Low-energy electron holography imaging of conformational variability of single-antibody molecules from electrospray ion beam deposition

Imaging of proteins at the single-molecule level can reveal conformational variability, which is essential for the understanding of biomolecules. To this end, a biologically relevant state of the sample must be retained during both sample preparation and imaging. Native electrospray ionization (ESI) can transfer even the largest protein complexes into the gas phase while preserving their stoichiometry and overall shape. High-resolution imaging of protein structures following native ESI is thus of fundamental interest for establishing the relation between gas phase and solution structure. Taking advantage of low-energy electron holography's (LEEH) unique capability of imaging individual proteins with subnanometer resolution, we investigate the conformational flexibility of Herceptin, a monoclonal IgG antibody, deposited by native electrospray mass-selected ion beam deposition (ES-IBD) on graphene. Images reconstructed from holograms reveal a large variety of conformers. Some of these conformations can be mapped to the crystallographic structure of IgG, while others suggest that a compact, gas-phase-related conformation, adopted by the molecules during ES-IBD, is retained. We can steer the ratio of those two types of conformations by changing the landing energy of the protein on the single-layer graphene surface. Overall, we show that LEEH can elucidate the conformational heterogeneity of inherently flexible proteins, exemplified here by IgG antibodies, and thereby distinguish gas-phase collapse from rearrangement on surfaces.

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

The thesis presents the development of a custom made atomic force microscope (AFM) conceived to image biological molecules at low temperatures in ultrahigh vacuum conditions, and its application to DNA single molecule studies. Starting from a first prototype working in contact mode, our aim was to implement non-contact AFM in order to reduce possible sample damaging due to strong tip interactions. The first chapter of the thesis discusses the technical solutions developed to improve the previous system. A complete setup for in situ exchange of samples and tips was designed and implemented. This permits a continuous operation of the microscope under the experimental conditions of UHV. Thermal instabilities and temperature differences between the tip and the sample were solved with the design of a movable shield to avoid heating by thermal radiation. The introduction of a new high voltage signal generator with variable frequency and the development of a user-friendly LabVIEW interface to communicate with this device notably improved the piezomotors driving. These piezomotors perform the micro-positioning of all the microscope parts and are driven through this interface. Finally, the previous tip-holder design was modified to incorporate a piezo actuator behind the cantilever, which provides the necessary excitation for the implementation of dynamic AFM modes. The installation of a Phase Lock Loop (PLL) circuit to lock the cantilever frequency and the replacement of old feedback electronics for a digital SPM Controller, with advanced options of frequency-shift feedback, was another requirement for the correct implementation of non-contact AFM. The second chapter deals with DNA sample preparation for high resolution imaging by AFM. Based on our studies, the advantages and drawbacks of the most commonly used techniques for DNA adsorption are discussed in detail. Our measurements in contact mode demonstrate the convenience of using hydrophobic substrates for high resolution DNA imaging. At hydrophilic surfaces, like mica, the presence of a thin water layer could be responsible of strong adhesion forces between the cantilever and the sample. In order to overcome this limitation we tested HOPG as hydrophobic substrate. HOPG, chemically modified with amphiphilic compounds, allowed us to deposit DNA without molecular damage or stress. The HOPG surface treatment was significantly improved. New protocols were developed to control the formation of dodecylamine disordered monolayers or lamella structures on the substrate. The lamella formation induces a subsequent self assembly of DNA single molecules. In the last chapter we discuss our results on DNA imaging obtained with the different operation modes implemented in our microscope: contact mode, tapping mode and frequency modulation (non-contact) AFM. We conclude that the last one provides higher signal stability and the necessary sensitivity to observe DNA details at a single-molecule level. We also present a study of frequency shift versus distance dependence (force spectroscopy experiments) performed in DNA samples. We discuss for the first time a plausible explanation to the origin of the atomic force contrast between the substrate and the DNA molecules. Force spectroscopy measurements performed along DNA molecules reveal two distinguishable types of interaction. This result suggests that the chemical composition of the minor-major groove and/or of the sugar-phosphate backbone along the molecule can be detected, opening numerous possibilities for further studies and applications of this technique.

EPFL2008

Ion Beam Deposition and STM Analysis of Macromolecules on Solid Surfaces in UHV

Nicha Thontasen

Organic and biological macromolecules have attracted great interest within nanotechnology research due to properties that can be useful in future devices. Interesting functionalities such as optical activity, host-guest binding and molecular recognition are based on effects at the single molecular level. To study the properties and functions of molecules at submolecular scale, an atomically defined environment with low contamination is of great advantage. This can for instance be provided on a clean surface in ultrahigh vacuum (UHV), where surface sensitive analytical techniques and advanced nanostructure fabrication can be applied. Atoms and small organic molecules are easily transferred to a surface in vacuum by thermal sublimation. Many large, functional molecules, however, are nonvolatile and disintegrate at an elevated temperature, which hinders their application and investigation within the UHV environment. To explore the properties of nonvolatile molecules adsorbed at surface as well as the nanostructure fabricated from such molecules, electrospray ion beam deposition (ES-IBD), a technique capable of transferring nonvolatile molecules intact to surfaces in UHV, and in situ scanning tunneling microscopy (STM) are combined. A home-built ES-IBD setup allows molecular beam deposition with an unprecedented level of control: the ion beam composition is monitored by time-of-flight mass spectrometry (TOF-MS) prior to the deposition, molecular flux and coverage controlled via the ion beam current and the beam energy can be measured and adjusted. The modified surfaces are characterized in situ by STM, which reveals structural and electronic properties with submolecular resolution. Moreover, ex situ analysis by atomic force microscopy (AFM) allows to access meso-scale structural formation. In addition, Laser Desorption Ionization (LDI), Matrix-Assisted Laser Desorption Ionization (MALDI) and Secondary Ion Mass Spectrometry (SIMS) are performed to check the chemical composition and purity of the adsorbed materials. This work first introduces the principles of our experiments and reviews of the literature on molecular ion beam deposition. In the following, the experimental method is characterized in detail using Rhodamine 6G, an organic dye, as a model system. It is shown that homogeneous layers of adsorbed, intact molecules are formed on the surface and single molecule adsorbates are observed by STM. An investigation by SIMS and LDI reveals that the kinetic energy of the molecular ions upon collision with the surface determines whether intact or fragment ions are deposited. Molecular layer growth was studied using cluster ion beams of the nonvolatile surfactant SDS, resulting in an arrangement of the molecules typical for surfactants in a head-to-head and tail-to-tail manner based on the structural motive. Crystalline structures are observed by AFM resembling inverted bilayer-membranes formed on graphite and SiOx surfaces. A similar structural feature revealed by STM is a flat-laying double row of the molecules on Cu(100) surface showing the robustness of the structural motive. The properties of single molecular functional systems are studied on two types of large functional molecules: macrocycle complexes and proteins. Macrocyclic host-guest complexes of the three cations: Na+, Cs+ and H+ trapped in the cavity of dibenzo-24-crown-8 (DB24C8) are formed in the electrospray solutions and transferred intact to the surface. Characterization by STM and density functional theory calculations shows the structure of the complexes with the alkali ion present in the cavity. The shape of the complex at the surface is also determined. Cytochrome c, an electron transfer protein is deposited on surfaces in UHV as an ion beam of high or low charge-state. STM reveals unfolded strands with distinguishable submolecular structural features after the deposition of high charge states. When low charge states are deposited, two significantly different structures are observed. Folded proteins appear as globular features of 2-3 nm diameter, while unfolded proteins are imaged as at strands. On Au(111) the unfolded strands fold into compact two-dimensional patches. Additionally, the variation in kinetic energy upon deposition and elevated surface temperature are also studied regarding the change in conformation of the adsorbed proteins. The results of this thesis open up new opportunities for the creation of functional nanostructures on surfaces. Both the preparation of layers and thin films as well as the controlled immobilization of single functional molecules were demonstrated with ES-IBD and characterized at submolecular resolution in situ by STM. A great variety of nonvolatile substances from small to large functional molecules with different binding motives like covalent, complex coordination or hydrogen bonds, is compatible with ESI and thus can be used as building blocks to construct new materials in a well-controlled manner.

EPFL2011

Electrospray Ion Beam Deposition of Complex Non-Volatile Molecules

Gordon Rinke

The rational synthesis of novel materials requires the control over the arrangement of matter in order to meet the desired properties for applications and devices. Ultimately, control means to define the place of each atom and determine its chemical state, as well as being able to confirm the result in a measurement. Control at the atomic level can be achieved with scanning tunneling microscopy (STM), both in imaging and atomic manipulation. The latter, however, can never be used in meso- or even macroscopic material synthesis. Self-ordering phenomena determines atomic control in material synthesis as well, observed for instance in protein folding or in molecular beam epitaxy (MBE) technology. In both cases the atomically determined arrangement of the atoms in the structure is controlled only by environmental parameters that steer the self-ordering phenomena. In this thesis I show that electrospray ion beam deposition (ES-IBD) or ion soft-landing is a material processing technique, which combines and even extends the level of control offered by MBE. It is demonstrated that a vast range of complex organic materials including peptides and proteins is now available to ultrapure vacuum processing and moreover completely novel schemes of controlling self-ordering processes are provided. In-situ STM is applied to investigate the structure formation of complex molecules deposited by ES-IBD on well-defined, clean, and atomically flat surfaces in ultrahigh vacuum at the greatest detail. This allows us to explore the novel control features that are intrinsic to the ES-IBD deposition process like online coverage monitoring, deposition energy control, and mass-selection to select charge states or reactive species. The molecules studied here include organic molecular salts, dye molecules, reactive polymer building blocks, and finally peptides and proteins. Crystalline layer-by-layer as well as island growth was observed and revealed the equivalence to conventional MBE growth. On the basis of these deposition experiments the crucial influence of clusters in the ion beam for high material flux was discovered. Furthermore, it was found that the chemical state of the molecule is a key factor for the deposition result as chemical reactions can be induced or the self-assembly behavior can be altered. Alongside the capability to handle also extremely reactive molecules, the feasibility to control chemical reactions occurring as a result of an external stimulus to a specifically selected reactive species is demonstrated. Employing the control parameters that are specific to ES-IBD like charge state selection and deposition energy to complex biomolecules opens up new perspectives for vacuum deposition. In addition to the self-assembly governed by the molecule-substrate and molecule-molecule interaction, it was possible to actively steer the structure formation of proteins by influencing their stiffness and their reactivity through their charge state as well as by the deposition energy, parameters intrinsic to the ES-IBD method. Hence, electrospray ion beam deposition is indeed a tool to prepare well defined surface coatings at highest level of control and complexity. To be relevant for industrial applications, the performance of the system has to be further improved, most importantly in terms of deposition rate. In general, ES-IBD enables new approaches for the growth of functional coatings but also serving as perfect sample preparation tool for the characterization of complex molecules on the atomic scale by scanning probe or more general surface science methods.

EPFL2013