John Harding University of Sheffield
P. Mark Rodger Warwick University
Laurie B. Gower University of Florida
Peter Vekilov University of Houston
NN1: Nucleation and Pre-Nucleation in Biomineralisation
Tuesday AM, November 29, 2011
Room 104 (Hynes)
9:30 AM - **NN1.1
Prenucleation Clusters and Non-Classical Nucleation.
Denis Gebauer 1 Show Abstract
1 Physical Chemistry, University of Konstanz, Konstanz Germany
Organisms form fascinating mineralized structures that reflect high levels of biological control over crystallization. This control appears to be based on bio(macro)molecules, which allow to tune and regulate crystal polymorphism, morphology, and orientation. It seems evident that the classical view on nucleation and growth of crystals is too simplified to account for such delicate self-organization both in biological and bio-inspired synthetic systems. In fact, so-called non-classical precursors and intermediates have been identified, which render sophisticated control patterns possible, and may be influenced by additives.In this talk, the non-classical concept of stable pre-nucleation clusters is introduced. Pre-nucleation clusters are agglomerates of ions that occur prior to nucleation and precede the formation of crystals. To date, the existence of such clusters has been verified for some minerals, and they appear to play a central role in the processes that underlie nucleation. The present talk will focus on calcium carbonate, which is the most abundant biomineral, but also of great geological and industrial importance.Physico-chemical characterization of cluster formation by means of equilibrium thermodynamics shows that they are stable and likely have distinct structures that may relate to different polymorphs. This notion is supported by analyses of nanoparticles of amorphous calcium carbonate (ACC) that were nucleated at different pH-values, and exhibit short-range structural analogies with calcite and vaterite. Based on these results, the presence of proto-crystalline structures in ACC is proposed, and the effect of distinct structures in additive-free ACC on the final crystalline polymorph is discussed. In conclusion, this leads to the formulation of a novel scenario for the course of the early stages of crystallization of calcium carbonate. In addition, recent modelling results are outlined that give novel insight into the nature of the pre-nucleation clusters.In the second part of the talk, the effect of different (macro)molecular additives on the crystallization process is outlined. The data show that additives can interact with pre-nucleation clusters and amorphous intermediates, thereby influencing the progress of crystallization in multiple ways. Indeed, different modes of interference can be categorized and the data allow for their quantification. This is an important step towards an understanding of the means by which crystallization may be controlled. For example, some additives interact specifically with one of the distinct precursor structures, thus determining the polymorphism of the resulting crystal phase. Different additives can act at different stages of crystallization. This allows for the identification of mechanisms underlying polymorph-control induced by additives, which may apply also for the (macro)molecules that are active during biomineralization.
10:00 AM - NN1.2
A Combined Theoretical and Experimental Study of the Nucleation of Amorphous CaCO3.
Raffaella Demichelis 1 , Paolo Raiteri 1 , Julian Gale 1 , David Quigley 2 , Denis Gebauer 3 Show Abstract
1 Chemistry, Curtin University, Perth, Western Australia, Australia, 2 Physics and Centre for Scientific Computing, University of Warwick, Coventry United Kingdom, 3 Chemistry, University of Konstanz, Konstanz Germany
The nature of the nucleation of amorphous calcium carbonate (ACC) is examined in the light of both recent experimental [1,2] and theoretical  results. While experiment demonstrates the existence of stable pre-nucleation clusters, followed by a nucleation event, computer simulations suggest that the free energy of adding ion pairs to amorphous calcium carbonate is exothermic regardless of size, which may indicate the absence of a barrier. How these two different sets of observations can be reconciled will be examined in this work through the use of molecular dynamics simulations of pre-nucleation calcium carbonate solutions.In this presentation we will explore the use of computer simulation methods to try to unravel the complexities of the nucleation and growth processes for calcium carbonate. Central to this is the development of a force field that is accurately calibrated against experimental free energies  since failure to do so can result in qualitative errors for interfacial properties. Based on this we have explored the stability of ACC versus crystalline nanoparticles while accounting for the variable water content in the amorphous structure . In the light of this, and new experimental results, we propose a model to explain the non-classical aspects of the nucleation mechanisms of calcium carbonate, the origins for which can be traced back to the interfacial properties. D. Gebauer et al. (2010), Angew. Chem. Int. Ed. 49, 8889. D. Gebauer et al. (2008), Science, 322, 1819. P. Raiteri et al. (2010), J. Phys. Chem. C, 114, 5997. P. Raiteri and J.D. Gale (2010), J. Am. Chem. Soc., 132, 17623.
10:15 AM - NN1.3
Multi-Step CaCO3 Nucleation and Precipitation: Formation of a Hydrated Condensed Phase Prior to Crystal Nucleation of CaCO3.
Mark Bewernitz 1 , Laurie Gower 2 Show Abstract
1 Biomedical Engineering, University of Florida, Gainesville , Florida, United States, 2 Materials Science and Engineering, University of Florida, Gainesville, Florida, United States
There have been many recent advances in understanding the mechanism of calcium carbonate crystallization. A prominent advancement proposes that calcium carbonate undergoes a two-step nucleation process where, at concentrations below supersaturation, calcium and carbonate ions first associate by means of an unknown mechanism into stable prenucleation clusters (PNCs) before eventually nucleating into the stable seed crystal. The properties of the intermediate PNCs play a crucial role in the final phase of solid calcium carbonate product, either by directly precipitating to a solid phase or by their interaction with polyelectrolytes which can induce or stabilize amorphous intermediates. We have investigated the properties of early stage CaCO3 cluster formation, in the absence of polyelectrolytes and at a pH of 8.5, using Ca2+ selective ion electrode, pH electrode, isothermal titration calorimetry (ITC), ultracentrifugation, nano-track particle size analysis and 13C diffusion ordered spectroscopy (DOSY) techniques. The results of the study suggest that an additional, previously unaccounted-for, condensed fluidic phase emerges at a critical bound Ca2+ concentration at this more neutral pH. This data suggests that the mechanism of calcium carbonate crystallization may be a multistep process with more than a single intermediate. The discovered intermediate phase may play an important role, through either direct precipitation or through interaction with charged polyelectrolytes (as in the polymer-induced liquid-precursor, PILP, mechanism), in directing the resulting phase and texture of the mineral product.
10:30 AM - NN1.4
Exploring the Onset of Order in Growing Metal Carbonate Clusters Using Replica-Exchange Molecular Dynamics.
Adam Wallace 1 , James DeYoreo 1 2 , Jillian Banfield 1 3 Show Abstract
1 , Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 , The Molecular Foundry, Berkeley, California, United States, 3 , University of California - Berkeley, Berkeley, California, United States
It is now widely recognized that the carbonate mineral constituents of many biomineralized tissues form by a multi-stage crystallization process that involves the nucleation and structural reorganization of transient amorphous phases. The structure of these intermediate phases remains elusive, as does the nature of the disorder to order transition. However, there is some spectroscopic evidence suggesting that even “amorphous” carbonates possess certain structural elements that are similar to their crystalline counterparts. While molecular simulations have been used to gain insights in these areas, studies of metal carbonate nucleation have been strongly inhibited by the presence of kinetic traps that prevent adequate sampling of the potential landscape upon which the growing clusters reside within timescales accessible by simulation. This research addresses this challenge by marrying the recent Kawska-Zahn (KZ) approach to simulation of crystal nucleation and growth from solution with replica-exchange molecular dynamics (REMD) techniques. REMD has been used previously to enhance sampling of protein conformations that occupy energy wells that are separated by sizable thermodynamic and kinetic barriers, and is used here to probe the initial formation and onset of order within hydrated calcium and iron carbonate cluster species. Results to date suggest that growing clusters initiate as short linear ion chains that evolve into two- and three-dimensional structures with continued growth. The planar structures exhibit an obvious 2d lattice, while establishment of a 3d lattice is hindered by incomplete ion desolvation. The formation of a dehydrated core consisting of a single ion is observed when the clusters are ~ 1.0 nm in diameter. At the same size a distorted, but discernible calcite-type lattice is also apparent. This size coincides with previous cryo-TEM measurements of the “pre-nucleation” cluster size distribution of calcium carbonate, suggesting that the appearance of the first fully desolvated ion is a critical bottleneck both on the energy landscape and for the establishment of order within the growing clusters.
10:45 AM - NN1.5
In Vitro Synthesis and Stabilization of Amorphous Calcium Carbonate (ACC) Nanoparticles within Liposomes.
Chantel Tester 1 , Steven Weigand 2 , Derk Joester 1 Show Abstract
1 Materials Science, Northwestern University, Evanston, Illinois, United States, 2 DND/CAT, Northwestern University, Argonne, Illinois, United States
Calcium carbonate is not only a model system for nucleation and growth, but is also a key biomineral, is used in many different industries, and control of its precipitation further affects water desalination and heat exchangers. Its metastable amorphous form (ACC), in recent years, went from being a curiosity in biomineralization to playing a central role in many processes in materials from bioglasses to concrete and from nutraceuticals to polymers.[1-3] The role of another amorphous biomineral precursor, amorphous calcium phosphate (ACP) in bone formation is just beginning to emerge and has substantial implications for biomedicine.[4-5] Despite their prevalence, biological control of these metastable phases remains poorly understood. In mineralizing cells, small vesicles are very likely involved in ACC synthesis, transport, and storage, adding an additional layer of complexity. The small size of the vesicles (30-300 nm), the lipid surface chemistry, and the high radius of curvature may each play an essential role in achieving manipulating the stability of amorphous precursors.
We recently introduced an in vitro system to investigate the stabilization and controlled crystallization of ACC within phospholipid bilayer membrane vesicles. In this system, aqueous calcium salts are encapsulated within unilamellar vesicles and precipitation is initiated by addition of ammonium carbonate. We will report on the use of simultaneous small- and wide angle X-ray scattering (SAXS/WAXS) in conjunction with cryo-electron microscopy to probe the nucleation, growth kinetics, and ordering of the resulting mineral phase. We show that confinement in liposomes leads to stabilization of ACC nanoparticles in the range from 10 to 300 nm - much greater than what has previously been observed. In contrast to bulk precipitation, isolated precipitation inside vesicles offers a unique platform with which to study the effects of particle size, membrane chemistry, and co-encapsulated additives on the stability of ACC. An understanding of how organisms use these parameters to control the amorphous to crystalline transition will inform the synthesis of shaped single crystals, stabilized far from equilibrium.
(1) E. M. Pouget, P. H. H. Bomans, J. Goos, P. M. Frederik, G. de With, N. Sommerdijk, Science 2009, 323, 1555-1458. (2) D. B. Wang, A. F. Wallace, J. J. De Yoreo, P. M. Dove, Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21511-21516. (3) D. Gebauer, A. Volkel, H. Colfen, Science 2008, 322, 1819-1822. (4) J. Mahamid, B. Aichmayer, E. Shimoni, R. Ziblat, C. H. Li, S. Siegel, O. Paris, P. Fratzl, S. Weiner, L. Addadi, Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 6316-6321. (5) J. Mahamid, A. Sharir, L. Addadi, S. Weiner, Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 12748-12753. (6) C. C. Tester, R. E. Brock, C.-H. Wu, M. R. Krejci, S. Weigand, and D. Joester, CrystEngComm 2011, 13, 3975-3978.
11:30 AM - **NN1.6
Transient Phases and Prenucleation Clusters in Biomimetic Calcium Phosphate Mineralization.
Nico Sommerdijk 1 Show Abstract
1 Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven Netherlands
The often astonishing materials properties of crystalline biominerals are generally related to the hierarchical assembly of specifically interacting organic and inorganic components. A yet unfulfilled dream of many scientists is to synthesize new materials with similar advanced properties applying Nature’s biomimeralization strategies. An absolute prerequisite for the design of such hybrid materials with predetermined structure and properties is to unravel the mechanisms of biologically and biomimetically controlled mineral formation.The in situ study of the development of mineral formation can make an important contribution to the understanding of the processes involved in biomineralization. CryoTEM has been demonstrated as a method to investigate the early stages of mineral formation without removing the developing particles from their aqueous environment. [3, 4] For calcium phosphate these investigations revealed that the formation of apatite is preceded by an amorphous phase, which itself is formed through the assembly and aggregation of what we have called “prenucleation clusters” [5,6]. Here we will discuss the precise structure of these “clusters” and their role in the formation of the subsequent amorphous and crystalline phases. It will be demonstrated that these recent insights re-unite classical and non-classical theories about mineral formation. N.A.J.M. Sommerdijk, H. Cölfen, MRS bulletin, 35, 116 (2010). A. Dey, G. de With, N.A.J.M. Sommerdijk, Chem. Soc. Rev. 92, 381, (2010). B.P. Pichon, P.H. H. Bomans, P.M. Frederik N. A. J. M. Sommerdijk, J. Am. Chem. Soc. 130, 4034 (2008). E.M. Pouget, P.H.H. Bomans, J.A.C.M. Goos, P.M. Frederik, G.de With, N.A.J.M. Sommerdijk, Science, 323, 1455 (2009). A. Dey, P.H.H. Bomans, F.A. Müller, J. Will, P.M. Frederik, G. de With and N.A.J.M. Sommerdijk, Nature Mater, 9 1010 (2010). F. Nudelman, K. Pieterse, A. George, P.H.H. Bomans, H. Friedrich1, L. J. Brylka1, P.A.J. Hilbers, G. de With N.A.J.M. Sommerdijk, Nature Mater, 9 1004 (2010)
12:00 PM - NN1.7
Effects of Heme on Sickle-Cell Hemoglobin Polymerization.
Peter Vekilov 1 , Veselina Uzunova 1 , Weichun Pan 1 , Oleg Galkin 1 Show Abstract
1 Department of Chemical and Biomolecular Engineering and Department of Chemistry, University of Houston, Houston, Texas, United States
Sickle cell anemia is a debilitating genetic disease, which affects hundreds of thousands babies born each year worldwide. Its primary pathogenic event is the formation of long fibers (with 14 molecules in the cross section) of a mutant, sickle cell, hemoglobin (HbS). Fiber formation is a first order phase transition, and, thus, sickle cell anemia is one of a line of diseases (Alzheimer’s, Huntington’s, prion, etc.) in which nucleation initiates pathophysiology. We have previously shown that the nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. In search of such components, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02 – 0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100 – 260 µM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than prior to dialysis. Towards an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at concentration 66 µM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor for the puzzling complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy. J. Mol. Biol. 365(2):425-439; Biophys. J. 93:902-91; Biophys. J. 92(1):267-277; Brit. J. Haematol. 139(2):173-184; J Mol Biol 377(3):882-888; Biopolymers 91(12):1108-1116; Biophys. J. 99(6):1976-1985.
12:15 PM - NN1.8
CaCO3 Polymorphs: Spherulitic Growth and Stabilization in Water-Ethanol Mixtures.
Karina Sand 1 , Juan Diego Rodriuez-Blanco 2 , Liane Benning 2 , Emil Makovicky 3 , Susan Louise Stipp 1 Show Abstract
1 Department of Chemistry, University of Copenhagen, Copenhagen Denmark, 2 School of Earth and Environment, University of Leeds, Leeds United Kingdom, 3 Geography and Geology, University of Copenhagen, Copenhagen Denmark
The precipitation of crystalline CaCO3 polymorphs and their morphology is affected by the presence of alcohol in alcohol-water mixtures. However, the crystal form, polymorph and rate of precipitation of such studies are sensitive to solution concentrations, calcium source, temperature, precipitation method and mixing speed. Solutions with a high ratio of organic molecules to water provide insight for biomineralization where the crystallization of hard parts takes place in the presence of organic molecules. An understanding of the growth of CaCO3 in alcohol-water mixtures is important because control of the precipitating polymorph and its morphology are two central parameters for succesful biomineralization of CaCO3. We used a simple homogeneous precipitation method, with a final concentration of CaCl2 and Na2CO3 of 25 mM and a temperature of 24 °C and varied the proportion of added alcohol (10 and 50%), the shaking speed (80 and 140 MOT), the type of alcohol (ethanol, 1-propanol and 2-propanol) and the reaction time (1 hour to 4 months). Vaterite and aragonite are stabilized by shaking speed and alcohol concentration where rapid shaking enhanced precipitation of vaterite. These two parameters also influenced the morphology of vaterite and gave rise to two distinct morphologies: a cauliflower shaped aggregate and a dendrite. Observation of the precipitate over time demonstrated that precipitates form and grow by classical ion by ion attachment from solution. Semi-hexagonal aragonite needles form by cyclic twin intergrowths and spherulitic growth explains the vaterite cauliflower shaped aggregates and aragonite sheafs. The results show that solutions with a low water activity act as a switch for controlling polymorph and morphology.
12:30 PM - NN1.9
Engineered Biomimetic Polymers as Tunable Agents for Controlling Rates of Crystal Growth and Tissue Mineralization.
Chun-Long Chen 1 , Jiahui Qi 1 2 , Adam Wallace 1 , Ronald Zuckermann 1 , James DeYoreo 1 Show Abstract
1 The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield United Kingdom
In nature, proteins are known to a play significant role in the control of mineral nucleation and growth, both to create functional tissues and prevent pathological mineralization. The research described here addresses the challenge of developing a class of compounds that mimic the mineralizing functions of proteins for use in the synthesis of functional crystalline materials and as therapeutic agents. Peptoids, or poly-N-substituted glycines, are a novel class of non-natural polymers recently developed to mimic both structures and functionalities of peptides and proteins, and bridge the gap between biopolymers and bulk polymers. As with peptides, sequence-specific peptoids can be efficiently synthesized by using automated solid-phase synthesis starting from a large number of chemically diverse amine building blocks. Moreover, peptoids exhibit much higher protease stability and thermal stability than peptides or proteins. Inspired by recent research that showed low concentrations of acidic peptides and proteins can significantly accelerate calcite growth, we designed and synthesized a suite of anionic peptoids and screened them for control over hard tissue minerals (e.g. calcite) morphology and growth rate. Results to date demonstrate both a high degree of morphological control and extreme levels of acceleration, with both characteristics observed to be highly dependent on peptoid hydrophobicity, number of carboxylic acid groups, peptoid sequence and concentration. At high concentrations (50 μM), calcite crystals formed in the presence of peptoid exhibit various unique shapes ranging from elongated spindles and twisted paddles to crosses and spheres. At low concentrations (<250nM), a number of the peptoids increased calcite growth rates by as much as a factor of 25, with the most effective being strongly amphiphilic. Based on previous research into peptide-induced acceleration, the energetic source of acceleration is likely to be a reduction in the activation barrier that controls the rate of solute addition at atomic steps on the calcite surface. Here we estimate the magnitude of the barrier and present a number of structural scenarios that could result in its reduction, including enhanced cation desolvation rates, disruption of the near-surface solution layer, and displacement of the waters believed to be strongly bound to calcite surfaces.
12:45 PM - NN1.10
Influence of near-Physiological Salines and Organic Matrix Proteins from Sternal ACC-Deposits of Porcellio Scaber on Calcium Carbonate Precipitation.
Sigrid Hennig 1 , Sabine Hild 2 , Helge Fabritius 3 , Christian Soor 1 , Andreas Ziegler 1 Show Abstract
1 Central Facility for Electron Microscopy, University of Ulm, Ulm Germany, 2 Institute of Polymer Science, Johannes Kepler Universität Linz, Linz Austria, 3 Microstructure Physics and Metal Forming, Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf Germany
Many living organisms use calcium carbonate as inorganic component in skeletal elements of their body. The mature biominerals are formed via amorphous precursors with short-range orders that resemble those of their prospective crystalline phases: calcite, aragonite and vaterite. In this respect the amorphous phase can be regarded as composed of randomly oriented nano-crystalline particles. Because of its high solubility, amorphous calcium carbonate (ACC) is unstable in vitro and must be stabilized to prevent spontaneous crystallization. It is known that in natural systems specific proteins, magnesium and phosphate play a role in the stabilization, and possibly also in the control of nucleation and short-range order of biogenic ACC. The mechanisms and the relative effects of these components, however, are poorly understood. Terrestrial isopods like Porcellio scaber use ACC for transient storage of cuticular calcium carbonate during their biphasic moult cycle. The ACC deposits consist of spherules that are formed in the exuvial gap in a medium whose ionic composition and pH are strictly controlled and contain an elaborate organic matrix consisting of at least seven different proteins that form radial and concentric elements. In order to elucidate the roles of the different factors involved in the formation of the deposits, we performed precipitation experiments using near-physiological salines with different compositions and soluble matrix proteins extracted from native ACC deposits and analysed structure, mineral phase and composition of the precipitates. The saline alone has no effect on the crystal phase, but leads to changes in crystal morphology due to magnesium content, which is even more pronounced in the presence of BSA that was used as control protein. The soluble matrix proteins lead to the formation of nanoscopic ACC granules that agglomerate in shapes mimicking the structure of native deposits.
NN2: Nanoparticles and Nanocomposites
Tuesday PM, November 29, 2011
Room 104 (Hynes)
2:30 PM - **NN2.1
In Situ Electron Microscopy Studies of Nanocrystal Growth and Assembly.
Paul Alivisatos 1 Show Abstract
1 , University of California, Berkeley, Berkeley, California, United States
In recent years, we have deployed a liquid cell for use in a transmission electron microscope and we have used it to monitor the growth and assembly of colloidal nanocrystals. Using this tool, we have observed unexpected mechanisms of growth and we have been able to observe many specific pathways for nanocrystal assembly that previously were not easy to anticipate.
3:00 PM - NN2.2
In Situ Synthesis of Gold Nanoparticles in Exponentially-growing Layer-by-Layer Films.
Liyan Shen 1 2 , Laetitia Rapenne 1 , Jian Ji 2 , Catherine Picart 1 Show Abstract
1 Department of Bioengineering, Grenoble Institute of Technology, Grenoble France, 2 Department of Polymer Science, University of Zhejiang, Hangzhu China
In situ synthesis of inorganic nanoparticles (NPs) in polyelectrolytes multilayers (PEMs) has since very recently received great attention. Due to the versatility of their composition, PEMs offer a unique opportunity to synthesize a variety of NPs. So far, mostly cationic precursors have been used and only few studies have studied the possibility to use amine groups to bind anionic precursors. Here, we take benefit of the large amount of amine groups present in exponentially growing poly(L-lysine)/hyaluronan (PLL/HA) films that are built in pH-amplified conditions to bind and sequester aurochlorate (AuCl4-) anions. By using unique assembly conditions to built micrometer thick PEM films and by varying solely the pH of the gold precursor solution from 3 to 9, we synthesized homogenous and well-dispersed gold NPs in mild conditions. The polypeptide-polysaccharide reactive template enabled the formation in a spatially confined environment of gold NP having a well-defined size range. Importantly, there was no particular effect of the film-ending layer (either PLL or HA), which suggested that the film bulk structure was very similar in both cases. The largest particles of about 9 nm were obtained at acidic pH of 3. When the pH was increased, smaller and more numerous NPs were synthesized. At pH 9, a very high density of extremely small NPs (about 1.7 nm in diameter) was synthesized. We finally propose a scheme for the mechanism of gold NPs formation, in which several groups of PLL and HA will contribute to the binding of gold ions, the nucleation and growth of NPs and finally to their stabilization in the “bulk” of the film.
3:15 PM - NN2.3
Probing Nucleation, Assembly, and Oriented Attachment with In Situ TEM.
Michael Nielsen 1 2 , Dongsheng Li 1 3 , Shermin Arab 3 , Frank Soberanis 3 , Cathrine Frandsen 1 4 , David Kisailus 3 , James De Yoreo 1 Show Abstract
1 , Lawrence Berkeley National Lab, Berkeley, California, United States, 2 , University of California, Berkeley, Berkeley, California, United States, 3 , University of California, Riverside, Riverside, California, United States, 4 , Technical University of Denmark, Lyngby Denmark
Probing the early events that determine the nucleation pathway and final crystal structureis one of the challenges in understanding templating, aggregation and oriented growth ofbiominerals. Despite the many advances in knowledge regarding these processes,important aspects remain poorly understood such as a the energetics of directednucleation, the structural evolution of incipient nuclei, and the mechanisms of orientedattachment. We are using in situ and ex situ TEM to investigate of crystal nucleation,oriented attachment and mesocrystal formation in a number of systems. Solutionimaging at nanometer scale and video rates is enabled by the combination of a customdesigned TEM stage and fluid cell. Significantly, the design of the cell and holderensures temperature and electrochemical control to initiate reactions of interest, such asthe onset of crystal nucleation. Here we show that calcium carbonate nucleates on a bareelectrode via metastable nanoparticles — most likely amorphous calcium carbonate —followed by consolidation and faceting of the crystalline material, and show how toextend the technique to observe oriented nucleation on organic templates. Growth ofranched rutile TiO2 nanostructures, obtained under hydro-solvothermal synthesisconditions, was investigated as a function of time using these techniques. Initially, aprimary wire forms with subsequent branching occurring via absorption of orientedanatase nanoparticles and their eventual transformation to rutile. We propose that thebranching in these nanostructures occurs via (101) twins. Iron oxide nanorods of theakaganeite phase grown hydrothermally self-assemble into high-aspect ratio ellipsoidalmesocrystals of the hematite phase. In situ imaging shows a progression of nanorodaggregation into disordered aggregates followed by gradual ordering into the finalmesocrystal. For all three systems, current work is focused on understanding themechanism and progression of the structural transformations.
3:30 PM - NN2.4
Polypeptide-Templated Synthesis of Calcite Nanorods.
Wolfgang Tremel 1 , Timo Schueler 1 , Jochen Renkel 2 , Michael Dietzsch 1 , Stephan Hobe 3 , Karl Fischer 4 , Martin Panthoefer 1 , Manfred Schmid 4 , Anja Hoffmann-Roeder 2 , Harald Paulsen 2 Show Abstract
1 Institut für Anorganische Chemie, Johannes Gutenberg-Universität, Mainz Germany, 2 Institut für Organische Chemie, Johannes Gutenberg-Universität, Mainz Germany, 3 Institut für Botanik, Johannes Gutenberg-Universität, Mainz Germany, 4 Institut für Physikalische Chemie, Johannes Gutenberg-Universität, Mainz Germany
The delicate mineral structures produced by organisms in the process of biomineralization are an inspiration for future materials science and nanotechnology because of their unique materials properties and their hierarchical order often over several length scales. Acidic proteins are believed to play a role in the synthesis of CaCO3 biominerals e.g. in nacre, sea urchins, or egg shells, produced under cellular control. Several proteins have been identified that are involved in the formation of CaCO3 structures observed in these organisms, and their addition in vitro results in the formation of CaCO3 under ambient conditions. We have extended these studies to show that synthetic peptides can also be used to mimic the calcification reactions that occur in living organisms using a combination of self-assembling and cell-surface recognizing protein building blocks with CaCO3 mineralizing peptides. To identify CaCO3 (calcite or vaterite) binding peptides, several rounds of phage display screening were carried out, employing heptapeptide and dodecapeptide libraries. After 3 rounds of panning, peptides with characteristic amino acid sequences were obtained. Besides the obvious prevalence of polar and ionic amino acids the comprehensive presence of proline indicates that high affinity towards CaCO3 surfaces depends on a short range beta-turn structure. Most of these structures are divided into two parts: a C-terminal end including polar, hydroxyl- and amide- amino (Ser, Thr, Tyr and Gln, Asn), and a N-terminal end including one or two unpolar amino acids and either an acidic or basic amino acid (Arg or Asp, Glu). The oligopeptides from each panning round were synthesized by solid phase peptide synthesis to carry out mineralization experiments either on carboxyl-terminated self assembled monolayers or using a contact-free ultrasonic levitator setup to avoid external templating effects. The crystallizations were monitored in situ by XRD, WAXS, SAXS and TEM. Crystallization experiments with two last-round heptapeptides (ATNPTDY and ASTQPLR) lead to the formation of highly anisotropic calcite nanorods with an aspect ratio > 100.
3:45 PM - NN2.5
Using Block Copolymers to Access Calcium Phosphate Nanocomposite Films with Tailored Structures and Properties.
Ruiqi Song 1 , Hiroaki Sai 1 , Ulrich Wiesner 1 , Lara Estroff 1 Show Abstract
1 , Cornell University, Ithaca, New York, United States
We show that the combination of sol-gel chemistry and evaporation-induced self-assembly leads to the synthesis of nanocomposite films from amorphous calcium phosphate (ACP) nanoparticles and poly(isoprene-block-dimethylaminoethyl methacrylate). The compatibility between organo-modified ACP nanoparticles and hydrophilic blocks enables ACP nanoparticles to swell the hydrophilic domains of the amphiphilic block copolymer, resulting in the formation of lamellar mesostructures. At high inorganic contents, nanocomposites with three dimensional network structures are formed. Compared to the neat polymer and lamellar nanocomposites, these composites exhibit significantly enhanced moduli (up to 818 MPa at 145 oC) and tailored glass transition temperatures of the polyisoprene segments in the range of -37.6 to -53.8 oC. Our results suggest that sol solvent-mediated formation of block copolymer micelles can serve as a colloidal template by spatially confining the assembly of ACP nanoparticles in the case of high inorganic contents, thereby providing a route to designing ACP-containing nanocomposites with tailored structures and tunable mechanical and thermal behavior.
4:30 PM - **NN2.6
Development of Self-Organized Inorganic/Organic Hybrid Materials through the Approaches Inspired by Biomineralization.
Takashi Kato 1 Show Abstract
1 Department of Chemistry and Biotechnology, School of Engeneering, The University of Tokyo, Tokyo Japan
Biominerals are organic/inorganic hybrids exhibiting versatile functions.[1,2] The hierarchical structures and formation processes of biominerals have inspired scientists to develop new functional hybrid materials under mild conditions. The interactions between organic and inorganic components are key to form the hybrids.[1,2] Here we report on our recent approaches to the development of new inorganic/organic hybrid materials. Thin-film hybrids with flat surfaces comprising calcium carbonate nanocrystals are prepared by using natural, semi-synthetic, and synthetic polymer templates by solution processes. The hybrids of calcium carbonate and organic polymers form the thin films having a wide variety of morphologies. Oriented calcium carbonate crystals[4,5] are obtained by using an acidic natural peptide isolated from the exoskeleton of crayfish, or in an ordered chitin matrix. Patterned thin-film and 3D complex structures of hybrids can be formed in hydrogel matrices. The self organized structures have been photo-imaged on the hybrid films. The amorphous transparent hybrids of calcium carbonate and acidic macromolecules are also formed under mild conditions. These approaches can be applied to the preparation of hybrid materials of a variety of inorganic crystals and organic functional macromolecules. In these bioinspired self-organization processes, the control of macromolecular structures and their interactions is essential. These self-organized hybrid materials have great potentials in a wide variety of fields of advanced technologies. T. Kato, T. Sakamoto, T. Nishimura, MRS Bulletin 35, 127 (2010). T. Kato, A. Sugawara, N. Hosoda, Adv. Mater. 14, 869 (2002). A. Sugawara, T. Ishii, T. Kato, Angew. Chem. Int. Ed. 42, 5299 (2003). A. Sugawara, T. Nishimura, Y. Yamamoto, H. Inoue, H. Nagasawa, T. Kato, Angew. Chem. Int. Ed. 45, 2876 (2006). T. Nishimura, T. Ito, Y. Yamamoto, M. Yoshio, T. Kato, Angew. Chem. Int. Ed. 47, 2800 (2008). T. Sakamoto, Y. Nishimura, T. Nishimura, T. Kato, Angew. Chem. Int. Ed. 50, 5856 (2011). Y. Oaki, S. Kajiyama, T. Nishimura, H. Imai, T. Kato, Adv. Mater. 20, 3633 (2008). Y. Oaki, S. Kajiyama, T. Nishimura, T. Kato, J. Mater. Chem. 18, 4140 (2008).
5:00 PM - NN2.7
Single-Crystal Calcite Nanorods from Amorphous Calcium Carbonate.
Yi-Yeoun Kim 1 , Nicola Hetherington 1 , Roland Kroeger 2 , Hugo Christenson 3 , Fiona Meldrum 1 Show Abstract
1 School of Chemistry, University of Leeds, LEEDS United Kingdom, 2 Department of Physics, University of York, York United Kingdom, 3 School of Physics and Astronomy, University of Leeds, Leeds United Kingdom
Amorphous calcium carbonate (ACC) is now recognised to be a common biomineral. While some organisms produce a stable ACC which remains amorphous for extended periods of time, the more interesting phase is arguably transient ACC, which acts as a precursor to calcite and aragonite and crystallises under biological control. Further, there is growing evidence that crystal nucleation and growth processes are often modified in confined volumes as compared with bulk solutions. This work provides a systematic study of the precipitation of calcium carbonate within well-defined volumes. In this work, we have explored the possibility of controlling the crystallisation of ACC through limiting contact of the mineral with the bulk solution, and demonstrate that remarkable control over the crystal product can be achieved using this templating methodology. Precipitation of ACC within the rod-shaped pores creates a system where the majority of the intra-membrane particle is in direct contact with the membrane walls and only the ends are in contact with the solution. ACC was precipitated within “50 nm” and “200 nm” diameter pores of polycarbonate track etch membranes both in the absence of additives, and in the presence of polyacrylic acid (PAA) or poly(aspartic acid) (pAsp). ACC precipitated in the presence of PAA or Pasp displays some fascinating properties and has been termed “PILP” (polymer-induced liquid precursor) due to the observation that it shows some liquid-like behaviour, including infiltration into pores. The current experiments therefore also enable us to investigate the mechanism of infiltration of PILP into porous media and provide strong evidence for capillary action.In the absence of additives, performing the experiment at this high supersaturation and low temperature (4 °C) results in the precipitation of ACC in the membrane pores, where the ACC is stable for 25 – 30 mins prior to crystallisation. Electron diffraction showed that they were single crystals of calcite, despite having a granular structure as viewed by TEM. Stabilisation of ACC with PAA, which produces a PILP phase, resulted in a marked increase in the yield of intra-membrane particles, and the vast majority of both the 50 nm and 200 nm pores now supported particle formation. The intra-membrane particles formed were again solid, and remarkably, most displayed extremely high aspect ratios as defined by complete filling of the membrane pores. Therefore, calcite nanowires with aspect ratios of ≈ 70 were produced in the 50 nm pores. The collected nanowires were amorphous phase at early stage and then transformed to calcite with time, which were confirmed using EXAFS, Raman microscopy and IR spectroscopy That such single crystal morphologies can be produced synthetically demonstrates the potential of this synthetic strategy, and it is envisaged that a similar approach could be applied to a wide range of materials which can be precipitated via amorphous precursor phases.
5:15 PM - NN2.8
Adaptive Resolution Coarse-Graining of Collagen-Hydroxyapatite Nanocomposites.
Patrick Kiley 1 , James Elliott 1 Show Abstract
1 Materials Science and Metallurgy, University of Cambridge, Cambridge United Kingdom
Bone displays a hierarchically organized composite structure of mineralized collagen nanofibrils that results in a material with greatly enhanced thermal and mechanical properties. However, much research over the last decade has demonstrated that the nucleation of inorganic, crystalline phases within this and similar biological systems follows a multi-step pathway involving the formation of a number of meta-stable intermediates and is therefore at odds with the comparatively simple ion-by-ion growth mechanism suggested by classical nucleation theory. Furthermore, many of the early events in this process happen on length- and time-scales that are difficult to access experimentally, and which are further complicated by the presence of biological molecules. As a result, computer modeling presents the possibility of revealing how these events unfold in a biological context. However, due to the hierarchical nature of these materials and the large size of the tropocollagen triple helix, it is challenging to create computational models that are neither too small to capture the diverse interactions underlying the material properties of collagen and bone nor too large to run for significant lengths of time. Here, we tackle these issues by using an adaptive resolution coarse graining method (AdResS). As a result, we are able to simulate specific collagen-collagen and collagen-solvent interactions in fully atomistic detail, in the presence and absence of a mineral phase. At larger distances from these interactions, the degrees of freedom of the solvent and collagen are dynamically reduced to a coarse-grained description that greatly enhances computational efficiency while fully retaining the hierarchical nature of the model. This approach reveals how specific interactions across the whole collagen molecule can lead to observed material properties.
5:30 PM - NN2.9
Synthesis and Characterization of Bioinspired Hierarchically Self-Assembling Organic-Inorganic Nanocomposites.
Xunpei Liu 1 2 , Surya Mallapragada 1 2 , Tanya Prozorov 2 , Yan-Yan Hu 3 2 , Lijun Wang 4 2 , Shuren Feng 4 2 , Adu Rawal 3 2 , Qinwen Ge 5 2 , Klaus Schimidt-Rohr 3 2 , Mufit Akinc 5 2 Show Abstract
1 Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States, 2 , Ames Laboratory, Ames, Iowa, United States, 3 Department of Chemistry, Iowa State University, Ames, Iowa, United States, 4 Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa, United States, 5 Department of Materials Science and Engineering, Iowa State University, Ames, Iowa, United States
Self-assembling hybrid materials with hierarchical order were synthesized using bioinspired bottom up approaches. Hierarchically self-assembling synthetic polymers were conjugated with biomineralization proteins/peptides, and were used as templates for the bioinspired synthesis of calcium phosphate, magnetite and zirconia. The inspiration for these were derived from the hierarchical structure in bone, and from magnetotactic bacteria, for the calcium phosphate and magnetite nanocomposites respectively. Self-assembled hydroxyapatite (HAp) nanocomposites were synthesized using amphiphilic triblock copolymer templates that self-assemble at the nanoscale and at the macroscale based on changes in temperature. Similar to native bone, citrate was added as a regulatory element in the in vitro synthesis to enable molecular control of the size and stability of HAp nanocrystals in the synthetic nanocomposites. The decrease of the HAp crystal size with increasing citrate concentration was confirmed by solid-state nuclear magnetic resonance (NMR) and wide-angle X-ray diffraction (XRD), and the shapes of HAp nanocrystals were determined by transmission electron microscopy (TEM). Uniform superparamagnetic magnetite nanocrystals with a size of about 30 nm, were synthesized in the presence of a recombinant Mms6 protein, which was derived from the magnetosomes of magnetotactic bacteria. Nanoparticle fabrication studies showed the nanoparticles synthesized by the wild type Mms6 differ greatly compared to the mutants, indicating that the number and placement of charged groups in the protein were very critical for its function. Zirconia nanocomposites were synthesized using lysozyme conjugated to block copolymer templates. Removal of the templates left behind a porous network of zirconia with a much higher surface area and thermal stability than those made without any template. Based on all of these studies, we developed a robust and modular method with control over the formation as well as placement of an inorganic phase in the nanocomposite structure, for a variety of different inorganic nanoparticles, and are able to design tailored functional organic templates for room-temperature bioinspired synthesis.
5:45 PM - NN2.10
Catalytic Peptides for Room-Temperature Inorganic Nanocrystal Synthesis Discovered through New Combinatorial Phage Display Library Approach.
Yoshiaki Maeda 1 , Zengyan Wei 1 , Hiroshi Matsui 1 Show Abstract
1 Department of Chemistry and Biochemistry, City University of New York, Hunter College, New York, New York, United States
Nature tends to find the easiest way to grow materials with high efficiency and high selectivity at room temperature, which conventional chemical synthesis methods cannot match. Biomimetic material growth is a potential path to break through room temperature inorganic nanocrystal synthesis because biocatalytic function of peptides exhibits high growth efficiency of materials in accurate structures at low temperature. Recently, several promising peptides and proteins were demonstrated to catalyze the growth of semiconductors. However, a successful discovery of the catalytic peptide sequences is strongly dependent on trial-and-error processes. Here, we report a novel evolutionary approach to identify catalytic peptides for the room-temperature growth of target semiconductor materials. The conventional phage display technique focuses on finding peptide sequences that bind the target substrate, however our combinatorial phage display library approach directly screens peptides that catalyze the target material growth. In this study, ZnO nanoparticle growth is targeted as a model. The phage displayed library was incubated with zinc precursor (10 mg/mL zinc nitrate solution) at room temperature. Among random peptide sequences displayed on phage, some of them catalytically grow ZnO particles. Then, target phages growing the ZnO nanocrystals on their displayed peptides were recovered from the solution by a simple centrifuge method. After unbound phages were removed by extensive washing, the residual phages are amplified. After three rounds of selection, the peptide sequences displayed on the phage viruses were analyzed and only two peptides were identified. Of these two peptides, ZP-1 peptide (GAMHLPWHMGTL) was determined to be the predominant sequence for the room-temperature growth. Atomic force microscopic images (AFM) and dynamic light scattering (DLS) analysis revealed that the ZP-1 peptides were aggregated into the spherical shape in aqueous solution. By mixing the ZP-1 peptide and zinc precursor, ZnO nanoparticles were synthesized, suggesting the catalytic property of ZP-1 peptide for the room-temperature synthesis of inorganic nanocrystal.The unique feature of this technique is the panning process of peptides that grow only target nanocrystals in precursor solutions. Our methodology provides a simple and convenient route to discover biomineralizing peptides for ZnO nanocrystal growth at room temperature. The broad impact is highly expected from this outcome since this novel screening technology can be applied to generate a wide range of catalysis.
NN3: Poster Session
Tuesday PM, November 29, 2011
Exhibition Hall C (Hynes)
9:00 PM - NN3.1
Biomimetic Template Directed Synthesis of One-Dimensional Inorganic Nanostructures.
Handan Acar 1 , Mustafa Guler 1 Show Abstract
1 , Materials Science and Nanotechnology, Ankara Turkey
Fabrication of one-dimensional nanostructures is one of the most attractive subjects in nanotechnology, due to their high aspect ratio and vast application areas. The biomineralization process is the exact process that made by the living organism to form stiff and hard tissues such as; shells, skeleton and teeth. Self-assembly of those nanostructures by mimicking the nature is also very popular and interesting. Herein, we demonstrated a new bottom-up approach for the fabrication of one-dimensional nanostructures that mimics the biomineralization process of nature and can yield wide opportunities for producing high-aspect-ratio inorganic nanostructures with high surface area. In this study, we synthesized a new peptide molecule, mimics amyloid fibers during its self-assembly into the nanofiber structure in ethanol by supramolecular interactions. We functionalized this peptide by chemically active special groups with the affinity to desired metal ions in the medium. With those special groups, we also enhance the catalytic character of the peptide molecule for biomineralization and polymerization. The materials produced by this method have vast potential in the field of catalysis, photovoltaic and semiconductors. We also demonstrated some of the most important physical properties of the produced one-dimensional nanostructures by this method.
9:00 PM - NN3.10
Exploring the Role of Primary Nucleation and Secondary Processes during Insulin Amyloid Fibril Formation.
Ryan Morris 1 , Kym Eden-Jones 1 , Rosalind Allen 1 , Cait MacPhee 1 Show Abstract
1 School of Physics & Astronomy, University of Edinburgh, Edinburgh United Kingdom
There has been immense interest in the assembly of amyloid fibrils due to their implication in many diseases such as Alzheimer’s Disease and Type-2 diabetes. Fibril formation has historically been explained via a nucleation-dependent process. A key feature of this model is the presence of an often long quiescent period preceding the appearance of fibrils known as the lag phase. This lag time has been attributed to the length of time needed to form a critical nucleus from which fibrils can grow. This phase of fibril assembly has garnered particular interest due to the observation that pre-fibril oligomeric species present during this time may be the cytotoxic agents responsible for amyloid associated pathologies. There has been much debate centered on the molecular mechanisms governing fibril assembly, particularly during the lag phase. Studies of the lag time in the past have been fraught with irreproducibility and the appearance of large variation in the measured lag time distributions. This subsequently has been attributed to the stochastic nature of nucleation. However, a recent model has been proposed which regards the primary nucleation event as a small contributor to the duration of the lag time. In this model, secondary processes, particularly fragmentation, dominate the formation and growth behavior of amyloid fibrils. By employing methods and materials which significantly reduce the previously noted variation in lag time, we have undertaken an extensive statistical investigation of amyloid fibril formation of bovine insulin via ThT fluorescence assays in the presence and absence of agitation. We observe the existence of two distinct concentration regimes where the lag time and growth of fibrils appear to be controlled by differing and competing processes. We attempt to explain this behavior via deterministic computational modeling.
9:00 PM - NN3.11
Synthesizing Bio-Minerals by Electrophoresis/Electro-Transport Approach.
Sean Morefield 1 , Mei Chandler 1 , Paul Allison 1 , Ruth Hidalgo-Hernandez 1 , Omar Rodriguez Negron 1 , Carlos Morales 1 , Philip Malone 1 Show Abstract
1 CF-M, US Army ERDC-CERL, Champaign, Illinois, United States
Biological materials such as mussel and scallop shells can include both calcite and aragonite in different layers of the same shell. The development of a hard shell in biological materials are principally composed of different polymorphs of calcium carbonate. In this paper, we investigate using an electrophoresis/electro-transport approach to create different polymorphs of calcium carbonate. Electromigrated calcium and carbonate ions nucleate and form calcium carbonate crystals in the pore spaces of porous polymer membranes. The nucleation and growth of the calcium carbonate crystals are examined as a function of varying applied electric voltage, charging time and addition of variable amounts of magnesium and strontium. The preliminary results show that varying the applied voltage across the test cell causes a shift in the ratio of the calcium carbonate polymorphs. This study will provide a basis for developing bio-inspired nanocomposites with controlled crystal morphology and chemistry.
9:00 PM - NN3.12
Hydroxyapatite Nucleation Using Biomimetic Peptides.
Joshua Padovano 1 , Sriram Ravindran 2 , Ana Bedran-Russo 3 , Anne George 2 Show Abstract
1 , University of Illinois at Chicago, Chicago, Illinois, United States, 2 Department of Oral Biology, University of Illinois at Chicago, Chicago, Illinois, United States, 3 Department of Restorative Dentistry, University of Illinois at Chicago, Chicago, Illinois, United States
Formation of calcified tissues is a cellular process. Several macromolecules orchestrate this event, however, type I collagen and noncollagenous proteins (NCP) are the key players. Dentin matrix protein-1 (DMP-1) is an acidic, NCP associated with the collagen matrix of mineralized tissues such as dentin and bone. DMP-1 has been shown to have collagen binding and HA nucleating domains that allow the protein to adhere to the collagen matrix and subsequently nucleate HA. In this study, we have engineered 2 designer nanopeptides containing the collagen binding and apatite nucleating domains of DMP1. The two peptides that were synthesized had molecular weights of 1726 (pA) and 2815 (pB) daltons, each containing both a single collagen binding and HA nucleating domain. Freshly extracted human third molars were sectioned to provide 3mm x 1.4mm x 250µm coronal dentin wafers that were subsequently demineralized in either 0.5M neutral buffered EDTA for 36hrs or 10% phosphoric for 12hrs. Complete demineralization was verified by X-ray analysis for radio opacity. The wafers from both demineralization methods were independently incubated over night in either pA or pB. Uncoated wafers, recombinant DMP1 and BSA coated wafers served as controls. Nucleation was carried out in a specialized nucleation chamber under physioilogical concentrations of calcium and phosphate ions. Scanning electron microscopy (SEM) showed the presence of calcium phosphate HA crystals associated with the collagen matrix of wafers incubated in both peptides pA and pB along with recombinant DMP1. Dentin wafers were also characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray diffraction. The data from our experiments suggest that our designer nanopeptides were able to bind to native 3-dimentional collagen scaffold and nucleate HA. Thus, biomimetic based nanopeptides could be utilized as a clinically relevant tool for remineralization.This work was supported by NIH DE 11657, 5T32DE018381 and the Brodie Endowment Fund.
9:00 PM - NN3.13
Mechanism of Orientation Control during Collagen Mineralization.
Haihua Pan 1 2 , Ruikang Tang 2 , Xiang Yang Liu 3 , Jinhui Tao 1 , Jim DeYoreo 1 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Lab, Berkeley, California, United States, 2 Chemistry, Zhejiang University, Hangzhou, Zhejiang, China, 3 Physics, National University of Singapore, Singapore Singapore
The formation mechanism governing hydroxyapatite (HAP) crystal alignment in bone has remained unknown for decades. In conflict with the classical template model, structural analysis demonstrates there is no epitaxial relationship between the mineral and collagen-I matrix. In this work, we investigate the forces that drive alignment of HAP crystallites during formation within the collagen matrix. We mimic bone formation by using simulated body fluid to precipitate amorphous calcium phosphate (ACP) onto type-I collagen fibrils assembled into the well-known D-band structure. Electron diffraction shows that subsequent transformation from ACP to HAP occurs with specific crystallographic alignment relative to the fibril axis, and the alignment of HAP crystallites along collagen fibril is improved further as the crystallites grow. Molecular dynamics (MD) simulations show that a non-specific force field, primarily due to electrostatic interactions and possessing a deep minimum along the axis of the assembled collagen fibrils, guides the oriented crystallization of HAP through minimization of free energy. The depth of this free energy trap increases with the size of the HAP crystallite and directs alignment of HAP along the fibril axis. This is predicted to give rise to increasingly better alignment of HAP crystallites as they grow, as found through electron diffraction measurements. In contrast, this observation does not support the conventional template model in which the alignment should be perfect and unchanged as epitaxial growth of the crystallite on the template progresses. Moreover, mineral-matrix structural mismatch in other biomineral and biomimetic systems suggests free energy traps resulting from non-specific forces may provide a general mechanism of orientation control.
9:00 PM - NN3.14
Picolitre Droplets Enable Study of the Early Stages of Crystallization of Calcium Carbonate.
Christopher Stephens 1 , Yi-Yeoun Kim 1 , Hugo Christenson 2 , Fiona Meldrum 1 Show Abstract
1 School of Chemistry, University of Leeds, LEEDS United Kingdom, 2 School of Physics and Astronomy, University of Leeds, Leeds United Kingdom
The heterogeneous nucleation and growth of CaCO3 was studied within regular arrays of picoliter droplets created on patterned self-assembled monolayers (SAMs). The SAMs provide well-defined substrates that offer control over CaCO3 nucleation, and these impurity-free droplet arrays were used to study crystal growth in spatially and chemically controlled, finite-reservoir environments.Here, SAMs of fluoroalkylthiols on Au were patterned using “deep” UV photolithography and backfilled with carboxylic acid-terminated alkylthiols, yielding circular hydrophilic regions with radii of 4−10 μm on a hydrophobic background. Supersaturated aqueous solutions of Na2CO3 and CaCl2 were then passed across the microengineered surfaces, trapping solution on the hydrophilic areas and leading to arrays of up to 20 000 independent droplets, which were stable for days at 100% humidity. Examination of crystallization in these arrays with time revealed a number of remarkable features. CaCO3 crystallization proceeded significantly more slowly in the droplets than in the bulk, allowing the mechanism of crystallization, which progresses via amorphous calcium carbonate, to be easily observed. In addition, the precipitation reaction terminated at an earlier stage than in the bulk solution, revealing intermediate growth forms. Confinement can therefore be used as a straightforward method for studying the mechanisms of crystallization on a substrate without the requirement for specialized analytical techniques. Furthermore, the results are also significant for biomineralization processes, where mineral formation occurs both within compartments and in association with organic matrices, showing that the environment in which a crystal forms can have a significant effect not only on its morphology and orientation but also on the rate of crystallization.
9:00 PM - NN3.16
Development of Amorphous Nanosilver-Chitosan-Collagen Biomaterial as an Antimicrobial Haemostatic Wound Healing Dressing.
Xingguo Cheng 1 Show Abstract
1 , Southwest Research Institute, San Antonio, Texas, United States
There is an urgent need to develop novel multifunctional wound healing dressing/bandage that is both haemostatic and antimicrobial due to severe burn, trauma, invasive surgery, and diseases (e.g., diabetic ulcers, venous wounds). This multifunctional wound dressing/bandage not only benefits warfighters, but also benefits the family members, other beneficiary of military health care, and even general public. Here we presented the fabrication of sliver-chitosan-collagen sheets which are suitbable to be used as antimcirobial haemostatic wound dressings. By using a novel, aqeous electrochemical process at low-voltage, collagen-chitosan sheets with controlled thickness, composition, and geometry were prepared. These chitosan collagen sheets were modified to be enriched with pending aldehyde groups. After simple immersion in silver nitrate soution, nanosilver was deposited onto the chitosan-collagen sheets. X-ray diffraction analysis indicated that the silver deposited is amorphous. SEM-EDX analysis indicated that silver nanoparticles (around 180 nm) as well as silver film was formed onto the chitosan collagen matrix. These amorphous nanosilver-chitosan-collagen sheets gradually released silver ions after immersion in physiological buffer, thus providing broad-spectrum antimicroibal activity. Since each component (collagen, chitosan, and silver) has been developed into wound care products and approved by FDA (e.g., SugiAid® Collagen Wound Dressing, Hem-Con® Chitosan bandage, Silver bandage), we expect that our novel amorphous nanosilver-chitosan-collagen biomaterial has the following advantages: 1) Collagen is haemostatic and promotes cell attachment and facilitates wound healing. 2) Chitosan is haemostatic, bacteria-static/bactericidal, and strong. 3) Nanosilver has improved broad-spectrum antibacterial properties compared with bulk silver due to the large surface area and high fraction of surface atoms. Silver NPs have strong bactericidal effect against multi-drug resistant bacteria commonly found in the battle field. Our future research will be directed towards developing and testing such novel biomaterial in a clincal setting. We expect amorphous nanosilver-chitosan-collagen wound dressing would help prevent complications from infections, reduce blood loss, and promote wound healing.
9:00 PM - NN3.17
Calcium Sulfate Dihydrate (Gypsum) Precipitates via Amorphous and Hemihydrate Intermediary Phases.
Yunwei Wang 1 , Yi-Yeoun Kim 1 , Hugo Christenson 2 , Fiona Meldrum 1 Show Abstract
1 School of Chemistry, University of Leeds, LEEDS United Kingdom, 2 School of Physics and Astronomy, University of Leeds, LEEDS United Kingdom
This work uses transmission electron microscopy and electron diffraction to investigate the nanoscale processes occurring during the early stages of precipitation of calcium sulfate from aqueous solution at room temperature. There is currently significant interest in non-classical crystallisation mechanisms and crystallisation via amorphous precursor phases, particularly in the bio-related calcium carbonate and calcium phosphate systems. This study makes steps towards determining the generality of this mechanism by investigating the possible existence of amorphous calcium sulfate (ACS). The experiments reveal a new crystallisation mechanism, where amorphous calcium sulfate and calcium sulphate hemihydrate are sequentially precipitated prior to calcium sulphate dihydrate (gypsum). This represents not only the first report of an amorphous calcium sulfate phase, but also of the synthesis of hemihydrate from a pure calcium sulphate solution at room temperature. Our results therefore highlight the possibility that many crystals may precipitate from solution via transient amorphous precursor phases, but that these are frequently overlooked due to their short lifetimes. Such in-depth understanding of the mechanisms by which crystals precipitate will contribute to the development new strategies leading to the control of crystal nucleation and growth.
9:00 PM - NN3.18
Metal Dependent Interactions of Mussel Adhesive Protein.
Dong Soo Hwang 1 2 , Hongbo Zeng 2 , Jacob Israelachvili 2 , Herbert Waite 2 Show Abstract
1 Ocean Science and Technology Institute, Pohang University of Science And Technology (POSTECH), Pohang Korea (the Republic of), 2 Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, California, United States
Adhesives and coatings from mussels have been investigated as a source of potential biomedical underwater adhesives because of their fascinating properties. These properties include strong cohesion, adhesion to various material substrates, water displacement, biocompatibility, and controlled biodegradability. Byssal threads in mussels (Mytilus sp.), extracellular connective tissues, enable strong underwater adhesion for mussels in the intertidal zone. Byssal thread is mainly composed of mussel foot proteins (mfps). Most mfps are responsible for adhesion and coating of mussel and contain Dopa (3, 4-dihydroxyphenylalanine) side-chains from 3 to 30 mole % depending on mfps type. Recently, Secondary Ion Mass Spectrometry (SIMS) and Energy dispersive X-ray (EDX) studies on byssal thread revealed byssal thread contains extraordinary levels of metal ions, and resonance Raman microscopy on byssal thread has directly confirmed the presence of DOPA-Fe(III) complexes in byssal thread. Therefore, metal mediated interaction between DOPA containing mfps using Surface force Apparatus (SFA) was investigated to understand and mimic of mussel adhesion mechanism. In the presence of metal ions, significant and reversible adhesion added when two mfp layers were brought into contact and separated at SFA. Among them, the cohesion of mfp-1 mediated by Fe (III) was reversible more than 10 times in the aqueous buffer and the magnitude of cohesion energy (~-20 mN/m) reached half the adhesion energy between biotin and avidin layers immobilized in the lipid bilayer (~- 40mN/m). Adhesions mediated by metal ions would be good example of adaptation of naturally occurring mussel adhesive to continuous stress exerted by seawater (i.e. tide and buoyancy) by means of an efficient energy dissipation mechanism. Although some of the adhesive bonds mediated by metal ions can be broken by mechanical stress exerted from the environmental change, they can be recovered without any requiring heat or high pressure. Furthermore, mimicking metal-mediated adhesion of mfps will be an important step for invention of wet industrial and biomedical underwater adhesives
9:00 PM - NN3.19
The Assembly of Gold Nanoparticles on DNA: Effect of Ligands Charge and Polarity.
Yaroslava Yingling 1 , Abhishek Singh 1 Show Abstract
1 Materials Science and Engineering, North Carolina State University, Raleigh , North Carolina, United States
DNA template can be used to trigger the self-assembly of metal or semiconductor nanoparticles into thin wires or predefined network for electronic devices. We performed atomistic molecular dynamics studies to investigate the effect of colloidal goldnanoparticle (GNP) ligands charge and polarity on the ability to bind DNA molecules. The end groups of GNP ligands were methyls (hydrophobic), amines (polar) and protonated amines (charged). We found that uncharged GNPs and GNPs with cationic ligand charge density of less than 10% can only bind to the minor groove of DNA. Whereas GNPs with ligands charge density of higher than 10% can bind to major or minor groove. Binding to major groove result in significant distortion and wrapping of DNA around the GNP. The extend of the DNA helical distortion strongly depends on the ligand charge density. We observed that by tuning the cationic charge density and polarity of GNP we can control the extend and efficiency of binding of NPs to DNA strands.
9:00 PM - NN3.2
Tooth Enamel Ultra-Structure: Correlation between Composition and Physical Properties.
Hazem Eimar 1 , Benedetto Marelli 1 , Showan Nazhat 1 , Samer Abinader 1 , Wala Amin 2 , Jesus Torres 3 , Rubens Albuquerque 1 , Faleh Tamimi 1 Show Abstract
1 Faculty of Dentistry, McGill University, Montreal, Quebec, Canada, 2 Faculty of Dentistry, The University of Jordan, Amman Jordan, 3 Department of Health Science III, Universidad Rey Juan Carlos, Alcorcon Spain
Tooth enamel is one of the hardest materials found in the animal kingdom. For this reason, understanding its unique properties is of special interest for the development of new synthetic materials. Enamel is a composite material that comprises an inorganic matrix composed of hierarchically organized carbonated hydroxyapatite (HA) crystals, and an organic matrix mainly composed of the protein amelogenin. This study was designed to investigate how variations in enamel ultrastructure and chemical composition (organic and inorganic) may affect tooth mechanical and optical properties. One hundred extracted sound teeth were collected from adult patients attending McGill Undergraduate Dental Clinic. Shade spectrophotometry, Vickers microindentation, FTIR, SEM-EDS and XRD were used to asses tooth shade (registered in universal shade parameters: Lightness, Chroma and Hue), enamel microhardness, chemical composition and crystallography (i.e. crystal size, lattice parameters of the crystal; a- and c- axes). The data obtained was analyzed for correlation, and statistical significance was set at P <0.05. Tooth enamel crystallographic structure, chemical composition, optical and mechanical properties varied dramatically within the studied population. Tooth enamel microhardness was affected by the size of HA crystals (R=-0.476, B=-0.028, P=0.001). Tooth shade hue was affected by enamel HA crystal size (R=-0.358, B=-0.866, P=0.007), tooth shade chroma was affected by enamel HA carbonization (R=-0.420, B=-99.06, P=0.0005), and tooth shade lightness was influenced by both the degree of HA carbonization (R=-0.266, B=-57.95, P=0.034), and HA crystal size (R=-0.313, B=-1.052, P=0.019). On the other hand, tooth enamel HA crystal size was inversely correlated to the relative protein content within tooth enamel (R=-0.352 B=-19.4, P=0.016). In the present study we have shown that variation in tooth enamel ultrastructure and chemical composition can affect its optical and mechanical properties. For instance, we have revealed that the protein content in teeth regulates enamel mechanical and optical properties by limiting the size of HA crystals. On the other hand, variation in the degree of enamel HA carbonization can also affect the tooth optical properties.
9:00 PM - NN3.23
Designed α-Helical Peptides for the Inhibition of Calcium Phosphate Growth.
Eric Chang 1 , Michael Ogawa 1 Show Abstract
1 Chemistry, Bowling Green State University, Bowling Green, Ohio, United States
The design of α-helical peptides based on the well understood coiled-coil motif serves as a rational means of developing supramolecular assemblies with defined structure and function. These systems have been tuned for a variety of applications including the binding of metals ions and the forming of fibrous networks, yet their effect on the growth of biologically relevant minerals such as calcium phosphates (CaP) remains largely unknown. Ideally, understanding how to design α-helical peptides to interact with growing CaP systems will allow them to be used in a biomimetic fashion to either nucleate or inhibit crystal growth. To this aim, our group seeks to understand how a series of α-helical, coiled-coil peptides with varying numbers of rigidly-arrayed acidic residues affects the growth of calcium hydrogenphosphate dihydrate (CaHPO4 x 2H2O, also called dicalcium phosphate dihydrate, DCDP) from a saturated solution of sodium phosphate and calcium acetate. Turbidity assays that monitor reaction kinetics have shown that increasing the concentration of these peptides increases the time needed for the DCDP crystals to form. This effect becomes stronger with increasing numbers of acidic residues and suggests an inhibitory mechanism. Scanning electron microscopy (SEM) and isothermal calorimetry (ITC) are being used to determine if the inhibition is due to the peptides interacting with growing DCDP crystal faces or binding to free calcium and/or phosphate ions. Ultimately, the findings of this study will allow our group to develop other α-helical peptides whose design can be used to rationally control the formation of important biological minerals.
9:00 PM - NN3.3
Kinetic Evolution of Surfactant Templated Calcium Phosphate Structures.
David Griffin 1 , Chris Ziegler 2 , Greg Tew 2 , Surita Bhatia 1 Show Abstract
1 Chemical Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States, 2 Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States
Bone tissue is a ubiquitous and ideal model for highly organized, hierarchical, naturally occurring biocomposites. In this study, calcium phosphate mineral formation in the presence of a templating triblock copolymer matrix was investigated as a construct for the natural mineralization process in bone. Though the exact thermodynamic and kinetic mechanisms by which the process of natural tissue mineralization occurs continues to be a topic of much debate, it has been shown multiple times that calcium phosphate mineral formation in a polymer template is preceded by an amorphous precursor. Favorable interactions between the precursor and polymer network act to retard the rate at which mineral forms and can induce the formation of unique crystal phases. This investigation uses time resolved X-ray diffraction (XRD) and small-angle X-ray scattering (SAXS) to characterize calcium phosphate mineral formation in a Pluronic® F108 polymer matrix. Solutions of F108 containing phosphate and calcium precursors were drop-cast on glass slides and allowed to dehydrate at 40 °C over a time of approximately 250 hours. Results indicate that phosphate ions are preferentially sequestered by the hydrophilic domain (PEO) of the surfactant. This interaction can lead to a lower rate of mineralization and, subsequently, a unique composite mineral composition. The degree to which hydrophilic polymer domains are saturated also plays an important role in determining final polymer microstructure.
9:00 PM - NN3.4
Understanding Hard-Soft Interfaces.
John Harding 1 , Steven Banwart 2 , James Elliott 3 , Tiffany Walsh 4 , Mark Rodger 4 , Fiona Meldrum 5 , Roland Kroger 6 , Dorothy Duffy 7 , Susan Stipp 8 Show Abstract
1 Dept. of Materials Science and Engineering , University of Sheffield, Sheffield United Kingdom, 2 Dept. of Civil and Structural Engineering, University of Sheffield , Sheffield United Kingdom, 3 Dept. Materials Science and Metallurgy, University of Cambridge, Cambridge United Kingdom, 4 Dept. of Chemistry and Centre for Scientific Computing, University of Warwick, Coventry United Kingdom, 5 School of Chemistry, University of Leeds, Leeds United Kingdom, 6 Dept. of Physics, University of York, York United Kingdom, 7 Dept. of Physics and Astronomy, University College London, London United Kingdom, 8 Nanoscience Center, University of Copenhagen, Copenhagen Denmark
The interface between minerals (hard) and organic molecules, arrays and scaffolds (soft) unites two kinds of problem. If we take the mineral surface as given and consider the adsorption of the soft interface upon it, we study problems of recognition and adaptation of molecules to surfaces. Examples include conformational folding induced by the surface and effects on molecular function. At longer length-scales we consider the binding of cells or bacteria to surfaces, reaching the areas of microbial growth and attachment and the formation of biofilms. On the other hand, if we take the soft side (particularly arrays and scaffolds) as given and focus on its ability to nucleate solids, we study the problem of biomineralisation — the process by which organisms make minerals. Understanding this process involves elucidating a range of strategies at several length and timescales: directed nucleation, templating, confinement, inhibition, and aggregation. The resulting biomaterials have complex hierarchical structures, often with distinct structural features at different length-scales, elaborate morphologies and distinctive properties. We present a number of examples from our recent work showing how a combination of theory and experiment can shed light on the fundamental mechanisms involved: from the atomic to the macroscopic level. Example systems include interfaces between soft matter and carbonates, phosphates, fused silica and quartz.
9:00 PM - NN3.5
Effect of Thermal Treatment on the Self-Assembly Behavior and Secondary Structure of Spider Silk-like Di-Block Copolymers.
Wenwen Huang 1 , Sreevidhya Krishnaji 2 3 , David Kaplan 3 , Peggy Cebe 1 Show Abstract
1 Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, United States, 2 Department of Chemistry, Tufts University, Medford, Massachusetts, United States, 3 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Biophysical studies have found evidence that different motifs are responsible for forming various secondary structures with corresponding influence on silk morphology, crystallinity, and mechanical properties. To obtain a fuller understanding of the origin of self-assembly behavior, and thus be able to control the morphology of biomaterials with well defined amino acid sequences for tissue regeneration and drug delivery, we have embarked on a program to create synthetic proteins inspired by the genetic sequences found in spider dragline silks (Nephila clavipes). We used these bioengineered spider silk block copolymers to study secondary structures and morphological features. In recent work, we synthesized a new family of silk-based block copolymers, HABn and HBAn (n=1,2,3,6), where B = hydrophilic block, A = hydrophobic block, and H is a histidine tag, and treated them with 1,1,1,3,3,3-Hexafluoro-2-propanol to obtain wholly amorphous samples. Samples crystallized by exposure to methanol were also studied as a control group. Thermal properties of these materials were determined by differential scanning calorimetry and thermogravimetry. Then we annealed the amorphous samples at 180°C, near their glass transition. In the Amide I region, the rate of beta-sheet crystal growth during annealing was monitored by real-time Fourier transform infrared spectroscopy. Beta sheets grew faster in samples with higher volume fraction of A-block and reached a higher crystallinity after annealing. The morphology can also be tuned by annealing. Using scanning and transmission electron microscopies, we observe fibrillar network structure in annealed samples, while in sample cast from methanol solution, we observed hollow micelles and fibrillar structures.Support was provided from the National Science Foundation, Division of Chemical, Bioengineering, Environmental, and Transport Systems, through CBET-0828028 and the MRI Program under DMR-0520655 for thermal analysis instrumentation.
9:00 PM - NN3.6
Surface-Directed Mineralization of Calcium Phosphate: Effects of Solution Composition and Surface Properties.
Rixiang Huang 1 , Boris Lau 1 Show Abstract
1 Geology, Baylor University, Waco, Texas, United States
In-vitro experiments immersing various substrates in simulated solutions have been frequently used to explore the mechanism behind the formation of biominerals containing calcium phosphate. However, most previous studies have yet to exclude the interference from homogeneous mineralization in the bulk solution and to provide direct evidences in determining whether the precipitates were mineralized by means of ion-based or amorphous precursors. In this project, we investigated the combined effects of surface and soluble organics on the biomineralization of calcium phosphate in order to provide information that are more relevant in in-vivo system. Specifically, simulated body fluid (SBF) and supersaturated solution containing polyaspartic acid (PAA) were thoroughly characterized and used in the surface-directed mineralization of calcium phosphate. It was shown that solutions with different compositions produced prenucleation clusters of different sizes and surface charges. Prenucleation clusters with hydrodynamic diameter of about 600 nm and zeta-potential of +4 mV were formed in the SBF. Stable prenucleation clusters with hydrodynamic diameter of about 300 nm and zeta-potential of -25 mV were formed in PAA. Substrate surfaces were prepared by coating quartz crystal sensors with different self-assembled monlayers: i) 11-mercaptoundecanoic acid (MUA, –COO- as terminal functional group for a negatively-charged surface), ii) 11-mercaptoundecanol (MUD, –OH as terminal functional group for a neutrally-charged surface), and iii) 11-amino-1-undecanethiol (AUT, –NH3+ as terminal functional group for a positively-charged surface). Surface deposition of the prenucleation clusters onto substrate surfaces were followed in real time by quartz crystal microbalance with dissipation (QCM-D). For both types of prenucleation clusters, the deposition kinetics onto the MUA surface was always faster than onto the MUD and AUT surfaces. Calcium phosphate mineralization seems to be directed by substrate surface and independent of the surface charge of the prenucleation clusters. It can also be confirmed that the surface-directed mineralization of calcium phosphate starts with the adsorption of amorphous prenucleation clusters instead of forming nuclei on the surface directly from ions in the solution. Surface-directed crystallization of the surface deposits is being characterized to reveal the detailed mineralization process and to identify the effects of surface and solution organics on this process.
9:00 PM - NN3.7
Mineralization of Chitosan-Based Biomaterials.
Nasreen Khan 1 , David Shields 1 , Amalie Donius 1 , Philipp Hunger 1 , Phuong Tran 1 , Ulrike Wegst 1 Show Abstract
1 Material Science and Engineering, Drexel University, Philadelphia, Pennsylvania, United States
Currently, artificial bone implants are made of inorganic materials that provide immediate relief to various bone replacement issues. However, long term replacements are limited due to mechanical and biological dissimilarities. That is why facilitating bone's natural ability to create a mineral component within a polymer matrix will greatly improve implant functionality. Specifically hydroxyapatite, a tricalcium phosphate derivative, can be incorporated into natural and biocompatible polymers with simple heating and desiccation techniques. Various methods for mineralization on other polymers such as PHEMA and agarose have been performed, leading way to different types of calcium based crystal structures. In this fashion, we have attempted two known techniques on the biopolymer chitosan, as an alternative to previous studies and mineral formations on other polymers. Using both heated urea bath and desiccation techniques, the growth of various hydroxyapatite and calcium carbonate crystal structures have been incorporated into chitosan. Greater crystallization within the substrate is possible by dissolving mineral directly into the chitosan polymer solution before processing. Controlling processing time and rate can also determine the size of crystal structures. With these techniques, the mineralization of 3-D chitosan and other biopolymer scaffolds is possible, allowing scaffolds and implants to possess a mineral phase that encourages the integration of bone cells such as osteoblasts to promote self-repair within the body. As a result, these implants are more similar to natural bone.
9:00 PM - NN3.8
Spectrum Imaging Analysis for Nanoparticles in Biological Specimens.
Hansoo Kim 1 , Ren Miao 2 , Paul Lindahl 2 Show Abstract
1 Microscopy and Imaging Center, Texas A&M, College Station, Texas, United States, 2 Chemistry, Texas A&M, College Station, Texas, United States
Spectrum imaging (SI) by an electron-microscope-spectroscopy system has been applied to analysis of materials. Various kinds of nanomaterials and bulk samples were characterized previously by SI method for component elements, physical and chemical properties, and so forth. In this study this method was successfully employed in biological specimens. It was found that due to the low atomic numbers of elements in cells metallic compounds with higher average atomic numbers can be directly observed down to a couple of nanometer in size by high angle annular dark field (HAADF) detector in scanning transmission electron microscopy (STEM). On top of that the component elements could be identified individually and quantified by energy dispersive x-ray spectroscopy (EDS) attached to STEM. Using the small 1-nm probe of STEM SI maps could be collected from extremely localized spots, which shows spatial distribution of the elements. By applying this method to biological specimens, Yah1p- and Atm1p-depleted Saccharomyces cerevisiae, local enrichment of specific elements in cells could be confirmed and its chemistry could be understood to some extent. By the HAADF detector Z-contrast images collected from highly scattered electrons show distinct brightness in the high Z (atomic number) area with high spatial resolution. In addition, the combination of STEM and EDX also allows compositional analysis of a nanometer-scale area. In fact, when an EDX spectrum was obtained Fe, P, and O elements were the only clearly detected elements other than carbon. To see the spatial distribution of each element, elemental maps for Fe, P, and O were obtained together after collection with a proper energy window assigned to the Kα X-ray fluorescent transition of each element.
9:00 PM - NN3.9
An Artificial Capsid Produced by Gold-Catalyzed Protein Remodeling.
Ali Malay 1 2 , Jonathan Heddle 1 , Satoshi Tomita 2 , Kenji Iwasaki 3 , Naoyuki Miyazaki 3 , Koji Sumitomo 4 , Hisao Yanagi 2 , Ichiro Yamashita 2 , Yakiharu Uraoka 2 Show Abstract
1 Advanced Science Institute, RIKEN, Wako, Saitama, Japan, 2 Graduate School of Materials Science, Nara Institute of Science & Technology, Ikoma, Nara, Japan, 3 Institute for Protein Research, Osaka University, Osaka Japan, 4 , NTT Basic Research Laboratories, Atsugi, Kanagawa, Japan
Bottom-up approaches in nanofabrication usually entail the creation of useful nanoscale structures through the rational modification of pre-existing building blocks, such as DNA or protein elements. In certain cases, however, new and unexpected nanostructures may be produced. In this study, we report the formation of a novel protein architecture from a stable precursor, in a reaction that is catalyzed by the addition of gold nanoparticles (NPs) under benign conditions. The starting material is a mutated thermostable ring-shaped protein with a diameter of 8 nm. Upon reaction with gold NPs at micromolar concentrations, the protein undergoes dramatic structural remodelling, resulting into hollow spheres reminiscent of viral capsids. Two distinct sizes of capsid-like structure (CLS) can be produced depending on the concentration of gold NPs. The resulting CLSs are highly thermostable yet the reaction is readily reversed by the addition of reducing agents. The mechanism behind this highly unusual remodeling of a stable protein multimer into a completely different structural form is discussed, and a catalytic protein unfolding-refolding effect of gold NPs is implicated. The resulting novel structures may have the potential to act as a nano-cargo carrier with tunable self-assembly properties. Finally, the implications of using gold NPs in nanomedicine with regard to toxicity will be considered.
John Harding University of Sheffield
P. Mark Rodger Warwick University
Laurie B. Gower University of Florida
Peter Vekilov University of Houston
NN4: Organic/Inorganic Interfaces
Wednesday AM, November 30, 2011
Room 104 (Hynes)
9:30 AM - **NN4.1
Crystalline-Molecular Interfaces: What is Hard and Soft?
Colin Freeman 1 , John Harding 1 , David Quigley 2 3 , Mark Rodger 3 4 , Jim DeYoreo 5 Show Abstract
1 Materials Science and Engineering, Univ Sheffield, Sheffield United Kingdom, 2 Department of Physics, University of Warwick, Coventry United Kingdom, 3 Centre for Scientific Computing, University of Warwick, Coventry United Kingdom, 4 Department of Chemistry, University of Warwick, Coventry United Kingdom, 5 The Molecular Foundry, Lawrence Berkeley National Labs, Berkeley, California, United States
In considering the interface between soft and hard matter we often think of the hard matter as rigid and immobile while the soft matter is readily deformed. In contrast, in the search for molecular templates for the growth of “hard” materials, the designer often thinks of the “soft” molecular shape as regular and the hard material as adaptable to this template. The interface is, however, very complex and frequently both the hard and soft materials can exert some influence upon each other, potentially in a complementary fashion.We present series of simulations on different systems where the importance of allowing flexibility within the system is examined. In the first scenario we consider the use of self-assembled monolayers (SAMs) to generate particular facets of calcite. SAMs have frequently been shown to be highly selective in producing a particular crystal surface although the reasons are still uncertain . We consider the effect of giving flexibility to the “soft” SAM template and how this influences the growth control . In the second scenario we examine the binding of the protein Ovocleidin-17 to an amorphous calcium carbonate interface. Without crystalline order this mineral interface behaves very differently to the calcite surface despite the same chemical components. In our third scenario we discuss the mineral surface. In biomineral simulations, this is frequently considered to be largely static and its function as a binder predominantly as an array of dangling bonds or charged points. We examine the effect of “freezing” this mineral or allowing it flexibility and how this influences the binding of molecules at the interface. Y.-J. Han, J. Aizenberg, Angew. Chem. Int. Ed. 42 (2003) 3668 D. Quigley, P.M. Rodger, C.L. Freeman, J.H. Harding, D.M. Duffy, J. Chem. Phys. 131 (2009) 094703
10:00 AM - NN4.2
Interfacial Energy of Calcite-MHA/MUA Self-Assembled Monolayers during Nucleation.
Qiaona Hu 1 2 , Mike Nielsen 2 , Udo Becker 1 , Jim De Yoreo 2 Show Abstract
1 geological sciences, U. of Michigan, Ann Arbor, Michigan, United States, 2 Molecular Foundry, Lawrence Berkeley National Lab, Berkeley, California, United States
Calcium carbonate (CaCO3) mineralization controlled by biogenetic activities, which occurs widely on the earth’s surface, is crucial for carbon cycling and ocean water chemistry. Evidences show that templated nucleation is a widespread strategy for controling the polymorph, morphology, and crystallographic orientation of CaCO3 precipitates. In the past decade, self-assembled monolayers (SAMs) of alkanethiols have been used as a simple model to reproduce the controls of organic substrates.Self-assembled monolayers of COOH-terminated 16-mercaptohexadecanoic acid (MHA) and 11-mercaptoundecanoic acid (MUA) monomers have been reported to strongly favor nucleation of calcite on the non-natural (012) and (013) faces, respectively (Han and Aizenberg 2003). However, no experimental work has been done to quantify the kinetic and thermodynamic barriers associated with templating on SAMs. Moreover, the requirement that nucleation proceed via an indirect pathway involving amorphous precursors remains unresolved. The objective of this study was to evaluate the interfacial energies associated with template-directed calcite on MHA and MUA, and to determine if there is a range of driving force at which nucleation occurs via the direct pathway. The results reveal that (1) MHA and MUA both significantly reduce the effective surface energy of calcite from about 97 mJ/m2 in solution to about 43.9 and 45.1 mJ/m2 respectively, providing a thermodynamic basis for the strong capacity of MHA and MUA to promote calcite nucleation; (2) the influence of the odd-even effect on the nucleation rate has little to do with differences in the thermodynamic barrier, but is due to unknown kinetic drivers; and (3) at solute activities below and slightly above the solubility limit of amorphous calcium carbonate, calcite forms directly.
10:15 AM - NN4.3
Positively Charged Additives Can Strongly Affect Calcium Carbonate Precipitation.
Bram Cantaert 1 , Yi-Yeoun Kim 1 , Henning Ludwig 1 , Fabio Nudelman 2 , Nico Sommerdijk 2 , Fiona Meldrum 1 Show Abstract
1 School of Chemistry, University of Leeds, LEEDS United Kingdom, 2 Laboratory of Materials and Interface Chemistry, Eindhoven University of Technology, Eindhoven Netherlands
Soluble macromolecules are essential to control over biomineral formation and properties. Following early studies where acidic macromolecules rich in aspartic and glutamic acid were extracted from nacre, negatively charged additives have been considered unique in their ability to control calcium carbonate precipitation. Here, we provide a dramatic demonstration that positively charged additives can be as important in controlling calcium carbonate precipitation and in doing so we challenge the current understanding that positively charged additives have little influence on CaCO3 growth. Calcium carbonate was precipitated on exposure of a 10 mM CaCl2 solution containing PAH (poly(allylamine hydrochloride, at concentrations of between 5 µg/ml to 2000 µg/ml to ammonium carbonate vapour. The results clearly demonstrate that poly(allylamine hydrochloride) (PAH) can stabilise amorphous calcium carbonate and direct the formation of thin films and fibres of CaCO3 analogous to those produced with poly(aspartic acid) via a so-called PILP (polymer induced liquid precursor) phase. These results are attributed to a microphase separation of PAH in the presence of carbonate ions rather than a strong interaction of the amine functional groups with the growing calcium carbonate crystals. In demonstrating for the first time that a system other than CaCO3 PAA/ PAsp can lead to such control over crystallisation, we highlight the opportunity for using such a counter-ion induced phase separation as a synthetic tool, and believe that this method can be used to create a wide range of minerals with non-crystallographic morphologies.
10:30 AM - NN4.4
The Influence of Biomolecules on the Dislocation-Driven Growth of 2D Nanoplates.
Audrey Forticaux 1 , Stephen Morin 1 , Song Jin 1 Show Abstract
1 Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States
The architectural diversity and complexity of crystals produced in natural biomineralization processes is far from being rivaled by synthetic nanomaterials. Understanding the role of matrix macromolecules in the fundamental crystal nucleation and growth processes that yield such biominerals can enable precise and efficient crystal morphology engineering of functional nanomaterials. Here we report the screw dislocation-driven growth of several two dimensional (2D) nanoplates from aqueous solutions under mild biomimetic conditions and propose a unified scheme general to any crystalline material that explains the growth of nanoplates as well as different dislocation-driven nanomaterial morphologies. Not only could these nanomaterials find application in energy storage, catalysis, and nanoelectronics, but their nanoplate morphology also provides a convenient platform for atomic force microscopy (AFM) and scanning electron microscopy (SEM) studies on how dislocation spirals and their associated step edges as well as the overall shape of the nanoplates are impacted by biomolecules such as β-peptides. To illustrate this, we will present preliminary results obtained from in situ fluid AFM investigations that show growth spiral evolution under the influence of select biomolecules. This study may eventually lead to a general understanding of how the cores and spiraling steps of screw dislocations are impacted by biomolecules applicable to biomineralization and other crystal growth processes.
10:45 AM - NN4.5
In Situ AFM Observation of Incorporation of Block Copolymer Micelles into Calcite Single Crystals.
Kang Rae Cho 1 , Yi-Yeoun Kim 2 , Haihua Pan 3 1 , Jolene Lau 1 , Qiaona Hu 4 1 , Debin Wang 1 , Raymond Friddle 5 , Fiona Meldrum 2 , James De Yoreo 1 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 School of Chemistry, University of Leeds, Leeds United Kingdom, 3 Department of Chemistry, Zhejiang University, Hangzhou China, 4 Department of Geological Sciences, University of Michigan, Ann Arbor, Michigan, United States, 5 Department of Hydrogen and Metallurgy, Sandia National Laboratories, Livermore, California, United States
Single crystals of biominerals often incorporate biopolymers, leading to enhancement of their mechanical properties. For example, sea urchin spines and plates, which are made of calcite single crystals incorporate cellular tissue networks and proteins, improving their fracture toughness. To gain insight into the incorporation process of biopolymers into single crystals of biominerals on the nano and micrometer scales, we observed the incorporation of a model biopolymer composed of micelles of a carboxylated block copolymer (PSPMA30-PDPA47) into growing calcite single crystals in real time using in situ atomic force microscopy (AFM). While at moderate pH values, the polymer adsorbed to surface as monomers and inhibited step growth, at higher pH where the micelles are stable, they adsorbed onto the calcite surface, but did not act as inhibitors of growth. Instead the steps closed around the adsorbed micelles without being inhibited and, through the continuous passage of steps, encapsulated the micelles into the crystal. In this way, a calcite-micelle single crystal composite was formed. Companion experiments on mica surfaces showed that the negatively charged micelles, which formed at the high pH of the growth experiments did not adsorb to negative (bare) mica but adsorbed to positive (polylysine-treated) mica. This suggests that the surface-exposed or background solution-calcium ions play a key role in establishing electrostatic adhesion of the micelles to the calcite surface.
11:30 AM - **NN4.6
Synthesis, Assembly, and Biological Properties of Calcium Phosphate Nanoparticles.
Lara Estroff 1 , Rui-Qi Song 1 , Debra Lin 1 , Siddharth Pathi 2 , Hiroaki Sai 1 , Claudia Fischbach 2 , Ulrich Wiesner 1 Show Abstract
1 Materials Science and Engineering, Cornell University, Ithaca, New York, United States, 2 Biomedical Engineering, Cornell University, Ithaca, New York, United States
Both bone and teeth are composed of nanoparticles of carbonated apatite assembled in an organic matrix. The materials properties of these particles (e.g., size, shape, crystallinity, composition, solubility, etc.) define, in part, the biological microenvironment in the tissue. In order to evaluate the effects of these different materials properties on biological systems, there is a need for synthetic methodologies that control nanoparticle formation and assembly. Here I will present two different strategies: 1) the incorporation of hydrothermally aged hydroxyapatite nanoparticles, with narrow size distributions and varying crystallinity, into gas-foamed/particulate leached poly(lactide-co-glycolide) scaffolds for studying breast cancer metastasis to bone, and 2) the use of amphiphilic diblock copolymers (e.g., poly(isoprene-block-dimethylaminoethyl methacrylate)) to structure-direct the assembly of sol-gel-derived amorphous calcium phosphate (ACP) nanoparticles into a range of mesostructures, including several types of continuous inorganic networks. Both of these strategies expand our ability to tailor the properties of calcium phosphate-based biomaterials and to evaluate the role of materials properties on biological function.
12:00 PM - NN4.7
Electrospinning of Partially Phosphorylated Hydrogel Polymers Designed to Promote Rapid Mineralization and Osteoblast-like-Cells Adhesion.
Pallab Datta 1 , Jyotirmoy Chatterjee 1 , Santanu Dhara 1 Show Abstract
1 School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India
Objectives: Interest in Phosphorylated Polymers (PP) is growing as designer materials to mimic the noncollagenous phosphoproteins in mediating matrix mineralization of hard tissues. We postulate electrospun nanofibers of phosphorylated polymers, which resemble the architecture of extracellular matrix of bone, can further provide templates and nucleating sites for controlled and ordered mineralization.Materials and Methods: PVA and Chitosan were selected as polymers of choice and phosphorylated with controlled degree of substitution. Parameters known to be critical for electrospinning of polymer solutions were evaluated. Finally developed nanofibers were characterized for chemical and mechanical properties, compared with non-phosphorylated polymers and their in vitro mineralization potential after treatment with SBF was observed by SEM, EDX and Alizarin red S staining.Results and Discussion: The chemical reaction adopted resulted in a polymer with acidic polymers containing the phosphate and phosphonic esters with chelation properties for calcium ions. The solution state and solid state physicochemical characterization of the polymers showed very widely different intermolecular bondings which was reflected in development of nanofibers based on phosphorylated polymers. Electrospun nanofibers of phosphorylated polymers remarkably accelerated the mineralization process compared to unmodified polymer nanofibers and also compared to the films/gels of phosphorylated polymers. MG 63 cells showed significantly higher adherence, higher proliferation rate and higher matrix mineralization.Conclusions: Phosphorylation of polymers was found to significantly accelerate the process of mineralization of nanofibrous matrix and also exhibit significant effects in increasing cell-material interactions inducing MG 63 cells matrix mineralization.
12:15 PM - NN4.8
Comparison of Mineralization Patterns in Juvenile and Mature Reef-Building Corals.
Renee van de Locht 1 , Anne Cohen 2 , Roland Kroeger 1 Show Abstract
1 Physics, University of York, York, North Yorkshire, United Kingdom, 2 , Wood Hole Oceanographic Institute, Woods Hole, Massachusetts, United States
One of the great challenges in the understanding of mineral formation in biological systems lies in the complexity of the interaction between the organic matter and the environment, which leads to the controlled precipitation of a crystalline phase by the organism. In reef building corals such as the Porites genus a rapid accretion of an aragonite carbonate phase provides a resilient exoskeleton required for the formation of reefs. Using both scanning and transmission electron microscopy (TEM), atomic force microscopy and Raman spectroscopy we investigated the exoskeleton of freshly formed juvenile coral skeletons in comparison to mature corals of the Porites genus. The juvenile corals show distinctly different growth patterns in that they accrete a base plate onto which the vertical structure is deposited after the coral larvae has settled on a surface. At an early stage of growth these structures reveal thorn-like protrusions around which the mineral phase is gradually deposited in form of acicular grains cladded around a center in a spiral fashion with their long sides approximately parallel to the direction of the protrusions. Raman spectroscopy shows that the tips of these protrusions are chemically different from the mineral phase surrounding it. This tip is most likely organic. Such protrusions could not be observed in the skeleton of the mature corals. This indicates a change in growth mode from the juvenile to the mature stage. Moreover, the investigation of polished and weakly etched cross-sections reveal a layer-type growth pattern with each layer consisting of acicular nanograins. The analysis of the etched polished cross-sections and the TEM analysis of the grain structure also indicate that there are at least two grain morphologies accreted by the organism: acicular and granular aragonite. These findings will be discussed in the context of existing models for coral skeleton growth.
12:30 PM - NN4.9
Biomimetic Mineral-Protein Composites Formed by an Automated Alternate Soaking Process.
Daniel Strange 1 , Oliver Armitage 1 , Michelle Oyen 1 Show Abstract
1 Department of Engineering, Cambridge University, Cambridge United Kingdom
There is considerable interest in producing mineralized materials using biomimetic methods due to their low energy costs and potential to create minerals more similar to those found in vivo. The alternate soaking process is a rapid method of forming hydroxyapatite and calcium carbonate coatings at room temperature and pressure. Substrates are alternately soaked in calcium- and phosphate- (or carbonate-) containing solutions, rinsing between each soak. However, the formed coatings are porous and brittle and it is difficult to form significant thicknesses of material. In this study, the alternate soaking process is modified and automated—molten gelatin is added to the soaking solutions in order to co-precipitate mineral-protein composites. Samples are investigated with Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive x-ray spectroscopy and nanoindentation. It is demonstrated that the resultant volume fraction of protein in the composite can be controlled by adjusting the concentration of protein in the soaking solutions. It is also shown that the composite coatings are mechanically robust. The modified process is used to fabricate a 2 mm thick hydroxyapatite-gelatin composite coating in 8.5 hours with a reduced modulus near to that of cortical bone. The mineral volume fraction of the composite was approximately the same as that of cortical bone (50%). Likewise, calcite-gelatin composites that contain a substantial mineral volume fraction (>90%) are deposited at a rate of approximately 50µm in 2 hours on de-mineralized eggshell membrane. This rate approaches that of egg-shell formation in vivo—although the process does not fully replicate the complex structure of natural egg-shell. This modified alternate soaking process opens up possibilities of rapidly creating biomimetic mineral-protein composites that more closely mimic the composition and structure-properties relationships of composites found in natural tissues.
12:45 PM - NN4.10
Citrate-assisted Growth of Hierarchical Hap Structures.
Maria Costa 1 , Catarina Santos 1 2 , Maria Almeida 1 Show Abstract
1 , University of Aveiro, Aveiro Portugal, 2 , Polytechnical Institute of Setubal, EST, Estefanilha, Setubal Portugal
The accurate manipulation of the building up of nano or microcrystals may precisely tune its morphology and hence the material properties which are intimately dependent on geometric factors, i.e. shape and size. These emerging engineered nanostructures are expected to bring a significant progress to the biomedical field, namely for diagnostics and therapies where new possibilities for achieving ideal drug delivery systems displaying a predictable behaviour are foreseen. Applications where the efficiency of a biologically active entity is conditioned by the dimensionality of its own carrier, will effectively benefit from nanoparticle able to penetrate selectively the target nanometer sized gaps. New synthesis methods enabling to produce nanoparticles with highly controlled morphology are thus currently needed. Developing such synthesis procedures and understanding their specific mechanisms at the early stage of particle nucleation and growth will prompt the design of nanoparticles with size and shape surgically tailored. Being unquestionable biomaterials for bone repair, osteologic implant coating, bone cements and scaffolds, calcium phosphate (CP) are being also exploited for novel drug delivery systems. The present work is focused on the study of Hydroxyapatite (Hap) synthesis, attempting to understand the role of citrate ion as a shaper tool of Hap particle. Citrate ion (Cit) is a biological friendly molecule with a well known complexing ability. Though its effectiveness on Hap morphology has already been reported, its effects at a high temperature at which Cit may undergo thermal decomposition were not clearly addressed so far. In this work a high-temperature, environmentally benign, solution-based approach containing calcium (Ca) and phosphate (P) precursors was selected for the preparation of Hap particles. The organic additive, i.e. Cit, was added to the precursor solution which was then autoclaved at T>150°C. The effects of the systematic variation of Cit amount, i.e of Cit/Ca ratio on the crystallinity, shape and size of the obtained Hap particles under specific conditions of temperature and pH are here reported for different growth times. Diferently sized particles ranging from nanometric to micrometric sized dimensions were precipitated. XRD and electronic microscopy analysis (TEM, SEM) of the precipitates reveal that crystalline hierarchical Hap structures comprising nanosized rods, hedgehog- and microsized fiber-like particles were engineered by the variation of Cit concentration. HPLC and FTIR analysis allowed accessing the products of Cit thermal decomposition and hence their contribution to modulate the nucleation step and also the shape evolution of Hap particles. The obtained results are discussed in the frame work of Hap nucleation and growth attempting to clarify the role of Cit-related species either as templating Hap nucleation either as limiting the Hap growth along along particular crystallographic directions.
NN5: Self-Assembly in Biological and Biomimetic Systems
Wednesday PM, November 30, 2011
Room 104 (Hynes)
2:30 PM - **NN5.1
Nucleation in Colloidal Systems Probed with Single-Particle Resolution.
Anthony Dinsmore 1 , Liquan Pei 1 , Derek Wood 1 , John Mergo 2 , John Savage 1 Show Abstract
1 Physics, University of Massachusetts, Amherst, Massachusetts, United States, 2 Physics, Cornell, Ithaca, New York, United States
Using colloidal spheres as model ‘atoms,’ we experimentally monitor the dynamics of freezing and melting in two dimensions, in the presence of controlled inter-particle interactions. Using optical microscopy, we track the motions of hundreds of individual microparticles with high resolution. Short-ranged attractive interactions between particles come from the depletion effect that arises from added micelles. The size and concentration of micelles changes strongly with temperature so that we can reversibly induce freezing and melting. The depletion attraction also confines the particles to a two-dimensional layer on a glass surface. We find that samples having relatively low particle concentration form crystallites via classical nucleation, as expected. By contrast, samples having intermediate concentration (area fraction approximately 0.20-0.30) follow a two-step process, in which liquid clusters appear transiently, then either break up or rapidly crystallize and then grow. By monitoring this two-step nucleation process, we measure the cluster energies and estimate line tension and chemical potential of the fluid and crystalline phases. From this data, we clearly identify a region in the phase diagram corresponding to metastable gas-liquid coexistence; this metastable g-l region leads to a greatly enhanced nucleation rate. At higher area fractions (> 0.5) we observe classical nucleation with no evidence of a second solid phase. Results will be compared to studies of nucleation of globular proteins in solution. We also explore nucleation in 2D systems having particle-size polydispersity, where the crystal phase is expected to be metastable. Finally, we explore nucleation in systems having a combination of short-range attraction and long-range repulsion, which has been predicted to cause a variety of new phases in equilibrium. Here we induce long-range repulsion by using paramagnetic particles and applying a magnetic field perpendicular to the particle layer. The role of metastable phases in determining the dynamics of phase transitions and the prospects for materials assembly will be discussed. We thank the NSF for support through grant DMR-0907195.
3:00 PM - NN5.2
Viral Capsid-Directed Assembly of Light-Harvesting Nanostructures.
Jolene Lau 1 , Debin Wang 1 , Stacy Capehart 2 , Matthew Francis 2 1 , James De Yoreo 1 Show Abstract
1 The Molecular Foundry, Lawrence Berkeley National Lab, Berkeley, California, United States, 2 Department of Chemistry, University of California, Berkeley, Berkeley, California, United States
Protein nanoparticles synthesized by cellular machinery display notable monodispersity and hierarchically assemble into structures at several length scales. When the coat protein of bacteriophage MS2 is recombinantly expressed in E. coli, 180 chemically identical subunits self-assemble into icosahedral virus-like particles. These hollow viral capsids, 27 nm in diameter, are readily modified with genetic or chemical methods and stable under a variety of aqueous conditions. The position and spacing of amino acid side chains are well-defined, symmetric, and dispersed across the capsid surface. As a result, the capsid can act as a template that holds functional molecules at defined locations. In this work, the interior of the MS2 capsid is modified with small molecule dyes, and the exterior surface is multivalently decorated with single-stranded DNA. These DNA-modified capsids bind non-covalently to surfaces or other capsids displaying complementary DNA strands. We patterned surfaces with DNA or passivating PEG molecules using self-assembly and top-down fabrication methods. Dye-loaded capsids were assembled on these surfaces and visualized with optical methods or probed with force microscopy. Altering the identity or length of the DNA sequences changes the capsid-capsid and capsid-surface interaction parameters, changing the rate of capsid adsorption or changing the patterns of nucleation of the deposited structures. Furthermore, dye-modified capsids brought into close proximity exhibit Förster resonance energy transfer (FRET). DNA hybridization thus mediates the formation of artificial light-harvesting structures comprised of MS2 capsids. By altering capsid modification and surface chemistry, we are investigating the interactions responsible for nucleation and growth of virus particles on 2-D patterns, as well as the correlation between organization and light harvesting efficiency.
3:15 PM - NN5.3
Self-Assembly of Hierarchically Organized Biomimetic Materials from a Photocurable Oil/Water/Surfactant System.
Jason Benkoski 1 , Melanie Morris 1 , Asmi Panigrahi 1 , Ryan Deacon 1 , Patrick Chan 1 Show Abstract
1 MERC, JHU/APL, Laurel, Maryland, United States
A defining feature of biological materials is a hierarchy of superimposed morphologies ranging from the macro- to nano-scale. In many cases, the hierarchical design paradigm avoids the normal tradeoffs between competing properties by independently optimizing the morphology for each property at separate levels of the hierarchy. Herein we describe a facile method that faithfully reproduces this hierarchical organization in synthetic polymers. The method bears many of the attractive characteristics of natural materials synthesis: self-assembly, aqueous processing, minimal waste, low energy, and low cost. It consists of a photocurable monomer and water. To this quasi-two-component system we add surfactants that sculpt the interface into the desired shape. The resulting structures are then solidified by crosslinking the monomer with UV light. Drawing from the rich phase behavior of oil/water/surfactant systems, we demonstrate complex biomimetic morphologies over many length scales. Quantitative image analysis reveals a superposition of distinct fractals that occur at discrete length scales from several mm down to 100 nm. The observed morphologies mimic a number of technologically interesting biological materials including honeycombs, lotus leaves, vascular structures, and bone.
3:30 PM - NN5.4
Self-Assembly of Helical Microtubules Directly Visualized by Optical Microscopy.
Hee-Young Lee 1 , Hyuntaek Oh 1 , Jae-ho Lee 1 , Srinivasa R Raghavan 1 Show Abstract
1 Department of Chemical & Biomolecular Engineering , University of Maryland, College Park, Maryland, United States
The assembly and growth of helical tubules by amphiphilic peptides or surfactants is a topic of great relevance to soft matter physics (e.g., the connection between molecular chirality and self-assembly) and to biological structures (e.g., tubules are believed to be intermediate structures in the formation of gallstones). The pathway for forming tubules from an initial state of vesicles or micelles has been studied theoretically, but has not been directly visualized in real-time. In the current study, we demonstrate the formation of tubule structures in aqueous systems consisting of the single-tailed diacetylenic surfactant, 10,12- pentacosadiynoic acid (PCDA) upon the addition of a short-chain alcohol. PCDA tubules can be conveniently prepared over a range of sizes from nano to micro. We focus on micron sized tubules because their assembly process can be directly captured by optical microscopy. The step-wise process involves a nucleation of helical microribbons from nanoscale vesicles. These ribbons then fold and re-arrange into closed tubules. In many cases, tubules further re-arrange into plate-like structures. A notable aspect of the above system is that the surfactant is achiral; yet, an overall chirality is observed in the tubules. Our studies offer new insights into tubule formation that will be valuable in clarifying and refining theoretical models for these fascinating structures.
3:45 PM - NN5.5
Understanding the Nucleation of Polypeptide Self-Assembly.
Cait MacPhee 1 , Ryan Morris 1 , Kym Eden 1 , Harriet Cole 1 2 , Rosalind Allen 1 , Perdita Barran 2 Show Abstract
1 School of Physics and Astronomy, The University of Edinburgh, Edinburgh United Kingdom, 2 School of Chemistry, The University of Edinburgh, Edinburgh United Kingdom
Amyloid fibrils are ordered aggregates of misfolded protein. These fibrils are of great interest because of their role in degenerative diseases including Alzheimer's and Type-2 diabetes. Their physical properties also make them potentially useful in the development of novel materials.It is well known that fibril formation occurs with "nucleation-like" kinetics in which a long lag phase is followed the rapid appearance of fibrils. However, despite much work, the molecular mechanisms responsible for fibril formation and growth remain unclear. This is particularly important because it is believed that pre-fibril oligomeric species present during the lag time may be the cytotoxic agents responsible for amyloid associated pathologies. Much recent debate has focussed on whether fibril formation is a stochastic nucleation process and the possible role of secondary processes such as fibril fragmentation.We have used a combination of mass spectrometry, high throughput fluorescence spectroscopy experiments and both stochastic and deterministic computer simulations to investigate in detail the kinetics of fibril formation by the protein bovine insulin. Our experiments reveal different kinetic behaviour in the regimes of high and low protein concentration, as well as stochasticity in the fibril growth rates. Using a series of computer simulation models with different early-stage fibril formation mechanisms and secondary nucleation processes, we show that this behaviour is not fully explained by any of the current models, but may point to the presence of multiple competing or sequential assembly processes during the apparent lag and growth phases of fibril formation. The fact that the apparent nucleation event and associated lag time is best explained by a mechanism that is independent of polypeptide concentration suggests that regarding polypeptide aggregation as a “nucleated” phenomenon may be incorrect. We further employ molecular dynamics simulations informed by ion mobility mass spectrometry experiments to map out the early stages of polypeptide self-assembly in atomistic detail.
4:30 PM - **NN5.6
Mesoscopic Aggregation in Protein Solutions.
Vassiliy Lubchenko 1 3 , Peter Vekilov 2 1 , Weichun Pan 2 , Ye Li 2 Show Abstract
1 Chemistry, University of Houston, Houston, Texas, United States, 3 Physics, University of Houston, Houston, Texas, United States, 2 Chemical Engineering, University of Houston, Houston, Texas, United States
Long-lived mesoscopic clusters of a dense protein liquid are observedin concentrated solutions of numerous proteins. These clusters are anecessary kinetic intermediate for the formation of solid aggregatesof native and misfolded protein molecules; the aggregates underliephysiological and pathological processes, and laboratory andindustrial procedures. We propose a novel physicochemical mechanism,whereby the clusters consist of an off-equilibrium mixture of singleprotein molecules and long-lived protein-containing complexes. Thepuzzling mesoscopic size of the clusters is determined by the lifetimeof the complexes and their diffusivity. We have predicted and observeda number of interesting kinetic and thermodynamic behaviors that areassociated with the mesoscopic clusters. These behaviors include (a)Ostwald-like ripening of the clusters (b) a crossover to long-rangedensity fluctuations at high concentrations; (c) a universal,diffusion-like scaling of the autocorrelation function of lightscattered of the protein solution; (d) non-trivial dependencies of thecluster size and volume fraction on the protein concentration in thesolution. The significance of anisotropic Coulomb interactions andpartial unfolding for the mechanism of complex formation will bediscussed. Our findings suggest novel ways to control proteinaggregation.
5:00 PM - NN5.7
Multiple Self-Assembly Functional Structures Based on Versatile Binding Sites of β-Lactoglobulin.
Elad Mentovich 1 2 , Netta Hendler 1 2 , Bogdan Belgorodsky 1 , Ludmila Fadeev 1 , Michael Gozin 1 , Shachar Richter 1 2 Show Abstract
1 Chemistry, Tel Aviv University, Tel Aviv Israel, 2 Nanoscience and Nanotechnology Institute, Tel Aviv University, Tel Aviv Israel
Amyloid-like fibrils are known for being associated with neurological diseases, such as Parkinson and Alzheimer. Furthermore, those fibers are used in nature for protein-based functional microbial coatings. However, in recent years, they gained a lot of attention for their ability of creation nanostructured such as nanotubes and nanorods and as a basis for functional materials and biofilms. Ideally, the combination of accurate self assembly and chemical versatility protein-based nanostructured films represent a novel path towards realizing new multifunctional materials built from the bottom up. However, most of the possibilities for chemical modulation are surface functionalization, or post assembly modulation beside several cases in which only specific modulators are inserted into specific proteins. The challenge of generalization of the species inserted into the protein, and by that adding new property for the nanostructures created from the protein building block remains unsolved.In this manuscript we try to face this challenge by mapping the binding site of β-lactoglobulin (β-Lg) and using this accurate positioning for self assembly nanostructures with chemical versatility of properties.We demonstrate that the β-Lg based fibril forms stable and well-defined complexes with linear retinoic acid, discose protoporphyrine IX, and spherical carboxyfullerene, exhibiting ligand-specific stoichiometries and modes of binding, which are saved in the fibril form.
5:15 PM - NN5.8
Mechanism of Collagen Assembly Studied by Dynamic Force Spectroscopy.
Jinhui Tao 1 , Raymond Friddle 1 , Debin Wang 1 , Jim De Yoreo 1 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States
Collagen represents the major structural protein of the extracellular matrix as well as bone and dentine. Elucidating its assembly mechanism is critical for understanding many cellular and medical processes as well as for hard-tissue engineering. In this work, the dynamics of collagen type I self-assembly on mica surfaces was studied by AFM. At acidic conditions, potassium ions critically affected the interaction between collagen and mica leading to ordered structures that varied dramatically with changing ion concentration. The structure evolved from random fibers to ordered fibers and finally to ordered bundles as potassium concentration increased from 100 mM to 200 mM and finally 300 mM. High-resolution AFM micrographs showed that these bundles were built by binding of single collagen triple-helices with each other at specific sites. In order to provide further insight into the mechanism of collagen assembly, the interactions between collagen-mica and collagen-collagen were measured directly with dynamic force spectroscopy (DFS) by using an AFM tip functionalized with full-length collagen. These measurements were complimented by molecular dynamics simulations of the structure of collagen and mica in the presence and absence of potassium ions, to identify the key interactions controlling orientation-specific binding of collagen on mica. At a single pulling speed, the forces of collagen-mica and collagen-collagen at 200 mM potassium ion concentration were 95 pN and 30 pN respectively, while the forces at 300 mM potassium ion concentration were 60 pN and 100 pN, respectively. From DFS data, we extracted the free energy of binding for these four cases. The observed reversal in the sequence of binding force between collagen-collagen and collagen-mica provides an answer to why highly organized bundle forms at the higher ion concentrations. The dependence of surface coverage on time was also determined under the range of ionic conditions described above, as well as under conditions for which assembly gave 2D films of the well-ordered D-band collagen structure. In the case of random fibrils (at low ionic strength), coverage followed a simple Langmuir adsorption curve, consistent with the observation that collagen-mica binding is stronger than collagen-collagen binding. However, for the D-band structure, assembly followed a highly non-linear time dependence in which the acceleration in coverage was correlated with the degree of D-band order. This result indicates growth proceeds in two steps with nucleation of a poorly ordered or disordered film followed by nucleation of the ordered structure.
5:30 PM - NN5.9
Sequence-Structure Correlations and Size Effects in Silk Nanostructure: Poly-Ala Repeat of N. Clavipes MaSp1 is Naturally Optimized.
Graham Bratzel 1 , Markus Buehler 2 Show Abstract
1 Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Deptartment of Civil & Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Spider silk is a self-assembling biopolymer that outperforms many known materials in terms of its mechanical performance despite being constructed from simple and inferior building blocks. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies under variations of genetic sequences have been reported. Here we report atomistic-level structures of the MaSp1 protein from the N. Clavipes spider dragline silk sequence, obtained using an in silico approach based on replica exchange molecular dynamics (REMD), applied to study the effects of a systematic variation of the poly-alanine repeat lengths on the resulting structure of silk at the nanoscale. Confirming earlier experimental and computational work, a structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly β-sheet crystal domains while disorderly regions are formed by glycine-rich repeats that consist of 310-helix type structures and β-turns. Our predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots and secondary structure content. The key result of the present study is our finding of a strong dependence of the resulting silk nanostructure depending on the poly-alanine length. We observe that the wildtype poly-alanine repeat length of six residues defines a critical minimum length that consistently results in clearly defined β-sheet nanocrystals. For poly-alanine lengths below six the β-sheet nanocrystals are not well-defined or not visible at all. These findings set the stage for understanding how variations in the spidroin sequence can be used to engineer the structure and thereby functional properties of this biological superfiber, and present a design strategy for the genetic optimization of spidroins for enhanced mechanical properties.