A Microfluidic Approach for Screening Submicroliter Volumes against Multiple Reagents by Using Preformed Arrays of Nanoliter Plugs in a Three‐Phase Liquid/Liquid/Gas Flow

Abstract

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.

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

“…To perform simultaneous hybrid screening and optimization, we used plugs [droplets surrounded by fluorinated carrier fluid and transported through microfluidic channels (24)]. Plugs have been used to perform the initial screening in a number of applications, including biochemical assays (25), chemical reactions (26), and crystallization of soluble proteins (22). In separate experiments, plugs have been used for optimization of protein crystallization conditions (23).…”

“…1): (i) To deliver many distinct reagents in the same experiment, as required for the initial screen, we used a preformed array of reagent plugs, each plug Ϸ120-140 nl in volume. Each pair of plugs in the array was separated by Ϸ40-nl spacer to ensure reliable transport of reagent plugs of different viscosities and surface tensions (22,27). As this array was flowed from the cartridge into the microfluidic device, each plug formed a long segment that could be manipulated as a continuous stream (Fig.…”

“…Technologies for miniaturization of crystallization experiments are needed, but handling solutions of membrane proteins is complicated by their low surface tension and high viscosity. To address these challenges, both robotic (18,19) and microfluidic (20)(21)(22)(23) technologies have been developed to screen crystallization conditions on a submicroliter scale for both soluble and membrane proteins, but no universally applicable technology has yet emerged. Given the complexity of the problem of crystallization of membrane proteins, it is unlikely that a single technology would provide a universal solution, but there is a clear need for miniaturized technologies sufficiently robust, simple, and inexpensive to be accessible to individual laboratories.…”

Emulsification in a microfluidic flow-focusing device: effect of the viscosities of the liquids

“…Since the generation of droplets with a predictable and reproducible size and size distribution determines their potential applications (including the synthesis of polymer colloids), several research groups have explored various aspects of the process of emulsification (Seo et al 2005a;Xu et al 2005;Zhang et al 2006;Cygan et al 2005;El-Ali et al 2005;Garstecki et al 2005a;Hudson et al 2005;Jensen and Lee 2004;Khan et al 2004;Song et al 2003;Zheng and Ismagilov 2005;Zheng et al 2003) . It has been observed that the size of droplets is controlled by the design of the microfluidic device (Sugiura et al 2002a, b;Tan et al 2006), the properties of liquids, and the rates of flow of two immiscible phases (Cramer et al 2004;GananCalvo 1998;Ganan-Calvo and Gordillo 2001;Garstecki et al 2004Garstecki et al , 2005aGarstecki et al , 2005bGarstecki et al , 2006Thorsen et al 2001;Serra et al 2007;Tice et al 2003;Ward et al 2005).…”

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

“…The compartmentalization of reactions in droplets provides rapid mixing of reagents, flexible control of sample volumes, and prevention of water evaporation and cross contamination between samples [1][2][3]. These advantages of droplet-based microfluidics have shown benefits to a wide range of chemical and biological applications, such as enzymatic kinetics [4][5][6], protein crystallization [7][8][9], polymerase chain reaction [10][11][12] and clinical diagnosis [13].…”