Investigating the Origins of
Protein-Surface Adsorption:
Experimental Results
Ellipsometry: A Macroscopic
Measure of Protein Surface
Adhesion
i
r
Ei = Rparallel / Rperpendicular
t
Ellipsometery measures the
Ratio of the electric fields of
The reflectid waves parallel
and perpendicular to the
Interface; from this you may
Extrapolate the thickness of
The interface
Alternatively: ellipsometry
Measures the abruptness of
Change 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
Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.
The experiment:
• 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, proteinSAM, and SAM-SAM
interactions compared
SAM-SAM & Protein-Protein
Interactions
Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.
Protein-SAM Interactions
Experimental results
Schematic of protein adhesion
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
Uout may be manipulated by varying N or s
Uin is primarily controlled by varying s
• Dense polymer
coatings (low s)
• Long polymer chains
(large N)
RaN
das
Effect of s and L on Surface
Interaction Forces
•
The polymer chains in a brush are not
fully extended:
Lequilibrium
Lcontour
•
 1 (0.6 for PMMA)
 s n (n  0.3 - 0.5)
There is a point at which the polymer
layer becomes incompressible: Do
D  D'  Do   s
where D’ = Dexperimental and
D
 1 (0.8 for high MW PMMA)
Leq
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.
The Effect of s and L on Protein
Adhesion to PEO
• At very high surface densities s, SAMs will
resist adsorption of all types of proteins, with
universal resistance achieved at lower s for
higher molecular weight (larger L) SAMs
• L is not as influential as s
• The highest L at optimum s 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 Hbonding
• 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. Am.
A. Chem.
Chem.Soc.
Soc.1999,
1999,121,
121,10134-10141.
10134-10141.
Tip-Surface Electrostatics: the
Effect of Ions in Solution
C16 tip + EG3
Si3N4 tip + EG3
DI H2O
PBS
Au
Ag
Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am.
A. Chem.
Chem.Soc.
Soc.1999,
1999,121,
121,10134-10141.
10134-10141.
Tip-Surface Electrostatics: the
Effects of Ionic Strength and
Molecular Weight
Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am.
A. Chem.
Chem.Soc.
Soc.1999,
1999,121,
121,10134-10141.
10134-10141.
Polymer Architectures
Linear
Comb
Star
Effect of Chain Architecture on
Protein Adsorption
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
Mayes, A. M.; Irvine, D. J.; Griffith, L. G. Mat. Res. Soc. Symp. Proc. 1998, 530, 73-84.
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:
Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).
F
 2kT
2R
per chain
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 compliancematched 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