Optimizing pesticide/herbicide formulations through non-invasive nanoscale imaging of live leaf surfaces

In a previous collaborative studentship between Syngenta (G. Bell) and School of Pharmacy, Nottingham (C.J. Roberts) we established a number of precedents in the application of surface analytical tools to fundamental issues in herbicide formulation and mode of action. In particular we demonstrated the ability of atomic force microscopy (AFM) to visualize a live leaf system exposed to various formulations and quantified the effects on the surface wax coating of the leaves - the main barrier to penetration of the active ingredients [Planta, 221 (2005) 123-134]. Subtle sub-micron thermal changes in the waxes were revealed directly on the leaf surface using scanning thermal microscopy [Applied Surface Science 243(2005) 158-165]. Amongst other advances was also the application of cryogenic secondary ion mass spectrometry to spatially and chemically map the dispersion of the formulation on a leaf surface and its penetration profile through the cuticular waxes [SIA, 39 (2007) 644-647 and Colloids and Surfaces B: Biointerfaces 67 (2008) 1-13]. These approaches provide valuable insight to aid the development and optimization of novel formulations. The proposed project will build on this previous work and will introduce a new complementary approach to the in situ real-time analysis of the formulation-leaf interface, namely Scanning Ion Conductance Microscopy (SICM). SICM exploits a sub-micron hollow pipette (typically 50-100 nm diameter) to non-invasively scan and visualize a surface in an electrolyte. It is a true non-contact imaging tool capable of dealing with soft, rough and changing surfaces in a manner AFM would find difficult. It represents a potentially valuable tool to Syngenta to aid optimisation and development of novel herbicides. In a wider context, SICM is set to revolutionize the study of the structure and function of live cells. The work of Klenerman (Cambridge) and Korchev (Imperial) have in particular revealed many new advances in this area, for example showing the ability to locally deliver materials to a cell surface and to make mechanical measurements [Nanomedicine 1 (2006) 107-114]. In proof of principle work we have locally introduced peptide nanotubes to a live cell and observed a cell blebbing response as the nanotubes were internalized. More recently, through imaging of hydrated tea-leaf surfaces in aqueous media, we have also investigated the effects of processing during tea production. To date SICM has seen little, if any, application outside mammalian/bacterial cell biology. Here we aim to significantly extend its application, and visualize live leaf-aqueous media interfaces onto which we will locally inject candidate pesticide/herbicide formulations through the SICM imaging pipette. The effect on leaf morphology and the physical properties of the leaf surfaces in relation to nature of the formulation and dosage will be studied. This, for example, will enable studies of changes in surface porosity and mechanical properties of the surface wax layer. In addition, through simultaneous imaging with optical/fluorescence microscopy, we will seek to obtain detailed information on the movement through the leaf structure, of both active ingredients and formulation adjuvants. Toxic effects on plant cells, and wherever possible, model 'pest' and mammalian cells will also be evaluated. These studies will be complemented, as appropriate, with experiments using a range of tools including particle sizing, AFM, SEM, TEM, XPS, Tof-SIMS, Raman and FT-IR. For example, to identify the distribution and state of the active, within the above, the PhD student will also be required to study and understand the structure and physicochemical properties of the formulations themselves. The ultimate goal of the PhD will therefore be to relate fundamental information at the nanoscale to the performance of the formulations, and aid in the optimization of the delivery of pesticides and herbicides.