In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces

1. In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces Andrew RichterValparaiso University

2. Acknowledgements Undergraduate Students Christopher McCay Jason Van de Walker Amanda Taticek Lawrence Selvy Josh Vredevoogd Sector 1 Jin Wang Peter Lee Sector 9 Ivan Kuzmenko Thomas Gog

3. Protein Adsorption Cell Membranes Stents www.endovasc.com/ images/graphics/stent.jpg Tissue Engineering Artificial Joints http://lifesci.rutgers.edu/~molbiosci/ProfessorPics/sponge_DTE_Chris_553.jpg http://www.orthosupplier.com/players/images/ionbond/medthin.jpg

4. Model Surfaces: Organic Films Self-Assembled Monolayers (SAMs) Octadecyltrichlorosilane (OTS) on silicon oxide  Hydrophobic interface (110º contact angle) (Can modify the “tail” of the molecule to create “functionalized surfaces”) http://www.barrettresearch.ca/teaching/ nanotechnology/nano07.htm

5. X-Ray Reflectivity 500 Å polymer film in air Total external reflection below θc. θc depends on density of film and what’s above it. • Reflected x-rays interfere with each other. • Creates a series of maxima and minima as a function of incident angle.

6. In Situ X-ray Reflectivity Benefit Problem

7. Proteins Studied Solvent: Potassium Buffered Saline Solution (PBS), pH 7.4 Concentration: 0.05 – 10 Temperature: 25 – 30 ºC Human and Bovine Serum Albumin (HSA, BSA) (Bovine) Immunoglobulin G (IgG) MW: 67 kDa MW: 146 kDa

8. Serum Albumin Results • Protein film develops very quickly and gives a clear signature. • BSA denatures extensively: • Forms a dense layer next to OTS (22% above water density) • Hydrophilic strands extend into solution. • Extent of film visible against water is 15 – 20 • There is a depletion layer of water above hydrophobic surface (Richter, APS 2005). • For most cases, the depletion layer persists after protein adsorption.

9. IgG Studies Ex situ ellipsometry suggested some time evolution over tens of minutes.

10. IgG X-ray Results • Film develops very quickly. • Like BSA, IgG denatures extensively: • Dense layer near OTS (16% above water density) • Extends into solution about 20

11. Comparison to Ex Situ Studies • IgG film clearly thicker than 20 • IgG density drops below water density about the same place as in situ. • Still see evidence for depletion layer.

12. Same Sample, in Solution • Looks very similar to in situ studied IgG films. • True extent of protein film gets masked by water.

13. Current Status • Protein films can be detected. • Can see high density layer next to surface. • Protein denatures extensively, with a slow decay of density into the solution. • Hinders complete analysis of film extent. • Time resolution currently elusive. • Protein films adsorb almost immediately. • Don’t see any conclusive long-term evolution.

14. Future Work • Try smaller, more compact proteins and peptides. • Develop faster reflectivity methods • Energy-dispersive • Ewald-sphere/linear detector • Play with solution parameters to change deposition rates and film completeness. • Use other functionalized surfaces. Thanks for your attention

15. X-ray Damage? Sample x-rayed during growth largely same as sample x-rayed after growth.

16. X-ray Damage? Purposeful damage experiments show little damage for less than 20 minutes exposure at 10% full beam intensity.

17. Depletion Layer Adelé Poynor, et al, "How Water Meets a Hydrophobic Surface," Phys. Rev. Lett. 97, 266101 (2006). Dosch, et al, “High-resolution in situ x-ray study of the hydrophobic gap at the water–octadecyl-trichlorosilane interface,” PNAS103, 18401-18404 (2006).