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Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection

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Abstract

Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. Among the 1,000 known cystic fibrosis-related mutations, DF508 is the most common, with a frequency varying between 50% and 70% according to geographical areas and population typology. In this work, we report the use of methylene blue as an electrochemical reporting agent in the discrimination of synthetic PCR analogue of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt). At optimum experimental condition, a discrimination factor between mutant and wild type of approximately 1.5-fold was found. The proposed assay was quantitative and linear in the range of 10–100 nM, exhibiting a limit of detection of 2.64 nM. Electrochemical studies at variable ionic strength conditions allowed further elucidation of the mechanism of the methylene blue (MB)–DNA interaction. To the best of our knowledge, this is the first report of detection of hybridisation solely via guanine-specific MB–DNA interaction simultaneously in MB solution, independent of electrostatic interaction as demonstrated in the ionic strength study. The introduction of formamide in the hybridization buffer, to improve discrimination, was also investigated. Finally, mutant wild type discrimination was demonstrated, at 10 nM concentration, with the use of a multi-sensor setup.

Electrochemical response before and after sensor regeneration

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Abbreviations

MB:

Methylene blue

MCH:

Mercaptohexanol

CF:

Cystic fibrosis

CFTR:

Cystic fibrosis transmembrane conductance regulator

PCR:

Polymerase chain reaction

MAPH:

Multiplex amplification and probe hybridisation

Mut ampl:

DF508 Cystic fibrosis mutation amplicon

Wt ampl:

Cystic fibrosis wild-type amplicon

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Acknowledgments

This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme ‘Isolation of foetal cells from maternal blood, SAFER, NEST-ADVENTURE 04977’. Dr. Valerio Beni kindly acknowledges the European Community’s, Seventh framework programme (FP7/2007-2013) under grant agreement nº (PIGF-GA-2008-220928) for the financial support. Hany Nasef wishes to thank Universitat Rovira I Virgili for a BRDI scholarship.

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Authors and Affiliations

  1. Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007, Tarragona, Spain

    Hany Nasef, Valerio Beni & Ciara K. O’Sullivan

  2. Institucio Catalana de Recerca i Estudis Avançats, Passeig Lluis Companys 23, 08010, Barcelona, Spain

    Ciara K. O’Sullivan

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  1. Hany Nasef
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  2. Valerio Beni
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  3. Ciara K. O’Sullivan
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Correspondence to Ciara K. O’Sullivan.

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Nasef, H., Beni, V. & O’Sullivan, C.K. Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection. Anal Bioanal Chem 396, 1423–1432 (2010). https://doi.org/10.1007/s00216-009-3369-5

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  • DOI: https://doi.org/10.1007/s00216-009-3369-5

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