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Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay

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Abstract

MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin–alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L−1 and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.

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Authors and Affiliations

  1. Dipartimento di Chimica “Ugo Schiff”, Università degli Studi di Firenze, Via della Lastruccia 3, 50019, Sesto Fiorentino (Fi), Italy

    Francesca Bettazzi, Ezat Hamid-Asl, Serena Laschi, Giovanna Marrazza, Marco Mascini & Ilaria Palchetti

  2. Istituto di Endocrinologia e Oncologia Sperimentale, CNR, 80138, Napoli, Italy

    Carla Lucia Esposito, Laura Cerchia & Vittorio De Franciscis

  3. Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80138, Napoli, Italy

    Cristina Quintavalle, Nello Formisano, Silvia Catuogno, Margherita Iaboni & Gerolama Condorelli

  4. Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandran, Babolsar, Iran

    Ezat Hamid-Asl

  5. Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II and Istituto di Endocrinologia e Oncologia Sperimentale, CNR, 80138, Napoli, Italy

    Silvia Catuogno & Gerolama Condorelli

Authors
  1. Francesca Bettazzi
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  2. Ezat Hamid-Asl
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  3. Carla Lucia Esposito
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  4. Cristina Quintavalle
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  5. Nello Formisano
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  6. Serena Laschi
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  7. Silvia Catuogno
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  8. Margherita Iaboni
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  9. Giovanna Marrazza
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  10. Marco Mascini
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  11. Laura Cerchia
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  12. Vittorio De Franciscis
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  13. Gerolama Condorelli
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  14. Ilaria Palchetti
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Corresponding author

Correspondence to Ilaria Palchetti.

Additional information

Published in the special issue Analytical Science in Italy with guest editor Aldo Roda.

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Bettazzi, F., Hamid-Asl, E., Esposito, C.L. et al. Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay. Anal Bioanal Chem 405, 1025–1034 (2013). https://doi.org/10.1007/s00216-012-6476-7

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  • DOI: https://doi.org/10.1007/s00216-012-6476-7

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