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Technical Notes Micromachined Hydrogel Stamper for Soft Printing of Biomolecules with Adjustable Feature Dimensions Amani Salim,†,‡ Zhenwen Ding,‡,§ and Babak Ziaie*,†,‡,| Weldon School of Biomedical Engineering, School of Electrical and Computer Engineering, Department of Physics, and Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana 47907 We report on the development of a hydrogel stamper with built-in reservoirs for soft-printing of biomolecules with adjustable feature dimensions. The stamper consists of potassium hydroxide (KOH)-etched silicon cavities onto which biomolecule-soaked low cross-linked density hydrogel with large pores and low mechanical strength is loaded. Application of weight to the top surface allows for a controllable protrusion of hydrogel from the opposite nozzles. Such protrusion combined with a suitable spacer between the stamper and a substrate provides a means for printing features with dimensions depending on the applied weight. Utilizing the above method, we successfully stamped bovine serum albumin conjugated with fluorescein isothiocyanate (BSA-FITC) model proteins on hydrophilic silicon substrates with a feature dimension ratio of 20:1 using a single stamper. Microscale patterning of biomolecules (DNA, antibody, enzyme, etc.) on solid surfaces is necessary for successful development of many biotechnological microdevices such as biosensors, microarrays (DNA and protein), tissue engineering scaffolds, and cell biology platforms.1-4 In recent years, many investigators have been moving toward using soft lithography, or microcontact printing with a poly(dimethylsiloxane) (PDMS) stamp, as a simple alternative to other more elaborate techniques.5,6 Among the problems associated with the PDMS stamper is the requirement for frequent re-inking and stamp surface treatments with oxygen * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (765)404-0726. † Weldon School of Biomedical Engineering. ‡ Birck Nanotechnology Center. § Department of Physics. | School of Electrical and Computer Engineering. (1) Nicu, L.; Leichle, T. J. Appl. Phys. 2008, 104, 111101. (2) Falconnet, D.; Csucs, G.; Gradin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–306. (3) Li, N.; Tourovskia, A.; Floch, A. Crit. Rev. Biomed. Eng. 2003, 31, 423– 488. (4) Schena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier, E.; Davis, R. W. Trends Biotechnol. 1998, 16, 301–306. (5) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. Annu. Rev. Biomed. Eng. 2001, 3, 335–373. (6) Weibel, D. B.; DiLuzio, W. R.; Whitesides, G. M. Nat. Rev. Microbiol. 2007, 5, 209–218. 10.1021/ac900169k CCC: $40.75 2009 American Chemical Society Published on Web 04/30/2009
plasma or grafting of hydrophilic molecules prior to printing.7,8 The surface treatment is required for hydrophilic molecules because of the poor wetting and inhomogeneous drying of such molecules on the PDMS surface. With their inherent softness and absorption/soaking capability, hydrogel stampers can overcome the above shortcomings. Simple capillary and molded hydrogel stampers (from silicon or PDMS masters) have been reported for delivering biomolecules to the surfaces.9-12 The capillaryloaded system requires sequential stamping and thus is not desirable for array patterning. It is also cumbersome to integrate the capillary with alignment tools. Molded hydrogel stamps are hard to handle and require highly cross-linked hydrogel for easier handling thus resulting in a lower soaking capability. In this article, we describe the development of a simple micromachined hydrogel stamper with built-in reservoirs that not only overcomes many of the aforementioned shortcomings but also allows a single stamper to be used for printing multiple feature dimensions. EXPERIMENTAL METHODS Figure 1a shows the top and bottom three-dimensional (3D) schematic of the hydrogel stamper. It consists of potassium hydroxide (KOH)-etched cavities in a 500 µm-thick silicon wafer onto which lightly cross-linked poly(methacrylic acid-co-acrylamide, m-AA-co-AAM) hydrogel is cast. Upon swelling in aqueous media, hydrogel protrudes from etched holes of the KOH cavity. For variable feature size printing, weights are used to provide uniform pressure from the top which results in reversible hydrogel protrusion from the bottom nozzles as long as the hydrogel does not deform beyond its elastic limit. The elastic modulus of the swelled hydrogel is measured to be 0.66 KPa as compared to 100 (7) Donzel, C.; Geissler, M.; Bernard, A.; Wolf, H.; Michel, B.; Hilborn, J.; Delamarche, E. Adv. Mater. 2001, 13, 1164–1167. (8) Delamarche, E.; Donzel, C.; Kamounah, F. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749–8758. (9) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971–3975. (10) Martin, B. D.; Brandow, S. L.; Dressick, W. J.; Schull, T. L. Langmuir 2000, 16, 9944–9946. (11) Coq, N.; Bommel, T. V.; Hikmet, R. A.; Stapert, H. R.; Dittmer, W. U. Langmuir 2007, 23, 5154–5160. (12) Majd, S.; Mayer, M. J. Am. Chem. Soc. 2008, 130, 16060–16064.
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Figure 2. (a) Superimposed confocal images showing the initial protrusion from the bottom nozzles of different sizes (50, 100, and 170 µm, the red signal is reflection from the silicon surface while the green signal is from BSA-FITC), (b) hydrogel protrusion vs applied weight.
Figure 1. (a) 3D schematic of the hydrogel stamper showing the top surface, bottom surface, and hydrogel protrusion with applied weights, (b) cross-section schematic of the biomolecule loading process.
KPa ∼ 1 MPa for PDMS, making it more deformable. Figure 1b is the schematic of the biomolecule loading process, which starts with KOH-etched cavities loaded with hydrogel [Figure 1b (i)], biomolecule absorption [Figure 1b (ii)], and protrusion of hydrogel after an overnight soak [Figure 1b (iii)]. The KOH cavities (3 × 3 to 5 × 5 arrays with the bottom-side spacings and openings of 50-1000 and 50 µm-1000 µm, respectively) were formed by etching through a 500 µm 〈100〉 p-type silicon wafer, coated with 3000 Å low stress nitride masking layer. Silicon etch was done in a 45% aqueous solution of KOH at a temperature of 85 °C for 8 h. After etching, the KOH-etched silicon cavities were rinsed in DI water for 10 min and blow-dried using N2. The remaining silicon nitride was completely removed by plasma reactive ion etching for 3 min (CF4/O2; 8:1; 100 W; 75 mTorr; etch rate 1000 Å/min). We should mention that alternatively one can use deep reactive ion etching (DRIE) to etch vertical holes through silicon. Although more expensive than KOH, DRIE can create smaller holes not limited by the crystallographic planes, thus increasing the printing resolution. After the silicon etch process, the cavities were treated with 10% solution (V/V) of γ-methacryloxypropyl trimethoxysilane in acetone for 1 h. This allows the creation of a covalent bond between the hydrogel and silicon cavities through methacrylate pendant groups.13 The pregel solution Poly(mAA-co-AAm, pH sensitive) was prepared by adding 334.5 mg of acrylamide (AAm, Sigma-Aldrich), 100.8 µL of methacrylic acid (mAA, Sigma-Aldrich, (13) Hilt, J. Z.; Gupta, A. K.; Bashir, R.; Peppas, N. A. Biomed. Microdevices 2003, 5, 177–184.
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Figure 3. (a) Confocal images showing the progression of stamped widths with various weights, (b) composite fluorescent image showing the increase in spot size from no weight, 13 g, and 52 g of applied weight. Substrate is 1 wt % NaOH-treated silicon wafer.
distilled after received), 4 µL of ethylene glycol dimethacrylate (EGDMA, cross-linker from Polysciences Inc.), and 50 µL of N,N,N′,N′-tetramethylethylenediamine (TEMED, accelerator from Sigma-Aldrich) to 1.2 mL of deionized (DI) water. This was referred to as solution A. Solution B consisted of 20 mg/mL ammonium persulfate (APS, initiator from Polysciences Inc.) in deionized water. Solution A and solution B were mixed in a volume ratio of 5.9:1 to make the final gel solution. The loaded KOH cavities were clamped in between two saranwrapped glass slides. After polymerization, the hydrogel filled silicon cavities were carefully removed from the glass slides and bathed overnight in a solution of 3 mL of 5 mg/mL BSA in 7.4 PBS solution, 20 µL glycerol anhydrous (Sigma Aldrich), and 20 µL Tween 20 (Sigma Aldrich). While polymerization of the pregel solution occurs after 15 min, the biomolecules were not loaded into the stamper until 7-10 h post polymerization. This was done to ensure that there was sufficient covalent binding between the
Figure 4. (a) 3 × 2 array of BSA-FITC printed spots using a stamper with 70 µm nozzle size, an 85 µm spacer, and 13 g applied weight, (b) printed IgGs using stampers with 1000 and 200 µm nozzles with 13 g applied weight.
loaded hydrogel and the treated silicon cavities. Glycerol prevents evaporation of BSA during the imaging and stamping processes while Tween 20 acts as a solubilizing agent helping in the transfer of molecules to the stamped substrate. For the substrates, we used silicon wafers treated with 1 wt % sodium hydroxide (NaOH) in deionized (DI) water at room temperature for 30 min. This enables the formation of hydrophilic OH- groups at the surface. RESULTS AND DISCUSSION The hydrogel’s ability to absorb biomolecules in its pores is related to the diffusivity of specific biomolecules in the hydrogel network. This, in turn depends on the pore size, the polymer gel composition, gel water content, and the nature and size of the biomolecule.14 The hydrogel stamper should have a pore size that is larger than the size of the diffusant. For such hydrogels the diffusivity depends on the porosity of the gel. To increase the diffusivity of proteins and other large molecules, one can also create artificial macropores in the hydrogel (at the expense of mechanical strength of the hydrogel).15 Our hydrogel has an approximate pore size of ∼10-15 nm16 (ref 16 discusses pore size calculation for poly(mAA) hydrogel at various pH values using a polymer network model), which is more than 3 times larger than the hydrodynamic radius of BSA and IgG molecules (∼3.5 and 5.5 nm, respectively). The transfer of biomolecules through the hydrogel network is also pH dependent.14 This is due to the effect of pH on the pore size (if the hydrogel is pH sensitive) and electrostatic interaction between the hydrogel and the biomolecules. Our experiments were carried out at pH 7.4, in which BSAFITC, and poly(mAA-co-AAm) network are negatively charged.17 Figure 2a shows the superimposed confocal images for initial protrusion of the hydrogel through the nozzles without the application of any weight for 50, 100, and 170 µm bottom nozzles (fluorescent because of the BSA-FITC molecules). As can be seen, larger nozzles result in greater protrusions (this is expected because of the isotropic nature of hydrogel swelling). Figure 2b is a plot of hydrogel protrusion versus applied weight for several bottom nozzle dimensions, showing a linear relationship between the two within the applied weight range. For a 100 µm bottom nozzle the initial protrusion was 114 µm increasing to 151 µm with (14) Khoury, C.; Adalsteinsson, T.; Johnson, B.; Crone, W. C.; Beebe, D. J. Biomed. Microdevices 2003, 5, 35–45. (15) Ford, M. C.; Bertram, J. P.; Hynes, S. R.; Michaud, M.; Li, Q.; Young, M.; Segal, S. S.; Madri, J. A.; Lavrik, E. B. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 2512–2517.
the application of 78 g weight. This is an increase of ∼30% with a linear protrusion coefficient of ∼0.5 µm/g. It is interesting to note that there is a slight increase in the linear protrusion coefficient (i.e., slope of the lines in Figure 2b) for larger nozzles. The amount of initial swelling can be adjusted by varying the cross-link density of the hydrogel. There is, however, a trade-off between the swelling ratio and the mechanical properties (stiffness) of the hydrogel (the higher the cross-link density the stiffer the hydrogel and the smaller the hydrogel pores). In our characterization, we limited the maximum weight to 78 g to prevent permanent deformation. After the removal of the largest applied weight (78 g) and subsequent soaking for 10 min in BSA-FITC solution, the hydrogel sprung back to its original protrusion. We anticipate that larger weights will deform the hydrogel beyond its elastic limit. Although we did not systematically investigate this to find the exact limit, we noticed that application of 225 g of weight to a 70 µm nozzle resulted in permanent deformation of the gel. For characterization of the printing process, we used a stamper with 70 µm nozzle size and weight increments of 13 g starting from zero to 78 g. Figure 3a shows the cross section of the hydrogel stamper including a spacer and the stamped silicon substrate. Stamping was conducted for 3 min contact time. Knowing the initial protrusion for a 70 µm bottom nozzle (∼85 µm), we used a SU8 spacer of 85 µm in thickness (spacer thickness was chosen to accommodate this protrusion thus preventing the hydrogel to touch the substrate before the application of weights). As can be seen (Figure 3a), by using a single stamper and changing the applied weights, one can easily adjust the width of the stamped area and exceed the resolution imposed by the dimensions of the KOH etched cavities. Figure 3b shows the composite top view of the stamped substrate for applied weights of 0, 13, and 52 g, indicating the progressive increase in the stamped dimensions of more than 20:1 (5 to 107 µm). Figure 4a shows a 3 × 2 array of BSA-FITC printed on treated silicon substrates using a stamper with 70 µm nozzle size with an 85 µm spacer and 13 g applied weight. We also stamped IgG biomolecules having larger hydrodynamic radius than BSA. Figure 4b shows printed IgGs using stampers with 1000 and 200 µm nozzle dimensions with 13 g applied weight. The hydrogel stamper is reusable if kept in a sealed Petri dish containing the biomolecules. We tested the stamper after 2 weeks storage time, and the results showed that the transfer of the biomolecules was still effective (see Supporting Information, Figure 5). Also, to achieve optimal transfer, we soaked the stamper Analytical Chemistry, Vol. 81, No. 11, June 1, 2009
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in the biomolecule solution for one minute prior to each stamping. We also anticipate that because of the porous nature of the hydrogel, multiple stamping can be achieved before re-inking is required (in our preliminary experiments, we were able to stamp 8 times on a hydrophilic silicon surface without re-inking). CONCLUSIONS In this paper, we demonstrated a microfabricated hydrogel stamper with built-in reservoirs and outlet protrusions that can be adjusted through the application of weight. This allows a single stamper to be used for patterning multiple feature dimensions. A single stamper was used to print fluorescent-labeled BSA on a silicon substrate with a 20:1 feature size ratio. The stamper is capable of printing over a large range of dimensions (mm-µm) (16) Lowman, A. M.; Peppas, N. A. Marcromolecules 1997, 30, 4959–4965. (17) Bohme, U.; Scheler, U. Chem. Phys. Lett. 2007, 435, 342–345.
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which can be controlled by several factors including (1) the size of the KOH-etched cavity, (2) the height of the spacer, (3) the applied weight, and (4) mechanical properties of the hydrogel. ACKNOWLEDGMENT The authors would like to thank Ms. Jennifer Sturgis at the Purdue University Bindley Bioscience Cytometry Imaging Lab for invaluable discussions and her assistance in confocal and fluorescence imaging. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 22, 2009. Accepted April 14, 2009. AC900169K
