SIMS2015 Session CT-TuP: Complementary Techniques and Multi-Technique Approaches Poster Session

The orientation of adsorbed proteins on surfaces has been shown to influence biological responses. Therefore, research and development of biotechnological applications such as sandwich ELISAs have focused on controlling the orientation of each protein layer. Characterizing protein orientation has been a challenge. Our goal is to address these challenges by developing methodology to study multilayer protein systems.

In this work, we aim to determine the orientation of protein G B1, an IgG antibody-binding domain of protein G, on various surfaces and the effect of orientation on antibody binding using a variety of surface-sensitive tools and simulations. We propose that binding selectivity will increase for well-ordered protein films due to high availability of binding domains. We used surface modification to control protein orientation, specifically utilizing four types of self-assembled monolayers (SAMs): N-Hydroxysuccinimide-terminated SAMs and dodecanethiol SAMs to immobilize protein G B1 in a random orientation and maleimide-terminated SAMs and bare gold to immobilize cysteine mutants of protein G B1 in a well-ordered orientation.

The surface sensitivity of ToF-SIMS enables us to detect different proteins and their orientation based on the amino acid concentrations. Previous ToF-SIMS methods have been developed for the analysis of single protein films. We intend to further develop this technique for the application of studying multilayer systems. We also use complementary techniques, such as XPS and quartz crystal microbalance with dissipation monitoring (QCM-D), to provide detailed information about the composition, coverage, and orientation of the adsorbed proteins.

Additionally, computational methods to predict the orientation of proteins on surfaces can complement and help interpret experimental techniques. In this work, we describe the development of a simulator to determine protein orientation on a surface using Monte Carlo (MC) simulations. To test the MC simulator, we used the simple peptide LKa14 because of its predictable structure and orientation on hydrophobic surfaces. Preliminary MC simulations have also predicted possible interactions between amino acid residues of protein G B1 and a graphene surface. We will extend the MC algorithm to predict the orientation of additional protein/surface combinations and validate using experimental results.

While the model systems explored in this study are less complex compared to biological systems of the real world, this is a first step in developing methodology using state-of-the-art tools that can be continuously improved to expand our knowledge and control of biomolecules on surfaces.

We have prepared a set of layered structures of Cu(In,Ga)Se₂, molybdenum and soda lime-glass which are a part of thin film solar cell structure. In the complete photovoltaic structure a layer of Cu(In,Ga)Se₂ is absorbing photons, molybdenum serves as a back contact and soda-lime glass is the substrate. Our absorbers were prepared in co-evaporation process from the elemental sources of copper, indium, gallium and selenium and they are 1.45 to 1.8 micrometer thick, the thickness of molybdenum layer is 650 nm. For this type of semiconducting material band gap value depends on the concentration of indium and gallium in the layer and can be easily controlled by the modification of fluxes of these elements during the co-evaporation process. Therefore, three of our samples have a flat distributions of indium and gallium which results in the same band gap across the layer. Only the total amount of indium and gallium is different, which gives different values of band gap between these samples. Remaining samples have graded indium and gallium profiles in order to improve carrier collection by built-in electric field resulting from the graded band gap. Graded profiles were obtained by the modification of indium and gallium flux during the co-evaporation of these elements. To modify fluxes we simply change temperature of crucibles filled with the materials to evaporate.

Two depth profile analytical techniques: secondary ion mass spectrometry (SIMS) and glow discharge mass spectrometry (GD-MS) were applied for analysis of the above mentioned structures. Aim of the study was to find optimal quantitative analytical procedures allowing us to monitor atomic concentrations of Ga and In and their gradient across the structure. Front side as well as backs side depth profiles were done. GD-MS analysis was performed in ~0.1 Torr Ar, 1.5 kV DC glow discharge and SMWJ-01 [1] spectrometer was used. 1.5mm diameter spots were analyzed. SIMS analysis was performed on SAJW-05 [2] apparatus equipped with Physical Electronics 06-350E ion gun and QMA-410 Balzers quadrupole analyzer. Sputtering with 5 keV Ar⁺, 100 µm diameter ion beam was performed over 1.6mm x 1.6mm raster area, with 10% electronic gate. Discussion of the results concerns different analytical parameters of the two techniques.

Acknowledgements:

Authors (M.M. and P.K.) thank Ministry of Science and Higher Education, Poland, for project no DI2013 013943 founded in years 2014-2018.

References

[1] P. Konarski, K. Kaczorek, M. Ćwil, J. Marks; Vacuum 81, 1323-1327 (2007).

[2] P. Konarski, A. Mierzejewska; Appl. Surf. Sci.203–204, 354–358 (2003).

The use of fluid injection for improved or enhanced oil recovery (EOR) is an often studied subject and results have been used in the production of hydrocarbons. New is the possibility of provoking mineral growth on a geological very short term during those injections with more possibilities for EOR and other related applications [1].

The first long-term test on chalk with the injection of MgCl₂ under reservoir conditions was realized. Outcrop chalk of Late Campanian age (Gupen Formation) from Liège (Belgium) was flooded for 516 days for improved/enhanced oil recovery purposes. We discover massive precipitation of secondary minerals, which changes the rock properties of the chalk fundamentally. These changes are taking place in non-geological times and on nano-scales, and are paramount for any EOR approaches, hence of highest importance for the hydrocarbon interested peer groups.

After flooding with MgCl₂ brine, the Liège chalk was sliced into 6 equal parts perpendicular to its long axis. It appears severely altered by the experience, changed in texture, exemplified by the absence of coccolith fragments and a more rough grain appearance, although angular, well-formed rhombic grains frequently occur. The first centimeters of the flooded chalk sample show an increase in MgO and a depletion of CaO by more than 70 %. These dramatic mineralogical and geochemical changes were observed with SEM-EDS [2]. However, the large spot-size of the SEM-EDS application (1-2 micron) did not allow in pinpointing the chemistry of the new growing mineral beyond doubt and the element distribution on a sub-micron scale. Therefore, we probed the phases in question by NanoSIMS.

The elements detected and mapped by NanoSIMS were the major and trace elements such as the carbone, chlorine and silicon. The analysis demonstrated the absence of CaO in the new grown mineral whereas chlorine and silica disseminated as nano-phase in the chalk matrix . The NanoSIMS results allowed to understand the distribution and mineralogical reactions and to gain reasoning for the precipitation of magnesite and to evaluate the dissemination of silica, Cl and other elements before and after flooding on a nano-scale for quantification purposes [2].

The formation of new secondary minerals seems to trigger an enhanced dissolution of the chalk matrix, which induced porosity changes. However, it is importance to test now compaction and other petrophysical proxies to model such long-term fluid injections on a larger scale.

[1] Hermansen, H. et al. J. of Petroleum Science and Engineering, v. 26, p. 11-18 (2000).

[2] Zimmermann et al. AAPG Bulletin, V. 99, No. 5, PP. 791 – 805 (2015)

ToFSIMS is an excellent technique for “label free” analysis of biological surfaces. In validating results when applied to new biomedical problems inevitably these results must be compared with conventional techniques such as staining and fluorescent labelling of structures. This is often more of a problem than it should be: biomedical collaborators have exactly the right tools to image staining or fluorescence using appropriate light microscopes, but there is always an issue of digital file format conversion and image registration that makes it difficult or uncertain to overlay this on the ToFSIMS image. This makes collaboration more time-consuming than it should be. Therefore instead we have implemented a hyperspectral imaging microscope in the entry chamber of our ToFSIMS system, so as to provide the best possible optical images of the region of interest.

The near-infrared region makes the instrument very powerful in the study of heterogeneous biological samples. Near-infrared (NIR) light penetrates many biological structures and materials, particularly lipids, and gives rise to diffuse scattering within. What is absorbed in the material is not reflected, so absorbance images can have significant contrast.

We have developed two Hyperspectral Imaging (HSI) systems, one for sample plattern imaging (centimetres in size) and a microscope version for feature imaging (millimetre regions of interest) based on a wide wavelength camera. An integrating sphere is used to ensure diffuse illumination and the absence of shadowing.

The determination of distribution of Li and B is of great interest in nuclear waste glass research [1-2]. During glass ceramic manufacture ions with different solubility are redistributed and form nano-sized droplets/crystals in multiple phases. The element distribution, especially alkali and boron, is very important to design the components of the glasses, optimize heating/cooling procedures, and parameterize many important corrosion mechanisms. It is challenging to use common analytical imaging tools, such as SEM and TEM, to image Li and B distribution in glass samples with nanoscale resolution. In this study, time-of-flight secondary ion mass spectrometry (ToF-SIMS), and nanoscale secondary ion mass spectrometry (NanoSIMS) and atom probe tomography (APT) were used to map the distribution of Li and B in a few representative glass samples. APT provides 3-dimensional imaging with spatial resolution (≤2 nm) for Li and B in glasses. ToF-SIMS and NanoSIMS provide ≤100 nm lateral resolutions for 2-dimensional imaging of Li and B in glass samples. Comparing these two techniques, 2-dimensional SIMS imaging is more sample-friendly, low time-cost, with flexible field-of-view (from 1 × 1 µm² to 500 × 500 µm², and image-stitching is feasible). Therefore, SIMS and APT are considered as complementary techniques for nanoscale imaging of Li and B in glass samples.

[1] S. Gin, J. V. Ryan, D. K. Schreiber, et al., 349–350, 99 (2013)

[2] J. Crum, V. Maio, J. McCloy, et al., 444, 481 (2014)

[3] This work was supported by the Department of Energy, Office of Nuclear Energy. This research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research located at Pacific Northwest National Laboratory.

Ion implantation of metal ions followed by annealing can be used for the formation of buried layers of metal nanoparticles in glasses with optical properties for perspective photonic materials. Results are presented from three samples of silica glasses implanted with Cu⁺, Ag⁺ or Au⁺ ions under the same conditions (energy 330 keV and fluence 1×10¹⁶ ions/cm²) and three silica glass samples implanted with the same metals and conditions but co-implanted subsequently by oxygen into the same depth. All the implanted glasses were annealed at 600°C, which lead to forming of metal nanoparticles. The depth profiles measured by RBS and SIMS indicated that the sequential implantation of oxygen followed by post-annealing caused the shift of Cu, Ag and Au aggregated into nanoparticles deeper into the glass substrate. Both RBS and SIMS are shown to be valuable complementary techniques.

Acknowledgement

A relationship between the surface composition of bacteria (N/P or N/C ratio) and surface electrical properties (isoelectric point and electrophoretic mobility) was observed for various sets of microorganisms [4]. XPS provides a good balance between qualitative information, quantification and surface selectivity, to investigate the spatial distribution of components and to understand properties and processes.

In the present study, we analyzed selected protein (fatal bovine albumin, BSA) and cell lines (normal and cancer; human and animal). For our experiments we choose human non-malignant (HCV29) and malignant cells (T24 and HT1376) of bladder cancer, human skin fibroblasts (HSF) and mouse embryonic fibroblasts (MEF). Cells were fixed and examined using optical and atomic force microscopy observation. Special attention was focused on XPS analyses. The chemical states of cells’ elements were examined by XPS spectra measurements (Scanning XPS Microprobe PHI 5000 Versa Probe II, ULVAC-PHI). Depth distribution of the elements and their chemical states were examined by sputtering with argon gas cluster ion beam (Ar-GCIB) with Zalar rotation. As a reference, the culture media and fatal bovine serum (FBS) atomic composition were analyzed. This very recent and unique technique allows to preserves chemical structure of organic materials under sputtering.

[1] P. G. Rouxhet, M. J. Genet, Surf. Interface Anal. 2011, 43, 1453

[2] M. Jayasundera, B. Adhikari, P. Aldred, A. Ghandi, J. Food Eng. 2009, 93, 266

[3] E. H. J. Kim, X. D. Chen, D. Pearce, J. Food Eng. 2009, 94, 182

[4] M. J. Genet, C. C. Dupont-Gillain, P. G. Rouxhet, Medical applications

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