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Handling of Picoliter Liquid Samples in a Poly(dimethylsiloxane)-Based Microfluidic Device Kazuo Hosokawa,*,† Teruo Fujii,‡ and Isao Endo
Biochemical Systems Laboratory, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
Transportation, metering, and mixing of picoliter-sized liquid samples were realized in a microfluidic device with a main working area of one square millimeter. The device was constructed by sealing microfabricated grooves on a chip made of poly(dimethylsiloxane) (PDMS). Two different samples were segmented into 600-pL droplets in a microchannel with a cross section of W (100 µm) × H (25 µm), and the droplets were merged together. For acceleration of the mixing, the merged droplet was shuttled back and forth. Recirculation in a moving droplet was proven to be effective for high-speed mixing in this diffusion-dominated scale. All the handling operations were carried out using air pressure transferred through microfabricated vent valves which have been newly developed. The demonstrated strategy, including fabrication, leads to high-performance and low-cost micro total analysis systems (µTAS). Research in the field of micro total analysis systems (µTAS)1,2 aims at miniaturization and integration of (bio)chemical instruments onto a microchip (“lab-on-a-chip”) for high-throughput analyses and/or portable systems. Within a few exceptions,3-6 most previously reported µTAS are single-step devices. A major hurdle in developing successful multistep µTAS is the liquidhandling technique, i.e., transportation, metering, and mixing in pico/nanoliter-scale. The difficulty stems from the change in hydrodynamic behavior caused by the scaling: surface tension and viscosity become dominant instead of gravity and inertia. Recently, pneumatic handling of liquid droplet (a liquid plug * To whom correspondence should be addressed. Fax: +81-298-58-7167. E-mail:
[email protected]. † Present address: Surface & Interface Technology Division, Mechanical Engineering Laboratory, AIST/MITI, 1-2 Namiki, Tsukuba, Ibaraki, 305-8564 Japan. ‡ Present address: Institute of Industrial Science, University of Tokyo, 7-22-1 Roppongi, Minato-ku, Tokyo, 106-8558 Japan. (1) van den Berg, A.; Bergveld, P., Eds. Micro Total Analysis Systems; Kluwer Academic Publishers: Dordrecht, 1995. (2) Harrison, D. J.; van den Berg, A., Eds. Micro Total Analysis Systems 98; Kluwer Academic Publishers: Dordrecht, 1998. (3) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (4) Anderson, R. C.; Bogdan, G. J.; Barniv, Z.; Dawes, T. D.; Winkler, J.; Roy, K. Proc. IEEE Transducers 97, Chicago, IL, June 16-19, 1997; pp 477480. (5) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (6) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science (Washington, D.C.) 1998, 282, 484-487. 10.1021/ac990571d CCC: $18.00 Published on Web 09/18/1999
© 1999 American Chemical Society
segmented by air)sa promising scheme because of its simplicity and minimal effect on (bio)chemistryswas demonstrated in microliter-scale4 and nanoliter-scale.6 We have succeeded in further miniaturization: two droplets of 600-pL volume were metered and mixed within an area of one square millimeter. This achievement leads to fully integrated and massively parallel µTAS for DNA analysis as well as combinatorial chemistry. The key component enabling this miniaturization is a microfabricated vent valve, which has been newly developed and named a hydrophobic microcapillary vent (HMCV).7,8 HMCV is inexpensively fabricated by a replica-molding technique of a silicone elastomer poly(dimethylsiloxane) (PDMS)9-11 and is operated at fairly large pressure. As shown in Figure 1A, the HMCV is an array of hydrophobic microcapillaries which connect the liquid and pneumatic channels. The cross sections of the microcapillaries are much smaller than that of the liquid channel. The hydrophobicity and the small cross sections endow the microcapillaries with resistance to intrusion of aqueous liquid, in contrast with the capillary action of a hydrophilic (e.g., glass) capillary. Therefore, keeping liquid confined in the liquid channel, we can freely inject or evacuate air through the HMCV. Although a single microcapillary works as a vent in principle, the array design has an advantage of reducing the resistance of total air flow. Positioning (Figure 1B) and metering (Figure 1C) of liquid can be carried out using HMCVs. Merging of two liquid droplets is realized by drawing off the air between the droplets. This is basically the reverse action of the metering. EXPERIMENTAL SECTION Design of the Microfluidic Device. On the basis of functions of the HMCV described above, we designed and fabricated the microfluidic device illustrated in Figure 2. It consists of a PDMS part and a poly(methyl methacrylate) (PMMA) substrate, both of which are transparent. The PDMS partsintrinsically hydrophobicswas molded against a microfabricated negative master. By sealing the grooves formed on the PDMS part, the PMMA (7) Hosokawa, K.; Fujii, T.; Endo, I. Proc. Micro Total Analysis Systems 98, Banff, Canada, Oct 13-16, 1998, pp 307-310. (8) Hosokawa, K.; Fujii, T.; Endo, I. Proc. IEEE Micro Electro Mechanical Systems 99, Orlando, FA, Jan 17-21, 1999, pp 388-393. (9) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science (Washington, D.C.) 1997, 276, 779-781. (10) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451-3457. (11) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.
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Figure 1. (A) Basic structure of the HMCV. It is an array of microcapillaries with cross section of w × h, which is much smaller than that of the liquid channel, W × H. The internal walls of the microcapillaries are hydrophobic. (B) Positioning operation. Liquid in the liquid channel is pulled by sucking the air from the pneumatic channel. The liquid-air interface is accurately positioned in front of the HMCV. (C) Metering operation. After the liquid positioning described in (B), an excessive amount of liquid can be cut by the air injected from the HMCV on the side wall of the liquid channel. The volume of remaining liquid is defined by the channel dimension. The merging of two droplets can be carried out in just the reverse of the way shown this figure.
Figure 2. Schematic of the microfluidic device. The main working region is magnified in the left square. Four independent pneumatic channels P1-P4 are connected to the side wall of the liquid channel via HMCVs. In parallel to the HMCVs, there are nonfunctional microcapillaries (light lines) for convenience in fabrication. Actual sizes of all the microcapillaries are identical.
substrate makes microchannels including HMCVs. The PMMA substrate has access holes for sample loading and pneumatic control. The main working region of this device consists of a liquid channel and four pneumatic channels with HMCVs. Liquid sample in the loading port is introduced to the liquid channel and manipulated by positive or negative air pressure supplied from the pneumatic channels. Mask Fabrication. For fabrication of the device, two masks were prepared in different ways. Both of them are 2.5-in-square quartz plates with patterned chromium thin films on them. One 4782 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 3. Simplified description of the fabrication process of the microfluidic device. (A) The pattern for the set of HMCVs was formed by reactive ion etching, for which a 0.3-µm SiO2 layer was used as a mask. (B) For the liquid and pneumatic channels, a 25-µm photoresist layer was coated and patterned. (C) A PDMS replica was molded against the negative master. (D) The PDMS part was peeled off, and it was pasted on the PMMA substrate with the access holes drilled in advance.
mask for fine pattern of the HMCVs was fabricated by a commercial supplier (Toppan Printing, Japan). The other mask is for a relatively coarse pattern of the liquid and pneumatic channels. It was fabricated in a more rapid and less costly way.11 Data drawn with graphic software (Adobe Illustrator) was printed out with a resolution of 4064 dpi on a transparent sheet by a desktop publishing shop. Using a contact mask aligner (PEM800; Union Optics, Japan), the pattern on the sheet was transferred to the positive photoresist layer on a blank mask, which had been purchased from ULVAC Coating, Japan. The blank mask has readily coated chromium and photoresist layers. After processing the photoresist, the exposed area of the chromium was etched by a wet process, and the photoresist was removed by O2 plasma in a reactive ion etching (RIE) machine (RIE-10NR; Samco International, Japan). Microfluidic Device Fabrication. The fabrication process of the microfluidic device is outlined in Figure 3. It is based on the replica-molding technique of PDMS.9-11 First, a negative master for molding was fabricated on a silicon wafer with a two-mask process. (1) A 0.3-µm-thick SiO2 layer was sputtered using a radio frequency sputtering machine (L-250S; ANELVA, Japan). (2) Using the contact mask aligner, the pattern for the set of HMCVs was transferred onto the wafer, which had been spin-coated with positive photoresist (OFPR-800; Tokyo Ohka Kogyo, Japan) in advance. (3) Four dry-etching processes were consecutively carried out using the RIE machine: after etching of the SiO2 layer, Si was anisotropically etched to a depth of 3 µm, then the photoresist and the SiO2 layers were removed (Figure 3A). (4) To obtain a 25-µm-thick structure for the pattern of liquid and pneumatic channels, ultrathick negative photoresist (SU-8 25; MicroChem, MA) was processed by following the manufacturer’s instructions. The contact mask aligner was used again for the UV exposure. After development, the wafer was baked at 150 °C for 5 min on a hot plate to reinforce the adhesion, and it was gradually cooled to room temperature over 1-2 h. (5) For mold release, the negative master was coated with a fluorocarbon layer polymerized by CHF3 plasma in the RIE machine (Figure 3B). Next, the PDMS part was molded against the master. (6) Prepolymer of PDMS (Sylgard 184; Dow Corning, A:B ) 1:10) was poured onto the master with a frame for holding the solution. (7) It was
cured in an oven at 65 °C for 60 min, followed by the second cure at 150 °C for 30 min (Figure 3C). (8) After curing, the PDMS part was peeled off from the master. (9) In parallel to the above processes, the PMMA substrate was fabricated. Five holes (four φ1.5 mm holes for the pneumatic ports and a φ3 mm hole for the sample loading port) were mechanically drilled through a 2-mmthick, transparent PMMA plate. Metal pipes for tube connection were glued to the pneumatic ports with epoxy adhesive. (10) The PDMS part was placed onto the PMMA substrate by hand (Figure 3D) under a stereomicroscope (SMZ-2; Nikon, Japan) for alignment. Since cured PDMS spontaneously adheres to various materials including PMMA, hermetically sealed microchannels were formed without an elaborate bonding technique or adhesive medium. Leakage Test. The HMCV endures a pressure difference without any liquid leakage to some critical value, which is called the “pressure barrier” hereafter. By overloading beyond the pressure barrier, the liquid-blocking ability of the HMCV is broken down, and liquid leaks at a rate which is determined by viscous friction. It is obvious that a large pressure barrier is desirable for the current purpose. The pressure barrier of an HMCV in the microfluidic device was tested as follows. The liquid channel was filled with deionized water and kept at the atmospheric pressure. We gradually decreased the air pressure in the pneumatic channel P1 (See Figure 2), which was connected to a vacuum pump (DA-5D; ULVAC Sinku Kiko, Japan) via a vacuum regulator (VR200-G; Koganei, Japan). The leakage of the water was observed from the PMMA side using a digital optical microscope (KH-2400 DP; Hirox, Japan). Handling of Liquid Samples. A series of liquid-handling operationsstransportation, metering, and mixingswas carried out in the microfluidic device. For visualization, two fluorescein aqueous solutions with different concentrations were prepared by dissolving fluorescein sodium salt (Sigma A6377) in deionized water. They are referred to as “concentrated solution” (0.2 mg/ mL) and “diluted solution” (0.02 mg/mL), respectively. Each pneumatic port was connected to an empty 1-mL syringe via an i.d. 1-mm silicone tube, and positive/negative air pressure was applied to the pneumatic channel by manually pushing/pulling the piston of the syringe. Motion of the liquid samples was monitored from the PDMS side using an inverted fluorescence microscope (DIAPOT-TMD; Nikon, Japan) and an ICCD camera (C2400-80; Hamamatsu Photonics, Japan). An image-processing system (ARGUS-50; Hamamatsu Photonics, Japan) was used for intensity data analysis.
deformation of the PDMS and/or roughness of the inner surfaces of the channel. The most important parameter dominating the pressure barrier is the contact angle θ. A negative value of cos θ (θ > 90°) is essential for an HMCV to work. In general, θ is affected by various factors such as surface roughness and contaminant adsorption. Especially, it is known that θ monotonically decreases with decreasing liquid surface tension γ.13 Since γ of water is reduced by a rise in temperature and the existence of surfactants, these factors (especially the latter) may significantly lower the pressure barrier. Further investigation is needed to ascertain the limitation of the HMCV in this respect. Handling of Liquid Samples. Figure 5 demonstrates the liquid-handling operations. First, the diluted solution was introduced (Figure 5B) and metered (Figure 5C) using the pneumatic channels P1 and P2, respectively. The volume of the metered droplet is calculated as about 600 pL (W(100 µm) × H(25 µm) × L(250 µm)). Similarly, another 600-pL droplet of the concentrated solution was metered using P3 and P4 (Figure 5D and E), and the two droplets were merged by sucking the air from P2 (Figure 5F). Finally, the merged droplet was pushed and pulled using P1 (Figure 5G and H) to promote mixing: this cycle is referred to as one “shuttling” hereafter. Mixing by Droplet Shuttling. Without shuttling, the mixing proceeds very slowly. In general, microfluidic mixing is dominated by diffusion because of a low Reynolds number.14 To overcome this notorious “mixing problem,” various micromixers have been devised for continuous-flow systems.15-20 On the basis of classic
RESULTS AND DISCUSSION Leakage Test. Results of the leakage test are summarized in Figure 4. The pressure barrier of this HMCV is concluded to be in the range of 30-35 kPa, which is sufficiently large for practical use. Theoretically, the pressure barrier can be estimated by -2γcosθ(1/w + 1/h), where γ and θ are surface tension of the liquid and the contact angle between the liquid and channel walls, respectively.8 In the case of our design (w ) 5 µm, h ) 3 µm) using the material properties of water and PDMS (γ ) 0.073 N/m, θ ) 110° 12), the pressure barrier is calculated as 27 kPa. We consider that the discrepancy between the theoretical and measured values of the pressure barrier may be caused by
(12) Murakami, T.; Kuroda, S.; Osawa, Z. J. Colloid and Interface Sci. 1998, 202, 37-44. (13) Fowkes, F. M., Ed. Contact Angle, Wettability, and Adhesion; American Chemical Society: Washington D.C., 1964; Chapter 1. (14) Brody, J. P.; Yager, P.; Goldstein, R. E.; Austin, R. H. Biophys. J. 1996, 71, 3430-3441. (15) Mensinger, H.; Richter, Th.; Hessel, V.; Do ¨pper, J.; Ehrfeld, W. Proc. Micro Total Analysis Systems, Enschede, The Netherlands, Nov 21-22, 1994; pp 237-243. (16) Branebjerg, J.; Gravesen, P.; Krog, J. P.; Nielsen, C. R. Proc. IEEE Micro Electro Mechanical Systems 96, San Diego, CA, Feb 11-15, 1996; pp 441446. (17) Evans, J.; Liepmann, D.; Pisano, A. P. Proc. IEEE Micro Electro Mechanical Systems 97, Nagoya, Japan, Jan 26-30, 1997; pp 96-101. (18) Miyake, R.; Tsuzuki, K.; Takagi, T.; Imai, K. Proc. IEEE Micro Electro Mechanical Systems 97, Nagoya, Japan, Jan 26-30, 1997; pp 102-107.
Figure 4. Leaking behavior of the HMCV with overload. The liquid channel is filled with deionized water. (A) When a negative pressure of -30 kPa is applied to the pneumatic channel P1, no leakage is observed. (B) At -35 kPa, several microcapillaries begin to have leakage. The water flows slowly. The image was taken 30 s after the pressure had become stable. (C) At -45 kPa, a significant amount of water flows through the microcapillaries.
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Figure 5. Video-clipped fluorescence images of handling operations for two fluorescein aqueous solutions with different concentrations. The experiment was carried out under an incomplete blackout to visualize the microstructure. (A) Initial state. (B) The diluted solution is introduced by sucking the air from the pneumatic channel P1. (C) By injecting air from P2, the solution is segmented into a 600-pL droplet and the rest, which is returned to the sample loading port. (D) After replacing the solution in the sample loading port with the concentrated solution, the new solution is introduced using P3. (E) The air from P4 makes another 600-pL droplet. (F) Two droplets are merged by drawing off the air from P2. Mixing starts by diffusion. (G) To promote the mixing, P1 is pressurized, and the merged droplet is pushed. (H) The droplet is pulled again to complete one “shuttling” cycle.
Figure 6. Schematic of recirculation pattern in a moving droplet. The coordinate system for the streamlines is located at the center of gravity of the droplet.
studies,21-23 it has been recently discussed that such mixers are unnecessary for segmented-flow systems because recirculation in a moving droplet (Figure 6) has a stirring effect.24 In the present work, we have experimentally proven this effect in picoliter-scale for the first time. Two experiments similar to that in Figure 5 were carried out using deionized water instead of the diluted solution. We measured fluorescence intensity distribution of the merged droplet, which had been shuttled three times (Figure 7A). Before measuring the intensity data, 64 (19) Fujii, T.; Hosokawa, K.; Shoji, S.; Yotsumoto, A.; Nojima, T.; Endo, I. Proc. Micro Total Analysis Systems 98, Banff, Canada, Oct 13-16, 1998; pp 173176. (20) Manz, A.; Bessoth, F.; Kopp, M. U. Proc. Micro Total Analysis Systems 98, Banff, Canada, Oct 13-16, 1998; pp 235-240. (21) Duda, J. L.; Vrentas, J. S. J. Fluid Mech. 1971, 45, 247-260. (22) Snyder, L. R.; Adler, H. J. Anal. Chem. 1976, 48, 1017-1022. (23) Snyder, L. R.; Adler, H. J. Anal. Chem. 1976, 48, 1022-1027. (24) Anderson, R. C.; Bogdan, G. J.; Puski, A.; Su, X. Proc. Micro Total Analysis Systems 98, Banff, Canada, Oct 13-16, 1998; pp 11-16.
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consecutive video frames (32 frames/s) were averaged for noise reduction using the image-processing system. As a result, we obtained a flat profile, which means that the droplet was fully mixed. Figure 7B summarizes the other experiment without shuttling. The mixing process was much slower than with the first case. Solvent Loss and Metering Accuracy. To evaluate the amounts of solvent loss and accuracies of metering in the above two cases, we measured the droplet lengths from the video images. Since the stage of the microscope was fixed in each case, the position of the channel end can be determined from the final (fully mixed) image. A reference point on the other end of the droplet (liquid-air interface) was chosen so that it gave the minimal length (e.g., the centers of the menisci in Figure 7B). Note that the design value of droplet length is 500 µm. The length of the stationary droplet (Figure 7B) decreased from its initial value of 485 µm by 2 and 15% at 30 and 180 s, respectively. The most probable explanation for this solvent loss is evaporation. In the case of the shuttled droplet (Figure 7A), the length decreased from 550 µm by 4.5% during the three cycles. The lost amount is greater than that for the corresponding time in the former case: 2%. Probably, a fraction of the lost amount was trapped on the side walls of the liquid channel, which have relatively rough profiles because of the inexpensive, transparencybased mask for fabrication. This type of solvent loss could be reduced by using a mask with higher precision. The metering
Figure 7. Proof of effectiveness of droplet shuttling in mixing acceleration. Droplets of deionized water and the concentrated fluorescein solution were merged in the same way as in Figure 5, except there was sufficient blackout for measurement. Fluorescence intensity profiles along the liquid channel are plotted. (A) After three shuttling operations. It took 30 s from merging of the droplets. (B) Without shuttling. It took 180 s to get the profile comparable to that shown in (A).
inaccuracies of the initial droplet lengths were -3% (stationary) and +10% (shuttled). We consider that the main cause of metering inaccuracy is uncertainty of the meniscus shape formed by air injection. This effect on the total volume can be reduced by improving the design of channel geometry, i.e., a bottleneck of the liquid channel should be made at the point of air injection. CONCLUSIONS In this paper, it was demonstrated that an HMCV-based liquidhandling scheme enables transportation, metering, and mixing of picoliter-sized liquid samples in a microdevice. Especially, a (25) Miura, K.; Shoji, S. Proc. Micro Total Analysis Systems 98, Banff, Canada, Oct 13-16, 1998; pp 85-88.
droplet-shuttling operation was proven to be effective for mixing contents in the droplet in this diffusion-dominated scale. The next step of our research is to develop a microvalve which can be actively opened or closed. It is needed for (i) controlling multiple droplets independently in branching channels, and (ii) preventing evaporation. Pneumatically driven microvalves4,25 are ideal for these purposes because of their compatibility with the HMCV-based liquid-handling scheme.
Received for review May 27, 1999. Accepted August 11, 1999. AC990571D
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