Investigating the Origins of Protein-Surface
Adsorption:Experimental Results
Ellipsometry: A Macroscopic Measure of Protein Surface Adhesion
i
t
rRparallel / RperpendicularEi =
Ellipsometery measures theRatio of the electric fields ofThe reflectid waves parallel and perpendicular to theInterface; from this you mayExtrapolate the thickness of The interface
Alternatively: ellipsometry Measures the abruptness ofChange in refractive index From the surrounding medium (air, e.g.) to the substrate, and from this extrapolates the film thickness
Force 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 hydrophilicity
Protein 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 compared
Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.
The experiment:
SAM-SAM & Protein-Protein Interactions
Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.
Protein-SAM Interactions
Schematic of protein adhesion
Experimental results
Kidoaki, 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 -COOH
Kidoaki, 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 conformation
Kidoaki, 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 N
Uout 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: Do
where D’ = Dexperimental and
PMMA)for (0.6 1contour
mequilibriu
L
L
PMMA)MW high for (0.8 1eqL
D
Yamaoto, 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'D
0.5)-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-dependent
Jeon, 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 hindered
Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.
Fibrinogen Adhesion
Mica
C16H33-Au
EG3-Au
EG3-Ag
Feldman, 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 + EG3 C16 tip + EG3
DI H2O PBS
Au
Ag
Feldman, 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 Weight
Feldman, 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 Architectures
Linear Comb
Star
Effect of Chain Architecture on Protein
Adsorption
Mayes, 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 achieved
Measuring Protein Adhesion with the Surface
Force Apparatus
Sheth, 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 dominate
Note: Derjaguin approximation: kT
R
F2
2
per chain
Sheth, 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, and even supramolecular structure of the polymer, b) the density of the polymer layer, c) the type of underlying substrate and its electrostatic properties, d) the identity of the targeted proteins, and e) the magnitudes of the forces the biomaterial will be subjected to in vivo