Un protocolo para la preparación de poli (acrilato del pentafluorophenyl) (granos de sílice poly(PFPA)) injertado se presenta. La superficie de poly(PFPA) funcionalizados es inmovilizada con anticuerpos y utilizada con éxito para la separación de proteínas por inmunoprecipitación.
Se requiere una suscripción a JoVE para ver este contenido. Inicie sesión o comience su prueba gratuita.
Artículo de método
Preparación de Poly(pentafluorophenyl acrylate) funcionalizados SiO2 cuentas para purificación de proteínas
En este artículo
Aviso de erratum
Important: There has been an erratum issued for this article. View Erratum Notice
Resumen
Resumen
Demostrar un método simple para preparar polivinílico (acrilato del pentafluorophenyl) (poly(PFPA)) injertado granos de sílice para la inmovilización de anticuerpos y aplicación posterior de inmunoprecipitación (IP). La superficie injertada poly(PFPA) es preparada mediante un proceso de dos pasos simples. En el primer paso, 3 aminopropyltriethoxysilane (APTES) se deposita como una molécula de vinculador en la superficie de la sílice. En el segundo paso, poly(PFPA) homopolímero, sintetizado mediante la adición reversible y fragmentación cadena transferencia (balsa) la polimerización, se injerta a la molécula del vinculador mediante la reacción de intercambio entre las unidades del pentafluorophenyl (PFP) en el polímero y los grupos de la amina de APTES. La deposición de APTES y poly(PFPA) en el sílice partículas son confirmadas por espectroscopia de fotoelectrones de rayos x (XPS), así como supervisadas por el cambio de tamaño de partícula medido por dispersión dinámica de la luz (DLS). (Amino-PEG) también se realiza para mejorar el hydrophilicity superficial de los granos, sustitución parcial de poly(PFPA) con poly(ethylene glycol) amine functionalized. La poly(PFPA) PEG-sustituido injertado silicona granos luego se inmovilizan con anticuerpos para el uso de IP. Para la demostración, se emplea un anticuerpo contra la proteína quinasa RNA-activado (PKR) y eficiencia IP se determina por borrar occidental. Los resultados del análisis muestran que los granos de anticuerpo inmovilizado de hecho pueden utilizarse para enriquecer PKR mientras que las interacciones entre proteínas no específicas son mínimas.
Introducción
Cepillos poliméricos reactivos han recibido mucho interés en los últimos años. Pueden ser utilizados para inmovilizar moléculas funcionales en materiales orgánicos o inorgánicos para crear superficies activadas con aplicaciones en áreas tales como detección y separación1,2,3,4, 5. Entre los polímeros reactivos registrados, los que contienen unidades de ester pentafluorophenyl son particularmente útiles debido a su alta reactividad con aminas y resistencia hacia la hidrólisis6. Un ta....
Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Protocolo
1. preparación de homopolímero de Poly(PFPA)
- Recristalización de AIBN
- 5 g de 2,2'-azobis(2-methylpropionitrile) (AIBN) se combinan con 25 mL de metanol en un matraz de 250 mL. Sumerja el vaso en un baño de aceite de 60 ° C, luego agitar vigorosamente la mezcla con una barra de agitación hasta que se haya disuelto completamente AIBN.
- Filtrar la solución caliente a través de papel filtro (retención de partículas de 5-8 μm) y guardar el filtrado a 4 ° C para permitir que los cristales que forma lentamente.
- Recoger el AIBN recristalizada por filtración. Combinar el producto recogido con 25 mL de metanol fresco y repita el proceso de....
Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Resultados
Un esquema para la preparación de poly(PFPA) había injertado SiO2 granos, con o sin PEG sustitución se muestra en la figura 1. Para controlar las APTES y poly(PFPA) proceso de pelado SiO2 granos, el injerto APTES funcionalizados SiO2 granos y poly(PFPA) injertado SiO2 granos se caracterizan por DLS (figura 2) y XPS (figura 3). Eficiencias IP de las cuentas .......
Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Discusión
La síntesis de poly(PFPA) injertado SiO2 cuentas se ilustra en la figura 1. Mediante el empleo de APTES como molécula de vinculador, cepillos de poly(PFPA) covalente injertados a SiO2 substrato pueden prepararse mediante un proceso de dos pasos simples. Aunque algunas de las unidades de la PFP son sacrificados por la reacción con APTES, se espera que un gran número de las unidades de la PFP permanecer disponible para la reacción posterior con amino-PEG o anticuerpos. Se.......
Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Divulgaciones
Los autores no tienen nada que revelar.
Agradecimientos
Este trabajo fue financiado por la Agencia para el desarrollo de defensa (Grant no. UD170039ID).
....Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Materiales
| Nombre | Empresa | Número de catálogo | Comentarios |
|---|---|---|---|
| 2,2-Azobisisobutironitrilo, 99% | Daejung Chemicals | 1102-4405 | |
| Alcohol metílico para HPLC, 99.9% | Duksan Pure Chemicals | d62 | |
| Solución de bromuro de fenilmagnesio 1.0 M en THF | Sigma-Aldrich | 331376 | |
| Disulfuro de carbono anhidro, ≥ 99% | Sigma-Aldrich | 335266 | |
| Bromuro de bencilo, 98% | Sigma-Aldrich | B17905 | |
| Éter de petróleo, 90% | Samchun Chemicals | P0220 | |
| Éter etílico, 99% | Daejung Chemicals | 4025-4404 | |
| Sulfato de magnesio anhidro, en polvo, 99% | Daejung Chemicals | 5514-4405 | |
| Acrilato de pentafluorofenilo | Santa Cruz Biotechnology | sc-264001 | contiene inhibidor |
| Óxido de aluminio, activado, básico, Brockmann I | Sigma-Aldrich | 199443 | |
| Cloruro de sodio (NaCl) | Daejung Chemicals | 7548-4400 | |
| Anísol anhidro, 99.7% | Sigma-Aldrich | 296295 | |
| Micropartículas de nanopartículas de sílice | GmbH | SiO2-R-0.7 | 5% c/v suspensión acuosa |
| 3-aminopropiltrimetoxisilano, >96.0% | Industria química de Tokio | T1255 | |
| Dimetilsulfóxido para HPLC, ≥ 99.7% | Sigma-Aldrich | 34869 | |
| Éter metílico de poli(etilenglicol) terminado en amino | Fuente | de polímero P16082-EGOCH3NH2 | |
| Tableta salina tamponada con 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 | |
| Inhibidor de la proteasa | Merck | 535140-1MLCN | |
| Bromo azul de fenol | Sigma-Aldrich | B5525-5G | |
| Tris-HCl (pH 6.8) | Biosolución | BT033 | |
| Dodecil sulfato de sodio | Biosolución | BS003 | |
| 2-Mercaptoetanol | Gibco | 21985-023 | |
| PKR Tecnología de señalización celular | anticuerpos | 12297S | |
| Anticuerpo GAPDH | Santa Cruz Biotechnology | sc-32233 | |
| Tecnología de señalización celular | IgG de conejo normal | 2729S | |
| HeLa | Banco de líneas celulares de Corea | 10002 | |
| Sonicador | DAIHAN Scientific | WUC-D10H | |
| Ultrasonido | BMBio | BR2006A | |
| Centrífuga I | Eppendorf | 5424 R | |
| Centrífuga II | LABOGENE | 1736R | |
| Rotador | FINEPCR Horno | ||
| vacío | DAIHAN Scientific | ThermoStable OV-30 | |
| Cromatografía de permeación en gel (THF) | Agilent Technologies | 1260 Infinity II | |
| Espectrómetro de fotoelectrones de rayos X | Thermo VG Scientific | Sigma Probe | |
| Dispersión dinámica de la luz | Malvern Instruments | ZEN 3690 |
Referencias
- 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.
Acceso restringido. Inicie sesión o comience una prueba gratuita para ver este contenido.
Reimpresiones y permisos
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).
