Formation of a Polymer Surface with a Gradient of Pore Size Using a

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Formation of a Polymer Surface with a Gradient of Pore Size Using a Microfluidic Chip Kristina Kreppenhofer,†,# Junsheng Li,‡,§,# Rodrigo Segura,∥ Ludmilla Popp,† Massimiliano Rossi,∥ Pavleta Tzvetkova,⊥ Burkhard Luy,⊥ Christian J. Kaḧ ler,∥ Andreas E. Guber,† and Pavel A. Levkin*,‡,§ †

Institute of Microstructure Technology, Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany § Department of Applied Physical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany ∥ Institute of Fluid Mechanics and Aerodynamics, Universität der Bundeswehr München, 85577 Neubiberg, Germany ⊥ Institute for Biological Interfaces, Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany ‡

S Supporting Information *

ABSTRACT: Here we demonstrate the generation of polymer monolithic surfaces possessing a gradient of pore and polymer globule sizes from ∼0.1 to ∼0.5 μm defined by the composition of two polymerization mixtures injected into a microfluidic chip. To generate the gradient, we used a PDMS microfluidic chip with a cascade micromixer with a subsequent reaction chamber for the formation of a continuous gradient film. The micromixer has zigzag channels of 400 × 680 μm2 cross section and six cascades. The chip was used with a reversible bonding connection, realized by curing agent coating. After polymerization in the microfluidic chip the reversible bond was opened, resulting in a 450 μm thick polymer film possessing the pore size gradient. The gradient formation in the microfluidic reaction chamber was studied using microscopic laser-induced fluorescence (μLIF) and different model fluids. Formation of linear gradients was shown using the fluids of the same density by both diffusive mixing at flow rates of 0.001 mL/min and in a convective mixing regime at flow rates of 20 mL/min. By using different density fluids, formation of a two-dimensional wedge-like gradient controlled by the density difference and orientation of the microfluidic chip was observed.

1. INTRODUCTION

However, so far, there are only few methods reported for the preparation of surfaces possessing a gradient of pore sizes. Collins et al.15 described a method for the preparation of gradient porous silicon films using electrochemical etching in hydrofluoric acid. The pore size of the prepared silicon surface ranged from 500 nm. Later, this method was used to prepare silicon surfaces with a gradient of pore sizes for studying neuroblastoma cell behavior23 and mesenchymal stem cell adhesion and differentiation.24 Woodfield et al.25 presented a 3D fiber deposition technique to produce polymer scaffolds mimicking the organization of an articular cartilage with pore sizes increasing from 200 to 1650 μm. Oh et al.26 used centrifugal forces to fabricate cylindrical scaffolds with pore sizes ranging from 88 to 405 μm. In the present work we develop a microfluidic chip enabling the fabrication of porous polymethacrylate films possessing gradients of pore sizes in the range from tens of nanometers to ∼1−2 μm. The method is based on the ability to precisely

Porosity, as a surface property, is essential for cell−surface interactions because it allows multidirectional exposure of cells and bacteria, growing on the surface, to soluble factors and nutrients. Most natural surfaces serving for attachment and growth of cells, such as basement membranes, are porous.1 Therefore, technical porous surfaces can provide substrates that better mimic the natural environment of cells in vivo. Gradient surfaces with continuously varying pore sizes along one direction offer the possibility to avoid difficulties associated with the one-sample-for-one-measurement approach and sample variations. Furthermore, they can increase the throughput of studies of cell surface interactions. There are a number of reports describing methods for making onedimensional (1D) gradients of surface chemistry.2−9 Several groups reported the preparation of 1D gradients of elasticity10−12 and surface morphology.8,13−22 Meredith et al. also prepared a surface possessing a two-dimensional (2D) gradient of roughness vs surface chemistry.20 Behavior of eukaryotic cells on gradients of morphology18,20,22 and on gradients of elasticity11,12 has also been studied. © 2013 American Chemical Society

Received: December 17, 2012 Revised: February 18, 2013 Published: February 21, 2013 3797

dx.doi.org/10.1021/la304997a | Langmuir 2013, 29, 3797−3804

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Figure 1. (A) A PDMS microfluidic chip for generating gradient polymer films. The chip contains a micromixer based on a cascade of zigzag microchannels (left side) and a reaction chamber (right side) for the continuous gradient and a glass slide sealing the microchannels. (B) Micromilled aluminum master structure with cascade micromixer (left side) and subsequent reaction chamber (right side). The frame allows for a reproducible thickness of the replicated PDMS parts. (C) Fabrication of a polymer gradient film using the microfluidic chip: (a) Polymerization mixtures are injected into the microfluidic chip by syringe pump building a gradient. The gradient is exposed to UV light for polymerization. (b) Removal of the PDMS part of the microfluidic chip. (c) Resulting gradient polymer film on the glass slide. the zigzag channels to the outlet. External dimensions of the PDMS part are standardized to the size of a glass slide (25 × 76 mm2). We used a poly(dimethylsiloxane) (PDMS; Sylgard 184 Silicone Elastomer Kit, Dow Corning) casting process against a master structure for simple and quick replication of the structured PDMS part. The master, as shown in Figure 1B, is micromilled from an aluminum blank. The two components, base and curing agent (Catalyst 87-RC, Dow Corning Corporation) of the Kit were mixed using the recommended mixing ratio of 10:1 (base:curing agent). The mixture was degassed in a desiccator and casted into the aluminum master. Homogeneous form filling was observed after a waiting time of 5 min. The PDMS was cured in an oven (WTB Binder Labortechnik GmbH) for 70−100 min at 65 °C and removed from the master. Inlets and outlet are punched open using precision dispensing tips (Nordson EFD). The frame around the structure in the master produces reproducible thick parts of 4 mm. To form a stable but reversible seal between the PDMS and glass, we coated the glass slide (Nexterion, Schott, Germany) with a curing agent.34,35 We used the Catalyst 87RC from the Dow Corning Corporation. A specially produced stainless steel roller was used to form a homogeneous and thin layer of the agent. The flexible PDMS part was unrolled onto the coated glass slide allowing air-inclusion-free bonding. Subsequently, the assembled chip was cured at 65 °C for 45 min in an oven (WTB Binder Labortechnik GmbH). Tubes were inserted into the punched holes. For the inlets we used a polyamide tube (inner diameter: 0.5 mm, Reichelt Chemietechnik GmbH & Co). The outlet was connected to a wider polysulfone tube (inner diameter: 1.5 mm, LHG-Laborgeräte Handelsgesellschaft mbH) for blockage prevention. Tubes were fixed with BEST Silicone 301 (BEST Klebstoffe GmbH & Co. KG). The microfluidic chips were left in an air-conditioned lab for 7 days to harden completely and obtain their final mechanical properties before usage.

control morphology of a porous polymethacrylate structure by varying the composition of porogenic solvents in the polymerization mixture.27 Using a microfluidic cascade micromixer,28−32 we create a thin film of the polymerization mixtures possessing a gradient of the porogenic component of the mixture. Subsequent UV-initiated polymerization of the film leads to the formation of a porous polymer surface with a gradient of pore sizes but with the same chemical composition. The potential of these micromixers to generate complex shaped gradients was reported elsewhere.28 Similar micromixers were used to analyze the influence of immobilized molecule gradients on cells2 and to produce gradient hydrogels.33 The gradient formation in the present micromixer was studied using the microscopic laser-induced fluorescence (μLIF) technique. The mixing of different model fluids in the reaction chamber was evaluated. Adapted flow rates were used to generate polymeric gradient films. The morphology of the surface and the bulk material of the polymer films were characterized by scanning electron microscopy (SEM).

2. MATERIALS AND METHODS 2.1. Manufacturing of the Microfluidic Chip. The microfluidic chip consists of (1) a microstructured poly(dimethylsiloxane) (PDMS) part containing a micromixer with two inlets and subsequent reaction chamber for creating the gradient and (2) a glass slide sealing the microchannels. The microfluidic chip is depicted in Figure 1A. The structured part of the microfluidic chip is made from PDMS and subsequently covered with a glass slide. All microstructures have a height of 680 μm. Zigzag channels of the micromixer have a width of 400 μm. The reaction chamber is 15 mm wide and 19 mm long from 3798


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The glass slides used for making the microfluidic chips were modified with 3-(trimethoxylsilyl)propyl methacrylate, as described before,36 to increase the adhesion between the porous polymethacrylate film and the glass surface. Briefly, glass slides were first activated by immersing in 1 M NaOH solution for 1 h, followed by immersing in 1 M HCl solution for 1 h. The activated glass was subsequently modified with a solution of 3-(trimethoxylsilyl)propyl methacrylate (20 vol %; pH value of the solution was adjusted to 5 with acetic acid) by placing a few droplets of the solution to one glass slide and covering it with another one for 30 min. 2.2. Polymerization Mixtures for Making the Surfaces. The polymerization mixtures we used are composed of photoinitiator (2,2dimethoxy-2-phenylacetophenone, DMPAP), monomers (butyl methacrylate, BMA), cross-linker (ethylene glycol dimethacrylate, EDMA), and porogens (cyclohexanol for mixture 1 and 1-decanol for mixture 2). The porogens are solvents for the monomer/cross-linker and used for generating pores during the polymerization. The ratio between the two porogens present in the polymerization mixture (1-decanol and cyclohexanol) defines the morphology of the porous structure, while keeping the same porosity, which is determined by the amount of the porogens.27,36 The two mixtures showed differing densities of 0.8537 g/cm3 (mixture 2) and 0.9160 g/cm3 (mixture 1). The composition of the two polymerization mixture is described as follows. Mixture 1: 30 wt % EDMA, 20 wt % BMA, 1 wt % (with respect to monomers) photoinitiator DMPAP, and 50 wt % cyclohexanol. Mixture 2: 30 wt % EDMA, 20 wt % BMA, 1 wt % (with respect to monomers) photoinitiator DMPAP, 50 wt % 1decanol, and 0.1 wt % Rhodamine B. All the chemicals for the mixtures are from Sigma-Aldrich (Germany). To check the effect of porogen diffusion on the gradient formation, the diffusion coefficients were measured with diffusion ordered NMR spectroscopy (DOSY) experiments using bipolar gradient pulses and stimulated echo with longitudinal eddy current delay (BPLED).37 Measurements were performed using a Bruker 600 MHz Avance III spectrometer with a TCI inversely detected 1H,13C,15N triple resonance cryogenically cooled probehead with actively shielded zgradient at 298 K. Spectrometer stability was controlled using a sealed acetone-d6 capillary, which also served as an internal reference for the diffusion coefficient. The measured reference of 4.32 × 10−9 m2/s is in excellent agreement with the literature value of 4.30 × 10−9 m2/s,38 and the corresponding diffusion coefficients of cyclohexanol in mixture 1 and 1-decanol in mixture 2 have been determined to be 260 and 290 μm2/s, respectively. 2.3. Making Polymer Surfaces with Gradient Morphology. The microfluidic chip was attached directly to 20 mL syringes (B. Braun Melsungen AG) filled with polymerization mixtures. A syringe pump (Harvard Apparatus PHD Ultra) was used to inject the polymerization mixtures in the microfluidic chip. Both syringes were added on one rider ensuring equal flow rates. The microfluidic chip was mounted below a UV lamp (OAI model 30 deep-UV collimated light source (San Jose, CA) fitted with a 500 W HgXe lamp) used for polymerization. The microfluidic chips were mounted with the PDMS part on the top. Stabilization of the gradient was ensured by visual observation. UV exposure (260 nm, 12 mW/cm2) started immediately after the pump was stopped. The exposure time was 15 min, ensuring complete polymerization even at the bottom of the microstructures. After polymerization, the PDMS part was manually removed from the glass slide. The surface of the polymer film was dried with a nitrogen stream. The glass slide with the polymer film was kept in methanol overnight for removing porogens before potential usage.

and subsequently getting polymerized by exposure to UV-light (Figure 1C(a)). After formation of the solid polymer film by polymerization, the PDMS part is removed from the glass slide (Figure 1C(b)), leaving the gradient polymer film on the glass slide serving as substrate (Figure 1C(c)). The prepared surfaces have external dimensions of 12 × 15 mm2 and a thickness of ∼450 μm. The deviation of the polymer thickness from the height of the microfluidic chamber (680 μm) is probably caused by the shrinkage of the porous polymethacrylate, which has been observed in other applications of the porous polymer in chromatography.39 Another possible reason for the reduced thickness of the polymethacrylate film could be the swelling of PDMS induced by the polymerization mixture. Absorption of some organic solvents by PDMS was reported before.40,41 The compatibility between PDMS and different solvents was predicted by comparing the solubility parameter of PDMS (∼7.3 cal1/2 cm−3/2) with that of the solvents. Generally, solvents that have solubility parameter (ranging from 7.3 to 9.5 cal1/2 cm−3/2) similar to that of PDMS were thought to be less compatible with PDMS.42 Here, we used a polymerization mixture composed of butyl methacrylate, ethylene glycol dimethacrylate, and 1-decanol (or cyclohexanol). Alcohols generally have a good compatibility with PDMS,42 and the reported solubility parameter for butyl methacrylate is 9.61 cal1/2 cm−3/2.43 Therefore, we expected that the polymerization mixtures that we used would have a good compatibility with PDMS. In order to produce a porous polymer surface possessing a gradient of pore sizes, two different polymerization mixtures (mixtures 1 and 2) were pumped into the chip from the two inlets. Mixture 1 containing cyclohexanol as a porogen forms a nanoporous polymer with pore diameters in the range of tens of nanometers after polymerization.36 The use of 1-decanol (mixture 2) as the sole porogenic solvent gives a microporous polymer with the average globule size in the range 1−2 μm.36 The two mixtures were pumped into the chip at the flow rate of 0.25 and 7.5 mL/min for 30 s to fill the whole chamber. All flow rate values reported in the article refer to the flow rate applied at one inlet of the micromixer. UV-initiated polymerization started immediately after stopping the syringe pump. A polymer film was formed in the microfluidic chamber after 15 min of polymerization. The chip was then opened, and a porous polymer film on the glass substrate was obtained. We analyzed the morphology of the porous structure by SEM at two different positions of the polymer film: at the surface (see Figure 2 and Figure S2) and at the cross section roughly 50 μm below the surface (see Figure S4). Images were taken at eight equidistant measuring points along the gradient 10 mm down the reaction chamber (see Figure S1). One set of surface SEM

3. RESULTS AND DISCUSSION 3.1. Preparation of Gradient Porous Polymers Using Microfluidic Chips. The microfluidic chip composed of a glass slide and PDMS chamber was used to generate a gradient in this study. The generation of a gradient film using the microfluidic chip is shown schematically in Figure 1C. Two different liquid polymerization mixtures are injected into the microfluidic chip, creating a gradient in the reaction chamber

Figure 2. Scanning electron microscopy (SEM) images of surface of a gradient porous polymer film generated with a flow rate of 0.25 mL/ min. Scale bar: 20 μm. 3799


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phenomena: (1) Diffusion: effective at small flow rates, when the rate of advection of the flow in the main direction is comparable with the rate of diffusion. (2) Forced convection: effective at larger flow rates, when transversal secondary flows arise as a consequence of the meandering shape of the zigzag channels.46,47 Turbulent mixing is prevented by the channel dimensions. To better understand the gradient formation mechanisms and to define the range of operational flow rates of the present micromixer, the μLIF technique48 was used to visualize the concentration gradients in the reaction chamber under different experimental conditions. The experimental procedure was described before.49 Briefly, a fluorescent marker is dissolved in one of the two fluids and images of the mixing fluids are taken using an epifluorescence microscope. The image intensity is proportional to the concentration of the marker in the fluid volume. For the present setup, the images were taken using a Leica M165 FC fluorescence microscope connected to an Imager sCMOS camera (LaVision GmbH). The flow was established by a syringe pump (Harvard PHD 2000), using the same flow rate at the two mixer inlets. Rhodamine B was used as a fluorescent marker and dissolved in one of the two fluids. We started with a characterization injecting only one test fluid into the micromixer. Isopropanol was injected in both inlets, having the fluid marked with Rhodamine B in the lower inlet. The resulting diffusion gradients for the respective flow rates of 0.001, 1, and 20 mL/min are shown in Figure 4. The

images (flow rate 0.25 mL/min) is shown in Figure 2. The SEM images show a highly porous polymer structure with a gradient of roughness from micro- to nanoscale (Figure 2A). We quantified both pore and polymer globule sizes using SEM micrographs. Three polymer films were analyzed for each flow rate (0.25 and 7.5 mL/min). The average globule and pore sizes are summarized in Table S1. For the polymer film produced using low flow rate, the mean globule sizes at the cross section (50 μm below the surface) range from 1.60 ± 0.37 to 0.16 ± 0.04 μm in a clear gradient shape. For the polymer film produced using high flow rate, the mean globule size at the cross section (50 μm below the surface) decreases from 1.61 ± 0.34 to ∼0.16 ± 0.04 μm in a step gradient, which is consistent with the reduced diffusion time in this case. The SEM micrographs obtained at the surface of the porous polymer film revealed a similar gradient of the porous structure along the width of the polymer films, which was also dependent on the flow rate (Figure 3 and Figure S3). Higher flow rate would lead

Figure 3. Summary of the pore and globule size measurements obtained from SEM images for three gradient polymer films produced using flow rate of 0.25 mL/min.

to the formation of a sharp step-like pore/globule size gradient while the low flow rate leads to the formation of a more shallow gradient. For some positions on the gradient surfaces (e.g., positions 2 and 3 for surfaces produced at 0.25 mL/min flow rate (Figure 2) and position 3 for the surface made at 7.5 mL/ min flow rate (Figure S2)), we observed formation of a hierarchical surface morphology with the incorporation of smaller polymer globules at the polymer surface. This phenomenon may be explained by a stronger absorption of 1-decanol (“microporogen”) by the PDMS layer leading to a localized increase of the concentration of cyclohexanol (“nanoporogen”) and a reduction of the overall amount of porogens in the prepolymer mixture at the PDMS interface. This could also explain the reduced average size (position 1: ∼500 nm; position 8: ∼200 nm) of the polymer globules at the surface (Figures 2 and 3; Figures S2 and S3; Table S1) in comparison to that in the bulk. It should be noted, however, that the superficial layer of the porous polymer film can be removed by using adhesive tape.44 3.2. Hydrodynamic Characterization of the Micromixer. To define the range of operational flow rates, we performed a hydrodynamic characterization of the micromixer using test fluids and the polymerization mixtures. The theoretical calculation of the concentration profile of a cascade micromixer is described elsewhere.28,30 Assuming homogeneous mixing in each zigzag channel, the concentration at the end would show a linear gradient profile. In the named literature homogeneous mixing was achieved by diffusion. In the present micromixer mixing can occur due to two

Figure 4. Isopropanol (upper inlet) and Rhodamine B diluted in isopropanol (lower inlet) injected in the micromixer (flow direction left to right) and observed by fluorescence microscopy. Diffusion based, no mixing, and convective mixing can be observed respectively with increasing flow rates. The red lines mark the boundaries of the chamber.

red lines mark the boundaries of the reaction chamber. The two different mixing phenomena can be clearly observed: a linear concentration gradient is present at 0.001 mL/min due to diffusive mixing and at 20 mL/min due to convective mixing. At 1 mL/min the flow is too fast for diffusive mixing and too slow for the secondary flows, resulting in practically no mixing at the end. To analyze the performance of the micromixer with two different fluids, we conducted a second experiment using two different fluids, namely isopropanol (from the upper inlet, marked with Rhodamine B) and water (from the lower inlet, no color). Results are shown in Figure 5. In this case, an apparent uniform mixing is observed for 0.001 mL/min and a linear concentration gradient for 1 mL/min. What is actually happening depends on the fact that the fluids have different densities (density ratio ∼1.3). In the horizontal orientation of the chip, the lower density isopropanol tends to slip over the 3800


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Figure 5. μLIF measurement (left) using isopropanol marked with Rhodamine B (upper inlet) and water (lower inlet) injected in the micromixer (flow direction left to right). Below the microscope images schemes show the arrangement of the two fluids in a cross section of the reaction chamber. The chip was used in a horizontal position (right). At 0.001 mL/min, water and isopropanol are stacked horizontally one on top of the other. At 1 mL/min the isopropanol slips over the water, resulting in a two-dimensional wedge-like gradient (see schemes at the bottom).

Figure 6. μLIF measurement (left) using isopropanol dyed with Rhodamine B (upper inlet) and water (lower inlet) injected in the micromixer (flow direction left to right). Below the microscope images schemes show the arrangement of the two fluids in a cross section of the reaction chamber. The chip was used in a vertical position (right). At 0.001 mL/min, water and isopropanol are again stacked one on top of the other, leading to a vertical stacking in the cross section (microscope image and scheme). Diffusive mixing of the two fluids can be observed in-between the two phases. At 1 mL/min the isopropanol slips again over the water.

gradients formed at 0.001 and 1 mL/min flow rates more resembled the situation with two fluids of the same density (Figure 4). The dependence of a gradient shape on the orientation of a microfluidic chip can be explained if the fluid density has an effect on the gradient formation, thereby confirming the hypothesis of the wedge-like gradient formation in Figure 5. An additional confirmation of the density driven wedge-like gradient formation was obtained by analyzing a cross-section of the gradient polymer film by SEM (see Figure

water during the propagation through the microfluidic channels. At 0.001 mL/min water and isopropanol are stacked one on top of the other, resulting in an apparent uniform concentration (compare scheme of the cross section). At 1 mL/ min there is less time for the less dense fluid to slip over the other, resulting in a 2D wedge-like arrangement of the two fluids (compare scheme of the cross section) that looks like a linear gradient from the top view of the μLIF visualization. This hypothesis was proved by repeating the experiment with a vertically oriented microfluidic chip (see Figure 6). In this case, 3801


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7). A study of a horizontally oriented chip with higher flow rates is shown in the Supporting Information (Figure S5).

dimensional wedge-like gradients was confirmed by both μLIF and SEM of polymer film cross sections. For the polymerization mixtures the micromixer enables convective effects and a gradient using the mentioned wedge-like distribution. Using a flow rate of 0.25 mL/min, we generated polymer films with a gradient of polymer globule sizes ranging from ∼100 to ∼500 nm. These results demonstrate a convenient way to generate thin porous polymer films with gradients of porous morphology which can be useful in a variety of biological applications.

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ASSOCIATED CONTENT

S Supporting Information *

Globule size and pore size distribution of the polymer surface prepared using flow rate of 0.25 and 7.5 mL/min; μLIF (microscopic laser-induced fluorescence) measurement of water (upper inlet) and isopropanol marked with Rhodamine B (lower inlet) injected in the micromixer (flow direction left to right, flow rate 20 mL/min) and observed by fluorescence microscopy; at 20 mL/min convective mixing starts to take place. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 7. Overall cross section scheme (A) and SEM images (B) of a polymer film showing the density driven wedge-like pore size gradient (scale bar: 1 μm).

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel +49-721608-29175; Fax +49721608-29040. Author Contributions

Finally, we repeated the experiment with the polymerization mixture 1 from the upper inlet and the polymerization mixture 2, dyed with Rhodamine B, from the lower inlet. In this case, the density ratio is smaller (∼1.1) and the two fluids are significantly more viscous than water and isopropanol. The apparent concentration gradient, corresponding to the wedgelike arrangement as discussed above, was observed already at 0.001 mL/min and up to 0.25 mL/min, as shown in Figure S6. Some convection effects can be observed at 10 mL/min although the fluids remain substantially unmixed. It was not possible to go to higher flow rates with the current syringe pump equipment although it would be in general possible to increase the flow rate with more powerful equipment. The burst pressure of the microfluidic chips is 0.36 MPa. It was measured using water and a pressure transducer (WIKA Alexander Wiegand SE & Co. KG). This value fits well in the range for so-called irreversible bonds described elsewhere.31 In conclusion, the μLIF measurements showed that the polymer film created using 0.25 mL/min possesses a pore size wedge-like gradient as a consequence of the density difference between the two fluids. The two-dimensional wedge-like gradient is confirmed by analyzing the polymer film’s cross section by SEM, shown in Figure 7.

#

K.K. and J.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.A.L. and J.L. are grateful to the Helmholtz Association’s Initiative and Networking Fund (grant VH-NG-621), BioInterfaces programme, and the Department of Applied Physical Chemistry (Prof. M. Grunze) for financial support. J.L. thanks the China Scholarship Council for a PhD scholarship. P.T. and B.L. thank the HGF programme BioInterfaces and the Fonds der Chemischen Industrie for financial support. The authors thank Elias Schipperges (Institute of Microstructure Technology, Karlsruhe Institute of Technology), Conrad Grehl, and Jürgen Brandner (both Institute of Fluid Mechanics and Aerodynamics, Karlsruhe Institute of Technology) for support with the measurement of the burst pressure. The authors thank Klaus Bade (Institute of Microstructure Technology, Karlsruhe Institute of Technology) for contentual support concerning the distribution of the polymer mixtures in the reaction chamber.

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4. CONCLUSION In this paper we demonstrated controlled and facile fabrication of thin polymer films possessing a gradient of pore sizes, using a microfluidic chip. The microfluidic chip contains a cascade micromixer to generate a gradient of two polymerization mixtures and a subsequent reaction chamber for the photopolymerization. We show that the concentration gradient obtained in the reaction chambers can be controlled by the flow rate, type of the used fluids, and the orientation of the microfluidic chip. The formation of density driven two-

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