We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates.

There are large number of proteins, the existence of which are known but not their crystal structure, because of difficulty in finding the exact condition for their crystallization. Heterogeneous nucleation on disordered porous substrates with small yet large distribution of pores is considered a panacea for this problem, but a universal nucleant suitable for crystallizing large variety of proteins does not really exist. To this end, we report here a nanowrinkled substrate which displays remarkable ability and control over protein crystallization. Experiments with different proteins show that on these substrates crystals nucleate even at very low protein concentration in buffer. A small number of very large crystals appear for precipitant concentrations varied over orders of magnitude, ~0.003-0.3 M; for some proteins, crystals appear even without addition of any precipitant, not seen with any other heterogeneous substrates. In essence, these substrates significantly diminish the influence of the above two parameters, thought to be key factors for crystallization, signifying that this advantage can be exploited for finding out crystallization condition for other yet-to-be-crystallized proteins.

In the context of protein crystallization, surface decorated with heterogeneous topographical features decreases the energy barrier for nucleation, thereby facilitating crystallization; a precipitant is, nevertheless, required to be used. Here we eliminate the need of such precipitant by using a combined effect of nanoscopic surface undulations and charges on a substrate. Using surface instabilities as a tool for generating such features on polymeric materials, we show that intrinsic curvature of nanofeatures (<10 nm) coupled with surface charges lead to spatial gradient in potential as high as 140 V•μm −1 , where curvature gets maximum. These surfaces show remarkable ability to induce nucleation not achieved by any other conventional process. They induce precipitantless nucleation of proteins, directed crystallization of a specific protein from a mixture of two or more species, and even simultaneous crystallization from a mixture of proteins. These results signify large scale molecular ordering at the bulk by effects initiated at the surface.

Crystallization of macromolecules such as proteins and peptides is known to be influenced by the topographical and chemical heterogeneity of the substrate. However, controlling the nucleation and the growth of crystal on such surfaces has been an issue. Here, we present systematic experiments carried out on hydrophilic elastomeric substrates topographically patterned by forming stretch induced surface wrinkles; the distance between the wrinkles, importantly the density of occurrence of defects between the wrinkles, is systematically varied. Furthermore, to maximize the effect of the substrates, the crystallization experiment is carried out between two such parallel substrates, the gap between which is maintained by using spacers. This process results in very controlled evaporation of the solvent. Experiments with two different model proteins: hen egg-white lysozyme and Thaumatin from Thaoumatococcus daniellii show that on surfaces with uniformly spaced wrinkles the crystals nucleate extensively but with insignificant growth. However, when a small number of defects are introduced into the patterns, fewer crystals nucleate, which grow to form large crystals. With further increase in the defect density, extent of nucleation increases again, but with decrease in the crystal growth. Thus, the crystal size attains maxima at an intermediate wavelength of the wrinkles and the defect density.

Nucleation of protein crystals has been shown to be facilitated by substrates decorated with both nano-to micro-scale hierarchical undulations and spatially varying surface potential. In fact, on such surfaces, several proteins were found to crystallize without having to use any precipitant in contrast to all other homogeneous and heterogeneous systems in which precipitant is an essential ingredient for nucleation. While these surfaces were so patterned whole through the area that was brought in contact with the protein solution, it was not clear exactly to what extent the surfaces were required to be patterned to trigger nucleation without use of any precipitant. Here we show that a simple incision may be enough on an otherwise smooth surface for this purpose. In particular, the substrate used here is a smooth silicone film with its surface plasma oxidized to create a thin crust of silica. An incision is then generated on this surface using a sharp razor blade. The silica crust being brittle leads to random nano-microscopic undulations at the vicinity of the incision. These undulations along with surface charge can induce protein crystal nucleation without precipitant.