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

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(CN6)4-/Fe(CN6)3- red-ox system, being able to quantitatively detect 1000 fg µL-1 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.

Electrochemical detection of plant virus using gold nanoparticle-modified electrodes.

Tristeza is one of the destructive diseases of citrus causing by citrus tristeza virus (CTV). Historically, CTV has been associated with serious outbreaks of quick decline of citrus, therefore CTV monitoring is important aspect for avoiding such re-emerging epidemics, which would threat citrus production through the world. In this context, we have designed for the first time a label-free impedimetric biosensor for the detection of nucleic acid of CTV. The sensing platform based on a screen-printed carbon electrode (SPCE) was modified by electrodeposited gold nanoparticles (AuNPs), which allowed to efficiently immobilizing thiolated ssDNA probes as well to enhance the electrode conductivity. The growth of AuNPs was optimized and characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We investigated the behavior of thiolated ssDNA probe layer and its hybridization with target DNA onto AuNP surfaces by EIS measurements in Fe(CN6)4-/Fe(CN6)3- red-ox system. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time were optimized so as to achieve the best performance. Impedance values of DNA hybridization increased with Citrus tristeza-related synthetic DNA concentration, showing a logarithmic relation in the range of 0.1-10 µM. The results also indicate that the biosensor was able to selectively detect CTV nucleic acids in the presence of other non-specific DNAs. Moreover, we have demonstrated the good performance of the system in a real plant sample matrix. In addition, the sensor reproducibility enhanced after the hybridization onto MCH/poly (AT) thiolated DNA probes which was confirmed by intra- and inter-day variability assays.

Biosensors for plant pathogen detection.

Infectious plant diseases are caused by pathogenic microorganisms such as fungi, bacteria, viruses, viroids, phytoplasma and nematodes. Worldwide, plant pathogen infections are among main factors limiting crop productivity and increasing economic losses. Plant pathogen detection is important as first step to manage a plant disease in greenhouses, field conditions and at the country boarders. Current immunological techniques used to detect pathogens in plant include enzyme-linked immunosorbent assays (ELISA) and direct tissue blot immunoassays (DTBIA). DNA-based techniques such as polymerase chain reaction (PCR), real time PCR (RT-PCR) and dot blot hybridization have also been proposed for pathogen identification and detection. However these methodologies are time-consuming and require complex instruments, being not suitable for in-situ analysis. Consequently, there is strong interest for developing new biosensing systems for early detection of plant diseases with high sensitivity and specificity at the point-of-care. In this context, we revise here the recent advancement in the development of advantageous biosensing systems for plant pathogen detection based on both antibody and DNA receptors. 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 also shown. Plastic and paper-based platforms have been used for this purpose, offering cheap and easy-to-use really integrated sensing systems for rapid on-site detection. Beside devices developed at research and development level a brief revision of commercially available kits is also included in this review.