Fast Electrochemical miRNAs Determination in Cancer Cells and Tumor Tissues with Antibody-Functionalized Magnetic Microcarriers

Abstract

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and diseases and constitute important targets in future medical diagnostics and prognostics. We report here a novel sensitive and rapid bioelectrochemical strategy for miRNA determination. This strategy involves the development of a sensing approach making use of magnetic beads (MBs) modified with a specific DNA-RNA antibody as capture bioreceptor and amperometric detection implying the H2O2/hydroquinone (HQ) system at disposable screen-printed carbon electrodes (SPCEs). The developed biosensor exhibits a dynamic range from 8.2 to 250 pM and a detection limit of 2.4 pM (60 amol) of a synthetic target without any amplification step in 2 h. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic cancer cell lines and human tumor tissues, which demonstrated its potential to perform determination of mature miRNAs in these complex samples. Moreover, the feasibility of the developed methodology to detect simultaneously the expression of two different miRNAs at dual SPCEs (SPdCEs) in one single experiment was also explored. The feasibility to capture and release target miRNAs make the developed methodology also an attractive tool to isolate, purify, and determine target miRNAs with great applicability in the clinical field.

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

“…Three miRNAs, miR‐21, let‐7a and miR‐31, associated with development and progression of cervical cancer in women , were selected as target miRNAs. The results obtained, after a suitable optimization of the methodology, showed that the new HCR‐based immunosensing strategy offers in a short assay time (120 min) a sensitivity 17 and 131 times higher than that previously reported using individually modified MBs with the same capture antibody without amplification or a HCR strategy implemented on the surface of MBs , respectively. The electrochemical platform was successfully employed for the simultaneous determination of three target miRNAs in RNA t extracted from various cancer cell lines and from cervical precancerous lesions of women suffering from the most severe grade of high‐grade squamous intraepithelial lesion (HSIL).…”

“…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 .…”

Magnetic Particles Coupled to Disposable Screen Printed Transducers for Electrochemical Biosensing

“…This approach involved the use of ProtG-MMPs modified with the antibody AbS9.6 specific for the DNA/RNA heteroduplexes as selective microcarriers to capture the hybrids previously formed in solution by homogeneous hybridization between the biotinylated complementary DNA probe and the target miRNA [45]. This immunosensing approach exhibited a dynamic range from 8.2 to 250 pM and a LOD of 2.4 pM (60 amol) of the synthetic target miRNA.…”

Improving Cancer Outcomes through Electrochemical Biosensing of Early Diagnosis/Prognosis Biomarkers in Human Biopsies

“…Within this context, the main characteristics of novel biosensing scaffolds for the determination of cancer related-miRNAs [1][2][3] and autoantibodies [4] against tumor associated antigens (TAAs), relevant biomarkers considered for both early diagnosis and prognosis, will be presented. The developed methodologies, based on the coupling of attractive bioreceptors and bioassay formats, functionalized magnetic microcarriers, and electrochemical detection at disposable transducers, have demonstrated practical applicability for the accurate determination of the endogenous concentration of the target analytes in solid (fresh and FFPE breast human tissues) and liquid (human serum from colorectal and ovarian cancer patients) biopsies.…”