Thermoelectric manipulation of aqueous droplets in microfluidic devices

This article describes a method for manipulating the temperature inside aqueous droplets, utilizing a thermoelectric cooler to control the temperature of select portions of a microfluidic chip. To illustrate the adaptability of this approach, we have generated an "ice valve" to stop fluid flow in a microchannel. By taking advantage of the vastly different freezing points for aqueous solutions and immiscible oils, we froze a stream of aqueous droplets that were formed on-chip. By integrating this technique with cell encapsulation into aqueous droplets, we were also able to freeze single cells encased in flowing droplets. Using a live-dead stain, we confirmed the viability of cells was not adversely affected by the process of freezing in aqueous droplets provided cryoprotectants were utilized. When combined with current droplet methodologies, this technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as freezing, temperature cycling, sample archiving, and controlling reaction kinetics.     

Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 microm wide by 3 microm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 microm wide by 50 microm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a approximately 10-fL droplet.     

Automated microfluidic screening assay platform based on DropLab

This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.     

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.     

Using a multijunction microfluidic device to inject substrate into an array of preformed plugs without cross-contamination: comparing theory and experiments

In this paper we describe a multijunction microfluidic device for the injection of a substrate into an array of preformed plugs carried by an immiscible fluid in a microchannel. The device uses multiple junctions to inject substrate into preformed plugs without synchronization of the flow of substrate and the array of preformed plugs of reagent, which reduces cross-contamination of the plugs, eliminates formation of small droplets of substrate, and allows a greater range of injection ratios compared to that of a single T-junction. The device was based on a previously developed physical model for transport that was here adapted to describe injection and experimentally verified. After characterization, the device was applied to two biochemical assays, including evaluation of the enzymatic activity of thrombin and determination of the coagulation time of human blood plasma, which both provided reliable results. The reduction of cross-contamination and greater range of injection ratios achieved by this device may improve the processes that involve addition and titration of reagents into plugs, such as high-throughput screening of protein crystallization conditions.     

Microfluidic and optical systems for the on-demand generation and manipulation of single femtoliter-volume aqueous droplets

This paper describes a fluidic and optical platform for the generation and manipulation of single femtoliter-volume aqueous droplets. Individual droplets were generated on-demand using a microfluidic chamber that confers environmental flow stability. Optical vortex traps were implemented to manipulate and transport the generated droplets, which have a lower refractive index than the immiscible medium in which the droplets are immersed and thus cannot be trapped using conventional optical tweezers. We also demonstrated the ability to shrink and increase the refractive index of the generated droplet, thereby permitting its facile fusion with another droplet using an optical tweezer. To illustrate the versatility of this platform, we have performed both fast (<1 s) and slow (>1 h) chemical reactions in these femtoliter-volume aqueous droplets.     

Microfluidic fabrication of asymmetric giant lipid vesicles

We have developed a microfluidic technology for the fabrication of compositionally asymmetric giant unilamellar vesicles (GUVs). The vesicles are assembled in two independent steps. In each step, a lipid monolayer is formed at a water-oil interface. The first monolayer is formed inside of a microfluidic device with a multiphase droplet flow configuration consisting of a continuous oil stream in which water droplets are formed. These droplets are dispensed into a vessel containing a layer of oil over a layer of water. The second lipid monolayer is formed by transferring the droplets through this second oil-water interface by centrifugation. By dissolving different lipid compositions in the different oil phases, the composition of each leaflet of the resulting lipid bilayer can be controlled. We have demonstrated membrane asymmetry by showing differential fluorescence quenching of labeled lipids in each leaflet and by demonstrating that asymmetric GUVs will bind an avidin-coated surface only when biotinylated lipids are targeted to the outer leaflet. In addition, we have demonstrated the successful asymmetric targeting of phosphatidylserine lipids to each leaflet, producing membranes with a biomimetic and physiologically relevant compositional asymmetry.     

Investigation of concentrically and eccentrically layered droplets by light scattering

Two pairs of immiscible liquid compounds are chosen to prepare levitated layered droplets with and without density difference between core and layer phases. The droplets are examined by light scattering along two orthogonal directions. A layered droplet without phase density difference is unambiguously identified as a concentric sphere by matching the observed scattering spectra with those calculated from the Aden-Kerker extension of Mie theory. For layered droplets with phase density difference, only the scattering spectrum from one of the scattering directions can be matched theoretically. These observations suggest that a static layered droplet is predominantly eccentric even though the embedded core is large by volume, as predicted from fluid mechanics. The consistency of the light-scattering characterization with the diffusion theory governing the evaporation of concentrically and eccentrically layered droplets is also established.     

Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient

In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the β-galactosidase-catalyzed hydrolysis of resorufin-β-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.     

Formation of Arrayed Droplets by Soft Lithography and Two-Phase Fluid Flow, and Application in Protein Crystallization

This paper presents an overview of our recent work on the use of soft lithography and two-phase fluid flow to form arrays of droplets. The crucial issues in the formation of stable arrays of droplets and alternating droplets of two sets of aqueous solutions include the geometry of the microchannels, the capillary number, and the water fraction of the system. Glass capillaries could be coupled to the PDMS microchannels and droplets could be transferred into glass capillaries for long-term storage. The arrays of droplets have been applied to screen the conditions for protein crystallization with microbatch and vapor diffusion techniques.     

Forming Sticky Droplets from Slippery Polymer Zwitterions

Polymer zwitterions are generally regarded as hydrophilic and repellant or “slippery” materials. Here, a case is described in which the polymer zwitterion structure is tailored to decrease water solubility, stabilize emulsion droplets, and promote interdroplet adhesion. Harnessing the upper critical solution temperature of sulfonium‐ and ammonium‐based polymer zwitterions in water, adhesive droplets are prepared by adding organic solvent to an aqueous polymer solution at elevated temperature, followed by agitation to induce emulsification. Droplet aggregation is observed as the mixture cools. Variation of salt concentration, temperature, polymer concentration, and polymer structure modulates these interdroplet interactions, resulting in distinct changes in emulsion stability and fluidity. Under attractive conditions, emulsions encapsulating 50–75% oil undergo gelation. By contrast, emulsions prepared under conditions where droplets are nonadhesive remain fluid and, for oil fractions exceeding 0.6, coalescence is observed. The uniquely reactive nature of the selected zwitterions allows their in situ modification and affords a route to chemically trigger deaggregation and droplet dispersion. Finally, experiments performed in a microfluidic device, in which droplets are formed under conditions that either promote or suppress adhesion, confirm the salt‐responsive character of these emulsions and the persistence of adhesive interdroplet interactions under flow.     

Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation

This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (s/n) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second.     

Microfluidic droplet-based liquid-liquid extraction

We study microfluidic systems in which mass exchanges take place between moving water droplets, formed on-chip, and an external phase (octanol). Here, no chemical reaction takes place, and the mass exchanges are driven by a contrast in chemical potential between the dispersed and continuous phases. We analyze the case where the microfluidic droplets, occupying the entire width of the channel, extract a solute-fluorescein-from the external phase (extraction) and the opposite case, where droplets reject a solute-rhodamine-into the external phase (purification). Four flow configurations are investigated, based on straight or zigzag microchannels. Additionally to the experimental work, we performed two-dimensional numerical simulations. In the experiments, we analyze the influence of different parameters on the process (channel dimensions, fluid viscosities, flow rates, drop size, droplet spacing, ...). Several regimes are singled out. In agreement with the mass transfer theory of Young et al. (Young, W.; Pumir, A.; Pomeau, Y. Phys. Fluids A 1989, 1, 462), we find that, after a short transient, the amount of matter transferred across the droplet interface grows as the square root of time and the time it takes for the transfer process to be completed decreases as Pe-2/3, where Pe is the Peclet number based on droplet velocity and radius. The numerical simulation is found in excellent consistency with the experiment. In practice, the transfer time ranges between a fraction and a few seconds, which is much faster than conventional systems.     

Spraying mode effect on droplet formation and ion chemistry in electrosprays

Depending on the spraying conditions and fluid properties, a variety of electrospray regimes exists. Here we explore the changes in ion production that accompany the transitions among the three axial spraying modes, the burst mode, the pulsating Taylor cone mode, and the cone-jet mode. Spray current oscillation and phase Doppler anemometry measurements, fast imaging of the electrified meniscus, and mass spectrometry are utilized to study the formation, size, velocity, and chemical composition of droplets produced in the three modes. High-speed images indicate that the primary droplets are produced by varicose waves and lateral kink instabilities on the liquid jet emerging from the Taylor cone, whereas secondary droplets are formed by fission. Dramatic changes in the droplet size distributions result from the various production and breakup mechanisms observed at different emitter voltages and liquid flow rates. We demonstrate that droplet fission can be facilitated by space charge effects along the liquid jet and in the plume. Compared to the other two regimes, a significantly enhanced signal-to-noise ratio, a lower degree of analyte oxidation, and milder fragmentation are observed for the cone-jet mode.     

On‐Site Formation of Emulsions by Controlled Air Plugs

Air plugs are usually undesirable in microfluidic systems because of their detrimental effect on the system's stability and integrity. By controlling the wetting properties as well as the topographical geometry of the microchannel, it is reported herein that air plugs can be generated in pre‐defined locations to function as a unique valve, allowing for the on‐site formation of various emulsions including single‐component droplets, composite droplets with droplet‐to‐droplet concentration gradient, blood droplets, paired droplets, as well as bubble arrays without the need for precious flow control, a difficult task with conventional droplet microfluidics. Moreover, the self‐generated air valve can be readily deactivated (turned off) by the introduction of an oil phase, allowing for the on‐demand release of as‐formed droplets for downstream applications. It is proposed that the simple, yet versatile nature of this technique can act as an important method for droplet microfluidics and, in particular, is ideal for the development of affordable lab‐on‐a‐chip systems without suffering from scalability and manufacturing challenges that typically confound the conventional droplet microfluidics.     

Experimental study on convergence of droplet streams under microgravity

Experiments on the convergence of two droplet streams have been carried out under microgravity in order to develop a technique for converging droplet streams under microgravity and to examine the behavior of droplets in a vacuum and under microgravity after the binary droplets collide with each other. The working fluid is silicone oil with a low vapor pressure. In this study, a method of orienting the droplet generators toward a convergence point has been tested. In all of the 68 experiments conducted under microgravity, it is confirmed that droplet streams are converged. It has been concluded that the method of orienting multiple droplet generators to a convergeing point is effective for converging droplet streams under microgravity. The behaviors of the colliding droplets under microgravity and in a vacuum have been classified into five types. The five types of behavior are mapped on a We (Weber number) — B (impact parameter) diagram. The range of Weber numbers in the experiments is from 200 to more than 3000.     

Pressure-driven laminar flow in tangential microchannels: an elastomeric microfluidic switch

This paper describes laminar fluid flow through a three-dimensional elastomeric microstructure formed by two microfluidic channels, fabricated in layers that contact one another face-to-face (typically at a 90 degree angle), with the fluid flows in tangential contact. There are two ways to control fluid flow through these tangentially connected microchannels. First, the flow profiles through the crossings are sensitive to the aspect ratio of the channels; the flow can be controlled by applying external pressure and changing this aspect ratio. Second, the flow direction of an individual laminar stream in multiphase laminar flow depends on the lateral position of the stream within the channel; this position can be controlled by injecting additional streams of fluid into the channel. We describe two microfluidic switches based on these two ways for controlling fluid flow through tangential microchannels and present theoretical arguments that explain the observed dependence of the flow profiles on the aspect ratio of the channels.     

Creating liquid droplets in gas bubbles by means of light beam controlled capillary convection

A new approach to obtaining liquid droplets for microfluidic devices is proposed. According to this method, a droplet with a volume of several tens of nanoliters is formed under the action of a light beam upon a film of wetting liquid surrounding an air bubble, which occurred within a thin layer of this liquid confined between two plates. The kinetics of growth of liquid droplets in air bubbles of various dimensions is studied, and the characteristic droplet size is estimated.     

Equations of the kinetics of droplet fragmentation in a high-speed gas flow

For the conditions of a high-speed gas flow, within the framework of the model of quasi-continuous fragmentation of a droplet due to the mechanism of gradient instability, a differential equation of mass loss has been obtained. Within the approximation of the droplet sphericity, the law of variation of its mass, which depends on droplet acceleration by the gas stream, as well as the conditions and time of complete fragmentation of the droplet, have been found. A differential equation for the quantity of torn off droplets, has been obtained. In the event of equality between the rates of dispersion and relaxation equalization of the droplet and gas flow velocities, the size distribution functions of the number and mass of torn off droplets, as well as the values of the modal radius and total number of torn off droplets, have been found.     

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.