Nanoparticle-based sensors for pathogen nucleic acid detection with interest for agriculture

Abstract

La presente tesis describe el desarrollo de sensibles, bajo coste y portátiles métodos de sensado basados en nanomateriales aplicados en la detección de ADN de patógenos relacionados a plantas. El trabajo se presenta avances significativos en el campo de los biosensores para el diagnóstico de las enfermedades en las plantas. En el Capítulo I se da una visión general de las aplicaciones llevabas a cabo y mejoras aportadas por parte del uso de nanomateriales diferentes en el campo de los biosensores, y las nuevas aplicaciones en la detección de enfermedad de las plantas en lo punto de atención. En las secciones siguientes se presentan tres estrategias de sensado para la detección de secuencias de ADN específicas para el virus del cidro (citrus tristeza virus (CTV)), un virus modelo. Los sistemas están basados en las técnicas electroquímicas y ópticas. El Capítulo III se presenta el uso de electrodos serigrafiados de carbono (SPCEs) como plataforma para hibridación y detección del ADN mediante impedancia. Esta plataforma se adaptó mediante la disposición de nanopartículas de oro (AuNPs), con el fin de obtener una estrategia más simple, sin marcas, menos costosa y más rápida. Del mismo modo, en el Capítulo IV se centra en el desarrollo de nuevos métodos para la amplificación y la detección de ADN de CTV en SPCE modificados con AuNPs. Este biosensor opera en modo libre de marcas y con límite de detección (LOD) conseguido está en el rango de 1000 fg μL-1. Finalmente, en el Capítulo V se presenta la tercera plataforma utilizada, que fue basada en papel y tiene el formato de un inmunoensayo de flujo lateral (LFIA, del inglés lateral flow immunoassays). En esta plataforma, las nanopartículas de oro se usan como marcas para obtener señal de color rojo, con el fin de realizar nuevas aplicaciones en diagnóstico de plantas, más rápido y simple

This thesis aims at developing sensitive, affordable and portable biosensors based on nanomaterials for the determination of nucleic acid related to plant pathogens. The work strives to contribute to the keeping up in the advancements of biosensing systems relevant to plant infection diagnostics which would be an essential solution in the future to the issues of plant disease monitoring and food security. Following Chapter I, state-of-the-art on the latest trends in the development of advantageous biosensors based on both antibody and DNA receptors for early plant disease detection, as well as the use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is given. The next sections of this dissertation will describe three diagnostic biosensing strategies for the detection of citrus tristeza virus (CTV) related nucleic acid using electrical and optical transducing techniques. The electrical sensing of CTV through DNA hybridization based approach and the in situ amplified nucleic acid method will be achieved on carbon sensing substrate modified with gold nanoparticles, while paper-based sensors will be operated in lateral flow format for the gold nanoparticle-based optical detection of CTV. Furthermore, all aspects of the developed biosensing systems, from the bioassay and biosensor design to their development and optimization are presented in which will be organized in the following manner: Chapter III will present highly specific DNA hybridization sensor based on AuNP-modified SPCE employing label-free impedance for the detection of the CTV-related nucleic acid, together with dedicating emphasis to the study of electrodeposition time of AuNPs, whose precise particle size and shape will be required for the enhancement of DNA hybridization rate. A set of voltammetric studies of deposited AuNPs will be discussed. Particular attention will be paid for assembling the thiolated DNA probe as sensing layer for biosensor construction. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time will be optimized, in order to precisely select the best working conditions for this diagnostic platform. Chapter IV will cover the whole process undertaken for preparation of in situ nucleic acid amplification on gold nanoparticle-modified sensor for sensitive and quantitative detection of CTV. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as relevant target for the demonstration of the proof-of-concept. This chapter will include two parts, the first one focuses on the design of RPA amplification assay, primers design, optimization of all essential bioassay aspects such as amplification temperature, volume and screening primers and finally the electrophoresis analysis for RPA products. The second part of this chapter will demonstrate label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Chapter V focuses on the application of isothermal nucleic acid amplification technology in simple lateral flow platform. The preparation of AuNP-based LFA for the highly sensitive direct detection of RPA amplified nucleic acid, the assembling of lateral flow step, the conjugation of AuNPs to the antibodies used for colorimetric detection, as well as the optimization of all working conditions and finally the analytical performance of the bioassay in LF will be explored. Moreover, aiming at truly achieving the point of care requirements of simple and affordable diagnostic technologies, the work here will present the possibility of amplifying nucleic acid without heat source and visual color detection. This approach would be of great potential as point of care diagnostics.