Name: taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics
Uploaded: 2014-11-10T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

We thank the Lindback Foundation for financial support, and Dr. Alexander Sidorenko and Elza Chu for fruitful discussions and technical assistance, and Professor Robert Smith for assistance with the C. elegans assays.

NOTE: In the procedures described below, laboratory safety guidelines must always be followed. Of particular importance is the safety when dealing with high voltage (&#62; 500 V) and handling chemicals.
1. Coating of a Conductive Substrate with Candle Soot
<ol>
	<li>Cut copper metal into rectangles (75 x 43 mm, 0.5 mm thick). Clean each copper substrate by immersion in copper etchant for about 30 sec, wash with tap water for about 20 sec, and dry with paper. 
	NOTE: If using Method 1 below, change the dimensions to 75 x 25 mm to fit into the machine.</li>
	<li>Sweep a lit paraffin candle under the copper substrate for 30&#8211;45 sec, to obtain an approximately uniform soot coating (about 40 &#181;m thick). Keep the substrate at ~1 cm inside the flame. Do not touch the fragile soot surface.</li>
</ol>
2. Protecting the Soot Layer with Coating
NOTE: The soot layer is very fragile, and must be coated for protection. Two simple alternatives (methods 1 and 2 below) are suggested here, but more robust protocols are currently under development.
<ol>
	<li>Method 1
	<ol>
		<li>Load the sample into the metal evaporator or sputtering system. Following the operation procedures of the system, evacuate the chamber, and start controlled deposition of gold onto the soot layer (150&#8211;200 nm). Let the device cool down to room temperature.</li>
		<li>Dip-coat the metalized substrate in a 1-dodecanethiol solution (1% v/v, in 95% ethanol, ACS/USP grade), for 10 min inside a chemical hood. Then, holding the device at an angle close to 60&#176;, gently wash the surface with several droplets of ethanol only. Let the devices dry, overnight.</li>
	</ol>
	</li>
	<li>Method 2
	<ol>
		<li>In a chemical hood, immediately after coating the substrate with soot and while the substrate is still warm from the candle flame, deposit some droplets of fluorinated liquid on one side of the substrate, and tilt the substrate to an angle close to 90&#176;. Deposit more droplets, and let them roll over the entire soot surface. 
		NOTE: When the droplet falls on a spot, soot will be washed away from that area. Let the droplets of fluorinated liquid spread as much as possible.</li>
		<li>Bake the substrate on a hot plate (160 &#176;C for 15 min) inside a chemical hood.</li>
		<li>Let the substrate sit overnight at room temperature before usage. Store indefinitely.</li>
	</ol>
	</li>
</ol>
3. Fabrication of Top Electrodes (Adapted from Abdelgawad et al.12)
<ol>
	<li>Draw the electrodes using graphic design software. Each electrode is 2 mm long, 0.3 mm wide, and the gap between electrodes is 0.3 mm. The gap between contacts (to snap into the connector, see below) is 2.3 mm (Figure 1).</li>
	<li>Trim a flexible copper laminate (35&#160;&#181;m thick) into the Monarch format (3.87 x 7.5 inches). Use other sizes if compatible with the printer. Load the laminate into the manual feed tray of a color printer.</li>
	<li>Make sure to use &#8220;rich black&#8221;, or &#8220;registration black&#8221;, when printing on the copper sheet (see Abdelgawad et al.12 for details) to allow a denser layer of black ink on the copper substrate, protecting the printed pattern during etching. Let the printed substrate dry completely, overnight.</li>
	<li>Inside a chemical hood, warm up (40 &#176;C) a beaker with 50 ml of copper etchant. Dip the printed laminate in the beaker, and gently shake it in the solution for about 10 min. Etching time depends on the copper etchant solution. Every few minutes, check the corrosion and see if the pattern is intact.</li>
	<li>Carefully wash the laminate with water, and remove the coating with acetone and ethanol in the chemical hood. Wash once again, and gently dry the laminate with paper towel.</li>
	<li>Carefully attach the laminate with electrodes to a glass slide (75 x 25 mm, ~1 mm thick), using double sided tape. Avoid air pockets.</li>
	<li>Attach a film of perfluoroalkoxy PFA to the electrodes using tape. This serves to prevent accidental contact of the electrodes with the droplet, which damages top electrodes due to short-circuit.</li>
</ol>
4. The Electronic Interface (Circuit in Figure 2)
<ol>
	<li>Solder the relays and the capacitors C to a universal circuit board.</li>
	<li>Assemble the remainder of the 10 relay drivers on a solderless breadboard for electronic circuits.</li>
	<li>Wire each relay driver&#8217;s input to a channel in the control board.</li>
	<li>Carefully snap the top electrodes into a connector (Figure 3). Wire each relay driver&#8217;s output to a top electrode, as shown in the figure. Note that there is a grounded connector contact between a pair of wires from relays, to minimize electrical noise. 
	NOTE: The connector sits on an adjustable platform to control the distance (0.1&#8211;0.5 mm) between top and bottom (soot-coated) substrate.</li>
	<li>Use a program to control timing for high voltage (HV) application (about 0.8 sec) to 4 electrodes at the same time, shifting 1 electrode in the direction of motion (i.e., for 0.8 sec, actuate 1234; then 2345, 3456, etc., 0.8 sec for each group, and then backwards, so droplet moves in the opposite direction as well).</li>
</ol>
5. Droplet Visualization and Handling
<ol>
	<li>To record droplet motion, use the visualization system, which is composed of a 24X &#8211; 96X magnification assembly combined with a CCD camera. Connect the video recorder to the camera using S-video.</li>
	<li>Pipette a 4 &#181;l droplet containing C. elegans in media on the bottom of the soot-coated substrate.</li>
	<li>Bring the top electrodes to ~0.3 mm above the droplet. The droplet should be close to the middle, just below the fifth electrode, for easier operation.</li>
	<li>Turn on the electronic interface and high voltage (500 VRMS), and adjust the top electrode distance to the droplet until it starts moving. Do not let the top electrodes touch the droplet.</li>
	<li>Collect data by recording the number of successful droplet transfers on the device in response to electric pulses. A successful experiment is characterized by at least 700 droplet transfers, i.e., one transfer after each electric pulse.</li>
	<li>Collect data continuously, until the droplet does not move anymore in response to 5 to 10 pulses. 
	NOTE: When the surface starts to degrade, motion could be restored by bringing the top electrodes closer to the droplet.</li>
</ol>

<ol>
	<li>Fair, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+is+a+true+lab-on-a-chip+possible.">Digital microfluidics: is a true lab-on-a-chip possible.</a> Microfluid Nanofluid. 3 (3), 245-281 (2007).</li><li>Gupta, S., Alargova, R. G., Kilpatrick, P. K., Velev, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Chip+Dielectrophoretic+Coassembly+of+Live+Cells+and+Particles+into+Responsive+Biomaterials.">On-Chip Dielectrophoretic Coassembly of Live Cells and Particles into Responsive Biomaterials.</a> Langmuir. 26 (5), 3441-3452 (2009).</li><li>Shih, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spot+analysis+by+digital+microfluidics+coupled+to+nanoelectrospray+ionization+mass+spectrometry.">Dried blood spot analysis by digital microfluidics coupled to nanoelectrospray ionization mass spectrometry.</a> Anal Chem. 84 (8), 3731-3738 (2012).</li><li>Gorbatsova, J., Borissova, M., Kaljurand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-on-dielectric+actuation+of+droplets+with+capillary+electrophoretic+zones+for+off-line+mass+spectrometric+analysis.">Electrowetting-on-dielectric actuation of droplets with capillary electrophoretic zones for off-line mass spectrometric analysis.</a> J Chromatogr. 1234, 9-15 (2012).</li><li>Qin, J., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+exploration+and+learning+in+C.+elegans.">Maze exploration and learning in C. elegans.</a> Lab Chip. 7 (2), 186-192 (2007).</li><li>Koc, Y., de Mello, A. J., McHale, G., Newton, M. I., Roach, P., Shirtcliffe, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-scale+superhydrophobicity:+suppression+of+protein+adsorption+and+promotion+of+flow-induced+detachment.">Nano-scale superhydrophobicity: suppression of protein adsorption and promotion of flow-induced detachment.</a> Lab Chip. 8 (4), 582-586 (2008).</li><li>Perry, G., Thomy, V., Das, M. R., Coffinier, Y., Boukherroub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+protein+biofouling+using+graphene+oxide+in+droplet-based+microfluidic+microsystems.">Inhibiting protein biofouling using graphene oxide in droplet-based microfluidic microsystems.</a> Lab Chip. 12 (9), 1601-1604 (2012).</li><li>Kumari, N., Garimella, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-Induced+Dewetting+Transitions+on+Superhydrophobic+Surfaces.">Electrowetting-Induced Dewetting Transitions on Superhydrophobic Surfaces.</a> Langmuir. 27 (17), 10342-10346 (2011).</li><li>Freire, S. L. S., Tanner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive-Free+Digital+Microfluidics.">Additive-Free Digital Microfluidics.</a> Langmuir. 29 (28), 9024-9030 (2013).</li><li>Deng, X., Mammen, L., Butt, H. -. J., Vollmer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candle+Soot+as+a+Template+for+a+Transparent+Robust+Superamphiphobic+Coating.">Candle Soot as a Template for a Transparent Robust Superamphiphobic Coating.</a> Science. 335, 67-70 (2011).</li><li>Kang, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Electrostatic+Fields+Change+Contact+Angle+in+Electrowetting.">How Electrostatic Fields Change Contact Angle in Electrowetting.</a> Langmuir. 18 (26), 10318-10322 (2002).</li><li>Abdelgawad, M., Watson, M. W. L., Young, E. W. K., Mudrik, J. M., Ungrin, M. D., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography:+masters+on+demand.">Soft lithography: masters on demand.</a> Lab Chip. 8 (8), 1379-1385 (2008).</li><li>Barbulovic-Nad, I., Yang, H., Park, P. S., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+cell-based+assays.">Digital microfluidics for cell-based assays.</a> Lab Chip. 8 (4), 519-526 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li></ol>

The authors declare that they have no competing financial interests.

The most critical step of the protocol is the protection of the soot layer, directly associated with the success in moving droplets. Metallizing the soot layer (method 1 above) allows close to 100% of fabrication success. However, the maximum operation time is about 10 min; possibly, droplet fractions are wetting the soot through holes in the metal layer. Coating the soot layer with the fluorinated liquid is the easiest and fastest alternative, and requires minimum resources, but only 40&#8211;50% of the fabricated substrates work (20 min maximum) &#8212; and the coating is not uniform. In fact, the soot layer is very fragile, and the viscous fluorinated liquid easily damages it. We are currently working on more robust alternatives to protect the soot layer, which would increase the operation time of the device. However, one important aspect is the adsorption of droplet contents to the surface. Previously,9 we quantified the amount of protein that attached to the surface during device operation, and a correlation was found between continued motion and reduced surface adsorption of bovine serum albumin (BSA). Notwithstanding, biofouling is a complex matter, and some authors even suggest that it might be impossible to completely suppress the effect; in theory, if only a single protein attaches to a surface, more will be attracted to this site. In fact, the maximum operation time reported for digital microfluidic devices (by other authors6) was about 40 minutes. Therefore, the robustness of the surface is a point of great importance and still a work in progress.
Note that, in electrowetting, the application of voltage often spreads the droplet with analytes on the surface, entirely hindering motion, unless additives are used. However, some additives can be toxic, or might only work within a range of analyte concentration in the droplet. Field-DW devices allow the transport of analytes ranging from proteins to single cells and entire organisms, without extra additives. In addition, device characteristics are largely independent of thickness, uniformity, and electrical properties of the soot layer (see Freire et al.9 for more information).
Therefore, the significance of the method described here is that it broadens the scope of applications for DMF, paving the way for the development of fully controlled lab-on-a-chip platforms working largely independently of droplet chemistry.
The dimensions of top electrodes are compatible with the resolution of the printer, and are not unique; narrower and closer electrodes could also work. In fact, other methods for the fabrication of printed circuit boards in electronics can be used as well. What matters is that the droplet is subjected to a non-uniform electric field, and it will move towards the region where the field is more intense. However, care should be taken in the design to keep the electric field between energized and floating electrodes below 3 MV/m to prevent sparking; here, the field is about 1.7 MV/m, without sharp edges.
The operation of the electronic circuits is as follows. Each top electrode, through a relay contact, is either connected to the output of a high voltage amplifier, or to a capacitor (C), to minimize electrical noise. The transistor T allows the low current outsourced by the control board, through the resistor R and the capacitor C1, to control the larger current required for the relay coil to operate. The diode D prevents circuit damage due to the variable current in the coil (see materials list for list of components). Only one control board allows individual addressing of all electrodes, and only one HV power supply is required (Figure 2), which output voltages (8&#8211;18 kHz, 500&#8211;660 VRMS) after amplifying the sine wave provided by a generator. Note that HV is kept as far away as possible from the control system, to minimize noise and possible circuit malfunction.
The assays reported here used 4 &#181;l droplets, simply due to the fact that smaller droplets containing C. elegans are more difficult to pipette. The culture of C. elegans will not be discussed here, and the reader should look for the protocols elsewhere (e.g., Brenner14).

The miniaturization of devices that work with liquids is of paramount importance for the development of &#8220;lab-on-a-chip&#8221; platforms. In this direction, the last two decades have witnessed a significant progress in the field of microfluidics, with a variety of applications.1-5 Contrasting with the transport of fluid in enclosed channels (channel microfluidics), DMF manipulates droplets on arrays of electrodes. One of the most attractive merits of this technique is the absence of movable parts to transport fluids, and motion is instantly stopped by turning off electric signals.
However, droplet motion is dependent on droplet contents, certainly an undesirable characteristic for a universal &#8220;lab-on-a-chip&#8221; platform. Droplets containing proteins and other analytes stick to device surfaces, becoming unmovable. Arguably, this has been the major limitation for broadening the scope of DMF applications;6-8 alternatives to minimize the unwanted surface fouling involve the addition of extra chemical species to the droplet or its surroundings, which could potentially affect droplet content.
Previously, our group developed a device to allow the transport of cells and proteins in DMF, without extra additives (Field-DW devices).9 This was achieved by combining a surface based on candle soot,10 with a device geometry that favors droplet rolling and leads to an upward force on the droplet, further decreasing droplet-surface interaction. In this approach, droplet motion is not associated with surface wetting.11
The goal of the detailed method described below is to produce a DMF device capable of transporting droplets containing proteins, cells, and entire organisms, without extra additives. The Field-DW devices pave the way for fully controlled platforms working largely independently of droplet chemistry.
Here, we also present simulations showing that, despite the high voltage required for device operation, the voltage drop across the droplet is a small fraction of the applied voltage, indicating negligible effects on bioanalytes inside the droplet. In fact, preliminary tests with Caenorhabditis elegans (C. elegans), a nematode used for a variety of studies in biology, show that worms swim undisturbed as voltages are applied.

<table><tbody><tr><td>Paraffin candle</td><td></td><td></td><td>Any paraffin candle</td></tr><tr><td>Sputtering system</td><td>Denton Vacuum, Moorestown, NJ</td><td></td><td>Sputter coater Desk V HP equipped with an Au target.&amp;nbsp;</td></tr><tr><td>1-dodecanethiol</td><td>Sigma-Aldrich</td><td>471364</td><td></td></tr><tr><td>Teflon</td><td>Dupont</td><td>AF-1600</td><td></td></tr><tr><td>Fluorinert FC-40</td><td>Sigma-Aldrich</td><td>F9755</td><td>Fluorinated liquid: Prepare Teflon-AF resin in Fluorinert FC-40, 1:100 (w/w), to create the hydrophobic coating.</td></tr><tr><td>Graphic design software -Adobe Illustrator</td><td>Adobe Systems</td><td></td><td>Other softwares might be used as well.</td></tr><tr><td>Copper laminate</td><td>Dupont</td><td>LF9110</td><td></td></tr><tr><td>Laser Printer</td><td>Xerox</td><td>Phaser 6360 or similar</td><td>Check for the compatibility with &quot;rich black&quot; or &quot;registration black&quot; (see text).</td></tr><tr><td>Copper Etchant</td><td>Transene</td><td>CE-100</td><td></td></tr><tr><td>Perfluoroalkoxy (PFA) film</td><td>McMaster-Carr</td><td>84955K22</td><td></td></tr><tr><td>Breadboard</td><td>Allied Electronics</td><td>70012450 or similar</td><td>Large enough to allow the assemble of 10 drivers.</td></tr><tr><td>Universal circuit board</td><td>Allied Electronics</td><td>70219535 or similar</td><td></td></tr><tr><td>Connector</td><td>Allied Electronics</td><td>5145154-8 or similar</td><td></td></tr><tr><td>Control board and control program (LabView software)</td><td>National Instruments</td><td>NI-6229 or similar</td><td></td></tr><tr><td>High-voltage amplifier</td><td>Trek</td><td>PZD700</td><td></td></tr><tr><td>Capacitors C and C1, 100 nF, 60 V</td><td>Allied&amp;nbsp;</td><td>8817183</td><td></td></tr><tr><td>Transistor T, NPN</td><td>Allied&amp;nbsp;</td><td>9350289</td><td></td></tr><tr><td>Diode D, 1N4007</td><td>Allied&amp;nbsp;</td><td>2660007</td><td></td></tr><tr><td>Relay&amp;nbsp;</td><td>Allied&amp;nbsp;</td><td>8862527</td><td></td></tr><tr><td>Visualization system</td><td>Edmund Optics</td><td>VZM 200i or similar</td><td>System magnification 24X &amp;#8211; 96X. It is combined with a Hitachi KP-D20B 1/2 in CCD Color Camera.</td></tr><tr><td>Recorder</td><td>Sony</td><td>GV-D1000 NTSC or similar</td><td>It is connected to the camera by an S-video cable.</td></tr><tr><td>Simulations</td><td>COMSOL Multiphysics</td><td>V. 4.4</td><td></td></tr></tbody></table>

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.

Previously, we have used Field-DW devices to allow the motion of proteins in DMF. In particular, droplets with bovine serum albumin (BSA) could be moved at a concentration 2,000 times higher than previously reported by other authors (without additives). This was due to the reduced interaction between droplet and surface; Figure 4 shows a droplet containing fluorescently-tagged BSA (see Freire et al.9 for more information on the experiments). The first picture on the left shows the droplet sitting on the soot-coated surface; the middle one, the effect of electric field, which, in addition to produce droplet rolling, also applies an upward force on the droplet, further reducing the interaction with the surface. Note the shear contrast (right) to a common alternative used in DMF, which is a surface coated only with fluorinated liquid (without candle soot); the strong interaction with the surface, indicated by the lower contact angle, quite often hinders motion.
Here, we use the experimental set-up (Figure 3) to continue the tests with these devices, now transporting droplets containing larger organisms, the worm C. elegans, a nematode used in a variety of biological assays.
Droplets with worms were successfully actuated on soot-coated substrates. In particular, Movie 1 shows a droplet moving in response to each voltage pulse (~0.8 sec interval) (note that the liquid fraction, stuck to a place with no soot, is out of the droplet pathway). Inspection after the experiments revealed that no worms, debris, or liquid residues, were left on droplets pathways after the experiments, indicating reduced interaction between droplet and surface.
The electronic interface (Figure 2) allows automation and better control of motion, since simultaneous actuation of groups of electrodes (Figure 1) increases the upward force, further reducing interaction with the surface.
Different experiments have shown that worms swim undisturbed as the droplet moves (20 min total actuation time), indicating that the high voltage (~500 VRMS) required for device operation is not harmful to the biological species being transported. This is supported by simulations, which have shown that the voltage drop across the droplet is an insignificant fraction (10-6%) of the voltage required for operation (Figure 5, potential difference between a point at the top and bottom of the plane in the middle of droplet); in fact, in droplets containing Jurkat T-cells, previous studies done by other authors13 suggest that such minimal voltage drops do not affect cell viability, proliferation, and biochemistry. For further validation, however, we are currently in the process of designing experiments to evaluate long term effects of the voltage on C. elegans. For the simulations described here, a ~2 &#181;l droplet was assumed to be of PBS (phosphate buffer saline), sitting on a 30 &#181;m thick layer of soot. Top and bottom electrodes were modeled as copper, and the applied voltage equal to 500 VRMS (For details on the simulations, see Freire et al.9).
<img alt="Figure 1" src="/files/ftp_upload/52091/52091fig1highres.jpg" /> 
Figure 1: Picture of top electrodes. Each one of the 10 is 2 mm long and 0.3 mm wide. The gap between 2 electrodes is also 0.3 mm, and the gap between contacts (bottom) is 2.3 mm.
<img alt="Figure 2" src="/files/ftp_upload/52091/52091fig2highres.jpg" /> 
Figure 2: Schematic of the controlling system for top electrodes, detailing 1 of the 10 relay drivers. Each top electrode is either subjected to voltage, or connected to a capacitor. On the right, a picture of the board with the relays. Note that the high voltage required for operation is kept away from the control board (white base) on the left. Droplet diagram (Middle) adapted with permission from Freire et al.9 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52091/52091fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52091/52091fig3highres.jpg" /> 
Figure 3: View of the experimental set-up. The distance between top and bottom (soot-coated) substrate is adjustable. The contacts from top electrodes are snapped into a connector. The wires from relays (shown here only 1, 2 and 3 of the 10 wires) are soldered to the connector, as indicated by the diagram on the right. Note that there is a grounded connector contact (e.g., connector contacts 2 or 4) between a pair of wires from relays (e.g., 1 or 3), to minimize electrical noise. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52091/52091fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52091/52091fig4highres.jpg" /> 
Figure 4: Droplets (4 &#181;l) with Fluorescently-tagged BSA (10 g/L). Left, sitting on a soot-based substrate; middle, one of the effects of the electric field is to apply an upward force on the droplet, further minimizing the interaction with the substrate; right, droplet on a surface coated only with fluorinated liquid (no soot). Adapted with permission from Freire et al.9
<img alt="Figure 5" src="/files/ftp_upload/52091/52091fig5highres.jpg" /> 
Figure 5: Simulations showing the voltage drop across a droplet in a Field-DW device. The potential difference between the top (point b) and bottom (point a) of the plane in the middle of the droplet is in the microvolt range, an insignificant fraction (10-6%) of the voltage for required operation.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52091/Freire - C-elegans.qt">Movie 1</a>. Droplet with C. elegans on a Field-DW device, moving in response to each voltage pulse (~0.8 sec interval). The liquid fraction shown on the bottom left of the video is not in the droplet pathway.

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

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; ...

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7.8K Views.  University of the Sciences. 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.

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

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 ...

Bioengineering

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

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 ...

Chemistry

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

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 ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...