We are thankful to the ACS PRF (grant 60302-UR9), Agrobio S.L. (contract #311325), and MCIN/AEI/10.13039/501100011033/FEDER, UE (grant No. PID2021-122369NB-I00).

<ol>
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The authors have nothing to disclose.

The protocol to fabricate three different glass-based devices has been described above. In the case of the device to generate simple drops, the flow rate and liquid properties are crucial to generate drops in a controlled manner. Drops will form at the tip in the dripping regime, or at the end of the jet in the jetting regime. The transition from dripping to jetting is parametrized by the dimensionless Weber number, We23. This number represents the ratio between inertial and surface tension forces, <img alt="Equation 1" src="/files/ftp_upload/63376/63376eq01.jpg" />), where &#961; is the density of the liquid,&#160;&#947; is the interfacial tension, Q is the flow rate and dtip is the diameter of the tip. When We &#60; 1, dripping occurs. For We &#62; 1, inertia forces overcome surface tension forces holding the drop at the tip, and a jet forms. Eventually, the jet will break into drops due to Rayleigh-Plateau instabilities. Thus, for a fixed liquid and experimental setup, the flow rate is the parameter that controls the transition from dripping to jetting. The dripping regime is characterized by resulting in nearly monodisperse drops, so it is desirable for drop generation, although production frequency is higher when drops are generated in the jetting regime.
For the coflow device, a square and a round capillary are used to make the two fluids coaxially&#160;flow in an easy and affordable manner. Note the tip size is much smaller than the size of the square capillary. The behavior in coflow is richer than that observed in the experiments described in step 1. A detailed discussion on dripping and jetting in coflow can be found in23. More details on drop size control can be found in24.
Adding a third liquid to the coflowing scheme would lead to what we called electro-coflow. Connecting a power supply to the metallic parts of the needles used for the inner and collector liquids allows creating an electric field in the region between them. Because the needles are in contact with conducting liquids (inner and collector liquids), these act as liquid wires setting the potential difference between the tip and the collector meniscus. Changing the properties of the outer fluid, like its viscosity or the flow rate, increases the richness and features of the modes relative to what is observed in standard electrospray22. For example, Figure 11 shows that the whipping mode has an ordered structure under certain experimental conditions17. This allows the study of its geometrical properties, which is not typically possible in electrospray.
The electro-coflow technique is able to overcome most of the problems that renders other electrically assisted techniques unsteady. One of the problems presented in electrically assisted techniques is that the emitted charged drops tend to discharge anywhere where the electric potential is lower than the one applied at the tip before they reach the counter-electrode. This is the reason 2 mm capillaries are suggested for our setup.&#160;The hydrophobic treatment of the square capillary avoids any drops from getting stuck on the walls allowing them to travel unperturbed until they reach the liquid collector, where they discharge. Using a liquid counter-electrode (see Figure 9) instead of the more typical metallic electrodes, eliminates charge accumulation and significant distortions in the electric field that would ultimately affect the drop production process and severely affect emulsion monodispersity.
An important practical detail related to the fabrication of the devices is the time it takes to build them. In all cases, the process takes a few hours (even less, if the glass treatment is done beforehand), but unfortunately, the epoxy needs about 10 h to cure. It is thus advisable to wait until the next day to test and use the devices.
One of the critical steps for the manufacturing of these three devices and ensuring reproducibility is the glass treatment. Glass should be rendered hydrophobic or hydrophilic depending on the liquids used. Avoiding wetting along the outside of the tip helps in achieving the steady-state production of drops.
An important question for all three devices pertains to how to pump the liquids: whether a syringe pump (fixed flow rate) or a pressure-driven setup (fixed pressure difference) is to be used. A syringe pump will allow the flow rate control of the liquids. A drawback for the syringe pumps is the introduction of vibrations in the system coming from the step size of the pump motor. For the pressure system, the drawback is the unknown flow rates of the liquids. The calibration of the system is an option, measuring the volume of the collected liquid for a given amount of time for different pressures. Some inconveniences of this method are that the dimensions of the tubing should remain constant every time they are changed, and the saturation of the filters in the lines (if used) could alter the calibration. An alternative is to calculate the flow rate of the inner liquid by measuring the drop production rate; measuring the size of the emitted drops during the dripping mode and its emission frequency will provide the flow rate. For the flow rate of the outer liquid, the volume of liquid collected during the time of the experiment can be measured. An inconvenience of doing this is that these flow rates are known a posteriori, and not during the execution of the experiments.
There are many applications38,39,40 of the technologies presented here in fields like cosmetics, food industry, and drug delivery among many others, like the use of the resultant emulsions as templates for gels applied in intensive agriculture. An upsurge application of microfluidic-related technologies&#160;is the development of innovative feeding systems for beneficial arthropods that will contribute to developing alternative regenerative agriculture. Nowadays, global food production systems are facing the challenge of satisfying the demands for increased productivity while maintaining their environmental and economic sustainability25. The release of mass-reared natural enemies, predators, and parasitoids of pests on crops has been shown to be a feasible and desired alternative to pesticide use from an environmental and economic perspective. Major achievements have been obtained in greenhouses introducing polyphagous predators13,27,34. The application of supplemental foods in crops promotes the early and long-term establishment of these predators when natural preys are scarce26,28,30, improving their resilience to different stressors. This is considered a valuable biological control supportive strategy that will optimize and expand biocontrol programs, both in protected and open field crops.
Bio producers of these predators have swiftly moved from an artisanal to a professional industry32, and the recent application of advanced analytical techniques with a holistic approach will allow us to better understand the nutritional requirements of the predators36. Although for some species commuting between different food sources can be beneficial31, most of the diets currently used are still based on a single factitious prey. Complementary artificial liquid diets should be considered to ensure a balanced diet. Liquid diets need to be encapsulated for their presentation. This strategy offers several benefits such as protection of the bioactive ingredients from abiotic factors of the environment (moisture, temperature, light, air, etc.), prevention of oxidation and evaporation loss, improvement of stability, and the increase of bioavailability, among others29,33. Some patents based on encapsulated artificial diets for feeding purposes of beneficial entomophagous insects have been reported (US Patent Nos. 5,799,607 and 6,129,935), but the commercial scale-up of these applications need to grow in parallel with the emerged knowledge of nutritional compositions of foods and predator requirements, together with microfluidic technologies adjusted for these in-crop field release conditions.

Controlled generation of drops in the micron and nanoscale with a narrow size distribution is a challenging task. These drops are of interest for the engineering of soft materials with many applications in science and technology1,2,3,4,5,6.
The most common devices for the high production rate&#160;of drops are mixers7 and ultrasound emulsificators8. These methods are simple and low cost, but they typically result in polydisperse drops with a wide range of sizes. Hence, additional steps are required to produce monodisperse samples. Microfluidic devices can be designed differently to provide an efficient way to drop formation. Additionally, the usually low flow rates involved (i.e., low Reynolds number) allow for great&#160;control over the fluid flow.
While microfluidic devices are commonly made using lithographic techniques with poly(dimethyl) siloxane (PDMS), this manuscript focuses on glass-based capillary devices. PDMS devices are usually chosen for their ability to design complex channel patterns and because of their scalability. Glass devices, on the contrary, are rigid and have greater solvent resistance than their PDMS counterparts. Additionally, glass can be modified to change its wettability, which allows controlling the generation of complex emulsions. Being able to independently treat the nozzle and channel walls enables the formation of drops in a controlled and reproducible manner, while assuring&#160;the stability of the resultant emulsions if the drops were to touch the walls9; otherwise the drops may coalesce and accumulate at the wall. Another difference between these two types of devices is that in glass-based devices, the flow is three-dimensional, while it is planar in conventional PDMS devices. This fact minimizes the drop contact with the channel walls so that the influence of contact lines can be neglected10, thereby protecting the stability of multiple emulsion drops.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63376/63376fig01.jpg" /> 
Figure 1: Different microfluidic device configurations. Sketches of (A) a T-junction, (B) a coflowing device, and (C) a flow-focusing device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63376/63376fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are three main geometries used, namely T-junction11, flow focusing12,13, and coflow14. In the T-junction geometry, the dispersed phase contained in the channel perpendicularly intersects the main channel which houses the continuous phase. The shear stress exerted by the continuous phase breaks the incoming dispersed liquid resulting in drops. The generated drops are limited in lower size by the dimensions of the main channel11. In the flow-focusing geometry, the two fluids are forced through a small orifice that is located in front of the injection tube. The result is&#160;the formation of a jet, which is much smaller than the injection tube12,13. Finally, the coflow geometry has a configuration characterized by the coaxial flow of two immiscible fluids14. In general, dripping and jetting can be observed depending on the operating conditions. The dripping regime occurs at low flow rates and the resulting droplets are very monodisperse and have a diameter proportional to the tip size. The drawback is its low production frequency. The jetting regime occurs at higher flow rates as compared to the dripping regime. In this case, the drop diameter is directly proportional to the diameter of the jet which can be much smaller than the diameter of the tip under the right conditions.
An alternative to these hydrodynamic approaches relies on the additional use of electric forces. Electrospray is a well-known and widely used technique for generating droplets. It is based on the principle that a liquid with a finite electrical conductivity will deform in the presence of a strong electric field. The liquid will eventually adopt a conical shape resulting from&#160;the balance between electric and surface tension stresses15. The process starts with the electric field inducing an electric current in the liquid that causes charges to accumulate at the surface. The presence of the electric field results in an electric force on these charges, which drags the liquid along, elongating the meniscus in the direction of the field. Under different conditions, the meniscus can either shed the charged drops or may emit one or several jets which then break into drops15. Although these electrically assisted microfluidic methods naturally allow the generation of small drops, they suffer from a lack of a steady-state operation that compromises the emulsion monodispersity. The resultant charged drops tend to discharge on the confining walls and/or anywhere in the device where the electric potential is lower than the imposed external voltage. Thus, the electrified meniscus becomes unstable, ultimately emitting drops in a chaotic way and causing their uncontrolled production and loss of monodispersity.
In electro-coflow, the electrical and hydrodynamic stresses are coupled in a coflow microfluidic device16 similar to the one used for generating double emulsions12. Two main features allow electro-coflow to be successful in reaching a steady-state emission regime: (i) the dispersed phase is ejected into another coflowing viscous liquid, and (ii) the use of a liquid counter-electrode or ground. Having a flowing outer liquid has proven to change the geometric properties of the drop emission process17. The liquid counter-electrode allows the discharge and extraction of the resultant drops, assuring the steady-state generation of drops. In addition, by exploiting the balance of electrical and hydrodynamic forces, the resultant drop sizes can potentially vary within a&#160;wider range than the sizes that can be covered by any of the previously mentioned techniques.
This detailed video protocol is intended to help new practitioners in the&#160;use and fabrication of glass-based microfluidics.

<table><tbody><tr><td>2-[methoxy(polyethyleneoxy)6-9propyl] trimethoxysilane.</td><td>Gelest</td><td>SIM6492.7</td><td></td></tr><tr><td>Ceramic tile</td><td>Sutter</td><td>CTS</td><td></td></tr><tr><td>Ethylene glycol</td><td>Fisher</td><td>BP230</td><td>These can be found at other companies like Sigma-Aldrich</td></tr><tr><td>Hexane</td><td>Sigma- Aldrich</td><td>34859</td><td>Available in other vendors</td></tr><tr><td>ITW Polymers Adhesives Devcon 5 Minute Epoxy Adhesive 25 mL Dev-Tube</td><td>Ellsworth adhesives</td><td>470740</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF 830</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter</td><td>P97</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher</td><td>12-544-1</td><td>Available in other vendors</td></tr><tr><td>Needle 20 Gauge, .0255&quot; ID, .0355&quot; OD, 1/2&quot; Long</td><td>McMaster</td><td>75165A677</td><td></td></tr><tr><td>SDS</td><td>Sigma-aldrich</td><td>428015</td><td>Surfactant</td></tr><tr><td>Silicone oil</td><td>Clearco</td><td>PSF-10cSt</td><td>The catalog number correspond to the 10cSt viscosity oil. Different viscosity oils can be found at this company</td></tr><tr><td>Span 80</td><td>Fisher</td><td>S0060500G</td><td>non-ionic surfactant</td></tr><tr><td>Square glass capillary 2mm ID (borosillicate 300 or 600 mm long)</td><td>VitroCom</td><td>S 102</td><td></td></tr><tr><td>Standard Glass Capillaries, 6 in., 2 / 1.12 OD/ID</td><td>World Precision instruments</td><td>1B200-6</td><td>These can be found at other companies like Sutter or Vitrocom</td></tr><tr><td>Syringe pump</td><td>Chemyx</td><td>FUSION 100-X</td><td>This model has a good quality/price ratio</td></tr><tr><td>Syringes (it will depend on the compatibility with the liquids)</td><td>Fisher</td><td></td><td>Catalog number will depend on the size</td></tr><tr><td>Trimethoxy(octyl)silane</td><td>Sigma- Aldrich</td><td>376221</td><td>Available in other vendors</td></tr><tr><td>Tubing ( it will depend on the compatibility with the liquids)</td><td>Scientific commodities</td><td>BB3165-PE/5</td><td>This reference is for polyethylene micro tubing. The size fits the needle size listed here</td></tr></tbody></table>

glass-based devices to generate drops and emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

In this manuscript, three different devices have been designed to generate drops. We have generated drops with a size of (3.29 &#177; 0.08) mm&#160;(Figure 4B) and (1.75 &#177; 0.04) mm&#160;(Figure 4C) using the device described in step 1. The emulsion drops can be generated using the coflow and the electro-coflow devices. For the latter, we show dripping in Figure 9, while cone-jet and whipping modes are shown in Figure 10 and Figure 11, respectively. In Figure 9 we show results using the same liquid as inner and collector liquids. If the goal of the experiments is collecting these drops, a different conducting liquid should be used as a collector (see&#160;18 for more details), as otherwise, the drops will merge with the collector as they touch.
The cone-jet and whipping modes are the most studied for their multiple practical applications; they are two of the many other modes that appear in electro-coflow19,20,21,22. For a more systematic review of the effect of the experimental parameters (flow rates and applied voltage), see the Discussion section and 22. These modes are steady in time when generated in the devices described in the manuscript. The steadiness of these modes allows their characterization using high-speed imaging with a microscope and related image processing.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63376/63376fig10.jpg" /> 
Figure 10: Cone-jet mode. Inner and collector liquid: ethylene glycol; outer liquid: 0.65 cSt silicone oil; inner flow rate is 16 &#181;L/h; outer flow rate is 30 mL/h; applied voltage is 750 V. The scale bar corresponds to 100 &#181;m. Microscope magnification is 20x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63376/63376fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63376/63376fig11.jpg" /> 
Figure 11: Whipping mode. Inner and collector liquid: ethylene glycol; outer liquid: 10 cSt silicone oil; inner flow rate is 240 &#181;L/h; outer flow rate is 20 mL/h; applied voltage is 1200 V. The scale bar corresponds to 100 &#181;m. Microscope magnification is 20x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63376/63376fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Glass-Based Devices to Generate Drops and Emulsions at JoVE.com

Glass-Based Devices to Generate Drops and Emulsions

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

glass-based-devices-to-generate-drops-and-emulsions

Nanyang Technological University

Research

JoVE Journal

Engineering

2.6K Views.  Augusta University. Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented. 

Video: Glass-Based Devices to Generate Drops and Emulsions

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 &#181;m/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

Chemistry

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Bioengineering

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...