PAPER IN FOREFRONT A novel zinc finger protein–based amperometric biosensor for miRNA determination 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 & Jose M. Pingarrón 1 Received: 21 September 2019 /Revised: 4 October 2019 /Accepted: 15 October 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 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 mag- netic capture of the resultant magnetic bioconjugates and H 2 O 2 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 (RNA t ) 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 Introduction The importance of detecting microRNAs (miRNAs or miRs) lies in their involvement in many biological processes implied in normal development, physiology, and diseases [1]. This large group of small non-coding RNAs, with critical regula- tory post-transcriptional gene expression functions, has poten- tial applications in diagnosis, prognosis, and therapy control of prevalent diseases including cardiovascular and neurologi- cal conditions and cancer [2]. Dysregulation of miRNAs has been shown to affect the hallmarks of cancer [3], covering evading growth suppressors, resisting cell death, activating invasion and metastasis, and inducing angiogenesis [4]. In fact, several studies have shown that altered miRNA expres- sion is a common feature of all human tumors [5, 6]. However, despite the high clinical relevance of miRNAs, several inherent and technical challenges remain regarding Published in the topical collection Euroanalysis XX with guest editor Sibel A. Ozkan. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-019-02219-w) contains supplementary material, which is available to authorized users. * Susana Campuzano susanacr@quim.ucm.es * Jose M. Pingarrón pingarro@quim.ucm.es 1 Faculty of Chemistry, Department of Analytical Chemistry, Complutense University of Madrid, Pza. de las Ciencias, 28040 Madrid, Spain 2 Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic 3 Cannan Research and Investment & Faculty of Medicine, Complutense University of Madrid, 28040 Madrid, Spain Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-019-02219-w