pubs.acs.org/Langmuir © 2010 American Chemical Society
Controlled Assembly of Protein in Glass Capillary Yuan Lin,†,‡ Zhaohui Su,† Elizabeth Balizan,‡ Zhongwei Niu,*,§ and Qian Wang*,‡ †
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China, ‡Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, and §Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100910, P. R. China Received May 4, 2010. Revised Manuscript Received June 24, 2010
By means of a slow drying process and the control of surface charge characteristics, protein stripe patterns were readily prepared on the luminal surface of a capillary. We systematically studied the effects of surface properties, pH, and protein concentration on pattern formation using optical microscopy, atomic force microscopy, and quartz crystal microbalance measurement. By balancing these parameters, a broad selection of proteins could be assembled within a capillary with well-defined stripe patterns. Neutravidin, one of the model proteins, was specifically chosen to demonstrate the bioactivity retained through the assembly process by interaction with fluorescently labeled biotin motifs. This technique therefore offers a facile approach for patterning proteins and other biomacromolecules in capillary tubes.
Introduction In this paper, we present a method for patterning different proteins by drying protein solutions in capillary tubes, while the proteins still retain their bioactivities. Controlling spatial arrangement of proteins is of great interest in biomedical and biotechnical applications, such as diagnostics, biosensing, probing cell-environment interactions, and drug discovery.1-3 Protein patterning has been investigated since 1970s, when MacAlear and Wehrung used photoresist technology to create patterns on an underlying compressed protein layer.4,5 So far, many methods have been developed for patterning proteins on surfaces, including inkjet printing,2,6 microscale direct writing,7 dip-pen nanolithography,8,9 microcontact printing,10 photolithography11 and soft lithography techniques.12 However, it is difficult to apply these techniques to the luminal surface of capillaries. We recently reported that bovine serum albumin (BSA) can be assembled in a capillary tube to form a stripe pattern.13 Smooth muscle cells (SMCs) cultured inside the BSA-patterned capillary tubes aligned perpendicular to the capillary tubes, which can potentially be used in blood vessel engineering. We envision this technique could be expanded to other types of *Corresponding authors. (1) Dubey, M.; Emoto, K.; Takahashi, H.; Castner, D. G.; Grainger, D. W. Adv. Funct. Mater. 2009, 19, 3046–3055. (2) Delaney, J. T.; Smith, P. J.; Schubert, U. S. Soft Matt. 2009, 5, 4866–4877. (3) Cretich, M.; Damin, F.; Pirri, G.; Chiari, M. Biomol. Eng. 2006, 23, 77–88. (4) MacAlear, J. M.; Wehrung, J. M. US Patent 4,103,073, 1978. (5) MacAlear, J. M.; Wehrung, J. M. US Patent 4,103,064, 1978. (6) Roth, E. A.; Xu, T.; Das, M.; Gregory, C.; Hickman, J. J.; Boland, T. Biomaterials 2004, 25, 3707–3715. (7) Mei, Y.; Cannizzaro, C.; Park, H. S.; Xu, Q. B.; Bogatyrev, S. R.; Yi, K.; Goldman, N.; Langer, R.; Anderson, D. G. Small 2008, 4, 1600–1604. (8) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30–45. (9) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702–1705. (10) Ruiz, S. A.; Chen, C. S. Soft Matter 2007, 3, 168–177. (11) Carrico, I. S.; Maskarinec, S. A.; Heilshorn, S. C.; Mock, M. L.; Liu, J. C.; Nowatzki, P. J.; Franck, C.; Ravichandran, G.; Tirrell, D. A. J. Am. Chem. Soc. 2007, 129, 4874–4785. (12) Coyer, S. R.; Garcia, A. J.; Delamarche, E. Angew. Chem., Int. Ed. 2007, 46, 6837–6840. (13) Lin, Y.; Balizan, E.; Lee, L. A.; Niu, Z. W.; Wang, Q. Angew. Chem., Int. Ed. 2010, 49, 868–872.
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proteins, which potentially will lead to applications in microseparation,14 biosensing,15 and blood vessel repairing.16 When a suspension solution dries on a surface, a ring-like structure is formed whenever the contact line is pinned. This phenomenon has widely been exploited to create hierarchical pattern structures.17 The contact line will stay pinned to form a single ring17,18 or shrink in a discontinuous manner to generate multiple rings.19-21 Different pattern formation using this phenomenon can be tuned on a planar surface or in a confined geometry.22-24 In general, the pinning and depinning (also called “stick-slip” motion) at the contact line governs such kind of pattern formation.25 Many basic theoretical studies have been reported to explain how different patterns are formed during the drying process. Spherical colloidal particle suspensions and polymer solutions are two broadly used systems for theoretical and experimental studies.18,23,26-28 Recently, carbon nanotubes and rod-like tobacco mosaic viruses were employed to create patterns with a drying process.29,13 For a protein drying inside the capillary tube, the evaporation of water triggers the pinning of (14) Davies, M. I.; Lunte, C. E. Chem. Soc. Rev. 1997, 26, 215–222. (15) Borisov, S. M.; Wolfbeis, O. S. Chem. Rev. 2008, 108, 423–461. (16) Hu, X. X.; Shen, H.; Yang, F.; Bei, J. Z.; Wang, S. G. Biomaterials 2008, 29, 3128–3136. (17) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827–829. (18) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E 2000, 62, 756–765. (19) Maheshwari, S.; Zhang, L.; Zhu, Y. X.; Chang, H. C. Phys. Rev. Lett. 2008, 100, 044503. (20) Shmuylovich, L.; Shen, A. Q.; Stone, H. A. Langmuir 2002, 18, 3441–3445. (21) Hong, S. W.; Byun, M.; Lin, Z. Q. Angew. Chem., Int. Ed. 2009, 48, 512–516. (22) Smalyukh, I. I; Zribi, O. V.; Butler, J. C.; Lavrentovich, O. D.; Wong, G. C. L. Phys. Rev. Lett. 2006, 96, 177801. (23) Xu, J.; Xia, J. F.; Hong, S. W.; Lin, Z. Q.; Qiu, F.; Yang, Y. L. Phys. Rev. Lett. 2006, 96, 066104. (24) Lin, Z. Q.; Granick, S. J. Am. Chem. Soc. 2005, 127, 2816–2817. (25) Adachi, E.; Dimitrov, A. S.; Nagayama, K. Langmuir 1995, 11, 1057–1060. (26) De Gennes, P. G. Rev. Mod. Phys. 1985, 57, 827–862. (27) Dimitrov, A. S.; Nagayama, K. Langmuir 1996, 12, 1303–1311. (28) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183–3190. (29) Hong, S. W.; Jeong, W.; Ko, H.; Kessler, M. R.; Tsukruk, V. V.; Lin, Z. Q. Adv. Funct. Mater. 2008, 18, 2114–2122.
Published on Web 07/07/2010
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the contact line (the “stick” process) due to the protein deposition, which makes the initial contact angle gradually decrease until reaching to a critical angle, at that point the capillary force (the depinning force) becomes bigger than the pinning force. This then causes the contact line to “slip” to a new position, thus forming a protein stripe. In this paper we report a systematic study of the effects of surface properties, pH, and protein concentration on pattern formation in order to better understand the protein selfassembly behavior in a confined environment.
Experimental Section Materials. Lysozyme (Lys) from chicken egg white was purchased from Rockland. Neutravidin (Neu) was purchased from Thermo. Human serum albumin (HSA) was purchased from Fluka. Bovine skin Type B gelatin (Gel B), R-chymotrypsinogen A (ChT A), concanavalin A (Type IV) (Con A), bovine pancreas ribonuclease A (Rib A), bovine serum albumin (g98% pure by gel electrophoresis) (BSA), and lipase (Lip) from Candida rugosa were purchased from Sigma-Aldrich Co. Fluorescein (FL) labeled biotin was purchased from Kirkegaard & Perry Laboratories. Poly(diallyldimethylammonium chloride) (PDDA) aqueous solution (Mw 100 000-200 000) and poly(styrene sodium sulfonate) (PSS) (Mw 70 000) were purchased from SigmaAldrich Co. All the chemicals were used as received without further purification. Ultrapure water was obtained from a Millipore Synergy UV system (18.2 MΩ 3 cm). The buffers were prepared following a standard protocol to maintain identical ionic strength, i.e., 10 mM dipotassium phosphate K2HPO4 (pH 9.5) and 10 mM monopotassium phosphate KH2PO4 (pH 4.4) were chosen to tune the different pH (4.4-9.5) of potassium phosphate buffer. For the pH 10.5 buffer, 10 mM NaOH was used to adjust the pH of a K2HPO4 solution. Unless otherwise noted, all experiments were performed using a 0.01 M pH 7.4 potassium phosphate buffer solution, further referred to as “buffer”. Characterization. Optical micrographs were acquired from an Olympus IX81 microscope using differential interference contrast. To image the protein stripes, the glass capillary was put on the sample holder surface, and then all the pictures were taken upon focusing on the lower surface. AFM images were obtained at ambient conditions on a NanoScope IIIA MultiMode AFM (Veeco) operated in tapping mode. The tube was broken first and the small pieces were picked up for the AFM measurement. The dynamic adsorption behavior of lysozyme was characterized with a Q-Sense E1 quartz crystal microbalance with dissipation monitoring (QCM-D). Substrate Preparation. Cylindrical capillary borosilicate glass tubes (length 2.2 cm and inner diameter 0.15 cm) from KIMBLE Co. were cleaned with a piranha solution (Caution!) (7:3 mixture of 98% H2SO4 and 30% H2O2) at 75 °C for 2 h. To provide different charge characteristics, the surface of a blank glass tube was modified via the layer-by-layer method using polyelectrolyte PDDA and PSS. In brief, the cleaned glass tube was submerged in a PDDA solution (1 mg/mL) for 1 h to allow complete coverage, removed and washed thoroughly with ultrapure water and dried with a stream of N2. Subsequently the positively charged surface was submerged in a PSS solution (2 mg/mL) for 20 min, and then washed thoroughly with ultrapure water and dried with N2. Three kinds of surfaces were used in this study: freshly cleaned capillary tubes, capillaries treated by layer-by-layer method with PDDA as the outmost layer, and capillaries treated by layer-by-layer method with PSS as the outmost layer. Protein Patterning and QCM-D Experiment. Each protein solution (25 μL) was injected into an individual capillary tube, and the tubes were maintained in a horizontal position on a flat bench at room temperature and 40-60% humidity for three days. To obtain a positively charged surface for QCM-D experiments, a SiO2-coated chip (QSX 303, silicon dioxide, 50 nm) was immersed in a 5:1:1 mixture solution of ultrapure water: ammonia (25%): 12804 DOI: 10.1021/la1017888
hydrogen peroxide (30%), heated to 75 °C, rinsed with ultrapure water, and dried with a stream of N2. The cleaned SiO2-coated chip was submerged in a PDDA solution (1 mg/mL) for 1 h, washed thoroughly with ultrapure water, and dried with N2. In order to obtain a negatively charged surface, a PDDA coated chip (positively charged surface) was submerged in a PSS solution (2 mg/mL) for 20 min, washed thoroughly with ultrapure water, and dried with N2. A modified chip (with either PDDA or PSS cap layer) was mounted in a QFM 401 chamber (Q-Sense, Gothenburg, Sweden) and buffer was injected. After a stable baseline was established, the protein solution (lysozyme in potassium phosphate buffer, pH 7.4) was injected into the chamber. In air or vacuum, if the added layer is rigid, and much thinner than the crystal, the frequency shift (Δf) is related to mass loaded (Δm) by the Sauerbrey equation30 Δm ¼ -
Fq lq Δf f0 n
where f0 is the fundamental frequency, n is the overtone number, and lq and Fq are the thickness and density of the quartz crystal, respectively. Bioactivity Test. A Neu solution (0.1 mg/mL, 25 μL) at pH 9.5 in a 0.01 M potassium phosphate buffer was injected into a cleaned uncoated glass capillary tube. Following the formation of the Neu patterns, FL-biotin (1.0 mg/mL, 25 μL) was injected to the tube. After 30 min, the tube was washed thoroughly with ultrapure water and dried with N2. The images of the samples were then taken on an Olympus IX81 fluorescent microscope.
Results and Discussion Principle of the Protein Patterning. When a protein solution of a certain concentration is dried in an open-ended horizontally laid capillary tube, a symmetrical stripe pattern is formed from both ends. Theoretically, this process is similar to that of drying colloidal and nano particle solutions in capillary tubes,31 and in theory can be employed for most proteins. As illustrated in Figure 1a, a thin meniscus is first formed at the liquid-vapor interface. As the water evaporates, convective flow drives proteins to move toward the contact line and deposit on the substrate. Surface roughness is generated by the deposition of proteins, producing a frictional force f, which together with liquid surface tension γf pins the position of the contact line.26 As the evaporation proceeds, water is progressively in contact with proteins only. Capillary force γL pulls the liquid inward, and the contact line is depinned. The contact line then slips and reaches another equilibrium position, where the water is back in contact with the glass surface (Figure 1b). Consequently, the process repeats periodically, and the resulting “stick-slip” motion forms a repeating pattern (Figure 1c). As the solution dried deeper within the capillary, the evaporation rate of water reduced causing the distance between the two pinning lines to gradually increase. Thus the stripe will become wider from the end of tube to the middle (Figure 2a). Concentration and properties of the protein, solution pH, and inner surface properties of the capillary tube are four key factors that govern the formation of different patterns. Patterns of Proteins. As shown in Table 1, ten different proteins were randomly selected for the controlled assembly experiments in our study. These proteins have different structures, surface properties, isoelectric points (pI) and molecular weights. All these proteins were able to form stripe patterns via the abovementioned process. Figure 2 shows optical images of stripe patterns (30) Sauerbrey, G. Z. Phys. A: Hadrons Nuclei 1959, 155, 206–222. (31) Abkarian, M.; Nunes, J.; Stone, H. A. J. Am. Chem. Soc. 2004, 126, 5978– 5979.
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Figure 1. Schematic illustration of protein stripe pattern formation in a capillary tube with a slow drying process. (a) Cartoon of a capillary tube containing a protein solution, gray arrows indicate drying directions. (b) Illustration of the thin meniscus formed at the contact line, indicated by black dashed square in the lower left corner of part a. Proteins are represented as green spheres. The red arrow represents direction of the frictional force f generated by deposition of proteins at the contact line. The gray arrow on right represents moving direction of proteins caused by convective force. Two thin black arrows represent surface tension γf and capillary force γL. The contact angle between water and substrate is θ. Yellow thin arrows represent evaporation of water. (c) Cartoon presentation of a protein stripe pattern (in green color) formed at contact line. Table 1. Proteins Selected for Controlled Assembly in Glass Capillaries
Figure 2. (a) Schematic illustration of the capillary tube with stripe protein patterns. (b-d) Optical images of stripe patterns of four proteins: human serum albumin (HSA), R-chymotrysinogen A (ChT A), concanavalin A (Con A), and ribonuclease A (Rib A), formed after slow drying within uncoated capillary tubes. All protein concentrations were 0.1 mg/mL in buffer. All images were taken at the same location related to the capillary tube, indicated by black square in the lower left corner of part a with the same scale bar. The protein stripe was located between two white arrows as shown in part b. The protein structures were generated using PyMol (www.pymol.org) with coordinates obtained from RCSB Protein Data Bank (www.pdb.org), and shown as insets of parts b-d.
of four different proteins, HSA, ChT A, Con A, and Rib A. Other proteins formed similar stripe patterns (data not shown). However, detailed characteristics of these protein stripes differ from one to another. As previously discussed, interaction between Langmuir 2010, 26(15), 12803–12809
protein
pI
Mw
concanavalin A (Con A) gelatin B (Gel B) bovine serum albumin (BSA) human serum albumin (HSA) lipase (Lip) neutravidin (Neu) R-chymotrysinogen A (ChT A) ribonuclease A (Rib A) avidin (Avi) lysozyme (Lys)
4.5 4.8 4.8 5.2 5.6 6.3 9.0 9.4 10.5 11.0
104 000 60 000 66 300 69 400 58 000 60 000 25 700 13 700 69 000 14 400
proteins, interaction between protein and substrate, protein concentration, pH, protein surface charge, and surface properties of the substrate all have big impacts on pattern formation. In the following discussion, we choose two typical proteins, Lys and Neu, to illustrate influences of these conditions. Influence of Protein Concentration. Lysozyme (Lys), known as muramidase or N-acetylmuramide glycanhydrolase, belongs to a family of enzymes which can damage bacterial cell walls. At pH 7.4, Lys (pI 11.0) carries positive charge, in order to reduce attractive electrostatic interaction between Lys and glass surface, the capillary was first cleaned with piranha solution and then coated with a thin layer of PDDA. Lys at different concentrations were then injected into capillary tubes. After three days drying at ambient condition, stripe patterns formed with Lys at a wide range of concentrations. As shown in Figure 3, when Lys concentrations were changed from 0.02 mg/mL to 0.3 mg/mL, the width of Lys stripe changed from 2.0 ( 0.2 μm to 20.6 ( 1.1 μm, and the height changed from 27.4 ( 1.0 nm to 113.4 ( 8.1 nm, measured by AFM. Considering the dimension of Lys is approximately 3 3 4.5 nm3,32 we conclude that multilayers of Lys (32) Imoto, T.; Johnson, L. N.; North, A. C. T.; Phillips, D. C.; Rupley, J. A. Enzymes 1972, 7, 665–868.
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Figure 3. Influence of the protein concentration on height and width of Lys (pH = 7.4) and Neu (pH = 4.4) stripes formed in PDDA-coated capillary tubes (9, height of Lys stripe; 2, width of Lys stripe; 0, height of Neu stripe; Δ, width of Neu stripe). The height and width of stripes were determined using AFM.
were deposited within the stripes. The protein concentration will determine the amount of proteins supplied to the stripe-growing region, which is directly related to the width and thickness of the stripes. For the concentration lower than 0.02 mg/mL there was not enough Lys supplied to the contact line, so no pattern was observed. Instead, a discontinuous film was observed. When the Lys concentration was greater than 0.3 mg/mL, the luminal surface of the capillary tube was fully covered with Lys, and no stripe pattern was found (data not shown). In order to further confirm the concentration effect on the protein pattern formation, neutravidin (Neu, pI 6.3) solutions with different concentrations were injected into the PDDA-coated capillary tubes at pH 4.4. As shown in Figure 3, when Neu concentrations were changed from 0.03 mg/mL to 0.5 mg/mL, the width of Neu stripe changed from 1.9 ( 0.2 μm to 18.7 ( 0.1 μm, and the height changed from 13.4 ( 2.9 nm to 102.7 ( 5.6 nm. Again, both width and height showed near linear increase with the increase of protein concentration. As the molecular weight of Neu (60 000) is larger than that of Lys (14 400), at the same concentration, compared to Lys there are less Neu being driven to the contact line, resulting in narrower and thinner Neu stripes in comparison with Lys. Influence of Surface Properties. For protein patterning, one key issue is to prevent nonspecific binding of protein onto the substrate. Charge-charge interaction and hydrophobic-hydrophobic interaction are two major driving forces for nonspecific adsorption of proteins. For conventional microfabrication or self-assembled monolayer techniques, polyethylene glycol (or oligoethylene glycol analogs) and blocking proteins such as BSA are commonly used to adsorb on bare surface and block other proteins from adhering.33-35 In our study, we try to minimize protein nonspecific binding by control of surface charge characteristics using simple layer-by-layer method. The isoelectric point (pI) of Lys is 11.0; therefore, at experimental conditions of pH 7.4, Lys is positively charged. Adsorption of Lys on different charged surfaces was monitored (33) Vogt, R. F.; Phillips, D. L.; Henderson, L. O.; Whitfield, W.; Spierto, F. W. J. Immunol. Methods 1987, 101, 43–50. (34) Bhatia, S. K.; Teixeira, J. L.; Anderson, M.; Shriverlake, L. C.; Calvert, J. M.; Georger, J. H.; Hickman, J. J.; Dulcey, C. S.; Schoen, P. E.; Ligler, F. S. Anal. Biochem. 1993, 208, 197–205. (35) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–698.
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Figure 4. Frequency shift during adsorbing Lys (0.1 mg/mL) in potassium phosphate buffer (pH 7.4) on PDDA and PSS coated surfaces monitored by QCM-D.
by a quartz crystal microbalance with dissipation monitoring (QCM-D) with a Q-Sense Flow Module (QFM 401) chamber. The sensor was made by an AT-cut piezoelectric quartz crystal sandwiched between two SiO2-coated electrodes, which was used as the sensing element. A SiO2-coated electrode was coated with a thin layer of PDDA (0.3 nm) by charge-charge interaction to give it a positively charged surface.36 To make an electrode negatively charged, a PDDA coated substrate was submerged to a solution of PSS (2 mg/mL) for 20 min, washed thoroughly with ultrapure water, and dried with N2. Adsorption of Lys on different charged electrodes showed great difference. As shown in Figure 4, adsorption of Lys on positively charged PDDA surfaces reached saturation after 5 min, and frequency decreased about 8 Hz, which corresponds to 47.2 ng of Lys calculated by Sauerbrey’s equation.30 However, on negatively charged PSS surfaces, adsorption of Lys reached saturation much faster (less than 1 min), and the frequency decreased 16 Hz, which corresponds to 94.4 ng of Lys. Upon rinsing with buffer for 200 s, physical adsorption of Lys could be removed from substrate and the adsorption of protein on the substrates reached another plateau. The frequency increased 5 Hz on both the negatively charged PSS surface and the positive charged PDDA surface, corresponding to 29.5 ng Lys was removed from both surfaces, which indicated very small amount of Lys (17.7 ng) left on the PDDA coated substrate. Change in adsorption behavior of Lys due to surface charge proves nonspecific adsorption of proteins can be modulated by surface charge characteristics. When Lys was dried within positively charged capillary tubes (PDDA coated luminal surface), positively charged Lys repelled the positively charged surface and ensured the stick-slip events to form the stripe pattern (Figure 5a). As shown in Figure 5c and 5e, there is no Lys adsorbed between two protein stripes (the white dash square in Figure 5c). However, when Lys was dried within negatively charged capillary tubes (either blank glass or PSS coated), positively charged Lys adsorbed significantly onto the negatively charged surface. As a result, although Lys will form similar stripe patterns (Figure 5b), there is a thin layer of Lys coated between two Lys stripes (Figure 5d and Figure 5f). Influence of the Solution pH. Clearly, electrostatic force plays an important role in guiding the adsorption of proteins on different surfaces. Since average surface charges of proteins are (36) Pfeiffer, I.; Seantier, B.; Petronis, S.; Sutherland, D.; Kasemo, B.; Zach, M. J. Phys. Chem. B 2008, 112, 5175–5181.
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Figure 5. Optical images of Lys (0.1 mg/mL) in potassium phosphate buffer (pH 7.4) dried in PDDA coated interior capillary surface (a) and PSS coated interior capillary surface (b). (c and d) AFM images of the square region enclosed by the white dashed line in parts a and b, correspondingly. (e and f) Enlarged view of the square region enclosed by the white dashed line in parts c and d.
determined by their structural features as well as the solution pH. In addition, the electrostatic interaction dictates the bioactivities of a protein when attached to a surface.37 To investigate the effect of solution pH to the assembly process, a series of 0.1 mg/mL Lys solutions at different pH were injected into the PDDA-coated capillaries. Figure 6 shows the relationship between pH and height of Lys stripes. Below pH 11, Lys carries net positive charges. The repulsion between Lys and PDDA-coated surfaces will guide most of Lys to deposit on the contact line and form stripe pattern. At pH 7.4 and 9.5, the heights of Lys stripe are 51.4 ( 0.9 nm and 74.6 ( 2.5 nm, respectively (Figure 6). At pH 4.4, the height of Lys stripe decreases to 38.5 ( 4.7 nm because the strong repulsion between Lys molecules prohibits further deposition of Lys on each stripe. At pH 10.5, the net charge of Lys decreases to near neutral, which results in much weaker repulsions among Lys molecules and between Lys and the PDDA-coated glass surface. Therefore, maximum adsorption of Lys on the PDDA-coated surface (between protein stripes) can be observed, reducing the amount (37) Kubiak-Ossowska, K.; Mulheran, P. A. Langmuir 2010, 26, 7690–7694.
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Figure 6. Influence of pH of Lys solution (0.1 mg/mL) on height of Lys stripes formed in PDDA-coated capillary tubes. The height profiles were determined using AFM. DOI: 10.1021/la1017888
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Figure 7. Optical images of neutravidin (0.03 mg/mL) at pH 7.4 (a) and 9.5 (d) in 0.01 M potassium phosphate buffer dried within uncoated capillary tubes. (b and e) AFM images of the square region enclosed by the white dashed line in parts a and d. (c and f) Height profiles for parts b and e, correspondingly. White lines in parts b and e indicate the scanning regions for the height profiles, as shown in parts c and f, respectively.
Figure 8. (a) Schematic illustration of Neu stripe pattern and Neu-biotin interaction. (b) Optical image of stripe pattern upon drying Neu (0.1 mg/mL) at pH 9.5 in the uncoated capillary tube. (c) Fluorescent image of the same stripe pattern in part b after interaction with fluorescein labeled biotin (FL-biotin) solution and washing. White arrows in parts b and c indicate Neu stripe before and after FL-biotin binding.
of available solution Lys in solution to form the stripe structures during the drying process. This is the reason why at pH 10.5, the height of Lys stripe is only 29.5 ( 7.3 nm (Figure 6). Neutravidin, a deglycosylated version of biotin binding protein avidin, with a mass of approximately 60 000 Da and a near-neutral pI (6.3), was also used to in this study. When drying Neu solution (0.03 mg/mL) in potassium phosphate buffer (pH 7.4) within glass capillary tubes (no PDDA or PSS coating), it was difficult to observe a stripe pattern using optical microscopy (Figure 7a). An enlarged AFM image showed the existence of residual proteins presented between two protein stripes (Figure 7b) and the height of 12808 DOI: 10.1021/la1017888
Neu stripe was only around 5 nm (Figure 7c). As the solution pH was changed to 9.5, a regular pattern was readily formed. AFM images showed much less protein adsorbed between stripes (Figure 7, parts d and e), and the height of the protein stripe was around 18 nm (Figure 7f). At pH 7.4, close to the pI of Neu, the charge density on the surface of Neu is relatively low, resulting in significant nonspecific binding of proteins,38 which is a much faster process in compared with the drying process. Additionally, after (38) Liu, B. L.; Cao, S. S.; Deng, X. B.; Li, S. J.; Luo, R. Appl. Surf. Sci. 2006, 252, 7830–7836.
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forming a thin layer of protein on top of the substrate, the surface contact angle will be greatly varied depending on protein properties, leading to the formation of shallower and ill-organized protein stripes. Increasing the solution pH (to pH 9.5) drastically increased the surface negative charges of Neu, which consequently reduced the nonspecific protein deposition and led to the formation of clear stripe structures. This is consistent with the above discussion about the relationship between pH and height of Lys stripes. Bioactivity. To test if Neu maintains bioactivity after the formation of stripe patterns, fluorescein modified biotin (FL-biotin) was used to test binding ability of Neu (Figure 8a). After stripe pattern was obtained (Figure 8b), a FL-biotin solution was injected into the capillary tube with the Neu stripe pattern. After 30 min incubation at room temperature, nonspecific binding of biotin was removed by rinsing with water thoroughly. As shown in Figure 8c, FL-biotin selectively recognized Neu stripes and showed bright green fluorescence. Between Neu stripes, very weak fluorescence could be detected. This indicates Neu can maintain its binding ability and selectivity with biotin units after forming stripe patterns in capillary tubes. Because certain percentage of Neu molecules (within the stripe) could be washed off during the assay, it is hard to quantitatively measure how much Neu still preserved their bind (39) Kim, E.; Kim, K.; Yang, H.; Kim, Y. T.; Kwak, J. Anal. Chem. 2003, 75, 5665–5672. (40) Lesaicherre, M. L.; Lue, R. Y. P.; Chen, G. Y. J.; Zhu, Q.; Yao, S. Q. J. Am. Chem. Soc. 2002, 124, 8768–8769.
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activities. Nevertheless, one can envision the potential applications of this method in biolabeling, bioimaging or biosensing.39,40
Conclusion In conclusion, we have developed a simple approach to prepare stripe-like protein patterns in glass capillary tubes with a slow drying process. The height and width of protein stripes were controlled by varying protein concentration. Well-defined stripe patterns were produced by balancing capillary tube internal surface charge and pH of protein solution. Stripe patterns of protein obtained by this self-assembly technique maintained the bioactivity of proteins, which was demonstrated by specific interaction between FL-biotin and Neu stripes in glass capillary. Our method affords an easy way to pattern different bioactive proteins, as well as protein assemblies and other biomacromolecules in capillary tubes, which will have potential applications in separation, microanalysis and biosensing. Acknowledgment. This work was supported by the US NSF (DMR-0706431, CHE-0748690), Alfred P. Sloan Scholarship, Camille Dreyfus Teacher Scholar Award, US-ARO-W911NF09-1-236, and W. M. Keck Foundation. Z.S. thanks the NSFC Fund for Creative Research Groups (50921062) for support. Z.N. expresses thanks for the support from the Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.
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