The authors would like to acknowledge the DFG for the support.

1. Hydrophobization of Magnetic Nanoparticles
<ol>
	<li>For the synthesis of the hydrophobic magnetic particles, add 0.85 g FeCl3 hexahydrate (3.14 mmol) and 0.30 g FeCl2 tetrahydrate (1.51 mmol) to 200 mL water/ethanol solution (4:1 v/v).</li>
	<li>To this mixture, add 0.20 mL 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PFOTES) (5.23 mmol) with vigorous stirring by a magnetic stirrer (500 rpm). Carry out the synthesis in an inert gas atmosphere (N2) by using a round-bottom flask with a cork in order to prevent the secondary oxidation of the magnetite particles.</li>
	<li>Adjust the solution drop wise with an ammonium hydroxide solution (1.5 M) to a pH of 8 (pH determined by a pH meter). Stir the solution for 24 h by using a magnetic stirrer.</li>
	<li>Separate the particles magnetically from the solution by placing the flask on a bar magnet (neodymium cuboid magnet 40 x 20 x 10 mm, adhesive force 25 kg). Pour out the solution while keeping the magnet attached to the bottom of the flask.</li>
	<li>Wash the particles three times with the water/ethanol solution while using a bar magnet as described in 1.4). Dry the particles at 60 &#176;C for 24 h (yield ca. 0.43 g).</li>
	<li>For analyzing the particles, use a Scanning Electron Microscope according to manufacturer&#39;s instructions.</li>
</ol>
2. Fabrication of Microreactors
<ol>
	<li>Grind all the dried particles slightly using a glass pestle and subsequently place all of them directly in a weighing pan (46 &#215; 46 &#215; 8 mm, polystyrene).</li>
	<li>Pipette 10 &#181;L reaction solution (composition as described in 5.1) onto all the particles and move the weighing pan slightly in a circular way for approximately 10 s (particle mass for a 10 &#181;L microreactor: ca. 3.2 x 10-7 kg 33). Store the remaining particles (particles which did not self-assemble around the reaction solution) at room temperature for further applications.</li>
	<li>For measuring the contact angle of the microreactor, build a 5 &#181;L microreactor with water as described in 2.2), place it on a teflon film and analyze the contact angle by using an optical contact angle measurement device according to manufacturer&#39;s instructions.</li>
</ol>
3. 3D-printing of the Coil Bodies
<ol>
	<li>Design the double coil bodies with a height of 16 mm (one chamber), a diameter of 10 mm and an inner diameter of ca. 4 mm by using a CAD software according to manufacturer&#39;s instructions.</li>
	<li>Print the coil bodies with a 3D printer according to the manufacturer&#39;s instructions by using materials such as polylactide filament. Wrap the bodies with a 0.08 mm copper wire to achieve 4,500 windings by using a computer controlled winding machine.</li>
</ol>
4. Fabrication of the Actuation Platform
<ol>
	<li>Arrange the double coils in a matrix (e.g. 3 x 3 matrix) on an electric board with a Peltier element underneath, screw on the double coils and connect them to a control via ribbon cable (Figure 3).</li>
	<li>Depending on the desired application, add an iron core (32 mm height, 4 mm diameter) or a neodymium magnet (12.5 mm height, 4 mm diameter, 1,035 kA m-1) to the coil body to gain a stronger magnetic field.</li>
	<li>To finish the platform place a plate, preferably quartz glass, with a maximum height of 1 mm on the coil matrix.</li>
	<li>Place a microreactor on the surface of the platform.</li>
	<li>To withdraw the upper particles from the reaction solution and thereby open a microreactor activate a coil with a neodymium magnet inside by using the control mentioned in 4.1). To close the microreactor again deactivate the coil.</li>
	<li>To merge two microreactors which are initially ca. 10 mm apart use neodymium magnets as described in 4.2). Lift up the magnet in the required coil bodies by activating the coils for ca. 25 s to open one microreactor (required distance between microreactor and magnet lies about 12 mm) and to move the other one to the same position on the platform.</li>
	<li>To cool down the reaction solution in the microreactor on the platform surface and to decrease the coil temperature switch on the Peltier element positioned beneath the coil matrix as described in 4.1).</li>
</ol>
5. Enzymatic Reaction by Merging Microreactors
<ol>
	<li>Dissolve horseradish peroxidase at a concentration of 0.1 &#181;g mL-1 in potassium phosphate buffer (0.1 M, pH 6.5). Dilute the substrate, 10-acetyl-3,7-dihydroxyphenoxazine (10 mM in dimethylsulfoxide (DMSO)) with potassium phosphate buffer (0.1 M, pH 6.5) to a concentration of 200 &#181;M.</li>
	<li>Use 10 &#181;L of each of these solutions to build two microreactors as described in 2.2). Merge the two microreactors by the mean of magnetic forces (neodymium cylinder magnet: 12.5 mm x 4 mm, 1,035 kA m-1) as described in 4.6) at 25 &#176;C.</li>
	<li>Detect the reaction by positioning a fluorescence probe (excitation wavelength: 570 nm, emission wavelength: 585 nm) ca. 10 mm directly above an opened microreactor before the merge.</li>
</ol>

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The authors have nothing to disclose.

For the successful use of microfluidic technologies, it is important to move the reaction volume corresponding to the requirements of the biotechnological synthesis and analyses. The actuation platform presented here makes it possible to move microfluidic droplets by magnetic force. The movement can be performed freely in two dimensions on a planar surface of a reaction platform by enclosing the liquid drop with a magnetic superhydrophobic shell. Thus an alternative system to predefined microfluidic channels with complex flow control installations, as used in conventional microfluidic systems, to pass solvents and samples is introduced. The automated actuation of small reaction droplets thus is a significant simplification of the known lab-on-a-chip platforms. Furthermore, the platform makes it possible to reversibly open a microreactor to add and remove reaction samples. In combination with a dispenser system, this can result in a high degree of automated reaction control and is the first step for in silico designable microreactions in a virtual lab-in-a-drop. The main limitation of this technique is that the microreactors can only be built with small volumes (up to ca. 30 &#181;L). A critical step in the protocol is the detection of the enzymatic reaction inside the microreactors because the fluorescence probe has to be adjusted properly. A further detection possibility may be UV/vis spectroscopy.
The development process of the platform showed that an electromagnetic coil with a diameter of 10 mm is sufficient for droplet movement. On the other hand, a double coil filled with air or an iron core is not able to induce the magnetic force which is needed to open a microreactor shell. Therefore, neodymium cores were chosen in the coils to perform this task. The resulting magnetic field gradients again can be varied by electromagnetical movement of the magnet vertical to the reaction platform. The pitch size of the platform is only limited by the control. The already existing control and software is designed and ready to use for a 10 x 10 matrix. The Peltier element is not needed for the described applications but may be necessary when the microreactor has to be fixed for a longer time and it will allow to cool down the reaction mixture above the platform surface.
In the future, the combination of the actuation platform with the liquid marbles can function as flexible lab-in-a-drop-systems for the implementation of screenings, fast optical analyses and complex enzyme cascade reactions with very small reaction volumes. In addition, the platform can be used for (bio-)chemical analyses such as PCR, electrophoresis or ELISA. Moreover, the screening of new, industrial relevant enzymes and aptamer amplifications are promising possibilities.

Technical applications with micro reactions are predominantly carried out in predefined microchannel chips. These systems are widely established and comprehensively described in the literature (inter alia 1,2,3). In 2011, the turnover of microfluidic technologies worldwide totaled 6.2 billion euro 4. In contrast, the use of freely movable micro reactor compartments was previously only examined and published to a limited extent. The most common method for moving aqueous micro droplets is electrowetting 5. Other methods for the motion of drops on surfaces are based on electric fields 6, magnetic force 7 or acoustic actuation 8. Due to their unfavorable surface to volume ratio, these droplet-based microreactor systems are exposed to strong evaporation effects. Thus, the drop motion is usually established as a liquid two-phase system, where the upper phase has a high boiling point protecting the aqueous phase from evaporation. Nevertheless, this approach involves a high risk of contaminating the reaction droplet by uncontrolled diffusion. This is a significant obstacle for the technical establishment of the mentioned systems.
Recent work is concerned with non-adherent liquid-solid phase transitions. A highly effective approach is the use of superhydrophobic surfaces, allowing the formation of spherical aqueous droplets. An extension of this reaction concept is the use of micro reaction compartments with a superhydrophobic surface or shell, which may for example consist of polytetrafluoroethylene (PTFE) particles 9. Their contact angles on surfaces are usually in the range of 160&#176; (depending on the surface roughness). The spherical compartments thus provide minimal resistance to movement on a surface and simultaneously provide protection against water evaporation.
Aqueous drops coated with micro sized PTFE particles may maintain their spherical shape up to a diameter of around 2 mm. At higher volumes, the hydrophobic shell is usually not completely closed anymore 10. The influence of other shell materials and the expansion of the field of application of the liquid marble to nonpolar solvents was implemented by Gao and McCarthy by using ionic liquids 12. For the formation of hydrophobic particle-based shells, so far particle diameters in sizes of 10 nm- 30 &#181;m have been described 11,14,16. New studies showed that hydrophobic nanoparticles as shell material are of even better use than that of microparticles 13. First stability studies confirmed an increase in stability when the particle size is reduced from ca. 600 nm to ca. 100 nm. This likely results from the denser particle distribution around the aqueous sphere 15.
The protection of aqueous reaction compartments by a hydrophobic shell and their designation as liquid marbles was first described in 2001 by Aussillous et al. and Mahadevan et al. 17,18. Since then, few applications of these defined reaction compartments have been described. For example, a gas sensor based on liquid marbles 19 and a detection method for water contamination based on an optically qualitative basis have been developed 20. The authors distinguish the advantages of high reaction rates and the low consumption of chemicals of their micro reaction systems. Recent publications deal with the production of pH-sensitive liquid marbles 16 or the representation of &#39;Janus particles&#39; with two different coatings of different functionality. For example, Bormashenko et al. could synthesize a microreactor with shells made of Teflon and semiconducting Carbon Black 21. Furthermore it was demonstrated that microreactors can efficiently and convenient synthesize polyperoxides by absorbing external oxygen as comonomer through the permeable gas-liquid interface 24. In another approach the shell of silica-particle-based liquid marbles provide the reactive substrate surfaces to regulate the classical silver mirror reaction 26. Current problems for research and development in the field of hydrophilic-core-hydrophobic-shell droplets are the particle size adjustment, the reproducible production of monodisperse droplets, the wettability of surfaces and the effect of a second hydrophilic shell on the micro reaction compartments 22, as well as a better control of the droplet trajectories, e.g. for the development of continuous microPCR-systems 4.
A magnetic actuation of these microreactors offers the advantage of relatively high movement ranges and a good selectivity of the force when working in biochemical systems. When using hydrophobic magnetite particles, they fulfil both the function of the magnetic force transmission to the movement of the microreactors, as well as the function of a hydrophobic shell. The magnetic movement of droplets with magnetic particles inside a droplet was postulated for the first time in 2006 by Lehmann et al. 23 and Shikida et al. 25, who used manually moved permanent magnets as actuators for the mobilization of a single droplet. Another approach to move a small amount of liquid was realized by Zhao et al., who used the hydrophobic Fe3O4 particles as magnetic shell. The shell of the magnetic liquid marble was opened on the upper side of the drop by a vertical reverse magnetic field 27. Based on this concept, Xue et al. were able to develop particles which form a microreactor with a surface tension of 20.1 dyne cm&#8722;1 28. Lin et al. fabricated novel cellulose-based micro/nano hierarchical spheres with both superparamagnetism and superhydrophobicity which provide god stability for magnetic liquid droplet transportation and manipulation 31.This was so far only released as a proof-of-principle study and not used for any application. The magnetic and electrical control of the liquid marbles is currently pursued in first approaches. Zhao et al. in 2010 15 and Zhang et al. 2012 29 were able to develop a droplet manipulation by the manual (hand-operated) movement of a permanent magnet beneath core-shell droplets. Bormashenko et al. 11 achieved the acceleration of a ferromagnetic liquid marble to a speed of 25 cm s-1 by approaching a neodymium magnet. The above mentioned principle studies were carried out exclusively by the manual movement of a small permanent magnet. As a next development step, Zhao et al. were recently able to estimate the required magnetic flux density for the movement of magnetic liquid marble by varying the distance of a permanent magnet 30. For a reaction control comparable to that of common lab-on-a-chip systems, it seems inevitable to provide the means of automated control of the discrete liquid volumes. To satisfy this need, we developed a new control system based on variable field gradients to fixate, move and open the magnetic microreactors.

<table><tbody><tr><td>3D-printer</td><td>FelixPrinters</td><td>Pro1</td><td></td></tr><tr><td>10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red)</td><td>Life Technologies</td><td>A12222</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>TU-KL</td><td>1072</td><td></td></tr><tr><td>CAD software</td><td>Siemens</td><td>Soled edge</td><td></td></tr><tr><td>Contact angle measuring device</td><td>Dataphysics</td><td>OCA 20</td><td></td></tr><tr><td>Cylinder magnet</td><td>Webcraft GmbH</td><td>S-04-13-N</td><td>https://www.supermagnete.de/stabmagnete-neodym-rund/stabmagnet-durchmesser-4mm-hoehe-12.5mm-neodym-n42-vernickelt_S-04-13-N</td></tr><tr><td>Dipotassium phosphate</td><td>Bernd Kraft</td><td>7758-11</td><td></td></tr><tr><td>Drying oven</td><td>Binder</td><td>FD 115</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>68-17-5</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt; tetrahydrate</td><td>TU-KL</td><td>1625</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt; hexahydrate</td><td>TU-KL</td><td>1622</td><td></td></tr><tr><td>Fluorescence probe</td><td>PerkinElmer</td><td>LS 55</td><td></td></tr><tr><td>Horseradish peroxidase</td><td>Carl Roth</td><td>9003-99-0</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Th.Geyer GmbH &amp;amp; Co</td><td>7722-84-1</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>Bernd Kraft</td><td>7778-77-0</td><td></td></tr><tr><td>Peltier element</td><td>Conrad&amp;nbsp;</td><td>193569</td><td></td></tr><tr><td>Perfluoroctyltriethoxysilane</td><td>Sigma-Aldrich</td><td>51851-37-7</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Helios NanoLab 650 DualBeam</td><td></td></tr><tr><td>Separation bar magnet</td><td>Webcraft GmbH</td><td>Q-40-20-10-N</td><td></td></tr><tr><td>Winding machine</td><td>IWT GmbH</td><td>FW122</td><td></td></tr></tbody></table>

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.

The shell particles have a diameter of around 640 nm. The magnetizable nanoparticles enclosed in this fluorosilane shell particles have diameters in a range between 22 nm and 37 nm. A 5 &#181;L microreactor with water as a liquid core had a contact angle of around 160&#176;.
The force needed to move a 10 &#181;L microreactor as described above is 1.34 &#177; 0.08 &#181;N. Figure 1 shows the electromagnetic force of a coil with 4,500 windings of copper wire and an inner iron core powered with 58 mA. The force distribution is estimated by a finite elements model (FEM) to determine the required coil properties to move a microreactor. As can be seen in Figure 1, the magnetic force conducted by the described coil is strong enough to move a microreactor to a distance farther than 10 mm away from the coil center.
The magnetic force to open a microreactor equals 0.85 &#177; 0.05 mN which is much higher than the force needed to move the droplet. As the magnetic force induced by the coil with the iron core within (Figure 1) is not strong enough to open a microreactor, a neodymium magnet was used in the coils. By powering the double coils in alternating current flow directions, the permanent magnet can be moved towards or away from the platform. Thereby, the microreactor can be opened or closed magnetically. If two microreactors with a completely intact shell lie side by side they do not merge as the surface tension inhibits their fusion. Therefore at least one must be opened.
Figure 2 shows the Michaelis-Menten kinetics resulting from merging a 10 &#181;L microreactor containing peroxidase with another 10 &#181;L microreactor containing the corresponding substrate (n = 3). By Lineweaver-Burk linearization the calculated Km value for the reaction within the microreactor is 86.85 &#181;M &#177; 10.95 &#181;M, the vmax value lies at 378.8 nmol L-1 s-1 &#177; 115.6 nmol L-1 s-1. As the Km value is in good correspondence with that given in literature, 81 &#177; 3 &#181;M 32, it can be assumed that the enzymatic reaction within the small-scale microreactor with the hydrophobic shell material is not influenced with respect to affinity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54643/54643fig1.jpg" /> 
Figure 1: Electromagnetic force simulation of a coil with 58 mA and 4,500 windings of copper wire in dependence of the distance of the microreactor to the coil center determined by FEM. The coil diameter is 10 mm, the diameter of the iron core is 4 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54643/54643fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54643/54643fig2.jpg" /> 
Figure 2: Michaelis-Menten kinetics of peroxidase measured by merging two 10 &#181;L microreactors. The temperature is 25 &#176;C and the buffer used is potassium phosphate (0.1 M) with a pH of 6.5. Three repetitions were carried out.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54643/54643fig3.jpg" /> 
Figure 3: Actuation platform. The actuation platform consists of a 3 x 3 matrix of double coils. One coil has 4,500 windings, a height of 16 mm (one chamber), a diameter of 10 mm and an inner diameter of ca. 4 mm. The height of the coils is predefined by the height of the used neodymium cylinder magnet. The diameter was chosen because previous studies showed that this is a reasonable distance for moving a microreactor with this kind of magnet. The number of the windings and the current was determined by FEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54643/54643fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation at JoVE.com

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

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 ...

aqueous-droplets-used-as-enzymatic-microreactors-their

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.3K Views.  University of Kaiserslautern. Lab-in-a-drop reactie systemen toestaan de veelzijdige uitvoering van complexe reacties in een schaal van microfluidic. Een automatische bediening platform bestaande uit een 3 x 3 matrix van elektromagnetische spoelen werd ontwikkeld en met succes gebruikt voor het samenvoegen van twee 10 µL microreactors en daardoor een enzymatische reactie in de resulterende vloeistof Friezen te initiëren.

Video: Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

Genetics

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

Engineering

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 ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Chemistry

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 ...

A Versatile Kit Based on Digital Microfluidics Droplet Actuation for Science Education

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a specific example. It also has fluorescent imaging capability and closed humidified enclosure based on an ultrasonic atomizer to prevent evaporation. The kit can be assembled within a short period of time and with minimal training in electronics and soldering. The kit allows both undergraduate/graduate students and enthusiasts to obtain hands-on experience in microfluidics in an intuitive way and be trained to gain familiarity with digital microfluidics.

We describe an educational kit that allows users to execute multiple experiments and gain hands-on experience on digital microfluidics.

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a ...

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.

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. ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

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 ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

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