Silk inverse opals from template-directed b-sheet transformation of regenerated silk fibroin{ Victoria M. Swinerd, a Andrew M. Collins, a Nicholas J. V. Skaer, b Tom Gheysens b and Stephen Mann* a Received 7th August 2007, Accepted 17th September 2007 First published as an Advance Article on the web 27th September 2007 DOI: 10.1039/b711975e We describe a novel and facile method for the fabrication of reconstituted silk monoliths with ordered interconnected air spheres based on intercalation and in situ b-sheet transforma- tion of regenerated silk fibroin solutions within the interstitial spaces of a sacrificial colloidal crystal template. The silk inverse opals are elastic and can withstand and recover from com- pressive loads of up to 112 MPa by reversible pore deforma- tion. They also exhibit super-hydrophobicity with water droplet contact angles of up to 140u due to periodic nanoscale protrusions associated with the surface texture of the inverse opal architecture. These properties indicate that silk inverse opals could have potential applications as biocompatible elastic scaffolds, storage–release, barrier and self-cleaning materials, and in the design of load-responsive microfluidic devices. Biologically derived materials are a resource and inspiration for the development of advanced functional polymers, composites and nanostructures. 1 In particular, there has been widespread interest in the use of silk, a protein-based polymer with remarkable mechanical properties, 2 for a variety of novel materials-based applications. For example, native silk fibres have been used to control the deposition of inorganic materials such as magnetite, gold and cadmium sulfide, 3 silica, 4 or titania and zirconia 5 to produce composite materials with multiple functionality. In addition, reconstituted silks in the form of fibres, 6 microspheres 7,8 and scaffolds for tissue engineering 9–11 have been prepared using regenerated or genetically engineered silk proteins (fibroins), but such procedures have not been exploited for the development of 3D patterned silk materials. Regenerated (redissolved) fibroins predominantly consist of partially degraded silk proteins in a-helical and random coil conformations, and as such appear to be promising starting materials for transformation into elastomeric silk matrices by promotion of the b-sheet conforma- tion. Formation of the b-sheet secondary structure, for example by treatment of fibroin solutions with solvents such as ethanol, results in enhanced inter-chain hydrogen bonding and nucleation of well-ordered crystalline domains separated by less crystalline a-helical regions that deform elastically. This approach is to some extent compromised by the mechanical properties of the reconstituted silks, which often do not match those of the native biomaterial due to chemical degradation of the constituent proteins during processing. 12,13 In general, it has proved difficult to produce patterned materials from regenerated silk proteins due to the inherent metastability of fibroin solutions during processing. Here, we present a method to produce three-dimensionally ordered macroporous reconstituted silk monoliths using the process of colloidal crystal templating. 14 Silk inverse opals were prepared by capillary infiltration of pre- formed colloidal crystals of 500 nm-diameter polystyrene micro- spheres with an aqueous 8%wt solution of regenerated silkworm (Bombyx mori ) silk fibroin.{ Opals prepared from polystyrene beads were used specifically to promote favourable interactions with the largely hydrophobic sequence domains of fibroin, 15 and slow capillary ingress undertaken to prevent premature shear force-induced b-sheet formation during fabrication. The infiltrated samples were air dried, and then treated with ethanol for one hour to induce b-sheet formation and concomitant deposition of a reconstituted silk matrix within the interstitial spaces of the colloidal crystal template. Removal of the polystyrene template by washing in toluene produced a monolithic replica in the form of a silk inverse opal (Fig. 1(a)). High magnification SEM images confirmed high fidelity replication of periodic arrays of spherical voids that were enclosed within thin walls of silk (Fig. 1(b)). The replicas consisted of domains of close-packed 400 nm-diameter spherical pores that were interconnected through 150 nm-wide apertures and surrounded by a continuous silk-matrix framework, ca. 50 nm in thickness. The pore diameter was slightly less than the mean size of the polystyrene beads, possibly due to contraction of the matrix when exposed to the vacuum of the SEM. Similar results were obtained using regenerated fibroin solutions at concentrations of 4 and 6%wt except that the wall thicknesses were reduced to approximately 40 nm (see ESI{, Fig. S1(a and b)). Replicas prepared using 2%wt fibroin solutions showed extensive fragmentation due to incomplete wall formation particularly at the contact edges between adjacent void spaces (see ESI{, Fig. S1(c)). Confirmation of b-sheet formation in the macroporous silk scaffold was determined by FTIR spectroscopy, which showed amide I (CLO str) and II (N–H bend) absorbance peaks at 1628 and 1526 cm 21 , respectively (Fig. 2). The presence of amide III (C–N str) peaks at 1233 cm 21 and 1260 cm 21 indicated that the reconstituted silk matrix contained a mixture of disordered a-helical and crystalline b-sheet domains. 16 This was also con- sistent with the presence of a shoulder peak at 1700 cm 21 close to the amide I absorbance, which was associated with a b-turn or intermediate b-sheet structure. 17,18 Elastic deformation properties of the silk inverse opal were determined by SEM imaging of samples maintained at different a Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol, UK BS8 1TS. E-mail: s.mann@bris.ac.uk b Oxford Biomaterials Ltd, Units 14-15 Galaxy House, New Greenham Business Park, Thatcham, UK RG19 6HR { Electronic supplementary information (ESI) available: SEM images of silk inverse opals prepared using regenerated fibroin solutions. See DOI: 10.1039/b711975e COMMUNICATION www.rsc.org/softmatter | Soft Matter This journal is ß The Royal Society of Chemistry 2007 Soft Matter, 2007, 3, 1377–1380 | 1377