In Situ Plant Virus Nucleic Acid Isothermal Amplification Detection on Gold Nanoparticle-Modified Electrodes
Khater, Mohga (Institut Català de Nanociència i Nanotecnologia)
De La Escosura-Muñiz, Alfredo (Institut Català de Nanociència i Nanotecnologia)
Altet Sanahujes, Laura (Vetgenomics)
Merkoçi, Arben (Institut Català de Nanociència i Nanotecnologia)

Date:	2019
Abstract:	Solid-phase isothermal recombinase polymerase amplification (RPA) offers many benefits over the standard RPA in homogeneous phase in terms of sensitivity, portability, and versatility. However, RPA devices reported to date are limited by the need for heating sources to reach sensitive detection. With the aim of overcoming such limitation, we propose here a label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle (AuNP)-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as a relevant target for demonstration of the proof-of-concept. RPA assay for amplification of the P20 gene (387-bp) characteristic of CTV was first designed/optimized and tested by standard gel electrophoresis analysis. The optimized RPA conditions were then transferred to the AuNP-modified electrode surface, previously modified with a thiolated forward primer. The in situ-amplified CTV target was investigated by EIS in a Fe(CN ) /Fe(CN ) red-ox system, being able to quantitatively detect 1000 fg μL of nucleic acid. High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated. Good reproducibility (RSD of 8%) and long-term stability (up to 3 weeks) of the system were also obtained. Overall, with regard to sensitivity, cost, and portability, our approach exhibits better performance than RPA in homogeneous phase, also without the need of heating sources required in other solid-phase approaches.
Grants:	Ministerio de Economía y Competitividad MAT2017-87202-P Ministerio de Economía y Competitividad SEV-2013-0295
Rights:	Tots els drets reservats.
Language:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Subject:	Citrus tristeza virus ; Electrochemical impedance ; Gel electrophoresis analysis ; Gold nanoparticle-modified electrodes ; Interfering species ; Isothermal amplifications ; Long term stability ; Modified electrode surfaces
Published in:	Analytical chemistry, Vol. 91, Issue 7 (April 2019) , p. 4790-4796, ISSN 1520-6882

DOI: 10.1021/acs.analchem.9b00340
PMID: 30843387

Postprint 23 p, 1.3 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Experimental sciences > Catalan Institute of Nanoscience and Nanotechnology (ICN2)
Articles > Research articles
Articles > Published articles