A Compact Disk-Like Centrifugal Microfluidic System for High

Medical & Biological Engineering & Computing 2013, 51 (5) , 525-535. DOI: 10.1007/s11517-012-1020-7. Q.L. Chen, K.L. Cheung, S.K. Kong, J.Q. Zhou, Y.W...
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Anal. Chem. 2010, 82, 4362–4369

A Compact Disk-Like Centrifugal Microfluidic System for High-Throughput Nanoliter-Scale Protein Crystallization Screening Gang Li,*,† Qiang Chen,† Junjun Li,† Xiaojian Hu,‡ and Jianlong Zhao† Nanotechnology Laboratory, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China, and Department of Physiology and Biophysics, Fudan University, Shanghai 200433, People’s Republic of China A centrifuge-based microfluidic system has been developed that enables automated high-throughput and lowvolume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control. With the advent of the post-genomic era, structural biology is becoming one of the fastest growing fields in current life science research.1-3 Determining the three-dimensional (3D) structures and functions of proteins and their complexes are key objectives of structural biology. In the determination of the 3D structures of proteins, X-ray crystallography is, by far, the most common technique. Despite high demands, several substantial hurdles still exist in obtaining a dataset of quality sufficient for structure determination, principally because of the difficulty in growing diffraction-quality crystals.4-6 Currently, protein crystals are * Author to whom correspondence should be addressed. Tel.: +86-2162511070-8703. Fax: +86-21-62511070-8714. E-mail: [email protected]. † Nanotechnology Laboratory, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences. ‡ Department of Physiology and Biophysics, Fudan University. (1) Teichmann, S. A.; Chothia, C.; Gerstein, M. Curr. Opin. Struct. Biol. 1999, 9, 390–399. (2) Burley, S. K. Nat. Struct. Mol. Biol. 2000, 7, 932–934. (3) Edwards, A. M.; Kus, B.; Jansen, R.; Greenbaum, D.; Greenblatt, J.; Gerstein, M. Trends Genet. 2002, 18, 529–536. (4) Berman, H. M.; Bhat, T. N.; Bourne, P. E.; Feng, Z.; Gilliland, G.; Weissig, H.; Westbrook, J. Nat. Struct. Mol. Biol. 2000, 7, 957–959. (5) Chayen, N. E. Trends Biotechnol. 2002, 20, 98–98.

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produced by trial-and-error methods,7 which are based on parallel and combinatorial mixing of hundreds of different solutions containing various salts, buffers, and precipitating agents with target protein samples. However, the availability of the protein essentially limits the number of conditions that can be screened. Conventional crystallization methods such as hanging-drop vapor diffusion, microbatch, dialysis, and free interface diffusion usually require microliter protein samples for each condition screened.7 Thus, it is often difficult to screen a complete set of initial crystallization conditions for most proteins, especially for eukaryotic proteins.8 Furthermore, using traditional methods, it is also labor-intensive and time-consuming to manually set up a large number of screens. The development of high-throughput, automatic, and miniaturized protein crystallization platforms is thus required. Over the past years, some efforts have been made to automate the manual setup of experiments and reduce the consumption of proteins. To ease this manual and time-consuming screening task, several crystallization robots have been developed.9-12 Although these machines can dispense volumes as small as 10 nL accurately, their cost and complexity make them unavailable to most crystallization laboratories. Moreover, because dispensing robots are sensitive to solution viscosity and surface tension, they must be properly calibrated for each working fluid and have difficulty dispensing small volumes of the highly viscous solutions (such as 2-methyl-1,3-propanediol (MPD) and polyethylene glycol (PEG), respectively, used in crystallography. Recently, advances of microfluidic technology offer new perspectives for high-throughput screening of the crystallization conditions.13-25 Microfluidic systems offer many advantages over conventional crystallization techniques: they provide convection-free environments,26 allow (6) Chayen, N. E. Curr. Opin. Struct. Biol. 2004, 14, 577–583. (7) Bergfors, T. Protein Crystallization: Techniques, Strategies, and Tips: A Laboratory Manual; International University Line Publishers: La Jolla, CA, 1999. (8) Hunte, C.; von Jagow, G.; Scha¨gger, H. Membrane Protein Purification and Crystallization: A Practical Guide; Academic Press: San Diego, CA, 2003. (9) Chayen, N. E.; Stewart, S.; Maeder, D. L.; Blow, D. M. J. Appl. Crystallogr. 1990, 23, 297–302. (10) Chayen, N. E.; Stewart, S.; Baldock, P. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 456–458. (11) Krupka, H. I.; Rupp, B.; Segelke, B. W.; Lekin, T.; Wright, D.; Wu, H. C.; Todd, P.; Azarani, A. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1523–1526. (12) Murthy, T.; Wang, Y.; Reynolds, C.; Boggon, T. J. J. Assoc. Lab. Autom. 2007, 12, 213–218. 10.1021/ac902904m  2010 American Chemical Society Published on Web 05/11/2010

automated screening of crystallization conditions in faster and lessexpensive manners, and can screen multiple variables, in a higherthroughput format, simultaneously. So far, there are two major types of microfluidic devices dedicated to biomolecule crystallization. The first one is a block of polydimethylsiloxane (PDMS) composed of several polymer layers prepared by multilayer soft lithography.13,16,20,21 It contains a complex network of channels for liquid handling or serving as actuation valves. Its commercial version dedicated to highthroughput screening can test 96 potential crystallization conditions with H cos θglass + cos θPDMS

(7)

Equation 7 reveals that the condition for spontaneous filling is highly sensitive to the aspect ratio of the microchannel. A wide and shallow channel is more favorable for water filling. The second (37) Adam, N. Nature (London, U. K.) 1957, 180, 809–810. (38) Blanco-Gomez, G.; Glidle, A.; Flendrig, L. M.; Cooper, J. M. Anal. Chem. 2009, 81, 1365–1370.

In the case of water, the contact angle corresponding to glass is ∼12°. Thus, the capillary pressure of eq 12 (Pc) is negative, which stops the liquid flow at the entrance of the chamber. When the external pressure is less than the calculated pressure resulting from eq 12, a microfluid is stably maintained in the microchannel, like the state of “valve-close (OFF)”. Conversely, to switch to the “valve-open (ON)” state, the external pressure must be higher than the pressure needed to push the fluid in a desired direction.

Figure 3. Schematic view of a capillary stop valve: (a) top view and side view of a capillary stop valve region and (b) simplified geometry of the liquid front at the edge of the capillary stop valve.

step is that liquid reaches the capillary stop valve consisting in a microchannel sudden enlargement, i.e., the junction of a metering channel and a reaction chamber (as shown in Figure 3a). At this point, the liquid meniscus must change its curvature to meet the equilibrium contact angle at the expansion walls, except at the bottom wall, which is made of glass; this phenomenon is because the meniscus seems to be pinned at the outlet edge of the PDMS walls but drawn along the bottom wall. In this situation, the surface tension turns out to be a retarding force that stops the fluid from moving forward. Assuming a simplified wedge-shaped liquid front, as schematically shown in Figure 3b, one can express the conditions as follows: dAsglass1 ) W dx

(8)

dAsPDMS1 ) 0

(9)

dA1a )

H dx W dx + cos θglass 2

(10)

WH dx 2

(11)

dV1 )

where dx is an arbitrary change in the wetted length of the bottom wall. Inserting these expressions into eq 5, one can get

[(

Pc ) γ1a

) ]

2 2 cos θglass - 1 1 H cos θglass W

(12)

EXPERIMENTAL SECTION Fabrication. The device is comprised of a hybrid PDMS/glass disklike microfluidic chip, formed by bonding a PDMS (Sylgard 184, Dow Corning) plate containing three-dimensional (3D) microchannel networks to a glass wafer (2 in. diameter and 2 mm thickness, Zhonghe Special Glass Co., Ltd.). The glass wafer was carefully cleaned before bonding with PDMS using a Piranha cleaning procedure: (1) Immerse in boiling Piranha solution (30% H2O2:98% H2SO4 ) 1:4, v/v) for 15 min. (Caution: Piranha solution is very dangerous, with the majority of its components being acidic and highly corrosive; it must be handled extremely carefully.) (2) Wash in flowing deionized (DI) water for 5 min. (3) Dry in a filtered nitrogen stream. The PDMS plate was fabricated from a two-level SU-8 master, using standard soft lithography techniques.31 Briefly, a two-level master was prepared from a multilayer SU-8 process. Sylgard 184 then was mixed in a 10:1 (w/w) ratio of resin to cross-linker and poured over the master to form structured microfluidic substrates. After curing, the PDMS was peeled off the master and punched for inlet/outlet ports. Finally, the PDMS was reversibly bonded to a glass substrate. Typical dimensions used in the device are as follows: precipitant feeding channel, 15 µm (height) × 140 µm (width); precipitant metering channel, 15 µm (height) × 140 µm (width) × 14.3 mm (length); protein metering channel (including a corresponding part of the common distribution channel), 15 µm (height) × 100 µm (width) × 20 mm (length); reaction chamber, 120 µm (height) × 1000 µm (length) × 1000 µm (width); air vent channel, 120 µm (height) × 30 µm (width); and oil channel, 120 µm (height) × 200 µm (width). To guarantee the reliability of the capillary stop valve, restriction openings for metering channels are designed to eliminate the curvature of a meniscus of a filling front and thus increase the pressure barrier of capillary stop valves (as shown in Figures 1 and 2). Crystallization Experiments. For the crystallization experiments reported in this work, two proteins were used to investigate the application of the microfluidic device in protein crystallization: chicken egg-white lysozyme (purchased from Sigma-Aldrich) (25 mg/mL in 0.1 M sodium acetate buffer, pH 4.6) and cyan fluorescent protein CyPet (obtained by purification from Escherichia coli strain Rosetta (DE3) pLysS, using the reported procedures39) (25 mg/mL in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.0, 150 mM sodium chloride, 2 mM β-mercaptoethanol). These two protein samples were provided as gifts. All crystallization solutions were prepared with distilled water and filtered before use. The standard proce(39) Zhou, Y.; Song, J.; Weng, L.; Hu, X.; Ding, Y.; Zhang, Z. Protein Pept. Lett. 2007, 14, 928–932.

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dure for growing crystals in the microfluidic disks consisted of three steps. First, protein solution and precipitant solutions were pipetted into the protein inlet and precipitant inlets, respectively. The liquid samples filled the feed channels and metering channels under the action of capillary forces, and stopped at the junction of microchannel and the reaction chamber, because of the capillary stop valve. The microfluidic disk then was mounted on a spincoater (KW-4A, Chemat Technology, Inc.), and protein solution and precipitant solutions were driven into the reaction chamber by high-speed centrifugation (>6000 rpm) for 1 min. After introducing and mixing the protein and the precipitant solutions, the microchannels connected to the reaction chambers were sealed with liquid paraffin to minimize the evaporation of solutions. After that, the device was preserved in a refrigerator at 4 °C for a microbatch crystallization. The chamber was examined by an inverted microscope (Model IX-51, Olympus, Tokyo, Japan) at regular intervals of 1 h. Optical micrographs were obtained using a CCD camera (Model DP70, Olympus, Tokyo, Japan). Crystal Extraction and Diffraction Studies. Prior to crystal extraction, the chips were placed inside a humidity hood, to avoid evaporation. The PDMS layer of the chip was peeled from the substrate, and 1 µL of cryoprotectant (25% glycol with mother liquor) was pipetted into the chambers, which contained crystals. The crystals then were picked up with a CryoLoop (Hampton Research) and flash-frozen in liquid nitrogen. X-ray diffraction (XRD) data were collected at 100 K on an X8 PROTEUM (PLATINUM 135 CCD detector with Microstar microfocus X-ray source, Bruker AXS, USA) with a 30-s exposure and 0.5° oscillation. RESULTS AND DISCUSSION Liquid Operation. Liquid operations, such as loading, metering, dispensing, and mixing, are indispensable for screening protein crystallization conditions. Often, surface effects are dominant in the microscale. Therefore, we took advantage of capillary action to control the water-phase liquid flow in the microfluidic disk, such as priming and valving. To demonstrate the functionality of the device, we first tested the ability of spontaneous priming and the reliability of capillary stop microvalves in the device. Here, two food dyes were used as samples to facilitate visualization. Red food dye represents the protein sample and blue food dye represents the precipitant. When using the device, the red food dye and the blue food dye were first introduced into the protein inlet port and the precipitant inlet ports, respectively, using pipets. Each port is connected to a microchannel with a width of 100 µm and a depth of 15 µm. Because of the hydrophilicity of the glass bottom wall (with a measured contact angle of ∼12°), such a crosssection can generate an adequate capillary pressure in the microchannel to drive the liquid flow. When dispensing a drop of sample in the inlet port, the liquid spontaneously entered the feed channels and the metering channels by capillary action until it reached the abrupt enlargement of the channel (as shown in Figure 4a). (Video given in the Supporting Information shows the typical self-priming process of the microfluidic CD.) The abrupt enlargement plays the role of a valve,35,40,41 which pins the interfacial meniscus of the liquid and alters the curvature of the meniscus. The capillary pressure in the liquid becomes negative and counteracts the external pressure (i.e., the capillary pressure at the inlet or the gravity of the stored liquid in the reservoir) to 4366

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Figure 4. Photographs of different stages during liquid operation: (a) priming and (b) metering and mixing. With priming, two food dye fluids spontaneously fill the metering channels and stop at the inlet of the reaction chamber. With metering and mixing, two metered food dye fluids are centrifuged into the reaction chamber, generating a mixed droplet.

finally stop the fluid moving forward. This type of passive valve, based on liquid-surface interactions (i.e., hydrophobic and hydrophilic) and specific geometric design, is very attractive for low-cost microfluidic devices, because it involves no moving parts and it is easy to fabricate and integrate seamlessly into the microfluidic devices. A centrifugal fluidic platform can easily achieve the valve “openings” by adjusting the rotation speed, according to the critical bursting pressure of the valve. After filling the feed channels and the metering channels with the protein sample and precipitants, the microfluidic disk was mounted on the spinstand and spun to split the sample and push them beyond the valve into the reaction chamber by the centrifugal force. In the microfluidic disk, the basic design of the (40) Cho, H.; Kim, H. Y.; Kang, J. Y.; Kim, T. S. J. Colloid Interface Sci. 2007, 306, 379–385. (41) Zimmermann, M.; Hunziker, P.; Delamarche, E. Microfluid. Nanofluid. 2008, 5, 395–402.

Figure 5. Optical micrographs showing the advancing menisci of three different types of crystallization solutions stop at the sudden opening of the capillary valve: (a) a typical salt solution (2 M NaCl/ 0.1 M NaAc pH 4.6, contact angle on glass ∼12°, contact angle on PDMS ∼108°), (b) a typical polymer solution (30% w/v PEG 8000/ 0.1 M (CH3)2AsO2Na pH 6.5/0.2 M AmSO4, contact angle on glass ∼18°, contact angle on PDMS ∼102°), and (c) a typical organic solution (70% v/v MPD/0.1 M HEPES pH 7.5, contact angle on glass ∼5°, contact angle on PDMS ∼64°). Dashed lines were added to show the menisci.

metering structure is based on a section of conduit (i.e., metering channel) combined with capillary stop valves (as shown in Figure 2). For each unit, one of the capillary stop valves is located at the outlet of the metering channel and the others, which are connected to air vents, are placed at the inward turning point of the microchannel. Based on these features, when the rotational speed of the disk exceeds a threshold value, the centrifugal force causes the protein liquid columns and the precipitant liquid columns, which are confined in the microchannel between valves, to burst into the corresponding reaction chamber at the same time, generating 24 discrete mixed liquid droplets with well-defined metering (see Figure 4b). Furthermore, the volume ratio of protein solution to precipitant solution can be varied by multiple dispensings of protein solution or precipitant solution. When the microfluidic disk is applied to the screening of protein crystallization conditions, the reliability of metered liquid volume must be ensured, because the variation of metered liquid volume can potentially affect the crystal results. To investigate the precision of the proposed fluidic metering structure, the microscopic images of the final metered droplet were captured using a CCD camera. The captured images were analyzed using an image analysis software (Image-Pro Plus) to determine the droplet area. The measured area was converted to a corresponding volume by multiplying the value of the reaction chamber depth, 120 µm. Five different microfluidic disks, containing 24 units on each CD, were tested. The mean volume of the liquid droplets in all reaction chambers was measured to be 31 nL with a coefficient of variation (CV) of 2.8%, which proved that our proposed microfluidic disk has reliable accuracy at the nanoliter scale. On the other hand, to evaluate the suitability of the microfluidic disk for dispensing real crystallization buffers, it was tested with 96 different precipitant solutions proposed in the literature (EasyXtal Classics Suite, Qiagen). These precipitants could be classified into three groups: salt solution group, polymer solution group, and organic solution group. The experiment results showed that there was no distinct difference among three group solutions during priming, and all three group solutions can spontaneously fill the feed channels, because of the capillary force after introduction. Nevertheless, it was observed that there were different front shapes and locations for the primed three group solutions while they were stopped at the entrance to the reaction chamber, because of the capillary valve (as shown in Figure 5). The meniscus of a stopped salt solution just encounters the valve edge, whereas the menisci of polymer solution and organic solution had

a slight spreading along the bottom of the valve. However, these variations are so small that they are considered negligible. As a result, droplets of all these solutions with precise volumes were successfully dispensed and introduced into the reaction chambers. Protein Crystallization. The proposed microfluidic disk provides a simple and economical platform for high-throughput screening. The fabrication of the microfluidic disk is simple and fast, and the crystallization process only uses a spinstand, without the need of syringe pumps, pressure controllers, or mechanical valves. As the microfluidic disk spins, a single injection of liquid can be simultaneously split into multiple nanoliter-sized aliquots and dispensed into an array of reaction chambers for different reactions. To evaluate the feasibility of the proposed microfluidic disk for high-throughput screening, we applied it to screening of protein crystallization conditions. The microfluidic disk proposed in this study can easily be used to establish a microbatch protocol for protein crystallization. With the parallel, discrete nanoliter microfluidic system, the traditional microbatch protocol was simplified: the protein sample is introduced by a single pipetting, and the protein sample and multiple precipitants are metered, dispensed, and mixed using a spinning, producing an array of droplets, with each droplet representing an independent crystallization experiment. Generally, the initial suitability evaluation of new protein crystallization platforms is usually conducted using proteins that crystallize readily. Following this tradition, we first monitored the crystallization of chicken egg-white lysozyme in the microfluidic disks under different eight conditions (triple runs for each condition). As a result, crystal growth was observed in the chips, and the different lysozyme crystal shapes were also obtained depending upon the precipitating salt species and the volume ratio of protein to precipitant. (Table S1 in the Supporting Information gives screening results for the crystallization of lysozyme.) Figure 6 shows four different crystal shapes obtained in the chips with different crystallization conditions. The tetragonal crystals were grown in 5% (w/v) NaCl at pH 4.5. The size of the crystals is typically 100 µm, large enough for XRD studies. Beside sample volume miniaturization, microfluidic systems present a supplementary advantage for crystal growth: smaller droplets also lead to more rapid equilibration times and allow for a fast rate of crystal formation. With the microfluidic systems, crystals 100 µm in size were observed at incubation times as short as 2 h, which is faster than that observed with the normal-scale droplets in a standard microplate.42,43 To further illustrate the suitability of the microfluidic disk, we then studied other proteins that might be more difficult to crystallize. Here, we examined the crystallization of the cyan fluorescent protein CyPet in the chips using the microbatch protocols and 12 different crystallization buffers (double runs for each condition). (Table S2 in the Supporting Information gives screening results for the crystallization of CyPet.) The CyPet protein also crystallized in the chip and exhibited different shapes (see Figure 7). To confirm the quality of the crystals obtained with the microfluidic CD crystallization platform, we analyzed them using (42) Santarsiero, B.; Yegian, D.; Lee, C.; Spraggon, G.; Gu, J.; Scheibe, D.; Uber, D.; Cornell, E.; Nordmeyer, R.; Kolbe, W. J. Appl. Crystallogr. 2002, 35, 278–281. (43) Fogg, M.; Wilkinson, A. Biochem. Soc. Trans. 2008, 36, 771–775.

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Figure 6. Optical micrographs of lysozyme crystals grown in the microfluidic CD under different conditions: (a) the tetragonal crystals growing with 5% (w/v) NaCl at pH 4.5, (b) the needlelike crystals growing with 0.5 M NaHCO3 at pH 4.5, (c) the platelike crystals growing with 3% (v/v) PEG 1000/5% (w/v) NaCl at pH 4.5, and (d) the rodlike crystals growing with 3% (v/v) PEG 6000/5% (w/v) NaCl at pH 4.5. Protein concentration was 25 mg/mL. Note that panels (a), (b), and (c) were obtained at a 1:1 volume ratio of protein to precipitant, whereas panel (d) was obtained in a 1:2 volume ratio of protein to precipitant. (Scale bar ) 50 µm.)

Figure 7. Optical micrographs of CyPet crystals grown in the microfluidic CD under different conditions: (a) the needlelike crystal clusters growing with 25% (w/v) PEG 4000/0.1 M NaAc (pH 4.6)/0.2 M AmSO4, (b) the sea urchin-like crystals growing with 30% (w/v) PEG 4000/0.1 M NaAc (pH 4.6)/0.2 M AmAc, (c) the needlelike crystals growing with 30% (w/v) PEG 4000/0.1 M Tris-HCl (pH 8.5)/ 0.2 M NaAc, and (d) the rodlike crystals growing with 30% (w/v) PEG 5000 MME/0.1 M MES (pH 6.5)/0.2 M AmSO4. Protein concentration was 25 mg/mL. All conditions were operated in a 1:1 volume ratio of protein to precipitant. Abbreviations: MME, monomethyl ether; MES, 2-(N-morpholino)ethanesulfonic acid. Scale bar ) 50 µm.

XRD. Figure 8 shows the diffraction patterns of a single lysozyme crystal grown in our microfluidic CD. Preliminary XRD studies revealed that the crystals obtained in the chips diffracted at least to 2.4 Å, which is the resolution limit of this X8 PROTEUM machine. We believe that it is possible for the crystals to allow better resolution data to be taken if using a synchrotron radiation 4368

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Figure 8. Example of a diffraction pattern from a lysozyme crystal obtained using the microfluidic CD. The crystal diffracted to the edge of the CCD, and the resolution ring of 2.40 Å was shown.

source. After collecting 20 images, the crystal space group was indexed to P422 with a cell constant of a ) b ) 78.8 Å, c ) 37.0 Å, which was in accordance with one of the reported lysozyme space groups.44 These data demonstrate that the crystals produced with this device are suitable for high-quality XRD analysis. In the present study, the reversible bonding of the microfluidic disk not only allows easy harvesting of the protein crystals from the reaction chamber, but it also allows the device to be reusable. To reuse the chip, we just disassemble it, clean the PDMS sheet and the glass substrate with ultrasound and piranha solution, respectively, and then bond them again. This significantly reduces the cost of screening the protein crystallization conditions. CONCLUSIONS In summary, we have developed a simple and economical highthroughput protein crystallization screening platform where protein crystallization experiments can be performed in a highly parallel manner with ultralow-volume protein solutions and nearly zero dead volume. Through the integration of capillary stop valves in a PDMS-glass hybrid microfluidic chip, this system could manipulate the motion of liquids, performing priming, metering, cutting, dispensing, and mixing by simply pipetting and spinning. Based on the combination of capillarity forces and centrifugation, protein sample and precipitants are spontaneously metered to the desired volumes and dispensed into reaction chambers for crystallization. The effectiveness of the proposed microfluidic system was successfully demonstrated by performing the crystallization of lysozyme and CyPet in chips. In applying this system to protein crystallization, we found that crystallization experiments in the chip result in faster crystal growth than conventional techniques. Furthermore, because of the reversible bonding of the microfluidic disk, the diffraction-quality crystals can be conveniently recovered for X-ray analysis. Overall, the present disk has greater feasibility and flexibility than current systems, because of its low sample consumption, small dead volume, and ease of (44) Blake, C. C. F.; Koenig, D. F.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757–761.

operation. It offers a simple, inexpensive, and reliable way to perform multiple nanoliter-volume reactions in parallel and should be ideally suitable for various applications such as enzyme assays, protein crystallization, drug discovery, and combinatorial chemistry. ACKNOWLEDGMENT The authors thank Prof. Xu Yan-hui (IBS, Fudan University) for the support of X-ray diffraction. This work was supported in part by a grant from the Major State Basic Research Development Program of China (Nos. 2005CB724305 and 2009CB918602), a grant from the National High Technology Research and Development Program of China (No. 2006AA02Z136),

and a grant from the National Natural Science Foundation of China (No. 60906055). SUPPORTING INFORMATION AVAILABLE Listings of the screening results of crystallization of lysozyme (Table S1) and CyPet (Table S2) (PDF). Movie showing a typical self-priming process of the microfluidic CD (Movie S1). (AVI file) This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 19, 2009. Accepted April 27, 2010. AC902904M

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