In Situ Plant Virus Nucleic Acid Isothermal Amplification Detection on Gold Nanoparticle-Modified Electrodes

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 relevant target for the demonstration of the proof-of-concept. RPA assay for the 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 Fe (CN6) 4-/Fe (CN6) 3red-ox system, being able to quantitatively detect 1000 fg μL-1 of nucleic acid. High selectivity against non-specific 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.

Role of different types of nanomaterials against diagnosis, prevention and therapy of COVID-19

“…SPCEs modification with AuNPs and thiolated nucleic acid immobilization were performed and reported by Khater et al 2017 ( Khater, Escosura-Muñiz, Altet, & Merkoçi, 2019 ). Firstly, the electrodes were pretreated by applying oxidative potentials in acetate buffer, and then rinsed and dried.…”

“…Probably, bigger nanoparticles proficiently cross-link virions, whilst ultra fine nanoparticles just decorate the surface of viruses ( Vonnemann et al, 2014 ). Computational models demonstrate that normal or disease cells grab the NPs to better predict the pharmacokinetic and pharmacodynamic characteristics of these nanomaterials ( Drain et al, 2014 ; Iranpour et al, 2018 ; Khater et al, 2019 ; Maltez-da Costa et al, 2012 ; Mullard, 2020 ; Nasrollahzadeh et al, 2020 ; Papp et al, 2010 ; Qiu et al, 2020a , 2020b ; Staroverov et al, 2011 ; Ulianas et al, 2014 ; Vonnemann et al, 2014 ; Xiang et al, 2020 ). For instance, Lunnoo and co-workers utilized a coarse-grained molecular dynamics (MD) simulation to perceive the internalization routes of different morphologies of gold nanoparticles ( Lunnoo, Assawakhajornsak, & Puangmali, 2019 ).…”

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

“…This on-site, rapid in-field assay for diagnosis of T. palmi is easy to use by non-expert personnel in plant biosecurity and pest management. RPA can also be combined with colloidal gold nanoparticles as probes for sensitive detection on disposable screen-printed carbon electrodes [ 200 , 201 ].…”

Isothermal Amplification of Nucleic Acids Coupled with Nanotechnology and Microfluidic Platforms for Detecting Antimicrobial Drug Resistance and Beyond

“…In an additional setting, Khater et al 146 developed a label-free highly integrated with in situ RPA amplification and detection system needing room temperature to perform the reaction. This method uses the advantages of AuNPs-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection.…”