Langmuir 2007, 23, 4465-4471
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Bioactive Surface Modification of Mica and Poly(dimethylsiloxane) with Hydrophobins for Protein Immobilization Ming Qin, Li-Kai Wang, and Xi-Zeng Feng* The Key Laboratory of BioactiVe Materials, Ministry of Education, College of Life Science, Nankai UniVersity, Tianjin 300071, PR China
Yan-Lian Yang, Rui Wang, and Chen Wang* National Center for Nanoscience and Technology, Beijing, 100080, PR China
Lei Yu, Bin Shao, and Ming-Qiang Qiao* Institute of Molecular Biology, College of Life Science, Nankai UniVersity, Tianjin 300071, PR China ReceiVed September 20, 2006. In Final Form: January 23, 2007 Bioactive surfaces with appropriate hydrophilicity for protein immobilization can be achieved by hydrophobin II (HFBI) self-assembly on mica and polydimethylsiloxane (PDMS) surfaces. X-ray photoelectron spectroscopy and water contact angle measurements illustrated that the surface wettability can be changed from superhydrophobic (PDMS) or superhydrophilic (mica) to moderately hydrophilic, which is suitable for protein (chicken IgG) immobilization on both substrate surfaces. The results suggest that HFBI assembly, one kind of hydrophobin from Trichoderma reesei, may be a versatile and convenient method for the immobilization of biomolecules on diverse substrates, which may have potential applications in biosensors, immunoassays, and microfluidic networks.
1. Introduction Modification of solid substrates and the subsequent immobilization of biomolecules are relevant to many areas in research applications. Surface-bound molecules are applied in biosensors, DNA microarrays, protein chips, cell culturing, biomolecule interaction investigations, and so forth. Mica1 and polydimethylsiloxane (PDMS)2 are the two main kinds of substrates in the patterning of biomolecules. Because it is the most readily available surface with atomic-scale flatness, freshly cleaved mica is a promising substrate for patterning applications. PDMS is a kind of soft polymer with attractive physical and chemical properties: elasticity, optical transparency, flexible surface chemistry, low permeability to water, low toxicity, and low electrical conductivity. Thus, it has been widely used in microfluidic devices and microcontact printing technology. Biomolecules, such as proteins, could not be immobilized on mica surfaces effectively without surface modification. Because they could retain protein activity for a long time and obtain less unspecific binding,3 hydrophilic surfaces are useful supports for protein immobilization, except for specific cases such as lipases.4 Hence, how to reduce the hydrophobicity of PDMS is a big obstacle for its application in biotechnology. Several methods have been reported to change the surface properties of mica and * Corresponding authors. (X.-Z.F.) E-mail:
[email protected]. Tel/ Fax: 86-22-23507022. (C.W.) E-mail:
[email protected]. Tel/Fax: 8610-62562871. (1) (a) Sarno, D. M.; Murphy, A. V.; DiVirgilio, E. S.; Jones, W. E.; Ben, R. N. Langmuir 2003, 19, 4740. (b) Pie´trement, O.; Pastre´, D.; Fusil, S.; Jeusset, J.; David, M. O.; Landousy, F.; Hamon, L.; Zozime, A.; Cam, E. L. Langmuir. 2003, 19, 2536. (c) Wang, L. K.; Feng, X. Z.; Hou, S.; Chan, Q. L.; Qin, M. Surface Interface Anal. 2006, 38, 44. (2) (a) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8. (b) Takayama, S.; Ostuni, E.; Qian, X. P.; McDonald, J. C.; Jiang, X. Y.; LeDuc, P.; Wu, M. H.; Ingber, D. E.; Whitesides, G. M. AdV. Mater. 2001, 13, 570. (c) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461. (3) Kusnezow, W.; Hoheisel, J. D. J. Mol. Recognit. 2003, 16, 165.
PDMS for protein immobilization, including surface oxidation and chemical modification.1b,c,5 The chemical modification of mica using a silanization reagent has shown great importance in AFM studies on biomolecules.6 Surface oxidation became a necessary pretreatment of PDMS when applied in microfluidic devices. However, some limitations existed in these methods; for example, the lower biocompatibility after chemical treatments is not always desirable in biodetections and immunoassays.2c,7 Here, one novel and simple method with hydrophobin II (HFBI) to modify both mica and PDMS surfaces for protein immobilization was developed, which might overcome these limitations. Hydrophobins,8 which are surface-active proteins, could improve the physical and chemical properties of solid supports and might immobilize proteins at surfaces simultaneously. Hydrophobins were first discovered in 1991 in filamentous fungi.9 They play vital roles in the growth and morphology of filamentous fungi, which are mostly linked to surface-chemical properties.10 For example, they can be coated on the surface of spores and (4) (a) Bastida, A.; Sabuquillo, P.; Armisen, P.; Ferna´ndez-Lafuente, R.; Huguet, J.; Guisa´n, J. M. Biotechnol. Bioeng. 1998, 58, 486. (b) Palomo, J. M.; Mun˜oz, G.; Ferna´ndez-Lorente, G.; Mateo, C.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M. J. Mol. Catal. B: Enzym. 2002, 19-20, 279. (c) Palomo, J. M.; Mun˜oz, G.; Ferna´ndezLorente, G.; Mateo, C.; Fuentes, M.; Guisan, J. M.; Ferna´ndez-Lafuente R. J. Mol. Catal. B: Enzym. 2003, 21, 201. (5) (a) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974. (b) Xiao, D. Q.; Zhang, H.; Wirth, M. Langmuir 2002, 18, 9971. (c) Lee, S.; Vo¨ro¨s, J. Langmuir 2005, 21, 11957. (6) Umemura, K.; Ishikawa, M.; Kuroda, R. Anal. Biochem. 2001, 290, 232. (7) Graham, D. J.; Price, D. D.; Ratner, B. D. Langmuir 2002, 18, 1518. (8) Kong, X. Q.; Grabitz, R. G.; Oeveren, W. V.; Kleec, D.; van Kootenb, T. G., Freudenthala, F.; Qing, Ma.; von Bernutha, G.; Seghayea, M. C. Biomaterials 2002, 23, 1775. (9) Wessels, J. G. H.; De Vries, O. M. H.; A Ä sgeirsdo´ttir, S. A.; Schuren, F. H. H. Plant Cell 1991, 3, 793. (10) (a) Wo¨sten, H. A. B. Annu. ReV. Microbiol. 2001, 55, 625. (b) Linder, M. B.; Szilvay, G. R.; Nakari-Seta¨la¨, T.; Penttila¨, M. E. FEMS Microbiol. ReV. 2005, 29, 877.
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mycelium.11 Furthermore, hydrophobins secreted from pathogenic fungi can help them attach to their hosts, such as on insect cuticles or plant leaves.12 Hydrophobins are fairly small (7-15 kDa) and share a characteristic pattern of Cys residues in their primary sequence.11b According to the difference in solubility of the aggregates, hydrophobins can be divided into classes I and II.13 Class I hydrophobins, such as SC3 from Schizophyllum commune, often form random patterns of nanoscopic rodlets and their bundles on surfaces.14 Class II hydrophobins, such as HFBI and HFBII from Trichoderma reesei,15 have been found to form highly ordered monolayer films and crystalline fibrils.15a,16 Besides, both class I and class II hydrophobins can interact with each other in their assemblies.17 Because of their surface adhesion and self-assembly abilities, hydrophobins are very attractive for adjusting the physiochemical properties of solid surfaces.18 Accordingly, they can be used as adhesion bindings for the immobilization of proteins onto solid substrates,14a,19 tags in fusion proteins for affinity purification,20 and coatings to improve the patterning of cells on the biomaterials.18a,21 Recently, hydrophobins have been studied as one kind of intermediate to immobilize proteins on solid surfaces. It is demonstrated that hydrophobins could cause enzymes to efficiently immobilize to hydrophobic surfaces covalently as a fusion protein19a or noncovalently and preserve the activity.22 Moreover, it has also been reported that lipase can be immobilized on agarose-bound hydrophobins, which results in increased lipase activity and stability.19b Thus, hydrophobins are probably an alternative method for protein immobilization on solid surfaces. It is still challenging to improve the wettability of PDMS and immobilize protein effectively on mica and PDMS surfaces. Here, we report a novel method to modify both the hydrophobic PDMS surface and the hydrophilic mica surface to moderate hydrophilicity for protein immobilization with hydrophobins II (HFBI). The surface modification of mica and PDMS with HFBI is investigated with X-ray photoelectron spectroscopy (XPS) (11) (a) Kershaw, M. J.; Talbot, N. J. Fungal Genet. Biol. 1998, 23, 18. (b) Wo¨sten, H. A., de Vocht, M. L. Biochim. Biophys. Acta 2000, 1469, 79. (12) Ebbole, D. J. Trends Microbiol. 1997, 5, 405. (13) Wessels, J. G. H. Annu. ReV. Phytopathol. 1994, 32, 413. (14) (a) Scholtmeijer, K.; Janssen, M. I.; Gerssen, B.; de Vocht, M. L.; van Leeuwen, B. M.; van Kooten, T. G.; Wo¨sten, H. A.; Wessels, J. G. Appl. EnViron. Microbiol. 2002, 68, 1367. (b) de Vocht, M. L.; Reviakine, I.; Wo¨sten, H. A.; Brisson, A.; Wessels, J. G.; Robillard, G. T. J. Biol. Chem. 2000, 275, 28428. (c) Paananen, A.; Vuorimaa, E.; Torkkeli, M.; Penttila, M.; Kauranen, M.; Ikkala, O.; Lemmetyinen, H.; Serimaa, R.; Linder, M. B. Biomacromolecules 2003, 4, 956. (d) de Vocht, M. L.; Scholtmeijer, K.; van der Vegte, E. W.; de Vries, O. M. H.; Sonveaux, N.; Wo¨sten, H. A. B.; Ruysschaert, J. M.; Hadziioannou, G.; Wessels, J. G. H.; Robillard, G. T. Biophys. J. 1998, 74, 2059. (15) (a) Torkkeli, M.; Serimaa, R.; Ikkala, O.; Linder, M. B. Biophys. J. 2002, 83, 2240. (b) Hakanpa¨a¨, J.; Paananen, A.; Askolin, S.; Nakari-Seta¨la¨, T.; Parkkinen, T.; Penttila¨, M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2004, 279, 534. (16) (a) Paananen, A.; Vuorimaa, E.; Torkkeli, M.; Penttila¨, M.; Kauranen, M.; Ikkala, O.; Lemmetyinen, H.; Serimaa, R.; Linder, M. B. Biochemistry 2003, 42, 5253. (b) Ritva, S.; Torkkeli, M.; Paananen, A.; Linder, M. B.; Kisko, K.; Knaapila, M.; Ikkala, O.; Vuorimaa, E.; Lemmetyinend, H.; Seecke, O. J. Appl. Crystallogr. 2003, 36, 499. (17) Askolin, S.; Linder, M. B.; Scholtmeijer, K.; Tenkanen, M.; Penttila, M.; de Vocht, M. L.; Wo¨sten, H. A. B. Biomacromolecules 2006, 7, 1295. (18) (a) Janssen, M. I.; van Leeuwen, M. B. M.; Scholtmeijer, K.; van Kooten, T. G.; Dijkhuizen, L.; Wo¨sten, H. A. B. Biomaterials 2002, 23, 4847. (b) Wessels, J. G. H. AdV. Microb. Physiol. 1997, 38, 1. (c) Kisko, K.; Torkkeli, M.; Vuorimaa, E.; Lemmetyinen, H.; Seeck, O. H.; Linder, M. B.; Serimaa, R. Surf. Sci. 2005, 584, 35. (19) (a) Linder, M. B.; Szilvay, G. R.; Nakari-Seta¨ la¨, T.; So¨derlund, H.; Penttila¨, M. Protein Sci. 2002, 11, 2257. (b) Palomo, J. M.; Penˇas, M. M.; Ferna´ndezLorente, G.; Mateo, C.; Pisabarro, A. G.; Ferna´ndez-Lafuente, R.; Ramı´rez, L.; Guisa´n, J. M. Biomacromolecules 2003, 4, 204. (20) Collen, A.; Persson, J.; Linder, M. B.; Nakari-Seta¨la¨, T.; Penttila¨, M.; Tjerneld, F.; Sivars, U. Biochim. Biophys. Acta 2002, 1569, 139. (21) Janssen, M. T.; van Leeuwen, M. B.; van Kooten, T. G.; de Vries, J.; Dijkhuizena, L.; Wo¨sten, H. A. B. Biomaterials 2004, 25, 2731. (22) Corvis, Y.; Walcarius, A.; Rink, R.; Mrabet, N. T.; Rogalska, E. Anal. Chem. 2005, 77, 1622.
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and water contact angle (WCA) measurements. The following protein immobilization is carried out on hydrophobin-coated surfaces using microcontact printing (µCP) technology with a PDMS stamp.23 The results suggest that HFBI assembly may be a versatile and convenient method for the immobilization of biomolecules on diverse substrates, which has potential applications in biosensors, immunoassays, and microfluidic networks. 2. Experimental Section 2.1. Materials. A PDMS elastomer kit (Sylgard 184, Dow Corning), curing agent, bovine serum albumin (BSA) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma. Chicken immunoglobulin (IgG) was used as an antigen. Fluorescein isothiocyanate (FITC)-labeled antichicken IgG, as an antibody, was developed in rabbits. Chicken IgG and FITC-labeled antichicken IgG were also purchased from Sigma. The details of the cultivation and isolation of hydrophobin II HFBI from Trichoderma reesei VTT D-98692 can be found in the Supporting Information. Aqueous solutions were prepared with redistilled water. 2.2. HFBI Assembly on Solid Substrates. 2.2.1. HFBI Assembly on Mica Surfaces. Mica that was freshly cleaved with a stainless steel razor blade24 was incubated for 20 min at 20 °C in an aqueous solution (100 µg mL-1) of hydrophobin HFBI, allowing the protein to adhere to the substrate surface. After blowing off the excess solution, the sheets were dried with nitrogen. 2.2.2. HFBI Assembly on PDMS Surfaces. Silicon elastomer and the curing agent (10:1 wt/wt) were poured onto the glass substrate after being mixed sufficiently and cured at 60 °C overnight. After being peeled off of the glass substrate, the PDMS substrate was rinsed with water several times and ultrasonicated in water for 10 min, followed by washing in ethanol solution (75 vol % ethanol in water) and drying with nitrogen.2a The HFBI assembly procedure is the same as that on the mica surface. 2.3. X-ray Photoelectron Spectroscopy (XPS). The XPS characterization of bare or hydrophobin-coated substrates was performed on an XPS apparatus (PHI-5300, Phi) employing a monochromatic Mg KR radiation source (1253.6 eV). The survey scan range was 0-1100 eV, and the electron takeoff angle was fixed at 45°. The energy resolution of the analyzer was 0.8 eV, and the sensitivity was 80-1600 kcps. The peaks in the elemental corelevel spectra were fitted using UNIX on an Apollo Domain series 3500. 2.4. Wettability Measurements. The water contact angle measurements were carried out with 5 µL water droplets at ambient temperature with an optical contact angle meter (Dataphysics Inc, OCA20). The WCA values were averaged from three measurements at different locations. 2.5. Microcontact Printing (µCP) of Proteins on the HFBICoated Substrates.1c,25 2.5.1. Fabrication of the PDMS Stamp. The PDMS stamp for µCP was prepared by curing a mixture of liquid PDMS and cross-linker (10:1 wt/wt) on the silicon master at 60 °C overnight. After being peeled off, flexible transparent stamps with patterns of about 100 µm were obtained by replicating the micropatterned silicon master.26 2.5.2. Chicken IgG Immobilization on Mica Surfaces. For mica, chicken IgG was immobilized on the HFBI-coated substrate by µCP, as shown in Scheme 1a. The PDMS stamp was cleaned several times with distilled water and then rinsed in 75% ethanol solution prior to use. The stamp surface was “inked” in chicken IgG solution for 2 min. Excess solution on the stamp was removed with a piece of lens paper. With the stamp gently pressed on the solid substrate, (23) (a) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551. (b) Quake, S. R.; Scherer, A. Science 2000, 290, 1536. (24) Wang, L.; Wang, E. K. Langmuir 2004, 20, 2677. (25) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. (26) (a) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. AdV. Mater. 2000, 12, 1067. (b) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225.
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Scheme 1. Schematic Illustration of the HFBI Coating on Different Surfaces and the Immunoassay on HFBI-Coated Surfacesa
a
HFBI coating and immunoassay with the µCP method on the mica surface and HFBI coating and immunoassay on the PDMS surface.
the chicken IgG molecules could be transferred directly from the PDMS stamp to the mica surface in a few seconds. 2.5.3. Chicken IgG Immobilization on the PDMS Surface. Because PDMS was not rigid enough and was not easy to metamorphose during µCP experiments, an alternative way was developed to pattern proteins. Cleaned copper TEM grids were pressed through a piece of glass to keep better contact between the grids and PDMS surfaces, as shown in Scheme 1b. The PDMS surface with grids was coated with HFBI using the same method as described in section 2.2.1. The copper TEM grids were carefully peeled off of the PDMS surface. This step must be done carefully so as not to destroy the pattern of chicken IgG on the surface. 2.5.4. FITC-Labeled Antichicken IgG Immobilization. The HFBImodified mica and PDMS surfaces with patterned chicken IgG immobilization were covered with BSA solution for 20 min to block the unprinted areas. Then the substrates were washed with distilled water about three to four times and dried with nitrogen. The surfaces with patterned antigens were incubated with FITC-labeled antichicken IgG for about 2 min and rinsed with water. After being dried with nitrogen, the printed stripes or dots could be observed with inverted fluorescence microscopy (Nikon TE2000-U, CCD-Rtke, Japan). Because the immunoassay on PDMS was almost the same as the traditional µCP method, except for the method of patterning antigens, it can be considered to be an alternative method of µCP. Image analysis was carried out with Labworks 4.5 software (UVP, Inc., Upland, CA). All proteins were dissolved in phosphate-buffered saline solution (PBS) at pH 7.4. The pH of the water used for washing was 7.4. The
concentrations of chicken IgG and FITC-labeled antichicken IgG were 200 µg/mL. The concentration of BSA used as a blocking reagent was 10 mg/mL.
3. Results and Discussion The pretreated mica and PDMS were incubated in HFBI solutions for surface modification, and the modified samples were characterized by XPS, WCA, and immunoassay using µCP. 3.1. Characterization of HFBI Coating by X-ray Photoelectron Spectroscopy. Figure 1a illustrates the full XPS spectra of the bare and HFBI-coated mica surfaces on the same scale, and Figure 1b illustrates the core XPS spectra of C 1s, N 1s, O 1s, and Si 2p on the bare and HFBI-coated mica. As shown in Figure 1a, a higher-intensity O 1s peak (534 eV) associated with lower-intensity peak series of Si 2s (157 eV), Si 2p (106 eV), Al 2s (123 eV), and Al 2p (78 eV) represents a characteristic XPS spectrum of the mica surface. The modification of the HFBI protein led to the intensity decrease of the Si 2s and Si 2p peaks and the signal increase of the N 1s peak (400 eV). Obviously, the element Si belongs only to the mica surface, whereas the element N belongs only to the amine group of the protein HFBI. Therefore, the increase in the N 1s signal and the decrease in the Si 2s and Si 2p signals confirm that HFBI has covered the mica surface to a greater extent. This can also be supported by the increase in the C 1s peak and decreases in the O 1s, Al 2s, and
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Figure 1. XPS spectra of mica surfaces before and after modification on the same scale: (a) full spectra and (b) XPS spectra of N 1s, C 1s, O 1s, and Si 2p.
Figure 2. XPS spectra of PDMS surfaces before and after modification on the same scale: (a) full spectra and (b) XPS spectra of N 1s, C 1s, Si 2p, and S 2p.
Table 1. XPS Determination of the Relative Atomic Compositions on the Mica, HFBI-Modified Mica, PDMS, and HFBI-Modified PDMS Surfaces sample type bare mica HFBI-modified mica bare PDMS HFBI-modified PDMS
C (%) N (%) O (%) Al (%) Si (%) S (%) 10.99 59.41 50.09 51.87
0.04 12.29 0.00 5.80
56.98 23.23 27.61 28.68
12.86 1.93
14.50 2.24 22.30 13.48
0.00 0.18
Table 2. Water Contact Angle (WCA)a on the Mica, HFBI-Modified Mica, PDMS, and HFBI-Modified PDMS Surfaces sample
unmodified (deg)
modified (deg)
mica PDMS
0 123.9
11.9 51.0
a n ) 3. The WCA values were averaged from three measurements at different locations.
Al 2p peaks, as seen in Figure 1b. Similar changes in peak intensity also occurred in the XPS spectra of bare and HFBI-coated PDMS surfaces, as shown in Figure 2. In Figure 2, the peak intensities of N 1s increased (from 0 to 5.80 %) whereas that of Si 2s decreased (from 22.30 to 13.48 %) after HFBI treatment. This can be ascribed to the HFBI modification of the PDMS surface because the element Si exists on the PDMS surface and the element N belongs only to the amine group of the HFBI protein. 3.2. Water Contact Angle Measurement. It has been reported that hydrophobins could be immobilized on different kinds of surfaces, including clean glass surfaces, Teflon, and
Figure 3. Photographs of 5 µL water droplets on (a) a mica surface, (b) an HFBI-modified mica surface, (c) a PDMS surface, and (d) an HFBI-modified PDMS surface.
polystyrene,19a,27 because of the amphiphilic nature of HFBI with different hydrophobic and hydrophilic parts. The introduction (27) (a) Lugones, L. G.; Bosscher, J. S.; Scholtmeyer, K.; de Vries, O. M.; Wessels, J. G. Microbiol. 1996, 142, 1321. (b) Wo¨sten, H. A. B.; Schuren, F. H. J.; Wessels, J. G. H. EMBO J. 1994, 13, 5848. (c) Lumsdon, S. O.; Green, J.; Stieglitz, B. Colloids Surf., B 2005, 44, 172
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Figure 4. Fluorescent images and cross-sections of patterned chicken IgG with FITC-labeled antichicken IgG on an HFBI-coated mica substrate. (The magnifications of the above FITC images in a and b are 40× and 100×, respectively.)
of HFBI can be expected to change the surface properties of mica and PDMS. Surface properties were investigated with WCA measurements on HFBI-coated solid supports, and WCA can be seen in Table 2. As shown in Figure 3a,b, the WCA of the bare mica surface was 0° whereas that of the HFBI-coated mica surface was 11.9°, which indicated that the wettability of the mica surface had been slightly decreased. However, on the PDMS surface, the HFBI coating led to a decrease in WCA from 123.9 to 51.0° as shown in Figure 3c,d, which showed the great increase in wettability of the PDMS surface. The WCA’s of modified surfaces were not to change even after storage for several days in air or in distilled water, which indicated the stability of the HFBI modifications. The changes in the WCA values are related to the amphiphilic properties of the HFBI molecules. Once coating the surfaces, the hydrophobic patches of the protein could face toward the hydrophobic surfaces, such as PDMS, which leads to a decrease in the WCA. When facing the hydrophilic surfaces, hydrophobic parts such as mica would turn outward, which would cause the WCA to increase. Thus, the surface wettability could be changed by the HFBI self-assembly on hydrophilic/hydrophobic interfaces. This also means that after coating with HFBI, hydrophobic surfaces such as PDMS could become moderately hydrophilic and the wettability of hydrophilic surfaces such as mica could decrease to some extent. The HFBI adsorption on both mica and PDMS compares favorably to previously reported results.19a,27c In the experiment, three factors might also affect the changes in WCA after modification with hydrophobin. First, the hydrophobin HFBI might only partially cover the support surfaces, which is caused by the small quantity of hydrophobins (50 µg) that could be immobilized on the solid surface.19b The changes in WCA and XPS results might be consequence of the HFBI coating and bare substrates. Second, the hydrophobin HFBI
structure reveals that a hydrophobic patch on the protein surface comprises only a small part of the total surface area. This means that although showing amphiphilic characteristic the major part of HFBI is hydrophilic.28 The protein structure may be related to the phenomena that the HFBI coating could make the hydrophobic surfaces (PDMS) greatly hydrophilic but would decrease the wettability of hydrophilic surfaces (mica) only slightly. Finally, the hydrophobin coating is strongly influenced by the composition of the surface, which might result in a difference in HFBI coverage on mica and PDMS surfaces. This may be one reason that the changes in WCA before and after modification vary according to different substrates. Those factors may explain why the HFBI coating could change the wettability of solid surfaces only partially. The HFBI modification has more significant virtues on the surface of PDMS. First, it can improve the wettability of PDMS greatly, which was always an obstacle for the application of PDMS in microfluidic networks. Second, its modification effect lasts longer than that of traditional plasma modification.5 Moreover, modification with hydrophobins has better biocompatibility and causes no physical damage to the surface. Thus, considering the increasing application of PDMS in the µCP and microfluidic networks, the appearance of this method seems more meaningful. 3.3. Fluorescent Image of Printed Protein. The above results have demonstrated that HFBI assembly on both surfaces could change the wettability from superhydrophobic (PDMS) or superhydrophilic (mica) to moderately hydrophilic. Because of the moderate hydrophilic nature of most proteins, HFBI-modified substrates may be an attractive candidate to immobilize functional (28) (a) Hakanpa¨a¨, J.; Linder, M. B.; Popov, A.; Schmidt A.; Rouvinena J. Acta Crystallogr. 2006, D62, 356. (b) Hakanpa¨a¨, J.; Szilvay, G. R.; Kaljunen, H.; Maksimainen, M.; Linder, M. B.; Rouvinen, J. Protein Sci. 2006, 15, 1.
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Figure 5. Fluorescent images and cross-sections of patterned chicken IgG with FITC-labeled antichicken IgG on an HFBI-coated PDMS substrate using TEM holey grids as masks. (The magnifications of the above FITC images in a and b are 100× and 200×, respectively.)
biomolecules. Linder et al.19a have reported that HFBI could improve the binding efficiency of the fusion protein onto hydrophobic surfaces such as silanized glass and Teflon. Palomo et al.19b also reported that the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) helped construct a support for the noncovalent immobilization and activation of lipases. Thus, it is reasonable to assume that the HFBI film can cause intermediate protein immobilization on solid surfaces. Here, we used microcontact printing technology to investigate protein immobilization on solid supports, and chicken IgG was used as a model protein. Microcontact printing, introduced by Whitesides and co-workers in 1993,25 was originally applied to pattern self-assembled monolayers of alkanethiols onto gold substrates. The authors used an elastomer, polydimethylsiloxane (PDMS), to form a patterned stamp, which locally transfers “ink” made up of molecules to gold surfaces. Later, biomolecules, such as protein and cells, could also be patterned on various solid surfaces using this method.26a,29 Verification and characterization of the µCP-deposited proteins were conducted using inverted fluorescence microscopy. Figures 4 and 5 show the fluorescent images and cross-sections of patterned chicken IgG with blocked FITC-labeled antichicken IgG on HFBI-coated mica and PDMS substrates, respectively. In Figures 4 and 5, the fluorescent patterns showed high contrast and resolution with a clean background. The characteristic dimensions of the fluorescent stripes are consistent with that of the PDMS stamps or TEM grid stamps. The images taken were then analyzed by Labworks software to generate intensity profiles. The intensity profiles showed high contrast between the signal in printed lines or dots and that in adjacent nonprinted regions,
which illustrated the efficacy of HFBI coating in protein adsorption. To suppress nonspecific binding, bovine serum albumin (BSA) was conventionally used in immunoassay to block all sites left available on a solid surface after patterning the desired proteins.26a BSA blocking enabled FITC-labeled antichicken IgG to bind only with patterned chicken IgG. Thus, the fluorescent patterns observed in Figures 4 and 5 confirmed that HFBI could be the linking layer for the immobilization of chicken IgG on both hydrophobic and hydrophilic surfaces. Moreover, after storing for more than 10 days, the fluorescent strips on the HFBImodified surfaces could still be available. Comparable experiments were conducted to immobilize IgG on mica surfaces with APTES treatment.1c,30 The fluorescent stripes obtained on the HFBI-coated mica surface have higher contrast and better resolution than those obtained on the APTESmodified mica surface. Furthermore, this modification procedure was easier and more rapid than chemical treatments. For PDMS, the HFBI modification was simpler than the chemical treatments, such as UV and plasma treatments. The nice fluorescent images indicated that the patterning of antigen using copper TEM grids was practical; this can also be applied to pattern proteins on other soft materials. Considering others’ work,17b,20 the mechanisms of protein (chicken IgG) immobilization on mica and PDMS might be different from each other. On the hydrophilic mica surface, because the hydrophobic patch of HFBI is exposed to the outside after coating, chicken IgG immobilization follows a mechanism named interfacial activation on hydrophobic surfaces. On the hydrophobic PDMS surface, the hydrophilic part of HFBI turns to the outside and can interact with the polar surface of chicken
(29) Hyun, J.; Ma, H. W.; Zhang, Z. P.; BeeBe, T. P., Jr.; Chilkoti A. AdV. Mater. 2003, 15, 576.
(30) Wang, H. D.; Bash, R.; Yodh, J. G.; Hager, G. L.; Lohr, D.; Lindsay, S. M. Biophys. J. 2002, 83, 3619.
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IgG through polar interactions, including electrostatic interaction, hydrogen bonding, and van der Waals forces. The results in Figures 4 and 5 proved that the HFBI-coated surfaces might be suitable for the immobilization of this protein class (e.g., chicken IgG), but whether HFBI could serve the same purpose for other kinds of proteins needs further verification. The antigenicity of the protein might also be affected by the adsorption process. Differences in the tertiary structure of proteins adsorbed to the hydrophobin and their influence on the adsorption behavior of proteins need further study. However, this method provides a potential for protein immobilization on solid surfaces. Combined with the improved immobilization of proteins on hydrophobin-modified solid surfaces through natural adhesion or fusion proteins19 and the improvement of fibroblast growth by the coating of genetically engineered SC3 hydrophobin,18a,21 the HFBI coating might be an effective method to implant biomolecules, such as proteins, on the surface of biomaterials for applications including clinical diagnostics, cell cultures, biosensors, and immunoassays. Another advantage of HFBI is its biocompatibility, which offers potential applications in the medical and food industries.
4. Conclusions In summary, a simple, convenient, rapid method was developed for the modification of mica and PDMS surfaces with HFBI
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assemblies. The XPS and WCA results illustrated that the wettability of the substrates can be changed from superhydrophobic (PDMS) or superhydrophilic (mica) to moderately hydrophilic by an HFBI coating, which is suitable for protein immobilization on both surfaces. The following immunoassays using µCP demonstrated that an HFBI coating was an effective way to immobilize proteins and preserve their bioactivity on both hydrophilic (mica) and hydrophobic (PDMS) substrates. In addition, an alternative method was developed to achieve protein patterning on the surfaces of soft materials (e.g., PDMS). This bioactive surface modification method will lead to protein immobilization on various surfaces for biodetection applications such as biosensors and microfluid devices. Acknowledgment. This work was supported by the National Natural Science Foundation of China (grant nos. 90403140, 20473097, 90406019, and 90406024) and the Tianjin Science Technology Research Funds of China (grant no. TJ043801111). We gratefully acknowledge Professor Jin Zhai for her help with the water contact angle measurements. Supporting Information Available: Strain, cultivation methods, characterization, isolation of HGBI from biomass, and results. This material is available free of charge via the Internet at http://pubs.acs.org. LA062744H
