Langmuir 2004, 20, 4299-4301
4299
Photodegradation Micropatterning of Adsorbed Collagen by Vacuum Ultraviolet Light
modifications of the proteins are necessary, the technique will be useful in various applications.
Yoshihiro Ito,*,† Masayuki Nogawa,† Hiroyuki Sugimura,‡ and Osamu Takai‡
Experimental Section
Kanagawa Academy of Science and Technology, KSP East 309, 3-2-1 Sakado, Takatsu-ku, Kawasaki, 213-0012 Japan, and Department of Materials Processing Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603 Japan Received November 1, 2003. In Final Form: February 18, 2004
Introduction Control of the organization of proteins on surfaces at a microscopic scale is important in the development of biosensors1 and protein microarrays2,3 and in the organization and control of the growth of cells on surfaces.4-7 Techniques for applying proteins to surfaces include photolithography,8-10 soft lithography,5,11 spot arraying,2 and direct writing using the tip of an atomic force microscope.12,13 Of the various patterning methods, photolithography is the most practical because it can transfer an entire pattern onto a photomask at one time. However, when the photolithographic method is applied to protein micropatterning, either proteins must be modified with photoreactive materials or reaction of proteins with the micropatterned surface is needed. Recently Sugimura et al.14,15 reported an effective method that could photopattern alkyl- and fluoroalkylsilane self-assembled monolayers (SAMs) by using vacuum ultraviolet (VUV) light, which has a wavelength much shorter than 200 nm. SAMs were effectively decomposed and successfully micropatterned under VUV irradiation at 172 nm through a photomask. In the present study, for the first time, protein micropatterning was carried out using photodegradation. Considering that the micropatterning technique can be applied even to physically adsorbed proteins, and that no † ‡
Kanagawa Academy of Science and Technology. Nagoya University.
(1) Gross, G. W.; Rhoades, B. K.; Azzazy, H. M. E.; Wu, M. C. Biosens. Bioelectron. 1995, 10, 553-567. (2) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (3) Houseman, B. T.; Mrksich, M. Trends Biotechnol. 2002, 20, 279281. (4) Ito, Y. Biomaterials 1999, 20, 2333-2342. (5) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (6) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227-256. (7) Groves, J. T.; Mahal, L. K.; Bertozzi, C. R. Langmuir 2001, 17, 5129-5133. (8) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu, W. S. Chem. Biol. 1997, 4, 731-737. (9) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1998, 14, 1927-1936. (10) Yang, Z. P.; Frey, W.; Oliver, T.; Chikoti, A. Langmuir 2000, 16, 1751-1758. (11) Ostuni, E.; Yan, L.; Whitesides, G. W. Colloid Surf., B 1999, 15, 3-30. (12) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S. H.; Mirkin, C. A. Science 1999, 283, 661-663. (13) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (14) Sugimura, H.; Ushiyama, K.; Hozumi, A.; Takai, O. Langmuir 2000, 16, 885-888. (15) Hong, L.; Sugimura, H.; Furukawa, T.; Takai, O. Langmuir 2003, 19, 1966-1969.
A collagen-coated glass plate was purchased from Asahi Techno Glass Co. Ltd. (Tokyo, Japan) and used without further modification. The plate was irradiated by VUV light of 172 nm in wavelength through a photomask using an apparatus for which details have been described elsewhere. An excimer lamp (Ushio Electric, UER20-172V, 10 mW/cm2) was used as a light source. In our VUV exposure system, a sample and a photomask were place in a vacuum at a pressure of 10 Pa. The photomask consisted of a 2 mm thick quartz glass plate with 93% transparency at 172 nm and a 0.1 µm thick chromium pattern. A 10 mm thick quartz glass plate was also placed on the photomask to attain satisfactory contact between the mask and the sample surface. The transparencies of the photomask and the quartz plate at 172 nm were 70% and 93%, respectively, and by use of a photometer for that wavelength (Ushio Electric, VUV-S172), the total light intensity at the surface was estimated to be 6.5 mW/cm2. Static water-contact angles of the sample surfaces were measured by a similar method16 at 25 °C in air using a contact angle meter (KRU ¨ SS GmbH, DAS10) based on the sessile drop method. All of the contact angles were determined by averaging values measured at five different points on each sample surface. The water contact angle error was about (1°. For the contact angle measurement, an unpatterned quartz glass plate was placed on the sample, to irradiate the sample entirely and to investigate surface states of the irradiated sample. Immunostaining of the sample surface was performed as follows. The sample was incubated with rabbit anti-calf collagen type I and IV (0.1 mg/mL, Sanbio b. v., http://www.sanbio.nl/) diluted 1:20 in phosphate-buffered saline (PBS) containing 1% BSA at room temperature for 2 h and washed with PBS. Subsequently, the sample was incubated with fluorescein isocyanate (FITC)-labeled donkey anti-rabbit immunoglobulin (0.5 mg/mL, Amersham Bioscience Corp., NJ) diluted 1:50 in PBS containing 1% BSA at room temperature for 30 min and washed with PBS. The stained sample was observed using a fluorescence microscope (Axiovert 200M, Carl Zeiss Co. Ltd., Oberkochen, Germany) equipped with a cooled CCD camera (Roper Scientific, Inc., Tucson, AZ). Excitation and emission wavelengths were 470 ( 25 and 545 ( 37.5 nm, respectively. Measurement of time-of-flight secondary ion mass spectrometry (TOF-SIMS) was performed using a TOF-SIMS TRIFTIII (Physical Electronics). The primary ion was 69Ga+, accelerating voltage of the ion gun was 25 keV, ion electric current was 2 nA, pulse width was 17 ns, pulse frequency was 10 kHz, range of mass was 0-1850 amu, and resolution of mass was M/∆M ) 700@C2H4+ and M/∆M ) 600@C2H-. Lateral force microscopic (LFM) observation was performed by using SPA-300HV and SPI-3800N (Seiko Instruments Inc.) equipped with Ultralever (Park Scientific Instruments).
Results and Discussion Figure 1 shows that the water contact angle monotonically decreased with increasing irradiation time. This result implies that the collagen layer is gradually degraded because of the VUV irradiation in the presence of oxygen. However, it took longer to degrade the collagen layer than the SAM. Previously Sugimura et al.14 reported that n-octadecyltrimethoxysilane SAM or heptadecafluoroalkylsilane SAM is degraded and completely hydrophilized within 5 min. However, in the present study, more than 10 min was needed for hydrophilization. Because the collagen layer was considered to be thicker than the SAM, more energy was needed for the degradation. (16) Hozumi, A.; Masuda, T.; Hayashi, K.; Sugimura, H.; Takai, O.; Kameyama, T. Langmuir 2002, 18, 9022-9027
10.1021/la036064f CCC: $27.50 © 2004 American Chemical Society Published on Web 04/13/2004
4300
Langmuir, Vol. 20, No. 10, 2004
Notes
Figure 1. Change in contact angle with irradiation time.
Figure 3. TOF-SIMS analysis of micropatterned surface: (a) surface distribution of positive ions,; (b) surface distribution of negative ions. VUV irradiation time was 20 min. Bars represent 100 µm.
Figure 2. (A) Photomicrographs of samples immunostained by the anti-collagen antibody. The VUV irradiation times were (a) 1, (b) 2, (c) 5, (d) 10, (e) 15, and (f) 20 min. Bars represent 100 µm. The bright spots in the photomicrographs are considered to be aggregates of adsorbed collagen. (B) Schematic illustration of immunostaining.
Figure 2 shows the result of immunostaining of the micropattern-photodegraded surface. The antibody did not recognize the surface that was exposed to VUV even for 1 min. This result demonstrates that the collagen that was partially degraded by the VUV irradiation was not recognized by the antibody. Recently, Hong et al.15 reported that oxidized products (that is, -COOH groups) form before the organic layer (SAM) has been completely decomposed and removed from the substrate. Considering that polyclonal antibody was used in the present study, it is hard to conceive that partial degradation induced molecular recognition by the antibody. In the present
Figure 4. LFM image of a micropatterned surface. VUV irradiation time was 10 h. Scanning area was 180 × 180 µm2, and the scanning speed was 1 Hz.
investigation, the surface of degraded collagen was covered with -COOH groups, as in the case of degraded SAM, and the negatively charged surface was considered to inhibit the recognition by the antibody, as illustrated in Figure 2B. Figure 2 also shows that there are some differences in the resolution and hole sizes of the patterns. Hong et al.17 (17) Hong, L.; Sugimura, H.; Takai, O.; Nakagiri, N.; Okada, M. Jpn. J. Appl. Phys. 2003, 42, L394-L397.
Notes
reported that the proximity gap between a sample substrate and a photomask affected the photodegradation and the patterning resolution. In the case of the alkylsilane self-assembled monolayer (SAM), the clearest contrast between the irradiated and masked regions was obtained when the SAM in the irradiated regions had been completely decomposed. The proximity gap was favorable for promoting the photodegradation reaction of SAM. However, on the other hand, it degraded the patterning resolution due to the geometric factors of the apparatus. By optimization of the exposure conditions, including the proximity gap, irradiation time, and geometric factors, the resolution and sizes of the protein pattern will also be precisely regulated. After irradiation for 20 min, the micropatterned surface was measured by TOF-SIMS (Figure 3). Na, Al, Si, K, Ti, Ca, and OH were present in the irradiated regions, and CN, CNO, Cl, and SO3 were present in the nonirradiated regions. It was considered that the collagen in the
Langmuir, Vol. 20, No. 10, 2004 4301
irradiated regions had been degraded and the Si, Al, and alkali ions from within the glass had been exposed. Figure 4 shows an LFM image. LFM works on a contactmode principle by measuring the interactions between the probe and the surface features of the sample. Wang et al. demonstrated that LFM is suitable for observing fine structures.18 Therefore, the micropatterned surface was observed by LFM. The LFM image also indicated micropatterning by VUV irradiation. The present study demonstrated that protein micropatterning can be conveniently performed by VUV irradiation without any modification of proteins. This method will be useful for preparation of various micropatterned surfaces. LA036064F (18) Wang, H.; Sun, Y.; Li, Z.; Wang, E.; Huang, B. Anal. Sci. 2000, 16, 1261-1264.
