Cytotoxicity of Metal and Semiconductor Nanoparticles Indicated by Cellular Micromotility
In the growing field of nanotechnology, there is an urgent need to sensitively determine the toxicity of nanoparticles since many technical and medical applications are based on controlled exposure to particles, that is, as contrast agents or for drug delivery. Before the in vivo implementation, in vitro cell experiments are required to achieve a detailed knowledge of toxicity and biodegradation as a function of the nanoparticles' physical and chemical properties. In this study, we show that the micromotility of animal cells as monitored by electrical cell-substrate impedance analysis (ECIS) is highly suitable to quantify in vitro cytotoxicity of semiconductor quantum dots and gold nanorods…
Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes.
We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from Escherichia coli (E. coli), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photoble…
Mammalian cell growth on gold nanoparticle-decorated substrates is influenced by the nanoparticle coating
In this work, we study epithelial cell growth on substrates decorated with gold nanorods that are functionalized either with a positively charged cytotoxic surfactant or with a biocompatible polymer exhibiting one of two different end groups, resulting in a neutral or negative surface charge of the particle. Upon observation of cell growth for three days by live cell imaging using optical dark field microscopy, it was found that all particles supported cell adhesion while no directed cell migration and no significant particle internalization occurred. Concerning cell adhesion and spreading as compared to cell growth on bare substrates after 3 days of incubation, a reduction by 45% and 95%, …
Dynamic force spectroscopy: analysis of reversible bond-breaking dynamics
The problem of diffusive bond-dissociation in a double well potential under application of an external force is scrutinized. We compute the probability distribution of rupture forces and present a detailed discussion of the influence of finite rebinding probabilities on the dynamic force spectrum. In particular, we focus on barrier crossing upon extension, i.e. under linearly increased load, and upon relaxation starting from completely separated bonds. For large loading rates the rupture force and the rejoining force depend on the loading rate in the expected manner determined by the shape of the potential. For small loading rates the mean forces obtained from pull and relax modes approach …
No Label Required: Protein Binding at Membrane Interfaces Visualized through Colloid Phase Transitions
Protein-membrane interaction probed by single plasmonic nanoparticles.
We present a nanosized and addressable sensor platform based on membrane coated plasmonic particles and show unequivocally the covering with lipid bilayers as well as the subsequent detection of streptavidin binding to biotinylated lipids. The binding is detected on membrane covered gold nanorods by monitoring the spectral shift by fast single particle spectroscopy (fastSPS) on many particles in parallel. Our approach allows for local analysis of protein interaction with biological membranes as a function of the lateral composition of phase separated membranes.
Surfactant-Induced Helix Formation of Cylindrical Brush Polymers with Poly(L-lysine) Side Chains
The complex formation of oppositely charged surfactants with some polypeptides is known to induce β-sheet or helix formation. Here, we report on the complex formation of cylindrical brush polymers with poly(L-lysine) side chains and sodium dodecylsulfate (SDS). With increasing amount of added surfactant the cylindrical polymers first adopt a helical conformation with a pitch of approximately 14-24 nm followed by a spherically collapsed structure before eventually precipitation occurs. CD measurements suggest that the helix formation of the cylindrical brush polymers is driven by the hydrophobicity of the ,8-sheets formed by the PLL side chain-SDS complexes.
Electrically induced deformation of giant liposomes monitored by thickness shear mode resonators.
Thickness shear mode resonators are capable of registering small changes in the thickness and viscoelastic properties of ultrathin films attached to their surface. It was found that it is possible to monitor the deformation of surface-bound giant liposomes by applying an electric field with small amplitudes. Changes in the apparent height of attached vesicles in the nanometer range were easily detected as a function of lipid composition. Increasing the bending modulus by adding cholesterol results in a significantly reduced deformation from 16.8 nm (5% cholesterol) down to 3.2 nm (20% cholesterol), rendering this new method a robust and sensitive tool to detect the bending elasticity of lip…
Melting and interdigitation of microstructured solid supported membranes quantified by imaging ellipsometry
The phase transition of individually addressable microstructured lipid bilayers was investigated by means of noncontact imaging ellipsometry. Two-dimensional membrane compartments were created on silicon substrates by micromolding in capillaries and the phase transition of supported dimyristoylphosphadiylcholine DMPC and dipentadecoylphosphatidylcholine DiC15PC membranes was determined measuring area expansion and thickness of the bilayer as a function of temperature, ethanol concentration, and cholesterol content. Apart from measuring the thermotropic behavior of DMPC on glass slides and silicon wafers, the authors were able to visualize the reversible induction of an interdigitated phase …
Mechanically interlocked calix[4]arene dimers display reversible bond breakage under force.
The physics of nanoscopic systems is strongly governed by thermal fluctuations that produce significant deviations from the behaviour of large ensembles1,2. Stretching experiments of single molecules offer a unique way to study fundamental theories of statistical mechanics, as recently shown for the unzipping of RNA hairpins3. Here, we report a molecular design based on oligo calix[4]arene catenanes—calixarene dimers held together by 16 hydrogen bridges—in which loops within the molecules limit how far the calixarene nanocapsules can be separated. This mechanically locked structure tunes the energy landscape of dimers, thus permitting the reversible rupture and rejoining of the individual n…
Mechanical properties of MDCK II cells exposed to gold nanorods
Background: The impact of gold nanoparticles on cell viability has been extensively studied in the past. Size, shape and surface functionalization including opsonization of gold particles ranging from a few nanometers to hundreds of nanometers are among the most crucial parameters that have been focussed on. Cytoxicity of nanomaterial has been assessed by common cytotoxicity assays targeting enzymatic activity such as LDH, MTT and ECIS. So far, however, less attention has been paid to the mechanical parameters of cells exposed to gold particles, which is an important reporter on the cellular response to external stimuli.Results: Mechanical properties of confluent MDCK II cells exposed to go…
Formation of layered titania and zirconia catalysed by surface-bound silicatein
Silicatein immobilised on self-assembled polymer layers using a histidine-tag chelating anchor group retains its hydrolytical activity for the formation of biosilica, and catalyses the formation of layered arrangements of biotitania and biozirconia.
Reversible hydrogen bond network dynamics: molecular dynamics simulations of calix[4]arene-catenanes.
We present detailed molecular dynamics (MD) simulations of mechanically interlocked calix[4]arene-catenanes under external force. Single-molecule force spectroscopy experiments revealed that the separation of dimers with two aliphatic loops results in reversible hydrogen bond breakage through an intermediate in a triple-well potential, while the tetra-loop species separates in a one-step manner (Janke, M.; et al. Nat. Nanotechnol. 2009, 4, 225). MD simulations show that calix[4]arenes interlocked by four loops (1) display a complete restructuring of the hydrogen bond network under mechanical force. All hydrogen bonds of the closed structure open, and new ones are formed in the extended stru…
Coiled-Coil Lipopeptides Mimicking the Prehairpin Intermediate of Glycoprotein gp41
In situ synthesis of lipopeptides as versatile receptors for the specific binding of nanoparticles and liposomes to solid-supported membranes.
A detailed study of the in situ coupling of small peptides such as CGGH6 (H6) and CGWK8 (K8) to maleimide functionalized phospholipid bilayers is presented. Individually addressable microstructured membranes are employed to unequivocally probe the conjugation. The in situ coupling of peptides via a terminal cysteine moiety to maleimide functionalized phospholipids is shown to be a convenient and versatile way to selectively fabricate peptide-modified phospholipid bilayers serving as specific receptor platforms for functionalized vesicles and nanoparticles. Specific binding of functional vesicles to the peptide-modified bilayers is achieved by either histidine complexation with Ni-NTA-DOGS c…
Phase selection of calcium carbonate through the chirality of adsorbed amino acids.
Monitoring the formation of biosilica catalysed by histidine-tagged silicatein.
Surface bound silicatein retains its biocatalytic activity, which was demonstrated by monitoring the immobilisation of silicatein using a histidine-tag chelating anchor and the subsequent biosilicification of SiO(2) on surfaces by surface plasmon resonance spectroscopy, atomic force microscopy and scanning electron microscopy.
New perspectives for the design of molecular actuators: thermally induced collapse of single macromolecules from cylindrical brushes to spheres.
Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis.
Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique…
Quantification of the Raf-C1 Interaction With Solid-Supported Bilayers
By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, wa…
Toxicity of gold-nanoparticles: Synergistic effects of shape and surface functionalization on micromotility of epithelial cells
Nanoparticle exposure is monitored by a combination of two label-free and non-invasive biosensor devices which detect cellular shape and viscoelasticity (quartz crystal microbalance), cell motility and the dynamics of epithelial cell-cell contacts (electric cell-substrate impedance sensing). With these tools we have studied the impact of nanoparticle shape on cellular physiology. Gold (Au) nanoparticles coated with CTAB were synthesized and studied in two distinct shapes: Spheres with a diameter of (43 ± 4) nm and rods with a size of (38 ± 7) nm × (17 ± 3) nm. Dose-response experiments were accompanied by conventional cytotoxicity tests as well as fluorescence and dark-field microscopy to v…
Quartz Crystal Microbalance for Bioanalytical Applications
The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid load have been developed, the QCM became accepted as a new powerful technique to monitor adsorption processes at solid/liquid interfaces in chemical and biological research rendering the method an attractive low-cost alternative for bioanalytic applications. In the last decade, adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of piezoelectric transducers comprising the interaction of DNA and RNA with complementary strands, specific recogn…
Equilibrium coverage fluctuations: a new approach to quantify reversible adsorption of proteins.
Liquid Crystals from Polymer-Functionalized TiO2 Nanorod Mesogens
In this work, we functionalized TiO2 nanorods with dopamine-functionalized diblock copolymers. After functionalization, they are well dispersible in organic solvents. Light scattering proves that t...
Cell motility probed by noise analysis of thickness shear mode resonators.
The quartz crystal microbalance (QCM) technique is an emerging bioanalytical tool to study the behavior of animal cells in vitro. Due to the high interfacial sensitivity of thickness shear mode (TSM) resonators it is possible to monitor the formation and breakage of cell-matrix interactions and changes in viscoelasticity of the cell bodies, as well as minute cell volume alterations by the time course of their resonance frequency even with millisecond time resolution. We found that mammalian MDCK-II cells grown on TSM resonators impose characteristic fluctuations on the resonance frequency, which are a quantitative indicator for dynamic activities of the cells on the surface and report on th…
Scanning force microscopy based rapid force curve acquisition on supported lipid bilayers: experiments and simulations using pulsed force mode.
In situ pulsed force mode scanning force microscopy (PFM-SFM) images of phase separated solid-supported lipid bilayers are discussed with the help of computer simulations. Simultaneous imaging of material properties and topography in a liquid environment by means of PFM-SFM is severely hampered by hydrodynamic damping of the cantilever. Stiffness and adhesion images of solid-supported membranes consisting of cholesterol, sphingomyelin, and 1,2-dioleyl-phosphatidylcholine obtained in aqueous solution exhibit contrast inversion of adhesion and stiff. ness images depending on parameters such as driving frequency, amplitude, and trigger setting. Simulations using a simple harmonic oscillator mo…
Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis
Inside Cover: Phase Selection of Calcium Carbonate through the Chirality of Adsorbed Amino Acids (Angew. Chem. Int. Ed. 29/2007)
Hierarchical Assembly of TiO2 Nanoparticles on WS2 Nanotubes Achieved Through Multifunctional Polymeric Ligands
Thefunctionalization of nanotubes is required in order to bene-fit from their outstanding properties, as any application inmaterials and devices is hindered by processing and manipu-lation difficulties. Only the attachment of appropriate chem-ical functionalities on the nanotube surface allows tailoringof the properties for the respective applications. As an ex-ample, the enhancement of the nanotube solubility is onemajor task since most pristine nanotubes are insoluble inboth water and organic solvents. Thus, the improvement ofthe solubility by chemical functionalization is an importantconcept for synthetic chemists and materials scientists. Tai-loring of the surface chemical bonds might a…
Label-Free Detection of Protein-Ligand Interactions by the Quartz Crystal Microbalance
In recent years the quartz crystal microbalance (QCM) has been accepted as a powerful technique to monitor adsorption processes at interfaces in different chemical and biological research areas. In the last decade, the investigation of adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of the QCM comprising the interaction of nucleic acids, specific molecular recognition of protein-receptor couples, and antigen-antibody reactions realized in immunosensors. The advantage of the QCM technique is that it allows for a label free detection of molecules. This is a result of the fact that the frequency response of the quartz resonator is propor…
Viscoelasticity of pore-spanning polymer membranes derived from giant polymersomes
We show how the viscoelastic properties of membranes formed from poly(butadiene)-block-poly(ethylene oxide) (PB130-b-PEO66) block copolymers can be locally accessed by atomic force microscopy. Polymer membranes are spread on microstructured porous silicon substrates from PB130-b-PEO66 vesicles by decreasing the osmotic pressure of the solution. Local viscoelastic properties of the pore-spanning polymer membranes were obtained from site-specific indentation experiments. Elastic moduli of these membranes were in the order of few MPa, while the elastic moduli of cross-linked membranes considerably increased to few GPa. Furthermore, the energy dissipation and velocity dependence of the hysteres…
Partially Reversible Adsorption of Annexin A1 on POPC/POPS Bilayers Investigated by QCM Measurements, SFM, and DMC Simulations
The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall are…
A new approach to assess gold nanoparticle uptake by mammalian cells: combining optical dark-field and transmission electron microscopy.
Toxicological effects of nanoparticles are associated with their internalization into cells. Hence, there is a strong need for techniques revealing the interaction between particles and cells as well as quantifying the uptake at the same time. For that reason, herein optical dark-field microscopy is used in conjunction with transmission electron microscopy to investigate the uptake of gold nanoparticles into epithelial cells with respect to shape, stabilizing agent, and surface charge. The number of internalized particles is strongly dependent on the stabilizing agent, but not on the particle shape. A test of metabolic activity shows no direct correlation with the number of internalized par…
Protein–Lipid Interactions. From Membrane Domains to Cellular Networks. Edited by Lukas K. Tamm.
Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes
AbstractThe specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at …
Scrutiny of the Failure of Lipid Membranes As A Function of Headgroups, Chain Length, and Lamellarity Measured by Scanning Force Microscopy
AbstractA fast, quantitative, and unambiguous screening of material properties of biomembranes using scanning force microscopy in pulsed force mode on lipid membranes is presented. The spatially resolved study of breakthrough force, breakthrough distance, adhesion, stiffness, and topography of lipid membranes as determined simultaneously by digitalized pulsed force mode provides new insight into the structure-function relationship of model membranes, which are systematically analyzed by varying chain length, lipid headgroup, and lamellarity. For this purpose, a novel unbiased analysis method is presented. A strong correlation between adhesion and breakthrough events is found on lipid bilaye…
Functionalized Calix[8]arenes, Synthesis and Self-assembly on Graphite
With the intention of building hollow tubular structures by self-assembly, we have designed and successfully synthesized a series of calix[8]arene derivatives. Their phenolic units were functionalized in p-position by various groups which are able to interact via hydrogen bonding or π−π stacking. Ethynyl, amide, urea, or imide links were chosen for the covalent attachment of these functional groups, to ensure the adjustment of an optimal distance for their interaction. Two different kinds of nanostructures self-assembled on a highly oriented pyrolytic graphite (HOPG) surface were found by scanning force microscopy: parallel aligned nanorods in which the calixarene molecules are adsorbed ed…
Rasterkraftmikroskopische In-situ-Detektion von molekularen Erkennungsreaktionen an mikrostrukturierten Lipidmembransegmenten
Scanning Force Microscopy of Artificial Membranes
Visualization of biological membranes by scanning force microscopy (SFM) has tremendously improved the current understanding of protein ‐ lipid interactions under physiological conditions. SFM is the only tool to directly image processes on surfaces in aqueous solution at molecular resolution. Besides being a supportive means to confirm results on lipid phases and domains obtained from fluorescence spectroscopy, calorimetry, and X-ray crystallography, SFM has contributed distinct aspects on the formation of 2D crystals of various membrane-confined proteins and morphological changes of membranes due to the interaction of peptides and proteins. This review will focus on recent results in SFM …
Protein-Lipid Interactions. From Membrane Domains to Cellular Networks. Herausgegeben von Lukas K. Tamm.
Innentitelbild: Phasenselektion von Calciumcarbonat durch die Chiralität adsorbierter Aminosäuren (Angew. Chem. 29/2007)
The non-bilayer lipid MGDG stabilizes the major light-harvesting complex (LHCII) against unfolding.
Abstract In the photosynthetic apparatus of plants a high proportion of LHCII protein is needed to integrate 50% non-bilayer lipid MGDG into the lamellar thylakoid membrane, but whether and how the stability of the protein is also affected is not known. Here we use single-molecule force spectroscopy to map the stability of LHCII against mechanical unfolding along the polypeptide chain as a function of oligomerization state and lipid composition. Comparing unfolding forces between monomeric and trimeric LHCII demonstrates that the stability does not increase significantly upon trimerization but can mainly be correlated with specific contact sites between adjacent monomers. In contrast, unfol…
Biosensors, Porous Silicon
Biosensors consist of a biologically active layer that responding to an analyte in solution and a powerful transducer that transforms and amplifies the reaction into a measurable signal. Biosensors can constantly measure the presence, absence, or concentration of specific organic or inorganic substances in short response time and ultimately at low cost. They are used commercially in health care, biotechnological process control, agriculture, veterinary medicine, defense, and environmental pollution monitoring. A common requirement of all of these applications is on-site chemical information—preferably in real time—on some dynamic or rapidly evolving process. Most biosensors are based on mol…
Force-clamp spectroscopy of reversible bond breakage.
We consider reversible breaking of adhesion bonds or folding of proteins under the influence of a constant external force. We discuss the stochastic properties of the unbinding/rebinding events and analyze their mean number and their variance in the framework of simple two-state models. In the calculations, we exploit the analogy to single molecule fluorescence and particularly between unbinding/rebinding and photon emission events. Environmental fluctuation models are used to describe deviations from Markovian behavior. The second moment of the event-number distribution is found to be very sensitive to possible exchange processes and can thus be used to identify temporal fluctuations of th…
Thermal Expansion of Microstructured DMPC Bilayers Quantified by Temperature-Controlled Atomic Force Microscopy
Mechanical Properties of Pore-Spanning Lipid Bilayers Probed by Atomic Force Microscopy
AbstractWe measure the elastic response of a free-standing lipid membrane to a local indentation by using an atomic force microscope. Starting point is a planar gold-coated alumina substrate with a chemisorbed 3-mercaptopropionic acid monolayer displaying circular pores of very well defined and tunable size, over which bilayers composed of N,N,-dimethyl-N,N,-dioctadecylammonium bromide or 1,2-dioleoyl-3-trimethylammonium-propane chloride were spread. Centrally indenting these “nanodrums” with an atomic force microscope tip yields force-indentation curves, which we quantitatively analyze by solving the corresponding shape equations of continuum curvature elasticity. Since the measured respon…
Microstructuring of phospholipid bilayers on gold surfaces by micromolding in capillaries
Microstructuring of lipid bilayers on gold surfaces was achieved by micromolding in capillaries employing chemically modified polydimethylsiloxane (PDMS). Microfluidic networks of PDMS were prepared by micromolding and functionalized with thiol end-groups using 3-mercaptopropyltrimethoxysilane. The PDMS stamps were firmly attached to the gold substrate via quasi-covalent linkage providing a tight seal, a prerequisite for establishing individual addressable capillaries. Bilayers composed of POPC/POPG were subsequently prepared on microstructured self assembly monolayers of 11-amino-1-undecanethiol via strong electrostatic interactions. This way it is possible to generate individually address…
The Nonbilayer Lipid MGDG and the Major Light-Harvesting Complex (LHCII) Promote Membrane Stacking in Supported Lipid Bilayers.
The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an in vitro model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleachi…
Multiple Site-Specific Binding of Fis Protein to Escherichia coli nuoA-N Promoter DNA and its Impact on DNA Topology Visualised by Means of Scanning Force Microscopy
Adhesion of liposomes: a quartz crystal microbalance study
Three different systems are presented, exploring the adhesion of liposomes mediated by electrostatic and lipid–protein interactions as well as molecular recognition of ligand receptor pairs. Liposomes are frequently used to gain insight into the complicated processes involving adhesion and subsequent events such as fusion and fission mainly triggered by specific proteins. We combined liposome technology with the quartz crystal microbalance (QCM) technique as a powerful tool to study the hidden interface between the membrane and functionalized surface. Electrostatic attraction and molecular recognition were employed to bind liposomes to the functionalized quartz crystal. The QCM was used to …
Persistence Length of Cylindrical Brush Molecules Measured by Atomic Force Microscopy
Mechanical properties of single cylindrical polymer brushes with polyisopropylacrylamide (PNIPAM) side chains deposited on mica were probed by atomic force microscopy. Visualization and stretching of individual molecules in aqueous solution clearly reveal the semiflexible nature of the cylindrical macromolecules. Imaging of the brushes on mica and inferring lp from a 〈R2〉 vs L plot results in an average persistence length of lp = 29 ± 3 nm, assuming the chains adopt their equilibrium conformation on the surface. Stretching experiments suggest that an exact determination of the persistence length using force extension curves is impeded by the contribution of the side-chain elasticity. Modeli…
Mechanical Properties of Single Molecules and Polymer Aggregates
This chapter deals with the mechanical properties of single polymer chains, aggregates, and supramolecular complexes. The topics discussed cover a broad range from fundamental statistical mechanics of the equilibrium elastic properties of single polymer chains to details of the behavior of binding pockets in biomolecular assemblies as observed by force spectroscopy. The first section treats the equilibrium mechanical properties of single polymer chains in various environments, investigated via extensive simulations employing coarse-grained models that have proven extremely successful in many branches of polymer physics, namely the bond-fluctuation model and the self-avoiding walk model. Apa…
The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots
In this work, cytotoxicity and cellular impedance response was compared for CdSe/ZnS core/shell quantum dots (QDs) with positively charged cysteamine–QDs, negatively charged dihydrolipoic acid–QDs and zwitterionic D-penicillamine–QDs exposed to canine kidney MDCKII cells. Pretreatment of cells with pharmacological inhibitors suggested that the uptake of nanoparticles was largely due to receptor-independent pathways or spontaneous entry for carboxylated and zwitterionic QDs, while for amine-functionalized particles involvement of cholesterol-enriched membrane domains is conceivable. Cysteamine–QDs were found to be the least cytotoxic, while D-penicillamine–QDs reduced the mitochondrial activ…
Statistics of reversible bond dynamics observed in force-clamp spectroscopy
We present a detailed analysis of two-state trajectories obtained from force-clamp spectroscopy (FCS) of reversibly bonded systems. FCS offers the unique possibility to vary the equilibrium constant in two-state kinetics, for instance the unfolding and refolding of biomolecules, over many orders of magnitude due to the force dependency of the respective rates. We discuss two different kinds of counting statistics, the event-counting usually employed in the statistical analysis of two-state kinetics and additionally the so-called cycle-counting. While in the former case all transitions are counted, cycle-counting means that we focus on one type of transitions. This might be advantageous in p…
Self-Assembled Polymers Based on bis-Tetra-Urea Calix[4]arenes Connected via the Wide Rim
Six double calixarenes were synthesised in which two tetra-urea calix[4]arenes are linked by a rigid spacer between the urea functions at their wide rim. The dimerisation of their tetra-urea parts leads to hydrogen-bonded polymeric assemblies in apolar solvents. The addition of the stoichiometric amount of a tetra-tosylurea calix[4]arene disrupts the polymeric structures due to the preferred formation of heterodimeric capsules between tetra-aryl and tetra-tosylurea calix[4]arenes. The existence of polymeric assemblies was further established by AFM studies on spin-coated samples.
Impact of compatible solutes on the mechanical properties of fibronectin: a single molecule analysis
The influence of ectoine and sarcosine on the mechanical properties of surface bound fibronectin has been investigated by means of force microscopy. Single molecule stretching experiments of fibronectin molecules reveal that ectoine and sarcosine increase the tendency of the polypeptide to coil, thus decreasing its apparent persistence length. This behavior can be explained by means of the preferential exclusion model implying that the osmolytes are expelled from the protein surface due to the increase in chemical potential of the denatured, i.e. stretched, state forcing the protein into a more compact structure. Detailed analysis of the unfolding forces, which are extracted from the succes…
From Defined Reactive Diblock Copolymers to Functional HPMA-Based Self-Assembled Nanoaggregates
This paper describes the synthesis of functional amphiphilic poly( N-(2-hydroxypropyl) methacrylamide)-block-poly(lauryl methacrylate) copolymers by RAFT polymerization via the intermediate step of activated ester block copolymers (pentafluoro-phenyl methacrylate). Block copolymers with molecular weights from 12000-28000 g/mol and PDIs of about 1.2 have been obtained. The amphiphilic diblock copolymers form stable super structures (nanoaggregates) by self-organization in aqueous solution. The diameters of these particles are between 100 and 200 nm and depend directly on the molecular weight of the block copolymer. Furthermore, we investigated the impact of these nanoaggregates on cell viabi…
Energy landscapes of ligand-receptor couples probed by dynamic force spectroscopy.
Playing a dominant role in many biochemical processes are the dynamic properties of molecular linkages; examples include cell adhesion, enzyme-catalyzed reactions, and molecular recognition by antibodies. Dynamic force spectroscopy, namely separating molecular bonds under external force ramps has rapidly become a powerful tool to study the rugged energy landscape of noncovalent ligand-receptor bonds. The picture shows a surface and tip-bound pair being pulled apart and the derived potential energy diagram.
Pathogen-Mimicking MnO Nanoparticles for Selective Activation of the TLR9 Pathway and Imaging of Cancer Cells
Here, design of the first pathogen-mimicking metal oxide nanoparticles with the ability to enter cancer cells and to selectively target and activate the TLR9 pathway, and with optical and MR imaging capabilities, is reported. The immobilization of ssDNA (CpG ODN 2006) on MnO nanoparticles is performed via the phosphoramidite route using a multifunctional polymer. The multifunctional polymer used for the nanoparticle surface modification not only affords a protective organic biocompatible shell but also provides an efficient and convenient means for loading immunostimulatory oligonucleotides. Since fluorescent molecules are amenable to photodetection, a chromophore (Rhodamine) is introduced …
Ein neuer Weg zu molekularen Aktuatoren: der thermisch induzierteÜbergang einzelner Makromoleküle von zylindrischen Bürsten zu kugelförmigen Objekten
ChemInform Abstract: Scanning Force Microscopy of Artificial Membranes
Visualization of biological membranes by scanning force microscopy (SFM) has tremendously improved the current understanding of protein ‐ lipid interactions under physiological conditions. SFM is the only tool to directly image processes on surfaces in aqueous solution at molecular resolution. Besides being a supportive means to confirm results on lipid phases and domains obtained from fluorescence spectroscopy, calorimetry, and X-ray crystallography, SFM has contributed distinct aspects on the formation of 2D crystals of various membrane-confined proteins and morphological changes of membranes due to the interaction of peptides and proteins. This review will focus on recent results in SFM …
Visualization of Molecular Recognition Events on Microstructured Lipid-Membrane Compartments by In Situ Scanning Force Microscopy This work was supported by the Deutsche Forschungsgemeinschaft (JA 963/1-2).
Lateral organization of G M1 in phase-separated monolayers visualized by scanning force microscopy
Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid m…
Hierarchical structure formation of cylindrical brush polymer-surfactant complexes.
The complex formation of cylindrical brush polymers with poly(l-lysine) side chains (PLL) and sodium dodecyl sulfate (SDS) can induce a helical conformation of the cylindrical brush polymer in aqueous solution (Gunari, N.; Cong, Y.; Zhang, B.; Fischer, K.; Janshoff, A.; Schmidt, M. Macromol. Rapid Commun. 2008, 29, 821-825). Herein, we have systematically investigated the influence of surfactant, salt, and pH on the supramolecular structure formation. The cylindrical brush polymers and their complexes with surfactants were directly visualized by atomic force microscopy in air and in aqueous solution. The alkyl chain length (measured by the carbon number, n) of the surfactant plays a key rol…
Modellmembranen auf Oberflächen. Verankert und doch mobil
Membranmodelle, die auf Festkorperoberflachen verankert sind, ermoglichen es, elektrische Verfahren sowie oberflachenanalytische und abbildende Methoden einzusetzen. So kann mit Membranen auf Oberflachen die laterale Organisation von Lipiden und Proteinen bis in den Nanometerbereich hinein visualisiert werden. Gleichzeitig sind diese Membranen sehr stabil und damit attraktiv fur verschiedene Bereiche der Biosensorik. Durch eine individuelle Adressierung kleinster mikrometergroser Membranareale mit verschiedenen Lipidkompositionen ist es moglich geworden, die Bindung von Proteinen an funktionale Membranen auf kleinstem Raum zu quantifizieren und zu parallelisieren. Fur die Untersuchung von T…
Phasenselektion von Calciumcarbonat durch die Chiralität adsorbierter Aminosäuren
Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis
Since their inception in 1985 by Tamm and McConnell [1], solid supported lipid bilayers have been widely used as model systems for cellular membranes [2]. They have been applied in fundamental and applied studies of lipid assemblies on surfaces, to study the structure of membranes and membrane dynamics, lipidreceptor-interactions and electrochemical properties of membranes [3-5]. Several attempts have been made to apply solid supported membranes (SSM) in biosensor devices [6]. Planar lipid membranes can be formed on various surfaces, i.e. glass, silicon, mica or metal surfaces such as platinum or gold. Surface attachment of the lipids is typically achieved following two different strategies…
Phase Transition of Individually Addressable Microstructured Membranes Visualized by Imaging Ellipsometry
The phase transition of individually addressable microstructured lipid bilayers was investigated by means of imaging ellipsometry. Microstructured bilayers were created on silicon substrates by micromolding in capillaries, and the thermotropic behavior of various saturated diacyl phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecoyl-sn-glycero-3-phosphocholine, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) bilayers as well as DMPC/cholesterol membranes was determined by measuring the area expansion and thickness of the bilayer as a function of temperature. We found an increase in the main phase transition temperature T(M) of 2-6 degrees C and a substa…
Reactive Polymers: A Versatile Toolbox for the Immobilization of Functional Molecules on TiO2 Nanoparticles
Membrane Stiffness of Animal Cells Challenged by Osmotic Stress
Facile synthesis and characterization of functionalized, monocrystalline rutile TiO2 nanorods.
Functionalized, monocrystalline rutile TiO2 nanorods were prepared from TiCl4 in aqueous solution under acidic conditions in the presence of dopamine, followed by aging and hydrothermal treatment at 150 degrees C. The surface-bound organic ligand controls the morphology as well as the crystallinity and the phase selection of TiO2. The presence of monocrystalline rutile TiO2 was confirmed by X-ray powder diffraction and HRTEM investigations. The as-prepared nanorods are soluble in water at pH3. The surface functionalization was analyzed by IR and 1H NMR, confirming the presence of dopamine on the surface. The surface amine groups can be tailored further with functional molecules such as dyes…
Supramolecular structures formed by calix[8]arene derivatives.
Octamethoxy calix[8]arenes substituted in the para position by amide, urea, and imide functions were synthesized from the octamethyl ether of tert-butylcalix[8]arene by ipso nitration, reduction, and acylation. Scanning force microscopy of spin coated samples on graphite suggests that these derivatives self-organize into tubular nanorods via hydrogen bonds between p-amide functions. A single-crystal X-ray structure reveals a centrosymmetric conformation for the octanitro derivative. [structure: see text]