Emerging Droplet Microfluidics - ACS Publications - American

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Emerging Droplet Microfluidics Luoran Shang, Yao Cheng, and Yuanjin Zhao* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ABSTRACT: Droplet microfluidics generates and manipulates discrete droplets through immiscible multiphase flows inside microchannels. Due to its remarkable advantages, droplet microfluidics bears significant value in an extremely wide range of area. In this review, we provide a comprehensive and in-depth insight into droplet microfluidics, covering fundamental research from microfluidic chip fabrication and droplet generation to the applications of droplets in bio(chemical) analysis and materials generation. The purpose of this review is to convey the fundamentals of droplet microfluidics, a critical analysis on its current status and challenges, and opinions on its future development. We believe this review will promote communications among biology, chemistry, physics, and materials science.

CONTENTS 1. Introduction 2. Microfluidic Chip Fabrication 2.1. Micromachining 2.2. Molding Replication 2.3. Modular Assembly 2.4. 3D Printing 2.5. Other Methods for Chip Fabrication 3. Dynamics of Microfluidic Droplets 3.1. Driving Force of Microfluidics 3.2. Droplet Formation by Passive Methods 3.2.1. Coflow 3.2.2. Cross-Flow 3.2.3. Flow-Focusing 3.3. Droplet Formation by Active Methods 3.3.1. Electrical Method 3.3.2. Magnetic Method 3.3.3. Thermal Method 3.3.4. Mechanical Method 3.4. Complex Droplet Formation in Microfluidics 3.4.1. Multicomponent Droplets 3.4.2. Double-Emulsion Droplets 3.4.3. Multiple-Emulsion Droplets 3.5. Droplet Manipulation in Microfluidics 3.5.1. Sorting 3.5.2. Coalescence 3.5.3. Splitting 3.5.4. Mixing 3.5.5. Trapping 4. Droplet Microfluidics for Bio(chemical) Analysis 4.1. Advantages of the Droplet Analysis Platform 4.2. Stability of Droplets in Analysis 4.3. General Analysis Strategies in Droplet Microfluidics 4.3.1. Reagent Encapsulation in Droplets © 2017 American Chemical Society

4.3.2. Droplet Indexing 4.3.3. Detection Approaches for Droplet Microfluidics 4.4. Biochemical Analysis in Droplet Microfluidics 4.4.1. Small-Molecule Detection 4.4.2. Biological Macromolecule Analysis 4.4.3. Cell Manipulations 4.4.4. Multicellular Organism Analyses 4.5. Applications of the Droplet Analysis Systems 4.5.1. Single-Cell Analysis 4.5.2. Medical Diagnostics 4.5.3. Drug Discovery 4.5.4. Other Applications 5. Droplet Microfluidics for Materials Generation 5.1. Nanoparticles from Droplet Microfluidics 5.1.1. Inorganic Nanoparticle Synthesis 5.1.2. Organic Nanoparticle Synthesis 5.1.3. Synthesis of Other Nanomaterials 5.2. Microparticles from Droplet Microfluidics 5.2.1. Polymer Microparticle Fabrication 5.2.2. Colloidal Material Assembly 5.3. Microcapsules from Droplet Microfluidics 5.3.1. Microcapsules from Single Emulsions 5.3.2. Microcapsules from Multiple Emulsions 5.4. Applications of the Droplet-Derived Materials 5.4.1. Drug Delivery 5.4.2. Tissue Engineering 5.4.3. Analytical Applications 5.4.4. Optical Devices 5.4.5. Imaging

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Received: December 29, 2016 Published: May 24, 2017 7964

DOI: 10.1021/acs.chemrev.6b00848 Chem. Rev. 2017, 117, 7964−8040

Chemical Reviews 5.4.6. Cell Mimics 5.4.7. Wettability 6. Conclusion and Perspective Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

Review

droplets with diverse morphologies and behaviors.26−30 In the aspect of applications, by virtue of such advances of theory and techniques, droplet microfluidics bears extensive value, including bio(chemical) analysis19,31 and nano- and microscale generation of materials.28,29,32 For bio(chemical) reactions and analysis, droplet reactors show enhanced mixing and mass transfer within short diffusion distances, and boundary effects (such as axial dispersion) are avoided.33−37 Thus, they possess several remarkable advantages. The first is miniaturization: droplet reactors are small, in the range of subnanoliter scale, making single-cell or molecular analysis possible. The second is compartmentalization: droplets generated in the microfluidic channels can be manipulated independently and therefore serve as individual units for reactions. The third is parallelization: droplets are monodispersed and identical, hence providing large-scale quantitative reaction platforms for high-throughput analysis. Therefore, droplet microfluidics offers a route to performing typical laboratory operations with a small volume of reagents in significantly less time. For materials fabrication, droplet microfluidics offers a versatile platform for generation of nano- or microsized particles.38−43 In nanoparticle (NP) synthesis, the superior control over reaction kinetics and thermodynamic parameters brings about NPs with a tunable size and crystal structure. In microparticle synthesis, as the droplets are generated in a highly controllable and reproducible manner, the production efficiency and particle monodispersity are much higher than those of conventional emulsification methods. Moreover, through geometric confinement, specific physical and chemical processes, and incorporaion of functional ingredients, the structures and compositions of the particles can be extremely flexible. Therefore, droplet microfluidics enables generation of materials with unprecedented features that would be difficult to obtain in other approaches. Because of the profound scientific and engineering standards of droplet microfluidics, several classic review papers on droplet microfluidics have emerged, which have usually been dedicated to certain aspects of its progress, such as chip fabrication,21,22,25 droplet physics,20 bio(chemical) analysis,32,34,35 or particle preparation.39,40,43 However, a comprehensive review on droplet microfluidics with emphasis on advancements in recent years remains lacking, and we believe that such a general and substantial review looking at all aspects of the droplet microfluidics would be of profound impact on this emerging, active, and pivotal field. Such a review should arouse a lot of inspiration from scientists with various research backgrounds. Therefore, we herein give an overall review on droplet microfluidics, covering its history to recent progress and future outlook. To begin, we will briefly summarize chip fabrication methods, as chip fabrication is the first and fundamental step in droplet microfluidics. Then, we will focus on the principle of droplet generation and manipulation, including physical mechanisms of droplet formation (either through passive hydrodynamic processes or by active external actuations), generation of droplets with various morphologies, and control of the droplet behaviors, such as coalescence or splitting. Following these two opening sections, much emphasis will be laid on the applications of the microfluidic droplets. Reactions in droplet microfluidic platforms will be discussed along with various analytical methods, and their functions in bio(chemical) analysis will be presented with focus on molecule detection, single-cell analysis, and other biomedical applications. The

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1. INTRODUCTION Microfluidics is the science and technology of systems with integrated channels on the microscale (tens to hundreds of micrometers), through which small quantities of fluids (usually 10−9 to 10−18 L) can flow in designed configurations that are controlled and manipulated systematically.1−3 The origin of microfluidics dates back several decades when it was motivated by the requirement of miniaturization and planarization in bio(chemical) analyses.4,5 Since then, the concept of “lab-on-achip” or micrometer-scale total analysis systems (μTASs) has been gradually set up.6,7 In the microfluidic world, as fluid dimensions shrink to the microscale, their specific surface area increases, thus bringing about behaviors divergent from those of macroscopic fluids, which can be characterized by three major phenomena: highly efficient mass−heat transfer, relative dominance of viscous over inertial forces, and significant surface effects.8−10 In addition, the high integration of microfluidic systems facilitates the coexistence and diverse interactions of multiple fluid phases.11 Such features have paved the way for miniaturized systematic control over and manipulation of individual fluids and fluid interfaces. Therefore, this fascinating area has been shedding new light on multidisciplinary research in the physical,12 chemical,13,14 biological,15,16 medical,17 and engineering18 fields. One subcategory of microfluidics is droplet microfluidics, which generates and manipulates discrete droplets through immiscible multiphase flows inside microchannels.19 Although at an early stage of development, microfluidics initially considered continuous flows of miscible fluid phases, droplet microfluidics has emerged from two complementary motivations: generating microscale flow reactors for μTAS studies and fabricating intricate droplet-based particles for materials research.20 In the past two decades, fostered by its great progress in both theoretical and technical aspects, droplet microfluidics has lived up to original expectations and is gradually becoming a significant branch of microfluidics, contributing to a huge scope of applications. The progress of droplet microfluidics in the past 20 years is embodied in the following respects. In the aspect of chip fabrication,2,21 benefiting from the constant introduction of new materials accompanied by novel fabrication techniques, droplet microfluidic chips have experienced a transitional development from simple two-dimensional (2D) microchannels22,23 to multifunctional three-dimensional (3D) systems.24,25 In the aspect of droplet dynamics, the principles of microfluidic droplet generation have been studied in depth. Droplets are usually generated either through passive hydrodynamic pressure26 or by active external actuations.27 An understanding of their unique fluid dynamics allows for precise control over droplets and their interfaces, and enables the novel design of systems for the generation and manipulation of 7965


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Figure 1. (A) Scheme of the transfer molding method using the PDMS replica as an intermediate, through which (i) μPSs and (ii) stencil-like μPSs are fabricated and (iii) microfluidic chips are further constructed after sealing and bonding. (B) Microfluidic circuit using one μPS sealed by a glass slide. (C) 3D microfluidic device made of three stacked μPSs, which has two inlets and one outlet. Adapted with permission from ref 68. Copyright 2007 Royal Society of Chemistry.

first-generation microfluidic chips emerged and were fabricated on silicon/glass substrates through standard micromachining methods.47 A typical micromachining process contains three steps.48−52 The first step is lithography.53,54 A substrate is first cleaned and spin-coated with a photoresist (PR) layer. Then the PR layer is covered by a mask with patterns of transparent and opaque areas. After exposure and development, the PR is selectively eliminated, and the geometric micropattern on the mask is thus transferred onto the substrate. The second step is etching.55,56 Under the action of a certain etchant, the substrate is selectively eliminated to generate microchannels with the desired geometry; then the PR is removed. The third step is bonding.57,58 This step is necessary to achieve closed channels rather than just open structures. Two micromachined substrates are aligned and made to cling to each other. Then, through thermal fusion or adhesive bonding, the two substrates are assembled to generate a sealed microfluidic chip. Generally, silicon and glass possess advantages in certain situations. For example, their thermal stability makes them feasible for some droplet-based chemical syntheses under high temperature. In addition, their solvent resistance is ideal for many droplet-based reactions in an organic environment. Besides, their specific optic properties, high thermoconductivity, and electroosmotic flow stability have allowed them to continue to play an important role in droplet microfluidics until now. In other aspects, however, their rigidity and gas impermeability features have restricted their applications under certain circumstances. For some biomedical analyses in an aqueous environment, glass or silicon is unnecessary. In addition, the high cost, demand for clean-room equipment, and considerable time consumption during the fabrication process are impediments for large-scale production.

applications of the droplet microfluidics for the synthesis of NP- and microparticle-based materials will also be described. Examples of the diverse materials and their unique functions will be enumerated. Finally, we will present a critical analysis on the current status and challenges, as well as opinions on the future development directions of droplet microfluidics.

2. MICROFLUIDIC CHIP FABRICATION The fabrication of chip microchannels is the first and fundamental step in droplet microfluidics. To generate and manipulate droplets and further achieve certain functions, the microchannels should be designed with a high degree of integration and specific geometric structures and physicochemical features, especially surface properties.21 This requires demand-driven selection of materials in coordination with appropriate fabrication methods. Currently, there are several kinds of major techniques for microfluidic chip fabrication, such as glass/silicon-based micromachining, polymer-based replica molding, commercially available modular assembly, newly developed 3D printing, and so on. Each of them has its own pros and cons and is suitable to different situations. 2.1. Micromachining

Microfluidics began to took shape when the progress of the silicon-based industry of microelectronics and microelectromechanical systems (MEMS) met with bio(chemical) requirements for μTAS applications.1−4 The advanced micromanufacturing techniques were thus directly applied to fabricating microchannels for gas or liquid chromatography44,45 and capillary electrophoresis.46 The miniaturized analytical ability with high sensitivity and resolution became a large motivation of early microfluidics.6,7 Under this background, the 7966


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Figure 2. Modular assembly of microfluidic chips using (A−C) capillaries and (D) commercial modules. (A) Scheme and optical images of the microfluidic chip fabrication through fiber embedment in PDMS and capillary insertion. Adapted with permission from ref 78. Copyright 2005 John Wiley & Sons. (B) Microfluidic chip fabricated by coaxial assembly of size-matching circular and square capillaries. (C) Microfluidic chip prepared by using a seven-bored capillary array as the injection tube. Adapted with permission from ref 81. Copyright 2014 Royal Society of Chemistry. (D) (i) Illustration of the “off-the-shelf” concept. Adapted with permission from ref 78. Copyright 2010 Royal Society of Chemistry. (ii) Microfluidic channels assembled by (iii) commercially available components. Adapted with permission from ref 86. Copyright 2013 Royal Society of Chemistry.

2.2. Molding Replication

monomer, different layers can be sealed after curing. The softness of the components allows the device areas to be reduced by more than 2 orders of magnitude. In addition, the high elasticity of PDMS makes it flexible to construct valves or pumps. This extension of soft lithography sparks the inspiration of constructing functional microfluidic systems.37,65 Given the gas permeability and nontoxicity of PDMS, such systems can be suitable for cellular applications.66,67 Even with the above-mentioned superiorities, PDMS also has its limitations. The organic incompatibility, small molecular absorption, and high-pressure intolerance restrict their applications in some certain situations. Therefore, attention has also been drawn to other kinds of polymer materials. As shown in Figure 1A, Studer et al. used structured PDMS stamps as replica intermediates to transfer the micropattern onto other polymers to construct the so-called micropatterned stickers (μPSs).68 By a following sealing and bonding procedure on a glass slide, microfluidic circuits were fabricated (Figure 1B). In addition, by stacking those stickers, 3D fluidic networks were prepared for droplet generation (Figure 1C). In fact, by using PDMS for transfer molding, many kinds of polymers, such as poly(urethane methacrylate) (PUMA)69 and commercial optical adhesives (NOAs),70 have been explored to generate microfluidic channels. Such chips compensate for the shortcomings of PDMS in some aspect while retaining the rapid prototyping process. The distinct physiochemical properties of the polymers contribute to different applications. Apart from PDMS soft lithography and PDMS-mediated transfer molding, there are other methods for rapidly prototyping microfluidic chips. Also, some thermoplastic materials can be applied to chip fabrication through hot embossing. As reported by Malaquin et al., a thermoplastic polymer (Dyneon THV) flat sheet was embossed in a heated hydraulic press between a cushion layer and a master layer of either PDMS or a silicon wafer.71 Then the replica was released and bonded by thermal treatment to generate the chips. Such a

The rapid progress of droplet microfluidics brings forward a demand for rapid prototyping and mass fabrication of the chips for both laboratory research and industrial production. In addition, the increasing biomedical and clinical application of droplet microfluidics requires the chips to be low-cost and thus disposable to avoid contamination.59 Therefore, the polymer replica molding technique has been applied to droplet microfluidic chip fabrication and is gradually gaining popularity.60,61 The most representative replica molding method is soft lithography, which was first proposed by Whitesides and coworkers. It refers to a set of techniques that use a soft elastomeric material replica, typically poly(dimethylsiloxane) (PDMS), to transfer structures or patterns from a master. The typical procedure of PDMS soft lithography includes the following steps.62 A transparent mask with a computer-aided design (CAD)-generated pattern is first prepared; then a photoresist layer (for example, SU-8) is used to transfer the pattern through photolithography and thus serves as the mold (master). Finally, PDMS prepolymer is cast on the mold and soon thermally cured to obtain the micropatterned channels. This process is remarkably faster and cheaper and can be carried out in mild conditions without using hazardous substances.63 Therefore, it has greatly promoted the progress of producing disposable chips for droplet-based biomedical and clinical applications. Besides, PDMS itself also shows some intrinsic advantages.22 Sealing of PDMS channels is much simpler without using a high temperature or pressure. Through air or oxygen-plasma oxidation, different components can be bonded conveniently. Therefore, multilayer stacking becomes much easier. This has also been demonstrated by Quake et al., who put forward the concept of “multilayer soft lithography”.64 They bonded multiple layers of PDMS and elastomers together through a covalent link. By simply adding an excess amount of the 7967


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Figure 3. 3D printed microfluidic chips. (A) Scheme of the SL fabrication process. Adapted with permission from ref 25. Copyright 2016 John Wiley & Sons. (B) SL manufacture of microfluidic standard components, including the connector, mixer, T-junction subcircuit, and flow-focus emulsifier. Adapted with permission from ref 95. Copyright 2014 National Academy of Sciences. (C) Microfluidic circuitry fabricated by the MJM method. Adapted with permission from ref 98. Copyright 2015 Royal Society of Chemistry. (D) (i) 3DTouch 3D printer. (ii) Microfluidic device generated through the FDM method. Adapted with permission from ref 100. Copyright 2012 Royal Society of Chemistry.

templates.78 The 3D axisymmetric flow-focusing geometry confined the droplets in the central axis of the channel and prevented contact with the walls. Weitz et al. set up a more explicit droplet microfluidic system that uses cylindrical and square glass capillaries as modules assembled on a glass slide (Figure 2B).24 The outer diameter of the cylindrical capillary and the inner diameter of the square tube match to ensure a coaxial geometry. The true 3D configuration of the microchannels can sheath the inner fluid; the chemical resistance of the glass capillary allows for generating droplets and microcapsules in either hydrophilic or hydrophobic systems. Since then, capillary microfluidics has been widely utilized and has shown versatility in generation of droplets and multiple emulsions.26 Besides, through an intricate geometric design, capillary channels can assemble into sequential insertion or multichannel injection regimes for hierarchical and parallel processing.79,80 It should be noted that, in the above-mentioned capillary microfluidic devices, a good alignment of the tubes is achieved manually. In contrast to this, Zhao et al. recently developed a novel device by inserting a commercially available, seven-bore annular capillary array into another capillary for single-step preparation of double emulsions (Figure 2C).81 The capillary array guarantees a fixed coaxial alignment without manual adjustment. Therefore, the innermost flow was sheathed radially by the middle flow and induced synchronous encapsulation of the inner and outer droplets. In addition, the multibore configuration of the capillary array provided parallel injection channels, thus facilitating precise preparation of multicomponent double emulsions. So far, capillary-based chip fabrication has been the most prevalent modular assembly method, and it has shown flexibility in droplet microfluidics, especially in particle-based materials fabrication and extended applications.43,82,83 In addition to capillary assembly, there are other types of modules for constructing microfluidic channels.84−89 For example, an “off-the-shelf” concept was put forward as using commercially available modules to construct microfluidic chips

device can withstand organic solvents and was used for dropletbased fluorescence detection. In addition, by using hot embossing or laser ablation, a variety of thermoplastics, including poly(methyl methacrylate) (PMMA), polycarbonate (PC), poly(vinyl chloride) (PVC), etc., become available provided that the surface roughness issue is well addressed.72−75 Besides, the thermoset polyester (TPE) can be applied directly to generating microfluidic chips by pouring the prepolymer onto the SU-8-coated silicon master and curing.76 Because TPE is gas impermeable, it is suitable for some oxygensensitive applications. Overall, polymer based replica molding, especially PDMS soft lithography have largely revolutionized microfluidic chip fabrication process as it enabled fast and inexpensive manufacturing with a broader range of materials options. The distinct properties of different polymers make it useful for different situations. So far, a large majority of microfluidic systems are based on such chips, and it has been applied to many aspects of droplet microfluidics including biochemical analysis and materials fabrication. 2.3. Modular Assembly

Modular assembly microfluidics emerges with the requirement for providing strict 3D symmetrical microchannels for stable and controllable microencapsulation, which is of great significance for biomedical and industrial areas. Conventional polymer chips are based on quasi-2D planar microchannels. This may lead to problems because the droplets are susceptible to contacting the channel walls, and thus, the droplet generation process becomes unstable. To circumvent this issue, spatial wettability modification of the channels with wellselected surfactants is needed,77 which is complicated and labor consuming. Therefore, researchers began to solve this problem from a geometric approach, i.e., construction of 3D droplet microfluidic channels. As presented in Figure 2A, a typical device could be fabricated by inserting cylindrical glass capillaries into PDMS channel molds, which were simply prepared using optical fiber 7968


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(Figure 2D).85 Standard fluidic fitting parts, such as Teflon tubes and microferrules, are assembled into a hydrodynamic focusing nozzle for droplet generation.86 Such individual components can be easily assembled, disassembled, and reassembled. Lapierre et al. demonstrated a faster and cheaper fabrication method using a micropipet tip and self-setting rubber.88 Chu et al. constructed microfluidic systems using glass slides as building blocks.89 The device is divided into three functional units respectively used for flow control, positioning, and connection. The easy assembly and reverse connection ability of the individual parts allow for further scale-up and functionalization research.

through two-photon absorption upon illumination to break through the optical diffraction limit. In addition to SL, there are other 3D printing techniques relevant to microfluidics.97−102 For example, Lin et al. used the multijet modeling (MJM, also known as PolyJet) method to construct microfluidic circuit components and operators (Figure 3C).98 This is a process of creating 3D structures with curable polymers by using inkjet printers. It has also been demonstrated that MJM enables resolution of tens of micrometers and a broader range of materials choices. In addition, the components could be integrated to achieve fluidic processors and networks. Besides, Cronin et al. used the fused deposition modeling (FDM) technique to build lab-on-a-chip reactors (Figure 3D) at low materials cost and fast speed.100 In this method, layers of thermoplastic polymers are deposited through a heated extrude nozzle to build up 3D structures. These techniques all together make 3D printing an increasingly important approach to fabricating 3D microfluidic chips.

2.4. 3D Printing

During the past several decades, droplet microfluidics has achieved rapid progress and shows great potential of outperforming traditional analysis methods. However, to make the potential value come true, it is essential for microfluidics to find a so-called “killer application” and promote its industrialization process.25,90−92 This calls for absolute 3D systems with integration of various components such as valves and pumps to achieve rapid, high-throughput performances and multiple functions. In soft lithography, the time and fabrication costs would increase dramatically with structural complexity; the multiple layer stacking process requires multistep procedures and thus is lower in automaticity. As an alternative, a brand new technology3D printinghas been introduced for microfluidic chip fabrication. 3D printing is a flourishing and burgeoning field which involves a class of techniques for directly generating 3D structures in a single step. It creates a real 3D structural object by building successive layers of materials and adding them together automatically under computer control. Apart from using masks, the 3D geometric information is stored in a CAD file and automatically processed by a computer-controlled printer system. This not only satisfies arbitrary structural design via a “mail-order” service, but also enables more accurate fabrication with high resolution and less time and labor costs. Besides, as an additive manufacturing method without removal of redundant parts, it is environmentally friendly. Moreover, such a fabrication route can be commercialized with integration of industrial-grade user interfaces and embedded control systems. These features may indicate a new trend of droplet microfluidic chip fabrication. Among the many types of 3D printing techniques, stereolithography (SL) is one of the best established methods and is most suitable for microfluidic chip fabrication.93 SL refers to a process of polymerizing photocurabe resin liquids layer by layer though laser irradiation, as sketched in Figure 3A.25 For fabricating microfluidic chips, the walls of the channel cavities are polymerized and the uncured precursor is soon drained. Bertsch et al. first applied SL to microfluidic chip fabrication and demonstrated that it is convenient and efficient in time and cost.94 Most importantly, it allows for generating small and complex channel structures that are unachievable using other methods. Recently, Malmstadt et al. employed the SL technique to generate a library of standard components and connectors to assemble into droplet emulsification circuits (Figure 3B).95 The self-alignment capacity and reconfigurable ability make it feasible for constructing hierarchical 3D configurations difficult to achieve by other modular technologies. It is worth noting that, by using the two-photon polymerization96 technique, the resolution can be improved

2.5. Other Methods for Chip Fabrication

The introduction of new technologies or materials always promotes new approaches for the microfluidic chip fabrication. For example, paper-based microfluidic chips103−106 have been fabricated through capillary wetting, photolithography, and embossing.105 These processes enable facile preparation, pumpfree operation, and surface-related reactions. In addition, by using “origami”,106 i.e., paper folding, 3D microfluidic chips can be made. Furthermore, with the intrinsic merits of paper materials, including abundant resources, porous structure, white background, and biocompatibility, such devices show value in diagnostics and chemical reaction. Besides, the bioprinting methodology was invented through controlled extrusion and deposition of hydrogel fiber templates for fabrication of microchannel networks and microfluidic constructs,107 which offered a perfect 3D environment for cellular studies and high potential for tissue engineering and organ-on-a-chip applications. On the other hand, by combining different fabrication techniques, materials of different physical and/or chemical properties can be composited into one chip. A prominent example is the construction of a hybrid glass−PDMS microdevice.108,109 The PDMS elastomer enables facile integration of pneumatic valves and pumps, and the glass substrate minimizes fluid−elastomer contact and thus provides precise control over the channel surface chemistry. Besides, the concept of “cofabrication” was applied to building sequentially aligned, multifunctional microchannels.110 By using multigroove PDMS molds, channels could be endowed with spatially heterogeneous functions through additive dropping. In addition, microfluidic chips could be combined with elements such as optical components and microheaters, which should realize multiple functions. Overall, with continuing interdisciplinary research and technical progress, the fabrication of the microfluidic chips would not be limited to the above technologies, and there will be more and more materials and approaches being employed.

3. DYNAMICS OF MICROFLUIDIC DROPLETS Understanding microfluidic flow behaviors is not only significant in fluid mechanics, but also instructive for exploiting its applications. In most cases, microfluidics deals with multiphase flows rather than single-phase flow according to the practical situations.111 Early on, stratified flows were 7969


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Figure 4. Driving force of microfluidics. (A) Photograph of a syringe pump with a pushing block, which drives the liquids in the syringes by electric control. (B) Compressed-air-driven microfluidic system. Compressed air passes through a valve to the main supply line, which branches into two control channels with sample arms and relief needle valves. Each of the control channels is attached to one of the two inlets of a microfluidic device through a piece of tubing. Adapted with permission from ref 114. Copyright 2010 Royal Society of Chemistry. (C) Gravity-driven microfluidic system: (i) scheme illustration; (ii) overall photograph of the system. Adapted from ref 115. Copyright 2013 American Chemical Society.

considered, in which streams of miscible fluid phases flow continuously along each other. Due to the miniaturized fluid dimensions and the resultant laminar flow condition, mixing is dominated by molecular diffusion, which is very slow.3 Although practical in some cases such as chemical deposition or interfacial studies, such stratified flow configuration is defective for initiating reactions, which requires rapid mixing.112 In droplet-based flows, however, immiscible flow phases are introduced, which generate a discrete volume of fluids and moving interfaces. The convective flow profile inside the droplets facilitates mixing, and the compartmentalization of the ingredients avoids contamination. Therefore, droplet microfluidics has been extensively studied both theoretically and practically ever since its advent. In general, the dynamics of microfluidic droplets are still governed by the linear Stokes equations, while nonlinear behavior occurs due to the deformable interfaces, the variable interfacial tension, and specific boundary conditions.20,113 Therefore, the fluid dynamic problems become more complicated. However, such complexities are compensated by offering flexible application platforms. On one hand, droplet microfluidics enables generation of well-calibrated drops in a reproducible and designable way, which is highly applicable in materials fabrication and related industry fields such as food, cosmetics, and pharmaceuticals.43 On the other hand, the compartmentalized droplet microreactors can be manipulated individually, thus facilitating bio(chemical) reactions and labon-a-chip applications.35

Generally, by using syringe pumps (Figure 4A), the forces applied to the syringe can be adjusted appropriately, and a specified volume flow rate can be maintained to provide measurable fluid pressures. Also, by using air pressures,114 fluids are pushed through a piece of tubing connecting the microfluidic device and the sample loading chamber, which were constructed by cutting and linking pipet tips. The static air pressures were monitored by pressure regulators, as shown in Figure 4B. Other than using such external mechanical forces, fluids can also be driven by their own gravity. As reported by Zhao et al., fluids fell via a vertically placed funnel into the microfluidic channels (Figure 4C). In this method, the sustainable and ultrastable driving was assured in two aspects: the fluids were drawn continuously through reservoirs, and the heights of the funnels were fixed with the help of an electromechanical controller.115 Once multiple fluid phases are introduced to meet in confined microchannels with designed geometries, the integration of the different phases and their interactions give rise to dynamic responses. Droplet generation is such a response that the interfaces deform and then pinch off. From an energetic perspective, the breakup of the continuously fed fluid and the generation of separate droplets is an energy input process because excess energy is provided and converted into the interfacial energy in the emulsions finally produced.27 This process can be categorized into passive and active methods. In the former case, the interface is deformed under certain flow fields; the pinch-off is naturally achieved as a result of interfacial instabilities, and thus, there is no exogenous energy supply. In the latter case, local actuations such as electrical, magnetic, thermal, and mechanical forces are exerted during the droplet breakup, and thus, external energy input is provided to account for the excess interfacial energy.

3.1. Driving Force of Microfluidics

Fluid phases are first introduced to flow into the microfluidic channels by different driving forces. To guarantee the performance of the overall system, the driving force should be measurable and stable. The most common driving force is pressure, which utilizes the relative pressure difference between the inlet and outlet of the flow channel to deliver the fluid. 7970


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Fischer et al.117 A capillary consisting of a nozzle was inserted into a rectangular flow channel. The dispersed phase was thus injected into the ambient continuous phase in a coaxial geometry. The authors provided comprehensive experimental data revealing that the sizes of the droplets were affected by the fluid properties and flow rates. The size decreased with the continuous flow rate, due to higher viscous shear stress; it increased with the dispersed flow rate, as a larger volume of dispersed phase fluid entered a droplet before breakup. Also, the size increased with reduction of the interfacial tension, as a result of lower resistance to droplet pinch-off. Compared with these parameters, the viscosity of the two phases had smaller effects on the droplet size. The authors also distinguished two different droplet regimes: the dripping regime, where droplets were generated next to the capillary tip, and the jetting regime, where an extending liquid jet formed before droplet breakup in the downstream. Furthermore, more detailed research was conducted on the transition process between dripping and jetting.118 Droplet behaviors were observed and characterized through state diagram analysis. At low flow rates of the two phases, dripping occurred. By individually increasing the continuous or disperse flow rates, a narrowing or widening jet was respectively generated before breakup, as illustrated in Figure 5B. The two classes of dripping to jetting transitions indicate different mechanisms with respect to different dominant forces. In the first class, when the continuous fluid rate was high, the viscous shear stress was dominant and the balance between viscous force and interfacial tension determined the transition of dripping to narrow jetting. This process could thus be characterized by the capillary number of the continuous phase, Cao. In the second class, when the dispersed flow rate was high, the inertial effect became dominant and the balance between inertial force and interfacial tension determined the transition of dripping to wide jetting. This process could thus be characterized by the Weber number of the dispersed phase, Wei. 3.2.2. Cross-Flow. In the cross-flow category, the dispersed and continuous fluids meet at an angle.27 The most common case is called the T-junction, where dispersed and continuous phases flow through orthogonal channels and meet at a crossjunction, as shown in Figure 6A. As the dispersed phase partially blocked the continuous phase, a shear gradient was established, and the dispersed phase elongated and eventually broke into droplets. The size of the droplets depended highly on the shear stress. Guillot et al. observed that the flow regimes varied with the flow rates, fluid viscosities, and aspect ratio of the channel cross-section. As depicted in the flow pattern diagram in Figure 6B, droplets could be generated at the Tjunction or at the downstream of the channel after a section of parallel flow.119 More explicitly, three distinct flow regimes were distinguished as squeezing, dripping, and jetting. As demonstrated by Stone et al.120 through a numerical study, in the squeezing regime when the value of Ca was low, the tip of the dispersed phase obstructed the junction and expanded into the continuous phase. The resultant increase of pressure thus squeezed the neck of the interface until it was pinched off. In the dripping regime, when the value of Ca was high enough, the interfacial tension was overcome by the viscous shear force, and thus, the droplet broke up before the dispersed phase blocked the channel. In the jetting regime, when the Ca value was further increased, a jet formed before the droplet broke up in the downstream of the channel.

3.2. Droplet Formation by Passive Methods

In passive, pressure-driven flow methods, immiscible dispersed and continuous fluids meet at a junction. The intricate geometrical design of the microchannel junction defines the local fluid fields and thus determines interface deformation and droplet breakup. According to the different channel geometries and fluid configurations, droplet formation can be classified into cof low, cross-f low, and f low-focusing categories.116 In all of these cases, the fluid behavior can be characterized through some important dimensionless numbers, which are calculated by the parameters of the fluid properties, flow condition, and geometric features. Such dimensionless numbers help to define the relative importance of different forces. The first is the Reynolds number, Re, which measures the inertial to viscous forces. Under normal flow velocities, Re is usually very small so that the inertial force becomes irrelevant. Generally, droplet formation is characterized by two competing effects: the extension and deformation of the interface induced by local shear stress and the resistance of deformation achieved through capillary pressure. Therefore, the most significant forces in droplet microfluidics are interfacial tension and viscous force. This thus brings up the capillary number, Ca, which compares the relative strength of viscous to interfacial forces. Ca is helpful in predicting droplet formation and its dimensions. The third number is the Weber number, We, which compares inertial to interfacial tension. Under high flow velocities, inertial effect matters play a role in the transition of discrete droplets into continuous jets. The above-mentioned three numbers and the relevant forces are the most important ones. However, under certain circumstances, several other forces also become relevant, such as gravity, buoyancy, and elastic effects, which can also be defined through corresponding dimensionless numbers. 3.2.1. Coflow. Coflow streams are achieved by using a set of coaxial microchannels. The dispersed fluid phase is introduced into an inner channel, and the continuous phase flows into an outer concentric channel in the same direction, as shown in Figure 5A. The first experimental coflow setup was reported by

Figure 5. Coflow droplet generation. (A) Scheme of coflow droplet microfluidics. Adapted with permission from ref 116. Copyright 2007 IOP Publishing. (B) Microscopic images of droplet generation in the (i) dripping regime, (ii) narrow jetting regime, and (iii) wide jetting regime. Adapted with permission from ref 118. Copyright 2007 American Physical Society. 7971


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phases were forced to pass a small orifice. Although there has been no simple model for droplet size prediction, a large number of experimental and theoretical studies show that the droplet formation is strongly affected by the channel geometry, flow rate, fluid viscosity, and addition of surfactants. Generally, this method allows for making small droplets as the breakup process is closely related to the size of the narrow contraction. Anna et al. further characterized the flow configurations into four regimes with the presence of surfactant, as shown in Figure 7B.123 In the “geometry-controlled breakup” regime with a small value of Ca, a dispersed phase finger flow squeezed through the narrow region, and was then pinched off into droplets due to the increasing hydrodynamic pressure. Increasing the value of Ca led to the dripping regime, where the dispersed phase finger was thinner and the droplets were smaller than the orifice diameter. A continued increase of the value of Ca led to the jetting regime, where the dispersed phase finger extended further and droplets broke up beyond the orifice outlet with larger size than in the dripping regime. When surfactant was added with a moderate concentration and the value of Ca was set between those of the first two regimes, a new regime called “thread formation” occurred, and a thin liquid thread was generated and further broke up into large droplets.

Figure 6. T-junction droplet generation. (A) Scheme of T-junction droplet microfluidics. Adapted with permission from ref 116. Copyright 2007 IOP Publishing. (B) Flow pattern diagram showing three distinct flow regimes with respect to different flow rates of the oil and water phases: (○) droplets were generated at the junction; (◇) a parallel flow formed; (●) droplets formed after the parallel flow was broken. Adapted with permission from ref 119. Copyright 2005 American Physical Society.

3.3. Droplet Formation by Active Methods

In active methods, droplet generation is activated or assisted by external forces to exert local actuation and achieve a fast response.27 According to the type of energy sources, active droplet generation can be classified into electrical, magnetic, thermal, and mechanical methods. 3.3.1. Electrical Method. Electrical control can be achieved by using a direct current (dc) or an alternating current (ac). Link et al. applied a constant dc voltage by incorporating electrodes into the flow-focusing device, as shown in Figure 8A.124 The water flow served as a conductor, and the oil stream acted as an insulator. Thus, the water−oil interface behaved like a capacitor. Free charges were accumulated on the interface after electrochemical reaction. Apart from the interfacial tension and viscous force, droplets breaking up were assisted by an additional electrical field force. The size of the droplets could be controlled precisely by tuning the electric field strength, which decreased at higher voltage. Besides, ac can be used for droplet generation through the electrowetting-on-dielectric (EWOD) effect.125 The EWOD mechanism lies in that an electrical field can be applied to reduce the contact angle between the conductive liquid flow and the channel.126 Therefore, through the controllable wettability switch of the channel, the dispersed phase liquid was first spread out, forming a liquid finger, and then retracted to break up into droplets. Besides, when the liquid flow is electrically neutral, an ac electric field can also be employed for droplet generation through another mechanism, which is called dielectrophoresis (DEP).19 The liquid polarizes in response to an electric field gradient, and the droplet can be prepared with the help of the DEP drawing force. 3.3.2. Magnetic Method. The noncontact, magnetic control can be actuated by using ferrofluids.127,128 Ferrofluids refer to suspensions of magnetic nanoparticles in an aqueous or oily carrier liquid. It can be treated as a continuum and serve as either a dispersed or a continuous phase in droplet microfluidics.127 Nguyen et al. applied a water-based ferrofluid to work as the dispersed phase, as shown in Figure 8B.128 They

It should be noted that, apart from the classic T-juction, there are other modified geometry designs for different purposes. For example, a “K-junction” device with an added leg provides an exit channel for the waste.121 In addition, a “Vjunction” device has been demonstrated to possess a high degree of operational flexibility.122 3.2.3. Flow-Focusing. In flow-focusing geometry, the dispersed and continuous phases flow coaxially through a contraction region and generate an elongation filament that eventually breaks into droplets, as shown in Figure 7A. The narrow region has the function of shear-focusing and thus contributes to uniform droplet generation. The early experimental apparatus was reported by using a planar flowfocusing geometry device, through which two immiscible fluid

Figure 7. Flow-focusing droplet generation. (A) Scheme of flowfocusing droplet microfluidics. Adapted with permission from ref 116. Copyright 2007 IOP Publishing. (B) Microscopic images of flowfocusing droplet generation in the (i) “geometry-controlled breakup” regime, (ii) “thread formation” regime, (iii) dripping regime, and (iv) jetting regime. Adapted with permission from ref 123. Copyright 2006 AIP Publishing LLC. 7972


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air, with the desired frequency and well-controlled size achieved by tuning the flow velocities, as shown in Figure 8D. Apart from using pneumatic/hydraulic elements, faster actuation can also be achieved by using piezoelectric elements,132 including surface acoustic wave (SAW) devices,133 to induce mechanical vibration for droplet generation. Besides, Ismagilov and coworkers invented a method called “SlipChip” for mechanical droplet generation without pumps or valves.134 Basically, the device consists of a top plate and a bottom plate, each containing wells with sample loading. To generate droplets, the wells are first aligned and then disconnected simply by slipping the chip. Thus, droplets could be generated in a multiplexed manner. 3.4. Complex Droplet Formation in Microfluidics

Droplet microfluidics offers a versatile route to precisely generate droplets one by one, and it is worth far more than just fabricating simple homogeneous droplets. Through the intricate design of fluid channels, complex droplets with the desired shape anisotropy and exceptional features can be prepared.43,80,83 By using parallel channels, Janus or multicompartmental droplets can be achieved; by combining the three categories of droplet-generating modules (coflow, crossflow, and flow-focusing), double or multiple emulsions can be generated. Such elaborate droplets can serve as templates for materials fabrication and have significant application values in related industry fields such as food, cosmetics, and pharmaceuticals. 3.4.1. Multicomponent Droplets. A straightforward design for rendering anisotropy to the droplets is to use parallel channel combinations to simultaneously emulsify different dispersed phases. For example, Nisisako et al. introduced a biphase flow-focusing microfluidic device.135 The main part of the device was a Y-shaped channel, through which two types of organic fluids were introduced individually from the two distinct branches. Then the two fluids merged into one biphase dispersed stream and broke up together into droplets by the shear sheath flow, as shown in Figure 9A. The droplets possessed Janus geometry with two distinct halves. It is worth noting that, due to the low value of Re in microfluidic systems, the streams were kept laminar, and the mixing of the two organic flows was dominated by diffusive rather than convective transport, which was rather slow. Thus, there existed a clear phase boundary, which guaranteed the feasibility of the method. In addition to such symmetry designs, the authors further presented an asymmetric biphase T-junction device with a similar Y-shaped channel, through which two organic dispersed fluids were injected.136 When the two immiscible dispersed phases M and S were emulsified by the continuous phase, two phases came into contact without mixing. They evolved into a configuration consistent with minimum interfacial energy. Quantitative analysis revealed that the interfacial tension between the two phases determined the spreading coefficients. In some cases, partial engulfing occurred, which gave rise to Janus droplets. This also occurred when the positions of the S and M streams were exchanged, as shown in Figure 9B. Yang et al. used a biphase coflow device which consisted of two inner capillaries and an outer capillary.137 The two dispersed fluids were not merged before emulsification, but rather broke up individually and coalesced into one droplet, as shown in Figure 9C. Moreover, such a parallel emulsification design can be extended to fabricate ternary or quaternary

Figure 8. Droplet generation mediated by active methods. (A) Charged droplet generation by applying a dc voltage: (i) scheme of the microfluidic device; (ii, iii) droplet generation at voltages of (ii) 600 V and (iii) 800 V. Adapted with permission from ref 124. Copyright 2006 John Wiley & Sons. (B) Ferrofluid droplet generation by applying a magnetic field: (i) scheme of the microfluidic device; (ii−v) formation of ferrofluid tips before droplet breakup at increasing value of the magnetic bond number, Bm. Adapted with permission from ref 128. Copyright 2011 Springer. (C) Thermally mediated droplet formation: (i) scheme of the microfluidic device; (ii, iii) droplet formation when the heater is (ii) turned off and (iii) turned on. Adapted with permission from ref 130. Copyright 2007 AIP Publishing LLC. (D) Mechanically mediated droplet formation by using active pneumatic choppers: (i) scheme of the microfluidic device; (ii−iv) droplet generation at increasing flow velocity ratio of the continuous and dispersed phases. Adapted with permission from ref 131. Copyright 2006 IEEE Xplore Digital Library.

demonstrated that the presence of a uniform magnetic field could exert an additional magnetic force that dragged the ferrofluid tip forward to form a longer thread before breaking up into droplets. The droplet behavior could be characterized by using a so-called magnetic bond number, Bm, with definition of the relative strength of the magnetic force to interfacial tension. 3.3.3. Thermal Method. The thermal control mechanism lies in the dependency of Ca on the temperature due to variations of fluid properties such as viscosity and interfacial tension. Heat can be introduced by heating the entire device or the junction or through localized laser irradiation.129,130 For example, a microheater and a temperature sensor were integrated into a flow-focusing device to mediate droplet formation, as shown in Figure 8C.130 They normalized the fluid viscosity and interfacial tension as functions of the temperature difference and demonstrated that the droplet regime and diameter can be well controlled by the temperature. 3.3.4. Mechanical Method. Mechanical forces can be exerted through hydraulic or pneumatic elements. They are thus often used in elastic devices where valves and pneumatic membranes can be easily integrated (as described before). Droplet generation and the droplet size can be controlled through adjustment of the pressure pulse. Lee et al. designed a 10-valve pneumatic chopper, which is integrated into the outlet of a flow-focusing junction.131 The focused dispersed stream is directly cut into droplets by the chopper through compressed 7973


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Figure 9. Microfluidic generation of Janus droplets. (A) Scheme and microscopic image of hemispherically colored droplet formation in a biphase flow-focusing device. Adapted with permission from ref 135. Copyright 2006 John Wiley & Sons. (B) Droplet formation in a biphase T-junction device: (i) the two organic phases M and S are located in the two branches of the Y-shaped channel, respectively; (ii) the positions of M and S phases are exchanged; (iii) alternate Janus droplet generation from two opposed T-junctions. Adapted with permission from ref 136. Copyright 2009 Springer. (C) (i) Scheme and (ii) microscopic image of Janus droplet formation at the tips of the paired injection capillaries of a coflow microfluidic device. Adapted with permission from ref 137. Copyright 2008 John Wiley & Sons.

Figure 10. Microfluidic fabrication of double emulsions. (A) W/O/W emulsion generation in a two-step T-junction device. Adapted from ref 138. Copyright 2004 American Chemical Society. (B) Double-emulsion generation in a sudden expansion channel: (i) scheme illustration of the microfluidic device with two junctions; (ii) oil droplets formed through flow-focusing at the first junction; (iii) double emulsions formed after water insertion at the second junction. Adapted with permission from ref 141. Copyright 2014 Elsevier. (C) Single-step generation of double emulsions in a device combining the coflow and flow-focusing configurations: (i) scheme illustration of the system; (ii, iii) double emulsion formation in the dripping and jetting regimes, respectively. Adapted with permission from ref 24. Copyright 2005 The American Association for the Advancement of Science. (D) Single-step generation of double emulsions in a capillary array injection device: (i) scheme illustration of the system; (ii, iii) microscope images showing the generation of (ii) double emulsions and (iii) multicomponent double emulsions. Adapted with permission from ref 81. Copyright 2014 Royal Society of Chemistry.

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Figure 11. Microfluidic fabrication of high-order multiple emulsions. (A) Generation of multiple emulsions in a stepwise flow-focusing device: (i−v) generation of single-, double-, triple-, quadruple-, and quintuple-emulsion droplets, respectively. Adapted with permission from ref 148. Copyright 2009 John Wiley & Sons. (B) Generation of multiple emulsions in a stepwise coflow platform: (i) scheme illustration of the system; (ii−iv) generation of single-, double-, and triple-emulsion droplets, respectively. Adapted with permission from ref 139. Copyright 2007 John Wiley & Sons. (C) (i) Scheme of single-step generation of quadruple emulsions. (ii, iii) Microscopic images of (ii) the generation process and (iii) the resultant quadruple-emulsion droplets. Adapted with permission from ref 149. Copyright 2011 John Wiley & Sons.

emulsions with multicomponent inner drops could also be generated conveniently.140 Apart from assembly of the basic droplet generators, Kim et al. introduced a two-step capillary-based device with a novel geometric design.141 The main part of the device consisted of a narrowing channel followed by a sudden expansion channel. In the first step, O/W emulsion droplets were generated by flowfocusing. In the second step, when the oil droplets flowed through the narrowing channel, the increase of the velocity and the accumulation of pressure accelerated droplet migration. Thus, the inertial effects became significant. The rear of the drop concaved inward in response to the parabolic flow field, whereas the front of the drop moved forward to enter the expanding channel and relaxed into a spherical shape again. Therefore, water from the continuous phase was inserted to form W/O/W double emulsions, as shown in Figure 10B. Double emulsions can also be generated in a single step through simultaneous convergence of the three phases into one point. Weitz et al. designed a capillary-based device which combined the coflow and flow-focusing configurations.24 The inner fluid and the immiscible middle fluid flowed coaxially, and an outer fluid that was immiscible with the middle fluid flowed in the opposite direction. When all three fluid phases were forced through a tapered orifice, double emulsions were generated in a single step as a result of the hydrodynamic focusing, as shown in Figure 10C. Kumacheva et al. reported a one-step flow-focusing device.142 It was different from the twostep flow-focusing device, where three phases flow through two cascade contraction regions and experience two successive emulsification processes. In this device, the three phases flowed into a single contraction region, and the inner and middle phases formed a liquid jet together. The jet further broke up due to capillary instability mechanisms, which enabled synchronized emulsification of the inner and middle phases.

droplets. Zhao et al. used a four-channel coflow device with a four-barrel capillary.79 Four different oil fluids were injected through each barrel as distinct dispersed phases, and a surrounding aqueous fluid served as the continuous phase and flowed in the same direction. It was observed that the four oil phases tended to coalesce at the outlet of the channel upon contact, therefore generating multicomponent droplets. As each fluid phase was injected separately, their flow rates could be adjusted independently through syringe pumps. 3.4.2. Double-Emulsion Droplets. Another method for adding complexity to the droplet geometry is to implement multilevel emulsification, through which double- or multipleemulsion droplets can be generated on demand. Such a hierarchical system contains smaller droplets inside and is ubiquitous in encapsulation and release-based application areas. Double emulsions can be generated by sequentially adopting the three fundamental droplet-generating modules. For this purpose, a two-step T-junction device was designed.138 In the first step, water-in-oil (W/O) emulsion droplets were generated in an upstream hydrophobic T-junction; in the second step, the droplets were encapsulated by another aqueous phase downstream through a hydrophilic T-junction, thus generating waterin-oil-in-water (W/O/W) double emulsions, as shown in Figure 10A. By changing the flow rates, the size and the number of inner drops could be controlled. Also, O/W/O emulsions can be generated by exchanging the wettability of the two junctions. Such a two-step design concept has also been applied to sequential coflow categories,139 and a large number of studies have demonstrated double-emulsion generation by these methods, through either 2D planar devices or 3D capillary devices. Besides, by using a hierarchical device with building blocks of a drop maker, a connector, and a liquid extractor, sequential emulsification was conducted, and double 7975


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Figure 12. Droplet sorting in a microfluidic device. (A) Passive, size-dependent droplet sorting: (i) scheme illustration of the method; (ii) microscopic image of the separation module. Adapted with permission from ref 152. Copyright 2009 AIP Publishing LLC. (B) Active, dielectrophoretic droplet sorting: (i) schematic view of the device; (ii) with no electric field, droplets fall into the waste channel; (iii) in the presence of an electric field, droplets fall into the collection channel. Adapted with permission from ref 153. Copyright 2006 AIP Publishing LLC.

the square capillary. The third-layer aqueous phase W3 flowed along the wall of the square capillary. These three phases flowed in the same direction. The fourth-layer oil phase O4 flowed through the interstice between the square capillary and the collection tube, and the fifth-layer aqueous phase W5 flowed along the wall of the square capillary. These two outer phases flowed in the direction opposite that of the above three inner phases. The fluid phases formed a liquid jet with multilayer interfaces through the orifice of the collection tube, which then broke up into W1/O2/W3/O4/W5 quadruple-emulsion droplets. The key to this design was the spatial confinement of the interface, which stabilized the multilayer liquid jet before it was broken up.

A similar concept was applied to single-step coflow design, as demonstrated by Zhao et al., who used an annular capillary array as the injection tube.81 The inner and middle fluids flowed coaxially through the inner layer and the sheath layer of the tube, respectively, into a surrounding outer fluid. When the three phases met at the tip of the injection tube, the inner and outer drops formed simultaneously and double emulsions were prepared. Moreover, by injecting multiple inner phases into the capillary array, multicomponent double emulsions with different inner cores could also be obtained from the same device, as shown in Figure 10D. The design of parallel and multilevel emulsification can be coupled. By using parallel channels for different inner or middle phase fluids, double-emulsion droplets with multiple cores81,143 or shells144 have been fabricated. It is worth noting that, in some cases, double-emulsion generation also needs to be mediated by external forces such as pneumatic control,145 electrical force,146 and mechanical vibration.147 3.4.3. Multiple-Emulsion Droplets. In addition to double emulsions, higher order emulsions with hierarchical onion-like configurations can also be fabricated, either through multiple combinations of the basic droplet generators or by one-step emulsification methods. For example, a multistep flow-focusing device was designed through a lithographic process.148 The basic module of the device was a flow-focusing drop generator with spatial wettability modification. Concatenating such modules enabled stepwise fabrication of single, double, triple, quadruple, and quintuple emulsions, as shown in Figure 11A. In addition, a more flexible three-step coflow device was invented by using consecutive capillary assemblies.139 The device consisted of an injection tube, two transition tubes, and a collection tube, which were sequentially inserted with good coaxial alignment. Thus, triple emulsions were generated in a highly controllable way as the size and number of drops at each level could be adjusted, as shown in Figure 11B. It was also anticipated that, by adding more transition tubes, higher order emulsions could be fabricated. Apart from these methods, highorder emulsions could be generated in a simpler device, which enabled single-step breakup of multilayer interfaces, as shown in Figure 11C.149 The device consisted of two tapered cylindrical capillaries as the injection and collection tubes, respectively. The two tubes were assembled end-to-end inside a square capillary. The first-layer aqueous phase W1 flowed through the injection tube. The second-layer oil phase O2 flowed through the interstice between the injection tube and

3.5. Droplet Manipulation in Microfluidics

Droplet microfliudics offers a way of fabricating monodisperse droplets. The droplets serve as individual microreactors when chemical and/or biological reagents are incorporated.34,35 To promote reactions, droplets need to be further manipulated after generation, to execute desired protocols including sorting, coalescence, splitting, mixing, trapping, etc. These manipulations are fundamental elements for integrated lab-on-a-chip applications. Generally, such manipulations can be conducted through passive hydrodynamic methods or by active methods through external triggers including mechanical, thermal, electric control, etc. 3.5.1. Sorting. Sorting is a process of transporting and distributing droplets into different channels downstream, and it is a necessary step for selection and segregation of droplets to conduct further analysis. Sorting can be classified into two types, passive sorting and active sorting. In passive sorting, droplets are guided to transport along a specific route under the hydrodynamic interactions between the channel geometries and the droplet properties.150−152 A typical example is to utilize the coupling effects between a specific flow profile and droplet size. For example, Griffiths et al. described such a size-dependent sorting system within a rectangular channel.152 Monodisperse droplets with larger and smaller dimensions were first generated separately in two distinct flow-focusing junctions. The two kinds of droplets were then mixed and collected to flow through one channel, the depth of which was set to be between the sizes of the two droplets. Therefore, the larger droplets flowed slowly and occupied most spaces of the channel, and the smaller droplets flowed faster and were trapped alongside the channel walls behind the larger droplets. Afterward, the channel 7976


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branched downstream into a widening central main channel and two narrowing side channels. Therefore, the larger droplets were released into the main channel, while the smaller droplets entered the side channels, as shown in Figure 12A. Apart from size-dependent sorting, droplets can also be separated by differentiation of other properties such as gravity and viscosity. Active sorting is achieved by external fields. One of the earliest and most extensively applied methods is electrophoretic manipulation. As shown in Figure 12B, a high-speed electrophoretic sorting platform was developed.153 Water droplets were generated in a flow-focusing channel and flowed downstream through a Y-shaped junction. The two branch channels were not equal in length. When no electrical field was added, the droplets tended to choose a route with lower hydraulic resistance. In analogy to Ohm’s law in electric circuits, the hydraulic resistance can be described by Poiseuille−Hagen’s law.154 Thus, droplets entered the shorter channel with lower resistance. When an electrical field was exerted, the drops were pulled into the longer channel under dielectrophoretic force. In addition to the electrophoretic method, droplet sorting or transport can be implemented through other effects. For example, magnetic drops can be guided under a magnetic field,155 an electrowetting-on-dielectric (EWOD)-induced wettability change can be used to direct drop movement into a certain path,156 a thermally induced surface tension gradient can be utilized for drop sorting through the Marangoni effect,157 and mechanical forces can also be applied to change the droplet route by using pneumatic valves158 or surface acoustic wave (SAW) devices.159 3.5.2. Coalescence. Controllable coalescence in a certain region is a crucial step for initiating reaction between droplets with different contents to conduct further applications. It can also be categorized into passive and active methods. The passive methods rely on the precise control over the velocity of individual droplets to cause relative motion of two droplets.160,161 The droplet pairs thus gradually approach each other and eventually coalesce. Such velocity control can be achieved through a designed channel geometry. It is worth noting that the contact of two droplets does not necessarily lead to coalescence, and the existence of surfactants needs to be considered. Bremond et al. introduced a coalescence method through droplet decompression.160 As shown in Figure 13A, a pair of droplets stabilized by a surfactant were first compressed and elongated in a narrow channel. Then the front droplet entered into an expanding channel. It was decelerated and constrained so that the rear droplet could catch up with it. At this stage, the two droplets did not coalesce. When the front drops continued to flow through a narrow channel, a separation between the two droplets induced interface instability and generated nipples on the contact points, which bridged the droplets together and promoted coalescence instantly. In other studies, similar functions of the nipples have also been achieved through drainage of the continuous phase, which brought the two droplets close enough. Apart from such an expanding channel design, droplet coalescence can also be initiated through other geometries, such as a junction, or by adjusting the droplet properties, such as viscosity and surface tension.161 Besides, active coalescence has been explored, and the most representative method is electrocontrol. As shown in Figure 13B, electrically conducting droplets with opposite charges could be guided to get close with the aid of electrostatic forces, and a high-frequency electric field acted to destabilize the interface and thus promote

Figure 13. Droplet coalescence in a microfluidic device. (A) Passive coalescence through a designed channel geometry: (i) complete microfluidic system; (ii) droplet coalescence process in a symmetrical coalescence chamber. Adapted with permission from ref 160. Copyright 2008 American Physical Society. (B) Active coalescence by electrocontrol: (i) scheme illustration of the method; (ii) with no electric field, droplets formed in the two nozzles flow independently and do not coalesce; (iii) when an electric field is applied, oppositely charged droplets come into contact and coalesce. Adapted with permission from ref 124. Copyright 2006 John Wiley & Sons.

coalescence.124 In addition, droplet motion and controllable coalescence could be achieved in a digital way through EWOD or DEP.162 Other methods, such as thermally triggered droplet destabilization163 and wettability-induced surface scratching,164 have also been confirmed feasible for coalescing droplets. 3.5.3. Splitting. Droplet splitting means to divide the droplets into smaller ones. It is a necessary protocol for highthroughput droplet generation, and the subdrops facilitate group studies. The most straightforward passive method for droplet splitting is by constructing a bifurcated channel with a Y-shaped junction or an obstruction.165 As shown in Figure 14A, droplets flowing through the junction or the obstruction would break up into smaller daughter droplets and enter into the subchannels. The volume of the daughter droplet is negatively correlated with the hydrodynamic resistance of the subchannel. Also, such a breakup process can be sequentially

Figure 14. Droplet splitting in a microfluidic device. (A) 2D splitting: (i) droplets split repeatedly through Y-shaped junctions; (ii, iii) scheme illustration and microscopic image of droplet splitting mediated by a square obstruction. Adapted with permission from ref 165. Copyright 2004 American Physical Society. (B) 3D splitting: (i) scheme of the splitting process; (ii−vi) microscopic images showing droplets entering the junction and split into two daughter droplets. Adapted with permission from ref 166. Copyright 2016 Royal Society of Chemistry. 7977


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Figure 15. Droplet mixing in a microfluidic device. (A) Passive mixing by virtue of a winding channel: (i) schematic illustration of the asymmetric flow pattern and (ii) enhanced mixing when droplets travel in a winding channel. Adapted with permission from ref 170. Copyright 2003 John Wiley & Sons. (B) Active mixing via thermocapillary effects: (i) scheme and microscopic image of the microfluidic device; (ii) numerical simulation snapshots showing the passive tracer particle location. When the laser alternates between the upper and lower halves of the droplet, particle “fingers” are formed, which penetrate into the opposite hemispheres of the drop and then advect to the rear of the drop. Adapted with permission from ref 175. Copyright 2009 IOP Publishing.

As shown in Figure 15A,170 droplet plugs were induced to flow through a winding channel. When the plugs passed the curved parts of the channel, the flow pattern within the plugs was asymmetric, and the axis of the counter-rotating recirculation vortices could be shifted periodically when the plugs traveled through each half-cycle of the channel. Therefore, cross-mixing was enhanced between the two halves of the inner regions. Similar to this design, rapid mixing has also been facilitated by other geometries such as zigzag- or composite-structured channels.171,172 Besides, mixing can be assisted actively by electric control through DEP or EWOD forces, local mechanical oscillation, or temperature-induced thermocapillary effects.173−175 For example, Baroud et al. demonstrated that, by using two stationary focused laser beams with appropriate switching frequencies, the symmetry flow pattern inside the drops would vanish, and chaotic flows could be produced due to the thermocapillary Marangoni effect. Therefore, the mixing of the two halves of the droplets could be enhanced, as shown in Figure 15B.175 3.5.5. Trapping. Droplet trapping means to locate droplets to a certain region for specific operations. The most facile method is passive geometric trapping. Hollfelder et al. designed a single-layer-structured PDMS device with geometric trap arrays using standard soft lithography techniques, as shown in Figure 16.176 Droplets were first generated and then directed to be entrapped within the trap by withdrawing the continuous-phase fluid. The droplet size was set between the size of the trap inlet and outlet. Therefore, the captured droplets blocked the exit and stopped the liquid flow, preventing another droplet from entering the trap. The captured droplets could reside in the traps for a certain time as desired and could also be released by reverse flow of the continuous-phase fluid. Besides, the trapping manipulations have also been mediated by active methods, in which external forces act to transport the droplets into the desired location, usually a geometrical trap or obstruction. For example, Hansen et al. developed a microfluidic system with integration of microchannels and pneumatic membrane valves.177 By using a row multiplexer and column

repeated through fractal structured channels, generating a series of droplets in high throughput. Although most passive splitting methods are based on planar 2D channels, 3D splitting in a capillary-based device has also been proposed. Droplets flowed into a θ-shaped capillary with identical subchannels and were then split into two daughter droplets, as shown in Figure 14B.166 Moreover, the 3D symmetrical configuration and the easier modification of channel wettability also facilitated splitting of double-emulsion droplets. Active splitting methods include usiing pneumatic valve actuation,167 an ultra-high-voltage electric field,168 and SAW devices.169 For example, Sung et al. developed an on-demand active splitting system assisted by SAWs. Unlike traditional SAW control methods, which act on the entire droplets uniformly, they applied a slanted-finger interdigitated transducer (SF-IDT) to emanate a narrow SAW beam by local activation. The droplets were thus partially exposed to the beam, and an acoustic radiation force (ARF) was generated on the drop interface as a result of the acoustic contrast between the dispersed- and continuous-phase fluids. Such a force acted like an acoustic knife to deform the droplet interface and eventually split it into two parts.169 Also, droplet splitting could be implemented in digital microfluidics.162 3.5.4. Mixing. Mixing of the reagents confined within the droplets is essential for reaction control and kinetic studies. In stratified microfluidic flow, due to the low value of Re, the flows are laminar and mixing is dominated by slow molecular diffusion. In droplets passing through a straight channel, although the special friction boundary condition results in a circulating pattern within the droplets, the flow pattern is symmetric, with two equal circulating flows distributed on each half of it.112 Therefore, the contents within each half can be mixed, but the mix between the two halves rarely occurs. As described before, there exists a clear phase boundary between the two adjacent miscible fluids even after they are emulsified into a single droplet. To foster homogenization of the contents, passive mix-enhancing methods have been proposed. Different channel geometries have been designed to induce chaotic flow and accelerate mixing by advection. 7978


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talization: reactions occur in isolated droplets, and the complete evolution process as well as the reaction outcomes are encapsulated to be monitored. Therefore, Taylor diffusion, including its resultant sample dilution and contamination, is eliminated, and the reactions can be localized and controlled accurately. Also, as the ingredients are protected by the carrierphase fluid, direct contact with channel walls is prevented. The second is miniaturization: the femtoliter to nanoliter scale of individual droplets largely reduces reagent consumption, and the accumulation of reaction products allows for rapid detection of reagents down to the single-molecule or singlecell level. The third is monodispersity: each droplet has an identical volume (often with polydispersity less than 1%) and experiences similar procedures, which enables quantitative comparison and screening applications. The fourth is high throughput: the high frequency of droplet generation and ultrafast manipulations (often with the aid of external forces) make it possible to produce, process, and analyze enough data with high resolution for both laboratory assays and industrial uses. The fifth is enhanced mixing: benefiting from the special convective flow profile inside the droplets, the mass transfer and heat transfer are improved, and hence, reactions are accelerated.

Figure 16. Droplet trapping in a microfluidic device. (A) Scheme of an individual trap and the flow profile when a drop approaches and resides in a trap. (B) Microscopic image of an array of traps, each of which contains one droplet. (C) Microscopic images showing the droplet release process. Adapted with permission from ref 176. Copyright 2008 Royal Society of Chemistry.

4.2. Stability of Droplets in Analysis

Although droplet generation does not necessarily rely on surfactants, the presence of surfactants in an emulsion system is required for droplets to serve as stable microreactors. A surfactant is a class of amphiphilic molecules containing both hydrophobic and hydrophilic groups. Along with the generation of droplet emulsions, surfactant molecules would diffuse and adsorb at the water−oil interfaces and decrease the interfacial tensions between the two immiscible phases. 192 The fundamental role of surfactants in droplet reactor systems is to prevent droplet coalescence. Without addition of surfactants, the emulsion system is unstable, and would eventually homogenize into separate phases due to minimum energy.193 On the contrary, the presence of surfactants acts to provide an energy to kinetically stabilize the system in a metastable thermodynamic state, making it possible for droplets to serve as durable reaction containers. In biochemical reactions, the whole droplet system must be biocompatible, as must the surfactants. This means that the surfactant should be carefully selected not to interact or exchange with the droplet ingredients. For example, many biochemical reactions in droplet microcontainers rely on the fluorocarbon oil as the carrier phase, as it provides several advantages, such as low solubility to biological compounds, less swelling of PDMS-based microfluidic channel walls, and gas permeability for cell-related reactions.194−196 However, the commercially available surfactants for this demand are limited. To solve this problem, Holtze et al. designed and synthesized perfluoropolyether (PFPE)−poly(ethylene glycol) (PEG)− PFPE triblock copolymers as nonionic surfactants in water-influorocarbon oil droplet emulsions.196 The good solubility of the outer block PFPE in fluorocarbon oils and the large steric repulsion played a stabilizing role, and the inert inner block PEG prevented adsorption of biological materials. Nowadays, such a surfactant has become the most commonly applied in fluorous-based droplet microfluidics. On the basis of this design, Haag et al. recently took a step further by introducing linear polyglycerol hydroxyl (LPG(OH)) and poly(methyl glycerol) methoxy (LPG(OMe)) to the triblock copolymer, as

valves, droplets could be selectively compartmentalized into 95 wells. Unlike using geometric traps, electrical or thermal methods help to locate droplets by creating an electrostatic potential well or a surface energy well, which serve as the energy trap.

4. DROPLET MICROFLUIDICS FOR BIO(CHEMICAL) ANALYSIS Since the 1950s, there have emerged several pioneering works demonstrating the basic idea of compartmentalizing reactions by isolating single molecules, cells, or microbes into droplets.35 However, the conception of using droplets to perform routine laboratory operations has taken almost half a century to come true, with the help of droplet microfluidics. The highly reproducible and on-demand fabrication of microfluidic chips allows for different reaction conditions.21 The comprehension of generation physics and manipulation techniques have paved the way for microfluidic droplets serving as miniaturized reactors.112 Also, the advance of modern analysis methods enabled qualitative and quantitative detection of droplet contents.178 Furthermore, the multiparallel and high-throughput platform is promising for better fulfilling the mass data requirement with advent of the -omics era.179 Therefore, droplet microfluidics has become a thriving research field, and tremendous work has been done to exploit its value in he analysis of small molecules, biomacromolecules, cells, and even multicellular organisms.180 Up to now, droplet microfluidics has achieved dramatic progress in various application fields, including single-cell analysis,181−183 medical diagnostics,184−186 drug discovery,187−189 food and feed industries,190 environmental monitoring,191 etc. 4.1. Advantages of the Droplet Analysis Platform

Compared with traditional reaction flasks, microfluidic droplets offer several attractive features.34,35 The first is compartmen7979


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Figure 17. (A) Biocompatible polyglycerol-based triblock surfactants: (i) chemical structure of PFPE−LPG(OH)−PFPE and PFPE−LPG(OMe)− PFPE; (ii, iii) microscopic images of (ii) the droplet generation process and (iii) droplets stored with no coalescence; (iv) scheme of the droplet interfacial area, which is stabilized by the surfactant. Adapted with permission from ref 197. Copyright 2015 Royal Society of Chemistry. (B) Molecular transport between droplets: (i) fluorescein is exchanged in several days; (ii) rhodamine 6G is exchanged over minutus; (iii, iv) two possible surfactant assembly models, i.e., (iii) micelle structure and (iv) vesicular structure. Adapted with permission from ref 199. Copyright 2016 Nature Publishing Group.

Figure 18. Reagent encapsulation in droplets. (A) Reagents are preloaded in nanoliter plug cartridges and then merged with the substrate-containing droplets. Adapted with permission from ref 201. Copyright 2006 Elsevier. (B) On-chip reagent dilution by using a buffer stream: (i) scheme illustration; (ii, iii) images of the channel junction in the blue rectangle of (i). By changing the flow rates of the buffer stream and the distinct reagent stream, the reagent concentrations are controlled. Adapted from ref 203. Copyright 2003 American Chemical Society. (C) Pico-injection of reagents by electric actuation. Adapted with permission from ref 204. Copyright 2010 National Academy of Sciences.

emulsion system.199 “Minimal emulsion” droplets were generated in microfluidics with precisely controlled parameters and microenvironments. Different fluorophores were applied as model molecules inside the droplets, and their transport was analyzed quantitatively by measuring the fluorescent intensity, which depicted the concentration. The results showed that the time scale of the transport varied between the distinct contents from several minutes to tens of hours, as shown in Figure 17B. The authors also put forward two possible structures of the surfactant supramolecular assemblies. Combined with mathematic model analysis, they concluded that the transport process was diffusion limited; the assembly of the surfactant molecules formed vesicles and acted as a nanoscopic medium to mediate

shown in Figure 17A.197 The synthesized PFPE−LPG−PFPE surfactant not only exhibited good biocompatibility, but also was feasible for additional chemical modification of the side chain, and thus was promising for creating functional inner surfaces of the droplets. Apart from biocompatibility, the possible molecular exchange is also a consideration. It is worth noting that droplets are not necessarily completely sealed, because small molecules can pass through the bilayers formed by the surfactants.198 The diffusion ability depends on the property of the surfactants and the encapsulated molecules. Baret et al. recently carried out a detailed study to unravel the molecular transport mechanisms in a PFPE−PEG−PFPE-stabilized, water-in-fluorinated oil 7980


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the solubility of the inner molecules in the carrier phase. In addition to solute exchange, solvents can also transport across the water−oil interface in response to the osmotic pressure difference, which should be considered for keeping the droplets intact.200

optical encoding of the droplets using dyes or quantum dots.205−207 To avoid interference between barcode signals and the reaction signals, droplet pairs can be generated alternately: one for reaction and another one following closely behind it for indexing. For example, droplet pairs containing fluorescent dyes were generated in a microfluidic device with two T-junctions on both sides of the main channel.207 Under appropriate flow conditions (e.g., viscosities, flow rates, etc.), droplets occurred in pairs without coalescence, as shown in Figure 19A. On-chip

4.3. General Analysis Strategies in Droplet Microfluidics

Once the system is set with appropriately selected surfactants and environmental parameters, the droplet analysis platform starts running, and the process can be roughly divided into the following steps. First, reagents are introduced to the droplets at a specific time and place. Then reaction is initiated by mixing or coalescence of liquids, and different manipulations are exerted according to specific scenarios. Last, the reaction outcomes are analyzed using different detection methods. As droplet manipulation techniques have been described in detail above, in this section, we will mainly cover reagent introduction and outcome detection. 4.3.1. Reagent Encapsulation in Droplets. The droplets are useful microreactors only when different reagents are introduced in a controlled manner. In some cases (e.g., screening), the same sample droplets are continuously targeted against a large amount of testing droplets (often called a droplet library) with different contents corresponding to various reaction conditions. The testing droplets can thus be stored within a capillary as a cartridge prior to reaction.201,202 Then the array of the testing droplets flows and sequentially coalesces with the sample droplets in a junction to conduct the screening process, after which they are stored and incubated in a downstream receiving capillary for detection, as shown in Figure 18A.201 In some other cases (e.g., kinetic studies), the amount of the reagents is not necessarily too great, but the reagent concentration needs to be adjusted within a certain range. Therefore, on-chip dilution is required. This can be achieved through laminar-flow mixing of the reagents before emulsification, with the help of a buffer flow.203 As shown in Figure 18B, two streams containing different reagents flow in parallel and are separated by a middle buffer stream, which serves as a dilution agent and prevents premature contact. The three streams are then emulsified into droplets either through a T-junction or through a flow-focusing device, and the concentration of the reagents can be determined by their relative flow rates. In cases of accurate control over the reagent addition order (e.g., multistep reactions), the sample should be injected directly into droplets at a specific time and location and in a fixed amount. For example, a robust technique was developed to inject reagents through a pico-injector into droplets by electric field actuation.204 A series of droplets flowed by the injector and energized the electrodes, which in turn destabilized the droplet interface and thus let in the reagents, as shown in Figure 18C. The injection was selectively performed by switching the electric field, and the added volume can be precisely adjusted by changing the drop velocity and the injection pressure. Moreover, combinatorial injections could also be achieved through independent control over multiple injectors. 4.3.2. Droplet Indexing. The reagent-containing droplets are information carriers that will evolve during the whole analysis process. Therefore, indexing of droplets is crucial to tracing their original identity, or to differentiating them, especially when a large amount of reagents are involved in one system. The most common strategy for indexing is by

Figure 19. Droplet indexing methods. (A) Optical indexing by using dyes. (i) Formation of alternating droplets. (ii, iii) Indexing concentrations by on-chip dilution. Fluids in channels that are linked by the dashed line are driven by the same pump to ensure the same flow rates. By changing the relative flow rates, droplet pairs are generated with (ii) low and (iii) high concentrations. Adapted from ref 207. Copyright 2004 American Chemical Society. (B) Synthesis of DNA barcodes in a “split-and-pool” method for droplet indexing. Adapted with permission from ref 36. Copyright 2015 Elsevier.

dilution through premixing with buffer solutions enabled generation of droplets with concentration gradients. By using the same syringe pump, the flow rates and thus the concentration of the drop pairs were correlated. Therefore, the concentration of the front reaction droplet could be indicated by real-time measurement of the fluorescence intensity of the back, indexing droplet. Although optical markers are very convenient and are widely applied, the amount of coding is limited due to the overlap of fluorescence signals. To further improve the encoding numbers, DNA barcodes were put forward by Merten et al. using DNA oligonucleotides as labels to barcode the droplet contents.187 This conception was then realized by Weitz and co-workers, who performed massive droplet indexing by encapsulating DNA-barcoded microbeads.36 The beads were synthesized using a cyclic “split-and-pool” process. During each cycle, a large number of beads were mixed together and divided into four groups with equal numbers, each of which was subjected to a distinct oligonucleotide synthesis reaction by adding different DNA bases (A, T, C, G). Then the beads were pooled together 7981


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Figure 20. Optical detection approaches in droplet microfluidics. (A) Bright-field microscopic imaging method for the identification of active catalyst. The steel tubing contains droplet arrays of reactors and indicators. Catalyst is identified through the color change of the indicator. Adapted from ref 209. Copyright 2010 American Chemical Society. (B) Fluorescence microscopic imaging method for the adsorption test of the fibrinogen adsorption to (i) Rf−CH2CH2OH and (ii) Rf−OEG. Adapted from ref 210. Copyright 2005 American Chemical Society. (C) Scheme of the FRET method for the analysis of streptavidin−biotin binding kinetics. Adapted from ref 214. Copyright 2007 American Chemical Society. (D) SERS spectra for (i) RB−AuNPs and (ii) the mixture of RB−AuNPs and 10 ppb mercury(II) ions. Adapted with permission from ref 221. Copyright 2009 Springer.

indicator, as shown in Figure 20A. When a mixture of methane and oxygen diffused into the droplet reactors in the presence of active catalysts, they reacted to generate methanol. Then, when the temperature was raised, methanol volatilized, entering the neighboring indicator droplet, and was oxidized by chromic acid. The valence-state transition of chromium caused a color change of the indicator droplet from yellow to purple, which indicated that the oxidation reaction occurred.209 Compared with bright-field microscopy, fluorescence microscopic imaging is more widely utilized because it allows for highly sensitive detection and quantitative measurement of droplet contents. By using a highly sensitive camera and filter equipment, the fluorescent images of a large number of droplets can be obtained at the same time, and their fluorescent intensities can be measured by image processing. Moreover, fluorescence microscopy provides localized information on droplet contents with high spatial resolution, and shows flexibility in characterizing interactions occurring at the droplet interface. For example, nonspecific protein adsorption at the liquid−liquid interface was studied in a water-in-fluorocarbon oil droplet microfluidic system.210 An aqueous solution of fluorescently labeled AlexaFluor−fibrinogen was emulsified by a fluorocarbon oil phase containing different surfactants. The direct visualization of the droplets revealed that the fluorescence signal increased at the droplet interface when noninert surfactant Rf−CH2CH2OH was present, which indicated adsorption. On the contrary, nonadsorption to the biocompatible surfactant Rf−OEG was indicated through a uniform fluorescence image, as shown in Figure 20B. The adsorption behavior was further determined quantitatively by kinetic measurement of enzyme using a fluorimeter.

again, and the whole process was repeated 12 times, after which each bead was labeled with a unique sequence of 12-bp DNA oligonucleotides indicating its synthesis path, as shown in Figure 19B. It can be estimated that the total possible coding number was 4,12 which is a huge amount for high-throughput indexing of droplets. 4.3.3. Detection Approaches for Droplet Microfluidics. It has been argued that, for bio(chemical) analysis, the product of a droplet microfluidic system is more like information than tangible substances.208 Such information is preserved during the whole reaction process and can be reflected in the dynamic variation of the droplet content properties. Therefore, it requires qualitative and quantitative detection of the droplet contents.178 In turn, the powerful detection techniques fulfill droplet analysis systems and promote their applications. Currently, there are many detection techniques, including optical methods, such as bright-field/ fluorescence microscopy, laser-induced fluorescence, and Raman spectroscopy. There are also nonoptical methods, such as electrochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, chemiluminescence, etc. Each method has its own advantages and is applicable to different situations. In some cases, these methods can also be coupled or combined to achieve better performance. Optical Methods. Imaging by using microscopy equipment is the most convenient and prevalent method in droplet content detection. By using bright-field microscopy, the physical properties of the droplets, such as size and color, can be directly visualized and dynamically monitored, providing useful information about the reaction status. For example, a chemical reaction was conducted in droplet reactors and detected instantaneously in an adjacent colorimetric droplet 7982


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system. For example, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been applied by depositing droplet reactors and the matrix on the MALDI plate before screening.223 It has shown high sensitivity of chemical detection. Besides, electrospray ionization mass spectrometry (ESI-MS) has received increasing attention in recent years because it enables both on-line and off-line analysis.224−226 To eliminate signal interference, the oil stream surrounding the droplets needs to be removed, and droplets need to be pre-extracted on-chip from the oil carrier phase to an aqueous stream by periodic electrical pulses. The aqueous flow is then delivered into the electrospray emitter for ESI-MS analysis. To overcome sample dilution in this procedure, droplet transfer can be finished in a reduced distance by using an integrated chip or just removed by directly coupling the droplets with a nanospray emitter. Huck et al. have recently made progress in ESI-MS detection of protein molecules at the femtomole level in individual droplets.227 As shown in Figure 21A, droplets containing α-chymotrypsinogen A were gen-

In cases of high-throughput screening and sorting, the use of fluorescence imaging is limited by the detection sensitivity and speed. Laser-induced fluorescence (LIF) spectroscopy offers an ultrahigh signal-to-noise ratio and detection sensitivity.211−213 Therefore, it has been widely applied in single-molecule detection within a confined illumination volume. In addition, the ultrafast response time meets the requirements of highthroughput analysis such as droplet polymerase chain reaction (PCR), single-cell assays, enzyme screening, etc. For the purpose of molecular dynamic monitoring, the fluorescence resonance energy transfer (FRET) technique has been demonstrated to be feasible by many researchers on biomolecular interactions within the droplets.214,215 For example, Edel et al. studied streptavidin−biotin binding kinetics in droplet microfludic systems on the basis of the FRET technique.214 As shown in Figure 20C, a FRET donor, Alexa 488, was conjugated directly with the streptavidin molecule, and an acceptor, Alexa 647, was used to label the biotin molecule using two complementary DNA strands as the medium. The binding of streptavidin and biotin molecules brought the two chromophores together, and energy was transferred from the donor to the acceptor, which resulted in a detectable FRET signal of red emission fluorescence. Combined with avalanche photodiode detectors, highly sensitive and fast measurement of the fluorescence intensity were achieved. In the above-mentioned fluorescence-based detection techniques, a fluorescent labeling procedure is needed, especially for detection of nonfluorescent analytes. In contrast to this, Raman spectroscopy offers a label-free strategy to provide a fingerprint of the molecule. In addition, its low sensitivity to water allows for noninvasive, on-chip detection of molecules in droplet microfluidic systems.216 The characteristic Raman shift helps to identify molecules, and the corresponding peak intensity gives quantitative information. As spontaneous Raman scattering is relatively weak, surface-enhanced Raman spectroscopy (SERS) has been developed. SERS is a highly sensitive technique which makes use of rough metal surfaces or nanostructures to enhance the Raman scattering signal through plasmonic and chemical effects.217 Initially, the SERS technique was coupled with microfluidics by adding metal nanocolloids in the flow channels for signal amplification.218 However, the aggregation of the colloids was problematic, and the undesired adsorption would cause the so-called “memory effect”.35,219 Droplet microfluidics can overcome this dilemma, as the mixture of sample and colloids can be confined in droplets, thus avoiding direct contact with channel walls.220 Choo et al. employed this strategy to detect mercury(II) ions in water.221 Mercury(II) ions and rhodamine B (RB)-adsorbed AuNPs were encapsulated within aqueous droplet reactors. Here, the RB served as a reporter molecule and generated SERS signals. The strong affinity of mercury(II) ions and the AuNPs caused partial release of RB molecules and hence decreased the SERS signal, as shown in Figure 20D. By measuring the signal change, the concentration of mercury(II) ions could be quantified. Apart from ion detection, SERS has also been used for analysis of various kinds of samples, including drugs, bacteria, etc.220 Nonoptical Methods. Mass spectrometry (MS) is another label-free detection method based on measurement of the mass-to-charge ratio. It allows for chemical structure identification from analysis of ionized molecular fragments and enables simultaneous detection of multiple samples.222 MS has been broadly incorporated into the droplet microfluidic analysis

Figure 21. Nonoptical detection approaches in droplet microfluidics. (A) Scheme of ESI-MS detection of protein molecule α-chymotrypsinogen A. Adapted from ref 227. Copyright 2013 American Chemical Society. (B) Electrochemical monitoring for H2O2 decomposition reaction kinetics: (i) the droplet approaches the first electrode; (ii) the droplet contacts the first electrode; (iii) the droplet connects the two electrodes; (iv) the droplet disconnects from the first electrode. Adapted from ref 230. Copyright 2009 American Chemical Society.

erated and stored off-chip. Then they were reinjected and directly sprayed at a flow rate within the so-called “nanoelectrospray ionization” regime, which gave high-quality mass spectra of individual droplets. It was worth noting that the surfactant was well-chosen not only for stable storage of the droplets, but also for compatibility with the MS system. Electrochemical detection relies on the interactions between electrical energy and chemical change in a reaction. It is costeffective and can be integrated into microfluidic chips 7983


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Table 1. Droplet-Based Analytes analyte

key process

function

ref

molecule

small molecules/ions small molecules DNA RNA miRNA protein

cell

DNA RNA protein

optical microscopy screening droplet PCR RT-PCR EXPAR IVTT enzymatic reaction screening PCR and sequencing RT-PCR and sequencing ELISA, immunoassays, screening, etc. protein screening and sequencing parameter measurement

molecule detection; reaction identification drug discovery; dose−response study gene detection gene expression analysis gene regulation analysis protein expression analysis molecule detection; kinetic study protein crystallization genomic analysis transcriptomic analysis protein detection; enzymatic kinetics; drug discovery enzyme function analysis, mutant selection metabolic analysis

238, 241, 307 302, 303 247−251 252, 253 254 256−258 260−264 207, 266, 267 288−290 36, 291, 292 273, 293−295, 301 296, 305 297−299

metabolites

Figure 22. (A) Droplet-based aqueous-phase titration reaction of argatroban. Adapted from ref 238. Copyright 2006 American Chemical Society. (B) Droplet-based organic-phase bromination reaction of alkenes: (i, ii) at different ratios of the flow rates of Br2 solution and styrene solution, respectively. Adapted from ref 241. Copyright 2005 American Chemical Society.

phenomenon to elucidate the structure of molecules. However, its sensitivity is relatively low. Therefore, it is often coupled with other analysis techniques such as liquid chromatography (LC) or MS, through which compound libraries can be identified in microfluidic droplet arrays with good sensitivity, rapid response, and high throughput.231 Besides, when droplets contain multiple active contents for detection, capillary electrophoresis (CE) comes in handy. With high separation speed and efficiency, CE has shown great significance in chemical separation. By coupling it with fluorescence-based analysis methods, it has also been applied in the droplet microfluidic analysis platform for the detection of biological samples such as amino acids.232 Separation of droplet contents is not efficient enough when each sample responds to distinct detection methods. To meet this requirement, droplets can be split into different downstream channels, each conducting independent analysis. Ismagilov et al. developed a sequential splitting device to generate four arrays of identical daughter droplets, which contained the same compound mixture for detection.233 Different additives were injected in each array for independent analysis. Specifically, Ca2+ ions were detected in the first array by fluorescence microscopy with the injection of a fluorescent indicator, insulin was detected using fluorescence correlation spectroscopy immunoassay in the second array with addition of anti-insulin antibody, glucose was analyzed by MALDI-MS through injection of Girard’s reagent T, and MPTS (a fluorescent dye) was detected in fluorescence microscopy as a control. These results were then recombined for global analysis. Similarly, Macarthur et al. utilized a 3D-printing microfluidic

conveniently. To some extent, it is complementary to optical imaging methods, because it can deal with opaque channels. Physical information on droplets such as size and velocity can be obtained through measurement of the capacitance change and impedance difference.228,229 Besides, chemical contents can also be analyzed. For example, Zheng et al. presented an electrochemical detection method for kinetic monitoring of a H2O2 decomposition reaction occurring in a droplet microfluidic system.230 As shown in Figure 21B, aqueous droplets containing H2O2 and catalase were generated in the microfluidic channel and passed by two microelectrodes, one a Pt working electrode and another a Ag/AgCl quasi-reference electrode. The two electrodes were spaced by a small gap and separately connected to a potentiostat to detect the electrochemical signals. When droplets approached the two electrodes and covered the gap, the electrochemical circuit was connected, which resulted in a current peak; when the droplets moved across the first Pt electrode, the circuit was cut off and the current signal disappeared. The reaction time was controlled by changing the flow rate and trajectory through pneumatic actuation. The reaction kinetics was monitored by the amperometric method within a single run. It was also anticipated that such detection methods would be applicable to many reactions, provided that there are electrochemically active ingredients generated. Combining Methods. In some specific situations, a single detection method is not enough, and thus, different techniques can be coupled to complement each other. For example, nuclear magnetic resonance (NMR) spectroscopy is a powerful technique which relies on the nuclear magnetic resonance 7984


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Figure 23. Droplet PCR platform. (A) Off-chip thermocycling platform for the detection of the mutated KRAS oncogene. Adapted with permission from ref 247. Copyright 2011 Royal Society of Chemistry. (B) Scheme of an on-chip thermocycling platform: (i) temperature profile; (ii) building blocks including the thermocycler, tubing, temperature sensors, and optical fibers. Adapted with permission from ref 249. Copyright 2013 Royal Society of Chemistry.

In addition to reactions in aqueous phases, droplet microfluidics also allows for organic-phase reactions. For this purpose, the materials for device fabrication need to be carefully selected to prevent possible deformation of the channels with the presence of harsh organic solvents. Beers et al. fabricated a microfluidic device through rapid prototyping using thiolenebased polymers, which show stronger organic stability than PDMS materials.241 Droplets of hexanes or toluene could be generated within an aqueous phase of sodium dodecyl sulfate (SDS) solution. They then used such a device to perform a bromination reaction by generation of droplets containing Br2, styrene, and benzene as the solvent. During the addition reaction of Br2 to the double bonds of styrene, the droplets faded gradually from orange to colorless, as shown in Figure 22B. 4.4.2. Biological Macromolecule Analysis. DNA Analysis. Manipulation and detection of DNA samples is vital to molecular biological analysis and has an extensive application value in biological sciences, diagnostics, etc. This process would be unpromising without PCR.35 PCR is one of the most robust and reliable techniques for DNA amplification. It was first introduced by Mullis in the 1980s, and the working principle relies on thermal cycling between three different temperatures for DNA denaturation, annealing, and elongation, respectively.242 The combination of PCR with the microfluidic technology can be traced back to the 1990s, with the reactions occurring in a continuous-single-phase microfluidic flow.243 Compared with the conventional bulk process, the total time was reduced due to enhanced heat transfer. Since then, microfluidic PCR has received wide research interest and achieved success in DNA analysis. However, the adsorption of the sample mixture to the channel walls and the nonuniform flow velocity profile bring about undesired contamination and signal noise.244 Thus, a novel technology, droplet PCR, emerged to overcome these problems. By using multiphase segmented flows, it offers compartmentalized chambers for separate amplification reac-

droplet splitter, through which norepinephrine and adenosine triphosphate (ATP) were detected by the electrochemical method and chemiluminescence method in the two subchannels, respectively.234 4.4. Biochemical Analysis in Droplet Microfluidics

An analyte is the object of interest during the analysis process. In the early stages, droplet microfluidics was aimed at molecular analysis.235 In addition to ions and small molecules, analysis of biological macromolecules such as nucleic acids and proteins has proved to better embody the flexibility of this platform. Meanwhile, droplet microfluidics also gave birth to revolutions on many standard molecular biology techniques, bringing a novel droplet platform for polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA), etc. Moreover, currently, droplet microfluidics has been focused more on cell-level analysis,183,236,237 and the typical cell operations can be performed in droplets. A selective summary of droplet-based analytes is given in Table 1. 4.4.1. Small-Molecule Detection. Microfluidic droplets act as miniaturized reactors for chemical reactions with precise control over local environments, and thus have been exploited in multiphase reactions including titration,238 precipitation,239 hydrolysis,240 etc. For example, a droplet microfluidic platform was developed for on-chip titration of the anticoagulant drug argatroban, and the clotting time was measured by the activated partial thromboplastin time (APTT) test to determine the appropriate dose of argatroban.238 As shown in Figure 22A, droplets containing a decalcified blood sample, argatroban, and the APTT reagent Alexin were first generated within a carrier fluid and flowed through a winding channel to enhance mixing. Then the droplets were incubated before merging with CaCl2 from a side channel. The generation of a fibrin clot was observed by bright-field microscopy, and thrombin was quantified by fluorescent microscopy. 7985


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alternating temperature zones has also been applied to other designs. For example, Hollfelder et al. developed a highthroughput droplet PCR device with radial channels of spatial temperature difference.250 Droplets flowed through the central and periphery zones alternately to complete a total of 34 cycles in 17 min. In other attempts, by using microfluidic chips with patterned chambers, droplet PCR was performed through onchip amplification, and data analysis was conducted subsequently.251 RNA Analysis. RNA detection is important for revealing the gene expression level. RT-PCR is a standard technique for RNA analysis. Derived from PCR, RT-PCR can be divided into two steps. First, a complementary DNA (cDNA) was created through reverse transcription. Then the cDNA was amplified and analyzed through the PCR process. As the droplet PCR technique has matured, droplet-based RT-PCR has also been widely accepted. Beer et al. developed an integrated microfluidic device for on-chip droplet generation, RNA isolation, reverse transcription, PCR amplification, and fluorescence realtime detection.252 As shown in Figure 24A, an aqueous mixture

tions. In addition, the sample could be sufficiently diluted so that each droplet contains no more than one copy of the DNA molecule. The ability to perform single-molecule DNA analysis enables detection of ultra-low-abundance sequences such as rare mutations, without being overlapped by high-abundance DNA sequence signals. Furthermore, large DNA fragments could also be isolated and amplified, without concerns for possible recombination with partially homologous fragments. Although the principle of isolated amplification was initially put forward by another technique, digital PCR,245,246 in which samples are partitioned in individual droplets positioned as microarrays, droplet PCR is superior with respect to highthroughput generation of monodisperse droplets and low-cost chip fabrication. Nowadays, droplet PCR has been widely applied in precise and sensitive quantification of DNA at the single-molecule level. There are also commercial droplet PCR platforms available from companies including Bio-Rad, RainDance, etc.184 Droplet PCR can be classified into two types, off-chip thermocycling and on-chip thermocycling. Griffiths et al. described an off-chip thermocycling droplet PCR platform for the detection of the mutated KRAS oncogene.247 As shown in Figure 23A, the complete process was divided into four steps: emulsification, thermocycling, reinjection, and detection. Specifically, an aqueous mixture of PCR reagents, primers, genomic DNA (gDNA), and two kinds of TaqMan probes was emulsified into uniform droplets, and the concentration of gDNA was low enough to ensure that each droplet contained at most one DNA. Then the emulsion was collected off-chip for DNA amplication. Next the emulsions were reinjected into the microfluidic device, and the droplets were detected by fluorescence microscopy. The two probes that were specific to the mutant and wild-type DNAs would generate green and red fluorescence signals, respectively, during amplification. Therefore, by counting the ratio of green droplets (positive signal) to red ones (negative signal), the number of mutations was calculated. In contrast to the relative quantification method by standard curve analysis, this strategy allows for absolute quantification of DNA molecules. Additionally, such an off-chip thermocycling method is convenient for other manipulations. For example, Yang et al. performed droplet PCR in agarose droplet reactors, which were then solidified upon a temperature decrease.248 Thus, the DNA amplicons were trapped within the gelation microbeads to better maintain the monoclonality. Apart from off-chip thermocycling, on-chip thermcycling has also been broadly explored. Hatch et al. developed a “conveyor belt” microfluidic platform with a flowing thermal cycler and portable detection apparatus, through which continuous droplet PCR reactions and real-time analysis were conducted.249 As shown in Figure 23B, the core of the whole system was a cylindrical thermocycler, which was composed of fluorinated ethylene propylene (FEP) tubes twining around two thermal blocks of different temperatures. Upstream droplets containing PCR reagents continuously flowed into the thermocycler via an inlet, passing through the two thermal blocks alternately, and went through cyclic procedures of melting, annealing, and extension. After 40 cycles, droplets flowed out eventually through an outlet. Real-time fluorescence monitoring was achieved through fiber optic coupling of a lightemitting diode (LED) source and photomultiplier tube (PMT). This method facilitated tracking of each droplet in the complete process and obtained dynamic data in good accuracy. A similar concept of letting droplets flow continuously through

Figure 24. Droplet platforms for RNA analysis. (A) RT-PCT platform, including on-chip droplet generation, RNA isolation, reverse transcription, PCR amplification, and fluorescence real-time detection. Adapted from ref 252. Copyright 2008 American Chemical Society. (B) EXPAR platform for miRNA detection, including droplet collection, EXPAR reaction, detection through a 3D particle counter, and digital data analysis. Adapted with permission from ref 254. Copyright 2015 Royal Society of Chemistry.

of viral sample and RT-PCR master mix was emulsified into droplets, and then the droplets were trapped through valve actuation. Therefore, the droplets were stopped to perform thermocycling for RT-PCR reactions, without arbitrary movement. Abate et al. conducted a sequential injection process of RT-PCR by adding reverse transcriptase into the reaction 7986


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mixture after droplet formation.253 Then the droplets were collected off-chip for amplification and absolute RNA quantification. It was also anticipated that heterogeneous samples could be analyzed by sequential injection of specific reagents. Despite its obvious efficiency, droplet-based RT-PCR also has shortcomings as the sample processing is complex and the nonlinear reverse transcription would bring quantification bias. Therefore, novel RNA analysis methods have been explored without being reliant on reverse transcription. Zhao et al. developed a droplet microfluidic system for absolute quantification of circulating microRNA (miRNA) molecules by specific isothermal exponential amplification (EXPAR).254 As shown in Figure 24B, a plasma sample and EXPAR reagents (A and B) were injected into the microfluidic channels and emulsified together into individual picoliter droplets. Then the droplets were collected off-chip for isothermal amplification. The amount of the target miRNA was measured by automatically counting the droplets showing a fluorescent signal. This platform enabled rapid and accurate quantification of circulating miRNA at ultralow concentration, shedding new light on miRNA-mediated gene regulation and medical diagnostic studies. Wang et al. presented another non-reversetranscription strategy for droplet-based RNA detection by virtue of enzyme-linked oligonucleotide hybridization assay (ELOHA).255 The target RNA was hybridized with a magnetic microbead-conjugated capture DNA and an enzyme-labeled detection DNA. The target RNA molecule was quantified by counting the fluorescent signals with the presence of the enzyme substrate. Protein Expression. As one of the most important biological molecules, proteins are involved in a vast majority of life activities. By using in vitro transcription/translation (IVTT) techniques, protein molecules could be expressed and purified. Such a linkage of genotype and phenotype is helpful for interrogating gene functions, and also for function-oriented mutant selection. Compared with in vivo expression, this process is faster and more flexible because the reaction conditions can be controlled with addition of different reagents. In this respect, droplet microfluidics provides an excellent platform, as the compartmentalized droplet containers mimic the natural cell boundary for molecular reactions. Dittrich et al. demonstrated an efficient droplet microfluidic system for in vitro expression of green fluorescent protein (GFP).256 As shown in Figure 25A,188 flows of protein expression compounds (including tRNAs, amino acids, enzymes, etc.) and gene templates were injected through two branch channels into the microfluidic device. They were then simultaneously emulsified and merged upon contact. The formed droplets then served as “artificial cells” and were stored off-chip for gene expression, during which the GFP gene was expressed and a chromophoric group was generated subsequently. Afterward, the droplets were reinjected into the device, and the identification of GFP was achieved by fluorescent microscopic observation and spectrum analysis. Following the same principle, a later work by Abell et al. addressed an integrated platform for on-chip incubation and protein expression.257 Detection of GFP was conducted on diluted “monoclonal droplets”, and the relatively stronger fluorescence signal depicted the mutant proteins compared to wild-type ones. In some cases, in vitro protein expression needs to be carried out after DNA amplification for efficient production of protein. Ryckelynck et al. reported a droplet microfluidic system for

Figure 25. (A) Scheme of in vitro protein evolution in a droplet microfluidic platform.188 Adapted with permission from ref 188. Copyright 2006 Nature Publishing Group. (B) In vitro expression of βalactosidase after isothermal hyperbranched rolling circle amplification of DNA: (i) HRCA droplet generation; (ii) HRCA droplet analysis; (iii) droplet pair formation and fusion; (iv) analysis of the fused droplets. Adapted from ref 258. Copyright 2009 American Chemical Society.

isothermal hyperbranched rolling circle amplification (HRCA) of DNA, followed by in vitro expression of β-alactosidase.258 The process was briefly divided into four steps, as shown in Figure 25B. First, droplets containing a plasmid DNA and HRCA mixture were generated in fluorinated carrier oil and incubated off-chip, and the process of HRCA amplification of the lacZ gene was conducted using a commercial kit. Second, the droplets were reinjected for fluorescence analysis of amplification products via PMTs. Next the HRCA droplets were electrocoalesced with droplets containing in vitro transcription reagents, including Escherichia coli extracts. The fused droplets were incubated for β-alactosidase expression. Afterward, the fused droplets were reloaded for measuring the β-alactosidase activity. It was confirmed through control experiments that the DNA amplification was a primary step as the in vitro transcription of a single lacZ gene did not yield an observable signal of β-alactosidase activity. On the other hand, the use of the HRCA technique simplified the DNA amplification process, compared with the thermocycling procedure involved in PCR. Protein Analysis. Enzymatic reactions play a significant role in protein analysis. With the presence of a target enzyme, the corresponding substrate is activated and thus generates signals (most commonly fluorescence) for detection.259 Definitely, droplet microfluidics offers an efficient platform for such reactions. Its virtue lies in that the concentration of enzyme molecules increases rapidly over the detection threshold along with the miniaturization of droplet reactors, thus bringing about 7987


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Figure 26. (A) Single-molecule detection of the prostate-specific antigen by droplet-based ELISA. (i) Scheme of the binding of the antibody-coated beads to the antigen and the formation of the sandwiched immunocomplex. (ii) Single beads are encapsulated in droplets containing substrate FDG and are trapped in the microfluidic chip for fluorescence accumulation. (iii−v) Three populations of droplets: (iii) droplets without the beads, (iv) droplets containing single bead without the immunocomplex, and (v) droplets containing single bead with the immunocomplex, which show fluorescent signals. Adapted from ref 261. Copyright 2013 American Chemical Society. (B) Droplet-based kinetic study of matrix metalloproteinases: (i) scheme illustration; (ii) enzyme accumulation by the concentrator; (iii) mixing of enzyme molecules and the substrate; (iv) encapsulation into monodisperse droplets; (v) monitoring of the enzymatic reactions by measuring the fluorescence signal. Adapted from ref 263. Copyright 2011 American Chemical Society.

reactions could also be used for substrate molecule sensing. As demonstrated by Ding et al., glucose detection was realized in a droplet microfluidic device, by measuring the enzymatic activity of glucose oxidase (GOx) through an electrochemical sensor.262 In addition to protein detection, droplet microfluidics also contributes to the enzyme kinetic study. Han et al. presented an integrated platform for activity assay of matrix metalloproteinases (MMPs), which are secreted enzymes involved in many physiological and pathological processes.263 Due to the ultralow abundance of MMPs in the physiological samples, the activity analysis of MMPs was quite difficult in conventional methods. In Han’s system, however, the MMP molecules were preconcentrated before emulsification and analysis. As shown in Figure 26B, the microfluidic device was composed of two parts: a molecule concentrator and a droplet generator. First, an aqueous stream containing MMPs and fluorescent tracer flowed through a main channel into the molecule concentrator, where a voltage was applied between the main channel and the polymer nanoporous junction. Thus, the negatively charged molecules in the main channel were pushed back, and the MMP molecules were trapped and accumulated into a plug when the electrokinetic force and the pressure-driven flow were balanced. Then the voltage was removed, and the plug was released to transport into the flow-focusing droplet generator, before which it was mixed with the substrate solutions from two side channels. After droplet generation, the reaction rate was monitored by measuring the increasing fluorescence intensity with time. By screening of the enzymatic turnover at different concentrations, the kinetic constant was calculated, following the Michaelis−Menten kinetic model. Moreover, the same group later conducted multiplexed activity assays on different proteases by sequentially injecting the protease mixture (obtained from a clinic sample) into the optical-barcoded droplet arrays containing different specific substrates through a pico-injector.264 Protein Crystallization. Elucidating a protein’s tertiary structure by X-ray diffraction is significant not only for providing structural information itself, but also for further comprehending its functions and better understanding the

higher signal-to-noise ratios and lower limits of detection. By measuring the signal intensity, enzyme molecules can be detected, or the enzymatic kinetics can be analyzed. In enzyme molecular detection, research has recently been focused on investigation of the single-molecule analysis, to uncover more information relative to conventional bulk experiments. Droplet microfluidic systems have been explored to meet this requirement. The enzyme can serve as the target molecule for direct detection. As reported, for example, Eijkel et al.260 used a micro- and nanofluidic network to generate femtoliter droplets containing a single β-glucosidase molecule. The enzyme activity was detected off-chip after incubation. Also, the enzyme molecules can serve as signal reporters for detection of nonenzyme proteins through specific molecular recognition. For example, Shim et al. introduced a droplet microfluidic platform for rapid detection of prostate-specific antigen (PSA) through ELISA.261 As shown in Figure 26A, single-molecule PSA was captured by a microbead with an antiPSA antibody coating; then the PSA was sandwiched by a secondary detection antibody, which was conjugated with the reporter enzyme β-galactosidase, through a biotin and streptavidin bridge. The beads with or without the immunocomplex were then singly compartmentalized into droplet reactors containing fluorogenic substrate fluorescein diβ-D-galactopyranoside (FDG). The droplets were then sequentially loaded and flushed out of a trap area with the aid of a valve system for on-chip incubation, during which the enzymatic turnover produced a fluorescence signal for real-time monitoring. The concentration of PSA could be quantified by numerical counting of the positive signals. This method enabled single-molecule protein detection down to 46 fM, which was well below the detection limit of ordinary ELISA. The key principle lies in the generation of droplet reactors down to the femtoliter scale, through specific design of the microfluidic channel geometry and appropriate selection of flow conditions. In that way, the concentration of a single enzyme molecule encapsulated in the droplet would be efficient for rapid accumulation of the catalytic product, to generate detectable fluorescent signals within a few minutes. Moreover, enzyme 7988


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Figure 27. Protein crystallization in droplet platforms. (A) Microbatch process: (i) scheme illustration; (ii) microfluidic device; (iii) thaumatin crystals formed in the droplets. (B) Vapor diffusion process: (i) scheme illustration; (ii) crystals yielded in the droplet of protein solution after water loss. (C) X-ray diffraction of the protein crystal in the capillary. Adapted with permission from ref 266. Copyright 2004 John Wiley & Sons. (D) Hybrid screening of protein crystallization: (i) scheme of the method; (ii, iii) scheme and microscopic images showing the generation of droplet arrays of different reagents separated by spacer plugs. Within each array, concentration gradients were generated by adjusting the flow rates. Adapted with permission from ref 267. Copyright 2006 National Academy of Sciences.

long-term stability and thus efficient crystallization. In the vapor diffusion process, droplets containing the reaction mixture (protein and precipitant) and a high-concentration NaCl solution were generated alternately through two junctions into the capillary channel and were separated by a waterpermeable oil poly((trifluoropropyl)methylsiloxane) homopolymer (FMS-121). Therefore, water in the protein-mixturecontaining droplets diffused into the adjacent salt droplets, and the protein crystallized until the osmotic pressure reached a balance, as shown in Figure 27B. This system enabled on-chip crystallographic analysis for evaluating the crystal quality by X-ray irradiation on the product in the capillary channel, as shown in Figure 27C. Moreover, a plug-based parallel approach was further invented for identifying the precipitant type and concentration simultaneously in one experiment.267 Droplet plug arrays containing different precipitants were generated in the carrier oil and separated by spacer plugs. Within each array, droplets contained the same precipitant but various concentrations by changing the flow rates. Therefore, the precipitant type was screened and the concentration was optimized simultaneously in one experiment, as shown in Figure 27D. In such a hybrid system, a large number of trials were conducted with small amounts of time and reagents. 4.4.3. Cell Manipulations. The microfluidic technique has revolutionized the method of cell-level analysis due to the ability of precise manipulation of fluids at length scales comparable with those of cells. Remarkably, droplet micro-

mechanisms involved in many physiological and pathological processes. Protein crystallization is a prerequisite step, which is a process of growing a protein crystal with high quality. Generally, protein crystallization occurs when the concentration of the protein exceeds its solubility in a precipitant. Also, this process can be affected by many other parameters such as temperature, pH, additives, etc. Therefore, finding the most appropriate conditions for crystallization is required.265 Fortunately, droplet microfluidic techniques provide an efficient platform for this demand, because a large number of parallel trials would be carried out in monodisperse droplets with small reagent consumption, and the local physicochemical parameters could be precisely controlled. Generally, protein crystallization methods can be classified into four types: vapor diffusion, microbatch, microdialysis, and free-interface diffusion. The former two methods have been incorporated into droplet microfluidic systems. For example, a PDMS/glass composite microfluidic device was developed for screening protein crystallization conditions within droplets.266 In the microbatch process, as shown in Figure 27A, aqueous flows of protein, precipitant, and additives were mixed and emulsified by a fluorinated oil phase to generate monodisperse droplets in the PDMS section of the channel. By varying the relative flow rates, the concentration of each reagent was precisely adjusted. The droplets were then transported into the capillary section for incubation and tested for identification of the appropriate crystallization conditions. In such a glass capillary channel, solution evaporation was best eliminated for 7989


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Figure 28. (A) Cell encapsulation and postsorting. (i) Scheme illustration. (ii) Droplet generation and cell encapsulation. The relatively large droplets marked by the red-dashed circles indicate the cell-containing drops. (iii) The cell-containing drops and the empty drops diverge into the observation chamber and the waste outlet, respectively, after passing the sorting region. (iv, v) Microscopic images of droplets containing (iv) a single cell and (v) no cell. Adapted with permission from ref 273. Copyright 2015 Elsevier. (B) Single-cell droplet encapsulation by using Dean flow in a curved channel: (i) scheme illustration; (ii) microscopic image of the cell ordering and deterministic cell encapsulation. Adapted with permission from ref 274. Copyright 2012 Royal Society of Chemistry.

Figure 29. (A) Droplet platform for algal cell culture: (i) scheme of the microfluidic device for cell encapsulation and incubation; (ii) bright-field microscopic images of the droplets containing cells over 6 days of culture; (iii) observation of cells dividing in the microdroplets. Adapted with permission from ref 275. Copyright 2011 Royal Society of Chemistry. (B) (i) Encapsulation of yeast cells in hPG−PEG premicrogel droplets. (ii) Scheme of the gelation process and confirmation of cell viability through green staining. Adapted from ref 276. Copyright 2012 American Chemical Society. (C) Encapsulation of human mesenchymal stem cells in double-emulsion droplets: (i) schematic illustration of cell encapsulation and spheroid formation; (ii, iii) phase image and live/dead staining of hMSC spheroids in droplets after 6 h, respectively. Adapted with permission from ref 278. Copyright 2013 Nature Publishing Group.

fluidics has recently been applied and shown high value for cellbased research.268,269 Prior to cell-based analysis (which will be further described in detail), the cell should be encapsulated efficiently, should be kept viable for a certain time, and should be compatible with various operations such as freezing, thawing, lysis, and gene delivery. Cell Encapsulation. Encapsulating a cell into each droplet is a prerequisite for the success of droplet-based cell assays. In a random process, an aqueous phase of cell suspensions was directly emulsified into droplets, and cells were thus encapsulated. By this means, the number of cells in each droplet follows the nonuniform Poisson distribution.270,271 That is to say, each droplet might contain one, two, three, or no cells, stochastically. Therefore, the cell suspension is usually highly diluted before encapsulation to ensure that most droplets contain no more than one cell. This would inevitably lead to reagent waste, and the throughput would be decreased, because a large majority of the droplets would contain no cell. To circumvent this obstacle, several methods have been

developed to achieve single-cell compartmentalization. For example, when using highly diluted cell suspensions, droplets with single-cell occupancy can be selected from blank ones by postsorting according to their different properties. Viovy et al. introduced a hydrodynamic sorting method based on jet formation by a cell-triggered Rayleigh−Plateau instability mechanism,272 which refers to a jet being destabilized and breaking into droplets consecutively by the passage of cells. Following this principle, Han et al. described a size-sorting method to enhance the single-cell encapsulation efficiency.273 As shown in Figure 28A, a microfluidic device was fabricated with a flow-focusing droplet generator and a hydrodynamic sorting channel. The sorting channel was composed of a deterministic lateral displacement (DLD) micropillar array. Under certain hydrodynamic parameters, droplet jetting was formed to produce large droplets containing cells and small droplets that were empty. As the critical dimension of separation (Dc) of the DLD channel was set between the sizes of the two kinds of droplets, the cell-encapsulated droplets 7990


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Figure 30. (A) Microscopic images showing the behavior of a B lymphoma cell: (i−iv) encapsulation in a droplet, (v) prior to freezing, and (vi) after freezing. Adapted from ref 279. Copyright 2007 American Chemical Society. (B) Sequential high-speed images showing single-cell photolysis within the droplet. Adapted from ref 280. Copyright 2005 American Chemical Society.

In addition, cells could be encapsulated in hydrogel materials through gelation reaction after encapsulation. As reported by Seiffert et al., yeast or mammalian cells were first encapsulated in droplets containing monomers of hyperbranched polyglycerol (hPG) and PEG, as shown in Figure 29B.276 Then the gelation occurred by radial-free Michael-type addition reaction without using any photoinitiator and ultraviolet (UV) irradiation, which are cytotoxic. Cells were thus laden in the hydrogel matrix, and the cell viability was tested in various monomer properties. Moreover, cells could be encapsulated within W/O/W double-emulsion droplets. Leong et al. studied bacterial activity in a double-emulsion system and demonstrated that the existence of the oil shell and the external aqueous phase offers several advantages, including limited desiccation, reduced organic fluid consumption, and greater control over the inner environment.277 Later, they applied a similar double-emulsion system for encapsulation of human mesenchymal stem cells (hMSCs) and observed rapid hMSC spheroid formation due to cell aggregation, as shown in Figure 29C.278 Cell Freezing, Thawing, and Lysis. For long-term storage of cells, the freezing and thawing of cells is required. Chiu et al. developed a microfluidic device with a thermoelectric cooler, by which the droplet temperature was manipulated through spatial thermocontrol.279 Briefly, a microscale thermoelectric cooler (TEC) was integrated into the microfluidic device, which generated highly localized cooling or heating by reversely applying a voltage on its two sides. Thus, by using aqueous solutions and oil fluids with different freezing points, the droplets and the encapsulated single cells could be selectively frozen and continued to flow, as shown in Figure 30A. Afterward, the cells were thawed before observation. By using a live/dead stain, the cell viability was confirmed to be unaffected by the freeze−thaw process when dimethyl sulfoxide (DMSO) was added to the cell media as a cryoprotectant. Cell lysis in droplets allows for release of cell contents for further analysis. This can be realized by physical or chemical methods. Physical methods can be conducted by laser irradiation, heating, or the osmotic pressure difference. Chemical lysis relies on the use of lysing agents. The virtue of droplet-based cell lysis lies in the confinement of the cell lysate within the intact droplet reactors. Therefore, cell content analysis could be conducted at the single-cell level. An example of the laser photolysis process is shown in Figure 30B, where a single cell was entrapped in an aqueous droplet.280 When a laser pulse was exerted, plasma formed and a shock wave was

deviated into an observation chamber, while the smaller empty droplets flowed in their original direction toward a waste outlet. In addition to such passive sorting methods, the presence of a cell brings about additional signal contrast for sorting by other approaches, such as fluorescence-activated droplet sorting. Apart from sorting, cell encapsulation strategies have also been improved by introducing hydrodynamic self-ordering of the cells before droplet generation. As demonstrated by Toner et al., when a high-density cell suspension flowed rapidly into a high-aspect-ratio microchannel, under inertial effects, cells showed self-ordering, and the rate of a cell entering the flowfocusing region matched the droplet formation rate.271 Therefore, each droplet contained exactly one cell. Instead of using straight channels, Kemna et al. described another geometric design for inertial ordering of cells by using a curved channel.274 As shown in Figure 28B, the curvature introduced a secondary Dean force, which arranged the cells into a single line and spaced them evenly. The single-cell encapsulation efficiency in this method reached 77%. Other examples of such hydrodynamic self-ordering can be seen by using pinched channel structures. By this means, cells were focused along the central line of the channel, as a result of balance between the lift forces and the contracting effects. Cell Culture. To guarantee the stability of the cell-loaded system in future analysis, the first thing to consider is to let cells survive a certain period of time. For in vitro cell culture, the environmental parameters should be set appropriately for sufficient exchange of substances, including nutrients, gas, and metabolites. The droplet microfluidic system meets this requirement, provided that the encapsulating media, the surfactant, and the carrier-phase fluid are all biocompatible. It also helps to identify the appropriate conditions for cellular growth by adjusting and assessing the environmental parameters. Up to now, different cell types, including prokaryotic and eukaryotic cells, have been successfully brought to culture in droplet microfluidic systems. For example, culture of different green microalgae species in droplet compartments over 10 days has been reported.275 The PDMS device was applied due to its gas permeability, and fluorinated oil was used as the carrier phase considering its biocompatibility and oxygen permeability. As shown in Figure 29A, algal cell suspension droplets were generated and then incubated. By using bright-field microscopy, cell growth and division were clearly observed. Thus, the growth curves were drawn to investigate the effects of droplet volume and initial cell number on cell viability. 7991


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Figure 31. Cell gene delivery in droplets. (A) Transfection of EGFP DNA into CHO cells by electroporation: (i) scheme illustration of the process; (ii) cell-containing droplets passing through the two electrodes; (iii) cell-containing droplets after electroporation. Adapted from ref 282. Copyright 2009 American Chemical Society. (B) Transformation of plasmid DNA into E. coli: (i) scheme illustration of the platform; (ii, iii) comparison of the transformation efficiency between (ii) droplet-based and (iii) conventional methods by transformant culture experiments. Adapted with permission from ref 283. Copyright 2011 Royal Society of Chemistry. (C) Bacteriophage-mediated transduction in E. coli: (i) scheme of the compartmentalization of phages into droplets containing bacteria; (ii) microscopic images of the bacteria and dividing bacteria, which are depicted by the arrowheads and the arrow, respectively. Adapted with permission from ref 284. Copyright 2010 John Wiley & Sons.

chamber with circulating warm water at 42 °C. Heat-shock transformation thus occurred, and the efficiency was comparable with that of traditional methods. Derda et al. described bacteriophage-mediated transduction by coencapsulation with host E. coli.284 As shown in Figure 31C, after random modification of the coating protein, a library of phage clones was generated and endowed with distinct growth features (depicted as S and R types, respectively). Then each phage was encapsulated into individual droplet compartments with E. coli as the growth medium. Thus, the phages were amplified and exempted from competition with each other. Therefore, The R/S ratio and the clones’ diversity were preserved against the selection pressure and elimination of inferior clones. 4.4.4. Multicellular Organism Analyses. Microfluidic droplets also allow for encapsulation of multicellular systems and provide a microscale environment for them to survive, proliferate, and even perform a complete life cycle. Köhler et al. encapsulated eggs from the zebrafish Danio rerio into microfluidic droplets and observed the embryonic development process.285 Another droplet microfluidic platform was developed for encapsulation of Caenorhabditis elegans.286 As shown in Figure 32A, eggs of the nematode C. elegans were encapsulated into large droplet compartments through a flowfocusing device and were continuously monitored under a microscope. After 2 days, hatched worms emerged. Then, after 4 days, each worm grew into adulthood and gave birth to the younger generation. Eventually, after 6−9 days, the worm died. Besides, Drevelle et al. presented a droplet-based platform for high-throughput screening of filamentous fungi.287 Briefly, single spores from a UV-mutated library of Aspergillus niger were encapsulated in microfluidic droplets containing fluorogenic substrate and were incubated for germination, growth of the mycelium, and enzyme secretion. Then the droplets were

generated, in association with cavitation bubbles. These effects acted to disrupt the cell. As the cell lysis was quite rapid, it allowed the cell state to be fixed at the moment of lysis, before any stress response occurred. Also, by using fluorescein di-β-Dgalactopyranoside, single-cell enzyme activity assay on intracellular β-galactosidase was realized. Cell Gene Delivery. Gene delivery is a process of introducing exogenous genetic information into host cells. It is a primary step for investigating gene expression and for improved gene therapy.281 It can be classified into three categories: transfection, transformation, and transduction. Transfection refers to introducing nucleic acids into eukaryotic cells by nonviral methods, transformation describes a similar nonviral gene introduction into bacterial cells, and transduction means a virus-mediated nucleic acid transfer through one bacterium to another. The droplet microfluidic platform offers a controllable and isolated strategy for such operations and is also possible for high-throughput processing. Lu et al. reported transfection of enhanced green fluorescent protein (EGFP) DNA into Chinese hamster ovary (CHO) cells via a plasmid.282 It was conducted within microfluidic droplets through electroporation, by which the cell membrane was breached and plasmid DNA was introduced. As shown in Figure 31A, cells and plasmid DNA were first encapsulated within conductive buffer droplets in an insulating oil fluid. Then the droplets flowed downstream through two microelectrodes, where a voltage was applied and cells were electroporated. By using fluorescence microscopy, the expression of plasmid vector DNA was validated. Wang et al. presented transformation of plasmid DNA into E. coli in microfluidic droplets via heat-shock treatment.283 As shown in Figure 31B, droplets containing an E. coli and plasmid DNA mixture were generated in a PDMS channel on an ice bath. Then the droplets were transported to a glass collection capillary, which was incubated in a heating 7992


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which caused undesirable coalescence before enzyme secretion. In nanoliter droplets, however, the area was large enough for hyphae to keep growing until the mycelial network formed and the enzyme was secreted, thus generating detectable fluorescent signals for screenings before coalescence, as shown in Figure 32B. 4.5. Applications of the Droplet Analysis Systems

As has been described above, droplet microfluidics has revolutionized conventional bulk bio(chemical) analysis processes by offering individual compartmentalized microreactors with highly controllable local environments and local manipulations. With these intrinsic superiorities, droplet microfluidics enables handling and analysis of a wide range of samples, including molecules and cells. Moreover, by virtue of the powerful modern detection technologies, various functions have been realized in such systems, including identification and quantification of certain contents, screening, and real-time dynamic monitoring. Therefore, microfluidic droplet analysis platforms have an extremely extensive application coverage. Here, we categorize their applications into five areas: single-cell analysis, medical diagnostics, drug discovery, food and feed industry, and environmental engineering. 4.5.1. Single-Cell Analysis. Single-cell analysis has been gaining increasing attention due to its capacity in revealing cell heterogeneity, which results from spatiotemporally differential gene expression. Besides, it is vital for interrogating life activities and pathologic development. However, assays on individual cells is difficult in bulk experiments, because signal variation between individual cells can be easily masked by cell group signals.268 Conventional single-cell analysis techniques, such as fluorescence-based flow cytometry, are capable of generating single-cell level signals while being difficult to use for monitoring dynamic responses over time.269 By contrast, the droplet microfluidic system enables single-cell manipulation

Figure 32. (A) Encapsulation and growth of nematode C. elegans in droplets: (i) egg encapsulation, which is depicted by the black arrow; (ii, iii) microscopic images taken after (ii) 2 and (iii) 4 days. The white arrows depict the larvae of the younger generation. Adapted with permission from ref 286. Copyright 2008 Elsevier. (B) Filamentous fungus growth in droplets. (i) Sequential microscopic images of a 250 pL droplet with a single germinating spore over 17 h. (ii, iii) Microscopic images of 18 nL droplets with a single germinating spore after (ii) 24 and (iii) 32 h, respectively. The red circles mark the hyphal tips extending outside the droplet. (iv) Fluorescence image of 18 nL droplets with a single germinating spore after 24 h for the identification of enzyme secretion. Adapted with permission from ref 287. Copyright 2016 Nature Publishing Group.

reinjected into a sorting device and were tested by measuring the fluorescence signal to determine the activity of the secreting enzyme α-amylase. The key to the success of this platform was that the droplet compartments were relatively large (∼10 nL), because in picoliter droplets (suitable for hosting bacteria), hyphae grew and expanded out of the droplet in a short time,

Figure 33. (A) Single-cell DNA analysis: (i) scheme of the encapsulation of the target cell and microbead into droplets containing the PCR mixture; (ii) scheme of PCR amplification and the attachment to the bead. Adapted from ref 288. Copyright 2008 American Chemical Society. (B) Single-cell multiplex sequencing by gel electrophoresis to confirm the amplification of t(14;18) and β-actin. Adapted with permission from ref 289. Copyright 2010 John Wiley & Sons. 7993


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Figure 34. Scheme of the drop-based ChIP-seq procedure for single-cell chromatin profiling and cell subpopulation clustering, which includes the following steps: cell encapsulation, lysis, and barcoding in a microfluidic-droplet-based platform, immunoprecipitation of the chromatin contents and DNA sequencing after droplet breakup, and single-cell chromatin profiling and subpopulation identification. Adapted with permission from ref 290. Copyright 2015 Nature Publishing Group.

buffer that contained proteinase K. This led to lysis of the cell and digestion of histone proteins, while the DNA molecules were retained. Afterward, the particles were re-emulsified with PCR reagents in a carrier oil phase, after which PCR was performed. Through separate PCR reactions and gel electrophoresis, two target gene loci, chromosomal translocation t(14;18) and the control gene β-actin, were isolated and sequenced, as shown in Figure 33B. In addition to obtaining static aspects of genomic information, a dynamic study on functional genomics is also very important. Cells in an organism show heterogeneity in the chromatin profile, which embodies functional genomic elements and gene regulation information. Chromatin immunoprecipitation sequencing (ChIP-seq) is a widely accepted technique for chromatic mapping at the genome level. However, it generally achieves ensemble profiles and is not accessible to cell−cell variation analysis. In view of this, the droplet-based ChIP-seq system has recently been developed by Bernstein et al. for single-cell chromatin profiling and cell subpopulation clustering, as shown in Figure 34.290 Cells were first encapsulated in droplets with detergent buffer for lysis and release of chromatin. Then such droplets with fragmented chromatins were merged with another flow of droplets containing DNA barcodes through electrocoalescence. With addition of DNA ligase solution, the barcode sequence was linked with the two ends of the nucleosomal DNA fragments, thus annotating the chromatin fragments and the corresponding cells throughout the whole process. Next the droplets were broken, and the chromatin contents were pooled together for antibody immunoprecipitation, followed by PCR amplification and next-generation DNA sequencing. Using this method, single-cell chromatin profiles were obtained. Moreover, by conducting a divisive hierarchical clustering algorithm on the profile data, groups of related cells were identified, which were further aggregated to different subpopulations. Single-Cell Transcriptomic Analysis. Single-cell RNA and transcriptomic studies help in interrogating the cell’s functions and the spatial-temporal variations of gene expression. They have been benefited from droplet-based RT-PCR technology. For example, single-cell RT-PCR within agarose droplets was demonstrated.291 Instead of using microbeads for nucleic acid

and dynamic monitoring in a highly controllable manner. In addition, the novel droplet-based molecular biological techniques have been employed to detecting cell contents including DNA, RNA, protein, and other metabolites. Moreover, the high-throughput processing ability provides a novel platform for next-generation sequencing, which has achieved an extensive impact and is seen as one of the greatest accomplishments in recent years. Overall, droplet-based single-cell analysis could be categorized into genomic, transcriptomic, protein, and metabolic analysis. Single-Cell Genomic Analysis. Genomic heterogeneity is one of the direct contributing factors to single-cell heterogeneity. It often occurs as a result of a random replication error or environmental stimuli. Single-cell gene analysis from a large group of cells is a significant method for subpopulation classification and mutation iedentification. This has been achieved in droplet microfluidics. As single cells could be compartmentalized into individual droplets, their single genome can also be encapsulated and further analyzed by PCR amplification and DNA sequencing. Mathies et al. described a single-cell genome amplification method.288 As shown in Figure 33A, to maintain single-cell genome fidelity, invividual cells were encapsulated with primer-conjugated microbeads and the PCR mixture in microfluidic droplets. Together with the initiation heating step, cells were lysed to release their genomic DNA for PCR amplification, and the fluorescently labeled amplicons were attached to the beads. When the amplification process was completed, the droplets were broken, and the beads went on for further analysis. Owing to the high-throughput capability, attomole-long DNA amplicons were generated, which facilitated Sanger sequencing. Using this method, single-cell analysis of the glyceraldehyde 3phosphate dehydrogenase (GAPDH) gene (from human lymphocyte cells) and gryB gene (from bacterial E. coli K12 cells) were demonstrated. In addition to monotarget gene analysis, Mathies et al. further reported single-cell multilocus sequencing within agarose droplet containers.289 Single lymphoblast cells were first encapsulated in agarose droplets, together with the primerfunctionalized microbeads. Then the droplets were cooled to form solid gel particles, which were then incubated in SDS 7994


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Figure 35. Single-cell RNA sequencing. (A) “inDrop” method. Adapted with permission from ref 292. Copyright 2015 Elsevier. (B) “Drop-seq” method. Adapted with permission from ref 36. Copyright 2015 Elsevier.

Single-Cell Protein Analysis. Single-cell protein analysis is an important aspect in characterizing cellular phenotype heterogeneity. Compared with that of nucleic acids, the concentration of proteins is usually very low and cannot be directly amplified. Conventionally, single-cell protein analysis was conducted by fluorescent flow cytometry. However, it is not applicable for detection of samples in ultralow abundance. Also, the lack of real-time monitoring and absolute quantification, as well as possible damage to cells, would be a drawback. Droplet microfluidics, however, have been demonstrated to be feasible for single-cell protein quantification, even at ultralow concentrations, providing a way for dynamic measurement without hampering the cells. For cell surface protein analysis, biomarker detection is vital in understanding the biochemical process and cell functions, as well as disease diagnosis. Joensson et al. reported a method for analyzing low-abundance biomarkers CCR5 and CD19 on the surface of single human monocytic cells through droplet ELISA assays.293 As shown in Figure 36A, cells were first labeled with biotinylated antibodies and then conjugated with streptavidin−β-galactosidase via biotin−streptavidin linkage. Then the cell suspension was injected into a microfluidic device and mixed with another flow that contained fluorogenic substrate FDG at a cross-junction, after which droplets were generated and single cells were encapsulated and incubated for sufficient enzymatic turnover. By using PMTs, high-throughput droplet analysis was processed. It was demonstrated that the signal resolution achieved in this method was higher than that of fluorescence-activated cell sorting (FACS). In addition to surface proteins, intracellular proteins could also be analyzed in the droplet microfluidic platform. Cooper et al. introduced an integrated system for quantification of intracellular proteins within droplets through immunoassays after electrical lysis.294 It was confirmed that this method achieved a faster response than conventional Western blots with less cell consumption. Moreover, proteins secreted from

pairing, the reverse primers were conjugated to agarose droplets through a covalent linkage. Therefore, the steric hindrance and charge repulsion were eliminated, and the amplification efficiency was increased. Using this method, expression variation of the EpCAM gene was demonstrated at the singlecell level between different cancer cells. Furthermore, two seminal works were recently published on large-scale, highthroughput droplet-based RNA sequencing methods, termed “inDrop” and “Drop-seq”, respectively. inDrop is a method for single-cell transcriptomics studies.292 As shown in Figure 35A, cells were first encapsulated into microfluidic droplets with lysis buffer, a reverse-transcription mixture, and hydrogel microspheres, which were loaded with DNA-barcoded primers. After cell lysis, the mRNAs were released and barcoded during cDNA synthesis. Then the droplets were broken, and the cDNAs were sequenced using the next-generation CEL-seq protocol. Using this method, mouse embryonic stem (ES) cells were analyzed before and after leukemia inhibitory factor (LIF) withdrawal. Through a large-scale investigation on cellular expression heterogeneity, rare subpopulations were identified, which would be inaccessible when just a few hundred cells are profiled. Similarly, Drop-seq is a method for single-cell expression profiling.36 As shown in Figure 35B, cells dissociated from a complex tissue were first encapsulated with microparticles that were loaded with DNA-barcoded primers. Then the cells were lysed, and the released mRNAs were captured on the microparticles. The single-cell transcriptomes were thus attached on the particles for subsequent reverse transcription, next-generation sequencing, and analysis, after droplet breakup. Using this method, highly parallel transcriptomic profiling of 44808 mouse retina cells was conducted, and 39 cell populations were ascertained. Overall, both of the methods have made breakthroughs in high-throughput processing on a large number of cells. 7995


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cells could also be confined in the droplets for analysis. Chen et al. presented a droplet-based method for single-cell multiplexed protease activity assay.295 As shown in Figure 36B, the detection principle lies in a computational approach to define the protease activity by measuring the fluorescent signals originating from cleavage of the FRET substrate by proteases. To recognize each protease, FRET substrates were modified with several pairs of fluorescent donors and quenchers corresponding to different excitation and emission wavelengths, respectively. Single cells were encapsulated with FRET substrates and lysis buffer in individual droplets. After sufficient reaction time, the substrates were all cleaved, and multiple fluorescent signals were detected simultaneously within a single droplet. Using this method, six protease activities could be measured through four kinds of cleavage reactions. The activity of certain proteases showed cellular heterogeneity within the same line. Moreover, the cell population distribution was determined by characterizing the protease profiles of three types of cells. Apart from protein detection and enzyme activity measurement, droplet microfluidics has also been applied to dissecting the enzyme’s function. For example, an ultra-high-throughput droplet system was developed to study the function of an enzyme with regard to its amino acid sequence.296 A library of E. coli cells carrying random gene mutagenesis (generated through error-prone PCR) were encapsulated individually into droplets together with fluorogenic enzyme substrate and lysis reagents. Then the droplets were incubated off-chip for enzymatic reaction, after which they were reinjected into a sorter device with on-line fluorescent detection. Next, the positive droplets (dictating the active enzyme variants) burst under an electric pulse to release the positive plasmids into an

Figure 36. (A) Single-cell detection of low-abundance surface biomarkers. Adapted with permission from ref 293. Copyright 2009 John Wiley & Sons. (B) Single-cell multiplexed protease activity assay: (left) scheme illustration of the FRET detection mechanism; (right) multiple fluorescent signals of different excitation and emission wavelengths are detected simultaneously in one droplet. Adapted with permission from ref 295. Copyright 2016 Elsevier.

Figure 37. Droplet-based β-glucosidase assay. (A) Microfluidic workflow. (B) Scheme of the fluorescence signal generation in droplets containing the active enzyme variant. (C) Microscopic images of the droplets containing wild-type (WT) Bgl3 and an inactive Bgl3 mutant. (D) Fluorescence signal detection through a PMT system. The three higher peaks and the remaining peaks indicate the WT Bgl3-containing drops and the empty drops, respectively. (E) Fluorescence intensity histogram of the Bgl3 random mutagenesis library. The red line is set as the sorting threshold. Adapted with permission from ref 296. Copyright 2012 National Academy of Sciences. 7996


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aqueous collection fluid for downstream DNA recovery and sequencing, as shown in Figure 37. This method combined droplet-based single-cell enzyme screening and next-generation sequencing, thus generating a detailed mapping of enzyme sequence−function relationships. By using glycosidase as the model enzyme, specific functional residues were discovered, and function-oriented beneficial mutations were selected in response to specific screening conditions. Single-Cell Metabolic Analysis. The cellular metabolome also embodies a cell’s life activities in relatively small molecules. Similar to cellular proteins, cell metabolites also exist at low abundance and are difficult to amplify directly. Conventional methods generally reflect an “averaged” metabolism level from a population of cells while neglecting cellular variation. Droplet microfluidics provides an advantageous technique for metabolite analysis and enables dynamic monitoring at the single-cell level. Boitard et al. developed a droplet platform for monitoring yeast metabolic activity at the single-cell level on the basis of the osmotic response of droplets that contained bioactivities. Briefly, single yeast Saccharomyces cerevisiae cells were encapsulated in droplets containing nutrients.297 The droplets were then placed at a fixed position for real-time observation. With sufficient glucose and a limited supply of other nutrients, cell division stopped while fermentation continued. Thus, glucose was converted into ethanol, carbon dioxide, and ATP. The continuous phase was specially selected so that the ethanol and carbon dioxide diffused out rapidly to dissolve in it, and contributed to net loss of biomass, which resulted in an osmotic change. Therefore, water flowed out of the cell-containing droplets, and the droplet volume decreased. By measuring such volume variation, the metabolic activity of individual cells was deduced and cellular diversity was explored. Apart from droplet volume measurement, Del Ben et al. reported single-cell metabolism analysis by measuring pH alternations in confined droplets.298 The principle lies in the “Warburg effect”, which refers to cancer cells’ predominant metabolic characteristic of glycolysis even with the presence of oxygen. By encapsulating a single cell in miniaturized droplet compartments, the rapid accumulation of lactate caused detectable pH variations in comparison with that of white blood cells. In addition, dynamic metabolism transformation has been identified by means of the droplet microfluidic analysis system. As demonstrated by Lindquist et al., [GAR+] is a prionlike element that induces an epigenetic switch in the S. cerevisiae metabolic feature from glucose specialists to multiple-carbonsource generalists.299 It can be induced in the yeast cell through a bacteria secreting factor. Such induction and the resultant metabolic conversion were demonstrated in the droplet microfluidic system. Briefly, yeast cells were labeled with mOrange and GFP to report the cell number and the prion status, respectively. Then the cells were encapsulated into individual droplets containing [GAR+] selection medium and incubated for 48 h. Through orthogonal fluorescent analysis, metabolic features of [GAR+]-loaded cells and [GAR−]-loaded cells were distinguished. In addition, to assess the prion induction level, [GAR−] cells were coencapsulated with E. coli as an inducing bacterium, and the fluorescence results showed that just a few bacterial cells are sufficient to achieve a high inducing efficiency. It is worth noting that, through this coencapsulation strategy, the cross-kingdom chemical communication and social interaction were demonstrated.

4.5.2. Medical Diagnostics. Medical diagnosis has long been a significant part of bio(chemical) analysis. Analytes in clinical samples often exist in small abundance and demand a short detection time. As described above, droplet microfluidics is capable of rapid and high-throughput detection of a wide variety of analytes down to the single-molecule or single-cell level. Therefore, it enables detection of various biomarkers from complex clinic samples and offers a possible way of revealing individual heterogeneity and promoting personalized medicine. Nucleic acids can be used as important biomarkers related to many kinds of diseases. For example, somatic gene mutations exist in patient samples in the form of rare tumoral DNA and can be treated as highly specific biomarkers in cancer diagnosis. Droplet PCR provides an absolute quantification method with ultrahigh sensitivity and reliability for detecting such rare mutations from a large amount of nonmutated counterparts. As demonstrated above, using droplet PCR, mutated KRAS genes could be detected from a 200000-fold excess of wild-type genes.247 Besides, miRNA plays an important role in apoptosis or necrosis. Circulating miRNA expression levels can reflect states of diseases such as neurodegenerative disorders and cardiovascular diseases and could be quantified in the plasma sample within a droplet-based EXPAR platform.254 In addition to nucleic acids, proteins are also significant indicators of disease status. For example, specific surface biomarkers that are expressed on cancer cells could be detected through single-cell droplet-based immunoassays. Also, MMPs, as an example of secreting proteins that are critical for tumor growth, invasion, metastasis, and angiogenesis, have been analyzed through FRET-based enzymatic reactions in droplet reactors.263 Moreover, for infectious disease diagnosis, such as bloodstream infections (BSIs), pathogenic microorganisms such as bacteria need to be detected at an early stage for treatment with proper antibiotics in time. This has been recently achieved in droplet microfluidic systems, through reactions between DNAzyme and the bacteria lysates in blood samples, which generated fluorescent signals for detection.300 4.5.3. Drug Discovery. Drug discovery is a process of investigating the interactions between potential drugs and targets. It depends on high-throughput screening trials with both qualitative and quantitative readouts. Conventional screening methods usually take a long time, and the number of clones to be tested during one experiment is quite small. By contrast, the microfluidic technique provides a highly parallel platform to meet this demand. Especially, droplet microfluidics possesses additional advantages such as compartmentalization, low contamination and dispersion, fast mixing, and low reagent consumption. Therefore, it has been widely applied in drug discovery with enhanced screening efficiency and throughput. In some cases, a compound library is screened to select the active species aimed at the specific target. In other cases, a dose−response screening is also conducted to determine the appropriate concentration of the compounds according to the target’s activity. Drug Component Screening. Monoclonal antibodies (mABs) are an important class of therapeutics for many diseases, including autoimmune, cancer, and infectious diseases. By employing a droplet platform, single-cell hybridoma screening of up to 300000 clones was achieved within 1 day.301 The “target” in this research was angiotensin-converting enzyme 1 (ACE-1), which is generally involved in treatment of hypertension and congestive heart failure. The “potential 7997


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Figure 38. Droplet-based drug discovery platform. (A) Scheme of the single-cell hybridoma screening principle. A mixture of hybridoma cells expressing either 4E3 or Elec-403 are encapsulated individually into droplets together with recombinant ACE-1 and are then screened by fluorescence detection. Adapted with permission from ref 301. Copyright 2009 National Academy of Sciences. (B) Workflow of the single-cell screening of molecular libraries. Adapted with permission from ref 302. Copyright 2012 National Academy of Sciences. (C) Scheme of the highresolution dose−response screening based on the Taylor−Aris dispersion phenomenon. Adapted with permission from ref 304. Copyright 2012 Nature Publishing Group.

Figure 39. Droplet-based screening of S. cerevisiae cells for higher secretion of α-amylase. (A) Mutant library generation. (B) High-throughput screening. (C) Confirmation of the α-amylase production ability of the sorted strains. (D) DNA sequencing of the selected strains. Adapted with permission from ref 305. Copyright 2015 National Academy of Sciences.

drugs”, i.e., the functional hybridoma cells, were screened for releasing mABs that inhibit ACE-1, which are called “4E3”. As shown in Figure 38A, a large population of hybridoma cells, expressing either 4E3 or Elec-403 (a noninhibitory antibody to ACE-1), were encapsulated into droplets with recombinant ACE-1. The droplets were then incubated for antibody accumulation, after which they were reinjected and added with fluorescent substrates of ACE-1 by passive droplet fusion. Finally, the droplets were detected, and those generating low fluorescence intensity were sorted.

In addition to hybridoma cell screening, molecular drug screening relies on generation of a library of droplets that differ in composition. In that case, the “target” was the cells, while the “potential drug” was the molecular library, which can be generated by parallel droplet generators and labeled with distinct barcodes, as shown in Figure 38B.302 Moreover, to enhance the therapy efficiency, combinatorial drugs could be screened by coalescence of droplets containing different compounds or premixing of the compounds before encapsulation. 7998


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observation of microscale flow phenomena in porous media. EOR has been confirmed in microfluidic models when nanoparticles were dispersed in the displacing phase.308 To analyze the effects of NPs on oil droplet trapping and mobilization, Zhu et al. developed a droplet microfluidic device with a pore−throat structure to stimulate the general scenario of oil-trapping in water-wet porous media.309 In brief, oil droplets were generated in the NP-dispersed aqueous phase and trapped in the throat region by a side flow. Next the droplets were pushed by the aqueous flow through the throat, and the critical flow rate was recorded in correlation with the droplet size. Through real-time microscopic monitoring, the authors figured out that the increasing NP concentration correlated with a smaller flow rate to push the oil droplets, which implied easier EOR. Also, through mathematic model analysis, they concluded that the mechanism is embodied in the role of NPs in increasing the capillary number, which is similar to the role of common surfactants. Overall, droplet microfluidics is becoming a powerful analytical platform. The use of droplets in such a highly controllable way facilitates a wide range of bio(chemical) analyses from the molecule level to the cell level. Its attainable advantages distinguish it from other conventional methods, especially in next-generation sequencing associated methods and single-cell analysis, which have made profound impacts in recent years. Nowadays, it has been widely adopted in a large number of world-class institutes and laboratories for biomedical research. Meanwhile, droplet-based analytical companies are on the rise. Most importantly, as a practical technique, there is much room for development. For example, how to find easier and more universal ways to break through the Poisson distribution limit and improve single-cell encapsulation efficiency is a major concern to avoid waste of the majority of empty drops. Seeing from this, there is no doubt that the droplet-based analytical system will continue to be a hot research field.

Dose−Response Screening. It has long been recognized that the biological effects of a chemical compound are closely related to its concentration. To determine the appropriate concentration of a drug, dose−response screening is required by means of potency evaluation. This could be achieved in the droplet microfluidic system, because the concentration of the component could be precisely adjusted by changing the relative flow rates (similar to the method used in protein crystallization). Link et al. reported another way of generating concentration gradients on the basis of the Taylor−Aris dispersion phenomenon, through combination of continuous and droplet-based microfluidic systems.303 As shown in Figure 38C, an aqueous compound flow was injected to spread out into a continuous buffer flow in a capillary channel.304 Due to the molecular diffusion and the parabolic flow velocity profile in the continuous flow, the compound concentration distribution was converted from an initial rectangular profile to a Gaussian profile over time. Then the compound−buffer flow was segmented into droplets together with enzyme and substrate solutions and was screened at different analysis points. Using this method, a total of 704 drugs with a concentration range of over 3 orders of magnitude was screened simultaneously for the inhibition of protein tyrosine phosphatase 1B (PTP1B), which is involved in treating cancer, type 2 diabetes mellitus, and obesity. 4.5.4. Other Applications. Although the application of the microfluidic droplet analysis system mainly focuses on biomedical fields, it also sheds new light on other fields, including food and feed industries. For example, recombinant enzymes can be produced by cell factories and can be used as industrial products. Droplet microfluidics provides a highthroughput way for rapidly optimizing the protein secreting efficiency. As shown in Figure 39, Nielsen et al. reported a platform for improving secretion of α-amylase by yeast S. cerevisiae cells through droplet-based screening and wholegenome sequencing.305 A library of yeast cells was first prepared through UV mutagenesis, which generated random mutants including those with improved α-amylase secretion ability. Then single cells were encapsulated into droplets together with fluorogenic substrate, after which they were incubated and reinjected for fluorescence-based screening. Next the sorted mutants underwent a secondary screening and a fermentation test to validate the secreting capacity, after which eight strains were eventually selected with the highest secreting ability. Through wholegenome sequencing, chromosome duplication and a total of 330 point mutations were identified. Such gene ontology analysis allows for better understanding of the protein secreting pathway and can also be helpful for reverse metabolic engineering. For environmental engineering applications, droplet microfluidics provides a platform for detection of analytes from soil or water samples. For example, Takeyama et al. reported a highthroughput platform for single-cell screening of a metagenomic library constructed from soil samples to select lipolytic enzymes.306 This offered a possible method for isolating novel enzymes to represent specific microbial communities in different ecological environments. In addition, Guo et al. employed a valve-based droplet microfluidic device to detect mercury(II) ion, through active fusion of sample droplets with probe droplets.307 Moreover, droplet microfluidics has been explored in enhanced oil recovery (EOR), due to its ability in direct

5. DROPLET MICROFLUIDICS FOR MATERIALS GENERATION Apart from the analytical reactions, the strengths of droplet reactors such as compartmentalization, miniaturization, monodispersity, and high throughput have also been embodied in synthetic reactions in the recent decades, through which nanomaterials could be generated in high quality.310,311 On the other hand, the droplet itself serves as a soft template that bears physical and chemical processes on the entirety or at the interface, enabling synthesis of microscale materials with flexible morphology, micro-nanostructure, and diverse components.32,43,80,83 Overall, droplet microfluidics provides powerful tools for synthesis of nano- and microscale materials with highly controllable physical and chemical properties. Therefore, these materials possess distinct functions and have found applications in many fields such as biomedicine, diagnostics, optoelectronics, etc. 5.1. Nanoparticles from Droplet Microfluidics

For a long time, materials scientists have been focusing on bulk substances. It was not until a few decades ago that materials on the 1−100 nm scale started to gain attention.312 Due to electric confinement and surface asymmetry effects, nanoparticles show distinct properties such as optical emission in semiconductor NPs313 and surface plasmon resonance in noble-metal NPs.314 Also, due to their scale being similar to that of biomolecules, 7999


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they can be tailored to coordinate with biological systems.315 Because of these excellent features and the resultant application values, there have emerged many techniques for NP synthesis, which can be classified into two main categories: “top-down” and “bottom-up”.316 The top-down strategy involves the microfabrication process of having NPs removed from large solid substances through milling or cutting, while the bottomup strategy seeks to obtain NPs through atomic or molecular assembly in a carrier phase. The bottom-up strategy has been widely adopted by virtue of its better control, and a number of solution-based approaches have been put forward through wet chemical reactions, including addition, oxidation, reduction, exchange, hydrolysis, etc.317−321 According to the classic theories proposed by LaMeR and Dinegar, the synthesis of NPs goes through four steps: supersaturation, nucleation, growth, and aggregation. In this process, chemical reactions result in the generation of solutes (atoms or molecules).322 When the concentration increases to a supersaturation degree, the atoms self-nucleate and form small nuclei. Then the nuclei grow into seeds and nanocrystals, and the solute concentration decreases to terminate further nucleation. The particles then aggregate and precipitate from the solution. From a technical perspective, the reaction environment during the complete synthetic course should be precisely controlled for two reasons. First, the reagent mixing, nucleation, and growth processes largely affect the size, shape, and crystal structure of the final products, which would determine the specific properties and functions of the NPs. For example, CdSe quantum dots show variable emissions with different particle sizes.323 The catalytic activities or optical features of metal NPs are also relevant to their size and facets and the surrounding medium.324 Second, for practical usage, scaling up the synthesis is needed at little or no cost of the NP quality. Therefore, the local environmental parameters such as temperature control should be taken seriously for producing NPs with uniform physical and chemical characteristics.325 Among those synthesis approaches, the microfluidic method stands out for its intrinsic advantages, including miniaturization, enhanced mass and heat transfer, and reduced time and reagent consumption.326 Especially, the droplet reactor offers additional fascinating strengths.39,327 For example, as the reaction is encapsulated in the confined droplets, toxic or volatile chemicals can be utilized, and the resultant NPs would not contact the channel walls, thus avoiding possible contamination and blocking. In addition, the advection flow field within the droplets accelerates the mixing even further, thus offering a well-defined starting point and an even residence time, which contribute to a narrower size distribution of the final NPs. Moreover, local control over the synthetic environments could be exerted on separate droplet reactors. Therefore, the reaction parameters scale up linearly, enabling homogeneous synthesis and quantity production. Typically, NPs can be synthesized in a homogeneous manner, which means that the seeds emerge in situ and grow in the droplet reactors, or they can be generated in a seed-mediated manner, where seeds are preformed and then added into the droplet reactors for growth. According to the reagent encapsulation methods, three modes can be identified. The reagents can be encapsulated all together in a single droplet or respectively placed in the droplets and the carrier phase. Also, reagents can be stored in distinct droplets and coalesced to initiate reaction. Such flexibility has enabled droplet microfluidic techniques to synthesize a variety of NPs, including inorganic, organic, and hybrid or complex nanoma-

terials, and to provide excellent control over the size, size distribution, shape, and crystal structure. A selective summary of droplet-based NP synthesis is given in Table 2. Table 2. Droplet-Based Synthesis of Nanoparticles component

example

metal

Au, Ag, Pd

ceramics

magnetic iron oxide, silica, hydroxides, and zeolites CdSe, PbS, Ag2S, CdTe

QDs nanocomposite organic MOFs

alloy, metal−silica, cesium lead halide perovskite, etc. PLGA, PMMA, DPA MOFs, nanomaterial assemblies

feature spherical/ nonspherical fast/uniform/ functionalized organic/waterbased hybrid components spherical/ core−shell porous/highorder structure

ref 328, 329 330−332 334−337 338−342 344−346 345, 349, 350

5.1.1. Inorganic Nanoparticle Synthesis. Metals. Metal NPs, especially noble metals, exhibit special optoelectronic properties and chemical activities, and have aroused much research interest in many application areas such as catalysis, sensing, photonics, etc.312,317,324 Synthesis of various metal NPs with controlled size and structure has been achieved in the droplet microfluidic platform. Generally, it is based on reduction reactions of the metal ion precursor with the presence of stabilizing ligands. For example, Brutchey et al. used a two-phase microfluidic droplet device to synthesize AuNPs and AgNPs.328 As shown in Figure 40A, a carrier oil phase of poly(chlorotrifluoroethylene) was injected through inlet 1. The two reaction reagents of metal salt precursor and the reductant flowed via inlets 2 and 4, respectively. Also, an ionic liquid stream of 1-butyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide (BMIM-Tf 2 N) flowed through inlet 3 and served as a stabilizer and also prevented

Figure 40. Droplet syntheses of metal nanoparticles. (A) Droplet synthesis of AuNPs and AgNPs: (i) scheme of the T-junction microfluidic device (the arrow marks the flow direction); (ii) TEM image of the synthesized AuNPs. Adapted from ref 328. Copyright 2012 American Chemical Society. (B) Droplet-synthesized PdNPs with different shapes and sizes: (i) 9 nm Pd cuboctahedra; (ii) 10 nm Pd cubes; (iii) 14 nm Pd cubes; (iv) 18 nm Pd cubes; (v) 37 nm Pd octahedra; (vi) 21 nm Pd concave cubes. Adapted from ref 329. Copyright 2014 American Chemical Society. 8000


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Figure 41. Droplet syntheses of ceramic nanoparticles. (A) Sequential microscopic images showing the formation of iron oxide precipitates after droplet coalescence. Adapted with permission from ref 330. Copyright 2008 John Wiley & Sons. (B) Synthesis of fluorescently functionalized silica NPs. Adapted with permission from ref 331. Copyright 2012 Royal Society of Chemistry. (C) TEM images of droplet-synthesized hydroxide and zeolite nanomaterials: (i) ZSM-5; (ii) γ-AlOOH; (iii) β-FeOOH. Adapted with permission from ref 332. Copyright 2012 Royal Society of Chemistry.

the reagent concentration, the size of the NPs could be adjusted, as shown in Figure 41B. Besides, Park et al. synthesized hydroxide and zeolite nanomaterials in an ionic liquid-assisted droplet system.332 Ionic liquids have been demonstrated to facilitate nanostructure construction and also for system stabilization in bulk process. In such droplet reactors, they were mixed with the inorganic precursors. After a short reaction time, ZSM-5 zeolite, γ-AlOOH nanobundles, and β-FeOOH nanorods were achieved with a narrow size distribution and uniform shape, as shown in Figure 41C. Due to the intrinsic advantages of droplet reactors, the production rate was much higher than in the bulk methods. Quantum Dots. Quantum dots (QDs) refer to semiconductor particles with a size of several nanometers. At this scale, the distinct effect of quantum confinement of the electrons emerges.313 This leads to fascinating electronic and photonic characteristics, which are highly dependent on the size and shape of the QDs. In the synthesis of QDs, pyrolytic reaction occurs, and thus, temperature should be precisely controlled to obtain high-quality products. Among various kinds of QD nanomaterials, CdSe is the most widely studied. Due to its tunable photoluminescence in the visible spectrum range, CdSe nanocrystals could serve as fluorescence probes and be used in biomedical as well as optical-electronic areas.323,333 Mathies et al. reported high-temperature synthesis of CdSe QDs in droplet reactors.334 Both the carrier fluid and the solvent of the cadmium and selenium precursors were selected to have physical-chemical stability under high temperature. Due to the temperature dependence of surface tension and viscosity, a stepped microstructure of the microchannel was designed to ensure that droplets could be generated at a high Ca value. Besides, lead chalcogenide QDs, including PbS NPs, were recently synthesized in droplet reactors by deMello et al., and the process was monitored in real time through nearinfrared (NIR) fluorescence spectroscopy to optimize the reaction conditions.335 Such NPs have small band gaps, and the emission is in the NIR region, thus hopeful for fabricating NIR lasers, as well as for other applications such as solar cells,

contact of the reaction reagents before droplet formation. When droplets were generated in the T-junction and kept in the dripping regime, the recirculating streamline induced a convective flow, which largely accelerated mixing of the two reaction reagents. This promoted a rapid nucleation burst and thus ensured a homogeneous synthesis environment. As demonstrated by the authors, the AuNPs and AgNPs generated by this means had a narrower size distribution than those synthesized in the batch stirring process. Apart from spherical NPs, Xia et al. reported droplet-based synthesis of PdNPs with nonspherical shapes.329 By adjusting the flow rates of the reducing and capping agents as well as the reaction temperature according to those used in batch production methods, the nucleation and growth procedures could be well-controlled to tailor the NP shape into cubes, cuboctahedra, octahedral, octahedral, and concave cubes, as shown in Figure 40B. Moreover, the seed-mediated method is more flexible and convenient for synthesis of metal NPs with nonspherical shapes such as octahedral shapes, rods, sharpedged shapes, etc.39 Ceramics. Droplet microfluidic reactors contribute to the synthesis of a variety of ceramics, including metal oxides, silica, hydroxides, zeolites, etc. Baret et al. synthesized magnetic iron oxide NPs within droplets through a precipitating reaction.330 As the synthesis process was very fast, the reagents should not come into contact before reaction. Thus, they were separately encapsulated in droplet pairs through hydrodynamic coupling. The droplets were then coalesced by applying an electric field. As shown in Figure 41A, the precipitate of iron oxide NPs was yielded in about 2 ms. Apart from metal oxides, Wacker et al. synthesized fluorescently functionalized silica NPs.331 The fluorescein isothiocyanate (FITC) dye was first linked with (aminopropyl)triethoxysilane (APTES) and mixed with tetraethyl orthosilicate (TEOS) to give the fluorescent precursor, which served as the silicon alkoxide solution (SA). Through hydrolysis reaction with the ethanol−ammonia hydrolyzing mixture after droplet emulsification, fluorescent silica NPs were synthesized. By changing the reaction time and 8001


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PLGA−DMSO and water droplets was formed in a carrier phase of silicon oil. Then, after droplet coalescence, DMSO was extracted out into water, and the PLGA nanospheres precipitated as a result of supersaturation. Due to their biocompatibility and biodegradability, the PLGA NPs were hopeful for biomedical applications. In addition, core−shell polymer NPs were generated through emulsion-assisted interfacial copolymerization. An organic phase containing methyl methacrylate (MMA) monomer was first emulsified into droplets within an aqueous carrier phase of hydrophilic cationic diallyldimethylammonium chloride (DADMAC) monomer. Then PMMA particles formed through thermally initiated radical polymerization reaction with the help of a thermal initiator, and DADMAC simultaneously polymerized into a soft shell along with the dynamic interfacial interactions.345 Apart from polymers, non-polymer-based organic synthesis has also been realized. For example, 2,2′dipyridylamine (DPA) NPs were generated in microfluidic droplets.346 As DPA is soluble in ethanol while unsoluble in water, its solubility in a water−ethanol mixture decreases with increasing of water content. On the basis of this antisolvent crystallization mechanism, DPA was first dissolved in a water− ethanol mixture close to saturation and emulsified into droplets. Then additional water was introduced through droplet coalescence. DPA NPs thus precipitated out due to supersaturation. In both of the examples, the droplet platform contributed to enhanced mixing of reagents. Therefore, rapid supersaturation and precipitation were realized to yield highquality NPs with a narrow size distribution. 5.1.3. Synthesis of Other Nanomaterials. Microfluidics has recently been employed for the synthesis of metal−organic framework (MOF) materials, which is one of the most fascinating groups of porous materials with an extremely broad application scope.347 Nanoscale MOF materials were achieved by Maspoch et al., through a microfluidic spray-drying strategy.348 It was demonstrated that the achieved nano-MOFs show size-dependent properties. Moon et al. synthesized MOFs in droplet reactors by confining the organic ligand and metallic salt precursors using a cosolvent solution within single droplets that were suspended in another immiscible phase.349 As shown in Figure 43A, MOF crystallization reaction occurred in three distinct mechanisms. In a solvothermal process, droplets containing N,N-dimethylformamide (DMF) and precursors were transferred into a heating zone for a specific heating residence time, which led to the crystallization reaction. On the basis of this method, HKUST-1, MOF-5, IRMOF-3, and UiO66 were synthesized. In a high-pressure hydrothermal process, aqueous droplets containing precursors flowed through a tube where a high back-pressure was applied to prevent evaporation. This method enabled generation of Ru3BTC2 crystals. Moreover, in a stepwise process, core crystals were first generated and then merged with a shell precursor solution in the secondorder reactor downstream, where heterostructured core−shell MOFs as well as Fe3O4@ZIF-8 were synthesized. Besides, Coronas et al. applied droplet microfluidics for the synthesis of MIL-88B type MOFs with uniform angular shapes.350 By changing the residence time, the size increased and the crystals gradually grew from round to more angular shapes, as shown in Figure 43B. Overall, droplet microfluidic methods facilitated continuous synthesis of nano-MOFs with rapid mixing and increased reaction kinetics. Therefore, products were generated in high quality.

sensors, etc. In addition to organic-based synthesis, watersoluble QDs such as Ag2S336 and CdTe337 have also been obtained in W/O droplet microreactors. Nanocomposites. Nanocomposites with hybrid components could be synthesized in droplet microreactors with tailored structures and thus functions. For example, through the seedmediated approach, alloyed NPs with complex structures were generated.338 As shown in Figure 42A, by using preformed Ag

Figure 42. Droplet synthesis of QDs. (A) TEM images showing the synthesized Au−Ag nanocages (i) with and (ii) without Triton X-100. Adapted from ref 338. Copyright 2014 American Chemical Society. (B) HRTEM images of (i) Au/Ag core/shell NPs and (ii) Au/Ag/Au double-shell NPs. Adapted with permission from ref 339. Copyright 2011 Elsevier.

cubic seeds as templates, Au−Ag nanoboxes or nanocages were synthesized via galvanic replacement reaction. It is worth noting that, in the reaction process, Triton X-100 was added as a neutral surfactant. The assembly of the surfactant mitigated the adsorption of Ag nanocrystals at the droplet interfaces while not affecting the ionic synthesis reactions. Therefore, the resultant NPs were uniform in size and shape, without generation of byproducts. Besides, core−shell nanocomposites have aroused wide research interests. For example, Kanuer et al. synthesized Au−Ag core−shell as well as Au−Ag−Au double-shell nanocomposites using a two-step method, as shown in Figure 42B.339 The synthesis was also a seed-mediated process based on the reduction reaction of HAuCl4 and AgNO3 by ascorbic acid. It was confirmed that the resultant products showed a narrower size distribution and thus an improved quality of optical spectra than those in the batch synthesis. The plasmonic resonance peak position could be tuned through deposition of metal shells, which is of significance for future optical-sensing applications. Besides the hybrid metals, nanocomposites of oxides, metal-coated silica, and fully inorganic nanocrystals of cesium lead halide perovskites have also been generated in droplet reactors.340−342 5.1.2. Organic Nanoparticle Synthesis. Although NP synthesis in droplet reactors mainly focuses on inorganic materials, organic NP synthesis has also been explored considering their potential usage in pharmaceutical formulations.343 For example, Lee et al. synthesized poly(lactide-coglycolide) (PLGA) nanospheres through a droplet-based solvent evaporation and extraction process.344 A mixture of 8002


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Apart from allowing simple chemical reactions to be conducted, the droplet platform provides a microchamber for nanomaterial assembly into high-order structures, which exhibit distinct features and potential application values. By using the single-phase flow-focusing regime or gas−liquid segmented flow regime, quantum dot−block copolymer colloids were assembled into “quantum dot compound micelles” (QDCMs).351 Besides, polymer-based nanoassembly particles could also be achieved in the droplets. Visaveliya et al. reported a droplet approach for the synthesis of core−shell PMMA− poly(DADMAC) NPs.345 By using these NPs as templates, three kinds of assembly behaviors were investigated, as shown in Figure 44. The first was the in situ particle−particle nanoassembly formed through controlled aggregation during the NP growth process. Due to the flexible properties of the core and shell polymer components, the soft NPs assembled into various anisotropic morphologies, which could be adjusted by changing the reaction conditions (Figure 44A). The second was the polymer−polymer nanoassembly formed through electrostatic binding of smaller hydrophobic NPs. The cationic poly(DADMAC) shell helped to entrap the PMMA NPs with the help of an ionic surfactant SDS. By changing the SDS concentration, the size of the formed surface NPs could be tuned (Figure 44B). The third was the polymer−metal nanoassembly through electrostatic adsorption of smaller

Figure 43. Droplet synthesis of nano-MOFs. (A) Scheme of the droplet microfluidic approaches: (i) solvothermal process; (ii) hydrothermal process; (iii) stepwise process for the generation of core−shell MOFs. Adapted from ref 349. Copyright 2013 American Chemical Society. (B) TEM images of the synthesized Fe-MIL-88BNH2. (i−iv) After different residence times, the particles gradually grow from round to more angular shapes. Adapted from ref 350. Copyright 2013 American Chemical Society.

Figure 44. Droplet syntheses of nanomaterial assemblies. (A) Polymer particle nanoassembly during ongoing polymerization: (i) scheme illustration of the assembly process; (ii, iii) SEM images of the nanoassemblies obtained at different flow rate ratios of the continuous to the dispersed phases; (iv, v) particle assemblies obtained at different time intervals in the SEM instrument; (vi, vii) particle assemblies obtained at different concentrations of DADMAC. (B) Particle nanoassembly through electrostatic interfacial interactions: (i) scheme illustration of the assembly process; (ii, iii) SEM images of the nanoassemblies at different concentrations of SDS. (C) Polymer−metal nanoassembly through electrostatic adsorption: (left) scheme illustration of the assembly process; (right) SEM image of the polymer/Au assembly. Adapted with permission from ref 345. Copyright 2015 John Wiley & Sons. 8003


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Table 3. Droplet-Based Fabrication of Microparticles and Microcapsules component polymer

geometry spherical

nonspherical

Janus multicompartmental porous surface-textured particles simple capsule

colloidal

liquid crystal

supramolecular vesicle

multicore capsule multishell capsule colloidal particle clusters colloidal crystal beads inverse opals surface-patterned hollow capsule core−shell cholesteric LCs nematic LCs hollow capsule simple capsule multicompartmental vesicle-in-vesicle

fabrication process

ref

chemical polymerization physical cross-linking solvent evaporation/diffusion channel confinement nonuniform solidification tuning interfacial energy biphase dispersed flows/drop pairs phase separation/solvent evaporation multiphase dispersed flows porogens/sacrificial templates phase separation interfacial instability chemical polymerization physical cross-linking solvent evaporation/diffusion phase separation wettability control multiple inner flows multiple middle flows evaporation-induced assembly evaporation-induced assembly direct polymerization sacrificial templates sacrificial templates interfacial assembly photopolymerization evaporation-induced assembly photopolymerization photopolymerization interfacial reaction/assembly solvent extraction/evaporation dewetting stepwise emulsification

356−361, 370, 373 362, 364−367 344, 368, 369 374 375, 376 377−379 135, 384 385−387 388, 389 390−393 394, 395 396, 397 81, 139, 419, 435, 436 437−439 440, 441 428 429 143, 443, 444 144 404 87, 382, 407−409 79, 410, 411 412−414 30, 417, 418 433, 434, 448−450 451 422 423 424, 425 430−432 456−459 460, 461 462

usually bring novel features. Herein, a selective summary of droplet fabrication of microparticles as well as microcapsules (as described in section 5.3) is given in Table 3. 5.2.1. Polymer Microparticle Fabrication. Polymer microparticles have always been of great interest in various application areas.43,352 For example, they could serve as substrates or microcarriers in biochemical analysis and tissue engineering.353 In addition, by embedding various ingredients, controlled release and delivery could be realized through a stimulus-responsive volume change.80,82 Conventionally, such particles within the range of tens to hundreds of micrometers can be synthesized by shear-induced emulsification, spray drying, coacervation, etc., most of which are specific to a certain type of material choice. Moreover, these methods suffer from insufficient control over particle uniformity in size and morphology. By contrast, the droplet microfluidic technique provides a universal platform by using monodisperse droplets as templates, which are entirely converted into solid particles through photoinduced/heat-induced polymerization or ionic reactions.354,355 In addition, it is efficient and flexible to render the microparticles with precisely regulated morphologies, micro-nanostructures, and novel features. Spherical Particles. Spherical particles are synthesized straightforwardly by solidification of the droplet templates, which involves a chemical or physical reaction process. Photopolymerization is one of the most prevalent chemical

negatively charged AuNPs on the surface. In addition, such a Au-rich surface further promoted additional AgNP deposition via a metal-catalyzed process (Figure 44C). These well-defined nanostructured particles showed high potential in biomedical areas such as controlled drug delivery, SERS-based bioanalysis, etc. 5.2. Microparticles from Droplet Microfluidics

Microparticles include a large category of particulate materials at the microscale, which have found important application values in various fields. The uniformity of microparticles is a vital factor for implementation of their functionalities. Therefore, it puts forward strict requirements to the synthesis approaches. Among various fabrication methods, droplet microfluidics fits the demand better because particles are synthesized directly by using monodispersed droplets as templates, which were generated precisely one by one. The droplets are converted into solid particles via distinct chemical and physical processes such as polymerization, self-assembly, dewetting, and gelation, according to the material composition. Through intricate adjustment of flow parameters and flexible design of fluid channels, droplets and the final particles could be endowed with the desired size and shape, including spherical, nonspherical, Janus, multicompartmental, or other complex morphologies. In addition, by adding specific ingredients in the droplets, micro-nanostructures could be introduced inside the particle or on the particle surface, which 8004


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Figure 45. Droplet fabrications of spherical polymeric microparticles. (A) Particle synthesis through UV-induced photopolymerization. Adapted from ref 356. Copyright 2005 American Chemical Society. (B) Synthesis of PNIPAm microparticles through redox-initiated polymerization. Adapted with permission from ref 361. Copyright 2007 John Wiley & Sons. (C) Synthesis of alginate microparticles through the physical cross-linking process. Adapted with permission from ref 362. Copyright 2007 John Wiley & Sons. (D) Synthesis of PLA microparticles through solvent diffusion. Adapted with permission from ref 368. Copyright 2011 Royal Society of Chemistry.

gelatin, chitosan, etc.362−365 Physical gelation can be mediated by thermal treatment, through which droplets are first prepared and then solidified by changing the temperature. This can be achieved off-chip, where droplets need to be collected after preparation. Also, by exerting a temperature gradient on the microfluidic device, on-chip continuous synthesis could be realized. Besides, physical gelation can be triggered by ionic reactions and generally involves a precursor polymer and a cross-linking agent. A typical example is the synthesis of alginate hydrogel particles, which resulted from instantaneous consolidation reactions of charged polysaccharide residues in contact with divalent cations. This could be achieved in three distinct manners. For example, in a coalescence-induced method, aqueous droplets of sodium alginate and CaCl2 were first generated at two branch channels, respectively. Then they were fused downstream in the central channel, and gelation occurred.366 Apart from using droplet pairs, the precursor and cross-linking agent can also be isolated beforehand in droplets and the continuous phase, respectively, and gelation could be triggered through diffusion of the Ca2+ ions from the oil phase to the droplets.367 Instead of preisolation, there is another strategy, which is called internal gelation. As demonstrated by Takeuchi et al., the precursor of sodium alginate and cross-linking agent of CaCO3 nanoparticles were coencapsulated in a single droplet, but in the inactive state.362 Acetic acid was added downstream in the continuous corn oil phase and diffused into the droplets. The reduction of the pH value resulted in the release of the Ca2+ ions, and thus triggered the gelation reaction, as shown in Figure 45C. For some polymers that are not applicable for polymerization or gelation reactions, solvent evaporation or diffusion could be applied to facilitate droplet solidification. This mechanism has been widely implemented in organic compounds dissolved in volatile solvent droplets for the fabrication of polymer particles. For example, PLGA microspheres were synthesized in dimethyl carbonate (DMC) droplets after rapid solvent evaporation.344 The DMC solvent was chosen for its immiscibility with water and compatibility with the PDMS channel walls. Ono et al. synthesized polylactide (PLA) microspheres continuously in a

processes because it enables in situ solidification and continuous fabrication in a fast response time, which helps to determine the particle location and to better control the size distribution. For example, a UV-polymerizable hydrogel solution was emulsified into droplets within an oil carrier phase, and solid particles were synthesized through chemical cross-linking upon exposure to UV light, as shown in Figure 45A.356 The size of the microparticles could be adjusted within the range of tens to hundreds of micrometers by changing the flow rates of the dispersed and continuous phases. The generation frequency was also tunable. This method has been extensively applied to synthesis of a large variety of microparticles by using precursors with unsaturated hydrocarbon chains. By using W/O droplet templates, hydrogel particles such as polyacrylamide (PAAm) and poly(ethylene glycol) diacrylate (PEGDA) can be fabricated.357,358 Also, organic polymer resin microparticles such as ethoxylated trimethylolpropane triacrylate (ETPTA) and tripropylene glycol diacrylate (TPGDA) can also be prepared through O/W emulsion templates.359,360 In addition to photoinitiated polymerization, microparticles could be synthesized via redox-initiated polymerization. For example, poly(N-isopropylacrylamide) (PNIPAm) microparticles were fabricated in situ in a sequentially aligned device.361 Briefly, silicon oil served as the continuous phase, and the middle aqueous phase contained NIPAm monomer and other additional agents. The inner fluid was also an aqueous solution, which contained ammonium persulfate (APS) as the initiator. Once the three phases were introduced to the channel, the pregel droplets were formed and the cross-linking reaction was initiated after sufficient mixing of the middle and inner fluids at room temperature, as shown in Figure 45B. By adding comonomers, amino groups were introduced and fluorescent dyes were labeled on the particles. Microgel particles synthesized via chemical gelation are mechanically strong. However, the use of polymerization initiators, as well as UV irradiation, is nonappropriate when considering their biotoxicity and biocompatibility. In those cases, physical gelation is more applicable and has been employed to synthesize many kinds of polymer microparticles from natural biological resources including alginate, agarose, 8005


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Figure 46. Droplet generation of nonspherical polymeric microparticles. (A) TPGDA microparticles with controllable morphologies through geometrical channel confinement: (i) spherical shape; (ii) disklike shape; (iii) rodlike shape. Adapted with permission from ref 374. Copyright 2004 John Wiley & Sons. (B) Tail-shaped alginate particle formation: (left) scheme illustration of the sedimentation process after droplet emulsification; (right) SEM image of the particles. Adapted with permission from ref 375. Copyright 2013 John Wiley & Sons. (C) SEM image of the PMMA toroidal particles, generated through phase separation and nonuniform solidification. Adapted with permission from ref 376. Copyright 2009 John Wiley & Sons. (D) HDDA particles with (i) a relatively convex surface and (ii) a concave surface. Adapted with permission from ref 377. Copyright 2007 John Wiley & Sons. (E) Biconcave-structured HDDA particles with a hole in the center. Adapted with permission from ref 378. Copyright 2014 John Wiley & Sons.

solvent diffusion mechanism.368 PLA was first dissolved in ethyl acetate (EA), an organic solvent of low toxicity. Then an O/W emulsion was prepared and transferred into an excess amount of water. Due to the relatively high solubility of EA in water, it diffused outside and thus solidified the droplets, as shown in Figure 45D. By changing the flow rates as well as the PLA concentration, the particle size was adjusted from 6 to 50 μm. As these methods were based on oil droplets, some organicsoluble ingredients could be encapsulated for specific functions. In addition to the O/W system, solvent diffusion has also been conducted in the W/O emulsion system to trigger sol−gel transition. Fang et al. used this method to synthesize silica microparticles.369 Silica sol emulsion droplets were dispersed in an organic fluid phase that was immiscible with and partially soluble in water. As the water in the droplets diffused gradually into the carrier phase, gel transition was triggered due to solvent loss, thus yielding silica particles with sizes of 7−50 μm. The above-mentioned particle synthesis methods are all based on multiphase flows of an aqueous phase and an organic phase, whereas gelation reactions can also be conducted in water-in-water (W/W) droplets. In recent years, aqueous twophase systems (ATPSs) have been implemented in the droplet microfluidic platform.147,370,371 In contrast to conventional droplet generation, which involves an aqueous phase and an oil phase, the ATPS consists of two aqueous solutions that are immiscible with each other by using incompatible polymers or salts as solvents. When the concentration of the polymer exceeds a certain value, the ATPS forms as a result of phase separation.

The most prominent feature of the ATPS is the whole aqueous environment, which provides a mild surrounding to protect the biomaterials from potential denaturation or destabilization.372 However, this also brings about negative effects, as the interfacial tension between the two immiscible aqueous phases is usually extremely smaller than in the water− oil systems, which hampers spontaneous generation of droplets. Therefore, active external forces such as mechanical vibration are often required to promote droplet generation and to implement a relatively wide range of flow rates.147 The most established ATPS in droplet microfluidics is the couple of dextran and PEG. Steijn et al. employed such system for synthesizing hydrogel microparticles through mechanical piezoelectric induced generation of W/W droplets.373 The dextran was functionalized with methacrylate groups for cross-linking reactions, and N,N,N′,N′ -tetramethylethylenediamine (TEMED) was added to accelerate the reaction. In the PEGrich phase, APS was introduced as the initiator. By using piezoelectric actuation, periodic mechanical disturbances were exerted and forced the droplets to break up, and microgel particles were synthesized through radical-mediated crosslinking reactions. Besides, a PEGDA−dextran system has also been created for direct particle synthesis through photopolymerization.370 Nonspherical Particles. Nonspherical microparticles have attracted broad research interests because they provide structural anisotropy and potential functional features. However, such particles are difficult to synthesize by conventional methods, because in the natural state the droplets tend to 8006


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Figure 47. Droplet fabrications of Janus and multicompartmental polymeric particles. (A) Bicolored polymeric particles. Adapted with permission from ref 135. Copyright 2006 John Wiley & Sons. (B) Hybrid Janus particles with two lobes. Adapted with permission from ref 384. Copyright 2009 John Wiley & Sons. (C) Formation of acorn-shaped Janus particles through solvent evaporation and phase separation: (i) scheme and (ii) microscopic images of the phase separation process; (iii) CLSM images of the acorn-shaped particles; (iv) set of red, green, and overlaid CLSM images and the fluorescence intensity profile. Adapted from ref 387. Copyright 2016 American Chemical Society. (D) Multicompartmental particles with (i) four and (ii) six compartments, obtained from quintuple- and septuple-barrelled capillaries, respectively. Adapted with permission from ref 389. Copyright 2012 John Wiley & Sons.

viscous deformation and the interfacial restoring forces, which resulted in the generation of alginate microparticles with teardrop or tail shapes. By changing the alginate viscosity and the Ca2+ concentration, the size and the morphology of the particles could be tuned. Besides, toroidal polymer microparticles have been synthesized through nonuniform solidification.376 Polymer was dissolved in DMF and was emulsified into droplets. With the diffusion of the solvent, the droplets shrank, and nonuniform solidification occurred due to phase separation, thus eventually yielding toroidal-shaped solid particles, as shown in Figure 46C. Using this method, microparticles have been synthesized through different polymers, such as poly(ether sulfone) (PES), polysulfone (PSF), PMMA, poly(vinylidene fluoride) (PVDF), etc. Apart from using single droplets as templates, particles could also be derived using more than one droplet, and thus with complex shapes. For example, acornlike or sharp-edged particles can be derived using two immiscible drops by tuning the wettability properties. When the drop pairs are emulsified together in a third carrier fluid phase (immiscible with either of the dispersed phases) and come into contact, an equilibrium structure forms in accordance with the spreading coefficient values and droplet volumes. By carefully controlling the interfacial tensions between two fluid phases using surfactants, the spreading coefficients could be adjusted so that partial

maintain a spherical shape for minimizing the total interfacial energy. In droplet microfluidics, additional forces could act on the droplets and thus change their shapes. One of the most convenient approaches is to exert spatial confinement. Kumacheva et al. described a versatile strategy of synthesizing TPGDA microparticles in various shapes such as spheres, disks, and rods, by controlling the droplet diameter (ds) and the cross-section geometry (height and width, h and w, respectively) of the microfluidic channel.374 As shown in Figure 46A, when both of the geometries were smaller than ds, the droplet maintained its original spherical shape, when w > ds and h < ds, that is, the droplet flowed into a wide channel, it changed to a disk shape, and when both of the geometries were smaller than ds, the droplet was confined into a rodlike shape. By illumination with UV light, these droplets were polymerized into solid particles and their shapes were retained. In addition to the particle shape being predetermined before solidification and preserved through rapid prototyping, the particle shape can also be tailored during the solidification process by adjusting the reaction parameters. For example, Yang et al. synthesized tail-shaped alginate microparticles in a slow cross-linking process.375 As shown in Figure 46B, sodium alginate droplets were first generated in a microfluidic device and then fell from the oil carrier phase into a CaCl2 solution under gravity. During the slow sedimentation process, the droplets were deformed under the competitive effects of 8007


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acrylamide monomer was then pushed to the other side and polymerized upon UV illumination. The resultant particles possessed Janus structures with one PNIPAm-rich hemisphere and another PAAm-rich hemisphere. Ardekani et al. adopted a similar route in an alginate/PNIPAm system and synthesized Janus particles through phase separation and ionic crosslinking.386 Besides, phase separation could be induced by solvent evaporation, through which Janus particles with two polymer components have been synthesized. For example, Kim et al. studied droplet evolution into different configurations with multiple domains under phase separation, which resulted from solvent depletion.387 Briefly, a pH-responsive cationic copolymer and another biodegradable polymer were dissolved in an organic solvent at a dilution state. When the solvent evaporated, the two immiscible polymers went through phase separation and concentrated distinctively. After several stages of development, including spinodal decomposition, coalescence, and phase inversion, two large domains would eventually emerge, and the final configuration of the two domains was determined by the materials and pH value. By using PLGA and poly(butyl methacrylate-co-2-(dimethylamino)ethyl methacrylate-co-methyl methacrylate) (p(BMA-co-DAMA-co-MMA)) polymers dissolved in chloroform at pH 10, the interfacial tension between the copolymer and continuous phase was large enough to yield a negative spreading parameter. Therefore, Janus particles evolved due to a dewetting mechanism of the two domains. The resultant particles exhibited a p(BMA-coDAMA-co-MMA)-rich nut and PLGA-rich cupule geometry, as shown in Figure 47C. Multicompartmental Particles. Similar to the Janus emulsions, multicompartmental droplets could be generated by emulsifying parallel multiphase flows into droplets at the same point. By using such droplets as templates, particles could be synthesized by rapid photopolymerization and thus imparted with heterogeneous compositions and multiple functions. Also, by merging immiscible droplet clusters, multicompartmental spherical particles could also be achieved. For example, ternary particles were synthesized through a microfluidic flow-focusing device.388 In the jetting regime, ternary droplets with three parallel monomer components were formed. This resulted from hydrodynamic and thermodynamic effects, determined by the interfacial energies and the corresponding spreading parameters. The liquid thread then broke up and was exposed to in situ UV irradiation, through which the ternary configuration of the droplets was preserved, and particles were synthesized with shape edges between the different compartments. By changing the flow rates, the volume ratio of each compartment could be adjusted. Moreover, by using a centrifuge-based droplet generator, multicompartmental alginate droplets were generated through a multibarrelled capillary and then solidified by CaCl2 solution, yielding multicompartmental particles accordingly, as shown in Figure 47D.389 Other Particles. Porous particles are valuable due to their distinct structure, which is applicable for drug delivery, adsorption, sensors, et al. By using droplet microfluidic techniques, porous particles could be synthesized by introducing templates into the droplet precursors and then removing them after solidification, thus generating void spaces in the particles. For example, Chu et al. synthesized open-cell porous PNIPAm microgel particles by using tiny oil drops as porogens.390 The oil drops were first embedded in the aqueous phase of NIPAm monomers. Such a mixture was then

engulfment between the two dispersed phases occurs in compliance with the minimum total interfacial energy. Therefore, the drop pairs form a dumbbell- or acorn-shaped configuration. By selective polymerization of one of the droplet pairs, particles with sharp edges could be synthesized. For example, Nisisako et al. constructed a triphase microfluidic system composed of photocurable monomer 1,6-hexanediol diacrylate (HDDA), a silicone oil phase, and an aqueous phase.377 Biphasic droplets were generated downstream at equilibrium, and microparticles were synthesized by photopolymerization of the monomer, as shown in Figure 46D. It was demonstrated that, with increasing fraction of the HDDA monomer, the particle shape varied from convex to planar and further became concave. Later, similar research was conducted by selective photopolymerization of ternary emulsion droplets.378 The acrylate monomer segment was sandwiched by two noncurable silicone oil segments from both sides, and was then polymerized to give microlens particles with a biconcave structure. By changing the relative flow rates of the three dispersed phases, the shape of the particles could be adjusted to be asymmetric or even to have a hole in the center, as shown in Figure 46E. Besides, Chu et al. used nanoparticle surface coating to control the interfacial properties, through which microparticles with an acorn-shaped configuration were obtained.379 Janus Particles. Janus particles, named after the ancient twofaced Roman god Janus, refer to particles with two hemispheres of different compositions.380,381 Such structural asymmetry brings about combination of physical and chemical features and thus functionalities. Janus particles have received increasing attention due to their application values in various areas. On the basis of the droplet microfluidic technique, a large variety of Janus particles have been fabricated with various aspects of anisotropies in color, electrical properties, magnetic properties, wettability, etc.79,135,382,383 Janus droplets serve as templates for direct synthesis of Janus particles through photoinduced or heat-induced polymerization, as well as ionic cross-linking. For example, bicolored microparticles were generated from Janus O/W droplets with two acrylic monomer phases containing pigments of carbon black and titanium oxide, respectively.135 By using parallel branch channels, biphase flows could be introduced independently and merged at a junction, after which they were emulsified simultaneously in the main channel to generate Janus droplets. Through thermally triggered polymerization, spherical Janus particles were yielded with black and white hemispheres, as shown in Figure 47A. Apart from solidifying droplets with two miscible components, Janus particles could also be derived from immiscible drop pairs that form a dumbbell- or acorn-shaped configuration.384 By polymerizing the entire droplets, hybrid particles with Janus features such as hydrophobic and hydrophilic properties were synthesized, as shown in Figure 47B. In addition to directly using heterogeneous droplets as templates, homogeneous emulsion droplets could be employed to generate Janus particles through phase separation. For example, PNIPAm−PAAm particles were synthesized through phase separation.385 An aqueous PNIPAm nanoparticle (about 500 nm in diameter) suspension containing acrylamide was first emulsified into droplets, together with a cross-linker and a photoinitiator. When heated above its transition temperature (32 °C), the PNIPAm hydrogel became hydrophobic and shrank to aggregate on one side of the droplets, due to a thermoresponsive volume-phase transition mechanism. The 8008


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Figure 48. (A) Droplet fabrication of porous PNIPAm microgels by using tiny oil drops as porogens: (left) scheme illustration; (right) SEM image showing the open-celled porous structure. Adapted from ref 390. Copyright 2014 American Chemical Society. (B) Porous NaPSS particle formation through solvent extraction and precipitation: (left) scheme illustration; (right) SEM image of the NaPSS particle. Adapted from ref 394. Copyright 2014 American Chemical Society. (C) SEM and TEM (inset) images of (i) budding particles and (ii) dendritic particles. Adapted with permission from ref 396. Copyright 2012 John Wiley & Sons. (D) SEM images of Janus particles with a wrinkled surface on (i) one side and (ii) both sides. Adapted with permission from ref 397. Copyright 2008 John Wiley & Sons.

precipitation. Finally, solid particles were yielded with a smooth surface and porous inner structures, as shown in Figure 48B. By conducting model analysis, the mechanism of pore generation was described as a result of the interactions between coarsening of the phase separation domains and the constrained solvent removal by the polymer skin. In addition, surface porous particles were also synthesized by phase separation. As demonstrated by Ono et al., droplets containing PLA, perfluorooctyl bromide (PFOB), and EA were first generated in an aqueous carrier phase. Then the droplets were transferred into water, and the EA solvent was extracted out. As PLA was insoluble in PFOB and soluble in EA, internal phase separation occurred and PFOB drop domains formed. At higher viscosity of the PLA solution, slow migration of the PFOB drops eventually led to precipitation on the droplet surface. Finally, the PFOB was eliminated through freeze-drying, and the resultant particles were decorated with a dimpled surface.395 Particles with surface micro/nanopatterns or roughness are promising as surface coatings, building blocks for self-assembly, and regulation of cell adhesion and growth through material− biological interactions. Such particles can be synthesized by droplet methods through interfacial instability mechanisms. For example, Zhu et al. prepared hierarchically structured microparticles by using O/W droplets containing organic solvent and amphiphilic block copolymers polystyrene−poly(ethylene oxide) (PS−PEO).396 After diffusion and evaporation of the solvent, the droplets shrank and the polymer was concentrated with increasing interfacial instability. Therefore, the droplets would no longer remain spherical. By using different compositions of the polymers, particles with a surface coating of buddings or dendritic surfaces were produced, as shown in

emulsified to produce W/O emulsion droplets, which were polymerized upon UV irradiation. Afterward, by adding 2propanol or by increasing the temperature, the embedded oil drops were squeezed out in response to volume shrinkage of the PNIPAm microgel, as shown in Figure 48A. The resultant microgel particles were accommodated with a large number of interconnected free channels and thus showed enhanced thermal response rates compared with normal hydrogels. Also, by using PS solid microbeads as sacrificial templates, hydrogel microparticles with spherical voids were synthesized after removal of the beads by using organic solvents.391 In addition, gas bubbles were also explored to serve as porogens. As described by Stone et al., gas-in-water-in-oil emulsions were first generated, and each droplet contained many bubbles by adjusting the flow rates and the gas pressure. Then porous particles were achieved by photopolymerization of the aqueous drops.392 Instead of direct injection of gas bubbles, Wen et al. employed another strategy of generating gas voids by post chemical reaction.393 They embedded hydrogen peroxide drops in the polymer monomer droplets. The liquid precursor polymerized, and H2O2 decomposed to release oxygen with UV irradiation, thus generating porous particles. Phase separation within droplets is another way of synthesizing porous particles. Cabral et al. prepared porous polyelectrolyte sodium poly(styrenesulfonate) (NaPSS) particles by solvent extraction.394 Briefly, droplets containing polymer and a solvent, S1, were generated and then transferred into another solvent, S2, in an external bath off-chip. S2 was selected to be miscible with S1 but immiscible with the polymer. The extraction of S1 into S2 thus induced internal phase separation in the droplets and caused polymer 8009


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Figure 49. Droplet-based generation of colloidal crystal beads. (A) CCB generation through evaporation-induced crystallization: (i) scheme illustration; (ii) SEM image of the bead surface; (iii) reflection spectra of different CCBs composed of silica NPs with different sizes. Adapted from ref 87. Copyright 2008 American Chemical Society. (B) Generation of magnetoresponsive Janus CCBs: (i) scheme illustration; (ii−iv) microscopic images of three kinds of particles with distinct diffraction colors. Adapted with permission from ref 382. Copyright 2013 Royal Society of Chemistry. (C) Molecular-analogue colloidal crystal clusters. “A” and “B” depict the magnetic part and colloidal crystal part of the Janus particles (the building blocks), respectively. Adapted with permission from ref 408. Copyright 2016 Royal Society of Chemistry. (D) Nonspherical CCBs derived from gas and colloidal suspension drop pairs. Adapted with permission from ref 409. Copyright 2014 Royal Society of Chemistry.

particles into ordered superstructures, including colloidal clusters composed of a finite number of particles and 3D spherical colloidal crystals with internal lattice arrangements. Remarkably, by using colloidal nanoparticles as building blocks, sacrificial templates, or interfacial stabilizers, colloidal crystal beads, inverse opals, and particles with surface patterns could be synthesized in a highly controlled way. Colloidal Particle Clusters. Colloidal assembly of a finite number of nanoparticles in droplets was studied by Pine et al. in an O/W emulsion system.403 Small numbers of hard nanospheres were first dispersed in an organic solvent and emulsified into an O/W emulsion. Due to surface tension effects, the nanospheres were attached and bound to the droplet interfaces. With the evaporation of the solvent, the generated capillary forces could pack the spheres into distinct polyhedral configurations, in compliance with the minimization principle of the second moment of mass distribution. They also applied a similar process in a microfluidic droplet platform and synthesized uniform colloidal assemblies with a narrow size distribution.404 W/O emulsions were first generated containing latex spheres. Then, as the droplets traveled along the microfluidic channel, the inner water was gradually absorbed by the oil phase, and the spheres were concentrated and eventually formed an ordered assembly. It was also observed that when the number of encapsulated spheres was relatively large, the uniformity of the resultant cluster was enhanced. Colloidal Crystal Beads. A colloidal crystal is a kind of 3D ordered superstructural material assembled through monodisperse colloidal nanoparticles. It is specially attractive because it represents an efficient bottom-up method for fabricating photonic crystals as a mimic of natural structural color materials. By using colloidal particles with size comparable to visible light wavelengths, a photonic band gap (PBG) would

Figure 48C. Later, the authors reported synthesis of surfacetextured polymer microparticles in a highly controllable way by adding 1-hexadecanol (HD) in the dispersed phase of the SDSstabilized O/W emulsion system, acting as a cosurfactant. Along with solvent removal, the presence of HD reduced the interfacial tension and caused SDS rearrangement by accumulation and interpenetration at the droplet interface. Therefore, the interfacial areas increased by generating folds and wrinkles due to large viscosities of the droplets. The resultant particles possessed textured surfaces, and the surface roughness could be accurately tailored by adjusting the HD concentration and the solvent evaporation rate.397 Moreover, by using immiscible polymer blends, Janus particles with tunable roughness on one side or on both sides were also fabricated, as shown in Figure 48D. In other attempts, for example, Cao et al. induced rapid hydrolysis of titanium tetraisopropoxide (TTIP) in a polymer droplets and synthesized PLGA/TiO2 hybrid microspheres with surface wrinkles.398 5.2.2. Colloidal Material Assembly. Self-assembly represents a unique method for bottom-up fabrication of materials. Through physical-chemical interactions between the basic building blocks, hierarchically ordered constructs with designed structures could be achieved.399 Such materials provide enhanced performance compared to the the unstructured counterparts and often bring about novel properties as well as functions.316,400,401 Colloidal particles are attractive building blocks because they could be synthesized with tunable size, uniformity, and surface decoration. In addition, their selfassembly process is relatively convenient, without using complex nanofabrication methods. Among various techniques for colloidal assembly, microfluidic droplets provide a geometric confinement402 to induce the organization of colloidal 8010


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Figure 50. (A) Droplet-based fabrication of non-close-packed CCBs. Adapted with permission from ref 410. Copyright 2009 Royal Society of Chemistry. (B) Multicompartmental CCBs: (i) four-compartmental particles; (ii, (iii) three-compartmental particles; (iv−vi) six-compartmental particles. Adapted from ref 79. Copyright 2013 American Chemical Society. (C) Nonspherical, anisotropic CCBs: (i) rodlike shape; (ii) cuboidlike shape; (iii) disklike shape. Adapted with permission from ref 411. Copyright 2014 Elsevier.

prepared by combining phase separation with evaporationinduced crystallization.382 As shown in Figure 49B, aqueous droplets containing silica and ultrafine magnetic nanoparticles were first generated and then dried under a magnetic field. During this process, the silica nanoparticles assembled symmetrically into a close-packed colloidal crystal structure, while the magnetic nanoparticles deposited unidirectionally into the bottom and infiltrated into the interstitial spaces between silica nanoparticles. The resultant particles possessed Janus structures and facilitated structural coloration as well as magnetically controllable movement. Besides, Janus CCBs could also be achieved in a microfluidic system with ternary immiscible phases. As demonstrated by Chen et al., an aqueous phase of PS suspension and an organic phase of photocurable monomer with magnetic NPs were emulsified to form stable biphasic droplets by adjusting the interfacial tensions.407 Then the PS compartment assembled into colloidal crystals via solvent evaporation, and the organic compartment was solidified upon UV irradiation, therefore generating magnetic Janus colloidal crystal particles. They further used such Janus building blocks to construct molecular-analogue polyhedral colloidal crystal particles by magnetically directed coalescence, as shown in Figure 49C.408 In recent years, a number of studies have been focused on preparing anisotropic colloidal crystals, for example, bowl-like particles, by solution-phase methods. Such particles could also be derived from the droplet microfluidic method. As demonstrated by Xu et al, nonspherical CCBs were derived from gas and colloidal suspension drop pairs.409 The attachment of a gas bubble on the liquid droplets generated a triphase interface and caused a “coffee-ring” effect, through which a strong concentration area was created that enhanced the quality of the colloidal assembly. By changing the flow rates, the relative volume of the gas bubble and colloidal suspension drop could be adjusted. After solvent extraction, the bubble was divided, and solid colloidal crystal particles were yielded in bowl-like or tomato-like shapes, as shown in Figure 49D. In addition to evaporation-induced crystallization, CCBs could also be synthesized by direct polymerization of droplets containing ordered nanoparticle crystallization arrays. A typical process is depicted in Figure 50A, where charged nanoparticles were first dispersed in a photocurable fluid phase, either an

arise due to the periodic modulation of the refractive index between the nanoparticles and the medium.405 Light of a certain wavelength located in this gap would be prohibited from propagating and thus would be reflected. Such features are extremely applicable in optoelectronic as well as biomedical application areas.406 The droplet microfluidic technique enables synthesis of spherical colloidal crystal materials, or colloidal crystal beads (CCBs), by exerting spherical confinement on the colloidal assembly process, which provides unique curvature elements and thus distinct optical phenomena.32,40 Generally, the most popular way of fabricating CCBs using droplet templates is evaporation-induced crystallization. As shown in Figure 49A, silica nanoparticles were first dispersed in an aqueous solution and then emulsified within an oil carrier phase to produce monodispersed droplets.87 After this, the droplets were transferred into a collection container with silicon oil for incubation. The evaporation of water caused concentration of the colloidal nanoparticles, and the capillarity provided a spherically symmetrical compressive force for particle arrangement. When the water was completely removed, the nanoparticles assembled into a close-packed pattern and stuck together by van der Waals attractions. To enhance the strength, a thermal sintering step was added so that the nanoparticles were slightly fused to be bridged together. The CCBs possessed an internal face-centered-cubic (fcc) arrangement and showed iridescent color corresponding to a characteristic reflection peak. According to the Bragg equation for a normal incident light on the (111) plane of the fcc structure, the main reflection peak position λ is estimated as follows:

λ = 1.633dnaverage where d represents the center-to-center distance between neighboring particles and naverage depicts the average refractive index. Therefore, by using silica particles of different diameters, a series of CCBs with different reflection peak positions were synthesized. In addition, by changing the flow rates and the initial silica concentration, the size of the CCBs could be adjusted. An the basis of this mechanism, CCBs with complex shapes have also been synthesized. For example, Janus CCBs with an anisotropic PBG structure and magnetic property were 8011


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Figure 51. Droplet-based syntheses of inverse opals. (A) Hydrogel-embedded inverse opal microspheres. Adapted with permission from ref 412. Copyright 2016 Royal Society of Chemistry. (B) Inverse opal spheres generated through evaporation-induced coassembly. Adapted with permission from ref 413. Copyright 2008 John Wiley & Sons. (C) Hybrid particles composed of a colloidal crystal core and an inverse opal shell through partial etching: (i) scheme illustration of the generation process; (ii−iv) three kinds of hybrid particles with distinct diffraction colors. Adapted with permission from ref 414. Copyright 2014 John Wiley & Sons.

flowed into the downstream channel with geometric confinement. By using cylindrical, square, or flat capillaries, rod-, cuboid-, or disklike droplets were formed, respectively. After in situ UV polymerization, the corresponding colloidal crystal particles were generated, as shown in Figure 50C. The rodlike particles showed an angle-independent optical property along the minor axes, due to their cylindrical symmetry. Inverse Opals. An inverse opal is a kind of photonic crystal material with distinct lattice topologies composed of periodically interconnected air holes. Compared with other photonic crystals, it is distinguished by high porosity, which is highly applicable for use in sorption and controlled release, catalysis, sensors, etc. Assembled colloidal nanoparticles could serve as sacrificial templates for synthesis of inverse opals, during which their periodic arrangement structure can be replicated. As shown in Figure 51A, for example, silica CCBs were first generated in droplet microfluidics through evaporation-induced crystallization. Then the CCBs were immersed into a pregel solution, which filled into the voids by capillary force, and were then polymerized upon UV irradiation. After this, the silica template was removed by the hydrofluoric acid etching process. The resultant inverse-opaline photonic beads showed iridescent colors and an ordered, interconnected porous structure.412 Instead of postperfusion of the hydrogel matrix as a replica, a coassembly method was put forward which is based on concurrent filling of the framework materials in the interstitial sites during CCB formation. For example, aqueous droplets of the mixture of PS nanoparticles and ultrafine silica nanoparticles (with size much smaller than the free voids) were first generated. After evaporation-induced coassembly, the PS nanoparticles formed ordered lattices, and the silica nanoparticles infiltrated into the void spaces. Finally, the PS was removed after calcination, and the inverse opal beads were generated, whose refection peaks depended on the size of the templating PS nanoparticles, as shown in Figure 51B.413 Apart from homogeneous inverse opal particles, hybrid counterparts with unique core−shell structures were also synthesized by

aqueous hydrogel solution or an oil resin, with high concentration. Due to the long-range attraction and electrostatic repulsion, the nanoparticles would self-assemble into a non-close-packed colloidal crystal array (CCA) structure, in compliance with the minimum energy configuration. Like the close-packed regime, such a non-close-packed arrangement also gave rise to a PBG property, and the characteristic reflection peak was determined by the refractive indexes of the hybrid components, as well as the nanoparticle concentration. The CCA suspension was then emulsified into droplets and polymerized rapidly upon UV illumination to generate solid CCBs, so that the periodic arrangement of the nanoparticles was permanently locked within the solid medium.410 Compared with the liquid environment, the stability of CCAs after polymerization was largely enhanced because it no longer relied on the electrostatic interactions, and they were therefore more resistant to external disturbances such as shocks or ionic impurities. Due to fast locking of the CCAs, this method enables generation of CCBs with complex morphologies by directly copying that of the droplet templates. For example, Zhao et al. fabricated Janus or multicompartmental CCBs using O/W droplet templates.79 Silica nanoparticles were first dispersed in an ETPTA resin to form a CCA suspension. By using different sizes of the silica particles at different concentrations, three kinds of CCA suspensions were prepared. These three phases, together with a fourth magnetically tagged phase, were introduced in parallel into a multibarrelled injection microfluidic device. By changing the flow rates of each phase, Janus or multicompartmental drops with controllable volume ratios were generated, and the corresponding CCBs were obtained through photopolymerization, as shown in Figure 50B. In addition, nonspherical colloidal crystal particles were generated from W/O droplet templates by exerting spatial confinement in the channels.411 Silica nanoparticles were dispersed into an aqueous solution of PEGDA precursor in high concentration. The formed CCA suspension was emulsified into droplets and 8012


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Figure 52. (A) Droplet-based preparation of hierarchically structured microspheres with silver-decorated hexagonal silica arrays on the surface. Adapted with permission from ref 417. Copyright 2010 Royal Society of Chemistry. (B) Droplet-based preparation of microparticles with dimpled surface morphology. Adapted with permission from ref 418. Copyright 2009 IOP Publishing.

Figure 53. Droplet-based fabrications of liquid crystalline particles. (A) Scheme of the chiral nematic structure. The helical pitch P corresponds to the repeating distance of a full 360° rotation along the axis of the mesogens. Adapted with permission from ref 420. Copyright 2014 John Wiley & Sons. (B) CNC self-organization under droplet confinement: (i) polarization microscopic image of the microfluidic generation of CNC droplets in oil carrier fluid; (ii) evaporation process of the CNC droplet; (iii) SEM image of a dry, buckled CNC particle; (iv) SEM image of the helicoidal assembly of the CNC.422 Adapted from ref 422. Copyright 2016 American Chemical Society. (C) Microscopic image of the polymerized CLC microparticles. Adapted with permission from ref 423. Copyright 2011 Royal Society of Chemistry. (D) Schematic illustrations and bright-field (BF) and polarized light (PL) images of spindle-shaped, spherical, spherocylindrical, and tear-shaped polymeric microparticles fabricated by using LC droplets as templates. Adapted from ref 425. Copyright 2017 American Chemical Society.

partial replication of the CCBs.414 In a similar process described above, an inadequate outside-in etching of the hydrogel-filled CCBs would give rise to hybrid particles composed of a colloidal crystal core and an inverse opal shell, as shown in Figure 51C. Due to the differentiation of the relative refractive indexes, the resultant hybrid particles had double reflection peaks. Surface-Patterned Particles. Colloidal nanoparticles could adsorb at the droplet interfaces to generate solid-particlestabilized emulsions, also known as Pickering emulsions.415 The colloidal particles spontaneously migrated to and irreversibly anchored on the interface due to significant reduction of the interfacial energy, which prevented thermal motion of the nanoparticles. Such particle-coated droplets have been demonstrated to facilitate fabrication of anisotropic supercolloidal particles with designed shapes through arrested coalescence.416 Also, they could serve as templates for

synthesizing particles with surface patterns. Moreover, by additional treatments, the particles could be tailored with more complex surface morphologies as well as hierarchical structural features. As shown in Figure 52A, for example, when silica-NPsuspended ETPTA fluid was emulsified into droplets, the silica particles protruded through the water−oil interface and assembled into a hexagonal array, due to the interfacial energy minimization mechanism.417 Under actions of electrostatic repulsions originating from the negative surface charge, the silica particles formed a non-close-packed pattern. Then the droplets were solidified under UV irradiation, thus generating solid microparticles with highly ordered surface patterns. Furthermore, by performing a silver mirror reaction, silver nanoparticles were selectively decorated on the silica arrays and the microparticles were imparted with hierarchical surface nanopatterns. This structure contributed to a large density of hot spots and high mobility of the microparticles. Besides, 8013


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Figure 54. (A) Phase diagram and schematic representation of the phase-separation cycles. Adapted with permission from ref 428. Copyright 2014 John Wiley & Sons. (B) Wetting-induced generation of double emulsions and microcapsules: (i) scheme illustration of the engulfing mechanism; (ii) scheme illustration of the formation of B/A1/C (W/O/O) and A2/B/C (O/W/O) double emulsions; (iii, (iv) SEM images of the microcapsules with (iii) an ultrathin shell and (iv) a relatively thick shell. Adapted with permission from ref 429. Copyright 2013 Royal Society of Chemistry.

work so that the cholesteric arrangement was fixed. Thus, the resultant particles had an opalescent appearance, as shown in Figure 53C. In addition to cholesteric LCs, by employing photocurable nematic LC monomers as the dispersed phase, liquid crystalline elastomeric particles, which combine the polymeric elasticity and LC self-organization properties, could be directly fabricated. With temperature variations, the particle shape would change reversibly due to phase transition, and therefore, these particles might serve as promising sensors and actuators.424 Wang et al. used nematic LC phase droplets as templates for polymerization reactions, through which polymer microparticles with different shapes were also synthesized.425 Droplets containing a mixture of reactive and nonreactive mesogens were first generated. By adjusting the surface anchoring through surfactant adsorption, the LC droplets showed various configurations of internal optical signatures, including bipolar, axial, preradial, and radial, with respect to different energy levels. Then the droplets were photopolymerized to confine the LC ordering in a cross-linked polymer network without significant perturbations. Afterward, the nonreactive mesogen was removed by ethanol, during which the polymerized LC droplets shrank anisotropically, yielding solid particles with complex geometries, including spindle, spherical, spherocylindrical, and tear shapes, as demonstrated in the bright-field and polarized light micrographs in Figure 53D.

microparticles with dimpled surfaces could be created by removal of the surface colloidal nanoparticles from the ETPTA matrix through wet-etching, thus leaving ordered arrays of cavities, as shown in Figure 52B. The resultant golf-ball-like particles were promising for interfacial wettability research.418 Cholesteric Liquid Crystalline Particles. Liquid crystals (LCs) are materials in a state combining the long-range order of crystal structures and the mobility of an isotropic liquid. LCs are highly polymorphic between different phase behaviors, depending on the molecular shape and the packing pattern of the mesogens. Among the different kinds of LCs, cholesteric liquid crystals (CLCs) are a fantastic phase and are often regarded as one-dimensional photonic crystals, because they exhibit a PBG structure due to the helical structure.419 As shown in Figure 53A, when the half-value of the helical pitch length (P) is comparable to the wavelength of visible light, the CLCs will exhibit opalescent colors.420 Circularly polarized light within the PBG would be selectively reflected. The characteristic reflection peak is determined by the P value and the refractive index. It has been demonstrated that, by using rodlike rigid particles, especially cellulose derivatives, as mesogenic units, CLCs could be derived through an evaporation-induced self-assembly process.420,421 Microfluidic droplets provide spherical confinement, which interacts with the assembly process in a way different from that of the planar geometry. Vignolini et al. monitored the self-organization of cellulose nanocrystals (CNCs) into CLC architectures under polarized optical microscopy.422 Aqueous droplets of diluted CNC suspensions were generated in an oil phase, during which an immediate radial ordering of CNCs occurred, as shown in Figure 53B. With the gradual water evaporation, a cholesteric shell formed outside-in, and buckled CNC microparticles were generated eventually after complete water loss. The cholesteric nature was maintained, as confirmed by the SEM image. Zentel et al. applied another droplet platform for fabricating CLC microparticles in combination with a photopolymerization process.423 Briefly, a lyotropic mixture of photocurable monomer and cellulose tricarbanilates was emulsified into O/ W droplets. The droplets flowed through a hot zone for temperature control and were irradiated by in situ UV light. The photopolymerization resulted in a semi-interpenetrating net-

5.3. Microcapsules from Droplet Microfluidics

Microcapsules are microscale materials composed of solid, liquid, or gas bubbles surrounded by a shell or coating. Due to its unique core−shell structure, and the high flexibility of material selection, microcapsules could be imparted with diverse properties and functionalities such as efficient encapsulation, controlled release, mass transfer, mechanical response, etc. Therefore, they have been extensively applied in an enormous range of areas, including optoelectronics, biomedicine, and pharmaceutical, foodstuff, and cosmetics industries.40,83,426 In conventional approaches such as shearinduced sequential emulsification, microcapsules are fabricated with polydispersity in size and shape, which is unfavorable for 8014


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Figure 55. (A) Droplet-based interfacial synthesis of hollow MOF capsules: (left) scheme illustration of the synthesis mechanism; (right) SEM image of the cross-sectional view of the capsule shell. The inset depicts the complete capsules. Adapted with permission from ref 430. Copyright 2011 Nature Publishing Group. (B) Droplet-based synthesis of supramolecular hollow microcapsules: (i) schematic illustration of the droplet generation process; (ii) schematic representation of the microcapsule formation in a dehydration-induced host−guest supramolecular assembly process. Adapted with permission from ref 432. Copyright 2012 The American Association for the Advancement of Science. (C) Droplet-based generation of magnetic hollow capsules. The sequential fluorescence images show the double-emulsion formation by self-emulsification and phase separation. Adapted from ref 434. Copyright 2016 American Chemical Society.

practical applications such as release kinetic research.427 Droplet microfluidics overcomes this dilemma due to its ability to fabricate uniform emulsion droplets with precisely controlled size and morphology. In a single-emulsion system, microcapsules could be derived through phase separation, wettability control, interfacial reaction, and assembly. Also, microcapsules could be conveniently derived from multiple emulsion templates via different shell solidification processes such as triggered polymerization, solvent evaporation, phase transition, and dewetting. Since a large variety of substances could be employed as the core and/or shell compositions, and many ingredients could be incorporated, such microcapsules could be tailored for specific usages. 5.3.1. Microcapsules from Single Emulsions. Phase Separation. As described above, internal physical processes, such as phase separation and wetting control, can act to remodel droplets into complex configurations.377,384,387 We herein focus on the emergence of core−shell configurations in this principle. Phase separation of a homogeneous droplet into high-order multiple emulsions has been studied by Haase et al.428 A ternary mixture of ethanol, water, and an oil, diethyl phthalate (DEP), was emulsified into an aqueous solution of a hydropholic surfactant. Internal phase separation occurred due to mass transfer, and the droplets went through compositional evolution. As mapped on the phase diagram in Figure 54A, selfsimilar evolution cycles occurred, starting with mass transfer, going through spinodal decomposition or nucleation, and finally coalescence into multiple layers. Also, by adding different ingredients, polymer microcapsules or vesicles were fabricated using such onion-like emulsion droplets as templates through solvent evaporation. This method provided an alternative way of generating microcapsules without using complex channels or constructing specific flow fields. Droplet Wettability. In addition to phase separation, wettability control provides another way of generating multiple emulsions from immiscible drop pairs and thus could be used for microcapsule fabrication. As shown in Figure 54B, when

two immiscible droplets, A and B, were suspended in a third carrier fluid, C, A would completely engulf B when the spreading coefficient SAB was greater than 0. Conversely, B would completely engulf A when SBA was greater than 0.429 Therefore, by adjusting the interfacial energies through addition of surfactants or other reagents, double emulsions were prepared and microcapsules were fabricated after photopolymerization. In addition, by changing the relative size of the precursor droplet pairs, the shell thickness of the capsules could also be adjusted. Interfacial Reaction and Assembly. Reactions occurring on droplet interfaces enable fabrication of capsules directly from single-emulsion templates. This is generally accomplished by using two reactive reagents, each dissolved in the dispersed phase and the continuous phase, respectively. One of the most representative cases is the synthesis of hollow MOF capsules.430,431 As reported by Vos and co-workers, the organic and inorganic precursors were first dissolved in two immiscible solvents.430 Then, when the droplets interfaces were generated in the microfluidic device, the metal ions and the ligands approached the growing MOF layer from the inner and outer sides in opposite directions, thus allowing for a self-completing growth of the MOF membrane into a 3D spherical configuration. During the synthesis process, the gaps between larger crystals could be self-sealed by newly formed crystallites due to faster rates of diffusion and reaction in such defect sites. Therefore, the resultant MOF film was uniform and defect-free, as shown in Figure 55A. Such hollow microcapsules were highly promising for use as microreactors, taking advantage of the porous feature of the MOF membranes. Moreover, as the reaction conditions were mild, encapsulation of biocompatible ingredients, such as enzymes, was realized. In addition, Abell et al. reported supramolecular hollow microcapsule fabrication in microfluidic droplet templates that exploited a host−guest system.432 Aqueous droplets were generated, which contained Cucurbit[8]uril (CB[8]) as the host, AuNPs modified with methyl viologen (MV2+) as the 8015


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Figure 56. (A) Droplet generation of polymeric-shell microcapsules with controllable core structures: (i, ii) formation of double emulsions with (i) two separate cores and (ii) a single Janus core; (iii, iv) polymerized particles with (iii) two separate cores of QDs and magnetic NPs and (iv) a single Janus core. Adapted from ref 443. Copyright 2011 American Chemical Society. (B) Rod-shaped microcapsules with multiple cores of different numbers and compositions: (i−iii) microscopic images; (iv) SEM image. Adapted with permission from ref 143. Copyright 2012 Nature Publishing Group. (C) Microcapsules with an interconnected microporous structure: (i, ii) microscopic and (iii, iv) SEM images of particles with three and four pores. Adapted from ref 444. Copyright 2015 American Chemical Society. (D) Microcapsules with surface nanopatterns: (i) scheme of the surfacepatterned colloidal silica arrays and surface functionalization capacity; (ii) SEM image of the outer surface; (iii) SEM image of the core−shell interface. Adapted with permission from ref 30. Copyright 2010 John Wiley & Sons.

electron-deficient first guest, and a water-soluble copolymer modified with naphthol (Np) as the electron-rich second guest. The droplets flowed through a winding channel for thorough mixing. A ternary complex was formed between the host molecule and the guest compounds through noncovalent interactions in the water environment. In a subsequent dehydration process, AuNPs and the copolymer self-assembled at the droplet interface into a network held together by the CB[8] ternary complexes, as shown in Figure 55B. Besides, colloidal assembly was explored to synthesize hollow colloidal photonic crystal capsules by combining microfluidic droplet formation and solvent extraction, which drove fast migration and assembly of the colloidal nanoparticles onto the droplet interfaces, with the presence of an appropriate extractant.433 Yao et al. applied another strategy of selfassembly, which was induced by combining the effects of solvent evaporation and phase separation.434 A mixture of dichloromethane, oleic acid-coated magnetic nanoparticles (MNPs), and PEG was emulsified into O/W droplets in the microfluidic device. Then a water core gradually formed, originating from reversed tiny droplets, as demonstrated by the fluorescent images in Figure 55C. This process resulted from self-emulsification and temperature-assisted phase separation. Afterward, dichloromethane evaporated slowly, and the MNPs aggregated to form a shell due to interparticle forces, yielding hollow spheres with solid walls. Such particles would fully capitalize on the geometric core−shell feature and the functional properties of MNPs. 5.3.2. Microcapsules from Multiple Emulsions. Polymer Shell Microcapsules. Microcapsules with conserved solid

shells could be derived by solidifying the shell of the multiple emulsions, especially double emulsions. The basic solidification approaches include polymerization,356 cross-linking,362 phase transition,385 self-assembly,382 etc., which have been described above. Here, some representative examples are listed to show that these methods are suitable for specific shell material compositions. For photocurable monomers, including some polymers and gels, photopolymerization is the most convenient method, through which a large variety of microcapsules have been created. By using W/O/W emulsions, capsules with hydrogel shells such as PEGDA419 and NIPAm139 have been widely explored. In addition, capsules with resin shells such as ETPTA,81 TPGDA,435 and HDDA436 can be derived from O/ W/O emulsions. By changing the flow rates, the sizes of the core and the shell, as well as the core numbers, could be adjusted. For gels or other polymers that are not photocurable, cross-linking reaction, electrostatic attraction, or evaporationinduced precipitation of the shell materials is performed to generate microcapsules. For example, O/W/O droplets containing sodium alginate in the shell were converted into microcapsules when they contacted calcium chloride solution in the downstream channel.437 Chitosan capsules could be fabricated by cross-linking with glutaraldehyde that was present in the inner and outer fluids.438 Polyelectrolyte (PE) microcapsules have recently been prepared through water-in-waterin-water (W/W/W) double emulsions by electrostatic attraction between oppositely charged polymers (PE+ and PE−) present in the shell and continuous fluid, respectively.439 PLA440 and PLGA441 capsules could be fabricated by removal of organic solvents such as chloroform and toluene. For some 8016


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Figure 57. Droplet fabrication of colloidal shell microcapsules. (A) Double-emulsion-templated colloidosome formation and SEM image of dried colloidosomes. The inset shows the colloidosome surface at higher magnification. Adapted with permission from ref 448. Copyright 2008 John Wiley & Sons. (B) Multicompartmental nonspherical colloidosome generation and SEM images of the colloidosomes with five and six internal voids. Adapted with permission from ref 449. Copyright 2009 John Wiley & Sons. (C) “Armored bubbles” with a surface coating of packed particles: (i) scheme of the microfluidic bubble generator; (ii) bubble generation and particle concentration; (iii, iv) microscopic images of the armored bubbles; (v) SEM image of the shell of the armored bubble. Adapted with permission from ref 450. Copyright 2009 John Wiley & Sons. (D) Microcapsules with a gel-immobilized CCA shell: (i) scheme illustration of the generation process; (ii−iv) gel-immobilized CCA shells with different colloidal particle concentrations and diffraction colors. Adapted with permission from ref 451. Copyright 2010 John Wiley & Sons.

formation process, yielding microcapsules with porous shells, hollow cores, or other anisotropic structures.445,446 Colloidal Shell Microcapsules. Microcapsules with this structure could be synthesized by inducing colloidal particle assembly in the shell. The most representative case is colloidosomes, which refer to hollow capsules with a shell of densely packed particles.447 For example, by dispersing silica nanoparticles in the middle phase during the generation of W/ O/W emulsions, the nanoparticles adsorbed at both the inner and the outer interfaces, playing a role in droplet stabilization. Then, after solvent removal, colloidosomes were formed with a dense packing of the nanoparticles, as shown in Figure 57A.448 Moreover, nonspherical colloidosomes were obtained in a similar way. Double-emulsion droplets were first prepared with multiple inner cores. During solvent evaporation, the outer interface deformed due to the buckling of the nanoparticle layers. Therefore, the resultant colloidosomes exhibited a multicompartmental structure with distinct peanutlike or polyhedral morphologies, as shown in Figure 57B.449 Apart from colloidosomes with nanoparticles at the liquid− liquid interfaces, there is another case where colloidal particles adsorbed at the gas−liquid interface. By this means, “armored bubbles” could be synthesized with a coating of packed particles.450 For example, monodisperse CO2 bubbles were dispersed in an aqueous NaOH solution containing PS-copoly(acrylic acid) (PAA) nanoparticles. Due to uniform gas dissolution and mass transfer, particles were adsorbed at the interface and assembled into a close-packed 2D crystalline shell, which was a monolayer thick, as confirmed in Figure 57C. Besides, non-close-packed colloidal shell capsules could be synthesized by using a CCA suspension as the shell material.451 By carefully adjusting the experimental parameters, O/W/O emulsion droplets were generated with a PS photonic crystal shell without disturbing the crystalline state. By adding

hydrocarbon molecules such as glycerides, the temperaturetriggered phase transition from a molten state into a solid phase helped to solidify the shell and thus create microcapsules.442 Compared with that of simple particles, the structure of microcapsules could be much more diversified by exerting dual control over the cores and the shell. For example, with the aid of certain surfactants, capsules with multiple core components can be synthesized by using several separate inner flows during emulsification. These inner cores are permanently separated by the solid shell polymer after UV irradiation. Interestingly, Zhao et al. reported a switch between double core and single Janus core droplets by temporarily turning on and off the inner and middle flows.443 In this method, capsules with double cores and Janus core structures could be fabricated within a single run, as shown in Figure 56A. In addition to Janus cores, capsules with Janus shells were also achieved by using parallel middle phase flows during emulsification.144 Apart from composition diversity, the shape of the microcapsules could also be controlled. By exerting geometric confinement of the double emulsions, capsules with a rodlike shape could be generated, as well as multicore capsules, as shown in Figure 56B.143 On the other hand, by adjusting the flow rates, densely confined core clusters would emerge and rearrange into polyhedron configurations, in compliance with minimum interfacial energy. Using such droplets as templates, capsules with interconnected microporous structure were obtained, as shown in Figure 56C. 444 To generate capsules with nanostructures, colloidal silica nanoparticles were dispersed in the middle phase. During the droplet generation process, they migrated and anchored at the core−shell interface as well as the droplet outer surface, providing ordered nanopatterns and silanol groups for chemical modification of functional molecules, as shown in Figure 56D.30 Besides, phase separation or dewetting mechanisms could also mediate the capsule 8017


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Figure 58. Droplet fabrication of vesicles. (A) Scheme representation of phospholipid vesicle preparation using double emulsions as templates. Adapted from ref 457. Copyright 2008 American Chemical Society. (B) Multicompartment polymersome generation: (i) scheme illustration of the generation process; (ii−iv) microscopic images of polymersomes with (ii) two, (iii) three, and (iv) four compartments. Adapted with permission from ref 460. Copyright 2011 John Wiley & Sons. (C) Generation of multicompartment liposomes loaded with distinct ingredients and the formation of liposome dimers with highly controllable configurations. Adapted from ref 461. Copyright 2016 American Chemical Society. (D) Photo- and thermoresponsive polymersomes. (i) Contrast between AuNP-doped and AuNP-free polymersomes. (ii) Fraction of intact polymersomes upon laser irradiation. The green and red lines correspond to AuNP-doped and AuNP-free polymersomes, respectively. (iii) Sequential confocal microscope images of a mixture of AuNP-doped (green) and AuNP-free (red) polymersomes upon laser irradiation. Adapted with permission from ref 464. Copyright 2012 John Wiley & Sons.

shown in Figure 58A, W/O/W emulsion droplets were generated with a shell of phospholipid in toluene and chloroform solvents.457 Then the organic solvents evaporated and forced the phospholipid to assemble into vesicles. The recent progress has seen the use of octanol as the solvent to assist liposome assembly, with the advantage of a faster evaporation rate.458 In addition to that of liposomes, polymersome synthesis has also been accomplished in a similar way through assembly of dissolved diblock copolymers.459 Multicompartmental vesicles have been constructed by virtue of a dewetting process.460 Double-emulsion droplets were first generated with multiple cores and an oil shell containing amphiphilic diblock copolymers dissolved in two solvents: a good solvent that was volatile and a poor solvent that was less volatile. The evaporation of the good solvent resulted in the attraction interactions and the reduction of solubility of the copolymers, which led to a dewetting process. Then, after oil removal, nonspherical polymersomes with controllable compartments were generated, as shown in Figure 58B. A similar dewetting mechanism was implemented for the fabrication of liposomes.461 By using additional surfactant with appropriate concentrations, the interfacial energies of the emulsion system were carefully controlled to achieve unilamellar and multicompartmental liposomes. In addition, by using distinct inner flows during droplet generation, different ingredients could be loaded into the distinct compartments with controlled numbers, as shown in Figure 58C. To enhance the practical performances, more complex vesicles have been fabricated. For example, multiple polymersomes, or polymersomes-in-polymersomes, were prepared for programmed release of different ingredients.462 Through stepwise emulsification, double and higher order polymersomes were generated sequentially, with a bilayer membrane being added in each step. Ingredients encapsulated in different levels could be released in a programmed manner by sequentially rupturing the multiple bilayer membranes. Multifunctional

photocurable monomer in the aqueous CCA suspension, the periodic arrangement of the PS particles was locked after UV irradiation, thus generating microcapsules with photonic crystal shells. By varying the colloidal particle concentrations, such capsules showed different diffraction colors, as shown in Figure 57D. Vesicles. Microcapsules with membranes of assembled layers from amphiphilic molecules are referred to as vesicles. The vesicle formation principle is generally considered a two-step process. The amphiphilic molecules first assemble into a bilayer structure, which then closes up to form vesicles.452 There are also other mechanisms being put forward which consider the generation of spherical micelles as an intermediate, and the micelles then evolve into vesicles by coalescence or molecule uptake.453,454 According to the composition of the layer membrane, it can be classified into liposomes, polymersomes, niosomes, peptosomes, etc.343 Also, it can be categorized into unilamellar or multilamellar vesicles with reference to the lamellarity. Vesicles are fascinating materials because smaller compounds could be encapsulated and isolated in the cores, in a way mimicking the natural biomembrane systems.455 This feature offers great opportunity for vesicular materials to be used as encapsulation and delivery vehicles in pharmaceuticals and cosmetics, as well as for investigating cellular physiological activities in response to various stimuli. The droplet microfluidic technique provides an excellent platform for fabrication of vesicles in a highly controlled way by using droplets as templates. For example, an aqueous phase was emulsified into a lipid phase containing phospholipids and oleic acid. The formed phospholipid-stabilized W/O emulsions were transferred into another aqueous mixture containing ethanol, which extracted the oleic acid rapidly, thus forcing another coating of phospholipids on the droplet surface, which promoted the assembly of lipid vesicles.456 To improve the flexibility, such a solvent removal principle has been further implemented in double-emulsion systems. As 8018


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Figure 59. Droplet-fabricated materials for controlled release. (A) Thermally triggered release behavior of PAAm−PNIPAm core−shell microgel capsules. In the first 10 s, the temperature is set above the LCST so that the capsules remain sealed. Then the temperature decreases, which triggers the release of the encapsulants due to the reswelling of the PNIPAm shells. Adapted from ref 480. Copyright 2010 American Chemical Society. (B) Chemical-triggered release of PS-membane capsules: (i) scheme illustration of the active release mechanism; (ii) sequential confocal images showing capsule release behaviors when exposed to 50 wt % toluene stimuli. Adapted from ref 482. Copyright 2013 American Chemical Society. (C) pHtriggered release of two drugs: (i) dissolution process of the PSi−PEI−PMVEMA@ASHF composite at pH 7.4; (ii) morphology and surface properties of the particles at different pH conditions. Adapted with permission from ref 491. Copyright 2011 John Wiley & Sons.

stimulus-responsive capacity. Therefore, they are highly applicable in extensive areas, including optical electronics, biomedicine, food and cosmetic industries, etc. Herein, we categorize their applications into several aspects. 5.4.1. Drug Delivery. Encapsulation and controlled release of active agents are of significant interest in developing advanced delivery vehicles for drugs, nutrients, fragrances, and cosmetics. Especially, for efficient drug delivery, the agents could be encapsulated within microparticles or microcapsules with desired doses and then released in a specific target location.465 Droplet-synthesized materials possess several advantages for this demand, which lie in the following aspects.466,467 First, droplet-synthesized particles are highly tunable and uniform in size, structure, and encapsulation efficiency. This provides a guarantee for maintaining a consistent release response, and thus for regulating the release rate. Second, the particle-based drug delivery system allows a wide range of material choices to be encapsulated, and multiple drugs could be loaded for investigating their synergetic effects. Third, by using different matrix materials, various release profiles such as sustained or burst release could be achieved under an external stimulus, which could be applicable for specific usages.

vesicles have been fabricated by embedding hydrophobic nanoparticles in the shell. For example, by introducing quantum dots or magnetic nanoparticles, the vesicles showed magnetic or fluorescent features in the membrane.463 In another attempt, stimulus-responsive polymersomes have been prepared for triggered release.464 In this system, the shell of the doubleemulsion droplets was an organic mixture of a thermoinsensitive copolymer, PEG-b-PLA, a thermosensitive copolymer, PNIPAm-b-PLGA, and dodecanethiol-stabilized AuNPs. After the diffusion-induced dewetting process, the two amphiphiles assembled into polymersomes with AuNPs located in the bilayers. The resultant polymersomes thus showed thermoresponsive release behavior when the temperature was raised above the lower critical solution temperature (LCST) of the PNIPAm-b-PLGA copolymer. Additionally, due to the photothermal feature of AuNPs, the polymersomes were endowed with a photoresponsive feature and encapsulant release capacity, as confirmed in Figure 58D. 5.4. Applications of the Droplet-Derived Materials

Droplet microfluidics enables synthesis of materials with uniform and highly controllable size and structures. By incorporating specific ingredients, such materials could be endowed with distinct physical and chemical properties such as optical features, mechanical strength, selective permeability, and 8019


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core, thus contributing to pulsed139 or squirting478,479 release behavior. Seiffert et al. fabricated PAAm−PNIPAm core−shell microgels for the controlled release of RITC-tagged dextran.480 When the temperature was above the LCST, the shell was dehydrated and became nonporous and impermeable, so the RITC−dextran was trapped in the cores. However, when the temperature decreased and the shell reswelled to became porous again, the mesoscopic RITC−dextran could penetrate the core and shell, therefore being released. During this process, the solid core remained spherical in shape without being affected by the shell responses, as shown in Figure 59A. Another strategy of using PNIPAm as the core material has also been explored. In this method, the PNIPAm−ethyl cellulose (EC) core−shell particles were fabricated with PNIPAm gates embedded in the shell. At lower temperature, the PNIPAm was swollen and the gate was closed so that the release of solute molecules depended solely on the diffusion through the hydrogel network. At elevated temperature, however, the PNIPAm shrank and the gate was open so that the solutes were expelled in the core and then released outward through the open channel, thereby showing a much faster release rate.481 In addition to the thermally triggered release behavior, other stimulus-triggered release behaviors have been investigated. Under some circumstances, when the external environment was changed, chemical reactions were triggered in the drug carrier, thereby accelerating the degrading process. The chemical reaction could be selective for a certain stimulus. For example, PS-based capsules were employed for active release of the cargo in response to a plasticizing stimulus.482 When the solidified capsules were in contact with the hydrocarbon oil, phase transition occurred to convert the PS membrane from a solid state to a plasticized or fluidized state. Then a localized defect emerged in the capsule shell, and the payloads were driven out through the defect, as shown in Figure 59B. Apart from burst release, sustained release could also be triggered by a chemical stimulus. For example, microcapsules with a shell containing the glucose-responsive phenylboronic acid (PBA) moiety have been fabricated and monitored under a physiological bloodmimicking environment. When the glucose concentration changed from 0.4 to 3.0 g/L, the release rate of insulin was much faster.483 In addition, magnetic response has been explored for triggered release by incorporating magnetic nanoparticles, and mechanic response has been applied by exerting external stress to induce brittle rupture of the shell.484 Apart from using solid particles or capsules, colloidosomes with aqueous cores were also utilized for active encapsulation and reversible release when exposed to external stimuli.485 By control of the pore morphology, the permeability of the shell could be adjusted. For example, a thermosensitive triblock copolymer was dissolved in the aqueous cores and adsorbed to the inner surface of the colloidosome shell to fill the interstices between the colloidal particles. At higher temperature, the polymers desorbed from the inner surface of the colloidosomes so that the pores were open, and the inner actives were released through the pores.486 Vesicles also play an important role in drug delivery systems. The osmotic pressure difference across the membrane is an important parameter. When the vesicles were subjected to a sudden change in osmotic pressure, they split or generated holes if the membrane was in a fluid phase.487,488 Alternatively, if the membrane were in a gel or glassy state that was not deformable, it would burst489 or buckle to generate cracks459 in response to hypotonic and hypertonic

Drug Encapsulation. Drug molecules could be encapsulated in particle and capsule-type carriers, generated through single or multiple emulsions, respectively. For example, in single emulsions, by directly adding drug molecules in the dispersed phase, the payloads could be embedded in the particles efficiently in a uniform manner.468 In addition to homogeneous particles, actives could be loaded into porous particles by being predispersing in the porogens.469 Also, by using double emulsions, drugs could be embedded in the cores442 or shells470 of the capsules, as well as trapped in vesicles464 and colloidosomes.471 By these means, a variety of drugs have been loaded in the carriers, including paclitaxel,470 lidocain,472 rifampicin,473 bupivacaine,468 fluorouracil (5-FU),474 etc. The carrier materials should be compatible with the drug molecules. Hydrophilic drugs were generally loaded into hydrogel-based carriers such as alginate and chitosan, while hydrophobic drugs could be loaded into polymer-based carriers.465 Especially, PLA and PLGA polymers have been approved by the U.S. Food and Drug Administration (FDA) due to their biocompatibility and biodegradability, and have been extensively explored in drug delivery system (DDS) development.466 In some cases, multiple drugs need to be encapsulated to study synergistic effects. For example, Santos et al. reported a nano-microcomposite system for oral dual delivery of a peptide (GLP-1) and an enzymatic inhibitor (DPP-4) through a single-emulsion platform.475 The DPP-4 was embedded in an enteric polymer matrix, and the GLP-1 was carried on the nanomatrix of PLGA and porous silicon nanoparticles. In addition, by using porous microparticle carriers, different actives could be loaded within the polymer matrix and inside the pores.469 Besides, Windbergs et al. fabricated microcapsule carriers in a double-emulsion platform, through which a hydrophilic drug (doxorubicin hydrochloride) and a hydrophobic drug (paclitaxel) were encapsulated in the aqueous core and the solid shell, respectively. The efficacy of such a synergistic anticancer drug system was confirmed in cancer cell lines.470 Controlled Release. Controlled release is vital in drug delivery systems. The release mode could be distinguished as passive and active, according to whether there is a response to external stimuli.465 In passive mode, drug release depends on diffusion and matrix degradation, and the release profile generally shows an initial burst step and a following sustained pattern.476 There are several important parameters affecting this process. For example, the release rate decreases with an increase of the particle size and increases with the particle monodispersity.468,474 Porous particles may exhibit a faster release rate due to their distinct structure, which contributed to more rapid degradation of the polymer matrix.469 Other parameters, including the type of matrix materials, the crosslinking agents, and the cross-linking methods, the releasing media, the possible additives, and the drug itself, all have impacts on the release rate.440 Besides the passive release, active release involves a response to external stimuli, including heat, chemical environment, pH, magnetic field, mechanical stress, etc. As a representative, PNIPAm has been widely explored in thermally controlled release studies. When the temperature was raised above the LCST of the PNIPAm hydrogel, it shrank rapidly and expelled the water. Therefore, hydrophilic agents embedded within the PNIPAm particles would be released in response to thermal treatment.477 Also, for microcapsules with a PNIPAm shell and an oil core containing actives, the thermal response would result in the break of the shell due to incompatibility of the oil 8020


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Figure 60. Droplet-fabricated materials for tissue engineering. (A) GelMA microgel beads for adhesion and proliferation of CSP cells on the surface. Adapted from ref 495. Copyright 2014 American Chemical Society. (B) Porous microcarriers for 3D cell culture and formation of spheroid aggregates. Adapted from ref 444. Copyright 2015 American Chemical Society. (C) Construction of the 3D tissue architecture. Adapted with permission from ref 499. Copyright 2011 John Wiley & Sons. (D) Skin-equivalent coculture system: (i, ii) evaluation of the type VII collagen (green fluorescence) secretion amount by (i) a monoculture group of beads coated by NHEKs and (ii) an NHEK/NHDF coculture group; (iii, iv) evaluation of the secretion of type IV collagen (red fluorescence) (iii) without or (iv) with chemical stimulation of TGF-β1 and ascorbic acid. Adapted with permission from ref 501. Copyright 2012 John Wiley & Sons.

environments, respectively. These changes would all contribute to the release of the inner actives. Also, the membrane would be degraded in response to external stimuli such as a change of pH.490 In some cases, release of multiple drugs with different physicochemical features is required, especially for treating complex diseases such as cancers. Regarding this point, Zhang et al. utilized a pH-responsive polymer hydroxypropyl methylcellulose acetate succinate (ASHF) to construct a triggered synergetic drug delivery system.491 O/W droplets of ASHF in a solvent of EA were first generated and then solidified by solvent diffusion at pH 5.5. During this process, a hydrophobic drug, celecoxib (CEL), was loaded directly by being dissolved in the EA solvent. Another drug, 5-FU, which was hydrophilic, was loaded inside a nanocomposite NPs and then encapsulated along with the NPs in the droplet. The NP composite was composed of a porous silicon (PSi), a mucoadhesive polymer, poly(methyl vinyl ether-co-maleic acid) (PMVEMA), and a linker polymer, polyethylenimine (PEI). When the pH was changed to 7.4, the PSi−PEI− PMVEMA@ASHF system could be dissolved. The embedded PSi−PEI−PMVEMA NPs (labeled with FITC) were thus released within 2 min, and the ASHF (labeled with TRITC) completely collapsed after 30 min, as demonstrated in Figure 59C. In addition, the composites exhibited pH-responsive properties, which were stable at pH ⩽ 6.5 and dissolved fast at pH 7.4. Since the loading degree of 5-FU could be adjusted by virtue of the PSi−PEI−PMVEMA NPs, the loading ratios of the two drugs could be controlled conveniently. Moreover, the

mucoadhesive properties helped to prolong the retention time and thus enhance the therapeutical efficiency. 5.4.2. Tissue Engineering. Tissue engineering is a combinative study on cell biology, material science, and engineering techniques, with a goal of improving or replacing biological tissues. For this demand, cells need to be grown in a matrix that mimics the living environment and maintains a 3D state, because the complex cellular interactions and the interactions between cells and the extracellular matrices (ECMs) may provide important cues for mediating cellular functions and proliferations.353 This proposed high requirements for the choice of materials as well as the processing technology. Hydrogel materials stand out due to their biocompatibility and biodegradability. Also, their physical and chemical properties could be regulated by control of behaviors such as swelling and degradation.492 Nowadays, many kinds of hydrogels have been widely exploited, including naturally derived and synthetic ones such as collagen, alginate, gelatin, PEG- and PAA-based hydrogels, etc.493 As for materials processing techniques, droplet microfluidics is well suited. Particle-based hydrogel biomaterials could be conveniently fabricated by using droplets as templates and could be imparted with a tailored size, component, and shape. Such materials could be employed as microcarriers for 3D cell culture. Also, cells could be encapsulated directly within the hydrogel matrix, and the cell−materials composites could serve as tissue building blocks. Moreover, 3D coculture systems could be constructed to act as tissue models for investigating physiological and pathological phenomena. 8021


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Figure 61. Droplet-fabricated materials for analytical applications. (A) Hydrogel particles with two-colored coding capacity through controllable ratios of two differently sized QDs. Adapted with permission from ref 503. Copyright 2011 Royal Society of Chemistry. (B) Photonic crystal microbubbles as suspension barcodes: (i, ii) scheme representation of (i) microfluidic droplet template generation and (ii) formation of the photonic crystal microbubbles by water-extraction-induced cavitation and colloidal assembly; (iii) microscopic images showing the evolution of the microbubbles at different stages. Adapted from ref 504. Copyright 2015 American Chemical Society. (C) Aptamer-functionalized PCBs for the specific capture of multiple types of CTCs. (i) Scheme illustration of the process. TD05, Sgc8, and Sgd5 are three kinds of aptamers, and two kinds of target CTC cells are stained green and blue. (ii) Optical, (iii) green fluorescent, and (iv) blue fluorescent images of the CTC-captured barcode particles. Adapted with permission from ref 506. Copyright 2014 John Wiley & Sons. (D) Hydrogel inverse opal particles used for label-free sensing. (i) Schematic illustration of the reflection peak shift after DNA molecular binding. (ii) DNA detection. Adapted with permission from ref 206. Copyright 2010 John Wiley & Sons. (iii) Protein detection. Adapted with permission from ref 510. Copyright 2009 John Wiley & Sons.

Cell Microcarriers. Hydrogel particles fabricated from droplet microfluidics with different sizes, shapes, and microstructures have been explored as cell microcarriers. Compared with conventional culture plates or flasks, such particles exhibited a larger surface-to-volume ratio. Also, the properties of the hydrogel were tunable to mimic physiological conditions. For example, methacrylated gelatin (GelMA) is a photocurable hydrogel composed of modified ECM components.494 It has excellent features, including biocompatibility, biodegradability, noncytotoxicity, and nonimmunogenicity. Khademhosseini et al. fabricated GelMA particles in droplet templates through photopolymerization.495 Cardiac side population (CSP) cells were cultured on the particles and proliferated over time, covering the entire particle surface, as shown in Figure 60A. The sizes of the particles were adjusted to be 100 μm to provide enough surface area. Other than simple spherical particles, microcarriers with complex shapes and microstructures were also designed. Inspired by the structure of stem cell niches, Zhao et al. reported a porous carrier with external−internal-connected pores for cell culture.444 The scaffold structure protected the

cells from shear forces, and the biopolymers inside the cores offered an ECM-mimicking environment for the formation of multicellular spheroid aggregates, as shown in Figure 60B. They also constructed surface-patterned microcarriers by infiltrating biocompatible hydrogel in the colloidal crystal beads.496,497 These particles showed an ordered hexagonal pattern of colloidal nanoparticles, which not only provided more surface area for further coating of collagen, but also served as a nanopatterned platform to improve cell adhesion and growth. Apart from seeding cells on the microcarriers, cells could also be embedded within the hydrogel matrix during the particle synthesis process. For example, there are many studies on cells being encapsulated in alginate droplets, which were then solidified by gelation reaction on contact with calcium ions.362 This method precluded using any thermo- or photoresponsive materials and thus eliminated possible damage to cells due to a sudden temperature change or chemical reactions. Recently, Werner et al. introduced another mild approach for generating cell-laden hydrogel beads through noncovalent cross-linking and avoided using calcium ions, because calcium ion is an important intracellular secondary messenger and regulates 8022


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convenience in both encoding and decoding. By simply dispersing fluorescent dyes into an organic solvent, fluorescence-encoded particles could be generated through droplet emulsification and solvent evaporation. QD-embedded particles could be fabricated in a similar way and serve as barcodes with a single excitation wavelength and narrow Gaussian emission line shapes.502 Also, by using a microfluidic device with a pyramidal channel network, single-colored barcodes with five-level stepwise concentration gradients of monochromatic QDs or double-colored barcodes with controllable ratios of two differently sized QDs were generated in parallel, as shown in Figure 61A.503 Apart from fluorescent barcodes, photonic crystal beads (PCBs) could be generated in droplet templates by evaporation-induced confined assembly of colloidal nanoparticles. Such particles exhibit structural colors, which serve as the coding element. As described above, by using colloidal nanoparticles of different diameters, the characteristic reflection peaks of the PCBs could be adjusted throughout the visible light range for coding.87 By molding hydrogel materials410 or constructing inverse opal structures,414 the reflection peak could be tuned by the relative refractive indices. By fabricating non-close-packed PCBs, the reflection peak could also be controlled by the initial nanoparticle concentration.79 Moreover, various multifunctional PCBs have been developed for enhancement of the barcode efficiency and functionality. For example, magnetoresponsive Janus PCBs were fabricated to enhance the controllable motion ability;382 multicompartmental PCBs79 or photonic crystal capsules143 were prepared to enlarge the coding amount. Recently, phonic crystal bubble barcodes were synthesized with tunable overall density.504 Briefly, microcapsules composed of a semipermeable solid shell and a colloidal suspension core were first generated. Then the particles were immersed in ethanol, and the inner water was extracted across the membrane. During this process, a bubble was generated due to a cavitation mechanism, and the colloidal particles were concentrated and assembled in the confined inner wall of the shell, thus generating encoded photonic crystal bubbles, as shown in Figure 61B. These particles could remain in suspension rather than depositing rapidly, and were also imparted with substantial coding levels as well as controllable movement capacity. Barcode particles have been widely applied in multiplex assays.406 For example, by measuring the fluorescent signals, the presence of target molecules and the binding reaction strength could be identified. Besides, PCBs show several advantages in multiplex assays. Because their color originates from the physical structure, the coding is extremely stable and the photobleaching or photoquenching could be avoided. In addition, the surface pattern, which originates from the ordered arrangement of the nanoparticles, provides more surface area for probe immobilization and target reaction. Therefore, PCBs have been widely employed in biomolecular assays as well as diagnostics.32 In this aspect, multiplex detection on different target tumor makers was realized by separately immobilizing several probe molecules on different PCB barcodes after simple chemical modification.505 Apart from molecular assays, PCBs have also been used for cell detection. For example, PCBs functionalized with dendrimer-amplified aptamer probes were developed for the capture and detection of multiple types of circulating tumor cells.506 Due to the specific binding affinity between the aptamers and the target molecules on the cancer cells, the cells would only be captured on the corresponding

plenty of cellular physiological activities. Instead, they introduced a system composed of conjugates of peptides with starPEG polymers and oligosaccharides. The encapsulated cells showed a high survival rate even after freezing.498 Tissue Building. Cell-laden hydrogels could serve as building blocks for constructing tissues. For example, collagen “cell beads” were generated and served as units for bottom-up building of macroscopic tissue architectures with desired geometric features.499 Briefly, collagen gel beads with cells seeded on the surface or encapsulated inside were first generated from droplet microfluidics. Then the microbeads were molded into a chamber. The surface cells formed cellular adhesions rapidly, and the nutrients could be supplied through the cavities between adjacent beads. Finally, the cells migrated and grew to form 3D tissues, as shown in Figure 60C. Also, by using arbitrarily designed molds, a millimeter-scale tissue assembly was constructed with a specific shape. Moreover, Segura et al. recently constructed a wound-healing scaffold for in situ tissue regrowth and regeneration.500 Microgel particles were generated with surface functionalities and annealed together to form an interconnected microporous scaffold. In vitro, the incorporated cells proliferated and formed a 3D network; in vivo, the cells migrated and rapid cutaneous-tissue regeneration was realized. The scaffold was also injectable and could be molded into arbitrary configurations. Another value of cell-laden hydrogels for tissue engineering is the coculture ability, which facilitated studies on cellular functions and stimulus response, through analysis of cell secretions. For example, microsized skin-equivalent tissues were constructed through a coculture system that was based on type I collagen beads.501 The collagen beads were first generated with human dermal fibroblasts (NHDFs) encapsulated inside. Then normal human epidermal keratinocytes (NHEKs) were seeded on the beads and gradually covered the bead surface, thus accomplishing the NHEK−NHDF cocultured system. This was a mimic of the hierarchical structure of skin tissues composed of dermal cells, epidermal cells, and the ECM. The dermal−epidermal interactions were demonstrated through an ELISA test on the secretion of type VII collagen. The results showed that the amount of secretion was higher in the coculture beads than in the monoculture beads solely coated by the NHEKs, therefore indicating the dermal−epidermal interactions. Moreover, the cellular response was investigated by using a chemical stimulus of TGF-β1 and ascorbic acid. The results confirmed the secretion of the type IV collagen, as shown in Figure 60D. Therefore, such a coculture system could serve as a well-defined skin tissue model. 5.4.3. Analytical Applications. Barcodes. The increasing requirements of high-throughput assays in biochemical analysis and medical diagnostics call for efficient strategies for multiplexing. The use of barcode particles provides a promising solution by carrying coding information that originated from their characteristic compositions and distinct features. Such barcodes serve as mobile carriers for probe attachment and target recognition, thus offering faster reaction kinetics and higher detection flexibility. For the synthesis of barcode particles, droplet microfluidics is an excellent technique due to precise control over the particle size, structure, and composition.40 Nowadays, a large variety of barcode particles have been achieved through droplet templates, and their encoding mechanisms are diverse, including graphical, electronic, optic, etc. Among these different types of barcodes, optic barcodes have been most extensively exploited due to the 8023


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PCB surface through a specific recognition reaction, as shown in Figure 61C. Similarly, by entrapping different plasma proteins such as phytohemagglutinin and fibronectin within the hydrogel inverse opal scaffolds, different kinds of blood cells could be recognized and captured, such as erythrocytes and platelets.412 Sensors. Particle-based sensors have been extensively studied. For example, AgNPs are attractive for SERS and biosensing applications, due to the high plasmonic efficiency and superior electromagnetic enhancement. Composite polymer/Au nanoassembly particles were synthesized in the droplet microfluidic platform. The presence of the metal-rich surface catalyzed the enforcement process of additional deposition of AgNPs. Such composite particles thus showed enhanced sensing abilities, as demonstrated by the SERS measurement of adenine analytes.345 Apart from nanoparticles, microparticles could also serve as important sensors in response to environmental parameter variations. For example, microcapsules with semipermeable shells could sense the surrounding osmotic conditions by mechanic responses such as buckling507 or by a change of the core content properties, such as structural colors.508 A temperature change was sensed by capsules containing cholesteric liquid crystals, reflected in a reversible shift in structural color and spectrum.419 In addition, by employing mesoporous CCB sensor elements, an optical nose could be developed to distinguish vapor species from the specific “fingerprint” of the spectrum shift.509 Significantly, particle-based sensors could be used for labelfree assays and diagnostics. In label-free assays, the target molecules do not need to be labeled by any tags, which is timeconsuming and expensive. Instead, they can be detected in their natural state without being disturbed. Also, dynamic monitoring of probe−target interactions could be realized by measuring the physicochemical changes on the sensors. In this aspect, silica inverse opal particles were used to detect tumor markers. The specific binding of the target molecules to the anticancer probes led to an increase of the average refractive index, which resulted in a red shift of the diffraction peak position.413 Besides, by constructing inverse opals using a DNA-responsive hydrogel206 or protein-imprinted polymers510,511 as the skeleton materials, the binding reactions would result in the shrinking or swelling of the polymer, thus changing the lattice spacing. Therefore, the binding events could be converted to detectable spectrum shifts, and the target molecule could be detected quantitatively, as shown in Figure 61D. 5.4.4. Optical Devices. Droplet-synthesized particles bearing optic properties have been utilized as basic units for constructing optical devices. For example, electroresponsive Janus particles with two hemispheres of black and white dyes have been utilized in twisting-ball displays.135 By reversing the electric field gradient, the particles could be flipped. Magnetoresponsive Janus particles containing magnetic nanoparticles and QDs in each hemisphere were prepared and then arranged in a panel of ordered arrays. The color switch was realized by altering the direction of the magnetic field, as shown in Figure 62A.512 When the magnetic hemispheres faced upward, the panel exhibited a red-brown color. Conversely, when the QD hemispheres faced upward, the panel displayed a white or blue color under daylight or UV irradiation, respectively. By incorporating colloidal photonic crystals, Janus particles with two hemispheres of black and structural color features have also been achieved.137 After being embedded in a PDMS film, these

Figure 62. Droplet-fabricated materials for constructing optical devices. (A) Magnetic-fluorescent Janus particles for display applications: (i) schematic representation of the fluorescent switch of the Janus particle arrays by changing the magnetic field direction; (ii−iv) the beads display images under (ii, iii) daylight and (iv) UV irradiation. Adapted with permission from ref 512. Copyright 2011 John Wiley & Sons. (B) “Photonic paper” with (i) wide viewing angles and (ii) rewritable ability. Adapted with permission from ref 513. Copyright 2013 John Wiley & Sons. (C) Microcapsules with CLC shells for constructing laser resonators: (i) scheme and (ii) microscopic image of the double-emulsion droplets. Adapted with permission from ref 515. Copyright 2013 John Wiley & Sons. (D) Polarizing microphotograph of magnetic-NP-doped CLC capsules showing the disclination line. Adapted with permission from ref 516. Copyright 2016 John Wiley & Sons.

particles rotated freely in response to an ac electric field, causing a color switch. Also, the film showed angle-independent reflection color. Furthermore, by using a PEG hydrogel to replicate the CCB assembled film, a novel “photonic paper” with wide viewing angles and flexible texture was developed.513 The rewritable ability of the paper was achieved due to the swelling response of the hydrogel inverse opal scaffold to the dripping of the ink, which resulted in an angle-independent color shift, as shown in Figure 62B. In addition to novel display devices, other optical devices have also been exploited. For example, the cholesteric liquid crystalline droplets could serve as an omnidirectional laser resonator due to radial arrangement of the helical axes.514 In this aspect, Uchida et al. fabricated a photonic structure with the CLC shell and an aqueous core containing dyes, as shown in Figure 62C.515 Three types of laser resonators (DFB, DBR, and WGM) were obtained, by changing the combination of the dyes and CLCs. The monodispersity contributed to a narrow lasing wavelength. They further demonstrated that magnetic nanoparticles would assemble into a needle-like aggregate along with the disclination line (Figure 62D) of the shell of the CLC capsules, which enabled magnetic control of the surface molecule orientation as well as the wave of the laser light. Such systems were expected to serve as units in magnetically 8024


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controllable laser arrays or optical combs.516 In addition, CLCs encapsulated in capsule cores have been confirmed to preserve their planar molecular arrangement and could serve as units for constructing flexible photonic devices.517 Besides, photoresponsive polymer-dispersed liquid crystal (PDLC) microspheres were prepared as imaging units to construct microlens arrays. The transmission of each microlens was switchable between transparent and opaque by UV or visible light irradiation.518 5.4.5. Imaging. Droplet microfluidics provides a way of synthesizing particles with precisely controlled size and uniformity, which could serve as contrast agents for enhancing the clarity and quality of the images in both optical research and clinic diagnosis. As a representative, microbubbles have been widely utilized for ultrasound sonography due to their high compressibility.519 Using droplet methods, microbubbles stabilized with particles,520 proteins,519 or solid shells521,522 have been fabricated, and their ability of acoustic contrast enhancement has been demonstrated.519 These bubbles could be generated by direct injection through an air-actuated membrane valve519 or by replacing the water in the capsule cores through a certain physical process such as drying521 or cavitation.522 In addition to acoustic imaging, BaSO4 crystallites were synthesized in situ within PNIPAm microgels for enhanced in vitro X-ray imaging. Compared with pure PNIPAm, the nanoBaSO4−PNIPAm image was much brighter.523 Moreover, biodegradable microparticles were developed for in vivo imaging applications. For the in vivo distribution of the microparticles, their diameter determines specific organ uptake, and the size distribution determines the extent of retention. Häfeli et al. fabricated FDA-approved PLA microspheres with a size of 9.0 μm and a high monodispersity. The particles were labeled with 99mTc through polymer-linked radiometal-specific chelators.524 After tail vein injection into mice, the particles were eventually retained and distributed homogeneously in the lung. By conducting micro-SPECT/CT (SPECT/CT = singlephoton-emission computed tomography/computed tomography) imaging experiments, these particles were demonstrated to serve as appropriate lung perfusion imaging agents. 5.4.6. Cell Mimics. The droplet microfluidic technique enables synthesis of micrometer-sized materials that mimic cell morphologies and functions, or in other words artificial cells. As the most representative aspect, vesicles with bilayers of amphiphilic molecule assembly have been employed as a mimic of natural cell membranes.525 A variety of studies have demonstrated the generation of liposomes for encapsulation of bioactive compounds (DNAs, proteins, etc.) and implementation of membranes functions.526,527 For example, Stachowiak et al. fabricated giant unilamellar vesicles (GUVs) with pore proteins that could mediate solute transportation.526 In addition, GUVs with controlled domains were expected to serve as models for studying the physical properties of the cell membrane.528 Apart from artificial membranes, hydrogel networks have been encapsulated in capsules529 or polymersomes530 as a mimic of cytoskeletal elements. Besides, the cell mechanical feature was mimicked by using solid particles as surrogates. For example, biotin-conjugated hydrogel particles were generated and constrained to flow through avidinconjugated microchannel constrictions. Compared with bare particles, the flow rate of the biotin-coated particles was reduced.531 This provided a model study of cell mechanical responses in confined channels.

5.4.7. Wettability. Amphiphilic particles have been employed to construct intelligent materials with special interfacial wettability features for liquid manipulation.532,533 Inspired by the ordered topological structures and the resultant fog manipulation features in some organisms, Zhao et al. fabricated Janus microparticles with a magnetic hemisphere and another photonic crystal hemisphere through photopolymerization of biphasic emulsion droplets.79 To render amphiphilic surface properties, the particles were first entirely treated with fluoroalkylsilane. Then they were magnetically orientated to allow the photonic crystal hemispheres to be selectively treated by oxygen plasma, as shown in Figure 63A.

Figure 63. (A) Janus particles with amphiphilic surface properties for liquid manipulation: (i) schematic illustration of the sequential partial surface treatment procedure; (ii) water droplets sitting on the Janus particle monolayer; (iii, iv) Janus-particle-coated liquid marbles on (iii) a glass slide and (iv) a water surface. Adapted from ref 79. Copyright 2013 American Chemical Society. (B) Synergistic self-assembly of amphiphilic Janus particles: (i, ii) scheme of the (i) assembly of the (ii) amphiphilic Janus particles at the water−air interface; (iii, iv) view and contact angle of the hydrophilic PVP side of the monolayer; (v, vi) view and contact angle of the superhydrophobic PS side of the monolayer. Adapted with permission from ref 534. Copyright 2016 John Wiley & Sons.

The resultant particles could form a monolayer on the water−air interface. Because the hemispheres facing the air had a hydrophobic nanostructured surface pattern, the monolayer was water-resistant. Also, such amphiphilic particles could be coated on water droplets to form liquid marbles, which could be further actuated to coalesce by a magnetic field. In another attempt, Yao et al. gained inspiration from natural molecular amphiphile assembly and prepared Janus amphiphilic particles as assembly units through diffusion-induced phase separation and magnetically driven dewetting. Such particles assembled at the water−air interface into a 2D amphiphilic monolayer and formed a hexagonal close-packed array, as shown in Figure 63B. The difference in chemical composition as well as surface roughness contributed to contrary wettability features of the 8025


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units need to be integrated into a compatible system and eventually automated and commercialized. The third issue concerns material fabrications. Generally, mass production is an inevitable topic to bring droplet microfluidics out of the laboratory as an industrialized technology. Fortunately, there is already some research on the scale-up of droplet microfluidic platforms.539−542 For example, three-dimensional ladderlike or treelike liquid delivery channels were designed in concert with droplet generators to achieve the parallelization of droplet formation.539,540 Moreover, to withstand high-pressure operation, the materials, the chip processing methods, and the bonding approaches of different layers have been prudently selected. Despite such promising explorations, there remain many challenges for commercial-scale manufacturing. First, when the scale expands to a certain degree, patterned wettability control of the complex channel needs to be achieved with high feasibility and accuracy. Second, one must consider the cost when pushing a technology to the commercial market. Regarding this, the development of low-cost microfabrication methods and materials is another prospect. Third, the current scaling up of droplet generation is restricted to single emulsions; while in practical applications, for example, to develop novel drug vehicles, multiple or complex droplet emulsions need to be generated, and progressively more novel interfacial reaction systems are expected to be incorporated. Fourth, compared with the microscale particles, the diversity and functionalities of the droplet microfluidics-derived nanoscale materials are still lacking. It is envisioned that, with further endeavors, droplet-based methods should also enable synthesis of nanomaterials with unprecedented features that would be difficult to obtain in other approaches. Thus, a lot of effort is still needed to overcome these challenges. In particular, indepth collaborative efforts and communication from different areas should be aimed to bridge the gap between material synthesis and applications. In summary, droplet microfluidics has evolved into a powerful technique and possesses application values cutting across multiple fields and disciplines. We hope that this review will inspire researchers from different backgrounds to work on addressing the issues described above and make contributions to push droplet microfluidics forward to become a widely reliable, truly industrial technology. We firmly believe that more exciting accomplishments will be achieved in droplet microfluidics.

opposite two sides, in analogy to similar features seen in some leaves.534 In addition to the main values mentioned above, the droplet -microfluidics-derived materials also have many other applications. With increasingly new substances and physical-chemical processes being implemented in droplet platforms,535−537 the resultant materials are finding a more significant role in widespread areas and will continue to cultivate new values in different fields.

6. CONCLUSION AND PERSPECTIVE In this review, we have presented a comprehensive summary of recent progress on droplet microfluidics, covering fundamental research from microfluidic chip fabrication and droplet generation to the application of droplets in bio(chemical) analysis and materials generation. Because of the deeply theoretical research and technological innovations, droplet microfluidics currently bears significant value in an extremely wide range of areas, including physical, chemical, biological, medical, and engineering fields. Despite the many exciting and compelling developments, there remain challenges that pose a gap between academic proof-of-concept studies and practical techniques for addressing real-world problems. Therefore, several important issues need to be solved to achieve the wide applicability of droplet microfluidics. The first issue should be focused on the basic research of the droplet microfluidics. There is still room for improvement of the existing droplet systems. Generally, the fluid composition is relatively simple in laboratory studies. However, for out-of-lab applications, especially in the fields of food and cosmetics, the ingredients are complex, often including various additives. Such impure components may have effects on the surface tension, interfacial stability, and rheological behaviors and hysteretic effects. Although droplet breakup dynamics and dripping-tojetting transition in a non-Newtonian, shear-thinning multiphase flow system have been explored,538 deeper investigations of the droplet generation mechanism and manipulation methods under more complex conditions are still expected. In addition, considering possible adsorption of some interfacial active ingredients such as protein molecules, temporospatial variations of interfacial parameters need to be investigated, which may hopefully be accomplished through advanced imaging techniques. The second issue concerns analytical applications, especially for high-throughput analyses. To make significant breakthroughs in bio(chemical) analysis, for example, in single-cell next-generation sequencing or in discovering new drugs, analytical instruments should be operated at the same high frequency as the signal generation and should not compromise the signal quality. This poses a challenge, but also direction, for the future development of analytical methods. Equally, to identify novel cellular physiological and pathological processes, multiplex assays are needed. To meet this demand, the analytical system should not only indicate whether a signal exists, but also be capable of recognition and quantification of distinct signals. In addition, the current droplet systems are usually run in specialized academic laboratories, which restrict the access to microfluidics for nonexperts. Therefore, much effort should be paid to the simplification and modularization of basic functional units, including sample preparation and treatment, specific reactions, signal detection, data processing, and active control units for droplet generation and manipulation. Moreover, these

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Yuanjin Zhao: 0000-0001-9242-4000 Notes

The authors declare no competing financial interest. Biographies Luoran Shang is a research scholar in Prof. David A. Weitz’s group in the School of Engineering and Applied Sciences of Harvard University. She received her bachelor’s degree from the School of Biological Science and Medical Engineering of Southeast University in 2013. She will receive her Ph.D. degree in 2017 under the supervision of Prof. Yuanjin Zhao at the State Key Laboratory of Bioelectronics of 8026


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Southeast University. Her current scientific interests are focused on droplet microfluidics and its biomedical applications. Yao Cheng is a postdoctoral researcher in the research group of Prof. Yuanjin Zhao at the State Key Laboratory of Bioelectronics of Southeast University. She received her bachelor’s degree from the School of Electronic Science and Engineering of Southeast University in 2010 and her Ph.D. degree from the School of Biological Science and Medical Engineering of Southeast University in 2016 under the supervision of Prof. Zhongze Gu and Prof. Yuanjin Zhao. Her current scientific interests are focused on microfluidic generation of biomaterials. Yuanjin Zhao received his bachelor’s degree in 2006 from the School of Clinical Medicine of Southeast University and his Ph.D. degree in 2011 from the School of Biological Science and Medical Engineering of Southeast University under the supervision of Prof. Zhongze Gu. In 2009−2010, he worked as a research scholar in Prof. David A. Weitz’s group in the School of Engineering and Applied Sciences of Harvard University. He was appointed as a full Professor in 2015 at Southeast University. His current scientific interests include microfluidic-based biomaterials fabrication, biosensors, and bioinspired photonic nanomaterials. He has published more than 80 research papers in international journals and applied for more than 60 related patents. His recent awards include the Young Chemist Award of the Chinese Chemical Society.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21473029 and 51522302), the NSAF Foundation of China (Grant No. U1530260), the National Science Foundation of Jiangsu (Grant No. BK20140028), the Program for New Century Excellent Talents in University, and the Scientific Research Foundation of Southeast University. REFERENCES (1) Whitesides, G. M. The origins and the future of microfluidics. Nature 2006, 442, 368−373. (2) Nge, P. N.; Rogers, C. I.; Woolley, A. T. Advances in microfluidic materials, functions, integration, and applications. Chem. Rev. 2013, 113, 2550−2583. (3) Stone, H. A.; Stroock, A. D.; Ajdari, A. Engineering flows in small devices: microfluidics toward a FFlab-on-a-chip. Annu. Rev. Fluid Mech. 2004, 36, 381−411. (4) Mark, D.; Haeberle, S.; Roth, G.; von Stetten, F.; Zengerle, R. Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications. Chem. Soc. Rev. 2010, 39, 1153−1182. (5) Sackmann, E. K.; Fulton, A. L.; Beebe, D. J. The present and future role of microfluidics in biomedical research. Nature 2014, 507, 181−189. (6) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Micro total analysis systems. 1. introduction, theory, and technology. Anal. Chem. 2002, 74, 2623−2636. (7) Haeberle, S.; Zengerle, R. Microfluidic platforms for lab-on-a-chip applications. Lab Chip 2007, 7, 1094−1110. (8) Squires, T. M.; Quake, S. R. Microfluidics: fluid physics at the nanoliter scale. Rev. Mod. Phys. 2005, 77, 977−1026. (9) Atencia, J.; Beebe, D. J. Controlled microfluidic interfaces. Nature 2005, 437, 648−655. (10) Darhuber, A. A.; Troian, S. M. Principles of microfluidic actuation by modulation of surface stresses. Annu. Rev. Fluid Mech. 2005, 37, 425−455. (11) Cheng, Y.; Zheng, F. Y.; Lu, J.; Shang, L. R.; Xie, Z. Y.; Zhao, Y. J.; Chen, Y. P.; Gu, Z. Z. Bio-inspired multicompartmental microfibers from microfluidics. Adv. Mater. 2014, 26, 5184−5190. 8027


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