This work supported has been supported in part by the National Science Foundation under Grant No. EFRI 0938047, and by the Virginia Tech Institute for Critical Technology and Applied Science (ICTAS). The authors would like to express appreciation to Dr. Eva Schmelz and Dr. Paul Roberts for their kind gift of MOSE-L cells. The authors acknowledge Angela Anderson for her assistance with cell culture, Caitlan Swaffar for her help with editing this document and preparing experiments, and all Bioelectromechanical Systems lab members.

1. Fabricating a Prototype cDEP Microfluidic Device: Overview
<ol>
	<li>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.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/50634/50634fig1.jpg" /> 
Figure 1. Schematic of a low frequency continuous sorting device. The sample channel runs left to right and is 500 &#956;m wide with sawtooth constrictions to 100 &#956;m. The two pairs of fluidic electrode channels compose the source and sink electrodes, respectively, and are separated from the sample channel by a 20 &#956;m thick PDMS barrier.
<img alt="Figure 2" src="/files/ftp_upload/50634/50634fig2.jpg" /> 
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 &#956;m deep features, and 5 min of wet etching by tetramethylammonium hydroxide (TMAH) 25% at 70 &#176;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 &#176;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.
<ol start="2">
	<li>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 &#176;C to avoid damaging the Teflon coating of the stamp. Allow the device to cool down.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig3highres.jpg" src="/files/ftp_upload/50634/50634fig3.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig3highres.jpg" target="_blank">Click here to view larger figure</a>.
2. Preparing a Cell Suspension in DEP Buffer
<ol>
	<li>Prepare a low conductivity (100 &#956;S/cm) buffer, which will be referred to as &#34;DEP buffer&#34; (8.5% sucrose [w/v], 0.3% glucose [w/v], 0.725% RPMI [v/v]).16</li>
	<li>Suspend cells in DEP buffer at 3 x 106 cells/ml. Here, cancerous late-stage mouse ovarian surface epithelial (MOSE-L) cells are used.</li>
</ol>
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.
<ol start="3">
	<li>Dye cells using a fluorescent membrane-permeable dye, such as the enzymatically-activated Calcein AM.</li>
	<li>Measure conductivity of the dyed cell suspension. The conductivity should be approximately 100 &#956;S/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.</li>
</ol>
3. Loading the cDEP Microfluidic Chip
<ol>
	<li>Place the microfluidic chip under vacuum for 30 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Draw cell suspension into syringe. Check that no bubbles are located in the tubing or syringe.</li>
	<li>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 &#956;m) insulating barrier, though it has been observed that the barrier can withstand a constant high throughput near 5 ml/hr without rupturing.</li>
	<li>To prime the electrode channels, quickly place empty pipette tips in one outlet of each fluidic electrode channel. Pipette 200 &#956;l 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.</li>
	<li>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.</li>
</ol>
4. Characterizing Crossover Frequency of Cells using cDEP Chip
<ol>
	<li>Position the chip on the stage of an inverted microscope equipped with digital camera (Figure 4).</li>
</ol>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50634/50634fig4highres.jpg" src="/files/ftp_upload/50634/50634fig4.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig4highres.jpg" target="_blank">Click here to view larger figure</a>.
<ol start="2">
	<li>Mount syringe on syringe pump and insert wire electrodes into fluidic electrode channel reservoirs.</li>
	<li>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/hr31,35 has been achieved in devices of other geometries.</li>
	<li>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.</li>
	<li>Upon achieving steady flow, apply the desired voltage at the desired frequency. In this experiment, voltage is 200 VRMS at frequencies between 5-60 kHz.</li>
	<li>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).</li>
</ol>
5. Image Processing for Characterizing Crossover Frequency
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Varying the Experiment to Perform Cell Sorting or Enrichment
<ol>
	<li>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 &#956;m fluorescing beads in DEP buffer. Perform the previously described steps to prepare a cDEP device.</li>
	<li>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 &#956;m beads, operate the microdevice above 11.90&#177;0.63 kHz. the first crossover frequency of MOSE-L cells recently reported by Salmanzadeh et al.33</li>
	<li>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.</li>
</ol>

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Dr. Rafael Davalos has a patent pending for contactless dielectrophoresis.

DEP is a powerful technique for determining dielectric properties of particles and directing particle motion towards sorting, isolation, or enrichment applications. Due to the detrimental effect of direct electrode contact with a sample, others have taken approaches similar to the previously presented method to avoid contact. For example, Bashir et al. developed contact-free DEP devices by using a microfluidic PDMS device separated from printed circuit board electrodes by a thin glass coverslip, and this technique is also made available in video format.23,36
Here, we have shown the fabrication of a cDEP chip and fluidic electrodes using a single PDMS substrate, and the experimental protocol for separating ovarian cancer cells from a mixture of cells and fluorescent beads. The presented technique has been successfully used for a variety of more complex and physiologically relevant applications, including sorting live and dead cells,28 tumor initiating cells from prostate cancer cells,30 cancer cells from dilute red blood cells,31,32 and differentiating among stages of breast cancer37 and ovarian cancer.29 cDEP has also been used for mixing particles.38 These applications suggest that by using the simple technique presented, diverse purposes can be accomplished simply by altering the design of the channel geometry.
DEP is useful on the microscale for manipulation of particles.13 For spherical particles, the translational dielectrophoretic force depends upon the size and electrical properties of the particle and its suspending medium, as well as the gradient of the electric field squared:
<img alt="Equation 1" src="/files/ftp_upload/50634/50634eq1.jpg" /> 
where &epsilon;m is the permittivity of the suspending medium, r is the radius of the particle, and Rc[k(&omega;)]is the real part of the Clausius-Mossotti (CM) factor. The CM factor is a measure of the relative polarizability of the particle compared to the suspending medium and determines the direction of the dielectrophoretic force. It is described as
<img alt="Equation 2" src="/files/ftp_upload/50634/50634eq2.jpg" /> 
where <img alt="Equation 3" src="/files/ftp_upload/50634/50634eq3.jpg" /> and <img alt="Equation 4" src="/files/ftp_upload/50634/50634eq4.jpg" /> are the complex permittivities of the particle and medium, respectively. The complex permittivity, <img alt="Equation 5" src="/files/ftp_upload/50634/50634eq5.jpg" />, depends upon conductivity (&sigma;) and frequency (&omega;). The CM factor of spherical particles is theoretically bound between -0.5 and 1. If the CM factor is negative, the particles experience nDEP because the medium is more polarizable than the particle, so particles move away from regions of high electric field gradients. If the CM factor is positive, the particles are more polarizable than the medium and they experience pDEP in which they move toward regions of high electric field gradients.
For biological particles that are nonhomogeneous in structure, such as cells, the Clausius-Mossotti factor can be determined from an effective value for the particle permittivity:
<img alt="Equation 6" src="/files/ftp_upload/50634/50634eq6.jpg" /> 
where <img alt="Equation 7" src="/files/ftp_upload/50634/50634eq7.jpg" /> and <img alt="Equation 8" src="/files/ftp_upload/50634/50634eq8.jpg" /> are the complex permittivity of the effective complex permittivity of the interior of the cell, such as the cytoplasm, and the plasma membrane, respectively; r is the radius of the cell, and d is the thickness of the plasma membrane.26
When DEP occurs with particles suspended in a fluid, the motion of the particle relative to the fluid will generate a drag force on the particle. This drag force must be considered when determining the overall force acting on the particle. For the situations of interest here, viscous forces dominate and particles are assumed spherical, small, and moving with relatively low velocity, so Stokes' drag law provides a good approximation for the drag force:
<img alt="Equation 9" src="/files/ftp_upload/50634/50634eq9.jpg" /> 
where &eta; is the viscosity of the fluid, up is the velocity of the particle, and uf is the velocity of the fluid, which may also be moving. Given the dielectrophoretic force, known fluid and particle properties, and a known flow velocity, the balance between the drag force and the dielectrophoretic force can be solved to estimate the particle velocity. The shear rate that the cells experience should be below the threshold at which cell lysing can occur.
Characterization of the electrical properties of particles is necessary to predict and control how they will respond under DEP them. In this work, we specifically utilized low frequency cDEP with MOSE-L cells to demonstrate the protocol for determining first crossover frequency of cells, and then showed continuous sorting of polystyrene beads and MOSE-L cells based on their opposing DEP responses.
Altering the geometry of the cDEP device will change the spatial gradients of the electric field, allowing devices to be designed for high frequency or low frequency operation, and for high selectivity and efficiency of sorting for a specified cell type. Additionally, high throughput devices can be developed by fabricating wider channels,30 channels in parallel,30 or by multilayer fabrication35, in which the electrode channels are vertically stacked above and below a relatively deep sample channel. Thin membranes form the barrier between layers. Preliminary testing with devices fabricated in polymethylmethacrylate (PMMA) and polycarbonate (PC) thin films have demonstrated DEP response of MOSE-L cells. Current efforts are underway to refine multi-layer high-throughput devices and to improve the peripheral system towards an eventual plug-and-play platform. To expand from the basic experimental technique presented, the applications and specifications of the device can be tailored to fit particular demands, such as sorting versus characterization, or adding sample reservoirs and a semi-automated system to the device.

Biological sample enrichment and particle sorting is often necessary for subsequent analysis.1 For instance, isolation of rare cells from body fluids has important applications in cancer detection and individualized medicine. 2,3 The most commonly used enrichment techniques are fluorescent activated cell sorting (FACS)4 and magnetic activated cell sorting (MACS),5 which rely upon expressed surface markers to differentiate cells. Other strategies include hydrodynamic6 or inertial7,8 sorting, optical tweezers,9 acoustophoresis,10 and dielectrophoresis.11,12 Dielectrophoresis is the movement of a polarized particle in the presence of a non-uniform electric field.13 DEP has been used for a wide range of applications,2,14 including sorting cells based on viability,15 characterizing bioelectrical properties of cells,16 and sorting upon induced changes in biophysical properties of cells.17,18 Traditional DEP utilizes planar electrodes patterned within a microfluidic channel to apply a voltage and induce a non-uniform electric field.13 While this is a powerful technique, challenges can arise, such as electrode delamination and electrolysis. Insulator-based dielectrophoresis (iDEP)19 has addressed the challenges of fouling, electrode delamination, and spatial degradation of the electrode field through patterning insulating structures that induce non-uniformities in a DC electric field. iDEP has been used to selectively separate live and dead bacteria cells,19 isolate bacterial spores,20 and manipulate DNA,21 among other applications. Joule heating can be a challenge because it can occur as a result of the high DC voltages often required. To ameliorate these challenges, contact-free DEP microdevices have been developed.22-24
The technique presented here utilizes contactless dielectrophoresis (cDEP), so named for the lack of direct contact between metallic electrodes and the sample channel.22 Unique to this strategy is the replacement of metallic electrodes with fluid electrode channels filled with a highly conductive solution. These fluid electrodes are capacitively coupled across a thin insulating barrier to the sample channel via an AC voltage. Eliminating sample contact with electrodes reduces issues associated with DEP-based methods such as electrolysis and bubble formation, sample contamination, and electrode delamination. As a result, cDEP is especially useful for biological samples because it supports viability of the cells in the sample. Importantly, cDEP can maintain sample sterility. A chip can be prepared in a cell culture hood and the experiment can be conducted without requiring sample contact to metal electrodes or requiring that the sample be open to the environment. A simple reservoir can be fixed to the chip outlet to facilitate sterile sample recovery. Additionally, the fabrication of fluid electrodes from the same biocompatible polymer material (PDMS) as the sample channel reduces the high cost incurred with custom patterning of metal electrodes, the time required for fabrication, and limits the need for specialized cleanroom equipment to the initial patterning of the reusable silicon wafer stamp.
Movement of particles due to DEP depends on the characteristics of the particle and the medium, as well as the spatial gradients of the electric field. A particle- and frequency-dependent factor, called the Clausius-Mossotti (CM) factor, takes a value in the range -0.5 to 1, and determines the direction of the DEP force. The frequency at which the CM factor is exactly zero is called the crossover frequency. This is the point at which no dielectrophoretic force is exerted on a particle and the CM factor changes sign. A single crossover frequency for inert solid microspheres occurs when the CM factor changes from negative to positive. 25 For mammalian cells in low conductivity buffer on the order of 0.01 S/m, a first crossover frequency indicating a transition from nDEP to pDEP exists near 10-100 kHz, and is influenced by the size, shape, cytoskeleton, and membrane properties of the cell.26,27 A second crossover frequency at a shift from the pDEP to nDEP regime is on the order of 10 MHz, and is influenced by the nucleus-cytoplasm ratio, cytoplasm conductivity, and endoplasmic reticulum.27 DEP force can be applied without the presence of fluid flow, but here we utilize a flowing fluid to achieve continuous sorting of suspended particles. The combined influence of the dielectrophoretic force and the Stokes' drag force dictate the translational motion of a particle.
We have developed devices for operation in two frequency ranges. Higher frequency devices (100-600 kHz) have operated using pDEP and achieved batch sorting of cells, such as prostate tumor initiating cells (TICs), murine ovarian surface epithelial (MOSE) cells, MDA-MB-231 breast cancer cells, or live THP-1 cells by selectively trapping cells of interest on insulating posts located in the sample channel.28-31 Lower frequency (5-100 kHz) devices operate continuously, and when operated at a frequency at which one population experiences pDEP while the background population experiences nDEP, can redirect particle trajectories to achieve sorting.32-34 These low frequency devices have been used to sort cancer cells from red blood cells, determine the changes in dielectric properties of a progressive MOSE cell line, and to elucidate the effects of non-toxic sphingolipid treatments on reversing aggressive characteristics of aggressive MOSE cells. Additionally, cDEP microdevices can be designed to operate at increased throughput, currently up to 1 ml/hr.31,35
As described, the flexibility and low cost of the fabrication process enables custom-designed device geometries, which allow the presented experimental procedure to be relevant for a wide range of applications. The long-term goal of cDEP is to realize label-free cell sorting and enrichment on a clinical level, with sample recovery for subsequent culture or processing. The technique presented here is a simple and inexpensive method, from fabrication to experimentation, which increases the accessibility of DEP. We demonstrate the preparation of a cDEP chip and the experimental protocol to achieve characterization and enrichment of ovarian cancer cells from fluorescent microspheres.

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning, Midland, MI,USA</td><td>Sylgard 184</td><td></td></tr><tr><td>Glass slides </td><td>The Microscope Depot</td><td>76079</td><td>2x3 inch-ground edges</td></tr><tr><td>Microbore PTFE Tubing</td><td>Cole-Parmer Instrument Co, Vernon Hills, IL, USA</td><td>EW-06417-31</td><td>Thin walled 20 gauge, 0.032&quot;ID x 0.056&quot;OD, 100 ft/roll</td></tr><tr><td>Luer-slip plastic syringes</td><td>National Scientific company</td><td>S7510-1</td><td></td></tr><tr><td>Needle tip</td><td>Howard Electronic instruments</td><td>JG20-1.0</td><td>20 Gauge 1.0&quot;, ID=0.025&quot; OD=0.036&quot;</td></tr><tr><td>D(+)-Sucrose</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>S3-500</td><td></td></tr><tr><td>D(+)-glucose, reagent ACS, anhydrous</td><td>Acros Organics N.V., Fair Lawn, NJ</td><td>AC410955000</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>Quality Biological Inc.</td><td>112-025-101</td><td></td></tr><tr><td>Calcein AM, Molecular Probes</td><td>Invitrogen Corp. (life technologies), Carlsbad, CA, USA</td><td>C3100MP</td><td>excitation wavelength 488/emission wavelength 516</td></tr><tr><td>Rhodamine B, O</td><td>Science Lab</td><td>SLR1465-100G</td><td>excitation wavelength 540/emission wavelength 625</td></tr><tr><td>Phosphate buffered saline (10X)</td><td>Gbiosciences, St. Louis, MO</td><td>RC-147</td><td></td></tr><tr><td>Leica, inverted light microscope </td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DMI 6000B</td><td></td></tr><tr><td>Leica DFC420, color camera</td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DFC420</td><td></td></tr><tr><td>Function generator </td><td>GW Instek, Taipei, Taiwan</td><td>GFG-3015</td><td></td></tr><tr><td>Wideband power amplifier </td><td>Amp-Line Corp., Oakland Gardens, NY, USA</td><td>AL-50HF-A</td><td></td></tr><tr><td>HFHV Output Transformer</td><td></td><td>AL-T50-V25/300-F100K-600K</td><td></td></tr><tr><td>High voltage amplifier</td><td>Trek</td><td>Model 2205</td><td></td></tr><tr><td>USB Modular Oscilloscope, 100 MHz</td><td>AgilentTechnologies</td><td>U2701A</td><td></td></tr><tr><td>Expanded Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-001/002 (115/230V)</td><td>air plasma</td></tr><tr><td>Scotch Magic tape</td><td>3M</td><td></td><td>any available width is sufficient</td></tr><tr><td>1.5 mm puncher </td><td>Harris Uni-Core</td><td>Z708836-25EA</td><td></td></tr><tr><td>.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>FluoSpheres Sulfate Microspheres</td><td>Invitrogen</td><td>F8858</td><td>4.0 &amp;mu;m, red fluorescent (excitation wavelength 580/emission wavelength 605)</td></tr><tr><td>AZ 9260 photoresist </td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>AZ 400 K developer</td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>Tetramethylammonium hydroxide (TMAH) 25% </td><td></td><td></td><td>provided by Virginia Tech cleanroom</td></tr><tr><td>Teflon coating</td><td></td><td></td><td>applied using DRIE machine</td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>452</td><td>100 mm diameter, 500 &amp;mu;m thickness, one side polished (SSP) </td></tr><tr><td>Deep Reactive Ion Etching (DRIE)</td><td>Alcatrel</td><td>AMS SDE 100</td><td></td></tr></tbody></table>

label-free isolation and enrichment of cells through contactless dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

DEP should be observed qualitatively in the sample channel to confirm the device is operational when the electric field is present. One method to test for the presence of DEP is to adjust the frequency to values far from the crossover frequency where strong pDEP and nDEP are predicted (roughly &gt;40 kHz to observe pDEP and &lt;10 kHz to observe nDEP for most mammalian cancer cells in a buffer with conductivity of 100 &mu;S/cm). It should be noted that inert microspheres exhibit pDEP below their crossover frequency and nDEP above their crossover frequency. For the sawtooth feature geometry presented here, pDEP should be seen for cells at the higher test frequency, indicated by the occurrence of pearl chaining and focusing of cells or particles at the sawtooth feature edge of the channel, while at the lower test frequency, nDEP should be apparent as cells are restricted to the region nearer the straight edge of the channel. At these low frequencies, cells may lyse due to electroporation, so the sample may appear to contain fewer cells, and those that are visible may appear enlarged or blurry if using an enzymatically-activated fluorescing dye. Verifying the frequencies at which pDEP and nDEP occur allows the determination of the crossover frequency as described in the protocol. For mammalian cancer cells, such as MOSE-L used here, we observed nDEP at 5 kHz (Figure 5a) and strong pDEP at 60 kHz (Figure 5b). MOSE-L cells had a diameter of 17.7&plusmn;3.3 &mu;m (n = 268 cells) and the beads have a 4 &mu;m nominal diameter. A full analysis of the dielectric properties of MOSE-L cells compared to MOSE cells in earlier stages of progression can be found in the recent work by Salmanzadeh et al.33
If pDEP and nDEP are not observed at these extremes, various problems could be occurring. The sample conductivity may be too high due to contaminants or cell lysing. Insufficient electric field may be generated due to bubbles trapped in the fluid electrode channels, which prevent conduction of current to the portion of the channels bordering the sample channel. Unsteady flow may be caused by a bubble trapped in the syringe or inlet tubing, bubbles resulting from not keeping the device under vacuum for a sufficient time or not quickly filling the device upon removing from vacuum, leakage due to delamination of poorly bonded PDMS to the glass slide, tears in the barrier membrane due to excessive force exerted when filling the channels, or flow rates at the extremes of the pump operating range.
In addition to determining the crossover frequency of cells, this technique can be used to sort a mixture, illustrated here by a mixture of MOSE-L cells and beads. This example takes advantage of the negative dielectrophoresis (nDEP) exhibited by 4 &mu;m beads at frequencies where MOSE-L cells experience positive dielectrophoresis (pDEP). We operated the device at 200 VRMS and 10 kHz (Figure 6a) and observed that both cells and beads were mixed as they experienced nDEP. At 50 kHz we observed movement of MOSE-L cells to the top of the channel, while beads were restricted to the lower portion of the channel, enabling separation of the mixture into two components (Figure 6b).
<img alt="Figure 5" src="/files/ftp_upload/50634/50634fig5.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig5highres.jpg"/> 
Figure 5. The position of MOSE-L cells is manipulated by changing the frequency of the applied electric field. The photos are taken at the final sawtooth feature in the sample channel and the fluidic electrodes are located at the corners on the right side. They are filled with PBS containing rhodamine B, which fluoresces to visualize the channels. (a) MOSE-L cells experience nDEP at 200 VRMS and 5 kHz. (b) MOSE-L cells experience pearl chaining and pDEP at 200 VRMS and 60 kHz. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig5highres.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 6" src="/files/ftp_upload/50634/50634fig6.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig6highres.jpg"/> 
Figure 6. A mixture of MOSE-L cells (green) and 4 &mu;m beads (red) flow through the final sawtooth feature in the sample channel of a low frequency cDEP device. (a) At 200 V and 10 kHz cells and beads are mixed. Arrows point to faintly appearing cells that likely have died due to the low frequency conditions. (b) At 200 V and 50 kHz cells experience pDEP and move to the feature edge of the channel, while beads experience nDEP and remain confined to the region nearer the straight edge. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig6highres.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis at JoVE.com

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

label-free-isolation-enrichment-cells-through-contactless

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.9K Views.  Virginia Tech. Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Video: Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Cancer Research

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Bioengineering

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 &#181;m x&#160;0.7 cm x&#160;2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 &#956;m diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 &#956;m polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 &#956;m micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

Engineering

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Developmental Biology

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Immunology and Infection

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.