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A novel zinc finger protein–based amperometric biosensor for miRNA determination

  • Eloy Povedano
  • Víctor Ruiz-Valdepeñas Montiel
  • María Gamella
  • Verónica Serafín
  • María Pedrero
  • Ludmila Moranova
  • Martin Bartosik
  • Juan José Montoya
  • Paloma Yáñez-Sedeño
  • Susana CampuzanoEmail author
  • Jose M. PingarrónEmail author
Paper in Forefront
  • 155 Downloads
Part of the following topical collections:
  1. Euroanalysis XX

Abstract

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin–horseradish peroxidase (Strep–HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (− 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA–RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP’s non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

Keywords

Zinc finger protein Screen-printed electrodes miR-21 

Notes

Funding information

The financial support of the CTQ2015-64402-C2-1-R (Spanish Ministerio de Economía y Competitividad) and RTI2018-096135-B-I00 (Ministerio de Ciencia, Innovación y Universidades) Research Projects and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged, and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (EP) and Universidad Complutense de Madrid (VRVM) are also gratefully acknowledged. VS was financially supported by a postdoctoral contract type Art 83. LOU with MiRNAx Biosens. S.L. company. MB and LJ would like to acknowledge financial support from the projects of Czech Science Foundation 17-08971S, MEYS – NPS I – LO1413, and MH CZ - DRO (MMCI, 00209805).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2219_MOESM1_ESM.pdf (584 kb)
ESM 1 (PDF 583 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Eloy Povedano
    • 1
  • Víctor Ruiz-Valdepeñas Montiel
    • 1
  • María Gamella
    • 1
  • Verónica Serafín
    • 1
  • María Pedrero
    • 1
  • Ludmila Moranova
    • 2
  • Martin Bartosik
    • 2
  • Juan José Montoya
    • 3
  • Paloma Yáñez-Sedeño
    • 1
  • Susana Campuzano
    • 1
    Email author
  • Jose M. Pingarrón
    • 1
    Email author
  1. 1.Faculty of Chemistry, Department of Analytical ChemistryComplutense University of MadridMadridSpain
  2. 2.Regional Centre for Applied Molecular Oncology (RECAMO)Masaryk Memorial Cancer InstituteBrnoCzech Republic
  3. 3.Cannan Research and Investment & Faculty of MedicineComplutense University of MadridMadridSpain

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