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

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.

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Funding

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).

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Correspondence to Susana Campuzano or Jose M. Pingarrón.

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Published in the topical collection Euroanalysis XX with guest editor Sibel A. Ozkan.

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Povedano, E., Ruiz-Valdepeñas Montiel, V., Gamella, M. et al. A novel zinc finger protein–based amperometric biosensor for miRNA determination. Anal Bioanal Chem 412, 5031–5041 (2020). https://doi.org/10.1007/s00216-019-02219-w

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Keywords

  • Zinc finger protein
  • Screen-printed electrodes
  • miR-21