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Langmuir 1998, 14, 5526-5531
Micropatterning of Biomolecules on Polymer Substrates Alexandra Schwarz,† Joe¨l S. Rossier,† Emmanuelle Roulet,‡ Nicolas Mermod,‡ Matthew A. Roberts,† and Hubert H. Girault*,† Laboratoire d’Electrochimie, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland, and Laboratoire de Biotechnologie Mole´ culaire, Institut de Biologie Animale and Centre de Biotechnologie UNIL-EPFL, Universite´ de Lausanne, CH-1015 Lausanne, Switzerland Received March 31, 1998. In Final Form: June 22, 1998 UV-excimer laser photoablation was used, in combination with surface blocking techniques, to pattern proteins on the surfaces of polyimide and poly(ethylene terephthalate). This technique involves physical adsorption of avidin through laser-defined openings in low-temperature laminates or adsorbed protein blocking layers. Visualization of biomolecular patterns were monitored using avidin and fluoresceinlabeled biotin as a model receptor-ligand couple. Adsorbed proteins could be shown to bind to UV-lasertreated polymer surfaces up to three times higher than on commercially available polymers. UV-laser photoablation was also used for the generation of three-dimensional structure, which leads to the possibility of biomolecule patterning within polymer-based microanalytical systems. The simplicity and easy handling of the described technique facilitate its application in microdiagnostic devices.
Introduction In this article we present techniques, based on UVlaser photoablation, for the patterning of biomolecules onto polymer surfaces. These techniques create ablated patterns in protein blocking layers which then allow the easy deposition of biomolecules onto underlying substrates from bulk solutions. Such patterns can be accurately positioned and produced in highly parrallel arrays. The resulting biopatterned surfaces may find application in the production of diagnostic devices or as components in various bioanalytical systems. Many recent advances in chemical analysis have involved the incorporation of biomolecules onto functional surfaces of new devices, which are capable of selective and high affinity binding to analytes of interest. Recently, there has also been intense activity in the miniaturization of chemical instrumentation.1 Some promising bioanalytical applications have already been demonstrated, which are based on immobilized receptors within microfabricated fluid handling platforms,2-4 and further advances in this field can be expected based on emerging techniques in the control and patterning of materials of biological origin such as enzymes, antibodies, and DNA.5 It is also noteworthy that a combination of patterning with microfluidics was recently demonstrated whereby immunoglobulins could be immobilized onto surfaces using a microfluidic network in order to spatially define the resulting pattern.6 It is clear that the precise surface patterning of functional molecules will be critical to the further development of many technologies. Immobilization has been † ‡
E Ä cole Polytechnique Fe´de´rale de Lausanne. Universite´ de Lausanne.
(1) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 244. (2) Murakami, Y.; Takeuchi, T.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2731-2735. (3) Manz, A. Chimia 1996, 50, 140-143. (4) Huang, X. L.; Walsh, M. K.; Swaisgood, H. E. Enzyme Microb. Technol. 1996, 19, 378-383. (5) Chrisey, L. A.; O’Ferrall, E. C.; Spargo, B. J.; Dulcey, C. S.; Jeffrey, C. M. Nucleic Acids Res. 1996, 24, 3040-3047. (6) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779-781.
accomplished in the past, mostly by application of bulk solution techniques and without the ability for careful spatial delineation of patterns.7 Recently, photoimmobilization8-10 and photolithography11-13 techniques, mimicking developments in microelectronics, have become central approaches to pattern proteins on surfaces. A patterning technique for covalent attachment of biomolecules onto silicon dioxide surfaces through the use of heterobifunctional cross-linking reagents was described by Ligler.14 In that report, UV-irradiation of surfaces, containing thiol, epoxy, or vicinal diol functionalities, was shown to be capable of patterning biomolecules with the use of a photomask. Recently, alternatives to photolithographic fabrication methods have also been explored such as the use of microcontact printing.15 Some authors have specifically reported on the micropatterning of the avidin receptor, citing its widespread use with various biotinylated reagents that have become commercially available. Electrostatic immobilization was reported to pattern avidin on polystyrene with submicrometer feature sizes.16 Kuhr et al.17 also described a maskless photolithography technique for patterning avidin on carbon electrodes. In this technique, photosensitive biotin was immobilized using the interference pattern of convergent UV-laser radiation. Five micrometer wide (7) Deshpande, S. S. Enzyme Immunoassays; Chapman and Hall: New York, 1996. (8) Hengsakul, M.; Cass, A. E. G. Bioconj. Chem. 1996, 7, 249-254. (9) Morgen, H.; Pritchard, D. J.; Cooper, J. M. Biosens. Bioelectron. 1995, 10, 841-846. (10) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (11) Flounders, A. W.; Brandon, D. L.; Bates, A. H. Biosens. Bioelectron. 1997, 12, 447-456. (12) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 1205012057. (13) Sundarababu, G.; Gao, H.; Sigrist, H. Photochem. Photobiol. 1995, 61, 540-544. (14) Ligler, F. S.; Bhatia, S.; Shriver-Lake, L. C.; Georger, J.; Calvert, J.; Dulcey, C.: US Patent 5391463, 1995. (15) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741-744. (16) Wybourne, M. N.; Mingdi, Y.; Keana, F. W.; Wu, J. C. Nanotechnology 1996, 7, 302-305. (17) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619-2625.
S0743-7463(98)00359-X CCC: $15.00 © 1998 American Chemical Society Published on Web 08/25/1998
Micropatterning of Biomolecules
patterns of cross-linked avidin and biotin-alkaline phosphatase were then produced; however, the proteins were not continuously immobilized over the whole pattern. It is known that the photoablation process is not only able to create structure within polymer substrates but also can change basic chemical properties of the resulting surfaces. Properties such as the hydrophobicity/hydrophilicity, charge, and/or formation of new functional groups can be altered under various ablative conditions.18 If sufficiently controlled, these changes could be engineered on the micrometer scale to affect protein adsorption (both positively or negatively) or to introduce functional groups which will allow easy covalent attachment of particular affinity reagents. Previous studies have noted differential chemical changes between the ablated substrate and ablatively ejected particles. Surfaces have been examined after ablation using XPS (X-ray photoelectron spectroscopy) and SIMS (secondary ion mass spectroscopy) techniques. Such studies have generally indicated an increased carbon/ oxygen ratio compared with the original substrate.19,20 In general this corresponds to a less charged, more hydrophobic surface. Also, enhanced surface roughness is characteristic for polymers after photoablation.20,21 In the present work, photoablated structures are first defined through a protective blocking layer, thereby opening the underlying substrate surfaces. This process can be used to pattern lines, holes, or entire networks of structures through the blocking layer. The critical dimensions of these patterns are fabricated here, in the range from 10 to 1000 µm. Two types of blocking layers are explored for patterning; low-temperature polymer laminates and protein (BSA) adsorbed layers. Either type is shown to be effective in blocking the adsorption of labeled proteins from bulk solution. But ablatively removed, it will allow such deposition in laser-defined regions. Avidin was adsorbed on the patterned polymer surface and visualized by fluorescence microscopy. Experimental Section Reagents. Poly(ethylene terephthalate) sheets (125 µm thick) were obtained from DuPont (Geneva, CH) under the name Mylar Type D. Polyimide sheets (50, 80, 100, and 125 µm thick) under the name Kapton were obtained from Goodfellow (U.K.). Poly(ethylene terephthalate) (PET), 35 µm thick with a 5 µm thick polyethylene (PE) adhesive layer on one side, was used as the laminating film (PET/PE) and was obtained from Morane (Oxon, U.K.). Bovine serum albumin (BSA), avidin (from chicken egg), fluorescein-labeled biotin, 14C-labeled BSA, tetramethylrhodamin-BSA (rh-BSA), phosphate buffer and magnesium chloride were obtained from Sigma (St. Louis, MO). UV-Excimer Laser Treatment and Fabrication of Patterns. The process of UV-excimer laser photoablation was performed as previously described.22 In brief, to create patterns, UV-laser pulses (193 nm), with a frequency of 10-50 Hz and a fluence per pulse of 1.8 J/cm2, were fired along an optical path consisting of a homogeinizer, a copper photomask, and a 10:1 telescope objective. The targeted sample stage was located approximately 5.5 cm from the objective. Lamination Technique. Before laser treatment, substrate sheets (approximately 5 cm × 5 cm) were cut off, washed with ethanol and water, dried under pressurized air and laminated with the PET/PE protective laminate at 125 °C using a standard (18) Srinivasan, R.; Braren, B. Chem. Rev. 1989, 89, 1303-1316. (19) Lazare, S.; Hoh, P. D.; Baker, J. M.; Srinivasan, R. J. Am. Chem. Soc. 1984, 106, 4288-4290. (20) Chan, C. M.; Ko, T. M.; Hiraoka, H. Surf. Sci. Rep. 1996, 24, 3-54. (21) Bolle, M.; Lazare, S. Appl. Surf. Sci. 1993, 69, 31-37. (22) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Girault, H. H. Anal. Chem. 1997, 69, 2035-2042.
Langmuir, Vol. 14, No. 19, 1998 5527 industrial lamination apparatus (Morane Senator (pneumatic control), Oxon, U.K.). The laminated substrates were then exposed to 250 laser pulses (50 Hz, 200 mJ). Rectangles (20, 40, 100, and 200 µm per 1 mm), holes (1 mm diameter), and a hole array (66 × 10 µm holes) were structured through the protective laminate using the corresponding copper masks. Distances of patterns were controlled either by the mask itself or by an X-Y table. Samples were mounted on the table which can be moved in 0.1 µm steps. After ablation, patterned sheets were incubated with the avidin solution (100 µg/mL in PBMS (100 mM phosphate, 1 mM MgCl2, pH ) 7.0)) for 2 h. After three washing steps with 200 µL PBMS the substrates were incubated with biotin-labeled fluorescein (100 µg/mL in PBMS) overnight at +4 °C or 2 h at room temperature. Careful washing with PBMS (3 times with 200 µL PBMS) followed and the substrates were dried in air. As last step the lamination was manually peeled off using a pair of tweezers. The whole biopatterning process is schematicaly shown in Figure 1a. BSA Blocking Technique. The polymer substrates were washed with ethanol and water and then dried under pressurized air. To ensure maximal substrate coverage, sheets were shaken in 10 mg/mL tetramethylrhodamin-BSA in PBMS for 3 h at room temperature and stored at +4 °C overnight. Before laser processing the substrates were washed in PBMS for 30 s and dried. The resulting substrates were exposed to 20 laser pulses (10 Hz, 200 mJ), which was more than sufficient to ablate the BSA layer. The patterned BSA-coated substrates were then treated with avidin and fluorescein-labeled biotin, as described above. This process is illustrated in Figure 1b. Fluorescence Microscopy. For fluorescence microscopy, substrates were mounted on glass slides and fluorescence images were made using a confocal microscope (Axiovert LSM 410, Zeiss, Germany). An Ar-ion laser (488 nm) for excitation and a 510525 nm bandpass filter was used for fluorescein detection and a He laser (543 nm) was used for rhodamin excitation with a 560 longpass filter, respectively. Line graphs were performed with the LSM 510 software from the original images without further treatment, then printed, scanned, and traced with FlexiTrace. SEM. PET was washed with ethanol and water and laminated. Laser ablation was carried out as described above using a 10 mm × 400 µm mask and a 66 hole array mask (hole diameter 100 µm) which produced a final line pattern of 1000 × 40 µm and 10 µm diameter holes, respectively. A total of 200 laser pulses (50 Hz, 200 mJ) were fired to completely ablate through the laminate. The laminate was then peeled off and the SEM picture was generated at 1 kV with 400× and 250× magnification. Quantification of Adsorbed Radio-Labeled Protein. One millimeter diameter hole patterns were produced by photoablation and lamination as described above, to carefully define the surface area for subsequent calculations. Untreated polymers were laminated on one side and cut off in approximately 3 mm × 6 mm pieces. Their dimensions were measured manually for the calculation of their individual size. Ablated holes were covered with 2 µL drops of 14C-labeled BSA solutions (10, 5 and 1 µg/mL in PBMS) and incubated for 2 h in a humidification chamber. Non-laser-treated surfaces were incubated with the corresponding protein solutions in plastic tubes. After incubation, the substrates were washed three times with PBMS and dried in air. Finally the protective lamination was removed. For calibration, three 2 µL drops of varying concentrations (0.25, 0.5, 1, 5, 10 µg/mL) were pipetted onto a polymer substrate and exposed to air until they were dried. All substrates were exposed for 22 h to an autoradiography screen (Phosphor screen, Molecular Dynamics), which was then scanned with a Storm Imager 840 (Molecular Dynamics).
Results and Discussion Before patterning experiments were conducted, native and laser-treated (more hydrophobic) polymer surfaces were tested for their protein adsorption characteristics. As BSA is known to strongly adsorb to hydrophobic surfaces,23 it was initialy studied in order to understand the adsorptive capacity on ablated surfaces. Many (23) Anzai, J.-i.; Guo, B.; Osa, T. Nucleic Acids Res. 1996, 40, 35-40.
5528 Langmuir, Vol. 14, No. 19, 1998
Schwarz et al. Table 1. Comparison for the Adsorption of 14C-Labeled BSA on Ablatively Treated and Unablated Polyimide Surfacesa 14C-BSA
a
Figure 1. (a) Schematic representation of the lamination technique (layer thickness is not to scale). Polymer substrates of interest are laminated with a 35 µm PE/PET protective layer. UV-excimer laser radiation ablates an opening in the protective layer where the mask is open to radiation, thereby producing the desired microstructure. Avidin was then adsorbed from bulk solution, leading to protein adsorption on the whole substrate. After washing, the lamination is peeled off and adsorbed avidin remains only in laser-defined areas. (b) Representation of the BSA blocking technique. Polymer substrates are first coated with BSA. Subsequent UV-excimer laser radiation ablates the protective protein where the mask is open to passage of radiation, thereby producing the desired micropattern. Ablatively defined BSA coated substrates are subsequently incubated with avidin. BSA prevents avidin from adsorption on a non-laser-defined surface. Avidin is patterned, adsorbing only in those regions defined by the laser.
proteins are also known to adsorb very strongly to hydrophobic surfaces;24 therefore, these findings might be further generalized. The techniques developed during these initial studies were then extended during patterning experiments to the avidin receptor as will be shown. Lamination Technique. Photoablation was used here to create cavities of defined size in a protective laminate through which protein adsorption could occur (see Experimental Section and Figure 1a). Laminated polyimide was treated with 250 laser pulses, which not only removed the protective laminate but also created 1 mm diameter hole patterns (14 µm deep) for protein adsorption. Auto-
(µg/mL)
PI ablated (ng/mm2)
PI unablated (ng/mm2)
1 5 10
78 ( 34 296 ( 182 592 ( 137
125 ( 40 203 ( 70
For more detail, see Experimental Section.
radiography experiments were carried out to investigate the adsorption behavior of 14C-labeled BSA on lasertreated polymer surfaces. These experiments showed that protein adsorption on ablated polyimide is increased (Table 1). Up to three times more protein was adsorbed in comparison to the non ablated surfaces (14C-labeled BSA >1 µg/mL). Ablated PET surfaces demonstrated a 2-fold increase in protein adsorption compared to nontreated PET (14C-labeled BSA >10 µg/mL), data not shown. The obtained maximal value of 600 ng/cm2 of adsorbed BSA fits reasonably well with expectations. It is reported that the adsorption of human serum albumin on contact lenses (hydroxyethyl methacrylate polymers) lies between 0.05 and 2 µg/cm2.25 Another study calculated that the value for a BSA monolayer should lie between 140 ng/cm2 for a parallel and 550 ng/cm2 for a perpendicular orientation of the ellipsoidal BSA molecule (dimensions 4 nm × 4 nm × 14 nm).23 Laser-treated polymer surfaces generally display enhanced roughness,20 and therefore, the surface area should be significantly increased compared with the original substrate material. Our observation of adsorptive capacity higher than monolayer concentrations is then to be expected. It should be noted that these results represent the total amount of proteins adsorbed to the surface, however, they give no conclusion about the activity of the adsorbed protein. Physical adsorption can decrease the activity of proteins of interest due to the fact that active centers can be denatured or hindered. Observations presented below of fluorescent protein patterns show that there remains sufficient avidin activity after adsorption in order to carry out receptor-ligand binding reactions. Protein Patterning via Lamination Technique. The lamination technique shown above was now extended to protein patterning. With this method, the initial unablated polymer substrate is completely blocked by first applying a protective laminate. The laminate is a 35 µm PET/PE layer and is thermally annealed to the substrate polymer (Figure 1a). The laser is used to create a defined pattern into the laminate by firing repeated pulses sufficient to eventually pierce the laminate and create structures of desired depth into the base polymer. An X-Y table allows the movement of samples in the submicrometer range so that distances of patterns can be easily modified. The resulting laser-defined laminate substrate structures were then immersed into an avidin solution (100 µg/mL). After washing steps, the substrates were incubated with the fluorescein labeled biotin. Finally the protective laminate is removed and only the receptorligand couple (avidin-biotin) remains in those regions that were defined by the UV-laser machining process. Scanning electron micrographs were obtained from a laser-defined PET pattern (no protein incubation) produced through the protective laminate; see Figures 2a and 3a. For SEM studies the lamination was peeled off, (24) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (25) Castillo, E. J.; Koenig, J. L.; Anderson, J. M.; Lo, J. Biomaterials 1984, 5.
Micropatterning of Biomolecules
Figure 2. (a) SEM picture of line-patterned PET. The substrate was laminated with the PE/PET protective layer and ablatively treated with 250 laser pulses. After creating the structure, the lamination was peeled off in order to visualize the PET substrate. (b) Fluorescein image from biotin-fluorescein bound to patterned avidin. Rectangles (40 µm × 1 mm) were produced with the lamination technique. These patterns were 20, 50, 100, and 200 µm apart (200 µm not shown). (c) Fluorescence intensities in function of the distance of the patterns (I to II).
to be able to study the polymer of interest. The patterned surface resulted in patterns with 1000 µm × 40 µm dimensions and 10 µm holes in a 66 hole array. It could be shown that the created patterns were very well defined. Enhanced roughness can be observed for the ablated PET
Langmuir, Vol. 14, No. 19, 1998 5529
surface, which is one explanation for enhanced protein adsorption in comparison to nontreated PET. The peel off process was very effective, leading to a homogeneous, flat PET surface around the ablatively defined patterns. Only for very small distances of line patterns (
