electronically conductive, they can be electrically charged by applying a voltage in an electrolyte solution [12, 13]. This voltagecharging phenomenon causes an electric field gradient to be developed down the length of the tubes spanning the membrane. We
have shown that this gradient can be used to electroporate E. coli as
the cells pass through the membrane. Importantly, the requisite
3.65 kV/cm needed to electroporated E. coli [7] can be achieved by
applying voltage pulses of only 4 V to the microtube membrane.
This is roughly three orders of magnitude smaller than used in
typical commercial electroporation devices. Furthermore, with
our device, 22% of the bacteria were determined to be reversibly
electroporated, which is more than an order of magnitude higher
than the efficiency obtained with a commercial electroporator.
Because our microtube membrane is flow-through, higher
throughput can be achieved compared to traditional batchwise
porators. A throughput of 30 million cells per minute was achieved
here, higher than any previously reported device.
2 Materials
Prepare all solutions using analytical reagents and purified water
obtained by passing house-distilled water through a water filtration
system. Diligently follow all waste disposal regulations when disposing of waste materials.
2.1 Gold Microtube
Membrane Preparation
(See Note 1) [14, 15]
1. Nucleopore polycarbonate track-etched membrane filters,
5 μm pore size, 47 mm diameter, 10 μm thickness and
5 Â 10
5 cm
‑2 pore density.
2. 0.026 M tin chloride solution: Weigh 0.50 g of anhydrous tin
(II) chloride and transfer to a 100 mL volumetric flask. Add
50 mL of methanol and around 40 mL of water. Pipet 0.5 mL
of trifluoroacetic acid, and dilute to the mark with water (see
Note 2).
3. 0.035 M silver nitrate solution: Weigh 0.60 g of silver
nitrate, transfer to a 100 mL volumetric flask, and dilute to
the mark with water. Transfer to a 200 mL beaker, and add
ammonium hydroxide (28–30% solution in water) dropwise
until the solution turns brown and then clear again (see
Note 3).
4. Gold plating solution: Weigh 0.070 g of sodium bicarbonate
and 0.53 g of sodium sulfite, and transfer both to a 100 mL
beaker. Add 30 mL of water and pipet 1.83 mL of formaldehyde solution (36.0–38.0% solution in water) in the beaker (see
Note 4). Add 0.83 mL of Oromerse, part B gold plating
solution (commercially available from Technic, Inc., Craston,
44
Juliette Experton et al.
have shown that this gradient can be used to electroporate E. coli as
the cells pass through the membrane. Importantly, the requisite
3.65 kV/cm needed to electroporated E. coli [7] can be achieved by
applying voltage pulses of only 4 V to the microtube membrane.
This is roughly three orders of magnitude smaller than used in
typical commercial electroporation devices. Furthermore, with
our device, 22% of the bacteria were determined to be reversibly
electroporated, which is more than an order of magnitude higher
than the efficiency obtained with a commercial electroporator.
Because our microtube membrane is flow-through, higher
throughput can be achieved compared to traditional batchwise
porators. A throughput of 30 million cells per minute was achieved
here, higher than any previously reported device.
2 Materials
Prepare all solutions using analytical reagents and purified water
obtained by passing house-distilled water through a water filtration
system. Diligently follow all waste disposal regulations when disposing of waste materials.
2.1 Gold Microtube
Membrane Preparation
(See Note 1) [14, 15]
1. Nucleopore polycarbonate track-etched membrane filters,
5 μm pore size, 47 mm diameter, 10 μm thickness and
5 Â 10
5 cm
‑2 pore density.
2. 0.026 M tin chloride solution: Weigh 0.50 g of anhydrous tin
(II) chloride and transfer to a 100 mL volumetric flask. Add
50 mL of methanol and around 40 mL of water. Pipet 0.5 mL
of trifluoroacetic acid, and dilute to the mark with water (see
Note 2).
3. 0.035 M silver nitrate solution: Weigh 0.60 g of silver
nitrate, transfer to a 100 mL volumetric flask, and dilute to
the mark with water. Transfer to a 200 mL beaker, and add
ammonium hydroxide (28–30% solution in water) dropwise
until the solution turns brown and then clear again (see
Note 3).
4. Gold plating solution: Weigh 0.070 g of sodium bicarbonate
and 0.53 g of sodium sulfite, and transfer both to a 100 mL
beaker. Add 30 mL of water and pipet 1.83 mL of formaldehyde solution (36.0–38.0% solution in water) in the beaker (see
Note 4). Add 0.83 mL of Oromerse, part B gold plating
solution (commercially available from Technic, Inc., Craston,
44
Juliette Experton et al.