Soft Matter Cite this: Soft Matter, 2011, 7, 9854
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Site specific protein immobilization into structured polymer brushes prepared by AFM lithography† Hendrik Wagner,‡a Yong Li,‡bc Michael Hirtz,d Lifeng Chi,*bc Harald Fuchsbc and Armido Studer*a Received 31st May 2011, Accepted 22nd August 2011 DOI: 10.1039/c1sm06013a
Protein immobilization into structured polymer brushes was achieved by AFM lithography. Dense polystyrene brushes with thicknesses larger than 40 nm show protein repellent properties whereas brushes with thicknesses smaller than 20 nm show the expected known protein adsorption behavior. Multi tip AFM lithography allows for large surface area patterning. Control of surface properties such as protein repellence, cell adhesion or corrosion protection by covering the surface with a thin polymer film is a heavily investigated research field.1–8 Properties of the surface can be tuned as a function of the molecular structures embedded in the film. In addition, surface properties can be further modified by structuring the surface with different patterning techniques. In particular structuring of polymer brushes has been studied along this line.9 Besides spin coating which leads to physiadsorbed polymer films showing restricted stability, stable polymer brushes can be prepared by covalent attachment of polymer chains to surfaces via either the ‘‘grafting from’’ or the ‘‘grafting to’’ approach.10 ‘‘Grafting from’’ processes, where polymerization is initiated by surface bound initiators, result in polymer brushes with high grafting densities. Due to its great functional group tolerance, surface initiated radical polymerization (SIP) has been often applied for the preparation of such brushes. In particular controlled radical polymerization which enables the preparation of dense polymer brushes with well defined brush thicknesses using the ‘‘grafting from’’ approach has been used.9 Different radical polymerization methods such as atom transfer radical polymerization (ATRP),11,12 reversible addition–fragmentation chain transfer (RAFT)13 and nitroxide-mediated polymerization (NMP)14–18 have been applied for the generation of polymer brushes
a
Organisch-Chemisches Institut, Westf€ alische Wilhelms-Universit€ at, Corrensstraße 40, 48149 M€ unster, Germany. E-mail: studer@ uni-muenster.de; Fax: +49-(0)251-83-36523; Tel: +49-(0)251-83-33291 b Physikalisches Institut, Westf€ alische Wilhelms-Universit€ at, WilhelmKlemm-Straße 10, 48149 M€ unster, Germany c Center for Nanotechnology (CeNTech), Heisenbergstraße 11, 48149 M€ unster, Germany. E-mail:
[email protected]; Fax: +49-(0)251-8333602; Tel: +49-(0)251-83-33651 d Institute of Nanotechnology, Karlsruhe Institute of Technology, and Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany † Electronic supplementary information (ESI) available: Experimental section. See DOI: 10.1039/c1sm06013a ‡ These authors contributed equally to this study.
9854 | Soft Matter, 2011, 7, 9854–9858
by SIP. As mentioned above, structuring of polymer brushes allows tailoring of surface properties.19 It is therefore obvious that different techniques for preparation of structured polymer brushes have been developed. Patterned polymer brushes can be generated by different top– down methods, such as photolithography,20 electron beam chemical lithography (EBCL),21–23 dip-pen nanolithography (DPN),24 microcontact printing (mCP)25,26 and bottom–up approaches such as Langmuir–Blodgett (LB) lithography.27 In addition, investigations on site selective mechanical tuning of polymer thin films have been performed. For example, nanoscratching, nanoshaving and nanowearing have been applied to create patterns on surface bound polymers.28–32 We recently presented a new ‘‘top–down’’ approach to prepare patterned polymer brushes applying mechanical nanoscratching by atomic force microscopy (AFM) lithography on polymer films.33 We showed that this method is well suited for structuring of polymer brushes prepared by surface initiated NMP (SINMP, Scheme 1), whereas moderate results were obtained by nanoscratching of spin coated polymer films. AFM lithography was also utilized to structure surfaces comprising conducting polymers in submicro/nanometer-scale.34 Polymer brushes have been used for the preparation of protein biochips, since proteins physiadsorb at hydrophobic polymers bound to surfaces.7,35,36 For example, structured protein biochips have been obtained by protein adsorption at polystyrene (PS) droplets, resulting from dewetting of PS thin films on poly(ethylene glycol) (PEG) modified surfaces.36 Moreover, AFM was utilized gaining protein patterns via DPN.37–42 Highly hydrophilic polymer brushes have been investigated as low-fouling biomaterials that show protein repellence.2,8 Size-exclusion effects of concentrated polymer brushes also play an important role in terms of protein repellence.3 Herein we report the site selective immobilization of proteins into nanostructured PS and poly(n-butyl acrylate) (PNBA) brushes generated by high-loading-force AFM-lithography. The polymer brushes used in the present study are readily prepared using SINMP on Si wafers containing a 300 nm oxide layer (Scheme 1). We will show that PS and PNBA brushes with brush thicknesses larger than 40 nm surprisingly exhibit protein repellent properties which is the basis for the site selective protein immobilization observed. For the generation of polymer brushes, initiator 1 was synthesized and subsequently immobilized at oxidized silicon surfaces by transsilyletherification resulting in wafers of type A (Scheme 1). This journal is ª The Royal Society of Chemistry 2011
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Scheme 1 Molecular structures of alkoxyamine initiators 1 and 2; TBTEP: 1-(tert-butyl)-3,3,5,5-tetraethylpiperazin-2-one (a). SIP by using NMP (b).
Details on the multistep synthesis of 1 and on the preparation of wafers A are provided in the ESI†. These functionalized silicon surfaces A were then used for polymerization. SINMP was conducted by placing the wafer A bearing alkoxyamine initiator based selfassembled monolayers (SAMs) in a neat monomer (styrene or nbutyl acrylate) in the presence of sacrificial alkoxyamine 2. PS brushes were prepared at 105 C, PNBA brushes at 125 C for 24 h. Addition of external alkoxyamine 2 was necessary to provide a sufficient concentration of nitroxide during SINMP which is necessary for a controlled nitroxide mediated polymerization.17 Moreover, with the help of 2 concomitant unbound polymer was formed, which was readily analyzed by gel permeation chromatography (GPC). This soluble polymer was used as a reference to estimate the chain length of the polymers bound to the surface (for details see the ESI†).17 The thickness of the polymer brushes was experimentally determined by AFM. The preparation of PS brushes using 0.063 mol% of 2 with respect to styrene resulted in PS brushes with a thickness of 50 nm. Surfaces with a PS thickness of 5 nm and 10 nm using wafers of type A were readily prepared by shortening the targeted chain length of the surface bound polymers by increasing the concentration of alkoxyamine 2. SIP in the presence of 0.4 mol% 2 provided dense PS brushes with around 5 nm thickness, a loading of 0.2 mol% 2 afforded 10 nm thick polymer brushes and 0.1 mol% 2 provided 22 nm thick brushes (see the ESI†). PS based surfaces have been widely used for nonspecific protein physiadsorption at surfaces.35,36 To study protein adsorption This journal is ª The Royal Society of Chemistry 2011
behavior of our surfaces, we treated the grafted PS brushes with buffered protein (fluorescence labeled) solution for 24 h at rt. After careful washing with water, samples were analyzed by fluorescence microscopy. First experiments were conducted on wafers bearing thick PS brushes (around 50 nm) and repeated at least two times. Initial studies were performed using a fluorescent dye tagged lectin (concanavalin A (Con A), rhodamine conjugate). To our surprise, we did not identify any protein adsorption at the PS-covered Si wafer (Fig. 1). Importantly, we also did not observe significant protein adsorption by fluorescence microscopy by switching from Con A to a fluorescent dye tagged streptavidin (Oyster-488 conjugate). This was surprising for us, since non-selective protein absorption to PS brushes is well established.35,36 We assumed that the high PS density in combination with the thickness of the PS layer might cause protein repellence. To further investigate this phenomenon, we analyzed protein adsorption on wafers bearing dense PS brushes with thicknesses of 5 nm, 10 nm and 40 nm, respectively. The 40 nm brushes also showed protein repellence. However, for the 5 and 10 nm systems, we observed protein adsorption for Con A as well as for streptavidin at the PS brushes, as expected for hydrophobic surfaces. We currently believe that protein repellence depends on the thickness of the PS brushes at least for the proteins studied herein. To further study this effect we rinsed wafers containing PS brushes with a thickness greater than 40 nm with a fluorescent dye tagged bovine serum albumin (BSA, rhodamine conjugate) and observed some adsorption for this particular protein. However, qualitative analysis shows that protein adsorption is far lower as compared to the results of adsorbing BSA on PS brushes with a thickness of 5 nm or 10 nm (Fig. 1). We believe that for PS brushes with thicknesses larger than 40 nm small amounts of water can enter the surface brush layer rendering the surface protein repulsive. Unfortunately water contact angle (CA) measurements of 40 nm and 10 nm PS brushes did not show a significant difference (43 nm PS brushes: CA(adv) ¼ 87.7 1.4 ; CA(rec) ¼ 77.4 0.9 ; 13 nm PS brushes: CA(adv) ¼ 86.5 1.4 ; CA(rec) ¼ 75.7 1.5 ), which can be explained generally by the poor
Fig. 1 Adsorption studies: adsorption of Con A on 5 nm PS brushes (a), on 10 nm PS brushes (b) and on 50 nm PS brushes (c). Adsorption of streptavidin on 5 nm PS brushes (d), on 10 nm PS brushes (e) and on 50 nm PS brushes (f). Adsorption of BSA on 5 nm PS brushes (g), on 10 nm PS brushes (h) and on 50 nm PS brushes (i). Scale bars for a–i: 10 mm.
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solubility of PS in water and the corresponding collapsed state of PS brushes in aqueous solution. Also FT–ATR–IR analyses of the surfaces did not reveal any changes in that regard. For PS brushes with thicknesses of 5 to 10 nm the chains at the surface layer are more densely packed and hence uptake of water (even small amounts) is likely more difficult: the hydrophobic surface then shows the expected and known protein adsorption properties. All polymer brushes, prepared for our study, show high grafting densities (see ESI, Table S1†), which should prohibit protein penetration into the polymer brushes.3 Therefore, recognition processes depending on the layer thickness is either due to interactions across the brush layer or due to a structural change of the interface of thicker PS brushes. Structural changes can be caused by radical termination reactions during the polymerization process, which are more likely for reduced external initiator concentrations or extended polymerization times as used for 40 nm PS brushes. We also performed protein adsorption studies as control experiments using spin coated PS at silicon surfaces bearing thicknesses of 110 nm and 300 nm respectively (see the ESI†). In both cases we observed the expected protein adsorption. As previously shown, AFM lithography on grafted polymer brushes allows for ready generation of nanostructured surfaces.33 Based on the results discussed above, we were encouraged to investigate site selective immobilization of proteins on structured PS brushes. As proteins belong to polar macromolecules they should show high adsorption to the scratched areas on the wafer which likely consists of polar SiOH moieties. Moreover, the brush areas, if thickness is large enough (>40 nm), should show protein repellence, thus leading to site selective protein adsorption at the nanoscale. This approach would represent an easy access to the preparation of biochips (Fig. 2). AFM lithography with single tips (silicon tapping mode cantilevers, k z 42 N m1) was performed on a Dimension 3000 AFM. Operating in contact mode and at a set point of 5 V yielded a loading force of about 22 mN and allowed for a reproducible and reliable lithography process (Fig. 2). We first treated structured PS brushes with the fluorescent dye labeled Con A, streptavidin and BSA for 24 h at rt. We used polymer brushes with a thickness greater than 40 nm to prevent adsorption to the polymeric regime as discussed above. Pleasingly, we observed site selective protein immobilization into the scratched out areas of the polymer brushes after extensive washing of the wafer. We tested different concentrations of Con A and washed the samples by ultrasonication or under mild rinsing conditions. All variations did not lead to a different outcome. However, selective immobilization was not achieved for structured PS wafers with a brush thickness of around 22 nm. Therefore, we assume that selectivity is mainly caused by the protein repellent properties of the thick PS brush area. In agreement with the results reported for non-structured wafers, the selectivity for immobilization of BSA into such nanostructures is not ideal since BSA also adsorbed at the polymeric PS regime (>40 nm) to some extent (Fig. 2c). Based on these results we sequentially conducted AFM lithography and protein immobilization. In a tandem process we first treated structured PS brushes with Con A and after a second AFM scratching we subsequently treated the resulting surface with streptavidin. We detected both proteins at the surface after two runs of protein adsorption showing that our approach allows for immobilization of two different proteins at the nanoscale (Fig. 2e and f). 9856 | Soft Matter, 2011, 7, 9854–9858
Fig. 2 Immobilization of Con A (a), streptavidin (b) and BSA (c) into PS nanostructures. AFM image of PS brush patterns (d). Sequential protein immobilization (e); scale bars: 2 mm. Schematic outline of protein immobilization into AFM structured polymer brushes (f).
We then decided to test whether selective immobilization of proteins can also be achieved on structured PNBA brushes. To this end, we prepared grafted PNBA brushes with a thickness greater than 40 nm (CA see the ESI†). After AFM lithography we placed the structured PNBA brushes in buffered solutions of Con A, streptavidin and BSA and observed selective protein immobilization into the nanostructures of PNBA brushes as well (Fig. 3). We did not see any detectible difference in immobilization of Con A and streptavidin by switching to the acrylate brushes. However, the PNBA brushes showed higher selectivity for site specific immobilization of the relatively unselective BSA as compared to BSA immobilization into structured PS brushes (compare Fig. 2c with Fig. 3c). Therefore, we recommend the use of more hydrophobic structured PNBA brushes for selective immobilization of more unselective proteins. Thanks to AFM lithography, basically any pattern can be written into the brush surface. In the case of the immobilization of streptavidin on PNBA brushes we created the logo of the University of M€ unster by AFM lithography with subsequent coloration by selective protein immobilization (Fig. 3b). To investigate protein–protein interactions and therefore the nativity of the immobilized proteins we treated patterned PNBA brushes first with non-fluorescent BSA for 24 h at rt and subsequently This journal is ª The Royal Society of Chemistry 2011
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adhesion was observed for brushes with thicknesses of 5 nm and 10 nm using Con A and streptavidin as model proteins. Further mechanistic investigations for a better understanding of the brush thickness dependent protein repellency is ongoing. Moreover, we successfully patterned PS and PNBA brushes (thickness > 40 nm) by AFM lithography. Patterned polymer brushes showed site specific adsorption of Con A, streptavidin and BSA into the scratched areas. BSA was additionally detected by FTIC conjugated sheep anti-BSA, as a proof for the nativity of the immobilized proteins within the nanopatterns. Our study represents an efficient novel approach to the generation of protein biochips. Furthermore, we presented a method for large area fabrication of nanostructures on polymer brushes. Parallel pattern writing was achieved by a cantilever array resulting in structured PS and PNBA polymer brushes that show highly selective protein adsorption over large areas (Fig. 3g–i).
Acknowledgements Fig. 3 Immobilization of Con A (a); scale bar: 2 mm, streptavidin (b); scale bar: 5 mm and BSA (c); scale bar: 2 mm, into PNBA nanostructures. Detection of BSA at PNBA patterns via FTIC (Fluorescein isothiocyanate) conjugated sheep anti-BSA (d); scale bar: 2 mm. FTIC conjugated sheep anti-BSA treated PNBA patterns (e: 2 h at 4 C; scale bar: 10 mm. f: 24 h at rt; scale bar: 2 mm). Immobilization of streptavidin in nanostructures generated by a cantilever array using AFM lithography towards PS brushes (g, h) and PNBA brushes (i); scale bars for g–i: 10 mm.
with FTIC conjugated sheep anti-BSA for 2 h at 4 C. After washing under mild rinsing conditions we observed the immobilization of the antibody into the nanostructures generated by AFM lithography (Fig. 3d). We incubated patterned PNBA brushes only with FTIC conjugated sheep anti-BSA for 2 h at 4 C as a control experiment. We did not observe any adsorption of the antibody in that case, which clearly shows that BSA remains in the native shape (Fig. 3e) and therefore protein–protein interactions are possible within the nanostructures at least to some extent in this particular case. As expected, incubating structured PNBA brushes under the general conditions (24 h at rt) with the FTIC conjugated sheep anti-BSA leads to protein immobilization into the patterns (Fig. 3f). Furthermore, we used a cantilever array for multi tip AFM lithography to generate biochips with nanopatterns over a large area. To this end, we utilized multiple tips of an AFM system (DPN 5000 system, NanoInk, Skokie, USA) to perform parallel writing. Since the cantilever arrays were designed for contact mode application with small constant k (0.5 N m1), we were able to apply a sufficient pressure to the tips to provide a reasonable force and therefore achieve an effective removal of the polymer brushes, relying on visual inspection of the cantilever deflection. We subsequently treated the prestructured polymer brushes with streptavidin for 24 h at rt and obtained various large area protein patterns at PS and PNBA brushes, which was readily confirmed by fluorescence microscopy (Fig. 3g–i).
Conclusion In conclusion we demonstrated that PS brushes as well as PNBA brushes prepared by NMP show either protein adsorption or repellence properties depending on their brush thickness: with a thickness larger than 40 nm they are protein repellent. However, protein This journal is ª The Royal Society of Chemistry 2011
We thank the Deutsche Forschungsgemeinschaft (DFG) and the European Science Foundation for funding our research within the SFB 858 and FP7-PEOPLE-2009-IRSES/247641. This work was partly carried out with the support of the Karlsruhe Nano Micro Facility (KNMF, www.knmf.kit.edu), a Helmholtz Research Infrastructure at Karlsruhe Institute of Technology (KIT, www.kit.edu)
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