FIB-milled plasmonic nanoapertures allow for long trapping times of individual proteins

We have developed a fabrication methodology for label-free optical trapping of individual nanobeads and proteins in inverted-bowtie-shaped plasmonic gold nanopores. Arrays of these nanoapertures can be reliably produced using focused ion beam (FIB) milling with gap sizes of 10-20 nm, single-nanometer variation, and with a remarkeie stability that allows for repeated use. We employ an optical readout where the presence of the protein entering the trap is marked by an increase in the transmission of light through the nanoaperture from the shift of the plasmonic resonance. In addition, the optical trapping force of the plasmonic nanopores allows 20-nm polystyrene beads and proteins, such as beta-amylase and Heat Shock Protein (HSP90), to be trapped for very long times (approximately minutes). On demand, we can release the trapped molecule for another protein to be interrogated. Our work opens up new routes to acquire information on the conformation and dynamics of individual proteins.

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

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

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Flavio Maurizio Mor

Life sciences have a constantly growing need for novel methodological approaches suitable to investigate the intracellular environment with increased temporal and spatial resolutions. Recently, optical near-field probes, such as laser-irradiated pointed metal, uncoated/metal-coated tapered optical fibers, as well as nano-emitters, such as single molecules or nanoparticles, have attracted increasing attention as key components of high-resolution microscopes. In parallel, super-resolution fluorescence microscopy, operating in far-field regime and overcoming the light diffraction limit, has markedly improved imaging resolution. Although both near- and far-field optical approaches drastically improve image resolution, they still represent numerous drawbacks, such as, technical difficulties concerning probe preparation (near-field) or potential damaging through very high light intensities (far-field). The aforementioned shortcomings of high-resolution optical microscopy can be overcome to a great extent by photonic force microscopy (PFM). PFM employs a strongly focused near-infrared (NIR) laser light to hold a dielectric or metallic particle as a local probe. Such an optical trap enables one to ‘cage’ a mesoscopic particle and track its three-dimensional (3D) thermal fluctuations in the surrounding environment, e.g. a viscous liquid. Therefore, besides offering near-field 3D imaging, PFM reports also on other important quantities concerning local mechanical properties, including force and viscosity as well as dynamical properties of the surrounding medium. This additional information is derived from the careful analysis of the jittery motion (so-called Brownian motion) of the trapped single-particle probe that collides with thermally-activated surrounding molecules. In this thesis, we first study in detail the Brownian motion of a single spherical particle that is confined within the harmonic potential of the optical trap. To this end, we optimize the NIR light path and electronic noise floor of our custom-built PFM set-up for detecting and quantifying resonances in the Brownian motion. These resonances arise from the coupling between the hydrodynamic memory and strong strength of the optical trap. Due to the high sensitivity of the short-time dynamics, the size of the particle can be measured by simultaneous fitting of the velocity autocorrelation function and power spectral density of its thermal fluctuations. In order to choose the best-suitable spherical probe for a given experiment in PFM, computational modeling based on a Matlab toolbox is performed. The generalized Lorenz-Mie theory is computed using the T -matrix method for various experimental conditions, including changes in the size and refractive index of the sphere, as well as different laser polarization states. The axial equilibrium position is examined to predict its location compared with the position of the laser focus. Optical forces acting on the sphere are investigated in 3D to highlight, in particular, the limits of the perfectly harmonic potential assumption. These limits are quantified and discussed. Subsequently, we identify different types of inorganic nano/micro-sized particles that can be trapped by the NIR laser of the PFM and used simultaneously as mechanical and near-field light sources. To this end, we take advantage of the visible light emission from single probes confined in the optical trap and excited by the NIR laser light. The emphasis is on particles revealing nonlinear optical properties. In particular, nonlinear optical field enhancement resulting from excitation of surface plasmon resonance (SPR) on trapped gold particles of 200 nm is demonstrated by measuring the emission of that second-harmonic generation (SHG). Furthermore, we show, under optical trapping conditions, SHG emission from single potassium niobate (KNbO3) nano/micro-sized particles. We also report on the multicolor upconversion luminescence (UCL), at the single particle level, for randomly shaped crystals of sodium yttrium fluoride codoped with ytterbium and erbium, NaYF4:Yb,Er (UCC), when they are held in the optical trap. In parallel, 3D Brownian fluctuations of a single trapped particle are quantified. Moreover, luminescence resonance energy transfer (LRET) between a KNbO3 particle or an UCC and molecules of an organic dye (rose bengal) adsorbed on their surface is highlighted and analyzed. Finally, randomly shaped UCCs and hexagonal nanoplates are characterized in detail at the single particle level. The hexagonal β phase in the polycrystalline and monocrystalline crystals is identified, in both randomly and hexagonally shaped particles by electron microscopy. UCL emission from the trapped crystal is quantified in terms of power and found to be dependent on the laser power density, size and shape of the particle as well as its orientation within the trap. Most importantly, the different colors in the UCL spectrum do not vary with the same trend for different orientations of the randomly or hexagonally shaped crystal upon trapping. This suggests the existence of crystalline anisotropy-dependent UCL. Thereby, we should be able to design the best-suited particle probe for LRET experiments with subwavelength resolution.

EPFL2013

Electrospray Ion Beam Deposition of Complex Non-Volatile Molecules

Gordon Rinke

EPFL2013

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

Nicha Thontasen

EPFL2011

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Flavio Maurizio Mor

EPFL2013