4. Cut the assembly into individual pieces following the microchannel patterns with a razor blade. After cutting, punch
through the assembly with a 4 mm puncher to make the cell
culture chamber. Make sure the hole is laid in the center of the
microchannel. Separate two layers which are ready for final
assembling described in the following steps.
5. The PC membrane has different roughness on two sides. The
smooth side gives more light reflection, which can be easily
identified with naked eyes. Place a piece of the PC membrane
on a cutting pad with the smooth side facing up. Cut the
membrane into circular pieces with a 6 mm puncher.
6. The top and bottom layers are placed under UV plasma for
cleaning (see Note 3).
7. Spin coat a clean silicon wafer with PDMS solution with a ratio
of 5:1 between base and curing agent under 3000 rpm
(766 Â g) for 30 s. Ink the side without microchannel of the
bottom layer by bringing the PDMS layer in direct contact with
the PDMS solution on the wafer. Place the bottom layer on
side with the inked side facing up. Place a PC membrane piece
on top of the bottom layer and make sure the center of the
membrane aligns with the center of the chamber and the
smooth side facing up.
8. Ink the top layer and bring the inked side in direct contact with
the inked surface of the bottom layer, such that the PC membrane is sandwiched in between.
9. Gently press the assembly with a tweezers to make sure the
contact is solid. Bake the final device at 65
C for 1 h.
10. Inlet and outlet are then introduced using a 2 mm and 1 mm
punchers, respectively.
3.3 Cell Culture and
Plasmid DNA
1. Cells in suspension are placed in the cell culture chamber of the
device.
2. The device is placed in an incubator at 37
C and 5% CO 2 for
24 h (see Note 4).
3. Cells should adhere to the PC membrane after 24 h. This can
be easily verified by microscope imaging.
3.4 Electroporation
1. 10 μL of a 3:1 (V/V) solution of PBS and GFP plasmid in
DMEM at the desired concentration was placed in the cell
culture chamber (see Note 5).
2. The electrodes are connected to the function generator, voltage amplifier, and oscilloscope.
3. The resistance is measured to test the electrical connection
(5–10 kΩ) (see Note 6).
Microfluidic Device for Localized Electroporation
95
through the assembly with a 4 mm puncher to make the cell
culture chamber. Make sure the hole is laid in the center of the
microchannel. Separate two layers which are ready for final
assembling described in the following steps.
5. The PC membrane has different roughness on two sides. The
smooth side gives more light reflection, which can be easily
identified with naked eyes. Place a piece of the PC membrane
on a cutting pad with the smooth side facing up. Cut the
membrane into circular pieces with a 6 mm puncher.
6. The top and bottom layers are placed under UV plasma for
cleaning (see Note 3).
7. Spin coat a clean silicon wafer with PDMS solution with a ratio
of 5:1 between base and curing agent under 3000 rpm
(766 Â g) for 30 s. Ink the side without microchannel of the
bottom layer by bringing the PDMS layer in direct contact with
the PDMS solution on the wafer. Place the bottom layer on
side with the inked side facing up. Place a PC membrane piece
on top of the bottom layer and make sure the center of the
membrane aligns with the center of the chamber and the
smooth side facing up.
8. Ink the top layer and bring the inked side in direct contact with
the inked surface of the bottom layer, such that the PC membrane is sandwiched in between.
9. Gently press the assembly with a tweezers to make sure the
contact is solid. Bake the final device at 65
C for 1 h.
10. Inlet and outlet are then introduced using a 2 mm and 1 mm
punchers, respectively.
3.3 Cell Culture and
Plasmid DNA
1. Cells in suspension are placed in the cell culture chamber of the
device.
2. The device is placed in an incubator at 37
C and 5% CO 2 for
24 h (see Note 4).
3. Cells should adhere to the PC membrane after 24 h. This can
be easily verified by microscope imaging.
3.4 Electroporation
1. 10 μL of a 3:1 (V/V) solution of PBS and GFP plasmid in
DMEM at the desired concentration was placed in the cell
culture chamber (see Note 5).
2. The electrodes are connected to the function generator, voltage amplifier, and oscilloscope.
3. The resistance is measured to test the electrical connection
(5–10 kΩ) (see Note 6).
Microfluidic Device for Localized Electroporation
95