Till Böcking

Demonstration of the importance of oxygenated species at the ends of carbon nanotubes for their favourable electrochemical properties

Definitive evidence is presented for the favourable electrochemical properties of carbon nanotube modified electrodes arising from the ends of SWNTs due to oxygenated carbon species in general, and carboxylic acid moieties in particular, produced during acid purification.

Peptide-modified optical filters for detecting protease activity.

The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological samples. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70‐facilitated disassembly

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J‐domain containing co‐chaperone, auxilin, associates with a freshly budded clathrin‐coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy‐chain‐binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6‐barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C‐terminus of the heavy chain, with a stoichiometry of about one per three‐fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J‐domain, splits ATP, it clamps firmly onto its heavy‐chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

Smart Tissue Culture: in Situ Monitoring of the Activity of Protease Enzymes Secreted from Live Cells Using Nanostructured Photonic Crystals

Monitoring enzyme secretion in tissue culture has proved challenging because to date the activity cannot be continuously measured in situ. In this Letter, we present a solution using biopolymer loaded photonic crystals of anodized silicon. Shifts in the optical response by proteolytic degradation of the biopolymer provide label-free sensing with unprecedented low detection limits (1 pg) and calculation of kinetic parameters. The enhancement in sensitivity relative to previous photonic crystal sensors constitutes a change in the sensing paradigm because here the entire pore space is responsive to the secreted enzyme rather than just the pore walls. In situ monitoring is demonstrated by detecting secretion of matrix metalloprotease 9 from stimulated human macrophages.

The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.

Introducing Distinctly Different Chemical Functionalities onto the Internal and External Surfaces of Mesoporous Materials

There is currently considerable interest in mesoporous materials for a diverse range of applications, such as catalysis, filtration and separation, molecular collection and storage, nanofluidics, medical imaging, drug delivery, and sensors. To instill functionality into the raw materials, there is often a need for modifying the outside differently than the inside (e.g. targeted drug delivery: external antibodies for cell targeting, internal therapeutics for delivery). Recently, Cheng and Landry demonstrated spatial chemical selectivity on mesoporous silica by exploiting the slow diffusion in the nanoscale pores to ensure that the exterior is modified preferentially to the interior. This technique is a significant advance but does not ensure complete separation of the external modification layer from the internal modification layer. Thus, a need still exists for a general, material-independent methodology that will allow the interior of mesoporous materials to be modified with a specific chemistry while allowing a completely different, well-controlled chemical landscape on the external surface. Herein we present a simple strategy towards achieving a sharp contrast in surface chemistry on the inside and outside of a mesoporous material. This approach relies on the combination of surface tension and capillarity to either prevent or promote the ingress of solution species inside the structure. As this method is dependent on surface tension, it relies on the hydrophobicity/hydrophilicity of the surface inside the pores relative to the solvent and on the pore size of the mesoporous structure and hence should be general for the modification of any mesoporous material. As a demonstration, we have used porous silicon (PSi) photonic crystals for two key reasons. Firstly, pore size is easily tuneable, and secondly, PSi can be engineered to exhibit a photonic band gap with high-quality optical properties. This latter feature is important, as it enables the chemical modification proceeding inside the structures to be monitored by shifts in reflectance spectra. To selectively derivatize the mesoporous PSi, the physical characteristics of a hydrophobic functional monolayer at the entrance of mesopores are exploited on a previously reported PSi photonic crystal (a rugate filter). After forming a dense alkyl monolayer throughout the structure, the hydrophobicity of the mesopores was found to completely prevent the ingress of water. Capitalizing on this observation, the tops of the optical filters can be derivatized with aqueous solutions while leaving the bulk of the crystal unmodified. To functionalize the interior, the PSi is prewet with ethanol to allow infiltration of aqueous solutions; alternatively, organic solvents can be used. To demonstrate the utility of this chemical strategy we modified the exterior of a rugate filter with the peptide Gly-Arg-Gly-Asp-Ser (GRGDS), containing the cell-adhesive tripeptide Arg-GlyAsp (RGD) for selective capture of cells, while modifying the interior with different chemical moieties (Figure 1). Cell adhesion was chosen as the test system, because some of the most promising applications for PSi photonic crystals are biophotonic sensing, “smart” implants, and targeted drug delivery, all of which require integration with live cells. Rugate filters, prepared by anodization of Si(100) using a sinusoidal current profile, with pore sizes of approximately 12 nm, display a narrow linewidth and high reflectivity resonance in the reflectance spectrum (see Figure S2 in the Supporting Information). First, to stabilize the freshly etched silicon and introduce reactive functional groups, a monolayer was formed by thermal hydrosilylation of 10-succinimidylundecenoate, resulting in a 93-nm shift in the filter resonance as air in the porous scaffold was replaced with the Si C linked monolayer (see Figure S2 in the Supporting Information). The PSi samples were immersed in water to evaluate the wetting behavior of the pores. The succinimide ester terminated monolayer produced a hydrophobic interface that completely prevents the infiltration of water, provided the surface was free of oxidized silicon (see Figure S1 in the Supporting Information). Next, the top surface was derivatized by incubation in an aqueous solution of the peptide GRGDS (Figure 1a, step 2a) to provide a surface that promotes cell adhesion. The reflectivity spectra after this step displayed no change in the position of the optical resonance relative to before the modification, which indicates that there was no significant derivatization inside the pores (see Figure S2 in the Supporting Information). Likewise, transmission FTIR spectroscopy, a technique only sensitive to internal chemical modifications, indicated no change (see Figure S3 in the Supporting Information), again consistent with only the external surface being modified and the interior remaining unaltered. Finally, as proof of principle for selective interior modification, the PSi structure containing RGD on the top was [*] Dr. K. A. Kilian, Dr. T. B cking, Prof. J. J. Gooding School of Chemistry, University of New South Wales Sydney 2052 (Australia) Fax: (+61)2-9385 E-mail: justin.gooding@unsw.edu.au

Spacing of Integrin Ligands Influences Signal Transduction in Endothelial Cells

The physical attributes of the extracellular matrix play a key role in endothelium function by modulating the morphology and phenotype of endothelial cells. Despite the recognized importance of matrix-cell interactions, it is currently not known how the arrangement of adhesive ligands affects the morphology, signal transduction processes, and migration of endothelial cells. We aimed to study how endothelial cells respond to the average spatial arrangement of integrin ligands. We designed functionalized silicon surfaces with average spacing ranging from nanometers to micrometers of the peptide arginine-glycine-aspartic acid (RGD). We found that endothelial cells adhered to and spread on surfaces independently of RGD-to-RGD spacing. In contrast, organization within focal adhesions (FAs) was extremely sensitive to ligand spacing, requiring a nanoscaled average RGD spacing of 44 nm to form lipid raft domains at FAs. The localized membrane organization strongly correlated with the signaling efficiencies of integrin activation and regulated vascular endothelial growth factor (VEGF)-induced signaling events. Importantly, this modulation in signal transduction directly affected the migratory ability of endothelial cells. We conclude that endothelial cells sense nanoscaled variations in the spacing of integrin ligands, which in turn influences signal transduction processes. Average RGD spacing similar to that found in fibronectin leads to lipid raft accumulation at FAs, enhances sensitivity to VEGF stimulation, and controls migration in endothelial cells.

Optical properties of II-VI colloidal quantum dot doped porous silicon microcavities

In this paper we report on the light emitting properties of mesoporous silicon vertical-cavity optical resonators with II-VI colloidal quantum dots selectively deposited in the cavity layer. Optical resonator structures exhibit reflectivity stop bands of several hundred nanometres and resonant modes with line-widths less than 3.5 nm. The observed modification of spectral and spatial emission properties of the quantum dots and tenfold enhancement at the resonance wavelength is consistent with cavity enhanced spontaneous emission. Using this hybrid fabrication approach we show that narrow band light emitting structures may be fabricated over a broad spectral region in the visible and near-infrared.

Hybrid lipid bilayers in nanostructured silicon: a biomimetic mesoporous scaffold for optical detection of cholera toxin

Cholera toxin levels are optically detected by affinity capture within hybrid lipid bilayer membranes formed in the nanostructures of porous silicon photonic crystals.