Nanoliter Dispensing Method by Degassed Poly(dimethylsiloxane

Anal. Chem. 2007, 79, 4924-4930

Nanoliter Dispensing Method by Degassed Poly(dimethylsiloxane) Microchannels and Its Application in Protein Crystallization Xuechang Zhou,† Lana Lau,† Wendy Wai Ling Lam,‡ Shannon Wing Ngor Au,‡ and Bo Zheng*,†

Department of Chemistry and Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

This paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannels were reversibly bound to arrays of microwells. The PDMS elastomer was predegassed and served as an internal vacuum pumping source. Various aqueous solutions were infused into arrays of microwells through the reversibly sealed PDMS microchannels. Microwells fabricated in PDMS, poly(methyl methacrylate) (PMMA), and glass were all compatible with this dispensing method. By removing the PDMS microchannels, arrays of droplets confined in the microwells were obtained. Multiplex reaction and screening at the nanoliter scale were carried out by binding two such arrays of microwells to form microchambers. We applied this method to screening the crystallization conditions of four known proteins. Long-term incubation of over 2 months was achieved by employing glass microwells. An unknown protein was then crystallized using the screening method in microwells. The crystals with sufficient size were harvested from the reversibly bound microwells. X-ray diffraction with a resolution of 3.1 Å was obtained. Liquid dispensers at the nanoliter scale offer the possibility of high throughput of chemistry and biology, allowing numerous experiments to be performed rapidly in parallel, while consuming little amounts of reagents. Many sophisticated methodologies of liquid dispensing have been developed, and some have been incorporated in robotic systems.1 For example, dispensers using micropistons or microsolenoid components have been commercialized and can pipet as little as 50 nL of liquid. Piezoelectric dispensing has been adopted for better accuracy and consistency, especially in inkjet printing systems.2-4 In pintool systems, tiny * To whom correspondence should be addressed. Phone: 852-2609-6261. Fax: 852-2603-5057. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biochemistry. (1) Persidis, A. Nat. Biotechnol. 1998, 16, 488-489. (2) Lemmo, A. V.; Rose, D. J.; Tisone, T. C. Curr. Opin. Biotechnol. 1998, 9, 615-617. (3) Schober, A.; Gunther, R.; Schwienhorst, A.; Doring, M.; Lindemann, B. F. BioTechniques 1993, 15, 324-329. (4) Onnerfjord, P.; Nilsson, J.; Wallman, L.; Laurell, T.; Marko-Varga, G. Anal. Chem. 1998, 70, 4755-4760.

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metal needles are dipped into the target liquid, and 2-500 nL of liquid can be delivered by capillary and surface tension forces.5 Acoustic dispensing6,7 has also been used to inject droplets of liquid from a reservoir.8 All these methods have achieved some success with notable limitations. For example, many of these methods suffer from inconsistent results from liquids of different viscosities or surface tensions, and nozzle obstructions or clogs might occur even in the mature dispensing technologies.9 In addition, these methods require sophisticated electronic and mechanical controlling systems and are costly in both manufacturing and maintenance. In contrast with the above-mentioned methods and commercial equipments, poly(dimethylsiloxane) (PDMS)-based microfluidic system offers a low-cost and simple platform for liquid dispensing at the nanoliter scale. Simply applying a pressure gradient between the two ends of the PDMS microchannel was found problematic due to the bubble formation inside.10 Yamada and Seki exploited the hydrophobicity of PDMS to control the movement of aqueous solutions.11 Nanoliter solution was infused into PDMS microchambers with accurate control of the volume. However, this method was limited to aqueous solutions and required precise control of pressure on the solution. On the other hand, the high solubility and permeability of air in PDMS was exploited in PDMS microfluidic channels to generate flow12,13 and to dispense nanoliter liquid into microwells or microchambers.14,15 Nuzzo and co-workers developed a channel outgas technique,10,16 in which the inlet of the microfluidic device was immersed in the target solution while the whole system was in vacuum. As the device was brought back to atmosphere, the target solution was infused into the PDMS (5) Wolcke, J.; Ullmann, D. Drug Discovery Today 2001, 6, 637-646. (6) Wood, R. W.; Loomis, A. L. Philos. Mag. 1927, 4, 417-436. (7) Elrod, S. A.; Hadimioglu, B.; Khuriyakub, B. T.; Rawson, E. G.; Richley, E.; Quate, C. F.; Mansour, N. N.; Lundgren, T. S. J. Appl. Phys. 1989, 65, 34413447. (8) Richard, E. Drug Discovery Today 2002, 7, s32-s34. (9) Felton, M. J. Anal. Chem. 2003, 75, 397A-399A. (10) Monahan, J.; Gewirth, A. A.; Nuzzo, R. G. Anal. Chem. 2001, 73, 31933197. (11) Yamada, M.; Seki, M. Anal. Chem. 2004, 76, 895-899. (12) Hosokawa, K.; Sato, K.; Ichikawa, N.; Maeda, M. Lab Chip 2004, 4, 181185. (13) Randall, G. C.; Doyle, P. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1081310818. (14) Eddings, M. A.; Gale, B. K. J. Micromech. Microeng. 2006, 16, 2396-2402. (15) Hansen, C. L.; Skordalakes, E.; Berger, J. M.; Quake, S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16531-16536. (16) Fosser, K. A.; Nuzzo, R. G. Anal. Chem. 2003, 75, 5775-5782. 10.1021/ac070306p CCC: $37.00

© 2007 American Chemical Society Published on Web 06/05/2007

Figure 1. Photograph of a PDMS microchannel patch on top of a patch of a 3 × 52 array of microwells, with 52 segments of Teflon tubing preloaded with the aqueous solution inserted into the inlets of the microchannels. The microwells were filled with Fe(SCN)x3-x aqueous solution via the degassed PDMS microchannels.

device due to the reduced pressure in the microchannel. This method consumed at least 20 µL of sample and was incompatible with solutions containing volatile solvents.16 Hosokawa et al. improved this method by degassing the PDMS device in vacuum and then adding the target solution to the inlet in atmosphere.12 Hansen et al. proposed a method of infusing liquid into nanoliter PDMS chambers by driving the air in the chamber out to atmosphere through PDMS with high external pressure on the liquid.15 The drawback of this method was the complex fabrication and operation of the PDMS device. These liquid-dispensing methods using the permeability of air in PDMS were effective for disposable devices for short-term (hours) analysis; however, long-term (days to weeks) storage, incubation, or reactions using these methods would be difficult due to the permeability of water and many other solvents in PDMS.13 Herein, we describe an alternative approach to dispensing nanoliter liquid using a PDMS microfluidic system. The dispensing setup consisted of a PDMS microchannel patch and a microwell patch (Figure 1). The PDMS microchannel patch was predegassed and served as an internal vacuum pumping source.12,16 Through the degassed PDMS microchannels, nanoliters of single or multiple reagents were aspirated into arrays of microwells. The microwell patch could be made of PDMS, PMMA, or glass, and the issue of the long-term storage or incubation of the reagents in PDMS microchannels could be solved. In this article, we carried out several investigations while implementing this idea: (1) characterizing the efficiency of the dispensing method; (2) generating arrays of reagent droplets confined in open microwells; (3) illustrating the method in protein crystallization. EXPERIMENTAL SECTION Fabrication of Microchannel Patches and Microwell Patches. PDMS microchannel and microwell patches were

fabricated using soft lithography.17,18 Briefly, a silicon wafer containing SU-8 (MicroChem) relief structure complementary to the microchannels or the microwells was fabricated using a photolithographic method. Microchannel and microwell patches with the thickness of 3 mm were then fabricated by casting a 10:1 (in weight) mixture of PDMS precursor and curing agent (Sylgard 184, Dow Corning) against the silicon master and curing the mixture at 60 °C for over 2 h. For microwell patches made in glass, glass microscope slides patterned by SU-8 photoresist with the thickness of 100 µm were first fabricated by photolithography. Only the microwell area was exposed. The glass slide was then soaked in 10% HF aqueous solution for 75 min, followed by rinsing with deionized water and drying with N2 flow. The SU-8 photoresist on the microscope slide was removed by soaking the slide in a solution of H2SO4 and H2O2 (v/v 3:1) for 10 min and then rinsing with deionized water. The depth of the glass microwell was measured by a profilometer (Tencor Alpha-step 500 surface profiler) as 110 µm. Poly(methyl methacrylate) (PMMA) microwells were fabricated by drilling PMMA slides using drill press with a cobalt microdrill bit with the diameter of 0.30 mm. Nanoliter Liquid Dispensing. A PDMS microchannel patch was reversibly bound with a microwell patch made in PDMS, PMMA, or glass, forming a microchip. The microchip was placed in a vacuum desiccator for 10 min of degassing at 6 kPa. After the microchip was brought back to atmosphere, a segment of Teflon tubing prefilled with the reagent was inserted into the inlet of the microchannel to start the dispensing. After the completion of the dispensing process, the microchip was covered with a layer of silicone oil (Fluid 5, Brookfield). The PDMS microchannel patch was then removed from the microwell patch. To prepare Teflon tubing prefilled with the reagent, a piece of Teflon tubing was attached to a 50 µL syringe (Hamilton). The tubing was then inserted into the reagent. By pulling back the plunger of the syringe, the reagent was aspirated into the tubing at the microliter scale. Finally the segment of the Teflon tubing containing the reagent was cut off and inserted into the PDMS microchannel for the dispensing. Screening Conditions of Protein Crystallization. Patches with 156 microwells were fabricated for screening conditions of protein crystallization. The 156 microwells were divided into 52 groups of 3 microwells (Figure 1). The microwells in each group were filled with the same precipitant solution. Four proteins were used for the screening test: chicken egg-white lysozyme (Wako, Japan) (60 mg/mL in 50 mM sodium acetate buffer, pH 4.5), thaumatin (Wako, Japan) (50 mg/mL in 0.1 M N-(2-acetamido)iminodiacetic acid buffer, pH 6.5), xylanase (Sigma-Aldrich) (10 mg/mL in 43% (w/v) glycerol/180 mM phosphate buffer, pH 7.0), and horseradish peroxidase (Sigma-Aldrich) (5 mg/mL in 0.1 M phosphate buffer, pH 7.2). The 50 precipitant reagents were from a commercial screening kit (Crystal Screen, Hampton Research). Microwell patches containing protein solution and precipitant reagents were prefabricated separately by using the dispensing method mentioned above and were covered with silicone oil. The two microwell patches were aligned under the microscope and bound together for mixing. The resulting microchip was incubated (17) McDonald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35, 491-499. (18) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.

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in a paraffin oil (BDH, England) bath at room temperature (21.522.5 °C). The crystals of the proteins were examined by using a polarized light microscope (Leica MZ 16, Germany) equipped with a CCD camera (SPOT Insight, Diagnostic Instruments). Crystallization and X-ray Diffraction of an Unknown Protein. An unknown protein, HP0753, was obtained by expressing the gene in E. coli followed by affinity purification. The final concentration of the protein was 6 mg/mL in 50 mM Tris buffer, pH 7.5/200 mM NaCl/0.5 mM EDTA/1 mM dithiothreitol/0.2 mM phenylmethanesulphonylfluoride/0.2 mM benzamidine. The precipitant reagents were from a commercial crystal screening kit (Wizard I, Emerald BioSystems). Two PDMS microwell patches that contained the protein solution and the precipitant reagents were fabricated and then bound. The microwell patches were placed in paraffin oil for incubation. After incubation for 24 h at room temperature (21.5-22.5 °C), the bound microwell patches were peeled apart while they remained in paraffin oil. 1 µL of glycol as the cryoprotectant was pipetted into the microwells which contained crystals. Then the crystals were picked up with a cryoloop (Hampton Research) and flash-frozen in liquid nitrogen. The X-ray diffraction data was collected at 100 K using a Rigaku MicroMax-007 X-ray generator and recorded by the R-AXIS IV++ IP detector. RESULTS AND DISCUSSION Dispensing Liquid by Degassed PDMS Microchannels. While the PDMS microchannel patch was placed in a vacuum chamber, the air dissolved in the PDMS was gradually depleted (Figure 2a). Once the PDMS patch was taken out and exposed to atmosphere, air slowly diffused back into the PDMS.12,19 The diffusion occurred both on the surface and through the PDMS microchannel (Figure 2b). When the reagent was placed at the inlet to block the air flow into the PDMS microchannel, the internal pressure in the microchannel decreased due to the continuous dissolving of air inside the microchannel into PDMS (Figure 2c). As a result, a pressure difference was generated between the internal and external parts of the microchip. The reagent was aspirated into the microchannel and gradually filled up the whole vacancy of the closed system, without the formation of air bubbles (Figure 2d). The dispensing method using degassed PDMS is compatible with microwells of different geometries, sizes, and substrate materials. Reagents of different chemical composition were successfully filled into arrays of microwells by using parallel microchannels (Figure 3a). The small dimension of the microchannel (50 µm in width and 10 µm in height) facilitated the alignment on top of the microwells of different volumes and geometries (Figure 3b). The elasticity of PDMS and the negative pressure in the microchannels allowed the PDMS microchannel patch to make conformal contact with many different substrates and to form leakfree reversible sealing. As a result, microwells fabricated in glass (Figure 3c) and PMMA (Figure 3d) were filled smoothly using degassed PDMS microchannels. Loading Liquid by Teflon Tubing. Instead of simply adding a droplet of liquid at the inlet of PDMS microchannels, the liquid was loaded into the inlet by inserting a segment of Teflon tubing (19) Merkel, T. C.; Bondar, V. I.; Nagai, K.; Freeman, B. D.; Pinnau, I. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 415-434.


Figure 2. Schematic illustration of the method of dispensing liquid into an array of microwells through the degassed PDMS microchannel: (a) the degassing of the PDMS patch in a vacuum chamber, (b) the redissolving of air into PDMS from atmosphere, (c) the aspiration of the liquid into the microchannel and the microwells after a segment of tubing preloaded with the liquid was inserted into the inlet, and (d) the completion of the dispensing process when the liquid filled up the whole vacancy.

Figure 3. (a) Micrograph of four arrays of circular-shaped microwells filled with aqueous solutions of Fe(SCN)x3-x (red), Cu(NH3)62+ (blue), NaCl (colorless), and KMnO4 (purple), respectively. (b) A micrograph of Fe(SCN)x3-x aqueous solution in rectangular-shaped microwells with decreasing volume. (c) A micrograph of Fe(SCN)x3-x aqueous solution in glass microwells. (d) A micrograph of Fe(SCN)x3-x aqueous solution in PMMA microwells. Scale bar: 2 mm.

that was prefilled with the liquid (Figures 1 and 2). No loss of liquid was observed during the process of prefilling and inserting the Teflon tubing. Due to the extremely small opening ( 36), the taverage increased almost linearly with n. Therefore, to achieve short average filling time and increase the throughput, parallel microchannels should be employed to fill a large number of microwells. We also studied the ttotal of nine microwells with liquids of different wettabilities and viscosities. The texposure was constant at 1 min. Liquids with various viscosities (up to 45 mPa s) and wettabilities were dispensed reliably through the degassed PDMS microchannels (Figure 4d). Removing the PDMS Microchannel Patch. In the present dispensing method, the PDMS microchannel patch was reversibly bound to the microwell patch. Such reversible sealing of the PDMS microchannel had a number of applications such as surface patterning,20-22 microfabrication,23 and cell analysis.24,25 In the (20) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408-2413. (21) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500-508. (22) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335-373. (23) Rodriguez, I.; Spicar-Mihalic, P.; Kuyper, C. L.; Fiorini, G. S.; Chiu, D. T. Anal. Chim. Acta 2003, 496, 205-215.


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Figure 5. (a) Schematic illustration of the process of removing the microchannel patch in the presence of silicone oil. (b) PDMS microwells filled with Fe(SCN)x3-x (red), Cu(NH3)62+ (blue), NaCl (colorless), and KMnO4 (purple) aqueous solutions, respectively. (c) PDMS microwells with a volume ratio of 1:2:4 filled with Fe(SCN)x3-x aqueous solutions. (d) Glass microwells filled with Fe(SCN)x3-x aqueous solutions. (e) PMMA microwells filled with Fe(SCN)x3-x aqueous solution. Scale bar: 2 mm.

present study, the PDMS microchannel patch was removed after the dispensing process to generate arrays of liquid droplets in the open microwells. However, directly peeling off the PDMS microchannel patch in air caused two problems: (1) the evaporation of the liquid in nanoliter volume was rapid; (2) the liquid in some microwells spilled over the surface of the microwell patch. To solve these problems, we placed the reversibly bound patches in a silicone oil bath before removing the PDMS microchannel patch. The evaporation of the liquid was effectively prevented by the oil layer on top. As the PDMS microchannel patch was lifted from the microwell patch, silicone oil filled the gap and the PDMS microchannels (Figure 5a). As a result, the liquid in the PDMS microchannels merged into the droplet in the nearest microwell, while all the droplets remained confined in the microwells (Figure 5a). By removing the PDMS microchannels presented in Figure 3, arrays of droplets with different reagents (Figure 5b) or with different volumes (Figure 5c) in PDMS microwells and droplets in glass microwells (Figure 5d) or in PMMA microwells (Figure 5e) were obtained. The volume of the liquid dispensed into each microwell was determined by the volume of the microwell. The PDMS microchannels were 10 µm in height, 50 µm in width, and 2 mm long between two neighbor microwells, while the typical microwells were 400 µm in diameter and 150 µm in height. The volume ratio (24) Khademhosseini, A.; Yeh, J.; Eng, G.; Karp, J.; Kaji, H.; Borenstein, J.; Farokhzad, O. C.; Langer, R. Lab Chip 2005, 5, 1380-1386. (25) Yu, H.; Alexander, C. M.; Beebe, D. J. Lab Chip 2007, 7, 388-391.


Figure 6. (a) Schematic illustration of the process of the mixing of reagents from two arrays of microwells. Different colors represent different reagents. (b) A micrograph of a 6 × 6 array of bound patches of microwells containing Fe(SCN)x3-x solutions with a concentration gradient.

of the microchannel to the microwell was 1:20; thus, the volume contribution of the liquid from the microchannel to the droplets in the microwell was negligible. Multiplex Reaction by Binding Two Microwell Patches. With the array of droplets in the open microwells, we were able to carry out multiplex reaction by binding two arrays of microwells containing the reactants to form an array of microchambers (Figure 6a). In our experiments, the two microwell patches were taken out of the silicone oil bath first, with the surfaces of the patches still covered by a thin layer of oil. With microwells of 400 µm diameter, we were able to align the two microwell patches by eyes. We envision that the addition of alignment posts and holes would facilitate the rapid and precise assembly of the two microwell patches.26,27 During the binding process, the silicone oil between the two microwell patches was pushed out gradually. The drainage of the oil film allowed the two microwell patches to approach each other slowly and facilitated the alignment by eyes. In addition, the oil film prevented the trapping of air bubbles between the two patches. Confined in the microwells and surrounded by silicone oil, the droplets had a convex-shaped surface, (26) Anderson, J. R.; Chiu, D. T.; Jackman, R. J.; Cherniavskaya, O.; McDonald, J. C.; Wu, H. K.; Whitesides, S. H.; Whitesides, G. M. Anal. Chem. 2000, 72, 3158-3164. (27) Lucchetta, E. M.; Lee, J. H.; Fu, L. A.; Patel, N. H.; Ismagilov, R. F. Nature 2005, 434, 1134-1138.

which protruded slightly above the patch surface and facilitated the coalescence of the droplets once the two patches were brought into contact (Figure 6a). Even for large arrays of microwells, the success rate of mixing was 100%, as demonstrated by mixing Fe(NO3)3 and KSCN in 6 × 6 array microwells (Figure 6b) and in the following crystallization screening experiments (3 × 52 array). Crystallizing Proteins in Paired Microwells. The arrays of the liquid droplets in microwells in the present work provided a simple platform for screening conditions of protein crystallization. Protein crystallization is a bottleneck in determining tertiary protein structures from the sequence data.28 The conditions of protein crystallization are usually identified by screening a large number of precipitants under the condition of microbatch or vapor diffusion.29 Recently, much progress has been achieved in developing microfluidic techniques for protein crystallization with the advantages of little sample consumption and high throughput.15,30-33 However, there are still a few issues of protein crystallization in microfluidic devices that impede their adoption in laboratories, including the complex device fabrication and operation15,30 and the requirement of sophisticated instrumentation for flow control.15,31,33 We addressed these issues by performing the protein crystallization through mixing the droplets of protein solution and the droplets of various precipitants prefabricated in two separate microwell patches (Figure 7a). The fabrication of the microwell and microchannel patches was simple and fast, and the crystallization process only used a vacuum desiccator, without the need of syringe pumps,31,33 pressure controllers,15,30 or valves.15,30 For each protein, we set up 150 screening trials. Each trial resulted from binding two microwells, which contained one droplet of the protein solution and one droplet of a precipitant reagent, respectively. The volume of each droplet was ∼20 nL. Only 5 µL of protein solution or less was loaded in the Teflon tubing in the beginning of the experiment for the 150 screening trials. The crystallization conditions of four known proteins were screened using the commercial screening kit, and the crystals were obtained with the optimal precipitants (Figure 7b-e). The screening results (Table S1 in the Supporting Information) were consistent with the previous reports.15,32 To prevent evaporation of the liquid in the microwells, the two bound microwell patches were placed in a paraffin oil during incubation. With the protection of the paraffin oil, PDMS and PMMA microwells allowed an incubation time up to 3 days without obvious loss of water (Figure 7b-d). For longer incubation time, glass microwells were used (Figure 7e), and no apparent loss of water was observed after incubation for over 2 months (Figure S2 in the Supporting Information). After the two microwell patches were bound, there existed an oil layer between the two patches. The oil layer was extremely thin, and no leakage or water diffusion between the droplets was observed during the incubation stage, whether the microwells were fabricated in PDMS, PMMA, or glass. (28) Chayen, N. E. Trends Biotechnol. 2002, 20, 98-98. (29) McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999. (30) Lau, B. T. C.; Baitz, C. A.; Dong, X. P.; Hansen, C. L. J. Am. Chem. Soc. 2007, 129, 454-455. (31) Li, L.; Mustafi, D.; Fu, Q.; Tereshko, V.; Chen, D. L.; Tice, J. D.; Ismagilov, R. F. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19243-19248. (32) Zheng, B.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2005, 44, 2520-2523. (33) Zheng, B.; Roach, L. S.; Ismagilov, R. F. J. Am. Chem. Soc. 2003, 125, 11170-11171.

Figure 7. (a) Schematic illustration of screening the conditions of protein crystallization by mixing protein solution and precipitants from two microwell patches. (b-e) Polarized light micrographs of the protein crystals grown in the microwells with the optimal precipitant. Scale bar: 200 µm. (b) Chicken egg-white lysozyme; precipitant: 0.1 M sodium acetate trihydrate buffer, pH 4.6/2.0 M sodium formate. (c) Horseradish peroxidase; precipitant: 0.2 M CaCl2/28% (v/v) PEG 400/0.1 M (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer, pH 7.5. (d) Xylanase; precipitant: 0.2 M CaCl2/ 28% (v/v) PEG 400/0.1 M HEPES buffer, pH 7.5. (e) Thaumatin crystals in the glass microwell incubated for 14 days; precipitant: 0.8 M potassium sodium tartrate tetrahydrate/0.1 M HEPES buffer, pH 7.5.

We further illustrated the application of the screening method by producing diffraction-quality crystals of an unknown protein, HP0753. HP0753 is an important protein in flagellar biosynthesis. Although there has been some success in the expression and purification of this protein, no crystal structure has been published. The protein crystals grew up to 200 µm after 24 h of incubation using the optimal precipitant (Figure 8a). The quality of the protein crystal could be evaluated by either cryocrystallography or in situ X-ray diffraction if the microwell patch was made of PDMS or glass.34-36 In the present study, the reversible binding of the two microwell patches allowed harvesting the protein crystals from the microwells. We were able to peel apart the two microwell (34) Hansen, C. L.; Classen, S.; Berger, J. M.; Quake, S. R. J. Am. Chem. Soc. 2006, 128, 3142-3143. (35) Yadav, M. K.; Gerdts, C. J.; Sanishvili, R.; Smith, W. W.; Roach, L. S.; Ismagilov, R. F.; Kuhn, P.; Stevens, R. C. J. Appl. Crystallogr. 2005, 38, 900-905. (36) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2004, 43, 2508-2511.


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as a passive pumping source is simple and fast for the end user. The method can be scaled up, and hundreds to thousands of microwells can be accommodated on one patch, allowing highthroughput screening. Microwells in various substrates, such as PDMS, PMMA, and glass, are compatible with the dispensing method. The microwell patches can be easily accommodated in commercial instrumentation, such as plate readers and robotic systems. A novel method was used to remove the PDMS microchannel patch from the microwell patch under the protection of an oil layer. Furthermore, the dispensing method offers a novel modular approach for screening and reaction by prefabricating droplets of multiple distinct reagents in the microwells and binding two microwells together. The reversible binding of the microwells facilitates harvesting the product from the microwells for further analysis. Currently the screening for crystallization conditions of proteins is limited to the microbatch condition, and it is under investigation whether this screening method can be implemented under vapor diffusion condition. Overall, the liquid dispensing and the associated screening methods offer a simple, inexpensive, and reliable way to store and use multiple nanoliter-volume distinct reagents in 96- or 384-well format and should be ideally suitable for individual laboratories for various applications such as enzyme assay, protein crystallization, cell analysis, and combinatorial chemistry.

Figure 8. Polarized light micrographs of the crystals of the unknown protein HP0753 in the bound microwells (a) and in the disassembled microwell (b), respectively. The precipitant was 20% (v/v) PEG 3000/ 0.2 M NaCl/0.1 M HEPES buffer, pH 7.5. Scale bar: 100 µm. (c) X-ray diffraction pattern with a resolution of 3.1 Å from a crystal of protein HP0753.

patches without disturbing the crystals in the microwells (Figure 8b), and then we slowly introduced the cryoprotectant into the microwells. The crystals were then mounted and subjected to X-ray diffraction studies. A diffraction pattern with a resolution of 3.1 Å was obtained (Figure 8c). CONCLUSION We developed a method of dispensing a nanoliter volume of liquid into arrays of microwells and illustrated the method by applying it to protein crystallization. The use of degassed PDMS

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ACKNOWLEDGMENT This work was supported by the Chinese University of Hong Kong and the Research Grants Council of Hong Kong. We thank Wen J. Li for the access to the cleanroom facility and Kam-Bo Wong for the generous gift of the crystallization kit. SUPPORTING INFORMATION AVAILABLE Table S1, listing the screening results of crystallization of the four known proteins, Table S2, listing the screening result of crystallization of HP0753, Figure S1, showing a typical process of dispensing liquid into a PDMS microwell, Figure S2, polarized light micrographs of thaumatin crystals in the glass microwell with different incubation durations. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 13, 2007. Accepted April 30, 2007. AC070306P

Nanoliter Dispensing Method by Degassed Poly(dimethylsiloxane

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