4 Notes
1. Because of the high secondary electron doses, lithographic
processes can be performed in a region in close proximity to
the milled surface, being effective in breaking carbon–carbon
bonding (Fig. 2a).
2. The insoluble resist remains attached to the membrane
(Fig. 2d), and represents an exact replica of the milled
structure.
3. Gold is used as the coating of the 3D nanoelectrodes for two
main reasons: first, it is biocompatible and a well-established
substrate for in-vitro cell growth [9, 11]. Second, gold
represents a suitable compromise between electrochemical
properties, chemical stability, manufacturability, and costeffectiveness.
4. The passivation strategy ensures that the entire planar surface is
electrically insulating and does not contribute to apply any
potential difference at the cell membrane.
5. This passivation allows membrane poration to happen only
where the cells are in direct contact with the nanostructure
tips, whereas the rest of the cells membrane remains in tight
adhesion with the passivation material. Therefore, any molecule flowing through the nanochannels can be intracellularly
delivered only to the attached cell, because the molecule movement is completely limited by the sealing of the cell membrane
on the passivation surface.
6. The final 3D hollow nanostructures are 1.8 μm high and are
coated with 60 nm of gold; the inner nanochannels have a
diameter of 250 nm, while the external diameter is around
400 nm. The top of the resulting hollow nanocylinders has a
toroidal shape with a sharp edge of approximately 80 nm.
7. Since the 3D nanoelectrodes are electrically connected
together by the gold layer deposited on the flat surface of the
nitride membrane and beneath the SU8, a single large electrode is formed which is wired to an external pulse generator.
References
1. Caprettini V, Cerea A, Melle G, Lovato L,
Capozza R, Huang JA, Tantussi F, Dipalo M,
De Angelis F (2017) Soft electroporation for
delivering molecules into tightly adherent
mammalian cells through 3D hollow nanoelectrodes. Sci Rep 7(1):8524. https://doi.org/
10.1038/s41598-017-08886-y
2. Chang LQ, Bertani P, Gallego-Perez D, Yang
ZG, Chen F, Chiang CL, Malkoc V, Kuang TR,
Gao KL, Lee LJ, Lu W (2016) 3D nanochannel
electroporation for high-throughput cell transfection with high uniformity and dosage control. Nanoscale 8(1):243–252. https://doi.
org/10.1039/c5nr03187g
3. Neumann E, Schaefer-Ridder M, Wang Y,
Hofschneider PH (1982) Gene transfer into
mouse glyoma cells by electroporation in high
electric fields. EMBO J 1:841–845
18
Jun Yin and Yang Li
1. Because of the high secondary electron doses, lithographic
processes can be performed in a region in close proximity to
the milled surface, being effective in breaking carbon–carbon
bonding (Fig. 2a).
2. The insoluble resist remains attached to the membrane
(Fig. 2d), and represents an exact replica of the milled
structure.
3. Gold is used as the coating of the 3D nanoelectrodes for two
main reasons: first, it is biocompatible and a well-established
substrate for in-vitro cell growth [9, 11]. Second, gold
represents a suitable compromise between electrochemical
properties, chemical stability, manufacturability, and costeffectiveness.
4. The passivation strategy ensures that the entire planar surface is
electrically insulating and does not contribute to apply any
potential difference at the cell membrane.
5. This passivation allows membrane poration to happen only
where the cells are in direct contact with the nanostructure
tips, whereas the rest of the cells membrane remains in tight
adhesion with the passivation material. Therefore, any molecule flowing through the nanochannels can be intracellularly
delivered only to the attached cell, because the molecule movement is completely limited by the sealing of the cell membrane
on the passivation surface.
6. The final 3D hollow nanostructures are 1.8 μm high and are
coated with 60 nm of gold; the inner nanochannels have a
diameter of 250 nm, while the external diameter is around
400 nm. The top of the resulting hollow nanocylinders has a
toroidal shape with a sharp edge of approximately 80 nm.
7. Since the 3D nanoelectrodes are electrically connected
together by the gold layer deposited on the flat surface of the
nitride membrane and beneath the SU8, a single large electrode is formed which is wired to an external pulse generator.
References
1. Caprettini V, Cerea A, Melle G, Lovato L,
Capozza R, Huang JA, Tantussi F, Dipalo M,
De Angelis F (2017) Soft electroporation for
delivering molecules into tightly adherent
mammalian cells through 3D hollow nanoelectrodes. Sci Rep 7(1):8524. https://doi.org/
10.1038/s41598-017-08886-y
2. Chang LQ, Bertani P, Gallego-Perez D, Yang
ZG, Chen F, Chiang CL, Malkoc V, Kuang TR,
Gao KL, Lee LJ, Lu W (2016) 3D nanochannel
electroporation for high-throughput cell transfection with high uniformity and dosage control. Nanoscale 8(1):243–252. https://doi.
org/10.1039/c5nr03187g
3. Neumann E, Schaefer-Ridder M, Wang Y,
Hofschneider PH (1982) Gene transfer into
mouse glyoma cells by electroporation in high
electric fields. EMBO J 1:841–845
18
Jun Yin and Yang Li