PacSurf2018 Session BI-MoM: 35 Years of NESAC/BIO I

Control and the ability to elicit specific responses from a biological system lies at the heart of most bioengineering. We want to immobilize proteins on biosensors but ask them to as sensitive as they are in solution or in the body, stimulate cells to assemble into tissues, reconstructing our bodily functions. We want methods that prevent bacteria forming biofilms and better still we would like them to stop bacteria attaching to surfaces full stop. But biology is soft and normally has lots of water associated with it, so how and why would you want to use vacuum based techniques to create coatings or characterise these systems?

This talk will explore how in my group and our collaborators, have tackled the challenges associated with interfacing vacuum deposited plasma polymers with water, proteins, lipids and cells to create a wide number of model systems and devices. At the same time, we have developed methods for chemically characterising these systems in vacuum, integrating XPS and ToF-SIMS with a range of other surface analytical and biological tools to gain insight into the materials we create and their interactions with biological systems.

ToF-SIMS is uniquely suited towards high spatial resolution imaging of surface modifications in materials with structure. While imaging 2D “flat” surfaces is relatively simple, working with three-dimensional (3D) “rough” surfaces is challenging due to the topography. This topography interferes with the ability to extract spatio-chemical differences in the sample and results in image shadowing and decreased mass resolution. In 3D depth profiling, topography can cause differential etching as the sputtering beam can impact the surface at different heights and angles. In many cases the combination of these factors prevents acquiring high quality imaging data since features associated with surface chemistry can be obscured. Multivariate image analysis methods have been used to assess the impact of topography on ToF-SIMS data, and AFM has been applied for topographical correction. However, the former method only assesses the impact, while the latter is time consuming and does not mitigate the effects of surface topography entirely. We propose an alternate methodology for imaging topographic samples with ToF-SIMS, demonstrating that through a simple polymer embedding methodology, topographic materials, ill-suited for ToF-SIMS analysis can be transformed into surfaces where topographical effects are minimized and high spatial resolution label free imaging of chemical modifications can be performed in topographic materials¹. Imaging surface modifications in the pores of biopolymer scaffolds will be presented, using a highly focused Bi₃⁺ LMIG beam for analysis in 2D, and the addition of an Ar₁₅₀₀⁺ sputter beam for depth profiling the pore in 3D.

FC (fluorocarbon) modification of PCL pores will be imaged, showing FC film deposits in the scaffold pore, and its distribution can be imaged in 3D. Water plasma hydrolyzed PCL scaffolds, modified with bovine serum albumin (BSA) through EDC/NHS bioconjugation chemistry can similarly be imaged, unlocking label free imaging of protein fragments at the scaffold/pore interface. We will also show that lipid imaging is possible in this challenging material class, extracting information in 2 and 3D from the pores of lipid modified poly(2-hydroxyethyl methacrylate)-co-methacrylic acid scaffolds.

1 M. J. Taylor, H. Aitchison, M. J. Hawker, M. N. Mann, E. R. Fisher, D. J. Graham and L. J. Gamble, Biointerphases, 2018, 13, 03B415.

ToF-SIMS data is complicated. Even a single spectrum can contain hundreds if not thousands of peaks. Each peak corresponds to a unique element, fragment or molecule from the surface analyzed. The relative intensity of these peaks can encode information about the chemistry, structure and composition of the surface. With modern ToF-SIMS instrumentation it is straight forward to collect multiple spectra across multiple samples resulting in large, complex data sets. To further add to the scale of the data one can also produce 2D and 3D ToF-SIMS images which can consist of millions of spectra and fill gigabytes of storage space. Since 1992 NESAC/BIO has lead the way in developing innovative tools that enable digestion of this smorgasbord of ToF-SIMS data. This included some of the first papers published applying multivariate analysis (MVA) methods to ToF-SIMS data. This effort has lead to the creation of the NBToolbox which contains a set of advanced tools to process and display ToF-SIMS spectra and images. Though the ToF-SIMS community is relatively small, the NBToolbox has over 300 users across 39 countries on 6 continents. It is regularly used in research presented in publications and presentations around the world. In this presentation I will highlight the developments spearheaded through the years by NESAC/BIO in ToF-SIMS data processing from spectra to 3D imaging. Examples will be presented from the early beginnings of “simple” controlled systems to current work with complex tissue samples in 2D and 3D.

Nanoparticles in a variety of forms continue to grow in importance for fundamental research, technological and medical applications, and environmental or toxicology studies. Physical and chemical attributes that lead to multiple types of particle instabilities complicate the ability to produce, appropriately characterize, and consistently deliver well-defined particles, frequently leading to inconsistencies, and conflicts in the published literature. In previous work examining 3-5 nm cerium oxide crystallites that had formed ~10 nm soft agglomerates in aqueous media we had observed chemical state changes (the ratio of Ce⁺³/Ce⁺⁴) and related optical absorption changes during particle formation and in response to environmental changes. The transformations have been further examined using micro-X-ray diffraction and Raman spectroscopy. We observed that in response to the environmental changes – adding H₂O₂ to the solution – these particles transformed from a ceria structure to an amorphous complex and returned to the crystalline phase upon solution aging. For comparison, 40 nm ceria nanoparticles were not observed to undergo this transformation and particles made up of crystallites of ~ 8 nm appeared to partially transform (or transform more slowly). We note that ceria nanoparticles of smaller size frequently have beneficial biological effects in comparison to the larger particles. The chemical state changes observed in ceria nanoparticles are usually assumed to be particle size dependent and to involve a change from cubic fluorite-type dioxide (CeO₂) to a hexagonal cerium sesquioxide (Ce₂O₃) with a continuous range of partially reduced CeO_2-x phases, where oxygen vacancies can be rapidly formed, arranged or eliminated. Our XRD and Raman data suggest that a much more complex transformation can occur for smaller ceria crystallites. Such changes were not readily identified by macroscopic in situ measurement such optical measurements or ex situ examination using TEM and XPS but were discovered by examination of ceria nanoparticles with molecularly and structurally sensitive methods with the particles in wet conditions (near in situ). Considering cerium oxide’s useful abilities to scavenge radicals, control the oxygen environment and provide regenerative oxidation state switching, it appears that the ease of ceria nanoparticles to transform between Ce⁴⁺ and Ce³⁺ rich phases is facilitated by small size, but is not constrained to be a transformation between defected and non-defected ceria phases.

Reference-free XRF analysis as primary method has been proven to be capable of underpinning all other applied methods since it yields the absolute mass deposition of the selected N,N’-ethylene-bis (stearamide) contaminant whilst X-ray absorption fine structure analysis determines the chemical species. Ambient vibrational spectroscopy and mass spectroscopy methodologies such as Fourier transform infrared, Raman, and secondary ion mass spectroscopy have been used in this systematic procedure providing an extensive range of complementary analyses.

The calibration procedure was developed using specially designed and fabricated model systems varying in thickness and substrate material. Furthermore, typical real medical devices such as both a polyethylene hip liner and a silver-coated wound dressing have been contaminated and investigated by these diverse methods, enabling testing of this developed procedure. These well-characterized samples may be used as calibration standards for bench top instrumentation from the perspective of providing traceable analysis of biomaterials and surface treatments. These findings demonstrate the potential importance and usefulness of combining complementary methods for a better understanding of the relevant organic materials.

[1] B. Pollakowski-Herrmann, A. Hornemann, A. M. Giovannozzi, F. Green, P. Gunning, Ch. Portesi, A. Rossi, Ch. Seim, R. Steven, B. Tyler, B. Beckhoff, Journal of Pharmaceutical and Biomedical Analysis 150, 2018, 308–317.

Proteins can act as Nature’s engineers at interfaces and manipulate both hard and soft tissue – they can shape biominerals, manipulate cell membranes and control water. Despite the apparent importance for chemists working in the fields of biomineralization, surface engineering and drug delivery the molecular mechanisms behind interfacial protein action have largely remained elusive. We use static and time resolved sum frequency generation spectroscopy combined with computer simulations to determine the structure and the mode of action by which these proteins interact with and manipulate interfaces. Here, I discuss our recent advances in the study of protein driven nucleation.

Taking clues from Nature we aim at understanding biomineralization processes at the molecular level to develop design rules for biogenic nanophase materials. Especially the high fidelity control of nanostructured silica within diatoms has been the envy of material scientists for decades. Where diatoms can grow silica using proteins at cell interfaces under ambient conditions, we still need high pH and harsh conditions to structure silica. Despite the apparent importance for physicists and chemists working in the fields of biomineralization, surface engineering, drug delivery, or diagnostics, the molecular mechanisms behind interfacial silica protein action have remained largely elusive. Our goal is to probe the structure and structural dynamics of such active proteins – in action at the surface. As a first step we study the diatom silica peptide R5 when interacting with silica. We use methods based on theoretical and experimental sum frequency generation spectroscopy combined with computer simulations to determine the structure and the mode of action by which these proteins interact with and grow extended 2D silica interfaces.

A particularly fascinating example of protein driven nucleation and phase transitions are ice-nucleating proteins. These proteins are used by specific bacteria to attack plants and cause frost damage by growing ice crystals at temperatures that would otherwise not allow ice formation. A recent survey by the NASA found that biogenic ice nucleators in the troposphere may affect global precipitation patterns. We have followed the interaction of biogenic ice seeding proteins with surrounding water to gain a detailed picture of protein-driven ice nucleation.