Identification of peptides that promote the rapid precipitation of germania nanoparticle networks via use of a peptide display library† Matthew B. Dickerson, a Rajesh R. Naik, b Morley O. Stone, b Ye Cai a and Kenneth H. Sandhage a a School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA. E-mail: ken.sandhage@mse.gatech.edu; Fax: +1 404 894 9140; Tel: +1 404 894 6882 b Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright Patterson AFB, Dayton, Ohio 45433, USA Received (in West Lafayette, IN, USA) 17th February 2004, Accepted 27th May 2004 First published as an Advance Article on the web 30th June 2004 Peptides that promote the rapid, room-temperature precipita- tion of amorphous germania nanoparticle networks from solution have been identified via use of a combinatorial peptide display library. Amorphous silicon dioxide (silica) is routinely produced under physiological conditions by biological organisms through the use of specialized proteins. 1 These organisms exert impressive control over the morphologies of three-dimensional (3D) silica structures. Spectacular examples of protein-mediated 3D assembly of silica nanoparticle structures are provided by diatoms (uni-cellular algae). 2 Each of the 10 4 –10 5 extant diatom species assembles a frustule (microshell) with a unique shape and with fine (submicron) features (e.g. pores, supporting ribs, channels, protuberances) that are organized into precise, species-specific patterns. 2 The spicules of certain sponges are also composed of amorphous silica. Indeed, glassy spicules produced by the sponge Euplectella possess refractive index profiles and waveguiding properties akin to man- made optical fibers. 3 The identification and functional analyses of proteins associated with in vivo silica formation, and the use of such proteins to direct the in vitro assembly of silica structures with tailored, non-natural shapes (biosculpting), are active areas of research. 1,4 Silica is the predominant constituent of a wide variety of commercial glasses, ranging from common window glasses to advanced glasses used in high-bandwidth optical fibers. In order to achieve an expanded range of properties, silica-based glasses can be alloyed with other glass-forming oxides, such as germanium oxide (germania). Silica–germania glasses possess higher re- fractive indices, and lower viscosities, than pure silica. 5 Fur- thermore, germania-based glasses possess reduced phonon energies and enhanced transmission at infrared wavelengths relative to silica-rich glasses. 6 As a result, germania-based glasses are receiving increased attention for use in integrated optical lasers, sensors, display devices, and amplifiers. 6 While natural silica-forming proteins have been identified by several authors, germania-forming proteins have yet to be found in nature. The objective of this paper is to identify peptides that promote germania precipitation, in order to allow for future in vitro biosculpting of germania-based assemblies with tailored shapes. A commercially available combinatorial peptide display library was used to identify peptides that exhibit strong binding to germania. 7,8 After several rounds of selective panning, 21 peptide-displaying phage clones with an enhanced affinity for germania were isolated. Preliminary experiments using a PCR method previously descri- bed 8e failed to yield any phage clones that remain attached to the germania particles after acid elution. This indicated that the acid elution was effective in releasing all of the germania-binding peptide displaying phages. The amino acid sequences of the displayed peptides were determined using DNA sequencing. The 3 most dominant peptides identified from the clones, labeled Ge2, Ge8, and Ge34 in Table 1, were chosen for further evaluation. In order to assess the ability of these germania-binding peptides to promote germania precipitation, the peptides were introduced (1 mg 2ml ) into a germanium alkoxide-bearing solution (0.135 M tetramethoxygermanium dissolved in methanol) at room tem- perature. Precipitation occurred rapidly upon introduction of either the Ge8 or Ge34 peptide into the alkoxide solution. In contrast, germania precipitation induced by the Ge2 peptide was difficult to detect by visual observation. Control experiments conducted with non-germania-binding peptides AG5 8a and AG-P28, 8e or in the absence of the germania-binding peptides, failed to yield germania precipitates from the alkoxide solution. The extent of germania precipitation (i.e. the germania precipitating activity) was quanti- fied by adapting the b-silicomolybdate colorimetric assay. 9 As shown in Fig. 1, the Ge8 and Ge34 peptides exhibited relatively high germania-precipitating activities, whereas the Ge2 peptide exhibited much lower activity. The control peptides, AG5 and AG- P28, showed no germania precipitating activity. The amino acids within peptides can provide molecular recogni- tion motifs for strong binding to specific inorganic surfaces. The molecular characteristics that result in strong binding of a particular peptide to a specific inorganic surface may also enable that peptide to enhance the precipitation of the inorganic solid from a solution. The germania-binding peptides that were particularly effective in promoting germania precipitation from an alkoxide solution, Ge8 and Ge34, possessed hydroxyl- and imidazole-containing amino acid residues. The Ge8 peptide possessed a more basic isoelectric point (pI), and a higher germania precipitating activity, than the Ge34 peptide. The germania-binding peptide with a low germania precipitating activity, Ge2, lacked histidine residues and possessed a more acidic pI. The failure of the control peptides AG5 and AG- † Electronic Supplementary Information (ESI) is available describing the biopanning, germania precipitation assay, and molybdenum blue assay procedures. See http://www.rsc.org/suppdata/cc/b4/b402480j/ Table 1 Amino acid sequences and calculated isoelectric points (pI) of peptides used in this study Peptide Amino acid sequence pI a Ge2 TSLYTDRPSTPL 5.50 Ge8 SLKMPHWPHLLP 8.51 Ge34 TGHQSPGAYAAH 6.61 AG5 SLATQPPRTPPV 9.47 AG-P28 SPLLYATTSNQS 5.24 a pI calculated using pI/mass program at www.expasy.ch. Fig. 1 Germania precipitating activity of germania-binding and control peptides (milligrams of germania formed per milligram of peptide). This journal is © The Royal Society of Chemistry 2004 DOI: 10.1039/b402480j 1776 Chem. Commun., 2004, 1776–1777