A strong adhesive block polymer coating for antifouling of large molecular weight protein☆

Xianling Meng,Xia Jiang,Peijun Ji*

Department of Chemical Engineering,Beijing University of Chemical Technology,Beijing 100029,China

1.Introduction

Biofouling is ascribed to the adsorption and accumulation ofproteins and micro-and macroorganisms on surfaces[1].Biofouling causes problems in medical implants,surgical devices,biosensors,food processing and packing,and marine ships[2-6].Biofouling starts with adsorbed proteins to create a conditioning film on the substrates,followed by colonization by adherent bacteria.The colonized bacteria result in the bacterial colonization is irreversible adhesion,and it is almost impossible to reduce biofouling once formed on a solid surface[8].Thus,it is necessary to develop effective coatings to protect the surface by preventing bacteria from adhering.For this purpose,extensive research has been carried out to develop antifouling materials and coatings against protein and bacterial adsorption.Hydrophilic polymers have been used for antifouling coatings.Glycocalyx-mimetic peptoids[9],zwitterionic polymers[10],polyelectrolyte[11],poly(ethylene glycol)(PEG)[12-15],and polysaccharides[16]have been extensively investigated to coat various substrates.A general explanation for hydrophilic polymers as antifouling materials is the formation of a strong hydration layeratpolymer/waterinterface preventing protein adsorption and further bacterial colonization.The hydration layer is due to hydrogen bonds between hydrophilic polymers and water and ionic solvation for zwitterionic polymers[9,10].

Poly(2-hydroxyethyl methacrylate)(PHEMA)is a hydrophilic polymer,it consists of both hydrogen acceptors and donors,facilitating the formation ofhydrogen bonds with watermolecules.PHEMAmembrane[16]and chitosan modified PHEMA[17]have been investigated for antifouling of protein and bacteria adsorption.The terminal hydroxyls of HEMA's pendent groups of PHEMA were modified with fluorinating moieties of different chain lengths,the resultant amphiphilic brushes can suppress significantly adsorption of proteins[18].More research work has been carried out for PHEMA based copolymers with antifouling properties,including a triblock copolymer of polyethylene glycol(PEG),HEMA,and 2-methacryloyloxyethyl phosphorylcholine(MPC)[19],poly(HEMA-co-glycidylmethacrylate)hydrogels with zwitterionic surfaces[20],copolymers composed of2-per fluorooctylethyl methacrylate and HEMA monomers[21],and poly(2-aminoethyl methacrylate hydrochloride-co-HEMA)[22].

PHEMA coated surface can resist the adsorption of small and modest molecular weight proteins,such as lysozyme and BSA.However,our results showed that the polymer-coated surface is not effective against the adsorption of large molecular weight proteins.Antifouling of large molecular weight proteins is ofsignificantimportance,as some proteins secreted by microorganisms have large molecular weights[23].Colonization of microorganisms starts with initial physically adsorbed proteins to create a conditioning film on the substrates[24].Hence effectively preventing the adsorption of large molecular weight proteins is essential.In the work reported herein we introduce a novel approach to prepare hydrophilic multilayer polymer coatings for antifouling of larger molecular weight proteins.As illustrated in Fig.1,the substrate is first coated with a hybrid polymer film,which is formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD).After grafting the macroinitiator 2-bromoisobutyryl bromide,the block polymer PMMA-b-PHEMA is grafted through surface-initiated atom-transfer radical polymerization.Three proteins,including recombinant monomeric green fluorescent protein(GFP),recombinant dimeric R-transaminase,and tetrameric catalase,were used to test the antifouling properties of the block polymer brushes.

Fig.1.Schematic illustration of the procedures for grafting copolymer PMMA-b-PHEMA.APTS:3-aminopropyltriethoxysilane;BIBB:2-bromoisobutyryl bromide.

2.Experimental

2.1.Raw materials

304 stainless steel sheet and 304 stainless steel sheet(#8 mirror)were purchased from Beijing Guangyanghuaxia Stainless Steel Co.Dopamine hydrochloride(98%)and 3-aminopropyltriethoxysilane were purchased from Sigma-Aldrich and used as received.Other chemicals were purchased from Sinopharm Chemical Reagent Co.The analytical grade chemicals were used as received without further purification.

2.2.Protein purification

The Escherichia coli expressing the proteins were obtained from the Department of Biochemical Engineering of Beijing University of Chemical Technology.The three proteins used in antifouling test are fusion proteins:green fluorescent protein(GFP)fusion with an elastin-like polypeptide(ELP)designated as GFP-ELP(54 kDa),GFP fusion with R-ω-transaminases and ELP designated as RTA-GFP-ELP(dimer,2×90 kDa),and catalase fusion with ELP designated as CATELP(tetramer,4×98 kDa).Protein expression and purification are described in Supplementary information.The purified proteins were analyzed by SDS-PAGE as depicted in Fig.S1(Supplementary information),which shows that high purity proteins have been obtained.

2.3.Fluorescein isothiocyanate(FITC)modification of catalase-ELP(CAT-ELP)

25 mg CAT-ELP was dissolved in 25 ml of 50 mmol·L-1NaH2PO4buffer(pH 8).350 μg FITC was added using a freshly prepared stock solution(1.2 mg·ml-1)in DMSO.The mass ratio of CAT-ELP to FITC was 1.0 mg/16.0 μg under the conditions.The reaction was carried out at 4°C in the dark.After 8 h,the reaction was quenched by addition of NH4Cl(50 mmol·L-1)at 4 °C.After 2 h,the solutions were dialyzed against 3.5 L of PBS.

2.4.Coating with polymers

The 304 stainless steel(SS)substrates were polished by P2500 silicon carbide papers.Then the SS samples were sonicated sequentially in deionized water,acetone,and ethanol for 30 min,followed by rinsing with deionized water for 10 min.The samples were activated by immersing in H2SO4/H2O2(3:1 by vol)[10].Fig.1 illustrates the procedures for grafting the block polymer PMMA-b-PHEMA on the substrate stainless steel.

2.5.Coating with the HPAPD film

The solution of dopamine hydrochloride was prepared by dissolving in ethanol/water(1:3 by vol)with a concentration of dopamine hydrochloride 1.5 mg·ml-1.Then 3-aminopropyltriethoxysilane was added to the solution,the molar ratio of 3-aminopropyltriethoxysilane to dopamine hydrochloride being 1:1.The solution pH was adjusted to be pH 10.5 by addition of NaOH(3 mol·L-1).By simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD),a hybrid polymer film was formed on the SS samples by immersing the SS substrates in the solution for 48 h.The SS-HPAPD samples were then rinsed thoroughly with deionized water,and dried through nitrogen-blowing.

For preparing SS-polydopamine,the solution of dopamine hydrochloride was prepared by dissolving in ethanol/water(1:3 by vol)with a concentration of dopamine hydrochloride 1.5 mg·ml-1.The solution pHwas adjusted to be pH 10.5 by addition ofNaOH(3 mol·L-1).Then SS was immersed in the solution,and the incubation was carried out for 2 h.

For measuring FTIR spectra,the samples were cut into small pieces.

2.6.Grafting the macro-initiator 2-bromoisobutyryl bromide

SS-HPAPD samples were placed in a triangular flask filled with triethylamine(1 ml)and dichloro methane(20 ml),and then 2-bromoisobutyryl bromide(1 ml)was added drop wise.The reaction was carried out at 0°C for 1 h,followed at room temperature for 24 h.After reaction,the obtained samples(referred as SS-HPAPD-Br)were rinsed sequentially with dichloro methane,acetone,and water,and then dried through nitrogen-blowing.

2.7.Grafting the block polymer PMMA-b-PHEMA

Grafting poly(methyl methacrylate)(PMMA)on SS-HPAPD-Br was carried out according to the ARGET ATRP method[25].20 ml of methyl methacrylate(MMA)was mixed with 20 ml of methanol solution(67%,v/v)in a flask.The mixture was deoxygenated through bubbling with dry N2gas for 15 min.4 mg CuBr2,354 mg sodium L-ascorbate,and 6 mg 2,2-dipyridyl were then added under N2atmosphere with a syringe.The mixture was magnetically stirred until all the chemicals were dissolved in the solution.During the period of dissolving chemicals,the solution was purged with dry nitrogen gas.Then the SS-HPAPD-Br samples were placed in the flask.The flask was purged with dry N2for 2 min,and then was sealed.The reaction was allowed to proceed at room temperature.After 24 h the samples were taken out and washed sequentially with acetone and water.The SS-HPAPD-PMMA samples were sonicated in acetone for 30 min,rinsed thoroughly with water and dried with nitrogen-blowing.

For further grafting poly(2-hydroxyethyl methacrylate)(PHEMA),20 ml of 2-hydroxyethyl methacrylate(HEMA)was mixed with 20 ml of methanolsolution(50%,v/v)in a flask.The mixture was deoxygenated through N2gas bubbling for 15 min.4 mg CuBr2,354 mg sodium L-ascorbate,and 6 mg 2,2-dipyridyl were then added under N2atmosphere with a syringe.The mixture was magnetically stirred until all the chemicals were dissolved in the solution.During the period of dissolving chemicals,the solution was purged with dry nitrogen gas.Then the SS-HPAPDPMMA samples were placed in the flask.The flask was purged with dry N2for 2 min,and then was sealed.The reaction was allowed to proceed at room temperature for 24 h.The samples were then taken out and washed sequentially with acetone and water.The SS-HPAPD-PMMA-b-PHEMA samples were sonicated in acetone for 30 min,rinsed thoroughly with water and dried with nitrogen-blowing.

For preparing the SS-HPAPD-PHEMA samples,starting with SS-HPAPD-Br,the same method was adopted for grafting PHEMA.

2.8.Surface characterization and measurement

2.8.1.X-ray photoelectron spectroscopy

XPS spectra were measured using an X-ray photoelectron spectrometer(Thermo VGESCALAB250).Using Mg K X-ray as the excitation source,the measurement was carried out at a pressure of 2×10-9Pa.Data analysis was carried out with Thermo Avantage XPS software.

2.8.2.FTIR spectra measurement

Infrared spectra were measured using a Fourier transform infrared spectrometer(Bruker TENSOR 27).Infrared spectra were obtained by collecting 128 scans per spectrum at a resolution of 2 cm-1.All spectra were corrected by subtracting the background.Ultrapure nitrogen gas was introduced at a controlled flow rate to purge water vapor.

2.8.3.Water contact angle measurement

The surface hydrophobicity and hydrophilicity of the polymer coatings were characterized using water contact angle measurements(OCA20;Data Physics Corp.,Bad Vilbel,Germany).The contact angles reported were the mean value from three substrates,with the value of each substrate obtained by averaging the contact angles from at least three surface locations.

2.8.4.Film thickness by ellipsometry

The ellipsometry measurement was performed on an SE200BA ellipsometer.The film thickness on SS surfaces was measured at angles 65°,70°and 75°and wavelengths from 400 to 800 nm.The refractive indices of the cleaned stainless steel(#8 mirror)substrates were measured prior to sample preparation.Three-layer optical model for SSHPAPD,SS-HPAPD-PHEMA and SS-HPAPD-PMMA-b-PHEMA was used to determine polymer film thickness.Ellipsometric data were fitted for thickness with the Cauchy layer model.A refractive index of 1.5 was assigned to the polymer brush layers[26].The refractive index of HPAPD was assumed to be 1.6,which was assigned for polydopamine film with good fitness[27].Five separate spots of each sample were measured to obtain mean film thickness and associated standard deviation.

2.8.5.Atomic Force Microscopy(AFM)

For preparing samples for AFM imaging,304 stainless steel sheet#8 mirror was used.AFM images were obtained on a SPM-9700(Shimadzu Inc.).A silicon microcantilever(spring constant 42 N·m-1and resonance frequency 320 kHz,Nanoworld AG,Switzerland)with a pyramid tip(radius of curvature～8 nm)was used for scan.The operating pointratio waschanged between 0.2 and 0.6 to acquire the bes tcontrast of phase image.

2.9.Coating stability test

Artificial seawater was prepared according to the article[28].The artificial seawater composition(perliter)was as follows:23.568 g ofNaCl,3.986 g of Na2SO4,0.197 g of NaHCO3,0.672 g of KCl,0.097 g of KBr,10.63 g of MgCl2·6H2O,1.471 g of CaCl2·6H2O,0.026 g of H3BO3,and 0.04 g of SrCl2·6H2O.The coupons were transferred to the exposure vessels containing deoxygenated artificial seawater.During the exposure periods deoxygenation was achieved by putting the exposure vessels in an anaerobic chamber(ELECTROTEK)under an atmosphere containing 5%H2,5%CO2,and 90%N2.The samples of pristine SS,SS-HPAPD,SS-HPAPD-PHEMA,and SS-HPAPD-PMMA-b-PHEMA were incubated in artificial sea water for 90 days,respectively,in triplicate for each exposure time.

2.10.Antibiofouling of protein adsorption

The green fluorescent proteins,GFP-ELP(54 kDa),RTA-GFP-ELP(dimer,2×90 kDa),and FITC modified CAT-ELP(tetramer,4×98 kDa)were dissolved separately in phosphate-buffered saline(PBS,pH 7.4)to prepare protein solutions with a concentration of 3 mg·ml-1.The SS,SS-HPAPD-PHEMA and SS-HPAPD-PMMA-b-PHEMA samples were tested for protein adsorption.The samples(2 cm×2 cm in area)were rinsed with PBS,and then were immersed in the protein solutions.The protein adsorption was allowed to proceed at room temperature.After incubation for 24 h,the samples were removed from the solution,gently washed with PBS,and then rinsed with distilled water.Then the samples were imaged with an Olympus BX60 fluorescence microscope(Olympus America Inc.,NY),equipped with a 495 nm excitation filter and a 525 nm emission filter.

3.Results and Discussion

3.1.Multilayer polymer coatings

The HPAPD film is formed on the stainless steel samples through hydrolytic polycondensation of3-aminopropyltriethoxysilane and polymerization of dopamine.In the hydrolytic polycondensation of 3-aminopropyltriethoxysilane,the transient silanol groups condense with other silanols[29].The polymerization ofdopamine is due to autoxidation of dopamine[30].The amines of the aminopropyl groups of 3-aminopropyltriethoxysilane react with the catechols of polydopamine[31].Thus,a hybrid polymer(HPAPD)network is formed.The HPAPD film was taken as a solid basis coating for further grafting polymers,as illustrated in Fig.1.Grafting bromine end groups on the HPAPD film is accomplished using the ARGET ATRP procedures[25].Polymerization of methyl methacrylate(MMA)and the chain propagation was initiated by coupling the MMA monomers with the Br end groups on SS-HPAPDBr.Similarly with the face-initiated atom transfer radical polymerization,polymerization of 2-hydroxyethylmethacrylate(HEMA)and chain propagation on SS-HPAPD-PMMA-Br is achieved.The obtained sample is designated as SS-HPAPD-PMMA-b-PHEMA.Surface color change is observed after coating the polymers,as shown in Table 1.It is shown that after the HPAPD coating,hydrophobicity of the SS-HPAPD surface decreases compared to SS.Further coating PHEMA makes the surface hydrophilic due to the OH and C=O groups from PHEMA.While for the sample SSHPAPD-PMMA-b-PHEMA,the hydrophilicity decreases compared to SS-HPAPD-PHEMA due to the presence of PMMA in the block polymer.

3.1.1.FTIR and XPS spectra

Fig.2a shows XPS spectra of the C 1s,O 1s,N 1s,Br 3d,Si2p and Si2s regions.In the wide scan spectra,the peaks for Si 2p and Si 2s were ascribed to the formation of HPAPD film.The content of Si is about 9.13%.The peak for Br 3d was ascribed to the grafted bromine end groups.Compared to SS-HPAPD-Br,the intensity of O 1s of SS-HPAPD-PHEMAwas relatively increased,and the intensity of C 1s of SS-HPAPD-PMMA-b-PHEMA was relatively increased.

Table 1Contact angle and thickness of the top layer for the samples

Fig.2b shows the FTIR spectra.The peaks at 3400 cm-1were ascribed to O--H and N--H stretching modes;aliphatic C--H stretching modes at 2940 and 2952 cm-1;and the peaks at 1650 and 1656 cm-1were attributed to ring C=O stretching modes from the quinone groups[32].Appearance of these peaks is consistent with the structure of dopamine[33,34].The peaks at 1572 and 1563 cm-1were assigned to ring C=C and ring C=N stretching modes from the aromatic amine species in the HPAPD film.The peaks at1149/1140 cm-1were assigned to C--N stretching vibration[32].The peak at 1730 cm-1was due to ester C=O stretching vibrations.The peaks at 766 cm-1were ascribed to Si--O--Si symmetric stretching[35,36].FTIR and XPS spectra confirm the coatings formed of HPAPD,HPAPD-Br,HPAPD-PHEMA,and HPAPD-PMMA-b-PHEMA.

3.1.2.AFM images

The morphology of the samples was examined with AFM.Fig.3 lists the AFM images for the SS,SS-HPAPD,SS-HPAPD-PHEMA,and SS-HPAPD-PMMA-b-PHEMA samples.There are scratches on the surface of the pristine SS sample(#8 mirror).After coating with the HPAPD film,the SS-HPAPD film became smooth.Aftergrafting the polymer PHEMA and block polymer PMMA-b-PHEMA,the RMS roughness for SS-HPAPD,SS-HPAPD-PHEMA and SS-HPAPD-PMMA-b-PHEMA are 1.84,1.14,and 2.10 nm,respectively.

3.1.3.Wettability and thickness

To determine the wettability and thickness,the multilayer polymer coatings were characterized by contact angle and ellipsometry measurements,respectively.Thickness of the top layer and contact angles are listed in Table 1.After coating HPAPD,the sample becomes hydrophilic,and the thickness of the film is about 45.36 nm.After grafting PHEMA,the thickness of the polymer is about 39.48 nm,the contact angle becomes smaller,indicating a more hydrophilic surface.After grafting the block polymer PMMA-b-PHEMA,the thickness is about 304.3 nm.It is indicated that the block polymer has a higher polymerization and growth efficiency than PHEMA on the SS-HPAPD surface.Possibly this is due to the factthatthe HEMA monomeris relatively larger than the MMA monomer,leading to a relatively lower efficiency for grafting PHEMA.Compared to the surface coating with PHEMA,the wettability after grafting PMMA-b-PHEMA was reduced,as the MMA monomer only contains hydrogen acceptors(O=C--O).

Fig.2.XPS(a)and FTIR(b)spectra for polymer coatings.

3.2.Coating stability

As illustrated in Fig.1,the HPAPD film was taken as a basis for further grafting polymer PHEMA and block polymer PMMA-b-PHEMA.We first investigated the coating stability of the HPAPD film by pull-off adhesion test.Adhesion strength of the HPAPD film coating on the stainless steel substrates was determined using the pull-off test method according to the ASTM D4541 procedure.A pull-off adhesion tester(De Felsko)was used.The aluminum dolly was glued on the HPAPD layer with Araldite 2015 epoxy adhesive(Huntsman advanced materials,Germany).Samples were then kept at room temperature for 24 h to ensure that the glue fully cured.The test started with pulling of the dolly.During pulling of the dolly,the glue on the SS-HPAPD sample broken off from the dolly surface as illustrated in Fig.4.The color and surface atthe places indicated by arrows look the same as that at other places outside the glue circles.It is indicated that the pull-off test has no damage to the HPAPD layer.Therefore we conclude that the HPAPD layer adheres strongly onto the stainless steel.

Fig.3.AFMimages of SS(a),SS-HPAPD(b),SS-HPAPD-PHEMA(c),and SS-HPAPD-PMMA-b-PHEMA(d).

The HPAPD film built a solid basis for grafting the block polymer.The hybrid polymer film is formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine.First we investigated the anticorrosion properties of the coatings by the HPAPD film and by polydopamine,respectively.The HPAPD-coated and polydopamine-coated samples were immersed in artificial seawater for 30 days.It is found that SS-HPAPD can resist the corrosion of the artificial seawater(Fig.5(a)),while a significant amountofsaltdeposits on the surfaces ofthe SS-polydopamine samples(Fig.5(b)).It is demonstrated that the SS-polydopamine samples cannot resist the corrosion of the artificial seawater as shown in Fig.5(b).The surface morphology of the SS sample shows severe corrosion after immersion for 30 days(Fig.5(c)).These images exhibit the advantage of SS-HPAPD over other samples.

Fig.4.Pull-off test for the sample of SS-HPAPD.

For investigating the coating stability of the polymers,ellipsometry and contactangle measurements were performed to determine changes of thickness and wettability by immersing the samples in artificial seawater for 7 and 90 days,respectively.For the sample SS-HPAPDPHEMA,the top layer thickness change is about 22.8%after 90 days of immersion,and the contact angle is increased to 50.36 from 39.48(Table 2),indicating that the PHEMA coating is not stable.The stability of SS-HPAPD-PMMA was tested as shown in Table S2.It was observed that after 90 days of immersing in artificial seawater,the thickness and contact angle had little changes.For the sample SS-HPAPDPMMA-b-PHEMA,the top layer thickness change was about 0.46%after 90 days of immersion,and the contact angle almost had no change during that period of time.The results demonstrated that the block polymer brushes are very stable.

3.3.Antifouling of proteins

For testing the antifouling of proteins adsorptions,we used three proteins,one is a modest molecular weight protein GFP-ELP,other two are large molecular weight proteins RTA-GFP-ELP and CAT-ELP.Both the samples of SS and SS-HPAPD-PMMA cannot prevent the adhesion of the proteins.The surface of SS-HPAPD-PHEMA is very hydrophilic(Table 1),and the samples can prevent the adsorption of GFP-ELP,as shown in Fig.6.However,SS-HPAPD-PHEMA cannot prevent the adhesion of large molecular weight proteins RTA-GFP-ELP and CAT-ELP.Possibly this is due to the multiple hydrogen bonding interactions occurring between the polymer brushes of PHEMA and the proteins.PHEMA consists of both hydrogen donors and hydrogen acceptors.RTA-GFP-ELP is a dimer protein and CAT-ELP is a tetramer one.Both proteins have large amount of amino,hydroxyl and carboxyl groups.Thus the multiple hydrogen bonding interactions may be strong enough to repel the water molecules out of the region in between the PHEMA brushes and the proteins,as explained by molecular simulation for the strong adsorption of lysozyme on the surfactant-coated surface[37],and the hydration layer may be destroyed.In a separate experiment,the SS-HPAPD-PHEMA samples were sonicated in cyclohexane for 20 min,and then were used to testthe antifouling ofprotein adsorption.It is found that the antifouling is improved as illustrated in Fig.S2(Supplementary information).The contact angle measurement demonstrates that after the sonication in cyclohexane,the contact angle is increased as shown in Table S1(Supplementary information),implying the decrease of hydrophilicity after sonication in cyclohexane.The sonication in cyclohexane makes a hydrophobic environment around the PHEMA brushes,facilitating the intro-hydrogen bonding interactions in between the PHEMA brushes as illustrated Fig.7.This reduced the amount of hydrogen donors and acceptors of PHEMA that are available to formhydrogen bondswith the proteins.Asa result,the antifouling effect is improved.

Fig.5.SEM images after immersing in artificial seawater for 30 days(a):SS-HPAPD;(b):SS-polydopamine;(c):SS.

The above analysis suggested that regulating hydrophilicity of the polymer brushes is helpful to improve the antifouling effect.Hence block polymerization of MMA with HEMA was carried out.Compared to the monomer HEMA,MMA is less hydrophilic.The formed PMMA-b-PHEMA brushes on the substrate have a much larger thickness than that of PHEMA,and the contact angle is also increased,indicating a decrease in hydrophilicity.Antifouling of proteins demonstrates that the adsorption of the proteins RTA-GFP-ELP and CAT-ELP on SSHPAPD-PMMA-b-PHEMA has been significantly reduced(Fig.6).Possibly this is due to the fact that the monomer MMA can only provide hydrogen acceptors(C=O),this decreases the hydrogen bonding interactions between the block polymer brushes and the proteins.This facilitates the formation of hydration layer in between the block polymer brushes and the proteins,preventing the direct contacting of the surface with the proteins.

Fig.6.Antifouling of protein adsorption by the samples.The scale bar in the photographs is 50 μm.

Fig.7.Schematic presentation for intro-hydrogen bonding interactions between PHEMA brushes after sonication in cyclohexane.(a)Sonication in water;(b)sonication in cyclohexane.

4.Conclusions

The HPAPD film results from simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine,and can strongly adhere onto SS.The HPAPD film was used as solid basis for grafting the block polymer PMMA-b-PHEMA.The multilayer polymer coatings exhibit stable stability.Three proteins were used for testing antifouling of protein adsorption.Recombinant green fluorescent protein(GFP)is a monomeric protein with a molecular weight about 54 kDa.Two large molecular weight proteins were also used for testing the antifouling properties.One is a dimer protein and each subunithas a molecular weightabout90 kDa,anotheris a tetramer protein and each subunit has a molecular weight about 98 kDa.It is demonstrated the coating formed of multilayer polymers has signi ficantly reduced the adsorption of the proteins.The approach to coating with the multilayerpolymers can be easily extended to other substrates,which means it has wide application potential.

Supplementary material

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cjche.2017.04.007.

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