Letter pubs.acs.org/NanoLett
Programmable Patterning of Protein Bioactivity by Visible Light Cordula Reuther,†,‡ Robert Tucker,† Leonid Ionov,† and Stefan Diez*,†,‡ †
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, 01307 Dresden, Germany
‡
ABSTRACT: The simple and quick patterning of functional proteins on engineered surfaces affords an opportunity to fabricate protein microarrays in lab-on-chip systems. We report on the programmable patterning of proteins as well as the local activation of enzymes by visible light. We successfully generated functional protein patterns with different geometries in situ and demonstrated the specific patterning of multiple kinds of proteins side-by-side without the need for specific linker molecules or elaborate surface preparation. KEYWORDS: Protein patterning, polyNIPAM, kinesin, microtubules, visible light
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proteins side-by-side by sequential processing without the need for specific linker molecules or elaborate surface preparations. Advantages of using visible light (instead of ultraviolet or infrared light) in the proposed photothermal patterning approach are that it is (i) inoffensive to proteins, (ii) versatile regarding the wavelength range, and (iii) easy to implement into a conventional fluorescence microscope. A schematic of our experimental setup is shown in Figure 1A. Our samples consisted of glass substrates coated with a “lightto-heat” converting layer. In order to enable an efficient conversion of light-to-heat, we decided on employing carbon, which (in contrast to other light absorbing materials like silicon or gold) possesses low reflectance and high absorption values over the whole visible wavelength range. For the pattering experiments, PNIPAM chains were grafted onto the carbon surfaces. The coated samples were assembled into flow-cells and mounted onto a Peltier element that enabled global temperature regulation. For local heating, the geometrical shapes of various diaphragms with different sizes were imaged onto the coated samples. The PNIPAM layer was characterized in detail by Ionov et al.24 In aqueous solution, the polymer chains are hydrated and assume an extended conformation below the lower critical solution temperature (LCST; 32 °C). In contrast, above the LCST the polymer chains are dehydrated and assume a collapsed conformation. The thickness H of the PNIPAM layer was about H = 6 nm in the collapsed state (35 °C) and increased about 8 times to H = 48 nm in the swollen state (25 °C). The polymer grafting density was optimized such that the average distance between the anchoring points of individual polymer chains D was about 4 nm. Therefore, the polymer layer in the swollen as well as in the collapsed state was in the brush regime (H > D).25 Moreover, below the LCST, PNIPAM
atterning functional proteins onto artificial substrates is of interest for advancing nanotechnology, tissue engineering, biosensor development, and cell biology.1−13 Toward this end, a number of chemical deposition methods based on optical lithography,14,15 atomic force microscopy (AFM),16,17 printing techniques,18 and chemical vapor deposition (CVD)19,20 have been applied recently. While each of these methods provides particular advantages, a general trade-off between spatial resolution, throughput, and maximum pattern size exists. For example, AFM-based techniques can be used to place small numbers of functional proteins with nanometer lateral resolution but are limited to low writing speeds and small pattern sizes. Optical methods, such as light-based activation of functional groups or ligands on surfaces,21 overcome most of the aforementioned limitations as they offer the potential for high through-put production combined with an often sufficient spatial resolution. However, when based on conventional lithography, expensive metal masks are needed, only predefined patterns can be created, and the high-energy of the ultraviolet radiation (often needed to trigger the photoactivation of proteins or protein-binding molecules) can be harmful for biological material.22 In addition to the localized deposition of proteins, for many applications it is important to externally control the function of surface-bound proteins in a spatiotemporal manner. Recently, ultraviolet-light based activation (similar to the protein-patterning approaches described above) has been used for the photochemical regulation of protein function, for example, when studying intracellular processes.23 Here, we report on the programmable patterning of proteins as well as the local change of enzyme accessibility (herein after referred to as local enzyme activation) using patterned widefield illumination by visible light. Specifically, the light locally heats the surface and switches the conformation of a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) polymer coating in which the proteins are adsorbed. We demonstrate the specific patterning of multiple kinds of © XXXX American Chemical Society
Received: April 24, 2014 Revised: May 29, 2014
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Figure 1. Light-induced heating on carbon-coated glass surfaces. (A) Experimental setup for local heating with visible laser light. The glass substrate to be optically patterned was coated with a “light-to-heat” converting carbon layer and was mounted to a Peltier element for temperature control on the upper side of a microscopic flow-cell. (B) Experimentally determined temperature distribution on a surface heated by a circular illumination pattern (diameter di = 20 μm). The point-to-point velocities of kinesin-driven microtubules gliding across the locally illuminated area were obtained by FIESTA.31 Applying a temperature-velocity calibration curve, the corresponding temperature values were determined and plotted as a function of the distance between the microtubule center and the center of the illuminated area (center of the illuminated area at x = 0). (C) Simulated temperature profiles across rectangular, longitudinally extended patterns (width of 20 μm) in dependence of the volumetric heat release rate of a 45 nm thick carbon layer upon absorption of light. (D) Simulated temperature profiles in dependence of the width of the illuminated rectangular patterns. The volumetric heat release rate was kept constant at 3 × 1013 W/m3.
constant at 23.5 °C. In a cyclic order the surface was locally illuminated by a 20 μm wide circular pattern for 2 s alternating with taking a fluorescence image of the microtubules within 100 ms exposure time. To avoid photobleaching of the Alexa 488labeled microtubules, we applied a wavelength of 561 nm for local light-to-heat conversion. For a relative laser intensity of 25% (total output power of the laser 3.6 mW) the point-topoint velocities of microtubules gliding across the locally illuminated surface were obtained by microtubule tracking based on the open-source software FIESTA.31 The local temperature values were determined according to the velocitytemperature calibration curve from ref 30 and are plotted in Figure 1B as a function of the distance between the microtubule center from the center of the illuminated circle. We found that the temperature in the illuminated area rose above 32 °C and reached values of up to 40 °C, which is below the denaturation temperature of most proteins. In the immediate vicinity of the illuminated area, the temperature dropped sharply to an average temperature of about 27 °C. Although due to heat dissipation this temperature is slightly higher than the Peltier temperature, it is still well below the LCST of PNIPAM. Without illumination, the surface temperature set by the Peltier element was verified by average microtubule velocities, which corresponded to 23 °C. When
chains repelled proteins, like kinesin-1 and streptavidin, from the surface and screened surface-bound proteins from solution. When the polymer chains collapsed above the LCST, small gaps opened up in-between them and allowed protein adsorption to the surface (primary adsorption).26,27 However, the polymer grafting density should be adjusted for molecules that differ significantly in size or shape in order to achieve the same temperature-induced switching of protein adsorption properties. To successfully pattern or activate functional molecules on a sample surface, it was important that the temperature in the illuminated area was higher than the LCST of PNIPAM but lower than the thermal denaturation temperature of proteins. Therefore, we characterized the light-induced heating on the carbon layers experimentally. We employed a biomolecular transport system to measure the local temperature distribution on the surface in situ. In particular, we performed in vitro gliding motility assays,28,29 where microtubules were propelled over a carbon-coated glass surface (without PNIPAM) covered with ATP-hydrolyzing kinesin-1 motor proteins. Because the enzymatic activity of kinesin-1 and thus the microtubule gliding velocity is temperature-dependent, the local temperature values could be determined using a velocity-temperature calibration curve.30 The temperature of the Peltier element was kept B
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Figure 2. Photothermal patterning of protein molecules. (A) Schematic illustration of photothermal protein patterning. Localized heating upon illumination (561 nm) causes the collapse of the thermoresponsive polymer, resulting in adsorption of proteins in the illuminated areas. When the illumination is removed, the photopatterned proteins remain entrapped in the polymer layer, and the swollen polymer chains prevent further protein binding. (B) Fluorescence images of Alexa Fluor 488 streptavidin patterned sequentially with increasing laser intensity (maximum power output 3.6 mW) and a circular illumination (di = 12.8 μm). The corresponding fluorescence intensity profiles across the center of each circle are depicted below. (C) Fluorescence images (left) and fluorescence intensity profiles (right) of Alexa Fluor 488 streptavidin patterned sequentially with different diaphragm sizes and size-adapted laser intensities. (Scale bars represent 20 μm.)
increasing the relative laser intensity to 35%, microtubules moving into the illuminated areas detached from the surface. This effect was presumably caused by irreversible denaturation of the kinesin-1 motor proteins evidenced also by the fact that microtubule gliding could not be recovered on these areas when the temperature was lowered later on. In contrast, control experiments on glass surfaces without carbon layers did not show any velocity increase. Thus, the temperature profile for a 20 μm wide circular pattern on carbon surfaces was ideal for PNIPAM switching and protein patterning when illuminated with a relative laser intensity of 25%.
To further investigate the potential of the photothermal patterning technique with respect to resolution and scalability, temperature distributions across illuminated, longitudinally extended rectangles were modeled using the Comsol software. Our simulations predict that the size of the “programmed” area, that is, the area where the local temperature was above the LCST of PNIPAM, increases with the volumetric heat release (Figure 1C), the width of the illuminated pattern (Figure 1D), and the Peltier temperature. The observed impact of the width of the illuminated pattern on the induced heating (Figure 1D) results from an increased relative heat-dissipation at the rims of C
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Figure 3. Sequential patterning of different proteins. (A) Streptavidin molecules labeled with three different dyes were consecutively patterned next to each other using laser light with λ = 561 nm (circular pattern, di = 12.8 μm). Fluorescence images were taken using the following excitation wavelengths and emission filters λex = 405 nm and BP 450/50 for Alexa Fluor 405; λex = 488 nm and BP 535/30 for Alexa Fluor 488; λex = 405 nm and BP 535/30 for Alexa Fluor 430. The right image is a pseudocolored overlay of the individual images. (B,C) Color overlays of fluorescence images showing patterned Alexa Fluor 488 streptavidin (green) with (B) microtubules (red) immobilized on the kinesin-1 pattern in the presence of the nonhydrolyzable ATP-analogue AMPPNP and (C) the maximum projection of moving microtubules (red) in the presence of ATP. (Scale bars represent 20 μm.)
smaller patterns. This also applies for different pattern shapes. Comparing the experimentally determined temperature distribution for a circle (Figure 1B) with the simulated distributions for rectangles in Figure 1C showed in the immediate vicinity of the illuminated pattern a steeper temperature drop for the circular pattern. Therefore, the laser power needs to be optimized for different pattern sizes and shapes. The dependence on the Peltier temperature may be instrumental in generating steeper temperature profiles by applying a lower Peltier temperature in conjunction with higher laser power. Moreover, our modeling revealed that steady-state conditions of local heating and heat dissipation are reached within 100 ms. As the conformational change of PNIPAM is faster than 10 ms,32 the illumination duration necessary for successful protein patterning can thus be optimized for the time necessary for sufficient protein adsorption onto the surface. We tested our approach by photothermally patterning fluorescent streptavidin molecules (labeled with Alexa Fluor 488, Figure 2). Experiments were performed by incubating streptavidin solutions with PNIPAM-layers grafted to carbonglass samples kept at low temperature (21 °C) such that the PNIPAM was in the extended, protein-repelling conformation (Figure 2A, left). For each pattern, the surface was illuminated for 1 min (again at 561 nm to avoid photobleaching of the fluorescently labeled streptavidin) in order to allow the
streptavidin molecules to bind out of solution (Figure 2A, middle). Unbound protein was washed out before imaging the generated patterns of protein irreversibly bound to the surface (Figure 2A, right). Protein molecules that possibly adsorbed to the polymer brush (ternary adsorption) during local heating by visible light were most likely released24,26 and washed out afterward (T < LCST) as well. In a first set of experiments, the sample was illuminated by a circular pattern (illuminated diameter di = 12.8 μm) with varying laser intensities. Between each illumination step the stage was shifted. The fluorescence images in Figure 2B show that the pattern size increased with increasing laser intensity. At a relative laser intensity below 30%, the induced heating was not sufficient to switch the conformation of the PNIPAM chains, and thus no streptavidin molecules bound to the surface. For a relative laser intensity of about 40%, the patterned circle had a diameter of about 12 μm, corresponding to the projected size of the diaphragm opening. Moreover, the whole area of the circle showed comparatively uniform fluorescence intensity. At higher laser intensities, the pattern size increased but also showed decreasing fluorescence intensity in the center. An increasing temperature, especially in the center of the pattern, probably caused this intensity decrease due to damage of the proteins or the fluorescent dye. In a second set of experiments, circular streptavidin patterns were created using different sizes of the illuminated patterns D
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Figure 4. Local activation of kinesin-1 by light-induced heating. (A) Schematic illustrations of the surface configurations during the experiment. Explanations and corresponding fluorescence images (all in the same field of view, depicted directly below the schematics) are shown in panels B to D. (B) Keeping the sample at 20 °C, PNIPAM chains in the extended conformation prevented microtubules (fluorescently labeled) in solution from binding to the surface-attached kinesin-1 molecules. (C) Localized illumination with laser light (561 nm) and subsequent conversion of light-to-heat caused the local collapse of the thermoresponsive polymer chains. The surface-bound kinesin-1 motors became accessible for microtubule gliding and were thus locally activated. The shown maximum projection of moving microtubules revealed that kinesin-1 molecules were only activated in the illuminated (i.e., heated) area and not on the surface around. (D) Global heating of the sample to 35 °C activated all surface-bound kinesin-1 motors and allowed microtubules to glide everywhere. (Scale bar represents 20 μm.)
proteins but also with different kinds of proteins, fluorescent streptavidin and unlabeled kinesin-1 motor proteins were patterned in a noncircular geometry (Figure 3B,C). First, fluorescent streptavidin (labeled by Alexa Fluor 488) was patterned by locally illuminating an elongated rectangular shape (sequential illumination of twice a rectangular shape with a = 3.2 μm and b = 12.8 μm with a 10 μm shift in y-direction in between). Afterward, unlabeled kinesin-1 molecules were patterned with three similar sequential illumination steps (using λ = 561 nm or λ = 488 nm, both generating patterns of similar quality). While the successful patterning of Alexa Fluor 488 streptavidin could be directly visualized (green area in the upper left corners of Figure 3B,C), microtubules were used to verify the successful patterning of functional kinesin-1 motors. Therefore, microtubules in a solution containing adenylyl-imidodiphosphate (AMPPNP, a nonhydrolyzable analogue of ATP) were added to the flow-cell. The temperature of the sample was then raised to 35 °C to globally collapse the PNIPAM chains on the entire surface and allow the microtubules to bind to the patterned kinesin-1 molecules. The immobilized microtubules (red signal in Figure 3B) visualize the position of the kinesin-1 pattern adjacent to the streptavidin pattern. After addition of an ATP-containing motility solution, the bound microtubules, as well as new microtubules binding from solution, were gliding across the patterned kinesin-1 molecules and were released at the edges of the pattern (Figure 3C). The robust gliding demonstrated that the photothermally adsorbed motor proteins kept their biological function and were not denatured during patterning.
(Figure 2C). The laser intensity in each illumination step was adjusted such that the resulting patterns had about the size of the illuminated area. Thus, relative laser intensities of 50, 80, and 100% were applied for di = 12.8, 7.1, and 4.8 μm, respectively. The circular protein pattern with the smallest size had a diameter of about 5 μm. However, smaller patterns may be realized by higher laser intensities. Both sets of experiments prove that proteins can be patterned reproducibly onto PNIPAM-coated carbon-glass samples via photothermal patterning. Moreover, the experimental results are in qualitative agreement with our theoretical predictions. To generate specific patterns of different kinds of proteins side-by-side, the possibility to sequentially pattern proteins is of importance. In one experiment, we tested whether streptavidin molecules labeled with three different dyes (Alexa 405, 488, and 430) could be patterned consecutively next to each other. For each of the three sequential patterning steps, the substrate surface was illuminated by laser light (561 nm) with a 12.8 μm wide circular pattern. In between, the protein solutions were exchanged and the microscope stage was shifted 20 μm in the ydirection. Afterward, using the corresponding excitation wavelengths and emission filters, monochromatic images were taken for each dye and overlaid in a three color image (Figure 3A). The result shows that each of the differently labeled protein was patterned highly specific just in one of the formerly illuminated areas. Thus, the patterned proteins must have been protected against further protein binding during subsequent incubation steps by the swollen PNIPAM chains. To demonstrate that this works not only with differently labeled E
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paths. It will thus enable the versatile and straightforward (re)configuration of directed transport. Moreover, applying our technique to pattern different types of motor proteins in a spatially separated manner will offer new possibilities for the biophysical investigation of intracellular mechano-systems in reconstituted in vitro environments. Materials and Methods. Carbon and PNIPAM Coating of the Glass Substrates. The glass substrates were coated with 45 nm thick layers of amorphous carbon by ion beam sputtering (performed by the Fraunhofer IWS (Dresden, Germany)). The method for grafting PNIPAM onto the carbon-coated glass surfaces was adapted from Ionov et al.36 Briefly, carbon-coated substrates were spin-coated (2000 rpm, 500 rpm/s, 30 s) with a 0.01% poly(glycidyl methacrylate) (PGMA, Mn = 65 000 g/ mol) solution in chloroform. The PGMA was annealed at 130 °C for 20 min in a vacuum oven. After annealing, the substrates were placed in hot chloroform (70 °C) in order to remove unbound PGMA. Poly(N-isopropylacrylamide) (PNIPAM, Mn = 45 000 g/mol) was dissolved in chloroform (1% solution). The surface of the substrates was then completely covered with a droplet of the PNIPAM solution. After the chloroform evaporated, the substrates were placed in the vacuum oven at 160 °C for 60 min to anneal the PNIPAM. Unbound PNIPAM was removed by washing the substrates in hot chloroform (70 °C). Flow-Cell Preparation. Protein patterning and activation assays as well as kinesin-1 gliding motility experiments were performed in 2 mm-wide flow-cells self-built from a carbon and/or PNIPAM coated sample on one side and a PEGylated coverslip37 on the other side. Pieces of parafilm were used as spacers. The back of the sample was colored with black permanent marker to block the autofluorescence of the thermal contact. The sample was mounted on a Peltier element38 with heat transfer compound for keeping it at a defined temperature. The Peltier was coupled to a power supply and a thermometer (Physitemp BAT-10). Kinesin-1 Gliding Motility Assays. Wild type kinesin-1 (full length Drosophila melanogaster) was expressed in Escherichia coli and purified applying a published protocol.39 Microtubules were polymerized from 5 μL of porcine brain tubulin40 (4 mg/ mL; labeled with different fluorophores as stated elsewhere) in BRB80 buffer (80 mM potassium PIPES, pH 6.9, 1 mM EGTA, 1 mM MgCl2) with 4 mM MgCl2, 1 mM Mg-GTP, and 5% DMSO at 37 °C. After 30 min, the microtubule polymers were stabilized and diluted 100-fold in room-temperature BRB80 containing 10 μM taxol. For gliding assays, a casein-containing solution (0.5 mg/mL in BRB80) was perfused into the flow-cell and allowed to adsorb to the surface for 5 min. Then a 10 μg/ mL kinesin-1 solution in BRB80CA (BRB80 buffer containing 1 mM Mg-ATP, 0.2 mg/mL casein, and 10 mM dithiothreitol (DTT)) was perfused. After 5−10 min, a microtubule containing solution (Motility solution: BRB80 with 10 μM taxol, microtubules (equivalent of 32 nM tubulin) 1 mM ATP, 40 mM D-glucose, 55 μg/mL glucose oxidase, 11 mg/mL catalase, 10 mM DTT) was added to the cell and imaging was started. Patterning of Proteins. The PNIPAM sample was kept at low temperature (15−25 °C) when the patterning solution (Streptavidin solution (0.125 mg/mL) containing a fluorescently labeled strepavidin conjugates (Alexa Fluor 405 or 430 or 488; Invitrogen) in BRB80 or kinesin-1 solution (20 μg/ mL) containing 0.5 mg/mL casein, 1 mM Mg-ATP, and 10 mM DTT in BRB80) was perfused into the flow-cell. The
Besides locally patterning functional proteins, we studied the possibility to locally control the activity of surface-bound enzymes by light-to-heat conversion. We therefore performed microtubule gliding assays on surfaces where kinesin-1 molecules were adsorbed everywhere on a PNIPAM coated glass-carbon surface at 35 °C (Figure 4). After adjusting the temperature to 20 °C, all microtubules were released from the surface, demonstrating the functionality of the PNIPAM layer (Figure 4A left panel and B). The Peltier element was then set to 24 °C and the surface was locally illuminated with laser light (561 nm) by a circular pattern (di = 20 μm). Microtubules started to land and glide exclusively in the photothermally heated area within less than 1 min of illumination (Figure 4A middle panel and C). The microtubules were gliding with an average velocity of 0.96 μm/s but also showed point-to-point velocities of up to 1.5 μm/s. These values correspond to temperatures of 32 and 40.5 °C, respectively. When the sample was globally heated to 35 °C by the Peltier all surface-bound kinesin-1 motors were activated and this allowed microtubules to glide everywhere (Figure 4A right panel and D). The activation was reversible and could be repeated. This experiment provides evidence that enzymes like kinesin-1 can be uniformly adsorbed to a substrate surface and then locally activated by light. In summary, we used patterned wide-field illumination by visible light to locally collapse a thermoresponsive polymer on carbon-coated glass substrates by light-to-heat conversion. Thereby, functional protein patterns with different geometries and sizes could be successfully generated in situ. As such, our method complements an approach recently published by Cheng et al.,33 who used a focused infrared laser to create two-dimensional protein micropatterns in a point-scanning manner. However, our results advance this technique by the possibility to use visible light patterned by various shapes of diaphragms. Visible light is inoffensive to proteins, versatile regarding the wavelength range, and makes our method widely applicable to most fluorescence microscopy setups. In contrast to the ring-shaped patterns created by a focused IR-beam, patterned wide-field illumination allowed to generate patterned areas showing a relatively homogeneous protein density within the micrometer range. Additionally, we demonstrated that it is possible to specifically pattern multiple kinds of proteins sideby-side on the same surface. Using other approaches this often is difficult because one needs a prestructured surface and different linkers or protein properties. Moreover, we showed on the example of the kinesin-microtubule transport system that patterns of enzymes can be reversibly activated and deactivated in a local and global manner on the time scale of seconds. This feature may, for example, be of interest for advanced nanotechnological applications of biomolecular transport systems.34 There, it will be beneficial to combine the local, optical activation of motor proteins with the guiding of filament-based shuttle systems in topographical channels. Locally confined pads of switchable polymers, integrated into topographical guiding structures, may act as gates or switchable junctions. Illuminating a specific gate will locally collapse the polymer chains, activate the motor proteins and allow the shuttles to pass. Compared to recent approaches achieving dynamically switchable tracks or gates by electrical heating,30,35 optical control (possibly improved by the use of digital lightprocessing technology) allows a very precise confinement of the heated area as well as programmable activation without the need for lithographically structuring any predefined conducting F
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(5) Sniadecki, N.; Desai, R. A.; Ruiz, S. A.; Chen, C. S. Nanotechnology for cell-substrate interactions. Ann. Biomed Eng. 2006, 34, 59−74. (6) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044−3063. (7) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric control of cell life and death. Science 1997, 276, 1425−1428. (8) McBeath, R.; Pirone, D. M.; Nelson, C. M.; Bhadriraju, K.; Chen, C. S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 2004, 6, 483−495. (9) Senaratne, W.; Andruzzi, L.; Ober, C. K. Self-Assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives. Biomacromolecules 2005, 6, 2427−2448. (10) Besson, E.; et al. A Novel and Simplified Procedure for Patterning Hydrophobic and Hydrophilic SAMs for Microfluidic Devices by Using UV Photolithography. Langmuir 2006, 22, 8346− 8352. (11) Babacan, S.; Pivarnik, P.; Letcher, S.; Rand, A. G. Evaluation of antibody immobilization methods for piezoelectric biosensor application. Biosens. Bioelectron. 2000, 15, 615−621. (12) Feng, C. L. Reactive microcontact printing on block copolymer films: Exploiting chemistry in microcontacts for sub-micrometer patterning of biomolecules. Adv. Mater. 2007, 19, 286. (13) Reuther, C.; Hajdo, L.; Tucker, R.; Kasprzak, A. A.; Diez, S. Biotemplated Nanopatterning of Planar Surfaces with Molecular Motors. Nano Lett. 2006, 6, 2177−2183. (14) Allen, R. D. Trends in patterning materials for advanced lithography. J. Photopolym. Sci. Technol. 2007, 20, 453−455. (15) Yamaguchi, M.; et al. Protein patterning using a microstructured organosilane layer fabricated by VUV light lithography as a template. Colloids Surf., A 2006, 284, 532−534. (16) Tinazli, A.; Piehler, J.; Beuttler, M.; Guckenberger, R.; Tampe, R. Native protein nanolithography that can write, read and erase. Nat. Nanotechnol. 2007, 2, 220−225. (17) Ginger, D. S.; Zhang, H.; Mirkin, C.A. The Evolution of DipPen Nanolithography. Angew. Chem., Int. Ed. 2004, 43, 30−45. (18) Xia, Y. N.; Whitesides, G. M. Soft lithography. Angew. Chem., Int. Ed. 1998, 37, 551−575. (19) Slocik, J. M. Site-specific patterning of biomolecules and quantum dots on functionalized surfaces generated by plasmaenhanced chemical vapor deposition. Adv. Mater. 2006, 18, 2095. (20) Jung, J. M.; Kwon, K. Y.; Ha, T. H.; Chung, B. H.; Jung, H. T. Gold-conjugated protein nanoarrays through block-copolymer lithography: From fabrication to biosensor design. Small 2006, 2, 1010− 1015. (21) Holden, M. A.; Cremer, P. S. Light Activated Patterning of DyeLabeled Molecules on Surfaces. J. Am. Chem. Soc. 2003, 2003, 8074− 8075. (22) Gerhardt, K. E.; Wilson, M. I.; Greenberg, B. M. Ultraviolet wavelength dependence of photomorphological and photosynthetic responses in Brassica napus and Arabidopsis thaliana. Photochem. Photobiol. 2005, 81, 1061−1068. (23) Young, D. D.; Deiters, A. Photochemical control of biological processes. Org. Biomol. Chem. 2007, 5, 999−1005. (24) Ionov, L.; Stamm, M.; Diez, S. Reversible Switching of Microtubule Motility Using Thermoresponsive Polymer Surfaces. Nano Lett. 2006, 6, 1982−1987. (25) Brittain, W. J.; Minko, S. A structural definition of polymer brushes. J. Polym. Sci., Polym. Chem. 2007, 45, 3505−3512. (26) Choi, S.; Choi, B. C.; Xue, C.; Leckband, D. Protein Adsorption Mechanisms Determine the Efficiency of Thermally Controlled Cell Adhesion on Poly(N-isopropyl acrylamide) Brushes. Biomacromolecules 2013, 14, 92−100. (27) Huber, D. L.; Manginell, R. P.; Samara, M. A.; Kim, B.-I.; Bunker, B. C. Programmed Adsorption and Release of Proteins in a Microfluidic Device. Science 2003, 301, 352−354.
sample was then locally illuminated with laser light (λ = 561 nm for streptavidin and kinesin-1 patterning; λ = 488 nm worked equally well for kinesin-1) through a 63× water immersion objective (Zeiss, numerical aperture NA = 1.2) for 1 min using a DirectFRAP unit (Zeiss) with a diaphragm wheel. Thereby, the desired pattern was illuminated with homogeneous intensity as an expanded laser beam was directed onto the diaphragm, located within the optical path of the used microscope. After patterning, nonadsorbed protein was removed by multiple perfusions with BRB80CA. For sequential patterning of different proteins, these steps were repeated. In the last step either antifade solution (BRB80 with 40 mM Dglucose, 55 μg/mL glucose oxidase, 11 mg/mL catalase, and 10 mM DTT) or microtubule containing solution (motility solution or microtubule solution: BRB80 with 10 μM taxol, microtubules (equivalent of 32 nM tubulin) 1 mM adenylylimidodiphosphate (AMPPNP), 40 mM D-glucose, 55 μg/mL glucose oxidase, 11 mg/mL catalase, 10 mM DTT) was added to the cell. Imaging. All experiments were performed on a Zeiss Axio Observer inverted optical microscope with a 63× water immersion objective (NA = 1.2). For data acquisition, a spinning disc scan head (Yokogawa CSU-X1) with two cameras (Zeiss AxiCam MRm and Roper evolve 515 EMCCD) was used in conjunction with AxioVision software (Zeiss). Images were acquired with an exposure time of 100 ms.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Addresses
(R.T.) Hansen Medical, Mountain View, CA 94043, U.S.A. (L.I.) Leibniz Institute of Polymer Research Dresden e.V., 01069 Dresden, Germany. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge Till Korten, Viktor Schröder, and Corina Bräuer for fruitful discussions and technical support. Special thanks go to Stefan Braun and Peter Gawlitza from the Fraunhofer IWS (Dresden, Germany) who realized the carbon coating of our samples. Moreover, we are grateful that the Light Microscopy Facility of the MPI-CBG provided equipment and assistance. The work was financially supported by the European Research Council (starting Grant 242933, NanoTrans), the Volkswagen Foundation (Grant I/ 82093), the German Research Foundation (Cluster of Excellence Center for Advancing Electronics Dresden, the Heisenberg Program, and grant IO 68/1-1).
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REFERENCES
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