Functionalised magnetic nanoparticles for nucleic acids separation in plant disease diagnostics

Magnetic nanoparticles (MNPs) can be employed for magnetic separation of complex mixtures in a wide range of biotechnology applications [1, 2]. Commercially available magnetic 'beads' for magnetic separation are generally expensive and not tailored to any specific application. The demand for beads with higher binding capacity and lower sedimentation rates encourages the development of smaller magnetic particles with higher surface area and tailored surface functional properties. Aim of this work is the production and characterization of functionalised magnetic nanoparticles for nucleic acid separation of plant pathogens and their comparison with commercially available products. For this purpose, superparamagnetic iron oxide nanocrystals were synthesized in an aqueous solution [3]. The magnetic and structural properties of the produced MNPs were characterized by means of magnetometry, X-ray diffraction and scanning electron microscopy. A thin silica layer was deposited on the MNPs surface and subsequently the functionalisation with probe oligonucleotides was attained [4]. The surface functionalisation was aimed at detection of phytoplasmas, unculturable, wall-less prokaryotes that cause disease in hundreds of plant species World-wide, often causing serious economic losses. Common detection techniques for phytoplasmas involve total DNA extraction from symptomatic tissues, amplification of phytoplasma DNA by Polymerase Chain Reaction (PCR) followed by a nested step of amplification, to increase sensitivity [5]. These procedures are very time expensive, labor intensive and have a high risk of false positives results due to sample contamination. Binding ability testing on the functionalised MNPs was performed by hybridization with specific phytoplasma PCR amplicons and a comparison with commercial magnetic separation products was made. The tests performed involved capture of single and double strand DNA fragments of different lengths. The preliminary results obtained show that custom-made MNPs efficiently capture single strand fragments up to 20 bases. At the moment both commercial and home-made functionalised magnetic particles are not suitable to capture specific double DNA strands of 100- 2000 base pairs. This may be ascribed to the steric hindrance of the MNPs that may interfere with the hybridization, since, in general, short oligonucleotides bind to complementary target faster and more efficiently than longer ones. Further activities, directed to test the binding ability of MNPs on single-strand templates, such as RNA of different lengths, will be presented.