In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces Andrew Richter Valparaiso University

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

Protein Adsorption Stents images/graphics/stent.jpg cs/sponge_DTE_Chris_553.jpg Cell Membranes Tissue Engineering /images/ionbond/medthin.jpg Artificial Joints

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

Method Comparison

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

In Situ X-ray Reflectivity Worse contrast—weaker features.  Little control over electron densities More background from solution and more absorption in solution.  Work at higher energies (i.e., 15 keV, 24 keV) More damage? But don’t have to remove from environment

In Situ X-ray Reflectivity Can work at liquid-solid interface: can study biomolecular interactions where they occur. Poor contrast between solution and film. Must use high energy (24 keV), high intensity sources (APS). Very high spatial resolutionMust limit exposure to lessen radiation damage. Label-freeLittle control over contrast Moderate time resolution (3 – 5 minutes) Only moderate time resolution BenefitProblem

In Situ Cell Teflon Cell 10 ml capacity 10 mm width Windows Kapton/Beryllium Transmission Design

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

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.

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

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

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.

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

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.

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

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

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

Depletion Layer Adelé Poynor, et al, "How Water Meets a Hydrophobic Surface," Phys. Rev. Lett. 97, (2006). Dosch, et al, “High-resolution in situ x-ray study of the hydrophobic gap at the water– octadecyl-trichlorosilane interface,” PNAS 103, (2006).