Johannes Preiner

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200-430 nm and a depth of 30-100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

The role of oxygen termination of nanocrystalline diamond on immobilisation of BMP-2 and subsequent bone formation

Medical implants are increasingly often inserted into bone of frail patients, who are advanced in years. Due to age, severe trauma or pathology-related bone changes, osseous healing at the implant site is frequently limited. We were able to demonstrate that coating of endosseous implants with nanocrystalline diamond (NCD) allows stable functionalization by means of physisorption with BMP-2. Strong physisorption was shown to be directly related to the unique properties of NCD, and BMP-2 in its active form interacted strongly when NCD was oxygen-terminated. The binding of the protein was monitored under physiological conditions by single molecule force spectroscopy, and the respective adsorption energies were further substantiated by force-field-calculations. Implant surfaces refined in such a manner yielded enhanced osseointegration in vivo, when inserted into sheep calvaria. Our results further suggest that this technical advancement can be readily applied in clinical therapies with regard to bone healing, since primary human mesenchymal stromal cells strongly activated the expression of osteogenic markers when being cultivated on NCD physisorbed with physiological amounts of BMP-2.

The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as cell growth support surfaces

Nanocrystalline diamond (NCD) films and nanoparticulate diamond powder (DP) are the two main representatives of diamond at the nanoscale. This study was designed to investigate the suitability of these biomaterials as cell growth supports and to determine surface characteristic properties best suited to cell attachment and proliferation. Surface topography, chemical termination and wetting properties of NCD- and DP-coated borosilicate glass substrates were correlated to attachment, proliferation and differentially regulated gene expression of human renal epithelial cells (HK-2 cell line) cultured on these surfaces. Hydrogen-terminated NCD (NCD-H) surfaces were shown to inhibit cell attachment, which indicates that the lack of functional polar groups prevents adherent cells from settling on a surface, whether nanostructured or not. In contrast to NCD-H, oxygen-terminated NCD (NCD-O) as well as DP surfaces demonstrated improved cell attachment, as compared to borosilicate glass, which is a commonly used material for cell growth supports. NCD-O not only revealed an increased cell attachment, but also a markedly increased proliferation rate. Finally, none of the investigated surface modifications appeared to cause adverse cellular reactions or markedly alter cellular phenotype.

Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns

Atomic force microscopy (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics of biomolecules by probing the interactions within individual molecules, or between a surface-bound molecule and a cantilever that carries a complementary binding partner. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 Å, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level.

Simultaneous topography and recognition imaging: physical aspects and optimal imaging conditions

Simultaneous topography and recognition imaging (TREC) allows for the investigation of receptor distributions on natural biological surfaces under physiological conditions. Based on atomic force microscopy (AFM) in combination with a cantilever tip carrying a ligand molecule, it enables us to sense topography and recognition of receptor molecules simultaneously with nanometre accuracy. In this study we introduce optimized handling conditions and investigate the physical properties of the cantilever-tip-sample ensemble, which is essential for the interpretation of the experimental data gained from this technique. In contrast to conventional AFM methods, TREC is based on a more sophisticated feedback loop, which enables us to discriminate topographical contributions from recognition events in the AFM cantilever motion. The features of this feedback loop were investigated through a detailed analysis of the topography and recognition data obtained on a model protein system. Single avidin molecules immobilized on a mica substrate were imaged with an AFM tip functionalized with a biotinylated IgG. A simple procedure for adjusting the optimal amplitude for TREC imaging is described by exploiting the sharp localization of the TREC signal within a small range of oscillation amplitudes. This procedure can also be used for proving the specificity of the detected receptor-ligand interactions. For understanding and eliminating topographical crosstalk in the recognition images we developed a simple theoretical model, which nicely explains its origin and its dependence on the excitation frequency.

The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues

Mobility of single-file water molecules determined by H-bonds. Channel geometry governs the unitary osmotic water channel permeability, pf, according to classical hydrodynamics. Yet, pf varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the pf of those channels and the diffusion coefficient of the single-file waters within them are determined by the number NH of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on NH is in line with the multiplicity of binding options at higher NH densities. We obtained high-precision pf values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

Fabrication of Highly Ordered Gold Nanoparticle Arrays Templated by Crystalline Lattices of Bacterial S-Layer Protein

Biomolecular self-assembly is emerging as a powerful tool for bottom-up approaches to the fabrication of functional nanoscale structures. Nanofabrication techniques based on protein and DNA self-assembly have been applied for nanoscale engineering of nanoparticle (NP) arrays. Bacterial cell surface layer (S-layer) proteins spontaneously self-assemble on various types of supports by forming two-dimensional crystals exhibiting different lattice symmetries (oblique, square or hexagonal) with lattice constants in the range from 3 to 30 nm. These excellent building blocks are usually composed of a single protein or glycoprotein species. Taking advantage of their spatially defined physical and chemical surface properties, S-layers offer an attractive approach for the fabrication of nanoparticle templates, and have been used successfully as biotemplates for the in situ nucleation of inorganic particles and for binding of gold NPs via electrostatic interaction. The possibility of genetically modifying S-layer proteins with different functional sequences pave the way for building a broad range of functional nanostructures through site-directed or covalent binding of molecules to S-layer templates. Herein, highly ordered arrays of 5 nm gold NPs were generated by using the repetitive pattern of a mutated S-layer protein as a binding template for well-organized arrangements. The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 with the ability to self-assemble into a square (p4) lattice is one of the most extensively studied S-layer proteins. In recent years, fusion proteins consisting of SbpA and various functional domains [e.g. the Fc-binding domain of protein A, core-streptavidin, the major birch pollen allergen (Bet v1), a hyper-variable region of a heavy-chain camel antibody, the enhanced green fluorescent protein (EGFP), and an enzyme of an extremophilic organism] have been successfully produced and studied. It was shown that the fusion does not interfere with the self-assembly properties and that the functional sequences are well accessible on the S-layer fusion protein lattice. The chimeric protein rSbpA–Cys is constructed by fusing a single cysteine residue to a C-terminally truncated form of the S-layer protein SbpA. Topographical images of the rSbpA–Cys mutant, recrystallized on a silicon surface, were obtained in PBS buffer using atomic force microscopy (AFM). As shown by the large-area scan in Figure 1A, the silicon surface is almost fully covered

Imaging and detection of single molecule recognition events on organic semiconductor surfaces.

The combination of organic thin film transistors and biological molecules could open new approaches for the detection and measurement of properties of biological entities. To generate specific addressable binding sites on such substrates, it is necessary to determine how single biological molecules, capable of serving as such binding sites behave upon attachment to semiconductor surfaces. Here, we use a combination of high-resolution atomic force microscopy topographical imaging and single molecule force spectroscopy (TREC), to study the functionality of antibiotin antibodies upon adsorption on pentacene islands, using biotin-functionalized, magnetically coated AFM tips. The antibodies could be stably adsorbed on the pentacene, preserving their functionality of recognizing biotin over the whole observation time of more than one hour. We have resolved individual antigen binding sites on single antibodies for the first time. This highlights the resolution capacity of the technique.

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells.

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.

High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.