Picoinjection of Microfluidic Drops Without Metal Electrodes

Summary

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.

Abstract

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.

Introduction

In droplet-based microfluidics, micron-scale aqueous droplets are used as "test tubes" 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. Combined, these properties allow millions of reactions with individual cells, nucleic acid molecules, or compounds to be performed in a matter of min with μl 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 "Faraday Moat" 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 techniques^4,5. Our approach is compatible with devices fabricated in other materials, like resins, plastics, and epoxies. The channels have heights and widths of 30 μm, which are optimal for working with droplets 50 μm 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.

Protocol

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 "sausage" shape and allows for more effective picoinjection. For our purposes, we designed 30 x 30 μm channels for droplets that were 50 μm in diameter.

1. Model picoinjection site(s) after those described by Abate et al.³ with the exception that the channels for the metal electrodes are removed, as they are unnecessary.
2. Add channels to serve as the Faraday Moat (Figure 1) that run between picoinjection site(s) and the upstream emulsion such that they shield the droplets from the electric field.
   Note: This prevents unintended merging.

2. Fabricate Devices Using Soft Photolithographic Techniques

1. Generate a transparency photolithography mask based on the CAD file using existing commercial services.
2. With the photomask, cure photoresist on silicon wafers to produce a device master, as described previously⁴.
3. Pour PDMS mixed with curing agent (11:1 ratio) over the device master contained in a 5 cm polystyrene Petri dish.
4. Place the master with PDMS in a vacuum desiccator for roughly 15 min to remove any air bubbles.
5. Cure the PDMS device by placing it in a 95 °C oven for 1 hr. Alternatively, the PDMS will cure at RT after 24 hr.
6. Remove the device by cutting around the perimeter with a surgical blade and carefully peeling the device from the master.
7. Punch inlet and outlet holes into the PDMS using a 0.5 mm biopsy punch.
8. Bond the device to a glass microscope slide using a plasma bonder⁴.

3. Prepare an Air Pressure Control Pump to Pressurize a Reservoir Containing the Fluid

1. Modify the pump output such that the pressurized air exits through a length of 2.7 mm inner diameter polyethylene tubing.
2. Construct it such that the tubing terminates at a luer-lock syringe tip by fitting the lumen over the nipple on the rear of the luer-lock.
3. Seal by filling the space between the luer-lock threads and the tubing with epoxy.
4. Attach a 27.5 G needle.

4. Prepare a Monodisperse Emulsion of Aqueous (Water-in-oil) Droplets Suspended in an Inert Fluorinated Carrier Oil with 2% (wt/wt) Dissolved Biocompatible Surfactant⁷

The specific reagents contained in these droplets depend on the application

1. In preparation for reinjection, load the emulsion into a 1 ml syringe with a 27.5 G needle.
2. Secure the syringe in a syringe pump and orient the pump vertically (needle upward).
   Note: This orientation causes the droplets to pack in a layer above the carrier oil. When the pump is started, the droplets will be pushed out of the syringe at high volume fraction by the oil layer under them.

5. Prepare Reagents for Introduction to the Microfluidic Chip

1. Punch three 0.5 mm holes into the cap of a 15 ml centrifuge tube (any container with a screw cap will suffice) using a biopsy punch, needle, or drill.
2. Insert a 0.5 mm diameter wire electrode and a ~20 cm length of PE-2 tubing through two of the holes so that they reach the bottom of the tube.
3. In the remaining hole, thread a ~2.5 cm of a ~20 cm length of PE tubing such that it will rest above the fluid level.
4. Seal any gaps on the top of the cap with UV-cured epoxy.
5. Fill the tube with the picoinjection fluid and screw on the cap.
6. Connect the output from the air-pressure control pump to the shorter length of tubing by inserting the needle into the lumen. The needle should fit snugly.
7. Fill a 1 ml syringe with 1 M NaCl to serve as the Faraday Moat.
8. Connect a 27.5 G needle and secure the syringe in a syringe pump.
9. Fill another 1 ml syringe with carrier/spacer oil, connect a 27.5 G needle, and secure it in a syringe pump.

6. Prepare the Microfluidic Device for Picoinjection

1. Connect the output tubing (longer length) from the injection fluid container to the inlet port of the picoinjection fluid on the microfluidic chip.
2. Connect the syringe containing the 1 M NaCl to the inlet port for the Faraday Moat on the microfluidic chip with a length of PE tubing.
3. Connect the syringe containing the carrier oil to the inlet port of the microfluidic chip with a length of PE tubing.
4. Insert PE tubing into the emulsion outlet port on the microfluidic chip. The tubing should terminate in an emulsion collection vessel, normally a 1.5 ml centrifuge tube.
5. Insert PE tubing into the outlet port for the Faraday Moat on the microfluidic chip. The tubing should terminate in a non-conducting and electrically isolated container to prevent a short circuit.
6. Connect the output of the high-voltage (HV) amplifier via alligator clip to the metal electrode submerged in the picoinjection fluid.
7. Connect the ground electrode of the HV amplifier via alligator clip to the metal of the syringe needle containing the 1 M NaCl.

7. Infuse Reagents into Microfluidic Chip

1. Introduce the 1 M NaCl (Faraday Moat) to the device at a rate of 100 μl/hr.
2. Introduce the droplet emulsion and carrier oil at rates suitable for the device dimensions. For our demo device, we introduce the drops and oil at 200 and 400 μl/hr, respectively. The flow rates should allow the droplets to pass the picoinjector at regular intervals separated by a gap of carrier oil.
3. Adjust the pressure applied to the picoinjection fluid such that the fluid pressure at the picoinjection orifice is in mechanical equilibrium with the droplet channel.
   Note: At this pressure (the Laplace pressure), the injection fluid should bulge into the droplet channel without budding off and forming its own drops (Figure 2). At these flow rates described above, we apply a pressure of ~13 psi to the injection fluid to achieve equilibrium at the injection site.

8. Begin Picoinjection

1. As droplets pass the injection orifice, apply a 0-10 V, 10 kHz, AC signal amplified 1,000x by the HV-amplifier (Figure 3).
2. Modulate the injection volume by changing the amplitude of the applied voltage.
   Note: Higher voltages should allow for more fluid to be introduced to the droplets. In our testing, we observe stable and consistent injection at voltages between 100 and 3,000 V using injection solutions of NaCl ranging from 10-500 mM (Figure 4).

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 ml Luer-Lok™ syringes	BD Medical	309628
LocTite UV-cured adhesive	Henkel	35241
PE-2 tubing	Scientific Commodities	BB31695-PE/2
Novec HFE-7500	3M	98-0212-2928-5
NaCl	Sigma Aldrich	S9888
1.5 ml centrifuge tubes	Eppendorf	22363531
BD Falcon 15 ml tube	BD Biosciences	352097
Air pressure control pump	Control Air Inc.		We recommend one under the control of DAQ and control software
Syringe pumps	New Era		Must be capable of holding 1 ml syringes and flowing at rates as low as 100 μl/hr
HV-amplfier			Must be capable of 1,000x amplification of signals between 0.01 and 10 V
Plasma bonder/cleaner	Harrick Plasma
3” silicon wafers	Sigma Aldrich	647535
PDMS	Dow Corning		Sylgard 184 with curing agent should be included
SU-8 photoresist	MicroChem		Viscocity depends on device dimensions