The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments

Harris, J. Milton

English

NASA Technical Reports Server

5j - 7
1987 N 8 8 - 15 6 2 _/_ 7.z /
NASA/ASEE SUMMER FACULTY RESEARCH FELLOWSHIP PROGRAM _ _.
MARSHALL SPACE FLIGHT CENTER
THE UNIVERSITY OF ALABAMA IN HUNTSVILLE
USE OF HYDROPHILIC POLYMER COATINGS FOR CONTROL OF ELECTROOSMOSIS
AND PROTEIN ADSORPTION
Prepared by:
Academic Rank:
University and Department:
NASA/MSFC:
Laboratory:
Division:
Branch:
NASA/Colleague:
Date:
J. Milton Harris
Professor
Univ. Alabama Huntsville
Chemistry
Space Science
Low Gravity Science
Biophysics
Robert Snyder
September 3, 1987
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https://ntrs.nasa.gov/search.jsp?R=19880006238 2020-03-20T07:37:45+00:00Z
ABSTRACT
The purpose of this project was to examine the utility of polyethylene
glycol (PEG) and dextran coatings for control of electroosmosis and protein
adsorption; electroosmosis is an important, deleterious process affecting
electrophoretic separations, and protein adsorption is a factor which needs
to be controlled during protein crystal growth to avoid multiple nucleation
sites. Performance of the project required use of X-ray photoelectron
spectroscopy to refine previously developed synthetic methods. The results
of this spectroscopic examination are reported. Measurements of
electroosmotic mobility of charged particles in appropriately coated
capillaries reveals that a new, one-step route to coating capillaries gives
a surface in which electroosmosis is dramatically reduced. Similarly, both
PEG and dextran coatings were shown by protein adsorption measurements to
be highly effective at reducing protein adsorption on solid surfaces.
These results should have impact on future low-g electrophoretic and
protein crystal growth experiments.
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INTRODUCTION
This work involves use of organic polymer coatings to control
electroosmosis and protein adsorption. Before giving details of the
experiments that were performed it is useful to provide a brief review of
some background concepts.
Electroosmosis can be described as movement of a conducting liquid
relative to a stationary charged surface, which results when an electric
field is applied to the liquid. Generally, clean surfaces will be
negatively charged, so that application of an electric field will produce
movement of the positive counterions toward the negative electrode. This
electroosmotic flow reduces the effectiveness of electrophoretic processes
and thus tends to eliminate the benefits achieved by performing
electrophoresis in low g where thermal and concentration driven convection
is eliminated.
We have previously shown that polyethylene glycol (PEG) coatings are
effective at reducing electroosmosis (1). Apparently this reduction
results from the hydrophilic polymer coating acting as a wetted viscous
layer (a "kelp forest") over the charged surface. The goal of the present
work is to simplify the coating chemistry by preparing a PEG-silane that
can be directly applied to glass surfaces. The previous technique required
three steps: preparation of an activated PEG, amination of the glass
surface, and coupling of the activated PEG to the aminated surface.
The second part of this project is concerned with examining the
ability of hydrophilic p.olymer coatings (PEG and dextran) for their ability
to reject proteins (i.e., their biocompatibility). The coatings are
tethered to the surface by a single covalent linkage (the kelp forest
analogy.) and would be expected to exclude proteins from the surface simply
by waving around. This phemonenon has been used to explain the ability of
covalently-linked PEG to render proteins nonimmunogenic. In this work we
have attached PEG and dextran to glass slides and measured the extent of
.fibrinogen 1-125 adsorption. Controlling protein adsorption is important
in protein electrophoresis and in protein crystal growth experiments where
it is desirable to avoid multiple nucleation sites.
An important aspect of all this work is utilization of X-ray
photoelectron spectroscopy (XPS) to characterize our surfaces. This
technique provides a direct elemental analysis of surfaces. By using it we
should have a more quantitative and direct means of following chemistry on
the surfaces. Previously, we have used wet chemical techniques that give
averages of surface chemistry. With XPS we can study surface homogeneity
and we can work with small slides having too little surface area to use the
wet chemical methods previously applied.
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OBJECTIVES
The objectives of this work are as follows:
(1) to prepare a PEG silane and couple it to glass
(2) to measure the electroosmotic mobility of a polystyrene
microsphere in a PEG-silane coated glass capillary
(3) to improve our glass amination procedures by use of X-ray
photoelectron spectroscopy
(4) to measure the extent of protein (fibrinogen) adsorption
on dextran- and PEG-coated glass using improved amination
procedures to coat the glass
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Results of X-Ray Photoelectron Spectroscopic (XPS) Surface Analysis
Our first goal was to use XPS to examine coating chemistry. The first
step in the standard coating procedure is to aminate the glass surface
using trimethoxyaminopropylsilane, 1. Previously we examined the
efficiency of this process by conducting the reaction on porous glass (with
a large surface area) and titrating the amine groups with acid. With XPS
we can directly determine the percentage of nitrogen on the surface of
slides actually used in the p.roteln adsorption experiments. Nitrogen has a
relatively small cross section, so we have fluorinated the surface by
reacting the aminated surface with pen.tafluorobenzaldehyde. Thus we can
check the nitrogen results by measuring the percentage of fluorine, an
element having a much larger cross section.
In a typical amination procedure the glass substrate is immersed in a
5% soluton of 1 in water for 24 hours at 80 degrees, and then cured dry at
110 degrees for four hours; after curing, the glass is exhaustively washed
with distilled water. Surfaces prepared in four different ways were
examined. The four routes are: (1) treatment with 1 followed by washing
with water before curing; (2) as in example 1, but done twice; (3)
treatment with 1 followed by curing without washing; (4) as in example 3,
but done twice. The results of these experiments, using two different XPS
instruments (a Perkin-Elmer at UAH and a Surface Science at University of
Washington), are given in Table 1.
Table 1.
slides (prepared four ways as described in text) treated
pentafluorobenzaldehyde on an Perkin-Elmer XPS and a Surface Science XPS.
Measurement of percent nitrogen and fluorine on aminated glass
with
Method
#1 #2 #3 #4
%N (Perkin-Elmer) 0.98 1.98 2.30 2.37
%N (Surface-Sci.) 1.66 1.95 2.75 5.09
4.49 5.45 4.17 7.17%F (Surface-Sci.)
The data show that higher percentages of nitrogen are obtained if the
surface is not washed before cunng. Also a higher nitrogen coverage is
obtained if the process is repeated. It is noteworthy that significant
different values for percent nitrogen were measured with the two
intstruments. The Perkin-Elmer machine is new and uncalibrated, so it is
likely that the Surface Science data are more trustworthy. Further
comparison of the two machines with known substrates will be done. Also,
the %F does not parallel the trends in %N. This result has two possible
explanations. First, it is known that the XPS probes the first 5 nm of the
surface, and the fluorines are applied directly onto the surface; so the
nitrogen and fluorine measurements are actually made on different layers of
surface. A second possibility is that the pentafluorobenzaldehyde liquid
alters the surface in some way. We are now exploring a vapor-phase
XX-3
fluorination procedure with trifluoroacetic anhydride to avoid this second
possibility.
RESULTS OF ELECTROOSMOSIS EXPERIMENTS
As noted above, the goal of this segment of the project is to attach
PEG to glass capillaries in a single step by reaction with a PEG-silane.
The PEG-silane was synthesized by reacting PEG with trimethoxysilylpropyl
isocyanate in methylene chloride. The reaction could be followed on IR bY
monitoring the isocyanate absorbance. Reaction of the PEG-silane with
guartz electrophoresis capillaries was performed as described above.
Electroosmotic mobilities were then determined for uncoated, standard two-
step coated, and silane coated capillaries; standard coating involves the
two-step process with amination and reaction with activated PEG described
above. In both cases, the PEG used was the monomethyl ether of PEG 5000.
Details on electroosmosismeasurementsare available in reference 1.
Measured electroosmotic mobilities (in um/s cm/V at pH 5.78, 7.5 mM
NaC1) were: uncoated, 1.7; standard coated, 0.3; and silane coated, 0.7.
This result shows that the sing!e-s.tep, silane coating is effective at
reducing electroomosis, although it xS not as effective as the standard
two-step procedure. It will be very interesting to follow up on these
results by doing XPS experiments to determine the extent of PEG coverage
using the two methods. Also it will be interesting to determine the pH
dependence of the two different coatings, since we earlier concluded that
the two-step procedure left some uncoupled amine groups which could show a
large response to pH variation.
RESULTS ON PROTEIN ADSORPTION
The final set of experiments were to measure the extent of protein
adsorption on PEG- and dextran-coated surfaces. The actual measurement was
extent of fibrinogen 1-125 adsorbed onto the materials. The results are
presented in Table 2.
Table 2. Fibrinogen 1-125 adsorption of PEG coated glass slides
substrate protein adsorbed (ug/cm 2)
glass 0.420
glass-amine 0.683
glass -PEG200 0.540
-PEG400 0.468
-PEG 1000 0.382
-PEG3000 0.206
-PEG 8000 0.104
-PEG20000 0.072
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As can be seen from Table 2, there is a dramatic drop in protein
adsorption with the PEG MW. Presumably the larger PEGs have a larger
exclusion volume and are thus better able to "sweep" the protein from the
surface. We have also examined dextran coatings. Although the experiments
are preliminary, we do have qualitative data showing that dextran is even
more effective than PEG at reducing protein adsorption. The dextran was
attached by reductive amination of aminated glass by the reducing end of
the polysaccharide.
CONCLUSIONS
The goals of the proposed work have been met. Polymer coating
chemistry was improved by use of XPS, and the PEG-silane was synthesized.
PEG coatings attached to electrophoresis capillaries by means of the PEG-
silane are effective at reducing electroosmosis, although they are not as
effective as those coatings applied by the standard two-step procedure.
Finally, PEG and dextran coatings are very effective at reducing protein
adsorption onto glass, and thus these coatings offer potential for
controlling nucleation sites in protein crystal .growth. Future work will
involve refinement of coating chemistry using fully calibrated XPS
spectrometers and examination of dextran coatings for control of protein
adsorption and electroosmosis.
REFERENCES
(1) B. J. Herren, S. G. Sharer, J. M. Van Alstine, J. M. Harris, and R. S.
Snyder, "Control of Electroosmosis in Coated Quartz Capillaries," J.
Colloid Interface Sci., llS, 46-55 (1987).
ACKNOWLEDGEMENTS
Assistance in obtaining electroosmosis measurements was provided by J.
M. Van Alstine and Todd Williams. Assistance in use of the XPS was
provided by Mike Edgell and Wayne Gombotz. Wayne Gombotz also provided
protein adsporption data. Financial assistance of this work by NASA is
gratefully acknowledged.
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Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

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