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

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.

[1]  S. Rajagopalan,et al.  Noncoding RNAs in Cardiovascular Disease: Pathological Relevance and Emerging Role as Biomarkers and Therapeutics. , 2018, American journal of hypertension.

[2]  D. Xing,et al.  Ultrasensitive Detection of MicroRNA in Tumor Cells and Tissues via Continuous Assembly of DNA Probe. , 2015, Biomacromolecules.

[3]  F. Ricci,et al.  A review on novel developments and applications of immunosensors in food analysis. , 2007, Analytica chimica acta.

[4]  María Pedrero,et al.  Non-Invasive Breast Cancer Diagnosis through Electrochemical Biosensing at Different Molecular Levels , 2017, Sensors.

[5]  A. Erdem,et al.  microRNA biosensors: Opportunities and challenges among conventional and commercially available techniques. , 2018, Biosensors & bioelectronics.

[6]  S. Yao,et al.  Carbon nanotube-based label-free electrochemical biosensor for sensitive detection of miRNA-24. , 2014, Biosensors & bioelectronics.

[7]  C. Esposito,et al.  SERS-active metal-dielectric nanostructures integrated in microfluidic devices for label-free quantitative detection of miRNA. , 2017, Faraday discussions.

[8]  Yan Zhang,et al.  Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth , 2011, Breast Cancer Research.

[9]  M. Mascini,et al.  A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs) , 2005 .

[10]  E. Giovannetti,et al.  MicroRNA-21 links epithelial-to-mesenchymal transition and inflammatory signals to confer resistance to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast cancer patients , 2015, Oncotarget.

[11]  Jing Zhang,et al.  An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA. , 2016, Biosensors & bioelectronics.

[12]  Liang Chen,et al.  miRNA Biomarkers in Breast Cancer Detection and Management , 2011, Journal of Cancer.

[13]  Muneesh Tewari,et al.  Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). , 2010, Methods.

[14]  Paul Bertone,et al.  Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. , 2010, RNA.

[15]  Miki Ohira,et al.  Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes , 2016, Journal of Human Genetics.

[16]  Terry J. Smith,et al.  Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay , 2017, Analytical and Bioanalytical Chemistry.

[17]  B. Nielsen,et al.  miR-21 Expression in Cancer Cells may Not Predict Resistance to Adjuvant Trastuzumab in Primary Breast Cancer , 2014, Front. Oncol..

[18]  Mohammad Hasanzadeh,et al.  Early stage screening of breast cancer using electrochemical biomarker detection , 2017 .

[19]  Erkang Wang,et al.  Electrochemical biosensors based on magnetic micro/nano particles , 2012 .

[20]  S. Campuzano,et al.  Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum , 2012 .

[21]  Nóra Varga,et al.  Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. , 2004, Nucleic acids research.

[22]  Wei Wang,et al.  Label-Free MicroRNA Detection Based on Fluorescence Quenching of Gold Nanoparticles with a Competitive Hybridization. , 2015, Analytical chemistry.

[23]  Susana Campuzano,et al.  Direct Determination of miR‐21 in Total RNA Extracted from Breast Cancer Samples Using Magnetosensing Platforms and the p19 Viral Protein as Detector Bioreceptor , 2014 .

[24]  S. Campuzano,et al.  Electrochemical miRNAs Determination in Formalin-Fixed, Paraffin-Embedded Breast Tumor Tissues Association with HER2 Expression , 2016 .

[25]  G. Yousef,et al.  Prognostic significance of metastasis-related microRNAs in early breast cancer patients with a long follow-up. , 2014, Clinical chemistry.

[26]  Ting Hou,et al.  Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay. , 2015, Analytical chemistry.

[27]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[28]  Suresh Shrestha,et al.  Bioluminescence-based detection of microRNA, miR21 in breast cancer cells. , 2008, Analytical chemistry.

[29]  J M Pingarrón,et al.  Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification. , 2016, Biosensors & bioelectronics.

[30]  Yang Li,et al.  An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe , 2005, Nucleic acids research.

[31]  M. Kassem,et al.  Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers , 2017, Cell Death & Disease.

[32]  Ke-Jing Huang,et al.  Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review. , 2018, Biosensors & bioelectronics.

[33]  Jianzhong Xi,et al.  Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation. , 2009, Analytical chemistry.

[34]  David Galas,et al.  Surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level. , 2010, Analytical chemistry.

[35]  María Pedrero,et al.  Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs , 2017, International journal of molecular sciences.

[36]  Zhizhong Han,et al.  An electrochemical microRNA biosensor based on protein p19 combining an acridone derivate as indicator and DNA concatamers for signal amplification , 2015 .

[37]  M. Marco,et al.  Electrochemical magneto immunosensing of antibiotic residues in milk. , 2007, Biosensors & bioelectronics.

[38]  P. Ferrari,et al.  Prognostic and predictive biomarkers in breast cancer: Past, present and future. , 2017, Seminars in cancer biology.

[39]  Gurman Singh Pall,et al.  Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot , 2007, Nucleic acids research.

[40]  Yun Chen,et al.  Quantification of microRNA by DNA-Peptide Probe and Liquid Chromatography-Tandem Mass Spectrometry-Based Quasi-Targeted Proteomics. , 2016, Analytical chemistry.

[41]  Zissimos Mourelatos,et al.  Microarray-based, high-throughput gene expression profiling of microRNAs , 2004, Nature Methods.

[42]  S. Campuzano,et al.  Magnetobiosensors based on viral protein p19 for microRNA determination in cancer cells and tissues. , 2014, Angewandte Chemie.

[43]  Susana Campuzano,et al.  Fast Electrochemical miRNAs Determination in Cancer Cells and Tumor Tissues with Antibody-Functionalized Magnetic Microcarriers , 2016 .