Amine-Reactive Poly(pentafluorophenyl acrylate) - ACS Publications

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Amine-Reactive Poly(pentafluorophenyl acrylate) Brush Platforms for Cleaner Protein Purification Hyunjoo Son,† Jayoung Ku,‡,§ Yoosik Kim,‡,§ Sheng Li,*,‡ and Kookheon Char*,† †

The National Creative Research Initiative Center for Intelligent Hybrids, School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Korea ‡ Department of Chemical and Biomolecular Engineering and §KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea S Supporting Information *

ABSTRACT: Reactive pentafluorophenyl acrylate (PFPA) polymer brushes grafted on silica particles were prepared using surface-initiated reversible addition and fragmentation chain transfer polymerization. The polymer brush was successfully immobilized with antibody, then used for protein separation. The immunoprecipitated proteins showed successful enrichment of target protein, with reduced nonspecific background and less contamination from eluted antibodies. To further improve protein recovery, the hydrophobic poly(PFPA) brush was modified with hydrophilic poly(ethylene glycol) (PEG). The partially PEG-substituted poly(PFPA) brush showed better dispersion in aqueous solution, leading to improved antibody immobilization efficiency. By optimizing both the brush molecular weight and the degree of PEG substitution, an optimal balance between surface hydrophilicity and number of available PFP units was found, leading to efficient target protein purification. This study shows that poly(PFPA) platform offers a versatile approach to prepare biomolecule-activated surfaces with tunable surface property, which has potential applications in protein separation and other areas.

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Immunoprecipitation (IP)26,27,33−35 is a bioseparation technique routinely performed to isolate a particular protein from a complex protein mixture. The protein separation is achieved using an antibody activated solid substrate. The antibody binds specifically to its target antigen, the bound antibody−antigen is then pulled down with the solid substrate, leading to the isolation of the target antigen via centrifugation. In the most common application, agarose resin or magnetic polystyrene beads are used as the solid support material. Antibody immobilization on the solid support is accomplished through the use of Protein A/G as a linker. Since Protein A/G binds to Fc fragment of the antibody, the antibody is attached with a specific orientation such that its antigen-binding site is exposed for subsequent interaction with the target antigen molecules. Consequently, the Protein A/G immobilized IP technology shows high antigen binding efficiency, and it is typically considered to be the current IP standard. However, this technology is not without its faults, with the most significant drawback being its high level of background signal due to nonspecific protein binding. Since Protein A/G is an efficient protein binder, it also interacts with other nontarget protein molecules in the mixture. After the agarose/polystyrene supports are pulled down, the target antigens are recovered along with a considerable amount of nontarget proteins. These

INTRODUCTION

Biomolecule activated organic and inorganic materials have become a new platform of advanced materials with applications in areas such as drug delivery,1−4 detection of biomolecules,5−13 and bioseparation.1,14−16 While the detailed methods used for the preparation of different biomaterials may differ, they all require the immobilization or attachment of a biomolecule of interest to a material surface.17−19 The immobilization of biomolecule can occur through physical adsorption20 via van der Waals forces and hydrogen bond formation. Immobilization via covalent bond formation has also been explored.4,5,7,10,21−25 In this case, immobilization is achieved by reaction between chemical functional groups expressed on material surface and complementary functionality (typically amine or thiol) present in biomolecules. Lastly, bioaffinity based immobilization1,26−28 schemes have also been reported. For example, Protein A/G contains binding domains that can selectively bind to the heavy chain within the Fc region of most immunoglobulins. This antibody-binding property makes Protein A/G an excellent linker material for immobilizing antibody onto material surfaces.26,29−31 Regardless of the mechanism of biomolecule immobilization, several common features are considered to be desirable for most applications.32 They are (1) high density of biomolecule on material surface; (2) full retention of biomolecule activity after immobilization; and (3) minimized nonspecific interaction between biomolecule-activated surface and nontarget molecules. © XXXX American Chemical Society

Received: December 11, 2017 Revised: February 4, 2018 Published: February 6, 2018 A

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Scheme 1. Schematic for the Preparation of Poly(PFPA) Brush via SI-RAFT Polymerization and (a) Subsequent PostModification to Immobilize Antibody; (b) Partial Substitution with Amino-PEG, Followed by Antibody Immobilization

molecules to create light-responsive polymer brushes.40 Additionally, since the poly(PFPA) chains were grafted from a solid substrate, they were found to exhibit improved chemical stability in comparison to polymer coating prepared through physical adsorption. Based on these results, we envision that poly(PFPA) brush can be a suitable material platform for antibody immobilization, with an emphasis on creating antibody-bound solid support surfaces for IP applications with enhanced separation efficiency. In this paper, we introduce poly(PFPA) polymer brush as a new material platform for antibody immobilization. Reactive poly(PFPA) polymer brushes grafted on silica particle surfaces are synthesized via surface initiated reversible addition and fragmentation chain transfer (SI-RAFT) polymerization. Amine-containing biomolecules such as antibodies then react with the PFP units leading to efficient immobilization of antibodies on the silica particle surfaces (Scheme 1a). To further fine-tune the surface condition, amino-terminated poly(ethylene glycol) (PEG) is also reacted with the same poly(PFPA) brush, creating silica particles with both PEG and antibody immobilized on the surface (Scheme 1b). Under optimized conditions, the antibody treated silica particles are successfully tested for IP experiment, and they show reasonable target protein binding efficiency as well as much reduced background from nonspecific interactions.

nontarget proteins contribute to nonspecific background, compromising the separation efficiency and accuracy of the technology. To resolve the Protein A/G related nonspecific binding problem, direct cross-linking of antibody to a solid support has been explored.12,22,23,25 For example, activated esters, such as N-hydroxysuccinimide (NHS) ester, can react with amine moieties abundantly present in most biomolecules to form amide bond. In one report, polyacrylamide gels were successfully used for enzyme immobilization via covalent bonding with copolymer of acrylamide and N-acryloxysuccinimide.21 In a more recent example, NHS-ester-functionalized poly(PEGMA) brush was used for immobilization of two proteins horseradish peroxidase and chicken immunoglobulin.23 However, NHS-ester based amide bond formation generally requires long reaction time with low reaction efficiency, giving rise to a solid support surface with low density of covalently bound antibody. Additionally, since the position of cross-linking is random,36 not every immobilized antibody has the correct orientation to accept an antigen, further decreasing the efficiency of the method. Recently, a new type of activated ester, pentafluorophenyl (PFP) ester, is receiving increasing attention due to its high reactivity with amines and resistance toward hydrolysis. Furthermore, polymers containing PFP, such as poly(pentafluorophenyl acrylates) and poly(pentafluorophenyl methacrylates) have been reported as an interesting class of reactive polymer precursors that can be readily functionalized by reaction with amine-containing moieties.37−44 In particular, previous studies published from our group examined surfaceinitiated polymerization of poly(pentafluorophenyl acrylate), or poly(PFPA), and reported the successful post-polymerization functionalization of the polymer with amino-containing

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MATERIALS AND METHODS

Materials. 2,2′-Azobis(2-methylpropionitrile) (AIBN) and other solvents were purchased from Sigma-Aldrich. The monomer, pentafluorophenyl acrylate (PFPA), and the RAFT chain transfer agent, benzyl dithiobenzoate (BDB), were synthesized according to procedures published before.37,43,45 AIBN was purified by recrystallization from methanol. Silica particles (0.255 μm, SD = 0.01 μm) in B

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Figure 1. Confocal images of poly(PFPA)-grafted silicon wafer surface after treatment with (a) fluorescent-tagged antibody; (b) excess amount of amino-PEG to saturate PFP units, then treatment with fluorescent-tagged antibody; (c) anti-GFP antibody, then amino-PEG to saturate unreacted PFP units, and finally incubation with GFP. aqueous suspension were obtained from Microparticles Gmbh. ωAmino-terminated poly(ethylene glycol) methyl ether (Mn = 550 g/ mol, PDI = 1.15) was purchased from Polymer Source. TAR RNAbinding protein (TRBP) antibody was purchased from AbFrontier, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology, green fluorescent protein (GFP) antibody was purchased from Sigma-Aldrich, and all other antibodies used were purchased from Cell Signaling Technology, Inc. Synthesis of Poly(PFPA) Brushes on Silicon Wafer. Poly(PFPA) brushes on silicon wafer were synthesized by SI-RAFT polymerization. The detailed procedures are reported in a previous publication.40 Immobilization of SI-CTA on Silica Particles (SiPs). The dithiobenzoic acid benzyl-(4-ethyltrimethoxylsilyl) ester, used as the SI-RAFT chain transfer agent (SI-CTA), was synthesized following literature procedures.46 Modification of SI-CTA on SiPs was performed through silane coupling reaction. A total of 1.20 mL (60.0 mg) of SiPs in aqueous suspension were repeatedly washed with ethanol, tetrahydrofuran (THF), and toluene, and separated by centrifugation. The recovered SiPs were then redispersed in 4 mL of anhydrous toluene in a Schlenk flask. A total of 0.030 g SI-CTA dissolved in 3.5 mL of anhydrous toluene was then added to the flask. The solution was stirred at 80 °C in an oil bath for 18 h. The modified SiPs were washed with toluene and dried in a vacuum oven at 80 °C overnight. Synthesis of Poly(PFPA) Brushes on Silica Particles via SIRAFT Polymerization. SI-CTA-modified SiPs (53.2 mg) were dispersed in anhydrous anisole. The dispersed particles were then charged into a Schlenk flask, along with 5 mg (0.0205 mmol) of BDB, 0.4 mg (0.00244 mmol) of AIBN, and 2.24 g (9.41 mmol) of PFPA. After three freeze−pump−thaw cycles, the flask was backfilled with nitrogen, then stirred in an oil bath at 70 °C for 43 h. The polymerization was terminated by cooling the reaction to room temperature. The poly(PFPA)-grafted SiPs were rinsed with toluene and THF to remove free poly(PFPA) chains formed by BDB, then dried in a vacuum oven. Post-Polymerization Treatment with Amino-Terminated PEG. Amino-terminated PEG dissolved in THF (0.5 mL) was added into a suspension of poly(PFPA)-grafted SiPs. The mixture was stirred at room temperature for 16 h. After the reaction, the PEG-substituted poly(PFPA) brushes were washed with THF several times then dried in vacuum oven. Antibody Immobilization. Antibody immobilization was performed on either silicon wafers or silica particles grafted with poly(PFPA) brushes. In both scenarios, the substrate material was placed in a desired solvent containing 5 μg of protein kinase R (PKR) antibodies. The mixture was incubated with rotation at 4 °C overnight. Immunoprecipitation Test. HeLa cell pellets were suspended in Tris-based lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM KCl, 0.5% NP-40, 10% glycerol, 1 mM DTT) supplemented with protease inhibitor cocktail (Calbiochem) and incubated on ice for 10 min. Cells

were then sonicated using Bioruptor and debris was separated by centrifugation. Lysates were then incubated with antibody-conjugated SiPs for 3 h at 4 °C. SiPs were washed three times with the lysis buffer, and SDS loading buffer was then added to elute the proteins attached to the antibody on the bead surface. Eluted protein was heated to 95 °C for 10 min and was subjected to further analysis using gel electrophoresis. Characterization. Gel permeation chromatography (GPC) was used to determine the molecular weight and the corresponding polydispersity index (PDI = Mw/Mn) of free poly(PFPA) chains generated using sacrificial free chain transfer agent in polymerization mixture to estimate the polymer brush molecular weight. GPC (YL9100, Young Lin Instrument Co. Ltd.) measurements were performed using polystyrene standards in THF with 5 mg/mL polymer sample concentration. The weight percent of poly(PFPA) brushes on SiPs was measured by thermogravimetric analysis (TGA, Q500, TA Instruments). The weight loss data were collected on heating from room temperature to 700 °C at a heating rate of 10 °C/ min under nitrogen flow. Poly(PFPA)-grafted SiPs were imaged by transmission electron microscopy (TEM, JEM1010, JEOL) under an acceleration voltage of 80 kV. The TEM samples were prepared by placing a few drops of a solution of 0.5 mg/mL of particles dispersed in THF on carbon-coated TEM grid. The surface morphology of poly(PFPA) brushes on silicon wafer was monitored by tapping-mode atomic force microscopy (AFM, Veeco, Innova), and the surfaces of functionalized poly(PFPA) brush on both silicon wafer and SiPs were examined in attenuated total reflectance (ATR) mode by Fourier transform infrared spectroscopy (FT-IR, TENSOR27, Bruker), both are shown in Supporting Information. The surface composition of SICTA-grafted SiPs, and functionalized and nonfunctionalized poly(PFPA)-grafted SiPs were also measured by X-ray photoelectron spectroscopy (XPS, AXIS-His, KRATOS), equipped with A1 monocromator anode and 18 mA/12 kV X-ray power. The dispersion property of the surface-modified SiPs was determined by dynamic light scattering (DLS), measured using Zetasizer Nano ZS90 (Malvern Instruments). The DLS samples were prepared by dispersing 2 mg of particles in 2 mL of water. The antibody concentration before and after incubation were measured using a photoluminescence spectrometer (PL, LS55, PerkinElmer; Supporting Information). For direct antibody visualization, glycerol based mounting solution was applied onto antibodyconjugated silicon wafer, then examined with Zeiss LSM 700 confocal microscope using C-Apochromat 40× lens with NA = 1.2. IP recovered proteins were examined using silver staining and Western blotting. For silver staining, the eluted proteins were size separated on a 10% SDS-PAGE gel, then subjected to EzWay Protein-Silver Staining Kit (Komabiotech) following the manufacturer’s instruction. Western blotting was performed using a 10% SDS-PAGE gel and transferred to PVDF membrane using Amersham semidry transfer system. PKR, TRBP, GAPDH, Prohibitin-1 (PHB1), Rab5, and Histone H3 were C

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Figure 2. (a) TGA curves of bare, SI-CTA-grafted, and poly(PFPA)-grafted SiPs. (b) XPS curves of SI-CTA-grafted and poly(PFPA)-grafted SiPs. (c) TEM images of bare SiPs (left, D ∼ 230 nm) and polymer brush-grafted particles (right). used as primary antibodies and horseradish peroxidase conjugated secondary antibodies were used to detect the primary antibodies.

Next, to further illustrate that the immobilized antibody retains its activity, in particular, its ability to bind with target protein, poly(PFPA) brush was first functionalized with antiGFP antibody, then treated with amino-PEG. The PEG treatment was used to saturate any unreacted PFP units in order to minimize reaction between PFP esters and amine groups of GFP directly. Following the surface treatment, the antibody immobilized surface was then incubated in GFP. Figure 1c shows the confocal image of the wafer surface following GFP incubation. Green fluorescent signals are clearly seen, and they can be mainly attributed to the binding of the GFP to the anti-GFP antibody immobilized on poly(PFPA) brush. Overall, the data shown in Figure 1 suggest that antibodies not only can be successfully immobilized on poly(PFPA) brush, they also retain their ability to interact with target protein. Synthesis of Poly(PFPA) Brushes Grafted on Silica Particles. While the silicon wafer substrate provides a convenient flat surface for characterization (more surface characterization data may be found in Supporting Information), for actual IP application, spherical particles with increased surface area to volume ratio would be a more suitable substrate choice. In fact, most conventional IP technologies, including the ones using Protein A/G for antibody immobilization, have micron-sized agarose beads or polystyrene beads as the solid substrate. The SI-RAFT polymerization was thus modified to prepare poly(PFPA) brushes on micron-sized spherical SiPs. Similar to the methodology40 reported for SI-RAFT polymerization on flat substrate, the same SI-CTA was prepared and immobilized on SiPs via silane coupling reaction. The modified surface was characterized by TGA (Figure 2a), and when compared to bare SiPs, the presence of material grafted to SiPs was confirmed. Additionally, XPS measurements were conducted and peaks associated with C−S and CS bonds of 2s

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RESULTS AND DISCUSSION Antibody Immobilization Using Poly(PFPA) Brushes. Previous studies on poly(PFPA) have shown that the PFP units are excellent leaving group that can readily undergo nucleophilic substitution reaction.37,40,43,47 In particular, poly(PFPA) brushes have been successfully functionalized by postpolymerization modification with primary amines.40 To demonstrate that the poly(PFPA) brush platform can be used for antibody immobilization, SI-RAFT polymerization was used to prepare poly(PFPA) brushes grafted from SI-CTA activated silicon wafer surfaces. Antibody immobilization was achieved by incubation of polymer brushes in PBS buffer. The PFP ester units present along the polymer chain react with amine units present in antibody, leading to amide bond formation, and consequently immobilizing the antibody on poly(PFPA)grafted substrate surface (Scheme 1a). To demonstrate antibody attachment, fluorescent dye conjugated antibodies were first functionalized on poly(PFPA)-grafted silicon wafer. After repeated washing to remove unbound antibodies, the wafer surface was examined under confocal microscope. As shown in Figure 1a, the presence of fluorescent signal on silicon wafer surface is clearly detected, confirming the successful functionalization of poly(PFPA) brush with antibody. The specificity of the fluorescent signal was examined by preincubating the poly(PFPA)-grafted silicon wafer with amine-functionalized PEG prior to antibody incubation. These PEG molecules are expected to quench most of the reactive PFP ester units. As shown in Figure 1b, the fluorescent signal from the wafer is decreased significantly as the fluorophore conjugated antibodies can no longer react with the PFP ester units. D

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Biomacromolecules sulfur originated from the dithioester groups on SI-CTA were observed (Figure 2b). Synthesis of poly(PFPA) brushes on SI-CTA-grafted SiPs was then carried out using SI-RAFT polymerization. The polymer brush molecular weight was estimated from the molecular weight of free poly(PFPA) chains generated using sacrificial free chain transfer agent, BDB, added to the polymerization mixture. It has been reported that the polymer brush molecular weight is actually comparable to or smaller than the molecular weight of the bulk polymerized homopolymer.48−50 However, the method is still useful in providing an upper limit estimation on brush molecular weight. The particular poly(PFPA) brush shown in Figure 2 was estimated to have a molecular weight of 33 kg/mol (PDI = 1.3). The presence of poly(PFPA) brushes on SiPs was further confirmed by TGA (Figure 2a). By comparing the weight loss curve of polymer brushes to that of SI-CTA-grafted SiPs, the weight percent of poly(PFPA) brush relative to the total mass of poly(PFPA)-grafted SiPs was determined to be 12.44 wt %, which corresponded to an estimated grafting density of 0.11 chains/nm2. The polymer brushes can also be visualized by TEM. As shown in Figure 2c, SiPs appear as dark spheres, surrounded by polymer brush shown with a lighter contrast. To further confirm that the polymer observed was a result of SIRAFT polymerization, XPS data for polymer brushes were obtained and compared to those of SI-CTA-grafted SiPs (Figure 2b; Detailed elemental XPS spectra are shown in Supporting Information). Following polymerization, S 2p peaks associated with SI-CTA decreased while F 1s peaks associated with PFPA units appeared, confirming that the polymer brush was indeed synthesized from the SI-CTA units attached on SiPs. IP Using Poly(PFPA)-Grafted Silica Particles. Following the successful synthesis of poly(PFPA)-grafted SiPs, antibodies were immobilized on the particle surfaces by incubation in a suitable solvent. Two different solvents, PBS and DMSO, were tested for antibody immobilization. PBS is the typical solvent used when dealing with biomacromolecules. However, poly(PFPA)-grafted SiPs are not well-dispersed in aqueous solution due to low surface energy, thus, DMSO is chosen as an alternative solvent to improve SiPs dispersion. The antibody immobilized SiPs, prepared in either PBS or DMSO, were used in IP experiments. In a typical setup, antibody immobilized SiPs are added to cell lysate containing total protein extracted from cells. The mixture is shaken to allow antibody-protein binding, and the bound proteins are then pulled-down along with the SiPs via centrifugation. The proteins are then separated from the solid substrate using an elution buffer and analyzed by gel electrophoresis. For the initial experiments, anti-PKR antibody was immobilized on poly(PFPA) brushes. Following IP, silver staining was used to visualize all proteins isolated. Figure 3 shows silver staining results for proteins immuoprecipitated using poly(PFPA)-grafted SiPs conjugated with antibody, as well as ones recovered using commercially available Protein A based IP kit. The most noticeable difference is the amount of proteins recovered via the two different methods. When Protein A based traditional IP is used, a large number of nontarget proteins are recovered in addition to the target. Since Protein A is an efficient protein binder, it is known to interact nonspecifically with a number of different proteins, leading to a high background. In comparison, when IP is performed using poly(PFPA)-grafted SiPs conjugated with antibody, the number

Figure 3. Silver staining results for proteins immunoprecipitated using poly(PFPA) based IP (lane 3) and conventional Protein A based IP kit (lane 5). Lane 1 shows the protein ladder. Lane 2 shows the input protein mixture before IP. Lane 4 shows the anti-PKR antibody immobilized on poly(PFPA) brush. The blue boxes indicate heavy and light chains of anti-PKR antibody.

of nontarget proteins recovered is significantly less, resulting in a much cleaner protein separation. Another observation we can make from the silver staining results is that protein bands located at ∼55 and ∼27 kDa are present in significant concentration in conventional IP recovered sample, but almost completely absent in poly(PFPA) based IP scheme. These bands correspond to the heavy and light chains of the antibody, respectively. A major shortcoming of Protein A/G based IP is that during protein elution, both the bound proteins and the immobilized antibodies are eluted. Therefore, the solid substrate loses attached antibodies after just one use. More importantly, the presence of eluted antibodies complicates data interpretation. If the target protein also eludes near 55 or 27 kDa, then to distinguish the target from the antibody is almost impossible. For the poly(PFPA) based IP scheme, antibodies are immobilized by covalent bond, thus they are not eluted during protein recovery, as confirmed by silver staining results. Consequently, the spectrum of the recovered protein is not complicated by extra bands from the immobilized antibodies. To determine poly(PFPA) based IP efficiency, the eluted protein sample was analyzed against three different antibodies via Western blotting: anti-PKR antibody was used to visualize the amount of PKR (target protein) immunoprecipitated; antiTRBP antibody was used to visualize protein TRBP, a known interactor with PKR, thus coimmunoprecipitate with PKR; and anti-GAPDH antibody was used as a negative control as GAPDH is an abundant protein that does not interact with PKR. Figure 4 shows the Western blot data of protein samples recovered using antibody-bound SiPs prepared in either PBS or DMSO solvent. When PBS is used as the solvent for antibody immobilization, despite the poor dispersion of SiPs, PKR enrichment is observed as indicated by the presence of PKR band and absence of GAPDH band. Furthermore, weak TRBP band is also seen, indicating that the degree of PKR enrichment is high enough such that its interactor protein can also be copurified. Surprisingly, when the IP experiment is conducted using antibody immobilized SiPs prepared in DMSO, no protein bands are observed. Despite the improved particle E

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conformation, which means antibody molecules cannot penetrate deep into the brushes to react with PFP units. In addition, the poly(PFPA)-grafted SiPs aggregate in aqueous solvent to reduce exposed surface area, further reducing the number of exposed PFP units available for reaction. It has been reported that the incorporation of ethylene glycol side chains to poly(PFPA) leads to enhanced polymer solubility in water;51 therefore, we chose to improve the surface hydrophilicity of poly(PFPA)-grafted SiPs by grafting short PEG chains to the polymer brushes (Scheme 1b). In particular, low molecular weight (Mn = 550 g/mol, PDI = 1.15) aminoterminated PEG polymers were used for this purpose. The PEG polymers contain amine functionality at the chain end, so they can be grafted to poly(PFPA) using the same PFP ester-amine reaction used for antibody attachment. The PEG treatment would necessarily reduce the number of PFP units available for further antibody immobilization; however, we hypothesize that by controlling the number of PEG substitution sites, the poly(PFPA) brush can still retain sufficient number of free PFP units for antibody attachment while significantly improving its surface hydrophilicity. Additionally, PEG is known for its ability to repel nonspecific protein binding, so its presence may further reduce background noise of our IP design. To prepare PEG-substituted poly(PFPA) brushes, the poly(PFPA)-grafted SiPs were combined with amino-PEG in THF. Assuming every PFP unit reacts immediately with every amine end group of amino-PEG, different concentrations of PEG solutions were prepared to yield 10%, 50%, and 100%

Figure 4. Western blot for proteins immunoprecipitated using poly(PFPA)-grafted SiPs treated with anti-PKR antibody in either PBS (lane 2) or DMSO (lane 3) solution. Lane 1 shows the input protein mixture before IP. The values of quantified Western blot signal intensity for PKR, TRBP, and GAPDH, normalized by input intensity, are presented below the respective bands.

dispersion, we conclude that DMSO is not a suitable solvent for antibody immobilization. IP Using PEG-Substituted Poly(PFPA) Brushes. To improve the efficiency of poly(PFPA)-grafted SiPs for IP applications, overcoming particle hydrophobicity is critical, especially since conducting antibody immobilization in organic solvent is shown to be not feasible. The PFP groups form unique low energy surfaces so poly(PFPA) brushes are not well solvated in water. The polymer brushes have collapsed

Figure 5. (a) Physical appearance of poly(PFPA)-grafted SiPs with different degrees of amino-PEG substitution when dispersed in water. (b) DLS measurements of poly(PFPA)-grafted SiPs with 0%, 10%, 50%, and 100% theoretical PEG substitution. The Z-average diameter and PDI of each sample are also reported. For the 0% and 10% PEG-substituted SiPs, partial aggregation is observed so the numbers reported are determined based on the peaks for nonaggregated particles only. F

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Figure 6. (a) Western blot on proteins immunoprecipitated using 10% PEG-substituted SiPs treated with anti-PKR antibody in PBS (lane 2). Lane 1 shows the input protein mixture before IP. The values of quantified Western blot signal intensity, normalized by input intensity, are presented below the respective bands. (b) Western blot on proteins immunoprecipiated using 10% PEG-substituted SiPs based IP (lane 2) and Protein A based IP (lane 3), using only secondary antibodies. Input (lane 1) is blank because no primary antibodies are used. (c) Western blot on proteins immunoprecipiated using 10% PEG-substituted SiPs based IP (lane 2) and Protein A based IP (lane 3). The proteins visualized are β-tubulin (rabbit or mouse), which are unlikely to interact with PKR. The weak input signal (lane 1) seen for β-tubulin (rabbit) is due to signal overexposure from Protein A based IP.

10% PEG-substituted SiPs to test because at even 10% PEG, evidence of brush layer swelling could be seen as the measured particle diameter increased from 335 to 409 nm. Although partial aggregation was still observed at this level of PEG substitution, we suspect there would be sufficient local poly(PFPA) solvation to improve antibody immobilization efficiency. Western blot experiment on a number of different proteins was conducted. In particular, several noninteracting proteins were visualized, including GAPDH, PHB1, Rab5, and Histone H3, to better illustrate IP separation efficiency. As shown in Figure 6a, the selective enrichment of the target PKR over nontarget proteins is confirmed, along with the successful coprecipitation of the PKR-interactor, TRBP. Furthermore, when comparing the amount of PKR immunoprecipitated using 10% PEG-substituted SiPs with those recovered using nonPEG-treated SiPs (Figure 4), an increase in IP efficiency is observed. This improvement can be mainly attributed to the increased number of immobilized antibody on 10% PEG-substituted SiPs. At 10% PEG substitution, although the poly(PFPA) brushes are not fully solvated, as evidenced by the observation of partial aggregation, local increase of poly(PFPA) solubility in an aqueous environment is still expected, which leads to an increased number of accessible PFP units for antibody reaction. While the concentration of antibody on SiPs is difficult to measure directly, it can be estimated by comparing the antibody concentration in buffer before and after incubation with poly(PFPA)-grafted SiPs. It was found that the concentration of the remaining antibody was significantly less for 10% PEG-

theoretical substitution of PEG relative to the total number of available PFP units on polymer brushes. The silica particles thus prepared are labeled x% PEG-substituted SiPs, where x represents the theoretical degree of PEG substitution. The PEG-substituted SiPs were then dispersed in water, and their particle size information was measured by DLS. The dispersion properties of poly(PFPA)-grafted SiPs with different degrees of theoretical PEG substitution are summarized in Figure 5. The nonsubstituted poly(PFPA)-grafted SiPs do not disperse well in water and the particles appear as large aggregates. As the degree of PEG substitution increases, particle dispersion is seen to improve significantly as the solution turns into a misty suspension. The hydrodynamic diameters of the particles are measured by DLS, and the Z-average values are reported. As the percent PEG substitution increases, particle size also increases. Since the silica core cannot expand, the increase in particle size suggests that the polymer brushes surrounding the silica particles are becoming more solvated thus more swollen in water. Note that for the 0% and 10% PEG-substituted SiPs, partial aggregation was observed, so the reported Z-average values were determined based on the nonaggregated population of SiPs. According to our hypothesis, a swollen poly(PFPA) brush conformation would increase the efficiency of antibody attachment, in comparison to a collapsed conformation, as the swollen brushes would allow a larger number of PFP units to be exposed for reaction. To test the hypothesis, the 10% PEG-substituted SiPs were used in an IP experiment, again having anti-PKR as the immobilized antibody. We chose the G

DOI: 10.1021/acs.biomac.7b01736 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules substituted SiPs, in comparison to 0% PEG-substituted SiPs, confirming a higher level of immobilized antibody on 10% PEG-substituted SiPs. The detailed data regarding antibody concentration measurements can be found in Supporting Information. To further quantify the efficiency of IP separation, IPenriched protein samples obtained using 10% PEG-substituted SiPs were compared to those obtained using conventional Protein A based IP. We performed Western blotting on sizeseparated protein membrane with only secondary antibodies. In principle, if the protein mixture does not contain any nontarget protein contaminants, such as the antibody used during IP, then no signal should be observed, as primary antibodies are not added. As shown in Figure 6b, while only a weak protein band is seen from sample recovered via poly(PFPA) based IP, multiple strong bands associated with anti-PKR antibody and antibody fragments can be seen from Protein A based IP. The presence of these contaminants can lead to misinterpretation of IP data and false identification of interacting protein targets. For example, using the same protein samples shown in Figure 6b, a control experiment was performed, where Western blotting was done on proteins that are unlikely to interact with PKR, such as β-tubulin. As shown in Figure 6c, strong signal associated with β-tubulin (rabbit) is observed only from the sample obtained from Protein A based IP. This is because the size of the β-tubulin (rabbit) overlaps with that of anti-PKR heavy chain. The poly(PFPA) based IP, on the other hand, is minimally contaminated by eluted anti-PKR antibody, thus, showing much improved accuracy in identifying target proteins from nontargets. Optimization of Poly(PFPA) Brush Platform for IP Applications. Effect of Poly(PFPA) Brush Molecular Weight. The molecular weight of poly(PFPA) brush is directly related to the number of PFP units available for functionalization, as well as polymer dispersion; therefore, control of poly(PFPA) molecular weight is thought to affect antibody immobilization, thus critically influences IP efficiency. Poly(PFPA) brushes of three different molecular weights were synthesized: low MW (12 kg/mol), medium MW (33 kg/mol), and high MW (72 kg/mol). The brush molecular weight was estimated based on the free poly(PFPA) homopolymer synthesized from sacrificial BDB. The grafting density for each polymer brush was then estimated based on the relative mass of poly(PFPA) and SiPs, determined via TGA analysis.52 Detailed information related to the grafting density calculation may be found in Supporting Information. The relevant polymer brush characterization data are summarized in Table 1. As expected, polymer brush molecular weight was found to have a considerable effect on SiPs dispersion. When the poly(PFPA)-grafted SiPs of different brush molecular weights were dispersed in aqueous solution, low-MW sample could be reasonably dispersed, while high-MW sample formed aggregates and precipitated out of the liquid phase. As the number of PFP units increases with brush molecular weight, the particle surface becomes increasingly more hydrophobic, leading to the observed differences in dispersion properties. The low-, medium-, and high-MW samples were each treated with PEG solution such that 10% theoretical PFP unit substitution was achieved. The PEG-substituted samples were then immobilized with anti-PKR antibody and tested for IP performance. For comparison, PEG-substituted SiPs without antibody attachment were also prepared, and they were labeled as “blank” samples. Six separate IP experiments involving

Table 1. Poly(PFPA)-Grafted SiPs with Low, Medium, and High MW poly(PFPA) brush on SiPs

Mna (g/mol)

PDI

low MW medium MW high MW

12250 32537 71715

1.16 1.27 1.32

b

wt % PPFPAc (%)

wt % SiPsc (%)

grafting densityd (chains/nm2)

6.86 11.82 16.89

88.97 83.91 76.81

0.15 0.11 0.07

a

Polymer brush Mn values are estimated based on molecular weight information on bulk poly(PFPA) synthesized from sacrificial BDB. b Polymer brush PDI values are estimated based on molecular weight information on bulk poly(PFPA) synthesized from sacrificial BDB. c Relative mass of the poly(PFPA) brush (wt % PPFPA) and the SiPs (wt % SiPs) are determined based on TGA weight loss data. dGrafting density is calculated according to eq S1 in Supporting Information.

poly(PFPA)-grafted SiPs of three different brush molecular weights were performed. The Western blot data showing the amount of immunoprecipitated target protein (PKR) and the amount of nontarget protein (GAPDH) from all six IP experiments are summarized in Figure 7. The blank samples, regardless of the poly(PFPA) brush molecular weight, show no noticeable protein recovery, indicating that the substrate material itself has minimal interaction with the protein mixture. For the low-, medium-, and high-MW brushes with antibody immobilized on the particle surface, selective concentration of the target protein over nontarget protein is confirmed for all three brushes. In particular, the low- and medium-MW samples show stronger PKR band than the high-MW sample, indicating more efficient target protein recovery. Although the lower molecular weight brushes contain fewer numbers of PFP units, their better dispersion property in aqueous solution more than compensates for this deficiency, such that the total number of PFP units accessible for antibody reaction is higher in the lower molecular weight samples. This then leads to a larger number of immobilized antibody in the lower molecular weight brush samples, and consequently the better IP performance. Effect of Degree of PEG Substitution. Besides controlling poly(PFPA) brush molecular weight, the PEG treatment also plays an important role in determining IP performance. As demonstrated in earlier sections, when using the same poly(PFPA)-grafted SiPs, a 10% PEG substitution improves particle surface hydrophilicity and leads to significant improvement in IP efficiency. However, every site of PEG substitution means that the site is no longer available for subsequent antibody immobilization. There must exist a threshold, beyond which PEG substitution would lead to decreased IP performance. Using the low-MW poly(PFPA) brushes, three different degrees of PEG substitution were examined: 1%, 10%, and 50%. The PEG-substituted SiPs were immobilized with anti-PKR antibody, then used for IP experiments. The resulting Western blot data are shown in Figure 8. Selective enrichment of the target PKR protein is seen for all three samples with different degrees of PEG substitution. The PKR recovery is highest for 10% PEG-substituted SiPs, and lowest for 50% PEG-substituted SiPs. At 50% PEG substitution, too many PFP units are sacrificed for PEG attachment. Even though the polymer brush is well solvated in aqueous solution, there are not enough PFP units remaining for antibody reaction. Additionally, at a high degree of PEG substitution, the density of PEG molecules on the particle surface is expected to be high. They might pose steric hindrance and prevent antibody, which is fairly large in H

DOI: 10.1021/acs.biomac.7b01736 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 7. Western blot for proteins recovered from IP using poly(PFPA) brushes of different molecular weights. Lane 1: input protein mixture before IP. Lane 2: IP using low-MW poly(PFPA) brush, with 10% PEG-substitution, followed by anti-PKR antibody incubation. Lane 3: IP using medium-MW poly(PFPA) brush, with 10% PEG-substitution, followed by anti-PKR antibody incubation. Lane 4: IP using high-MW poly(PFPA) brush, with 10% PEG-substitution, followed by anti-PKR antibody incubation. Lane 5: IP using low-MW poly(PFPA) brush, with 10% PEGsubstitution, no antibody treatment. Lane 6: IP using medium-MW poly(PFPA) brush, with 10% PEG-substitution, no antibody treatment. Lane 7: IP using high-MW poly(PFPA) brush, with 10% PEG-substitution, no antibody treatment. (b) Quantification of Western blot signal intensities of low-, medium-, and high-MW polymer brush (Lanes 2−4) for PKR and GAPDH, normalized by input intensity. The relative intensity values for nonantibody treated SiPs (IP: Blank) are negligible. The calculated intensity ratios are also presented below their respective bands.

Figure 8. Western blot for proteins recovered from IP using low-MW poly(PFPA)-grafted SiPs treated with different amino-PEG substitution. Lane 1: input protein mixture before IP. Lane 2: IP using 1% PEG-substituted SiPs, followed by anti-PKR antibody incubation. Lane 3: IP using 10% PEGsubstituted SiPs, followed by anti-PKR antibody incubation. Lane 4: IP using 50% PEG-substituted SiPs, followed by anti-PKR antibody incubation. (b) Quantification of Western blot signal intensity for PKR and GAPDH, normalized by input intensity. The calculated intensity ratios are also presented below their respective bands.

target protein, with reduced background due to nonspecific protein interaction. Furthermore, since the antibodies were immobilized through covalent bond formation, reduced protein contamination from antibody elution was also confirmed. To further improve IP efficiency, the surface hydrophilicity of the particles was improved through sacrification of PFP units to attach amino-functionalized PEG. The partially PEG-substituted SiPs were found to have poly(PFPA) brushes adopting a more swollen conformation, thus, allowing more PFP units to be exposed for antibody reaction, ultimately leading to significant improvement in protein recovery efficiency. To optimize the surface conditions for antibody immobilization, different molecular weight polymer brushes as well as different degrees of PEG substitution were examined. An optimized balance between surface hydrophilicity and number of available PFP units was achieved at 10% PEG substitution in combination with a relatively low molecular weight poly(PFPA) brush. This study demonstrates that poly(PFPA)grafted SiPs can be an effective alternative to traditional IP for providing clean protein separation. Beyond protein purification,

size (2−10 nm), to access the free PFP units remaining on the particle surface. At moderate degree of PEG substitution (∼10%), the poly(PFPA) brushes are locally solvated, while the PEG layer is not too dense, thus, allowing sufficient number of PFP units to be available for antibody reaction. We conclude that by reaching an optimal balance between surface hydrophilicity and number of accessible PFP units, efficient protein separation with reduced nonspecific background can be achieved using poly(PFPA) based IP schemes.

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CONCLUSIONS Reactive poly(PFPA) brushes grafted from SiPs were prepared using SI-RAFT polymerization. The PFP ester units react readily with amine moieties present in protein molecules, leading to the successful immobilization of antibodies on SiPs. The antibody immobilized polymer brush substrate was successfully used for protein purification, demonstrating itself as an effective alternative to traditional Protein A/G based IP technology. IP experiments using poly(PFPA)-grafted SiPs conjugated with antibody showed successful enrichment of I

DOI: 10.1021/acs.biomac.7b01736 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules the poly(PFPA) brush platform is also expected to find applications in many different areas requiring biomolecule immobilization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01736. 1. Characterization of functionalized poly(PFPA) brush grafted on silicon wafer. 2. Characterization of functionalized poly(PFPA)-grafted SiPs. 3. Estimation of grafting density of poly(PFPA) brush on SiPs. 4. Antibody concentration measurement for poly(PFPA)-grafted SiPs with different degrees of PEG substitution (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Kookheon Char: 0000-0002-7938-8022 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Research Foundation of Korea (NRF) funded by the Korea Ministry of Science, ICT and Future Planning (MSIP), The National Creative Research Initiative Program for “Intelligent Hybrids Research Center”, No. 2010-0018290), and the BK21 Plus Program in SNU Chemical Engineering.

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DOI: 10.1021/acs.biomac.7b01736 Biomacromolecules XXXX, XXX, XXX−XXX