Article pubs.acs.org/Langmuir
Patterning Nanoparticles in a Three-Dimensional Matrix Using an Electric-Field-Assisted Gel Transferring Technique Xiaoshu Dai, Sarah A. Knupp, and Qiaobing Xu* Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States S Supporting Information *
ABSTRACT: In this paper, we describe an electric-fieldassisted gel transferring technique for patterning on two- and three-dimensional media. The transfer process starts with the preparation of a block of agarose gel doped with charged nanoparticles or molecules on top of a screen mask with desired patterns. This gel/mask construct is then brought into contact with the appropriate receiving medium, such as a polymer membrane or a piece of flat hydrogel. An electric field is applied to transfer the doped charged nanoparticles or molecules into the receiving medium with a pattern defined by the screen mask. This printing method is rapid and convenient, the results are reproducible, and the process can be done without using expensive micro/nanofabrication facilities. The capability to pattern structures such as arrays of nanoparticles into three-dimensional hydrogels may find applications for positioning cell signaling molecules to control cell growth and migration.
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INTRODUCTION In this study, we demonstrated an electric-field-assisted gel transferring (EFAGT) technique for patterning of nanoparticles in three-dimensional (3D) media. This patterning method is based on a blotting technique routinely used in molecular biology to identify target molecules through an electric-fieldassisted molecule transferring process dependent on the charge and size. Utilizing the blotting technique and a simple screen mesh for a pattern, the proposed EFAGT technique provides a means by which complex, multidimensional arrays can be patterned without using expensive and arduous microfabrication approaches. The fabrication of periodic micro/nanoparticle arrays is of great interest due to their size-dependent properties leading to a wide range of applications.1−3 For example, in tissue engineering, patterning with adhesion signals that mimic spatial cues to guide cell attachment and function is used to control cell behavior and induce structural and functional tissue formation on surfaces.4−8 Although photolithography is a technique that is comprehensively developed for patterning, the high costs associated with equipment and the need for access to clean rooms make this technique inconvenient for biologists.9 Soft lithography consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric material for pattern transfer.10,11 Dip-pen nanolithography (DPN), another contact printing method utilizing a scanning probe for printing, can pattern biomolecules in submicrometer resolution.12−14 In contrast, inkjet printing is a noncontact method to generate patterns.15,16 Both soft lithography and inkjet printing have © 2012 American Chemical Society
been used to pattern extracellular matrix molecules on various 2D surfaces to control cell adherence and growth.6,7,17−19 However, it remains challenging to pattern molecules or nanoparticles into 3D media using either method. Two-photoninitiated photopolymerization20−22 and digital micromirror device projection printing23 have been demonstrated to fabricate precise 3D microstructures; however, these processes rely on expensive equipment and special expertise and are hard to adapt by most laboratories. In this study, we adapted a blotting technique routinely used in molecular biology to identify target molecules and extended its application in creating patterned molecules and nanoparticles in various media. This printing method is rapid, convenient, and reproducible and can be conducted without using expensive micro/nanofabrication facilities.9,11,24,25 Additionally, the transferred patterns are not limited to common, commercially available meshes, as patterns with diverse and customized geometry can be easily generated through microfabrication methods.9,24 Thus, this proposed method provides a facile and useful technique to pattern complex structures such as arrays of nanoparticles into the 3D hydrogels, which are difficult and often expensive to fabricate using traditional methods. This capability may find applications for positioning cell signaling molecules to control cell growth and migration in tissue engineering. Received: November 1, 2011 Revised: January 5, 2012 Published: January 12, 2012 2960
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Figure 1. Schematic illustration of the procedure for patterning by EFAGT. (a) A piece of gel doped with charged materials is formed on one side of a screening mesh. After the tape on the other side of the mesh is peeled off, the gel/mesh construct is brought into contact with a receiving medium. The gel/mesh/receiving medium is sandwiched between two electrode plates. By applying voltage, charged materials in the gel can be transferred into the receiving medium into a pattern defined by the screen mask. (b) 3D illustration showing the transfer result.
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box was half-immersed in an ice bath to prevent overheating. A 150 mA current was used for pattern transfer. Optical, Fluorescence, and Confocal Microscopy. The visible light and fluorescence patterns (green fluorescent protein (GFP), excitation/emission 470 nm/520 nm) were observed using an inverted light microscope (Leica Microsystems Inc., Buffalo Grove, IL). A Leica confocal microscope (DMIRE2 63x NA1.2, excitation/emission 488 nm/500−550 nm) (Leica Microsystems) was used to analyze the 3D structure printed in the hydrogel. ImageJ was used to reconstruct a 3D image. Scanning Electron Microscopy (SEM) Imaging. Scanning electron microscopy was performed on a Zeiss UltraPlus analytical field emission scanning electron microscope (Carl Zeiss, Thornwood, NY) to determine the transferred pattern.
EXPERIMENTAL SECTION
Materials. SimplyBlue SafeStain (contains Coomassie G-250) and poly(vinylidene fluoride) (PVDF) membranes (0.2 μm pore size) were purchased from Invitrogen (Carlsbad, CA). The cell strainers (40 μm woven nylon mesh) were purchased from Fisher Scientific (Pittsburgh, PA). Agarose was purchased from Sigma (St. Louis, MO). Fluoresbrite microspheres (0.05 μm, 2.5% solid latex) were purchased from Polysciences, Inc. (Warrington, PA). TAE (50×) buffer was purchased from Boston BioProducts (Worcester, MA). A nylon mask was generously provided by Industrial Netting (Minneapolis, NM). A porous glass membrane (Incom, Inc., Charlton, MA) was generously provided by Prof. David Walt at Tufts University. Preparing the Charged Particle Doped Gel. The agarose gel was prepared by dissolving the desired amount of agarose powder in water with the help of a microwave. The dye solutions that contain the charged molecules/particles were mixed in after complete dissolution of the gel powder. To obtain Coomassie G250 doped gel, 0.2 g of agarose powder was mixed with 5 mL of deionized (DI) water. The solution mixture was heated using a microwave for 30 s. The solution mixture was cooled at room temperature for 2−3 min, and then 5 mL of SimplyBlue SafeStain was added. The resulting homogeneous solution mixture was then poured onto a mesh. The bottom side of the mesh was tightly taped to prevent the solution from leaking through. The tape was carefully pealed after the gel solidified. To prepare a fluorescence nanoparticle doped gel, approximately 50 μL of the nanoparticle suspension was added into 5 mL of a warm 2% agarose/ water mixture. The same procedure was used when casting the gel on top of a glass fiber. Fabrication of the Gel Cartridge. The desired agarose gel/dye mixture was directly poured into a cell strainer. The bottom and the side meshes of the cell strainer were taped to prevent leakage. Obtaining a flat top and bottom gel surface is important to ensure a tight contact between the transferring gel and the receiving medium. EFAGT Setup. An XCell II blot module (Invitrogen, Carlsbad, CA) was used for gel transfer. The gel/mask/membrane, assembled as shown in Figure 1, was sandwiched between several pieces of sponges presoaked with transfer buffer (0.5 M Tris−acetate−EDTA (TAE) buffer) and then loaded into a gel cassette. After the cassette was locked into position, 1× TAE buffer was used to fill in the upper buffer chamber. DI water was used to fill the lower buffer chamber. The gel
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RESULTS AND DISCUSSION Process of EFAGT. As shown in Figure 1, we adjusted the existing conventional blot technique for pattern transfer. We sealed the bottom of the screen mask with Scotch tape and cast a block of agarose gel doped with charged molecules or nanoparticles on the top of the mask. Here for the purpose of illustration, we used commercially available polymer meshes with open squares (40−700 μm) and porous glass membranes with 20 μm open pores as the screen mask. After the gel was cured on the screen mask, the resulting gel/mask construct was then brought into contact with the appropriate receiving medium, such as a PVDF membrane or a piece of flat agarose hydrogel. An electric field was then applied to transfer the doped, charged nanoparticles or molecules onto the receiving medium with the pattern defined by the screen mask. To demonstrate the feasibility of the electric-field-assisted gel transferring technique for patterning, we used Coomassie G250 stain as the “ink” to dope a 2% agarose gel. By dissolving 0.2 g of agarose powder in 5 mL of water and 5 mL of SimplyBlue SafeStain (50% concentration), a gel with uniformly distributed blue color was cast on top of a nylon mesh 500 μm in size, as shown in Figure 2a, inset. A 40 V potential was applied, allowing the negatively charged dye to transfer onto a PVDF membrane. The transfer time was limited 2961
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A 2% agarose gel doped with SimplyBlue SafeStain to transfer a pattern at 150 mA for 10 min to a PVDF membrane yields an average concentration of 0.33 ± 0.008 μg/mm2. The concentration of dye stain in each printed spot was determined by fitting the gray scale histogram value to a calibration curve. The method used to obtain a calibration curve is discussed in detail in the Supporting Information. The pattern transfer efficiency also correlates with the porosity of the transferring gel. Under the same intensity of electric field, the rate of dye transfer from 0.5% agarose gel is about 1.5 times faster than that from 2% agarose gel (Supporting Information, Figure S3), but generates a less defined boundary due to the possible increased diffusion of the dye molecules. Thus, the conditions used for gel transfer should be optimized on the basis of the feasibility of the setup (gel stiffness, easy to handle), the diffusion velocity of the dye molecules, the electric field, and the transfer time. The ink-doped agarose gel acts as a reusable cartridge and can be used to print multiple times. As shown in Figure 2c, we cast a block of 2% agarose gel doped with Coomassie G-250 within a cell strainer, which is made of woven nylon mesh (mesh size 40 μm, Figure 2c, inset) and a commonly used device in cell biology to obtain a single cell suspension. One gel cartridge can be repeatedly used to print a pattern into the receiving membrane more than 10 times (we stopped at 10, but it could be more). Figure 2d shows the fourth repeated transfer optical image of the dye transferred to the PVDF membrane using 40 V of potential for 10 min by EFAGT. The boundary of the pattern is not very sharp. We believe that the nonflat surface of the woven screen mesh from the cell strainer (Figure 2c, inset) and the small size of the dye caused the fast diffusion of the dye into the boundary. Nanoparticle Printing by EFAGT. Similar to the previously described process for preparing the dye-doped gel cartridge, we doped the agarose gel with fluorescently labeled charged nanoparticles and demonstrated that we are able to produce patterned charged nanoparticles using EFAGT. To prepare the nanoparticle-loaded agarose gel, we mixed 50 μL of a 50 nm negatively charged fluorescent polystyrene nanoparticle suspension (w/w, 2.5%) with every 5 mL of the gel mixture (2% w/v). We assembled the nanoparticle-doped gel cartridge and receiving PVDF membrane in a process shown in Figure 1. Then we applied the potential (40 V) for 20 min to
Figure 2. Digital images of (a) the polypropylene mesh (inset: SEM image of the nonwoven polypropylene mesh with ∼500 μm mesh size) and (b) the patterned PVDF membrane with Coomassie G-250 stain using an electric-field-assisted gel transferring process. (c) Digital image of the gel cartridge of a cell strainer loaded with Coomassie G250 stain (inset: SEM image of the woven nylon mesh with 40 μm pores on the bottom of the cell strainer). (d) Light microscope image showing the Coomassie G-250 pattern on the PVDF membrane printed using the gel cartridge. The blue region indicates the area with dye, and the bright region is the area without the dye.
to 5 min to prevent overtransfer since Coomassie G-250 molecules have a relatively small molar mass of 854.02 g/mol. After 5 min a uniform pattern appeared on the PVDF membrane, as shown in Figure 2b. The transferred pattern is stable and remains sharp for months. The successful pattern transfer proves the feasibility of applying this technique for transferring larger and more functional particles and molecules.
Figure 3. (a) Fluorescence microscope image of the fluorescent nanoparticle pattern on a PVDF membrane by EFAGT using a gel cartridge loaded with fluorescent polymer nanoparticles. (b) SEM image of the PVDF membrane after being printed with Fluoresbrite nanoparticles. The highlighted region shows the presence of the nanoparticles (d = 50 nm). 2962
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Figure 4. (a) SEM image of the porous glass membrane used as a screen mask. (b) Fluorescence microscope image of fluorescent nanoparticles patterned on PVDF membranes by EFAGT using the porous glass membrane as a mask.
Figure 5. (a) Fluorescence microscope image showing the surface structure of the fluorescent nanoparticles printed into a piece of agarose gel. (b) Reconstructed confocal fluorescence image showing the 3D structure of the transferred gel. The cross-section profile is also shown in (c).
Using a Glass Mesh as the Screen Mask. We used a hollow glass mesh (∼1 mm thick) comprising an array of uniform 20 μm pores as the mask for EFAGT and patterned the nanoparticles in an array of circular geometry. Negatively charged fluorescent nanoparticles were doped in 2% agarose gel. After the particle-containing agarose gel was heated at 100 °C, we cast it on top of a glass mesh. The gel solution penetrates into the porous glass membrane and solidifies after cooling to room temperature. We then used the resulting glass membrane as a screening mask and transferred the nanoparticles into a PVDF membrane with 40 V of potential for 20 min. We imaged the resulting patterns of nanoparticles using a fluorescence microscope, as shown in Figure 4b. Compared with the corrugate surface of polymer screen meshes, the bottom surface of the porous glass membrane is flat, allowing a very close contact at the glass fiber membrane/PVDF interface.
allow the nanoparticles to migrate onto the PVDF membrane. A longer transfer time (1−2 h) could also be used without significant cross-diffusion since the velocity of relatively large size nanoparticle (50 nm) migration through the gel is slow. After transfer, we used a fluorescence microscope to observe the patterned formation from the transferred nanoparticles. As shown in Figure 3a, the periodic array of nanoparticles on the PVDF membrane confined into a pattern corresponds well to the original nylon mesh screen mask (Figure 2c, inset). Compared with the diffusive pattern observed from Coomassie blue dye patterning (Figure 2d), the lower diffusion of the nanoparticles is attributed to their large size and small diffusion rate. The SEM image (Figure 3b) revealed that the polymer nanoparticles were successfully transferred onto the porous PVDF membrane from the agarose gel under an electrical field. 2963
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ACKNOWLEDGMENTS This research was supported by Tufts University. Q.X. also acknowledges the Tufts Faculty Research Awards Committee (FRAC) for an award. We performed the SEM analysis at Harvard University, Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation (NSF) under NSF Award ECS-0335765. We acknowledge Antonio Varone and Nikos Fourligas for help with confocal microscope imaging.
Thus, circular patterns with well-defined edges were obtained. Even though we directly utilized commercially available meshes for patterning using EFAGT, we should be aware that more complicated patterns could be obtained from patterned membranes using microfabrication and soft lithography.10,24 3D Printing into the Hydrogel. Compared with other patterning techniques, one of the advantages of EFAGT lies in that the transferrable materials, e.g., a molecule or a nanoparticle, are able to penetrate deep into the recipient medium to generate a pseudo-3D pattern. Figure 5a shows the nanoparticle patterns on a flat sheet of 1% agarose gel (∼500 μm thick) as the receiving medium. We used the porous glass membrane filled with agarose gel doped with fluorescent nanoparticles (prepared as previously described) as the reagent “reservoir” for patterning. We utilized a confocal microscope to illustrate the arrangement of the nanoparticles in the gel, as shown in the tilted 3D image of the patterned gel (Figure 5b). Figure 5c shows a closer image of the nanoparticles organized into columnlike structures in the receiving 1% agarose gel and at a depth of the nanoparticles of ∼16 μm after traveling for 2 h under a 40 V potential.
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CONCLUSION
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ASSOCIATED CONTENT
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REFERENCES
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Patterning technology has been used in the biology field for years toward creating biosensors and improving tissue engineering and in various biological assays. However, many of the current methods of fabrication can be expensive, require clean room access, and have difficulty in generating 3D patterns. In this study, we demonstrated the ability to use electrophoresis and blotting technology together (EFAGT) for patterning molecules and nanoparticles. The simplicity and convenience make this technique useful for general laboratory applications, in particular for biologists who are interested in materials patterning but not able to access micro/nanofabrication facilities. As with any technique, EFAGT has its disadvantages. For example, it is currently restricted to patterning charged materials, and may not be suitable for directly patterning biomolecules with little net charge. This challenge could be solved by formation of charges on the desired molecules, either by binding with a negatively charged dye (e.g., native polyacrylamide gel electrophoresis)26 or by encapsulation into charged micro/nanoparticles before EFAGT. Generating true 3D patterning with arbitrary geometry remains a challenge with EFAGT, even though we successfully demonstrated the pseudo-3D patterning in the hydrogels. However, in combination with the capability of separation of different molecules by electrophoresis, this technique could be potentially useful to generate a sequential array of stacked molecules in the receiving gel which is difficult or impossible to make by other methods.
S Supporting Information *
Additional information on the experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 2964
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