Paper BI+AS+BA+NS+SS-ThA11
Immobilization of Peptide-Based Stimuli-Responsive Biomolecules on Silica Surfaces

Thursday, October 31, 2013, 5:20 pm, Room 102 B

The immobilization of stimuli-responsive biomacromolecules onto silica surfaces is often performed the development of silica-based biosensors, protein microarrays and supramolecular assemblies. The R5 silaffin peptide, derived from Cylindrotheca fusiform, is of current interest because of its capacity to induce and regulate silica precipitation at ambient conditions. In this study, we found that a fusion protein comprised of a synthetic silaffin R5 peptide and elastin-like polypeptide (ELP) bound reliably to silica particles and flat silica-based surfaces. ELPs are a class of stimuli-responsive polypeptides that undergo a reversible lower critical solution temperature (LCST) phase transition. In silaffin-ELP fusion proteins, the R5 peptides serve as silica-binding domains that immobilize ELPs onto silica, allowing its surface properties to be modulated upon change in temperature through the LCSTs of the ELPs. The attachment of silaffin-ELP to silica particles was confirmed by temperature- and time-dependent turbidity, zeta potential, and dynamic light scattering measurements. As demonstrated through zeta potential measurements, the positively charged silaffin-ELPs neutralized the negative charge on the silica particles, confirming the binding of silaffin-ELPs. Dynamic light scattering experiments revealed an increase in particle size after surface modification. The sizes of surface-modified particles also changed in response to temperature. We also investigated the absorption of silaffin-ELP on oxidized silicon wafers. The elemental composition of the protein-modified surfaces was characterized by X-ray photoelectron spectroscopy. We also used ellipsometry and atomic force microscopy (AFM) to test the thickness and roughness of the protein bound surfaces. Contact angle measurements were performed to examine the temperature-responsive nature of the surfaces. Furthermore, we demonstrated that GFP-ELP fusion protein can be adsorbed to silaffin-ELP modified silica surface through co-aggregation above their LCSTs. A thermally triggered aggregation behavior of fluorescently-labeled silica particles was also visualized using confocal fluorescence microscopy. The results of this study demonstrated that a silaffin tag can be used to immobilize ELPs on silica surfaces such as silica particles, silicon wafers and glass slides, and that these protein-modified surfaces can be used to capture and immobilize ELPs and ELP-fusion proteins reversibly onto their surfaces. This system has potential uses in bioseparations, biomaterials, and biosensors.