Investigation on Human Serum Protein Depositions Inside Polyvinylidene Fluoride-Based Dialysis Membrane Layers Using Synchrotron Radiation Micro-Computed Tomography (SR-μCT)

Hemodialysis (HD) membrane fouling with human serum proteins is a highly undesirable process that results in blood activations with further severe consequences for HD patients. Polyvinylidene fluoride (PVDF) membranes possess a great extent of protein adsorption due to hydrophobic interaction between the membrane surface and non-polar regions of proteins. In this study, a PVDF membrane was modified with a zwitterionic (ZW) polymeric structure based on a poly (maleic anhydride-alt-1-decene), 3-(dimethylamino)-1-propylamine derivative and 1,3-propanesultone. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and zeta potential analyses were used to determine the membrane’s characteristics. Membrane fouling with human serum proteins (human serum albumin (HSA), fibrinogen (FB), and transferrin (TRF)) was investigated with synchrotron radiation micro-computed tomography (SR-μCT), which allowed us to trace the protein location layer by layer inside the membrane. Both membranes (PVDF and modified PVDF) were detected to possess the preferred FB adsorption due to the Vroman effect, resulting in an increase in FB content in the adsorbed protein compared to FB content in the protein mixture solution. Moreover, FB was shown to only replace HSA, and no significant role of TRF in the Vroman effect was detected; i.e., TRF content was nearly the same both in the adsorbed protein layer and in the protein mixture solution. Surface modification of the PVDF membrane resulted in increased FB adsorption from both the protein mixture and the FB single solution, which is supposed to be due to the presence of an uncompensated negative charge that is located at the COOH group in the ZW polymer.


Membrane Surface Modification
The procedure of PVDF membrane surface modification is described in our previous paper [38]. The bare PVDF membrane was vacuum-dried before being introduced into a 2:1:2 ratio suspension consisting of a premixed 90:10 chloroform-ethanol mixture and Membranes 2023, 13, 117 3 of 13 50% PMAL ® -C8. The membrane was completely immersed within this mixture and stirred (100 rpm at 25 • C). Approximately 25 µL of the synthesized polymer solution was utilized in modifying the membrane. The membrane was then removed from the polymer solution and washed in ethanol (50%) for 10 min and ultrapure deionized water for 10 min in order to remove the adhering polymer solution. Subsequently, the membrane was completely immersed in a propanesultone and methanol mixture at 40 • C for 60 min in order to complete the ring-opening polymerization reaction with PMAL ® -C8. Finally, ethanolwashing and vacuum-drying procedures were carried out before storage. The chemical structure of the resultant membrane is given in Figure 1. The modified membrane is referred to here and subsequently as the PVDF-ZW membrane.

Membrane Surface Modification
The procedure of PVDF membrane surface modification is described in our previous paper [38]. The bare PVDF membrane was vacuum-dried before being introduced into a 2:1:2 ratio suspension consisting of a premixed 90:10 chloroform-ethanol mixture and 50% PMAL ® -C8. The membrane was completely immersed within this mixture and stirred (100 rpm at 25 °C). Approximately 25 μL of the synthesized polymer solution was utilized in modifying the membrane. The membrane was then removed from the polymer solution and washed in ethanol (50%) for 10 min and ultrapure deionized water for 10 min in order to remove the adhering polymer solution. Subsequently, the membrane was completely immersed in a propanesultone and methanol mixture at 40 °C for 60 min in order to complete the ring-opening polymerization reaction with PMAL ® -C8. Finally, ethanol-washing and vacuum-drying procedures were carried out before storage. The chemical structure of the resultant membrane is given in Figure 1. The modified membrane is referred to here and subsequently as the PVDF-ZW membrane.

Human Serum Protein Solution Preparation
Human serum protein solutions were prepared using a saline and phosphate buffer solution with resultant pH = 7.4. The concentrations of individual proteins and in their mixtures were as follows: HSA, 50 mg/mL; FB, 2 mg/mL; and TRF, 3 mg/mL. Each protein was conjugated with corresponding gold NPs before being mixed. The resultant solutions were injected into PVDF and PVDF-ZW membranes, with further analysis of the membranes using the X-ray based synchrotron technique.

In Situ Synchrotron Advanced Imaging Techniques at BioMedical Imaging and Therapy (BMIT) Beamline
Visualization of protein adsorption was conducted using a monochromatic beam at 20 keV energy. A beam monitor, AA-40 (500 μm LuAG scintillator, Hamamatsu, Japan), coupled with a high-resolution camera, PCO Dimax HS (PCO, Germany), providing a pixel size of 5.5 μm and a field of view (FOV) of 4.4 mm × 2.2 mm, was used to record the X-ray radiographs. A high photon flux allows for very detailed observation of particle deposition in microscopic layers of the membrane. CT projections were recorded at 20 keV at the 05ID-2 beamline of the BioMedical Imaging and Therapy (BMIT), at the Canadian Light Source (CLS), as presented in Figure 2a. The resulting radiographs were converted into graphical images using Avizo software. Further image analysis was performed using imageJ software, National Institutes of Health, BSD2. Gold nanoparticles conjugated with proteins produced the brightest spots on the image, thus providing quantitative

Human Serum Protein Solution Preparation
Human serum protein solutions were prepared using a saline and phosphate buffer solution with resultant pH = 7.4. The concentrations of individual proteins and in their mixtures were as follows: HSA, 50 mg/mL; FB, 2 mg/mL; and TRF, 3 mg/mL. Each protein was conjugated with corresponding gold NPs before being mixed. The resultant solutions were injected into PVDF and PVDF-ZW membranes, with further analysis of the membranes using the X-ray based synchrotron technique.

In Situ Synchrotron Advanced Imaging Techniques at BioMedical Imaging and Therapy (BMIT) Beamline
Visualization of protein adsorption was conducted using a monochromatic beam at 20 keV energy. A beam monitor, AA-40 (500 µm LuAG scintillator, Hamamatsu, Japan), coupled with a high-resolution camera, PCO Dimax HS (PCO, Germany), providing a pixel size of 5.5 µm and a field of view (FOV) of 4.4 mm × 2.2 mm, was used to record the X-ray radiographs. A high photon flux allows for very detailed observation of particle deposition in microscopic layers of the membrane. CT projections were recorded at 20 keV at the 05ID-2 beamline of the BioMedical Imaging and Therapy (BMIT), at the Canadian Light Source (CLS), as presented in Figure 2a. The resulting radiographs were converted into graphical images using Avizo software. Further image analysis was performed using imageJ software, National Institutes of Health, BSD2. Gold nanoparticles conjugated with proteins produced the brightest spots on the image, thus providing quantitative information about the protein amount adsorbed at each scanned layer (see Figure 2b). In case of adsorption from multiprotein solutions, each protein was detected and analyzed on the basis of the specific shape of nanoparticles used for conjugation with each protein.
Thus, spherical particles were used for conjugation with HSA, rods (sphericity ratio 0.85) were used for conjugation with FB, and cylinders (sphericity ratio 0.91) were used for conjugation with TRF. Detailed information about membrane imaging and radiograph representation with membrane division into layers and regions is described in our recent papers [38][39][40][41][42]. Membrane thickness was modeled with 10 regions of interest (ROIs), as shown in Figure 2c. Region 1 represents the very top membrane surface. The middle layers represent the internal membrane structure. The bottom membrane parts are located in Region 10. In order to ensure the accuracy of the data, four measurements were carried out for each sample. The data presented in the discussion are an average of the measurements.
information about the protein amount adsorbed at each scanned layer (see Figure 2b). In case of adsorption from multiprotein solutions, each protein was detected and analyzed on the basis of the specific shape of nanoparticles used for conjugation with each protein. Thus, spherical particles were used for conjugation with HSA, rods (sphericity ratio 0.85) were used for conjugation with FB, and cylinders (sphericity ratio 0.91) were used for conjugation with TRF. Detailed information about membrane imaging and radiograph representation with membrane division into layers and regions is described in our recent papers [38][39][40][41][42]. Membrane thickness was modeled with 10 regions of interest (ROIs), as shown in Figure 2c. Region 1 represents the very top membrane surface. The middle layers represent the internal membrane structure. The bottom membrane parts are located in Region 10. In order to ensure the accuracy of the data, four measurements were carried out for each sample. The data presented in the discussion are an average of the measurements.

Fourier Transform Infrared Spectroscopy ATR-FTIR Analyses
Prior to characterization, these membrane specimens were appropriately vacuumdried for one day at 30 °C. They were then placed on a Renishaw-inVia Raman Microscope (Renishaw, UK) stage and were ready for surface chemical analysis using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Both bare and ZWcoated PVDF membranes were probed for membrane-bound chemical groups with IR spectra, all recorded in transmittance mode.

Scanning Electron Microscope (SEM)
The comparative surface morphologies of both ZW-coated and bare membranes were recorded with the aid of a Hitachi SU8010 Field-Emission Scanning Electron Microscope, Hitachi, Ibaraki, Japan. All SEM images were recorded at 3 kV accelerating voltage in order to avoid burning of the Cr-coated membrane samples (Q150T ES).

Zeta Potential Analyzer
A Zetasizer-Nano (Malvern Instruments Ltd., UK) was used to measure the HD membranes surface charge (zeta potential) with a precision of ±0.01 mV. The membrane sample preparation and software steps are detailed in our recent study [43].

Fourier Transform Infrared Spectroscopy ATR-FTIR Analyses
Prior to characterization, these membrane specimens were appropriately vacuumdried for one day at 30 • C. They were then placed on a Renishaw-inVia Raman Microscope (Renishaw, UK) stage and were ready for surface chemical analysis using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Both bare and ZW-coated PVDF membranes were probed for membrane-bound chemical groups with IR spectra, all recorded in transmittance mode.

Scanning Electron Microscope (SEM)
The comparative surface morphologies of both ZW-coated and bare membranes were recorded with the aid of a Hitachi SU8010 Field-Emission Scanning Electron Microscope, Hitachi, Ibaraki, Japan. All SEM images were recorded at 3 kV accelerating voltage in order to avoid burning of the Cr-coated membrane samples (Q150T ES).

Zeta Potential Analyzer
A Zetasizer-Nano (Malvern Instruments Ltd., Malvern, UK) was used to measure the HD membranes surface charge (zeta potential) with a precision of ±0.01 mV. The membrane sample preparation and software steps are detailed in our recent study [43]. Figure 3 shows the ATR-FTIR spectra of both zwitterionic-polymer-modified (PVDF-ZW) and unmodified (PVDF) membranes. As expected, the spectrum of the bare membrane shows peaks corresponding to C-F vibration, 1410 (bending) and 1199 (stretching) cm −1 , as well as C-H vibrations at 2978 (stretching) and 1383 (deformation) cm −1 [43]. The alteration in PVDF peak intensities and positions for the PVDF-ZW sample attests to the surface polymer adsorption, especially the suppressed peaks at 2978 cm −1 (C-F bending) and 1383 cm −1 (C-F stretching) [44][45][46]. The PVDF membrane was polymerized with the zwitterionic polymer, and the effect of this process could be identified by ATR-FTIR analysis. Evidence of polymer adsorption on PVDF for the PVDF-ZW membrane can be seen in the absorption peaks related to stretching vibrations of N-H, C-N, and C-C around 3000, 1471, and 1000 cm −1 , respectively. There are still characteristic absorption bands for N-H wagging at 764 cm −1 . These are the main chemical groups of PMAL-C8 moiety on the zwitterionic polymer. It is important to note that these peaks are completely absent from the spectrum of the unmodified membrane [47]. The ring-opening polymerization reaction of 1,3-propanesultone after reaction with the tertiary amine on PMAL ® -C8 yields new IR vibration peaks at 1619 cm −1 corresponding to N-H (bending). This marks the acylation of propanesultone ring in the presence of methanol. The presence of sulfonate SO − 3 (stretching) chemical groups as well as that of CH 3 and CH 2 (bending) could also be seen at 1039, 1381, and 1462 cm −1 , respectively [47]. The symmetric and asymmetric stretching sulfonate vibrations that would have been seen at 1055 and 1238 cm −1 , respectively, are not observed, which is due to the overlapping CF 2 peak around 1182 cm −1 . The band at 1664 cm −1 could be assigned to amide C=O stretching, whereas the one at 1524 cm −1 is N-H stretching of the O=C-NH groups in the polymer chain [48]. Compared with virgin PVDF, a new peak at 953 cm −1 appeared in the zwitterionic sulfobetaine PVDF membrane, which can be attributed to the quaternary amine groups. The observed evidence of ZW on the PVDF membrane shows that the chemical modification process via dip coating was successful. This indicates that the polymer coated the PVDF membrane. Tiny shoulder peaks consistent with the C-H bond are common for both membranes at 2900 cm −1 .

Results and Discussion
3.1. Zwitterionic-Polymer-Modified and Unmodified PVDF Membrane Characteristics Figure 3 shows the ATR-FTIR spectra of both zwitterionic-polymer-modified (PVDF-ZW) and unmodified (PVDF) membranes. As expected, the spectrum of the bare membrane shows peaks corresponding to C-F vibration, 1410 (bending) and 1199 (stretching) cm −1 , as well as C-H vibrations at 2978 (stretching) and 1383 (deformation) cm −1 [43]. The alteration in PVDF peak intensities and positions for the PVDF-ZW sample attests to the surface polymer adsorption, especially the suppressed peaks at 2978 cm −1 (C-F bending) and 1383 cm −1 (C-F stretching) [44][45][46]. The PVDF membrane was polymerized with the zwitterionic polymer, and the effect of this process could be identified by ATR-FTIR analysis. Evidence of polymer adsorption on PVDF for the PVDF-ZW membrane can be seen in the absorption peaks related to stretching vibrations of N-H, C-N, and C-C around 3000, 1471, and 1000 cm −1 , respectively. There are still characteristic absorption bands for N-H wagging at 764 cm −1 . These are the main chemical groups of PMAL-C8 moiety on the zwitterionic polymer. It is important to note that these peaks are completely absent from the spectrum of the unmodified membrane [47]. The ring-opening polymerization reaction of 1,3-propanesultone after reaction with the tertiary amine on PMAL ® -C8 yields new IR vibration peaks at 1619 cm −1 corresponding to N-H (bending). This marks the acylation of propanesultone ring in the presence of methanol. The presence of sulfonate SO (stretching) chemical groups as well as that of CH3 and CH2 (bending) could also be seen at 1039, 1381, and 1462 cm −1 , respectively [47]. The symmetric and asymmetric stretching sulfonate vibrations that would have been seen at 1055 and 1238 cm −1 , respectively, are not observed, which is due to the overlapping CF2 peak around 1182 cm −1 . The band at 1664 cm −1 could be assigned to amide C=O stretching, whereas the one at 1524 cm −1 is N-H stretching of the O=C-NH groups in the polymer chain [48]. Compared with virgin PVDF, a new peak at 953 cm −1 appeared in the zwitterionic sulfobetaine PVDF membrane, which can be attributed to the quaternary amine groups. The observed evidence of ZW on the PVDF membrane shows that the chemical modification process via dip coating was successful. This indicates that the polymer coated the PVDF membrane. Tiny shoulder peaks consistent with the C-H bond are common for both membranes at 2900 cm −1 .   Figure 4a. The magnified image in Figure 4b shows distinct microporous structures with different sizes (approximately 0.1 μm max). It is clear that the morphology of the pristine PVDF is quite different from that of the modified membrane, as can be seen in Figure 4b,d. The zwitterionic polymer significantly altered the PVDF membrane but

Human Serum Protein Adsorption from Protein Mixture Solution
Based on the SR-μCT analysis, we detected a difference in the protein composition in the adsorbed layer on the membrane surface in comparison with the protein composition in the initial solution (see Figure 5). Thus, the content of HSA in the protein mixture in the initial solution is 90%, whereas this value drops to less than 80% in the protein mixture layer adsorbed by membranes. Moreover, the HSA content for the PVDF membrane is 4% higher than that for the PVDF-ZW membrane. Interestingly, the HSA content tends to decrease with membrane thickness (see Figure 6a) for both membranes.

Human Serum Protein Adsorption from Protein Mixture Solution
Based on the SR-µCT analysis, we detected a difference in the protein composition in the adsorbed layer on the membrane surface in comparison with the protein composition in the initial solution (see Figure 5). Thus, the content of HSA in the protein mixture in the initial solution is 90%, whereas this value drops to less than 80% in the protein mixture layer adsorbed by membranes. Moreover, the HSA content for the PVDF membrane is 4% higher than that for the PVDF-ZW membrane. Interestingly, the HSA content tends to decrease with membrane thickness (see Figure 6a) for both membranes.

Human Serum Protein Adsorption from Protein Mixture Solution
Based on the SR-μCT analysis, we detected a difference in the protein composition in the adsorbed layer on the membrane surface in comparison with the protein composition in the initial solution (see Figure 5). Thus, the content of HSA in the protein mixture in the initial solution is 90%, whereas this value drops to less than 80% in the protein mixture layer adsorbed by membranes. Moreover, the HSA content for the PVDF membrane is 4% higher than that for the PVDF-ZW membrane. Interestingly, the HSA content tends to decrease with membrane thickness (see Figure 6a) for both membranes.    It is to be noted that the change in HSA content is compensated by the corresponding increase (by more than 12%) in FB content predominately; i.e., no significant change in TRF content is observed, and its content remains on the same level for both the initial solution and the adsorbed protein layer. Notably, the FB content for the PVDF-ZW membrane is the highest (3% higher than that for PVDF membranes), indicating a higher interaction of the PVDF-ZW membrane with this protein. As well as for overall FB content, FB It is to be noted that the change in HSA content is compensated by the corresponding increase (by more than 12%) in FB content predominately; i.e., no significant change in TRF content is observed, and its content remains on the same level for both the initial solution and the adsorbed protein layer. Notably, the FB content for the PVDF-ZW membrane is the highest (3% higher than that for PVDF membranes), indicating a higher interaction of the PVDF-ZW membrane with this protein. As well as for overall FB content, FB content inside the membrane also tends to increase (see Figure 6b), compensating the corresponding decrease in HSA content (see Figure 6a).
TRF content across membrane thickness possesses the same tendency to increase as in the case of FB, although the absolute change in TRF content is less than 1%, whereas FB content changes within 3-4%.
The protein-protein and protein-membrane interactions induced a gain of free energy, so the protein molecule underwent conformational changes and spread on the membrane surface as a part of the third stage. Depending on the degree of interaction, a large protein can dislocate a small one in a phenomenon called the Vroman effect. This dynamic interaction and equilibrium lead to progressive changes in the composition of the protein cake layer, affecting the filtration performance and biocompatibility profile of the surface [49][50][51][52][53].
Considering adsorbed protein distribution (see Figure 7), not quite uniform distribution could be observed for both membranes. Proteins were shown to tend to locate more at the bottom of the membrane (high index regions), whereas both membranes have a similar pattern in protein distribution.
Membranes 2023, 13, x 8 of 13 content inside the membrane also tends to increase (see Figure 6b), compensating the corresponding decrease in HSA content (see Figure 6a). TRF content across membrane thickness possesses the same tendency to increase as in the case of FB, although the absolute change in TRF content is less than 1%, whereas FB content changes within 3-4%.
The protein-protein and protein-membrane interactions induced a gain of free energy, so the protein molecule underwent conformational changes and spread on the membrane surface as a part of the third stage. Depending on the degree of interaction, a large protein can dislocate a small one in a phenomenon called the Vroman effect. This dynamic interaction and equilibrium lead to progressive changes in the composition of the protein cake layer, affecting the filtration performance and biocompatibility profile of the surface [49][50][51][52][53].
Considering adsorbed protein distribution (see Figure 7), not quite uniform distribution could be observed for both membranes. Proteins were shown to tend to locate more at the bottom of the membrane (high index regions), whereas both membranes have a similar pattern in protein distribution.

FB Adsorption from Its Single Solution
Besides the adsorption of the protein mixture, FB adsorption from its single solution was also studied, since this protein is believed to play a major role in the blood clotting process. Figure 8 demonstrates the difference in FB adsorption ratios when FB is adsorbed from its single solution and from a protein mixture. For both membranes, an increased FB adsorption ratio was observed when FB was adsorbed from the protein mixture, which can be explained by the Vroman effect. Thus, HSA has a concentration of 50 mg/mL, which results in its adsorption on the membrane surface and its further replacement with FB. Without HSA, the adsorption rate of FB seems to be reduced, resulting in a lower adsorption ratio. Moreover, the PVDF-ZW membrane possesses a slightly higher FB adsorption, which correlates with increased FB content in the adsorbed protein layer (see Figure 5).
Membranes 2023, 13, x 9 of 13 Besides the adsorption of the protein mixture, FB adsorption from its single solution was also studied, since this protein is believed to play a major role in the blood clotting process. Figure 8 demonstrates the difference in FB adsorption ratios when FB is adsorbed from its single solution and from a protein mixture. For both membranes, an increased FB adsorption ratio was observed when FB was adsorbed from the protein mixture, which can be explained by the Vroman effect. Thus, HSA has a concentration of 50 mg/mL, which results in its adsorption on the membrane surface and its further replacement with FB. Without HSA, the adsorption rate of FB seems to be reduced, resulting in a lower adsorption ratio. Moreover, the PVDF-ZW membrane possesses a slightly higher FB adsorption, which correlates with increased FB content in the adsorbed protein layer (see Figure 5). The possible explanation for the increased FB adsorption by the PVDF-ZW membrane could be related to ZW polymer structure and charge distribution (see Figure 9). In addition, FB illustrated an anisotropic charge distribution of fibrinogen by computing the local charges of each domain [54,55]. Thus, it can be stated that this polymer contains an uncompensated negative charge located at the COOH groups. Although the COOH group possess weak acidic properties, this seems to be enough to affect protein adsorption, causing some increase in electrostatic interaction between the membrane surface and positive moieties in the FB molecule.  The possible explanation for the increased FB adsorption by the PVDF-ZW membrane could be related to ZW polymer structure and charge distribution (see Figure 9). In addition, FB illustrated an anisotropic charge distribution of fibrinogen by computing the local charges of each domain [54,55]. Thus, it can be stated that this polymer contains an uncompensated negative charge located at the COOH groups. Although the COOH group possess weak acidic properties, this seems to be enough to affect protein adsorption, causing some increase in electrostatic interaction between the membrane surface and positive moieties in the FB molecule.
Membranes 2023, 13, x 9 of 13 Besides the adsorption of the protein mixture, FB adsorption from its single solution was also studied, since this protein is believed to play a major role in the blood clotting process. Figure 8 demonstrates the difference in FB adsorption ratios when FB is adsorbed from its single solution and from a protein mixture. For both membranes, an increased FB adsorption ratio was observed when FB was adsorbed from the protein mixture, which can be explained by the Vroman effect. Thus, HSA has a concentration of 50 mg/mL, which results in its adsorption on the membrane surface and its further replacement with FB. Without HSA, the adsorption rate of FB seems to be reduced, resulting in a lower adsorption ratio. Moreover, the PVDF-ZW membrane possesses a slightly higher FB adsorption, which correlates with increased FB content in the adsorbed protein layer (see Figure 5). The possible explanation for the increased FB adsorption by the PVDF-ZW membrane could be related to ZW polymer structure and charge distribution (see Figure 9). In addition, FB illustrated an anisotropic charge distribution of fibrinogen by computing the local charges of each domain [54,55]. Thus, it can be stated that this polymer contains an uncompensated negative charge located at the COOH groups. Although the COOH group possess weak acidic properties, this seems to be enough to affect protein adsorption, causing some increase in electrostatic interaction between the membrane surface and positive moieties in the FB molecule. Figure 9. Charges distribution in ZW polymer used for PVDF modification. Figure 9. Charges distribution in ZW polymer used for PVDF modification.
When FB distribution across membrane thickness is considered (see Figure 10), it can be observed that PVDF and PVDF-ZW membranes possess different behaviors. Thus, FB is uniformly distributed across PVDF-ZW membrane thickness, whereas unmodified PVDF holds FB predominately in the middle regions.
Membranes 2023, 13, x 10 of 13 When FB distribution across membrane thickness is considered (see Figure 10), it can be observed that PVDF and PVDF-ZW membranes possess different behaviors. Thus, FB is uniformly distributed across PVDF-ZW membrane thickness, whereas unmodified PVDF holds FB predominately in the middle regions. Figure 10. Comparison of FB distribution across membrane (PVDF and PVDF-ZW) thicknesses when adsorbed from a single-protein solution.

Conclusions
PVDF and PVDF-ZW membranes possess similar behaviors in protein adsorption. Thus, both membranes tend to adsorb more FB, resulting in a change in protein composition in the adsorbed layer in comparison with the protein mixture solution; i.e., FB content in the adsorbed protein layer increased from 3.6% (protein mixture solution) to 15% for PVDF and 18% for PVDF-ZW membrane. This increase in FB content was compensated by the corresponding decrease in HSA content, indicating the major role of HSA in the Vroman effect. At the same time, TRF content was not affected and remained approximately the same for the protein mixture solution and adsorbed protein layer for both membranes. This assumption was confirmed by comparing the FB adsorption ratio of the FB single solution and FB adsorption from the protein mixture solution. FB adsorption from the protein mixture solution was higher for both membranes. Notably, the PVDF-ZW membrane adsorbed more FB compared with the unmodified membrane. This difference is supposedly due to the structure of the ZW polymer used for modification. The presence of the uncompensated charge located on COOH groups resulted in a stronger interaction with the FB molecule, causing increased FB adsorption.  Institutional Review Board Statement: The principal investigator of the project, Amira Abdelrasoul, received Research Ethics Approval and the Operational Approval to conduct research in Saskatchewan Health Authority, Canada. She was responsible for the regulatory approvals that pertain

Conclusions
PVDF and PVDF-ZW membranes possess similar behaviors in protein adsorption. Thus, both membranes tend to adsorb more FB, resulting in a change in protein composition in the adsorbed layer in comparison with the protein mixture solution; i.e., FB content in the adsorbed protein layer increased from 3.6% (protein mixture solution) to 15% for PVDF and 18% for PVDF-ZW membrane. This increase in FB content was compensated by the corresponding decrease in HSA content, indicating the major role of HSA in the Vroman effect. At the same time, TRF content was not affected and remained approximately the same for the protein mixture solution and adsorbed protein layer for both membranes. This assumption was confirmed by comparing the FB adsorption ratio of the FB single solution and FB adsorption from the protein mixture solution. FB adsorption from the protein mixture solution was higher for both membranes. Notably, the PVDF-ZW membrane adsorbed more FB compared with the unmodified membrane. This difference is supposedly due to the structure of the ZW polymer used for modification. The presence of the uncompensated charge located on COOH groups resulted in a stronger interaction with the FB molecule, causing increased FB adsorption.

Institutional Review Board Statement:
The principal investigator of the project, Amira Abdelrasoul, received Research Ethics Approval and the Operational Approval to conduct research in Saskatchewan Health Authority, Canada. She was responsible for the regulatory approvals that pertain to this project and for ensuring that the authorized project was conducted according to the governing law. All the experimental protocols involving humans were conducted according to the governing law. All the participants in this study, from the hemodialysis center at St. Paul's Hospital, signed a written informed consent form, approved by the Biomedical Research Ethics Board (Bio-REB).

Data Availability Statement:
The raw/processed data obtained at the Canadian Light Source (CLS), required to reproduce these findings of this study are available from the corresponding author (Amira Abdelrasoul, A.A.) on reasonable request.