3.1. XPS Analysis
As shown in Fig. 1, the main peaks of C1s, O1s, S2s and S2p appear at 284.80 eV, 532.17 eV, 231.19 eV and 168.30 eV as the modified base membrane PSf. PSf-OH membrane is obtained by chloromethylating the polysulfone membrane and then hydroxylation. According to the XPS pattern of PSf-OH, a new Cl2p peak appeared in 200.04 eV, indicating that the -Cl group was successfully grafted to the membrane surface. As shown in Table.1, the content of hydroxylated polysulfone membrane Cl increased from 0–0.36%, while its O content increased from 15.68–16.01%. This indicates that the -OH group is successfully grafted to the surface of the polysulfone membrane. In order to obtain the PSf-COOH and PSf-SO3H membranes, we aminated the PSf-Cl membranes, and a new peak appeared in the XPS pattern of the PSf-COOH and PSf-SO3H membranes, that is, a N1s peak appeared in 399.90 eV, indicating that the -NH2 group successfully replaced the -Cl group. Successfully grafted onto chloromethylated polysulfone membrane. After carboxylation of PSf-NH2 membrane, the oxygen content on the surface of PSf-COOH membrane increased from 15.68–21.02%, while the content of N and Cl decreased respectively. It can be seen that the -COOH group was successfully grafted to the surface of the polysulfone membrane. After sulfonic ylation of PSf-NH2 membrane, the sulfur content of PSf-SO3H membrane increased from 3.14–4.02%, and the peak intensity of S2s and S2p at 231.19 eV and 168.30 eV increased, indicating that -SO3H group was successfully grafted to the surface of the polysulfone membrane.
Table 1
The elemental surface compositions of PSf, PSf-OH, PSf-COOH and PSf-SO3H
Samples | | Elemental(%) |
C | O | N | Cl | S |
PSf | 81.18 | 15.68 | 0 | 0 | 3.14 |
PSf-OH | 78.63 | 16.01 | 2.10 | 0.36 | 2.90 |
PSf-COOH | 74.93 | 21.02 | 1.30 | 0.11 | 2.64 |
PSf-SO3H | 73.44 | 20.91 | 1.63 | 0 | 4.02 |
3.2. Water Contact Angle Analysis
The static contact Angle is a direct method of evaluating wettability and decreases with increasing hydrophilicity to indicate the hydrophilicity of the film surface. As shown in Fig. 2, the contact angles of the membranes, the contact angles of PSf, PSf-OH, PSf-COOH and PSf-SO3H membranes were 86.2°, 69.5°, 40.1° and 38.2°, respectively, while the contact angles of PSf and PSf-SO3H membranes were significantly reduced to 86.2° to 38.2° after grafting -SO3. Therefore, it can be concluded that the hydrophilicity of PSf-OH, PSf-COOH and PSf-SO3H modified film has been greatly improved.
3.3. Adsorption of Protein
According to the thrombosis principle of surface contact with blood, the amount of protein adsorbed on the membrane surface is considered to be an important index to evaluate the compatibility of the membrane to blood. There is a competitive process in the adsorption behavior of proteins. Adsorption of clotting associated proteins may lead to thrombosis, while competitive adsorption of other proteins may have the opposite effec. As shown in Fig. 3, the adsorption capacity of different modified polysulfone membranes for proteins was significantly different, among which the adsorption capacity of BSA in PSf membrane was 326 µg/cm2. The BSA adsorption capacities of PSf-COOH and PSf-SO3H films modified by different functional groups were 221 µg/cm2, 13 5µg/cm2 respectively. According to the test results, the functionalized polysulfone membrane has specific anti-protein adsorption effect, and its anti-protein performance is as follows: PSf < PSf-OH < PSf-COOH < PSf-SO3H.
3.4. MD simulates the initial system state
Bovine serum albumin (BSA) belongs to transport albumin and is one of the most commonly used blocking proteins in biological detection, especially in the detection of adsorbed proteins in blood compatible membrane materials. In addition, BSA molecule has a complete all-atomic structure with high resolution, which is an ideal model protein for detecting protein adsorption correlation simulation methods. In order to study the mechanism of protein adsorption on the surface of membrane materials, BSA was used as the protein model. The model was BSA dimer, whose crystal structure was derived from the RCSB protein database, with a total of 1166 amino acid residues, molecular weight of 133.28 KDa, electric dipole moment of 1819.31 D, and static charge of -32. During the simulation process, the BSA was placed at the upper 0.5nm distance from the surface model, as shown in Fig. 4. The initial velocity of the atom is given by the Maxwell-Boltzmann distribution at 300 K, the simulated step size is 2 fs, and the coupling time is 0.5 ps.
3.5. Influence of BSA dimer backbone stability
The Rg (Gyrate radius) and Root mean square deviation (RMSD) of simulated proteins were calculated to investigate the effects of different end functional groups on the stability of protein main chain[26].
The Rg of a protein is the root-mean-square distance of the equivalent particle with respect to the rotation coordinates of all atoms of the protein backbone, assuming that the protein molecular mass is concentrated at a single point. The Rg value of protein is positively correlated with the volume change of protein. The larger the Rg value of protein is, the larger the volume of protein is. The simulation formula is as follows:
$$Rg={\left(\frac{{\sum }_{i}{‖{r}_{i}‖}^{2}{m}_{i}}{{\sum }_{i}{m}_{i}}\right)}^{\frac{1}{2}}$$
1
where\({r}_{i}\)represents the position of atom i with respect to the molecular center of mass, \({m}_{i}\) represents the mass of atom i.
The variation of cycloidal radius (Gg) during the adsorption of BSA dimer in PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H membrane systems was obtained through simulation, as shown in Fig. 5. The results show that the main chain molecular structure rotation radius of BSA dimer still has the largest variation range in the PSf-CH3 system, indicating that the volume of protein has changed greatly in the range of charge interaction, and the volume of protein in the PSf-CH3 system has been changing during the time-limited simulation time. In the PSf-COOH system, the main chain molecular structure of BSA dimer has the smallest change in its protein cycloidal radius, and the volume change of the protein tends to be stable after 20 ns, indicating that the protein configuration is not easy to change in the PSf-COOH system, and the probability of variability is low. What is more special is that the change law of protein in PSf-OH system is that the volume change of protein is small in the initial 20ns, and the volume change of protein is larger in the time range of 20 ns to 40 ns, and then the volume change of protein tends to be stable. From the perspective of protein rotation radius, the order of the volume change from large to small was PSf-CH3 > PSf-OH > PSf-SO3H > PSf-COOH. This indicates that BSA changes the least in the PSf-COOH system, and the volume of BSA is not prone to repeated folding when the end functional group of the surface of the polysulfone membrane is grafted with -COOH, and in a sense, the protein denaturation of the protein in contact with the membrane is effectively reduced.
Root mean square deviation (RMSD) refers to the coordinate deviation of the atomic coordinates in the protein main chain molecule relative to the reference structure, which is fitted by the least square method. It is used to indicate that with the change of the simulated time protein configuration, the larger the RMSD value fluctuation range, indicating that the protein configuration is prone to change, and the larger the RMSD value, the higher the probability of protein mutation [27].
The main chain atoms N, Cα and C were selected for fitting calculation of RMSD of protein molecule. The simulation formula is as follows:
$$\text{R}\text{M}\text{S}\text{D}({t}_{1},{t}_{2})={\left[\frac{1}{M}\sum _{i=1}^{N}{m}_{i}{‖{r}_{i}\left({t}_{i}\right)-{r}_{i}\left({t}_{2}\right)‖}^{2}\right]}^{\frac{1}{2}}$$
2
Where \({r}_{i}\left(t\right)\) represents the position of atom i at time t.
The changes of RMSD in the main chain of BSA dimer during the adsorption of PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H membrane systems were obtained through simulation, as shown in Fig. 6. The results show that the RMSD value of the BSA dimer backbone chain in the PSf-CH3 system has the largest change, and its change range is the largest at 10 ns, and the maximum change value is 0.7 nm. In the subsequent 10 ns to 60 n, it has been in a large fluctuation range, and it becomes stable after 55 ns. This is mainly due to the negative charge at the end of the BSA dimer and the electrostatic interaction with the positively charged PSf-CH3, resulting in serious changes in the BSA chain configuration. The greater the change of RMSD, the higher the probability of irreversible structural changes of the surface protein molecules, and even the denaturation of proteins. The fluctuation range of RMSD of BSA dimer backbone is small in the PSf-COOH and PSf-SO3H systems, especially in the PSf-SO3H system, the RMSD value tends to fluctuate stable after 30 ns, compared with that in the PSf-SO3H system. The RMSD value fluctuates relatively late in the PSf-OH and PSf-COOH systems, and becomes stable after 45 ns. Therefore, BSA in the PSf-COOH and PSf-SO3H systems has a small conformational change in contact with the membrane and good conformational stability. After 30 ns, its molecular structure deformation pressure region is relieved and it can exist in the system stably. According to the change rule of RMSD of BSA dimer main chain, the conformational change of its protein is easy to occur in the order of PSf-CH3 > PSf-OH > PSf-COOH > PSf-SO3H. These results indicated that the changes of BSA in PF-SO3H system were minimal. When the terminal functional group of the surface of the polysulfone membrane was grafted with SO3H, BSA was not prone to irreversible non-specific protein changes, which effectively reduced the protein denaturation in contact with the membrane.
3.6. Influence of BSA dimer flexibility and motility
By simulating RMSF (Root mean square fluctuation) of a protein and SASA (Solvent accessible surface area) of a residue in a protein molecule, The influence factors on the flexibility and motility of BSA dimer in PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H membrane systems were investigated through the changes of RMSF and SASA[27, 28].
The root mean square fluctuation (RMSF) of protein refers to the standard deviation between the atomic coordinates of the protein structure after the conformation of the reference frame and the current frame. It reflects the displacement of a single atom in the protein molecular chain in a specified period of time, and describes the movement of the atom[29].
The formula of simulation calculation is as follows:
$$\text{R}\text{M}\text{S}\text{F}={\left(\frac{{\sum }_{i}{\left({r}_{t}-{r}_{ref}\right)}^{2}}{T}\right)}^{\frac{1}{2}}$$
3
where T represents the number of frame capture points in time.
The change rule of RMSF of the main chain of BSA dimer during the adsorption process of PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H membrane systems was obtained through simulation, as shown in Fig. 7. In the PSf-CH3 system, the RMSF fluctuation range of BSA residues is very small and basically in a relatively stable state, because the peptide chains of protein molecules have been mostly adsorbed on the membrane surface due to adsorption on the membrane surface, and the dynamic range of their peptide chains in solution is small. However, RMSF of BSA residues in the PSf-COOH and PSf-SO3H systems remained in the fluctuation range, indicating that most of the protein molecular peptide chains were not adsorbed to the membrane surface and were in a relatively active state.
The solvo-accessible surface area (SASA) of residues in protein molecules is calculated using a bicubic dot matrix, and its simulation formula is as follows:
$$A=4{\pi }\sum _{i}{r}_{i}^{2}\frac{{m}_{acc}\left(i\right)}{m}$$
4
where \({m}_{acc}\left(i\right)\) represents the number of points at which atom i is not closed by surrounding atoms, and sums the accessible areas of the atoms used in the protein molecular chain.
The SASA of BSA dimer residues has a great relationship with the hydrophilicity of the system, and the hydrophilicity also further affects the position state of the BSA dimer atoms. In other words, the greater the change value of SASA, the greater the surface energy of the membrane surface in contact with BSA. Since the molecular structure of macromolecule BSA is flexible, the protein has been folded, and under the folding action, some residues are wrapped in other molecules and cannot directly contact with the membrane surface, so the SASA value of the residues wrapped in the interior is relatively low.
As shown in Fig. 8, the SASA changes of BSA residues in PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H membrane systems. In addition to the hydrophobic PSf-CH3 system, the residue SASA of BSA dimer has a large range of changes, indicating that the BSA dimer has a higher surface energy after contact with PSf membrane. In the hydrophilic PSf-OH, PSf-COOH and PSf-SO3H membrane systems, the surface of the membrane material is too small to cause protein folding, which further indicates that the smaller the surface energy of the membrane surface, the smaller the probability of irreversible folding and denaturation of the protein during the adsorption process with the membrane surface.
3.7. Influence of interface interaction energy
The free energy of the interface between PSf-CH3, PSf -OH, PSf -COOH and PSf -SO3H grafted membranes and BSA was simulated, including the free energy of van der Waals interaction (\({E}_{vdw}\)) and electrostatic potential energy (\({E}_{elect}\)). Table 2 shows that the adsorption of BSA proteins on the surface of PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H grafted membranes is mainly caused by the synergistic force of van der Waals interaction free energy (\({E}_{vdw}\)) and electrostatic interaction. Through simulation data analysis, The van der Waals interaction free energy (\({E}_{vdw}\)) is much smaller than the electrostatic potential energy (\({E}_{elect}\)), indicating that the membrane protein interaction mainly relies on the electrostatic interaction, that is, the dominant force of protein adsorption on the surface of the charged material is the electrostatic potential energy. The electrostatic interaction between BSA protein and PSf-CH3, on the positively charged surface is smaller than that between BSA protein and PSf-OH, PSf-COOH and PSf-SO3H on the negatively charged surface. The higher the density of negative charge on the membrane surface, the higher the electrostatic potential energy (\({E}_{elect}\)), and the higher the electrostatic interaction between the membrane and protein interface.
Table 2
Interaction energy of different graft membrane-BSA interface
Interaction interface | \({E}_{vdw}\) | \({E}_{elect}\) |
PSf-CH3-BSA(KJ·mol-1) | -36 | -270 |
PSf-OH-BSA(KJ·mol-1) | -108 | -330 |
PSf-COOH-BSA(KJ·mol-1) | -156 | -480 |
PSf-SO3H-BSA(KJ·mol-1) | -253 | -253 |
3.8. Orientation distribution and adsorption sites of BSA dimer
The adsorption orientation of protein on the surface of the material has a decisive effect on the activity efficiency of protein. Orientation Angle (θ) is defined as the Angle between protein dipole moment and normal vector. The adsorption orientation distribution curves of bovine serum albumin (BSA) dimer in different systems during the stabilization stage are shown in Fig. 9. The cosine values corresponding to the orientation peak distribution of BSA in the PSf-OH, PSf-SO3H and PSf-COOH systems are all negative values, which are about − 0.075, -0.125 and − 0.175, respectively. The corresponding orientation angles are θ = 94.01°, θ = 97.18o and θ = 100.08o, respectively. Compared with the PSf-CH3 system, the orientation distribution peaks of PSf-OH and PSf-COOH are narrower. This indicates that the surface adsorption of BSA in these systems is affected by the repulsion force, and the adsorption orientation is relatively stable under the continuous repulsion force. In the PSf-CH3 system, the orientation distribution peak of the BSA dimer is wider, and the bimodal phenomenon appears. The adsorption orientation of the surface protein fluctuates greatly under the action of electrostatic adsorption, and the activity efficiency of the protein changes after contact with the membrane surface.
As shown in Fig. 10, the adsorption orientation and adsorption sites of BSA dimer in PSf-CH3, PSf-OH, PSf-COOH and PSf-SO3H systems. In electrically neutral systems, where there is a lack of long-range electrostatic interaction forces, the interaction between the BSA dimer and the PSf membrane surface occurs only through adsorption or attraction to solvent molecules and ions. In the positively charged PSf-CH3 system, the positively charged groups on the surface of the PSf-CH3 membrane interact with both the A and B chains of the BSA dimer, resulting in the adsorption of both the A and B chains of the BSA dimer to the PSf-CH3 membrane. There are more binding sites at the end of the main chain of the BSA dimer. Moreover, the orientation distribution of BSA dimer is consistent with the direction of electrostatic adsorption. In the negatively charged systems of PSf-OH, PSf-COOH and PSf-SO3H, especially in the systems of PSf-COOH and PSf-SO3H, there are fewer binding sites between the end of the main chain of BSA dimer and the membrane material, and no direct binding occurs between them.