Un protocole pour la préparation de poly (acrylate de pentafluorophényl) (perles de silice greffée de poly(PFPA)) est présentée. La surface fonctionnalisés poly(PFPA) est ensuite immobilisée avec des anticorps et utilisée avec succès pour la séparation de la protéine par immunoprécipitation.
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Article de méthode
Préparation de Poly(pentafluorophenyl acrylate) fonctionnalisés SiO2 perles pour la Purification de protéines
Dans cet article
Avis d’erratum
Important: There has been an erratum issued for this article. View Erratum Notice
Résumé
Résumé
Nous démontrons une méthode simple pour préparer les poly (acrylate de pentafluorophényl) (poly(PFPA)) greffés perles de silice pour immobilisation des anticorps et l’application ultérieure immunoprécipitation (IP). La surface greffées poly(PFPA) est préparée par un processus en deux étapes simples. Dans un premier temps, 3-aminopropyltriethoxysilane (APTES) se dépose comme une molécule de l’éditeur de liens sur la surface de la silice. Dans la deuxième étape, poly(PFPA) homopolymère, synthétisé par l’addition réversible et polymérisation de transfert (RAFT) chaîne fragmentation, est greffée à la molécule de l’éditeur de liens par l’intermédiaire de la réaction d’échange entre les unités de pentafluorométhyl (PPP) sur le polymère et les groupes amine sur APTES. Les dépôts de APTES et poly(PFPA) sur la silice particules sont confirmées par spectrométrie de photoélectrons (XPS), ainsi que surveillés par le changement de taille de particules mesurées via la diffusion de la lumière dynamique (DLS). Pour améliorer la surface hydrophilicité des perles, substitution partielle de poly(PFPA) avec poly(ethylene glycol) amine fonctionnalisés (amino-PEG) est également réalisée. Le poly(PFPA) PEG-substitués greffés silice perles sont alors immobilisés avec des anticorps pour l’application de la propriété intellectuelle. Pour démonstration, un anticorps dirigé contre la protéine kinase activée par RNA (PKR) est employé, et efficacité de la propriété intellectuelle est déterminée par Western Blot. Les résultats d’analyse montrent que les perles d’anticorps immobilisé en effet peuvent servir à enrichir PKR, tandis que les interactions entre protéines non spécifiques sont minimes.
Introduction
Brosses de polymère réactif ont reçu beaucoup d’intérêt ces dernières années. Ils peuvent être utilisés pour immobiliser des molécules fonctionnelles sur les matières organiques ou inorganiques pour créer des surfaces activées avec des applications dans des domaines tels que la détection et la séparation1,2,3,4, 5. Parmi les polymères réactives signalés, ceux contenant des unités pentafluorophényl ester sont particulièrement utiles en raison de leur forte réactivité avec des amines et résistance envers l’h....
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Protocole
1. préparation des Poly(PFPA) homopolymère
- Recristallisation de AIBN
- Mélanger 5 g de 2,2'-azobis(2-methylpropionitrile) (AIBN) avec 25 mL de méthanol dans un bécher de 250 mL. Plonger le bécher dans un bain d’huile de 60 ° C, puis remuer vigoureusement le mélange avec une barre de remuer jusqu'à AIBN est entièrement dissous.
- Filtrer la solution chaude sur papier filtre (5-8 μm rétention de particules) et conserver le filtrat à 4 ° C pour permettre les cristaux à forme lentement.
- Recueillir l’AIBN recristallisé par filtration. Combiner le produit recueilli avec 25 mL de méthanol frais et répéter le processus de recristallisation.....
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Résultats
Un schéma pour la préparation des poly(PFPA) greffés SiO2 perles, avec ou sans PEG substitution est illustrée à la Figure 1. Pour surveiller les APTES et poly(PFPA) processus, nu SiO2 perles, la greffe selon fonctionnalisés SiO2 perles et poly(PFPA) greffés SiO2 perles sont caractérisés par des listes de distribution (Figure 2) et XPS (Figure 3). Efficacité.......
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Discussion
La synthèse de poly(PFPA) greffés SiO2 perles est illustrée à la Figure 1. En employant selon comme une molécule de l’éditeur de liens, brosses poly(PFPA) greffés par covalence à SiO2 substrat peuvent être préparés grâce à un processus en deux étapes simples. Même si certaines unités PPP sont sacrifiés pour la réaction avec APTES, un grand nombre des unités de PPP est censé rester disponible pour une réaction plus tard avec amino-PEG ou anticorps. Les groupes de PPP son.......
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Déclarations de divulgation
Les auteurs n’ont rien à divulguer.
Remerciements
Ce travail a été soutenu par l’Agence pour le développement de défense (Grant No. UD170039ID).
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Matériaux
| Nom | Entreprise | Numéro de catalogue | Commentaires |
|---|---|---|---|
| 2,2-Azobisisobutyronitrile, 99 % | Daejung Chemicals | 1102-4405 | |
| Alcool méthylique pour HPLC, 99,9 % | Duksan Pure Chemicals | d62 | |
| Solution de bromure de phénylmagnésium 1,0 ; M dans THF | Sigma-Aldrich | 331376 | |
| Disulfure de carbone anhydre, &ge ; 99 % | Sigma-Aldrich | 335266 | |
| bromure de benzyle, 98 % | Sigma-Aldrich | B17905 | |
| éther de pétrole, 90 % | Samchun Chemicals | P0220 | |
| éther éthylique, 99 % | Daejung Chemicals | 4025-4404 | |
| Sulfate de magnésium anhydre, poudre, etc. 99 % | Daejung Chemicals | 5514-4405 | |
| Acrylate de pentafluorophényle | Santa Cruz Biotechnology | sc-264001 | contient un inhibiteur |
| Oxyde d’aluminium, activé, basique, Brockmann I | Sigma-Aldrich | 199443 | |
| Chlorure de sodium (NaCl) | Daejung Chemicals | 7548-4400 | |
| Anisole anhydre, 99,7 | Sigma-Aldrich | 296295 | |
| Nanoparticules de silice | Microparticles GmbH | SiO2-R-0.7 | 5 % p/v suspension aqueuse |
| 3-Aminopropyltrimethoxysilane, >96.0 % | Tokyo Chemical Industry | T1255 | |
| Dimethyl sulfoxyde pour HPLC, &ge ; 99,7 % | Sigma-Aldrich | 34869 | |
| Éther méthylique poly(éthylène glycol) à terminaison aminographique | Source de polymère | P16082-EGOCH3NH2 | |
| Comprimé salin tamponné au phosphate | Takara | T9181 | |
| Tween-20 | Calbiochem | 9480 | |
| Tris-HCl (pH 8,0) | Invitrogen | AM9855G | |
| KCl | Invitrogen | ||
| NP-40 | VWR | E109-50ML | |
| Glycérol | Invitrogen | 15514-011 | |
| Dithiothreitol | Biosesang | D1037 | |
| Inhibiteur de protéase | Merck | 535140-1MLCN | |
| Bromo phenol blue | Sigma-Aldrich | B5525-5G | |
| Tris-HCl (pH 6,8) | Biosolution | BT033 | |
| Dodécylsulfate de sodium | Biosolution | BS003 | |
| 2-Mercaptoethanol | Gibco | 21985-023 | |
| Technologie de signalisation cellulaire d’anticorps | PKR | 12297S | |
| Anticorps GAPDH | Santa Cruz Biotechnology | sc-32233 | |
| de signalisation cellulaire | IgG de lapin normal | 2729S | |
| HeLa | Korea Cell Line Bank | 10002 | |
| Sonicator | DAIHAN Scientific | WUC-D10H | |
| Ultrasonateur | BMBio | BR2006A | |
| Centrifuge I | Eppendorf | 5424 R | |
| Centrifugeuse II | LABOGENE | 1736R | |
| Rotator | FINEPCR | ROTATOR/AG | |
| Four à vide | DAIHAN Scientific | ThermoStable OV-30 | |
| Chromatographie par perméation de gel (THF) | Agilent Technologies | 1260 Infinity II | |
| Spectromètre photoélectron à rayons X | Thermo VG Scientific | Sigma Probe | |
| Diffusion dynamique de la lumière | Malvern Instruments | ZEN 3690 |
Références
- 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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Réimpressions et autorisations
Erratum
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).
