Article pubs.acs.org/Langmuir
Modification of Surfaces by Chemical Transfer Printing Using Chemically Patterned Stamps Christian Wendeln, Oliver Roling, Christian Schulz, Carsten Hentschel, and Bart Jan Ravoo* Organic Chemistry Institute and Center for Nanotechnology (CeNTech), Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, 48149 Münster, Germany S Supporting Information *
ABSTRACT: The preparation of well-defined molecular monolayers and their patterning on the microscale and nanoscale are key aspects of surface science and chemical nanotechnology. In this article, we describe the modification of amine-functionalized surfaces using a new type of contact printing based on chemically patterned, flat PDMS stamps. The stamps have discrete areas with surface-bond tetrafluorophenol (TFP) groups, which allow the attachment of carboxylic acids in the presence of coupling agents such as diisopropylcarbodiimide (DIC). The generated active esters can be reacted by placing the stamps in contact with aminefunctionalized surfaces. The process leads to the transfer of acyl residues from the stamp to the substrate and therefore to a covalent attachment. Patterning occurs because of the fact that reaction and transfer take place only in areas with TFP groups present on the stamp surface. Different types of amine-decorated surfaces were successfully modified, and the transfer was visualized by fluorescence microscopy. To the best of our knowledge, the covalent transfer printing (CTP) of an immobilized molecular monolayer from one surface to another surface is unprecedented.
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INTRODUCTION
Besides the study of ink-transfer processes and the investigation of chemical reactions that are applicable in the field of microcontact chemistry, much effort was made to develop new types of polymeric stamps with advantageous properties. Some of the achievements are hydrophilic stamps for the printing of polar inks,14,15 porous stamps with an inkreservoir capacity,16 flat stamps with topographical ink diffusion barriers,17,18 and catalytically active stamps.19−21 It has been demonstrated that flat, catalyst-modified stamps and flat stamps with ink diffusion barriers can be used for submicrometer patterning because stamp deformation effects22 such as pairing, buckling, and roof collapse are circumvented. Another category of stamps has a modified surface that allows loading with defined substances by highly specific interactions. A striking example is affinity contact printing, which was initially reported by Bernard et al.23,24 In their work, PDMS stamps were modified by the attachment of different types of antibodies and antigens. The prepared affinity stamps could be used to “fish” specific binding partners out of a protein mixture in solution. The protein-modified stamps were dried and subsequently brought into contact with a substrate. The printing led to the transfer of the proteins onto the surface. The affinity stamps could be reused for several cycles of printing.
Within the past decades, a variety of techniques have been developed for the patterning of surfaces. Prominent methods are based on electron beam lithography,1 dip-pen nanolithography,2 photolithography,3 and Langmuir−Blodgett patterning.4 Among these, many different approaches that rely on printing have been described. In particular, microcontact printing5 is nowadays a broadly applied method and used in different fields of science. It benefits from several advantages, including low cost, simplicity, high versatility, and high quality patterning with resolution down to the micrometer and even submicrometer scale. In its original version, patterning by microcontact printing is achieved with polymeric stamps that have a relief structure and are made from an inversely structured master. For printing, the stamps are inked with the substance of choice (“ink”) and brought into conformal contact with a substrate. After removal of the stamp, the ink remains on the contacted areas. Ink transfer is in every case driven by a concentration gradient and may additionally be supported by attractive interactions between ink and substrate. In many cases, hydrophobic and hydrophilic interactions, electrostatic interactions,6,7 chemisorption,8−10 and host−guest interactions11,12 play an important role. Moreover, the ink can be covalently attached to a chemically modified surface by a reaction that takes place in the area of contact (reactive microcontact printing or microcontact chemistry).13 © XXXX American Chemical Society
Received: December 18, 2012 Revised: January 30, 2013
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Preparation of Hexenyl-Modified Stamps. Oxidized PDMS stamps (1 h oxidation time) were put in a round-bottomed flask, and a little glass jar with a drop of 5-hexenyltrichlorosilane was added. The flask was evacuated by an oil pump and closed. After 1 h of incubation, the stamps were removed and stored for 8 to 12 h in air to allow for the complete hydrolysis of the silane. Preparation of Tetrafluorophenol-Modified Stamps (TFP Stamps). Hexenyl-modified, flat PDMS stamps were patterned by photochemical microcontact printing of TFP-thiol (1) using oxidized PDMS stamps (55 min oxidation time). To this end, a solution of 2,2′dimethoxyphenylacetophenone (DMPA, 30 mM) and TFP-thiol (1) (60 mM) in ethanol was dropped on the surface of an oxidized stamp (1−3 drops). After 60 s of incubation, the stamps were dried in a stream of argon and carefully placed on a piece of hexenyl-modified PDMS. Subsequently, the PDMS “sandwich” was irradiated for 3 min with a high-power UV-LED. The distance between the LED and the contact area of the two stamps was approximately 2 cm. Finally, the TFP-modified stamps were washed with Milli-Q water and ethanol and dried in a stream of argon. Preparation of N-Acetylcysteamine-Modified Stamps. NAcetylcysteamine-modified stamps were prepared following the same procedure as described for the TFP stamps with the difference that a solution of N-acetylcysteamine (60 mM) in ethanol (+30 mM DMPA) was used for printing. Preparation of N-Acetylcysteamine/TetrafluorophenolModified Stamps. The stamps were prepared by printing Nacetylcysteamine (60 mM + 30 mM DMPA) in lines (10-μm-wide lines spaced by 5 μm) and then TFP-thiol (1) (60 mM + 30 mM DMPA) in the perpendicular direction (10-μm-wide lines spaced by 5 μm) on flat, hexenyl-modified PDMS. The stamps were washed with Milli-Q water and ethanol and dried in a stream of argon. Preparation of Topographically and Chemically Structured TFP Stamps. In the first step, hexenyl-modified PDMS stamps with a relief structure were prepared as described for the synthesis of flat, hexenyl-modified PDMS but with the difference that topographically patterned PDMS (10-μm-wide lines spaced by 5 μm) was used as the substrate. In the following step, TFP-thiol (1) was printed on the alkene-modified PDMS by using an oxidized PDMS stamp with a relief structure (dots with a diameter of approximately 10 μm). The printing was carried out as described for the preparation of flat TFP stamps. Finally, the obtained stamps were washed with Milli-Q water and ethanol and dried in a stream of argon. Formation of Silanol Groups on Glass and Silicon Surfaces by Piranha Activation. Glass and silicon substrates were cut into pieces of 2.6 × 1.4 cm2 and cleaned by sonication in pentane, acetone, and Milli-Q water. Subsequently, the substrates were immersed in a freshly prepared piranha solution (H2SO4/H2O2 3:1). After 30 min, the substrates were carefully washed with Milli-Q water and dried in a stream of argon. The surfaces were immediately used for further modification by silylation. Preparation of Aminopropyl-Modified Substrates. Aminopropyl-terminated glass was prepared similarly to a reported procedure.30 Piranha-activated substrates were immersed in a freshly prepared, stirred solution of 3-aminopropyltriethoxysilane (APTES) in toluene (2 vol %). After 90 min, the substrates were washed with ethanol, sonicated in ethanol (3 min), and dried in a stream of argon. The surfaces were positioned in a flask, and the flask was evacuated. The substrates were baked for 10 min at a temperature of 120 °C and then immediately used or stored under an atmosphere of argon. Preparation of PAMAM G4-Dendrimer-Decorated Substrates. Piranha-activated glass surfaces were immersed in a stirred solution of methyl-11-trichlorosilylundecenoate in toluene (0.1 vol %). After 40 min, the surfaces were washed with Milli-Q water and ethanol. After being dried in a stream of argon, the ester-terminated surfaces were hydrolyzed for 2 h by 2.5 M HCl at 85 °C. The substrates were washed with Milli-Q water and dried. The obtained carboxylic acid-modified substrates were converted to NHS-esterterminated substrates by reaction with a solution of dicyclohexylcarbodiimie (1.0 M) and N-hydroxysuccinimide (1.0 M) in DMF (peptide synthesis grade).31 After 1 h, the surfaces were washed with
The concept was also later applied to the printing of DNA by DNA-decorated stamps.25,26 Another type of receptor-functionalized stamp was developed by Sadhu et al.27 They synthesized structured PDMS stamps that are modified with a layer of covalently attached βcyclodextrins. The stamps were inked with adamantanemodified, fluorescently labeled molecules by exposure to a solution of the molecules or by using an ink pad. Adamantane is known to bind strongly to the cavity of β-cyclodextrin due to the formation of inclusion complexes. The loaded stamps were subsequently brought into conformal contact with surfaces that are functionalized with a monolayer of β-cyclodextrin. During contact, adamantane-modified molecules are transferred to the surface until an equilibrium distribution is reached. After the removal of the stamp, patterned surfaces were obtained. The immobilization of fluorescent molecules could successfully be visualized by fluorescence microscopy. In this article, we report a novel printing method in which a molecular monolayer that is immobilized on a donor surface can be transferred by reaction to an acceptor substrate surface. We describe the chemical patterning of amine-functionalized substrates by the covalent transfer of acyl residues from a stamp to a substrate surface. The stamps have discrete regions with tetrafluorophenol (TFP) groups that allow the formation of reactive active esters by coupling to carboxylic acids. The active esters can be reacted by contact with nucleophilic groups such as amines. The use of TFP for the formation of active esters was found to be advantageous over the use of other electronwithdrawing alcohols such as N-hydroxysuccinimide and Nhydroxybenzotriazole because the resulting esters show a higher stability toward humidity. TFP-active ester resins have been applied for the efficient synthesis of peptides in organic solvents and even in water.28,29 To the best of our knowledge, the covalent transfer printing (CTP) of an immobilized molecular monolayer from one surface to another surface is unprecedented.
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EXPERIMENTAL SECTION
General. 5-Hexenyltrichlorosilane and 10-undecenyltrichlorosilane were obtained from ABCR. D-Biotin, 6-heptynoic acid, (3aminopropyl)triethoxysilane (APTES), PAMAM G4 dendrimer solution (1,4-diaminobutane core, 10% in methanol), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. DMF (peptide synthesis grade) was supplied by Iris Biotech. Tetramethylrhodamine isothiocyanate (TRITC)-labeled streptavidin was delivered by Thermo Fisher Scientific. Silicon wafers (B-doped, 100 orientation, 20−30 Ω cm) were kindly donated by Siltronic AG (Burghausen, Germany). Glass substrates were prepared from IDL microscope slides (Interessengemeinschaft der Laborfachhändler) by cutting them into pieces (2.6 × 1.4 cm2). Surface cleaning procedures were carried out using absolute ethanol and Milli-Q water. The Milli-Q water was prepared from distilled water by a PureLab UHQ deionization system (Elga). Photochemical microcontact printing by thiol−ene chemistry was carried out using a high-power UV-LED (P8D236, Seoul Semiconductior, 365 nm peak wavelength, 18 nm spectrum half width, 90 mW optical power) supplied by Conrad Electronics. Preparation of Poly(dimethylsiloxane) (PDMS) Stamps. PDMS stamps were prepared by casting a 10:1 (v/v) mixture of Sylgard 184 silicone elastomer base and curling agent (Dow Corning) on a fluorinated silicon master (flat or patterned). The prepolymer was cured overnight in an oven at 80 °C, and the obtained polymer block was cut into small pieces. For the oxidation of the PDMS surface, the stamps were incubated in a UV-ozonizer (PSD-UV, Novascan Technologies) and subsequently stored under water. B
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Figure 1. Principle of chemical transfer printing (CTP) and preparation of tetrafluorophenol (TFP)-modified stamps. (A) A TFP stamp is chemically loaded by esterification with a carboxylic acid. (B) The active ester stamp is brought into conformal contact with (C) an aminefunctionalized surface. Because of aminolysis, which takes place exclusively in the area of contact, acyl residues are transferred onto the amines of the substrate, and this leads (D) to a patterned surface with newly generated amides. (E) TFP stamps can be prepared from conventional PDMS stamps. In the first step, the PDMS surface is oxidized by ozone and subsequently exposed to 5-hexenyltrichlorosilane to give (F) hexenyl-modified PDMS. In the following step, TFP residues are attached by photochemical microcontact printing of TFP-thiol (1). (G) The obtained TFP stamps have (H) a clear, transparent appearance. (I) The hydrophilic TFP-modified areas can be visualized under an optical microscope by the selective condensation of water droplets on the surface. DCM and dried in a stream of argon. Subsequently, each substrate was covered with 100 μL of a PAMAM G4 dendrimer solution (10 wt % in methanol) and covered with a microscopy cover slide. After 2 h, the glass substrates were washed with NEt3 in ethanol (5 wt %) and with ethanol and dried in a stream of argon. The surfaces were used immediately or stored under an atmosphere of argon. Preparation of Ethylene Glycol Amine-Terminated Substrates. Carboxylic acid-terminated glass substrates were prepared as described for the PAMAM dendrimer-decorated substrates. The substrates were covered with a few drops of a freshly prepared solution of diisopropylethylamine (0.2 M), O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluroniumtetrafluoroborat (TBTU, 0.16 M) and Boc-monoprotected 2,2′-(ethylenedioxy)bis(ethylamine)32 (0.2 M) in DMF (peptide synthesis grade). After 2 h, the surfaces were washed with Milli-Q water and ethanol and dried in a stream of argon. Removal of the tert-butylcarbamate protective groups was carried out immediately before printing on the glass substrates. Therefore, the substrates were incubated for 5 h in a solution of trifluoroacetic acid (TFA) in DCM (30 vol % TFA). Finally, the surfaces were washed with Milli-Q water, NEt3 in ethanol (5 vol %), and ethanol. The surfaces were dried in a stream of argon and used immediately. Preparation on Undecenyl-Modified and TetrafluorophenolModified Silicon Substrates. 11-Undecenyl-modified substrates were prepared as described elsewhere.33 Subsequent microcontact printing of TFP-thiol (1) with flat, oxidized PDMS stamps led to TFPmodified substrates. The printing was carried out as described for the preparation of TFP stamps. Chemical Transfer Printing. A freshly prepared solution of DMAP (0.01 M), diisopropylcarbodiimide (0.1 M), and a carboxylic acid (0.1 M in the case of heptynoic acid and 0.05 M in the case of biotin) in DMF (peptide synthesis grade) was stirred for 5 min under argon. The surfaces of the TFP stamps were covered with 3−5 drops of the prepared solution and incubated under argon. After 4 h, the stamps were washed with DMF (peptide synthesis grade), dried in a stream of argon, and carefully placed on amine-modified substrates. The printing time was 1 to 2 min. Subsequently, the stamps were again removed and the glass surfaces were thoroughly washed with Milli-Q water and ethanol and dried in a stream of argon. For cleaning, the substrates were sonicated in a solution of triethylamine (5 vol %) in ethanol and dried. Remaining amines were acetylated by incubating the substrates in a solution of acetic anhydride in pyridine (5 vol %). After 1 h, the substrates were again cleaned by intensive washing with ethanol and dried. Repetitive Chemical Transfer Printing. Repetitive transfer printing by reusing a stamp was carried out as described for single printing procedures with the difference that the stamp was again
exposed to the loading solution (DMAP, DIC, and acid) and subsequently used for printing. The reloading and printing were carried similarly as described in the paragraph above. Up to three surfaces were successfully patterned with the same stamp. Streptavidin Binding Studies. Substrates were incubated in a solution of bovine serum albumin (BSA, 3 wt %) in PBS (1× PBS, pH 7.5) and washed with the same buffer without BSA (2 × 5 min). Subsequently, the substrates were covered with a solution of TRITClabeled streptavidin (c = 100 nM) in PBS (pH 7.5) at room temperature. After 15 min, the surfaces were washed with PBS, rinsed with distilled water, and carefully dried with a tissue. Analysis was carried out by fluorescence microscopy. Detection of Surface-Attached Alkynes by CuAAC with Lissamine Rhodamine B Azide (4). A freshly prepared Cu(I) solution (20 vol % of the used DMF), which was prepared by dissolving 5 mg of CuSO4·5H2O and 20 mg of sodium ascorbate in 1 mL of water, was added to a 10 mM solution of lissamine rhodamine B azide (4) in DMF. Alkyne-modified substrates were added under argon, and the mixture was heated to 70 °C for 4 h. Finally, the surfaces were washed thoroughly with Milli-Q water and ethanol and dried in a stream of argon.
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RESULTS AND DISCUSSION The concept of chemical transfer printing (CTP) using TFPmodified stamps (TFP stamps) is described in Figure 1. In the first step, a TFP stamp (Figure 1A) is loaded by exposure to a solution of dimethylaminopyridine (DMAP), diisopropylcarbodiimide (DIC), and the respective carboxylic acid in DMF. The reaction leads to the formation of surface-attached active esters (Figure 1B). After washing and drying, the stamp is brought into conformal contact with an amine-functionalized surface (Figure 1C). Because of aminolysis, which takes place in the contact area between the stamp and substrate, acyl residues are transferred onto the substrate. Removal of the stamp leads to a chemically patterned surface (Figure 1D) under recovery of the TFP stamp (Figure 1A). The observation that the aminolysis of active esters can occur between two surfaces was reported by Ouellet et al., who successfully glued amine-functionalized PDMS onto a monolayer of NHS esters on gold.34 To investigate CTP, an effective method for the preparation of TFP-modified stamps was developed. The synthesis was carried out starting from flat PDMS stamps, which were prepared by curing a mixture of the commercially available C
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Figure 2. Analysis of hexenyl-modified stamps and homogeneously covered TFP stamps. (A, B) The static contact angles of hexenyl-modified PDMS show a significant decrease after the immobilization of the hydrophilic TFP groups. (C) XPS analysis revealed that no fluorine could be detected in the case of alkene-modified stamps, (D) whereas TFP stamps showed the presence of a significant F 1s peak. (E, F) AFM topography measurements indicated that the surfaces of both stamps are very smooth.
surface areas (advancing, 65 ± 3°; static, 60 ± 3°; receding, 26 ± 5°) (Figure 2A,B). The change in hydrophilicity could also be visualized by selective water condensation on the structured regions of the immobilized TFP linker (Figure 1I). The attachment of TFP-thiol (1) by photochemical microcontact printing was additionally confirmed by X-ray photoelectron spectroscopy (XPS). In the case of the hexenylmodified PDMS stamps, no signal could be detected in the F 1s region (Figure 2C). After the immobilization of 1 by printing with a flat stamp, the surface showed a strong fluorine signal in the F 1s region (Figure 2D). The signal can easily be explained with the covalent attachment of TFP groups on the surface. The general proof of the covalent immobilization by thiol−ene chemistry was demonstrated by the successful modification of a 10-undecenyl monolayer on silicon. The printing of TFP-thiol (1) was carried out similarly to that carried out on hexenylmodified PDMS. The covalent attachment of the TFP groups was verified by XPS. Detailed analysis showed the presence of fluorine atoms by a signal in the F 1s region (Figure SI-1B), nitrogen atoms by a signal in the N 1s region (Figure SI-1C), and sulfur atoms by a peak in the S 2s region (Figure SI-1D). Additionally, the C 1s carbon peak verified the photochemical immobilization of 1 by visualizing photoelectron emissions
Sylgard kit on a flat silicon master (Figure 1E). The stamps were oxidized by UV-ozone for 1 h in order to produce silanol groups on the surface of the PDMS. Then, the stamps were reacted with 5-hexenyltrichlorosilane from the gas phase to give alkene-modified, flat stamps (Figure 1F). The oxidation level was found to be very crucial because prolonged exposure to ozone leads to a glassy stamp surface that does not adhere to the substrate. In addition, the silanization conditions were found to be important because long exposure to trichlorosilane leads to milky stamps that similarly do not stick to surfaces. For the attachment of TFP groups to the alkene-modified, flat PDMS stamps, TFP-thiol (1) was immobilized via photochemical microcontact printing.33 To this end, another structured stamp was inked with a solution of the thiol and brought into conformal contact with the PDMS. The radical addition of the thiol to the alkene groups was induced by UV light. Removal of the stamp and intensive washing of the flat PDMS surface led to the transparent TFP stamp (Figure 1G,H). The attachment of the hydrophilic tetrafluorophenol linker to the surface of the alkene-modified PDMS (advancing, 93 ± 3°; static, 87 ± 2°; receding, 77 ± 3°) was accomplished with a significant decrease in the water contact angles and therefore to a higher hydrophilicity of the TFP-decorated D
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Figure 3. Chemical transfer printing (CTP) of D-biotin on (A) aminopropyl-functionalized, (B) on PAMAM dendrimer-decorated, and (C) on ethylene glycol amine-terminated surfaces. (D−F) Fluorescence microscopy images of TRITC-streptavidin immobilized on biotin-modified surfaces, prepared by CTP using (D) aminopropyl-terminated, (E) PAMAM dendrimer-modified, and (F) ethylene glycol amine-modified glass substrates. (F) A single TFP stamp can be used for three printing procedures of biotin, as detected by streptavidin binding.
from carbons in a C−F/CO (approximately 288 eV) and in a C−O/C−N environment (approximately 286 eV) (Figure SI1A). Besides the effective modification of PDMS with TFP groups, the surface flatness is another factor that has a significant influence on the transfer printing efficiency. To investigate the surface topography of the PDMS, atomic force microscopy (AFM) measurements were performed. It was found that the surfaces of hexenyl-modified stamps and also of TFP stamps are very smooth. In both cases, a root-mean-square roughness of less than 1 nm (0.995 nm in the case of the hexenyl-modified stamp and 0.631 nm in the case of the TFP stamp) was determined. The values should be sufficient to allow an effective transfer of molecules from PDMS to a substrate. In the first experiments, CTP of D-biotin (2) was carried out on aminopropyl-modified glass (Figure 3A), on PAMAM G4 dendrimer-modified substrates (Figure 3B), and on ethylene glycol amine-terminated surfaces (Figure 3C). The attachment of biotin to the TFP stamps could be achieved by covering the surfaces with a solution of the acid (0.05 M), DMPA (0.01 M), and DIC (0.1 M) in DMF. After 4 h, the stamps were washed with DMF, dried in a stream of argon, and carefully placed on the amine-modified surfaces. The contact time was 1 to 2 min. After the substrates were carefully cleaned, remaining amine groups were acetylated by acetic anhydride in pyridine. Detection of the immobilized biotin residues was carried out by incubation with a solution of rhodamine-labeled streptavidin and analysis by fluorescence microscopy. Streptavidin is a tetrameric protein that strongly binds to biotin with high selectivity.
Fluorescence microscopy analysis of an aminopropylterminated surface that was modified by printing biotin with a chemically structured, flat TFP stamp (dots with a diameter of about 10 μm) and exposed to streptavidin proved the immobilization of the protein in the expected pattern (Figure 3D). Further investigations concerning the reproducibility of the printing were carried out by repeating the experiment with three different TFP stamps. In every case, a clear, strongly fluorescent pattern was observed (Figure SI-2A). If the printing was carried out with stamps that were modified with Nacetylcysteamine (dot pattern) instead of TFP-thiol (1) no fluorescent pattern was found (Figure SI-2B). Only in some small areas were extremely weak patterns found that were not comparable to the results that were achieved by using TFP stamps. The experiments clearly demonstrate that the replacement of the TFP group by a methyl group leads to a vanishing of the pattern, and this strongly supports the concept of CTP. Loading the stamp with D-biotin in the absence of coupling agent DIC was also attempted. Subsequent printing on an amine-decorated surface, exposure to streptavidin, and fluorescence microscopy analysis indicated no binding. This clearly demonstrates that coupling to the stamp is a prerequisite for the transfer of biotin. In addition to CTP on aminopropyl-terminated surfaces, PAMAM G4-dendrimer-modified substrates were modified by printing biotin with a flat TFP stamp (5-μm-wide lines spaced by 15 μm). The surfaces were prepared by the attachment of PAMAM G4 dendrimers to a monolayer of undecylcarboxylic acid NHS esters (details in Experimental Section). Exposure of the surface to streptavidin and subsequent fluorescence microscopy revealed that biotin was transferred with good resolution (Figure 3E). The high fluorescence intensity can be E
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Figure 4. CTP of heptynoic acid (3) on (A) aminopropyl-modified glass. (B) The procedure leads to a patterned surface with terminal alkynes. (C) Reaction of the alkynes with lissamine rhodamine B azide (4) by CuAAC shows the successful printing by causing a fluorescent pattern.
Figure 5. CTP with complex TFP stamps. (A) Schematic illustration of a chemically pattered, flat TFP stamp with acetamide- and TFP-modified surface areas. (B, C) Fluorescence microscopy images of TRITC-streptavidin immobilized on a biotin pattern, which was prepared by printing the molecule with the bifunctionalized TFP stamp described in image A. (D) Schematic illustration of a chemically and topographically pattered TFP stamp. (E, F) Fluorescence microscopy images of lissamine rhodamine B azide (4) attached to a pattern of terminal alkynes. The pattern was prepared by CTP of heptynoic acid (3) using a TFP stamp described in image D.
explained by an optimal transfer efficiency that might be explainable by the flexibility of the dendrimer amines on the surface. Additionally, biotin was printed with TFP stamps (10-μmwide TFP lines that are spaced by 5 μm) in lines on ethylene glycol amine-modified surfaces (Figure 3C). Fluorescence microscopy after streptavidin binding verified the immobilization of the protein to the functionalized surface areas (Figure 3F). To investigate the reusability, the stamps were again loaded by exposure to a solution of the ink, washed with DMF, dried, and pressed onto another ethylene glycol-modified substrate. The procedure was repeated so that up to three printings with the same stamp were carried out. Fluorescence microscopy revealed that the resolution and the pattern quality decreased with every printing (Figure 3F). Moreover, the reproducibility of the printing results decreased. These observations can be explained by polymer surface rearrangements that take place during exposure to DMF in the loading step and by the mechanical stress that takes place on the polymer surface during printing. We also observed the increasing appearance of cracks with every printing. Nevertheless, the reusability of the stamps is possible, and patterns with an acceptable accuracy can be achieved even in the third print. Besides the CTP of D-biotin, heptynoic acid (3) was printed with TFP stamps (dots with a diameter of 10 μm). The printing procedure was carried out as in the case of biotin with the difference that an acid concentration of 0.1 M was used in the loading step. The successful immobilization of the alkyne on an
aminopropyl-terminated surface was verified by the attachment of lissamine rhodamine azide (4) via copper-catalyzed azide− alkyne cycloaddition (CuAAC) and subsequent fluorescence microscopy analysis. The appearance of the fluorescence pattern verified the successful printing and also the attachment of the fluorophore to the surface. After the investigation of the printing results with monofunctionalized, chemically patterned TFP stamps, bifunctionalized TFP stamps were prepared and used for printing studies. For instance, a stamp was prepared by first printing Nacetylcysteamine on hexenyl-modified PDMS (10-μm-wide lines spaced by 5 μm) and then TFP-thiol (1) in the perpendicular direction (10-μm-wide lines spaced by 5 μm). The procedure led to a flat stamp with TFP-modified rectangles (5 μm × 10 μm) that are spaced by N-acetylcysteaminemodified squares (10 μm × 10 μm) (Figure 5A). This pattern was obtained because the printing of N-acetylcysteamine in the first step led to a complete consumption of alkenes in the contacted areas so that TFP-thiol (1) could not be immobilized in these regions during the second printing. CTP of biotin with the obtained stamp on an aminopropyl-functionalized surface and subsequent streptavidin binding showed that biotin was successfully transferred in TFP-modified areas (Figure 5C,D). The results again prove the general suitability of the concept of CTP. Finally, CTP was also investigated by using a doublepatterned TFP stamp. The used stamp had a topographical surface structure that was simultaneously modified by a pattern of immobilized TFP groups (Figure 5D). Preparation of the F
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ACKNOWLEDGMENTS We thank Siltronic AG (Burghausen, Germany) for the donation of silicon wafers and Prof. Martin Winter (Westfälische Wilhelms-Universität Münster) for access to the XPS system. The Deutsche Forschungsgemeinschaft is acknowledged for the financial support of this work (grant Ra 1732/21).
stamp was carried out by printing TFP-thiol (1) with an oxidized PDMS stamp (dots with a diameter of about 10 μm) on a structured, hexenyl-functionalized PDMS stamp (10-μmwide lines spaced by 5 μm). CTP of heptynoic acid (3) with this type of stamp on aminopropyl-modified glass and the subsequent attachment of lissamine rhodamine B azide (4) led to a complex fluorescence pattern (Figure 5E,F). Intense fluorescence was found in areas where the TFP groups of the stamp came into conformal contact with the substrate. This observation verifies the chemical transfer of acyl residues from TFP groups to the amines of the surface. Nevertheless, some fluorescence was found in all contacted areas. This can be explained by residual amounts of coupling agent and heptynoic acid that diffuses out of the PDMS during printing and leads to background peptide coupling in all contacted surface areas. Another observation with topographically patterned TFP stamps was that the achieved patterning was not very accurate because the contact areas of the stamps were deformed (Figure 5E,F). The deformation was caused by the swelling of the polymer during exposure to DMF. It must be concluded that CTP should be carried out with flat, chemically patterned stamps and not with topographically patterned stamps if solvents are involved that lead to a significant swelling of the stamp polymer.
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CONCLUSIONS We have shown that TFP-modified PDMS stamps can be used for the chemical transfer printing (CTP) of carboxylic acids on amine-functionalized surfaces. The acids were attached covalently by the formation of stable amide bonds. The transfer was achieved by contacting the active ester-modified stamps with the amine SAMs. The chemical transfer of a single monolayer of immobilized molecules by a covalent reaction at the interface of two surfaces has to our knowledge not been reported so far. Nevertheless, all printing experiments and subsequent fluorescence microscopy analysis indicated the general suitability of the concept. Furthermore, the reusability of the stamps was demonstrated, and up to three printings could successfully be carried out with the same stamp. Chemically patterned, flat stamps were found to give better printing results than topographically structured stamps. Because of the fact that ink diffusion and stamp deformation can be prevented in CTP, submicrometer resolution should be achievable. The possibility to immobilize the TFP residues on the surface of the stamps by photolithography opens the way for the preparation of nanostructured stamps. Nanopatterning by TFP stamps in combination with an investigation and optimization of the transfer efficiency will be central elements in further studies. ASSOCIATED CONTENT
S Supporting Information *
Synthesis and analysis of inks and adsorbates. Fluorescence microscopy experiments. XPS data. This material is available free of charge via the Internet at http://pubs.acs.org.
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