CHAPTER FIVE
Discussion of results
The results presented in chapter four suggest that the various factors such as
plasma film thickness, protein concentration, plasma input power, pH of the
buffer used and most importantly the surface chemistry of the film affect the
adsorption of the proteins in one way or the other.
Considering the surface chemistry of the plasma polymer films investigated
using FTIR, both films before and after extraction showed an adsorption peak
at 3309 cm-1 (N-H stretching), which indicated the presence of amine groups
with the films deposited at low plasma input powers showing a broader peak.
It was also observed that the amine peak for the films after extraction was
broader compared to the freshly deposited films, which suggested a high
retention of the amine groups after film extraction in PBS. As the plasma input
power increased from 10 to 90W the intensity of this peak decreases for both
films relative to the multiple peaks at 2967cm -1, 2935 cm-1 and 2867 cm-1
which are as a result of the C-H stretching of aliphatic groups. This effect is
more clearly seen for the films after extraction. This suggests that at low
plasma input powers, the amine functionality is retained to a greater extent
than in high plasma input powers. This high retention of amine groups at low
plasma input powers has also been observed in various works done by the
group with allylamine as the monomer.4,10
Krishnamurthy and co-workers
also observed this high retention of amine at low plasma input powers when
they investigated plasma polymerised allylamine using FTIR33 .
49
The FTIR results are also in conformation to the water contact angle data of
the films. From figure 4.3, the contact angle increases with increasing plasma
input power suggesting a linear dependency of the contact angle on the
plasma input power. The differences in contact angles for films before and
after extraction can be attributed to the variations in cross linking densities
and different proportions of the amine groups retained before and after film
extraction in PBS as already shown in the FTIR spectra (see section 4.1.1
and 4.2). Another possible cause may be the surface roughness. At high
plasma input powers, there is an increase vapour phase initiation of
polymerisation with an increased residue deposition which leads to a
particulate like rough texture on the surface and may result in higher values
for wettability.33 There is also the possibility of surface etching caused by
ablation at high plasma Input powers. This was also confirmed by the
increase in the average surface roughness value obtained for the plasma
polymer films by AFM measurements with films deposited at higher input
power showing the higher roughness values. Z. Zhang in her work with
plasma polymerised maleic anhydride and di- (ethylene-glycol) vinyl ether
also observed an increase in surface roughness of the films with increasing
plasma input power.10 This observation can be said to be general for all
plasma polymer films.
SPR kinetic results of the films measured in PBS showed a decrease in the
percentage reflectivity with films deposited at low plasma input power showing
the highest percentage decrease. This decrease can be thought of as the
dissolution of low molecular weight and non-covalently bonded materials from
50
the polymer network which is observed as a decrease in film thickness; and
the removal of deposited films as a result of insufficient adhesion of the film to
the substrate.(10,15) Considering also the effect of input power on the degree of
cross-linking, at high input powers, the films are highly cross-linked and
contains relatively small amount of low molecular weight particles. At low
energy input, only relatively small amounts of free radical species are present
at the surface of the growing film. Due to this, plasma films polymerised under
such conditions contain lower molecular weight material, which dissolve from
the polymer network in solution. There is also a high mobility of the polymer
chains in solution and materials that are not covalently bonded to the
substrate tend to be washed away.
Protein adsorption results from SPR kinetic measurements showed an
increase in adsorption with decreasing plasma input powers (see fig 5.1 and
5.2). There are several factors believed to be contributing to this effect.
The first of these factors is the plasma conditions applied and surface
chemistry. As already shown by the FTIR results, at low plasma input powers,
there is a high retention of the amine groups, which also makes the surface
more hydrophilic. This observation is in agreement with the FTIR results of
Zhihong Zhang10, V. Krishnamurthy et al33 and other works done by the group.
From the adsorption kinetics, there is a clear dependence of the adsorbed
thickness on the plasma input power applied, with the film deposited at low
plasma input power with high retention of amino groups showing the highest
adsorption. The retention of amine groups at the surface can be considered to
51
be a contributing factor to the amount proteins adsorbed on the surface of the
film.
Protein adsorption process is known to be driven by a combination of factors
such as surface functionality, hydrophobic interactions and electrostatic
interaction. Considering the plasma polymer film, PPAA films are known to be
positively charged in PBS buffer at a pH of 7.4. At this pH BSA with an
isoelectric point of 4.4 is also expected to posses a negative charge. Hence
the negative charge proteins are strongly adsorbed by electrostatic
interactions. The higher amino density of the plasma polymer films retained at
low plasma input power leads to a stronger electrostatic interactions between
the proteins and the film surface. On the other hand, IgG with IP value
between 6.5-8 at pH of 7.4 is expected to be slightly positive or neutral. Due
to the almost neutral character of the protein and the surface of the film, there
is a high repulsion, which is believed to cause the hydrophobic part of the
protein to be exposed and react with the film surfaces. That is hydrophobic
interaction is more pronounce. There is also the dependence of the plasma
film thickness on the adsorption with higher film thickness showing higher
adsorption. Z. Zhang10 observed the same phenomena with fibrinogen
adsorption on plasma polymerised allylamine films whereby she observed an
increase in fibrinogen adsorption with increasing polymer thickness.
52
Initial adsorption
retained
0.01
11
90W
0.110
Concentration (mg/ml)
19
8
0.017
50W
0.16
15
4
0.013
10W
0.12
11
0
2
4
6
8
10
12
14
16
18
20
22
24
Protein thickness (nm)
Fig. 5.1 effects of plasma input power and IgG concentration on its adsorption onto PPAA
films
53
26
Initial adsorption
Retention
11
0.01
90W
0.1
10
Concentration (mg/ml)
19
8
0.017
50W
0.16
15
4
0.013
0.1
10W
2
11
0
2
4
6
8
10
12
14
16
18
Protein thickness (nm)
Fig. 5.2 effects of plasma input power and BSA concentration on its adsorption onto PPAA
films
A second factor is the protein concentration and the orientation of the protein.
From the graphs presented above, there is an obvious dependence of the
protein adsorption affinity on the concentration. This observation has already
been explained as to be due to the dynamic phenomena that take place
during protein adsorption as illustrated in figure 2.2 where there is the
possibility of exchange reaction between already adsorbed protein molecules
and molecules from the solution. This exchange reaction is believed to go on
until the adsorbed protein molecules develop its contact points with the
54
20
surface. Once this happens, Subsequent increase in concentration may
increase the amount of adsorbed molecules. Also, considering the
orientations of the proteins at an interface and the molecular dimensions of
the proteins, IgG has a molecular weight of 156,000 Daltons with a height
approximately 12nm, BSA on the other hand has a molecular weight of
68,460 Daltons with dimensions of approximately 8nm by 4nm.10 (see fig. 4.7)
Comparing the adsorbed thickness at the various concentrations, it can be
seen that at the same concentration, IgG adsorption is higher than that of
BSA. This is mainly due to the differences in the molecular weight and
dimensions of both proteins. Considering the adsorbed protein thickness at
the various concentrations, it can be assumed that at high concentrations,
both proteins adsorb into one or more layers on the surface of the film. BSA
especially can be assumed to adsorb in two layers end-on, whereas IgG
adsorbs in one layer head-on for about the same polymer thickness. The
concept of multilayer adsorption can be assumed because, proteins are
known to under go conformational changes which are likely to expose more
sites which may provide a new environment for bulk proteins that approach
the initial adsorbed layer. If this layer results in attractive interactions between
the adsorbed and bulk molecules, additional layers will begin to form at the
interface. 38
One other important factor observed to affect the adsorption of the proteins is
the pH of the buffer used. It was observed for both proteins, that, after the
introduction of the buffer with pH 4, the percentage reflectivity decreases
55
which indicates the loss of some protein molecules from the surface of the film
(fig. 4.1.7and 4.1.8). This can be due to the fact that at pH of 4, both proteins
obtain a positive charge, which induces the release of less-well bound
proteins.36 This loss was confirmed by the decrease in the protein thickness
from 27.5nm to 21.7nm and 17.2nm to13.4nm for BSA and IgG respectively
after washing with PBS. The opposite was observed after the introduction of a
higher pH buffer (9.5). As can be seen from figures 4.1.9 and 4.2, there is an
increase of about 10% in the percentage reflectivity indicating an increase in
the adsorbed protein thickness. A possible cause might be due to the fact that
at high pH values, the proteins obtain large negative charges, which are
known to cause unfolding and even aggregation. There maybe other
contributing factors to this observation, which are not yet known and are
subject for further investigation.
5.1
Conclusions
The adsorption of proteins on plasma polymerised allylamine was investigated
using SPR. The results presented demonstrate that plasma polymerised
allylamine films can be used as surfaces to influence the adsorption of
proteins. The result showed that there is no single property of the surface that
dominates the protein adsorption behaviour and that protein adsorption is
significantly affected by the surface functional groups, the concentration of the
protein in solution as well as the polymerisation condition. Among the three
plasma conditions applied, the affinity of proteins on the low plasma input
power film was higher than that deposited at higher plasma input power due
56
to the low degree of cross-linking, as well as the high retention of the amine
groups at the surface.
57
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