Rajesh R. Naik Air Force Research Laboratory
Carole C. Perry Nottingham Trent University
Kiyotaka Shiba Japanese Foundation for Cancer Research
Rein Ulijn University of Manchester
T1: Bioinspired Materials
Tuesday AM, April 10, 2007
Room 2006 (Moscone West)
9:00 AM - T1.1
Biogenic Nanostructured Silica Formation in Diatoms: Proteins, Genes, and Structure.
Mark Hildebrand 1 Show Abstract
1 , Scripps Institution of Oceanography, UCSD, La Jolla, California, United States
Diatoms make cell walls containing three-dimensional nano- and micro- scale silica structures, with a complexity and organization exceeding that possible with current synthetic approaches. Diatom silica structure formation occurs dynamically inside an expandable and moldable membrane-bound intracellular compartment called the silica deposition vesicle, with control occurring at multiple levels. Understanding the molecular details of the process requires identifying structural intermediates and correlating their formation with genes and proteins involved. We have applied molecular, genomic, and microscopic approaches to examine diatom cell wall synthesis. During characterization of structural intermediates, we identified three scales and two distinct stages in structure formation, and observed a correlation between the formation process and optimization of the final structural property required. Using synchronized cultures, we used microarrays to screen all genes in the genome of Thalassiosira pseudonana for a characteristic expression pattern identified in other cell wall genes. One hundred and four genes were identified, and categorized according to cellular role or possible function. We are determining the intracellular localization of proteins encoded by these genes to evaluate their involvement in cell wall formation. Correlation of structure formation with the genes and proteins involved will be essential to understand how diatom genetic information is translated into active chemical moieties that ultimately control the formation of the solid material. These approaches, coupled with genetic manipulation tools, will enable elucidation and manipulation of the intracellular systems involved in biogenic nanostructured silica synthesis, facilitating applications of diatoms in materials science.
9:45 AM - T1.3
Lessons from Diatoms: Formation of nanopatterned functional ceramics under ambient conditions
Nils Kroger 1 2 , Nicole Poulsen 1 , Ken Sandhage 2 , Matt Dickerson 2 , Gul Ahmad 2 Show Abstract
1 Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, United States, 2 Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
The direct syntheses of functional organic-inorganic hybrid materials are severely restricted by the incompatible thermal and/or chemical conditions often required to form desired organic and ceramic phases. Recently, biomineral-forming organisms like diatoms (see figure) and sponges have been recognized as inspirational sources of new strategies for directly synthesizing such hybrid materials under environmentally benign conditions. Here we present novel in vitro and in vivo approaches to produce functional ceramics employing molecules from diatoms as well as using living diatoms themselves.The in vitro approach relies on diatom genes that encode proteins (termed silaffins) involved in biosilica formation. We have designed recombinant silaffins that are able to induce the rapid formation of silica and titania from aqueous precursor solutions. Recombinant silaffin rSilC enabled the synthesis of hierarchically nanopatterned rutile microparticles at ambient temperature and neutral pH. Due to its high refractive index, rutile is highly desirable material for photonic applications, yet previous rutile syntheses require extreme reaction conditions (600-800C, or strongly acidic, hydrothermal conditions). Using an in vivo approach we have developed a unique method for the silica immobilization of functional proteins. The bacterial enzyme hydroxylaminobenzene mutase (HabB) was expressed as a silaffin-fusion protein in the diatom Thalassiosira pseudonana, which resulted in targeted incorporation of the fusion protein into the T. pseudonana silica cell wall. The diatom silica bound HabB was enzymatically active, and remained functional as well as associated with the diatom silica during isolation.
10:00 AM - T1.4
Silicification Biomimetic Studies in Confined Media.
Thibaud Coradin 1 , Clementine Gautier 1 2 , Pascal Lopez 2 , Myriana Hemadi 1 , Jacques Livage 1 Show Abstract
1 Chimie de la Matiere Condensee, CNRS-UMR 7574, Université Paris VI, Paris France, 2 Diatom Signaling and Morphogenesis, CNRS-FRE 2910, Ecole Normale Supérieure, Paris France
10:15 AM - **T1.5
The Bioclastic and Shape-preserving Inorganic Conversion (BaSIC) Route to Chemically Tailored Three-Dimensional Nanostructured Microassemblies
Kenneth Sandhage 1 2 , Samuel Shian 1 2 , Michael Weatherspoon 1 2 , Zhihao Bao 1 2 , Phillip Graham 1 2 , David Lipke 1 2 , Eric Ernst 1 2 , Matthew Dickerson 1 2 , Shawn Allan 1 2 , Ye Cai 1 2 , Gul Ahmad 1 2 , Michael Haluska 1 2 , Robert Snyder 1 2 , Laura Sowards 3 , Rajesh Naik 3 Show Abstract
1 Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States, 2 Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, United States, 3 Biotechnology Group, Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio, United States
Appreciable worldwide R&D effort is underway to develop new fabrication routes to three-dimensional (3-D) nanostructured micro-assemblies for advanced devices. Such processes must be capable of: i) precise 3-D fabrication on a fine (down to nanometer) scale, and ii) mass production on a large scale. These often-conflicting requirements can be addressed with a hybrid fabrication paradigm that merges biological self-assembly with synthetic chemistry: Bioclastic and Shape-preserving Inorganic Conversion (BaSIC)*. Nature provides numerous examples of organisms that assemble biominerals into complex 3-D (bioclastic) microscale structures. For example, diatoms (unicellular planktonic algae) assemble intricate 3-D microshells (frustules) comprised of silica nanoparticles. Each of the tens of thousands of extant diatom species forms a microshell with a distinct shape and pattern of fine features. Sustained reproduction of a given diatom species can yield enormous numbers of identical frustules (e.g., 40 reproduction cycles can yield >1 trillion replicas!). Such massively parallel, genetically precise, and environmentally benign 3-D self-assembly is highly attractive for device manufacturing, and has no man-made analog. However, the natural silica-based chemistry of diatom frustules inhibits their use in a variety of devices. With BaSIC, these biogenic assemblies can be converted into a wide range of new functional chemistries (TiO2, F-doped TiO2, ZrO2, Cl-doped ZrO2, MgO, BaTiO3, Eu-doped BaTiO3, Zn2SiO4, Mn-doped Zn2SiO4, polymers, etc.), while preserving the 3-D frustule morphologies. Various chemical conversion approaches (gas/solid reaction, liquid/solid reaction, conformal coating, reaction and coating, coating and reaction) utilized in the BaSIC process to generate shape-preserved replicas will be described. Such shape-preserving chemical alteration has also been applied to nanostructured synthetic silica preforms. The optical and chemical properties of such chemically-altered nanostructured micro-assemblies will be discussed. Future advances in the genetic engineering of diatoms (to allow for tailoring of frustule shapes), coupled with shape-preserving chemical modifications via BaSIC, offer the exciting promise of 3-D Genetically Engineered Micro/nanodevices (3-D GEMs).*K. H. Sandhage, “Shaped Microcomponents via Reactive Conversion of Biologically-derived Microtemplates,” U.S. Patent No. 7,067,104, June 27, 2006.
10:45 AM - T1.6
The activity of Diatom inspired synthetic polyamines in Silicification
Carole Perry 1 , David Belton 1 , Vadim Annenkov 2 , Siddharth Patwardhan 1 , Elena Danilovtseva 2 Show Abstract
1 Biomedical and Natural Sciences, Nottingham Trent University, Nottingham United Kingdom, 2 , Limnological Institute of Siberian Branch of Russian Academy of Sciences, Irkutsk Russian Federation
In nature many organisms are able to capture and use monosilicic acid in the formation of highly functional and sometimes elaborate siliceous structures. Much effort has been invested in attempts to determine the underlying synthetic principles involved in this chemical manipulation, not least into investigations involving the unicellular algae diatoms. Within the frustule considerable levels of polyamines have been found either as post translational modifications on the side chain of lysine groups in the peptides known as silaffins or as isolated molecules. Silaffins and the polyamines are proposed to play a significant role in diatom biosilicification producing diverse nano-structured silica valves. The isolated polyamines influence the condensation of silica from condensing systems based on alkoxysilanes or sodium silicate precursors, producing dense silica spheres that are not produced under similar conditions in their absence. The presence of polyamines in nature is not an unusual occurrence with cellular compounds such as spermine, and spermidine, common and the formation of these much longer diatom polyamine species is likely to be a serendipitous extension of their biosynthetic process. The polyamines so far isolated from diatoms have been shown to be very species dependant with varying methylation levels of the amine functionality from primary to quaternary and it is thought that this species dependency ultimately determines the final structural form of the diatom thecae.Here we report on the novel stepwise synthesis of a group of linear methylated propylamines and their activity in silica formation in vitro. Measurements were conducted to determine kinetic effects, the solution state of the polyamines, the surface area and porosity of the siliceous products and the gross morphologies. Comparison is made with the naturally occurring polyamines and the influence of chain length and amine separation assessed both as amine solution species and in the nature of the siliceous materials produced. Their effects on the condensation of a model silicic acid system is reported in terms of kinetic changes through pH and amine concentration as well as characterisation of the novel bimodal (dense hollow microporous spherical and thin walled vesicular) silicas produced. The charge states of the amines during addition to the condensation process were found to affect their solution chemistry and to be critical to the nature of the materials produced, with these charge states being in turn highly affected by amine length. The influence of the level of primary, secondary and tertiary amine functionality has also been investigated. These results show that rates of proton transfer are important in terms of flux from amines to precursor. Finally we show how siliceous materials with tailored properties can be produced through control of pH, amine concentration, chain length and ionic strength.
11:30 AM - T1.7
Alkylation is crucial to enhancement of silica condensation rate by diamines.
David Robinson 1 , Blake Simmons 1 , Judith Rognlien 1 Show Abstract
1 , Sandia National Laboratories, Livermore, California, United States
11:45 AM - T1.8
Formation of Porous Hydroxyapatite
Deepa Khushalani 1 Show Abstract
1 Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai India
Most biological materials with predominantly mechanical function have a hierarchical structure consisting of several different length scale levels (angstrom to millimeter) and incorporate a composite structure where an inorganic component is epitaxially linked to an organic component. In this way, tough materials are designed by nature based on a complex configuration where size and morphology of component and its intricate link to the other species present in the composite all play a pivotal role in creating an impressive 3D organization with multifaceted function. In bone for example, the organization is an elaborate design based on collagen fibrils reinforced with nanosized inorganic mineral particles (hydroxyapatite). These nanosized mineral platelets are able to sustain a large tensile stress whereas the protein layer between them sustain sheer stress. This is an excellent example of how morphology of the inorganic component plays a pivotal role in the function of the overall composite. To this aim, both soft-condensed matter and solid inorganic porous supports have been studied in formation of porous hydroxyapatite. The work carried out using (1) a novel micoremulsion involving reverse micelles of calcium bis(2-ethylhexyl)phosphate (Ca(DEHP)2) and (2) Porous membrane supports of alumina and polycarbonate will be detailed. Products such as nanoporous porous hydroxyapatite with intricate tubular morphology will be presented along with their characterization involving microscopy, diffraction, and their uses in bioactive studies will also be evaluated.
12:00 PM - T1.9
Directed Evolution of Hydroxyapatite-Associated Protein Using Phage Display
Seung-Wuk Lee 1 2 4 , Jing Hang Huh 3 2 , Eddie Wang 1 4 , Emily Perttu 1 4 , Yue Zhao 1 Show Abstract
1 Bioengineering , University of California, Berkeley, Berkeley, California, United States, 2 Physical Bioscience Division, Lawrence Berkeley National Lab, Berkeley, California, United States, 4 , UCSF and UCB Joint Graduate Group in Bioengineering, Berkeley, California, United States, 3 Chemical Biology, University of California, Berkeley, Berkeley, California, United States
The formation of natural bone is thought to occur by the templated mineralization of HA by the surrounding proteins, which include collagen and highly acidic phosphoproteins attached to the collagen scaffold. It has been proposed that the acidic groups serve as binding sites for calcium ions and align them in an orientation that matches the HA crystal lattice, but the biological mineralization process is not understood at the molecular level. Using directed evolution process (phage display), we identified specific binding peptides for single crystal hydroxyapatite in various pH ranges and study their interactions between HA binding peptides and crystal surfaces. Remarkably, the consensus HA binding peptides resulted in characteristic tripeptide repeat (Pro-X-Y) at pH 7.5 and (Ser-Ser-Asp) at pH 5. These sequences are similar to the major repeats of type I collagen and dentin phosphoproteins respectively. Using a panel of synthetic peptides, we defined the structural features required for binding and mineralizing activity of HA. We also incorporated these short HA-binding peptides to construct three-dimensional bone-like materials.
12:15 PM - **T1.10
Mollusk shell nacre- and prismatic sequence - directed crystal design.
John Evans 1 , Sebastiano Collino 1 , Jennifer Giocondi 2 , Christine Orme 2 , Il Kim 1 , Katya Delak 1 Show Abstract
1 , New York University, New York, New York, United States, 2 Biophysical and Interfacial Sciences, Lawrence Livermore National Laboratories, Livermore, California, United States
The biomineralization environment of the nacre and prismatic layers of the mollusk are undoubtedly complex, consisting of many proteins present at the same or at different times in the extracellular matrix, and it is likely that numerous polypeptides jointly manage the overall polymorph selection process. Similarly, it has been postulated that Mg (II) also participates in calcium carbonate mineralization, although the exact role of this metal ion is not yet defined. Up until now, our in vitro mineralization studies of individual nacre- and prismatic-specific polypeptides have omitted this important feature of complexity, i.e., the participation of different polypeptides and Mg (II) in the calcium carbonate formation and the polymorph selection process. Obviously, if Nature employs multiple agents to effect net control over crystal formation, then material science might also need to adopt similar approaches to “tailor” inorganic - based materials with specific properties. To address this issue, we have explored the combinatorial effects of nacre and prismatic polypeptide sequences and the presence of “catalytic” levels of Mg (II)(1:10 Mg : Ca) on in vitro crystal growth. We find that It appears that low levels of Mg (II) can affect the mineralization activity of certain calcium biomineralization sequences in selective ways. In particular, we observe either subtle effects on crystal morphology or surprising alterations that deviate from normal polypeptide activity patterns. Moreover, Mg (II) can “switch” the function of a set of peptides to mimic one another with regard to selective adsorption, pinning, or growth patterns in calcite dislocation hillocks. We believe that Mg (II) can catalytically alter the outcome of protein-mediated biomineralization. With regard to combinatorial mixtures of mollusk shell polypeptides, there is clearly a synergistic effect when specific biomineralization protein sequences are combined together within in vitro assays settings. At this point we do not know if the observed morphologies are du