) to develop different competitive electrochemical biosensing platforms for the quantification of miRNAs. Nevertheless, most of these platforms involve different preparation steps and require the use of expensive affinity reagents [ 26 , 27 , 28 , 29 , 30 , 31 ] and multiple probes in connection with amplification strategies [ 28 , 32 ].…”

Recent Advances on Electrochemical Biosensing Strategies toward Universal Point‐of‐Care Systems

“…This robust electrochemical biosensing strategy was also demonstrated through high-sensitivity,r apid, simultaneous detection of miRNAs directly from metastatic cancer cells and human tumor tissues with ap ico-molar sensitivity. [29] Similar sandwich setup based on magnetic separation and enzyme labeling was demonstrated by the detection of exosomes,i ndicating the versatility of this strategy. [30] Furthermore,b uilding on the same design logic involving the nucleic acid hybridization and amplification system, ac omparison study demonstrated 11 different methods for nucleic acid detection, confirming the diversity and generality of this electrochemical sensing strategy for high-sensitivity nucleic acid detection.…”

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

“…With the aim of overcoming some of these limitations, attractive EC biosensors have recently been proposed for the determination of miRNAs using novel bioreceptors such as tombusvirus protein p19, with a high affinity for the determination of RNA‐RNA homohybrids of limited length or commercial antibodies with a high affinity for RNA‐DNA heteroduplexes of any length . Moreover, to minimize the disadvantages of conventional amplification strategies, the use of an isothermal amplification known as hybridization chain reaction (HCR) has recently been proposed for the determination of miRNAs .…”

“…However, the development of new strategies with improved sensitivity while maintaining the simplicity and assays time would be tremendously relevant for the determination of other miRNAs with fewer copies or in more challenging clinical samples which require higher sensitivity. It is also important to note that the strategies developed so far have only been applied to the individual or simultaneous detection of two target miRNAs .…”