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Functional Binary Micropattern of Hyperbranched Poly(ether amine) (hPEA-AN) Network and Poly(ether amine) (PEA) Brush for Recognition of Guest Molecules Xiaolu Ye,† Xuesong Jiang,*,†,‡ Bing Yu,† Jie Yin,† and Philipp Vana‡ †

School of Chemistry & Chemical Engineering, State Key Laboratory for Metal Matrix Composite Materials, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China ‡ Institut für Physikalische Chemie, Georg-August-Universität Göttingen, Tammannstr. 6, D-37077 Göttingen, Germany ABSTRACT: A binary micropattern of anthracene-contained hyperbranched poly(ether amine) (hPEA-AN) network and poly(ether amine) (PEA) brush on gold surface was developed and explored. First, a micropatterned hPEA-AN network array on gold surface was fabricated by photolithography via photodimerization of anthracene moieties, and a PEA brush was subsequently immobilized on the remaining free gold surface areas by chemical adsorption of thiol groups. The patterned hPEA-AN network exhibits selectivity with respect to the adsorption of hydrophilic dyes: Methyl orange is strongly adsorbed, but rhodamine 6G is not, as indicated by the fluorescence response. The PEA brush domain exhibits excellent protein adsorption repellency, whereas the hPEA-AN network layer readily adsorbs protein. These characteristics make the binary hPEA-AN network and PEA brush array sensitive to different kinds of dyes and proteins, which open up pathways to potential applications as microsensors, biochips, and bioassays.

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INTRODUCTION Large-scale and high-throughput patterned polymer surfaces are of fundamental importance to many areas of modern technology such as the printing technology,1 microfluidics,2 sensors,3−5 microchannels,6 bioassays,7 and so on. Among the most studied polymers, polymer brushes and polymer networks are widely used in patterning surfaces. Because of its robustness, its ability to modify surface properties on a molecular scale and the variation of chemical and mechanical properties, patterned polymer brushes provide a pathway to control the surface properties according to the functionality of polymer, which is immobilized on the surface.7−10 The other widely studied patterned polymers are networks, which constitute an important class of biomaterials.11−13 The patterned networks are frequently used in the development of biochips,14 cell control and manipulation,15,16 and protein microarrays.17,18 Despite the numerous applications of patterned polymer brushes and networks, patterned surface arrays combining a mixture of polymer brushes and networks are less explored. A binary patterned polymer brush and network array, however, can provide a structured surface with more abundant functions.4,19−28 In the present work, we demonstrate the fabrication of a binary micropatterned polymer surface consisting of a polymer brush and a network film structure on gold surface (Scheme 1). Gold has been widely used in a number of bioanalytical devices and bioassays due to its high conductivity, easy surface-modification, and excellent resistance to oxidation.9,18,29−33 The control of the surface properties of gold is one of the key factors to the performance of these © 2011 American Chemical Society

Scheme 1. (a) Chemical Structure of Anthracene-Contained Hyperbranched Poly(ether amine) (hPEA-AN) and ThiolContaining graft Poly(ether amine) (gPEA-SH) and (b) Strategy for the Fabrication of the Micropatterned Binary Surface

biomedical devices.8,29,34−36 Many research efforts have thus been focused on the modification of gold surfaces with networks and polymer brushes.7,14,29,37−39 In this work, the micropatterned network film was first fabricated on the gold surface via photolithograpy, which is one of the well-established technologies for the preparation of micropatterns and has found wide applications in microReceived: November 16, 2011 Revised: December 25, 2011 Published: December 30, 2011 535

dx.doi.org/10.1021/bm201614y | Biomacromolecules 2012, 13, 535−541

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then washed in chloroform by ultrasonication to remove physically adsorbed polymer, leaving the covalently immobilized PEA brush.39 Dyes Adsorption Experiments. The gold substrate with hPEAAN network was immersed in the R6G or MO/Mili-Q water (0.5 mg/ mL) for 30 min and then washed extensively with pure water running for 10 min. To get a better comparable result, we dipped part of the substrate into the dye solution. The part of the micropattened hPEAAN network can adsorb dye molecules, whereas the other part cannot. The fluorescence imaging was carried out with the laser scanning confocal microscope (LSCM) with the excitation wavelength set to 405 nm, and the related fluorescence intensity of the fluorescence pattern was evaluated with Leica Software. Protein Adsorption Experiments. The gold substrates with hPEA-AN network or the binary surface (hPEA-AN@PEA) were dipped in BSA-FITC/phosphate buffer solution (PBS) (2 mg/mL, pH 7.4) for 6 h and then washed extensively with pure water running for 30 min. The protein adsorption fluorescence imaging was performed by LSCM with a 40× objective, and the excitation wavelength was set at 488 nm. Characterization. Besides gold substrates, hPEA-AN layer was also prepared on the quartz slides for UV−vis and fluorescence spectra. The quartz plates with hPEA-AN network covered were prepared by the same way mentioned above. (The quartz plates were spin-coated with hPEA-AN chloroform solution (5 wt %). After drying at 80 °C for 30 min, the hPEA-AN layer was irradiated with 365 nm light.) The UV−visible spectra of the hPEA-AN network on the quartz slides with different irradiation energy were checked by a UV-2550 spectrophotometer (Shimadzu). The influence of irradiation energy change on fluorescence intensity of hPEA-AN network was measured by QM/TM/IM steady-state and time-resolved fluorescence spectrofluorometer (PTI Company). The excitation wavelength was set at 381 nm, and the fluorescence emission spectra (FLES) were recorded between 400 and 650 nm. The quartz slides covered with hPEA-AN network (exposure dose is 800 mJ/cm2) were also addressed with R6G and MO solution. Then, the UV−visible and fluorescence spectra of the network before and after dye adsorption were recorded. The surface topography of the micropatterned binary hPEA-AN network and PEA brush were observed by profile micrometer (VF7510 KEYENCE) with VF-125 objective and by atom force microscopy (AFM). (AFM images were taken by SII Nanonavi Esweep under ambient conditions.) The AFM was operated in contact mode by using silicon nitride cantilevers with a force constant of 0.12 N m−1. Fluorescence images of the micropatterned hPEA-AN network were viewed with an LSCM (Leica TCS-SP5, Leica, Wetzlar, Germany) equipped with UV lasers. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) experiments were carried out on a PHI-5000C ESCA system (Perkin-Elmer) with Al Kα radiation (hn = 1486.6 eV). In general, the X-ray anode was run at 250 W, and the high voltage was kept at 14.0 kV with a detection angle at 54°. The pass energy was fixed at 46.95 eV to ensure sufficient sensitivity. The base pressure of the analyzer chamber was ∼5 × 10−8 Pa. The sample was directly pressed to a self-supported disk (10 × 10 mm) and mounted on a sample holder and then transferred to the analyzer chamber. The whole spectra (0−1200 eV) of all elements were recorded with high resolution. The data analysis was carried out by using the RBD AugerScan 3.21 software provided by RBD Enterprises or XPS Peak 4.1 provided by Raymund W. M. Kwok. Water Contact Angle Measurements. Water contact angle (WCA) measurements were measured with a contact angle meter (model CAM Micro) at room temperature. The precision of the angle measurement was ±0.1°. Contact angles were averaged from at least three different spots for each sample.

electronics because of its potentials for large-scale production and easy processing.15,40,41 Anthracene-containing hyperbranched poly(ether amine) (hPEA-AN) was chosen for the fabrication of micropatterned networks because of several advantages: hPEA-AN can be cross-linked by photodimerization of anthracene upon UV-light exposure,42,43 which means that the patterned network can be fabricated by photolithography without addition of photoinitiator. Very recently, we found that the cross-linked hPEA-AN nanogels exhibit strong fluorescence and can encapsulate hydrophilic guest molecules in water; after encapsulation of methyl orange (MO), the fluorescence of cross-linked hPEA-AN nanogels was almost completely quenched.43 These novel characteristics motivated us to fabricate the micropatterned hPEA-AN networks upon surfaces, which may find applications as a microsensor. Thiol-containing graft poly(ether amine) (gPEASH) was immobilized on the gold surface by chemicaladsorption of the thiol group.29,44 gPEA-SH can form a thick polymer brush with graft density of ∼0.8 chain/nm2 on gold surface in less than 5 min, which exhibits excellent performance in terms of protein-resistance with long-term stability.39 After confirmation of the formation of the binary patterned hPEAAN network and PEA brush array on the gold surface, we demonstrated that the obtained patterned polymer surface was sensitive to guest molecules such as dyes and proteins. On the binary patterned surface, proteins were oriented adsorbed in the predetermined sections. These characteristics may potentially be exploited in microsensors and bioassays.7,10,11,45

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EXPERIMENTAL SECTION

Materials. Anthrance-containing hyperbranched poly(ether amine) (hPEA-AN) and thiol-containing graft poly(ether amine) (gPEA-SH) were synthesized as previous report.43,44 The molar ratio of between the hydrophobic PPO and the hydrophilic PEO monomer is 2/1 in the synthesis of hPEA-AN. The molar ratio of the hydrophobic PPO and the hydrophilic L100 is 1/2 in the synthesis of gPEA-SH. (111)Oriented single-crystal silicon wafer was purchased from Shanghai Risen. The silicon wafers were cut into square chips of 1 cm × 1 cm in size. Rhodamine 6G (R6G, Aldrich) and MO (Sinopharm Chemical Reagent) were both used without further purification. Bull serum albumin conjugated with fluorescein isothiocyanate (BSA-FTIC) was bought from Beijing Zoman Biotechnology. Chloroform, ethanol, and other chemicals are of analytical grade, except as noted. Gold Substrates Preparation. The silicon wafers were first treated with a strong acidic oxidizing solution of concentrated sulfuric acid and hydrogen peroxide (H2SO4/H2O2 (30%) 3/1 v/v) for 1 h to remove the native organic layer (Caution: Be caref ul using “piranha” solution as it is very dangerous), then were washed thoroughly with pure water and dried. The silicon wafers were coated with a chromium (40 nm) adhesion layer, followed by sputtering of the 100 nm gold layer (SPF-312, ANEIVA). The obtained gold surface was cleaned by a mixture of hydrogen peroxide/ammonium hydroxide/water 1/1/5 at 80 °C for 5 min to remove any carbonaceous contamination, then washed thoroughly with Milli-Q water and dried in a vacuum oven. Fabrication of the Micropatterned Binary hPEA-AN Network and PEA Brush. The processes for fabrication of the patterned hPEAAN network and PEA brush surface were shown in Scheme 1. The clean and dry gold substrate was spin-coated with hPEA-AN chloroform solution (5 wt %). After drying at 80 °C for 30 min, the thickness of hPEA-AN layer was ∼180 nm. The hPEA-AN layer was covered by a mask and then irradiated with 365 nm light of 800 mJ/ cm2. The substrate was then developed in chloroform for 5 min to remove the un-cross-linked hPEA-AN. The gold substrate with the patterned hPEA-AN network was immersed in 1% gPEA-SH chloroform for 1 h. The gold substrate was

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RESULTS AND DISCUSSION

Fabrication and Characterization of the Micropatterned Binary Polymer Surface (hPEA-AN@PEA). The micropatterned binary polymer surface array on gold was prepared by fabricating the patterned hPEA-AN network 536


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light, resulting from the dimerization of AN. Taking both crosslinking reaction and fluorescence intensity of hPEA-AN layer into consideration, the exposure dose of UV-light was chosen as 800 mJ/cm2 for the generation of the patterned hPEA-AN network. Upon the exposure dose of 800 mJ/cm2, the dimerization degree of AN is ∼62%, which guaranteed both enough cross-linking intensity of hPEA-AN to form network and strong fluorescence of the resulted patterned network. After the patterned hPEA-AN network was obtained, the gold substrate was immersed in gPEA-SH chloroform (1 wt %) for 1 h. Because of the strong coordination between −SH groups and Au atoms,29,39 gPEA-SH was immobilized on the area of gold surface, which was uncovered by the previous hPEA-AN network. To confirm the formation of PEA brushes, we used XPS to analyze the patterned surface. In the XPS spectrum of the gold substrate with hPEA-AN network (Figure

through photolithography, followed by the immobilization of gPEA-SH chains on the uncovered area of the gold surface via chemical adsorption. The whole strategy to fabricate the micropatterned binary polymer surface on gold substrate is illustrated in Scheme 1. hPEA-AN chloroform solution was first spin-coated on the gold substrate to obtain hPEA-AN layer with thickness of ∼180 nm. The hPEA-AN layer was covered by a mask and was then irradiated by UV light. Because anthracene can undergo photodimerization upon irradiation with UV light (λ> 300 nm), the hPEA-AN layer of the exposure area can be photo-cross-linked. After being developed in chloroform, the polymer in the unexposed area was removed, leaving the cross-linked hPEA-AN network pattern behind. The topography and the fluorescence micrograph of the obtained hPEA-AN pattern are shown in Figure 1. The network pattern

Figure 1. (a) Surface topography and (b) fluorescence micrograph of the micropatterned hPEA-AN layers on the gold substrate. Scale bars: (a) 80 μm and (b) 100 μm.

on the surface was found to be neat and smooth, indicating an excellent pattern quality. Fluorescence images were taken by LSCM and revealed that the cross-linked hPEA-AN network exhibited strong blue fluorescence, which can be ascribed to the emission of residual AN groups in hPEA-AN. To determine the exposure dose of UV-light, we measured the UV−vis spectra of hPEA-AN layer on the quartz slide after the exposure of UV light (Figure 2). The characteristic absorption assigned to AN groups decreased with the increasing exposure dose of UV, which can be ascribed to the photodimerization of AN groups upon the exposure of UV light. The photodimerization of AN groups leads to the crosslinking of hPEA-AN to form network. The dimerization degree of AN can be estimated according to the change of AN characteristic absorption in UV−vis spectra (Figure 2a).43 Upon the exposure of UV light, the emission fluorescence spectra of hPEA-AN layer were also recorded. The fluorescence intensity decreased with the increasing exposure dose of UV

Figure 3. XPS spectra of (a) gold substrate with the micropatterned hPEA-AN layer and (b) gold substrate with the binary micropatterned hPEA-AN layer and PEA brush (hPEA-AN@PEA).

3a), the signals at 285, 532, and 399 eV were observed, which could be ascribed to C, O, and N of hPEA-AN, respectively. The appearance of the strong signals at 84 and 350 eV assigned to Au (Au 4f, Au 4d) indicated that hPEA-AN network did not cover the whole surface of gold. After the deposition process in gPEA-SH chloroform solution, the signals at 89 and 350 eV almost disappeared (Figure 3b), indicating that PEA brush was grafted to the gold surface and fully covered the bare area, which was not overlapped by the patterned hPEA-AN network. Water contact angle measurement was used to trace the processes for the formation of the patterned binary polymer surface (Figure 4). WCA on the bare gold surface is ∼88°, whereas WCA on the patterned hPEA-AN network is 72°. The

Figure 2. (a) UV−vis spectra and (b) fluorescence emission spectra of hPEA-AN network films with different exposure energy. 537


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exhibits a fluorescent pattern with strong blue emission (Figure 6a) before immersion. As shown in Figure 6b, the part of pattern, which was immersed in MO solution, was almost dark, whereas the rest of pattern exhibits very strong blue fluorescence emission. Compared with the immersed area, the fluorescent emission intensity of the nonimmersed area was 10 times stronger (Figure 6d,e). This can be explained by the fact that the fluorescence emission of hPEA-AN network, which originates from the excited AN, was quenched by the adsorbed MO molecules. To further understand the interaction between the hPEA-AN network and the dye MO in water, we prepared an hPEA-AN network on a quartz slide, then recorded its UV− vis and fluorescence spectra before and after the adsorption of MO. It can be seen in Figure 6g that the absorption around 430 nm, which can be ascribed to the absorption of MO, is observed in the UV−vis spectrum of the hPEA-AN network after the immersion, which suggests the encapsulation of MO in the hPEA-AN network. As shown in Figure 6h, the fluorescence intensity of the hPEA-AN network was greatly decreased after the absorption of MO, which can be attributed to the quenching of the excited anthracene by the MO guest molecule. The hPEA-AN networks are fluorescent as a result of the blue emission from AN. MO molecules have a relatively strong visible absorption in the range of 400−550 nm. When MO is adsorbed by the hPEA-AN network, the distance between MO molecules and anthracene moieties becomes so small that the blue emission from the hPEA-AN network can instantaneously be absorbed by MO. As a result, the blue emission intensity of the hPEA-AN network decreases greatly after the adsorption of MO. We also immersed the hPEA-AN network in aqueous solution of the hydrophilic dye R6G. Interestingly, we found that the hPEA-AN network could not adsorb R6G. As shown in Figure 6i, the UV−vis spectra of hPEA-AN network kept almost the same before and after the immersion, suggesting that no encapsulation of R6G in the hPEA-AN network took place. This was further confirmed by the fluorescence image of the patterned surface (Figure 6c). No apparent change was found after the patterned hPEA network was immersed in R6G aqueous solution as well as by the relative fluorescence intensity (Figure 6f). On the basis of these results from the dye adsorption experiments, we can conclude that the micropatterned hPEA-AN network can adsorb specific hydrophilic dyes selectively, which provides pathways to potential applications as microsensor.

Figure 4. Water contact angle on the different surfaces (a) gold, (b) patterned hPEA-AN network, and (c) binary patterned hPEA-AN@ PEA.

changed WCA can be explained by the fact that parts of the hydrophobic gold surface were covered by hPEA-AN network, resulting in the decreased WCA. After the chemical-adsorption of gPEA-SH on the uncovered area of the gold surface, WCA on the binary patterned hPEA-AN@PEA is ∼61°, indicating the successful immobilization of the hydrophilic PEA brush on the gold surface. The surface morphology of PEA-AN network and PEA brush was revealed by AFM images (Figure 5), which showed the micropattern with high quality. This is in good agreement with the results from the profile micrometer. Compared with the smooth and homogeneous gold surface (Figure 5b), the surface displayed small “mushroom-like” states after chemical adsorption of gPEA-SH, indicating the presence of PEA brush (Figure 5d). The thickness of the hPEA-AN network and PEA brush was determined according to the sectional profile images of AFM. As shown in Figures 5c,f, the height of the single patterned hPEA-AN network was ∼174 nm, whereas the height difference between the hPEA-AN layer and PEA brush is ∼146 nm, indicating that the thickness of PEA brush is ∼28 nm. Selective Adsorption of the Hydrophilic Dyes. Our previous research has shown that core-cross-linked nanoparticles of poly(ether amine) can selectively encapsulate hydrophilic dyes in water,43,46 and the fluorescence of hPEAAN nanoparticle can be quenched quantitatively by such adsorbed dyes.43,46 It is very attractive to know whether our micropatterned hPEA-AN network can also selectively encapsulate hydrophilic dyes because the patterned network surfaces might be used as the platform for microsensors. To probe this idea, we carefully immersed the part of gold substrate with the patterned hPEA-AN network in aqueous MO solution for 1 h, and it was subsequently by pure water to remove adsorbed dye on the surface of the network. The micropatterned hPEA-AN network before and after this procedure was evaluated by LSCM. The hPEA-AN network

Figure 5. AFM pictures of (a) gold substrate with the micropatterned hPEA-AN layer and (d) gold substrate with micropatterned hPEA-AN layer and micropatterned PEA brush and the corresponding sectional-profile images (c,f). The AFM image b shows the bare gold substrate, and panel e is the section covered with the PEA brush. 538


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Figure 6. Fluorescence micrographs of (a) the micropatterned hPEA-AN layer, (b) the micropatterned hPEA-AN layer partially disposed with MO (left half of the picture), and (c) the micropatterned hPEA-AN layer disposed with R6G and the corresponding relative intensities (d−f). (g) UV− vis spectra and (h) fluorescence emission spectra of the hPEA-AN layer before and after MO adsorption. (i) UV−vis spectra of the hPEA-AN layer before and after R6G adsorption. Scale bars: 250 μm in panels a−c.

Protein Adsorption on the Binary Pattern (hPEA-AN@ PEA). Motivated by the excellent performance of the patterned hPEA-AN network with respect to selective response to dye guest molecule, we evaluated the interaction between the binary patterned hPEA-AN@PEA and BSA protein conjugated with FTIC (BSA-FTIC) by fluorescence microscopy. The adsorption of BSA-FTIC was observed both on micropatterned hPEAAN network and on bare gold, which was proved by a bright green fluorescence micrograph (Figure 7a). Compared with the very small green dots on the bare gold surface, however, which could be caused by the aggregation of adsorbed BSA-FTIC, the hPEA-AN network exhibited much stronger and very homogeneous green emission, suggesting that BSA was not only adsorbed on the surface but also encapsulated inside the hPEA-AN network. Figure 7b shows an LSCM image of the binary patterned hPEA-AN@PEA after absorption of BSAFTIC. The area of the PEA brush is almost dark, whereas the area of hPEA-AN network exhibits a strong green emission. The dense green dots on the bare gold disappeared after growth of PEA brush, indicating that no adsorption of BSA took place and that the PEA brush exhibits excellent resistance

Figure 7. Fluorescence micrograph for BSA-FITC adsorbed onto a gold substrate with micropatterned hPEA-AN layer only (a) and both micropatterned hPEA-AN layer and micropatterned gPEA-SH brush (b). Scale bars: 75 μm in panels a and b.

against nonspecial protein adsoprtion. The strong fluorescence contrast demonstrates the formation of the regular protein microassays on the binary hPEA-AN@PEA surface. 539


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The hPEA-AN network and PEA brush exhibit rather contrary interaction to BSA-FTIC, which is the key factor for the formation of well-defined protein microassays. The interaction between hPEA-AN layer and BSA is proposed to include two parts: one is the electrostatic interaction and the other is the hydrophobic interaction. BSA is electronegative in PBS (pH 7.4) and can be adsorbed on the hPEA-AN layer through electrostatic interaction because of the existence of amino groups in hPEA-AN. Because of the large amount of the hydrophobic AN moieties and short PPO chains in hPEA-AN, hydrophobic domains can be formed in the hPEA network, resulting in the adsorption of BSA through hydrophobic interaction. Taking the structure of PEA brush into consideration, the hydrophobic PPO chains containing −SH groups are closed to the surface of gold, whereas the hydrophilic PEO chains stretch outside to water, which provide the excellent resistance against BSA-FTIC.7,47,48

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CONCLUSIONS We developed a functional binary micropattern of hPEA-AN network and PEA brush (hPEA-AN@PEA) on gold surface, which was fabricated by combining photolithography of hPEAAN and chemical-adsorption of PEA-SH. The patterned hPEAAN network exhibited selective interaction and noninteraction with the hydrophilic dyes MO and R6G, respectively, with respect to the fluorescence response. The PEA brush domains exhibit excellent resistance against BSA-FTIC adsorption, whereas the hPEA-AN layer adsorbs BSA-FTIC deliberately. Consequently, a well-defined protein microassay was formed on the surface of hPEA-AN@PEA. These characteristics of the micropatterned hPEA-AN network and gPEA brush open up potential applications in the field of (i) microsensors, as the micropatterned binary surface possesses selective response to specific guest molecules, such as dyes and proteins, and in the field of (ii) bioassays, as proteins are properly directed to defined regions by the patterned hPEA-AN layers and formed protein microarray on the surface, which would be useful for some point-of-care diagnostics.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-21-54743268. Fax: +86-21-54747445.

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ACKNOWLEDGMENTS We thank the National Nature Science Foundation of China (21174085), Science & Technology and Education Commission of Shanghai Municipal Government (11QA1403100, 12ZZ020), and the Shanghai Leading Academic Discipline Project (B202) for their financial support. X.J. also acknowledges support by the Alexander von Humboldt Foundation. P.V. acknowledges receipt of a Heisenberg-Professorship by the German Research Foundation (DFG).

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REFERENCES

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