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The structure, orientation and formation of amphiphilic α-helix model peptide films on fluorocarbon surfaces has been monitored with sum frequency generation (SFG) vibrational spectroscopy, near edge X-ray absorption fine structure (NEXAFS) spectroscopy and X-ray photoelectron spectroscopy (XPS). The α-helix peptide is a 14-mer of hydrophilic lysine and hydrophobic leucine residues with a hydrophobic periodicity of 3.5. This periodicity yields a rigid amphiphilic peptide with leucine and lysine side chains located on opposite sides. XPS composition analysis confirms the formation of a peptide film that covers about 75% of the surface. NEXAFS data are consistent with chemically intact adsorption of the peptides. A weak linear dichroism of the amide π* is likely due to the broad distribution of amide bond orientations inherent to the α-helical secondary structure. SFG spectra exhibit strong peaks near 2865 cm−1 and 2935 cm−1 related to aligned leucine side chains interacting with the hydrophobic surface. Water modes near 3200 cm−1 and 3400 cm−1 indicate ordering of water molecules in the adsorbed--peptide fluorocarbon surface interfacial region. Amide I peaks observed near 1655 cm−1 confirm that the secondary structure is preserved in the adsorbed peptide. A kinetic study of the film formation process using XPS and SFG showed rapid adsorption of the peptides followed by a longer assembly process. Peptide SFG spectra taken at the air–buffer interface showed features related to well ordered peptide films. Moving samples through the buffer surface led to the transfer of ordered peptide films onto the substrates.

Surface trimer crystallization on poly (ethylene terephthalate) studied by timeofflight secondary ion mass spectrometry J. Vac. Sci. Technol. A 13, 1217 (1995); 10.1116/1.579864 Timeofflight static secondary ion mass spectrometry of additives on polymer surfaces J. Vac. Sci. Technol. A 9, 1307 (1991); 10.1116/1.577617 MASTIF: Mass analysis of secondaries by timeofflight technique. A new approach to secondary ion mass spectrometry Rev. Sci. Instrum. 60, 3188 (1989); 10.1063/1.1140550 Timeofflight secondary ion mass spectrometry of polymer materialsThis study presents the static time-of-flight secondary ion mass spectrometry ͑TOF-SIMS͒ spectra of 15 poly͑amino acids͒, solvent or spin cast onto either mica or silicon substrates. These poly͑amino acid͒ spectra are useful for interpreting the complex static TOF-SIMS spectra obtained from adsorbed protein films and peptide-functionalized surfaces. Previous studies have reported poly͑amino acid͒ spectra acquired with a quadrupole SIMS instrument. The spectra obtained with a TOF-SIMS instrument in this study have significantly higher sensitivity and mass resolution, which are essential for producing good, high-quality reference spectra.

The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can an accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C2H5S+ intensity, a secondary ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK310) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide’s lysine side chains were ordered on the quartz surface, while the peptide’s leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK310 film and the orientations of amide bonds within the LK310 peptide.

The adherence and activation of primary human monocytes was investigated on a polyelectrolyte multilayer film containing hyaluronic acid (HA) and poly-L-lysine (PLL). The sequential layer-by-layer deposition of the multilayer film was characterized by surface plasmon resonance. Eight alternating bilayers displayed an effective thickness of 16.15 nm with a total polymer coverage of 2.10 microg/cm2. For cell studies, HA-PLL multilayers were constructed on tissue culture polystyrene (TCPS) substrates and characterized by time of flight second ion mass spectrometry (ToF-SIMS) analysis. Principal component analysis of the ToF-SIMS spectra resolved no significant difference in surface chemistry between PLL-terminated and HA-terminated multilayer surfaces. Monocyte adhesion on PLL- and HA-terminated surfaces was measured by the lactate dehydrogenase assay and showed a significant decrease in cell adhesion after 24 h incubation. Cell viability measured by Live/Dead fluorescent staining showed significant cell death in the adherent cell population over these 24 h. Tumor necrosis factor-alpha (TNF-alpha) production, a measure of monocyte activation, was quantified by ELISA and normalized to the number of adherent monocytes. The activation of monocytes on PLL-terminated and HA-terminated surfaces was nearly identical, and both surfaces had TNF-alpha levels that were 8-fold higher than TCPS. These results demonstrate that sufficient PLL had diffused into the surface layer to direct monocyte adherence and to induce cytokine activation and cell death on the HA-terminated multilayer films. The diffusion of the second multilayer component to the coating surface should, thus, be taken into account in the design of polyelectrolyte-based biomaterial coating strategies.

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In the evaluation of tissue adhesives, freshly harvested tissues are the most relevant to clinical application; however, it is difficult to obtain and test experimental samples immediately following surgery. The aim of the present investigation is to elucidate the effect of storage conditions on the ability of preserved tissue samples to mimic freshly harvested tissue. Porcine small intestine was used as a model tissue for the experiments. The treatment conditions were freshly harvested, 24-h aged in phosphate-buffered saline at 5 degrees C and formalin-fixed tissue. Adhesive performance of cyanoacrylate and a newly synthesized tissue adhesive on the three substrates was characterized by T-peel testing. Regardless of the adhesive used, the fresh tissue samples had the weakest bond strength. The 1-day aged tissue produced a bond that was approximately twice as strong, and the formalin-fixed intestine substrate had intermediate bond strength. Comprehensive tissue characterization using attenuated total reflection infrared (ATR-IR) spectroscopy, captive-bubble contact angle, Raman spectroscopy, bulk tissue water content measurement and histology were employed. The results from the different characterization techniques show that storage of tissue samples causes interfacial and chemical changes, limiting their ability to mimic freshly harvested tissue. In particular, the results show the importance of water detected by ATR-IR in mediating adhesion to the tissue samples.