Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

This paper describes a microfluidic system to screen and optimize organic reaction conditions on a submicrogram scale. Optimization of reaction conditions is required to achieve high efficiency and selectivity in organic reactions. Combinatorial methods 1 and high-throughput screening 2 are powerful tools for optimization. To perform solution-phase synthesis, typical microtiter plates or reaction blocks for parallel synthesis run reactions on the scale of mL/ reaction 1 and are less applicable to precious substrates (e.g., products of long synthetic sequences and natural products that can be isolated only in small quantities). To address this problem, one approach used arrayed micro-wells in combination with a robotic liquid sampler on the scale of ~125 nL per reaction. 3 To reduce the use of robotics and to minimize evaporation, others used microchannels 4-6 to perform reactions, including synthesis of pyrazoles with UV detection (5 μL per reaction) 6 and optimization of glycosylation conditions 5 (~2 mg reagent per reaction).Here, we report a screening method that consumes substrates on the scale of less than 1 μg per reaction. The system uses discrete droplets (plugs) 7 as microreactors 8 separated and transported by a continuous phase of a fluorinated carrier fluid. Such approach is not limited to microfluidics-fluorinated fluids were used previously to segment samples in NMR 9 and PCR 10 in tubes to prevent dispersion of sample solutions. Previously, we demonstrated the use of a microfabricated PDMS plug-based microfluidic system to perform assays and crystallization experiments in aqueous solutions with optical detection. 11 Here, we developed an approach that does not require microfabrication of microfluidic devices, 12 is applicable to synthetic reactions in organic solvents, and uses detection by MALDI-MS.The system consisted of three components: preformed cartridge 11,13 of reagent plugs, a PEEK Tee, and a receiving tubing (Figure 1). A cartridge is an array of discrete plugs surrounded by fluorinated carrier fluid; each plug is composed of a solution of a different reagent. The cartridge was prepared by serially aspirating the reagents into a piece of Teflon tubing prefilled with carrier fluid. A commercially available PEEK Tee connected the cartridge and the inlet tubing, containing as little as a submicroliter volume of a solution of the substrate. Fluorinated carrier fluid (FC-70) was used to fill the two syringes ( Figure 1) and the connecting tubing, enabling no-loss manipulation of submicroliter volumes of solutions. FC-70 has low miscibility with organic reagents and reasonably low viscosity (Supporting Information). To perform the reactions, the flow was induced with the two syringes, and the reagent plugs were sequentially merged with the substrate solution. After all resulting plugs flowed out of the Tee into the receiving tubing, the flow was stopped, and the receiving tubing was sealed. After incubation, E-mail: r-ismagilov@uchicago.edu. Supporting Information Available: Materials and meth...

This paper analyzes the effect of mixing on nucleation of protein crystals. Nucleation is an important aspect of protein crystallization. 1 In-depth research has been conducted leading to insights into the mechanism of crystal nucleation as well as novel methods to control it 1,2 (e.g., work on nucleation at the liquid-liquid-phase boundary and on levitated droplets), but the effect of several factors affecting nucleation is not well understood. Mixing is proposed to be responsible for uncertainties in batch protein crystallization. 3 Mixing is also important in the nucleation of small molecule crystals 4 and selectivity of organic reactions, 5 but its effect on nucleation of protein crystals has not been studied experimentally. One barrier to this study is the difficulty of controlling and monitoring mixing, especially when using manual or robotic pipets. Crystal nucleation is a stochastic process, and to obtain statistically significant data, studies of nucleation necessitate a large number of experiments. The amount of the protein and labor required presents another barrier.Here we show that a plug-based microfluidic system 6 is suitable for observing mixing effects in crystal nucleation. The system is capable of setting up hundreds of crystallization experiments in a short period of time, 7 requiring little labor and ~1 μL samples of protein solutions. It relies on nL-volume aqueous plugs in a water-immiscible, fluorinated carrier fluid, where each plug acts as a microreactor in which a crystallization trial takes place. Mixing by chaotic advection in the system is well-controlled and characterized. 8 Chaotic advection has been pioneered in single-phase microfluidic devices. 9 The use of two-phase flows in plugs is attractive because it can transport solids, 10 such as precipitates that might arise during crystallization experiments.Mixing experiments were carried out by combining protein (thaumatin) and precipitant (2 M KNaC 4 H 4 O 6 ) solutions in a PDMS microfluidic channel. The solutions were separated by a thin stream of buffer to avoid contact 6 before forming plugs (Figure 1a). Mixing was changed by varying the total flow rate (higher flow rate corresponding to more rapid mixing in a winding channel). 8 The flow rate ratios between protein, buffer, and salt solutions were kept constant. Mixed plugs were collected in a glass capillary, 7 which was sealed and incubated at 18 °C. The number of crystals in each plug was monitored over time.Nucleation was sensitive to flow rate and mixing. Rapid nucleation took place at low flow velocities: i) precipitate was visible as the plugs were being mixed, and ii) these plugs yielded precipitation or showers of microcrystals after incubation for 8 h, indicating many nucleation events per plug. At high flow velocities, no precipitation was visible, and only a few large crystals grew after 8 h (Figure 1) of incubation, representing only a few nucleation events in each plug.E-mail: r-ismagilov@uchicago.edu. Supporting Information Available: Experimental details and a...