Srabani Kar and Santra Et Al.

Journal of Micromechanics and Microengineering
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Single Cell Electroporation-Current Trends, Applications and Future

prospects
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Page 1 of 57 AUTHOR SUBMITTED MANUSCRIPT - JMM-103716.R1
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4 Single-Cell Electroporation: Current Trends, Applications and Future
5 Prospects
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9 Srabani Kar 1 , L. Mohan 1 , Koyel Dey 1 , Pallavi Shinde 1 , Hwan-You Chang 2 , Moeto Nagai
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11 , Tuhin Subhra Santra 1, *
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Department of Engineering Design, Indian Institute of Technology-Madras, India 600 036
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16 Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30013
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Department of Mechanical Engineering, Toyohashi University of Technology, Toyohashi,
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20 Japan 441-8580
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23 Abstract
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26 The ability to deliver foreign molecules into a single living cell with high transfection
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efficiency and high cell viability is of great interest in cell biology for the applications in
therapeutic development, diagnostics and drug delivery towards personalize medicine. Many
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31 chemical and physical methods have been developed for cellular delivery, however most of
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33 these techniques are bulk approach, which are cell-specific and low throughput delivery. On
34 the other hand, electroporation is an efficient and fast method to deliver exogenous
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36 biomolecules such as DNA, RNA, oligonucleotides into target living cells with the advantages
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38 of easy operation, controllable electrical parameters and avoidance of toxicity. The rapid
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development of micro/nanofluidic technologies in last two decades, facilitates to focus an
41 intense electric field on the targeted the cell membrane to perform single cell micro-nano-
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43 electroporation with high throughput intracellular delivery, high transfection efficiency and
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45 cell viability. This review article will emphasize the basic concept and working mechanism
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associated with electroporation, single cell electroporation and biomolecular delivery using
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48 micro/nanoscale electroporation devices, their fabrication, working principles and cellular
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50 analysis with their advantage, limitation, potential applications and future prospect will also be
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52 elaborated.
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54 Keywords: electroporation, single cell micro-nano-electroporation, micro-nano fluidic
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56 devices, intracellular delivery, transfection efficiency, cell viability.
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* Corresponding author;
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AUTHOR SUBMITTED MANUSCRIPT - JMM-103716.R1 Page 2 of 57
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3 E-mail address: tuhin@iitm.ac.in, santra.tuhin@gmail.com (Dr. Tuhin Subhra Santra)
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6 1. Introduction
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8
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9 Treating critical diseases in recent years demands significant research on intracellular delivery
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11 in the field of medical science and cell biology[1,2]. Efficient and safe intracellular delivery of
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several kinds of bio-molecules such as large plasmids for gene expression and oligonucleotides
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14 (antisense DNA or interfering RNA) for protein regulations play a key role in cell biology
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16 research and understandings, such as mutagenesis and genetic engineering of microorganisms
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18 [3,4]. Most of the biological compounds of medical interest such as dyes, drugs, DNA, RNA,
19 proteins, peptides, and amino acids are polar and hence are unable to cross the cell membranes
20
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21 [5]. Though nucleic acid-based therapeutics can have the promising potential, challenges arise
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23 to deliver highly negative charged biological molecules. In this regard, gene silencing got
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significant attention for transferring small sized interfering RNAs (siRNAs) as specific
26 targeting therapeutic reagents [6,7]. Further progress was assisted with the unique idea of
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packaging DNA or RNA inside nanoscale viral vector. Though this technique offers very high
30 transducing efficiency, its application in human body is a question in terms of safety concerns
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[8,9] and encourage non-viral vectors mediated delivery of bio-molecules though being less
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33 efficient than the former one [10]. The non-viral methods include chemical treatment [11],
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35 utilization of a biolistic gun [12], ultrasound [13], microinjection [14,15], optoporation or
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37 photoporation [16–18], microwave [19–21], hydrogel technique[22] etc. However, most of
38 these techniques are used for bulk electroporation and often limited by low efficiency of
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40 plasmid delivery into different cell type due to toxicity, plasmid degradation, and immune
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42 response[23,24]. In contrary, electroporation has been often elucidated to be a more convenient
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way to deliver several kinds of biomolecules from nano to macro-size, and many efforts have
45 been devoted to improve the efficiency [25].
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48 In 1756 electroporation was first demonstrated on human and animal skin by applying electrical
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sparks though the phenomenon was not identified as electroporation [26]. Later throughout 18th
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and 19th centuries, several sets of experiments were conducted on different kind of bio-systems
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53 such as dead frog, muscle-nerve, red blood cells etc. to understand electrical effects on
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55 biological materials [27]. It was 1936, maybe for the first time, G.M. McKinley [28] proposed
56 the impact of the electric field itself on cell membrane, beyond its thermal effects. The
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58 phenomena were strongly supported by A.L. Hodgkin [29] in 1951 who proposed the
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60 breakdown of insulating cell membrane “under the influence of the abnormally high potential
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3 difference” which is now named as “irreversible electroporation”. In the context of
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5 biomolecule-cell interaction studies, reversible electroporation, where the cell membrane
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7 reseals again after switching off the field, is requisite instead of irreversible electroporation. In
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the present review, we have stressed more on reversible single cell electroporation (SCEP)
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10 rather than the irreversible one.
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13 Traditional or conventional electroporation is bulk electroporation (BEP) where millions of
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cells are exposed to an external homogeneous electric field with amplitude in the order of a
16 few kV/cm [30]. The inter-electrode distance varies from millimeter to centimeter range. A
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18 few hundreds of volts are applied at the electrodes to achieve proper electric field needed to
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20 accomplish electroporation [31,32]. It is, therefore, a technique to transfect millions of cells
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together. However, studying intracellular behavior of individual cell requires further control
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23 over cell distribution, and electric field distribution and hence the concept of (single cell
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25 electroporation) SCEP arises which offers an in-depth characterization of cells. Furthermore,
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heterogeneity in single cells, even if they are originated from same mother cell, has already
been shown to be important in cancer diagnostic, classification, and treatment [25,33]. Also,
30 cross contamination between individual cell contents can be prevented if cells are isolated.
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32 Furthermore, analyzing cellular omics at bulk population provides average information which
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cannot reveal the details about molecular dynamics at single cell level [25,34]. Despite the fact
35 that several high-resolution strategies exist to detect, photograph, and analyze [35–40], just a
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37 few method exist for controlling and manipulating the biochemical nature of the contents of
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39 single cells and subcellular organelles [41,42]. At present, it is difficult, to transfect an
40
individual cell with a gene without transfecting its adjacent neighbor. These challenges were
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42 readily accepted by the micro-nano fluidic research community around twenty years back, and
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44 consequently publishing research articles with significant improvement in this field [43,44].
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46 There are few steps of SCEP: a) trap a single cell from the population, b) apply a highly focused
47 intense electric field amplitude and pulse only to the targeted cell without affecting others. To
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49 accomplish the first step, researchers had invoked small population of cells instead of dense
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51 distribution. For example, in 1997 Teruel et al. [45] used only 1 l of the sample to load
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53 adherent cells into a 10 mm2 surface area. Further research interests grew on the miniaturization
54 of electroporation device giving many additional advantages such as negligible cell damage,
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56 dosage control capabilities with single-cell resolution, avoiding the use of expensive high-
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58 voltage pulse generators and complicated microchamber mounts etc. Several approaches have
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been made to reduce the device size to micron scale and nanoscale [25,44,46] including
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3 modification of chip structures with varieties of micro/nano-electrode arrangements,
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5 introducing varieties of electrode materials [46], improving transfection by applying ac
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7 voltages with different waveforms and frequencies [47], improving the image quality in single
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cell limit etc. Apart from micro/nanochip-based techniques, solid micro-electrodes, micro-
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10 capillaries, micro-pipette, nanofountain probe, nanochannel and nanoelectrode based
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12 techniques also become popular for SCEP [43,48–53]. All those techniques are very special in
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14 the sense of transfecting a target cell without affecting the adjacent one. Moreover, sub-cellular
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organelles can be targeted providing a greater control and opportunity to study beyond single
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17 cell limit [54]. Thus the emerging field of single cell electroporation has great impact on a vast
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19 area of research from cell biology to biophysics, single cell analysis to therapeutic applications
20
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21 towards personalize medicine.
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23 Till now there are a few reviews in single cell electroporation: one by Olofsson et al.[55] in
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25 2003 on basis of probe types and by Wang et al. [56] in 2010 and Santra et al.[31] in 2013
26
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presenting the guidance with all important research articles in this topic. Beside those articles
which mainly focuses on SCEP techniques, there are reviews on micro/nanofluidic devices
30 for electroporation irrespective of bulk or a single cell [57,58]. However, all those reviews
31
32 overlook the progress of theoretical studies which are essentials to frame up the entire progress
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of this field. In this review, we have discussed so far progress of SCEP including proposed
35 devices, their challenges and benefits, applications and future prospectives. First, we started
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37 with the theoretical understanding of cellular behavior in an external electric field. Then the
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39 next section discussed the parameters and their impact on cellular uptake. The third section
40
presented the so far studies, recent trends, the comparative analysis of different methods and
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42 challenges of SCEP. The fourth or the final section presented the drawback and future
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44 perspective.
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47 2. Theory of electroporation
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There are several reviews on the theory of electroporation [59–62] combining so far theoretical
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understandings in the literature. A number of theoretical models have been presented to explain
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53 electroporation [63]. In this review, we briefly outline the mostly accepted aqueous pore
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55 formation model explaining the interaction of externally applied electric fields with cell
56 membrane. In general, the structure of cell membrane is a phospholipid bilayer membrane and
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58 is typical ~5 nm in thickness [64]. Each layer of phospholipid contains a polar head and non-
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60 polar hydrophobic hydrocarbon tails arranged in such a way that the polar head of the two
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3 layers face opposite to each other and hydrophobic tails remain at the middle part of membrane
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5 structure, making them amphiphilic [64]. In the context of electroporation (EP), this structure
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7 is of particular importance because of the potential and dipolar electric field distribution across
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the membrane. The conductivities of the cytoplasm (~0.3 S/m) and extracullular medium (~1.2
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10 S/m) are much higher than cell membrane (~3×10-7 S/m) [63]. When an external voltage pulse
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12 is applied, the cell membrane acts as dielectric capacitor. The field is highly intense across the
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membrane usually in the order of 103-108 V/m depending on the experimental condition
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[48,65]. As a result, a strong electrical force acts on membrane transferring a Maxwell stress
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17 leading to pore formation and membrane integrity [66–68]. This electric field inducs a position-
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19 dependent modulation of the membrane potential difference, named as transmembrane
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21 potential (𝑉𝑚 ) which plays the key role in electroporation. The correlation between the
22 transmembrane potential and pore formation was thoroughly investigated by experimental
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24 measurements and theoretical predictions which showed that pore formation occurs only to the
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26 membrane area where 𝑉𝑚 is above a critical threshold (~0.2 V-1.0 V) [65,69]. A heuristic
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overview of pore formation and evolution was presented by Debruin et al. [70] in 1998. The
paper assumed that two kinds of pores exist - hydrophobic or non-conducting pores and
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31 hydrophilic or conducting pores [70]. Initially the pores are hydrophobic and most of them are
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33 quickly destroyed by lipid fluctuations. The creation and stability of pores are determined by
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change of membrane free energy due to pore creation which includes pore edge energy,
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36 membrane surface energy, steric repulsion of lipid heads lining the pore, and the energy
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38 induced by transmembrane potential. When hydrophobic pores of radius r > r* (~0.5 nm) are
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40 created, they convert spontaneously to long-lived hydrophilic pores, which make the membrane
41 permeable. The pore radius r* corresponsds to the size at which it converts from hydrophobic
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to hydrophilic [65]. The value of r* is determined by the meeting-point of membrane free

44
45 energy curves for hydrophobic and hydrophilic pores. The hydrophilic pores within a small
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range of radii just above r* immediately expand to the minimum-energy radius rm to minimize
48 free energy. The abrupt increase of membrane conductance occurs when hydrophilic pores are
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50 created due to onset of charge trasnport through the pores. The explicit expression of temporal
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52 evolution of pore denity N is given by ordinary differential equation [64,70]

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54 𝑑𝑁 2 𝑁 2 (1)
55 = 𝑒 𝛽𝑉𝑚 (1 − 𝑒 −𝑞𝛽𝑉𝑚 ),
56 𝑑𝑡 𝑁0
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5
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3 where 𝑁0 is the pore density per unit area when 𝑉𝑚 = 0 V, , 𝛽, and 𝑞 are constants. After the
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5 voltage pulse being applied, the pore density starts to increase, reaches equilibrium pore density
6 2
7 𝑁𝑒𝑞 = 𝑁0 𝑒 𝑞𝛽𝑉𝑚 . After removal of voltage pulse when 𝑉𝑚 drops to zero , the pore density starts
8
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9 to decrease. The pores start to shrink rapidly to the minimum-energy radius of 0.8 nm, where
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11 they persist until resealing takes place [65]. The experimentaly observed long lived permeable
12 state of cells can be explained by the shrinkage of pores to the minimum-energy radius [65].
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14 While pore shrinkage occurs at few tenths of microseconds, resealing takes from a few seconds
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16 to minutes depending on experimental parameters [65,71,72]. The temporal evolution of pore
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18
radius is determined by the rate of change of membrane energy with pore radius i.e., the
19 advection velocity. The schematic of pore creation and evolution is shown in Figure 1. Thus
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Figure 1. Hypothetical structural rearrangements of a lipid bilayer membrane dur ing

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53 electroporation.
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55 the stages of electroporation are composed of the follwing steps: The ‘‘induction step’’
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57 corresponds to the charging of cell membrane and nucleation of pores at cell membrane (~a
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59 few hundreds of nanoseconds), the ‘‘expansion step’’ corresponds to thse pore evolution, the
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3 ‘‘stabilization step’’ corresponds to the postpulse shrinkage of pores (~at few tenths of
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5 microseconds), and the ‘‘resealing step’’ corresponds to the resealing of small pores (a few
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7 seconds to minute) [65].
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9 Mathematical model of transmembrane potential - When a spherical cell with non-conducting
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11 membrane is placed in an externally applied uniform electric field 𝐸𝑎 , the field distribution can
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13 be obtained by Laplace equations, ∇2 𝜑 = 0, which can be solved analytically giving the so
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15 called generalized Schwan equation for transmembrane potential
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17 𝑉𝑚 = 1.5𝑟𝑐𝑒𝑙𝑙 𝐸𝑎 cos 𝜃 (1 − exp(−𝑡/𝜏)), (2)
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20 where 𝑟𝑐𝑒𝑙𝑙 is cell radius and  is angle between the normal to the membrane and the direction
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22
of the field. τ is the charging time of the cell to reach maxima of 𝑉𝑚 . τ depends on the dielectric
23 properties of the membrane, the conductivities of cytoplasm (𝜎𝑖𝑛 ) and external buffer (𝜎𝑜𝑢𝑡 ).
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25 Considering the membrane to be pure dielectric, the simplified expression of τ is given by
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𝜏 = 𝑟𝑐𝑒𝑙𝑙 𝐶𝑚𝑒𝑚𝑏𝑟𝑛𝑒 (𝜎𝑖𝑛 + 2𝜎𝑜𝑢𝑡 )/(2𝜎𝑖𝑛 𝜎𝑖𝑛 ), (3)
30 where 𝐶𝑚𝑒𝑚𝑏𝑟𝑛𝑒 is membrane capacitance. The value of τ is in the order of few millisecond,
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32 checked experimentally and calculated theoretically [73]. In order to obtain permeable cell
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34 membrane, the value of Vm has to be larger than a critical threshold voltage (~0.2 V-1.0 V).
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36 Here in the context, of SCEP, it is imporatnt to mention that most of the cases the targated
37 single cell does not experience homogeneous electric field. Instead, the recent micro-fluidic
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39 devices are designed in such a way that the field distribution is preferably intense at the cell
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41 trapping sites [74–77]. Thus aformentioned Schwan equation is no longer valid and requires
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numerical dervation of field distributions and transmembrane potential.
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45 During electroporation, an electrical current flows through the cell medium and results in Joule
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47 heating in the system due to the resistive nature of the medium. As a consequence temperature
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49 of the system can increase. In a homogeneous planner system of electrical conductivity , the
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Joule heating rate per unit volume is given by 𝑄 = 𝜎|−∆𝜑|2 due to an applied electrical
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52 𝜎
potential 𝜑 and the corresponding temperature rise is ∆𝑇 = 𝜌𝐶 |∆𝜑|2 ∆𝑡, when ∆𝑡 is electric
53 𝑝
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55 pulse duration, 𝜌 is mass density, and 𝐶𝑝 is specific heat [78]. These relations assumes no
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fringe effect on electrodes and no heat dissipation in-between the pluses. However, the Joule
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58 heating can be controlled by the applied voltage and the electrical properties of the system
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60 including its proper design. The side effect of Joule heating include local burn in the cells and
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3 hence should be minimal while conducting the electroporation. These side-effect can be
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5 reduced by applying low field, short pulse and using a low ionic content pulsing buffer.
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7 Miniaturization of the electroporation device can meet all those requirements and single cell
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electroporation is the straightforward consequence of device miniaturixzation. The impact of
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10 temperature on electroporation is described in detail in the next section.
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13 3. Electroporation parameters
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16 The extent of experimental success for electroporation is mathematized by two technical words
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18 – transfection efficiency and cell viability, obtained by analyzing experimental data. There are
19 a lot of factors playing important roles to achieve electroporation with high transfection
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21 efficiency and high cell viability. A significant improvement in the experimental outcomes is
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23 only possible by proper optimization of different electroporation protocols. Briefly, we have
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discussed the important experimental parameters and their interlinks.
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3.1. Electric field distribution
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30 Electric field distributions play a crucial role to obtain high cell viability because only the part
31 of membrane experience electroporation where the transmembrane voltage is above the
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33 threshold value. As shown by Kotnik et al. [69,79] intracellular uptake only occurred at the
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35 poles of the cell. For transmembrane potential high above the threshold value causes
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irreversible pore formation and eventual cell death [80–82]. Thus the concept of localized
38 electroporation came in which cell is partially exposed to external fields to induce local
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40 membrane deformation. Our previous work has shown to achieve ~90-95% cell viability by
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42 localized single cell electroporation (LSCEP) [71,83,84].
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45 3.2. Electric pulse
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47 The electroporation experiments, started with DC voltages, showed distinguished response for
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49 AC voltages. Several experiments were carried out to reveal the impacts of pulse duration,
50 voltage amplitude and repetition rate on transfection efficiency and cell viability [72]. When
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52 single pulse was considered millisecond pulse was seen to create larger pore size with higher
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54 resealing time compare to microsecond pulse [85]. The pore size extends with pulse duration
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while the pore density increases with field intensity [65].
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3 3.3. Electrode material
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6 A metal electrode in electroporation chamber accomplishes transduction of the ionic current
7 into the electrical current [86]. The metal particles with a size of a few hundreds of nanometre
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9 are released from the electrodes after electric pulses being applied and create local
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11 enhancements of the electric field leading to the occurrence of electrochemical reactions at the
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electrode/electrolyte interface [87,88]. Though various kinds of metal like aluminum [87], gold
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14 [30], silver [89], platinum [53,90] have been used as electrode materials, aluminum is mostly
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16 used materials as electrodes because of high conductivity, ease of manufacturing and low-cost.
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18 However, the major drawback of using aluminum is that generated aluminum ions and
19 aluminum oxides are dissolved at both the anode and cathode after electroporation
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21 contaminating the electrolyte causing toxic effects and a conglomeration of cells and aluminum
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23 complexes and therefore can reduce on cell viability after electroporation being conducted [91–
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93]. One more issue is the direct imaging of cells during electroporation since any metal
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an
electrode like gold or aluminum will impede the cell-view from microscope objective. It is,
therefore, important to use transparent electrodes instead of metal or design the chip in such a
30 way that the electrodes would not disturb the view. One such approach was using optically
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transparent indium tin oxide (ITO) electrode amenable to optical inspection of cells during
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33 electroporation and subsequent effects, facilitating precise measurement of the following
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35 processes after electric field being applied to the cells [48,77]. However, there are few
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37 limitations of ITO such as low conductivity, easy degradation in the ionic electrolyte at high
38 voltage, high risk of damage due to Joule heating in the system etc. Researchers also use carbon
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40 fiber as electrode materials especially in micropipette based electroporation which also shows
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42 good results [87].
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44 One more critical concern is the performance of electrode for AC voltage instead of DC. The
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46 low impedance, highly stable metal electrodes are required, particularly in micropipette
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48 method, to operate them at high-frequency voltages for recording rapid signals [86]. Though
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Pt and Ag/AgCl electrodes are attractive for high conductivity, Pt electrodes are expensive and
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Ag/AgCl electrodes must maintain a layer of AgCl, responsible for the reversible redox
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53 reaction at the electrode surface and stable half-cell potential in electrolytes containing Cl as
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55 the main anion [86]. It is also important to note that excessive coating of AgCl tends to increase
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the resistance of the electrode demanding precise optimization on its thickness to minimize the
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58 electrode-electrolyte interface impedance [94]. One intermediate approach was coating an Ag
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60 wire followed by removing a portion of the initial deposit by reversing the voltage bias,
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1
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3 resulting in electrodes with a lower and more stable electrode impedance [95]. It is also
4
5 important to keep the electrode-electrolyte junction potential to be stable during an experiment
6
7 to avoid offset artifacts.
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10 3.4. Electroporation buffer
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12 Selection of appropriate buffer solution is a serious and inevitable job for high yield
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14 electroporation. The choice of the buffer depends on cell type [96,97]. An appropriate buffer
15 with low ionic content can enhance transfection efficiency [96]. Usually, buffer with high ionic
16
17 strength (high conductance) such as phosphate buffered saline (PBS), HEPES buffer, serum-
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19 free growth media are used in electroporation of mammalian cells [97]. Also, the buffer pH is
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as important as buffer compositions to mimic the cytoplasm of the cell [96,97].
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3.4. Temperature
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26 Ambient temperature has a great effect on electroporation and requires to be optimized before
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and after electroporation in order to control membrane permeabilization, transfection, pore
resealing, and cell viability. Because temperature acts on lipid fluidity; consequently pore
30
31 resealing time is found to be shortened at a higher temperature. Fast resealing decreases
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33 transfection efficiency and increases cell viability for example cells were found to be permeable
34 for up to 4 h at 4 °C, while at 37 °C the membrane resealing occurs in less than 5 min [98]. In
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36 addition, the critical voltage for electropermeabilization was found to be strongly dependent
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38 on temperature, for example, it increases by a factor of 2 with decreasing temperature from
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40 37C to 5C in Madin-Darby canine kidney epithelial cells [99]. The experimental studies also
41 showed that the rise of temperature can reduce the critical potential for electroporation. The
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critical voltage at 5C was seen as much as two times the voltage needed at 37C [100].
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46 Nowadays the use of polymeric electroporation chip is very popular because of their easy to
47
fabricate, low cost, easy visualization characteristics. Mostly electro-kinetically driven
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49 microfluidic devices are based on polydimethylsiloxane (PDMS), and hybrid PDMS/Glass.
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51 However, their low conductivities give rise to localized Joule heating at the microchannel
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53 leading to a significant temperature rise. For example, a nearly fivefold increase in the
54 maximum buffer temperature has been seen in the PDMS/PDMS chips over the PDMS/Glass
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56 systems at high potential field strengths [101].
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3 4. Why SCEP?
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6 Compare to traditional bulk electroporation (BEP), where thousands of volts are applied across
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8 millions of cells with little control over the permeabilization of individual cells, single-cell
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electroporation (SCEP) technique offers acute transfection of individual cells having flexibility
11 on targated cell numbers (from single cell to high throughput) with low-voltage-pulse (a few
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13 volts) induced highly focused electric field. SCEP techniques also offer manipulation on the
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15 cell positioning, cell concentration and reduce reagent consumption. BEP undergoes excess
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heat generation due to Joule heating because of resistance in the systems, local pH variation,
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18 electric field distortion, metal ion dissolution from metallic electrodes, and irreversible
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20 electroporation caused by excessive voltage, resulting in low delivery efficiency and low cell
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22 viability. Real-time monitoring of intracellular delivery in SCEP helps to establish optimal
23 electroporation protocols helps to achieve very high transfection efficiency with high cell
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25 viability. Moreover, delivering well-defined amounts of materials into cells is important for
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various biological studies and therapeutic applications. SCEP allows qualitative and
quantitative dosage control to conduct cell-autonomous studies of protein function, not possible
30 in BEP [51]. Moreover, the contents of individual cells can be extracted and analyzed, critical
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32 for investigating the distribution of various critical parameters between cells, instead of average
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34 data analysis from a population of cells in BEP [102,103]. In this context, it is important to
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mention that even though the cells which are identical in geometry, contents and overall
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37 behavior, genetically exhibit marked variations in cellular response and gene expression even
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39 when the population's characteristics are predictable, implying that BEP is not sufficient to
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41 describe the precise cellular function [104]. Furthermore, the complexity of neurons and
42 neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of
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44 single cells [105]. In this context, SCEP has been shown to be useful for in vivo fluorescent
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46 imaging of neuronal morphology, and connectivity[105]. In order to manipulate and analyze
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the cellular machinery, access to its content is often required [106]. The capability to study the
49 properties of intracellular organelles is a challenging task in cell biology and its therapeutics
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studies. In this context SCEP can be a prospective technique yet far from direct application.
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53 Both batch-type and flow-through designs have been made to face several existing challenges
54 in current electroporation systems.
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3 5. Single cell electroporation: Intracellular delivery and analysis
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6 In conventional electroporation technique, an electric field is applied on aggregations of
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8 millions of cells located between the electrodes, called bulk electroporation. There is no control
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over individual cells in the medium and thus it blocks precise bio-physical studied in single
11 cell limit in addition with adverse side effects like uncontrolled Joule heating, considerable pH
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13 change of solutions, lack of precise targeting etc.
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With the recent advancement of micro/nano-technologies, micro-needle injection offers precise
17 control over single cell drug/gene delivery but demands high skilled practitioners, special
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19 equipment, controlled environment. In contrary, single-cell electroporation techniques become
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21 more popular in research community for its simple and convenient working principle to study
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any kind of cells for understanding bio-physics properties allowing unprecedented spatial and
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24 temporal control on cargo delivery. It was first proposed by Huang et al. in 1999 who designed
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26 a microchip having facility to capture and transfect individual cells [107,108]. Here we will
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briefly discuss the proposed devices and their performances for SCEP. So far demonstrated
methodologies of single cell electroporation platforms can be divided into seven categories:
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32 5.1. Micropipette based single cell electroporation and configurations
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While a flexible highly localized single cell electroporation technique is considered with
36 multiplex solution delivery, microfluidic pipette is one of the best approaches. It is of particular
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38 importance because of its special capabilities to obtain high-resolution spatial control very
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40 close to targeted single cell or subcellular organelles such as the somatic, dendritic, or axonal
41 region of an adherent neuron, because the exposer can be handled freely in three dimensions
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by using a micromechanical positioner (Figure 2) [52,109]. Though it was initiated with the
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45 concept of generating blebs across the membrane of targeted single cell or bunch of cells to
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allow bio-molecules to get in, later multipurpose micropipette was combined with electrodes
48 to use them in electroporation [105,106].
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37 Figure 2. (a-c) Schematic view of the circulating liquid tip. (b-f) Photographic images of the
38 microfluidic pipette. (g) Illustration of a microfluidic pipette in a typical experimental setup.
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40 (h) Working principle of the microfluidic pipette. Pressure difference was created between the
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42 channels to completely recirculate the solution into the pipette. The volume of the recirculation
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zone (green sphere in panel h) was comparable to the size of the single cell of diameter 10 µm
45 (red sphere in panel h). “Reprinted with the permission from [109]. Copyright (2010) American
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47 Chemical Society”.
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To apply the electric field two strategies of electrode arrangements were proposed by Ainla et
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al. [106] : one, the electrodes are arranged externally (with respect to the device) (Figure 3A),
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53 and two, electrodes are fabricated into the pipette enabling solution delivery and targeted field
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55 to be applied together on one device facilitating the fluidic and electrode probes to be aligned
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unitedly (Figure 3B) unlike the one by one positioning for the former case. Mostly carbon-fiber
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58 microelectrodes are used in this platform.
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32 Figure 3. (A,B) Schematic view of the pipette tip with two configurations of electrodes. The
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35 representation of a two-channel superfusion pipette, allowing multiplexing between two
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37 chemical environments:𝐶𝑎2+ for chemical stimulation and Trypan blue for a subsequent
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39 viability test. “Reproduced from [106] with permission of The Royal Scociety of Chemistry.
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41 Steinmeyer et al. [105] demonstrated front loaded micropipette to deliver multiple genetic
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43 vectors and reagents (indicated by different colors in Figure 4 ) into targeted cells within the
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45 same brain tissue. They achieved high-throughput, efficient, reliable, and combinatorial
46 delivery with single-cell accuracy. The schematic of the step by step process is shown in Figure
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48 4. They performed multi-colored labeling of adjacent cells with different fluorescent reporter
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50 plasmids and obtained very high ransfection rate. They were also able to transfect neurons
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within the same brain tissue with different plasmids encoding different protein isoforms for
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53 analysis of their effects on linear dendritic spine density.
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19 Figure 4. Schematic of sequential operations and complete setup. (a) The micropipette was
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21 front-loaded from a multiwell plate containing reagents, (b) before being rapidly transferred
22 and positioned at a fixed location in the field of view of the microscope objective. (c,d) The
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24 three-axis stage was used to hold the slice bath/manipulation chamber for moving the sample
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26 and bringing cells into contact with the stationary micropipette, allowing cells to be
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transfected. (e) The micropipette tip was automatically transferred to a cleaning solution bath
and a rinsing reservoir for washing and finally loading sample from a different well of the
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31 multiwell plate, allowing the cycle to continue. “Reproduced from [105]. CC BY 4.0”
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Ohmachi et al. [110] also utilized this method to deliver a variety of biomolecules such as
35 plasmids, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent
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37 oligonucleotide (ECHO) RNA probes into individual living E. gracilis cells showing very high
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39 transfection efficiency and cell viability. This technique was also used to deliver siRNA into
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GFP-expressing Golgi and Purkinje cells in cerebellar cell cultures [111] and into adult sensory
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42 neurons [112] in primary cultures. Also, oligonucleotides and plasmid DNA were reported to
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44 be delivered in neurons of organotypic rat hippocampus and mouse cortex slice cultures with
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46 transfection efficiency being as high as ~80% [113]. Small oligonucleotides were found to
47 enter the cell immediately during pulse application while large plasmids were detected to
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49 remain localized at the cell membrane for more than 10 min before they enter the cell. This
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51 method of SCEP was found to serve no effect on the electrophysiology of transgene-expressing
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cells. Expression of several GFP-tagged neuronal proteins demonstrates that the method is
54 beneficial to analyze subcellular targeting and/or signaling of transgenic proteins in
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56 physiologically intact neurons [113]. In vivo electroporation of Xenopus laevis tadpole brains
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58 were also transfected with DNA, dextrans, morpholinos and their combinations by using
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modified patch electrodes and common electrophysiological equipment [114] and micropipette
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3 [115]. In vivo -targeted electroporation was performed on single tectal neurons within the
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5 albino Xenopus laevis tadpole using a pipette electrode filled with a solution of the delivery
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7 molecules and with a tip diameter much smaller than the width of the target cell [116]. A similar
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method was utilized for longitudinal imaging of synaptic structure and function in the adult
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10 mouse neocortex in vivo [117].
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13 To obtain acute control over electroporation area, Iwata et al. [118] et al. proposed nanopipette
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based non-contact approach for SCEP where the probe and cell surface distance can be
16 controlled by using a scanning ion conductance microscope (SICM) allowing low-invasive
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18 electroporation of a single cell. The schematic diagram is shown in
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35 Figure 5. An electrode, acting as an inner electrode, is inserted within the nanopipette and outer
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37 electrode is fabricated at its outer edge. When an external voltage is applied between those
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39 electrodes, a local-focused electric field is created at the edge/tip of the nanopipette transfecting
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the targeted single cell. The nanopipette is positioned in the vicinity of the target cell by
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42 detecting the ion current using the SICM technique and the SICM unit is put on a sample stage
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44 of an inverted optical microscope for direct observation of cell manipulation and experiments
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46 were conducted on Hela cell to deliver potassium iodide (PI) dye [118]. Though there are a lot
47 of works using micropipettes, the major disadvantage is the fact that the tip has to be repeatedly
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49 repositioned and aligned in a sequential manner to target different regions requiring
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51 considerable skill and expertise.
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19 Figure 5. Nanopipette for single-cell electroporation. A local electric field is formed at the
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21 edge of the nanopipette by applying an electrical voltage between the outer electrode and
22 inside electrode of the nanopipette. “Reproduced from [118]. Copyright (2014) The Japan
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24 Society of Applied Physics”.
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30 The patch-clamp technique facilitates to record ionic currents flowing either through single ion
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32 channel or through whole cell membrane [119]. There are two operational configurations - cell-
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attached mode and whole-cell mode [119]. The whole-cell mode is mostly utilized for single
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35 cell electroporation [82,120,121]. Here we discuss the approach presented by Ryttsén et al.
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37 [120] who used patch-clamp in combination with microelectrodes and fluorescence
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39 microscopy to facilitate the recording, labeling and genetic manipulation of single cells. The
40 schematic of the technique is shown in Figure 6. A few micron-sized carbon fiber
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42 microelectrodes, controlled by high-graduation micromanipulators, were enclosed in a plastic
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44 tip filled with electrolyte which is in contact with a metal (silver) wire to apply the voltage.
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The targeted cell/cells were patch clamped and the electrodes are positioned on opposite sides
47 of the cell (Figure 6). The electrode tips were positioned 2–5 m from the outer cell surface at
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49 an angle of 0–20°, and 160–180° with respect to the object plane, and 90° with respect to the
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51 patch pipette. This method enables probing of membrane properties, important for studying
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53 membrane dynamics, throughout the electroporation in the targeted single cell by measuring
54 the current response during and after the exposer of pulsed external voltage. This technique
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56 was also used for transfecting neurons to deliver plasmid DNA, leading to stable transgene
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58 expression [120,121]. Rae et al. [122] also used the modified patch-clamp technique to
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electroporate single cell and deliver mostly several kinds of DNAs.
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solution containing the solutes to be introduced into the cell and the electrodes were arranged
55 in a linear fashion across the cell opposing each other, at a 90° angle with respect to the patch
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5 “Reprinted with permission from [120]”.
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10 Kitamura et al. [121] (2008) performed patch-clamp recordings from single neurons in vivo
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12 which was visualized by two-photon microscopy. A patch electrode was used to capture
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15 fluorescent dye. The same electrode was then placed on the cell to allow the formation of a
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17 gigaseal. Thus the method was shown to be useful for studying transgene expression after
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19 single-cell electroporation of plasmid DNA into identified cell types, without the need to pre-
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label neuronal populations in vivo [121].
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5.3. Micro/Nano- electrode based devices
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26 Among many transfection techniques to deliver biomolecules into cells, the micro-electrode
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based devices are very popular for the minimal cellular disturbance and their enough size for
single cell analysis. Micro-pattern electrodes with reduced inter-electrode distance permit a
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31 considerable reduction in operating voltage to reach the same electric field strength (V cm−1)
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33 and have become one of the main concepts of micro-electroporation systems in the last decade.
34 This kind of device was first proposed by Lundqvist et al. [46] in 1998. Two carbon fiber solid
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36 microelectrodes with diameter ~5 µm were placed on two side of a selective adherent
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38 progenitor cell by using high graduation micromanipulators. The schematic setup is shown in
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Figure 7. A gap of 2-5 micron was maintained between the electrode tip and cell surface to
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41 avoid electrochemical transformation of proteins and other membrane-associated structure.
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43 Since the size of the electrodes were much less than average cell dimension and could be placed
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45 precisely (in steps of 0.2 micron) by a micromanipulator, the high spatial resolution was
46 achieved. Thus this method facilitated to select a particular cell from a randomly distributed
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48 cell patterns cultured on a planner substrate to apply high voltage without affecting adjacent
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50 cells. Two factors are important for successful electroporation: one, the cell should be isolated
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from adjacent cell and two, the electric field should be well focused near the targeted cell
53 membrane which requires cell trapping on specific positions of the device.
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31 Figure 7. i. Schematic setup for single-cell electroporation. ii. Selective electroporation of
32 single cell. (A and C) Bright-field images of all cells. The arrow shows the targeted one. (B
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34 and D) Fluorescence showing electroporation is localized preferentially to targeted cells. iii.
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36 Time sequencing of electroporation with three 1-ms pulses of ~0.5 V. “Reprinted from [46],
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Copyright (1998), with permission from National Academy of Sciences, U.S.A”.
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3 In 2015, Bürgel et al. [123] presented a microchip where sets of facing Pt electrodes of
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Figure 8. The microsystem is combined with electrical impedance spectroscopy (EIS) offering
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35 sensitive to the cell passing between the electrodes. The current change is being measured to
36 determine the dielectric properties of the cell. The electrodes were temporarily switched on
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35 cavity geometry on the microchannel of the microfluidic chip, can induce electroporation
36 around the contact area of cells and cavity resulting high-yield electro-fusion in Myoblast cells.
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38 A theoretical analysis was carried out along with experiments to understand pore distribution
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40 for different configurations of electrode micro-cavity structures. He et al. [30] fabricated a
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MEMS-based micro electroporation chip containing three-dimensional (3D) gold electrodes to
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determine the optimal physical parameters for the uptake of biomolecules in HeLa cells.
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46 In our previous works [71,125] we demonstrated a unique approach for microelectrode based
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single cell nano-electroporation. In this configuration, transparent ITO electrodes were used
49 instead of metallic one allowing direct imaging. The device structure is shown in Figure 9. The
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electrodes were covered with a dielectric passivation layer of SiO2 to prevent direct touch of
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53 electrodes to the cells so that entire cell surface does not experience the intense electric field;
54 instead area of permeabilization can be controlled in addition with the facility of cell-selective
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56 electroporation and eventually improved viability [126]. Furthermore, it also avoids bubble
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58 generation, reduces chemical reaction, and control over Joule heating [72]. To construct sub-
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60 micron range electroporation region the final ITO electrodes and SiO2 layer were cut by using
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5 holes in the sub-micro-channel from the bottom of the device facilitating the user to keep the
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7 molecules and dye solution separate from cell suspension with cell viability ~60-80 %. In the
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next work, we modified the electrode structure from rectangular edge to triangular edge
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10 [74,77,127] to achieve ultra-localized the electric field with higher field strength resulting in
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12 an improved cell viability (~90-95%) for plasmid delivery.
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Figure 9. (a) Schematic representation of localized single cell nano-electroporation chip,

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45 where an intense electric field affects a small region of the cell membrane to permeate drug
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from outside to inside of the cell. “Reprinted with permission from [48]. Copyright (2013)
48 American Institute of Physics”. (b) Upper left Figure: Optical microscope image of patterned
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50 ITO electrode (20X). Lower left Figure: the SEM image of ITO electrode with micro
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52 channel. Upper right Figure: Working principle of electroporation and intracellular delivery.
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“Reprinted with permission from [125]. Copyright (2012) Springer Publisher Ltd”. (c)
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55 Scanning electron microscopy (SEM) image of nano-electrode tip with 60 nm electrode gap
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and 40 nm tip diameter. “ [2014] IEEE. Reprinted with permission from [77]”.
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3 In the previously described micro/nano- devices, the cell membrane is electroporated only
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5 across the fraction of cell surface which is perpendicular to the electric field distribution.
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electroporation area of a cell. To address this limitation, Zheng et al. [128] designed pinch flow
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10 technique based microfluidic device which can control cell rotation allowing to expose the
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12 perpendicular electric field all over the cell surface. The device consists of a three/two-inlet
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14 microfluidic channel decorated with a pair of micro-electrodes (inter-electrode distance ~ 410
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microns). The corresponding schematic diagram is shown in
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10 Electroporation buffer or pulsing buffer solution was infused from top and bottom (only
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18 5.4. Flow-through microfluidic microhole array
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In 2001, Huang et al. [107] for the first time demonstrated microfluidic device for SCEP which
22 was indeed a flow-through a device having a microhole to capture single cell. The chip
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24 consisted of three fabricated silicon chips as the top layer, middle layer and bottom layer, glued
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high electric field applied by using two translucent polysilicon electrodes. The chip had the
facility to capture a cell into micron size hole where the cell would experience the dense electric
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31 field. However, the chip had little control over cell and drug dosage. Later, the same group
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34 individual cells can be controlled precisely by measuring real-time electrical currents that flow
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36 through the cell. The schematic of chip structure is shown in
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Figure 11a). The upper chamber contained a micro-channel containing a micro-hole having
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58 to deliver the desired genetic molecules and then released by pressure difference to be replaced
59 by the next cell. Around ~100% gene transfer rate had been reported. To avail the visual
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35 layout of micro-hole, micro-channel and integrated electrodes. “Reprinted with permission
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from [129]”.
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39 Essentially at the same year of 2003, Khine et al. [89] developed a PDMS chip to selectively
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41 immobilize and locally electroporate single cells. This chip did not require any chip attached
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electrodes unlike previously discussed micro/nano- electrode based techniques or glass
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44 pipettes, unlike micro-pipette based electroporation techniques. The electrodes were placed at
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46 a distance from the cell eliminating toxic effect to the cells arising from Joule heating.
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48 Moreover, this device not only allows parallel single cell electroporation but also facilitate
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direct imaging of cells due to transparent PDMS and monitor the current change during
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electroporation. Figure 12 shows the chip layout, displaying wide inlet and outlet channels
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53 along with attached syringes for cell trapping by applying negative pressure. Experiments were
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55 conducted on Hela cell to deliver Trypan blue showing characteristic ‘jumps’ in current
56 corresponding to drops of cell resistance for an applied voltage <1 volt which indicate
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15 Figure 12. (a) The image of chip layout. The wide channels are for cell input and output. An
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20 and working principle. (c) An image showing trapped cell in the channel. The cell extends
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22 into the channel leaving a tail-like structure in the channel. “Reproduced from [89] with
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In 2007, Valero et al. [130] demonstrated silicon-glass microfluidic device capable of trapping
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32 operation and the schematic device structure is shown in Figure 13. The device contained two
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34 parallel microchannels (one have with ~50 m and the other one ~20 m) connected by nine
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36 micro-holes (width ~4 m) acting as trapping sites. The arrangements of the platinum
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electrodes are shown in Figure 13a which allow selective addressing of each microhole to
39 achieve control over cell selective electroporation. The cells flowed through the upper wider
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41 channel and other channel was used to create under pressure to trap the cells at the microholes.
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43 Once the cells have been trapped, the pressure was stopped and top and bottom reservoirs were
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loaded with DNA construct which had to be delivered into the cells.
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Figure 13. a. Working principle of cell trapping and electroporation. b. Microfluidic chip
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5.5. Micro/nano-channel based microfluidic electroporation devices
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delivery of various transfection agents into living cells. Nano-channel based device was
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35 connected by a nanochannel integrated with optical tweezers to manipulate cell position close
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37 to the nanochannel in one microchannel, as shown in Figure 14. The transfection agent was
38 loaded in the other microchannel. An intense electric field was delivered over a very small area
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40 on the cell membrane across the nanochannel, allowing a precise dose of biomolecules to be
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42 electrophoretically driven through the nanochannel, the cell membrane and into the cell
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cytoplasm, without affecting cell viability. The applied voltage duration and number of pulses
45 were adjusted to control the dose. The method could create a very large pore or several large
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47 pores in the cell membrane adjacent to the nanochannel thus reduced the affected area and
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49 provided a spatial resolution of 200 nm. Further experiments were carried out showing the
50 precise delivery ability and intracellular RNA analysis ability of molecular beacons without
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52 cell lysis or fixation [53]. In addition, a precise dosage of genes or other types of stimulus could
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54 be controlled into individual cells before molecular beacons-based living cell interrogation
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56 which is a very important feature for rare cell analysis. The platform also could handle a cell
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59 High transfection efficiency was found for large genes which are indeed a challenge for
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3 conventional EP techniques. Also, it offered greater flexibility than PCR and
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5 immunocytochemistry-based methods. Also, OSKM plasmid of induced pluripotent stem cell
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7 (iPSC) was injected into mouse embryonic fibroblasts cells showing high delivery dosage in a
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much more uniform manner compared with similar gene transfection carried out by the
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10 conventional bulk electroporation (BEP) method [76].
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Figure 14. (a) Optical image of the micro-nano channel. . “Reproduced from Ref. [53] CC BY
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37 4.0”. (b) Design of cover lid and entire package system to encapsulate NEP chip. (I) Gold-
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39 coated DNA nanowire (SEM). (II) Imprinting with 300 nanochannels. (III) NEP device with
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41 MNM array in the central area. (IV) Device package case. (V) Ready for cell and gene loading.
42 (VI) Multi-location spinning disc. “Reprinted with permission from [76]. Copyright (2014)
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48 5.6. Field effect transistor-based devices for SCEP
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50 In 2013, Jokilaakso et al.[132] utilized nanowire and nanoribbon channel field effect transistor
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52 for single cell electroporation. They used silicon nanowires with diameters in the range 50-100
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nm or nanoribbon with width ~2 m as transistor channel allowing them to obtain highly
55 localized electric field at the nanometre scale above the device surface. The schematic of the
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19 Figure 15. In this device magnetic beads were used to move the cells into the desired position
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and thus overcomes the need for any trapping center; any transparent substrate required for
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22 optical tweezer; and high voltage with low medium conductivity in the dielectrophoresis-based
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24 trapping of cells. The device surfaces were passivated using an organosilane monolayer to
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26 prevent unwanted adhesion with the cell. In this study, pulsed voltages at 10 MHz repetition
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rate and amplitudes up to 900 mVpp were applied between the shorted source-drain and the
back gate of the transistor. They also confirmed that the cell only at the top of the transistor
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cell transfection. 100 % cells were seen to be lysed at 900 mVpp voltage for 1 s.
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Figure 15. (a,b) The schematic device structure of FET. (c,d) The SEM image of the grouping
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3 and nanoribbons. “Reproduced from [132] with permission of The Royal Society of
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5.7.Nanofountain probe SCEP
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13 The schematic of the nanofountain probe SCEP set up is shown in Figure 16. The device
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contains twelve cantilevered probes with an average aperture size ~750 nm (Figure 16a) which
16 connected to two independent microreservoirs through sealed microchannels. The molecular
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18 solution to be delivered was stored in the on-chip microreservoirs to load into the
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20 microchannels by using capillarity or an externally applied pressure. A nanomanipulator or
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21 AFM was used to control the position of interest of the targeted cell in an extremely small step
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23 size of 40 nm. The onset of contact of the probe-tip on targeted area of a specific cell was
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25 detected either by optical observations of the cell morphology or by monitoring the change of
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electrical resistance due to the sealing between probe tip and cell. After the contact being
established, an electric pulse was applied between Ag/AgCl wire and conducting coverslip
30 acting as electrodes as shown in Figure 16. This technique of SCEP using nanofountain probe
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32 has recently (2018) been utilized by Yang et al.[133] for generation of monoclonal cell lines.
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In this approach, arrays of cell colonies (with each colony has ~10-20 cells) were patterned by
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35 microstamping. An indivisual cell of each colony was transfected with a plasmid encoding GFP
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37 and resistance to the antibiotic Zeocin, by using nanofountain probe method. When the colony
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39 of GFP-positive cells proliferated from the initial transfected cell, the stable clonal cell lines
40 were selected using Zeocin. Thereafter GFP-expressing colonies were harvested as clonal cell
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42 lines with stable transfection. Thus this method conferred a significant advantage over slower
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Figure 16. (a) Working principle of nanofountain probe. (b) Schematic of the package for
single cell electroporation. “Reprinted with permission from [51]. Copyright (2013)
30 Americal Chemical Society.
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36 5.8.Parallel single cell electroporation
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38 The bulk or individual electroporation techniques of cells are limited either by limited
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40 selectivity for bulk or low throughput for individual electroporation. So far demonstrated
41 techniques have few issues. For example, microelectrode EP technique offers high
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throughput electroporation allowing single cell selectivity through individual addressing of the
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45 microelectrodes. Indeed, this is not a direct method of cell selection. Microstructures of
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electrodes were also designed on microfluidic devices to apply focused electric field on the
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48 particular trapped cell without affecting the surroundings, also resulting in high throughput
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50 [130,134]. Micropipette based technique, though allow to select a particular cell of
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52 experimentalist’s choice from a population and allow different drugs to be injected into
53 different cells with accurate dosage control, which none of the other techniques allow for, is a
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55 very slow approach to porate cells one by one and requires a skilled user and is, generally, low
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3 Beside those mechanisms in situ parallel single cell electroporation technique was first
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5 demonstrated by Valley et al. [135] in 2009 which allows an array of single cells to be
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7 transfected simultaneously with controlling over selectivity on each individual cells. The
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10 schematic device layout and the cell-selective electroporation is shown in Figure 17.
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35 showing experimental setup and mechanism of electroporation. (c) Optical patterns cause
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electric field concentration across illuminated cells resulting in selective electroporation.
38 “Reproduced from [135] with permission of The Royal Society of Chemistry”.
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In 2010 Fei et al. [136] demonstrated micronozzle array structures to improve non-uniform
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45 electric field distribution on a cell population, which is indeed parallel single cell
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47 electroporation, though not mentioned by the author, allowing manipulation of cell
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49 distributions and controlled electroporation of bunch of cells. The device consists of a pair of
50 cross channels (~500 µm width and depth), one act as top and other as the bottom, connected
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52 by a center hole as shown in Figure 18. The top channel intersects with a 1 cm diameter
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54 reservoir located at the center of the device where microporous membranes can be fixed.
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Femtosecond pulsed laser ablation technique was adapted to form a pattern of
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59 terephthalate (PET) membraned coated with 2-3 µm thick gelatin, acting as cell binding
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3 membrane. The device was fabricated by using a poly(methyl methacrylate) (PMMA). A 45
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5 µm thick PMMA film enclosing the bottom channel was used at the back side of the device
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7 allowing top access via reservoirs at the ends. The shape of the pore could be tuned from micro-
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nozzle to straight microchannel by changing the laser energy. Experiments were performed on
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10 mouse embryonic stem cells to transfect with plasmids gWiz SEAP as reporter genes, and the
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12 transfection of on the 100 × 100 array was evaluated 24 h after electroporation. The
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because the electric field is more concentrated at the small-end of the micronozzle, resulting in
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17 better-localized electroporation resulting ~75% cell survival.
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39 Figure 18. (a) Schematic of disk structure and working principle. (b) Phase contrast (left)
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and fluorescent image (right) of cells trapped in 10 × 10 micronozzle array. “Reprinted with
42 permission from [136]. Copyright (2010) American Chemical Society”.
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47 At the essentially the same year 2010, Wang et al. [137] independently demonstrated single-
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49 cell electroporation chip that can locally electroporate array of cells with an operating voltage
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51 as low as ~1 Volt. The chip consists of one bottom metal electrode on the top of a substrate, a
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53 sandwiched dielectric polymer layer with thickness ~10 µm patterned with an array of
54 microwells, and a conducting top lead as another electrode. The distance between these two
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56 electrodes was 60 µm, the diameter of the microwell was ~10 µm, nearly same to average cell
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58 diameter. The schematic configuration of the device is shown in
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19 Figure 19. When an AC voltage was applied to the electrodes, the microwell arrays create non-
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21 uniform electric fields to generate positive dielectrophoresis forces to capture one single-cell
22 at each microwell. After cell capturing, the AC voltage was shifted to a DC pulse voltage to
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24 generate an intense electric field across the membranes of the captured cells leading to
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26 electroporation. The field distribution, shown in Fig. 19 , showed the strong electric field was
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generated on the cell membrane in contact with the microwell, and decay quickly upwards,
allowing localized electroporation at relatively low voltage. Human lymphoma cells in an 8 ×
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high field strength implementing both DEP trapping and cell electroporation with the same
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6 High throughput “3D Nanochannel electroporation chip” (NEP) was introduced by Chang et
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8 al. [138] in 2016 which can parallely electroporate ~40,000 cells per cm2 of the chip. This
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9 system consists of four parts, as shown in Figure 20, one 3D electroporation chip, a support
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11 platform, PDMS spacer, a pair of electrodes. The top of 3D chip is a PDMS chamber to load
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13 cells to be electroporated. The molecules to be delivered are filled into the bottom chamber,
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separated from the top chamber by the 3D chip (Figure 20). During cell loading, a vacuum was
16 applied underneath the 3D chip to drive the cells towards the nanopores for alignment and
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18 enhancing the contact with the pores. High voltage pulses were applied between the top and
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20 the bottom electrode. Voltages with different amplitudes (15-140 V), pulse widths (5,10, and
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30 ms) and number of pulses were varied to study the impact on delivery efficiency. The device
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23 showed around >90% of cell viability and ~90% of transfection efficiency when only the cells
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25 sited on the nanopores are counted. This approach also offers precise control over dosage with
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high throughput. The results of NEP were compared with microchannel electroporation chip
(MEP) with pore diameter of ~5 m. The device set up was same as for NEP, the only
30 differences are the micro-scale pore size and using magnetic micro-beads for cell alignment in
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32 the MEP system [139]. In their next work, they modified the NEP chip with micro-cap array
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34 to align the cells on nanochannel more precisely [140]. They achieved a high-throughput
35 massively parallel delivery of genetic cargo (microRNA, plasmids) into mouse primary
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37 cardiomyocytes with transfection efficiency ~ 90-100% in a controllable fashion.
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Figure 20. (a) The schematic set up of 3D electroporation chip. (b) The schematic of
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34 Guo et al. [141] demonstated micro-electrode array chip having facility of cell positioning and
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real time impedance monitoring for cell selective parallel electroporation at single cell level.
37 They patterened array of quadrupole-electrodes unit (named positioning electrodes) for cell
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39 positioning and pair of planer electrodes (named center electrodes) located at the centers of
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41 each quadrupole-electrode unit for performing electroporation (Figure 21). Based on negative
42 dielectroforesis by using the positions electrodes the cells were trapped and positioned onto the
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44 center electrodes. Finally, the cells were electroporated by applying voltage to the center
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Figure 21. (a) Schematic diagram of electrical electrical arrangement for the center
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19 Results of selective electroporation in array dyed by Propidium iodide. “Reprinted with
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In order to conduct single cell electroporation, cell positioning is the foremost required step
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because only target cell has to experience the intense electric field required to break down the
32 bilipid membrane. Many methods of cell positioning have been demonstrated such as creating
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34 pressure difference at trap centers, optically-induced-dielectrophoresis (ODEP), electrically
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36 induced dielectrophoresis, and micro-fluidic structures [142]. Dielectrophoresis (DEP) is a
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movement of polarized particle or cell due to translational force by a non-uniform electric field.
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39 It is one of the most versatile methods to control cell movement and positioning due to its
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41 capability to integrate with in-situ cellular measurements. Also, the cells under DEP-based
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43 manipulation could maintain good viability [142]. Here is the table listing the methods adapted
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47 Year/ Group Trapping configuration
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1. Huang et al. Applying a negative pressure to pull towards and captured
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[107,129,143] by a microhole
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53 (2000,2001,2003).
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3 2. Khine et al. [89] (2005), A negative pressure had been applied to pull cell towards
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5 Valero el al. [130] trapping channel or microhole structures.
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7 (2007).
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3. 2008; Ionescu- Zanetti Cells are trapped in the capillary channel by slight
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11 et al. [144] transient negative pressure
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(2009), Boukany et al. optical tweezers)
17 [75] (2011), Valley et
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19 al. [135] (2009).
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5. Fei et al. [136] (2010), A vacuum was used to trap the cells
23 Chang et al. [138] on the support membrane. (PSCEP)
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25 (2016).
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[137] Electrically assisted DEP force
(2010), Bertani et al.
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31 [145] (2015), Wang et
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35 7. Jokilaakso et al. [132] Magnetic beads
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37 (2013).
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43 7. Applications
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45 Precise control on drug/gene delivery with minimal external applied voltage irrespective of cell
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47 and cargo types make SCEP an attractive platform for biomedical and therapeutic research.
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49 SCEP is widely applicable to investigate cell to cell variation with their intracellular
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characterization [55]. The selective cell to cell fusion can be achieved with this technique which
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52 might be useful for single-cell cloning and in-vitro fertilization [146]. Due to advantage of
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54 micro/nano- fabrication and miniaturization of electrodes at nanoscale level, it is possible for
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56 selective organelles manipulation within a cell [147]. SCEP is useful to record action potential
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and transmembrane potential from electroactive cells and tissue, which is potentially applicable
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59 for biomedical research such as electrophysiological study during cell development and
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3 pharmaceutical screening [90]. Most exciting achievement is to maintain cell viability for long
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5 time after performance of electroporation, enabling real-time study of interaction between
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7 impermeable molecules in the living cell. Several studies have been conducted in last two
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decades including intracellular delivery of RNA, DNA, proteins and their complexes. In recent
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10 years, researchers extensively focus on their direct biomedical applications such as adoptive
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12 immunotherapy, gene/RNA-based therapies, cell reprogramming, and intracellular bio-
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14 interrogation of living cells etc. Here we have discussed recent progress in this field. The
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uptake of small molecules (e.g., dyes), and RNA has been successfully tested on a variety of
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17 cell types, including human cells, hamster cell, yeast, and bacteria [53,75,131]. Also, plasmid
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19 DNA or macromolecules like quantum dots have been successfully delivered in vitro [75] as
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21 well as in vitro on a living tadpole brain on single cell level [114]. Nanochannel electroporation
22 device has been used to analyze a different kind of the RNAs such as messenger RNA(mRNA),
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24 micro RNA (miRNA) in single cell [53].
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Mutations of the GATA2 gene have been associated with leukemia. Bertani et al. [145] used
3D NEP chip to drive GATA2 molecular beacons into human immortalized myclogeneous
30 leukemia cell via electroporation. The molecular beacon opens up and binds to GATA2 present
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32 within cell, detected via fluorescence. Thus it shows a greater prospect to study the
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hematopoietic stem cells important for leukemia disease analysis and drug discovery in future.
35 Electroporation technique is highly applicable for transdermal drug delivery, where insulin-
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37 loaded nanocarriers are delivered into the blood for diabetes treatment [148]. However, for
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39 diabetes diagnosis, the glucose concentration variations are needed to detect under ambient
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condition. Thus it is necessary to characterize beta cells accurately at single cell level, which
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42 produce insulin [149]. Moreover, transdermal drug delivery by electroporation is by far not
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44 limited to insulin.
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SCEP technique has some drawback. As for example, when fabricated electrodes are dipped
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48 far apart in the media, the current flows through the entire solution resulting in Joule heating
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50 and chemical cross contaminations due to electrode reactions at the electrode-solution interface
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52 and the formation of an electric double layer at the surface of the electrodes leading to reduction
53 of applied electric field strength. Although whole-cell patch-clamp methods [6, 7] overcome
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55 many of the electrophysiological problems of sharp electrode recording, they are laborious and
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sequentially obtaining physiological and morphological data from multiple cells.
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3 8. Future prospects
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6 Single cell electroporation techniques were introduced in the past two decades for biological
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8 and therapeutic research. It enables to study cell-to-cell variation in a population; to investigate
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and manipulate intra-cellular chemistry of cells. Despite all new approaches,
11 their delivery performance and reliability have not yet been approved to exhibit their full
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13 potential to biological researchers and users. This can be assigned to several drawbacks
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15 associated with the current SCEP systems: (a) relatively complicated operational steps for
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direct use in biological fields; (b) each configuration has its own advantages and limitations
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18 regarding the cost and performance; and (c) inadequate database for clinically important
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20 molecular probes and cells to verify the relevance.
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The commercialization of the aforementioned new approaches demands easy-to-use strategies
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24 along with their excellent performance and reliability. In this regards, user-friendly high-level
25
26 automation is highly encouraged and expected by the end users in this twenty-first century.
27
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29
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Furthermore, the whole operating system should be well integrated into one single system to
utilize the full potential of SCEP. For example, in situ imaging of single cell analysis, which is
30
31 a well-established technique, has to be well integrated with electroporation device for gene
32
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33 transfection. High-throughput parallel single cell electroporations approach with high
34
35
uniformity can have interesting future prospects for studying gene delivery and subsequent
36 protein expressions [135,136,150].
37
38
39 So far demonstrated SCEP devices utilize Lab-on-a-Chip or bio-micro electro-mechanical
40
41
systems (Bio-MEMS) technologies for device fabrication. The most common concerns of those
42 devices are that they require many expensive and complicated equipment. Furthermore, the
43
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44 outputs are mostly stochastic increasing the number of trials and repetition of the same
45
46 expensive steps and lot of chemicals. Therefore, optimization only expanse a lot of intellectual
47
effort besides the equipment and steps. Further concerns rely on unwanted leakage, non-
48
49 repeatability of operational outcome, post-delivery unwanted issues such as channel blockage
50
51 by cells, corrosion of device surfaces and electrodes due to chemical reaction etc. These issues
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52
53 have to be handled in a serious manner to envision future ready-to-use SCEP systems. So far
54 demonstrated SCEP devices, the utilization of micro/nano-fluidic and Lab-on-a-chip
55
56 technology integrated with micro total analysis systems (μ-TAS) can provide high throughput
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58 single cell data with minimum reagent consumption [151]. The bioinformatics technique can
59
60
42
1
2
3 employ to study this large ensemble of SCEP data to find answers of unresolved questions in
4
5 past centuries.
6
7
8 In the light of recent progress, in particular, the high throughput parallel single cell
pt
9 electroporations techniques, we can say that we are not very far from our envision of utilizing
10
11 those devices for human resources. We need to face few more challenges. For example, most
12
13 of the newly fabricated devices use RNA, DNA plasmid for demonstrating the device
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14
15
performance, basically testing delivery efficiency and cell viability. But, there are very few
16 studies to utilize those devices for understanding the cytosol interaction with molecules of
17
18 medical interests which are very useful for disease diagnostic and therapeutic applications.
19
20 Such utilization demands extensive collaborative efforts among engineers, biologists,
us
21
physicists, chemists, and medical practitioners. At the present stage of progress in this
22
23 interdisciplinary field, more attention should be dedicated on the collective efforts for
24
25 collecting a database of important molecules in parallel with improving the device
26
27
28
29
quick concept demonstration.
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performances and using them for studying meaningful intracellular delivery instead of just
30
31 9. Conclusions
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33
34 This review has discussed the evolution of the different techniques for single cell
35
36 electroporation from its origins to its future prospects for cellular therapy and drug discovery.
37
There are several approaches presented in more than a few hundreds scientific papers to study
38
39 electro-physio properties from cellular to sub-cellular level. The basic need of this method is
40
41 to extend the electro-cellular research in more controlled and precise way down to nanometer
42
43 scale and analyse sub-cellular biological structures by utilizing the advance bio-
pte
44 nanoengineering techniques. The attempts of applying nanoscience and nanoengineering have

45
46 already an impact and, proven its importance and still, a research area needed to be improved
47
48 more. However, methods like micro-pipette based electroporation techniques permit
49
50
simultaneous delivery of mutiple reagents into individual cell with high transfection efficiency
51
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and viability. The nanopiller elctroporation help to record intracelluar action potentials, that
52
53 play key role in nervous system and cellular process. Smart designs of integrated nanofluidic
54
55 channels offer to study ion channel dynamics throughout the electroporation by measuring
56 current. The recents progress of high throughput parallel single cell delivery can have high
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57
58 impact on biological science towards personalized medicine. The advent of micro-total analysis
59
60 systems integrated with Lab-ob-a-Chip devices will magnify its impact in future. Hopefully,
43
1
2
3 this review has presented sufficiently detailed description of the state-of-the-art to inspire the
4
5 readers and propose new solutions to analyse subcellular biological insights that remain to be
6
7 overcome.
8
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9
10 Acknowledgement
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12 The authors greatly appreciate for the financial support from Science and Engineering Research
13
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14 Boards (SERB), Department of Science and Technology (DST), Government of India, under
15
16 grant number ECR/2016/001945. We acknowledge all the publishers to obtain the copyright
17 permission.
18
19
20 Conflicts of Interest
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21
22
23 The authors declared no conflicts of interest.
24
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Journal of Micromechanics and Microengineering

Single Cell Electroporation-Current Trends, Applications and Future

Manuscript version: Accepted Manuscript

This Accepted Manuscript is © 2018 IOP Publishing Ltd.

View the article online for updates and enhancements.

This content was downloaded from IP address 157.89.65.129 on 07/10/2018 at 05:04

to hydrophilic [65]. The value of r* is determined by the meeting-point of membrane free

52 evolution of pore denity N is given by ordinary differential equation [64,70]

Figure 1. Hypothetical structural rearrangements of a lipid bilayer membrane dur ing

Figure 9. (a) Schematic representation of localized single cell nano-electroporation chip,

30 A micro/nanofluidic based single-cell analysis platform offers to control precise amounts of

44 John Wiley & Sons Ltd”.

44 and labor-intensive dilution procedures.

57 micropore/micronozzle array with pore radius ~0.4 µm on nanoporous polyethylene

57 Figure 19. Schematic device configuration of electroporation in the non-uniform distribution

44 by SCEP researchers for cell positioning.

44 nanoengineering techniques. The attempts of applying nanoscience and nanoengineering have

44 a review Mil. Med. Res. 4 24

52 the optimal physical parameters in the uptake of biomolecules in HeLa cells

Stat. Physics, Plasmas, Fluids, Relat. Interdiscip. Top. 59 3471–82

Nanochannel electroporation delivers precise amounts of biomolecules into living cells

57 Electrotransfection of Cells Using Carbon-Based Electrodes Cell. Mol. Bioeng. 9 538–

[109] Ainla A, Jansson E T, Stepanyants N, Orwar O and Jesorka A 2010 A microfluidic

57 O 2016 A modified single-cell electroporation method for molecule delivery into a

electroporation Adv. Healthc. Mater. 3 682–9

44 [140] Chang L, Gallego-Perez D, Chiang C L, Bertani P, Kuang T, Sheng Y, Chen F, Chen

30 [148] Rastogi R, Anand S and Koul V 2010 Electroporation of polymeric nanoparticles: An

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