**3. Single cell electroporation**

### **3.1. Prospect of SCEP over Bulk Electroporation (BEP)**

For single cell electroporation (SCEP), the electric field parameters can be controlled to avoid cell death. In SCEP, where an inhomogeneous electric field is applied locally surrounding the single cell adhesion or suspension, whereas in bulk electroporation (BEP), a homogeneous electric field is applied to suspension of millions of cells together. Fig.1. shows two types of conventional bulk electroporation(BE) chamber, to apply electric field with suspension of millions of cells together for vitro experiment. Both figures has shown the cross sectional view with two metal electrodes.

Table-1, illustrates the five steps where "Induction step" describes the field induced mem‐ brane potential increase which provides local defects, when it reached to a certain critical value (above 200mV). Here mechanical strength of the cell membrane depends upon buffer composi‐ tion.The"Expansionstep"comeswhenfieldpresentswithastrengthlargerthanacriticalvalue. Inthiscaseelectromechanicalstresspresent."Stabilizationstep"indicates,fieldintensityislower than threshold value, a stabilization process will take place in a few milliseconds. As a result membrane will be permeabilized for small molecules. "Resealing step" demonstrates a slow resealing on a scale of seconds and minutes. The "Memory effect" comes due to some changes of the membrane properties for longer time, such as an hours, but cell behavior is still normal [48-50].Table-2demonstrate electroporationconditions ofvarious celltypes [51], where electric field strength, pulse length, no of pulses, time between two pulses vary in each different type of

**(µS) Number of pulses (sec)**

HMSC 700V 90 5sec 0.1 75,000 HUVEC 250V 150 - - 75000 RPTEC 300V 300 - 0.1 75000

NHDF-neo 900V 70 5sec 5 75000 PC-12 450V 200 - - 75000 Rat astrocytes 300V 90 0.1sec 0.1 75000 NHA 450V 120 0.1sec 0.1 75000 K562 350V 130 0.1sec 0.1 150000

**Table 2.** Electroporation conditions for various cell types. Permission to reprint obtained from RNA society [51].

For single cell electroporation (SCEP), the electric field parameters can be controlled to avoid cell death. In SCEP, where an inhomogeneous electric field is applied locally surrounding the single cell adhesion or suspension, whereas in bulk electroporation (BEP), a homogeneous electric field is applied to suspension of millions of cells together. Fig.1. shows two types of conventional bulk electroporation(BE) chamber, to apply electric field with suspension of millions of cells together for vitro experiment. Both figures has shown the cross sectional view

300V 400 - 0.1 200000

**Time between pulses (second)**

**Number of cells**

cells.

**Cell type Voltage**

Human T-Cells

**(Volt)**

**3. Single cell electroporation**

with two metal electrodes.

**3.1. Prospect of SCEP over Bulk Electroporation (BEP)**

**Pulse length**

66 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

**Figure 1.** Bulk electroporation apparatus for vitro experiment. Two types of electroporation chamber, to apply an ex‐ ternal electric field into the suspension of millions of cells together. Each chamber (a.b) consists cross sectional view of cuvette with two metal electrodes. Figure has redrawn with reprint permission [8].

Fig.2. demonstrates the single cell electroporation technique, where an external electric field is applied across the single cell membrane surface.

**Figure 2.** Single cell electroporation (SCEP) (a) Electric field was applied outside of the cell membrane (b) When exter‐ nal electric field reaches to a certain threshold value of the cell membrane, then cell membrane can permeabilized to deliver drug/biomolecules inside the single cell. After electroporation cell membrane reseal again.

When an external electric field beyond the certain threshold value of the cell membrane, then cell membrane canpermeabilizedtodeliver biomolecules inside the single cell.The success rate like surviving cell for single cell electroporation is far better compared with bulk electropora‐ tion (BEP). This technique is faster and easy to perform with less toxicity and technical difficul‐ ty for application of wider tissues and cells. By this electroporation technique, the specific cell membrane region with small volume can be targeted to deliver the drugs, which can help to preserve expansive gene or molecules. Due to small volume of electroporation, different gene can be transferred in different electroporated time without cell damage. SCEP technique can provide precise temporal and spatial gene or dye delivery inside the cell. These processes are affordable methods for fluorescently labeled and genetically manipulated individual cells [52]. This level of electroporation study is more convenient to understand molecular and genetic mechanisms with their biological functions and SCEP has ability to control temporally molecu‐ lar biology of the cell, which was challenging task for transgenic model systems [52]. For bulk electroporation the required voltages are very high (103 V) and this technique has little control of individual cell resulting in suboptimal parameters [53], as a result it is difficult to achieve reversible electroporation of all cells [54]. Moreover in single cell electroporation, there is good opportunity to observe the single cell response with specific cell size, shape, status and orienta‐ tion of the electric field. SCE is useful for primary culture and heterogeneous culture such as brain tissue culture [55].

The first single cell electroporation has been demonstrated by using two carbon fiber micro‐ electrodes [46], where the electrodes (2 µm to 5 µm) was positioned from the boundary of the cell surface at an 0-20° angle and 160-180° angle with respect to the objective plane. The patch clamp technique demonstrated the single cell electroporation (SCE), where patch-clamp pipette was sealed on the cell at a 900 angle with respect to the microelectrodes [56]. Using this technique, from transmembrane current response, it was possible to determined electric field strength for ion permeable pore formation and kinetics of pore opening, closing as well as pore opening times [56]. The electrolyte-filled capillary (EFC) coupled with a high-voltage power supply has been used for single cell experiment [57]. For application of a large voltages across EFC, It causes the formation of pores in the cell membranes which induces an electroosmotic flow of electrolyte. Micropipettes filled with DNA or other molecules stimulated by electric field have been electroporate the single cell at the tip of the pipette and successfully deliver the molecules inside single cell [58]. Microfabricated chip was used to incorporated the biomolecules into live biological cells for single cell experiment [59]. To achieve successful single cell electroporation, cell must be isolated from its population or inhomogeneous electric field must be focused on a particular cell, leaving neighboring cells unaffected [60]. Microfab‐ ricated devices can fulfil both isolated single cell and focused the electric field on particular single cell. Also this technology can offer other functionalities into the chip. Nowadays, SCEP research is growing on rapidly for biomedical application in vivo and in vitro. However to allow selective manipulation of single organelles within a cell, the electrode size must be reduced to nanoscale level. Nanoelectrode can provide less toxicity with high cell viability during electroporation experiment. Thus the localized single cell membrane electroporation concept has come in several years [61]. Fig.3. shows the localized single cell membrane electroporation (LSCMEP) process, where electric field is applied very short region of the cell membrane.

As a result, due to permeabilization of the cell membrane, drug/biomolecules can be delivered precisely (through sub micrometer to nanometer region of the cell membrane surface) inside the single cell. By this technique selective manipulation of organelles and biochemical effects can be analyze more precisely of the individual cell and this technique have more advantage compared to SCEP. Also the cell rapture and cell death can be minimize because electric field can intense in localized region of the cell membrane compared to SCEP. But this technology is now in underdeveloped stage. Recently Boukany et al. suggested nanochannel electropo‐

affordable methods for fluorescently labeled and genetically manipulated individual cells [52]. This level of electroporation study is more convenient to understand molecular and genetic mechanisms with their biological functions and SCEP has ability to control temporally molecu‐ lar biology of the cell, which was challenging task for transgenic model systems [52]. For bulk

of individual cell resulting in suboptimal parameters [53], as a result it is difficult to achieve reversible electroporation of all cells [54]. Moreover in single cell electroporation, there is good opportunity to observe the single cell response with specific cell size, shape, status and orienta‐ tion of the electric field. SCE is useful for primary culture and heterogeneous culture such as

The first single cell electroporation has been demonstrated by using two carbon fiber micro‐ electrodes [46], where the electrodes (2 µm to 5 µm) was positioned from the boundary of the cell surface at an 0-20° angle and 160-180° angle with respect to the objective plane. The patch clamp technique demonstrated the single cell electroporation (SCE), where patch-clamp

technique, from transmembrane current response, it was possible to determined electric field strength for ion permeable pore formation and kinetics of pore opening, closing as well as pore opening times [56]. The electrolyte-filled capillary (EFC) coupled with a high-voltage power supply has been used for single cell experiment [57]. For application of a large voltages across EFC, It causes the formation of pores in the cell membranes which induces an electroosmotic flow of electrolyte. Micropipettes filled with DNA or other molecules stimulated by electric field have been electroporate the single cell at the tip of the pipette and successfully deliver the molecules inside single cell [58]. Microfabricated chip was used to incorporated the biomolecules into live biological cells for single cell experiment [59]. To achieve successful single cell electroporation, cell must be isolated from its population or inhomogeneous electric field must be focused on a particular cell, leaving neighboring cells unaffected [60]. Microfab‐ ricated devices can fulfil both isolated single cell and focused the electric field on particular single cell. Also this technology can offer other functionalities into the chip. Nowadays, SCEP research is growing on rapidly for biomedical application in vivo and in vitro. However to allow selective manipulation of single organelles within a cell, the electrode size must be reduced to nanoscale level. Nanoelectrode can provide less toxicity with high cell viability during electroporation experiment. Thus the localized single cell membrane electroporation concept has come in several years [61]. Fig.3. shows the localized single cell membrane electroporation (LSCMEP) process, where electric field is applied very short region of the cell

As a result, due to permeabilization of the cell membrane, drug/biomolecules can be delivered precisely (through sub micrometer to nanometer region of the cell membrane surface) inside the single cell. By this technique selective manipulation of organelles and biochemical effects can be analyze more precisely of the individual cell and this technique have more advantage compared to SCEP. Also the cell rapture and cell death can be minimize because electric field can intense in localized region of the cell membrane compared to SCEP. But this technology is now in underdeveloped stage. Recently Boukany et al. suggested nanochannel electropo‐

V) and this technique has little control

angle with respect to the microelectrodes [56]. Using this

electroporation the required voltages are very high (103

68 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

brain tissue culture [55].

membrane.

pipette was sealed on the cell at a 900

**Figure 3.** Localized single cell membrane electroporation (LSCMEP) technique, where drug/biomolecules can deliver precisely inside the single cell (a) Electric field was applied in a very small region of the cell membrane area (Localized way) (b) After electric field application, due to permeabilization of the cell membrane, drug/biomolecules can success‐ fully deliver inside the single cell. Permission to reprint obtained from Springer [63].

ration with precise amount of biomolecules delivery by LSCMEP process. Where single cell has been positioned in one microchannel by optical tweezers and transfection agent was loaded to another microchannel. Two microchannel were connected by one nanochannel. Due to application of voltage between two microchannels, transfection agent was delivered through nanochannel using electrophoretically driven process and finally drugs delivered inside single cell through a very small area of the cell membrane [62]. Nawarathna et al. demonstrated localized electroporation technique using atomic force microscopy (AFM). Where modified AFM tip (0.5 µm) was used as a nanoelectrode, which was produced localized electric field into the cell membrane [61]. Fig 4.(a-h) shows the results of LSCMEP technique using AFM tip for electroporation process and Fig.4(i) demonstrated the AFM tip, which was positioned on top of the single cell for LSCMEP process.

**Figure 4.** (a) Bright field image of AFM tip where the cell in the electroporation medium (cell A is electroporated while cell B and C are about 20 µm away from cell A). (b) Fluorescence image of rat fibroblast cell after electroporation. (c) Confocal fluorescence image of an electroporated cell. (d)-(h) Sequence of real time confocal fluorescence images of rat fibroblast cell after electroporation. (i) Calculated spatial distribution of electric field in the vicinity of the cell being electroporated. Permission to reprint obtained from American Institute of Physics (AIP) [61].

Chen et al. demonstrated localized single cell membrane electroporation (LSCMEP) by using microfluidic device. Where ITO thin film was used as microelectrode with 1 µm gap between two micro-electrodes. The ITO microelectrode with 100 nm thickness and 2 µm width intense electric field much more in between two microelectrode gap [63]. Fig.5. shows the device fabrication for localized electroporation experiment.

**Figure 5.** Fabrication process of ITO microelectrode based localized single cell electroporation chip. (a) Fabrication process step (b) Optical microscope image of patterned ITO microelectrodes. (c) SEM image of ITO microelectrodes with micro channel (FIB etch), Permission to reprint obtained from Springer [63].

**Figure 6.** After application of 8Vpp 20 ms pulse, cell survival fluorescence image of HeLa cell at different time scale, Permission to reprint obtained from Springer [63].

According to the results, 0.93 µm electroporation regions were achieved successfully with 60% cell viability for 20 microsecond pulse. Fig.6. demonstrates the cell survival fluorescence image of HeLa cell at different time scale during LSCMEP process.
