Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 wafer on a spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,750 rpm, providing a layer thickness of 80 µm.
3. Remove the wafer and bake on a 135 °C hotplate for 30 min. Allow the wafer to cool to RT before moving on to the next step.
4. Expose the coated wafer to the 1^st layer mask under a collimated 190 mW, 365 nm LED for 3 min. After exposure, place the wafer on a 135 °C hotplate for 1 min, then cool to RT before proceeding to the next step.
5. Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 5,000 rpm, resulting in a layer that provides an additional thickness of 40 µm.
6. Remove the wafer and bake on a 135 °C hotplate for 30 min, then cool to RT before moving to the next step.
7. Align the 2^nd layer mask onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 °C hotplate for 4 min 30 sec, then cool to RT before proceeding to the next step.
8. Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,000 rpm, resulting in a layer that provides an additional thickness of 100 µm.
9. Remove the wafer and bake on a 135 °C hotplate for 30 min, then cool to RT before moving to the next step.
10. Align the 3^rd layer mask onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 °C hotplate for 9 min, then cool to RT before proceeding to the next step.
11. Develop the masks by immersing in a stirred bath of propylene glycol monomethyl ether acetate for 30 min. Wash the wafer in isopropanol and bake on a 135 °C hotplate for 1 min. Place the developed master in a 100 mm Petri dish for polydimethylsiloxane (PDMS) molding.

2. PDMS Device Fabrication

1. Prepare 10:1 PDMS by combining 50 g of silicone base with 5 g of curing agent in a plastic cup. Mix the contents with a rotary tool fitted with a stir stick. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.
2. Pour the PDMS to give a thickness of 3 mm over the master and place back into the desiccator for further degassing. Once all bubbles are removed, bake the device at 80 °C for 80 min.
3. Cut the device from the mold using a scalpel and place on a clean surface with the patterned side up and punch the fluidic inlets and outlets with a 0.75 mm biopsy punch. The device must be cut so that the 120 µm and 220 µm tall geometries are accessible from the side of the device.
4. Plasma treat the device, with feature side up, along with a pre-cleaned 2 inches by 3 inches glass slide at 1 mbar O₂ plasma for 20 sec in a 300 W plasma cleaner. Bond the PDMS device by placing the patterned side of the PDMS device onto the plasma-treated side of the glass slide. Place the device in a 80 °C oven and bake the assembled device for 40 min.
5. Render the channels hydrophobic by using a syringe to flush the device with a fluorinated surface treatment fluid. Immediately bake the device at 80 °C for 10 min to evaporate the solvent.

3. Preparation of Optical Components

1. Prepare the laser excitation fiber by removing the insulation from the last 5 mm of a 105 µm core, 125 µm cladding, NA = 0.22 optical fiber.
2. Prepare the 2^nd color laser excitation fiber by repeating 3.1 for a 2^nd identical fiber.
3. Prepare the fiber for collecting the fluorescence signal by repeating 3.1 for a 200 µm core, 225 µm cladding, NA = 0.39 optical fiber.
4. Inspect the tips of all the fibers — if the tips do not end in a flat surface, re-cleave the ends with a fiber scribe.
5. Attach a laser fiber coupler to a 50 mW, 405 nm laser and attach one of the 105 µm core fibers to the laser. Direct the stripped end at the sensor of the laser power meter, and use the fine adjustments of the laser coupler to maximize the laser power.
6. Repeat 3.5 for a 50 mW, 473 nm laser.
7. Mount a 446/510/581/703 nm quad bandpass filters on the PMT using lens tubes to block laser light and transmit emitted fluorescence. Attach fiber coupler so that light travels through the filters before hitting the PMT.
8. Attach the fiber from 3.3 to the unit assembled in 3.7.

4. Offline Mixed Emulsion Generation

1. Obtain a flow focus microfluidics dropmaker device with a 60 µm x 60 µm orifice.
2. Fill a 1 ml syringe with HFE 7500 oils with 2% ionic fluorosurfactant ⁹.
3. Fill a series of 1 ml syringes with the following combinations of fluorescein (FITC) and dextran-conjugated blue dyes (CB) in PBS: 1 nM FITC/10 nM CB, 10 nM FITC/10 nM CB, 1 nM FITC/100 nM CB, and 10 nM FITC/100 nM CB.
4. Mount the dropmaker device on the stage of an inverted microscope and the aqueous and oil syringes on syringe pumps. Couple the syringes to the devices using PE-2 tubing.
5. Running syringe pumps at 500 µl/hr for the oil and 250 µl/hr for the aqueous solutions, create a mixture of 80 µm droplets containing the solutions from 4.3. For each type of aqueous sample, use a fresh dropmaker device to eliminate cross-contamination. Collect ~200 µl of emulsion from each dye combination into an empty syringe.
6. After the 4 droplets types have been collected in a single collection syringe, mix the emulsion by repeatedly rotating the syringe.
7. Repeat steps 4.2-4.6 with aqueous solutions containing 0.1, 1, 10, and 100 nM FITC in PBS.

5. Optical Fiber Insertion

1. Place the microfluidic chip fabricated in step 2 on the stage of an inverted microscope coupled with a digital camera capable of < 100 µsec shutter speeds.
2. Working carefully from the side, insert the fiber coupled to the 473 nm laser into the farthest upstream 120 µm channel, taking care not to puncture through to the main flow channel.
3. Insert the fiber coupled to the 405 nm laser into the farthest downstream 120 µm high side channel, providing fiber spacing of 300 µm.
4. Insert the PMT-coupled fiber into the 220 µm tall channel normal to the two laser excitation fibers.

6. Fluorescence Detection of Mixed Emulsions 

1. Mount a 5 ml syringe filled with HFE 7500 with 2% ionic fluorosurfactant to the spacer oil inlet of the detection device with PE-2 tubing.
2. Mount the syringe containing the mixed FITC/CB emulsion on a vertically oriented syringe pump and couple to the device's droplet reinjection inlet using PE-2 tubing. Connect a length of PE-2 tubing from the device exit to a waste container.
3. Prime the device by running each of the pumps at 1,000 µl/hr until both oil and droplets are seen to be regularly combining in the device and flowing downstream.
4. Adjust the flow rates such that the spacer oil runs at 6,000 µl/hr and the droplets at 100 µl/hr, providing significant spacing between droplets traveling through the detection region.
5. Turn on the lasers, start the data acquisition program, and adjust the PMT gain to provide signals that are more than 100x the baseline noise floor. Adjust the laser power so that all of the doublet peaks are clearly visible on a single, linearly scaled timetrace.
6. Acquire 60 sec of the PMT voltage timetrace at at least 20 kHz. Import the data into computing program such as Matlab and measure the heights of peaks of the recorded doublets using the custom computing program script.
   NOTE: This is provided as supplementary information.
7. Repeat steps 6.2-6.7 using the FITC-only mixed emulsion created in 4.7, with the 405 nm laser turned off.

Results

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...

Discussion

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...

Materials

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 μm core / 225 μm cladding optical fiber with one stripped end and one FC/PC connector	Thorlabs	Custom
Patch cable with 105 μm core / 125 μm cladding optical fiber with one stripped end and one FC/PC connector	Thorlabs	Custom
125 μm fiber stripping tool	Thorlabs	T08S13
225 μm 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