Supporting Materials

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Supplementary Material
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Dielectrophoretic discrimination of cancer cells on a microchip
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Chengjun Huang1, 2, *, Chengxun Liu1, *, Bart Minne1, 3,
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Juan Enrique Ramirez Hernandez1, 3, Tim Stakenborg1, Liesbet Lagae1,3,
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IMEC, Kapeldreef 75, B-3001, Leuven, Belgium
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Current affiliation: Institute of Microelectronics, Chinese Academy of Sciences,
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No.3, Bei-Tu-Cheng West Road, Beijing, 100029, China.
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Also at: Department of Physics and Astronomy, KU Leuven, Celestijnenlaan, 200d,
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B-3001, Leuven, Belgium
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*Corresponding author:
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Dr. Chengjun Huang
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Institute of Microelectronics, Chinese Academy of Sciences
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No.3, Bei-Tu-Cheng West Road, Beijing, 100029, China.
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Tel: +86-10-82995743.
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Fax: +86-10-82995684.
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Email: [email protected]
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and
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Dr. Chengxun Liu
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IMEC, Kapeldreef 75, B-3001, Leuven, Belgium
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Tel: +32 16288953.
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Fax: +32 16281097.
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Email: [email protected]
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Experimental protocols
1. DEP crossover frequency device fabrication and DEP medium preparation
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A microchip with interdigitated gold microelectrodes was used, which were fabricated on a
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silicon wafer using standard photolithographic patterning process. The width of the electrode and
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the spacing between two neighboring electrodes were both 80 µm. A plastic “O-ring” with 5 mm
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in diameter and 1 mm in height was glued on the microchip as a cell sample reservoir.
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To perform the DEP crossover frequency measurements, DEP mediums with different
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conductivities were prepared. Herewith, 8.5% (w/v) sucrose solution and 150 mM NaCl solution
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were mixed with different volume ratios to obtain the desired medium conductivities ranging
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from 2 µS/cm to 1440 µS/cm. The conductivities of the mixture were examined using a
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conductivity meter (Hanna HI8733, Hanna instruments, USA) and the osmolalities (around 300
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mmol/kg) of the mixture were monitored to maintain the proper osmosis.
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2. DEP crossover frequency measurements
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The measurement setup is illustrated in Fig. S1. To measure the DEP crossover frequency of
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different cells, 100 µL (i.e., ~ 103cells) of the prepared cancer cells or PBMCs suspensions were
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added into the sample reservoir on the DEP device and covered with a glass slip to prevent
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evaporation. A bright field microscope (Nikon Bright Field/Dark Field Microscope LV150, Nikon
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Corp., Japan) connected with a camera (ThorLabs USB2.0 Digital Camera) was used to visualize
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the cell movements. The device was powered with a function generator (Tetronix AFG3022B
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Dual Channel) and an AC voltage of Vpp=4 V (peak-to-peak) was applied between the two
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adjacent microelectrodes. Individual cells, located between the two adjacent electrodes, were
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randomly selected and examined. The voltage frequency was slowly swept and the cell
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movements driven by the induced DEP force were observed. The crossover frequency at which
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the cell experienced no DEP movement was recorded. The diameter of each cell was measured
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from the captured images. At least 30 individual cancer cells were measured for each of the
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studied conditions. In addition, over 300 individual PBMCs were measured. All measurements
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were performed at room temperature.
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Fig. S1 Schematic of the used measurement setup to determine the DEP crossover frequency
of cells
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3. Cancer cells and blood cells preparation
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Four different cancer cell lines, MCF-7, MDA-MB-231, SKOV-3, and LnCap were cultured
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in house according to the American Type Culture Collection (ATCC) guidelines. Prior to DEP
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crossover frequency measurements, the cell lines were harvested in their native cell culture
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mediums with a cell density of around 106 cells/mL, then washed three times and finally
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re-suspended in the above-prepared DEP mediums for the measurements.
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Whole blood samples were received from the Belgian Red Cross and the PBMCs were
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prepared using standard Ficoll-Paque density gradient centrifugation.1 Similar to cancer cells, the
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isolated PBMCs were also washed for three times before re-suspending in DEP mediums with the
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desired conductivities for the measurements. In another set of experiments, the different purified
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subpopulations of PBMCs, such as monocytes, T cells, B cells, and NK cells were received from
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Johnson and Johnson (Johnson and Johnson, NJ, USA) and used for the DEP crossover frequency
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measurements after washing in DEP mediums.
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4. Cell treatments
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4.1 Cell fixation and cell permeabilization
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To prepare fixated MCF-7 cells, 1 mL freshly harvested MCF-7 cells were centrifuged and
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re-suspended in the cell fixation solution containing 250 µL of PBS and 250 µL of Insider Fix
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(Miltenyi Biotec, Germany) with 3.7% of formaldehyde. The sample was incubated for 20 min at
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room temperature. Afterwards, the cells were washed 3 times with the prepared DEP mediums
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and their crossover frequencies were measured. To permeabilize the cells, 1 mL freshly harvested
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MCF-7 cells were centrifuged and re-suspended in the cell permeabilization solution containing
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10 µL of PBS and 90 µL of Inside Perm with 0.05% azide and detergent (Miltenyi Biotec,
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Germany). The sample was incubated for 20 min at room temperature and washed 3 times with
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the prepared DEP mediums for further use.
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4.2 Antibody coupling of cancer cells
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To couple MCF-7 cells with antibody, 1 mL freshly harvested MCF-7 cells were centrifuged
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from the cell culture medium and re-suspended in 100 µL PBS containing 10 µL of monoclonal
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CD326 (EpCAM) antibody conjugated to FITC (Miltenyi biotec, Germany). The sample was
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incubated for 10 min, washed 3 times and re-suspended in the DEP mediums for the
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measurements. The antibody coupling efficiency was examined with a confocal fluorescence
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microscope (Carl Zeiss LSM 5 PASCAL, Carl Zeiss GmbH, Germany). More than 98% of the
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total observed cells showed fluorescently green, indicating a good antibody coupling efficiency.
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References:
1M.
Cristofanilli, De G. Gasperis, L. Zhang, M.C. Hung, P.R.C. Gascoyne, G.N. Hortobagyi,
Clinical Cancer Research, 8, 615 (2002).
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