Surface attachment of protein fibrils via covalent modification strategies.

Chemical control of surface functionality and topography is an essential requirement for many technological purposes. In particular, the covalent attachment of monomeric proteins to surfaces has been the object of intense studies in recent years, for applications as varied as electrochemistry, immuno-sensing, and the production of biocompatible coatings. Little is known, however, about the characteristics and requirements underlying surface attachment of supramolecular protein nanostructures. Amyloid fibrils formed by the self-assembly of peptide and protein molecules represent one important class of such structures. These highly organized beta-sheet-rich assemblies are a hallmark of a range of neurodegenerative disorders, including Alzheimer's disease and type II diabetes, but recent findings suggest that they have much broader significance, potentially representing the global free energy minima of the energy landscapes of proteins and having potential applications in material science. In this paper, we describe strategies for attaching amyloid fibrils formed from different proteins to gold surfaces under different solution conditions. Our methods involve the reaction of sulfur containing small molecules (cystamine and 2-iminothiolane) with the amyloid fibrils, enabling their covalent linkage to gold surfaces. We demonstrate that irreversible attachment using these approaches makes possible quantitative analysis of experiments using biosensor techniques, such as quartz crystal microbalance (QCM) assays that are revolutionizing our understanding of the mechanisms of amyloid growth and the factors that determine its kinetic behavior. Moreover, our results shed light on the nature and relative importance of covalent versus noncovalent forces acting on protein superstructures at metal surfaces.

Quantitative approaches for characterising fibrillar protein nanostructures

Polypeptide sequences have an inherent tendency to self-assemble into filamentous nanostructures commonly known as amyloid fibrils. Such self-assembly is used in nature to generate a variety of functional materials ranging from protective coatings in bacteria to catalytic scaffolds in mammals. The aberrant self-assembly of misfolded peptides and proteins is also, however, implicated in a range of disease states including neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. It is increasingly evident that the intrinsic material properties of these structures are crucial for understanding the thermodynamics and kinetics of the pathological deposition of proteins, particularly as the mechanical fragmentation of aggregates enhances the rate of protein deposition by exposing new fibril ends which can promote further growth. We discuss here recent advances in physical techniques that are able to characterise the hierarchical self-assembly of misfolded protein molecnles and define their properties. © 2010 Materials Research Society.

Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions.

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

Probing protein aggregation with quartz crystal microbalances.

The supra-molecular self-assembly of peptides and proteins is a process which underlies a range of normal and aberrant biological pathways in nature, but one which remains challenging to monitor in a quantitative way. We discuss the experimental details of an approach to this problem which involves the direct measurement in vitro of mass changes of the aggregates as new molecules attach to them. The required mass sensitivity can be achieved by the use of a quartz crystal transducer-based microbalance. The technique should be broadly applicable to the study of protein aggregation, as well as to the identification and characterisation of inhibitors and modulators of this process.

Nucleated polymerization with secondary pathways. I. Time evolution of the principal moments.

Self-assembly processes resulting in linear structures are often observed in molecular biology, and include the formation of functional filaments such as actin and tubulin, as well as generally dysfunctional ones such as amyloid aggregates. Although the basic kinetic equations describing these phenomena are well-established, it has proved to be challenging, due to their non-linear nature, to derive solutions to these equations except for special cases. The availability of general analytical solutions provides a route for determining the rates of molecular level processes from the analysis of macroscopic experimental measurements of the growth kinetics, in addition to the phenomenological parameters, such as lag times and maximal growth rates that are already obtainable from standard fitting procedures. We describe here an analytical approach based on fixed-point analysis, which provides self-consistent solutions for the growth of filamentous structures that can, in addition to elongation, undergo internal fracturing and monomer-dependent nucleation as mechanisms for generating new free ends acting as growth sites. Our results generalise the analytical expression for sigmoidal growth kinetics from the Oosawa theory for nucleated polymerisation to the case of fragmenting filaments. We determine the corresponding growth laws in closed form and derive from first principles a number of relationships which have been empirically established for the kinetics of the self-assembly of amyloid fibrils.

Electrostatic effects in filamentous protein aggregation.

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules.

Hierarchical orientation of crystallinity by block-copolymer patterning and alignment in an electric field

Electron and hole conducting 10-nm-wide polymer morphologies hold great promise for organic electro-optical devices such as solar cells and light emitting diodes. The self-assembly of block-copolymers (BCPs) is often viewed as an efficient way to generate such materials. Here, a functional block copolymer that contains perylene bismide (PBI) side chains which can crystallize via π-π stacking to form an electron conducting microphase is patterned harnessing hierarchical electrohydrodynamic lithography (HEHL). HEHL film destabilization creates a hierarchical structure with three distinct length scales: (1) micrometer-sized polymer pillars, containing (2) a 10-nm BCP microphase morphology that is aligned perpendicular to the substrate surface and (3) on a molecular length scale (0.35-3 nm) PBI π-π-stacks traverse the HEHL-generated plugs in a continuous fashion. The good control over BCP and PBI alignment inside the generated vertical microstructures gives rise to liquid-crystal-like optical dichroism of the HEHL patterned films, and improves the electron conductivity across the film by 3 orders of magnitude. © 2013 American Chemical Society.

Mechanics of capillary forming of aligned carbon nanotube assemblies.

Elastocapillary self-assembly is emerging as a versatile technique to manufacture three-dimensional (3D) microstructures and complex surface textures from arrangements of micro- and nanoscale filaments. Understanding the mechanics of capillary self-assembly is essential to engineering of properties such as shape-directed actuation, anisotropic wetting and adhesion, and mechanical energy transfer and dissipation. We study elastocapillary self-assembly (herein called "capillary forming") of carbon nanotube (CNT) microstructures, combining in situ optical imaging, micromechanical testing, and finite element modeling. By imaging, we identify sequential stages of liquid infiltration, evaporation, and solid shrinkage, whose kinetics relate to the size and shape of the CNT microstructure. We couple these observations with measurements of the orthotropic elastic moduli of CNT forests to understand how the dynamic of shrinkage of the vapor-liquid interface is coupled to the compression of the forest. We compare the kinetics of shrinkage to the rate of evporation from liquid droplets having the same size and geometry. Moreover, we show that the amount of shrinkage during evaporation is governed by the ability of the CNTs to slip against one another, which can be manipulated by the deposition of thin conformal coatings on the CNTs by atomic layer deposition (ALD). This insight is confirmed by finite element modeling of pairs of CNTs as corrugated beams in contact and highlights the coupled role of elasticity and friction in shrinkage and stability of nanoporous solids. Overall, this study shows that nanoscale porosity can be tailored via the filament density and adhesion at contact points, which is important to the development of lightweight multifunctional materials.

Capillary bending of Janus carbon nanotube micropillars.

We present a scalable process for the fabrication of slanted carbon nanotube micropillar arrays by inclined metal deposition and capillary self-assembly. Local control of the micropillar angle from vertical to nearly horizontal is achieved, and is explained using a finite element model. These structures may be useful for microscale contacts and anisotropic smart surfaces.

Engineering of micro- and nanostructured surfaces with anisotropic geometries and properties

Widespread approaches to fabricate surfaces with robust micro- and nanostructured topographies have been stimulated by opportunities to enhance interface performance by combining physical and chemical effects. In particular, arrays of asymmetric surface features, such as arrays of grooves, inclined pillars, and helical protrusions, have been shown to impart unique anisotropy in properties including wetting, adhesion, thermal and/or electrical conductivity, optical activity, and capability to direct cell growth. These properties are of wide interest for applications including energy conversion, microelectronics, chemical and biological sensing, and bioengineering. However, fabrication of asymmetric surface features often pushes the limits of traditional etching and deposition techniques, making it challenging to produce the desired surfaces in a scalable and cost-effective manner. We review and classify approaches to fabricate arrays of asymmetric 2D and 3D surface features, in polymers, metals, and ceramics. Analytical and empirical relationships among geometries, materials, and surface properties are discussed, especially in the context of the applications mentioned above. Further, opportunities for new fabrication methods that combine lithography with principles of self-assembly are identified, aiming to establish design principles for fabrication of arbitrary 3D surface textures over large areas. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Corrugated carbon nanotube microstructures with geometrically tunable compliance

Deterministic organization of nanostructures into microscale geometries is essential for the development of materials with novel mechanical, optical, and surface properties. We demonstrate scalable fabrication of 3D corrugated carbon nanotube (CNT) microstructures, via an iterative sequence of vertically aligned CNT growth and capillary self-assembly. Vertical microbellows and tilted microcantilevers are created over large areas, and these structures can have thin walls with aspect ratios exceeding 100:1. We show these structures can be used as out-of-plane microsprings with compliance determined by the wall thickness and number of folds. © 2011 American Chemical Society.

Non-destructive characterization of structural hierarchy within aligned carbon nanotube assemblies

Understanding and controlling the hierarchical self-assembly of carbon nanotubes (CNTs) is vital for designing materials such as transparent conductors, chemical sensors, high-performance composites, and microelectronic interconnects. In particular, many applications require high-density CNT assemblies that cannot currently be made directly by low-density CNT growth, and therefore require post-processing by methods such as elastocapillary densification. We characterize the hierarchical structure of pristine and densified vertically aligned multi-wall CNT forests, by combining small-angle and ultra-small-angle x-ray scattering (USAXS) techniques. This enables the nondestructive measurement of both the individual CNT diameter and CNT bundle diameter within CNT forests, which are otherwise quantified only by delicate and often destructive microscopy techniques. Our measurements show that multi-wall CNT forests grown by chemical vapor deposition consist of isolated and bundled CNTs, with an average bundle diameter of 16 nm. After capillary densification of the CNT forest, USAXS reveals bundles with a diameter 4 m, in addition to the small bundles observed in the as-grown forests. Combining these characterization methods with new CNT processing methods could enable the engineering of macro-scale CNT assemblies that exhibit significantly improved bulk properties. © 2011 American Institute of Physics.

Local Frustration Determines Molecular and Macroscopic Helix Structures

The effect of displaying cytochromes from an amyloid fibre is modelled as perturbation of -strands in a bilayer of helical -sheets, thereby explaining the spiral morphology of decorated amyloid and the dynamic response of morphology to cytochrome conformation. The morphology of the modelled fibre, which consists of minimal energy assemblies of rigid building blocks containing two anisotropic interacting units, depends primarily on the rigid constraints between units rather than the soft interactions between them. The framework is a discrete version of the bilayered frustration principle that drives morphology in Bauhinia seedpods. We show that self-assembly of frustrated long range structures can occur if the building blocks themselves are internally frustrated, e.g. amyloid morphology is governed by the conformation of the misfolded protein nucleating the fibre. Our model supports the idea that any peptide sequence can form amyloid if bilayers can form first, albeit stabilised by additional material such as chaperones or cytochromes. Analysis of experimentally derived amyloid structures supports our conclusions and suggests a range of frustration effects, which natural amyloid fibres may exploit. From this viewpoint, amyloid appears as a molecular example of a more general universal bilayered frustration principle, which may have profound implications for materials design using fibrous systems. Our model provides quantitative guidance for such applications. The relevance to longer length scales was proved by designing the morphology of a series of macroscopic magnetic stacks. Finally, this work leads to the idea of mixing controlled morphologically defined species to generate higher-order assembly and complex functional behaviour. The systematic kinking of decorated fibres and the nested frustration of the Bauhinia seed pod are two outstanding examples.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues.

Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.