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Bioengineering

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Published: May 5th, 2016

DOI:

10.3791/54010

1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco

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 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.

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 l....

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1. SU8 Master Fabrication

  1. 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 µm 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.
  2. Place a pre-cleaned 3 inch diameter silicon waf.......

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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 µm 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 µm layer of SU-8 is spun onto the wafer, and patterned using a second mask to create features that will form 120 µm tall laser fiber i.......

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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.......

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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.

....

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

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