1. Fabricating a Prototype cDEP Microfluidic Device: Overview

1. Follow previously reported procedures²² using photoresist and Deep Reactive Ion Etching (DRIE) to etch the desired channel design (Figure 1) into a silicon wafer (Figure 2). Deposit a thin layer of Teflon to improve the release of the microdevice from the wafer.

Figure 1. Schematic of a low frequency continuous sorting device. The sample channel runs left to right and is 500 μm wide with sawtooth constrictions to 100 μm. The two pairs of fluidic electrode channels compose the source and sink electrodes, respectively, and are separated from the sample channel by a 20 μm thick PDMS barrier.

Figure 2. The fabrication process of a cDEP device. (a) For 60 sec, UV light selectively reacts with photoresist exposed through a mask pattern of the cDEP device geometry. (b) Photoresist is removed using developer. (c) Deep Reactive Ion Etching (DRIE) is used to etch 50 μm deep features, and 5 min of wet etching by tetramethylammonium hydroxide (TMAH) 25% at 70 °C is used to reduce roughness on the side walls. (d) A Teflon coating is added to improve device release from the wafer. (e) PDMS is poured onto the re-usable wafer stamp after degassing and cured for 45 min at 100 °F. (f) PDMS is removed from the wafer. PDMS and a clean glass slide are exposed to air plasma for 2 min and bonded together.

2. Wrap the wafer with aluminum foil to prevent spill-over. Mix polydimethylsiloxane (PDMS) in 10:1 ratio of elastomer to curing agent, de-gas for approximately 20 min, and pour onto the wafer. Heat for 45 min at 100 °C to avoid damaging the Teflon coating of the stamp. Allow the device to cool down.
3. After cooling, remove aluminum foil, trim PDMS using a surgical blade, and punch access holes at channel inlet/outlet using a blunt puncher suited to the size of tubing. Here, a 1.5 mm blunt puncher is used.
4. Clean a glass microscope slide using a rinse of soap and water, ethanol, DI water, and drying with air, respectively. Clean the PDMS device using Scotch Magic tape. Examine under microscope to be sure channels are clean. Expose the slide and PDMS device to air plasma for 2 min. Firmly press channel side of device to glass slide, avoiding formation of air bubbles between the device and the slide (Figure 3).

Figure 3. (a) The mask used for photolithography during the silicon wafer stamp fabrication, and (b) the finished PDMS-glass device with the sample and fluid electrode channels filled with green and red food coloring, respectively. See Figure 1 for dimensions. Click here to view larger figure.

2. Preparing a Cell Suspension in DEP Buffer

1. Prepare a low conductivity (100 μS/cm) buffer, which will be referred to as "DEP buffer" (8.5% sucrose [w/v], 0.3% glucose [w/v], 0.725% RPMI [v/v]).¹⁶
2. Suspend cells in DEP buffer at 3 x 10⁶ cells/ml. Here, cancerous late-stage mouse ovarian surface epithelial (MOSE-L) cells are used.

Note: Cell viability analysis by the trypan blue dye exclusion method showed a slight decrease in viability (from 95.1-91.6%) of early-stage MOSE (MOSE-E) cells suspended in DEP buffer at room temperature after 5 hr. It is predicted that a similar slight decrease in viability occurs for MOSE-L cells, indicating that cells should not be stored in DEP buffer long-term.

3. Dye cells using a fluorescent membrane-permeable dye, such as the enzymatically-activated Calcein AM.
4. Measure conductivity of the dyed cell suspension. The conductivity should be approximately 100 μS/cm. If the conductivity measurement is too high, spin the sample down, resuspend in DEP buffer at 3 x 10⁶ cells/ml and repeat the conductivity measurement.

3. Loading the cDEP Microfluidic Chip

1. Place the microfluidic chip under vacuum for 30 min.
2. Meanwhile, cut a piece of flexible tubing to span the distance from the syringe pump to the microscope stage where the microfluidic chip will be located. Here, a 6 inch length of tubing is required. Fit tubing to needle tip on syringe.
3. Estimate the required length for outlet tubing by dividing the target collected sample volume by the cross-sectional area of the tube. Cut the require length of tubing.
4. Draw cell suspension into syringe. Check that no bubbles are located in the tubing or syringe.
5. Upon removing chip from vacuum, insert the outlet tubing and prime the sample channel quickly to avoid trapped air bubbles in the channels. Gently tap the syringe when priming to avoid rupturing the thin (20 μm) insulating barrier, though it has been observed that the barrier can withstand a constant high throughput near 5 ml/hr without rupturing.
6. To prime the electrode channels, quickly place empty pipette tips in one outlet of each fluidic electrode channel. Pipette 200 μl of PBS containing a small amount of rhodamine B and gently tap to introduce the fluid into the opposite end of the electrode channel. Maintain gentle pressure until PBS with rhodamine B visibly progresses up the tip of the empty pipette tip. Repeat for each electrode channel. Rhodamine B is used only to fluorescently visualize the fluidic electrode channels.
7. After priming, fill each pipette tip reservoir with PBS with rhodamine B. Avoid bubble formation in the pipette tip reservoirs, and remove any visible bubble by pipetting gently up and down. Detach the outlet tubing and discard appropriately, then insert a fresh outlet tubing reservoir to collect the target volume.

4. Characterizing Crossover Frequency of Cells using cDEP Chip

1. Position the chip on the stage of an inverted microscope equipped with digital camera (Figure 4).

Figure 4. (a) Overall experimental setup for cDEP experiments. The cDEP chip is located on the platform of the inverted microscope. A camera sends images to the computer for recording. The syringe pump maintains a constant inlet flow. The function generator generates a signal which is stepped up by the high voltage amplifier and connected to the cDEP chip by wire electrodes. The oscilloscope monitors the signal sent to the chip. (b) Detail view of the cDEP chip and fluidic and electronic connections. Click here to view larger figure.

2. Mount syringe on syringe pump and insert wire electrodes into fluidic electrode channel reservoirs.
3. Pump fluid through at 1 ml/hr to ensure good contact between the syringe pump and the syringe. Visually inspect the channel length to ensure unimpeded flow of cells and the absence of bubbles or leaks. Reduce flow rate to 0.005 ml/hr.
   Note: Throughput as high as 1 ml/hr^31,35 has been achieved in devices of other geometries.
4. For safety, test the electronics by powering on the function generator, high voltage amplifier, and oscilloscope, and adjusting to desired voltage and frequency. Here these parameters are 200 V and 20 kHz. Upon verification of acceptable performance, power off. Connect electrodes to high voltage amplifier.
   Note: Observe appropriate electrical safety procedures when operating the electronics at the high voltages used for these experiments.
5. Upon achieving steady flow, apply the desired voltage at the desired frequency. In this experiment, voltage is 200 V_RMS at frequencies between 5-60 kHz.
6. Record video files of the cell response with image processing techniques (step 5) to quantify cell distribution in the channel and to characterize the crossover frequency. To do this, maintain a constant voltage and vary the frequency systematically. At the start and end of the experiment, as well as randomly between experimental runs, record the control cell distribution, the distribution of the cells without applying any electrical field. Estimate the amount of time required for cells to flow from the inlet to the monitored location (here, 5 min). After setting a new frequency, wait this amount of time, then begin recording (here, a 2 min video of cell distribution).

5. Image Processing for Characterizing Crossover Frequency

1. Using a computational script, analyze each video frame by frame to determine the relative y-position (where the z-direction is the vertical depth of the channel, the y-direction is the channel width, and x-direction is the channel length) of each fluorescing cell or particle at a specified x-coordinate downstream from the region of high DEP force in the channel. Create a graph of the relative distribution.
2. Compare the centerline of the distribution at each voltage and frequency with the centerline of the control distribution. For a given voltage and varying frequencies, determine the crossover frequency, where the centerline matches the y-position of the control.

6. Varying the Experiment to Perform Cell Sorting or Enrichment

1. Suspend desired mixture in DEP buffer at a total concentration of 3 x 10⁶ particles/ml. Here, this mixture is MOSE-L cells with 4 μm fluorescing beads in DEP buffer. Perform the previously described steps to prepare a cDEP device.
2. To sort or enrich cells from a mixture, determine the crossover frequencies of the target cells and the background particles as previously described. Operate the experiment at a frequency between the two crossover frequencies of the target cells and the background particles so that the two populations of particles experience DEP forces in opposite directions. To sort MOSE-L cells and 4 μm beads, operate the microdevice above 11.90±0.63 kHz. the first crossover frequency of MOSE-L cells recently reported by Salmanzadeh et al.³³
3. To collect the contents of a sorted sample, remove the outlet tubing upon collecting the target volume, by placing a glove finger over the outlet opening and gently tugging on the tubing. Pour the collected target volume into a reservoir for further analysis. Note: Other sample recovery methods could include attaching a permanent reservoir to the chip and removing the sample by simple pipetting.