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Determination of miRNAs in serum of cancer patients with a label- and enzyme-free voltammetric biosensor in a single 30-min step

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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 6-ferrocenylhexanethiol (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.

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Funding

The Tunisian Ministry of Higher Education and Scientific Research (Lab. LR99ES15). NR thanks the Tunisian PRF program for financial support (NanoFastResponse ref. PRF2017-D4P1, SmartBioSens ref. PRFCOV19-D2P2 and COVID-PP ref. PRFCOV19-D3P1) and Prof. Besma Loueslati (University of Tunis El Manar, Biology department) for providing the serum samples used in this study. Ministerio de Ciencia e Innovación Project, PID2019-103899RB-I00 and the TRANSNANOAVANSENS−CM Program from the Comunidad de Madrid (Grant S2018/NMT−4349) are gratefully acknowledged.

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Authors and Affiliations

  1. Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), Chemistry Department, University of Tunis El Manar, 2092, Tunis El Manar, Tunisia

    Mohamed Zouari & Noureddine Raouafi

  2. Institut Pasteur de Tunis, 13 place Pasteur, 1002, Tunis, Tunisia

    Mohamed Zouari

  3. Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E–28040, Madrid, Spain

    Susana Campuzano & José M. Pingarrón

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  1. Mohamed Zouari
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  2. Susana Campuzano
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  4. Noureddine Raouafi
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Correspondence to Noureddine Raouafi.

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The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Zouari, M., Campuzano, S., Pingarrón, J.M. et al. Determination of miRNAs in serum of cancer patients with a label- and enzyme-free voltammetric biosensor in a single 30-min step. Microchim Acta 187, 444 (2020). https://doi.org/10.1007/s00604-020-04400-w

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