Manipulation and Deposition of Complex, Functional Block Copolymer Nanostructures Using Optical Tweezers.

Block copolymer self-assembly has enabled the creation of a range of solution-phase nanostructures with applications from optoelectronics and biomedicine to catalysis. However, to incorporate such materials into devices a method that facilitates their precise manipulation and deposition is desirable. Herein we describe how optical tweezers can be used to trap, manipulate, and pattern individual cylindrical micelles and larger hybrid micellar materials. Through the combination of TIRF imaging and optical trapping we can precisely control the three-dimensional motion of individual cylindrical block copolymer micelles in solution, enabling the creation of customizable arrays. We also demonstrate that dynamic holographic assembly enables the creation of ordered customizable arrays of complex hybrid block copolymer structures. By creating a program which automatically identifies, traps, and then deposits multiple assemblies simultaneously we have been able to dramatically speed up this normally slow process, enabling the fabrication of arrays of hybrid structures containing hundreds of assemblies in minutes rather than hours.

Structural features distinguishing infectious ex vivo mammalian prions from non-infectious fibrillar assemblies generated in vitro.

Seeded polymerisation of proteins forming amyloid fibres and their spread in tissues has been implicated in the pathogenesis of multiple neurodegenerative diseases: so called "prion-like" mechanisms. While ex vivo mammalian prions, composed of multichain assemblies of misfolded host-encoded prion protein (PrP), act as lethal infectious agents, PrP amyloid fibrils produced in vitro generally do not. The high-resolution structure of authentic infectious prions and the structural basis of prion strain diversity remain unknown. Here we use cryo-electron microscopy and atomic force microscopy to examine the structure of highly infectious PrP rods isolated from mouse brain in comparison to non-infectious recombinant PrP fibrils generated in vitro. Non-infectious recombinant PrP fibrils are 10 nm wide single fibres, with a double helical repeating substructure displaying small variations in adhesive force interactions across their width. In contrast, infectious PrP rods are 20 nm wide and contain two fibres, each with a double helical repeating substructure, separated by a central gap of 8-10 nm in width. This gap contains an irregularly structured material whose adhesive force properties are strikingly different to that of the fibres, suggestive of a distinct composition. The structure of the infectious PrP rods, which cause lethal neurodegeneration, readily differentiates them from all other protein assemblies so far characterised in other neurodegenerative diseases.

Real-time tracking of metal nucleation via local perturbation of hydration layers.

The real-time visualization of stochastic nucleation events at electrode surfaces is one of the most complex challenges in electrochemical phase formation. The early stages of metal deposition on foreign substrates are characterized by a highly dynamic process in which nanoparticles nucleate and dissolve prior to reaching a critical size for deposition and growth. Here, high-speed non-contact lateral molecular force microscopy employing vertically oriented probes is utilized to explore the evolution of hydration layers at electrode surfaces with the unprecedented spatiotemporal resolution, and extremely low probe-surface interaction forces required to avoid disruption or shielding the critical nucleus formation. To the best of our knowledge, stochastic nucleation events of nanoscale copper deposits are visualized in real time for the first time and a highly dynamic topographic environment prior to the formation of critical nuclei is unveiled, featuring formation/re-dissolution of nuclei, two-dimensional aggregation and nuclei growth.Electrochemical deposition is important for industrial processes however, tracking the early stages of metallic phase nucleation is challenging. Here, the authors visualize the birth and growth of metal nuclei at electrode surfaces in real time via high-speed non-contact lateral molecular force microscopy.

Uniform patchy and hollow rectangular platelet micelles from crystallizable polymer blends.

The preparation of colloidally stable, self-assembled materials with tailorable solid or hollow two-dimensional (2D) structures represents a major challenge. We describe the formation of uniform, monodisperse rectangular platelet micelles of controlled size by means of seeded-growth methods that involve the addition of blends of crystalline-coil block copolymers and the corresponding crystalline homopolymer to cylindrical micelle seeds. Sequential addition of different blends yields solid platelet block comicelles with concentric rectangular patches with distinct coronal chemistries. These complex nano-objects can be subject to spatially selective processing that allows their disassembly to form perforated platelets, such as well-defined hollow rectangular rings. The solid and hollow 2D micelles provide a tunable platform for further functionalization and potential for a variety of applications.

Transformation and patterning of supermicelles using dynamic holographic assembly.

Although the solution self-assembly of block copolymers has enabled the fabrication of a broad range of complex, functional nanostructures, their precise manipulation and patterning remain a key challenge. Here we demonstrate that spherical and linear supermicelles, supramolecular structures held together by non-covalent solvophobic and coordination interactions and formed by the hierarchical self-assembly of block copolymer micelle and block comicelle precursors, can be manipulated, transformed and patterned with mediation by dynamic holographic assembly (optical tweezers). This allows the creation of new and stable soft-matter superstructures far from equilibrium. For example, individual spherical supermicelles can be optically held in close proximity and photocrosslinked through controlled coronal chemistry to generate linear oligomeric arrays. The use of optical tweezers also enables the directed deposition and immobilization of supermicelles on surfaces, allowing the precise creation of arrays of soft-matter nano-objects with potentially diverse functionality and a range of applications.

Conductive-AFM Patterning of Organic Semiconductors.

Using a conductive atomic force microscope (c-AFM) redox-writing technique, it is shown that it is possible to locally, and reversibly, pattern conducting, and nonconducting features on the surface of a low molecular weight aniline-based organic (semi)-conductor thin film using a commercial c-AFM. It is shown that application of a voltage between the tip and sample causes localized redox reactions at the surface without damage.

Cell paintballing using optically targeted coacervate microdroplets.

We present a new approach for the directed delivery of biomolecular payloads to individual cells with high spatial precision. This was accomplished via active sequestration of proteins, oligonucleotides or molecular dyes into coacervate microdroplets, which were then delivered to specific regions of stem cell membranes using a dynamic holographic assembler, resulting in spontaneous coacervate microdroplet-membrane fusion. The facile preparation, high sequestration efficiency and inherent membrane affinity of the microdroplets make this novel "cell paintballing" technology a highly advantageous option for spatially-directed cell functionalization, with potential applications in single cell stimulation, transfection and differentiation.

Self-assembling cages from coiled-coil peptide modules.

An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.

Opportunities in high-speed atomic force microscopy.

The atomic force microscope (AFM) has become integrated into standard characterisation procedures in many different areas of research. Nonetheless, typical imaging rates of commercial microscopes are still very slow, much to the frustration of the user. Developments in instrumentation for "high-speed AFM" (HSAFM) have been ongoing since the 1990s, and now nanometer resolution imaging at video rate is readily achievable. Despite thorough investigation of samples of a biological nature, use of HSAFM instruments to image samples of interest to materials scientists, or to carry out AFM lithography, has been minimal. This review gives a summary of different approaches to and advances in the development of high-speed AFMs, highlights important discoveries made with new instruments, and briefly discusses new possibilities for HSAFM in materials science.

Shear response of nanoconfined water on muscovite mica: role of cations.

By monitoring the thermal noise of a vertically oriented micromechanical force sensor, we detect the viscoelastic response to shear for water in a subnanometer confinement. Measurements in pure water as well as under acidic and high-ionic-strength conditions relate this response to the effect of surface-adsorbed cations, which, because of their hydration, act as pinning centers restricting the mobility of the confined water molecules.

Force spectroscopy of an elastic peptide: effect of D2O and temperature on persistence length.

This study explores the mechanical unfolding of elastic protein analogues as a function of temperature, in both H2O and D2O, using atomic force microscopy (AFM) force spectroscopy in a specially constructed AFM liquid cell. This represents the first time that the effect of D2O on protein flexibility has been investigated at the single molecule level by this technique. Model elastic peptides, R6, were encoded from synthetic genes expressed in Escherichia coli. The peptides possess short N- and C-terminal domains with central repetitive domains containing 13 repeats of the motif PGQGQQ-plus-GYYPTSLQQ. These sequences mimic those in native high molecular weight subunit glutenin proteins which confer elasticity to bread dough. Fitting single molecule stretching events to the worm-like chain model, allows determination of the molecular persistence length under various experimental conditions. The effect of increasing the temperature is to increase the persistence length of the peptide in both H2O and D2O, consistent with the expected "thermal softening" effect. However, the effect is significantly enhanced in D2O, in which the persistence length at 45°C is ∼25% greater than the value measured in H2O at the same temperature. Stronger intrapeptide H-bonding due to isotopic substitution of hydrogen for deuterium is the most likely cause of the enhanced backbone rigidity.

Insulin signaling to the glomerular podocyte is critical for normal kidney function.

Diabetic nephropathy (DN) is the leading cause of renal failure in the world. It is characterized by albuminuria and abnormal glomerular function and is considered a hyperglycemic "microvascular" complication of diabetes, implying a primary defect in the endothelium. However, we have previously shown that human podocytes have robust responses to insulin. To determine whether insulin signaling in podocytes affects glomerular function in vivo, we generated mice with specific deletion of the insulin receptor from their podocytes. These animals develop significant albuminuria together with histological features that recapitulate DN, but in a normoglycemic environment. Examination of "normal" insulin-responsive podocytes in vivo and in vitro demonstrates that insulin signals through the MAPK and PI3K pathways via the insulin receptor and directly remodels the actin cytoskeleton of this cell. Collectively, this work reveals the critical importance of podocyte insulin sensitivity for kidney function.

Direct real-time imaging of protein adsorption onto hydrophilic and hydrophobic surfaces.

Atomic force microscopy has been used to follow in real time the adsorption from solution of two of the gliadin group of wheat seed storage proteins onto hydrophilic (mica) and hydrophobic (graphite) surfaces. The liquid cell of the microscope was used initially to acquire images of the substrate under a small quantity of pure solvent (1% acetic acid). Continuous imaging as an injection of gliadin solution entered the liquid cell enabled the adsorption process to be followed in situ from zero time. For omega-gliadin, a monolayer was formed on the mica substrate during a period of approximately 2000 s, with the protein molecules oriented in parallel to the mica surface. In contrast, the omega-gliadin had a relatively low affinity for the graphite substrate, as demonstrated by slow and weak adsorption to the surface. With gamma-gliadin, random deposition onto the mica surface was observed forming monodispersed structures, whereas on the graphite surface, monolayer islands of protein were formed with the protein molecules in a perpendicular orientation. Sequential adsorption experiments indicated strong interactions between the two proteins that, under certain circumstances, caused alterations to the surface morphologies of preadsorbed species. The results are relevant to our understanding of the interactions of proteins within the hydrated protein bodies of wheat grain and how these determine the processing properties of wheat gluten and dough.

The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity.

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.

Glycopolymer charge density determines conformation in human ocular mucin gene products: an atomic force microscope study.

Atomic force microscopy (AFM) has been applied to the study of heterogeneity in the structure and function of individual biopolymers with complex structures such as glycoproteins, polysaccharides and nucleic acids. In this work we describe experiments which shed light on the heterogeneity of human ocular mucin gene products. By separating samples of native human ocular mucins on a caesium chloride density gradient, at least three populations consisting predominantly of products of the gene MUC5AC can be identified. Separation on the caesium chloride density gradient is governed by molecular architecture and charge density, and thus provides a route to the discrimination between different glycoforms within a glycoprotein sample. AFM images of these populations show that each is characterised by different conformational properties and polymer diameters, both of which can be attributed to differences in the degree and nature of glycosylation. These differences in glycosylation are likely to be the result of post-translational processing and may also have functional consequences. The AFM's ability to examine the composition of a predominantly single gene product population at the level of the single molecule allows the consequences of post-translational process heterogeneity to be examined at high resolution.