Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes

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Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality Zhen Lei,†,‡ Jiaxue Gao,†,‡ Xia Liu,† Dianjun Liu,† and Zhenxin Wang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China. ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2hydroxyethyl methacrylate (HEMA) monomers via surfaceinitiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMAHEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL−1 for MMP-2 and 5.7 pg mL−1 for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained. KEYWORDS: polymer-brush substrate, poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) P(GMA-HEMA), protein microarray, peptide microarray, binding of human IgG with rabbit antihuman IgG antibody, matrix metalloproteinase

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INTRODUCTION

Currently, the commonly used microarray substrates consist of 2D planar glass slides that are modified by amine,7 aldehyde,8 epoxide,7 or poly-L-lysine9 to produce a monolayer with reactive groups. The reactive groups (active sites) on the 2D glass slide are strongly restricted by the limited surface area of the slide, and the immobilized probes may lie flat on the surface, resulting in poor accessibility to the targeted analytes.10 In comparison to the 2D surfaces, the 3D substrates exhibit higher binding capacity and simultaneously provide more space between immobilized biomolecules, which makes the interaction of the immobilized probes with their target in solution more free.6,11 Various 3D substrates have been used to fabricate microarrays including porous nitrocellulose membrane,12 hydrogel-based microstructures,13 and porous silicon (PSi) patterns.14 Polymeric coating is a simple method for preparing 3D microarray substrates.6 Among these coatings, polymer brushes are attractive 3D substrates because the polymer brushes have several advantages including facile in situ

Microarrays have been employed as powerful tools with extensive applications in biomedical and bioanalytical fields because microarrays allow high-throughput analysis of a very great deal of different analytes at the same time and on a small scale.1−3 With the tremendous success of DNA microarrays, protein/peptide microarrays used for the quantification of proteins as well as investigation of their functions and interactions have received much attention in recent years.2−5 However, the sensitivity of a protein/peptide microarray still limits their broad application due to the lack of efficient amplification methods like polymerase chain reaction (PCR) applied in DNA microarrays and high nonspecific protein adsorption. The amount of immobilized biomolecules within a single spot typically determines the analytical performance (e.g., sensitivity, dynamic range, and analysis speed) of microarraybased assays.6 Therefore, the efficient immobilization of protein or peptide probes on a nonfouling substrate with high loading capacity and good accessibility to analytes is an alternative strategy for improving the sensitivity of microarray-based assays. © XXXX American Chemical Society

Received: January 28, 2016 Accepted: April 6, 2016

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DOI: 10.1021/acsami.6b01156 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Preparation of P(GMA-HEMA) brushes via SI-ATRP on a glass slide and fabrication of protein and/or peptide microarray on the P(GMAHEMA) brush substrate.

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synthesis, high biomolecule immobilization capacity, and accessible scaffolds with sufficient space for biomolecules binding.15−18 For instance, aldehyde-functionalized poly(2hydroxyethyl methacrylate) (PHEMA) brushes were employed as platforms for the immobilization and hybridization of oligonucleotides.19 Then PHEMA brush nanocone arrays with various morphologies and periods were prepared as substrates for DNA detection, which are rapid, visible, and multiplex.20 However, both the works require subsequent functionalization after polymerization. Hu et al. proposed an antigen microarraybased competitive mycotoxins immunoassay on a POEGMAco-GMA brush substrate. The polymer brushes were synthesized by copolymerization of oligo(ethylene glycol) methacrylate (OEGMA) and GMA via SI-ATRP. Here, the antibodies were directly covalently immobilized on the brushes through the anchoring site of GMA, and the nonspecific protein adsorption can be suppressed due to OEGMA.21 Klok et al. prepared PGMA-co-PDEAEMA copolymer brushes on a Ta2O5 optical waveguide chip via SI-ATRP for use as a substrate in protein microarray. In comparison to PGMA brushes, the incorporation of 2-(diethylamino)ethyl methacrylate (DEAEMA) in the brushes can accelerate the immobilization of protein and increase protein binding.22 However, few examples of the preparation of polymer brush-based peptide microarrays have been reported. As protein microarray analogue, peptide microarrays have been widely used for studying enzyme functionality and inhibition, drugs screening, and cell−cell communication.5,23,24 In this study, a 3D P(GMA-HEMA) brush substrate was prepared for fabricating protein and peptide microarrays that could be applied for detection of protein and evaluation of enzyme activity. The polymer brush-grafted substrate exhibited relatively higher protein and peptide immobilization capacities than those of a conventional 2D substrate. In particular, the P(GMA-HEMA) surface can effectively resist adsorption of proteins and provide good accessibility to the analytes. In two different proof-of-principle experiments, the results demonstrated that the P(GMA-HEMA)-based microarrays were able to sensitively and selectively detect rabbit antihuman IgG antibodies and assay activities of matrix metalloproteinases.

EXPERIMENTAL SECTION

Reagents. Glycidyl methacrylate (GMA, ≥97%) and 2-hydroxyethyl methacrylate (HEMA, 97%) were received from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Triethylamine (TEA, >99.0%) and 2-bromoisobutyryl bromide (BIB, >98.0%) were purchased from TCI Co., Ltd. (Shanghai, China). (3-Aminopropyl)triethoxysilane (APTES, 98%), copper(I) bromide (CuBr, 99.0%), and 2,2′-bipyridyl (bipy, ≥ 99.0%) were purchased from Aladdin Co., Ltd. (Shanghai, China). Human IgG, rabbit antihuman IgG/Cy3, and avidin-Cy3 were received from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The peptide substrates (i.e., CAEGFFSARGHRPLK (biotin), named S0; CGGKGPRSLSGK (biotin), named S1; CGGKGPLGVRGAK (biotin), named S3; and CGGRGDSP) were ordered from ChinaPeptides Ltd. (Shanghai, China). The latent formats of recombinant human matrix metalloproteinases (proMMP-2 and proMMP-9) were obtained from Sino Biological Inc. (Beijing, China). 4-Aminophenyl mercuric acetate (APMA) was supplied by GenMed Medical Science and Technology Ltd. (Shanghai, China), and bovine serum albumin (BSA) was provided by GEN-VIEW Scientific Inc. (USA). Phorbol12-myristate-13-acetate (PMA) was offered from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The microscope slides, commercial 2D epoxy slides and polytetrafluoroethylene (PTFE) grids were obtained from CapitalBio Ltd. (Beijing, China). All of the other chemicals were analytical grade, and all aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm). Characterization. The attenuated total reflection−Fourier transform infrared spectroscopy (ATR-FTIR) of obtained polymer brushes were conducted with a Bruker Vertex 70 FTIR spectrometer equipped with an ATR unit at a resolution of 4 cm−1 with 32 scans (Bruker Co., Germany). X-ray photoelectron spectra (XPS) were recorded on a VG ESCALAB MKII spectrometer (VG Scientific Ltd., UK). The morphologies of the substrate surfaces were obtained by a Multimode 8 atomic force microscopy (AFM) system (Veeco Co., USA). The water contact angles (WCAs) of the slides were obtained with a contact angle goniometer (Krü ss GmbH, Germany) at room temperature. The thickness of the polymer brushes was determined by spectroscopic ellipsometry (UVISEL, Horiba Jobin Yvon). Preparation of Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) P(GMA-HEMA) Brushes on a Glass Slide. The synthesis procedure for the P(GMA-HEMA) brushes on the glass slide was based on the literature methods with a slight modification (shown in Figure 1).17,25 First, the plain microscope glass slides (7.5 cm × 2.5 cm × 0.1 cm) were immersed into 3 M potassium hydroxide (KOH) solution at ambient temperature for 2 h to introduce hydroxyl groups. After being washed with water and dried by centrifugation, the glass B


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ACS Applied Materials & Interfaces slides were further activated using a piranha solution (H2SO4 (98 wt %)/H2O2 (30 wt % in H2O) = 3:1 (v:v)) for an additional 1 h. After washing and drying, the activated slides were soaked in 5% (v/v) APTES in ethanol for 1 h. After the thorough washing with ethanol and water, the silanized slides were annealed under vacuum condition at 120 °C for 2 h. Subsequently, the amino-modified slides were immersed into dichloromethane (CH2Cl2) containing 1% (v/v) BIB and 1% (v/v) TEA at 0 °C for 15 min and then at 25 °C for another 2 h. For preparing the SI-ATRP growth solution, 30 mL of methanol/ H2O (50% (v/v)) containing GMA (1%, v/v) and HEMA (10%, v/v) were mixed with 150 mg of CuBr and 312 mg of bipy under ultrasonication. After being degassed with pure Ar for 40 min, the SIATRP growth solution was applied to three initiator-modified slides in a sealed box and allowed to react at 30 °C for 9 h. Finally, the functionalized slides were rinsed thoroughly with ethanol and water, dried by centrifugation, and stored at 4 °C for further use. The asprepared P(GMA-HEMA) brush modified slide is referred to as the P(GMA-HEMA) brush substrate. Protein/peptide Microarray Fabrication and Application. According to our previous studies, proteins and peptides were spotted on the P(GMA-HEMA) brush substrates and commercial 2D epoxy glass slides with a SmartArrayer 136 system using a standard contact printing procedure (see the Supporting Information for details), respectively.26,27 To study protein binding, various concentrations of rabbit antihuman IgG/Cy3 in phosphate buffered saline (PBS (pH 7.5, 50 mM PB, 0.15 M NaCl)) supplemented with 1% (w/v) BSA were incubated with the human IgG microarrays at 37 °C for 1 h. Then the microarrays were washed with PBS containing 0.05% (v/v) Tween-20 for 5 min (3 times), PBS for 5 min (3 times), and water for 3 min (3 times), followed by drying via centrifugation. For assaying the enzyme activities, the interactions of MMPs (pure MMPs or cell-secreted MMPs) with peptide substrates on the microarrays were evaluated according to our previously reported assay conditions with a slight modification (see the Supporting Information for details).28 For the on-chip cultured cells, MDA-MB-231 cells were directly plated on the subarrays spotted with a mixture consisting of MMP peptide substrates and RGD-containing peptides with various densities, cultured, and treated as described in the off-chip experimental procedure (see the Supporting Information for details). The culture medium in the subarray was abandoned, and the cells were removed from the substrates by 0.02% ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) digestion. Subsequently, 50 nM avidin-Cy3 was applied to each subarray followed by the treatment described previously. Data Acquisition and Analysis. The fluorescence signals were acquired by scanning the microarrays with a LuxScan-10K fluorescence microarray scanner (CapitalBio Ltd., China) with a green channel. Prior to evaluation, the background signals stemming from the microarray were recorded and subtracted. Then, 6 (for pure protein and MMPs detection) or 10 (for on-chip cell-secreted MMPs detection) replicate spots per sample were used to calculate the average values and standard deviations. The relative change of fluorescence intensity (ΔF% = (F0 − F)/F0 × 100%) was employed to evaluate the MMP activities, where F and F0 are the average fluorescence intensities of 6 or 10 spots on the same microarray treated with and without MMP, respectively.

polymerization. HEMA is easily polymerized by ATRP, producing the corresponding polymer brushes that can effectively prevent nonspecific adsorption of biomolecules. In addition, these brushes exhibit good biocompatibility, making the polymer brushes useful for cell experiments. The successful stepwise surface modification of the glass slide can be monitored by the water contact angle (WCA). As shown in Figure S1a (Supporting Information), a relatively hydrophilic glass slide with a WCA of 35.9° was generated after cleaning with KOH and piranha solution. Upon aminosilanization, the WCA increased to 52.6° due to the hydrophobic sequence (propyl chain) of APTES. The successful immobilization of the initiator was based on the increase in the WCA to 76.6°. After the SI-ATRP of GMA and HEMA, the WCA decreased to 46.9°. This WCA falls in between that of the PGMA (57°)29 and PHEMA (37°)30 polymer brushes, indicating that the P(GMA-HEMA) brush coating provides a hydrophilic surface. The successful synthesis of P(GMA-HEMA) brushes was further confirmed by ATR-FTIR analysis of the silica particles grafted with the polymer brushes. The results in Figure S1b (Supporting Information) indicate that after APTES functionalization, the characteristic absorption peaks attributed to the C−H stretching vibrations of the alkyl group appeared at 2976 and 2898 cm−1. For the initiator (BIB) immobilization, the characteristic peaks corresponding to CO stretching at 1637 cm−1 (amide I) and N−H bending at 1535 cm−1 (amide II) were observed, demonstrating that the amide linkages were formed between the carbonyl groups of BIB and the terminal amino groups of APTES. In the final step, a sharp absorption band attributed to the CO stretching vibration in the ester of HEMA and GMA appeared at 1726 cm−1. This result indicates the successful growth of P(GMA-HEMA) brushes on the surface. The stepwise preparation of the polymer brushes was also confirmed by XPS analysis. As shown in Figure S2b (Supporting Information), after aminosilanization, a new peak at the binding energy of 400.0 eV was observed which was due to nitrogen. The high-resolution C1s peak of the APTESfunctionalized slide (Figure S2e (Supporting Information)) included a C−C peak at 284.9 eV and a C−N peak at 286.5 eV. For the initiator-immobilized slides, a new Br3d peak was observed at the binding energy of 71.2 eV, and the expected C−Br peak (286.9 eV) and CO moiety (288.2 eV) appeared in the high-resolution C1s peak, indicating the formation of initiator monolayer (Figures S2c, S2f (Supporting Information)). After the SI-ATRP growth of P(GMA-HEMA) brushes, the XPS survey spectrum demonstrated that bromine was still present on the surface (Figure S2d (Supporting Information)), which is consistent with the reported “living” reaction.29 The high-resolution C1s signal (Figure S2g (Supporting Information)) can be fitted with five peaks that represent five different carbon atoms of the P(GMA-HEMA) brushes as follows: the aliphatic carbon atoms (C−C/C−H) at 284.8 eV, the carbon atoms neighboring to the ester groups (C−CO) at 285.1 eV, the carbon atoms of C−O species at 286.2 eV, and those of the oxirane groups (C−O−C) at 286.8 eV and the carbonyl groups (CO) at 288.8 eV.30,31 In addition, the high-resolution O1s spectrum (Figure S2h (Supporting Information)) can be decomposed into four curves: the O−CO peak at 533.6 eV, the C−O−C peak at 533.2 eV, the C−O−H peak at 532.5 eV, and the O−CO peak at 531.8 eV. In addition, XPS analysis was also employed to determine the composition of the polymer brushes, which was estimated based on the fitted peak

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RESULTS AND DISCUSSION Preparation and Characterization of Polymer Brushes. As shown in Figure 1, the P(GMA-HEMA) brushes were coated on glass slides at 30 °C via SI-ATRP in the methanol/water mixture with CuBr (I) as the catalyst and 2,2′bipyridyl (bipy) as the ligand. GMA was chosen as the comonomer because the oxirane group of GMA can serve as an active site for covalent immobilization of biomolecules, resulting in simplification of the functionalization step after C


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Figure 2. Fluorescence intensities and images of microarrays as a function of the concentrations of anti-IgG-Cy3 (a) and peptide (b) in the spotting solution.

Figure 3. Dose-dependent responses for immunoassay (a) and MMP-9 cleavage (b) on P(GMA-HEMA) brush substrate and 2D epoxy slide. The concentrations of IgG and peptide S1 were both 0.1 mg mL−1 in the spotting solutions.

area ratio of [C−O−H]/[C−O−C].32 In the SI-ATRP growth solution, the monomers (i.e., HEMA and GMA) were added at a molar ratio (%) of 91.8:8.2. As expected, the surface composition (%) of the grafted brushes after the copolymerization was 89.2:10.8 (HEMA:GMA), suggesting that the composition of the polymer brushes was in good agreement with the monomer feed composition in solution. Furthermore, the surface of the polymer brush-grafted slide was characterized by AFM in tapping mode (as shown in Figure S3 (Supporting Information)). The surface roughness of the slide increased after the formation of the P(GMA-HEMA) brushes, and the polymer brushes film was uniform on the microscopic level.

The thickness of the grafted polymer brushes measured by an ellipsometer was ca. 23.6 nm. Loading Capacity of the P(GMA-HEMA) Brush Substrate. Because the oxirane groups of GMA residues can provide anchoring sites for covalent immobilization of biomolecules through the spontaneous reaction with amine groups under mild conditions, the P(GMA-HEMA) brush substrate can be used to immobilize proteins and peptides and fabricate the corresponding protein and peptide microarrays, respectively. The loading capacity of P(GMA-HEMA) brush substrate was evaluated by spotting different concentrations of rabbit antihuman IgG/Cy3 and peptide. Figure 2 shows the D


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was developed for the simultaneous detection of MMPs. MMPs are a family of endopeptidases that are capable of degrading a variety of extracellular matrix proteins,33 and their overexpression may be closely related with some pathological processes, such as arthritis, inflammation, especially tumor invasion, metastasis, and angiogenesis.33−36 As shown in Figure 1, the biotinylated peptide substrates were spotted on the P(GMA-HEMA) brush substrate, and these substrates can bind to avidin-Cy3, generating an intense fluorescence signal. After MMP cleavage, the peptide fragments with biotin moieties of corresponding peptide substrates were released from the surface, leading to decrease of the fluorescence signal. The phenomenon is dependent on the activities of MMPs, higher enzymatic activity results in lower fluorescence intensity. In this proof-of-principle experiment, the interactions of three peptide substrates (S0, S1, and S3) with two MMPs (MMP-2 and MMP-9) were studied. Here, S0 is a control peptide which is not cleaved by MMPs, S1 is a specific substrate of MMP-9, and S3 is a universal substrate of MMP-9 and MMP-2.28,37−39 The enzymatic cleavage reactions were carried out using our previously reported optimum experimental conditions except for the enzymatic cleavage time and peptide substrate concentrations in the spotting solutions.28 As expected, specific cleavage of peptide substrates by MMPs is clearly observed (as shown in Figure S6 (Supporting Information)). In particular, satisfactory ΔF% was obtained at 2 h activated MMPs cleavage with 0.2 mg mL−1 peptide substrates in the spotting solution (as shown in Figure S7 (Supporting Information)). The assay time and peptide substrate concentrations were less than those of our previously reported commercial aldehyde 3D peptide microarray-based fluorescence assay (4 h reaction time with 0.5 mg mL−1 S1 and 1.0 mg mL−1 S3 in spotting solution).28 Under the optimum conditions, various concentrations of activated MMP solutions were incubated with the microarray. As shown in Figure 4, the ΔF% was proportional to the logarithm of the MMP concentration. For single MMP detection, the dynamic ranges were 10 pg mL−1 to 5.0 ng mL−1 for MMP-9, and 10 pg mL−1 to 50 ng mL−1 for MMP-2, respectively. And the limits of detection were 6.2 pg mL−1 for MMP-9 and 8.0 pg mL−1 for MMP-2. For the simultaneous detection of MMPs in the enzyme mixture, the dynamic ranges were 10 pg mL−1 to 5.0 ng mL−1 for MMP-9 and 10 pg mL−1 to 10 ng mL−1 for MMP-2, and the limits of detection were 5.7 pg mL−1 for MMP-9 and 6.0 pg mL−1 for MMP-2, respectively. Furthermore, the sensitivities (the slopes of the lines) of the MMPs detection on the P(GMA-HEMA)-brush substrate were higher (2 times) than those of our previously reported results on aldehyde 3D substrate.28 In addition, the detection limits were even lower than those of previously reported methods, such as FRET, gel electrophoresis, and SPR, etc.40−45 The activities of cellularly secreted MMPs were also determined by the P(GMA-HEMA) brush substrate-based peptide microarray. A highly invasive breast carcinoma cell line, MDA-MB-231, was chosen as a model cell line due to its heavy secretion of MMP-2 and MMP-9.46,47 As a specific activator of the ERK pathway, PMA is used to stimulate the secretion of MMPs by the MDA-MB-231 cells.48 For the off-chip cultured cells, the peptide microarray was directly incubated with the collected conditioned medium because the MMPs are typically secreted out of the cells and released into the culture media. As shown in Figure 5a, ΔF% of substrate S1 and S3 are increased by increasing the PMA concentration in the cell culture medium, indicating that the cells secret high level of MMPs

covalent coupling of the protein and peptide on P(GMAHEMA) brush substrate compared to that on the commercial 2D epoxy slide. The fluorescence intensity increased as the concentration of rabbit antihuman IgG/Cy3 increased on both substrates (as shown in Figure 2a). However, the fluorescence intensity of P(GMA-HEMA) brush substrate was 2 times higher than that of the 2D slide. For peptide immobilization, as shown in Figure 2b, the saturated fluorescence intensity of the P(GMA-HEMA) brush substrate was also much higher than that of the 2D slide. The obtained results demonstrated that the P(GMA-HEMA) brush substrate has high binding capacity for proteins and peptides. The high binding capacity for biomolecules was due to the P(GMA-HEMA) brush substrate providing a 3D accessible scaffold. In addition, the oxirane groups of GMA along the backbone provided abundant binding sites for biomolecule attachment. Reaction Efficiencies of Biomolecules on the P(GMAHEMA) Brush Substrate. The reaction efficiencies of the immobilized proteins/peptides were investigated based on the immunoassay performance of immobilized human IgG with rabbit antihuman IgG/Cy3 and enzymatic cleavage of immobilized peptide S1 by MMP-9. As displayed in Figure 3a, the fluorescence intensity of the P(GMA-HEMA) brush substrate-based IgG microarray increased sharply as the concentration of the target protein increased. In the presence of 100 ng mL−1 rabbit antihuman IgG/Cy3, the fluorescence intensity of the P(GMA-HEMA) brush substrate-based IgG microarray was approximately five times higher than that of the 2D IgG microarray. The experimental results indicate that immobilized human IgG possesses better binding ability on the P(GMA-HEMA) brush substrate than that on the 2D slide. The excellent immunoassay performance of the P(GMAHEMA) brush substrate-based IgG microarray may attribute to the high protein loading capacity, good surface hydrophilicity (46.9° (P(GMA-HEMA) brush substrate) vs 73.1° (2D slide)), and minimized nonspecific protein adsorption (as shown in Figure S4 (Supporting Information)).11 Figure 3b shows that the cleavage efficiency of S1 by MMP-9 was as high as 53% on the P(GMA-HEMA) brush substrate and only 20% on the 2D slide. This result also indicates that the P(GMA-HEMA) brush substrate has higher reaction efficiency. In comparison to the 2D slide, the 3D brush-like accessible scaffold of the P(GMA-HEMA) brush substrate allows MMP-9 to easily penetrate into the gaps of the brush and hydrolyze the peptide. Stability of the P(GMA-HEMA) Brush Substrate. The long-term stability of the prepared polymer-brush substrate is of importance for its practical application. After stored at 4 °C for 3 months, the substrates were evaluated to determine the loading capacity of protein and peptide and the reaction efficiencies with the counterpart. As shown in Figure S5 (Supporting Information), no significant differences were observed between the performances of the stored and freshly prepared slides (used in Figures 2 and 3) with respect to both the loading capacity and reaction efficiencies. In comparison to the commercial aldehyde 3D slides, which have a shelf life of 6 months under vacuum according to the instructions, the prepared P(GMA-HEMA) brush-grafted slides are sufficiently stable for long-term use as substrates for microarrays. P(GMA-HEMA) Brush Substrate-Based Peptide Microarray for Simultaneous Detection of MMPs. To confirm the practical applicability of the as-prepared P(GMA-HEMA) brush substrate, a peptide microarray-based fluorescence assay E


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cellular secretion of MMPs can be inhibited at high cell densities.49 As expected, the cellular secretion levels of both MMP-2 and MMP-9 gradually increased as the cell density decreased, reached a maximum at 5000 cells/well and then decreased with decreasing cell density (as shown in Figure 5b). In addition, the cell-secreted MMPs can be determined as low as the cell density of 10 cells/well. In a final experiment, the secretion of MMP-2 and MMP-9 by MDA-MB-231 cells cultured on the P(GMA-HEMA) brush substrate was evaluated. As a hydrophilic and biocompatible polymer, PHEMA is commonly used as nonfouling surface to resist protein adsorption and cell adhesion.50 However, the oxirane groups of PGMA can be used as active sites for the immobilization of extracellular matrix proteins or RGD-based peptides to facilitate cell adhesion. In this case, RGD-containing peptide (CGGRGDSP) is used to improve the growth activity of cells because the cells cultured on the blank P(GMAHEMA) brush substrate exhibit relatively low viability and poor cell morphology (as shown in Figure S8 (Supporting Information). Considering the high peptide loading capacity of the P(GMA-HEMA) brush substrate, 0.2 mg mL−1 S1/S3 together with 2 mg mL −1 RGD-containing peptide (CGGRGDSP) are spotted on the slides to determine the cell-secreted MMP-2 and MMP-9 on chip. Figure 6a exhibits the relative activities of MMP-2 and MMP-9 upon PMA stimulation. The cellular secretion levels of two MMPs are increased with increasing concentration of PMA in the culture medium. In particular, compared to densely grown cells, the sparsely plated cells secrete high level of MMP-2 and MMP-9 with good proteolytic activities (Figure 6b). The phenomenon is consistent with that of off-chip cultured cells.

Figure 4. (a) Data analysis (ΔF% of substrate S1 cleaved by MMP-9, ΔF% of substrate S3 cleaved by MMP-2, and ΔF% of substrate S1 or S3 cleaved by MMPs mixture), and the corresponding fluorescence images (S1 cleaved by MMP-9 (b), S3 cleaved by MMP-2 (c), and S1 and S3 cleaved by MMPs mixture (d)).

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CONCLUSIONS Nonfouling P(GMA-HEMA) brushes were successfully prepared on a glass surface by copolymerizing GMA and HEMA monomers via SI-ATRP. In the brushes, PGMA provides abundant active sites for covalent immobilization of proteins or

after PMA stimulation. Furthermore, the effect of cell density on MMP-2 and MMP-9 secretion was investigated because

Figure 5. Data analysis (a,b) and the corresponding fluorescence images of subarrays (c,d) of the effects of PMA stimulation (a,c) and cell density (b,d) on the cellular secretion of MMPs. The MDA-MB-231 cells were cultured in a 48-well plate and the subarrays were incubated with cell conditioned medium. F


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Figure 6. Data analysis (a,b) and the corresponding fluorescence images of subarrays (c,d) of the effects of PMA stimulation (a,c) and cell density (b,d) on the cellular secretion of MMPs. The MDA-MB-231 cells were seeded on the P(GMA-HEMA) brush substrate spotted with S0/S1/S3 and RGD-containing peptide. The peptide substrates were directly cleaved by the cellularly secreted MMPs.

Notes

peptides, while PHEMA can effectively resist nonspecific protein adsorption and cell adhesion. The as-prepared P(GMA-HEMA) brushes have excellent stability and high loading capacity for proteins and peptides and were employed as substrate for fabricating protein and peptide microarrays. The results indicate that the as-fabricated protein/peptide microarrays exhibit reasonably high sensitivity for detecting protein binding and enzyme activity. After coimmobilization with a RGD-based short peptide, the P(GMA-HEMA) brush substrate-based peptide microarray facilitated cell adhesion and allowed the determination of the activities of MMP-2 and MMP-9 secreted by cells cultured on chip. Although the asprepared microarray substrates have been evaluated by two well-known biomolecular recognition systems as proof-ofprinciple experiments, the P(GMA-HEMA) brush substratebased protein/peptide microarrays show great potential for high-throughput screening of protein binding and protease functionality.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (grant no. 21475126 and 21127010) for financial support.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b01156. Details regarding the fabrication of protein/peptide microarray and cell staining. characterization of asprepared polymer-brush substrate (WCA, ATR-FTIR, XPS, AFM), resistance to protein adsorption, comparison of the performance between the freshly prepared P(GMA-HEMA) brush substrates and the ones stored for 3 months, optimization of experimental conditions, and fluorescence images of stained cell (PDF)

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Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes

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