本文介绍了一种制备聚五氟苯基丙烯酸酯（poly(PFPA)）接枝二氧化硅微球的方案。该聚(PFPA)功能化表面可固定抗体，并已成功用于通过免疫沉淀实现蛋白质分离。
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本文内容
勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
我们展示了一种简便的方法，用于制备聚（五氟苯基丙烯酸酯）（poly(PFPA)）接枝的二氧化硅微球，以实现抗体固定化并进一步应用于免疫沉淀（IP）。poly(PFPA)接枝表面通过一个简单的两步法构建。第一步，将3-氨基丙基三甲氧基硅烷（APTMS）作为连接分子沉积在二氧化硅表面。第二步，通过可逆加成-断裂链转移（RAFT）聚合合成的poly(PFPA)均聚物，通过聚合物上的五氟苯基（PFP）单元与APTMS上的氨基之间的交换反应，接枝到连接分子上。APTMS和poly(PFPA)在二氧化硅颗粒表面的沉积通过X射线光电子能谱（XPS）进行确认，并通过动态光散射（DLS）测量的颗粒尺寸变化进行监测。为了提高微球表面的亲水性，还进行了部分poly(PFPA)与氨基功能化的聚乙二醇（amino-PEG）的取代反应。随后，将PEG取代的poly(PFPA)接枝二氧化硅微球用于抗体的固定化，以进行IP应用。作为示例，采用针对RNA激活蛋白激酶（PKR）的抗体，并通过Western blotting测定IP效率。分析结果表明，固定化抗体的微球确实可用于富集PKR，同时非特异性蛋白相互作用极低。
引言
近年来，反应性聚合物刷引起了广泛关注。它们可用于将功能分子固定在有机或无机材料上，从而制备出具有检测和分离等应用的活化表面1,2,3,4,5。在已报道的反应性聚合物中，含有五氟苯基酯单元的聚合物因其对胺类具有高反应活性且耐水解而尤为有用6。其中一种聚合物是聚(PFPA)，它可在聚合后通过含有伯胺或仲胺的分子进行简便的功能化修饰7,8,9,10。例如，聚(PFPA)刷可与氨基螺吡喃反应，制备出具有光响应性的表面7。
聚(PFPA)的制备及其应用已在多项先前发表的研究中有所描述6,7,....
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方案
1. 聚(PFPA)均聚物的制备
- 偶氮二异丁腈（AIBN）的重结晶
- 在250 mL烧杯中加入5 g 2,2’-偶氮双(2-甲基丙腈)（AIBN）和25 mL甲醇。将烧杯浸入60 °C油浴中，用磁力搅拌子剧烈搅拌，直至AIBN完全溶解。
- 将温热的溶液通过滤纸（5–8 μm颗粒截留）过滤，并将滤液置于4 °C下静置，使晶体缓慢析出。
- 通过过滤收集重结晶后的AIBN。将收集的产物与25 mL新鲜甲醇混合，重复重结晶过程。
- 将经过两次重结晶的AIBN在室温（RT）下于真空烘箱中干燥过夜。将产物避光保存于< -10 °C条件下。
- 苯甲基二硫代苯甲酸酯的合成25
- 准备一个500 mL三颈圆底烧瓶，配备磁力搅拌子、回流冷凝管、滴液漏斗和橡胶隔膜。通过回流冷凝管将烧瓶连接至氮气管线，用氮气吹扫烧瓶内部空气。将温度计插入隔膜中。通过注射器经同一隔膜加入41 mL（0.041 mol）四氢呋喃（THF）中1 M苯基溴化镁溶液。
- 将苯基溴化镁溶液在油浴中加热至40 °C，然后通过滴液漏斗缓慢加入3.1 g（0.041 mol）二硫化碳，保持溶液温度在40 °C。
- 在15分钟内通过滴液漏斗向所得混合物中加入7.1 g（0.042 mol）溴化苄。将反应温度升至50 °C，并....
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结果
聚(PFPA)接枝SiO2微球的制备示意图（可选是否进行PEG取代）如图1所示。为监测APTMS和聚(PFPA)的接枝过程，采用动态光散射（DLS）（图2）和X射线光电子能谱（XPS）（图3）对裸SiO2微球、APTMS功能化的SiO2微球以及聚(PFPA)接枝的SiO2微球进行了表征。微球的免疫沉淀效率通过Western blotting测定。图4展示了使用1% PEG取代的聚(PFPA)接枝微球进行免疫沉淀的Western blotting结果，其中微球分别与无抗体、非特异性抗体或抗PKR抗体共同孵育。图5展示了使用0%和1% PEG取代的聚(PFPA)接枝微球进行免疫沉淀的Wester.......
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讨论
聚(PFPA)接枝SiO2微球的合成过程如图1所示。通过使用APTM作为连接分子，可通过一个简单的两步法在SiO2基底上共价接枝聚(PFPA)刷状聚合物。尽管部分PFP单元在与APTM反应中被消耗，但仍预期有大量PFP单元可保留下来，用于后续与氨基-PEG或抗体的反应。已知PFP基团会形成低表面能，因此聚(PFPA)刷在水中溶胀性较差28。在免疫沉淀（IP）应用中，抗体需固定在聚(PFPA)刷上，该置换反应在水相缓冲溶液中进行，以保持抗体的活性。正如我们先前发表的研究所述，用亲水性分子（如氨基功能化的PEG）部分取代PFP单元，可提高表面亲水性，从而提升抗体固定效率18。在本研究中，还制备了部分PEG取代的聚(PFPA)，再通过相同的APTM连接分子将其接枝到SiO2表面。总体而言， 图1所示的方法可用于制备具有不同PEG取代程度的聚(PFPA)接枝.......
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披露
作者无任何利益冲突需要披露。
致谢
本工作由国防发展局（资助编号：UD170039ID）提供支持。
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 2,2-偶氮二异丁腈，99% | Daejung Chemicals | 1102-4405 | |
| 用于高效液相色谱的甲醇，99.9% | Duksan Pure Chemicals | d62 | |
| 苯基溴化镁 THF 溶液，1.0 M | Sigma-Aldrich | 331376 | |
| 无水二硫化碳，≥99% | Sigma-Aldrich | 335266 | |
| 溴化苄，98% | Sigma-Aldrich | B17905 | |
| 石油醚，90% | Samchun Chemicals | P0220 | |
| 乙醚，99% | Daejung Chemicals | 4025-4404 | |
| 无水硫酸镁，粉末， 99% | Daejung Chemicals | 5514-4405 | |
| 五氟苯基丙烯酸酯 | Santa Cruz Biotechnology | sc-264001 | 含抑制剂 |
| 氧化铝，活化，碱性，Brockmann I | Sigma-Aldrich | 199443 | |
| 氯化钠 (NaCl) | Daejung Chemicals | 7548-4400 | |
| 无水苯甲醚，99.7% | Sigma-Aldrich | 296295 | |
| 二氧化硅纳米颗粒 | Microparticles GmbH | SiO2-R-0.7 | 5% w/v 水悬浮液 |
| 3-氨基丙基三甲氧基硅烷，>96.0% | Tokyo Chemical Industry | T1255 | |
| 用于高效液相色谱的二甲基亚砜，≥99.7% | Sigma-Aldrich | 34869 | |
| 氨基封端的聚(乙二醇)甲醚 | Polymer Source | P16082-EGOCH3NH2 | |
| 磷酸盐缓冲液片剂 | Takara | T9181 | |
| 吐温-20 | Calbiochem | 9480 | |
| Tris-HCl (pH 8.0) | Invitrogen | AM9855G | |
| KCl | Invitrogen | AM9640G | |
| NP-40 | VWR | E109-50ML | |
| 甘油 | Invitrogen | 15514-011 | |
| 二硫苏糖醇 | Biosesang | D1037 | |
| 蛋白酶抑制剂 | Merck | 535140-1MLCN | |
| 溴酚蓝 | Sigma-Aldrich | B5525-5G | |
| Tris-HCl (pH 6.8) | Biosolution | BT033 | |
| 十二烷基硫酸钠 | Biosolution | BS003 | |
| 2-巯基乙醇 | Gibco | 21985-023 | |
| PKR 抗体 | Cell Signaling Technology | 12297S | |
| GAPDH 抗体 | Santa Cruz Biotechnology | sc-32233 | |
| 正常兔 IgG | Cell Signaling Technology | 2729S | |
| HeLa | 韩国细胞库 | 10002 | |
| 超声破碎仪 | DAIHAN Scientific | WUC-D10H | |
| 超声波处理器 | BMBio | BR2006A | |
| 离心机 I | Eppendorf | 5424 R | |
| 离心机 II | LABOGENE | 1736R | |
| 旋转混合仪 | FINEPCR | ROTATOR/AG | |
| 真空干燥箱 | DAIHAN Scientific | ThermoStable OV-30 | |
| 凝胶渗透色谱（THF） | Agilent Technologies | 1260 Infinity II | |
| X射线光电子能谱仪 | Thermo VG Scientific | Sigma Probe | |
| 动态光散射仪 | Malvern Instruments | ZEN 3690 |
参考文献
- Johnsson, B., Löfås, S., Lindquist, G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Analytical Biochemistry. 198 (2), 268-277 (1991).
- Kurzawa, C., Hengstenberg, A., Schuhmann, W.
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重印与许可
勘误
Formal Correction: Erratum: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification
Posted by JoVE Editors on 4/30/2019. Citeable Link.
An erratum was issued for: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification. Throughout the article, the term "3-aminopropyltriethoxysilane" has been replaced with "3-aminopropyltrimethoxysilane", and "APTES" with "APTMS".
The Keywords were updated from:
Poly(pentafluorophenyl acrylate), 3-aminopropyltriethoxysilane, reactive polymer brush, post-polymerization functionalization, antibody immobilization, immunoprecipitation
to:
Poly(pentafluorophenyl acrylate), 3-aminopropyltrimethoxysilane, reactive polymer brush, post-polymerization functionalization, antibody immobilization, immunoprecipitation
The Abstract was updated from:
We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltriethoxysilane (APTES) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTES. The deposition of APTES and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.
to:
We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.
The fourth paragraph of the Introduction was updated from:
In this contribution, we report an alternative method to prepare poly(PFPA) grafted surface for antibody immobilization and IP application. In a simple two-step process, as illustrated in Figure 1, an APTES linker molecule is first deposited onto the silica surface, then the poly(PFPA) polymer is covalently attached to the linker molecule through the reaction between the PFP units on the polymer and the amine functions on APTES. This preparation method allows for the permanent crosslinking of poly(PFPA) to a substrate surface, but avoids the many complications associated with SI-CTA synthesis and SI-RAFT polymerization of poly(PFPA) brushes. Partial substitution of the PFP units with amino-PEG can still be performed, allowing fine-tuning of the polymer brush surface properties. We show the poly(PFPA) grafted silica beads thus prepared can be immobilized with antibodies and used for protein enrichment via IP. The detailed bead preparation procedure, antibody immobilization, and IP testing are documented in this article, for readers interested in seeking an alternative to conventional Protein A/G based IP.
to:
In this contribution, we report an alternative method to prepare poly(PFPA) grafted surface for antibody immobilization and IP application. In a simple two-step process, as illustrated in Figure 1, an APTMS linker molecule is first deposited onto the silica surface, then the poly(PFPA) polymer is covalently attached to the linker molecule through the reaction between the PFP units on the polymer and the amine functions on APTMS. This preparation method allows for the permanent crosslinking of poly(PFPA) to a substrate surface, but avoids the many complications associated with SI-CTA synthesis and SI-RAFT polymerization of poly(PFPA) brushes. Partial substitution of the PFP units with amino-PEG can still be performed, allowing fine-tuning of the polymer brush surface properties. We show the poly(PFPA) grafted silica beads thus prepared can be immobilized with antibodies and used for protein enrichment via IP. The detailed bead preparation procedure, antibody immobilization, and IP testing are documented in this article, for readers interested in seeking an alternative to conventional Protein A/G based IP.
Step 2.1 of the Protocol was updated from:
Treatment of SiO2 beads with APTES
to:
Treatment of SiO2 beads with APTMS
Step 2.1.1 of the Protocol was updated from:
SiO2 particles are available in the form of a 5% (w/v) aqueous suspension. Combine 0.8 mL of SiO2 suspension with 40 mg of APTES and 8 mL of methanol in a 20 mL scintillation vial equipped with a stir bar.
to:
SiO2 particles are available in the form of a 5% (w/v) aqueous suspension. Combine 0.8 mL of SiO2 suspension with 40 mg of APTMS and 8 mL of methanol in a 20 mL scintillation vial equipped with a stir bar.
Step 2.1.3 of the Protocol was updated from:
Transfer the solution to a conical tube. To isolate the APTES functionalized SiO2 beads, centrifuge the solution at 10,000 x g for 5 min, then remove the supernatant. Wash the beads by re-dispersing them in 3 mL of fresh methanol. Shake the tube by hand for mixing, but if necessary, improve the dispersion by sonication in a water bath for a few seconds. Centrifuge the beads at 10,000 x g for 5 min. Remove the supernatant and repeat the wash step one more time.
to:
Transfer the solution to a conical tube. To isolate the APTMS functionalized SiO2 beads, centrifuge the solution at 10,000 x g for 5 min, then remove the supernatant. Wash the beads by re-dispersing them in 3 mL of fresh methanol. Shake the tube by hand for mixing, but if necessary, improve the dispersion by sonication in a water bath for a few seconds. Centrifuge the beads at 10,000 x g for 5 min. Remove the supernatant and repeat the wash step one more time.
Step 2.1.4 of the Protocol was updated from:
Combine the methanol washed SiO2 beads with 3 mL of dimethyl sulfoxide (DMSO). Shake the mixture by hand, or if necessary sonicate for a few seconds, until the beads are fully dispersed in DMSO. Centrifuge the beads at 10,000 x g for 5 min, then remove the supernatant. Repeat the step to ensure complete solvent exchange from methanol to DMSO.NOTE: The final suspension contains the APTES functionalized SiO2 beads dispersed in 4 mL of DMSO.
to:
Combine the methanol washed SiO2 beads with 3 mL of dimethyl sulfoxide (DMSO). Shake the mixture by hand, or if necessary sonicate for a few seconds, until the beads are fully dispersed in DMSO. Centrifuge the beads at 10,000 x g for 5 min, then remove the supernatant. Repeat the step to ensure complete solvent exchange from methanol to DMSO.
NOTE: The final suspension contains the APTMS functionalized SiO2 beads dispersed in 4 mL of DMSO.
Step 2.2 of the Protocol was updated from:
Grafting poly(PFPA) to APTES functionalized SiO2 beads
to:
Grafting poly(PFPA) to APTMS functionalized SiO2 beads
Step 2.2.2 of the Protocol was updated from:
Add 1 mL of APTES functionalized SiO2 beads suspended in DMSO (from Step 2.1.4) to the poly(PFPA) solution. React at RT for 1 h with vigorous stirring.
to:
Add 1 mL of APTMS functionalized SiO2 beads suspended in DMSO (from Step 2.1.4) to the poly(PFPA) solution. React at RT for 1 h with vigorous stirring.
Step 3.4 of the Protocol was updated from:
To prepare APTES functionalized SiO2 beads suspended in DMSO, follow the same steps shown in Step 2.1. Transfer 1 mL of the bead suspension into the PEG-substituted poly(PFPA) solution prepared in Step 3.3. Allow the grafting between poly(PFPA) and APTES functionalized SiO2 beads to proceed at RT for 1 h with vigorous stirring.
to:
To prepare APTMS functionalized SiO2 beads suspended in DMSO, follow the same steps shown in Step 2.1. Transfer 1 mL of the bead suspension into the PEG-substituted poly(PFPA) solution prepared in Step 3.3. Allow the grafting between poly(PFPA) and APTMS functionalized SiO2 beads to proceed at RT for 1 h with vigorous stirring.
The first paragraph of the Representative Results was updated from:
A schematic for the preparation of poly(PFPA) grafted SiO2 beads, with or without PEG substitution is shown in Figure 1. To monitor the APTES and poly(PFPA) grafting process, bare SiO2 beads, APTES functionalized SiO2 beads, and poly(PFPA) grafted SiO2 beads are characterized by both DLS (Figure 2) and XPS (Figure 3). IP efficiencies of the beads are determined by Western blotting. Figure 4 shows the Western blotting results for IP using 1% PEG-substituted poly(PFPA) grafted beads, where the beads are incubated with no antibody, a non-specific antibody, or anti-PKR antibody. Figure 5 shows the Western blotting results for IP using 0% PEG-substituted poly(PFPA) grafted beads and 1% PEG-substituted poly(PFPA) grafted beads, both incubated with anti-PKR antibodies.
to:
A schematic for the preparation of poly(PFPA) grafted SiO2 beads, with or without PEG substitution is shown in Figure 1. To monitor the APTMS and poly(PFPA) grafting process, bare SiO2 beads, APTMS functionalized SiO2 beads, and poly(PFPA) grafted SiO2 beads are characterized by both DLS (Figure 2) and XPS (Figure 3). IP efficiencies of the beads are determined by Western blotting. Figure 4 shows the Western blotting results for IP using 1% PEG-substituted poly(PFPA) grafted beads, where the beads are incubated with no antibody, a non-specific antibody, or anti-PKR antibody. Figure 5 shows the Western blotting results for IP using 0% PEG-substituted poly(PFPA) grafted beads and 1% PEG-substituted poly(PFPA) grafted beads, both incubated with anti-PKR antibodies.
Figure 1 was updated from:
Figure 1: Schematic for the preparation of poly(PFPA) grafted SiO2 beads using APTES as a linker molecule. (a) Poly(PFPA) grafted beads. (b) Partially PEG-substituted poly(PFPA) grafted beads.
to:
Figure 1: Schematic for the preparation of poly(PFPA) grafted SiO2 beads using APTMS as a linker molecule. (a) Poly(PFPA) grafted beads. (b) Partially PEG-substituted poly(PFPA) grafted beads.
Figure 2 was updated from:
Figure 2: DLS measurements for (a) bare SiO2 beads (SiO2), (b) APTES functionalized SiO2 beads (APTES-SiO2), and (c) poly(PFPA) grafted SiO2 beads (poly(PFPA)-SiO2), dispersed in DMSO. The Z-average diameter (d) and polydispersity index (PDI) of each sample are reported.
to:
Figure 2: DLS measurements for (a) bare SiO2 beads (SiO2), (b) APTMS functionalized SiO2 beads (APTMS-SiO2), and (c) poly(PFPA) grafted SiO2 beads (poly(PFPA)-SiO2), dispersed in DMSO. The Z-average diameter (d) and polydispersity index (PDI) of each sample are reported.
Figure 3 was updated from:
Figure 3: XPS spectra for bare SiO2 beads (SiO2), APTES functionalized SiO2 beads (APTES-SiO2), and poly(PFPA) grafted SiO2 beads (poly(PFPA)-SiO2). The peaks examined correspond to (a) Si 2p, (b) O 1s, (c) N 1s, and (d) F 1s.
to:
Figure 3: XPS spectra for bare SiO2 beads (SiO2), APTMS functionalized SiO2 beads (APTMS-SiO2), and poly(PFPA) grafted SiO2 beads (poly(PFPA)-SiO2). The peaks examined correspond to (a) Si 2p, (b) O 1s, (c) N 1s, and (d) F 1s.
The first and second paragraphs of the Discussion were updated from:
The synthesis of poly(PFPA) grafted SiO2 beads is illustrated in Figure 1. By employing APTES as a linker molecule, poly(PFPA) brushes covalently grafted to SiO2 substrate can be prepared via a simple two-step process. Although some of the PFP units are sacrificed for the reaction with APTES, a large number of the PFP units are expected to remain available for later reaction with either amino-PEG or antibodies. The PFP groups are known to form low energy surfaces so poly(PFPA) brushes do not solvate well in water28. For IP application, the antibodies need to be immobilized on the poly(PFPA) brushes, and this exchange reaction is done in aqueous buffer solution in order to preserve the activity of the antibodies. As reported in our previous publication, partial substitution of the PFP units with hydrophilic molecules such as amine-functionalized PEG can improve surface hydrophilicity, leading to increased antibody immobilization efficiency18. In this study, partially PEG substituted poly(PFPA) is also prepared, then grafted to the SiO2 surface using the same APTES linker molecule. Overall, the methods illustrated in Figure 1 allow the preparation of poly(PFPA) grafted surfaces with different degrees of PEG substitution. These polymer brushes with tunable surface properties provide an ideal platform for antibody immobilization and subsequent IP application.
The bead preparation process is monitored by both DLS and XPS. The DLS results for various functionalized SiO2 beads in DMSO are summarized in Figure 2. The bare SiO2 beads exhibit hydrodynamic diameter of 666 nm, in agreement with the manufacturer reported bead size (0.676 μm; SD = 0.03 μm). After APTES treatment, the bead diameter increases to 740 nm; and with poly(PFPA) treatment, the bead diameter further increases to 1889 nm. It is important to point out that the polydispersity index (PDI) for the poly(PFPA) grafted beads is rather large (PDI = 0.76), which is indicative of poor quality sample containing large aggregates. Although the DLS curve only shows one nano-sized peak, small amount of aggregates may be present in the suspension. The functionalized SiO2 beads are also examined by XPS to determine surface composition (Figure 3). Following APTES treatment, N 1s peak associated with the amine groups on APTES is detected. And, following poly(PFPA) treatment, F 1s peak associated with the PFP units on the polymer is detected. Together these data show the successful functionalization of the SiO2 surface, first with APTES, then with poly(PFPA).
to:
The synthesis of poly(PFPA) grafted SiO2 beads is illustrated in Figure 1. By employing APTMS as a linker molecule, poly(PFPA) brushes covalently grafted to SiO2 substrate can be prepared via a simple two-step process. Although some of the PFP units are sacrificed for the reaction with APTMS, a large number of the PFP units are expected to remain available for later reaction with either amino-PEG or antibodies. The PFP groups are known to form low energy surfaces so poly(PFPA) brushes do not solvate well in water28. For IP application, the antibodies need to be immobilized on the poly(PFPA) brushes, and this exchange reaction is done in aqueous buffer solution in order to preserve the activity of the antibodies. As reported in our previous publication, partial substitution of the PFP units with hydrophilic molecules such as amine-functionalized PEG can improve surface hydrophilicity, leading to increased antibody immobilization efficiency18. In this study, partially PEG substituted poly(PFPA) is also prepared, then grafted to the SiO2 surface using the same APTMS linker molecule. Overall, the methods illustrated in Figure 1 allow the preparation of poly(PFPA) grafted surfaces with different degrees of PEG substitution. These polymer brushes with tunable surface properties provide an ideal platform for antibody immobilization and subsequent IP application.
The bead preparation process is monitored by both DLS and XPS. The DLS results for various functionalized SiO2 beads in DMSO are summarized in Figure 2. The bare SiO2 beads exhibit hydrodynamic diameter of 666 nm, in agreement with the manufacturer reported bead size (0.676 μm; SD = 0.03 μm). After APTMS treatment, the bead diameter increases to 740 nm; and with poly(PFPA) treatment, the bead diameter further increases to 1889 nm. It is important to point out that the polydispersity index (PDI) for the poly(PFPA) grafted beads is rather large (PDI = 0.76), which is indicative of poor quality sample containing large aggregates. Although the DLS curve only shows one nano-sized peak, small amount of aggregates may be present in the suspension. The functionalized SiO2 beads are also examined by XPS to determine surface composition (Figure 3). Following APTMS treatment, N 1s peak associated with the amine groups on APTMS is detected. And, following poly(PFPA) treatment, F 1s peak associated with the PFP units on the polymer is detected. Together these data show the successful functionalization of the SiO2 surface, first with APTMS, then with poly(PFPA).
