Imaging secondary structure of individual amyloid fibrils of a ß2-microglobulin fragment using near-field infrared spectroscopy.

Amyloid fibril diseases are characterized by the abnormal production of aggregated proteins and are associated with many types of neuro- and physically degenerative diseases. X-ray diffraction techniques, solid-state magic-angle spinning NMR spectroscopy, circular dichroism (CD) spectroscopy, and transmission electron microscopy studies have been utilized to detect and examine the chemical, electronic, material, and structural properties of amyloid fibrils at up to angstrom spatial resolution. However, X-ray diffraction studies require crystals of the fibril to be analyzed, while other techniques can only probe the bulk solution or solid samples. In the work reported here, apertureless near-field scanning infrared microscopy (ANSIM) was used to probe the secondary structure of individual amyloid fibrils made from an in vitro solution. Simultaneous topographic and infrared images of individual amyloid fibrils synthesized from the #21-31 peptide fragment of ß(2)-microglobulin were acquired. Using this technique, IR spectra of the amyloid fibrils were obtained with a spatial resolution of less than 30 nm. It is observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum. The near-field images of the amyloid fibrils exhibit much lower scattering of the IR radiation at approximately 1630 cm(-1). In addition, the near-field images also indicate that composition and/or structural variations among individual amyloid fibrils were present.

Phospholipid membrane encapsulation of nanoparticles for surface-enhanced Raman scattering.

Lipid-encapsulated surface-enhanced Raman scattering (SERS) nanoparticles, with promising applications in biomedical diagnostics, were produced. Gold nanoparticles, 60 nm in diameter, were coated with a ternary mixture of DOPC, sphingomyelin, and cholesterol. The lipid layer is versatile for engineering the chemical and optical properties of the particles. The stability of the lipid-encapsulated particles is demonstrated over a period of weeks. The versatility of the layer is demonstrated by the incorporation of three different Raman-active species using three different strategies. The lipid layer was directly observed by TEM, and the SERS spectrum of the three dye species was confirmed by Raman spectroscopy. UV-vis absorption and dynamic light scattering provide additional evidence of lipid encapsulation. The encapsulation is achieved in aqueous solution, avoiding phase transfer and possible contamination from organic solvents. Furthermore, when fluorescent dye-labeled lipids were employed in the encapsulant, the fluorescence and SERS activity of the particles were controlled by the use of dissolved ions in the preparation solution.

The control of marine biofouling on xerogel surfaces with nanometer-scale topography.

Mixtures of n-octadecyltrimethoxysilane (C18, 1-5 mole-%), n-octyltriethoxysilane (C8) and tetraethoxysilane (TEOS) gave xerogel surfaces of varying topography. The 1:49:50 C18/C8/TEOS xerogel formed 100-400-nm-wide, 2-7-nm deep pores by AFM while coatings with ≥3% C18 were free of such features. Segregation of the coating into alkane-rich and alkane-deficient regions in the 1:49:50 C18/C8/TEOS xerogel was observed by IR microscopy. Immersion in ASW for 48 h gave no statistical difference in surface energy for the 1:49:50 C18/C8/TEOS xerogel and a significant increase for the 50:50 C8/TEOS xerogel. Settlement of barnacle cyprids and removal of juvenile barnacles, settlement of zoospores of the alga Ulva linza, and strength of attachment of 7-day sporelings were compared amongst the xerogel formulations. Settlement of barnacle cyprids was significantly lower in comparison to glass and polystyrene standards. The 1:49:50 and 3:47:50 C18/C8/TEOS xerogels were comparable to PDMSE with respect to removal of juvenile barnacles and sporeling biomass, respectively.

Insights into the composition, morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus.

To date, the calcareous tubes of serpulid marine worms have not been studied extensively in a biomineralization context. The structure and composition of the tube shell and adhesive cement of the marine tubeworm Hydroides dianthus were studied using a variety of characterization techniques, including powder XRD, FTIR, SEM, EDX, and AFM. The tube and cement were determined to be inorganic-organic composite materials, consisting of inorganic aragonite (CaCO(3)) and Mg-calcite ((Ca(0.8)Mg(0.2))CO(3)) crystals, and both soluble and insoluble organic matrices (SOM and IOM). SEM imaging revealed a variety of crystal morphologies. AFM nanoindentation of the inorganic components yielded Young's moduli of approximately 20GPa in the wet state, and approximately 50GPa in the dry state. Amino acid analysis of the SOM indicated substantial amounts of acidic and non-polar neutral amino acids. Part of the insoluble organic tube lining was identified as being composed of collagen-containing fibres aligned in a criss-crossed structure. The SOM and organic tube lining were found to contain carboxylated and sulphated polysaccharides. In an artificial seawater solution, the SOM and the organic tube lining mediated CaCO(3) mineralization in vitro.

The role of surface energy and water wettability in aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel surfaces in the control of marine biofouling.

Xerogel films with uniform surface topogrophy, as determined by scanning electron microscopy, atomic force microscopy (AFM), and time-of-flight secondary ion mass spectrometry, were prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl- containing precursors. Young's modulus was determined from AFM indentation measurements. The xerogel coatings gave reduced settlement of zoospores of the marine fouling alga Ulva compared to a poly(dimethylsiloxane) elastomer (PDMSE) standard. Increased settlement correlated with decreased water wettability as measured by the static water contact angle, theta(Ws), or with decreased polar contribution (gamma(P)) to the surface free energy (gamma(S)) as measured by comprehensive contact angle analysis. The strength of attachment of 7-day sporelings (young plants) of Ulva on several of the xerogels was similar to that on PDMSE although no overall correlation was observed with either theta(Ws) or gamma(S). For sporelings attached to the fluorocarbon/hydrocarbon-modified xerogels, the strength of attachment increased with increased water wettability. The aminopropyl-modified xerogels did not follow this trend.

Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability.

Previous work has shown that organosilica-based xerogels have the potential to control biofouling. In this study, modifications of chemistry were investigated with respect to their resistance to marine slimes and to settlement of barnacle cyprids. Adhesion force measurements of bovine serum albumin (BSA)-coated atomic force microscopy (AFM) tips to xerogel surfaces prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl-containing precursors, indicated that adhesion was significantly less on the xerogel surfaces in comparison to a poly(dimethylsiloxane) elastomer (PDMSE) standard. The strength of adhesion of BSA on the xerogels was highest on surfaces with the highest and the lowest critical surface tensions, gamma(C) and surface energies, gamma(S), and duplicated the 'Baier curve'. The attachment to and removal of cells of the diatom Navicula perminuta from a similar series of xerogel surfaces were examined. Initial attachment of cells was comparable on all of the xerogel surfaces, but the percentage removal of attached cells by hydrodynamic shear stress increased with gamma(C) and increased wettability as measured by the static water contact angle, theta(Ws), of the xerogel surfaces. The percentage removal of cells of Navicula was linearly correlated with both properties (R(2) = 0.74 for percentage removal as a function of theta(Ws) and R(2) = 0.69 for percentage removal as a function of gamma(C)). Several of the aminopropylsilyl-containing xerogels showed significantly greater removal of Navicula compared to a PDMSE standard. Cypris larvae of the barnacle B. amphitrite showed preferred settlement on hydrophilic/higher energy surfaces. Settlement was linearly correlated with theta(Ws) (R(2) = 0.84) and gamma(C) (R(2) = 0.84). Hydrophilic xerogels should prove useful as coatings for boats in regions where fouling is dominated by microfouling (protein and diatom slimes).

Water-stable diblock polystyrene-block-poly(2-vinyl pyridine) and diblock polystyrene-block-poly(methyl methacrylate) cylindrical patterned surfaces inhibit settlement of zoospores of the green alga Ulva.

Nanopatterned surfaces with hydrophobic and hydrophilic domains were produced using the diblock copolymer polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA). The PS-b-P2VP diblock copolymer, mixed with the cross-linker benzophenone and spin-coated onto silicon wafers, showed self-assembled cylindrical structures, which were retained after UV treatment for cross-linking. The thin films displayed cylindrical domains after immersion in water. This study shows that pattern retention in water is possible for a long period of time, at least for two weeks in pure water and three weeks in artificial seawater. The PS-b-PMMA diblock showed self-assembled cylindrical structures. PS-b-P2VP and PS-b-PMMA cylindrical patterned surfaces showed reduced settlement of zoospores of the green alga Ulva compared to unpatterned surfaces. The copolymers were investigated using atomic force microscopy and X-ray photoelectron spectroscopy.

Nanoscale structures and mechanics of barnacle cement.

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of beta-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1-100 nm length scale.

Aqueous-Based Fabrication of Low-VOC Nanostructured Block Copolymer Films as Potential Marine Antifouling Coatings.

The ability to fabricate nanostructured films by exploiting the phenomenon of microphase separation has made block copolymers an invaluable tool for a wide array of coating applications. Standard approaches to engineering nanodomains commonly involve the application of organic solvents, either through dissolution or annealing protocols, resulting in the release of volatile organic compounds (VOCs). In this paper, an aqueous-based method of fabricating low-VOC nanostructured block copolymer films is presented. The reported procedure allows for the phase transfer of water insoluble triblock copolymer, poly(styrene-block-2 vinylpyridine-block-ethylene oxide) (PS-b-P2VP-b-PEO), from a water immiscible phase to an aqueous environment with the assistance of a diblock copolymeric phase transfer agent, poly(styrene-block-ethylene oxide) (PS-b-PEO). Phase transfer into the aqueous phase results in self-assembly of PS-b-P2VP-b-PEO into core-shell-corona micelles, which are characterized by dynamic light scattering techniques. The films that result from coating the micellar solution onto Si/SiO2 surfaces exhibit nanoscale features that disrupt the ability of a model foulant, a zoospore of Ulva linza, to settle. The multilayered architecture consists of a pH-responsive P2VP-"shell" which can be stimulated to control the size of these features. The ability of these nanostructured thin films to resist protein adsorption and serve as potential marine antifouling coatings is supported through atomic force microscopy (AFM) and analysis of the settlement of Ulva linza zoospore. Field trials of the surfaces in a natural environment show the inhibition of macrofoulants for 1 month.

Self-seeding in one dimension: a route to uniform fiber-like nanostructures from block copolymers with a crystallizable core-forming block.

One-dimensional micelles formed by the self-assembly of crystalline-coil poly(ferrocenyldimethylsilane) (PFS) block copolymers exhibit self-seeding behavior when solutions of short micelle fragments are heated above a certain temperature and then cooled back to room temperature. In this process, a fraction of the fragments (the least crystalline fragments) dissolves at elevated temperature, but the dissolved polymer crystallizes onto the ends of the remaining seed fragments upon cooling. This process yields longer nanostructures (up to 1 µm) with uniform width (ca. 15 nm) and a narrow length distribution. In this paper, we describe a systematic investigation of factors that affect the self-seeding behavior of PFS block copolymer micelle fragments. For PI(1000)-PFS(50) (the subscripts refer to the number average degree of polymerization) in decane, these factors include the presence of a good solvent (THF) for PFS and the effect of annealing the fragments prior to the self-seeding experiments. THF promoted the dissolution of the micelle fragments, while preannealing improved their stability. We also extended our experiments to other PFS block copolymers with different corona-forming blocks. These included PI(637)-PFS(53) in decane, PFS(60)-PDMS(660) in decane (PDMS = polydimethylsiloxane), and PFS(30)-P2VP(300) in 2-propanol (P2VP = poly(2-vinylpyridine)). The most remarkable result of these experiments is our finding that the corona-forming chain plays an important role in affecting how the PFS chains crystallize in the core of the micelles and, subsequently, the range of temperatures over which the micelle fragments dissolve. Our results also show that self-seeding is a versatile approach to generate uniform PFS fiber-like nanostructures, and in principle, the method should be extendable to a wide variety of crystalline-coil block copolymers.

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 role. While helical structures were formed with n=10, 11, and 12, no helices were observed with n<10 and n>13. Addition of salt destroys the helical structures as do pH conditions below 4 and above 6, most probably because the polymer-surfactant complexes start to disintegrate. Circular dichroism was utilized to monitor the PLL side chain conformation and clearly revealed that beta-sheet formation of the side chains induces the helical conformation of the atactic main chain.

Nanomechanical fingerprints of individual blocks of a diblock copolymer chain.

Atomic force microscopy (AFM)-based single polymer chain pulling experiments have been used to study the structural transitions of individual homopolymer chains in water. Polystyrene (PS) showed a three-regime force-extension profile exhibiting a force plateau reminiscent of a first-order transition, as predicted theoretically, whereas poly(methyl methacrylate) (PMMA) showed a characteristic saw-tooth pattern reminiscent of multidomain disassembly behavior. The two distinct structural transtions provide fingerprints for the individual homopolymers, which can be used to identify individual blocks of symmetric and asymmetric PS- b-PMMA diblock copolymer chains.

Chemical imaging of the surface of self-assembled polystyrene-b-poly(methyl methacrylate) diblock copolymer films using apertureless near-field IR microscopy.

The nanoscale chemical composition variations of the surfaces of thin films of polystyrene- b-poly(methyl methacrylate) (PS- b-PMMA) diblock copolymers are investigated using apertureless near-field IR microscopy. The scattering of the incident infrared beam from a modulated atomic force microscopy (AFM) tip is probed using homodyne detection and demodulation at the tip oscillation frequency. An increase in the IR attenuation is observed in the PMMA-rich domains with a wavenumber dependence that is consistent with the bulk absorption spectrum. The results indicate that even though a small topography-induced artifact can be observed in the near-field images, the chemical signature of the sample is detected clearly.