Name: picoinjection of microfluidic drops without metal electrodes
Uploaded: 2014-04-18T18:00:00
Description: Watch this Scientific Journal Video about Picoinjection of Microfluidic Drops Without Metal Electrodes at JoVE.com

This work was supported by the Department of Bioengineering and Therapeutic Sciences at UCSF, the California institute for Quantitative Biosciences (QB3), and the Bridging the Gap Award from the Rogers Family Foundation.

1. Design Device Dimensions and Topologies Based on Experimental Needs Using Computer Aided Design (CAD) Software
Note: Select emulsion channel diameters smaller than those of the spherical droplets. This forces the droplets into a cylindrical or &#34;sausage&#34; shape and allows for more effective picoinjection. For our purposes, we designed 30 x 30 &#956;m channels for droplets that were 50 &#956;m in diameter.
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	<li>Model picoinjection site(s) after those described by Abate et al...

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	<li>Kritikou, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It's+cheaper+in+the+Picolab.">It's cheaper in the Picolab.</a> Nat. Rev. Genet. 6 (9), (2005).</li><li>Ahn, K., Agresti, J., Chong, H., Marquez, M., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocoalescence+of+drops+synchronized+by+size-dependent+flow+in+microfluidic+channels.">Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels.</a> Appl. Phys. Lett. 88 (26), (2006).</li><li>Abate, A. R., Hung, T., Mary, P., Agresti, J. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+injection+with+microfluidics+using+picoinjectors.">High-throughput injection with microfluidics using picoinjectors.</a> Proc. Natl. Acad. Sci. U S A. 107, 19163-19166 (2010).</li><li>Harris, J., 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=Fabrication+of+a+microfluidic+device+for+the+compartmentalization+of+neuron+soma+and+axons.">Fabrication of a microfluidic device for the compartmentalization of neuron soma and axons.</a> J. Vis. Exp. (7), (2007).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+microfluidic+systems+in+poly(dimethylsiloxane).">Rapid prototyping of microfluidic systems in poly(dimethylsiloxane).</a> Anal. Chem. 70 (23), 4974-4984 (1998).</li><li>O'Donovan, B., Eastburn, D. J., Abate, 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=Electrode-free+picoinjection+of+microfluidic+drops.">Electrode-free picoinjection of microfluidic drops.</a> Lab on a Chip. 12 (20), 4029-4032 (2012).</li><li>Holtze, 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=Biocompatible+surfactants+for+water-in-fluorocarbon+emulsions.">Biocompatible surfactants for water-in-fluorocarbon emulsions.</a> Lab on a Chip. 8 (10), 1632-1639 (2008).</li><li>Chung, C., Lee, M., Char, K., Ahn, K., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+dynamics+passing+through+obstructions+in+confined+microchannel+flow.">Droplet dynamics passing through obstructions in confined microchannel flow.</a> Microfluid. Nanofluid. 9, 1151-1163 (2010).</li><li>Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamical+instability+of+thin+liquid+films+between+conducting+media.">Dynamical instability of thin liquid films between conducting media.</a> Phys. Rev. Lett. 83 (12), 2359-2361 (1999).</li><li>Priest, C., Herminghaus, S., Seemann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+electrocoalescence+in+microfluidics:+Targeting+a+single+lamella.">Controlled electrocoalescence in microfluidics: Targeting a single lamella.</a> Appl. Phys. Lett. 89 (13), 134101-134103 (2006).</li><li>Florent, M., Siva, A. V., Hao, G., Dirk, E., Frieder, 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-controlled+droplet+generation+in+a+microfluidic+flow-focusing+device.">Electrowetting-controlled droplet generation in a microfluidic flow-focusing device.</a> J. Phys: Condens. Matter. 19 (46), (2007).</li><li>Eastburn, D. J., Sciambi, A., Abate, 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=Picoinjection+enables+digital+detection+of+RNA+with+droplet+rt-PCR.">Picoinjection enables digital detection of RNA with droplet rt-PCR.</a> PLoS ONE. 8 (4), (2013).</li></ol>

The relationship between injection volume and applied voltage is dependent on many factors including device dimensions, length of the tubing carrying the picoinjection fluid to the device, molarity of picoinjection fluid, and the velocity of the droplets as they pass they injector. For this reason we recommend that the volume/voltage relationship be characterized before each run of picoinjection by measuring injection volumes at the edges of the working ranges of voltage and molarity. Additionally, at higher voltages and...

In droplet-based microfluidics, micron-scale aqueous droplets are used as &#34;test tubes&#34; for biological reactions. The advantage to performing reactions in the tiny droplets is that each drop uses only a few pl of reagent and, with microfluidics, the drops can be formed and processed at kilohertz rates1.&#160;Combined, these properties allow millions of reactions with individual cells, nucleic acid molecules, or compounds to be performed in a matter of min&#160;with &#956;l&#160;of total material.
To use drops for applications like these, techniques are needed for adding controlled volumes of reagents to the drops; such operations are analogous to pipetting into test tubes. One method for accomplishing this is electrocoalescence, wherein a drop of reagent is merged with the target drop by applying an electric field. The electric field disrupts the arrangement of surfactant molecules on the interfaces of the drops, inducing a thin-film instability and triggering coalescence in emulsions that are otherwise stable2. Electrically-induced merging is also exploited in the design of the picoinjector, a device that injects reagents into drops as they flow past a pressurized channel3. To apply the electric field, picoinjector devices utilize metal electrodes, but the integration of metal electrodes into microfluidic chips is often a complex and error-prone process as the liquid-solder wires are easily compromised by air bubbles or dust and other debris in the channel, as well as fractures from stress or bending during device setup.
Here we present a method to perform picoinjection without the use of metal electrodes, making the fabrication simpler and more robust. To trigger picoinjection, we instead use the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. We also add a &#34;Faraday Moat&#34; to shield sensitive regions of the device and act as a universal ground (Figure 1). The moat electrically isolates the droplets upstream of the picoinjection site by providing a ground, preventing unintended droplet merger. An added benefit of our technique is that the volume injected into the drops depends on the magnitude of the applied voltage, allowing it to be adjusted by tuning the applied signal.
We fabricate our devices in poly(dimethylsiloxane) (PDMS) using soft photolithographic techniques4,5. Our approach is compatible with devices fabricated in other materials, like resins, plastics, and epoxies. The channels have heights and widths of 30&#160;&#956;m, which are optimal for working with droplets 50 &#956;m in diameter (65 pl). We introduce reagents via polyethelene tubing (0.3/1.09 mm inner/outer diameter) inserted into ports created during device fabrication with 0.50 mm biopsy punches, similar to methods described previously5. The exact makeup of the injection fluid depends on the specific application. The fluid need only contain dissolved electrolytes at concentrations high enough to yield sufficient conductivity for the electrical signal to be transmitted to the picoinjector. In bench testing, we have found that ionic concentrations greater than 10 mM should suffice6, though this value and fluid conductivities depend on the specific device dimensions and magnitude of the applied voltage.

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			<td height="21" style="height:21px;width:283px;">1 mL Leur-Lok&#8482; syringes</td>
			<td style="width:143px;">BD Medical</td>
			<td align="right" style="width:160px;">309628</td>
			<td style="width:415px;"></td>
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			<td height="21" style="height:21px;">LocTite UV-cured adhesive</td>
			<td style="width:143px;">Henkel</td>
			<td align="right">35241</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:283px;">PE-2 Tubing</td>
			<td style="width:143px;">Scientific Commodities</td>
			<td>BB31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Novec HFE-7500</td>
			<td>3M</td>
			<td style="width:160px;">98-0212-2928-5</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:283px;">NaCl</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:160px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">1.5 mL centrifuge tubes</td>
			<td style="width:143px;">Eppendorf</td>
			<td align="right" style="width:160px;">22363531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">BD Falcon 15 ml tube</td>
			<td style="width:143px;">BD Biosciences</td>
			<td align="right" style="width:160px;">352097</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:283px;">Air Pressure Control Pump</td>
			<td style="width:143px;">Control Air Inc.</td>
			<td style="width:160px;"></td>
			<td style="width:415px;">We recommend one under the control of DAQ and control software</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Syringe Pumps</td>
			<td>New Era</td>
			<td style="width:160px;"></td>
			<td>Must be capable of holding 1ml syringes and flowing at rates as low as 100 uL/hr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">HV-Amplfier</td>
			<td style="width:143px;"></td>
			<td style="width:160px;"></td>
			<td>Must be capable of 1000x amplification of signals between 0.01 and 10 V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma Bonder/Cleaner</td>
			<td style="width:143px;">Harrick Plasma</td>
			<td style="width:160px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">3&#8221; silicon wafers</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td align="right" style="width:160px;">647535</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PDMS</td>
			<td style="width:143px;">Dow Corning</td>
			<td style="width:160px;"></td>
			<td>Sylgard 184 with curing agent should be included</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">SU-8 Photoresist</td>
			<td style="width:143px;">MicroChem</td>
			<td style="width:160px;"></td>
			<td>Viscocity depends on device dimensions</td>
		</tr>
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picoinjection of microfluidic drops without metal electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.

Microscopic images taken at the picoinjection site show that electrification of the picoinjection fluid is enough to trigger injection (Figure 2). The injected volume can be controlled by modulating the amplitude of the applied voltage, with higher voltages allowing for higher injection volumes. We plot the injection volume versus the magnitude of the applied voltage for three representative molarities of injection fluid in (Figure 3). To demonstrate the speed our method, we selectively ...

Watch this Scientific Journal Video about Picoinjection of Microfluidic Drops Without Metal Electrodes at JoVE.com

Picoinjection of Microfluidic Drops Without Metal Electrodes

We have developed a technique for picoinjecting microfluidic drops that does not require metal electrodes. As such, devices incorporating our technique are simpler to fabricate and to use.

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of ...

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