© Copyright 2005 American Chemical Society
JUNE 7, 2005 VOLUME 21, NUMBER 12
Letters Protein Micropatterning Using Surfaces Modified by Self-Assembled Polystyrene Microspheres Fung Ling Yap Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117576
Yong Zhang* Division of Bioengineering and Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore 117576 Received February 21, 2005. In Final Form: April 22, 2005 A technique for micropatterning of proteins on a nonplanar surface to improve the coverage and functionality of biomolecules is demonstrated. A nonplanar microstructure is created by the self-assembly of polystyrene microspheres into an array of microwells on a silicon wafer to allow the integration of a nonplanar spot on a planar chip. After the microspheres were deposited into the microwells, they were conjugated with proteins. The curve surfaces of the microspheres present more surface area for attaching biomolecules which will increase the density of biomolecules and, hence, the sensitivity for detection. Moreover, proteins immobilized on a curved surface can retain their native structures and function better than on a planar surface because of a smaller area of interaction between the protein and the substrate. Patterning of biomolecules was tested with two model fluorescent proteins. The results show that precise patterning of biomolecules on a nonplanar spot can be achieved with this technique.
Introduction The attachment of biomolecules within designated regions on solid surfaces while preventing nonspecific adhesion at other regions is the basis of micropatterning. Micropatterning of biomolecules plays an important role in the development of biosensors,1 tissue engineering,2 and fundamental studies of cell biology.3 Tremendous effort has been put in by many research groups to develop techniques that are compatible for patterning biomolecules on the micrometer scale. The most widely used techniques * To whom correspondence should be addressed. E-mail: biezy@ nus.edu.sg. (1) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180-1218. (2) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. FASEB J. 1999, 14, 1883-1900. (3) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335.
are photoresist lithography4 and soft lithography3 which involve attaching biomolecules on a planar substrate. While it is possible to achieve a high molecular density on a planar substrate, for example, via thiols-based selfassembld monolayers on a gold substrate,5 the density can be improved by using a nonplanar microstructure to increase the surface area available for attachment of biomolecules In this paper, we present a new technique of micropatterning which allows the integration of a nonplanar spot on a planar chip. An array of microwells was fabricated on a silicon chip using photoresist lithography techniques. A nonplanar microstructure is created by assembling polystyrene microspheres in the microwells. (4) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609. (5) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75, 1-68.
10.1021/la050454f CCC: $30.25 © 2005 American Chemical Society Published on Web 05/06/2005
5234
Langmuir, Vol. 21, No. 12, 2005
Letters
Scheme 1. Procedure for Micropatterning of Biomolecules on a Nonplanar Surface via Assembly of Polystyrene Microspheres
Biomolecules can then be selectively adsorbed onto the surfaces of the microspheres by ensuring that the regions outside are grafted with poly(ethylene glycol) (PEG). The procedure for patterning is outlined in Scheme 1. Experimental Section [3-(2-Aminoethylamino)propyl]trimethoxysilane (amino-silane), bovine serum albumin conjugated to fluoresceinisothiocyanat (BSA-FITC), sheep anti-rabbit immunoglobulin G Cy3 conjugate (IgG-Cy3), and phosphate buffered saline solution (PBS) were purchased from Sigma-Aldrich. 2-[Methyoxy(polyethylenoxy)propyl]trimethoxysilane (molecular weight 460-590, PEG-silane) was purchased from Gelest, Inc. The 0.454-µm carboxylated polystyrene microspheres (solid content 2.59%) were purchased from Polysciences, Inc. Positive photoresist AZ4620 and AZ 400K Developer were purchased from Clariant. All reagents were used as received. First, the silicon wafer was modified with PEG-silane to create a nonfouling surface according to the method reported previoulsy.6 The wafer was rinsed with deionized water prior to use. Oxidation of the wafer was carried out by treatment with hot piranha solution (30% hydrogen peroxide with 98% sulfuric acid in a ratio of 1:4, v/v) at 100 °C for 10 min. The mixture should be used with extreme caution because of its high oxidizing power and risk of explosion. The wafer was then rinsed with a copious amount of deionized water and dried. Grafting was done by a solution of 3 mM PEG-silane in toluene (with 0.8 mL of concentrated HCl/L) for 18 h at room temperature. Next, the wafer was rinsed with toluene and deionized water and sonicated for 2 min to remove nongrafted material. Next, microwells were fabricated on the silicon wafer using standard photoresist lithography techniques. A positive photoresist AZ4620 was spin-coated on the PEG modified wafer at 2000 rpm for 30 s. The resist was soft baked at 95 °C for 1 min. A transparency mask adhered to a blank photomask was placed in contact with the resist under a mercury lamp (EVG EV620 Mask Aligner). The photoresist was exposed for 60 s at 10 mW/ cm2. The resist was developed with AZ 400K Developer/deionized water (1:4) for 10 min and rinsed with deionized water. This was followed by a post-exposure bake at 95 °C for 10 s. Next, the photoresist pattern was subjected to reactive ion etching (Phantom Trion). The wafer was exposed to oxygen plasma (100 W, oxygen at 100 mTorr) for 10 s to remove the PEG layer. The unit was then operated under a mixture of CF4 and oxygen gas (100 W, CF4 at 18 mTorr, and oxygen at 2m Torr) for 16 cycles of 30 s, with an interval of 1 min between each cycle to allow the wafer/photoresist to cool. Next, the microwells were modified with amino-silane according to similar methods reported previously.7 The microwells were immersed in 2% amino-silane in deionized water for 4 min at room temperature in a vacuumed environment to remove air bubbles within the microwells so that amino-silane can enter. The wafer was rinsed with deionized water and baked for 10 min (6) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 14571460. (7) Petri, D. F. S.; Wenz, G.; Schunk, P.; Schimmel, T. Langmuir 1999, 15, 4520-4523.
at 120 °C. The photoresist was then removed by rinsing with acetone and deionized water. About 30 µL of 0.454-µm carboxylated polystyrene microspheres (solid content 0.5%, pH 4.3) was dispensed on the array of microwells and incubated for 1 h to allow it to dry. The chip was then rinsed with deionized water to remove excess particles. After the beads were deposited in the microwells, proteins were attached to the microspheres through conjugation with the carboxyl groups. Micropatterning of biomolecules was performed with two model proteins, that is, BSA-FITC and IgG-Cy3. The chip was incubated overnight in a 0.1 mg/mL protein solution (PBS, pH 7.4) and rinsed with deionized water.
Results and Discussion The diameter of the microwell is 25 µm with a centerto-center spacing of 100 µm. Each sample is made up of an array of 50 × 50 microwells. The depth of the microwell is approximately 700 nm as determined by the Veeco NT3300 profiler system. Figure 1A shows the optical micrograph of the microwells fabricated on the silicon wafer.
Figure 1. Optical Micrographs of (a) empty microwells and (b) microwells filled with microspheres.
Polystyrene microspheres were assembled into the microwells by dispensing a suspension of the microspheres
Letters
Langmuir, Vol. 21, No. 12, 2005 5235
Figure 2. SEM images of (a) an array of microwells filled with microspheres and (b and c) self-assembled microspheres in a microwell.
(pH 4.3) onto the array of microwells and allowing it to dry. During the incubation period, the solvent evaporated slowly and the microspheres were pushed along the substrate by capillary force.8 As the evaporation boundary receded over the background region, the surface was dewetted and no microspheres were deposited on it. However, as the evaporation boundary passed over the microwells, capillary force and electrostatic attraction caused the deposition of the microspheres into the wells. At pH 4.3, the -NH2 groups on the surface of the microwells and the -COOH group on the polystyrene microspheres were ionized into -NH3+ and -COO-, respectively.9 Figure 1B shows the optical micrograph of the microwells after they were filled with microspheres. Figure 2A shows the scanning electron microscopy (SEM) image of an array of microwells filled with microspheres. The microspheres were deposited inside the microwells while the region outside the wells is free of particles. Figure 2B shows a typical image of microspheres assembled within the microwells. A monolayer of closely packed microspheres covered the surface of the microwell. Figure 2C is an enlarged SEM image to depict the hexagonal arrangement of microspheres inside the microwells. The coverage of particles in the microwells may be improved by several factor methods, for example, increasing the concentration of the amino-silane and the reaction time and the concentration of particles. After the beads were deposited in the microwells, there were two surfaces on the silicon wafer; one has PEG, and the other has carboxylic groups (on the microspheres). Proteins were attached to the microspheres through conjugation with carboxyl groups. Micropatterning of biomolecules was performed with BSA-FITC and IgGCy3. We observed that immersion of the chip in the protein solution did not result in an observable disturbance to the initial microsphere pattern. The microscopic fluorescence images for patterning BSA-FITC and IgG-Cy3 are depicted in Figure 3. Both images confirm that protein molecules were selectively adsorbed onto the carboxylated polystyrene microspheres. The PEG modified regions between the microwells had resisted protein adsorption, resulting in precise deposition of protein on a nonplanar surface created by the microspheres. Although the method is demonstrated using proteins, this technique can be easily extended to patterning other biomolecules by ensuring that the microspheres and substrate have the relevant functional groups for adhesion and resistivity, respectively. We have demonstrated a new micropatterning technique which involves immobilization of biomolecules on a nonplanar surface at a designated area via assembly of polystyrene microspheres. The advantages of our proposed (8) Xia, Y.; Gates, B.; Yin, Y.; Lu, Y. Adv. Mater. 2000, 12, 693-713. (9) Saito, N.; Maeda, N.; Sugimura, H.; Takai, N. Langmuir 2004, 20, 5182-5184.
Figure 3. Fluorescent images of polystyrene microspheres assembled in microwells adsorbed with (a) BSA-FITC and (b) IgG-Cy3.
method are manyfold. First, the curve surfaces of the microspheres assembled on a planar surface increase the surface area for immobilization of biomolecules compared to a planar surface. The amount of increase in the surface area can be calculated by considering that each closely packed microsphere (with a radius r) occupies a hexagonal area on the surface (Figure 4).
area of planar hexagonal area ) 6r2/x3
(1)
curve surface area created by microspheres ) 2πr2 (2) ratio of curve surface area/planar area ≈ 1.81
(3)
As derived above, the curve surface area created by microspheres closely packed on a planar surface is approximately 1.81 times higher than a planar surface. A partially filled microwell will have an increase in surface area ranging between 1 and 1.81 times. This increment
5236
Langmuir, Vol. 21, No. 12, 2005
Figure 4. Schematic diagram to show that each microsphere occupies a hexagonal area on the substrate.
is independent of the size of the particles; however, the size of the particles will affect the functionality of protein adsorbed on it (discussed in the next paragraph). The increment in the surface area will subsequently increase the density of biomolecules that can be immobilized and, hence, the sensitivity for detection. As the demand for sensitive biosensors continues,10 this technique may be used as an immobilization strategy in biosensor devices to enhance the signal for detection. More recently, patterning of biomolecules with nano-resolution has been made possible with the support of techniques such as dip pen lithography11 and e-beam patterning.12 As the size of the pattern becomes smaller, it is crucial that sensitivity is not compromised due to a reduction in surface area. Our method of using microspheres for patterning can resolve the conflict between size of feature and sensitivity. Apart from having a good coverage of biomolecules at the designated region, it is also crucial for biomolecules, especially proteins, to retain their native function. It has been well-documented that immobilization of protein on surfaces typically affects its conformation and, hence, the biological function.13 Recently, two independent research groups14,15 investigated the effect of surface curvature upon which protein is immobilized on protein conformation and functionality. Vertegel et al. studied the enzymatic activity (10) Mehrvar, M.; Abdi, M. Anal. Sci. 2004, 20, 1113. (11) Piner, R. D.; Zhu, J.; Xu, J.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661-663. (12) Eck, W.; Stadler, V.; Geyer, W.; Zharnikov, M.; Golzhauser, A.; Grunze, M. Adv. Mater. 2000, 12, 805-808. (13) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233-244. (14) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20, 6800-6807. (15) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Langmuir 2004, 20, 10639-10647.
Letters
of lysozyme adsorbed on silica nanopraticles with sizes ranging from 4 to 100 nm while Lundqvist et al. investigated the adsorption of human carbonic anhydrase on silica nanoparticles with diameters ranging from 6 to 15 nm. Both groups came to the same conclusion that a surface of greater curvature (nanoparticles with a smaller diameter) affects the porotein’s secondary structure to a smaller extent. This is because larger particles result in a larger interaction area between the protein and the particle and will affect the secondary structure of protein more predominantly than smaller particles.14,15 From here, we can infer that protein immobilized on a highly curved surface can retain its native structure and function better than on a planar surface (with zero curvature). Furthermore, our method of micopatterning via assembly of microspheres introduces a more versatile system for performing surface modification. The conventional method of micropatterning via photoresist lithography involves carrying out surface modification on a substrate carrying the photoresist pattern. Because of the presence of the photoresist, surface modification has to be limited to using inorganic solvent as the medium for reaction. This is because some organic solvent will dissolve the photoresist. By using microspheres, surface modification can be carried out on the particles instead, prior to assembly on the silicon wafer. Therefore, this method can bypass the need of performing surface modification directly on the patterned photoresist and do away with the restrictions that are inherent with the conventional method. Conclusions In summary, we have presented a new method of micropatterning of biomolecules on a nonplanar spot integrated on a planar substrate via assembly of polystyrene microspheres. Various components of the system can be modified to cater to the intended applications, for example, the size and type of particles used for assembly. This method is applicable for patterning a variety of biomolecules including antibodies, antigens, enzymes, receptors, peptides, oligonucleotides, DNA, and many more. Acknowledgment. This work was supported by a NUS research grant. LA050454F
