pubs.acs.org/Langmuir © 2010 American Chemical Society
Fabrication of Submicrometer Biomolecular Patterns by Near-Field Exposure of Plasma-Polymerized Tetraglyme Films Claire R. Hurley,† Robert E. Ducker,†,§ Graham J. Leggett,*,† and Buddy D. Ratner*,‡ †
Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, U.K., and ‡University of Washington Engineered Biomaterials, University of Washington, Box 355061, N330J William H. Foege Building, 1705 NE Pacific Street, Seattle, Washington 98195. §Present address: Centre for Biologically Inspired Materials & Material Systems, Department of Mechanical Engineering & Materials Science, Duke University, Durham, North Carolina 27708. Received January 25, 2010. Revised Manuscript Received March 12, 2010 Plasma-polymerized tetraglyme films (PP4G) have been modified by exposure to ultraviolet (UV) light from a frequency-doubled argon ion laser (244 nm) and characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS data indicated that the ether component of the C 1s spectrum declined after UV exposure, while components due to carbonyl and carboxylate groups increased. The film was physically eroded by UV exposure: after 100 s the rate of erosion reached a steady state of 0.05 nm s-1. The coefficient of friction, measured by friction force microscopy (FFM), increased substantially following exposure to UV light, reaching a limiting value after 10 min exposure, in agreement with the time taken for the ether and carboxylate components in the C 1s spectrum to reach a limiting value. Samples exposed to UV light through a mask yielded excellent frictional contrast. When immersed in solutions of proteins and protein-functionalized nanoparticles labeled with fluorescent markers, selective adsorption occurred onto the exposed regions of these samples. Excellent fluorescence contrast was obtained when samples were characterized by confocal microscopy, indicating that the exposed areas become adhesive toward proteins, while the masked areas remain resistant to adsorption. Submicrometer structures have been formed by exposing PP4G films to UV light using a scanning near-field optical microscope coupled to a UV laser. Structures as small as 338 nm have been formed and used to immobilize proteins. Again, excellent contrast difference was observed when labeled proteins were adsorbed and characterized by confocal microscopy, suggesting a simple and effective route to the formation of submicrometer scale protein patterns.
Introduction “Fouling”, or the deposition of unwanted biological matter, is a ubiquitous problem in biomaterials science.1 Proteins, in particular, adhere strongly (i.e., they form monolayers that are irreversibly bound) to most surfaces. The development of nonfouling surfaces is thus an important goal.2 In molecular diagnostics, including the development of chip-based methods for the high-throughput analysis of biomolecules, spots of immobilized biomolecules are formed and used to probe for specific complementary partners (for example, in a protein chip, antibodies are deposited in arrays; each antibody is effectively a probe for a specific antigen, or complementary partner).3 Fouling, in the form of nonspecific adsorption onto the regions between spots, is a major problem. For example, if fluorescence-based methods are subsequently used to interrogate the array, false positives may result. Consequently, for the successful fabrication of biochips, including protein chips, it is necessary to deploy an effective method for the inhibition of nonspecific adsorption between locations in the array. Poly(ethylene glycol) (PEG) is perhaps the best known and most extensively studied nonfouling material.4 In vitro, it exhibits exceptional resistance to the adsorption of proteins. *Corresponding authors. E-mail: (G.J.L.)
[email protected]; (B.D.R.)
[email protected]. (1) Chan, R.; Chen, R. J. Membrane Sci. 2004, 242, 169. (2) Dalsin, J.; Messersmith, P. Mater. Today 2005, 8, 38. (3) Wegner, G.; Lee, H.; Marriot, G.; Corn, R. M. Anal. Chem. 2003, 75, 4740. (4) Harris, J. M. Poly(Ethylene Glycol) Chemistry: Biochemical and Biomedical Applications; Plenum: New York, 1992.
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The mechanisms by which PEG and related materials resist protein adsorption remain contentious. Explanations have included steric effects,5 the nature of the interaction between water molecules and the surface,6,7 and electrostatic effects.8 However, its effectiveness is well-attested.9-12 Self-assembled monolayers (SAMs) terminating in oligo(ethylene glycol) (OEG) units have been shown to replicate this protein resistance and they have subsequently attracted a great deal of interest.13,14 However, while monolayers can be very effective in some applications, they are subject to significant limitations (for example, a highly specific choice of substrate) that restrict their applications. There has thus been interest in other strategies, for example the grafting of PEG side-chains onto polymers and monolayers.15 PEG brush (5) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (6) Wang, R. L. C.; kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767. (7) Feldman, K.; Haehner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (8) Dicke, C.; Haehner, G. J. Phys. Chem. B 2002, 106, 4450. (9) Zhou, F.; Huck, W. T. S. Phys. Chem. Chem. Phys. 2006, 8, 3815. (10) Ekblad, T.; Bergstr€om, G.; Ederth, T.; Conlan, S.; Mutton, R.; Clare, A.; Wang, S.; Liu, Y.; Zhao, Q.; D’Souza, F.; Donnelly, G.; Willemsen, P.; Pettitt, M.; Callow, M.; Callow, J.; Liedberg, B. Biomacromolecules 2008, 9, 2775. (11) Nagahama, K.; Hashizume, M.; Yamamoto, H.; Ouchi, T.; Ohva, Y. Langmuir 2009, 25, 9734. (12) Pan, J.; Zhao, M.; Liu, Y.; Wang, B.; Mi, L.; Yang, L. J. Biomed. Mater. Res. A 2009, 89, 160. (13) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12. (14) Ostuni, E.; Chapman, R. G.; Holmlin, E. R.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (15) Patel, S.; Thakar, R.; Wong, J.; McLeod, S.; Li, S. Biomaterials 2006, 27, 2890.
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structures have been shown to exhibit exceptional stability and effectiveness in inhibiting nonspecific adsorption.16-19 Plasma polymerization is an extremely attractive method for the control of surface chemistry. It remains a low-cost, rapid method for the deposition of thin, conformal layers of material onto a variety of substrates.20 It provides a convenient means of functionalizing three-dimensional structures whose topologies render them difficult to coat with other film deposition methods. Lopez et al.21 first reported the plasma deposition of polymer films that replicated the protein resistance of PEG. They prepared films by the radio frequency plasma deposition of tetraethylene glycol dimethyl ether (tetraglyme) (PP4G). Protein adsorption on these surfaces was found to be lower than 10 ng cm-2 and as a result it was found that blood platelet and monocyte adhesion was significantly reduced in vitro.22 However, patterning of a plasma deposited thin film is difficult. Several authors have utilized methods based around deposition through a mask.23 This approach typically yields a resolution on the order of tens of μm. There are currently no methods for the patterning of plasma polymers on submicrometer length scales. Here we report a simple, new method for patterning the PP4G films, based upon degradation of the plasma polymer film following exposure to UV light. In a single step, the protein-resistant, nonfouling material is transformed into one that binds proteins. We have found that by utilizing a scanning near-field optical microscope (SNOM) as the light source, it is possible to fabricate structures significantly smaller than 1 μm and to use these to control the adsorption of proteins in a spatially resolved fashion. Because of the simplicity of the fabrication process, its exquisite spatial resolution and the versatility of plasma-polymerized films, the method promises to have very widespread utility.
Experimental Section Plasma-polymerized tetraglyme films (PP4G) were prepared by RF-plasma deposition in a capacitively coupled reactor that has been described.24 Exact deposition conditions are: First, argonetch the samples at 350 mT, 40 W, for 5 min. Then, tetraglyme deposition at 350 mT, 80 W, for 1 min followed by 10 W deposition for 20 min. Finally, samples are quenched in tetraglyme vapor with the power turned off for 5 min. After deposition, samples were washed in reverse osmosis water (Elgar) for 3 24 h to remove loosely adsorbed tetraglyme. Samples were then dried in a gentle stream of nitrogen and exposed to laser UV light. A frequency-doubled argon ion laser (Coherent Innova FreD 300C) was used for the UV exposure, with a wavelength of 244 nm at a power of 100 mW (measured at the sample surface). The laser beam cross-sectional area was increased by passing it through two divergent lenses and reflecting it off a mirror onto the sample surface producing a spot of approximate size 1 cm2. Patterns were fabricated by exposure of the samples through a mask composed of a copper electron microscope grid attached to the underside of a quartz slide. Exposure times ranged from 0 to 20 min. X-ray photoelectron spectroscopy (XPS) measurements were made using a Kratos Axis Ultra X-ray photoelectron spectro(16) Ma, H. W.; Hyun, J. H.; Stiller, P.; Chilkoti, A. Adv. Mater. 2004, 16, 338. (17) Ma, H.; Wells, M.; T, P. B., Jr.; Chilkoti, A. Adv. Funct. Mater. 2006, 16, 640. (18) Ma, H.; Textor, M.; Clark, R. L.; Chilkoti, A. Biointerphases 2006, 1, 35. (19) Hucknall, A.; Rangarajan, S.; Chilkoti, A. Adv. Mater. 2009, 21, 2441. (20) Luo, H.; Sheng, J.; Wan, Y. Appl. Surf. Sci. 2007, 253, 5203. (21) Lopez, G. P.; Ratner, B. D.; Tidwell, C.; Haycox, C.; Rapoza, R.; Horbett, T. J. Biomed. Mater. Res. 1991, 26, 415. (22) Shen, M.; Wagner, M.; Castner, D.; Ratner, B.; Horbett, T. Langmuir 2003, 19, 1692–1699. (23) Hartley, P.; Thissen, H.; Vaithianathan, T.; Griesser, H. Plasmas Polym. 2000, 5, 47. (24) Haque, Y.; Ratner, B. D. J. Appl. Polym. Sci. 1986, 32, 4369.
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meter (Kratos Analytical, Manchester, U.K.). Samples were prepared 24 h in advance and kept in low light conditions overnight. Prior to XPS analysis samples were kept under a vacuum of 10-8 mbar for approximately 2 h. A minimum of two XPS spectra were obtained on different samples for each exposure time. Resulting XPS spectra were analyzed using the CasaXPS program (Casa, http://www.casaxps.com, U.K.). Survey scan and narrow scan spectra of C 1s and O 1s peaks were obtained and curve resolution conducted using a linear baseline. Peaks were fitted using combinations of Gaussian (30%) and Lorentzian (70%) curves. The fwhm of all components was constrained to be equal to that of the aliphatic carbon, but all other parameters were left free to vary during fitting. Quantitative friction force microscopy (FFM) measurements were made using a Digital Instruments Nanoscope IIIa Multimode atomic force microscope (Veeco UK, Cambridge, U.K.) operating in contact mode. The tips were silicon nitride Nanoprobes (Veeco UK) with nominal force constants of 0.06 N m-1. Spring constants were determined from measurements of the thermal spectra using the method of Hutter and Bechoeffer.25 Experiments were performed under ethanol using a liquid cell fitted with a silicone O-ring. Measurements were taken at three positions on three sample surfaces for each exposure time; from these an average friction coefficient was obtained. Friction loops were acquired with a maximum photodetector deflection of 2 V, reducing the load until the tip pulled free of the surface, in steps of 0.4 V. Areas of 5 μm2 were scanned with 256 scan lines at a frequency of 1 Hz. Tapping mode topographical measurements were also made in air using the same microscope. Silicon cantilevers with a spring constant of 50 N m-1 (Veeco) were used. FFM images were acquired using a ThermoMicroscopes Explorer AFM (Veeco UK) using silicon nitride tips with nominal force constants of 0.06 N m-1. Images were taken, in phase and z piezo modes, of samples of PP4G exposed through copper electron microscope grids with periodicities of 600 squares per inch. The forward and reverse phase images were subtracted and the resulting contrast was measured in nA. Near-field photolithography was carried out using a ThermoMicroscopes Aurora 3 shear-force mode scanning near-field optical microscope (SNOM) coupled to the same frequencydoubled argon ion laser used to carry out micropatterning. Probes were prepared by etching fused silica optical fibers in hydrogen fluoride, and coating them with aluminum. The probes were attached to tuning forks for mounting in the microscope. The surface was first exposed to 244 nm UV light through a copper “finder” grid (one marked with a variety of clearly distinguished numbers and letters; Agar, Cambridge, U.K.) for 2 min to produce macroscopic features that would allow easy relocation of lithographic features for AFM analysis after SNP was performed. The sample was then mounted on the sample stage and a SNOM tip positioned over an unexposed region of the surface, masked by during the initial exposure but close to a readily identifiable feature. The tip was lowered to be in a close proximity with the sample surface and put into feedback. A predefined pattern of lines was then “written” onto the surface at 4, 5, and 10 mW and 0.1 and 0.2 μm s-1. It should be noted that the power quoted here is the power of the beam coupled to the optical fiber probe; there is no reliable means of measuring the power in the near-field at the sample surface. Lines were relocated on the surface and imaged in tapping mode, using silicon probes with nominal spring constants of 42 N m-1, and also imaged using confocal microscopy. Antisheep IgG (Sigma, labeled with FITC conjugate) and FluoSphere NeutrAvidin (Invitrogen Molecular Probes, 0.04 μm labeled microspheres, 505-515 nm) were made up to 5 μg mL-1 solutions with phosphate buffered saline (0.1 mol dm-3) and reverse osmosis water, respectively. Samples were placed in (25) Hutter, J. L.; Bechhoeffer, J. Rev. Sci. Instrum. 1993, 64, 1868, 3342.
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Figure 1. (a-c) Change in the C 1s spectrum with exposure for PP4G films exposed to UV light (244 nm at 100 mW cm-2). (d, e) variation in the relative size of the components of the C 1s spectrum as a function of UV exposure time. Lines are added in part d to guide the eye.
sample vials, immersed in the protein solution and left for 1.5 h before washing repeatedly in reverse osmosis water. After this time a high contrast in the nonfouling property of the films was observed via confocal microscopy. Confocal microscopy was carried out using two instruments, a Carl Zeiss Vision LSM and a Witec AlphaSNOM. Samples were fixed to glass slides with a drop of water and placed face down on the sample stage. The microscope was focused on the surface in reflection mode and laser light of a suitable wavelength (488 nm) was then used to excite fluorescence. Light was passed through a 505-530 nm filter during acquisition of fluorescence images.
Results and Discussion X-ray Photoelectron Spectroscopy. The effect of UV irradiation at 244 nm (100 mW cm-2) on PP4G films was studied using X-ray photoelectron spectroscopy (XPS). Figure 1 shows the development of the XPS C 1s spectrum with exposure to UV light for PP4G films. For the virgin material (Figure 1(a)), the spectral envelope can be appropriately fit with four peaks at 285.0, 286.5, 287.8, and 289.3 eV, attributed to -C-C-C-, -C-C-O-, -C-CdO (or -O-C-O-) and -O-CdO (or -O-C-(OR)2), respectively. Their relative compositions were 15.4, 77.6, 5.3, and 1.7% respectively. The tetraglyme monomer contains a single carbon bonding environment, the ether carbon. However, plasma polymers typically have structures that are more complex than the monomers from which they are formed, because of the variety of reaction pathways that are available. The observation of four carbon bonding environments in Figure 1 is therefore not surprising and is in agreement with previous studies.26-28 (26) Johnston, E.; Bryers, J.; Ratner, B. D.; Tidwell, C. Langmuir 2005, 21, 870. (27) Palumbo, F.; Favia, P.; Vulpio, M.; d’Agostino, R. Plasmas Polym. 2001, 6, 163. (28) Horcas, I.; Fernandez, R.; Gomez-Rodriguez, J.-M.; Colchero, J.; GomezHerrero, J.; Baro, A. M. Rev. Sci. Instrum. 2007, 78, 013705.
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After exposure of the tetraglyme films to UV light, there was a reduction in the area of the ether carbon component compared to the other peaks (Figure 1, parts a and b), the areas of which were all observed to increase. The change appears to be modest, because the XPS sampling depth (ca. 10 nm) is significantly greater than the near-surface region modified by UV irradiation. However, a quantitative analysis of the C 1s spectra made the effects of UV exposure clear (Figure 1, parts d and e). The areas of the four components in the C 1s spectrum have been plotted as a percentage of the total C 1s peak area. The area of the peak corresponding to the ether carbon decreased substantially with exposure (Figure 1e), to ca. 63% of the C 1s peak area after 15 min, while all three of the other components increased in magnitude with the exposure time. The rate of decrease of the ether component slowed with time, approaching a limiting value after ca. 10 min, and corresponding to this, the areas of the peaks corresponding to the aliphatic carbon and the carboxylate carbon also approached a limiting value after a similar period of time. The behavior of the carbonyl carbon appeared to be a little different, continuing to increase up to 20 min exposure. The decrease observed in the relative composition of the ether component (∼14%) was matched by increases in both the carbonyl and carboxylate components (∼10 and 3% respectively) after 15 min exposure. It is clear from the XPS data that the surface structure of the PP4G film is rapidly modified by exposure to UV light. Critically, the protein-resistant polyether linkages formed during the plasma deposition process are substantially degraded to the much more adhesive carbonyl and carboxylate functions. It appears that irradiation at a comparatively short wavelength is necessary for photodegradation of the plasma polymer film to occur, because when illumination was carried out at 325 nm (using light from a HeCd laser), there was no evidence for degradation. DOI: 10.1021/la100362q
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Figure 2. (a) Friction load plots for PP4G films exposed to UV light. (b) Variation in the normalized coefficient of friction as a function of UV exposure time for PP4G films.
Friction Force Microscopy. UV-modified PP4G films were characterized by FFM29 in order to test whether this technique could be used to measure the rate of change of PP4G surface structure at high spatial resolution following patterning. Previous work30 has demonstrated that Amontons’ law, FF ¼ μFN where FF is the friction force, FN is the load, and μ is the coefficient of friction, may be used when experiments are performed with the sample immersed in ethanol. In air, studies of PET suggested that single asperity contact mechanics applied, but that the tipsample interaction is intrinsically less well-defined.30 As a consequence, it was decided to carry out measurements with the sample immersed under ethanol. Figure 2a shows the variation in the photodetector response (proportional to the friction force) with load for a PP4G film exposed to UV light for four different time periods. It can be seen that the relationship between the friction force and the load is linear, indicating conformity to Amontons’ law. Figure 2b shows the variation in the friction coefficient, μ, as a function of the exposure time. The values of μ, normalized to values measured after a very long (effectively infinite) exposure, were found to rise from an initial value of 0.35 ( 0.12 to 1.00 ( 0.10 over a period of 10 min. This exposure time correlates closely with the time at which the relative areas of the ether, ester and aliphatic carbon components in the C 1s spectrum reached limiting values, indicating that changes in μ may be an effective means of measuring changes in the surface composition following photodegradation.
(29) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. Xiao, X.; Hu, J.; Charych, D.; Salmeron, M. Langmuir 1996, 12, 235. Li, S.; Cao, P.; Colorado, R.; Yan, X.; Wenzl, I.; Shmakova, O. E.; Graupe, M.; Lee, T. R.; Perry, S. S. Langmuir 2005, 21, 933. Beake, B. D.; Leggett, G. J. Langmuir 2000, 16, 735. Shon, Y.-S.; Lee, S.; Colorado, R.; Perry, S. S.; Lee, T. R. J. Am. Chem. Soc. 2000, 122, 7556. Clear, S. C.; Nealey, P. F. J. Chem. Phys. 2001, 114, 2802. Harrison, J. A.; Mikulski, P. T. J. Am. Chem. Soc. 2001, 123, 6873. Brewer, N. J.; Foster, T. T.; Leggett, G. J.; Alexander, M. R.; McAlpine, E. J. Phys. Chem. B 2004, 108, 4723. Mikulski, P. T.; Herman, L. A.; Harrison, J. A. Langmuir 2005, 21, 1219. Mikulski, P. T.; Gao, G.; Chateauneuf, G. M.; Harrison, J. A. J. Chem. Phys. 2005, 122, 024701. Yang, X.; Perry, S. S. Langmuir 2003, 19, 6135. Kim, H. I.; Houston, J. E. J. Am. Chem. Soc. 2000, 122, 12045. Brewer, N. J.; Beake, B. D.; Leggett, G. J. Langmuir 2001, 17, 1970. Marti, A.; Hahner, G.; Spencer, N. D. Langmuir 1995, 11, 4632. Vezenov, D.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006. (30) Hurley, C. R.; Leggett, G. J. Langmuir 2006, 22, 4179. Colburn, T. J.; Leggett, G. J. Langmuir 2007, 23, 4959.
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Patterned Samples. Samples of PP4G were exposed to UV light through a mask (an electron microscope grid) and characterized by AFM. Figure 3a shows a tapping mode topographical image of a sample exposed for 360 s, together with a cross section through the image (Figure 3b). A clear height difference is evident between the masked regions (bars) and the exposed regions (squares), indicating removal of material during the degradation process. The height difference between the exposed and masked regions, as a function of exposure time, is shown in Figure 3c. Initially, the rate of change was rapid, but subsequently the height difference increased at a decreasing rate, with a steady state rate of change of 0.05 nm s-1 being reached after 100 s, when nearly 15 nm of material had been eroded. FFM images acquired under ethanol and in ambient conditions exhibited different contrast. When imaged under ambient conditions (Figure 3d), samples yielded darker contrast in exposed areas than in masked ones, indicating that the coefficient of friction was smaller there, while samples imaged under ethanol yielded brighter contrast for the exposed areas than for the masked ones, indicating a stronger frictional interaction following UV exposure. Previous studies of the contact mechanics of polymer films have shown that the friction-load relationship is different under ethanol (where Amontons’ law is obeyed) and under ambient conditions (where the strong adhesion due to the capillary that forms between the tip and the sample has a dominant influence). It is suggested that the UV exposure process yields low molecular weight material at the sample surface that is soluble in ethanol. This is consistent with the high rate of sample erosion indicated by the data in Figure 3. This material dissolves into the organic liquid, leaving the modified plasma polymer (as opposed to the soluble debris from surface degradation) at the surface. In air, in contrast, the low molecular weight degradation products remain at the surface and lubricate the tip-sample contact, by providing a weak interface, and the tip slides easily across the surface. Despite these problems, it proved possible to conduct a semiquantitative analysis of the extent of modification in exposed regions of patterns by measuring line sections through FFM images. Following earlier work31 the friction signals from exposed and masked areas were determined from line sections (Figure 3e), and used to calculate the friction force in the exposed regions relative to that measured for the unmodified material. The relative (31) Chong, K. S. L.; Sun, S.; Leggett, G. J. Langmuir 2005, 21, 2903. Whittle, T. J.; Leggett, G. J. Langmuir 2009, 25, 9182.
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Figure 3. (a) Topographical image of a patterned sample following 360 s exposure. The square regions have been exposed to UV light and the bars have been masked. (b) Mean line section through the rectangular region marked in part a. (c) Variation in the mean height difference between the masked and exposed areas as a function of time. (d) Representative friction image and line section. (e) Calculation of the relative friction difference between the masked and exposed areas, χt. (f) Variation in χt as a function of exposure time.
friction difference between the masked and exposed areas, χt, as a function of time is shown in Figure 3f. It can be seen that after ca. 10 min, the friction contrast difference changes little, indicating that although erosion continues at longer times (Figure 3c), the composition changes comparatively slowly relative to the rate of change at shorter exposures. This finding is consistent with the other data for unpatterned materials. Protein Adsorption Studies. PP4G films exhibit exceptional resistance to protein adsorption.32 The possibility of using UV exposure to selectively render PP4G adhesive to proteins was explored, by exposing samples to light from the laser source and then immersing them in a solution of FITC-labeled anti-IgG. The time of exposure to the UV source was varied between 0 and 10 min, and the samples characterized, following immersion in the antibody solution, by confocal microscopy. Parts a-d of Figure 4 show representative data. After 30 s UV exposure, there was already a clear pattern, with no fluorescence being detected from the masked areas where the film remained intact, and a strong signal being detected from the square regions where photodegradation had occurred leading to a reduction in the protein-resistance of the surface. As the UV exposure time increased, the contrast difference between the exposed and masked areas increased, with the optimum contrast being observed after ca. 10 min, in agreement with all of the XPS and FFM data above. There was no evidence of adsorption to the masked areas, and strong fluorescence from exposed regions, indicating sharp differentiation between nonfouling PP4G films and photodegraded areas. As an alternative test for the degree of discrimination between masked and exposed areas, samples were exposed to UV light and immersed in a solution of FluoSphere Neutravidin. This reagent consists of polymeric nanoparticles (0.04 μm diameter) conjugated to neutravidin. The beads are labeled with a fluorescent dye. (32) Shen, M. C.; Martinson, L.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. J. Biomater. Sci. Polym. Ed. 2002, 13, 367.
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Figure 4. (a-d) Confocal microscopy images of photopatterned samples of PP4G after exposure to UV light for differing time periods and immersion in a solution of FITC-labeled anti-IgG. (e and f) Confocal microscopy images of patterned samples following immersion in a solution of Neutravidin nanoparticles. The scale bars are all 10 μm, except in part e, where it measures 100 μm. Streaking in parts b and c results from interference during the measurement.
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Figure 5. (a) Tapping mode AFM image of lines fabricated in PP4G films using near-field exposure. (b) Average line section through the lines in part a. (c) 8080 μm2 Tapping Mode AFM image and (d) confocal microscopy image of microscopic bars and lines fabricated by near-field lithography in a PP4G film, following immersion in a solution of FITC-labeled anti-IgG. The box in part c identifies the region imaged in part d.
surface such as PP4G. Parts e and f of Figure 4 show fluorescence images of a sample exposed for only 2 min (i.e., far from complete modification) after immersion in FluoSphere Neutravidin. The contrast is sharp and strong, indicating excellent differentiation between the masked and exposed areas. Nanopatterning. In an effort to fabricate smaller protein patterns, exposure was carried out using an optical fiber-based SNOM coupled to a frequency-doubled argon ion laser. PP4G thin films were first patterned using a grid for 2 min to create micrometer-scale surface features and enable easy identification of a specific region in which nanolithography could be executed. A suitable position was then located on the grid in an unexposed region. The SNOM tip was brought into feedback in close proximity to the sample surface and a series of lines exposed by tracing the SNOM probe across the sample. Samples were imaged using tapping mode AFM. Figure 5a shows tapping mode AFM images of a series of lines written at a rate of 0.2 μm s-1 and with a laser power of 4 mW. Line sections revealed that the full width at half-maximum height (fwhm) of the lines was 338 nm, and the depth was ca. 10 nm, indicating substantial removal of material from the exposed regions. In a SNOM, the sample is brought within a few nm of a nanometric asperity (typically g50 nm diameter). The sample interacts with the evanescent field associated with the aperture. Recently, a SNOM has been used to expose photoactive organic monolayers to UV light leading to patterning with a resolution as good as 9 nm.33 However, a key criterion in those high-resolution studies was the use of a photoactive functionality distributed with monolayer coverage on a solid surface. In previous studies where photoactive polymers have been used, the resolution has been less (33) Sun, S.; Chong, K. S. L.; Leggett, G. J. J. Am. Chem. Soc. 2002, 124, 2414. Sun, S.; Leggett, G. J. Nano Lett. 2004, 4, 1381. Leggett, G. J. Chem. Soc. Rev. 2006, 35, 1150.
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good than has been realized using monolayer resists34 because the electric field associated with the SNOM probe is known to diverge rapidly in the dielectric film beneath the aperture.35 The line width is probably further increased in the present case by the process of degradation that accompanies UV exposure of PP4G films, leading to the erosion of material underneath the SNOM probe in addition to surface chemical modification. Nevertheless, the line width demonstrated here represents a very significant advance over other methods previously used to pattern plasma polymer films. To explore the potential utility of near-field methods in protein patterning, lines were fabricated in PP4G films adjacent to micrometer-scale structures fabricated using mask-based methods, and the samples exposed to solutions of FITC-labeled antiIgG. The micrometer-scale structures were formed to serve as registry features, enabling the execution of lithography at a readily identified location, and subsequent imaging of the same location using a confocal microscope. The resulting features were imaged both by tapping mode AFM and by confocal microscopy. Figure 5 shows an example. In the AFM image (Figure 5c), a cross-shape is observed where bars in the mask have protected the PP4G film from UV exposure. The contrast is brighter in these regions than in the rest of the surface because UV exposure has eroded the polymer in exposed regions leading to a height reduction. Four bars are clearly visible where the sample has been exposed to light from the SNOM. Despite the adsorption of protein, these remain visible as depressions because the depth eroded during exposure exceeds the height of the protein molecules. The surface of the exposed, larger regions in Figure 5c appears “spotty”. The origin of this is not clear, although the most likely explanation is probably the adsorption of material (e.g., salts) from the buffer solution. The fluorescence microscopy image (Figure 5d) shows the four bars fabricated by near-field exposure at slightly higher magnification, with the corner of a 10 μm squared region formed in the mask-based process evident in the upper left quarter. The modified areas now exhibit bright contrast, and the intact regions dark contrast, because the protein does not adsorb to PP4G but does adsorb to the regions exposed to UV light. There is excellent differentiation between the contrast observed in the two different regions. The lines formed by nearfield lithography were at the resolution limit of the optical microscope used here to acquire fluorescence images; as a consequence, the features in Figure 5d are significantly broadened and thus appear to be broader than the corresponding features in Figure 5c.
Conclusions The spatial organization of biological molecules is important in many technologies. There is thus a great deal of interest in the development of new biological patterning techniques. While the literature contains many reports of methods for micrometer-scale patterning of biomolecules, many of these are substrate-specific (for example, methods based on the patterning of alkanethiols). The capability offered by plasma polymerization for application to a very wide range of substrates renders it an attractive method for the control of interfacial biological interactions. However, the patterning of plasma-polymer materials presents problems. While methods for deposition through masks have been reported, (34) Krausch, G.; Mlynek, J. Microelectron. Eng. 1995, 32, 219. Krausch, G.; Wegscheider, S.; Kirsch, A.; Bielefeldt, H.; Meiners, J. C.; Mlynek, J. Opt. Commun. 1995, 119, 283. (35) Riehn, R.; Charas, A.; Morgado, J.; Cacialli, F. Appl. Phys. Lett. 2003, 82, 526.
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the spatial resolution has previously been limited because of the propensity of plasma-phase species to under-cut the mask. The method reported here offers significant advantages. Not only is it possible to generate patterns rapidly and conveniently from a preformed film, but the resolution, even for the maskbased process, is very good and yields sharp edges. By using a scanning near-field optical microscope to undertake the exposure, the resolution is clearly very much enhanced and is significantly better than 1 μm. Other methods, for example far-field exposure using a high numerical aperture lens, may also yield submicrometer resolution. For a plasma polymer substrate, this represents a significant step forward in capability. In a more general context, however, this method would free researchers from the constraints associated with particular substrates and substrate geometries that are associated with the monolayerbased methods that have previously attracted the most interest in biomolecular patterning. There is significant scope to improve the resolution achieved during near-field patterning. Reduction of the thickness of the
Langmuir 2010, 26(12), 10203–10209
Article
plasma deposited film would lead to reduced divergence of the electric field, for example, better confining the excitation. The use of increased writing rates may also be beneficial: in the present studies high rates of sample degradation were observed, indicating extensive modification and significant scope for reduction of exposure by the application of high writing speeds. Such measures may yield a resolution closer to the aperture size. Acknowledgment. The authors thank Winston Ciridon (University of Washington) for preparation of the plasma-deposited tetraglyme films, Tracie Whittle (University of Sheffield) for acquiring XPS spectra and Shuqing Sun and Robert Manning (University of Sheffield) for acquiring confocal micrographs. C.R.H. thanks the Engineering and Physical Sciences Research Council (EPSRC) for a research studentship. B.D.R. thanks University of Washington Engineered Biomaterials (UWEB)(NSF Grant EEC-9529161) for support. G.J.L. thanks RCUK (Grant EP/C523857/1), EPSRC, and the RSC Analytical Chemistry Trust Fund for support.
DOI: 10.1021/la100362q
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