Ein Protokoll für die Zubereitung von Poly (Pentafluorophenyl Acrylat) (poly(PFPA)) gepfropft Kieselsäure Perlen wird vorgestellt. Die poly(PFPA) funktionalisierten Oberfläche ist dann mit Antikörper immobilisiert und die Protein-Trennung durch Immunopräzipitation erfolgreich eingesetzt.
Methodenartikel
Vorbereitung der Poly(pentafluorophenyl acrylate) funktionalisiert SiO2 Perlen Proteinreinigung
Wird in einem neuen Tab geöffnet
In diesem Artikel
Erratum-Hinweis
Important: There has been an erratum issued for this article. View Erratum Notice
Zusammenfassung
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Zusammenfassung
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Wir zeigen eine einfache Methode um Poly (Pentafluorophenyl Acrylat) vorzubereiten (poly(PFPA)) gepfropft Kieselsäure Perlen für Antikörper Immobilisierung und nachfolgende Immunopräzipitation (IP) Anwendung. Die poly(PFPA) veredelte Oberfläche ist über einen einfachen Schritten bereit. Im ersten Schritt wird 3-Aminopropyltriethoxysilane (APTES) wie ein Linker-Molekül auf der Oberfläche Kieselsäure hinterlegt. In einem zweiten Schritt poly(PFPA) Homopolymer, über die reversible Addition und Fragmentierung Kette Transfer (FLOß) Polymerisation synthetisiert ist gepfropft an die Linker-Molekül durch die Reaktion der Austausch zwischen den Pentafluorophenyl (PFP) Einheiten auf die Polymer und Amin Gruppen auf APTES. Die Abscheidung des APTES und poly(PFPA) auf die Kieselsäure Teilchen sind durch Röntgen-Photoelektronen-Spektroskopie (XPS) bestätigt, sowie durch die Änderung der Größe der Partikel überwacht über dynamische Lichtstreuung (DLS) gemessen. Verbesserung der Oberfläche Hydrophilie der Perlen, partielle Substitution von poly(PFPA) mit Poly(ethylene glycol) Amin funktionalisiert wird auch (amino-PEG) durchgeführt. Die PEG ersetzt poly(PFPA) gepfropft Kieselsäure, die Perlen mit Antikörpern für IP-Anwendung dann immobilisiert sind. Zur Demonstration ein Antikörper gegen Proteinkinase RNA aktiviert (PKR) beschäftigt, und IP-Effizienz wird durch Western blotting bestimmt. Die Ergebnisse der Analyse zeigen, dass die Antikörper immobilisiert Perlen in der Tat lässt sich PKR zu bereichern, während unspezifische Protein-Interaktionen minimal sind.
Einleitung
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Reaktive Polymer Bürsten haben viel Interesse in den letzten Jahren erhalten. Sie können verwendet werden, um funktionelle Moleküle auf organische oder anorganische Materialien für aktivierte Oberflächen mit Anwendungen in Bereichen wie Erkennung und Trennung1,2,3,4zu immobilisieren, 5. Unter die reaktive Polymere berichtet sind jene mit Pentafluorophenyl Ester Einheiten besonders aufgrund ihrer hohen Reaktivität mit Aminen und Beständigkeit gegenüber Hydrolyse6. Eine solche Polymer....
Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Protokoll
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Vorbereitung des Poly(PFPA) Homopolymer
- Rekristallisation der Havariekommission
- Kombinieren Sie 5 g 2,2'-azobis(2-methylpropionitrile) (Havariekommission) mit 25 mL Methanol in einem 250 mL Becherglas. Tauchen Sie den Becher in ein Ölbad 60 ° C, dann rühren Sie kräftig die Mischung mit Stir Bar bis Havariekommission vollständig aufgelöst ist.
- Filtern Sie die warme Lösung durch Filterpapier (5-8 μm Partikel Retention) und speichern Sie das Filtrat bei 4 ° C langsam erlauben die Kristalle zu bilden.
- Sammeln Sie die umkristallisiert Havariekommission durch Filtration. Kombinieren Sie das gesammelte Produkt mit 25 mL frischem Met....
Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Ergebnisse
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Ein Schaltplan für die Zubereitung von poly(PFPA) gepfropft SiO2 Perlen, mit oder ohne PEG Substitution ist in Abbildung 1dargestellt. Zur Überwachung der APTES und poly(PFPA) Prozess, nackten SiO2 Perlen, Pfropfung APTES funktionalisiert SiO2 Perlen und poly(PFPA) gepfropft SiO2 Perlen von DLS (Abbildung 2) und XPS (Abbildung 3) gekennzeichnet sind. IP-Eff.......
Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Diskussion
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Die Synthese von poly(PFPA) gepfropft SiO2 Perlen in Abbildung 1dargestellt ist. Durch den Einsatz von APTES als Linker Molekül, können poly(PFPA) Bürsten kovalent gepfropft, SiO2 Substrat über einen einfachen Schritten zubereitet werden. Obwohl einige der PFP-Einheiten für die Reaktion mit APTES geopfert werden, sollen eine große Anzahl von PFP-Einheiten für spätere Reaktion mit amino-PEG oder Antikörper verfügbar bleiben. Die PFP-Gruppen sind dafür bekannt, niederener.......
Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Offenlegungen
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Die Autoren haben nichts preisgeben.
Danksagungen
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
Diese Arbeit wurde von der Agentur für Defense Development (Grant Nr. unterstützt. UD170039ID).
....Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Materialien
| Name | Unternehmen | Katalognummer | Kommentare |
|---|---|---|---|
| 2,2-Azobisisobutyroniril, 99% | Daejung Chemicals | 1102-4405 | |
| Methylalkohol für die HPLC, 99,9% | Duksan Pure Chemicals | d62 | |
| Phenylmagnesiumbromidlösung 1,0 M in THF | Sigma-Aldrich | 331376 | |
| Schwefelkohlenstoff wasserfrei, ≥ 99% | Sigma-Aldrich | 335266 | |
| Benzylbromid, 98 | %Sigma-Aldrich | B17905 | |
| Erdölether, 90% | Samchun Chemicals | P0220 | |
| Ethylether, 99% | Daejung Chemicals | 4025-4404 | |
| Magnesiumsulfat wasserfrei, Pulver 99% | Daejung Chemicals | 5514-4405 | |
| Pentafluorphenylacrylat | Santa Cruz Biotechnology | sc-264001 | enthält den Inhibitor |
| Aluminiumoxid, aktiviert, basisch, Brockmann I | Sigma-Aldrich | 199443 | |
| Natriumchlorid (NaCl) | Daejung Chemicals | 7548-4400 | |
| Anisol wasserfrei, 99,7% | Sigma-Aldrich | 296295 | |
| Siliziumdioxid-Nanopartikel | Mikropartikel GmbH | SiO2-R-0,7 | 5 % w/v wässrige |
| Suspension 3-Aminopropyltrimethoxysilan, >96,0 % | Tokyo Chemical Industry | T1255 | |
| Dimethylsulfoxid für die HPLC, ≥ 99,7% | Sigma-Aldrich | 34869 | |
| Aminoterminiertes Poly(ethylenglykol)- | methylether Polymerquelle | P16082-EGOCH3NH2 | |
| Phosphat gepufferte Kochsalztablette | Takara | T9181 | |
| Tween-20 | Calbiochem | 9480 | |
| Tris-HCl (pH 8,0) | Invitrogen | AM9855G | |
| KCl | Invitrogen | ||
| NP-40 | VWR | E109-50ML | |
| Glycerin | Invitrogen | 15514-011 | |
| Dithiothreitol | Biosesang | D1037 | |
| Proteaseinhibitor | Merck | 535140-1MLCN | |
| Bromo phenol blue | Sigma-Aldrich | B5525-5G | |
| Tris-HCl (pH 6,8) | Biosolution | BT033 | |
| Natriumdodecylsulfat | Biosolution | BS003 | |
| 2-Mercaptoethanol | Gibco | 21985-023 | |
| PKR Antikörper | Zellsignaltechnologie | 12297S | |
| GAPDH Antikörper | Santa Cruz Biotechnologie | sc-32233 | |
| Normale Kaninchen-IgG-Zellsignaltechnologie | 2729S | ||
| HeLa | Korea Cell Line Bank | 10002 | |
| Ultraschallgerät | DAIHAN Scientific | WUC-D10H | |
| Ultraschallgerät | BMBio | BR2006A | |
| Zentrifuge I | Eppendorf | 5424 R | |
| Zentrifuge II | LABOGENE | 1736R | |
| Rotator | FINEPCR | ROTATOR/AG | |
| Vakuumschrank | DAIHAN Scientific | ThermoStable OV-30 | |
| Gelpermeationschromatographie (THF) | Agilent Technologies | 1260 Infinity II | |
| Röntgen-Photoelektronenspektrometer | Thermo VG Scientific | Sigma Sonde | |
| Dynamische Lichtstreuung | Malvern Instruments | ZEN 3690 |
Referenzen
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
- 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.
Zugriff eingeschränkt. Bitte melden Sie sich an oder starten Sie eine Testversion, um diesen Inhalt anzuzeigen.
Nachdrucke und Genehmigungen
Genehmigung beantragen, um den Text oder die Abbildungen dieses JoVE-Artikels zu verwenden
Genehmigung beantragenErratum
Loading...
$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
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).