Determination of miRNAs in serum of cancer patients with a label- and enzyme-free voltammetric biosensor in a single 30-
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ORIGINAL PAPER
Determination of miRNAs in serum of cancer patients with a labeland enzyme-free voltammetric biosensor in a single 30-min step Mohamed Zouari 1,2
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Susana Campuzano 3
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José M. Pingarrón 3
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Noureddine Raouafi 1
Received: 3 April 2020 / Accepted: 17 June 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO2H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Keywords miRNA-21 . Electrochemical biosensor . Label-free . Serum samples . Nanomaterial . Breast cancer
Introduction Dysregulation of microRNAs (miRNAs), naturally occurring small (about 22 nucleotides long) RNA transcripts that are not transcribed into proteins but regulate the transcription, stability, or translation of protein-coding genes in the mammalian genome [1], is associated with cancer initiation, development, and metastasis as well as with tumor response to treatments. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-020-04400-w) contains supplementary material, which is available to authorized users. * Noureddine Raouafi [email protected] 1
Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), Chemistry Department, University of Tunis El Manar, 2092 Tunis El Manar, Tunisia
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Institut Pasteur de Tunis, 13 place Pasteur, 1002 Tunis, Tunisia
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Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E–28040 Madrid, Spain
The apparent stability of miRNAs in archived patie
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