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,

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycolcapped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS micro-fluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption. This paper describes the biocompatibility of a plug-based microfluidic system obtained through control of surface chemistry the aqueous-fluorous interface. This microfluidic system uses multiphase flows of immiscible fluorous and aqueous liquids to form droplets (plugs) and to transport them with rapid mixing and no Taylor-like dispersion. 1 We have previously used the system to measure enzyme kinetics 2 and perform protein crystallization 3-6 using submicroliter volumes of sample.

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.

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,