This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization trial. Crystallization trials were set up inside 7.5-nL aqueous droplets. These droplets, each containing solutions of protein, precipitants, and additives in variable ratios, were formed in the flow of immiscible fluids inside microfluidic channels. 1,2 We have used the system to set up hundreds of trials at a rate of several trials per second under computer control. The goal of this Communication is to quantify this approach and validate it by crystallizing correct polymorphs of several common watersoluble proteins.New methods of protein crystallization are becoming especially important because of the success of genome sequencing projects. Crystallization is a bottleneck in determining tertiary protein structures from sequence data. 3 Protein crystallization occurs in the labile region of the crystallization phase diagram, a narrow region where nucleation but not precipitation can occur. 4 The phase diagram is multidimensional and complex, and, despite progress in theory, 5 concentrations of the protein and the reagents (precipitants, buffers, and additives) that place the solution into the labile region are usually determined by screening. Minimal volumes of the protein solution should be used during screening, because many proteins are only available in very small quantities. 6 Manual screening by mixing stock solutions in many ratios is time-consuming and requires at least 100 nL of the protein solution per trial. To overcome these limitations, robotic systems have been developed that can perform automated mixing of stock solutions, and which can set up crystallization trials with volumes from 1 µL down to 100 nL, 7 consuming as little as 10 nL of individual solutions. 8 These robotic systems are expensive and have not yet seen wide adoption in individual laboratories.Microfluidic systems are useful for experiments that require minimal use of reagents. 9 Microfluidic platforms, therefore, are an attractive choice for macromolecular crystallization, 6 as was clearly demonstrated by Hansen et al. 10 These authors have crystallized proteins on a microfluidic device by free interface diffusion, a method that was previously possible only in microgravity. 10 Only ∼10 nL of the protein solution was used for each of 144 trials, which were conducted inside microfabricated chambers controlled by pressure-operated valves.The system described here (Figure 1) used three steps to crystallize proteins inside droplets, implemented using PDMS microfluidic devices with channels of 150 × 100 µm 2 crosssectional dimensions: 1,2 (1) Aqueous stock solutions were loaded into syringes, and syringes were connected to the convening channels of a microfluidic device. Only one stock solution and one syringe were required for each reagent or protein. A syringe containing water-immiscible fluorinated oil was connected to a perpendicular channel. (2) The flow of the aque...

This paper reports a composite microfluidic system for performing protein crystallization trials in nanoliter aqueous droplets inside capillaries suitable for X-ray diffraction and the evaluation of the quality of the crystals directly by on-chip X-ray diffraction. Crystallization conditions can be screened with both microbatch and vapor-diffusion techniques by eliminating evaporation of solutions and by controlling diffusion of water between droplets.Growing of high-quality crystals of proteins and other macromolecules plays an important role in structural biology. Crystallization conditions are usually identified by performing a large number of trials in which variable ratios of solutions of a protein, precipitants, and additives are pipetted together by hand (≈1 μL droplets) or with a robotic dispenser (≈100-10 nL droplets). [1] This process could be improved by a system that minimizes the amount of protein sample consumed, reduces labor, and is simple and economical enough to be implemented in individual laboratories. Micro-fluidics could serve as a platform for such a system because it allows sophisticated handling of small volumes (nL-pL) of reagents in a potentially simple, inexpensive format. [2] In addition, microfluidics may provide an opportunity to perform experiments that are difficult to do on larger scale-for example, Hansen et al. have crystallized proteins on a polydimethylsiloxane (PDMS) microfluidic device by free interface diffusion. [3] Previously, we screened crystallization conditions [4] on a PDMS microfluidic chip by using nanoliter droplets [5] formed in microfluidic channels.In this work, we rely on the same droplet-based platform, but take three steps that advance this work beyond our previous report. 1) We implemented the microbatch technique, in which we completely eliminated problems of uncontrolled evaporation through PDMS. PDMS is waterand oil-permeable, and the crystallization of proteins in PDMS, at least in our hands,

For screening the conditions for a reaction by using droplets (or plugs) as microreactors, the composition of the droplets must be indexed. Indexing here refers to measuring the concentration of a solute by addition of a marker, either internal or external. Indexing may be performed by forming droplet pairs, where in each pair the first droplet is used to conduct the reaction, and the second droplet is used to index the composition of the first droplet. This paper characterizes a method for creating droplet pairs by generating alternating droplets, of two sets of aqueous solutions in a flow of immiscible carrier fluid within PDMS and glass microfluidic channels. The paper also demonstrates that the technique can be used to index the composition of the droplets, and this application is illustrated by screening conditions of protein crystallization. The fluid properties required to form the steady flow of the alternating droplets in a microchannel were characterized as a function of the capillary number Ca and water fraction. Four regimes were observed. At the lowest values of Ca, the droplets of the two streams coalesced; at intermediate values of Ca the alternating droplets formed reliably. At even higher values of Ca, shear forces dominated and caused formation of droplets that were smaller than the cross-sectional dimension of the channel; at the highest values of Ca, coflowing laminar streams of the two immiscible fluids formed. In addition to screening of protein crystallization conditions, understanding of the fluid flow in this system may extend this indexing approach to other chemical and biological assays performed on a microfluidic chip. This paper characterizes a method of forming stable alternating droplets of two sets of aqueous solutions in microchannels. In addition to characterization, we describe an application of this method to indexing concentrations of the solutes in the droplets used for assays. Protein crystallization under microbatch conditions was used as an example. Indexing here refers to measuring the concentration of a solute by addition of a marker, either internal or external.Droplets in microfluidic channels allow transport in microfluidic channels with no dispersion 1 and allow rapid mixing using chaotic advection. 1-3 In this paper, we use the word "droplet" to describe a plug-an aqueous droplet that is surrounded by an immiscible fluid and is in apparent contact with all four walls of a hydrophobic microchannel. 4 Such droplets in microfluidic channels are being used as microreactors 5-8 in chemical kinetics, 9 chemical amplification, 10 chemical and biological analysis, 11,12 and protein crystallization. 13,14 Tens to hundreds of crystallization trials or assays can be performed in the same channel inside droplets. We have recently used alternating aqueous droplets formed in a water-permeable carrier fluid to perform protein crystallizations under vapor diffusion conditions and to concentrate nanoliter volumes of solutions. 14 In all of these experiments, each droplet co...

Plugging a gap in screening-Arrays of nanoliter-sized plugs of different compositions can be preformed in a three-phase liquid/liquid/gas flow. The arrays can be transported into a microfluidic channel to test against a target (see schematic representation), as demonstrated in protein crystallization and an enzymatic assay.Keywords crystal growth; enzymatic arrays; microreactors; screening methods; three-phase system Herein, we describe a simple, economical microfluidic method of screening a small volume (down to submicroliter volumes) of a solution against a large number of reagents on the nanoliter scale. The use of microfluidics to miniaturize chemical and biological screening is an important and active area of research in such diverse areas as biochemical assays, protein crystallization, and combinatorial chemistry. [1][2][3][4][5][6][7] Nanoliter aqueous plugs (droplets) transported through microchannels in an immiscible liquid have been used in a liquid/liquid flow system and allow miniaturization while eliminating dispersion, [8,9] accelerating mixing, [10] and providing control over the surface chemistry. [11] Applications of such systems to protein crystallization, [3,12] kinetic measurements, [10] assays, [13,14] DNA analysis, [8] and chemical synthesis [15] have been demonstrated. Such plug-based microfluidic systems have been especially attractive for applications in which the concentrations of several reagents had to be varied. The concentrations were varied by rapidly changing the flow rates of the reagent streams as the droplets were formed. [3,12] Plugbased methods of that type require equipment for varying flow rates, and even though such equipment could be as simple as a few computer-controlled syringe pumps, this requirement presents a barrier to many potential users in chemical and biochemical laboratories. In addition, to increase the number of reagents that can be screened, both the number of the microfluidic channels in the device and the number of flow control devices have to be increased proportionally. Herein, we implement a complementary approach that uses pre-formed arrays of plugs to simplify the experiment for the user, relies on a liquid/liquid/gas three-phase flow system to ensure robustness, and allows a much larger number of reagents to be tested in a scalable fashion.

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