Porous Multilayer-Coated PDMS Stamps for Protein Printing | Langmuir

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Porous Multilayer-Coated PDMS Stamps for Protein Printing† Huaping Xu,*,‡,§, Alberto Gomez-Casado,‡ Zhihua Liu, David N. Reinhoudt,‡ Rob G. H. Lammertink,*,§ and Jurriaan Huskens*,‡ Molecular Nanofabrication Group and §Membrane Technology Group, MESAþ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500AE Enschede, The Netherlands, and Department of Chemistry, Tsinghua University, Beijing 100084, China )

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Received February 12, 2009. Revised Manuscript Received June 19, 2009 A polyelectrolyte multilayer was assembled on top of a patterned PDMS stamp employing the layer-by-layer (LbL) assembly technique. By post-treatment with a base and further cross-linking, a porous multilayer-coated PDMS composite stamp was obtained. With the pore structures acting as an ink reservoir, the multiple printing of proteins was successfully achieved without the need to re-ink the stamp.

Introduction The transfer of biomolecules onto surfaces in a controlled way has become particularly interesting to both material scientists and biologists.1 Microcontact printing (μCP) is a potentially biocompatible method for patterning biomolecules directly onto a large surface.2 A potentially powerful concept involving μCP is the combination with inkjet spotting onto a stamp, followed by contact printing, in order to make multiple copies of the spotted array.3 A prerequisite for the success of this strategy is that an inked stamp should be usable for multiple printing steps without re-inking. Only then is efficient use made of the main advantages of spotting (multiplexing, i.e., the positioning of different biomolecular inks onto the same substrate) and μCP (pattern replication). So far, this concept has been shown for DNA transfer 3 but not for proteins. For μCP, poly(dimethylsiloxane) (PDMS) elastomer has been widely used as a stamp material to generate motifs and structures ranging from about 100 nm to micrometers, owing to advantages such as conformal contact with solid substrates, optical transparency, and chemical inertness.4 In the past few years, a lot of work has been done to improve the low Part of the “Langmuir 25th Year: Self-assembled monolayers: synthesis, characterization, and applications” special issue. *Corresponding authors. (H.X.) Tel: þ86-10-62798700. Fax: þ86-1062792406. E-mail: [email protected]. (R.G.H.L.) Tel: þ31-534892063. Fax: þ31-53-4894611. E-mail: [email protected]. (J.H.) Tel: þ31-53-4892995. Fax: þ31-53-4894645. E-mail: [email protected]. †

(1) Sauer, S.; Lange, B. M. H.; Gobom, J.; Nyarsik, L.; Seitz, H.; Lehrach, H. Nat. Rev. Genet. 2005, 6, 465–476. (2) (a) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. Adv. Mater. 2000, 12, 1067–1070. (b) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225–2229. (3) Rozkiewicz, D. R.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2007, 129, 11593. (4) (a) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550. (b) Gates, B. D.; Xu, Q. B.; Stewart, M.; Ryan, D.; Willson, C. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1171. (c) Michel, B.; Bernard, A.; Bietsch, A.; Delamarche, E.; Geissler, M.; Juncker, D.; Kind, H.; Renault, J.-P.; Rothuizen, H.; Schmid, H.; Schmidt-Winkel, P.; Stutz, R.; Wolf, H. IBM J. Res. Dev. 2001, 45, 697–719. (5) Schmid, H.; Michel, B. Macromolecules 2000, 33, 3042. (6) Bietsch, A.; Michel, B. J. Appl. Phys. 2000, 88, 4310–4318. (7) Menard, E.; Bilhaut, L.; Zaumseil, J.; Rogers, J. A. Langmuir 2004, 20, 6871– 6878. (8) Geissler, M.; Bernard, A.; Bietsch, A.; Schmid, H.; Michel, B.; Delamarche, E. J. Am. Chem. Soc. 2000, 122, 6303–6304. (9) Sharpe, R. B. A.; Burdinski, D.; Huskens, J.; Zandvliet, H. J. W.; Reinhoudt, D. N.; Poelsema, B. J. Am. Chem. Soc. 2005, 127, 10344.

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Figure 1. UV-vis spectroscopy of the LbL assembly of PVPy/PAA on TPEDA-functionalized PDMS. The inset shows the growth of the UV-vis absorbance at 256 nm versus the number of bilayers.

mechanical stability of PDMS and to solve the ink diffusion problem.5-12 Unlike the printing of small apolar inks such as alkanethiols, which can be absorbed inside the PDMS stamp and then transferred upon contact between the stamp and substrate, the printing of heavy inks such as proteins with PDMS is much more challenging because of their larger size. The μCP of proteins has been shown by various research groups,13 but in most cases, stamps need to be re-inked after each transfer step. Proteins cannot be readily absorbed by the stamps but merely adhere to the stamps’ outer surfaces. Therefore, the protein inks are transferred from the stamp’s surface to the substrate upon contact, which necessitates re-inking after every step. Some hydrophilic materials with a so-called “ink reservoir” function have been fabricated by different groups, for instance, by (10) Liebau, M.; Huskens, J.; Reinhoudt, D. N. Adv. Funct. Mater. 2001, 11, 147–150. (11) Li, X. M.; P
eter, M.; Huskens, J.; Reinhoudt, D. N. Nano Lett. 2003, 3, 1449–1453. (12) Perl, A.; P
eter, M.; Ravoo, B. J.; Reinhoudt, D. N.; Huskens, J. Langmuir 2006, 22, 7568. (13) (a) Tan, J. L.; Tien, J.; Chen, C. S. Langmuir 2002, 18, 519. (b) Jang, C.; Tingey, M. L.; Korpi, N. L.; Wiepz, G. J.; Schiller, J. H.; Bertics, P. J.; Abbott, N. L. J. Am. Chem. Soc. 2005, 127, 8912. (c) Ross, E. E.; Joubert, J. R.; Wysocki, R. J.; Nebesny, K.; Spratt, T.; O'Brien, D. F.; Saavedra, S. S. Biomacromolecules 2006, 7, 1393.

Published on Web 07/13/2009

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Figure 2. (A) C 1s XPS spectra of a multilayer film of 20 PVPy/PAA bilayers on NH2-PDMS before (I) and after (II) 1 h of base immersion. (B) AFM height image of a 20-bilayer PVPy/PAA film on NH2-PDMS after base immersion. (C) AFM section analysis of image B. (D) AFM height image of a TPEDA-coated Si substrate in contact with a 20-bilayer un-cross-linked PVPy/PAA film. (E) AFM section analysis of image D.

Figure 3. Br 3d XPS spectra of a porous 20-bilayer PDMS stamp after cross-linking with BrC3H6Br (A) and NH2-PDMS after reaction with BrC3H6Br (B). Scheme 1. Schematic Representation of the Fabrication of Porous LbL PDMS Stampsa

a O2 plasma and functionalization with TPEDA (a), LbL assembly of PVPy/PAA on TPEDA-functionalized PDMS (b), and treatment with NaOH and crosslinking by BrC3H6Br (c).

chemical attachment of a silane or hydrophilic polymer layer on a PDMS surface that was treated with oxygen plasma or UV/ ozone.14-19 The thus-formed hydrophilic polymer layer on the stamps was sufficient to absorb small polar inks. Micropatterned (14) Olander, B.; Wirsen, A.; Albertsson, A.-C. J. Appl. Polym. Sci. 2004, 91, 4098–4104. (15) Efimenko, K.; Walace, W. E.; Genzer, J. J. Colloid Interface Sci. 2002, 254, 306–315. (16) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2002, 74, 4117–4123. (17) Delamarche, E.; Donzel, C.; Kamounah, S. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749–8758. (18) Sadhu, V. B.; Perl, A.; P
eter, M.; Rozkiewicz, D. I.; Engbers, G.; Ravoo, B. J.; Reinhoudt, D. N.; Huskens, J. Langmuir 2007, 23, 6850. (19) Azzaroni, O.; Moya, S. E.; Brown, A. A.; Zheng, Z.; Donath, E.; Huck, W. T. S. Adv. Funct. Mater. 2006, 16, 1037–1042.

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hydrogels such as agarose and polyacrylamide were recently reported to transfer controllable amounts of materials onto various substrates. However, only small ionic inorganic species were shown to be absorbed by these hydrogel stamps.20-23 Whether large entities such as proteins can be taken up by the hydrogel stamps is yet unclear. Furthermore, agarose is likely to shrink in dry air, which may lead to difficulties in μCP. Amino dendrimer-modified PDMS stamps (so-called dendri-stamps) were used to transfer DNA onto substrates recently.3 However, the dendrimers were not chemically bonded onto the PDMS surface and were also transferred during printing. For the fabrication of stable, elastomeric stamps for multiple printing of proteins, new methods need to be developed. Since the first report by Decher et al.,24 layer-by-layer (LbL) assembly has attracted increasingly more attention as an effective (20) Fialkowski, M; Campbell, C. J.; Bensemann, I. T.; Grzybowski, B. A. Langmuir 2004, 20, 3513. (21) Klajn, R.; Fialkowski, M.; Bensemann, I. T.; Bitner, A.; Campbell, C. J.; Bishop, K. J. M.; Smoukov, S.; Grzybowski, B. A. Nat. Mater. 2004, 3, 729. (22) Mayer, M.; Yang, J.; Gitlin, I.; Gracias, D. H.; Whitesides, G. M. Proteomics 2004, 4, 2366. (23) Coq, N.; van Bommel, T.; Hikmet, R. A.; Stapert, H. R.; Dittmer, W. U. Langmuir 2007, 23, 5154–5160. (24) (a) Decher, G. Science 1997, 277, 1232. (b) Decher, G.; Hong, J. D. Macromol. Chem. Symp. 1991, 46, 4232.

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Figure 4. AFM height (A) and phase images (B) of a porous 20-bilayer PDMS stamp after cross-linking with BrC3H6Br. AFM height image (C) of cross-linked porous LbL PDMS after 5 min of sonication in PBS buffer (pH 7.5).

Experimental Section Materials and Substrate Preparation. Poly(4-vinylpyridine)

Figure 5. UV-vis spectroscopy of a porous 15-bilayer PDMS stamp before cross-linking (A), after cross-linking and sonication in pH 4.0 buffer solution for 5 min (B), and without cross-linking and sonication in pH 4.0 buffer solution for 5 min (C).

method of fabricating ultrathin films in both theoretical and experimental fields. The popularity of this method arises from its simplicity, versatility, and systematic control over the structure and thickness of the resulting film. Exposure of the LbL multilayer to solutions with different pH values or ionic strengths sometimes leads to the formation of micro/nanoporous structures.25-28 The aim of this work is to grow a porous ultrathin film on the surface of PDMS that can act as an “ink reservoir” without sacrificing the conformal contact of PDMS. A multilayer is assembled onto a patterned PDMS stamp via the LbL technique. By post-treatment with a base and further crosslinking, a porous LbL PDMS composite stamp is obtained. With the pore structures acting as an “ink reservoir”, multiple printing of proteins without re-inking is studied as well as the stability of the multilayer architecture on the stamp upon printing. (25) (a) Fu, Y.; Bai, S. L.; Cui, S. X.; Qiu, D. L.; Wang, Z. Q.; Zhang, X. Macromolecules 2002, 35, 9451. (b) Bai, S. L.; Wang, Z. Q.; Zhang, X. Langmuir 2004, 20, 11828. (26) Mendelsohn, J. D.; Barrett, C. J.; Chan, V. V.; Pal, A. J.; Mayes, A. M.; Rubner, M. F. Langmuir 2000, 16, 5017. (27) Fery, A.; Sch€oler, B.; Cassagneau, T.; Caruso, F. Langmuir 2001, 17, 3779. (28) Kim, B. Y.; Bruening, M. L. Langmuir 2003, 19, 94.

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(PVPy) (Mw=50 000, Polysciences, Inc.), poly(acrylic acid) (PAA, 50 wt %, solution in water, Mw = 5000, Acros), N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEDA, Aldrich), and 1,3-dibromopropane (BrC3H6Br, Aldrich) were used as received. Fluorescein (Fc) ChromPure Human IgG, Fc fragment (HIgGFc), was bought from Jackson Immuno Research Europe. Prior to use, SiO2/Si and glass slides were cleaned and activated by immersion in piranha (3:1 conc H2SO4/33 wt %H2O2; CAUTION! Piranha solutions should be handled with great care in open containers in a fume hood. Piranha is very corrosive, toxic, and potentially explosive!) for 30 min and rinsing with substantial amounts of water. Stamp Fabrication. PDMS stamps were prepared from commercially available Sylgard-184 poly(dimethyl siloxane) (Dow Corning). The curing agent and the prepolymer were manually mixed in a 1:10 volume ratio and cured overnight at 60 °C. After being peeling off of the master, the PDMS stamps were oxidized by exposure of the surface to a Tepla 300E microwave oxygen plasma for 30 s. A TPEDA layer was assembled onto the newly oxidized PDMS stamp by vapor deposition, yielding NH2-tailored PDMS (NH2-PDMS). For UV measurements, a very thin layer of PDMS was dip-coated onto quartz slides. For LbL-coated stamps, PAA (50 wt %, solution in water) was first freeze-dried and redispersed in methanol. The LbL film was fabricated by alternating the immersion of the NH2-PDMS stamp in PVPy (1 mg/mL) and PAA (1 mg/mL) solutions in methanol for 10 min each while rinsing with methanol three times (30 s each) after each step. The resulting multilayer-covered PDMS stamp was immersed in aqueous NaOH (0.1 M) for 1 h to generate porous structures. After rinsing with water and drying thoroughly in a nitrogen stream, the porous structure was crosslinked by a vapor-phase reaction with BrC3H6Br for 8 h under reduced pressure in a desiccator, followed by rinsing with water and PBS buffer. μCP of HIgG-Fc. Several drops of an aqueous solution of HIgG-Fc were added to the surface of porous LbL PDMS or NH2-PDMS. After 30 min of inking, the excess amount of solution was blown away by a nitrogen stream. The inked stamp was put into contact with the substrate for 2 min. Analysis. UV-vis measurements were carried out on a Varian Cary 3E UV-spectrophotometer. AFM analyses were carried out with a NanoScope III (Veeco/ Digital Instruments, Santa Barbara, CA) multimode atomic force microscope equipped with a J scanner in tapping mode by using Si Langmuir 2009, 25(24), 13972–13977

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Figure 6. Fluorescence microscopy images (221 μm  166 μm) and intensity profiles (insets) of fluorescein-labeled HIgG-Fc printed on TPEDA-functionalized glass slides in the first (A) and second (B) prints using an NH2-PDMS stamp.

Figure 7. Fluorescence microscopy images (221 μm  166 μm) and intensity profiles (insets) of HIgG-Fc printed on TPEDA-functionalized glass slides in the first through fourth prints(A-D, respectively) using a porous LbL PDMS stamp.

cantilevers (Nanosensors) with a nominal spring constant of about 42 N m-1. Fluorescence microscopy was performed using an Olympus IX71 inverted research microscope equipped with a U-RFL-T mercury burner as a light source and an Olympus DP70 digital camera (12.5 million pixel cooled digital color camera) for image acquisition. X-ray photoelectron spectroscopy (XPS) was performed on a QuanteraSXM (Physical Electronics).

Results and Discussion Scheme 1 shows the preparation procedure of the porous LbL PDMS stamps. Amino-coated PDMS was prepared by O2 plasma treatment followed by gas-phase deposition of N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEDA). The LbL multilayer assembly of PVPy/PAA on NH2-PDMS was achieved from methanolic solutions based on hydrogen bonding between the pyridine and carboxylic acid groups. Although LbL films are commonly constructed on the basis of electrostatic attraction between two polymers, other weak interacLangmuir 2009, 25(24), 13972–13977

tions such as hydrogen bonding have also been employed as the driving force for LbL assembly.29 The assembly process was monitored by UV-vis spectroscopy. The UV-vis curves (Figure 1) indicated the steady growth of LbL multilayers on PDMS. The absorption band at 256 nm in the UV region is assigned to the presence of PVPy. From the insets of Figure 1, it can be seen that the absorbance at 256 nm versus the number of bilayers increases in a nonlinear fashion. This is in contrast to the linear increase observed on hard substrates such as quartz or silicon, as reported previously by Zhang and coworkers.25a We ascribe this to a less-dense TPEDA layer on the PDMS surface. When the LbL multilayer-assembled PDMS was immersed in a basic aqueous solution for 1 h, PAA was removed from the multilayer. The dissolution is clearly confirmed by XPS spectra (29) (a) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (b) Wang, L. Y.; Wang, Z. Q.; Zhang, X.; Shen, J. C.; Chi, L. F.; Fuchs, H. Macromol. Rapid Commun. 1997, 18, 509.

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Figure 8. AFM height images of HIgG-Fc printed on TPEDA-functionalized Si in the first (A) and second (B) prints using a porous LbL PDMS stamp (insets show larger scan areas).

Figure 9. Fluorescence microscopy image (221 μm  166 μm) of printed HIgG-Fc on TPEDA-functionalized glass by contacting an HIgG-Fc-inked and rinsed porous LbL PDMS stamp.

obtained before and after base immersion. (Figure 2 A) The distinct peak at 288.4 eV corresponding to C 1s of the carboxylic acid groups in PAA disappeared in the spectrum of the sample after base treatment. However, the release of PAA did not destroy the whole film on top of the PDMS surface. PVPy, the other component of the LbL film, remained on the substrate because of its poor solubility in the basic aqueous solution. The remaining PVPy led to the formation of a porous film on the surface of PDMS (Figure 2B,C). The diameter of the pores was around several tens of nanometers up to 200 nm, which is smaller than observed on hard substrates.25 This can be attributed to the lower molecular weight of PVPy used here. The thickness of a porous 20-bilayer film on PDMS was around 30 nm as indicated by SEM (not shown). Without cross-linking of the porous structure, the film can be completely or partially transferred to substrates upon microcontact printing. As seen from Figure 2D, the un-cross-linked porous LbL film was transferred to an NH2-modified silicon substrate 13976 DOI: 10.1021/la901797n

upon contact, which is in agreement with a previous study by Hammond et al. dealing with multilayer transfer by “polymer-onpolymer” stamping.30,31 Thus, the stabilization of the porous structure is important for the fabrication of robust, porous LbL PDMS stamps. In order not to destroy the porous structures, the cross-linking of PVPy with BrC3H6Br was performed in the gas phase instead of in solution. The XPS peaks of Br 3d appearing at 65.6 and 68.8 eV (Figure 3A) are ascribed to free and PVPy-bound bromide, respectively.32 AFM images (Figure 4A,B) did not show any morphological change after cross-linking. Cross-linking was probed by verifying an enhanced stability relative to an untreated film. Both the cross-linked and un-cross-linked films were immersed in a pH 4.0 buffer solution and sonicated vigorously for 5 min. As can be seen clearly from Figure 5, the absorbance at 256 nm decreased dramatically for the un-cross-linked film, which indicates the desorption of PVPy from NH2-PDMS. However, the absorbance of the cross-linked film remained unchanged, which indicates a successful cross-linking reaction. Stability tests were also carried out by sonication in a pH 7.5 PBS buffer solution. Also, in this case the absorbance of the cross-linked film remained constant before and after sonication. AFM imaging showed that the porous structures remained intact on the PDMS substrate (Figure 4C). It should be pointed out that the cross-linking reaction happens not only between porous multilayers but also between the porous multilayer and the NH2 functional groups on the PDMS surface. XPS showed a clear Br 3d signal (Figure 3B) after the vapor-phase reaction of NH2-PDMS with BrC3H6Br for 8 h and subsequent rinsing with ethanol. This covalent binding of the multilayer to the PDMS surface is important in preventing multilayer transfer during microcontact printing. Indeed, contacting a cross-linked (30) Jiang, X. P.; Zheng, H. P.; Gourdin, S.; Hammond, P. T. Langmuir 2002, 18, 2607. (31) Zheng, H. P.; Rubner, M. F.; Hammond, P. T. Langmuir 2002, 18, 4505. (32) http://srdata.nist.gov/xps

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multilayer-coated stamp onto an amino-coated Si substrate did not lead to any transfer of polymer whereas without cross-linking transfer was clearly observed (Figure 2D). The static contact angle of cross-linked porous LbL PDMS is around 64°, showing that the stamp is hydrophilic. The porous LbL PDMS stamp was used to investigate the possibility of multiple printing of a protein without re-inking. For visualization by fluorescence microscopy, the fluorescein-labeled Fc fragment of a human immunoglobulin (HIgG-Fc) was used as the ink (1 μM in PBS buffer, pH 7.5). An amino-terminated SAM of TPEDA was formed on a glass slide so that electrostatic interaction between the proteins and the positively charged substrate provides affinity between the ink and the substrate. As a reference, experiments were also carried out with a TPEDA-functionalized PDMS stamp (NH2-PDMS). In the latter case, patterns can be clearly seen upon the first printing (Figure 6A). The fluorescence intensity decreased dramatically upon the second printing step (Figure 6B). In this case, patterns can be barely seen, indicating that most of the ink has been transferred to the substrate during the first printing step. When using a porous LbL-PDMS stamp, line patterns were clearly seen on the glass substrate upon the first printing step (Figure 7A), giving direct evidence of the transfer of HIgG-Fc from the stamp to the substrate. The fluorescence intensity was much higher than that of the first print using an NH2-PDMS stamp. With the porous LbL PDMS stamp, even multiple printing steps without re-inking were possible, as indicated by the fluorescence microscopy images of the second and third prints (Figure 7B,C). Only for the fourth print was the intensity significantly lower than that of the first print using NH2-PDMS. This indicates that in the case of the porous stamps the proteins are trapped inside the pores of the stamps and are transferred to the substrate upon contact, probably mediated by residual water. As seen from the AFM image (Figure 8A), a thick layer of proteins was transferred to the substrate upon the first print. Aggregates of proteins were also found. Upon the second print, a thin layer of around 1 nm, approximately a monolayer, was transferred (Figure 8B), and aggregates appeared to be absent. Because the protein aggregates are observed only for the first print, their occurrence is most likely due to the aggregation of proteins on the outside of the stamp during inking. These aggregates are removed upon the first print so that subsequent prints reflect the transfer of proteins that have resided inside the porous structures. Assuming that about 1-3-nm-thick layers of proteins are transferred from the stamp’s interior for each print for three consecutive prints and considering the porous layer thickness of 30 nm (see above), at least about 20% of the volume of the porous structure can be filled with proteins and emptied again. This holds prospects for increasing the number of consecutive successful prints by increasing the thickness of the porous multilayer on the stamp.

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The ink reservoir function of the porous stamps was further confirmed by rinsing an already HIgG-Fc-inked porous LbL PDMS stamp with plenty of PBS buffer. Upon contact, the fluorescent patterns were clearly observed (Figure 9), which means that the ink was absorbed by the stamp and migrated out during contact with the substrate.

Conclusions Porous LbL-PDMS stamps were successfully fabricated by multilayer formation on top of PDMS via hydrogen bonding followed by base treatment and subsequent cross-linking. The pore structures act as an ink reservoir for the absorption of protein inks, which facilitates the multiple printing of HIgG-Fc without the need to re-ink and could not have been achieved by printing with normal NH2-PDMS. The main achievement here is that stamps have been created by which proteins are transferred from the inside of the stamp, reminiscent of versatile alkanethiol printing by regular PDMS and surpassing the mostly observed mechanism of the surface transfer of large molecules such as proteins by regular and oxidized PDMS. Because the inking is done from water or buffer and because the porous stamps remain hydrated, the proteins are likely to remain bioactive. Nevertheless, this remains to be investigated in future studies. With the approximately 30-nm-thick multilayer structure used here, multiple prints resulting in protein layers a few nanometers thick can be produced, indicating significant porosity and the use of porosity for the reversible loading of proteins. Because the pore diameter and the thickness of the porous films can be tuned by the variation of polymer concentration, the number of bilayers, the pH value of the base solution, and the base immersion time,24 the size of the reservoir of the porous stamp can be well controlled and thus the number of prints may also be controlled. It will be interesting to study the effect of pore size and ink size on the transfer yield. Moreover, by using polyelectrolytes with recognition properties, selective ink recognition and transfer can be anticipated. Because of the high flexibility of the LbL process, it can also be envisaged that the contact layer of the stamp can be equipped with a polyelectrolyte that suppresses the now observed protein aggregation. Moreover, we have shown recently that the same LbL and cross-linking procedure can also be implemented successfully in a dip-pen nanolithography procedure allowing submicrometer patterning with proteins.33 All in all, these results indicate that the rational design of stamps and tips for nanofabrication with proteins is within reach. Acknowledgment. We are grateful for financial support from the Strategic Research Orientation Nanofabrication of the MESAþ Institute for Nanotechnology, University of Twente, The Netherlands. (33) Wu, C.-C.; Xu, H.; Otto, C.; Reinhoudt, D. N.; Lammertink, R. G. H.; Huskens, J.; Subramaniam, V.; Velders, A. H. J. Am. Chem. Soc. 2009, 131, 7526.

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