Principal Supervisor: Prof Rachel O’Reilly (Chemistry)
Co-supervisor: Prof Richard Napier (Life Sciences)
PhD project title: Self-assembling nanobiosensors for imaging hormone gradients in plants
University of Registration: Warwick
The design, synthesis and evaluation of self-assembling, fluorescent polymer nanodots displaying DNA aptamers sensitive to indole-3-acetic acid for application in imaging of morphogenic gradients in plants.
Background: Quantum dot (QD)-based fluorescent reporters based on heavy metal nanocrystals are well known imaging reagents. Based on cadmium and selenium (CdSe) the core crystals range in size from 2.5-6 nm. However, leakage of the heavy metal causes toxicity and limits their application in plant sciences. Recent advances in materials chemistry and polymer science have delivered a multitude of possibilities for generating fluorescent nanoparticles (FnPs) from carbon-based polymers (Robin and O’Reilly, 2014). These have bright emissions due to multiple encapsulated fluorophores, biocompatibility due to their carbon-based chemistry and fluorophores may be incorporated within defined monomers to maximise stoichiometry and brightness. Through functionalisation of these polymer nanoparticles they can be tailored towards applications as vehicles for utilisation in plant sciences. In particular through the display of DNA aptamers they can be developed for use as nanobiosensors in plants.
The target for the nanobiosensors will be the morphogenic hormone indole-3-acetic acid (IAA). The wonderful diversity of shape, size and form of plants depends on correct and reproducible concentration gradients of this hormone, yet methods for the quantitation of its concentrations in vivo are lacking. This project will develop the auxin-sensitive fluorescent nanobiosensors designed to provide the first quantitative measurements of dynamic hormone gradients. Such detailed understanding of hormonal control will underpin advances in modern food production through development of a new understanding of developmental control and hormone flux in living plants.
Methodology: To develop the nanobionsensors for analyte detection, suitable DNA aptamers specific for IAA must be developed. This will be achieved using selective elution from novel, immobilised analogues. The aptamer binding affinity and selectivities will be characterised using a range of biophysical techniques such as SPR, thermal melt and fluorescence spectroscopy. The polymer nanoparticles (of diameters 10-20 nm) will be prepared using standard controlled radical polymerisation methods, with varying parameters such as chain length, degree of cross-linking, size and valency. The particles will also be labelling with the bright aminobromomaleimide probe developed by the host group. Once prepared these constructs will be characterised using fluorescence spectroscopy, light scattering and microscopy. Then click chemistry will be utilised to couple the aptamers to the scaffold and evaluate their efficacy as IAA nanosensors, comparing their performance to that of the free aptamers. To explore the application of these constructs as nanobiosensors for plants confocal microscopy will be used to evaluate their penetrance into live tissues and record IAA concentrations as they change during tropic growth responses.
BBSRC Strategic Research Priority: Food security
Techniques that will be undertaken during the project:
- Synthetic chemistry (polymer nanoparticle synthesis and characterisation)
- Analytical chemistry (size exclusion chromatography, light scattering, electron microscopy, fluorescence spectroscopy etc)
- Biophysics (surface plasmon resonance, isothermal titration calorimetry etc)
- Cell biology (confocal microscopy, toxicology, physiology)
- Depending on ability, Matlab routines for data analysis