Investigating the Origins of Protein-Surface Adsorption:Ellipsometry: A Macroscopic Measure of Protein Surface AdhesionForce Microscopy: A Microscopic Measure of Protein AdhesionProtein vs SubstrateSAM-SAM & Protein-Protein InteractionsProtein-SAM InteractionsNot All Proteins Were Created EqualThe Importance of ConformationSlide 9Effect of s and L on Surface Interaction ForcesThe Effect of s and L on Protein Adhesion to PEOThe Effect of the Substrate on the SAM ConformationFibrinogen AdhesionTip-Surface Electrostatics: the Effect of Ions in SolutionTip-Surface Electrostatics: the Effects of Ionic Strength and Molecular WeightPolymer ArchitecturesEffect of Chain Architecture on Protein AdsorptionMeasuring Protein Adhesion with the Surface Force ApparatusCompression Leads to Protein-Surface BindingConclusionsInvestigating the Origins of Protein-Surface Adsorption:Experimental ResultsEllipsometry: A Macroscopic Measure of Protein Surface AdhesionitrRparallel / RperpendicularEi = Ellipsometery measures theRatio of the electric fields ofThe reflectid waves parallel and perpendicular to theInterface; from this you mayExtrapolate the thickness of The interfaceAlternatively: ellipsometry Measures the abruptness ofChange in refractive index From the surrounding medium (air, e.g.) to the substrate, and from this extrapolates the film thicknessForce Microscopy: A Microscopic Measure of Protein Adhesion •Protein is covalently attached to the probe tip•Adhesion is measured on various substrates/SAMs of different degrees of hydrophilicityProtein vs Substrate•Three different blood plasma proteins studied: Albumin (Alb), Immunoglobulin G (IgG), and Fibrinogen (Fib)•Four different SAMs studied; in order of increasing hydrophilicity: -CH3, -OH, -NH2, -COOH •Protein-protein, protein-SAM, and SAM-SAM interactions comparedKidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.The experiment:SAM-SAM & Protein-Protein InteractionsKidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.Protein-SAM InteractionsSchematic of protein adhesionExperimental resultsKidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.Not All Proteins Were Created Equal•In order of increasing SAM affinity for each protein:–Alb, IgG: -CH3 >> (-OH, -NH2) > -COOH–Fib:-CH3 >> -OH > -NH2 > -COOH–Fib > Alb, IgG on all surfaces except -COOHKidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.The Importance of Conformation* The extent of protein interaction depends not only on the type of SAM, but also on the SAM conformationKidoaki, S.; Nakayama, Y.; Matsuda, T. Langmuir 2001, 17, 1080-1087.Re: Methods for Counteracting Protein-Surface Interaction with Polymer Coatings•Dense polymer coatings (low )•Long polymer chains (large N)d R  NUout may be manipulated by varying N or  Uin is primarily controlled by varying Effect of  and L on Surface Interaction Forces•The polymer chains in a brush are not fully extended: •There is a point at which the polymer layer becomes incompressible: Dowhere D’ = Dexperimental andPMMA)for (0.6 1contourmequilibriuLLPMMA)MW high for (0.8 1eqLDYamaoto, Shinpei; Muhammad, Ejaz; Yoshinobu, Tsujii; Matsumoto, Mutsuo; Fukuda, Takeshi. Macromolecules 2000, 33, 5602-5607.Yamaoto, Shinpei; Muhammad, Ejaz; Yoshinobu, Tsujii; Fukuda, Takeshi. Macromolecules 2000, 33, 5608-5612. oD D'D0.5)-0.3(n nThe Effect of  and L on Protein Adhesion to PEO•At very high surface densities , SAMs will resist adsorption of all types of proteins, with universal resistance achieved at lower  for higher molecular weight (larger L) SAMs•L is not as influential as  •The highest L at optimum  is most effective at protein resistance•Adhesion is temperature-dependentJeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid and Interface Sci., 142 (1), 149-158 (March1, 1991).Jeon, S. I.; Andrade, J. D. J. Colloid and Interface Sci., 142 (1), 159-166 (March 1, 1991).Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc., 1993, 115 10714-10721.The Effect of the Substrate on the SAM Conformation•PEO on gold in aqueous solution is predominantly in a helical conformation stabilized by H-bonding•On silver, however, the binding sites are so close that the helix is sterically hinderedFeldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.Fibrinogen AdhesionMicaC16H33-AuEG3-AuEG3-AgFeldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.Tip-Surface Electrostatics: the Effect of Ions in SolutionSi3N4 tip + EG3C16 tip + EG3DI H2OPBSAuAgFeldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.Tip-Surface Electrostatics: the Effects of Ionic Strength and Molecular WeightFeldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.Polymer ArchitecturesLinear CombStarEffect of Chain Architecture on Protein AdsorptionMayes, A. M.; Irvine, D. J.; Griffith, L. G. Mat. Res. Soc. Symp. Proc. 1998, 530, 73-84.In contrast to linear polymers, the center mass for star polymers lies at some distance away from the surface.This results in a much more energetically favored state for protein adhesion at the surface, once diffusion through the polymer layer is achievedMeasuring Protein Adhesion with the Surface Force ApparatusSheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).Compression Leads to Protein-Surface Binding•A: Protein resistance still observed at low compressive loads ( <4kT )•B: Under sufficient compressive loads ( >4kT ) attractive interactions dominateNote: Derjaguin approximation: kTRF22per chainSheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).Conclusions•Design of biomaterials is challenged by the complicated, interrelated factors involved in of achieving biocompatibility: i.e. protein resistance vs cell specificity•Because they are easily tailored to meet specific chemical needs, polymers are often used as coatings on compliance-matched devices•Optimization of polymer coatings is a delicate balance among a) size, architecture,