Name: multicolor fluorescence detection for droplet microfluidics using optical fibers
Uploaded: 2016-05-05T14:00:00
Description: Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

This work was supported by DARPA grant number 84389.01.44908, an NSF CAREER award (DBI-1253293), an NIH exploratory/developmental research grant (CA195709), and NIH New Innovator Awards (HD080351, DP2-AR068129-01), and a New Directions grant from the UCSF resource allocation program.

1. SU8 Master Fabrication
<ol>
	<li>Design the microfluidic structures for three layer fabrication using design software and have the designs printed by a vendor on circuit board film with 10 &#181;m resolution. The details of device design are given in an attached reference 6 and the channel geometries are shown in Figure 1. The layers should include alignment marks to help collocate features from each fabrication layer 8.</li>
	<li>Place a pre-cleaned 3 inch diameter silicon waf...

<ol>
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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><li>Cole, R. H., de Lange, N., Gartner, Z. 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=Compact+and+modular+multicolour+fluorescence+detector+for+droplet+microfluidics.">Compact and modular multicolour fluorescence detector for droplet microfluidics.</a> Lab Chip. 15 (13), 2754-2758 (2015).</li><li>Guo, F., Lapsley, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+droplet-based,+optofluidic+device+for+high-throughput,+quantitative+bioanalysis.">A droplet-based, optofluidic device for high-throughput, quantitative bioanalysis.</a> Anal Chem. 84, 10745-10749 (2012).</li><li>. Lithography Available from: <a target="_blank" href="https://www.memsnet.org/mems/processes/lithography.html">https://www.memsnet.org/mems/processes/lithography.html</a> (2015)</li><li>DeJournette, C. J., Kim, J., Medlen, H., Li, X., Vincent, L. J., Easley, C. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+completely+in+vitro+ultrahigh-throughput+droplet-based+microfluidic+screening+system+for+protein+engineering+and+directed+evolution.">A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution.</a> Lab Chip. 12, 882 (2012).</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=Ultrahigh-Throughput+Mammalian+Single-Cell+Reverse-Transcriptase+Polymerase+Chain+Reaction+in+Microfluidic+Drops.">Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops.</a> Anal Chem. 85 (16), 8016-8021 (2013).</li><li>Martini, J., Recht, M. I., Huck, M., Bern, M. W., Johnson, N. M., Kiesel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+encoded+multicolor+fluorescence+detection+in+a+microfluidic+flow+cytometer.">Time encoded multicolor fluorescence detection in a microfluidic flow cytometer.</a> Lab Chip. 12 (23), 5057-5062 (2012).</li><li>Bliss, C. L., McMullin, J. N., Backhouse, C. 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Fiber optic detection requires the alignment of optical fibers with respect to fluid channels. Because our device utilizes guide channels fabricated with multilayer photolithography, placement of masks with respect to each other is of great importance. If the fiber guide channels are too close to the fluid channel, there is a potential for fluid leakage; if the guide channels are located too far away or misaligned, the fluorescence signal gathered by the detection fiber may be significantly diminished. Proper alignment c...

Droplet microfluidics provide a platform for high throughput biology by compartmentalizing experiments in a large number of aqueous droplets suspended in a carrier oil 1. Droplets have been used for applications as varied as single cell analysis 2, digital polymerase chain reaction (PCR) 3, and enzyme evolution 4. Fluorescent assays are the standard mode of detection for droplet microfluidics, as their bright signals and fast time response are compatible with detecting sub-nanoliter droplet volumes at kilohertz rates. Many applications require fluorescence detection for at least two colors simultaneously. For instance, our lab commonly performs PCR-activated droplet sorting experiments that use one detection channel for the result of an assay, and uses a secondary background dye to make assay-negative droplet countable 5.
Typical detection stations for droplet microfluidics are based on epifluorescence microscopes, and require complicated light manipulations schemes to introduce excitation light from free space lasers into microscope to be focused on the sample. After fluorescence is emitted from a droplet, the emitted fluoresced light is filtered so that each detection channel utilizes one photomultiplier tube (PMT) centered on a wavelength band. Epifluorescence microscope-based optical detection systems provide a barrier to entry due to their expense, complexity, and required maintenance. Optical fibers provide the means to construct a simplified and robust detection scheme, since fibers can be manually inserted into microfluidic devices, removing the need for mirror-based light routing, and allowing light paths to be interfaced using optical fiber connectors.
In this paper, we describe the assembly and validation of a compact and modular scheme to perform multicolor fluorescence detection by utilizing an array of optical fibers and a single photodetector 6. Optical fibers are coupled to individual lasers and are inserted normal to an L shaped flow channel at regular spatial offsets. A fluorescence collection fiber is oriented parallel to the excitation regions and is connected to a single PMT. Because a droplet passes through the laser beams at different times, data recorded by the PMT shows a temporal offset that allows the user to distinguish between the fluorescence emitted after the droplet is excited by each distinct laser beam. This temporal shift eliminates the need to separate emitted light to separate PMTs using a series of dichroic mirrors and bandpass filters. To validate the efficacy of the detector, we quantitate fluorescence in droplet populations encapsulating dyes of different color and concentration. The sensitivity of the system is investigated for single color fluorescein detection, and shows the ability to detect droplets with concentrations down to 0.1 nM, a 200x sensitivity improvement as compared to recent fiber based approaches reported in the literature 7.

<table>
	<tbody>
		<tr>
			<td>Photomasks</td>
			<td>CadArt Servcies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; silicon wafers, P type, virgin test grade</td>
			<td>University Wafers</td>
			<td>447</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3035</td>
			<td>Microchem</td>
			<td>Y311074</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>Microchem</td>
			<td>Y111072</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Krayden</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringes</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>27 gaugue needles</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>PE 2 polyethylene tubing</td>
			<td>Scientific Commodities, Inc.</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec 7500</td>
			<td>Fisher Scientific</td>
			<td>98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr>
			<td>Ionic Krytox Surfactant</td>
			<td></td>
			<td></td>
			<td>Synthesis instructions in ref #10</td>
		</tr>
		<tr>
			<td>Dextran- conjugated cascade blue dye</td>
			<td>Life Technologies</td>
			<td>D-1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma</td>
			<td>28803</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/581/703-25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Thorlabs</td>
			<td>PMM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber port</td>
			<td>Thorlabs</td>
			<td>PAFA-X-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>lens tube</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 200 um core / 225 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 105 um core / 125 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>125 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T08S13</td>
			<td></td>
		</tr>
		<tr>
			<td>225 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T10S13</td>
			<td></td>
		</tr>
		<tr>
			<td>laser fiber adapter</td>
			<td>OptoEngine</td>
			<td>FC/PC Adapter</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MDL-III-405</td>
			<td>Distributor for CNI lasers</td>
		</tr>
		<tr>
			<td>473 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MLL-FN-473-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

multicolor fluorescence detection for droplet microfluidics using optical fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Fabrication of a PDMS device that allows for the insertion of optical fibers requires a multistep photolithography procedure to create channels of varying height (Figure 1). First, an 80 &#181;m tall layer of SU-8 is spun onto a silicon wafer and patterned using a mask to create the fluid handling geometry. Next, an additional 40 &#181;m layer of SU-8 is spun onto the wafer, and patterned using a second mask to create features that will form 120 &#181;m tall laser fiber i...

Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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