High-Antifouling Polymer Brush Coatings on Nonpolar Surfaces via

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High-antifouling Polymer Brush Coatings on Nonpolar Surfaces via Adsorption-Crosslinking Strategy Leixiao Yu, Yong Hou, Chong Cheng, Christoph Schlaich, Paul-Ludwig Michael Noeske, Qiang Wei, and Rainer Haag ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13515 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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ACS Applied Materials & Interfaces

High-antifouling Polymer Brush Coatings on Nonpolar Surfaces via Adsorption-Crosslinking Strategy Leixiao Yu,1 Yong Hou,1 Chong Cheng,1 Christoph Schlaich,1 Paul-Ludwig Michael Noeske,3 Qiang Wei,* 1,2,4 Rainer Haag *1,4 1

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195, Berlin, Germany

2

Department of Department of Cellular Biophysics, Max-Planck Institute for Medical

Research, Heidelberg; Postal address: Heisenbergstr. 3, 70569, Stuttgart, Germany 3

Fraunhofer Institute for Manufacturing Technology and Advanced Materials (IFAM),

Wiener Str. 12, 28359 Bremen, Germany 4

Multifunctional Biomaterials for Medicine, Helmholtz Virtual Institute, Kantstr. 55,

14513, Teltow-Seehof, Germany

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Abstract: A new “adsorption - crosslinking” technology is presented to generate a highly dense polymer brush coating on various nonpolar substrates, including the most inert and low-energy surfaces of polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE). This prospective surface modification strategy is based on a tailored bifunctional amphiphilic block copolymer with benzophenone units as the hydrophobic anchor/chemical cross-linker and terminal azide groups for in situ post modification. The resulting polymer brushes exhibited long-term and ultra-low protein adsorption and cell adhesion benefiting from the high density and high hydration ability of polyglycerol blocks. The presented antifouling brushes provided a highly stable and robust bioinert background for biospecific adsorption of desired proteins and bacteria after secondary modification with bioactive ligands, e.g., mannose for selective ConA and E. coli binding.

Keywords: polymer brush coating, adsorption - crosslinking, low-energy surface modification, low fouling, biospecific surface

INTRODUCTION With the fast development and diversification in biomaterial science, there is an increasing utilization of polymeric materials, especially for implant devices, blood contacting devices, and biosensors.1 Polymeric biomaterials used for these purposes are mostly selected on the basis of their bulk mechanical properties, rather than the suitability of their surface properties. However, the adsorption of blood proteins on the surfaces initiates a cascade of biological response and also hinders the effectiveness of the internal body attached sensoric or implant devices.2-4 Therefore,


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surface modification of polymeric biomaterials has been of intense recent interest. Some types of surface modification only require a relatively low density of immobilized functional groups, for example, triggering cell spreading just needs a minimum density as low as 1 fmol cm-2 adhesive ligands.5 Unlike those, a protein resistant surface strictly requires high coverage ratio of the coatings allowing very limited defect densities or rather no defects.6 Ideally, the coatings should be thin, smooth, and colorless to maintain the bulk or sensoric properties of the materials to the least possible extent. Polymer brushes, which are ultrathin and consist of polymer chains that are tethered with one chain end to an interface,7 are extremely suitable to become protein resistant coatings. Although there is a vast body of literature regarding strategies for material surface modification,8 it is still difficult to construct dense polymer brush coating on nonpolar polymeric surfaces, e.g., polyolefines, such as polystyrene (PS) and extremely unreactive surfaces, i.e., PDMS and PTFE, due to the lack of functional surface groups. State of the art methods include direct brush formation via plasma irradiation,9 physisorption of amphiphiles,10 and activation of the substrate by denatured proteins,11 or a layer of dopamine/polyphenol coating12,13 followed by brush generation. However, there are still unresolved challenges from the point of view of technical applicability. Plasma treatment may not only tailor the properties of thin coatings but may also change the surface properties of the underlying substrates, and it requires sophisticated equipment. Also, the traditional amphiphilic coatings suffer from stability problems when exposed to liquid medium for several days. The



so-called universal polydopamine/polyphenol or catecholic coatings are not stable enough on nonpolar surfaces and they also dramatically increase the thickness and roughness of the substrates, while denatured proteins often undergo degradation in physiological environments. Moreover, the greatest challenge in constructing polymer brushes is obtaining a high grafting density, which results in lateral steric repulsion to stretch back-folded polymer chains into a brush conformation. All of the above-mentioned technologies often fail to generate brush conformation and hence do not reach a high protein resistance.

Scheme 1. (a) Synthesis of bifunctional amphiphilic block copolymer PG-BPh. (b) PG-BPh brush coatings fabricated via “adsorption - crosslinking” approach based on a sequence of versatile photo-initiated C-H insertion crosslinking (Scheme S1) steps. Benzophenone (BPh) groups serve as hydrophobic domains in the first “Adsorption”


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step and further contribute to surface anchoring and/or intralayer crosslinking during UV irradiation in the second “Crosslinking” step. The ω-N3 terminal groups in the bifunctional amphiphilic block copolymer serve as in situ modification sites.

Herein, we report a new polyglycerol (PG)-based bifunctional amphiphilic block copolymer with reactive anchors/crosslinkers (Scheme 1) that rapidly generate a stable polymer coating on various nonpolar surfaces in a non-invasive manner. The hydrophobic property of benzophenone (BPh) was highlighted herein besides it well-known aliphatic C-H insertion chemistry. Benzophenone (BPh) has been widely used as surface treatment reagent to tether polymer chains onto surfaces14,15 and photo initiator to induce “grafting from” polymerization16,17 as well as crosslinker to generate surface gels.18,19 However, all of these applications only take place on the surfaces contain aliphatic C-H groups or require extra anchors, e.g. siloxane, to pre-immobilize BPh on the substrates. Overall, BPh has never been recognized as a direct anchor on pristine surfaces. In this study, multiple BPhs were incorporated into the block copolymer as the crosslinkable hydrophobic block, which induced the hydrophobicity-based physisorption on various nonpolar substrates regardless of C-H groups

and

resulted

in

polymer

brushes

with

high

density

even

on

polytetrafluoroethylene (PTFE). In the second step, the BPh groups were covalently linked with substrates and/or neighboring polymer chains via photo-induced grafting and crosslinking (Scheme 1). The intralayer crosslinking combined with the weak hydrophobic interaction at the interface facilitated to obtain a polyvalent anchoring on



the substrates that lack aliphatic C-H groups (i.e., PTFE). The resulting PG-BPh coatings showed excellent protein and cell resistance, which required and reversely indicated the high density of the polymer brushes as described above. The antifouling performance was maintained for at least one year in the physiological buffer, which benefited from the stably anchoring and high thermal and oxidative stability of the polymer backbone. Although we have designed a set of mussel-inspired universal coatings in previous studies,20,21 the protein resistance on coated hydrophobic nonpolar substrate (e.g., PS) surface never reached the same low level as on TiO2 surface. The ongoing challenge of achieving such low fouling on nonpolar surfaces was only mastered in the present study, which allows the application of these antifouling coatings. Moreover, the PG-BPh coating is “ready-to-use” for post modification profiting from the defined ω-terminal groups. Purposely-designed N3 terminals were in situ functionalized with glyco-ligands, i.e., mannoses, that, on the one hand, specifically adsorb concanavalin A (ConA), a kind of lectins by multivalent protein carbohydrate interactions and, on the other hand, still prevent the nonspecific adsorption of other proteins.

EXPERIMENTAL SECTION Materials. All chemicals and solvents were reagent or HPLC grade, used as received and purchased from Sigma (Steinheim, Germany) unless stated otherwise. The deionized water used was purified using a Millipore water purification system with a minimum resistivity of 18.0 MΩ·cm. Allyl glycidyl ether (AGE) was purified by


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stirring over CaH2, distillation in vacuum before used and storage over molecular sieves.

Ethoxyethyl

glycidyl

ether

(EEGE)

was

synthesized

from

2,

3-epoxypropan-1-ol (glycidol) and ethylvinyl ether according to a reference.22 It was purified by stirring over CaH2, distillated in vacuum before use and stored over molecular sieves. The bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (BCN) was prepared according to previously reported protocol.23 The synthesis of coating polymers and BCN modified α-D-mannose are listed in the Supporting Information.

Coating. Polystyrene (PS), Polytetrafluoroethylene (PTFE), Polypropylene (PP), Polyvinyl chloride (PVC), Polyethylene terephthalate (PET), Polyurethane (PU), and Polydimethylsiloxane (PDMS) slides (1 × 1 cm2) were cleaned ultrasonically in isopropanol and water. All the QCM chips were cleaned according to the standard cleaning protocol from LOT (LOT-Quantum Design GmbH, Darmstadt, Germany). To prepare PG brush coatings, the cleaned slides were immersed into a solution of 1 mg/mL PG-BPh in Milli-Q water at room temperature for 2h. After that, the slides were thoroughly rinsed with Milli-Q water and then dried by N2 stream. The coated slides were put under a LED UV lamp (PLS-0365-010-11-C, 365 nm, 30 mW/cm2) and irradiated for 30s. For the surface’s post-modification, crosslinked coating slides were dipped into a 10 mM Man-BCN methanol solution and shaken for 3 h at room temperature. The slides were thoroughly rinsed with methanol and Milli-Q water and then dried by N2 stream. It is important to note that the concentration of the polymer solutions for coating was always 1.0 mg/mL, which was far smaller than the critical



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micelle concentrations (CMC) of PG-BPh polymer (> 100 mg/mL). It has been shown that if the concentration of amphiphilic block copolymer was higher than the CMC, the block copolymer would form micelles in the selective solvent and the adsorbed surface layer itself may have a micellar structure instead of a smooth monolayer brush coating. The coating on 3D architectural surface was conducted in a similar operation as the coating on planar surface. Flow the FITC isothiocyanate modified PG-BPh aqueous solution (5 mg/mL) into a PE tube and a microfluidics chip and kept them in the dark at room temperature for 2 h. The coating on platelet storage bags was performed by immersing a piece of a platelet storage bag inside of FITC modified PG-BPh solution and stored in dark at room temperature for 2h. After that, we thoroughly washed them with methanol and Milli-Q water and then dried them by N2 stream. A LED UV lamp (365 nm; 42 mW/cm2) was used to initiate the crosslinking of benzophenone and the further immobilized the coating onto 3D architecture surface. Quartz

Crystal

Microbalance

(QCM)

with

Dissipation.

Quartz

crystal

microbalance (QCM, Q-Sense E1, Sweden) with dissipation was used to test the adsorption on the sensor surfaces. QCM allows the monitoring of changes in resonance frequency (∆f) and dissipation (∆D) of a piezoelectric quartz crystal as a function of time. f and D were recorded at the fundamental frequency (4.95 MHz) and its 3rd, 5th, 7th, 9th, 11th, and 13th overtones. Only the third overtone was shown in the sensor grams. The frequency response of QCM includes the contributions from both polymers and the water molecules that were bound to the polymer chains.


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Energy dissipation (D) changes represent the rigidity of the coatings, which is related to the hydration ratio and the conformations of the adsorbed polymers. 24,25 For the adsorption of PG-BPh, the cleaned sensor chip was inserted into flow chamber (QFM 401, Q-Sense, Sweden, internal volume of 40 µL) and incubated in Milli-Q water with a flow rate of 0.1 mL/min. After baseline equilibration, a solution of PG-BPh (1 mg/mL in Milli-Q water) was pumped into the flow chamber (0.1 mL/min). After 1 h of dynamic online adsorption, the flow chamber was alternately rinsed with Milli-Q water, aqueous solution of deconex 1% (w/w, Borer Chemie AG, Switzerland), and Milli-Q water (0.1 mL/min). The whole measurement was performed at 25 °C. The Sauerbrey equation was used to calculate the mass of the adsorbates (∆m = C × ∆f, where ∆m is the change in mass, C is the mass sensitivity constant of the quartz crystal (-17.7 ng·cm-2·Hz-1), and ∆f is the overtone-normalized frequency change). For the online monitor of mannose surface immobilization, a pre-coated PS chip with PG-BPh was inserted into the flow chamber (QFM 401, Q-Sense, Sweden, internal volume of 40 µL) and incubated in MeOH with a flow rate of 0.05 mL/min. After baseline equilibration in the MeOH, a solution of Man-BCN (50 mg/mL in MeOH) was pumped into the flow chamber (0.05 mL/min). After 30min of dynamic online adsorption, the flow chamber was rinsed with MeOH (0.1 mL/min). To ensure the completely reaction of the SPAAC, Man-BCN (50 mg/mL in MeOH) was pumped into the flow chamber (0.05 mL/min) again for another 30 min and then followed with the buffer rinse. The whole measurement was performed at 25 °C. The conversion of



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this azide-alkyne cycloaddition at the interface was calculated from the equation:

Conv. =

mMan / MMan mcoating / MPG − BP

The mcoating and the mMan are calculated from the Sauerbrey equation. The

MPG-BPh and the MMan are the molecular weight of PG-BPh and Man-BCN respectively. The protein adsorption was measured similarly. The coated sensors were inserted into the

flow

chamber

and

incubated

in

pH

7.4

N-(2-hydroxyethyl)

piperazine-N’-(2-ethanesulfonic acid) (HEPES, 0.1 mol/L, pH 7.4, and 150 mmol/L NaCl) buffer. After baseline equilibration, deconex (1% w/w in HEPES buffer) was pumped into the flow chamber for 10 min and followed by rinsing with HEPES buffer for 15 min. Then the protein solution (1 mg/mL) was pumped into the flow chamber. After 30 min, the surface was rinsed with HEPES buffer again for another 15 min. The flow rate used for all experiments was 0.1 mL/ min, and the temperature was 25 °C. The Voigt model for viscoelastic layers was used to calculate the mass of adsorbed proteins with the help of the software package Q-tools (version 3.0.15.553, Q-Sense, Sweden), because the D/f ratio is not small enough to consider the surfaces as rigid surfaces. The density of the adsorbed protein layer was assumed to be 1200 kg m-3, the fluid density to be 1000 kg m-3, and the fluid viscosity to be 0.001 kg ms-1. The protein used to test coating resistance were fibrinogen (Fib, 450 kDa, from bovine plasma), bovine serum albumin (BSA, 66 kDa), lysozyme (14.3 kDa, from chicken egg white), concanavalin A (Con A, 102 kDa, from Canavalia ensiformis), human blood plasma and full fetal bovine serum (FBS) without dilution.


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Cell Resistance. Cell resistant experiments on PG-BPh coated PP slides, PVC slides and tissue culture polystyrene (TCPS) wells were done with adherent NIH-3T3 murine fibroblast cells (ACC no. 59, DSMZ, Braunschweig, Germany). Cells were collected from Petri dishes by incubation in trypsin (dilution 1:250) for 5 min at 37 °C. The trypsin was removed from cell suspension by centrifugation, the top layer was removed, and the remaining cells were re-suspended in a fresh medium. The PP and PVC slides were incubated with 10,000 cells in 1 mL of cell medium (cell number was determined via a Neubauer chamber) for 3 days at 37 °C and 5% CO2. The wells of the 24-well cell culture plates were coated to test the coatings on TCPS. 1 mL cell medium with 10,000 cells was added into each coated/uncoated well. The incubated time was the same as the experiments with PP and PVC slides. The adhering cells were observed by microscope directly (TELAVAL 31, Zeiss, Germany), without removing the medium or rinsing the slides with PBS buffer. Bacterial specific adhesion. For the adhesion experiments, a suspension of E. coli (DH5α) in CASO broth with an optical density of 0.5, corresponding to about 6 × 108 bacteria/cm3 at the beginning of the exponential growth phase, was used. The control and modified PS and PTFE slides 1.2 × 1.2 cm2 were washed with 70% ethanol, which was followed by five sterile PBS washes and then fitted in the bottom of a sterile 24-well plate. 1 mL of 1×106 CFU/mL of E. coli. in a lysogeny broth (LB) medium was added to each well and incubated at 37 °C for 10 h without shaking. Each slide was then gently rinsed by immersion in sterile PBS four times. The bacterial adhered onto the substrate was stained by Syto 9 solution. After 10 minutes,



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the sample was gently immersed with fresh PBS buffer and observed after drying under confocal laser scanning microscopy (CLSM, Leica DMI6000CSB SP8) with an FITC filter. Protein adsorption from mixed proteins. The protein adsorption experiments on bare PS surface, PG-BPh coatings and Man-PG-BPh coating surface were performed with BSA and ConA. The BSA was labeled with fluorescein isothiocyanate (BSA-FITC)

and

ConA

was

labeled

with

Rhodamine

B

isothiocyanate

(ConA-Rhodamine B). The testing surfaces were incubated in the mixed solution of 1 mg/mL BSA-FITC and 1 mg/mL ConA-RB in PBS buffer for 3h at room temperature and then were gently rinsed with PBS buffer. The adsorption proteins on surfaces were directly observed by fluorescent microscope (Axio Scope.A1, Zeiss, Germany) with FITC filter and DsRed filter. Coating kinetics study. The PS substrates used for the coating kinetics study were prepared by spin coating a homogeneous PS layer onto silicon wafers. Several drops of polystyrene dissolved in DMF (1% w/v) were dropped onto a clean silicon wafer and spin coated at 4000 rmp for 2 min. The thickness of the PS layer was 45 ± 0.2 nm determined by ellipsometry. The prepared PS substrates were then immersed into the PG-BPh polymer solution (1mg/mL in H2O) for a set time. After rinsing with sufficient MilliQ water and drying under high vacuum, the coating thickness was measured. According to the hypothesis of Ligoure and Leibler,26 the coating rate in the initial tethering regime (diffusion regime) was predicted to be controlled by the polymer


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chain diffusion from solution. The change of the coating density (below σ*) was proposed to follow the relationship below: 1/ 2

 Dt  2  a 

σ (t) ~ 

(φ0 / N ) Equation 1

where σ(t) is the polymer graft density, D is the diffusion coefficient of the polymer, and ϕ0 is the volume fraction of monomer in solution. The σ* is denotes the grafting density that the tethered chains just start to overlap (σ = σ* ≈ N-1). Above the overlap concentration, a further adsorption requires some stretching of the chains. There is a potential barrier to oppose the penetration of the chains. The barrier becomes an essential obstacle and the diffusion of free chains does not control anymore the adsorption kinetics. The cross-over surface coverage σ are defined as27: s1/3e

γ N σ eq 2/3 s 2/3

≅

2 N σ eq 2/3T 3

Equation 2

where s = σ(t)/σeq and T = t/τ are reduced variables, τ is a short characteristic time, and γ is a constant. In the terminal kinetic regime, polymer chains must penetrate a preexisting brush layer to reach the surface to tether onto the substrate. The change of grafting density in this regime is penetration-limited and predicted to the following relationship: 

t    τ ex   

σ ( t ) = σ eq 1 − exp  − 

Equation 3

τex is exponential relaxation regime time (the penetration-limited regime).

RESULTS AND DISCUSSION Synthesis of amphiphilic block copolymer. The PG-BPh bifunctional



amphiphilic block copolymer was synthesized via the ring-opening anionic polymerization of ethoxyethyl glycidyl ether (EEGE) and allyl glycidyl ether (AGE), followed by acetal deprotection, thio-ene amination with cysteamine and BPh linking by amide formation. The amount of the grafted BPhs was verified to guarantee an optimal coating density and brush conformation of the adsorbed polymers from aqueous solution (1 mg/mL). With the increase of BPh content in the polymer chain, the thickness of the resulting coatings on PS surface gradually increased and reached a plateau value at approx. 3.2% (in mole ratio) BPh content, corresponding to ~ 4 BPh units per polymer chain. At this BPh content, the water contact angle (WCA) plot exhibited an inflection point. The surface hydrophobicity decreased before reaching this point and increased hereafter (Figure S1). This indicates that a change of the internal layer structure occurs at this point (water contact angle of 63° ± 1°) and suggests that the layer termination is dominated by PG based chains. We conclude that a certain amount of BPh was required to generate enough hydrophobicity at the interface, but too many BPh units would increase the steric effect and result in a less dense coating. Therefore, in the following the block copolymer with 4 BPh units was utilized unless otherwise specified. In a block-selective solvent (H2O), which is good for the PG block but poor for the BPh block, pristine substrate surfaces were exposed, and an adsorbate layer was formed. Quartz crystal microbalance (QCM) with dissipation was employed to analyze the adsorption of the polymers on PS sensors. The results show that the hydrated mass of the adsorbed PG-BPh on the sensor surfaces was about 950 ng/cm2


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(Figure 1). However, the polymers only weakly adsorbed onto the PS substrate and were easily rinsed away by surfactant solutions. Nearly 65% of the adsorbed polymers detached after rinsing with 1% w/w deconex aqueous solution. This result was expected and agreed with the properties of adsorbates formed by traditional amphiphilic polymers. However, when the freshly deposited coatings were exposed to UV-irradiation (365 nm, 42 mW/cm2), the obtained coatings maintained stable upon rinsing with surfactant solutions (Figure 1a). Under UV irradiation, the BPh groups underwent a n - π* transition into a triplet state and abstracted a hydrogen atom from neighboring aliphatic C-H groups, i.e., from either the substrate or the neighboring polymer chains,28 which resulted in a covalent C-C bonding with the substrate or the neighboring polymer chain (Scheme 1, Scheme S2). The optimized UV-irradiation time was identified by a decrease in WCA and increase in coating stability under subsequent rinsing with deconex (Figure S2). Only 30 seconds of irradiation was sufficient to covalently immobilize the polymers onto PS substrate and/or completely crosslink the polymer chains. The BPhs were only located in the hydrophobic block of the copolymers, thus the intralayer crosslinking did not affect the brush conformation of the hydrophilic PG blocks which were oriented towards the substrate surface. The first step of polymer diffusion onto the solid substrates allowed a very fast layer preparation/formation. As indicated by ellipsometry (Figure 1b), the dry thickness reached a plateau value with approx. 3.5 nm in 3 min. This value did not increase even after immersion times up to 10 hours, which matched the change of hydrated thickness obtained by Voigt fitting from QCM curves (Figure S3). We concluded that



the respective surface coverage corresponds to the equilibrium surface grafting density. The grafting density of polymer chains was calculated according to the following equation:29

σ =(h ρ Na ) / Mn where h is dry the thickness of the coating, ρ is the bulk density of the coating polymer (assumed to be 1.1 g/cm3 for PG-BPh), Na is Avogadro number and Mn is the number average molecular weight of the polymer. The resulting equilibrium coating density was about 0.2 chains/nm2, which was in the scale of a dense monolayer brush coating and significantly higher than previously reported “grafting to” brush coatings.30,31

Figure 1. (a) QCM frequency (f) shift as a function of time during the adsorption of PG-BPh on a PS sensor surface. The black curve shows buffer rinsing. The blue curve indicates physisorption of amphiphilic block copolymer and the red curve demonstrate the surfactant washing. (b) Ellipsometric thickness of PG-BPh polymers on PS substrates with the evolution of the coating time.

As mentioned above, the generated radicals from BPhs can randomly insert any


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neighboring aliphatic C-H group and form covalent C-C bonds, regardless of the coating polymer and the substrate. Therefore, the PG-BPh coatings displayed substrate independent properties. The static water contact angles of all the tested surfaces, significantly decreased after the coating as expected (Figure S4). Following the UV-treatment, the PG-BPh polymers were even effectively tethered onto chemical inert PDMS and PTFE, which are otherwise hard to coat. The stability of the PG-BPh coatings was investigated on selected substrates, namely PS, PP and PTFE. The contact angles of the PG-BPh coated surfaces did not obviously increase after incubation in a SDS solution (2%, w/w) for 30 min under sonication (Figure S5). The chemical composition of PG-BPh coatings on PS substrates was confirmed by X-ray photoelectron spectroscopy (XPS) analysis (Table S1). The deconvoluted S2s spectra for PG-BPh coating and the corresponding pristine PS substrate is shown in Figure 2a. The significant S2s peaks, ascribed to the sulfur of sulfide bond, were only detected in case of substrates covered with PG-BPh polymer coatings but not in case of pristine substrates (Table S1), this finding confirms the successful polymer coating. The corresponding N1s spectra was shown in Figure S6. The nitrogen peaks at 399.0 ± 0.1 and 401 ± 0.1 eV were attributed to the nitrogen (N-H, amide) and cationic ammonium nitrogen in the polymer chain, respectively. It was assumed that the only one peak at ∼ 399 ± 0.1 eV in the pristine substrate spectrum related to the N-H bond of some additive in PS. The surface morphology of the coatings and the respective bare substrates was investigated by atomic force microscopy (AFM) (Figure S7). On the coated



substrates, island-like structures could be clearly observed, and this morphology was construed typical of monolayer polymer brush coating.32 The monolayer coatings were ultra-smooth and ultra-thin. Both the average roughness (Ra) and root-mean-square roughness (Rq) of the coatings were slightly smaller than those of the bare PS substrate (Figure 2b). We assume that the coating levels the grooves of the substrates. The coating thickness under ambient condition was 3.6 ± 1.0 nm as measured by AFM and correlated well with the ellipsometry measurements (Figure 1b). Besides the coating on 2D planar surfaces, the polymers can also be efficiently used for the complex 3D systems including PDMS microfluidic chips (Figure 2c-2d), polyethylene microtubes, as well as PVC blood platelet storage bag (Figure S8) by simple dip-coating. Besides, PG-BPh coatings were also successful on polar inorganic surfaces (including gold, titania, and silica) because of the crosslinkable BPh anchors (Table S2, Figure S9).


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Figure 2. (a) The deconvoluted XPS S2s signal curves of PG-BPh coatings on PS substrate and the corresponding curves of pristine PS substrate. (b) The average surface roughness (Rq and Ra) for bare and coated PS surfaces, which was calculated from 5 AFM images (2 × 2 µm2). (c) the image of microfluidic chip (PDMS) and (d) the corresponding fluorescence image after PG-BPh coating. The PG-BPh polymer used here was covalently modified with FITC isothiocyanate (2% to all OH groups in the backbone) prior to coating. Kinetics of Brush Formation. The adsorption of polymer chains from dilute solution to the impenetrable solid surface to form a monolayer of polymer brushes has been extensively studied.27,33,34 According to the hypothesis of Ligoure and Leibler,26 the initial tethering regime (diffusion regime) for an end-functionalized polymer



attached to a substrate surface should be diffusion controlled and exhibit a t0.5 dependence on the tethering time (Equation 1). The diffusion of polymer chains from solution to the substrate surface is comparably fast. The tethering rate of PG-BPh onto a PS substrate surface in the diffusion regime corresponded well to the prediction as shown in Figure 3a. During the first 1.3 min, the surface grafting density (σ) exhibited a linear relationship with t0.5. The characteristic time τ1 is obtained from the inflection point of the fitting curve and indicates the end of the first kinetic regime. The corresponding areal density of polymer chains on the substrate surface is σ1. The average distance between tethering sides on the substrate was defined as d, which can be calculated from the experimental values of σ following the equation: d = (σπ/4)-1/2.33 The value of d at the end of the diffusion regime was 7.7 nm, which is far larger than the gyration radius (Rg) of PG-BPh polymers (that is 1.7 ~ 1.8 nm for polyglycerol with molecular weight 10 kDa35). According to the commonly used criterion:33 it may be expected that for d > 2Rg the tethered polymer chains have sufficient lateral space on the substrate surface and should be in an expanded coil or mushroom conformation. Therefore, a layer of nonoverlapping and relaxed polymer chains covered the substrate in the first coating regime. When the grafting density further increased, the tethering rate progressively slowed down and was proportional to ln(t) due to the increased energy barrier (Figure 3b). This coating regime was controlled by the diffusion of free polymers through the already tethered chains to reach the substrate surface. This slow tethering was expected to continue until saturation. At this point, the energy/enthalpy benefits of


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adsorbing polymer chains to the surface would be offset by the entropic cost of chain stretching.26,36 The grafting density σ in this regime is defined in Equation 2. In this period, the d gradually decreased and finally was lower than 2Rg. The gradually increased areal density of chains on the substrate surface would result in lateral compression of the adsorbed polymer chains and stretching them away from the surface to avoid an overlap.26 Therefore, there was an obvious conformation transition of the tethered polymer chains from mushroom to brush. At the end of regime two (σ2 = 0.2, d = 2.24 nm < 2Rg = 3.5 nm), most of the tethered polymer chains were in a moderately-dense and extended conformation (Figure 3c).

Figure 3. (a) Grafting density of PG-BPh polymers as a function of t0.5 in the diffusion-controlled region. (b) ln(t) dependence of the grafting density of PG-BPh in the crossover regime. (c) Natural logarithm of the normalized grafting density of



PG-BPh as a function of time in the terminal regime (d) ∆D - ∆f plots of the adsorption of PG-BPh on PS sensor surfaces (60 min online coating). The starting point of the dynamic coating process was defined as ∆D = ∆f = t =0.

Following the conformation transition, there was a terminal kinetic regime, where only a small amount of polymers could be tethered onto the surface due to the penetration barrier. The polymers tethering kinetics exhibited a penetration-limited behavior. As shown in Figure 3c, the natural logarithm of the normalized tethering rate, ln[1 - σ(t)/σequil], displayed an almost linearly progressive declination with time as predicted for a penetration barrier (Equation 3).26 The declining rate was associated with a progressive increase in the penetration barrier during the tethering procedure and a dense polymer brush layer built up. Thus, the pre-attached polymer chains were further stretched up because of an increased chain density. Finally, a dense monolayer brush coating was generated. In summary, the adsorption kinetics of the PG-BPh block copolymers to the PS substrate exhibited three regimes: (i) the diffusion-controlled regime where σ(t) was dependent on t0.5; (ii) regime where σ(t) slowly increased with ln(t) and was accompanied with a conformation transition of the adsorbed polymers from mushroom to brush, and finally (iii) penetration-limited regime where the areal density of polymers displayed a linearly progressively increase with time to obtain the dense brush coatings. Due to the limited time interval, only 3-5 data points were collected in each kinetic coating regime for a linear regression approach. It is probably not so accurate to state that all the data have a good linear


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fitting, but they display a clear tendency to support our hypothesis. The QCM-D testing can simultaneously measure the kinetics of changes in the resonant frequency, f (c.f. the adsorbed mass), and the dissipation factor, D (c.f. rigidity viscoelastic properties). To gain more information on the adsorption behavior, ∆D is plotted against ∆f in a ∆D - ∆f plot was introduced by Hӧӧk, et al.24 This way of plotting eliminates the time as an explicit parameter, which can be used to obtain additional information on the adsorption processes via an analysis of the slopes ∂D/∂f.24,25,37 The ∆D - ∆f plot could reveal the adsorption behavior of PG-BP polymers on substrate, which was not directly seen in common ∆f-t plot and ∆D-t plot. The different slopes in the ∆D - ∆f plots indicate different kinetic processes during coating. Herein, it was used to study the coating kinetics of PG-BPh on hydrophobic surface together with ellipsometry data. The ∆D versus ∆f plot of the polymer adsorption displayed three phases with significantly different slopes (Figure 3d). The slope of the first phase (|∂D/∂f1| = 0.12) was significantly greater than the second phase (|∂D/∂f2| = 0.06). This indicated that loosely bound coatings that were formed in the first phase with flexible polymer chains were accompanied by a large amount of hydrodynamically coupled water.24 The inflection point between these two phases was about -31.6 Hz, which corresponded to t = 1.3 min (τ’1) and exactly matched the end time for the diffusion-controlling regime. Following the second phase, a third phase with a smaller slope (|∂D/∂f3| = 0.02) appeared where ∆D only slightly increased with an increase of |∆f|. The adsorbed polymers in this phase were likely to compact the coating in such a way that the trapped water was replaced by additional polymer



chains,25 which resulted in highly dense coatings. The break point (τ’2) between these two phases was at 22 min, which coincided with the onset time of the penetration-limited regime as well. Antifouling Performance. After successful monolayer characterization, the protein resistance of the PG-BPh coating was evaluated by single proteins including fibrinogen (Fib), bovine serum albumin (BSA), and lysozyme (Lys), as well as by the complex protein environment of undiluted fetal bovine serum (FBS) and human blood plasma. As indicated by QCM studies, the coatings exhibited an extremely high protein resistance (Figure 4a, Table S3). The coated PS surface successfully repelled > 97% of the adsorbed single proteins relative to the bare PS surfaces. More notably, Fib, a large protein presents in relatively large quantities in the blood and strongly adsorbs to hydrophobic surfaces, was repelled till < 0.2%. Only < 4 ng/cm2 Fib (including the mass of the associated water) was adsorbed onto the coated surfaces, which is comparable with the benchmark protein-resistant system that was generated by surface polymerization on gold surfaces and evaluated by surface plasmon resonance (SPR, the mass of the associated water was excluded).38 Most impressively, the proteins in FBS and blood plasma were effectively repelled as well. Only 2.8% adsorbed on the PG-BPh coated PS, whereas, more than 10% adsorbed on an universal monolayer20 and even more than 20% adsorbed on multilayer coated PS21 in our previous studies. It is known that the highly complex protein mixture in serum or plasma strongly fouled many types of “protein resistant” polymer coatings.39 The excellent protein resistance of our coating can be explained by the highly hydrated


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and densely packed PG brushes, which exhibited highly flexible aliphatic polyether backbone with multiple hydrophilic functional groups. This polar PG-block has been proven to have high affinity with water molecules to generate a hydration layer.20,21 The hydration layer serves as an efficient barrier to non-specific protein adsorption.6,40 Moreover, the coatings were very stable over time and they still resisted > 95% of the proteins from FBS even after incubation in physiological buffer for 1 year (Figure 4b), which benefited from the stable anchoring and high thermal and oxidative stability of the polymer backbone. Mammalian cell adhesion on surface is known to be mediated by the adsorption of extracellular matrix (ECM) proteins.41 Therefore, the protein-resistant surfaces are likely to be cell-resistant as well. Cellular resistance of the PG-BPh coated surfaces was evaluated by NIH3T3 mouse fibroblasts, which are involved in the tissue responses upon implantation into living tissue.42 NIH3T3 cells were cultured on the coated PS (Figure 4c), PP, and PVC (Figure S10) slides as well as the related bare references. Cells grew to confluence on the bare surfaces (PS, PP, and PVC) after only three days, whereas, only a few and evidently unspread cells sedimented to the coated surfaces before rinsing, and almost no cells (< 1) attached after gentle rinsing with PBS.



Figure 4. (a) Protein adsorption on PG-BPh coated PS surfaces. (b) Protein adsorption from FBS to the coated PS surfaces before and after incubation in PBS buffer for 1 month, 2 months, 6 months and 1 year. (c) NIH3T3 cell adhesion on the PG-BPh coated PS and the pristine surfaces after 3 days of cell culture. Scale bar: 50 µm.

Biospecific protein adsorption and bacteria capture. The presented nonspecific antifouling surfaces provided an excellent bioinert background for the biospecific adsorption of desired proteins. α-D-mannose (Man), which is a key component of the glycochain on the cellular surface,43 was grafted onto PG-BPh coatings via in situ strain-promoted azide-alkyne cycloaddition (SPAAC)44 between


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the azide terminal groups on the brush chain and a cyclo-alkyne functionalized mannose. The yield of the in situ SPAAC on the coated surface was very high, whereby at least 95% of the azide terminal groups of PG-BPh brushes were reacted with Man-BCN (Figure S11). After immobilization, about 890 ng/cm2 concanavalin A (ConA), which contains a Man binding site,45 was conjugated onto the coatings and measured by QCM (Figure 5). By comparison, only about 6 ng/cm2 (148 folds lower) adsorbed on the coatings without mannose (Figure 5b). Additionally, the immobilized mannose did not obviously increase the adsorption of BSA. Furthermore, the specific protein adsorption from mixed proteins was performed with the 1:1 mixture of fluorescein labeled BSA (FITC-BSA) and Rhodamine B labeled ConA (Rhodamine B-ConA). As shown in Figure 5c-5h, both proteins were adsorbed on bare PS surfaces with strong fluorescent intensities but not on the PG-BPh coated surfaces. However, only Rhodamine B-ConA could be detected on the Man-PG-BPh coated surfaces. These results indicated the good selectivity of the functional coatings.



Figure 5. (a) In situ mannose functionalization onto PG-BPh coatings via SPAAC. (b) Specific adsorption of ConA and unspecific adsorption of BSA on Man-modified PG-BPh coatings investigated by QCM. The protein adsorption from the 1:1 mixture of fluorescein labeled BSA (FITC-BSA) and Rhodamine B labeled ConA (Rhodamine B-ConA) onto the (c, d) bare PS surfaces, (e, f) PG-BPh coated surfaces, and (g, h) Man-PG-BPh coated surfaces. Scale bare indicates 100 µm.

The E. coli biofilm development is a complex process that is important for disease and for engineering applications.46 The conjugated Man ligand can mediate and promote the specific adhesion of mannose-specific (MS) bacteria such as E. coli, Klebsiella pneumoniae, and Salmonella spp.47 This attachment is mediated by


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bacterial surface lectins in the form of type 1 fimbriae, which are terminated by an α-D-mannose specific lectin named FimH.48 These type 1 fimbriae are expressed in several hundred copies on the bacterial cell surface to achieve tight adhesion through multivalent protein carbohydrate interactions. After incubating with E. coli suspension for 10 h, a large number of bacteria attached and formed a monolayer biofilm onto the Man-PG-BPh modified PS (Figure 6a-b) and PTFE (Figure 6c-d) surfaces, while only a few cells could be observed on the bare and PG-BPh coated surfaces. Therefore, this E. coli specific capture coating could be utilized in many biotechnological fields, including applications in filtration, degradation of wastewater, and in biotechnological processes, such as the production of bulk and fine chemicals, as well as biofuels microbial fuel cells.49

Figure 6. (a) Biospecific capture of E coli on bare, PG-BPh coated and Man-PG-BPh coated PS surface. Bacteria were cultured on the test surface for 10h. Scale bar: 20 µm. (b) Number of E coli that adhere on bare, PG-BPh coated, and Man-PG-BPh coated PS. (c) Biospecific capture of E coli on bare, PG-BPh coated and



Man-PG-BPh coated PTFE surface. Bacteria were cultured on the test surface for 10 h. Scale bar: 20 µm. (d) Number of E coli that adhere on bare, PG-BPh coated, and Man-PG-BPh coated PTFE surfaces.

CONCLUSION In summary, a dense bifunctional and long-term stable monolayer brush coating forms on various nonpolar surfaces, including the highly inert materials, PDMS and PTFE by a simple “adsorption-crosslinking” technology based on a bifunctional amphiphilic block copolymer with benzophenone (BPh) as the reactive anchor. The optimized hydrophobicity of the BPh functional block enabled BPh to be a direct anchor on pristine surfaces, which largely extended the use of BPh for material surface modification. The adsorbed BPhs initiated the unselective chain insertion crosslinking reaction under short UV irradiation to immobilize the polymer chains either on the substrates presented aliphatic C-H groups via covalent bonding or on the other substrates by multivalent adsorption and covalent crosslinking. This process resulted in an ultrathin, smooth, and highly stable monolayer brush coating. Besides the coatings on 2D planar surfaces, the PG-BPh polymers can also be used to coat complex 3D systems, e.g., microfluidics channels, which extended its potential application to a lab on chip. The modified nonpolar surfaces exhibited outstanding antifouling properties and were stable in physiological buffer for at least one year. After post-modification with biospecific ligands, e.g., mannose, these nonspecific antifouling surfaces were converted to highly biospecific protein adsorption and bacteria capture coatings via multivalent protein carbohydrate interactions. Therefore,


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this highly stable monolayer coating provides a new platform for universal material surface modification and can be used in a wide range of biointerface applications. We thus believe our work opens up new avenues for the modification of nonpolar material surfaces and in situ immobilization of a wide variety of selective biomolecules.

AUTHOR INFORMATION Corresponding Authors * [email protected], *[email protected] Notes The authors declare no competing ﬁnancial interest.

ASSOCIATED CONTENT Supporting Information. Synthetic details, used characterization methods, supplementary contact angle data, supplementary XPS data, supplementary AFM images, supplementary cell adhesion images on coated surfaces, coating stability testing, coating on polar surfaces, coating on 3D architectures, in situ surface click reaction and any associated references are available in the Supplementary Information.

ACKNOWLEDGMENTS



This work was supported by the Chinese Scholar Council, the SFB 765 of the German Science Foundation, and Helmholtz Virtual Institute on “Multifunctional Biomaterials for Medicine”. We thank Dr. Pamela Winchester for proof-reading this manuscript.

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TOC Graphic 251x190mm (300 x 300 DPI)


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High-Antifouling Polymer Brush Coatings on Nonpolar Surfaces via

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