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. 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