A Facile Bifunctional Strategy for Fabrication of Bioactive or Bioinert

Article pubs.acs.org/IECR

A Facile Bifunctional Strategy for Fabrication of Bioactive or Bioinert Functionalized Organic Surfaces via Amides-Initiated Photochemical Reactions Zhengfang Wu,† Dehui Wang,† and Peng Yang*,† †

Key Laboratory of Applied Surface and Colloids Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xìan, 710119 China S Supporting Information *

ABSTRACT: The excellent potential of organic polymeric materials in the biomedical field could be exploited if their interfacial problem could be fully resolved. A necessary prerequisite to this purpose often involves the simple but effective synthesis of a bioactive surface to endow polymer surfaces with high reactivity toward efficient biomolecules conjugation and a bioinert surface to prevent nonspecific adsorption of nontarget biomolecules. Although the corresponding research has been an important topic, actually few strategies could pave the way to comprehensively and simply tackle both of the bioactive and bioinert surfaces preparation issues. Herein we report an extremely simple and integrative bifunctional method that could efficiently tailor an organic material surface toward both bioactive and bioinert functions. This method is based on the use of an amides-initiated photochemical reaction in a confined space, which depending on the type of solutes used, results in the incorporation of primary amine groups or surface carbon radicals on an inert polymer surface. The grafted amine group could be used as a highly reactive site for biomolecule conjugation, and the surface carbon radical could be used to initiate radical graft polymerization of antifouling polymer brushes. We expect this simple but powerful method could provide a general resolution to solve the interfacial problem of organic substrate, offering a low-cost practical approach for real biomedical applications.

1. INTRODUCTION Both bioactive and bioinert organic surfaces are widely utilized in the biomedical field. On a bioactive surface, functional biomolecules and materials could be conjugated to provide key biofunctions,1−5 while the bioinert surface is designed to prevent nonspecific biomolecules/cells adsorption.6−9 These two types of surfaces require a delicate surface modification strategy to finely tailor the interfacial chemical and physical structures. Although the corresponding research has been an important topic and some excellent approaches have been reported,10−20 a simple, low cost, and efficient strategy to achieve this aim, especially with the ability to synthesize both bioactive and bioinert organic surfaces integrated in one-step systematical multipurpose approach, is limited and remains challenging.21 In sharp contrast to the classical methods, the present work proposed a systematical strategy that integrated double functions to prepare both bioactive and bioinert organic surfaces via an extremely simple but effective method. For both bioactive and bioinert surface preparations, the setup and handling process in this newly proposed method is almost same, while the only difference is the use of distinctive compounds for each respective purpose. This method is based on the use of photosensitivity of amides under UV irradiation, as found by us in a previous work.22 In that study, we just demonstrated that N,N-dimethylformamide (DMF) could be photosensitively dissociated under UV to form a dimethyl amine molecule, which could subsequently attack the ester bond on polyester substrates to introduce a tertiary amine group [R−C-N(CH3)2]. However, the method has three noticeable limitations. First, the substrate was limited to the © 2014 American Chemical Society

use of polyesters while more common polymeric materials such as polyolefins without ester functionalities contained were excluded. Second, the introduced tertiary amine group [R−CN(CH3)2] could not undergo classical conjugation chemistry with electrophilic groups (e.g., COOH, CHO) because of the absence of a reactive N−H bond in the introduced tertiary amine group. Third, the extra function of the preparation of the bioinert surface was not imparted to this method. Therefore, the method reported in a previous study22 was actually not a general and applicable strategy for the preparations of both bioactive (aminated) and bioinert polymer surfaces. In contrast, the present work has tackled the above limitations through the use of rationally selected new chemistries. The newly found experimental results and underlying reaction mechanism as shown herein demonstrate that a new kind of modification could be generally applicable to (1) polyolefin substrates containing only alkyl chains; (2) the introduction of standard reactive primary amine groups (R-C-NH2) that are eligible to undergo classical conjugation reaction with biomolecules (i.e., bioactive surface preparation) and (3) photograft polymerization of nonfouling monomers (i.e., bioinert surface preparation). This novel bifunctional chemical path is illustrated in Scheme 1. For the preparation of the bioactive surface, the carbonyl group in amides could be photodissociated to form corresponding amine radicals (a), which could further abstract Received: Revised: Accepted: Published: 9401

March 11, 2014 May 6, 2014 May 13, 2014 May 13, 2014 dx.doi.org/10.1021/ie501058f | Ind. Eng. Chem. Res. 2014, 53, 9401−9410

Industrial & Engineering Chemistry Research

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Scheme 1. Proposed Mechanism for the Bifunctional Strategy on Polymer Surface Functionalization to Prepare Bioactive and Bioinert Surfaces

method could provide a general and systematical solution toward the challenging interfacial problem of organic substrate, and thus afford a low-cost practical approach for real biomedical applications of polymeric materials.

the hydrogen atom from the polymer surface to form corresponding amine molecules and a surface carbon radical (b). The incorporation of amines with photocontrolled site precision was then achieved by both of the radical recombination reaction between the surface carbon radical and amine radical (c1) and the in situ surface aminolysis reaction between amine molecules and ester bonds (if possible) on the surface (c2). Via the use of amides containing primary amine moiety, the primary-aminated polymer surface is thus formed as a bioactive substrate that could be further conjugated with other functional molecules or materials (d) based on versatile reactivity of primary amines to other reactants including isocyanates, aryl halides, or azides, hydroxysuccinimide-ester or imdoesters, sulfonyl cholorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, carbodiimides, anhydrides,23 and transglutaminase,24,25 as well as coordination with gold nanopaticles.26 If monomers are supplied into such an amide-based photochemical system, the surface-initiated graft polymerization of monomers could be instantly achieved to prepare the bioinert surface. This was because as mentioned above, the in situ formed reactive amine radical (a) would attack the C−H bond on the polymer surface to produce a surface carbon radical, which could consequently initiate the graft polymerization of monomers (b).27 We thus utilized several classical antifouling monomers, for example, oligo(ethylene glycol) methacrylate (OEGMA), 2-hydroxyethyl methacrylate (HEMA), N-(2-hydroxypropyl)methacrylamide (HPMA), and carboxybetaine acrylamide (CBAA), to successfully construct excellent bioinert regions on commodity polymer substrates that could prevent nonspecific adsorption of proteins, in sharp contrast to nonspecific adsorption of proteins on pristine polymer surface without nonfouling polymer grafts covered. The combinational bifunctional strategy proposed in the present work has great potential for the synthesis of bioactive and bioinert surfaces on polymeric substrates in one extremely simple and integrative method. Following the modification on the polymer surface by this bifunctional strategy, the experimental results shown in the present work on the target protein immobilization and micropatterning as well as antifouling test demonstrate that this facile and powerful

2. RESULTS AND DISCUSSION 2.1. Fabrication of a Primary Amine Array on a Polymer Surface. The key point to achieve a primary amine array on a polymer surface was the utilization of an acetone solution of special amide reagent, N-acetylethylenediamine (AED). The experimental process was extremely facile and outlined as simply sandwiching the acetone solution of AED between two polymer films where the top film was A UVtransparent film [e.g., biaxially oriented polypropylene (BOPP)], and the bottom film was any polymeric material [e.g., polyethylene terephthalate (PET)], followed by transferring this sandwich setup under a UV lamp for the UV irradiation at a given time. Subsequently, the final ready-to-use grafted substrate (any top/bottom polymer substrate) could be obtained after routinely washing and drying the substrate. The commercially available AED reagent contains both the photosensitive amide group and reactive primary amine site, and accordingly, by the photochemical reaction of amide moieties in AED, the reactive ethylenediamine species could be in situ obtained, which subsequently attacked the C−H and COO (ester) sites along the polyester chains on the polymer surface based on the combinational reactions of c1 and c2 in Scheme 1, as supported by the following experimental results. In the reaction path c1, the radical transfer was developed from the ethylenediamine radical to the polymer surface by hydrogen abstraction from the C−H bond, and then the subsequent radical coupling between the resultant surface carbon radical and ethylenediamine radical directly created the primary amine array. In the reaction patch c2, a surface aminolysis reaction on the ester bonds of the polymer substrate was taking place, and the result of this reaction was that the ester bond was replaced with the amide structure, and the primary amine group that was connected as a distal group to the amide moiety was consequently introduced on the exposed polymer surface. 9402

dx.doi.org/10.1021/ie501058f | Ind. Eng. Chem. Res. 2014, 53, 9401−9410


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Figure 1. Qualitative and quantitative characterizations on the aminated PET surface by ninhydrin test (the ninhydrin testing solution was formulated as 6% ninhydrin ethanol solution containing a determined amount of water and pyridine at the ratio of water/pyridine = 1:49 v/v): (a) qualitative characterization to show the color contrast between the virgin (left) and aminated (right) surfaces in the ninhydrin testing solution; (b) quantitative measurement of the surface grafting density of amine groups by correlating the ultraviolet absorption of the ninhydrin testing solution at different irradiation time (c) with the calibration curve from the following reference: Y. Zhu, C. Gao, X. Liu, J. Shen, Biomacromolecules 2002, 3, 1312−1319.

to the polymer surface by hydrogen abstraction from C−H bond and subsequent radical coupling between the surface carbon radical and ethylenediamine radical (c1). A gradual increasing weight percentage of primary amine (C-NH2), with the prolonged irradiation time, indicated that during the entire amination reaction, the reaction c1 progressively dominated the whole process. Besides N 1s spectrum, the C 1s spectrum on the modified surface also supported the grafting of primary amines (f), showing a peak centered at 286.1 eV assigned to the superposition result of three finer carbon substructures as O C−N (amide) at 287.4 eV, C−O at 286.6 eV, and amino (CNH2) at 285.8 eV.34−36 The above hypothesis was further supported by direct amination on inert polyolefins without any ester bonds (Supporting Information, Figure S3). The effective incorporation of amine groups on BOPP and LDPE (low density polyethylene) surfaces revealed by XPS in Figure S3 strongly reflected that even without the contribution from the ester bond-based aminolysis reaction by the ethylenediamine molecules (c2), the single process c1 in Scheme 1, as the radical transfer from the ethylenediamine radical to the polymer surface by hydrogen abstraction from the C−H bond and subsequent radical coupling between the surface carbon radical and ethylenediamine radical, could still lead an effective amination. Besides ATR-FTIR and XPS measurements, the introduction of primary amine groups was also qualitatively indicated by the standard ninhydrin test (Figure 1),37,38 reflecting a quick appearance of purple blue color in ninhydrin solution with the immersed aminated surface (Figure 1a). The ninhydrin analysis method was further employed to quantitatively detect the amount of NH2 groups on the aminated PET film (Figure 1b). The blue reaction product of ninhydrin with free NH2 on the PET surface has a maximum absorbance at 538 nm in the solvent of 1,4-dioxane/2-propanol (1:1) (Figure 1c). Through correlation of the corresponding absorbance intensity with the calibration curve obtained by plotting the absorbance intensity of ninhydrin test solution with different amounts of 1,6hexanediamine in 1,4-dioxane/isopropane (1:1, v:v) added, the experimental surface density of grafted amines could be estimated based on the experimental amine concentration divided by the surface area of the substrate. The resultant grafted amine density on the modified surface was thus plotted with the irradiation time (Figure 1b), and showed the maximum NH2 density yielded at 8 min as 5 × 10−7 mol/ cm2. From this result one could calculate that the average area

The incorporation of primary amine groups on a polyester PET surface was first checked by attenuated total reflectanceFourier transform infrared (ATR-FTIR) (Supporting Information, Figure S1) and X-ray photoelectron (XPS) spectroscopies (Supporting Information, Figure S2). The ATR-FTIR spectra evidenced the characteristic peak around 1650 cm−1 assigned to possible primary amine (1650−1580 cm−1) and amide (O C−N) I (1630−1695 cm−1) structures,28,29 indicating the possibility of the surface aminolysis reaction to replace the ester bond with amide structure and introduce primary amines. With increasing irradiation time, the intensity of this absorption band was enhanced, which reflected that more amide/amine structures were formed. To explicitly characterize the fine chemical structure on the modified surface, the functionalized substrate was further characterized by XPS. As compared with that of the virgin PET substrate (a), the N 1s signal remarkably appeared on the modified surface (Supporting Information, Figure S2b). Timeresolved XPS (Supporting Information, Figure S2c) showed that the nitrogen atomic concentration on the modified surface was enhanced with an increase in the irradiation time, and reached a plateau after 8 min as 1.8% based on the calculation in the XPS survey spectra, indicating that a large number of amine groups were introduced after the modification (c.f. the following surface grafting density calculation of amine groups). The separated N1s peak could be further deconvoluted by curve fitting into two distinct binding energies with different assignments (Supporting Information, Figure S2d): 399.0 eV for the N atom in the primary amine and 400.5 eV for the N atom in the amide group.20,30−33 The ratio of these two assignments (C-NH2/OC−N) increased with prolonging the irradiation time (Supporting Information, Figure S2e), and at the optimized irradiation time (8 min), the two assignments have an atomic ratio of 85:15 which is much higher than the calculated value of 1:1 if it is supposed that the grafted molecular structure has the form of R−(CO)−N−CH2− CH2−NH2. This indicated that besides the surface aminolysis forming the expected structure of R−(CO)−N−CH2− CH2−NH2, another large proportion of nitrogen atom was introduced onto the surface as the form of free primary amine without the amide structure as the bridging group. This result is well consistent to the reaction pathways proposed in Scheme 1, where besides the surface aminolysis by the ethylenediamine molecules (c2), there was another predominant process involving the radical transfer from the ethylenediamine radical 9403



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Figure 2. AFM scanning on the PET surfaces before (a) and after (b) the amination reaction. The irradiation time was 8 min. The corresponding 3D images with scale bars for each surface were inserted in the panels as the insets.

Figure 3. Fluorescence microscope image (a) and the corresponding fluorescence intensity profile (b) on the patterned aminated PET surface after FITC-SAv immobilization.

per amino-terminated chain was 0.03 Å2, supposing that the grafted surface was absolutely smooth and all the amino groups laid as a single layer. This is obviously unreasonable because instead of the hypothesis that the absolutely smooth and single graft layer, the actual surface roughness and the possible multilayer grafting should be considered. Atomic force microscopy (AFM) measurements showed that the aminated PET surface had a root-mean-square (rms) of 6.72 nm with a densely packed and homogeneously distributed protrusion topography made by the occurrence of limited surface etching due to the chain scission (Figure 2), which was in contrast to a lower rms of 3.36 nm on the control virgin PET surface. Obviously, the elevated roughness as reflected by the higher rms and fluctuant surface would increase the practical surface area accounting for the lowering of actual amine density. Another factor is that as found in a previous report, the reactants may penetrate to a certain depth from the superficial outer surface due to the swelling ability of organic solvent on the PET substrate,27 and accordingly the formation of stratified multilayer amine groups may regulate the exceptionally high amine density to a reasonable value. Anyway, the above measurement reflected an effective amine grafting on the polymer surface with high grafting density, which along with

the other features of this method including simplicity, rapidity, and photocontrolled site-selectivity was encouraging for practical biomaterial application.39 The above evidence in ATR-FTIR and XPS spectra reflected that the amide photochemical reaction, especially c2 in Scheme 1 led to a chain scission on the substrate from ester bond sites. Therefore, a possible way to directly monitor this photochemical reaction process is to follow the mass change (decrease) of the exposed film with the reaction proceeding. As shown in Supporting Information, Figure S4, the mass loss of the exposed film was enhanced with prolonged irradiation time and increased UV intensity. At the latter stage of the irradiation (e.g., 16 min), the mass loss grew slowly and approached a maximum value around 0.8%. With the same irradiation time, the mass change also was enhanced with increased UV intensity and varied slowly after the intensity reached 10000 μW/cm−2. The optimized mass loss yielding around 8 min was consistent with the change of nitrogen atomic concentration obtained by the aforementioned XPS measurements, indicating the modification reaction was approaching a balance after 8 min. The low mass change reflected that instead of the surface aminolysis resulting in the chain scission (c2 in Scheme 1) and consequent mass loss, the 9404



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Figure 4. Optical microscope image of the patterned aminated PET surface after HRP immobilization. The immobilized HRP could not be seen before AEC staining (a) and in contrast, a micropatterned HRP array could be revealed after AEC staining by showing a red color after the AECHRP biochemical reaction to produce red products on the circular array (b).

mass change−free radical coupling reaction between the polymer surface radical and ethylenediamine radical (c1 in Scheme 1) should dominate the reaction process, which was in accordance with the judgment concluded from the deconvoluted N1s XPS analysis (Supporting Information, Figure S2e). After the detailed characterization on the aminated polymer surface, the reactivity of introduced amine groups on the surface was utilized for site-selective protein immobilization through a classical aldehyde−amine condensation reaction.40 Through incubating the aminated surface in glutaraldehyde (GA) solution, the surface primary amine group was efficiently transformed to an aldehyde structure by the aldehyde−amine condensation. The resultant surface with immobilized GA could further rapidly react with amino, thiol, phenol, and imidazole groups in target biomolecules, providing high biomolecules immobilization stability (often multipoint binding) and low reactivity loss.41−46 To evaluate the ability of sitespecifically capturing target biomolecules, the micropatterned aminated surface was first fabricated by the photomaskcontrolled amination reaction. Such a micropatterned aminated surface was incubated in GA solution, and sequentially incubated in a model protein, FITC-SAv solution. As shown in Figure 3, FITC-SAv could be effectively immobilized onto the aminated area through the amine (in the protein)− aldehyde condensation reaction, showing an obvious fluorescent pattern on the micropatterned amine grafting regions. The background fluorescence was from the cocontributions of nonspecific adsorption of proteins on the hydrophobic polymer background during the incubation and the self-fluorescence of PET substrate itself due to the existence of some photoactive additives (e.g., photostabilizers) in commercial polymeric films.47,48 To more clearly reveal the protein immobilization on the modified surface, HRP that presents red color after its substrate AEC staining was further immobilized on the micropatterned aminated surface by incubating sequentially the micropatterned aminated surface in GA and HRP solution. As shown in Figure 4, AEC as the specific HRP substrate could effectively stain the micropatterned aminated surface after the step of incubation in HRP solution, producing a micropatterned red circular array that corresponds well to the immobilized HRP circular array. In contrast, no visible red color was observed on the micropatterned circular aminated array before AEC staining, indicating that the AEC staining is an effective specific

enzyme−substrate reaction to reveal the regions of HRP microarray.49 2.2. Fabrication of Antifouling Surface on a Polymer Substrate by DMF-Initiated Photografting Polymerization. The second function of the amide photochemical system, photoinitiator-free photografting polymerization of monomers on untreated virgin polymer surface, was achieved by simply using a DMF solution of monomers without any other reagents added and arbitrary polymer films. The experimental process was extremely facile and outlined as simply sandwiching the DMF solution of the target monomer between two polymer films where the top film was UVtransparent film (e.g., BOPP) and the bottom film was any polymeric material (e.g., PET), followed by transferring this sandwich setup under a UV lamp for the UV irradiation for given time. Subsequently, the final ready-to-use grafted substrate (any top/bottom polymer substrate) could be obtained after routinely washing and drying the substrate. The advantage of this chemical design is on the one hand, avoiding the use of any photosensitizers, photoinitiators, or coinitiators in the reaction solution except for utilization of mere common organic solvent and monomers, and on the other hand, excluding the need for prefunctionalization or prepriming on a polymer substrate that is usually required by conventional surface-initiated grafting polymerization methods. Similar to AED used for the amination on a polymer substrate, DMF, besides possessing good solvation function, is also one kind of amide reagents that would dissociate to form highly active dimethyl amine radical under UV irradiation.22 It has been reported that such a radical could subsequently attack a C−H bond on a polymer surface to form a surface carbon radical for further initiating radical graft polymerization of vinyl monomers.27 This unique photoinitiator-free graft polymerization50,51 excludes interference from common issues in the conventional graft polymerization process, for example, the stability and toxicity of a graft layer containing residual initiators/sensitizers/catalysts/ligands, and complicated multistep procedures for prefunctionalization/prepriming on a target polymer substrate. Particularly, with regards to the construction of an antifouling polymer coating on the organic material surface for biomedical application, recently developed methods to achieve this aim often involve the use of atom transfer radical polymerization (ATRP),10−12,52 or a dihydroxyphenylalanine (DOPA)-mediated coating,53 etc. In contrast to these methods, we believe the present photoinitiator-free photografting 9405



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Figure 5. Effect of DMF on the photografting polymerization of POEGMA on PET surface. (a) 3D optical image of the irradiated sample surface by only using the bulk OEGMA without the DMF added as the reaction solution; (b) 3D optical image of the irradiated sample surface by using the mixture of DMF and OEGMA as the reaction solution.

Figure 6. AFM scanning on the micropatterned POEGMA-grafted PET surface: (a) micropatterned surface scanning on the sample with the irradiation time being 8 min; (b) regional scanning on the graft polymer area to study the surface topography of the graft layer; (c) thickness change of POEGMA grafting layer on PET surface with different irradiation time. The thickness data were directly read out by AFM profile scanning on the micropatterned POEGMA-grafted PET substrate (the inset in panel a).

S5 and S6, the ATR-FTIR and XPS characterizations on the grafted surface precisely revealed the desired chemical structure of graft polymer chains without the notable appearance of side reaction products. This is because (1) the polymerization speed in our method was relatively fast (∼8 min) so that the degradation of the monomer and substrate was not obvious (also vide infra, in the gravimetric analysis part) and (2) the confined polymerization microenvironment from the sandwich setup used in this method suppressed to some extent the occurrence of side reactions.27 Accordingly, with these features included, the conventional shortages in radical polymerization may not be a big problem in the present method. Similar to the modulation ability of conventional methods on the morphology of a graft polymer layer, the DMF-initiated photografting method could also impact good control on the morphology of a graft polymer layer. In general, surfaceinitiated photografting polymerization is often used to afford a high density and probably even photo-cross-linked polymer graft layer.54 As shown in Figure 6, the graft layer thickness obtained by this method could be conveniently controlled in the range of nanometers by simply tuning the irradiation time to provide an ultrathin polymer film, and on the grafted sample surface, a flat topography with low roughness (RMS 1.66 nm) was acquired. The dense, homogeneous, and flat graft layer observed in Figure 6 implied that the polymer grafting density on the modified substrate was high enough to cover fully the underlying polymeric substrate so that the resultant surface property could be majorly determined by the top polymer graft layer, which ensured the acquisition of robust nonfouling behavior from the modified surface. The change of graft layer thickness presented a slow growing at the initial stage, which might be attributed to the inhibition effect of oxygen dissolved

polymerization could provide a much simpler but similarly powerful protocol to prepare a well-defined antifouling polymer coating on an arbitrary organic substrate with its intrinsic advantage of having the capability of direct photoinduced micropatterning on polymer surfaces without the use of complex photolithography procedures.36 By using OEGMA as a representative monomer,10,11 the first evidence for successful POEGMA grafting was provided by ATR-FTIR and XPS spectra characterizations. It was clearly revealed that after applying a DMF solution of OEGMA onto a BOPP surface under UV irradiation, the characteristic peaks for the ester bond of the POEGMA polymer chain were found in the ATR-FTIR spectra (Supporting Information, Figure S5). With prolonged irradiation time, the peak intensity of ester bond (CO and C−O−C) in ATR-FTIR increased gradually, indicating the reaction time-controlled growth of graft chains. The XPS spectra on the POEGMA-grafted BOPP surface (Supporting Information, Figure S6a,b) further supported the successful grafting, presenting a substantial increase of surface oxygen atomic concentration. The deconvoluted C 1s and O 1s (Supporting Information, Figure S6 panels c and d, respectively) could be assigned to the characteristic structure of ester bonds contained in POEGMA. It was further checked by a control experiment that such photografting strongly relied on the photosensitivity of DMF, because bulk photografting polymerization of OEGMA monomers without DMF added could not produce an effective graft polymer layer and patterning (Figure 5), but the same mixture with DMF added produced a patterned polymer graft layer with high quality. This sharp difference reflected the possibility that the selfinitiating graft polymerization of the monomers50,51 could be excluded. As also reflected in Supporting Information, Figures 9406



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Figure 7. Optical images of the patterned antifouling polymer-grafted BOPP surface before and after HRP immobilization. (a) Optical image of POEGMA micropatterns on BOPP surface. (b) Optical image of the BOPP substrate with patterned POEGMA graft layer after the incubation in HRP solution and sequentially AEC staining. (c and d) Optical images of the BOPP substrate with patterned poly(HEMA) (c) and poly(CBAA) (d) graft layer after the incubation in HRP solution and sequential AEC staining. For comparison, a 3D optical image of the poly(HEMA) micropatterns grafted on BOPP surface before HRP immobilization was shown in the inset of panel c.

Figure 8. Cy5-SAv adsorption on the POEGMA-grafted PET surface. (a) Optical image of the surface with patterned POEGMA graft layer before the adsorption; (b) corresponding self-fluorescent image of panel a. (c) Fluorescent image of the surface with patterned POEGMA graft layer after incubation in Cy5-SAv solution; (d) corresponding line profile of gray value in panel c. (e) Overlay image of panels a and c. In the overlay image (e), the observed red spots within the POEGMA grafted array were not from the nonspecific adsorption of Cy5-SAv, but from the self-fluorescence of UV-exposed substrate itself, as already demonstrated in panel b. This kind of self-fluorescent pattern from the grafted substrate (b) had low contrast inside and outside the circular POEGMA graft regions, which was largely enhanced after incubation in the Cy5-SAv solution (c) due to the adsorption of Cy5-SAv outside the circles (POEGMA graft layer).

in the solution on the radical graft polymerization. Because of side reactions, for example, photodegradation induced by longterm irradiation, a longer exposure time than 8 min resulted in a decrease of graft layer thickness. This process can be further supported by the following graft polymerization kinetics analysis. For this aim, the mass increase after photografting on a polymer substrate could be followed to monitor the

photografting process in a quantitative way. The corresponding photografting parameters based on the mass change such as grafting percentage (GP), grafting efficiency (GE), and monomer conversion of polymerization (CP), as well as monomer conversion of grafting (CG), could be simply controlled and adjusted by formulating the photografting conditions, such as UV irradiation time and the ratio of DMF 9407



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multiple proteins by using a more stringent practical biological mixture. For example, as shown in Supporting Information, Figure S9, the XPS measurements on the pristine PET and POEGMA-grafted PET surfaces after immersion in undiluted serum clearly demonstrated that excellent nonfouling behaviors were obtained on the POEGMA-grafted surface, as reflected by comparing that the N 1s signal from the protein molecules was observed on the naked PET surface without POEGMA grafts, but did not appear on the POEGMA-grafted PET surface.

to monomer added in the system (Supporting Information, Figure S7). Generally, all four parameters increased with prolonged irradiation time, and the optimized grafting conditions could be determined as the irradiation time being 8 min and the ratio of DMF to OEGMA being the lowest (0.5:4.5) of all four experimental entries. Under the optimized condition, the GP could reach 8% with GE, CP, and CG approaching 45%, 65%, and 52%, respectively. Irradiation times longer than 8 min did not result in a significantly increased GP and GE, because as found in previous work,27 long irradiation times may facilitate the occurrence of some side reactions that would deteriorate the grafted polymer chains. This judgment was also supported by a serious aging phenomenon observed on the film after 16 min irradiation. Regarding the ratio of DMF to OEGMA monomer, obviously a low content of DMF was better for the grafting polymerization because, similar to that of a general polymerization law, a high concentration of the initiators (i.e., DMF in present work) would lead to the quick formation of a large number of primary radicals at the early stage of polymerization, which consequently induced side reactions to influence the final grafting. By similar characterization methods, we further demonstrated a series of classical functional antifouling polymers; for example, poly(CBAA), 15−18 poly(HEMA), 7 and poly(HPMA),7 could be also effectively photografted on polymer surface, and the grafting reaction parameters for each polymer have been depicted (Supporting Information, Figure S8). The resulting bioinert antifouling substrate was eventually evaluated by the resistance test toward protein nonspecific adsorption. As a representative example, the micropatterned antifouling polymers including POEGMA, poly(HEMA), and poly(CBAA)-grafted BOPP substrate was incubated in HRP solution. After AEC staining, it was clearly seen that HRP only adsorbed nonspecifically onto the unexposed hydrophobic background to show the red color of the biochemical reaction product between AEC and HRP outside the circular antifouling graft polymer regions (Figure 7). In another fluorescent method, by incubating micropatterned POEGMA grafted polymer substrate (Figure 8a) in Cy5-SAv solution, it was easy to find that the bright red fluorescent patterns were selectively retained on the surrounding background circular POEGMA regions (Figure 8c,d), presenting an excellent protein-repelling behavior from the POEGMA graft layer. Similar to that in the aforementioned discussions, a fluorescent background from the circular POEGMA-grafted regions was detected (Figure 8b) and from the self-fluorescence of PET substrate itself owing to the existence of some photoactive additives (e.g., photostabilizers) in the commercial polymeric films.47,48 Moreover, the overlay image (Figure 8e) of the optical image of the patterned graft layer before the incubation in the protein solution and the fluorescent image of the patterned graft layer after the incubation in the protein solution further showed that the regular POEGMA pattern could accurately repel the nonspecific adsorption of Cy5-SAv with microscale-precision. The reason why the Cy5 fluorophore was used to replace the FITC used in the first section 2.1 was that first Cy5 possessed more stable photostability and brighter fluorescent signal than FITC, and second the self-fluorescence of PET substrate under the excitation wavelength (i.e., 633 nm) used for Cy5 was much weaker than that under the excitation wavelength (i.e., blue light excitation) used for FITC (data not shown). Besides testing a single protein in buffer solution, we further tested the resist ability toward nonspecific adsorption of

3. CONCLUSIONS We have developed a set of novel amides-based photochemical methods that could provide bifunctions for the preparation of both bioactive and bioinert surfaces. The resultant bioactive surface is a type of active aminated polymer surface that could directly conjugate with target molecules through the bioconjugation reactions of primary amine groups. By solely using a DMF solution of functional monomers, bioinert antifouling polymers, such as POEGMA, poly(HEMA), poly(HPMA), and poly(CBAA) could be simply but effectively grafted onto an arbitrary polymeric substrate to afford a fast conversion from a biofouling to an antifouling surface on an organic polymeric material. We expect that the features of this method, such as simplicity, effectiveness, efficiency, generality, and fastness, finally lead to the development of a low-cost practical strategy for industrial and benchtop applications. The major limitation in the present work was the issue of self-fluorescence from the UV-exposed polymeric substrate, which elevated the background signal under a fluorescent microscope. Since this kind of background signal was from the commercial substrate itself, a further improvement on this method could be expected by replacing the commercial substrate with a synthesized pure organic layer/film without any extra additives. As the present method has demonstrated that the mere precondition for such an organic basement is its abundance of saturated C−H bonds for amination and grafting polymerization (as outlined in Scheme 1), the candidates suitable for such a bifunctional modification are very flexible. Accordingly, our next target is to implant such a method onto a variety of C−H enriched organic molecular thin films including self-assembling monolayers (SAMs), chemical vapor deposited (CVD) organic thin film, spin-/dip-deposited organic coating, and biomolecular thin film (e.g., lipid/peptide/protein films). Even the potential for performing this modification directly on inorganic substrates such as glass, quartz, and C−H enriched diamond surface desrves to be explored, because recent studies have shown that the surface reactivity on these inorganic substrates is able to undergo similar UV-initiated surface photochemical modification.55,56

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ASSOCIATED CONTENT

S Supporting Information *

Full experimental details and characterization methods, additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9408



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(19) Li, C.; Glidle, A.; Yuan, X.; Hu, Z.; Pulleine, E.; Cooper, J.; Yang, W.; Yin, H. Creating “Living” Polymer Surfaces to Pattern Biomolecules and Cells on Common Plastics. Biomacromolecules 2013, 14, 1278−1286. (20) Siow, K. S.; Britcher, L.; Kumar, S.; Griesser, H. J. Plasma Methods for the Generation of Chemically Reactive Surfaces for Biomolecule Immobilization and Cell ColonizationA Review. Plasma Process. Polym. 2006, 3, 392−418. (21) Kang, S. M.; Hwang, N. S.; Yeom, J.; Park, S. Y.; Messersmith, P. B.; Choi, I. S.; Langer, R.; Anderson, D. G.; Lee, H. One-Step Multipurpose Surface Functionalization by Adhesive Catecholamine. Adv. Funct. Mater. 2012, 22, 2949−2955. (22) Yang, P.; Zhang, X. X.; Yang, B.; Zhao, H. C.; Chen, J. C.; Yang, W. T. Facile Preparation of a Patterned, Aminated Polymer Surface by UV-Light-Induced Surface Aminolysis. Adv. Funct. Mater. 2005, 15, 1415−1425. (23) Hermanson, G. T. Bioconjugation Chemistry, 2nd ed.; Elsevier, Academic Press: New York, 2008. (24) Griffin, M.; Casadio, R.; Bergamini, C. M. Transglutaminases: Nature’s Biological Glues. Biochem. J. 2002, 368, 377−396. (25) Josten, A.; Meusel, M.; Spener, F.; Haalck, L. Enzyme Immobilization via Microbial Transglutaminase: A Method for the Generation of Stable Sensing Surfaces. J. Mol. Catal. B: Enzym. 1999, 7, 57−66. (26) Yang, P.; Yang, W. Hydroxylation of Organic Polymer Surface: Method and Application. ACS Appl. Mater. Interfaces 2014, 6, 3759− 3770. (27) Yang, P.; Deng, J.; Yang, W. Surface Photografting Polymerization of Methyl Methacrylate in N,N-Dimethylformamide on Low Density Polyethylene Film. Macromol. Chem. Phys. 2004, 205, 1096− 1102. (28) Rück-Braun, K.; Petersen, M. Å.; Michalik, F.; Hebert, A.; Przyrembel, D.; Weber, C.; Ahmed, S. A.; Kowarik, S.; Weinelt, M. Formation of Carboxy- and Amide-Terminated Alkyl Monolayers on Silicon(111) Investigated by ATR-FTIR, XPS, and X-ray Scattering: Construction of Photoswitchable Surfaces. Langmuir 2013, 29, 11758−11769. (29) Jackson, M.; Mantsch, H. H. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit. Rev. Biochem. Mol. Biology 1995, 30, 95−120. (30) Gabriel, G.; Sauthier, G.; Fraxedas, J.; Moreno-Mañas, M.; Martínez, M. T.; Miravitlles, C.; Casabo, J. Preparation and Characterization of Single-Walled Carbon Nanotubes Functionalised with Amines. Carbon 2006, 44, 1891−1897. (31) Jansen, R. J. J.; van Bekkum, H. XPS of Nitrogen-Containing Functional Groups on Activated Carbon. Carbon 1995, 33, 1021− 1027. (32) Xu, G.; Hibino, Y.; Aoki, M.; Awazu, K.; Tanihara, M.; Imanishi, Y. Chemical Changes of Polymeric Surfaces after Modification with Multi-Source Cluster Deposition. Colloids Surf., B 2000, 19, 249−256. (33) Kónya, Z.; Vesselenyi, I.; Niesz, K.; Kukovecz, A.; Demortier, A.; Fonseca, A.; Delhalle, J.; Mekhalif, Z.; Nagy, J. B.; Koós, A. A.; Osváth, Z.; Kocsonya, A.; Biró, L. P.; Kiricsi, I. Large Scale Production of Short Functionalized Carbon Nanotubes. Chem. Phys. Lett. 2002, 360, 429− 435. (34) Vesel, A.; Mozetic, M. Modification of PET Surface by Nitrogen Plasma Treatment. J. Phys.: Conference Ser. 2008, 100, 012027:1−4. (35) Vesel, A.; Mozetic, M.; Zalar, A. XPS Study of Oxygen Plasma Activated PET. Vacuum 2008, 82, 248−251. (36) Hucknall, A.; Simnick, A. J.; Hill, R. T.; Chilkoti, A.; Garcia, A.; Johannes, M. S.; Clark, R. L.; Zauscher, S.; Ratner, B. D. Versatile Synthesis and Micropatterning of Nonfouling Polymer Brushes on the Wafer Scale. Biointerphases 2009, 4, FA50−FA55. (37) Soto-Cantu, E.; Cueto, R.; Koch, J.; Russo, P. S. Synthesis and Rapid Characterization of Amine-Functionalized Silica. Langmuir 2012, 28, 5562−5569. (38) Zhu, Y.; Gao, C. Y.; Liu, X.; Shen, J. C. Surface Modification of Polycaprolactone Membrane via Aminolysis and Biomacromolecule

ACKNOWLEDGMENTS P.Y. thanks the following for his financial support: National Natural Science Foundation of China (NSFC) for Grant 21374057 and Grant 51303100; the National Science Changjiang Scholars and Innovative Research Team in University (IRT 1070), the Fundamental Research Funds for the Central Universities (GK201301006), the Start-up Funding from Shaanxi Normal University and “100 Talents Program” from Shaanxi Province (801045).

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REFERENCES

(1) Börner, H. G., Lutz, J.-F. Eds. Bioactive Surfaces. In Advanced Polymer Science; Vol. 240, Springer: Berlin, Germany, 2011. (2) Wischerhoff, E.; Badi, N.; Lutz, J.-F.; Laschewsky, A. Smart Bioactive Surfaces. Soft Matter 2010, 6, 705−713. (3) Goddard, J. M.; Hotchkiss, J. H. Polymer Surface Modification for the Attachment of Bioactive Compounds. Prog. Polym. Sci. 2007, 32, 698−725. (4) Yu, Q.; Zhang, Y.; Wang, H.; Brash, J.; Chen, H. Anti-fouling Bioactive Surfaces. Acta Biomater. 2011, 7, 1550−1557. (5) Xu, F. J.; Neoh, E. G.; Kang, E. T. Bioactive Surfaces and Biomaterials via Atom Transfer Radical Polymerization. Prog. Polym. Sci. 2009, 34, 719−761. (6) Hucknall, A.; Rangarajan, S.; Chilkoti, A. In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins. Adv. Mater. 2009, 21, 2441−2446. (7) Rodriguez-Emmenegger, C.; Houska, M.; Alles, A. B.; Brynda, E. Surfaces Resistant to Fouling from Biological Fluids: Towards Bioactive Surfaces for Real Applications. Macromol. Biosci. 2012, 12, 1413−1422. (8) Matsumoto; Fukuda, T.; Miura, Y. Bioinert Surface to Protein Adsorption with Higher Generation of Dendrimer SAMs. Colloids Surf. B Biointerf. 2011, 84, 280−284. (9) Hou, S.; Burton, E. A.; Wu, R. L.; Luk, Y.-Y.; Ren, D. Prolonged Control of Patterned Biofilm Formation by Bio-inert Surface Chemistry. Chem. Commun. 2009, 10, 1207−1209. (10) Ma, H.; Hyun, J.; Chilkoti, A. “Non-Fouling” Oligo(ethylene glycol)-Functionalized Polymer Brushes Synthesized by SurfaceInitiated Atom Transfer Radical Polymerization. Adv. Mater. 2004, 16, 338−341. (11) Ma, H.; Li, D. J.; Sheng, X.; Zhao, B.; Chilkoti, A. ProteinResistant Polymer Coatings on Silicon Oxide by Surface-Initiated Atom Transfer Radical Plymerization. Langmuir 2006, 22, 3751−3756. (12) Vaisocherová, H.; Yang, W.; Zhang, Z.; Cao, Z.; Cheng, G.; Piliarik, M.; Homola, J.; Jiang, S. Ultralow Fouling and Functionalizable Surface Chemistry Based on a Zwitterionic Polymer Enabling Sensitive and Specific Protein Detection in Undiluted Blood Plasma. Anal. Chem. 2008, 80, 7894−7901. (13) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study. Anal. Chem. 1999, 71, 777−790. (14) Frey, B. L.; Corn, R. M. Covalent Attachment and Derivatization of Poly(L-lysine) Monolayers on Gold Surfaces as Characterized by Polarization-Modulation FT-IR Spectroscopy. Anal. Chem. 1996, 68, 3187−3193. (15) White, A. D.; Nowinski, A. K.; Huang, W.; Keefe, A. J.; Sun, F.; Jiang, S. Decoding Nonspecific Interactions from Nature. Chem. Sci. 2012, 3, 3488−3494. (16) Cao, Z. Z.; Brault, N.; Xue, H.; Keefe, A.; Jiang, S. Manipulating Sticky and Non-sticky Properties in a Single Material. Angew. Chem., Int. Ed. 2011, 50, 6102−6104. (17) Chen, S.; Jiang, S. An New Avenue to Nonfouling Materials. Adv. Mater. 2008, 20, 335−338. (18) Keefe, A. J.; Jiang, S. Poly(zwitterionic) Protein Conjugates Offer Increased Stability without Sacrificing Binding Affinity or Bioactivity. Nat. Chem. 2012, 4, 59−63. 9409



Article

Immobilization for Promoting Cytocompatibility of Human Endothelial Cells. Biomacromolecules 2002, 3, 1312−1319. (39) Bùi, L. N.; Thompson, M.; Mckeown, N. B.; Romaschin, A. D.; Kalman, P. G. Surface Modification of the Biomedical Polymer Poly(ethylene terephthalate). Analyst. 1993, 118, 463−474. (40) Gan, S.; Yang, P.; Yang, W. Photoactivation of Alkyl C−H and Silanization: A Simple and General Route to Prepare High-Density Primary Amines on Inert Polymer Surfaces for Protein Immobilization. Biomacromolecules 2009, 10, 1238−1243. (41) Wine, Y.; Cohen-Hadar, N.; Freeman, A.; Frolow, F. Elucidation of the Mechanism and End Products of Glutaraldehyde Crosslinking Reaction by X-ray Structure Analysis. Biotechnol. Bioeng. 2007, 98, 711−718. (42) Batalla, P.; Fuentes, M.; Mateo, C.; Grazu, V.; FernándezLafuente, R.; Guisán, J. M. Covalent Immobilization of Antibodies on Finally Inert Support Surfaces through their Surface Regions Having the Highest Densities in Carboxyl Groups. Biomacromolecules 2008, 9, 2230−2236. (43) Lorente, G. F.; Palomo, J. M.; Mateo, C.; Munilla, R.; Ortiz, C.; Cabrera, Z.; Guisán, J. M.; Fernández-Lafuente, R. Glutaraldehyde Cross-Linking of Lipases Adsorbed on Aminated Supports in the Presence of Detergents Leads to Improved Performance. Biomacromolecules 2006, 7, 2610−2615. (44) López-Gallego, F.; Betancor, L.; Mateo, C.; Hidalgo, A.; AlonsoMorales, N.; Dellamora-Ortiz, G.; Guisán, J. M.; Fernández-Lafuente, R. J. Enzyme Stabilization by Glutaraldehyde Crosslinking of Adsorbed Proteins on Aminated Supports. Biotechnol. 2005, 119, 70−75. (45) Spitznagel, T. M.; Jacobs, J. W.; Clark, D. S. Random and SiteSpecific Immobilization of Catalytic Antibodies. Enzyme Microb. Technol. 1993, 15, 916−921. (46) Walt, D. R.; Agayn, V. I. The Chemistry of Enzyme and Protein Immobilization with Glutaraldehyde. Trends Anal. Chem. 1994, 13, 425−430. (47) Schwarz, A.; Rossier, J. S.; Roulet, E.; Mermod, N.; Roberts, M. A.; Girault, H. H. Micropatterning of Biomolecules on Polymer Substrates. Langmuir 1998, 14, 5526−5531. (48) Rossier, J. S.; Bercier, P.; Schwarz, A.; Loridant, S.; Girault, H. H. Topography, Crystallinity and Wettability of Photoablated PET Surfaces. Langmuir 1999, 15, 5173−5178. (49) Renshaw, S. Ed. Immunohistochemistry: Methods Express; Scion Publishing Ltd: Bloxham, UK, 2007. (50) Deng, J. P.; Yang, W. Auto-initiating Performance of Styrene on Surface Photografting Polymerization. Macromol. Rapid Commun. 2001, 22, 535−538. (51) Deng, J. P.; Yang, W. Self-Initiating Performance of Maleic Anhydride on Surface Photografting Polymerization. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 3246−3249. (52) Jiang, H.; Xu, F.-J. Biomolecule-Functionalized Polymer Brushes. Chem. Soc. Rev. 2013, 42, 3394−3426. (53) Lee, B. P.; Messersmith, P. B.; Israelachvili, J. N.; Waite, J. H. Mussel-Inspired Adhesives and Coatings. Annu. Rev. Mater. Res. 2011, 41, 99−132. (54) For a review, see: Yang, P.; Yang, W. Surface Chemoselective Phototransformation of C−H Bonds on Organic Polymeric Materials and Related High-Tech Applications. Chem. Rev. 2013, 113, 5547− 5594. (55) Cao, X.; Zhang, T.; Deng, J.; Jiang, L.; Yang, W. An Extremely Simple and Effective Strategy to Tailor the Surface Performance of Inorganic Substrates by Two New Photochemical Reactions. ACS Appl. Mater. Interfaces 2013, 5, 494−499. (56) Zhong, Y. L.; Loh, K. P. The Chemistry of C−H Bond Activation on Diamond. Chem.Asian J. 2010, 5, 1532−1540.

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