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

Lead Research Organisation: University of Southampton
Department Name: School of Physics and Astronomy

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description With this grant award we generated significant new knowledge which was published to the top Journals of the field worldwide and it was presented in international conferences and universities (please see the section publications). Our research led to the development of a new type of sensor that can detect nucleotides 100 -1000 times better than conventional sensors, Most important the sensor is portable and it has been used for detection of mRNAs relevant to the health of crops in the field. Furthermore, new research questions have been opened as to how this sensor can be modified to detect different targets at the same solution at the same time and how we can expand its range of detection to other biomolecules that are associated with DNA and it is critical to monitor. We would be very excited to carry on our research and modify our sensors to detect antibiotics in waste water as well as toxins and molecules of interest in food. For this reason we look for the best platform to fund future research of our work with the aim to also bring our sensor to commercialization.
An important aspect of our work was that our project was interdisciplinary. Therefore this resulted to new collaborations between chemistry, biology and physics. Most significant was that the postdocs, postgraduates and undergraduate students involved in this program gained unique skills across the different disciplines which they will bring forward .An increased research capability was developed which is the scientific "fuel" for maximizing the impact of our work an initiating new research pathways.

In summary the funding, although of small duration resulted in new knowledge and high impact publications in top Journals and contributions to the academia such as :
1) ACS Nano, 2018, 12 (6), pp 6273-6279 DOI: 10.1021/acsnano.8b03261
2) ACS Nano, 2018, 12 (4), 3333-3340 DOI:10.1021/acsnano.7b08620
3) ACS Sensors 2017, 2 (1), 52-56 DOI: 10.1021/acssensors.6b00651
4) Chemical Reviews 2019,119(8),4819-4880 doi:10.1021/acs.chemrev.8b00733
5) Plant, cell & environment, 40 (11), pp. 2754-2770
Conferences:
1) Proc. SPIE, 10507, Colloidal Nanoparticles for Biomedical Applications XIII; 2018, 105070U doi: 10.1117/12.2282413
2) Proc. SPIE, 10892, Colloidal Nanoparticles for Biomedical Applications XIV; 2019,108920A doi: 10.1117/12.2502724
3) Keynote talk, GOLD 2018, Paris
4) Invited talk, Changchun Institute of Applied Chemistry, August 2019, Changhun, China
5) Invited talk, Beijing University of Chemical Technology August 2019, Beijing, China
Exploitation Route During this grant award we developed a new DNA sensor based on 2D materials and upconversion nanoparticles which is able to optically sense nucleotides at 100 times smaller concetrations than conventional sensors. Furthermore we made our sensor portable which means that can be used in a field of action. Our research publications were very succesful attracting recognition from the scientific community, which was translated to invited talks in international conferences and Universities. Future directions that will be pursued by us and others will be a) to perform multiplex detection, meaning to be able to detect different targets within the same sample and b) to expand their range of detection to other systems than DNA, for example antibiotics and toxins which may be found in food and water. The main advantages of our sensor is that it is simple to make, it can detect the target to very low conentrations and it is portable and transformative. Therefore we hope to identify companies that have an interest in sensing biomolecules associating with DNA so that we bring our work to commercialization.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The invited presentation that I gave at the SPIE Photonics West interantional conference attracted the interest of BioRad-a leading company, which sells biological kits. We had local discussions and later on a skype meeting with technical details related to the development of our sensor. The talks are ongoing exploiting the potential of our senosr for commercialization.
First Year Of Impact 2019
Sector Healthcare
Impact Types Societal,Economic

 
Description Collaboration with Oxford-Prof. Tom Brown group 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Apart from the participants in thi sgrant we have also collaborated with the Department of Chemistry, University of Oxford (Tom Brown group), whose team synthesized the oligonucleotide sequences neccessary to our project.
Collaborator Contribution Our partners synthesized all the oligonucleotides sequences neccessary for coating the nanoparticles.
Impact The outcomes are the publications assigned to this project.
Start Year 2017