Reviews
`
`R. F. Ismagilov et al.
`
`Droplet-Based Microfluidics
`Reactions in Droplets in Microfluidic Channels
`Helen Song, Delai L. Chen, and Rustem F. Ismagilov*
`
`DOI: 10.1002/anie.200601554
`
`Keywords:
`analytical systems · interfaces ·
`microfluidics · microreactors ·
`plugs
`
`Angewandte
`Chemie
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`7336
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`www.angewandte.org
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` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
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`Microfluidics
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`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 compart-
`mentalizing 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 micro-
`fluidics 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.
`
`Angewandte
`Chemie
`
`From the Contents
`
`1. Introduction: Reactions in
`Droplets
`
`2. Criteria for Performing
`Reactions in Droplets
`
`3. Applications of Droplet-Based
`Microfluidics
`
`4. Conclusions and Outlook
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`1. Introduction: Reactions in Droplets
`
`This Review discusses the use of droplets in microfluidic
`channels as chemical microreactors for performing many
`reactions on a small scale (Figure 1).[1] Microreactors in
`
`Figure 1. Droplets formed within microfluidic channels can serve as
`microreactors. In this example, the reactions are performed within
`aqueous droplets, which contain reagent A, reagent B, and a separat-
`ing stream containing buffer. The droplets are encapsulated by a layer
`of a fluorinated carrier fluid and transported through the microchan-
`nels. Reprinted from reference [1].
`
`general[2] and bulk micellar systems[3] have been covered in
`recent review articles in Angewandte Chemie and thus will not
`be covered herein. We describe new techniques that have
`been developed to perform chemical reactions within droplets
`and reactions that have been studied by using droplets. We
`also discuss how droplet-based microfluidics can lead to new
`scientific methods and insights.
`
`1.1. Reaction Control for High Throughput
`
`A number of applications require multiple reactions to be
`performed in parallel, for example, drug discovery, gene-
`expression analysis, and high-throughput assays. For these
`
`applications, it is only feasible to perform reactions on a
`microscale, because reagents can be expensive or only
`available in limited amounts. In other applications, multiple
`reactions need to be carried out to characterize stochastic
`processes, such as the nucleation of crystals.
`Microfluidics can be used to handle small volumes of
`liquid and is a promising method to form microreactors.[2, 4, 5]
`Benefits from miniaturization include low consumption of
`reagents and high-throughput fabrication of devices. For
`example,
`soft
`lithography with poly(dimethylsiloxane)
`(PDMS) can be used for the rapid and inexpensive fabrica-
`tion[6, 7] and modification[8–10] of devices.
`
`1.2. Parallel or Serial Compartmentalization of Multiple
`Reactions
`
`To perform many reactions in high throughput, each
`reaction condition must be uniquely addressable or indexed.
`The reactions can be indexed if each reaction condition is
`compartmentalized either in parallel or in series[11] (Figure 2).
`Reaction conditions can be compartmentalized in parallel by
`using a well plate (Figure 2 a). The final result of the reaction
`is indexed as a function of the spatial location of the well.[12–14]
`Reaction conditions can also be compartmentalized in series
`as in flow injection analysis (Figure 2 b). The final result of the
`reaction is indexed as a function of the elution time. Chemical
`indexing in combinatorial chemistry has also been achieved
`by using molecular tags,[15] labeled beads,[16, 17] and encoded
`particles.[18, 19]
`
`[*] H. Song, D. L. Chen, Prof. Dr. R. F. Ismagilov
`Department of Chemistry and Institute for Biophysical Dynamics
`The University of Chicago
`5735 South Ellis Avenue, Chicago, IL 60637 (USA)
`Fax: (+ 1) 773-702-0805
`E-mail: r-ismagilov@uchicago.edu
`Homepage: http://ismagilovlab.uchicago.edu/
`Supporting information (three movies showing droplets forming and
`mixing in microfluidic channels, and a movie of the mixing of
`modeling clay by chaotic advection) for this article is available on the
`WWW under http://www.angewandte.org or from the author.
`
`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
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` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`at a range of velocities in the microchannel. Taylor
`dispersion describes the transport and broadening of a
`pulse of a solute within a solution that is flowing
`through a tube.[29, 30] As a result of dispersion, local-
`ization of the reaction and accurate control of the
`reaction time is difficult. Cross-contamination can occur
`between pulses of different reagents that are traveling
`through the same tube. Furthermore, reagents are in
`direct contact with the solid microchannel wall, so the
`surface chemistry of the wall must be controlled. The
`control of surface chemistry is especially critical for
`devices that use electroosmotic flow to solve the
`problem of Taylor dispersion.[31, 32] Dilution of
`the
`sample solution can occur through diffusion within a
`single-phase flow, especially with prolonged incubation
`times. This broadening of the sample pulse occurs
`regardless of whether the flow is driven by pressure or
`electroosmotic forces.
`
`1.4. Compartmentalization in Nanoliter-Sized Droplets
`
`Nanoliter-sized droplets can serve as compartments
`for reactions. Multiple reactions can be performed by
`varying the reaction conditions within each droplet. The
`problems of evaporation, complicated fluid handling, disper-
`sion, and diffusion can be overcome by using multiphase flows
`of immiscible liquids to form droplets in microfluidic channels
`(Figure 1).[1] Evaporation can be controlled for long-term
`incubation experiments by transporting droplets into glass
`capillaries.[33, 34] Complicated fluid handling is minimized, as
`uniformly sized droplets form spontaneously in a cross-stream
`flow of two immiscible liquids.[1, 35–39] The flow of fluids within
`the microchannels can be used to manipulate droplets and
`arrays of droplets in a controlled manner.[1, 37, 38, 40–42] Disper-
`sion due to convection and diffusion is eliminated because the
`reagents are encapsulated within droplets. Furthermore,
`surface chemistry can be easily controlled at the liquid–
`liquid interface between the immiscible phases of the reagent
`fluid and the carrier fluid.[43] Mixing within droplets can be
`achieved by chaotic advection.[1, 37, 40, 44, 45]
`To obtain compartments that do not move (as analogues
`of well plates), droplets can be transported into capillaries
`and incubated for up to one year. To obtain compartments
`that move (as analogues of flow injection analysis), flow can
`be used to transport droplets continuously through the
`
`Figure 2. Comparison of reactions compartmentalized in parallel and in series. a) Paral-
`lel reactions performed by using well plates: The reagents are localized within wells,
`and the target sample is delivered to the well through a multipipettor. The reaction
`products are detected by scanning over the samples, and each reaction is indexed as a
`function of the spatial location of each well. b) Serial reactions performed by using flow.
`Reactions are localized within pulses and separated by a buffer solution between each
`reagent. These pulses are transported through the reaction tube by the flow, and the
`target sample is delivered to the reagents. The pulses are transported by the flow past a
`stationary detector to analyze the reaction products. Each reaction is indexed as a
`function of the elution time.
`
`1.3. Control of the Compartmentalization
`
`For performing reactions in parallel, the handling of the
`fluid and evaporation within the compartments needs to be
`controlled. PDMS devices with enclosed chambers and valves
`allow many reactions to be performed by multiplexing
`methods.[20–23] This system has been applied to protein
`crystallization,[24–26] bacterial chemostats,[27] and the synthesis
`of radiolabeled probes.[22] The valves used to handle fluids
`require multilayer fabrication. As PDMS is permeable to the
`vapor of organic and aqueous solutions, eliminating evapo-
`ration through PDMS requires special measures (although
`this permeability was controlled and used as an advantage in
`some applications,[28] such as the filling of dead-end chan-
`nels).[25]
`For reactions performed in series in a single-phase flow,
`dispersion can lead to cross-contamination of reaction con-
`ditions and dilution of samples. Pressure-driven flow of a
`single-phase solution through a microchannel is laminar and
`displays a parabolic velocity profile.[29, 30] As a result of the
`parabolic flow profile, the reagents in solution are transported
`
`Helen Song received her BSc and MSc
`degrees in chemistry at the University of
`Chicago. She started her work in developing
`techniques for droplet-based microfluidics
`and using this method to study enzyme
`kinetics at the University of Chicago in 2002
`under the supervision of Prof. Rustem Ismag-
`ilov. She successfully defended her PhD
`thesis in 2005.
`
`Delai Chen received his BSc in chemistry at
`Peking University and his MSc in chemistry
`at the University of Chicago. He started his
`work on using droplet-based microfluidics to
`study stochastic processes in protein crystal-
`lization and to optimize conditions in
`organic reactions at the University of Chi-
`cago in 2004 under the supervision of Prof.
`Rustem Ismagilov.
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`7338
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` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
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`Microfluidics
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`microchannel in the device. By using droplets, it is possible
`both to compartmentalize reactions and control the reaction
`time accurately.
`
`1.5. Scope of the Review
`
`In this Review, we discuss reactions that occur within
`segmented flows in a microfluidic system. Segmented flows
`are composed of at least two immiscible phases: one dispersed
`phase and one continuous phase. The droplet consists of the
`dispersed phase, and the continuous phase encapsulates the
`droplet and preferentially wets or coats the surface of the
`microchannel.
`For this Review, we differentiate between two types of
`segmented flows on the basis of the phase in which the
`reaction takes place. In the first type, discrete liquid droplets
`are encapsulated by a carrier fluid that wets the microchannel
`(Figure 3 a).[1] These droplets, termed “plugs” herein, form
`the dispersed phase in which the reactions occur. Systems that
`use plugs differ from segmented-flow injection analysis in that
`reagents in plugs do not come into contact with the micro-
`
`Figure 3. Reactions can be studied in two types of segmented flows in
`microfluidic channels. a) Discrete liquid plugs are encapsulated by an
`immiscible continuous phase (for example, a fluorocarbon-based
`carrier fluid). Reactions occur within the dispersed phase (within the
`plugs). Owing to the surface properties of the microchannel walls,
`these walls are preferentially wet by the continuous phase. b) Aqueous
`slugs are separated by another immiscible phase (for example, discrete
`gas bubbles). Reactions occur within the continuous phase (i.e.,
`within the slugs).
`
`Rustem Ismagilov received his PhD in 1998
`at University of Wisconsin, Madison under
`the direction of Prof. Stephen F. Nelsen and
`was a postdoctoral fellow with Professor
`George M. Whitesides at Harvard University.
`He began his independent career at the
`University of Chicago in 2001 and was
`promoted to Associate Professor in 2005.
`His current research involves using micro-
`fluidics to control complex chemical and
`biological systems in space and time.
`
`Angewandte
`Chemie
`
`channel wall and are transported without dispersion. In the
`second type of segmented flow, liquid “slugs” are separated
`by discrete gas bubbles (Figure 3 b).[46–51] In this case, reac-
`tions occur within the slugs that form the continuous phase;
`reagents are exposed to the walls of the channels and some
`dispersion occurs. This second system is similar to segmented-
`flow injection analysis.[52–56] For the remainder of the Review,
`the terms “plugs” and “slugs” are used to differentiate these
`two types of segmented flows.
`We do not cover reactions in bulk systems that use
`multiple immiscible phases to form micelles, emulsions, and
`droplets. Emulsions take advantage of only some of the
`spatial control available by compartmentalization. Extensive
`review articles cover reactions in micelles,[3] colloids,[57, 58]
`miniemulsions,[59] and multilayer microcapsules.[60] Emulsions
`and vesicles have been used for in vitro compartmentaliza-
`tion,[61, 62] and vesicle reactors can also act as synthetic
`cells.[63–65] Emulsions formed by microfluidics[66–68] have been
`used to synthesize permeable colloids[69] and to fabricate
`monodisperse capsules by using steady coaxial jets.[70]
`The area of “digital microfluidics”, in which droplets are
`manipulated by an array of electrodes[71–75] (“active control”)
`rather than through a continuous flow (“passive control”), is
`also not covered. The principles of electrowetting-based
`actuation and the automation of this technique have been
`reviewed.[76–78] Digital microfluidic devices have been used for
`analyzing proteins and peptides with matrix-assisted laser
`desorption/ionization mass-spectrometry (MALDI MS),[79, 80]
`performing PCR with optical detection,[81] measuring glucose
`concentration in droplets with optical detection,[82] perform-
`ing a luciferase-based assay,[83] and synthesizing anisotropic
`particles.[84]
`Microfluidic systems that use multiphase flow but do not
`report reactions will not be discussed in detail. We define
`reactions broadly and include the interconversion of chemical
`species[85] and phase transitions, such as crystallization and the
`formation of particles. Methods of forming and manipulating
`droplets are being continuously developed to take advantage
`of novel physical principles, such as electrowetting,[86–88]
`magnetic fields,[89, 90] optically induced Marangoni effects,[91, 92]
`acoustic waves,[93] and surface chemistry.[94, 95] Recent innova-
`tions on the generation of gas bubbles in microfluidic devices
`include the use of segmented flow in microchannels as
`multiphase monolith reactors,[96] the formation of monodis-
`perse gas bubbles by flow focusing,[97, 98] the study of nonlinear
`dynamics of a flow-focusing bubble generator,[99] and charac-
`terization of the transport of bubbles in square channels.[100]
`The flow of immiscible fluids within microchannels may
`result in continuous laminar flow rather than the formation of
`droplets.[101] These continuous-laminar-flow systems, though
`suitable for conducting chemical
`reactions and bioas-
`says,[102–104] as well as for patterning and microfabrication
`within microchannels,[105, 106] are not discussed herein.
`This Review summarizes recent developments in the use
`of droplets in microfluidics as chemical reactors for many
`reactions. We introduce techniques for conducting reactions
`and discuss examples of reactions in droplet-based micro-
`fluidic systems. Furthermore, we examine how microfluidics
`can open up new research areas.
`
`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
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` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`2. Criteria for Performing Reactions in Droplets
`
`To perform reactions within microfluidic devices, at least
`two criteria should be met. First, the microfluidic tool should
`be able to perform typical procedures that are conducted for
`reactions on the macroscale. These procedures include the
`controlled addition of reagents to a reaction mixture, the
`thorough mixing of reagents, control of the reaction time, the
`combining and splitting of reaction mixtures for multiple-step
`reactions, and analysis over the course of a reaction. Second,
`the microfluidic tool should provide a characteristic advant-
`age, for example, the ability to perform more reactions under
`more reaction conditions. As with any high-throughput
`screening technique, there must be a method for organizing
`and indexing each reaction condition. Likewise, there must be
`an efficient method for assaying many different conditions
`and also for optimizing a particular condition. These methods
`should be scalable, straightforward, and simple. In this
`section, we discuss techniques that were developed for
`droplet-based microfluidics to fulfill the two criteria de-
`scribed above.
`
`Figure 5. Formation of droplets by flow-focusing.[111] Modified from
`reference [111]. a) A schematic of the device. The rectangle outlines
`the field of view in (b). Copyright 2003 American Institute of Physics.
`
`For both methods, surfactants are often added to the
`continuous phase to stabilize the fluid–fluid interfaces of the
`droplets.[1, 35, 111] The conditions under which monodisperse
`droplets form have been well documented.[96, 118–123] The
`droplets are not useful as general microreactors, however,
`unless methods of introducing reagents into the droplets are
`also developed, as is discussed below.
`
`2.1. Formation of Droplets within Microfluidic Channels
`
`2.2. Introduction of Reagents into Droplets
`
`Droplet formation in two-phase systems has been exten-
`sively studied for both liquid–liquid flows[107–109] and gas–
`liquid flows.[110] Although many methods are available for
`making bulk emulsions, droplets in microfluidic channels
`have been generated mostly by two techniques: T junc-
`tions[1, 35–39] and flow-focusing.[36, 97, 98, 111–115] In T junctions, the
`disperse phase and the continuous phase are injected from
`two branches of the “T”. Droplets of the disperse phase are
`produced as a result of the shear force and interfacial tension
`at the fluid–fluid interface (Figure 4; see also Figure 1). The
`
`Figure 4. Formation of droplets within a T junction of a microfluidic
`device.[35] In this case, the oil is a mixture of hydrocarbon and the
`surfactant Span80, and the channels are made of polymerized acry-
`lated urethane. Reprinted with permission from reference [35]. Copy-
`right 2001 American Physical Society.
`
`phase that has lower interfacial tension with the channel wall
`is the continuous phase.[37] To generate droplets in a flow-
`focusing configuration,
`the continuous phase is injected
`through two outside channels and the disperse phase is
`injected through a central channel
`into a narrow orifice
`(Figure 5).[111] This geometry is only slightly more difficult to
`implement than a T junction and may facilitate the formation
`of droplets, especially small or viscous droplets.[45, 97, 111, 115–117]
`
`Different techniques have been developed to introduce
`reagents into droplets for different droplet-based microfluidic
`applications. For high-throughput screenings, one target
`sample must be tested against a large number of different
`reaction conditions. Each reaction condition may be com-
`posed of different reagents or a different combination of a set
`of reagents. For measuring kinetics or optimizing reaction
`conditions, only a few reagents need to be incorporated within
`the droplet, but the concentration of these reagents are
`varied. For multiple-step reactions, the addition of a reagent
`should occur at a specific time during the course of the
`reaction. Each of these applications requires a different
`method for introducing the reagents.
`
`2.2.1. Cartridge Technique
`
`For screening a large number of reaction conditions
`against one target sample, preformed cartridges can be
`produced to store an array of plugs (Figure 6 a,b).[42] Each
`plug contains a different reaction condition of different
`reagents. For example, an array of 48 plugs was formed in
`which each plug contained 15 nL of a different reagent.[42] The
`target sample can be introduced into the preformed plugs by
`using a microchannel T junction (Figure 6 c). Reagent car-
`tridges can be stored inside sealed capillaries for months
`without evaporation or exposure to the ambient environ-
`ment.[42]
`Reagent cartridges can be used for applications that
`require parallel screening of one target sample against many
`different reagents or reaction conditions. By using a repeated
`splitting device, 16 reagent cartridges (each containing
`approximately 20-nL plugs) were formed in parallel by
`splitting one array containing large droplets (ca. 320 nL).[125]
`Such parallel preparation of reagent cartridges accelerates
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`Figure 7. Controlling the concentrations of reagents within plugs by
`on-chip dilution.[44] a) Experimental setup; the blue rectangle shows
`the field of view for microphotographs shown in (c). b) A graph
`quantifying the on-chip dilution method. The concentrations measured
`from the fluorescence intensity of plugs traveling through the micro-
`channel are plotted as a function of theoretical concentration calcu-
`lated from the flow rates of the streams containing reagent A, the
`dilution buffer, and reagent B. c) The concentrations of the reagents
`were controlled by the relative flow rates of the reagent streams
`(values in parentheses, in nL s1). Reprinted with permission from
`reference [44]. Copyright 2003 American Chemical Society.
`
`allows rapid switching of reaction conditions without stopping
`the experiment or wasting valuable reagents. This approach is
`not limited to the geometries shown in Figure 7; it is also
`compatible with “flow focusing” (Figure 5) for forming
`droplets with controlled concentrations of reagents.
`
`2.2.3. Direct Injection of Reagents into Droplets
`
`For multiple-step reactions, a reaction mixture is allowed
`to react for a certain time and then another reagent is added
`to the mixture. In droplet-based microfluidics, the first step of
`the reaction can be contained within a plug that is transported
`within the microchannel. Then, a reagent for the second step
`of the reaction can be injected into the plug through a side
`channel further along the microchannel network.
`A reagent flowing through a side channel can be injected
`directly into droplets through a T junction (as shown in
`Figure 6 for the injection of a target sample into an array of
`plugs). However, if the T junction is preferentially wetted by
`the carrier fluid (e.g., with a hydrophobic junction and
`aqueous reagents), then the injection of the reagent into a
`plug is favorable only at low values of the capillary number
`(Ca 0.01).[129, 130] At higher values of Ca (for example, at
`higher flow rates), the injection can be accomplished by
`mechanical agitation of the PDMS channel.[129]
`Another injection method was developed with a side
`channel that is preferentially wetted by the reagent fluid (e.g.,
`with a hydrophilic channel and aqueous reagents; Fig-
`ure 8 a).[131, 132] As a droplet is formed from the injection of
`reagent fluid from a side channel into the main channel,
`wetting of
`the side channel prevents this droplet
`from
`breaking off until a plug passes by the side channel, and this
`droplet is injected into the plug.[131] With this injection
`method, the volume of the reagent injected into the plug
`increases linearly with the flow rate of the reagent stream in
`
`Figure 6. Preformed cartridges of plugs enable the combination of a
`large number of reagents with a sample in sub-microliter vol-
`umes.[42, 124] a,b) Four different reagents stored as an array of plugs in a
`capillary. The plugs are separated by a fluorocarbon carrier fluid, as
`well as air bubbles (in b), to prevent cross-communication between
`the plugs. Scale bars: 200 mm. c) Merging of plugs from a preformed
`cartridge with a target sample stream through a T junction. The
`resulting array of plugs is transferred into a receiving capillary and the
`trials are collected. d) Photograph of the T junction. Reprinted from
`reference[42] (a,b) and with permission from Elsevier from refer-
`ence [124] (c,d).
`
`high-throughput screening. Reagent cartridges have already
`been used in a range of applications: screening of protein
`crystallization conditions and in enzyme assays,[42] screening
`of the reaction conditions for an organic reaction,[126] and for
`immunoassays (using a liquid–air two-phase cartridge).[127]
`
`2.2.2. Variation of Reagent Concentrations
`
`For some reactions, rate constants or optimal reaction
`conditions are determined by varying the concentrations of
`several reagents. In one case, laminar flow was used to dilute
`two reagents within plugs on a chip.[44] The plugs were formed
`by using two streams of reagents with a buffer stream in the
`middle to prevent premature mixing of the two reagent
`streams (Figure 7). Varying the relative flow rates of the three
`aqueous streams varies the concentration of the reagents
`within the plug (Figure 7 c). For example, a higher flow rate of
`the stream containing reagent A (green stream, 45 nL s1)
`results in a larger proportion of the green reagent in the plug
`(Figure 7 c, left).
`This technique was also used to obtain an array of plugs,
`whereby each plug contained a unique composition of four
`reagents.[128] The composition of reagents within each plug
`was determined by the relative flow rates of the reagent
`streams, which were controlled by a computer. This capability
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`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
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` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`Figure 8. Injection of a CaCl2 solution into a plug (blood) through a
`hydrophilic side channel.[132] a) Time-lapse microphotographs of the
`injection process. b) The injection volume is controlled by the flow
`rate (mL min1) of the CaCl2 stream. Each data point on the graph
`denotes measurements for 10 plugs (y = 24.947 x0.2312,
`R2 = 0.9849). Reprinted with permission from reference [132]. Copy-
`right 2006 American Chemical Society.
`
`(Figure 8 b).[132] Cross-contamination
`channel
`side
`the
`between plugs at the T junction was quantified by fluores-
`cence measurements and found to be minimal.[131]
`For gas–liquid slugs, the reagents need to be injected into
`the continuous phase, as reactions occur within this phase.
`Such injection is easy, as this continuous phase is always in
`contact with the microchannel wall. However, contamination
`between slugs can result, especially in rectangular micro-
`channels. The merging of a tracer dye into the continuous
`phase was demonstrated for slugs within microchannels.[48]
`
`2.3. Controlling Mixing by Chaotic Advection
`
`The control of mixing is important for reactions and
`autocatalytic processes.[133, 134] Rapid mixing of reagents is
`necessary to determine the starting time of a reaction
`accurately. Droplet-based microfluidics allows rapid mixing
`and the extent of mixing can be quantified.[1, 40, 135] It has been
`reported that for a single-phase microfluidic flow, a reagent
`can be mixed by hydrodynamic focusing with a large excess of
`a second reagent with mixing times of only 10 ms.[136] The
`mixing was sufficiently rapid that the time resolution of
`kinetic measurements was limited by dispersion and not by
`mixing. Chaotic advection in a staggered herringbone
`mixer[137] was used to achieve complete mixing of
`two
`reagents in milliseconds and to reduce dispersion. This idea
`of chaotic advection (see Figure 9 and a movie in the
`Supporting Information) was
`implemented with drop-
`lets.[1, 40, 46] Chaotic advection[137–139] relies on repeated folding
`and stretching of the two fluids to achieve layers of fluids
`(striations) that become exponentially thinner and thinner
`(Figure 9) until mixing by diffusion becomes rapid. Mixing in
`droplets by chaotic advection can be achieved in sub-milli-
`second times[40] without dispersion and is especially useful
`when both mixing on a short timescale and dispersion over a
`longer timescale need to be controlled.
`
`Figure 9. Model for the mixing of two reagents by chaotic advection at
`low values of the Reynolds number; photographs of two layers of
`modeling clay being stretched and folded. Images are courtesy of
`Joshua D. Tice.
`
`Droplets traveling through a microchannel experience
`internal recirculation, which has been used to enhance mixing
`in plugs[37, 140–143] and slugs.[46, 50, 144–146] In straight channels, two
`symmetric vortices form on the left and right halves of a plug
`(in the direction of plug movement, Figure 3). The mixing
`occurs by convection within each half and mainly by diffusion
`between the two halves of the plug. In winding channels
`(Figure 10), the interface between the two halves of the plug
`
`Figure 10. Mixing by chaotic advection in a plug moving through a
`winding channel. The interfaces between the red and blue fluids are
`reoriented, stretched, and folded as the plug moves through the
`corners and straight sections of the channel. Reprinted with permis-
`sion from reference [40]. Copyright 2003 American Institute of Physics.
`
`is reoriented from the direction of plug movement and is
`stretched and folded by recirculations (Figure 10). This
`technique greatly enhances mixing (see two movies in the
`Supporting Information for a comparison of mixing in straight
`and winding channels).
`The extent of mixing is dependent on the number of
`winding turns and can be quantified by analyzing fluorescence
`images (Figure 11 b). By using a “bumpy” mixer (Figure 12)
`to generate oscillating shear within plugs, even viscous
`biological samples containing high concentrations of bovine
`serum albumin or hemoglobin can be mixed within milli-
`seconds.[45] The striations (Figure 12 c) observed in this mixer
`
`7342
`
`www.angewandte.org
`
` 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`Angew. Chem. Int. Ed. 2006, 45, 7336 – 7356
`
`7
`
`

`
`Microfluidics
`
`Angewandte
`Chemie
`
`tions in fluids can be controlled by microfluidics.[119] These
`interactions can be used to concentrate solutes within the
`droplets,[147] to maximize efficiency of a catalyst embedded on
`the surface of a microchannel,[104] and to synthesize coated
`particles by interfacial reactions.[112, 117] A high surface-area-
`to-volume ratio ensures rapid heat transfer between plugs and
`the carrier fluid and allows rapid switching between different
`temperatures. This rapid switching is essential for DNA
`amplification,[148] in vitro protein expression in plugs,[149] and
`DNA analysis.[51]
`Interactions at solid–liquid and liquid–liquid interfaces in
`a microchannel can be advantageous for certain applications
`yet detrimental for others. Therefore, these interactions need
`to be controlled. For plugs, the reaction occurs within the
`dispersed phase, which does not come in contact with the solid
`microchannel wall but is encapsulated by a layer of the carrier
`fluid. Therefore, reactions within plugs will be affected by the
`surface chemistry at the liquid–liquid interface. For slugs, the
`reaction occurs within the continuous phase, which is in
`contact with both the solid microchannel and the dispersed
`phase. Therefore, reactions within slugs will be affected by the
`surface chemistry at both the solid–liquid and liquid–gas
`interfaces.
`To form plugs in microchannels, the surface of channels
`should be treated to ensure that the carrier fluid (and not the
`aqueous phase) preferentially wets the channel wall. The
`surface tension between the aqueous phase and the carrier
`fluid should be lower than the surface tension between the
`aqueous phase and the channel wall. Surfactants can be used
`within the carrier fluid to lower the surface tension between
`the aqueous phase and the carrier fluid. However, this surface
`tension should not be lowered too far as the capillary number
`Ca of the flow must be low to favor the formation of plugs;[38]
`the capillary number is defined as Ca = mU/g, in which m
`(kg m1 s1) is the viscosity of the fluid, U (m s1) is the flow
`rate and g (N m1) is the interfacial tension between the
`aqueous phase and the carrier fluid.
`Perfluorinated liquids are optimal carrier fluids for the
`formation of plugs. They are considered to be chemically and
`biologically inert and have been used as blood substitutes,[150]
`for liquid ventilation of fetuses,[151] for diagnostic ultrasound
`imaging,[152] for cell cultures,[153, 154] and in drug delivery.[155]
`Many types of fluorocarbons and fluorinated surfactants are
`commercially available (however,