Um protocolo para a preparação de poli (acrilato de pentafluorophenyl) (poly(PFPA)) enxertados grânulos de sílica é apresentado. A superfície funcionalizados poly(PFPA) é então imobilizada com anticorpos e usada com sucesso para a separação de proteínas através da imunoprecipitação.
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Artigo de método
Preparação de Poly(pentafluorophenyl acrylate) acrescida SiO2 grânulos para purificação de proteínas
Neste artigo
Aviso de errata
Important: There has been an erratum issued for this article. View Erratum Notice
Resumo
Resumo
Vamos demonstrar um método simples para preparar poli (acrilato de pentafluorophenyl) (poly(PFPA)) enxertados grânulos de sílica para imobilização de anticorpo e aplicação subsequente da imunoprecipitação (IP). A superfície enxertados poly(PFPA) é preparada através de um processo de duas etapas simples. Na primeira etapa, 3-aminopropyltriethoxysilane (APTES) é depositado como uma molécula de vinculador na superfície da sílica. Na segunda etapa, poly(PFPA) homopolímero, sintetizado através da adição reversível e polimerização de transferência (balsa) de cadeia de fragmentação, é enxertado à molécula de vinculador através da reação de troca entre as unidades de pentafluorophenyl (PFP) sobre o polímero e os grupos amina na APTES. A deposição de APTES e poly(PFPA) sobre a sílica, partículas são confirmadas por espectroscopia de fotoelétron de raios x (XPS), bem como monitoradas pela alteração de tamanho de partículas medido através de difusão dinâmica da luz (DLS). Para melhorar a superfície Hidrofilia dos grânulos, substituição parcial de poly(PFPA) com poly(ethylene glycol) acrescida de amina (amino-PEG) também é executada. O PEG-substituídos poly(PFPA) enxertados sílica grânulos então são imobilizados com anticorpos para aplicação de IP. Para demonstração, um anticorpo contra uma proteína quinase RNA-ativado (PKR) é empregado, e eficiência IP é determinada pela mancha ocidental. Os resultados da análise mostram que os grânulos de anticorpo imobilizado na verdade podem ser usados para enriquecer PKR enquanto interações não específicas da proteína são mínimas.
Introdução
Escovas de polímero reativo tem recebido muito interesse nos últimos anos. Eles podem ser usados para imobilizar moléculas funcionais em materiais orgânicos ou inorgânicos para criar superfícies registradas com aplicações em áreas como a deteção e a separação1,2,3,4, 5. Entre os polímeros reativos relatados, aqueles que contêm unidades de éster de pentafluorophenyl são particularmente útil devido a sua alta reatividade com aminas e resistência em direção a hidrólise6. Um tal polímer....
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Protocolo
1. preparação de homopolímero de Poly(PFPA)
- Recristalização de AIBN
- Combine 5 g de 2,2'-azobis(2-methylpropionitrile) (AIBN) com 25 mL de metanol em um copo de 250 mL. Mergulhe o béquer em banho de óleo 60 ° C e, em seguida, agitar vigorosamente a mistura com uma barra de agitação até AIBN é totalmente dissolvido.
- Filtrar a solução quente através de papel de filtro (5-8 μm retenção de partículas) e armazene o filtrado a 4 ° C, para permitir que os cristais de forma lenta.
- Recolha o AIBN recrystallized por filtração. Combinar o produto coletado com 25 mL de metanol fresco e repetir o processo de recristalização.
- O 2 x ....
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Resultados
Um esquema para a preparação de poly(PFPA) enxertado SiO2 grânulos, com ou sem PEG substituição é mostrada na Figura 1. Para monitorar o APTES e poly(PFPA) enxertia processo, desencapado SiO2 grânulos, APTES acrescida de SiO2 grânulos, e poly(PFPA) enxertados SiO2 miçangas caracterizam-se por DLS (Figura 2) e XPS (Figura 3). Eficiências IP dos grânulos são .......
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Discussão
A síntese de poly(PFPA) enxertados SiO2 grânulos é ilustrado na Figura 1. Empregando APTES como uma molécula de vinculador, escovas poly(PFPA) covalentemente enxertadas de SiO2 substrato podem ser preparadas através de um processo de duas etapas simples. Embora algumas das unidades de PFP são sacrificadas para a reação com APTES, um grande número de unidades a PFP deverão permanecer disponível para posterior reação com anticorpos ou amino-PEG. Os grupos PFP são conhecid.......
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Divulgações
Os autores não têm nada para divulgar.
Agradecimentos
Este trabalho foi financiado pela Agência para o desenvolvimento de defesa (Grant no. UD170039ID).
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Materiais
| Nome | Empresa | Número de catálogo | Comentários |
|---|---|---|---|
| 2,2-Azobisisobutironitrila, 99% | Daejung Chemicals | 1102-4405 | |
| Álcool metílico para HPLC, 99,9% | Duksan Pure Chemicals | d62 | |
| Solução de brometo de fenilmagnésio 1,0 M em THF | Sigma-Aldrich | 331376 | |
| Dissulfeto de carbono anidro, ≥ 99% | de benzila 335266 | Sigma-Aldrich | |
| , 98% | Éter de petróleo Sigma-Aldrich | B17905 | |
| , 90% | Samchun Chemicals | P0220 | |
| Éter etílico, 99% | Daejung Chemicals | 4025-4404 | |
| Sulfato de magnésio anidro, pó, 99% | Daejung Chemicals | 5514-4405 | |
| Pentafluorofenil acrilato | Santa Cruz Biotechnology | sc-264001 | contém inibidor |
| Óxido de alumínio, ativado, básico, Brockmann I | Sigma-Aldrich | 199443 | |
| Cloreto de Sódio (NaCl) | Daejung Chemicals | 7548-4400 | |
| Anisol anidro, 99,7% | Sigma-Aldrich | 296295 | |
| Micropartículas de nanopartículas de sílica | GmbH | SiO2-R-0.7 | 5% p/v suspensão aquosa |
| 3-Aminopropiltrimetoxissilano, >96.0% | Tokyo Chemical Industry | T1255 | |
| Dimetilsulfóxido para HPLC, ≥ 99,7% | Sigma-Aldrich | 34869 | |
| Éter metílico de poli (etilenoglicol) amino-terminado | Fonte de polímero | P16082-EGOCH3NH2 | |
| Comprimido de solução salina tamponada com fosfato | Takara | T9181 | |
| Tween-20 | Calbiochem | 9480 | |
| Tris-HCl (pH 8,0) | Invitrogen | AM9855G | |
| KCl | Invitrogen | ||
| NP-40 | VWR | E109-50ML | |
| Glicerol | Invitrogen | 15514-011 | |
| Dithiothreitol | Biosesang | D1037 | |
| Inibidor de protease | Merck | 535140-1MLCN | |
| Bromo fenol azul | Sigma-Aldrich | B5525-5G | |
| Tris-HCl (pH 6,8) | Biosolution | BT033 | |
| Dodecil sulfato de sódio | Biosolution | BS003 | |
| 2-Mercaptoethanol | Gibco | 21985-023 | |
| PKR Anticorpo | Celular | 12297S | |
| GAPDH Anticorpo | Santa Cruz Biotecnologia | SC-32233 | |
| Normal Rabbit IgG | Cell Signaling Technology | 2729S | |
| HeLa | Coreia Cell Line Bank | 10002 | |
| Sonicator | DAIHAN Scientific | WUC-D10H | |
| Ultrasonicator | BMBio | BR2006A | |
| Centrífuga I | Eppendorf | 5424 R | |
| Centrífuga II | LABOGENE | 1736R | |
| Rotador | FINEPCR | ROTATOR/AG | |
| Forno a vácuo | DAIHAN Cromatografia | ThermoStable OV-30 | |
| (THF) | Agilent Technologies | 1260 Espectrômetro | |
| Thermo VG Sonda | |||
| Espalhamento de luz dinâmico Malvern | Instruments | ZEN 3690 |
Referências
- 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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Reimpressões e permissões
Errata
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).
