Development of a graphene oxide nanosensor for fast detection of mRNA-related sequences in crops.

This project exploits the use of nanotechnology to design a system that will have uses in crop science and also has potential for wider applications. Nanotechnology is the engineering of functional systems at the molecular level and this project aims to develop a sensor that can be used as a tool to improve key aspects of crop research. The sensor that we will develop is based on the use of nanoparticles and graphene oxide. Graphene itself is an atom thick, hexagonal lattice, nanomaterial made of carbon atoms and graphene oxide has a number of useful properties that are amenable to its development as a biosensor. Our aim is to develop a sensor that will efficiently detect changes in gene-expression levels of various nutrient transporters in response to micronutrient stress in crops. Insufficient amounts of micronutrients such as zinc (Zn) in crop tissues has a detrimental effect on crop yield and on grain nutritional quality thus impacting food security. Membrane transporter proteins are responsible for the transport of key micronutrients in plants and under nutrient stress conditions their gene expression can be up-regulated to ensure more efficient uptake and use of the available nutrients. Our sensor is based on detecting these changes in gene expression by a process involving binding to specific oligonucleotides for the gene in question. Nucleotides serve as the basis of nucleic acids like DNA and RNA and our sensor will be designed to monitor changes in the levels of the RNA that encodes these nutrient transporters. We will focus on barley and wheat cereal crops where we have shown that under Zn stress particular members of the ZIP transporter family are up-regulated. These Zn-transporting ZIPs are proposed to increase the efficiency of Zn uptake and allocation under conditions of low Zn supply and thus can be used as markers of nutritional stress. Currently, gene expression analysis makes use of PCR-based techniques, which are time consuming and can be costly and technically challenging. A kit enabling rapid detection of nutrient deficiencies in crops by monitoring changes in marker gene expression will improve fundamental plant research and enable real world applications in crop-health monitoring.
This project idea is timely for future applications and aligned with the urgency for the improved nutrition value and performance of crops across the globe. This is the first time that this newly discovered sensor based on highly advanced nanotechnological developments will be applied in crop science.

Real-time quantitative reverse transcriptase PCR (QRT-PCR) is the most sensitive technique routinely used for monitoring gene expression in plant biology research. In this method, complementary DNA (cDNA) is first produced from mRNA through a reverse transcription process and this is used to quantify the gene of interest (in relative or absolute terms). For quantification, two types of detection chemistries are used, both requiring the use of fluorescence dyes. While the sensitivity of the technique is undeniable, there are limitations associated with it including: amount of time and effort required to prepare samples; sensitivity to contaminants; cost of specialised equipment, chemicals and consumable. These tend to limit its application range to specialized studies in a research environment. Next to RT-PCR, in-situ PCR is a technique capable of measuring mRNA distributions inside biological tissue. In-situ techniques generally are limited by intrinsic variations in uptake and transport of molecules within the cell tissue, and require careful control experiments.
There is therefore a need for new methods for detection of oligonucleotide specific sequences that can complement existing techniques in sensitivity with additional benefits of speed, ease of use, and availability outside the research laboratory. Recently, we have demonstrated a new technique for sensitive detection and quantification of a poly-Thymine sequence based on upconversion nanoparticles and graphene oxide. In this project, we aim to further develop this technique toward detection of more exploratory oligonucleotide sequences in plants without the additional amplification steps used in PCR. With a sensitivity range down to zeptomoles, we will explore Zn-deficiency responses in barley and wheat, a key area of research with important agricultural relevance.

Plants are the basis of all foodstuffs that we ingest; therefore understanding the processes of plant micronutrient nutrition is highly relevant not only to those researchers working on plants but also those in animal nutrition and there is burgeoning interest at the interdisciplinary interface between plant and animal/human nutrition. On the other hand, improving plant nutrition is an important biotechnological goal and the generation of crops with superior nutritional content would be very valuable. Our project will develop a portable nanosensor that will be able to rapidly detect oligonucleotides sequences related to expression of nutrient transport proteins in response to micronutrient stress in crops. This development will allow an easier access to information related to the question of how efficiently plants uptake various types of micronutrients. The development of this new tool, will enhance the knowledge economy and bring tangible value that will benefit the applicability of the sensor not only in plant biology but also in other fields.