Surface Properties and Protein Adsorption Performance of Fluorinated

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Surface Properties and Protein Adsorption Performance of Fluorinated Amphiphilic Polymers Benfeng Zhu, Jingjing Yang, Jiao Liu, Yanbin Meng, Xiaoqing Du, Chao Cai, and Zhao Zhang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.9b01069 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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The Journal of Physical Chemistry

Surface Properties and Protein Adsorption Performance of Fluorinated Amphiphilic Polymers Benfeng Zhu a, Jingjing Yang a, Jiao Liu b, Yanbin Meng a, Xiaoqing Du c, Chao Cai d, Zhao Zhang a,* a Department

b

of Chemistry, Zhejiang University, Hangzhou, Zhejiang, 310027, China

College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, 410082,

China c

School of Materials Science and Energy Engineering, Foshan University, Foshan, Guangdong,

528000, China d College

of Chemical Engineering, Ningxia University, Yinchuan, Ningxia, 750021, China

ABSTRACT: To obtain amphiphilic polymers, fluorinated groups were successfully embedded in polyacrylates.

The

amphiphilic

polymers

were

synthesized

by

free

radical-initiated

copolymerization using hydrophilic acrylate monomers and hydrophobic 2-perfluorooctylethyl methacrylate (FMA). 1H NMR spectroscopy and Fourier transform infrared results indicated that the fluorinated monomer FMA was successfully embedded in the polymers. The surfaces of the amphiphilic polymers were subsequently characterized by contact angle measurement, atomic force microscopy and X-ray photoelectron spectroscopy, showing that a well-designed amphiphilic structure with fluorinated groups in the polymers was fabricated with the increase of the number of FMA units (n). Protein adsorption experiments indicated that, in addition to the surface chemical composition and roughness of the polymer, the fluorinated degree of the bulk/internal polymer layer (k) also plays a significant role in its bioadhesion behavior via adjusting the charge-state of the 1

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polymer surface. With the decrease of k, the critical factor determining the protein adsorption gradually turns from the polymer’s surface to its internal area, and the minimum adsorption for both BSA and HFg on the synthesized polymer surface occurs when k is approximately 12.

INTRODUCTION The undesired accumulation of foulants (such as proteins, bacteria, cells and microalgae) on solid surfaces has led to great problems.1 It is meaningful to study, understand and limit the adsorption of proteins or other fouling organisms on solid surfaces such as biomedical implants,2 food packaging3 and even marine ship hulls.1, 4-5 Therefore, more efforts are being focused on the exploitation of new environmentally friendly materials with antifouling surfaces to prevent undesired bioadhesion. Various strategies have been tested for constructing antifouling surfaces and these strategies have been somewhat successful. Hydrophilic materials such as poly(ethylene glycol) (PEG)6-7 are beneficial for enhancing antifouling performance because many fouling organisms are hydrophobic.8 Hydrophobic polymer surfaces based on siliconpolymers9-10 or fluoropolymers11-13 with low surface energies have also been investigated in detail. The hydrophobic surfaces can significantly reduce the adhesion of fouling organisms or release the fouling organisms from the surface.14 Recently, there has been great interest in the development of amphiphilic polymer materials with broad-spectrum antifouling properties, which contain hydrophobic and hydrophilic components.15-20 Dimitriou et al.21 fabricated a polystyrene-block-poly[(ethylene oxide)-stat-(allyl glycidyl ether)] [PS-b-P(EO-stat-AGE)] statistical diblock terpolymer by varying the incorporation of allyl glycidyl ether (AGE) in the poly(ethylene oxide) block from 0 to 17 mol %. The AGE repeat 2


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units

were

subsequently

functionalized

by

thiol-ene

chemistry

with

1H,1H,2H,2H-

perfluorooctanethiol. The amphiphilic surface based on a fluorinated hydrophobic component and PEG hydrophilic component exhibited better protein adsorption resistance than the corresponding hydrophilic or hydrophobic surface. Huang et al.22 designed V-shaped fluorinated amphiphilic polymers by grafting 2-perfluorooctylethylmethacrylate (FMA) to the end of PEG42-b-PAA11-bPMMA38. The chemical composition and domain size were controlled by altering the number of FMA units (y). The results indicated that the surface with y = 8 showed an alternating phase separation structure and exhibited desirable protein-resistant performance. The chemical incompatibility of hydrophobic and hydrophilic components in the amphiphilic polymers resulted in the tailored formation of nano- or microdomains due to phase segregation.22 Therefore, it is possible that amphiphilic surfaces can be fabricated by varying the ratio of hydrophobic and hydrophilic components. However, the reported studies focused on the effect of the surface composition of amphiphilic polymers on protein adsorption, ignoring the role of the composition in the bulk. Inspired by these successful strategies, we fabricated amphiphilic polymers that are resistant to protein adsorption and investigated the effect of the surface and bulk composition of these polymers on protein adsorption performance. Polyacrylates have been used as antifouling materials due to their good corrosion resistance23 and excellent mechanical robustness.24-25 Amphiphilic polymers were directly obtained by polymerization of the hydrophobic fluorinated acrylate and hydrophilic acrylates. Zhao et al.26 obtained a series of amphiphilic copolymers composed of 2perfluorooctylethyl methacrylate and 2-hydroxyethyl methacrylate by radical polymerization. When the percentage of hydrophilic and hydrophobic groups ranged from 4% to 7% and 4% to 14% on 3



the surface, respectively, the fluorinated amphiphilic copolymers possessed better antifouling properties than the homopolymers. In this study, fluorinated amphiphilic polymers composed of hydrophilic acrylate and hydrophobic 2-perfluorooctylethyl methacrylate were synthesized by two-step free radical-initiated copolymerization. The composition of polymers was controlled by varying the ratio of FMA and hydrophilic acrylate monomers. The amphiphilic polymer films were made by spraying and characterized by contact angle measurement, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Bovine serum albumin (BSA) and human plasma fibrinogen (HFg) were used as model proteins to evaluate the protein adsorption properties of the amphiphilic polymer films. EXPERIMENTAL SECTION Materials 2-perfluorooctylethyl methacrylate (FMA) and diiodomethane were analytical reagents obtained from Aladdin Reagents. Methyl methacrylate (MMA), butyl acrylate (BA), styrene (st), acrylic acid (AA), vinyltrimethoxysilane (VTMS) and xylene were analytical reagents obtained from Sinopharm Chemical Reagent (Shanghai, China). Human plasma fibrinogen (HFg) was obtained from Hefei Bomei Biotechnology Co., Ltd. Second Branch. Bovine serum albumin (BSA) and benzoyl peroxide (BPO) were obtained from Macklin Reagents, and the BPO was recrystallized with chloroform before use. Preparation of fluorinated amphiphilic polymers A representative preparation procedure was as follows: xylene (10.0 mL) was added into a four-necked flask and heated to 90 °C under continuous mechanical stirring. Subsequently, a mixed 4


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solution containing VTMS (0.5 g, 3.4 mmol), MMA (6.2 g, 62.0 mmol), BA (3.8 g, 29.7 mmol), st (1.0 g, 9.6 mmol), AA (0.2 g, 2.8 mmol) and BPO (0.13 g, 0.5 mmol) was added into the flask dropwise under a nitrogen atmosphere within 2 h. Then, FMA (2.8 mmol, 1.5 g) and BPO (0.02 g, 0.08 mmol) were sufficiently dispersed in 7.0 mL xylene and added into the stirred solution drop by drop at 90 °C under a nitrogen atmosphere within 1 h. The resultant solution was stirred for another 3 h at 90 °C. Preparation of fluorinated amphiphilic polymer films Steel was used as the substrate in this work, and the steel substrates were cleaned three times with ethanol and dried with nitrogen. The polymers were sprayed on the horizontally placed steel substrates using a 0.8-mm nozzle spray gun at a constant pressure of approximately 5 bar. The distance between the substrate and spray gun was maintained at ~ 10 cm with an angle of approximately 60°. OC4H9

OCH3

+

O

+

OH

+

Si(OCH3)3

O

O O

+

BPO

BPO, xylene

y

O

3 CO

z

l O

n

mO OH

O

H

3

3C

Si

H OC OCH 3

O

O

H

x

O

(CF2)7CF3

O

Xylene

C 4H 9

CF 3 F 2) 7

(C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Preparation of the fluorinated amphiphilic polymers.

Characterization The structure of the polymers was characterized by Fourier transform infrared (FT-IR) spectra recorded in KBr discs on a Nicolet iS10 spectrometer. 1H NMR spectra were recorded on a Brulcer 5



DMX-400 NMR instrument in CDCl3 with tetrame thylsilane (TMS) as an internal standard. The molecular weight of the polymers was determined by gel permeation chromatography (GPC) using a D40 (TSK CO.) with tetrahydrofuran (THF) as the eluent at a flow rate of 1.0 mL/min at 40 °C. The GPC chromatograms were calibrated with polystyrene (PS) as a standard. The composition of polymers was determined by fluorine elemental analysis (F-EA). F-EA was carried out by flask combustion and the concentration of fluoride ion was measured by a fluoride ion selective electrode with a saturated calomel electrode (SCE) as the reference. The contact angles (CAs) were measured using a contact angle instrument (JC2000DF, China) at room temperature. Each of the water contact angles were the averages of at least five measurements obtained at different surface positions using 5.0 µL droplets of water. According to Owens and Wendt’s theory,27 the surface free energies were calculated from the corresponding water and diiodomethane contact angles of the samples. X-ray photoelectron spectroscopy (XPS) was performed usingan Escalab 250Xi system with an Mg Ka X-ray source at 250 W (140 kV) under a 1.0 × 10-8 Torr vacuum with a takeoff angle of 30°. The C1s speak of the C-C bond at 284.6 eV was used to calibrate the spectra. The phase images of the polymers were obtained by atomic force microscopy (AFM, Multimode-8, Bruker Nano Inc.) in tapping mode at 1.5 Hz. The zeta potentials of the polymer films were measured by electrokinetic analyzer (SurPASS Anton Paar, Gmb H, Austria) with KCl (1 mmol/L) solution as the electrolyte solution, and the pH was adjusted with KOH and HCl solutions. Protein adsorption measurement The protein adsorption of the polymers was evaluated using BSA and HFg as model proteins. The experiments were performed according to the literature.28-29 Briefly, after immersion in 6


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phosphate buffer saline (PBS, pH = 7.4) for 30 min, the samples were incubated with 0.1 mg mL-1 BSA (or HFg) in PBS buffer at room temperature for 2 h. Then, the samples were thoroughly rinsed with PBS buffer solution and deionized water, followed by drying in a stream of nitrogen gas. Samples adsorbed with BSA (or HFg) were measured by XPS and calculated by integration of the peak area of the N1s peak from the corresponding XPS spectra.21, 28-29 RESULTS AND DISCUSSION Synthesis and characterization of the amphiphilic polymers The synthetic route of the amphiphilic polymers is outlined in Scheme 1. To obtain the target heterogeneous amphiphilic polymers with various compositions, different molar concentrations of FMA were added. However, it is difficult to characterize the FMA concentration by GPC or 1H NMR due to the intrinsic association30-31 of the FMA units, as well as the low concentration of FMA in the polymers. Thus, the fluorinated monomer content was determined by fluorine elemental analysis.28 The number of FMA units (n) in amphiphilic polymers was calculated according to the following equation: WF (%) = (17 × 19 × n) / Mn

(1)

where WF is the fluorine elemental content, Mn is the molecular weight of the amphiphilic polymer, and 17 and 19 represent the number of fluorine atoms in FMA and the molar mass of the fluorine atom, respectively. The amphiphilic polymer samples of the calculated n of 0.5, 1.1, 2.5, 4.2, 5.4 and 7.6 are designated as Pk (k=1, 2, …, 6), respectively, and their chemical structures are listed in Table 1. For comparison, the polymer sample of no FMA units (n = 0) was designated S0. The embedded FMA units in the amphiphilic polymers (Pks) were confirmed by FT-IR (Figure S1 (Supporting Information)). When compared to S0, a new absorbance at approximately 660 cm-1 that 7



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has been ascribed to the vibrations of CF2 groups32-34 appears for all Pks. Figure S2 (Supporting Information) shows the 1H NMR spectra of the S0 and P4 samples (P4 as an example here), where the small peak at approximately 4.23 ppm for P4 demonstrates the existence of the protons of OCH2CH2(CF2)7CF3 in the FMA unit.32 These results indicate that a series of amphiphilic polymers with various FMA content were successfully obtained. Table 1. Characteristics of amphiphilic polymers.

a

Sample

Mn×10-4 a

PDI b

WF(%) c

nd

P1

1.78

1.69

0.87

0.5

P2

1.87

1.96

1.91

1.1

P3

1.92

1.85

4.17

2.5

P4

2.02

1.81

6.70

4.2

P5

2.04

1.83

8.60

5.4

P6

2.09

1.79

11.78

7.6

Determined by GPC. b Polydispersity index (PDI). c Obtained by fluorine elemental analysis. d Calculated from

GPC and fluorine elemental analysis.

Surface structure of the amphiphilic polymer films The performance of amphiphilic polymer films in antifouling applications is primarily determined by their surface characteristics and behavior in aquatic environments.21 Thus, their surface characteristics were first characterized by contact angle measurements and XPS. The contact angles and surface free energies of the polymer films with different FMA content are illustrated in Figure 1. The water contact angle of S0 is approximately 85°. With the increase of n, the water contact angle of amphiphilic polymers rapidly increases to 114°, which is close to the theoretical 8


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value of PFMA.35 The corresponding surface free energy decreases from 38.3 mN/m to 11.9 mN/m as n increases from 0 to 7.6. S0 has a hydrophilic carboxyl group and FMA contains a hydrophobic long fluorinated alkyl chain. Thus, the chemical composition of the fluorinated polymers will be altered by varying the number of FMA units in the polymers. The surface chemical composition and orientation of the fluorinated alkyl groups in the polymers were probed by XPS (Figure 2). The F/C ratio on the surface obtained from XPS rapidly increases from 0.45 to 0.84 when n is 0.5 to 4.2, and then increases little when n > 4.2. The F/C ratio in polymer bulk almost increases linearly with n. Therefore, it is obvious that the F/C ratio on the surface is much larger than the corresponding F/C ratio in the bulk, which indicates that the fluorinated species are enriched on the polymer surface due to the increase of n and tend to be saturated when n reaches 4.2.

Figure 1. (a) Contact angles and (b) Surface free energies of polymers S0 and P1-P6.

9



Figure 2. F/C ratios of polymers S0 and P1-P6 on the surface and in the bulk.

To further investigate the orientation of fluorinated alkyl groups on the surface of amphiphilic polymers P1-P6, high resolution C1s XPS spectra (Figure S3 (Supporting Information)) were recorded at a takeoff angle of 30°. Each discrete peak was attributed to the corresponding carbonbased functional group, and the composition ratio was calculated from the relative peak area of each carbon component. The C1s XPS spectra were resolved into five Gaussian fitted peaks (Si was not detected on the surface of the samples): the peak of CC at approximately 284.6 eV, COC=O at approximately 286.4 eV, C=O at approximately 288.4 eV, peaks near 294.0 and 291.5 eV that represent CF3 and CF2, respectively. The peak assignments agree with previously reported values.33 The calculated surface compositions of polymers P1-P6 are listed in Table 2, and clearly indicate that when n increases from 0.5 to 7.6, the CF3/CF2 ratio increases to 0.40 and the surface fluorination degree CF, S% (defined as the ratio of the (CF3+CF2)/Ctotal in the surface layer, calculated from XPS, hereinafter) reaches 0.36 (Figure 3). It is widely reported that when the CF3/CF2 ratio is larger than 0.142, the fluorinated groups gradually form micelles and are perpendicularly oriented on the surface of the amphiphilic polymers22, 36-38 and the fluorinated component is easily enriched on the surface, which results in significant phase-segregated domains due to the incompatibility between the nonfluorinated and fluorinated components even though the fluoride content in the polymer is very low.39-40 Therefore, it can be rationally deduced that, a special amphiphilic surface with incompatible (hydrophobic and hydrophilic) regions is formed when n is greater than or equal to 2.5 for the fluorinated polymers, which is also demonstrated by the AFM phase images of the Pks’ surfaces (Figure 4). Table 2. Surface element and composition of P1-P6 polymer films.

10


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Surface composition (%) Sample CC/CH

COC=O

C=O

CF2

CF3

P1

60.1

14.9

14.9

9.5

0.6

P2

60.9

10.9

10.9

15.4

1.9

P3

41.7

12.4

12.4

25.5

8.0

P4

39.5

12.8

12.8

25.4

9.5

P5

38.5

13.1

13.1

25.3

10.0

P6

36.2

13.9

13.9

25.7

10.3

Figure 3. CF3/CF2 and CF,S% of the amphiphilic polymers as a function of the number of FMA units.

Surface phase image can distinguish the soft materials (dark domain) and hard materials (bright domain) in the nanoscale.41 Compared to S0 (Figure 4a), FMA is a hard feature and presents as a bright spot. With increasing FMA content in the polymers, the fluorinated groups are gradually enriched on the polymer surface (Figures 2-3) and form micelles (Figure 4), resulting in assembly of the bright spots and their phase-segregation with the antipathic nonfluorinated components. When the number of FMA units in the Pks is greater than or equal to 2.5, the alternating of the bright and dark moieties of larger area is distinct on the surface (Figures 4d-f), which indicates that an

11



amphiphilic surface with both hydrophilic and hydrophobic regions is almost formed. The strengthened phase-segregation of thus incompatible (hydrophobic and hydrophilic) regions enhances the instability of a water drop on a polymer surface, which results in the increase of the contact angle with n (Figure 1).

Figure 4. AFM tapping phase images of sample films: (a)-(f) S0, P1, P2, P3, P4 and P6, respectively.

Protein adsorption on the amphiphilic polymer surface Sample S0 and the Pks were used to investigate the connection between the polymer composition and its protein adsorption performance. When no nitrogen atom exists in the sample, XPS is a simple and effective method to test the protein adsorption behavior based on measurement of the 400 eV peak for N1s.15,

29, 42-43

Here, bovine serum albumin (BSA) and human plasma

fibrinogen (HFg) are used as the model proteins to evaluate the protein adsorption performance of the polymers. The relative intensities of the N1s XPS peaks (Figure S4 (Supporting Information)) clearly show that the adsorbed amount of BSA and HFg on the polymer surface is significantly influenced by n, and the fluorinated polymers exhibit higher protein resistance than the hydrophilic 12


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S0, except for HFg adsorbed on P6. Baier found that there is a foul-release zone at the critical surface energy of 20-30 mN/m,44 which has been attributed to the formation of weak boundary layers between the polymer surface and adhesive proteins.45 It is unexpected that the protein adsorption experiments here are inconsistent with the “Baier curve”; therefore, the relationship of the amount of BSA or HFg adsorbed on the sample surface versus surface free energy was plotted (Figure S5 (Supporting Information)). However, Figure S5 (Supporting Information) undoubtedly shows that P4, with a surface energy of 11.93 mN/m, has the minimum adsorption value for both BSA and HFg. When the surface free energy decreases from 38.29 mN/m to 11.93 mN/m, the corresponding N1s% (the atom percent of N1s on the surface) decreases from 5.63% to 0.66% for BSA and from 3.43% to 1.02% for HFg. N1s% subsequently increases sharply when the surface energy decreases from 11.93 mN/m to 11.91 mN/m. It is widely accepted that the surface chemical composition and surface roughness are the main factors accounting for bioadhesion.46 Ober et al.47 prepared surface active triblock copolymers (SABC) by blending polyethylene glycol (PEG) and two different semifluorinated alcohol side chains (F10H10 and F8H6) with a soft thermoplastic elastomer (TPE), polystyrene-blockpoly(ethylene-ran-butylene)-block-polystyrene (SEBS). The longer semifluorinated chain (F10H10) is more efficient at segregating to the surface than the shorter semifluorinated chain (F8H6), which results in better fouling-release performance compared to the F8 chain. However, in this study, the ratio of the fluorinated groups on the polymer surface remains almost constant when n is greater than 4.2 (Figures 2-3), and the AFM results also show that the surface roughness of all samples is less than 2 nm, and its effect on protein adsorption is negligible.31 These results indicate that factors 13



other than the surface chemical components and roughness determine the protein adsorption. To further examine the factors that dominate the protein adsorption, the bulk fluorination degree CF, B% of Pks was also calculated (defined as the ratio of the (CF3+CF2)/Ctotal in the whole polymer, calculated from monomer ratio of the polymer), and plotted with CF, S% versus the corresponding adsorption amount of BSA and HFg (Figure 5). With the increase of n and when its value is less than 2.5, CF, B% slowly increases from 0.5% to 2.3% and CF, S% sharply increases from 10.1% to 33.6% whereas the corresponding N1s% drastically reduces from 3.87% to 1.05% for BSA and from 2.57% to 1.28% for HFg. These results indicate that when the ratio of the fluorinated groups in the polymers is very low, the fluorinated groups tend to be enriched on the surface and the newly formed amphiphilic surface structure dominates the surface protein adsorption. Conversely, when n is larger than 4.2, N1s% increases as n from increases 0.66% to 2.04% for BSA and from 1.02% to 3.61% for HFg, whereas the corresponding CF, B% increases from 3.4% to 6.5% and the CF, S% is almost invariant. This suggests that when the ratio of fluorinated groups in the polymers is very large, the crucial factor determining the protein adsorption is the fluorination degree in the bulk (CF, B%) rather than that on the surface (CF, S%). Moreover, it is interesting that the polymer surfaces of the minimum adsorption for both BSA and HFg possess the same chemical composition. This result is different from the amphiphilic copolymers composed of FMA and HEMA reported previously26, where the adsorbed BSA and HFg achieved their minimum adsorption at the (bulk) fluorinated group content of 9% and 4%, respectively.

14


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Figure 5. Plot of BSA and HFg adsorbed on amphiphilic polymer surface with different degrees of fluorination on the surface and in the bulk.

Scheme 2 shows the relationship of the fluorination degree on the surface (CF, S%) and in the internal region (CF, B%) with n in the polymers. With the increase of n, the fluorinated groups are gradually enriched on the surface to form an amphiphilic (hydrophilic-hydrophobic) surface structure, which has been shown to exhibit better protein-resistant performance than individual hydrophobic or hydrophilic surfaces.48-49 The ratio of fluorinated groups in the internal layer continuously increases, which has a significant effect on the amount of the protein adsorption after the fluorinated groups are saturated on the surface.

Scheme 2. The fluorination degree on the surface and internal layer versus the number of FMA units in the polymers.

According to the above analyses, it can be concluded that the CF, S% and the CF, B% do not 15



individually dominate the surface protein adsorption, and the minimum adsorption value of proteins (BSA and HFg) on polymers should be synergistically determined by both CF, S% and CF, B%. Therefore, the fluorinated degree (or the enrichment degree of the fluorinated groups) of the bulk polymer k (k = CF, S% / CF, B%) was calculated and tentatively plotted versus the adsorption amount of the proteins (Figure 6). Interestingly, k has a significant effect on the protein adsorption, and the lowest amounts for both BSA and HFg are deposited in the region where k is approximately 12. When k is larger than 14.4, both CF, B% and CF, S% are very low and only a molecular surface layer is enriched with the fluorinated groups (Scheme 2). The surface rearrangement of the fluorinated polymers has been demonstrated as the main factor responsible for the loss of the anti-protein adsorption property31, 47, which results in the easy adsorption of the proteins on the polymer surface. When k is less than 7.9, CF, B% is large enough and the polymer surface is saturated with the fluorinated groups. This situation is similar to the composition of the hydrophobic perfluorocarbon polymers that have been attested to be poorly resistant to protein adsorption.46

Figure 6. Plot of protein adsorbed on the surface of polymers P1-P6 with different values of k.

It is widely reported that the adsorption of proteins on the polymer surface mainly proceeds 16


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via their mutual electric attraction50 and the configuration transformation51 of the proteins. Both BSA and HFg are negatively charged in PBS buffer (pH = 7.4) as their isoelectric points are 4.9 and 5.8, respectively, the zeta potentials of the polymer films with different fluorination degrees (P1P6) were determined and are plotted in Figure S6 (Supporting Information). These polymer films are also negatively charged when immersed in PBS buffer (pH = 7.4), and the zeta potential (Figure 7) shifts negatively with the increase of the polymer’s fluorine content. When k is less than 10 (n > 4.2) or after the polymer surface is saturated with the fluorinated groups (Figure 3), the zeta potential still significantly negatively shifts (Figure 7), which indicates that CF, B% also plays an important role in the surface charge state of the polymers. In PBS buffer (pH = 7.4), both the polymer and the proteins used are negatively charged, which leads to two results. First, when the polymer’s zeta potential is not too negative (k > 14.4 or n < 4.2), the repelling of the same charges decreases the protein adsorption amount on the polymers with the increase of the fluorination degree, and reaches its adsorption minimum when the surface fluorine content is saturated (Figure 6). Second, the adsorption process of BSA and HFg should initiate from the electrostatic attraction of their partially positively charged (chain) end with the negatively charged polymer surface. When the zeta potential drifts negatively, the above electrostatic attraction and the repulsive force between the polymer surface with the protein’s partially negatively charged (chain) end increase, the distance between the above partially oppositely charged (chain) ends also increases, and the negatively charged ionic cloud on the proteins gradually deviates. Consequently, when the zeta potential is sufficiently negative (k < 10 or n > 4.2), the electrostatic attraction is high enough to arrange the protein molecules directionally by acting as a surfactant, which increases the protein adsorption amount on the polymers (Figures 5-6). This phenomenon of the increasing 17



protein adsorption on the polymer surface with the negative drift of the polymer’s potential has also been attributed to the charge-adjust of the proteins from negative to positive.52-53

Figure 7. The zeta potential of polymers P1-P6 with different values of k.

According to the above analyses, it can also be concluded that the polymer’s surface charge state is simultaneously determined by the polymer’s surface and internal composition, which further influences the protein’s adsorption behavior. With the increase in the number of FMA units in the polymers, the determining factor gradually turns from the fluorination degree of the surface to the internal layer, and there is a minimum point for proteins adsorbed on polymers under the synergy of the fluorination degree on the polymer surface and in its internal layer. CONCLUSIONS In this work, a series of amphiphilic polymers were prepared by free radical-initiated copolymerization using hydrophilic acrylate monomers and hydrophobic FMA with diverse ratios of FMA in the polymers. The surfaces of the amphiphilic polymers and the adsorption behaviors of BSA on the polymer surfaces were investigated. With the increase in the number of FMA units in the amphiphilic polymers, the water contact angle initially drastically increases and rapidly reaches a relatively stable value of 114° whereas the surface free energy of the polymer changes oppositely. 18


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Almost all the fluorinated polymers exhibit better protein resistance than the corresponding fluorinefree polymer. P4 is the best polymer against both BSA and HFg, with a surface free energy of ca. 11.93 mN/m, which is much lower than that of Baier’s foul-release zone (20-30 mN/m). In addition to the surface chemical composition and roughness, k (k = CF, S% / CF, B%) also plays an important role in the polymer’s bioadhesion behavior. With the decrease in k, the critical factor determining the protein adsorption gradually changes from the polymer’s surface to its internal layer, especially when k < 10 or n is larger than 4.2 in this study. As k decreases, the fluorinated groups are gradually enriched on the polymer surface to form perpendicularly oriented micelles and are finally saturated while their content in the internal polymer layer continuously increases. That drifts the zeta potential of the polymer negatively and affects its electric attraction with the proteins. Therefore, the adsorption of both BSA and HFg on the polymer surfaces initially decreases and then increases, and the largest protein resistance occurs when k is approximately 12.

ASSOCIATED CONTENT Supporting Information The Supporting Information includes the FT-IR spectra of S0 and P1-P6, the 1H NMR spectra of S0 and P4, the XPS C1s core level spectra of amphiphilic polymers with different numbers of FMA units, the XPS spectra of N1s of the S0 and P1-P6 surfaces after protein adsorption and plot of the atom percent of N1s versus the surface free energy for samples surface.

AUTHOR INFORMATION Corresponding Authors: 19



E-mail: [email protected] Acknowledgements The work was supported by the National Natural Science Foundation of China (Project 51771173, 21273199, 51741107). The authors wish to acknowledge support from the Zhejiang Yuxi Corrosion Control Corporation.

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Table of Contents graphi

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Surface Properties and Protein Adsorption Performance of Fluorinated

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