Microneedles for DNA extraction in plant studies

Abstract

The urgency for sustainable life is growing due to the increase in global population and over-consumption. Preserving the agricultural areas, which are among the most basic needs of this lifestyle, is of great importance. In this regard, rapid and effective sampling techniques for plant studies (such as specific gene analysis and genetically modified organism (GMO) determination) and pathogen detection play a significant role in both enhancing the crop yield and preventing the economic losses. Specifically, early pathogen detection prevents their spread by employing correct pesticides and quarantine measures. In plant studies, the conventional cetyltrimethylammonium bromide (CTAB) is widely used for DNA extraction. However, the hazardous chemical consumption, long-time duration (3-4 h) and high-cost requirements necessitate new DNA extraction methods. To address this, in this thesis, chemical-free DNA extraction was achieved using polymeric microneedles (MNs) in 10 seconds. For that, hydrophilic poly (vinyl alcohol) (PVA) and resin-based 3D-printed MNs were utilized as a DNA extraction tool for both plant studies and pathogen detection. In this process, the effect of concentration on mechanical strength and hydrophilicity of PVA MNs were evaluated. On the other hand, same optimizations were carried out for 3D-printed MNs fabricated using different printing angles (0o, 45o, and 75o) and curing times (10, 20, and 30 min). In plant studies, first, DNA extraction from Oryza sativa and Gossypium hirsutum was completed using MNs, and their DNA extraction performances were assessed using polymerase chain reaction (PCR). In pathogen detection, the extracted DNA of Puccinia triticina was first amplified using loop-mediated isothermal amplification (LAMP). The presence of that pathogen was controlled by naked-eye using lateral flow assay (LFA). Finally, a portable heater system was developed, enabling the in-field pathogen detection within 40 minutes. In summary, we anticipate that the combination of optimized MNs for high DNA extraction and portable detection systems will provide significant benefits to sustainable agriculture and life.