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Electrochemical genosensor for the direct detection of tailed PCR amplicons incorporating ferrocene labelled dATP

I. Magriñá, A. Toldrà, M. Campàs, M. Ortiz, A. Simonova, I. Katakis, M. Hocek, CK. O'Sullivan,

. 2019 ; 134 (-) : 76-82. [pub] 20190330

Language English Country England, Great Britain

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc19034654

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.

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$a An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.
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$a Campàs, Mònica $u IRTA, Ctra. Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Tarragona, Spain.
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$a Ortiz, Mayreli $u Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
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$a Simonova, Anna $u Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, CZ-16610, Prague, Czech Republic; Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843, Prague 2, Czech Republic.
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$a Katakis, Ioanis $u Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
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$a Hocek, Michal $u Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, CZ-16610, Prague, Czech Republic; Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843, Prague 2, Czech Republic. Electronic address: michal.hocek@uochb.cas.cz.
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$a O'Sullivan, Ciara K $u Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010, Barcelona, Spain. Electronic address: ciara.osullivan@urv.cat.
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