Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

C.A. Crot, D.G. Schultz, M. Meron, A. Kilislioglu, P.D. Edirisinghe, K.A. Skinner-Nemec, L. Hanley 1 Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA 60607-7061 2 ChemMatCARS, Argonne National Laboratory, 9700 S Cass Ave, Argonne, IL, USA 60439 3 Istanbul University, Department of Chemistry, Avcilar 34320, Istanbul, Turkey 4 Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave, Argonne, IL, USA 60439

Long-period X-ray standing wave fluorescence (XSW) and X-ray reflectivity techniques are employed to probe the conformation of a Br-poly(ethylene glycol) (PEG)-peptide adsorbate at the hydrated interface of a polystyrene substrate. The Br atom on this Br-PEG-peptide construct serves as a marker atom allowing determination by XSW of its position and distribution with respect to the adsorption surface with angstrom resolution. Adsorption occurs on native or ion-beam-modified polystyrene films that are spin-coated onto a Si substrate and display either nonpolar or polar surfaces, respectively. A compact, oriented monolayer of Br-PEG-peptide can be formed with the peptide end adsorbed onto the polar surface and the PEG end terminating with the Br tag extending into the aqueous phase. The 108-141 A distance of the Br atom from the polystyrene surface in this oriented monolayer is similar to the estimated approximately 150 A length of the extended Br-PEG-peptide. This Br-polystyrene distance depends on adsorption time and surface properties prior to adsorption. Incomplete multilayers form on the polar surface after sufficient adsorption time elapses. By contrast, adsorption onto the nonpolar surface is submonolayer, patchy, and highly disordered with an isotropic Br distribution. Overall, this combination of X-ray surface scattering techniques with a novel sample preparation strategy has several advantages as a real space probe of adsorbed or covalently bound biomolecules at the liquid-solid interface.

Chemical derivatization of peptides allows efficient F2 laser single photon ionization (SPI) of Fmoc-derivatized peptides covalently bound to surfaces. Laser desorption photoionization mass spectrometry using 337-nm pulses for desorption and 157.6-nm pulses for threshold SPI forms large ions identified as common peptide fragments bound to either Fmoc or the surface linker. Electronic structure calculations indicate the Fmoc label is behaving as an ionization tag for the entire peptide, lowering the ionization potential of the complex below the 7.87-eV photon energy. This method should allow detection of many molecular species covalently or electrostatically bound to surfaces.

Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture. Peptides covalently bound to silicone surfaces are examined here for the enhancement of cellular adhesion during in vitro dynamic flexing. A procedure is described for the chemical modification of medical grade silicone membranes with covalently bound GRGDSP peptides. The conditions for mechanical studies of cardiac cell cultures are then duplicated and it is demonstrated that the peptide layers survive 48 h of mechanical flexing in vitro. Specifically, mechanical flexing in vitro of the 30 pmol/cm2 peptide-modified silicone membranes has no significant effect on the amount of peptides that remains bound to the surface. Cardiac fibroblasts display enhanced adhesion to these peptide-bound silicone membranes for at least 24 h of growth, compared with native silicone or tissue culture polystyrene. The effects of serum versus serum-free media on fibroblast growth are also examined.