James J. DeYoreo

Peptides Enhance Magnesium Signature in Calcite : Insights into Origins of Vital Effects

Studies relating the magnesium (Mg) content of calcified skeletons to temperature often report unexplained deviations from the signature expected for inorganically grown calcite. These “vital effects” are believed to have biological origins, but mechanistic bases for measured offsets remain unclear. We show that a simple hydrophilic peptide, with the same carboxyl-rich character as that of macromolecules isolated from sites of calcification, increases calcite Mg content by up to 3 mole percent. Comparisons to previous studies correlating Mg content of carbonate minerals with temperature show that the Mg enhancement due to peptides results in offsets equivalent to 7° to 14°C. The insights also provide a physical basis for anecdotal evidence that organic chemistry modulates the mineralization of inorganic carbonates and suggest an approach to tuning impurity levels in controlled materials synthesis.

Reversed calcite morphologies induced by microscopic growth kinetics: Insight into biomineralization

Abstract This experimental investigation of calcite growth quantifies relationships between solution supersaturation and the rates of step advancement. Using in situ fluid cell atomic force microscopy (AFM), we show that the movement of monomolecular steps comprising growth hillocks on {10 1 4} faces during the growth of this anisotropic material is specific to crystallographic direction. By quantifying the sensitivity of step growth kinetics to supersaturation, we can produce spiral hillocks with unique geometries. These forms are caused by a complex dependence of step migration rates, vs+ and vs−, upon small differences in solution chemistry as they grow normal to the conventional fast ([ 4 41]+ and [48 1 ]+) and slow ([ 4 41]− and [48 1 ]−) crystallographic directions. As solute activity, a, decreases, vs+ and vs− converge and growth hillocks express a pseudo-isotropic form. At still lower supersaturations where a approaches its equilibrium value, ae, an inversion in the rates of step advancement produces hillocks with unusual reversed geometries. Comparisons of the kinetic data with classical theoretical models suggest that the observed behavior may be due to minute impurities that impact the kinetics of growth through blocking and incorporation mechanisms. These findings demonstrate the control of crystallographic structure on the local-scale kinetics of growth to stabilize the formation of unusual hillock morphologies at the near-equilibrium conditions found in natural environments.

Kinetics of Silica Nucleation on Carboxyl- and Amine-Terminated Surfaces: Insights for Biomineralization

An in situ, atomic force microscopy- (AFM-)-based experimental approach is developed to directly measure the kinetics of silica nucleation on model biosubstrates under chemical conditions that mimic natural biosilica deposition environments. Relative contributions of thermodynamic and kinetic drivers to surface nucleation are quantified by use of amine-, carboxyl-, and hybrid NH(3)(+)/COO(-)-terminated surfaces as surrogates for charged and ionizable groups on silica-mineralizing organic matrices. The data show that amine-terminated surfaces do not promote silica nucleation, whereas carboxyl and hybrid NH(3)(+)/COO(-) substrates are active for silica deposition. The rate of silica nucleation is approximately 18x faster on the hybrid substrates than on carboxylated surfaces, but the free energy barriers to cluster formation are similar on both surface types. These findings suggest that surface nucleation rates are more sensitive to kinetic drivers than previously believed and that cooperative interactions between oppositely charged surface species play important roles in directing the onset of silica nucleation. Further experiments to test the importance of these cooperative interactions with patterned NH(3)(+)/COO(-) substrates, and aminated surfaces with solution-borne anionic species, confirm that silica nucleation is most rapid when oppositely charged species are proximal. By documenting the synergy that occurs between surface groups during silica formation, these findings demonstrate a new type of emergent behavior underlying the ability of self-assembled molecular templates to direct mineral formation.

Engineered Biomimetic Polymers as Tunable Agents for Controlling CaCO3 Mineralization

In nature, living organisms use peptides and proteins to precisely control the nucleation and growth of inorganic minerals and sequester CO(2)via mineralization of CaCO(3). Here we report the exploitation of a novel class of sequence-specific non-natural polymers called peptoids as tunable agents that dramatically control CaCO(3) mineralization. We show that amphiphilic peptoids composed of hydrophobic and anionic monomers exhibit both a high degree of control over calcite growth morphology and an unprecedented 23-fold acceleration of growth at a peptoid concentration of only 50 nM, while acidic peptides of similar molecular weight exhibited enhancement factors of only ∼2 or less. We further show that both the morphology and rate controls depend on peptoid sequence, side-chain chemistry, chain length, and concentration. These findings provide guidelines for developing sequence-specific non-natural polymers that mimic the functions of natural peptides or proteins in their ability to direct mineralization of CaCO(3), with an eye toward their application to sequestration of CO(2) through mineral trapping.

In situ AFM Study of Amelogenin Assembly and Disassembly Dynamics on Charged Surfaces Provides Insights on Matrix Protein Self-Assembly

Because self-assembly of matrix proteins is a key step in hard tissue mineralization, developing an understanding of the assembly pathways and underlying mechanisms is likely to be important for successful hard tissue engineering. While many studies of matrix protein assembly have been performed on bulk solutions, in vivo these proteins are likely to be in contact with charged biological surfaces composed of lipids, proteins, or minerals. Here we report the results of an in situ atomic force microscopy (AFM) study of self-assembly by amelogenin--the principal protein of the extracellular matrix in developing enamel--in contact with two different charged substrates: hydrophilic negatively charged bare mica and positively charged 3-aminopropyl triethoxysilane (APS) silanized mica. First we demonstrate an AFM-based protocol for determining the size of both amelogenin monomers and oligomers. Using this protocol, we find that, although amelogenin exists primarily as ~26 nm in diameter nanospheres in bulk solution at a pH of 8.0 studied by dynamic light scattering, it behaves dramatically differently upon interacting with charged substrates at the same pH and exhibits complex substrate-dependent assembly pathways and dynamics. On positively charged APS-treated mica surfaces, amelogenin forms a relatively uniform population of decameric oligomers, which then transform into two main populations: higher-order assemblies of oligomers and amelogenin monomers, while on negatively charged bare mica surfaces, it forms a film of monomers that exhibits tip-induced desorption and patterning. The present study represents a successful attempt to identify the size of amelogenin oligomers and to directly monitor assembly and disassembly dynamics on surfaces. The findings have implications for amelogenin-controlled calcium phosphate mineralization in vitro and may offer new insights into in vivo self-assembly of matrix proteins as well as their control over hard tissue formation.

Tuning calcite morphology and growth acceleration by a rational design of highly stable protein-mimetics

In nature, proteins play a significant role in biomineral formation. One of the ultimate goals of bioinspired materials science is to develop highly stable synthetic molecules that mimic the function of these natural proteins by controlling crystal formation. Here, we demonstrate that both the morphology and the degree of acceleration or inhibition observed during growth of calcite in the presence of peptoids can be rationally tuned by balancing the electrostatic and hydrophobic interactions, with hydrophobic interactions playing the dominant role. While either strong electrostatic or hydrophobic interactions inhibit growth and reduces expression of the {104} faces, correlations between peptoid-crystal binding energies and observed changes in calcite growth indicate moderate electrostatic interactions allow peptoids to weakly adsorb while moderate hydrophobic interactions cause disruption of surface-adsorbed water layers, leading to growth acceleration with retained expression of the {104} faces. This study provides fundamental principles for designing peptoids as crystallization promoters, and offers a straightforward screening method based on macroscopic crystal morphology. Because peptoids are sequence-specific, highly stable, and easily synthesized, peptoid-enhanced crystallization offers a broad range of potential applications.

Surface-Directed Assembly of Sequence-Defined Synthetic Polymers into Networks of Hexagonally Patterned Nanoribbons with Controlled Functionalities

The exquisite self-assembly of proteins and peptides in nature into highly ordered functional materials has inspired innovative approaches to the design and synthesis of biomimetic materials. While sequence-defined polymers hold great promise to mimic proteins and peptides for functions, controlled assembly of them on surfaces still remains underdeveloped. Here, we report the assembly of 12-mer peptoids containing alternating acidic and aromatic monomers into networks of hexagonally patterned nanoribbons on mica surfaces. Ca(2+)-carboxylate coordination creates peptoid-peptoid and peptoid-mica interactions that control self-assembly. In situ atomic force microscopy (AFM) shows that peptoids first assemble into discrete nanoparticles; these particles then transform into hexagonally patterned nanoribbons on mica surfaces. AFM-based dynamic force spectroscopy studies show that peptoid-mica interactions are much stronger than peptoid-peptoid interactions, illuminating the driving forces for mica-directed peptoid assembly. We further demonstrate the display of functional domains at the N-terminus of assembling peptoids to produce extended networks with similar hierarchical structures. This research demonstrates that surface-directed peptoid assembly can be used as a robust platform to develop biomimetic coating materials for applications.

Subnanometer Replica Molding of Molecular Steps on Ionic Crystals

Replica molding with elastomeric polymers has been used routinely to replicate features less than 10 nm in size. Because the theoretical limit of this technique is set by polymer-surface interactions, atomic radii, and accessible volumes, replication at subnanometer length scales should be possible. Using polydimethylsiloxane to create a mold and polyurethane to form the replica, we demonstrate replication of elementary steps 3-5 Å in height that define the minimum separation between molecular layers in the lattices of the ionic crystals potassium dihydrogen phosphate and calcite. This work establishes the operation of replica molding at the molecular scale.

Steric and Electrostatic Complementarity in the Assembly of Two-Dimensional Virus Arrays

A highly ordered assembly of biological molecules provides a powerful means to study the organizational principles of objects at the nanoscale. Two-dimensional cowpea mosaic virus arrays were assembled in an ordered manner on mica using osmotic depletion effects and a drop-and-dry method. The packing of the virus array was controlled systematically from rhombic packing to hexagonal packing by modulating the concentrations of poly(ethylene glycol) surfactant in the virus solutions. The orientation and packing symmetry of the virus arrays were found to be tuned by the concentrations of surfactants in the sample solutions. A phenomenological model for the present system is proposed to explain the assembly array morphology under the influence of the surfactant. Steric and electrostatic complementarity of neighboring virus capsids is found to be the key factors in controlling the symmetry of packing.

Scanning Probe‐based Fabrication of 3D Nanostructures via Affinity Templates, Functional RNA, and Meniscus‐mediated Surface Remodeling

Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns on surfaces is one of the central challenges in the field of nanotechnology. Here we review three applications of the atomic force microscope (AFM) that address this challenge. In the first, we use two-step nanografting to create patterns of self-assembled monolayers (SAMs) to drive the organization of virus particles that have been either genetically or chemically modified to bind to the SAMs. Virus-SAM chemistries are described that provide irreversible and reversible binding, respectively. In the second, we use similar SAM patterns as affinity templates that have been designed to covalently bind oligonucleotides engineered to bind to the SAMs and selected for their ability to mediate the subsequent growth of metallic nanocrystals. In the final application, the liquid meniscus that condenses at the AFM tip-substrate contact is used as a physical tool to both modulate the surface topography of a water soluble substrate and guide the hierarchical assembly of Au nanoparticles into nanowires. All three approaches can be generalized to meet the requirements of a wide variety of materials systems and thereby provide a potential route toward development of a generic platform for molecular and materials organization.