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We report successful and selective dual protein assembly on patterned bifunctional βCD-Ni(ii)NTA surfaces, using red fluorescent protein variants with hexahistidine-tags and teal fluorescent protein variants conjugated with a peptide containing three adamantyl groups. We show that dual protein patterns can only be assembled, when opposing supramolecular interactions have been optimized and nonspecific interactions have been sufficiently suppressed.

Extracellular microenvironment is highly dynamic where spatiotemporal regulation of cell-instructive cues such as matrix topography tightly regulates cellular behavior. Recapitulating dynamic changes in stimuli-responsive materials has become an important strategy in regenerative medicine to generate biomaterials which closely mimic the natural microenvironment. Here, light responsive liquid crystal polymer networks are used for their adaptive and programmable nature to form hybrid surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness at the same time to direct cell migration. This study shows that the cell speed and migration patterns are strongly dependent on the height of the (light-responsive) micrometer scale topographies and differences in surface nanoroughness. Furthermore, switching cell migration patterns upon in situ temporal changes in surface nanoroughness, points out the ability to dynamically control cell behavior on these surfaces. Finally, the possibility is shown to form photoswitchable topographies, appealing for future studies where topographies can be rendered reversible on demand.

The modulation of the hierarchical nucleated self-assembly of tri-β3-peptides has been studied. β3-Tyrosine provided a handle to control the assembly process through host-guest interactions with CB[7] and CB[8]. By varying the cavity size from CB[7] to CB[8] distinct phases of assembling tri-β3-peptides were arrested. Given the limited size of the CB[7] cavity, only one aromatic β3-tyrosine can be simultaneously hosted and hence CB[7] was primarily acting as an inhibitor of self-assembly. In strong contrast, the larger CB[8] can form a ternary complex with two aromatic amino acids and hence CB[8] was acting primarily as cross-linker of multiple fibers and hence promoting the formation of larger aggregates. General insights on modulating supramolecular assembly can lead to new ways to introduce functionality in supramolecular polymers.

Supramolecular chemistry provides an attractive entry to generate dynamic and well-controlled bioactive surfaces. Novel host–guest systems are urgently needed to provide a broader affinity and applicability portfolio. A synthetic strategy to carborane–peptide bioconjugates was therefore developed to provide an entry to monovalent supramolecular functionalization of ß-cyclodextrin coated surfaces. The ß-cyclodextrin·carborane–cRGD surfaces are formed efficiently and with high affinity as demonstrated by IR-RAS, WCA, and QCM-D, compare favourable to existing bio-active host–guest surface assemblies, and display an efficient bioactivity, as illustrated by a strong functional effect of the supramolecular system on the cell adhesion and spreading properties. Cells seeded on the supramolecular surface displaying bioactive peptide epitopes exhibited a more elongated morphology, focal adhesions, and stronger cell adhesion compared to control surfaces. This highlights the macroscopic functionality of the novel supramolecular immobilization strategy.

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host–guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N-terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N-terminus is more efficient than through protein side-chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site-specific protein immobilization under mild conditions in a reversible manner.

Item does not contain fulltext, We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Supramolecular control of adhesion of cells is demonstrated using synthetic integrin binding RGD peptide-ferrocene conjugates that were immobilized via host-guest chemistry onto cucurbit[7]uril coated gold surfaces.

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit[7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Item does not contain fulltext, PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

1 december 2011, Item does not contain fulltext, Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

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We present time-resolved photoluminescence measurements on two series of oligo-p-phenylenevinylene materials that self-assemble into supramolecular nanostructures with thermotropic reversibility in dodecane. One set of derivatives form chiral helical stacks, while the second set form less organized "frustrated" stacks. Here we study the effects of supramolecular organization on the resonance energy transfer rates. We measure these rates in nanoassemblies formed with mixed blends of oligomers and compare them with the rates predicted by Förster theory. Our results and analysis show that control of supramolecular order in the nanometer length scale has a dominant effect on the efficiency and dimensionality of resonance energy transfer.

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High magnetic fields were used to deform spherical nanocapsules, self-assembled from bolaamphiphilic sexithiophene mols. At low fields the deformation-measured through linear birefringence-scales quadratically with the capsule radius and with the magnetic field strength. These data confirm a long standing theor. prediction [W. Helfrich, Phys. Lett. A 43, 409 (1973)], and permit the detn. of the bending rigidity of the capsules as (2.6+-0.8)*10-21 J. At high fields, an enhanced rigidity is found which cannot be explained within the Helfrich model. We propose a complete form of the free energy functional that accounts for this behavior, and allows discussion of the formation and stability of nanocapsules in soln.

The electrical properties of supermolecular assemblies of oligo(p-phenylene vinylene) were studied. These materials self-assemble into well-defined cylindrical structures in solution with lengths in the range of 100 nm–10 µm and diameters between 5 and 200 nm. Atomic force microscopy showed that by adjusting the concentration, either individual molecular wires or a dense film could be deposited. The molecular wires showed poor electrical conduction. Several tests were performed that show that it was the molecular wires themselves, not the contacts, that limit the conductivity.

The electrical properties of supermolecular assemblies of oligo(p-phenylene vinylene) were studied. These materials self-assemble into well-defined cylindrical structures in solution with lengths in the range of 100?nm–10??m and diameters between 5 and 200?nm. Atomic force microscopy showed that by adjusting the concentration, either individual molecular wires or a dense film could be deposited. The molecular wires showed poor electrical conduction. Several tests were performed that show that it was the molecular wires themselves, not the contacts, that limit the conductivity., Kavli Institute of Nanoscience, Applied Sciences

The film morphology of pi-conjugated oligomers has been controlled by self-assembly in solution. To this end supramolecular hydrogen-bonded systems of oligo(p-phenylenevinylene) (OPV) carrying ureido-s-triazine hydrogen-bonding groups are used. Neutron scattering experiments in dodecane solutions show that columnar stacks are formed. Films with thicknesses on the order of 100 nm are made that have a supramolecular organization resembling the organization present in solution. Uniform rodlike morphological domains range over several hundreds of nanometers as shown by atomic force microscopy. The rodlike morphology of the OPVs was also preserved when blended with a C-60 derivative, producing stable photovoltaic devices.

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We present time-resolved photoluminescence measurements on two series of oligo-p-phenylenevinylene (OPV) materials that are functionalized with quadruple hydrogen-bonding groups. These form supramolecular assemblies with thermotropic reversibility. The morphology of the assemblies depends on the way that the oligomers are functionalized; monofunctionalized OPVs (MOPVs) form chiral, helical stacks while bifunctionalized OPVs (BOPVs) form less organized structures. These are therefore model systems to investigate the effects of supramolecular assembly, the effects of morphology, and the dependence of oligomer length on the radiative and nonradiative rates of -conjugated materials. The purpose of this work is to use MOPV and BOPV derivatives as model systems to study the effect of intermolecular interactions on the molecular photophysics by comparing optical properties in the dissolved phase and the supramolecular assemblies. A simple photophysical analysis allows us to extract the intrinsic radiative and nonradiative decay rates and to unravel the consequences of interchromophore coupling with unprecedented detail. We find that interchromophore coupling strongly reduces both radiative and intrinsic nonradiative rates and that the effect is more pronounced in short oligomers.

Exciton dynamics in chiral helixes of an oligo-p-phenylenevinylene deriv. monofunctionalized with a ureido-s-triazine (MOPV) were studied. The latter functional group leads to oligomer dimerization by hydrogen bonding in dodecane soln., resulting in reversible supramol. assembly by solvophobic and p-p interactions. MOPV is a model system to study exciton transfer dynamics as it is possible to study the effect of intermol. interactions by comparing optical properties in the dissolved phase (above the transition temp.) and the supramol. assemblies. The model allows to ext. microscopic informations such as the av. exciton hopping annihilation rates. It is believed that this model also allows the quant. description of the exciton dynamics on supramol. architectures from subpicosecond to nanosecond timescales.

Femtosecond transient absorption measurements were carried out on p-conjugated supramol. assemblies in a high-pump-fluence regime. Oligo(p-phenylenevinylene) monofunctionalized with ureido-s-triazine (MOPV) self-assembles into chiral stacks in dodecane soln. below 75 Deg at a concn. of 4 * 10-4 M. Exciton bimol. annihilation was obsd. in MOPV stacks at high excitation fluence, indicated by the fluence-dependent decay of 11Bu-exciton spectral signatures and by the sublinear fluence dependence of time- and wavelength-integrated photoluminescence (PL) intensity. These two characteristics are much less pronounced in MOPV soln. where the phase equil. is shifted significantly away from supramol. assembly, slightly below the transition temp. A mesoscopic rate-equation model is applied to ext. the bimol. annihilation rate const. from the excitation fluence dependence of transient absorption and PL signals. The results demonstrate that the bimol. annihilation rate is high with a square-root dependence in time. The exciton annihilation results from a combination of fast exciton diffusion and resonance energy transfer. The supramol. nanostructures have electronic properties that are intermediate between mol. aggregates and polymeric semiconductors

We demonstrate a novel method to visualize individual molecular chains in the surface layer of organic semiconducting polymer films of arbitrary thickness. The method is based on scanning force microscopy and employs a combination of a sensitive phase detection system and metal coated tips with a low (similar to1 N/m) spring constant and a relatively high Q factor (similar to200). The molecularly resolved morphology is observed in phase images that are taken simultaneously with the topography. Surprisingly, we found that, when the tip apex radius exceeds the intermolecular spacing in the surface layer, surface aggregates -when present- are visualized. In both cases, the phase contrast is shown to result from van der Waals interaction between the conjugated backbone of the polymer chains and the metallic tip, and can quantitatively be described by a simple harmonic oscillator model. We have employed this method to study the morphology of poly(p-phenylene vinylene) (PPV) derivatives with different substitutions.

The morphology of films of PPV derivatives is studied with molecular (single chain) resolution by phase-imaging scanning force microscopy. It is found that the symmetry of substitution is directly related to surface morphology and aggregation behavior. The molecular resolution in the phase contrast is shown to result from van der Waals interaction between the conjugated backbone of the polymer chains and the metallic tip, and can quantitatively be described by a simple harmonic oscillator model.