3. DEP is applied across the bottom and top electrodes to position single cells over the nanopores (see Note 7).
4. DEP is switched off and immediately afterward the cells are
transfected.
5. The cells are then removed from the chip and placed in the
regular cell culture medium (see Note 8).
3.4 Thin Film
Microfluidics
1. A microcap array (SU8 photoresist) is patterned over nanochannel array on a silicon chip).
2. Each microcap consists of a “U-shape” feature that contains a
front-side cup for cell entry (see Note 9).
3. To enhance cell flow, the chip surface is pretreated to be hydrophilic using piranha solution and oxygen plasma.
4. For precise cell positioning, the chip, with the “U-shape” of
microcap facing up, is vertically dipped in (~5 min) and out of a
cell suspension (~10
6 cells/ml) in PBS.
5. Gravity and hydrodynamic forces modulate cell trapping within
the microcaps, directly over top of the nanochannel outlets
[19, 20].
6. Tailoring the geometries of the microcap array to be slightly
larger than the cell size can achieve single-cell capture in a
single cup, resulting in a clear cellular array on the chip
surface [21].
7. Trapped by the microcap, the cell contacts with the nanochannel so that it can be effectively electroporated while target
biomolecules are delivered into cytosol.
3.5 3D NEP
Electroporation
1. The scenario of negative charged cargo: In the Bio-Rad system,
a red cable is used as the positive end from the power supply
unit and generally connects to the top electrode of the chip
assembly; a black cable is used as the negative end from the
power supply unit and generally connects to the bottom electrode of the chip assembly (see Note 10).
2. Turn on the Bio-Rad electroporation power system and apply
the appropriate settings. Typical electroporation voltage range
for 3D NEP is within 50–200 V. The optimum condition
should be experimentally calibrated in terms of delivery
efficiency.
3. Select the desired signal, voltage, pulse length, number of
pulses, and pulse interval (see Note 10).
4. Optimal settings may vary from cell type to cell type.
5. Apply the voltage from the Bio-Rad machine by selecting the
“Pulse” button.
6. Disconnect electrodes from the chip and turn off the Bio-Rad
machine.
38
Lingqian Chang et al.
4. DEP is switched off and immediately afterward the cells are
transfected.
5. The cells are then removed from the chip and placed in the
regular cell culture medium (see Note 8).
3.4 Thin Film
Microfluidics
1. A microcap array (SU8 photoresist) is patterned over nanochannel array on a silicon chip).
2. Each microcap consists of a “U-shape” feature that contains a
front-side cup for cell entry (see Note 9).
3. To enhance cell flow, the chip surface is pretreated to be hydrophilic using piranha solution and oxygen plasma.
4. For precise cell positioning, the chip, with the “U-shape” of
microcap facing up, is vertically dipped in (~5 min) and out of a
cell suspension (~10
6 cells/ml) in PBS.
5. Gravity and hydrodynamic forces modulate cell trapping within
the microcaps, directly over top of the nanochannel outlets
[19, 20].
6. Tailoring the geometries of the microcap array to be slightly
larger than the cell size can achieve single-cell capture in a
single cup, resulting in a clear cellular array on the chip
surface [21].
7. Trapped by the microcap, the cell contacts with the nanochannel so that it can be effectively electroporated while target
biomolecules are delivered into cytosol.
3.5 3D NEP
Electroporation
1. The scenario of negative charged cargo: In the Bio-Rad system,
a red cable is used as the positive end from the power supply
unit and generally connects to the top electrode of the chip
assembly; a black cable is used as the negative end from the
power supply unit and generally connects to the bottom electrode of the chip assembly (see Note 10).
2. Turn on the Bio-Rad electroporation power system and apply
the appropriate settings. Typical electroporation voltage range
for 3D NEP is within 50–200 V. The optimum condition
should be experimentally calibrated in terms of delivery
efficiency.
3. Select the desired signal, voltage, pulse length, number of
pulses, and pulse interval (see Note 10).
4. Optimal settings may vary from cell type to cell type.
5. Apply the voltage from the Bio-Rad machine by selecting the
“Pulse” button.
6. Disconnect electrodes from the chip and turn off the Bio-Rad
machine.
38
Lingqian Chang et al.