Anal. Chem. 2006, 78, 6433-6439
Microfluidic and Optical Systems for the On-Demand Generation and Manipulation of Single Femtoliter-Volume Aqueous Droplets Robert M. Lorenz, J. Scott Edgar, Gavin D. M. Jeffries, and Daniel T. Chiu*
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700
This paper describes a fluidic and optical platform for the generation and manipulation of single femtoliter-volume aqueous droplets. Individual droplets were generated ondemand using a microfluidic chamber that confers environmental flow stability. Optical vortex traps were implemented to manipulate and transport the generated droplets, which have a lower refractive index than the immiscible medium in which the droplets are immersed and thus cannot be trapped using conventional optical tweezers. We also demonstrated the ability to shrink and increase the refractive index of the generated droplet, thereby permitting its facile fusion with another droplet using an optical tweezer. To illustrate the versatility of this platform, we have performed both fast (1 h) chemical reactions in these femtoliter-volume aqueous droplets. The field of microfluidics1-6 has grown tremendously since its inception due to the attributes of reduced reagent consumption, shorter reaction times, parallel processing, and increased detection sensitivity and has most notably been implemented for capillary electrophoresis (CE) separation7-13 and cytometry applications.14,15 Microfluidic systems afford the means to investigate minute fluid volumes,16-19 as well as provide a high degree of control of the chemical and physical environment,20 and, as such, have become * To whom correspondence should be addressed. E-mail: chiu@ chem.washington.edu. (1) Delamarche, E.; Juncker, D.; Schmid, H. Adv. Mater. 2005, 17, 29112933. (2) Fiorini, G. S.; Chiu, D. T. BioTechniques 2005, 38, 429-446. (3) Squires, T. M.; Quake, S. R. Rev. Mod. Phys. 2005, 77, 977-1026. (4) Pihl, J.; Karlsson, M.; Chiu, D. T. Drug Discovery Today 2005, 10, 13771383. (5) Marle, L.; Greenway, G. M. TrAC, Trends Anal. Chem. 2005, 24, 795-802. (6) Stone, H. A.; Stroock, A. D.; Ajdari, A. Annu. Rev. Fluid Mech. 2004, 36, 381-411. (7) Thorslund, S.; Lindberg, P.; Andren, P. E.; Nikolajeff, F.; Bergquist, J. Electrophoresis 2005, 26, 4674-4683. (8) Kelly, R. T.; Li, Y.; Woolley, A. T. Anal. Chem. 2006, 78, 2565-2570. (9) Kamei, T.; Toriello, N. M.; Lagally, E. T.; Blazej, R. G.; Scherer, J. R.; Street, R. A.; Mathies, R. A. Biomed. Microdevices 2005, 7, 147-152. (10) Kelly, R. T.; Woolley, A. T. Anal. Chem. 2005, 77, 96A-102A. (11) Sandlin, Z. D.; Shou, M.; Shackman, J. G.; Kennedy, R. T. Anal. Chem. 2005, 77, 7702-7708. (12) Cellar, N. A.; Burns, S. T.; Meiners, J.-C.; Chen, H.; Kennedy, R. T. Anal. Chem. 2005, 77, 7067-7073. (13) Buettgenbach, S.; Wilke, R. Anal. Bioanal. Chem. 2005, 383, 733-737. (14) Huh, D.; Gu, W.; Kamotani, Y.; Grotberg, J. B.; Takayama, S. Physiol. Meas. 2005, 26, R73-R98. (15) Wu, H.; Wheeler, A.; Zare Richard, N. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12809-12813. 10.1021/ac060748l CCC: $33.50 Published on Web 07/27/2006
© 2006 American Chemical Society
a popular choice of researchers when developing new small-scale technologies. Recently, a number of methods utilizing droplets in microfluidic channels21-24 have demonstrated practical applicability as biological and chemical reaction vessels.25 Droplet chip systems embody the flexible nature of microfluidics that mixes a high level of integrated functionality with precise control26-33 and are being used in such diverse areas as protein crystallization,34-38 biological assays,25,39,40 small-scale reactions,41-46 fabrication,47 and double emulsions.48 (16) Hatakeyama, T.; Chen, D. L.; Ismagilov, R. F. J. Am. Chem. Soc. 2006, 128, 2518-2519. (17) 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. (18) Foquet, M.; Korlach, J.; Zipfel, W. R.; Webb, W. W.; Craighead, H. G. Anal. Chem. 2004, 76, 1618-1626. (19) Hong, J. W.; Quake, S. R. Nat. Biotechnol. 2003, 21, 1179-1183. (20) He, M.; Sun, C.; Chiu, D. T. Anal. Chem. 2004, 76, 1222-1227. (21) Ward, T.; Faivre, M.; Abkarian, M.; Stone, H. A. Electrophoresis 2005, 26, 3716-3724. (22) Garstecki, P.; Fuerstman, M. J.; Stone, H. A.; Whitesides, G. M. Lab Chip 2006, 6, 437-446. (23) Link, D. R.; Anna, S. L.; Weitz, D. A.; Stone, H. A. Phys. Rev. Lett. 2004, 92, 054503/054501-054503/054504. (24) Anna, S. L.; Bontoux, N.; Stone, H. A. Appl. Phys. Lett. 2003, 82, 364366. (25) He, M.; Edgar, J. S.; Jeffries, G. D. M.; Lorenz, R. M.; Shelby, J. P.; Chiu, D. T. Anal. Chem. 2005, 77, 1539-1544. (26) Tan, Y.-C.; Cristini, V.; Lee, A. P. Sens. Actuators, B: Chem. 2006, B114, 350-356. (27) Tan, Y.-C.; Lee, A. P. Lab Chip 2005, 5, 1178-1183. (28) Tan, Y.-C.; Fisher, J. S.; Lee, A. I.; Cristini, V.; Lee, A. P. Lab Chip 2004, 4, 292-298. (29) Velev, O. D.; Prevo, B. G.; Bhatt, K. H. Nature 2003, 426, 515-516. (30) Chatterjee, D.; Hetayothin, B.; Wheeler, A. R.; King, D. J.; Garrell, R. L. Lab Chip 2006, 6, 199-206. (31) Wheeler, A. R.; Moon, H.; Kim, C.-J.; Loo, J. A.; Garrell, R. L. Anal. Chem. 2004, 76, 4833-4838. (32) Wheeler, A. R.; Moon, H.; Bird, C. A.; Ogorzalek Loo, R. R.; Kim, C.-J.; Loo, J. A.; Garrell, R. L. Anal. Chem. 2005, 77, 534-540. (33) Moon, H.; Cho, S. K.; Garrell, R. L.; Kim, C.-J. J. Appl. Phys. 2002, 92, 4080-4087. (34) Chen, D. L.; Gerdts, C. J.; Ismagilov, R. F. J. Am. Chem. Soc. 2005, 127, 9672-9673. (35) Zheng, B.; Gerdts, C. J.; Ismagilov, R. F. Curr. Opin. Struct. Biol. 2005, 15, 548-555. (36) Zheng, B.; Roach, L. S.; Tice, J. D.; Gerdts, C. J.; Chen, D.; Ismagilov, R. F. Spec. Publ.-R. Soc. Chem. 2004, 297, 145-147. (37) Zheng, B.; Tice, J. D.; Ismagilov, R. F. Adv. Mater. 2004, 16, 1365-1368. (38) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2004, 43, 2508-2511. (39) Abkarian, M.; Faivre, M.; Stone, H. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 538-542. (40) Kotz, K. T.; Gu, Y.; Faris, G. W. J. Am. Chem. Soc. 2005, 127, 57365737.
Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 6433
Although these droplet methods have demonstrated remarkable results, they suffer from some common limitations: (1) These systems are restricted to the generation of a continuous stream of droplets. Droplets are generated on the hertz to kilohertz scale with speeds from micrometers to centimeters per second. This presents a challenge for direct chemical analysis of specific droplets in real time, such as CE analysis of droplet contents, because it is difficult to decouple flow in one part of the fluidic system (e.g., droplet generation) from another (e.g., CE separation). (2) Chemical reaction times are limited by chip design; a long reaction time would require either a chip that provided a longer reaction loop at the cost of size or a reduction in flow rate that would affect droplet generation and stability. (3) Chip architecture and reaction conditions are not easily changed due to an optimum flow balance that must be maintained, especially in the case of programmed downstream droplet merging. Consequently, while flow-based droplet generation is simple and rapid, these methods pose difficult challenges in device integration, especially those that aim to chemically analyze the droplet contents. To overcome the existing complications set by streaming droplet generation, we have developed a microfluidic platform for the on-demand generation and optical manipulation of single femtoliter-volume aqueous droplets. In contrast to continuous stream droplet generation, droplets in our system are generated and addressed in a reaction chamber that is isolated from flow. Enhanced stability and control are conferred from the reduced flow environment, which facilitates direct droplet manipulation and detection, and allows long-term reactions to proceed undisturbed. The platform takes advantage of the low cost and high yield enabled by the established method of rapid prototyping, as well as the potential for implementation and integration into other microfluidic designs for enhanced functionality of simple chips or into more complicated configurations for highly parallel or combinatorial work. EXPERIMENTAL SECTION Fabrication of Microfluidic Chamber and In-Channel Generation of Droplets. Silicon masters patterned with SU-8 photoresist were fabricated using photolithography as described in detail elsewhere.25 Briefly, the two-layer patterns were generated using two different thicknesses of SU-8 (2.5-µm-thick SU-8 2002 and 80-µm-thick SU-8 2050) with two-step photolithography. The thin (2.5 µm) layer pattern was generated first, after which we spin-coated onto the one-layer master, a second thicker (80 µm) layer of SU-8. After alignment and exposure, the second layer was developed and the patterned master was silanized overnight with tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to facilitate (41) Hung, L.-H.; Choi, K. M.; Tseng, W.-Y.; Tan, Y.-C.; Shea, K. J.; Lee, A. P. Lab Chip 2006, 6, 174-178. (42) Shestopalov, I.; Tice, J. D.; Ismagilov, R. F. Lab Chip 2004, 4, 316-321. (43) Xu, S.; Nie, Z.; Seo, M.; Lewis, P.; Kumacheva, E.; Stone, H. A.; Garstecki, P.; Weibel, D. B.; Gitlin, I.; Whitesides, G. M. Angew. Chem., Int. Ed. 2005, 44, 724-728. (44) Zheng, B.; Tice, J. D.; Ismagilov, R. F. Anal. Chem. 2004, 76, 4977-4982. (45) Millman, J. R.; Bhatt, K. H.; Prevo, B. G.; Velev, O. D. Nat. Mater. 2005, 4, 98-102. (46) Chang, S. T.; Velev, O. D. Langmuir 2006, 22, 1459-1468. (47) Yi, G.-R.; Thorsen, T.; Manoharan, V. N.; Hwang, M.-J.; Jeon, S.-J.; Pine, D. J.; Quake, S. R.; Yang, S.-M. Adv. Mater. 2003, 15, 1300-1304. (48) Nisisako, T.; Okushima, S.; Torii, T. Soft Matter 2005, 1, 23-27.
6434 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
Figure 1. Schematic outlining the experimental setup used to generate, manipulate, and image droplets. The CW Nd:YAG laser was used for trapping, either with an optical tweezer or with an optical vortex trap. The CW Ar+ laser was used to excite fluorescence.
the release of the poly(dimethylsiloxane) (PDMS) replica from the silicon master. To form the microchannels, the pattern on the master was replicated in PDMS and then sealed with oxygen plasma to a coverslip with a thin layer of spin-coated PDMS. It was necessary to coat the coverslip with PDMS because the generation of aqueous droplets required channels with four hydrophobic walls to prevent wetting by the aqueous phase of the walls of the channel. The layer of PDMS on the coverslip (thickness of 130160 µm) must be very thin (
