Self-Assembly of Virus Particles on Flat Surfaces via Controlled

Nov 24, 2010 - Sabine Eiben , Claudia Koch , Klara Altintoprak , Alexander ... Petia Atanasova , Insook Kim , Bingling Chen , Sabine Eiben , Joachim B...
0 downloads 0 Views 3MB Size
Download PDF
pubs.acs.org/Langmuir © 2010 American Chemical Society

Self-Assembly of Virus Particles on Flat Surfaces via Controlled Evaporation† )

Yuan Lin,‡ Zhaohui Su,*,‡ Guihua Xiao,‡ Elizabeth Balizan,§ Gagandeep Kaur,§ Zhongwei Niu, and Qian Wang*,§

)

‡ State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China, §Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, United States, and Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100910, PR China

Received September 29, 2010. Revised Manuscript Received November 4, 2010 Dynamic self-assembly of nonvolatile solutes via controlled solvent evaporation has been exploited as a simple route to create a variety of hierarchically assembled structures. In this work, two glass slides were used to form a confined space in which a solution of a rodlike nanoparticle, tobacco mosaic virus (TMV), was evaporated to create large-scale stripe patterns. The height and width of the stripes are dependent on the TMV concentration. The large-scale-patterned surfaces can be applied to control surface hydrophobicity and direct the growth of bone marrow stromal cells. We systematically studied the effects of stripe width and height on surface hydrophobicity using optical microscopy, atomic force microscopy, and contact angle measurements. This technique offers a facile approach to form 2D patterns on a large surface from a wide range of proteins as well as other biomacromolecules.

Introduction The self-assembly of biomacromolecules to form hierarchically ordered structures promises new opportunities in developing novel biomaterials. In particular, controlling the surface arrangement of proteins is very desirable for many applications such as microarray development, sensing and diagnosis, and tissue engineering. In this regard, several elegant approaches for protein patterning based on lithography technology have emerged.1-3 We recently reported the formation of stripe patterns from drying solutions of proteins and virus particles in glass capillary tubes.4,5 We found that a variety of proteins could be patterned on the luminal surface of a capillary tube with their bioactivities retained.4 Furthermore, when rodlike tobacco mosaic viruses (TMV) were used, the orientation of TMV could be readily controlled by varying the particle concentration and surface properties of the capillary interior surface.5 The basic principle of this assembly is the so-called “coffee ring” effect in which when drying a sessile drop containing polymers, colloidal particle suspensions, or carbon nanotubes, a number of concentric coffee rings through the repetitive stick-slip motion of Part of the Supramolecular Chemistry at Interfaces special issue. *Corresponding authors. E-mail: [email protected]; [email protected]. †

(1) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30–45. (2) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702–1705. (3) Coyer, S. R.; Garcia, A. J.; Delamarche, E. Angew. Chem., Int. Ed. 2007, 46, 6837–6840. (4) Lin, Y.; Su, Z.; Balizan, E.; Niu, Z.; Wang, Q. Langmuir 2010, 26, 12803–12809. (5) Lin, Y.; Balizan, E.; Lee, L. A.; Niu, Z. W.; Wang, Q. Angew. Chem., Int. Ed. 2010, 49, 868–872. (6) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E 2000, 62, 756–765. (7) Xu, J.; Xia, J. F.; Hong, S. W.; Lin, Z. Q.; Qiu, F.; Yang, Y. L. Phys. Rev. Lett. 2006, 96, 066104. (8) Degennes, P. G. Rev. Mod. Phys. 1985, 57, 827–863. (9) Dimitrov, A. S.; Nagayama, K. Langmuir 1996, 12, 1303–1311. (10) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183–3190. (11) Hong, S. W.; Jeong, W.; Ko, H.; Kessler, M. R.; Tsukruk, V. V.; Lin, Z. Q. Adv. Funct. Mater. 2008, 18, 2114–2122.

1398 DOI: 10.1021/la103917x

a three-phase contact line can be formed.6-11 In such a process, the contact line either stays pinned to form a single ring6,12 or shrinks in a discontinuous manner to generate multiple rings.13-15 This phenomenon has widely been exploited to create a variety of hierarchically assembled structures by designing the geometry of the confinement space.8-10,15-18 In this article, we report the use of two glass slides to form a confined space in which to study the self-assembly process of rodlike TMV via a controlled evaporation method. Proteins and virus particles showed similar self-assembly behavior in both capillary tubes and two stacked glass slides. In our previous paper, the capillary tube was used to mimic a human blood vessel.5 In the current work, we further extend the study to a much larger space (i.e., two stacked glass slides were used to create largescale ordered structures), which may have more extensive implications. As an example, we demonstrate that the patterned surfaces can be applied to control surface hydrophobicity and direct the growth of bone marrow stromal cells.

Experimental Section Materials. Tobacco mosaic virus (TMV) was isolated according to previously reported methods.19 Excess salts were removed by several rounds of dialysis against deionized ultrapure water. Ultrapure water was obtained from a Millipore Synergy UV (12) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827–829. (13) Maheshwari, S.; Zhang, L.; Zhu, Y. X.; Chang, H. C. Phys. Rev. Lett. 2008, 100, 044503. (14) Shmuylovich, L.; Shen, A. Q.; Stone, H. A. Langmuir 2002, 18, 3441–3445. (15) Hong, S. W.; Byun, M.; Lin, Z. Q. Angew. Chem., Int. Ed. 2009, 48, 512– 516. (16) Hong, S. W.; Xia, J. F.; Lin, Z. Q. Adv. Mater. 2007, 19, 1413–1417. (17) Byun, M.; Hong, S. W.; Qiu, F.; Zou, Q. Z.; Lin, Z. Q. Macromolecules 2008, 41, 9312–9317. (18) Byun, M.; Laskowski, R. L.; He, M.; Qiu, F.; Jeffries-El, M.; Lin, Z. Q. Soft Matter 2009, 5, 1583–1586. (19) Niu, Z. W.; Bruckman, M. A.; Li, S. Q.; Lee, L. A.; Lee, B.; Pingali, S. V.; Thiyagarajan, P.; Wang, Q. Langmuir 2007, 23, 6719–6724.

Published on Web 11/24/2010

Langmuir 2011, 27(4), 1398–1402

Lin et al.

Article

Figure 1. (a-c) Schematic illustration of TMV stripe pattern formation between two glass slides by a slow drying process. A 0.1 mg/mL solution of TMV (in 0.01 M potassium phosphate buffer, pH 7.4) was used in the experiment. (a) Glass slides and spacers. (b) Illustration of the TMV solution (orange) constrained between two slides. (c) Upon drying, the TMV stripe pattern (cartoon in orange) formed. The red point indicates the starting position of the contact angle measurement (Figure 4); the black arrow indicates the direction of the x axis of Figure 4. (i-iii) Optical micrographs of stripe patterns formed in the regions indicated in part c: (i) the outermost region, (ii) the middle, and (iii) the innermost region. (iv, v) AFM images of the square region enclosed by the white lines in i and ii, respectively. (vi) Enlarged view of the square region enclosed by the white lines in iv. Scale bars are 50 μm for i-iii, 10 μm for iv and v, and 400 nm for vi.

system (18.2 MΩ cm). All experiments were performed using a pH 7.4 potassium phosphate buffer solution. Substrate Preparation. Glass slides from VWR Co. (2.5  7.5 cm2, 1.2 mm thick) were cleaned in a piranha solution (7:3 98% H2SO4/30% H2O2) at 75 °C for 2 h, washed thoroughly with ultrapure water, and dried with N2 gas. Two horizontally stacked glass slides were laid on a flat surface and separated by two glass spacers (length, 2.5 cm; width, 0.5 cm; height, 0.12 cm), and then 1.5 mL of a TMV solution was injected between the two glass slides at room temperature and 40-60% humidity. After one and a half days, these two glass slides were separated and washed thoroughly using ultrapure water to remove the potassium phosphate salt completely. The slides were dried with nitrogen gas before tests were conducted. Cell Culture. Primary BMSCs were isolated from the bone marrow of young adult 80 g male Wister rats (Harlan SpragueDawley, Inc.). The procedures were performed in accordance with the guidelines for animal experimentation by the Institutional Animal Care and Use Committee, School of Medicine, University of South Carolina. Cells were maintained in growth medium (Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), gentamicin (50 mg/mL), and amphotericin B (250 ng/mL)) and passaged no more than four times after isolation before use in experiments. Immunofluorescence Staining. Cells were fixed with 4% paraformaldehyde in PBS. After being washed three times with PBS, the fixed cells were permeabilized with 0.1% Triton-X100 in PBS for 5 min and then blocked with 1% BSA in PBS for 1 h at room temperature. BSA (1%) in PBS containing 0.5 μg/mL DAPI and 0.2 μg/mL FITC-phalloidin was added to each well to stain the cell nucleus and cytoskeletal structure. The samples were then mounted and sealed with clear nail polish before images were taken on an Olympus IX81 fluorescence microscope. Characterization. Optical micrographs were acquired from an Olympus IX81 microscope using differential interference contrast. AFM images were obtained under ambient conditions using a NanoScope IIIA MultiMode AFM (Veeco) in tapping mode. Langmuir 2011, 27(4), 1398–1402

Contact angle data were collected on a VCA-Optima goniometer (AST Products, Inc.).

Results and Discussion Self-Assembly of TMV on a Confined Flat Surface. As shown in Figure 1, two horizontally stacked glass slides (length, 7.5 cm; width, 2.5 cm) were laid on a flat surface and separated by two glass spacers (length, 2.5 cm; width, 0.5 cm; height, 0.12 cm), which formed a confined space in which to control the drying process of TMV solutions. In a typical experiment, a solution of TMV (0.1 mg/mL in 0.01 M potassium phosphate buffer, pH 7.4) was first injected between the two glass slides. At room temperature and 40-60% humidity, the solvent (water) was slowly evaporated over 1 day before two glass slides were separated. Under an optical microscope, the patterns on the two slides were mirror images of each other and displayed tetramerous radial symmetry as schematically illustrated in Figure 1c. In addition, on each side of the slide, the stripes became wider as they approached the longitudinal axis in the center (Figure 1, i-iii). However, the spacing between stripes became narrower. Atomic force microscopy (AFM) was used to quantify the width and height of the stripes. Figure 1(iv, v) contains two representative AFM images corresponding to the marked positions in Figure 1(i,ii), showing that each stripe is composed of two layers of TMV particles. An enlarged image (Figure 1, vi) indicates that the particles form a close-packed structure. As the solution dried to a deeper level, the reduced water evaporation rate caused the distance between the two pinning lines to increase gradually, which led to an increase in the width of stripes (data not shown). In the center of the slide, complete drying of the TMV solution formed potassium phosphate salt crystals together with TMV deposits. The crystallization of salt destroyed the TMV patterning and weakened the interactions between TMV and the glass surface. Upon washing, the center part of the TMV pattern as well as the salt DOI: 10.1021/la103917x

1399

Article

Lin et al.

Figure 2. AFM images of TMV patterns formed by drying a TMV solution (in 0.01 M potassium phosphate buffer, pH 7.4) between two glass slides with TMV concentrations of (a) 0.01, (b) 0.1, (c) 0.3, and (d) 1.0 mg/mL. All images have the same scale bar of 10 μm. All images were taken at position ii in Figure 1c. The cross-sectional analysis was generated by AFM software and is shown as insets in panels a-c.

could be completely washed away, exposing the bare glass surface (data not shown). The formation of TMV stripes follows the same principle that we previously reported for a capillary tube system. Confined by two glass slides, a thin meniscus is formed at the solid-liquid-vapor interface (contact line) of the liquid. As the water evaporates, convective flow drives TMV particles to the contact line, which then deposit onto the surface. Surface roughness is generated by the deposition of TMV particles, producing a frictional force f that together with the liquid surface tension γf pins the contact line. As evaporation proceeds, water is progressively in contact with only TMV. The capillary force γL pulls the liquid inward, and the contact line becomes depinned. The contact line slips and reaches another equilibrium position where water is back in contact with the glass surface. Consequently, this process is repeated periodically, and the stick-slip motion results in a periodic pattern.5 During the drying process, the solution volume becomes smaller and the contact line recedes deeper toward the medial line of the glass slides. Concurrently, the evaporation rate of the solution decreases, causing the distance between pinned lines to increase gradually as shown in Figure 1(i-iii). Moreover, to maximize the interfacial coverage per particle, individual TMV particles orient parallel to the plane of the interface, as demonstrated in our previous works.5,20,21 The concentrations of both TMV and salt will determine the number of particles supplied to the growing stripe region, which is directly related to the width and thickness of the stripes.4,5 At low TMV concentrations (0.01 mg/mL < [TMV] < 0.07 mg/mL), monolayer stripes were formed (Figure 2a) and the stripe width increased linearly with particle concentration (data not shown). (20) He, J. B.; Niu, Z. W.; Tangirala, R.; Wan, J. Y.; Wei, X. Y.; Kaur, G.; Wang, Q.; Jutz, G.; Boker, A.; Lee, B.; Pingali, S. V.; Thiyagarajan, P.; Emrick, T.; Russell, T. P. Langmuir 2009, 25, 4979–4987. (21) Kaur, G.; He, J. B.; Xu, J.; Pingali, S. V.; Jutz, G.; Boker, A.; Niu, Z. W.; Li, T.; Rawlinson, D.; Emrick, T.; Lee, B.; Thiyagarajan, P.; Russell, T. P.; Wang, Q. Langmuir 2009, 25, 5168–5176.

1400 DOI: 10.1021/la103917x

The height of the TMV monolayer was 14.5 ( 0.7 nm, consistent with the diameter of TMV particles (i.e., 18 nm). Higher concentrations of TMV solution drive more TMV particles to the tip of the meniscus, where the particle film is growing, resulting in thicker and wider stripes. At higher TMV concentrations (0.1 mg/mL < [TMV] < 0.7 mg/mL), multilayer TMV stripe patterns were formed (Figure 2b,c). For TMV concentrations of 0.1, 0.3, and 0.5 mg/mL, the heights of the multilayer stripes were around 25.5 ( 1.7, 48.7 ( 3.6, and 58.7 ( 2.3 nm, respectively. When the TMV concentration is higher than 0.7 mg/mL, a continuous TMV film (multilayers) is formed on the glass surface but no clear stripe pattern could be observed. The orientation of most TMV particles remained parallel to the contact line (Figure 2d). Increasing salt concentration enhances the screening of the surface charge of TMV, which can drive more TMV to deposit on the contact line. As shown in Figure 3, when the concentration of TMV was fixed at 0.01 mg/mL, different patterns from stripe monolayers to stripe multilayers to continuous films were obtained when the potassium phosphate concentration was varied from 10 to 500 mM. However, if the TMV concentration was too low (100 mM) stripe patterns could be obtained only near the edges of the slide because of the limited amount of TMV available. No viral particle was observed in the rest of the slide. Wettability of a TMV-Patterned Surface. It is well known that the topography of a surface can greatly influence its hydrophobicity/hydrophilicity.22,23 We therefore investigated the wettability variation along the direction normal to the stripe pattern. A 0.1 mg/mL solution of TMV was used in our initial study, which formed stripe patterns as shown in Figure 1. As shown in Figure 4, from the top to the bottom of the slide, water contact angle first decreases and then increases, corresponding to the (22) Checco, A.; Hofmann, T.; DiMasi, E.; Black, C. T.; Ocko, B. M. Nano Lett. 2010, 10, 1354–1358. (23) Drelich, J.; Wilbur, J. L.; Miller, J. D.; Whitesides, G. M. Langmuir 1996, 12, 1913–1922.

Langmuir 2011, 27(4), 1398–1402

Lin et al.

Article

Figure 3. (A-C) Optical images of patterns formed on glass slides after the drying of TMV solutions (0.01 mg/mL, pH 7.4) with different salt concentrations: (A) 10, (B) 100, and (C) 500 mM. (D-F) AFM images of enlarged views of the square regions in A-C, respectively.

Figure 4. Contact angles at different positions on the glass slide surface after the formation of TMV stripe patterns upon the drying of a TMV solution (0.1 mg/mL, in 0.01 M potassium phosphate buffer, pH 7.4) between two slides. The x axis indicates the distance along the longitudinal axis indicated by the black arrow in Figure 1c; the zero point is at the topmost edge indicated by the red point in Figure 1c.

symmetry of the pattern. At positions noted in Figure 1c(i-iii), the contact angles were 72.4 ( 3.2, 58.1 ( 4.0, and 48.3 ( 3.4°, respectively. For reference, the contact angle of a pure TMV film (θTMV) was around 45.4 ( 2.7°, and that of a clean glass surface (θglass) was 10.2 ( 1.5°. Water contact angle on a composite surface may be calculated according to the standard CassieBaxter equation:24 cos θ ¼ fA cos θA þ fB cos θB Here, θA and θB are the contact angles the drop makes with uniform surfaces A and B, and fA and fB are the area fractions of the A and B surfaces beneath the drop. If a drop of water wets the heterogeneous surface completely, then its contact angle on the stripe-patterned surface should be in the range of θglass < θ < θTMV. However, for position I (Figure 1c), the contact angle was ∼72°, which is much higher than that for a pure TMV film (∼45°). We concluded that water does not penetrate a significant percentage (24) Cassie, A. B. D.; B., S. Trans. Faraday Soc. 1944, 40, 546.

Langmuir 2011, 27(4), 1398–1402

Figure 5. Influence of TMV concentration (in 0.01 M potassium phosphate buffer, pH 7.4) on the contact angle. Area I is a monolayer TMV stripe, area II is multilayer TMV stripe, and area III is a continuous TMV film. Contact angles were measured at position i in Figure 1c.

of the grooves (the part between two TMV stripes) and air is trapped in the grooves.25 Although it is hard to describe quantitatively the drop penetration depth in the grooves, the surface contact angle is clearly correlated with the TMV pattern (i.e., the width and thickness of the TMV stripes as well as the spacing between adjacent TMV stripes). Because all monolayer samples have the same stripe thickness, this resulted in an equally sized meniscus and thus equally sized adjacent spacing.5 Therefore, at low concentrations (0.01 mg/mL < [TMV] < 0.07 mg/mL), monolayer stripe TMV patterns were formed and the surface contact angle increased almost linearly with the initial protein concentration (Figure 5, area I). This is consistent with the above relationship between the monolayer stripe width and concentration. The thickness of a TMV monolayer was 14.5 ( 0.7 nm on the basis of AFM measurements. In this height range, the water drop can penetrate and touch the glass surface completely, and the wettability of the TMV-glass composite surface can be described using the Cassie-Baxter equation. For example, the area fraction of the monolayer TMV stripe patterns produced at a 0.01 mg/mL TMV concentration was 13.3 ( 2.4% according to AFM data (Figure 2a), and from the Cassie-Baxter equation, the contact angle was estimated to be 18.8 ( 1.6°, which nicely agrees with the experimental value of 20.3 ( 3.2°. For the multilayer stripes, following the height increase, more air is likely trapped in the grooves, so the contact angle increases accordingly (Figure 5, area II).22 In this case, it is impossible to obtain the theoretical values because of air entrapment and structural complexity. As the concentration increased to 0.7 mg/mL, as more TMVs were driven to the contact line, the bottom layer of the TMV stripes became a nearly continuous film and the relative height of the TMV stripe layers decreased accordingly. Thus, the contact angle decreased to 59.5 ( 3.6°. When the TMV concentration was further increased to 1.0 mg/mL, the whole glass slide was fully covered by TMV, no stripe pattern could be observed (Figure 2d), and the contact angle was 47.4 ( 4.3° (Figure 5, area III), consistent with that of the pure TMV film (45.4 ( 2.7°). We could further modulate the surface hydrophobicity by varying the surface chemistry.26-28 For example, when bovine (25) (26) (27) (28)

Marmur, A. Langmuir 2003, 19, 5956–5959. Berim, G. O.; Ruckenstein, E. Langmuir 2009, 25, 9285–9289. Extrand, C. W. Langmuir 2003, 19, 3793–3796. Marmur, A. Langmuir 2003, 19, 8343–8348.

DOI: 10.1021/la103917x

1401

Article

Lin et al.

Figure 6. Fluorescent optical images of BMSCs cultured at different positions on a TMV stripe-patterned surface with the same 100 μm scale bars. The inset (center) highlights the positions where images a-d were taken.

serum albumin (BSA) was employed as the starting material instead of TMV, a similar stripe pattern could also be formed following the same protocol (data not shown). At 0.1 mg/mL, at position i noted in Figure 1c, a contact angle of 112 ( 2.7° was obtained, compared to 68 ( 3.1° for the pure BSA film and 72.4 ( 3.2° for the TMV striped film. Therefore, this technique offers a robust method for assembling other types of biomacromolecules on a flat surface, which gives it great potential in controlling surface hydrophobicity. Surface TMV Pattern-Directing Cell Growth. Surface chemistry, topography, and hydrophobicity play very important roles in altering cell adhesion, migration, and cytoskeletal organization, which are critical to cell growth and development.29-33 To demonstrate potential application of protein stripe patterns in regulating cell growth, bone marrow stromal cells (BMSCs) were cultured on a TMV stripe-patterned glass substrate. Previous results showed that the presence of TMV on the substrate significantly upregulated the osteogenesis of BMSCs.34,35 As shown in Figure 6a-c, BMSCs cultured on the TMV stripepatterned substrate are elongated and spread on these templates following the TMV stripe direction. In comparison, Figure 6d (29) Mendonca, G.; Mendonca, D. B. S.; Aragao, F. J. L.; Cooper, L. F. Biomaterials 2008, 29, 3822–3835. (30) Liu, Q. H.; Cen, L.; Yin, S.; Chen, L.; Liu, G. P.; Chang, J.; Cui, L. Biomaterials 2008, 29, 4792–4799. (31) Castellanos, T.; Ascencio, F.; Bashan, Y. FEMS Microbiol. Ecol. 1997, 24, 159–172. (32) Bashur, C. A.; Dahlgren, L. A.; Goldstein, A. S. Biomaterials 2006, 27, 5681–5688. (33) Stevens, M. M.; George, J. H. Science 2005, 310, 1135–1138. (34) Kaur, G.; Valarmathi, M. T.; Potts, J. D.; Jabbari, E.; Sabo-Attwood, T.; Wang, Q. Biomaterials 2010, 31, 1732–1741. (35) Kaur, G.; Wang, C.; Sun, J.; Wang, Q. Biomaterials 2010, 31, 5813–5824.

1402 DOI: 10.1021/la103917x

shows the irregular spreading of BMSCs at position d, where no TMV stripes were formed and the contact angle was 13.2 ( 2°, the same as for the glass surface. Because BMSCs possess a multilineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors,36,37 we will further investigate how surface morphology and hydrophobicity impact cell differentiation and gene expression in a future study.

Conclusions We have developed a simple approach to the fabrication of stripe patterns on glass slides by drying TMV solutions under confinement. The height and width of the TMV stripes were controlled by varying the TMV concentration. Stripe patterns obtained by this technique can be used to control surface hydrophobicity and direct the directionality of cell adhesion and spreading. We envision that this method will afford a facile way of patterning different biomacromolecules on flat surfaces and will have great potential in many biomedical applications. Acknowledgment. This work was supported by the NSF (DMR-0706431, CHE-0748690), the ARO (W911NF-09-1-236), an Alfred P. Sloan scholarship, a Camille Dreyfus Teacher Scholar Award, and the W. M. Keck Foundation. Z.S. and Z.N. thanks the NSFC Fund for Creative Research Groups (50921062) and the NSFC Fund (21074143) for support. (36) Ferrari, G.; Cusella-De Angelis, G.; Coletta, M.; Paolucci, E.; Stornaiuolo, A.; Cossu, G.; Mavilio, F. Science 1998, 279, 1528–1530. (37) Kaur, G.; Valarmathi, M. T.; Potts, J. D.; Wang, Q. Biomaterials 2008, 29, 4074–4081.

Langmuir 2011, 27(4), 1398–1402