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Fabrication of Microwell Arrays Based on Two-Dimensional Ordered Polystyrene Microspheres for High-Throughput Single-Cell Analysis Chuansen Liu, Jiangjiang Liu, Dan Gao, Mingyu Ding, and Jin-Ming Lin* The Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China This paper describes a method of fabricating rounded bottom microwell arrays (MA) in poly(dimethylsiloxane) (PDMS) by molding a monolayer of ordered polystyrene (PS) microspheres. PS microspheres were self-assembled on a glass slide and partially melted mainly from the bottom at 240 °C to increase adhesive force with the substrate. The partially melted PS arrays were used as master to generate MA. Microwell sizes are tunable in the 10-20 µm range with rounded bottoms; such a 3D structure is not readily obtainable through conventional soft lithography. Both adherent and nonadherent cell types can be retained in the microwells with high efficiency. As a demonstration of the advantage of real-time cell screening with this MA, single cell enzyme kinetic analysis was also carried out on trapped single cells. The PDMS MA may find applications in high-throughput drug screening, guided formation of cell clusters, and multicellular communication. Traditional cell based biological assays are usually performed with bulk techniques by virtue of the availability and simplicity of well established equipment. Such analysis provides averaged temporal responses of cells exposed to environment stimuli and, thus, overlooks the heterogeneity among large population of cells and even leads to misunderstanding about the time-dependent cellular process.1 In contrast, single cell experiments could avoid the loss of information resulting from ensemble averaging and supply statistical rich data to reveal the distribution of responses among a large population of cells. Single cell level analysis is also important in the fundamental study of cell-cell and cell-surface interactions which deepen our understanding in the proliferation process of cells.2 To fulfill such kinds of analysis, long-term rather than instant monitoring of single cells in a high-throughput manner is always desirable.3 Therefore, positioning of single cells in an isolated manner deserves our investigation. In recent years, microfluidic platforms for single cell analysis becomes the hub of scientific study as it shows great potentials for wide applications * To whom correspondence should be addressed. E-mail: jmlin@ mail.tsinghua.edu.cn. Fax/Tel: +86 10 62792343. (1) Svahn, H. A.; van den Berg, A. Lab Chip 2007, 7, 544–546. (2) Di Carlo, D.; Lee, L. P. Anal. Chem. 2006, 78, 7918–7925. (3) Lindstrom, S.; Larsson, R.; Svahn, H. A. Electrophoresis 2008, 29, 1219– 1227.
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in genetic analysis,4,5 tissue engineering,6,7 and drug screening.8,9 Microfluidic approaches possess the advantages of feasibility on cell manipulation together with reagents handling and real-time monitoring capabilities.10-15 Nevertheless, such methods often rely on complex multilayer soft lithography or advanced surface modification.16-18 Fabrication methods such as microcontact printing and conventional soft lithography techniques are two of the most widely adopted techniques for fabricating single-cell analysis platforms. Microcontact printing19 was utilized for patterning substrates with well-defined microscale “cell-friendly” and “cell-unfriendly” regions for the trapping of single adherent cells on site. Such a patterning method is limited to single adherent cell analysis only. Other developments on single cell trapping platforms based on conventional soft lithography showed that the single cell trapping efficiency can be improved (>80%) using poly(dimethylsiloxane) (PDMS) microwells20-23 or microweirs24-26 with a feature size comparable to single cells. In these cases, cells entered the blank (4) Ryley, J.; Pereira-Smith, O. M. Yeast 2006, 23, 1065–1073. (5) Thomas, C. H.; Collier, J. H.; Sfeir, C. S.; Healy, K. E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1972–1977. (6) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2480–2487. (7) Bruzewicz, D. A.; McGuigan, A. P.; Whitesides, G. M. Lab Chip 2008, 8, 663–671. (8) Wang, Z. H.; Kim, M. C.; Marquez, M.; Thorsen, T. Lab Chip 2007, 7, 740–745. (9) Manbachi, A.; Shrivastava, S.; Cioffi, M.; Chung, B. G.; Moretti, M.; Demirci, U.; Yliperttula, M.; Khademhosseini, A. Lab Chip 2008, 8, 747–754. (10) Li, X. J.; Ling, V.; Li, P. C. H. Anal. Chem. 2008, 80, 4095–4102. (11) Wheeler, A. R.; Throndset, W. R.; Whelan, R. J.; Leach, A. M.; Zare, R. N.; Liao, Y. H.; Farrell, K.; Manger, I. D.; Daridon, A. Anal. Chem. 2003, 75, 3581–3586. (12) Lee, P. J.; Hung, P. J.; Shaw, R.; Jan, L.; Lee, L. P. Appl. Phys. Lett. 2005, 86, 223902. (13) Huang, B.; Wu, H. K.; Bhaya, D.; Grossman, A.; Granier, S.; Kobilka, B. K.; Zare, R. N. Science 2007, 315, 81–84. (14) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–411. (15) Li, P. C. H.; de Camprieu, L.; Cai, J.; Sangar, M. Lab Chip 2004, 4, 174– 180. (16) Carrico, I. S.; Maskarinec, S. A.; Heilshorn, S. C.; Mock, M. L.; Liu, J. C.; Nowatzki, P. J.; Franck, C.; Ravichandran, G.; Tirrell, D. A. J. Am. Chem. Soc. 2007, 129, 4874–4875. (17) Chandra, R. A.; Douglas, E. S.; Mathies, R. A.; Bertozzi, C. R.; Francis, M. B. Angew. Chem., Int. Ed. 2006, 45, 896–901. (18) Jin, L. H.; Yang, B. Y.; Zhang, L.; Lin, P. L.; Cui, C.; Tang, J. Langmuir 2009, 25, 5380–5383. (19) Wilbur, J. L.; Kumar, A.; Biebuyck, H. A.; Kim, E.; Whitesides, G. M. Nanotechnology 1996, 7, 452–457. (20) Rettig, J. R.; Folch, A. Anal. Chem. 2005, 77, 5628–5634. 10.1021/ac102094r 2010 American Chemical Society Published on Web 10/19/2010
sites which were not occupied by cells. After flushing away the freestanding cells outside the microstructures, subsequent stimulating reagents were perfused onto the cells for single cell analysis. Cell trapping on microwell arrays (MA) has two notable benefits: imaging processing is simplified and microenvironment of each cell is controlled. Therefore, such MA is suitable for enzyme kinetics analysis, drug screening, and basic cell biology studies at the cellular level. The arrayed microstructures for cell trapping are generally fabricated on PDMS by soft lithography. Soft lithography, a collection of techniques based on printing, molding, and embossing with an elastomeric stamp, is widely used for fabricating microstructures and nanostructures.27 Soft lithography has the capability to generate prototypes in PDMS replica using a master with patterned microstructures. At the current stage of development, the master is usually generated using photolithography. The desired pattern is drawn by CAD software programs and transferred to a transparency film or chrome mask. The patterns on the photomask are projected by UV light onto a thin film of photoresist spin coated on a silicon wafer. At last, the master is obtained by removing the unexposed photoresist. It is an expensive and time-consuming process, especially when the feature size is less than 20 µm, which requires cost-ineffective chrome photomasks for photolithography. Otherwise, the resolution limitation of the transparency photomask makes it only applicable to fabricate microstructures for large single cell or multicell trapping. Conventional photolithography usually requires a high cost and at least half day to produce a master. Deutsch28 reported a miniature cell retainer fabricated on a glass substrate patterned with MA using a chrome mask. This fabrication process requires photolithography and etching of glass substrates; thus, it is relatively complex and time consuming. In this work, we propose and develop a simple method of fabricating masters for generating MA with feature sizes less than 20 µm for single cell trapping. By self-assembling polystyrene (PS) microspheres on glass substrate and partially melting the microspheres, we achieved masters for PDMS replication. Honeycomb MA on PDMS were created by replica molding the masters and used for large scale single cell trapping and analysis. Our method is a facile, fast, and inexpensive approach to fabricate MA with tunable sizes of 10-20 µm which is difficult to get through conventional soft lithography. To demonstrate its advantages, single cell enzyme activity analysis29 was carried out on the trapped cells with real-time monitoring. This MA may find applications in high-throughput cell screening, cell based sensor arrays, and multicellular communication. (21) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828–2834. (22) Ferrell, N.; Gallego-Perez, D.; Higuita-Castro, N.; Butler, R. T.; Reen, R. K.; Gooch, K. J.; Hansford, D. J. Anal. Chem. 2010, 82, 2380–2386. (23) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 9855–9862. (24) Di Carlo, D.; Aghdam, N.; Lee, L. P. Anal. Chem. 2006, 78, 4925–4930. (25) Di Carlo, D.; Wu, L. Y.; Lee, L. P. Lab Chip 2006, 6, 1445–1449. (26) Skelley, A. M.; Kirak, O.; Suh, H.; Jaenisch, R.; Voldman, J. Nat. Methods 2009, 6, 147–152. (27) Qin, D.; Xia, Y. N.; Whitesides, G. M. Nat. Protoc. 2010, 5, 491–502. (28) Deutsch, M.; Deutsch, A.; Shirihai, O.; Hurevich, I.; Afrimzon, E.; Shafran, Y.; Zurgil, N. Lab Chip 2006, 6, 995–1000. (29) Peng, X. Y.; Li, P. C. H. Anal. Chem. 2004, 76, 5282–5292.
EXPERIMENTAL SECTION Chemicals and Materials. The 10% (wt) aqueous suspension of polystyrene (PS) microspheres with a relative standard deviation (RSD) of
