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Droplets for Ultrasmall-Volume Analysis Daniel T. Chiu, Robert M. Lorenz, and Gavin D. M. Jeffries University of Washington Seattle By using methods that permit the generation and manipulation of ultrasmall-volume droplets, researchers are pushing the boundaries of ultrasensitive chemical analyses. (To listen to a podcast about this feature, please go to the Analytical Chemistry Web site at pubs.acs.org/ ancham.) What are ultrasmall volumes, and why would one want to perform chemical analyses in them? The next question might be, how does one go about doing chemistry and chemical analysis in ultrasmall volumes? The goal of this article is to clarify the first two questions and to offer various approaches to address the third. Our intent is to introduce concepts and challenges of ultrasmall volume analysis and describe the advances being made in the field. Because of the limited amount of space, we had to confine our referencing to highlight only a few select areas and recent advances. The motivations to conduct chemistry in small, confined volumes are diverse. The chemistry of life takes place in a small, enclosed volume that we call a cell; it’s a volume of picoliters, and it is further subdivided into even smaller volumes, from femtoliters to attoliters, that are defined by subcellular organelles. To understand how chemistry takes place in these tight spaces, it is often beneficial to re-create these very small volumes, such as with the use of lipid vesicles,1-3 so we can perform controlled experiments to understand the effects of molecular confinement. Likewise, to conduct chemistry on a single cell or subcellular structure and analyze its molecular contents requires the ability to create similarly small volumes to overcome diffusion and to prevent dilution.4 Another motivation to downsize volumes is the need to create large, dense arrays of parallelized small volumes for high-throughput screening, combinatorial chemistry and biology, or chemical synthesis.5 SMALL VOLUMES AND ULTRASMALL VOLUMES Because of the diverse set of motivations and applications that require small, confined volumes, the range of volumes involved varies just as widely, from microliters6 and nanoliters7 to femtoliters and attoliters.1,4,8-13 At first glance, a nanoliter might seem 10.1021/ac900306q CCC: $40.75 2009 American Chemical Society Published on Web 06/09/2009
rather small, but it is actually 6 orders of magnitude larger than a femtoliter. Correspondingly, the technologies used to control a femtoliter volume can be quite different than those used to manipulate a nanoliter volume; the resultant chemistry also can be quite different, because of the large differences in the surfaceto-volume ratios between these two volume scales. For example, the rate of change in concentration caused by evaporation scales as the fifth power of the surface-to-volume ratio,14 and thus can be extremely rapid for a femtoliter volume but relatively slow when nanoliters are involved. Similarly, detection technologies used to probe femtoliters need to be ∼1 million× more sensitive than those used for nanoliters: a solution containing 1 µM of dissolved species corresponds to ∼600 million molecules in a nanoliter but only ∼600 molecules in a femtoliter. As a result, powerful measurement techniques that have been successfully adapted for the analysis of nanoliters, such as NMR7,15 and MS,16 currently lack the needed sensitivity for the analysis of femtoliter volumes. A clear distinction exists, therefore, in the challenges and approaches used to manipulate and probe nanoliters and femtoliters. To distinguish these two regimes, we refer to ultrasmall volumes as volumes that are 10 pL or less; this is comparable to or smaller than a typical mammalian cell. Most ultrasmall volumes we describe here will be in the femtoliter range. In contrast, small volumes are in the range of nanoliters to hundreds of picoliters; these volumes are used in most traditional microfabricated devices. Small volumes are too small to be pipetted in air with micropipettes (which go down to 1-2 µL) but large enough to be stably manipulated in air using surface tension, such as by electrowetting,17 in through-hole well plates,18 or in enclosed microchannels.19 Although evaporation is a concern, it is still possible to manipulate small volumes for a short period of time in air. However, ultrasmall volumes must always be immersed in, but separated from, another fluid by a physical barrier. To illustrate, a 1-nL droplet takes about 20 seconds (s) to evaporate in air, whereas a 1-fL droplet would evaporate in ∼5 ms. Similarly, it is possible to define a small volume or a nanoliter plug in a microfluidic device even when the volume of interest and its surrounding solutions are in direct contact without any physical Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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Figure 1. Chemistry in ultrasmall volumes. Images before (ai, aii) and after (aiii, aiv) electrofusion of a fluo-3-containing vesicle (left) and a Ca2+-containing vesicle (right); these vesicles were a few tens of femtoliters in volume. (av) A five-container network with differentiated interior contents (red and green fluorescent dyes). (bi) Pneumatically actuated microchambers and microfluidic channels in PDMS for single-cell analysis, which show the cell manipulation section on the left and the molecule-counting section on the right. (bii, biii) Detection of the activity of single β-galactosidase molecules as they hydrolyze fluorescein-di-β-D-galactopyranoside to fluorescein. A low concentration of the enzyme was dispersed and enclosed in an array of 30-fL chambers. (bii) By pressing a small area of a patterned PDMS sheet, successive rounds of opening and closing could be performed, allowing the exchange of the contents of each chamber. (biii) Fluorescent images of the enzymatic activity in the chambers. (ci and cii) Aqueous droplets for use as defined chemical volumes. (ci) Five droplets generated simultaneously on demand, visualized using different food-dye colorings (scale bar represents 25 µm). (cii) Schematic and images demonstrating how two aqueous droplets can be fused using optical vortex trapping for the desired modification of the chemical contents of the droplet, which is shown by an increase in fluorescence signal (scale bar represents 5 µm). Panels adapted with permission from (ai-aiv) Ref. 1;(av) Ref. 3;(bi) Ref. 23; (bii and biii) Ref. 24; (ci) Ref. 41; and (cii) Refs. 12, 13, and 29.
partition,20 because diffusional broadening is nonlinear and is ∼10,000× slower in a small volume than in an ultrasmall volume. It takes 36 s for a small molecule like fluorescein to diffuse across 215 µm (diameter of a 10-nL sphere) but a mere 3.6 ms to diffuse across 2.15 µm (diameter of a 10-fL sphere), assuming Brownian motion (x2 ) 2Dτ, where x is the distance traveled, D is the diffusion coefficient, and τ is the time) and an aqueous diffusion coefficient of 0.64 × 10-9 m2/s for fluorescein. The manipulation and analysis of “small volumes” has been well covered in the literature, because of the trend that drove the development of miniaturized devices and assays over the past two decades. Approaches vary from microwells and lab-on-a-chip devices19 to large droplets manipulated on surfaces17 and segmented flows (e.g., aqueous plugs sandwiched between oil or air) routed in microchannels.21,22 CREATING ULTRASMALL VOLUMES The problem to be studied will determine the best approach for forming ultrasmall volumes. Figure 1 illustrates three complementary techniques. The use of lipid vesicles to define a small volume has the advantages of being biomimetic and a first-order approximation of the biological cell.1,3 Exquisite techniques for generating complex networks of lipid vesicles connected by lipid nanotubes have been developed,2,3 and these are especially suited for applications aimed at understanding the dynamics of lipid membranes or the biochemical reaction environments inside cells. Figures 1ai-aiv show the initiation of a simple chemical reaction by fusing two lipid vesicles. Figure 1av shows a network of five lipid containers linked by lipid nanotubes; the vesicles contain red and green fluorescent dyes, demonstrating how the contents can be varied and controlled by limiting diffusional mixing. The use of lipid vesicles to create an ultrasmall volume represents a bottom-up approach, in which the membrane that 5112
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defines the volume is formed by self-assembly. For applications that require a top-down engineering approach, the use of prefabricated chambers might be more appropriate.23 Here, a large array of microchambers can be designed to specification and fabricated in parallel using photolithography.24 Figure 1bi shows microchambers formed using pneumatic valves, and in Figure 1bii an array of femtoliter chambers fabricated in PDMS can repeatedly confine volumes when pressed against a coverslip containing a dilute enzyme and substrate solution (Figure 1biii). The ability to design and draw out each and every microchamber and fluidic element on a computer is attractive and gives this approach an engineering advantage. The third method to create ultrasmall volumes is with droplets. This approach merges the top-down characteristics of microfabrication and the bottom-up features of self-assembly. Microfabrication is used to precisely define the network of channels that form and manipulate droplets, whereas the formation of droplets and the layer of surfactant molecules at the droplet interface relies on self-assembly. Figure 1c shows the simultaneous on-demand generation of droplets with varying contents, as well as the optical manipulation of droplets for fusion and the resultant change in the encapsulated chemical environment. The microfluidics approach to making droplets offers accurate control, flexibility, and high throughput. As with lipid vesicles, the droplet interface can be tuned with surfactant molecules, and each droplet can be manipulated with high precision using different physical mechanisms, such as optical vortex trapping (Figure 1cii). In fact, these attractive features offered by droplets have driven the explosive growth of droplet microfluidics over the past 5 years.5,25
BIOLOGICAL ASSAYS, SINGLE CELLS, ORGANELLES, AND MOLECULES IN DROPLETS Droplets have emerged in the past few years as a platform for a wide range of applications; some use monodisperse droplets generated by microfluidics,5,11-13,22,25-33 and others use emulsion systems.9,34 Some droplet- and plug-based systems employ nanoliter volumes,5,17,25,35 whereas others are in the picoliter or femtoliter regime.10,12,13,29,35 We will emphasize biomedical applications. However, droplets that are generated using microfluidics also are finding use in materials science because they are highly monodisperse in size, which in turn imparts homogeneity to micro- or nanoparticles that are being synthesized. Our lab and others have been developing droplet platforms for biological assays,4,5,8,10,13,21,26,36-42 in particular, ultrasmallvolume droplets as nanolabs for single-organelle and single-cell studies. For such applications, chemical transformations of the biological molecules within droplets are required. For example, the limited number of molecules present in a small subcellular compartment will require sensitive methods of detection, such as fluorescence, in which case the biomolecules must be reacted and labeled with fluorophores. The motivations for studying and analyzing single cells and single subcellular organelles are diverse. Some biological problems occur at the single-cell level; examples include the detection of stem cells and the rare cancer cells that circulate in blood. At the subcellular level, almost all biochemical studies are performed in bulk; this requires thousands or millions of organelles. Yet, the composition of a given type of subcellular organelle can be highly heterogeneous within the same cell. For example, the properties (e.g., membrane potential and calcium level) of mitochondria can depend on their precise spatial position inside the cell. In diseases that trace themselves to mitochondrial malfunction, both normal and abnormal mitochondria coexist in a given cell. Bulk assays that average over millions of mitochondria will necessarily mask the distinctness of individual mitochondria.43 The volumes of droplets used for these single-cell and singleorganelle studies are typically comparable to the dimension of the cellular target (femtoliters to low picoliters). COMPARTMENTALIZATION Aqueous droplets effectively compartmentalize many different chemical reactions among millions of discrete reaction vessels. As such, droplets have found usage in a wide range of applications that take advantage of this feature. A current advance in DNA sequencing technology is based on droplets,9 because of their stable controlled environment, prolific generation, and ease of parallel handling. Here, the genome of interest is first broken into fragments that are individually contained in water droplets through emulsification; the aqueous phase already contains the appropriate reagents needed for PCR. Because of the confined environment created by the individual droplets, ∼1000 discrete PCR microreactions per microliter are possible; this highly parallel nature clonally amplifies each genome fragment rapidly, thereby serving the important goal of creating the target library needed for sequencing by ligation. The emulsion droplets used are polydisperse and tend to be ∼65 fL in volume. In directed evolution and gene enrichment, desired functional variants of enzymes, ribozymes, or binding proteins are selected
for by screening large gene libraries of potential variants. As opposed to traditional screening techniques, in vitro compartmentalization links the gene and its product by co-compartmentalization in aqueous emulsions.34 Emulsified droplets contain one gene per droplet, together with the necessary biomolecules for expression of the gene as well as a screening substrate. This technique provides high-throughput screening of the variants in parallel, and once chosen, the corresponding gene is simultaneously collected. The polydisperse droplets used so far are usually ∼4 fL in volume. Droplet- and plug-based microfluidics has become an attractive platform for carrying out protein crystallization because of the ability to conduct many parallel crystallization experiments and to handle the small amounts of protein samples that are available.5,33 In addition, the ability to form a stream of droplets or plugs with various contents at different concentrations is well suited to testing many possible conditions for crystallization. Because of the need to form crystals of usable size, enough reagents must be contained within each droplet, and therefore, the droplets usually need to be in the nanoliter range. DROPLET SIZE As evidenced by the above examples, applications such as growing a crystal5,33 or sequencing a gene44 require large nanoliter droplets. However, applications such as analyzing a single organelle and controlling single-molecule reactions demand femtoliter droplets to prevent dilution. In the case of directed evolution,45 high-throughput DNA sequencing,9 and digital PCR,36 small volumes increase the density of the droplets per unit volume. Nanoliter droplets and plugs tend to be quite stable and easy to manipulate in the traditional microfluidic format, whereas femtoliter droplets often require more sensitive methods of droplet manipulation and analysis. Bulk emulsions have been an established area of research for many decades, and thus, a large body of knowledge exists on these systems. For example, emulsions have found extensive use and application in petroleum processing and mining, agriculture and food production, personal care and cosmetics, materials manufacturing, environmental cleanup, and drug delivery. Most of this research has focused on the physical stabilities and properties of the emulsions. Only in recent years have emulsions been used as nanoreactors, in which it is possible to control individual droplets (as opposed to bulk emulsions) and the reaction conditions in each droplet precisely. These new uses of droplets as nanoreactors will demand a better understanding of droplet chemistry in the context of these applications, and much development lies ahead in this exciting new area. DROPLET FORMATION To utilize droplets as ultrasmall reaction containers, the first step is to develop methods for forming droplets of the desired sizes and composition. Droplets created by emulsification are highly heterogeneous, but those formed using microfluidic techniques are generally very monodisperse in size. The two most common microfluidic methods of forming droplets are continuous-stream droplet generation based on a T-channel geometry5,32,46 or with a flow-focusing design.5,30,46 In the T-channel or T-junction geometry (Figure 2a), a perpendicular flow of the continuous oil phase meets the inlet of the dispersed aqueous phase, where Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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Figure 2. Droplet generation. Images showing (ai) the formation of droplets and (aii) segmented plugs using cross flow at a T-channel. The aqueous plugs in (aii) contained red and green food dyes; the inset shows an enlarged picture of the flow pattern in the plug, and arrows emphasize the difference in contact angles of the green and red dyes with PDMS. The scale bar represents 100 µm. (bi-biii) Schematics showing the use of 2D and 3D flow focusing for droplet generation; cross-sectional views are to the right of each device. With 3D flow focusing, issues pertaining to surface wetting by the aqueous phase can be avoided because the aqueous phase does not make contact with the walls. The dimensions for flow-focusing devices range from the tens to hundreds of micrometers, with the final orifice in the range of tens of micrometers. (ci and ciii) Schematics showing the on-demand generation of single droplets using (ci and cii) a pulsed electric field and (ciii and civ) pressure. (cii) Images showing the electrogeneration of a 420-fL droplet with a single electric pulse (pulse amplitude: 1 kV; pulse duration: 10 ms; orifice dimension: 6 (D) × 6.7 (W) × 63 (L) µm3; scale bar represents 10 µm). (civ) A sequence of images showing the generation of a 65-fL aqueous droplet. The scale bar represents 4 µm. Panels adapted with permission from (ai) Ref. 32, (aii) Ref. 22, (b) Ref. 30, (ci and cii) Ref. 27, and (ciii and civ) Ref. 13.
droplets or plugs are generated. The three primary phenomena at play are viscous shear stress, interfacial tension, and an upstream increase in pressure as the emerging interface moves into the main channel. Droplets produced with diameters smaller than the width of the main channel are dependent on the capillary number (Ca) of the system, whereas plugs are dependent on the flow rate ratio, as expressed by: L/w ) 1 + RQdis/Qcon, where L is the length of the plug, w is the width of the channel, Qdis/ Qcon is the ratio of the aqueous flow rate to the oil flow rate, and R is a constant on the order of 1 that depends on the geometry of the T-junction. The flow-focusing method (Figure 2b) relies on three parallel flows, in which the aqueous phase is sandwiched between two oil-phase flows; these three flows converge on an orifice to break the aqueous flow into droplets. In the dripping regime, in which droplets break off one characteristic diameter from the orifice, the droplet diameter is smaller than the orifice diameter, and it decreases as a function of increasing Ca and decreasing flow rate ratios. In the jetting regime, droplets break off at least three orifice diameters downstream and have diameters that are proportional to the jet diameter. The advantages of these continuous-stream methods are their ease of use and extremely high throughput: thousands of droplets (from nanoliter to femtoliter size) can be generated easily per second. In contrast to continuous-stream droplet generation, the formation of single droplets on demand, together with high-precision single-droplet manipulation, offers the potential for precise control over each droplet formed.12,13,26,29 Many droplet analysis techniques, such as ones that involve chemical separation of the droplet content, do not possess the throughput needed to analyze a fast-flowing stream of droplets. Therefore, for applications that require a detailed chemical analysis of each droplet, the use of single droplets formed on demand may be more appropriate. As shown in Figure 2c, individual droplets can be created on demand by either of two methods. The first requires the application of a pulsed electric field,11,27 whereas the second uses a 5114
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source of pulsed pressure.10,13,41 Figure 2ci shows the schematic of the experimental setup for the electrogeneration of single droplets. Figure 2cii shows the formation process of a single water droplet, in which a short (ms) and intense (kV) electric pulse was used to deflect the aqueous/oil interface, resulting in the formation of an aqueous jet that breaks off into a droplet because of Rayleigh instability.11,27 The size of the droplet formed is determined by the amplitude and duration of the pulse and the dimension of the microchannel. Figure 2ciii shows a schematic13 for generating droplets using sudden changes in applied pressure. In this design, the main reaction chamber is surrounded by small inlets, and each inlet contains a different solution or chemical reagent. Figure 2civ illustrates the formation of a single droplet with this method. This fluidic design is flexible: more inlets can be added with very little change to the conditions in the reaction chamber, and the process can be easily automated using electrically driven micrometers and injectors. In summary, to achieve throughput, it is best to use steadystate methods (e.g., T-channel or flow-focusing) to generate a continuous stream of fast-flowing droplets. To retain control over individual droplets and for applications that require detailed chemical analysis of each droplet, the generation of single droplets on demand will likely be most appropriate. The volume of most continuous-stream droplets formed using steady-state methods lies in the nanoliter to mid-picoliter range, because formation of smaller droplets tends to require higher shear rates and flow rates; this makes the droplets more difficult to control and manipulate downstream. Droplets formed by using pulsed electric fields or pressures are mostly in the femtoliter range. DROPLET MANIPULATION Once formed, droplets can be manipulated with a wide range of physical mechanisms, such as pressure, dielectrophoresis,47 magnetic fields,48 optical fields,29,49 and thermal gradients.50,51 Some methods can be used to manipulate a fast-flowing stream of droplets in a high-throughput format, whereas others are best
Figure 3. Droplet manipulation. (ai and aii) Passive hydrodynamic manipulation of droplets. (ai) Droplets were sorted by size, and (aii) three droplets were fused using a flow-rectifying design. (bi and bii) Active manipulation of droplets with applied electric field. (bi) Droplets were injected with an electrostatic charge of opposite sign, which caused them to fuse when they were brought into close proximity (scale bar represents 100 µm). (bii) Sorting of charged droplets using electric field (scale bar represents 100 µm). (ci-civ) Thermoelectric (TE) manipulation of droplets. (ci) Schematic showing the placement of the TE cooler with respect to the layout of the microchannels; (cii) bright-field image showing unfrozen droplets (scale bar represents 50 µm); and images of a stream of droplets over the TE cooler (ciii) before and (civ) after freezing (scale bar represents 50 µm for both iii and iv). Labels in panel (ci) show the positions along the channel where the images shown in (cii-civ) were taken. (di-diii) Modulation of chemical concentration in vortex-trapped droplets. (di) Fluorescence images of an aqueous droplet containing Alexa 488 dye as the droplet went through one cycle of shrinkage and expansion (scale bar represents 3 µm); (dii) plot of normalized fluorescence intensity per unit volume versus the respective reciprocal volume, showing species conservation during droplet shrinkage and expansion (insets illustrate the change in the trapping position of the droplet in the axial direction as the droplet changed in volume); (diii) images showing three consecutive cycles of droplet shrinkage and expansion (scale bar represents 5 µm). Panels adapted with permission from (ai and aii) Ref. 31, (bi) Ref. 28, (bii) Ref. 52, (ci-civ) Ref. 42, and (di-diii) Ref. 12.
suited for controlling individual droplets. Assuming Stokes drag for the spherical droplets (Fd ) 6πµRV where Fd is the drag force, µ is the dynamic viscocity, R is the radius of the sphere, and V is the velocity of the sphere), the forces needed to move droplets are on the order of a few piconewtons to tens of nanonewtons, depending on droplet size and the viscosity of the oil phase. Ultrasmall droplets can be manipulated with any of the aforementioned methods, but small droplets are most commonly manipulated with hydrodynamic pressure. Figure 3 provides some examples to illustrate the range of possibilities in manipulating droplets. Figure 3a shows the passive sorting (3ai) and fusion (3aii) of droplets flowing in a continuous stream using pressure.31 Figure 3b offers an example of electric-field-mediated droplet fusion28 (3bi) and sorting52 (3bii). In addition to directing the motion of droplets using pressure or electric fields, the temperature of droplets can be controlled precisely in a high-throughput flowthrough format (Figure 3c). Here, an important and useful feature is the vastly different freezing points for aqueous solutions and immiscible oils. The freezing points of commonly used immiscible oils range from -10 °C (e.g., perfluorodecalin) to ∼ -100 °C (e.g., silicone oils). In fact, the viscosity of AS 4 silicone oil does not change significantly even down to ∼-50 °C, and AS 4 is thermostable up to 200 °C.42 As a result, the temperature of the aqueous phase can be varied over a wide range while maintaining flow in the immiscible phase. Figures 3cii-civ show the freezing of a flowing droplet stream using thermoelectric cooling. This technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as controlling reaction kinetics, on-chip continuous-flow PCR, and the on-chip freezing and storage of cellular samples.42
It is also possible to dynamically tune the concentration of molecules contained within femtoliter-volume droplets without changing the droplet temperature significantly, a feat that is difficult to achieve using a macroscopic container.12,14,49 In Figure 3d, a droplet is held by an optical vortex trap,12,13,49,53 shrinks in volume, and upon release from the trap or when the power of the trap is lowered, becomes larger again. An optical vortex trap is formed using a class of laser beam called the Laguerre-Gaussian beam, which is characterized by a helical phase distribution across its wave front that results in the formation of a stably propagating dark core. In an optical vortex trap, the aqueous droplet is trapped in this dark core because an aqueous solution is usually of a lower refractive index than its surrounding oil. The ring of light around the dark core thus acts as a light cage that confines the droplet to the vortex core. This ring of laser light also overlaps slightly with the droplet interface, which leads to localized heating (e 1 K) of the droplet and a localized solubility increase for water in the surrounding oil phase. The end result is the increased dissolution of water from the droplet into the oil surrounding the droplet and the corresponding decrease in the droplet’s volume. When the power of the vortex trap is decreased or turned off, the water dissolved in the surrounding oil phase returns to the droplet, thus leading to droplet expansion. This process is reversible (Figure 3di), and in fact it is possible to expand the volume of one droplet (target droplet) beyond its original size by accumulating the water released during the shrinkage of an adjacent droplet (donor droplet). For subpicoliter droplets, the temperature change during this process of droplet shrinkage is e 1 K, because the small size of such droplets ensures efficient heat conduction away from the droplet and prevents temperature buildup in the droplet.49 Because most biomolecules (e.g., Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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proteins, nucleic acids, metabolites, and ions) are charged and often are large, they do not partition into the oil phase and thus can become highly concentrated in the droplet as the water molecules exit the droplet during shrinkage. Dynamic control over the concentrations of dissolved species in a nanoscale reaction vessel provides a degree of control that was previously difficult to achieve in a macroscopic chemical system. IN-DROPLET ASSAYS Droplet generation and manipulation are at the front end of a droplet-based analytical platform. The types of applications made possible by using droplets also depend critically on the availability of sensitive readout methodologies. This constraint is especially pertinent for ultrasmall-volume droplets. Currently, the most sensitive and applicable technique is based on fluorescence detection, which offers detection limits down to the singlemolecule level.10,54 In fact, few alternatives exist, given the minute amounts of sample present in ultrasmall-volume droplets; for example, NMR and most common modes of electrochemistry lack the requisite sensitivity to interface with femtoliter-volume droplets. MS is another sensitive technique that has been used to analyze single-cell and organelle samples, but this method usually requires larger initial samples than optical fluorescence detection techniques do. As shown in Figure 4, four types of in-droplet assays are based on fluorescence. The simplest approach is to employ a fluorogenic substrate that turns into a fluorescent product in the presence of a catalyst, such as an enzyme (Figure 4a). In addition to encapsulating single cells, we have developed the ability to encapsulate single organelles. This method utilizes laser surgery to excise the organelle, combined with an optical trap to move the organelle to the droplet-generation region.8,55 The fluorescent detection approach can be made sensitive enough to monitor a single molecule over time in a droplet (Figure 4c). Of more immediate pertinence to cellular studies is the ability to carry out real-time PCR in droplets by using fluorescent reporters38 (Figure 4b). In addition to measuring fluorescence intensity from droplets, subcellular organelles and nanoparticles in droplets can be sized by using fluorescence correlation spectroscopy (Figure 4d); this technique measures the diffusion coefficient of molecules and thus their hydrodynamic size. Here, the use of droplets effectively confines the diffusion of the contained molecules so their trajectories can be repeatedly measured to extract an accurate diffusion coefficient.40 Figure 4dii shows sizing of synaptic vesicles in a droplet. This capability is particularly suited to the sizing of single subdiffraction-limited objects in free solution, a task that is currently difficult to accomplish with existing techniques. SEPARATION AND ANALYSIS OF DROPLET CONTENTS The use of fluorescence readout alone is highly limiting, especially for droplets that contain a complex mixture of biological molecules. To address this shortcoming, separation techniques for analyzing the contents of ultrasmall droplets have been developed.26,56 Figures 5a and 5b shows two examples, in which the contents of droplets are first emptied into a separation channel filled with aqueous buffer. This release of the droplet’s contents is achieved by fusing the droplet with the immiscible partition 5116
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Figure 4. Fluorescence-based readout of droplet content. Singlecell enzymatic assay within an aqueous droplet in soybean oil. (ai-aiv) A mast cell was encapsulated in an aqueous droplet that contained the fluorogenic substrate fluorescein di-β-D-galactopyranoside. (aiii) After laser-induced cell lysis, (aiv) β-galactosidase catalyzed the formation of the product fluorescein, which caused the droplet to become highly fluorescent. (bi) Images of the PCR chip showing the overall channel and flow configuration, droplet generation at the T-junction, and monodisperse droplets formed in the downstream channel. (bii) Real-time PCR data from picoliter droplets. Droplets were identified from the bright-field image and then monitored at each cycle to generate real-time fluorescence curves. (ci) Schematic of hydrosomes inertially injected on demand into an immiscible fluorinated oil matrix. Single molecules in the hydrosomes are optically excited, and fluorescence is detected. (cii) Single-molecule fluorescence from sulforhodamine B isolated in hydrosomes. The arrows indicate when the excitation is switched on. (di) Fluorescence correlation spectroscopy uses point detection with a confocal probe volume to detect burst events from contained particles at the center of a water droplet, thus measuring the diffusion time and size of the particles. (dii) Normalized fluorescence correlation curves of synaptic vesicles encapsulated within a droplet (solid dark gray) and in bulk solution (dashed light gray). Panels adapted with permission from (ai-aiv) Ref. 8, (bi and bii) Ref. 38, (ci and cii) Ref. 10, and (di and dii) Ref. 40.
that separates the aqueous phase in the separation channel with the immiscible phase contained within the droplet-generation and manipulation region of the microfluidic system. Once the contents have been emptied into the separation channel, a high voltage is applied to carry out CE separation. CE is particularly suited for the separation and analysis of ultrasmall-volume droplets because of its high separation efficiency, speed, and sensitivity. Because of the minute amounts of sample contained within an ultrasmall-volume droplet, we have developed a two-beam lineconfocal detection geometry for measuring the electrophoretic mobility of individual molecules undergoing continuous-flow CE
Figure 5. Chemical separation and analysis of droplet contents. (ai, aii, and b) Analysis of droplet contents by CE. (ai) A sequence of images showing the generation and transport of a single aqueous droplet to the CE separation channel; (aii) electropherogram showing the separation of fluorescein isothiocyanate (FITC) and FITC-labeled glycine, glutamate, and aspartate from a femtoliter-volume droplet. (aiii and aiv) Continuous-flow single-molecule CE. (aiii) Simulation showing the distribution of laser intensity across the width of a 2-µm channel for two-beam cross-correlation spectroscopy and continuousflow CE, and (aiv) measurement of the electrophoretic mobility of FITC, FITC-labeled glycine, and FITC-labeled glutamate (the inset shows the same data as a function of migration velocity). (bi-biii) Sampling of droplet contents with CE. (bi) Microfluidic design highlighting the K interface where sampling of droplet contents occurs, and (bii) a series of images showing the sampling of a 12-nL droplet (the sampled volume was ∼20 pL). (biii) Electropherogram that resulted from the sampling of plugs containing 1-µM FITC-labeled serine. (c) Droplet compartmentalization of CE separation. (ci) Schematic illustrating the technique for encapsulating the outflow of a chemical separation into individual droplets, which can be docked and stored on-chip for further analysis; (cii) electropherogram showing the separation of a mixture of amino acids; (ciii) a blowup of the glutamate peak, showing that the separated band is encapsulated into a series of droplets. (d) MS analysis of nanoliter volumes. (di) Analysis of 30-nL sample plugs with MALDI MS (in a dihydroxybenzoic acid matrix). (diii) TOF-SIMS (positive-ion mode with a C60+ source and a 1 × 1 mm2 imaging area) analysis of the contents of picoliter vials. (dii) Scanning electron microscopy image showing the tapered walls and flat bottom of a ∼100-pL volume vial etched in a Si wafer; (diii) positive-ion SIMS images of phosphatidylglycerol (red), phosphatidylcholine (green), cholesterol (white), and sulfatide (yellow) in four picoliter-volume vials. Panels adapted with permission from (ai and aii) Ref. 26, (aiii and aiv) Ref. 54, (bi-biii) Ref. 56, (ci-ciii) Ref. 39, (di) Ref. 16, (dii) Ref. 57, and (diii) Ref. 58.
separation (Figures 5aiii and aiv). Continuous-flow CE is particularly suited for analyzing ultrasmall-volume samples because of the rapid dilution that occurs when the droplet’s contents are emptied into the CE separation channel and when the contained molecules are no longer confined by the immiscible boundary. Within 10 ms, for example, a 1-fL droplet containing 1 mM of analyte will be diluted to a volume of 1 pL with 1 µM of analyte.
After 1 s, this concentration drops to 1 nM. Figure 5aiv shows our use of two-beam fluorescence cross-correlation spectroscopy to measure the electrophoretic mobility of single amino acids tagged with fluorescein, in which we demonstrated an overall detection efficiency of >94% for single dye molecules flowing in a 2-µm-wide channel. Overall, the ability to use CE for droplet analysis greatly expands the current repertoire of techniques used to study the chemistries that occur within aqueous droplets. CE is beneficial for the analysis of droplets, but in return droplets also can facilitate CE analysis of ultrasmall volumes. In Figure 5ci, droplets are used to compartmentalize the bands separated by CE, thus preventing the dilution and loss of the separated components and facilitating their downstream manipulation and analysis; each peak in Figure 5ciii is a droplet. The droplet-compartmentalized band might contain unresolved analytes, in which case the droplets might be removed for a seconddimension separation.39 Alternatively, the droplets can be docked and stored on-chip for a range of additional analysis. This approach should be particularly useful in the analysis of complex cellular components. The analysis of ultrasmall-volume droplets will be greatly enhanced by the use of high-information-content measurement techniques such as NMR15 and MS.16,57,58 Unfortunately, these methods currently lack the needed sensitive interface for most ultrasmall-volume droplet applications. The utility of MS, however, has the potential to be greatly improved by more efficient ionization techniques and sample transfer from ambient pressure into the mass spectrometer. Figure 5d shows the analysis of 30nL plugs with MALDI and 100-pL microvials with TOF-SIMS. New applications will undoubtedly emerge when MS can be used for the routine chemical analysis of ultrasmall volumes. OUTLOOK Like earlier miniaturized techniques, ultrasmall-volume droplets are an enabling technology. Their ultimate impact in biology and analytical chemistry will be determined by many factors, including ease of use, robustness, and the spectrum of methods available for manipulating and analyzing such small volumes. The intense research activity in this area during the past 5 years has resulted in a range of methods for forming and manipulating ultrasmall droplets, and this aspect of droplet research is beginning to mature. Much less developed are sensitive separation and readout technologies for analyzing the potentially complex but extremely limited samples that are contained within droplets. Fluorescencebased single-molecule CE and real-time PCR are powerful methods for analyzing the molecular contents of droplets, but the range of biological problems that can be attacked using these techniques is likely narrower than one would like. Therefore, pushing the limit of sensitivity of MS and other high-information-content analytical techniques for droplet analysis will greatly expand the range of droplet applications. Given the fact that biological interactions in cells inherently take place in ultrasmall volumes, there always will be a need to interface with biochemical reactions at these small scales. From this perspective, the future of ultrasmall-volume chemistry is exciting, and developments going forward will undoubtedly enhance our ability to understand and control how biological systems function. Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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ACKNOWLEDGMENT We are grateful for support provided by the National Institutes of Health (EB005197), the National Science Foundation (CHE0844688), the Keck Foundation, and the Sloan Foundation. Daniel T. Chiu is a professor of chemistry at the University of Washington Seattle. He is a member of the Center for Nanotechnology and the Neurobiology and Behavior Program at the University of Washington, as well as a member of the Fred Hutchinson Cancer Research Center’s Cancer Consortium. His current research is focused on developing new methods for probing complex biological processes at the single-cell and singlemolecule levels and on applying these new techniques for addressing pressing biological problems. Robert M. Lorenz and Gavin D. M. Jeffries are research associates in the Department of Chemistry at the University of Washington. Lorenz’s research focuses on the development of optical and microfluidic techniques for droplet-based single-cell and organelle studies. Jeffries’s research interest lies in the development of new optical trapping, single-molecule detection, and droplet manipulation techniques. Address correspondence about this article to Chiu at chiu@ chem.washington.edu.
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