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Molding a Silver Nanoparticle Template on Polydimethylsiloxane to Efficiently Capture Mammalian Cells Hai-Jing Bai, Hong-Lei Gou, Jing-Juan Xu, and Hong-Yuan Chen* Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Received July 21, 2009. Revised Manuscript Received September 1, 2009 Herein, a functional template made up of in situ synthesized silver nanoparticles (AgNPs) is prepared on polydimethylsiloxane (PDMS) for the spatial control of cell capture, where the residual Si-H groups in the PDMS matrix are used as reductants to reduce AgNO3 for forming AgNPs. In virtue of microfluidic system, a one-dimensional array pattern of AgNPs is obtained easily. Further combining with plasma treatment, a two-dimensional array pattern of AgNPs could be achieved. The obtained PDMS-AgNPs composite is characterized in detail. The PDMS-AgNPs composite shows good antibacterial property in E. coli adhesion tests. The patterns possess hifi and high resolution (ca. 8 μm). Cell patterns with high efficiency and spatial selectivity are further formed with the aid of H-Arg-Gly-Asp-Cys-OH (RGDC) tetrapeptide which is grafted on the AgNPs template. Cells immobilized on the template show a good ability for adhesion, spreading, migration, and growth.
Introduction The spatial control of mammalian cell adhesion, spreading, and growth plays a crucial role in the development of many research fields ranging from tissue engineering, medical diagnostics, drug screening, and biosensing to fundamental studies of cell biology. Two strategies have been exploited to capture cells in designated areas on cell culture substrates:1,2 one is the usage of physical barriers, and the other is the construction of identification-based cell addressable regions in which physical adsorption and surface chemistry are widely used. However, many limitations of physical adsorption have been pointed out penetratingly.3,4 For example, it is difficult to assess the effective density of adsorbed proteins which are available for binding to cells. Conformation and orientation of adsorbed proteins as well as the dynamic nature of protein-adsorbed substrate are considered to be responsible for the above questions. Compared to physical adsorption, surface chemistry has been regarded as a more powerful tool in this field. And it has been perfectly developed to be a collection of techniques containing self-assembly, layerby-layer assembly, plasma treatment, and so on. It is noteworthy that gold substrate has been acting as a superstar for a long time in the application of self-assembly technique for spatial control of cells. However, also a member of the noble metals, silver has rarely been concerned in this field. As we know, silver exhibits good antibacterial and anti-inflammatory properties, yet is relatively nontoxic to mammalian cells, and some medical devices such as wound dressing and urinary catheters have adopted silver to inhibit the incidence of infections *To whom correspondence should be addressed. Telephone/Fax: +86-2583594862. E-mail:
[email protected] (1) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227–256. (2) Tourovskaia, A.; Barber, T.; Wickes, B. T.; Hirdes, D.; Grin, B.; Castner, D. G.; Healy, K. E.; Folch, A. Langmuir 2003, 19, 4754–4764. (3) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267–273. (4) Yap, F. L.; Zhang, Y. Biosens. Bioelectron. 2007, 22, 775–788. (5) Gabriel, M. M.; Sawant, A. D.; Simmons, R. B.; Ahearn, D. G. Curr. Microbiol. 1995, 30, 17–22. (6) Dowling, D. P.; Donnelly, K.; McConnell, M. L.; Eloy, R.; Arnaud, M. P. Thin Solid Films 2001, 398, 602–606.
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and to improve healing rate.5-10 In the past few years, silver nanoparticles (AgNPs) have been reported to be a more robust form of silver which could provide substantial and sustained contact with the bacterial cells and is not easily quenched by other salts and proteins due to its high surface to volume ratio.11,12 Therefore, combining AgNPs with substrate could not only keep the feasibility of self-assembly but also append special biological effect to the substrate. In the past decade, polydimethylsiloxane (PDMS) has been proved to be one of the most popular polymers for bio-MEMs and microfluidic devices due to its well-known advantages such as chemical inertness, flexibility, optical transparency, permeability, and simple machining.13,14 In addition, its biocompatibility is commendable in biological and medical usage; for example, it could act as a substrate for cell culturing directly.15 Recently, various PDMS composites emerged to reinforce PDMS for special applications: carbon nanotube-PDMS composite with strengthened mechanical behavior,16 ZnO-PDMS composite with high fluorescence and sensitivity to temperature changes,17 nickel nanowire-PDMS composite with magnetic response,18 (7) Saint, S.; Elmore, J. G.; Sullivan, S. D.; Emerson, S. S.; Koepsell, T. D. Am. J. Med. 1998, 105, 236–241. (8) Greenfeld, J. I.; Sampath, L.; Popilskis, S. J.; Brunnert, S. R.; Stylianos, S.; Modak, S. Crit. Care Med. 1995, 23, 894–900. (9) McLean, R. J. C.; Hussain, A. A.; Sayer, M.; Vincent, P. J.; Hughes, D. J.; Smith, T. J. N. Can. J. Microbiol. 1993, 39, 895–899. (10) Bishop, J. B.; Phillips, L. G.; Mustoe, T. A.; Vanderzee, A. J.; Wiersema, L.; Roach, D. E.; Heggers, J. P.; Hill, D. P.; Taylor, E. L.; Robson, M. C. J. Vasc. Surg. 1992, 16, 251–257. (11) Baker, C.; Pradhan, A.; Pakstis, L.; Pochan, D. J.; Shah, S. I. J. Nanosci. Nanotechnol. 2005, 5, 244–249. (12) Pal, S.; Tak, Y. K.; Song, J. M. Appl. Environ. Microbiol. 2007, 73, 1712– 1720. (13) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H. K.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27–40. (14) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563–3576. (15) Mi, Y. L.; Chan, Y. N.; Trau, D.; Huang, P. B.; Chen, E. Q. Polymer 2006, 47, 5124–5130. (16) Dyke, C. A.; Tour, J. M. J. Phys. Chem. A 2004, 108, 11151–11159. (17) Zhou, J. H.; Yan, H.; Zheng, Y. Z.; Wu, H. K. Adv. Funct. Mater. 2009, 19, 324–329. (18) Keshoju, K.; Sun, L. J. Appl. Phys. 2009, 105, 023515-1–023515-5.
Published on Web 09/21/2009
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and so on have been reported successively. For obtaining both the merits of PDMS and AgNPs, the fabrication of AgNPs-PDMS composite has been attempted. Wen et al. have reported the fabrication of microheaters based on a conducting composite of silver microparticles embedded in PDMS.19 Sepaniak et al. have fabricated AgNPs-PDMS nanocomposite substrates with nanotransfer printing technique for surface-enhanced Raman spectroscopy research.20 Aiming to develop conductive photodefinable PDMS composite, Pan et al. have attempted to adulterate benzophenone and silver powder into the PDMS matrix, and the prepared PDMS composite has been demonstrated to possess excellent antibacterial performance.21 Here, we use an in situ synthesis method which was founded by our research group previously22 combining microfluidic system to build a AgNPs template on PDMS for efficiently directing cell-anchoring with spatial selectivity. Compared with the bulk adulteration adopted by ref 21 and the nanotransfer printing technique employed by ref 20, this in situ synthesis method is more low-cost and convenient, in which residual Si-H groups in the PDMS matrix are used as a reductive agent alone to make the formation of AgNPs. The prepared PDMS-AgNPs composite substrate takes advantage of the properties of both constituents: biocompatibility, optical transparency, permeability and simple machining for PDMS, and antibacterial property and apt to be grafted with self-assembly technique for AgNPs. In virtue of microfluidic control and plasma treatment, various AgNPs patterns with high resolution and high fidelity are constructed easily. On the PDMS substrate with the patterned template made up of AgNPs, a synthetic tetrapeptide shows perfect selectivity to bind with AgNPs and induce cell patterning successfully.
Experimental Section Materials and Reagents. The Sylgard 184 (including PDMS monomer and curing agent) was purchased from Dow Corning (Midland, MI). RGDC peptide was synthesized by GenScript (NJ). Acridine orange (AO) was from Amresco (Solon, OH). AgNO3 was purchased from Shanghai Shenbo Chemical, Co., Ltd. (Shanghai, China). Glass plate coated with chromium and photoresist for chip fabrication were obtained from Shaoguang Microelectronics Corp. (Changsha, Hunan, China). Phosphate buffer solution (PBS) (pH 7.4) was made up of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4. Milli-Q grade water (Millipore Inc., Bedford, MA) was used for solutions and cleaning steps. All other chemicals were of analytical grade. UV-vis Measurement. PDMS monomer and curing agent were mixed at a mass ratio of 10:2 (η 0.2) and then placed in a vacuum desiccator for degassing and cured at 80 °C for 90 min. Prepared PDMS slices were immersed and incubated in 15 mM AgNO3 solution for different times at 25 °C. The slices were then rinsed with water thoroughly and dried with nitrogen gas. Capacitance Measurement. Bulk gold electrodes (2 mm diameter) were abraded with fine SiC paper and polished carefully with 0.3 and 0.05 μm alumina slurry and then sonicated in water and absolute ethanol. A mixture of PDMS monomer and curing agent (η 0.2) was dropped on the surface of the electrodes, followed with spin coating at 1500 rpm for 5 min. The electrodes were cured at 80 °C for 90 min, followed by rinsing thoroughly with water and drying with nitrogen gas. The electrodes were then inserted into 15 mM AgNO3 solution for incubation for 1 day at 25 °C. For capacitance measurements, the gold electrodes with (19) Liu, L. Y.; Peng, S. L.; Niu, X. Z.; Wen, W. J. Appl. Phys. Lett. 2006, 89, 223521-1–223521-3. (20) Abu Hatab, N. A.; Oran, J. M.; Sepaniak, M. J. ACS Nano 2008, 2, 377– 385. (21) Cong, H. L.; Pan, T. R. Adv. Funct. Mater. 2008, 18, 1912–1921. (22) Zhang, Q.; Xu, J. J.; Liu, Y.; Chen, H. Y. Lab Chip 2008, 8, 352–357.
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coating were used as working electrode, and a conventional Ag/ AgCl electrode as the reference electrode. A DC polarized voltage of 300 mV accompanied with an AC voltage of 20 mV at 1 M Hz was applied for the measurement of capacitance at room temperature in PBS. Circuit diagram is omitted. Antibacterial Performance. The PDMS-AgNPs composite film was prepared by incubating PDMS (η 0.2) with 15 mM AgNO3 solution for 3 days. Then it was cut into pieces and placed in a tissue culture plate and cultured with 108 cells mL-1 of Escherichia coli (E. coli) (donated by Gulou Hospital, Nanjing, China) in 0.9% NaCl solution at 37 °C. The native PDMS was handled as a control group. The appearance of bacteria adhesion on different materials was observed for varying time periods up to 24 h. Molding AgNPs Template on PDMS. Microfluidic glass chips with different micropatterns were made with traditional photolithography and wet chemical etching techniques. The glass plate was covered by a mask with designed micropatterns for UV exposure, followed by developing with 0.5% NaOH solution and etching with 1 M HF/NH4F solution. The glass chip was then sealed reversibly with a clean PDMS slice (η 0.2) in which two holes had been punched in advance at regions corresponding to both ends of the micropattern in the glass chip. AgNO3 solution (15 mM) was injected into the channels and reacted with PDMS for 3 days at 25 °C. For the two-dimensional array pattern, air plasma pretreatment was applied to the microfluidic system composed of the glass chip and PDMS slice for 10 min. The PDMS slice was then peeled off and sealed with the glass chip orthogonally, followed by incubating channels with 15 mM AgNO3 solution for 3 days at 25 °C. Freshly prepared PDMS substrate with AgNPs template was rinsed with PBS thoroughly. In the process, to keep the channels always filled with AgNO3 solution is the key to achieve a perfect copy of the patterns on PDMS. Cell Patterning. PDMS substrate with AgNPs template was immersed in a solution of 2 mM RGDC dissolved in 50% ethanol overnight for grafting RGDC on AgNPs efficiently. Prepared RGDC-grafted substrate was rinsed with 50% ethanol to remove unreacted RGDC. Human liver carcinoma SMMC-7721 cells (from Gulou Hospital, Nanjing, China) were maintained in RPMI medium 1640 (Gibco Invitrogen Corp., U.S.A.) supplemented with 10% fetal bovine serum (Gibco Invitrogen Corp., U.S.A.) at 37 °C and 5% CO2 atmosphere. Confluent SMMC7721 cells were trypsinized, washed, and then resuspended in RPMI medium 1640 supplemented with 10% fetal bovine serum, and directly seeded on the RGDC-grafted PDMS substrate at a density of ca. 5 105 cells mL-1. The cell number was determined with a hemocytometer. After incubation in a CO2 incubator for 2 h, the substrate was washed with PBS moderately before observation. The viability of cells immobilized on the AgNPs template was investigated using AO fluorescence dye. Imaging and Image Analysis. A DMIRE2 inverted fluorescence microscope (Leica, Germany) equipped with DP71 CCD (Olympus, Japan) was used for microimaging (bright field, phase contrast, and fluorescent images). Image-Pro Plus (IPP) software was employed for image analysis. Instruments. Scanning electron micrographs were obtained on a FEI Sirion 200 field emission scanning electron microscope (FEI, U.S.A.) at an accelerating voltage of 5 kV. Ultraviolet and visible absorption spectra (UV-vis) were acquired with a UV-3600 spectrophotometer (Shimadzu, Kyoto, Japan). Atomic force micrographs (AFMs) were obtained in tapping mode in air and at room temperature with an Agilent 5500 atomic force microscope (Agilent Technologies, Inc., U.S.A.). X-ray photoelectron spectroscopy (XPS) measurement was taken with a Thermo ESCALAB 250 (Thermo, U.S.A.) instrument equipped with a monochromatic Al KR source operated at 150 W. The capacitance measurement was performed with a Keithley 4200-SCS semiconductor characterization system (OH, U.S.A.). A semiconductor thermostat (Jingie Industry and DOI: 10.1021/la902683x
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Figure 1. (a) UV-vis spectra of PDMS-AgNPs composites with different synthesis times. (b) Ag 3d core-level XPS spectrum of PDMS-AgNPs composite. (c) SEM image of native PDMS. (d) SEM image of PDMS-AgNPs composite. Inset in (d) is the magnified AgNPs cluster. Scale bars in (c) and (d) are 500 nm and 1 μm, respectively.
Trading Co., Ltd., Tianjin, China) was employed for precise temperature control.
Results and Discussion In Situ Synthesis of AgNPs on PDMS. Cured PDMS presents a network structure which is built through the crosslinking reaction between vinyl groups in the monomer and silicon hydride groups (Si-H) in the cross-linker.21-24 Here, the reducibility of residual Si-H groups distributed in the PDMS network is used to react with AgNO3. As expected, the PDMS slice became yellow gradually with the increase of the soaking time of PDMS in AgNO3 solution. This dynamic in situ synthesis process of AgNPs was investigated by UV-vis spectrometry in detail. In Figure 1a, the symmetrical plasmon resonance absorption band centered at 421 nm is indicative of the formation of AgNPs,25 and the increase of absorption intensity implies the increasing amounts of formed AgNPs. From Figure 1a, both an obvious red shift and a minor broadening of the plasmon band could be observed as the incubation time increases, which might result from the increasing number of AgNPs and their variation in size. The Ag 3d core-level XPS spectrum of PDMS-AgNPs composite is shown in Figure 1b. The strong signals of Ag at 368.0 eV (3d5/2) and 374.0 eV (3d3/2) further confirm the presence of the silver in its metallic state.26 The surface morphologies of native PDMS and PDMS-AgNPs composite with a preparation time of 72 h are shown in Figure 1c and d. AgNPs coating on PDMS present irregular cluster-shaped agglomeration. This might result from a higher mass ratio of the cross-linker to the monomer (η 0.2), and a quick reaction between AgNO3 and the Si-H groups on the PDMS surface results in a great collection of AgNPs on the (23) Simpson, T. R. E.; Tabatabaian, Z.; Jeynes, C.; Parbhoo, B.; Keddie, J. L. J. Polym. Sci., Part A: Polym. Chem 2004, 42, 1421–1431. (24) Bhagat, A. A. S.; Jothimuthu, P.; Papautsky, I. Lab Chip 2007, 7, 1192– 1197. (25) Mock, J. J.; Barbic, M.; Smith, D. R.; Schultz, D. A.; Schultz, S. J. Chem. Phys. 2002, 116, 6755–6759. (26) Stathatos, E.; Lianos, P.; Falaras, P.; Siokou, A. Langmuir 2000, 16, 2398– 2400.
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Figure 2. (a) Model for capacitance measurement to prove the generation of AgNPs on PDMS and its equivalent-circuit diagrams. (b) Actual measuring values before and after incubation of electrode.
surface and the block for the entrance of AgNO3 into the network of PDMS. In addition, AgNPs clusters in Figure 1d are separate from each other. The magnified image of AgNPs clusters (inset in Figure 1d) suggests that a single AgNPs cluster comprises tens of relatively independent AgNPs. Every silver nanoparticle has its unique shape and size, and the shape of nanoparticle changes from roundness to polygon as the size of nanoparticles increases. A simple capacitance measurement method27 is also used to testify the generation of AgNPs on PDMS. The actual measuring values for PDMS capacitance before and after incubation with AgNO3 solution are 22.40 ( 1.02 pF and 16.28 ( 0.57 pF, respectively (Figure 2a). There is an obvious decrease, which can be illustrated by the model and the corresponding equivalent-circuit (27) Krause, C.; Mirsky, V. M.; Heckmann, K. D. Langmuir 1996, 12, 6059– 6064.
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Figure 3. Antibacterial performance of PDMS-AgNPs composite. Culture time of E. coli was 24 h. (a) Phase contrast image of E. coli adhesion on native PDMS. (b) Phase contrast image of E. coli adhesion on PDMS-AgNPs composite.
diagrams presented in Figure 2b. The change of capacitance value could be expressed as follows: C1 ¼ CP S 0 C2 ¼
ð1Þ
C P C Ag S 0 þ C P 2 ðS 0 - S 1 Þ C P þ C Ag
ð2Þ
CP 2 S 1 >0 C P þ C Ag
ð3Þ
ΔC ¼ C 1 - C 2 ¼
where CP, CAg, S0, and S1 are the apparent specific capacitance of native PDMS and AgNPs, surface area of electrode, and the area coated by AgNPs, respectively. Antibacterial Performance. In the medical and health care fields, AgNPs are prevalent in that they could act as catalysts to attack the bacteria by several approaches, such as poking bacteria membranes, destroying metabolic enzymes, denaturing proteins, disrupting bacteria division and proliferation process, and so on, and thereby give rise to excellent antibacterial properties.21 In order to evaluate the antibacterial performance for our prepared material, bacteria adhesion tests were performed using E. coli. The microscopic images in Figure 3 represent the states of E. coli adhesion on native PDMS and PDMS-AgNPs composite. After 24 h incubation, E. coli adhered to PDMS firmly, but there is a dramatic decrease in the number of adhering cells on the PDMSAgNPs composite. The phenomenon suggests that the PDMSAgNPs composite has excellent antibacterial performance in comparison with native PDMS. Microfabrication of AgNPs Template on PDMS. In the later part of the last century, rapid development of micromachining techniques encouraged cell patterning studies greatly. A series of methods such as photolithography,28-30 soft photolithography,31-33 laser-scanning lithography,34,35 laser ablation,36,37 (28) Bhatia, S. N.; Yarmush, M. L.; Toner, M. J. Biomed. Mater. Res. 1997, 34, 189–199. (29) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 9855–9862. (30) Hui, E. E.; Bhatia, S. N. Langmuir 2007, 23, 4103–4107. (31) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551–575. (32) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335–373. (33) Suh, K. Y.; Seong, J.; Khademhosseini, A.; Laibinis, P. E.; Langer, R. Biomaterials 2004, 25, 557–563. (34) Miller, J. S.; Bethencourt, M. I.; Hahn, M.; Lee, T. R.; West, J. L. Biotechnol. Bioeng. 2006, 93, 1060–1068. (35) Lee, S. H.; Moon, J. J.; West, J. L. Biomaterials 2008, 29, 2962–2968. (36) Liu, Y. M.; Sun, S.; Singha, S.; Cho, M. R.; Gordon, R. J. Biomaterials 2005, 26, 4597–4605. (37) Iwanaga, S.; Akiyama, Y.; Kikuchi, A.; Yamato, M.; Sakai, K.; Okano, T. Biomaterials 2005, 26, 5395–5404.
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and hot-embossing imprint lithography38 have been founded. Here, a microfluidic system containing a glass chip with pattern prepared by the most conventional lithography is employed for microfabricating one-dimensional AgNPs array on PDMS directly. The schematic diagram of this strategy is shown in Figure 4a. However, a limitation that cannot be ignored is that a two-dimensional array pattern with independent micropatterns is difficult to prepare by the microfluidic system alone. Plasma treatment is widely used to modify material surfaces with high versatility and precision, in a cost-effective way. The most important advantage of such a process is the possibility to change the chemistry and/or topography of the surfaces with high control, without altering the bulk properties of the materials.39 And the surface of oxidized PDMS is known to recover its hydrophobicity due to the migration of free PDMS chains from the bulk phase to the surface.40,41 Thus, it is a desirable strategy: eliminating Si-H groups locally via plasma treatment to build invisible barriers for prohibiting the production of AgNPs. And the dynamic nature of these invisible barriers suggests that there is not a remarkable influence to the substrate for succeeding cell culture. As shown in Figure 4b, double employments of the glass chip in which the direction of channels is orthogonal lead to a successful two-dimensional array pattern with high precision. Compared with microcontact printing (μCP), the most versatile technique in soft lithography, the approach we present here provides some advantages. First, the synthesis of nanoparticles and the formation of a nanoparticle pattern on PDMS surface are synchronized. Second, the proposed method could be further developed to pattern different metal nanoparticles (such as Au and Ag nanoparticles) on a PDMS surface simultaneously by injecting different precursors into different microchannels. As we know, a polymer such as PDMS is the selected material for soft lithography, as it can make conformal contact with nonplanar surfaces.4 This property of PDMS is critical to hifi microfabrication, which is demonstrated by the good match of linear optical density spectra of panels (a) and (b) in Figure 5. Spatial resolution of patterns is an important parameter to evaluate the level of micropatterning technique. It can be looked as a function of the biomaterials (e.g., cells, proteins, bacteria) or the detection limit of the analyzing technology.42 Figure 5d shows (38) Charest, J. L.; Eliason, M. T.; Garcia, A. J.; King, W. P. Biomaterials 2006, 27, 2487–2494. (39) Sardella, E.; Favia, P.; Gristina, R.; Nardulli, M.; d’Agostino, R. Plasma Processes Polym. 2006, 3, 456–469. (40) Fritz, J. L.; Owen, M. J. J. Adhes. 1995, 54, 33–45. (41) Makamba, H.; Kim, J. H.; Lim, K.; Park, N.; Hahn, J. H. Electrophoresis 2003, 24, 3607–3619. (42) Barron, J. A.; Wu, P.; Ladouceur, H. D.; Ringeisen, B. R. Biomed. Microdevices 2004, 6, 139–147.
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Figure 4. Schematic diagram for the microfabrication of AgNPs template. (a) Microfluidic architecture is used for molding one-dimensional AgNPs array template directly. By injecting AgNO3 solution into the microchannels and incubation for a required time, the AgNPs template which is the copy of the pattern in glass chip is formed on PDMS. (b) Combing plasma treatment, complex two-dimensional AgNPs array template is prepared. This process comprises three steps. First, the microfluidic architecture is treated in plasma cleaner in whole, and invisible barriers in the region of channels are formed after plasma treatment. Second, the PDMS slice is peeled from the glass chip and then coated with the same glass chip in the orthogonal direction. Third, filling the microchannels with AgNO3 solution is employed for the production of twodimensional array pattern. All scale bars in the images are 200 μm.
that the best spatial resolution of the stripe pattern achieved by the in situ synthesis method can reach 8 μm. The high resolution also benefits from the excellent leak tightness of PDMS. Figure 5e is an AFM image of the border between the AgNPs stripe and PDMS. An obvious stair at the border corresponds to the jump in the linear optical intensity spectrum of the stripe pattern in Figure 5c. RGDC-Induced Cell Anchoring. Eukaryote cells of high organisms interact with materials in a complex manner. The adsorption of extracellular matrix (ECM) molecules on the substrate is the first process, which is followed by a ligand-acceptor interaction between ECM molecules and integrin receptors on the cell membrane to mediate cell adhesion.43 Arg-Gly-Asp (RGD) peptide which is found in fibronectin (a type of ECM) is a ligand for several integrin receptors. Many previous works have made use of this peptide to tailor cell-adhesive surface of patterns and enhance the attachment and growth of cells.3,35,44-49 In our experiment, it has been demonstrated that PDMS substrate with AgNPs template cannot afford spatial selectivity for cell capture (the result is not shown). Via self-assembly technique, RGDC peptide has been successfully grafted onto AgNPs template, which has been proven in the cell adhesion test. After culture for 2 h in medium supplemented with serum, SMMC-7721 cells only adhered to the AgNPs template grafted with RGDC (Figure 6a and b). The results show that the fall of ca. 20 nm (estimated value according to Figure 5e) between raised template and PDMS can be completely neglected in cellular adhesion, and the self-assembled monolayers (SAMs) of RGDC are attractive enough to gather cells on them. In addition, there is not any negative effect on the spatial control of cell capture in our work, (43) Sordel, T.; Kermarec-Marcel, F.; Garnier-Raveaud, S.; Glade, N.; SauterStarace, F.; Pudda, C.; Borella, M.; Plissonnier, M.; Chatelain, F.; Bruckert, F.; Picollet-D’hahan, N. Biomaterials 2007, 28, 1572–1584. (44) Ruoslahti, E. Annu. Rev. Cell Dev. Biol. 1996, 12, 697–715. (45) Massia, S. P.; Hubbell, J. A. J. Cell Biol. 1991, 114, 1089–1100. (46) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A 2001, 98, 5992–5996. (47) Hahn, M. S.; Miller, J. S.; West, J. L. Adv. Mater. 2006, 18, 2679–2684. (48) Koh, W. G.; Itle, L. J.; Pishko, M. V. Anal. Chem. 2003, 75, 5783–5789. (49) Kalinina, S.; Gliemann, H.; Lopez-Garcia, M.; Petershans, A.; Auernheimer, J.; Schimmel, T.; Bruns, M.; Schambony, A.; Kessler, H.; Wedlich, D. Biomaterials 2008, 29, 3004–3013.
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Figure 5. Microfabrication with high fidelity and resolution. (a) Microscopic image of microchannels on glass chip. Scale bar is 200 μm. (b) Microscopic image of AgNPs stripes on PDMS copying from (a). Scale bar is 200 μm. (c) Linear optical density spectra of images (a) and (b). Line scan is along the white line in (a) and (b). (d) Microscopic image of stripe pattern with a minimum spatial resolution of 8 μm. (e) AFM image of the border between AgNPs stripe and PDMS.
although it is generally recognized that seeding in serum-containing medium results in destruction of ECM patterns due to nonselective adsorption of serum proteins onto the polymeric surface50,51 and PDMS is strong to adsorb the hydrophobic biomolecules.41,52 In a microarray pattern with six independent 150 150 μm2 corrals (Figure 6b), the number of cells captured in a single corral is calculated manually. They are 36, 37, 35, 40, 38, and 38 cells. The average value for captured cells in an individual corral is 37 ( 2 cells. At 5 h after seeding, most cells are observed (50) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388–392. (51) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 561–567. (52) Liu, J. K.; Lee, M. L. Electrophoresis 2006, 27, 3533–3546.
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Figure 6. Bright field images of SMMC-7721 cells patterned within 280 μm lines or on corrals array for different culture times: 2 h (a), 2 h (b), 5 h (c), and 18 h (d). The size of a single corral in (b) is about 150 150 μm2.
to elongate in the axial direction upon spreading (Figure 6c). At 18 h after seeding, cells migrated out to the PDMS region (Figure 6d). Thus, the results illuminate two features of the RGDC-grafted AgNPs template: one is the efficient and quick capture to cells; the other is that a cell pattern with high quality can be formed even in the presence of serum.
Conclusions
template with high efficiency and quality in virtue of self-assembly technique. It can be concluded that the method presented here holds the following advantages: low cost and convenience for the preparation of PDMS-AgNPs composite and AgNPs template with high precision which contributes to an excellent cell pattern. We believe this PDMS-AgNPs composite and the provided method will be useful in many other fields such as microfluidics, immunoassay, and so on.
In this work, a PDMS-AgNPs composite with prominent antibacterial properties is prepared by an in situ synthesis method and successfully used for the spatial control of cell capture. High resolution and hifi AgNPs template used to direct cell patterning is molded with a smart combination of in situ synthesis, microfluidic control, and plasma treatment. Cells are captured on the
Acknowledgment. This work was supported by the National Natural Science Foundation of China (Nos. 20890021, 20775033), the National Natural Science Funds for Creative Research Groups (20821063), and the 973 Program (2007CB936404).
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