PacSurf2024 Session BI1-TuM: Biomaterials/Interfaces - Biointeractions

Supercritical Angle Raman Microscopy (SAR-M) emerges as a transformative technique for the in-depth study of molecular conformations at surfaces, providing unparalleled spatial resolution and sensitivity. This presentation explores the application of SAR-M to investigate the structural dynamics of amyloid and synuclein proteins, which are pivotal in neurodegenerative diseases such as Alzheimer's and Parkinson's. Utilizing the unique capabilities of SAR-M, we demonstrate its proficiency in capturing subtle conformational changes and aggregations of these proteins at the nanoscale, which are critical to understanding their pathological roles as well as the role of ions like Calcium.

Amyloid and synuclein proteins are known for their propensity to misfold and aggregate, forming insoluble fibrils that are toxic to neuronal cells. Traditional microscopy techniques often fall short in providing the necessary resolution and chemical specificity to study these proteins' surface interactions and early aggregation stages. SAR-M overcomes these limitations by exploiting the supercritical angle fluorescence to enhance Raman scattering signals, thereby achieving superior surface sensitivity.

Through a series of experiments, we detail the conformational mapping of amyloid-beta peptides and alpha-synuclein at different aggregation stages. The results reveal distinct molecular signatures and structural transitions, offering new insights into the mechanisms driving protein misfolding and aggregation. Additionally, SAR-M's capability to monitor these processes in real-time opens avenues for investigating the effects of potential therapeutic agents aimed at inhibiting or reversing protein aggregation.

Serrano D, Seeger S, Light: Science and Applications (2017) 6, e17066

Dubois A, Serrano D, Zhang X, Seeger S, Analytical Chemistry (2020) 4963

Münch NS, Das S, Seeger S, PCCP (2024), in press

Spatial and temporal variability of leave surfaces modulates plant-microbe and microbe-microbe interactions, creating diverse microenvironments for microbial colonizers such as bacteria and fungi. Mimicking leaf complexity on artificial surfaces greatly aids in the study of microorganisms residing on plant leaf surfaces [1]. Over the years, a number of surrogate surfaces aiming to replicate leaf surface topography have been proposed, ranging from simple nutrient agars to complex casts [1]. These surrogate surfaces are often used to deconstruct leaf surfaces into individual aspects, as this enables bio-interactions to be studied in separation [2].

In this paper I will discuss ongoing efforts to develop biomimetic leaf surfaces as a platform technology to study bio-interactions. In particular, I will focus on work related to bacterial colonization [2-4] and invasion by pathogenic rusts [6]. To date, this has involved Arabidopsis thaliana [2,3], as well as wheat, poplar, eucalyptus and mānuka mimics [5]. Incorporating properties such as leaf topography or hydrophobicity, these mimics all aim to promote colonizer survival in the absence of a living plant host. Characterizing agarose, polydimethylsiloxane (PDMS) and gelatin, we have determined PDMS to be one of the most suitable materials for leaf replicas [6]. Diffusion of water and nutrients to the surface of PDMS can be optimized by addition of fillers [7]. Increasing permeability, we have been able to demonstrate the possibility of delivering fructose to the surface, thus allowing division and distribution of bacteria to be affected [2]. Such leaf replicas have since also enabled us to culture in-vivo biotrophic rusts, normally considered “un-culturable” on artificial substrates due to the need for a living host [5], as well as helped to demonstrate that RNAi can be used to inhibit infections by these rusts [8].

1. Doan, H.K., Leveau, J.H.J., Phytopathology 105:1036-1042, 2015.
2. Bernach, M., PhD Thesis. 2024, University of Canterbury: Christchurch.
3. Soffe, R., et al., Sci. Rep. 9:14420, 2019.
4. Soffe, R., et al., Small 2002035, 2022.
5. Sale, S., PhD Thesis. 2024, University of Canterbury: Christchurch.
6. Soffe, R., et al., PLoS ONE 14:e021810, 2019.
7. Bernach, M., et al., Jpn. J. Appl. Phys. 58:SDDK01, 2019.
8. Degnan, R.M., et al., Mol. Plant Pathol. 24:191-207, 2022.