- Email: [email protected]
Controllable cell electroporation using microcavity electrodes
Sensors and Actuators B 240 (2017) 434–442
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Controllable cell electroporation using microcavity electrodes Xiaoling Zhang a,b , Ning Hu a,∗ , Xi Chen a , Ting Fan a , Zhenyu Wang c , Xiaolin Zheng a , Jun Yang a,∗ , Shizhi Qian d,∗ a
Key Laboratory of Biorheological Science and Technology, Ministry of Education, Chongqing University, Chongqing, 400030, PR China Key Laboratory for Optoelectronic Technology and Systems, Ministry of Education, Chongqing University, Chongqing, 400030, PR China College of Biomedical Engineering, Chongqing Medical University, Chongqing, 400016, PR China d Institute of Micro/Nanotechnology, Old Dominion University, Norfolk, VA, 23529, USA b c
a r t i c l e
i n f o
Article history: Received 21 June 2016 Received in revised form 23 August 2016 Accepted 30 August 2016 Available online 31 August 2016 Keywords: Electro-fusion Transmembrane potential Microelectrode Microcavity Myoblast cell
a b s t r a c t Cell electro-fusion includes four steps, cell alignment, cell electroporation, reconstruction of cytomembrane and cytoplasm exchange. The cell alignment and electroporation steps are highly related to the intensity and distribution of the electric field, which depend on the applied voltage as well as the microelectrode structure. The microelectrode structures were first evaluated based on the numerical analysis of the electric field and the transmembrane potential induced on biological cells when the cell electroporation and electro-fusion were performed based on different designs of microelectrodes. Microelectrodes in a micro-cavity geometry can induce electroporation around the contact area of the paired cells for high-yield electro-fusion. Microfluidic chips with co-planar microelectrodes and microelectrodes within micro-cavities have been fabricated and tested for electro-fusion of Myoblast cells, and the experimental results confirmed the numerical analysis. © 2016 Published by Elsevier B.V.
1. Introduction When cells are subject to an external electric field, a voltage difference is induced across the cell membrane, which is termed as the transmembrane potential (TMP). If TMP exceeds the membrane breakdown voltage, which is about 1 V for most cells, nanopores are created on the cell membrane, which is called electroporation or electropermeabilization [1,2]. The created nanopores can be reversible or irreversible, decided by the duration and the intensity of the external electric field. For the irreversible electroporation, the TMP required is around twice as high as the reported reversible electroporation potential [3]. One of the most important applications of the cell reversible electroporation is cell electro-fusion, which uses a high-strength electric field to induce cell alignment/pairing, membrane permeabilization, and fusion of paired cells to form hybrid cells [4]. Since the formed hybrid cells contain the genetic materials from parent cells, cell electro-fusion provides a feasible technology and approach to understand and study genetics [5,6], immunology [7,8], hybridization, and crossbreeding [9,10]. Compared with the virus- [11] and PEG-induced cell
∗ Corresponding authors. E-mail addresses: [email protected] (N. Hu), [email protected] (J. Yang), [email protected] (S. Qian). http://dx.doi.org/10.1016/j.snb.2016.08.172 0925-4005/© 2016 Published by Elsevier B.V.
fusion techniques, electro-fusion attracts high attention due to its non-toxicity, wide adaptability to cell types, easy implementation and high reproducibility [4,12,13]. In addition, the electro-fusion efficiency is much higher than the other methods. Generally, the electro-fusion process can be divided into four sequential steps: cell alignment (pairing), reversible electroporation on cell membrane, reconstruction of cytomembrane, and cytoplasm exchange between two cells [4]. Cell alignment and reversible electroporation are the most important two steps, which highly depend on the local electric field intensity as well as its gradient. Typically, cell alignment is achieved under a low-amplitude (around 100–300 V cm−1 ) and high-frequency (around 1–3 MHz) sinusoidal alternating current (AC) field, while a series of short-duration (around 10–50 s) and high-strength (around 1–10 kV cm−1 ) electric pulses is applied to induce reversible electroporation [14]. For a successful cell electro-fusion, the reversible electroporation should take place only at the contact area of the paired cells, which requires that the TMP distribution should be well controlled to induce electroporation at the contact area. The TMP distribution is controlled by the electric field around the cell, which depends on the geometry of the microelectrodes as well as the microfluidic channel. In the last decade, although many kinds of microelectrode/microfluidic structures have been developed for cell electroporation [15–18] and electro-fusion [19–22], there are few studies focused on the controllable electroporation.
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Techaumnat and Washizu [23] conducted a numerical analysis of the TMP on biological cells with the effects of an orifice plate in electroporation and electro-fusion. Electric field is constricted at the orifice, where the magnitude and localization of the TMP can be controlled, and yields high electro-fusion efficiency. Rems [24] reported that nanosecond pulses can induce selective electroporation at the contact areas of paired cells, regardless of the cells’ sizes. In the nanosecond range, cell membranes are still in the charging phase, and electric field at the contact area reaches the highest value, yielding selective contact electroporation. Previously we developed and tested continuous protruding microelectrode array [25–27] for cell electrofusion. As the most important process of cell electrofusion, the location and intensity of reversible electroporation are highly dependent on the electric field distribution around the cells. To obtain desirable cell alignment and reversible electroporation efficiency, two kinds of discrete microelectrode arrays, 3D thin film microelectrode [28] and discrete co-planer vertical sidewall microelectrode [29], have been developed to optimize the electric field distribution. Although the cell electrofusion efficiency is improved by this structure, we found that irreversible electroporation occurring at the contact point between discrete microelectrodes and their adjacent cells. Therefore, the existing microelectrode structures still need to be optimized to achieve controllable electroporation. In addition to cell electrofusion, electroporation has been widely used in other applications such as cell lysis [30] and gene transfection [31]. Depending on the application, the desirable position for cell electroporation is different. In addition, most previous studies related to cell electroporation focused on experimental investigation using different structures of microelectrodes and/or microchannel, and there is no appropriate design tool for the design and optimization of the device for achieving selective cell electroporation at the desirable position. This study develops a mathematical model for the electroporation process, and is further
435
Fig. 1. Configuration for the discrete co-planar microelectrode array (A) and the micro-cavity structure (B).
validated by experimental results. Based on the distribution of the formed nanopore density predicted from the validated model, one can easily modify and optimize the design for controlling the position of cell electroporation, which would improve the efficiency of the electroporation applications.
Fig. 2. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array with one cell, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with one cell, (C) The absolute value of TMP of P1 and P2 for the case of single cell electroporation in the discrete co-planar microelectrode and in the micro-cavity structure.
436
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Table 1 Values of constants and parameters used in the simulations. Parameter
Value
Microchannel size (H × W × L) Microelectrode length (l) Cavity size (Lca × Wca ) Cell radius (a) Medium conductivity (out ) Medium permittivity (εout ) Cytoplasmic conductivity (in ) Cytoplasmic permittivity (εin ) Cell membrane thickness (dm ) Cell membrane conductivity (m0 ) Cell membrane permittivity (εm ) 0 creation rate coefficient (˛) equilibrium pore density when = 0 (N0 ) pore creation rate (q) characteristic voltage of electroporation (VEP ) pore radius (rp ) conductivity of solution inside the pore (p ) relative entrance length of pores (n) energy barrier within pore (w0 ) Faraday’s constant (F) gas constant (R) absolute temperature (T)
30 m × 80 m × 60 m 20 m 20 m × 20 m 7.5 m 0.012 S m−1 80 0.3 S m−1 70 5 nm 5e-7 S m−1 4.5 8 V for single cell electroporation 10 V for cell electro-fusion 1e9 m−2 s−1 1.5e9 m−2 2.46 258 mV 0.76e-9 0.16 S m−1 0.15 2.65 9.65e4 Cmol−1 8.314 J K−1 mol−1 295 K
Reference
2. Materials and methods
Fig. 1 shows schematic diagrams of the microchannels with a pair of discrete co-planar microelectrode (A) and microelectrodes within the micro-cavity structure (B), respectively. The purple faces in Fig. 1 denoted the microelectrodes, which are part of the vertical side walls of the microchannel. In the co-planar structure, the planar electrodes of length l are on the same plane of the corresponding vertical side walls. In the 2nd design with micro-cavities, the size of which are Lca × Wca and determined based on the size of the cells to be fused in the experiments. Microelectrodes can be fabricated along one side wall of the micro-cavity (i.e., b = 0) as well as part of the other two side walls with the length of b. The entire vertical side walls of the micro-cavity will become the microelectrode when b = Wca . We consider spherical cells of radius a suspended in a buffer solution with conductivity out , which fills the microchannel of height H, width W and length L. To investigate the electric field distribution and TMP, we assume a potential 0 is imposed on one electrode and the opposite electrode is ground. For the case of single cell electroporation, only one cell was fixed in the vicinity of a microelectrode. For electroporation in cell electrofusion, two cells were aligned as cell-cell pair and fixed near the microelectrode. We also considered a chain of three cells to investigate the case of electro-fusion of multiple cells. The position of the pole at the microelectrode was denoted by P1 , and the first and second contact point of the paired three cells are denoted as P2 and P3 , respectively. Between the paired cells, we inserted a small separation of 0.001 m so that cells are not completely contiguous. The electric potential inside (in ) and outside (out ) the cell can be described by the following electrostatic equations [32]:
∂ (∇ ˚in ) ∂t
−∇ (out ∇ out ) − ε0 εout ∇
[7] [33] [33] [33] [33] [33] [32] [33] [33]
The required voltage for electroporation/electro-fusion (0 ) was applied via the embedded discrete electrodes. Thus, the boundary condition on electrodes was assumed as
2.1. Mathematical model
−∇ (in ∇ in ) − ε0 εin ∇
Eppendorf Co. [35] [36] [7] [33]
= 0,
∂ (∇ ˚out ) ∂t
(1)
= 0 or0.
(3)
Electrical insulation boundary condition was imposed on the rest walls of the microchannel. Since the cell membrane is very thin, it was not physically included in the model. To take into account the thin membrane, a current density flowing through the membrane was used [33]: J (t) =
m (t) (˚in − out ) ε0 εm ∂ (˚in − out ) + . dm dm ∂t
(4)
The first and the second terms on the right-hand side of Eq. (4) represent the conductive and the capacitive components of the electric current through the membrane, respectively. Here, J (t) is the current density; εm and dm are the relative permittivity and the thickness of the membrane, respectively; m is the conductivity of the membrane; in and out are, respectively, the potentials at the cytoplasm/membrane and membrane/medium interfaces. Note that the membrane conductivity varies during the electroporation process, and is modeled by m (t) = m0 + N (t) p rp2 A.
(5)
In the above, m0 is the membrane conductivity without electroporation; rp is the formed pore radius; p is the conductivity of solution inside the pore; N is the pore density governed by following equation [32,33] with an initial condition of N = N0 : 2 dN (t) = ˛e(˚(t)/VEP ) dt
1−
N (t) −q(˚(t)/VEP )2 e N0
,
where N0 is the pore density in the non-electroporated membrane; the parameters ˛, q, and VEP describe the characteristics of the electroporation process; and the induced transmembrane potential (TMP = ) is defined as (t) ≡ in (t) − out (t)
= 0.
(2)
Here, ε0 is dielectric permittivity of the vacuum; εi and i are, respectively, the relative dielectric permittivity and conductivity of the cytoplasm (i = in) and the medium (i = out).
(6)
(7)
The parameter A in Eq. (5) is given by [33] e vm − 1 . A= v e m (w0 ew0 −nvm − nvm ) / (w0 − nvm ) − (w0 ew0 +nvm + nvm ) / (w0 + nvm )
(8)
In the above, vm = F/RT with F, R, and T being the Faraday’s constant, the gas constant, and absolute temperature of the suspension,
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
437
respectively; n is the relative entrance length of pores; and w0 is the energy barrier within pore. In this current study, we assume that all pores have equal and time-independent radii, which is about 0.8 nm [32,34]. The timedependent Eqs. (1) and (2) are simultaneously solved subject to the boundary conditions, Eqs. (3) and (4) in which m is calculated from Eqs. (5)–(8), with the commercial finite element package, COMSOL, using the values of parameters listed in Table 1. 2.2. Cell culture and cell preparation In our experiments, Myoblast cells (Third Military Medical University, Chongqing, China), which were labelled by green fluorescence (pAdTrack-GFP), were used to monitor the process of electroporation. Myoblast cells were cultured in F10 supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin. They were incubated at 37 ◦ C in humidified atmosphere containing 5% CO2 and 95% air. Before the experiment, cells were trypsinized by using EDTA (0.25/0.02%; Sigma) and collected in a small tube. After collecting the cells, the electro-fusion medium (Osmolaity: 90 ± 10 mOsmol/kg, conductivity: 0.012 S/m, Eppendorf Co., electro-fusion buffer for eukaryotic cell, German) was used to wash the cells three times and suspend them as a concentration of 1.0 × 106 (5.0 × 106 ) cells/mL for single cell electroporation (cell electro-fusion). 2.3. Experiments protocol The microfluidic chips, which integrated discrete co-planar microelectrodes and micro-cavity microelectrodes respectively, are designed and fabricated on SOI wafer [29,37]. The discrete microelectrodes and micro-cavity microelectrodes are designed based on the optimized microelectrode structures from the numerical simulation of the electric field inside the microchannel. To fabricate the side wall electrodes with a certain length of b, an insulator made by SiO2 -Ployisilicon-SiO2 with length of Lca -b is fabricated first based on the method described by the previous work [29,37]. Then the highly doped silicon is etched by the inductively coupled plasma technique to form the designed micro-cavity electrode. In addition, these fabricated microfluidic chips are packaged to connect the external power generator for experiments. Before experiments, the microfluidic chips were rinsed by the electro-fusion buffer for 10 s. A droplet of the suspending solution was pipetted into the microfluidic chips. And then AC electric pulses (Vp-p : 2–3 V, frequency: 1 MHz, duration: 30 s) were applied to the microelectrodes. Due to the positive DEP under the considered conditions, cells are attracted to the microelectrodes. A little higher voltage of DC pulse signal (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses number: 5) was applied to induce cell irreversible electroporation. The fluorescence of the dye in the cell leaked out after the cell was electroporated, which was monitored with a CCD camera (Motic 3000, China) through an Olympus BX 61 microscope (Olympus Optical Co. Ltd., Japan). 3. Results and discussion 3.1. Single cell electroporation For cells suspended in an infinite domain and exposed to a uniform electric field of Ee , if the nanopores are not created, TMP () is given by the Schuwan equation [38]: = 1.5Ee a cos ,
(9)
where a is the radius of the cell, and is the angle between the direction of electric field Ee and the outward normal from the center of the cell to the site on the cell membrane. TMP is symmetrical
Fig. 3. Pore density along cell membrane for a single cell in the discrete co-planar microelectrode array (A) and the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
on both sides of the cell, and reaches its maximum value at the poles ( = 0 and 180◦ ). For the cell confined in a microchannel as shown in Fig. 1, the distribution of TMP is highly dependent on the spatial distribution of the electric field, which is decided by the structure of the microelectrode and microchannel. Fig. 2 depicts the spatial distribution of the electric field strength in the microchannel with discrete co-planar microelectrode array (Fig. 2A) and the micro-cavity microelectrode with b = 0 (Fig. 2B) in the presence of one cell in the vicinity of the electrode. For the case of co-planar microelectrode, the electric field strength near the microelectrode (i.e., the pole P1 ) is the highest, and decreases with increasing the distance from the microelectrode. In contrast, the electric field is concentrated within the micro-cavity, however, the electric field at the poles of the cell, P1 and P2 , is not the highest. The electric field in the area between the cell and the side walls of the microcavity is the highest. Since the distance between the two opposite microelectrodes in the micro-cavity is larger than that of co-planar case, under the same voltage 0 , the maximum magnitude of the
438
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Fig. 4. Single cell electroporation using the discrete co-planar microelectrode array (A) and the micro-cavity structure (B). The cells were exposed to a high-voltage electroporation pulse (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses: 5).
Fig. 5. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array with two cells, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with two cells, (C) The absolute value of TMP of P1 and P2 for the case of the cell electro-fusion in the discrete co-planar microelectrode and in the micro-cavity structure.
electric field in the microchannel with co-planar microelectrodes is higher than that with the micro-cavity microelectrodes. Due to the same reason, the steady-state TMP shown in Fig. 2C in micro-cavity structure is lower than that in co-planar structure at the same position. Fig. 2C shows that the TMP at P1 located in the vicinity of the microelectrode is always higher than that at P2 in the discrete coplanar microelectrode array. In the micro-cavity structure, the TMP
at P1 is a little higher than that at P2 at the beginning, and is significantly lower than that at P2 when it reaches the steady state. If we also extend the microelectrode to cover part of the side walls inside the micro-cavity, the steady-state TMP at P1 is considerably lower than that at P2 , as shown by the results for b = 7.5 m in Fig. 2C. The results suggest that we can achieve the highest TMP at P2 , where cell fusion occurs, and relatively low TMP at P1 to avoid cell rupture
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
439
Fig. 6. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array microfluidic channel with three cells, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with three cells, (C) The absolute value of TMP at P1 and P2 for the case of three cells in the discrete co-planar microelectrode (no symbols) and in the micro-cavity structure (with symbols).
occurring there by adjusting the length of the microelectrodes on the side wall of the micro-cavity. When TMP exceeds the threshold voltage, many nanopores are induced along cell membrane and cell electroporation occurs. Fig. 3 shows the pore density of one cell induced along the cell membrane at 40 s in the discrete co-planar microelectrode array (Fig. 3A) and in the micro-cavity structure with b = 0 (Fig. 3B) and b = 7.5 m (Fig. 3C). The purple lines in Fig. 3 denoted as the microelectrode and the black ones denoted as the insulting material. Membrane areas with the pore density ranging from 1/10 Max to Max were marked with color legend and Max represents the corresponding maximum pore density. For the discrete co-planar microelectrode array, the highest pore density occurs near the microelectrode, and very limited pores formed at the other pole, P2 , since the TMP at P1 is much higher than that at P2 , as shown in Fig. 2C. For the microcavity structure with b = 0, the highest pore density also occurs near P1 , but there were also a large number of pores formed near P2 . In contrast, when b = 7.5 m, the highest pore density occurs near P2 instead of P1 . These pore density results are consistent with the results of the TMP shown in Fig. 2C. Because the distance between two electrodes in the micro-cavity structure is longer than that in the discrete co-planar structure, the intensity of the electric field is lower, yielding lower TMP and accordingly lower pore density, as shown in Figs.2C and 3. To validate the above theoretical predictions, we conducted experiments on single cell electroporation in the microchannels with discrete co-planar microelectrode array and the micro-cavity microelectrode with b = 0. Fig. 4 depicts a time-sequential fluorescence images, which show single cell was electroporated and finally ruptured in the discrete co-planar microelectrode array (Fig. 4A) and the micro-cavity microelectrode (Fig. 4B). Fig. 4A shows that cell electroporation and rupture occur near the microelectrode in the co-planar microelectrode structure. Cytoplasm leaked into the microchannel from the formed nanopores. Because cells carried
green fluorescence, it can be seen that cytoplasm diffused in fluid medium within the microchannel. When the leakage of cytoplasm reached to a certain extent, the cells dissolved away. This is consistent with our previous simulation results shown in Figs.2C and 3. Fig. 4B demonstrates that cell rupture occurs near the pole, P2 , instead of near P1 , which is consistent with the theoretical results shown in Figs.2C and 3. Therefore, one can use different microelectrode structures to control the electroporation positions, which will have three main applications: cell lysis (releasing the subcellular contents of the cells), cell transfection (inserting DNA or other molecule into the cells), and cell electro-fusion (paired cells merged into a hybrid cell). For cell lysis, it would be easier to collect the subcellular contents released from the cells away from the microelectrode, and one can control the electroporation occurring near the pole of P2 . For cell transfection, DNA or other molecule could be first concentrated near the microelectrode, and electroporation occurring near the microelectrode will improve the transfection efficiency. For successful cell fusion, the membrane breakdown should take place only near the contact area of the paired cells (i.e., near the pole of P2 ) and avoid cell rupture at other parts of the cell membrane. 3.2. Electroporation in cell electro-fusion In this section we will control the electroporation for the application of cell electro-fusion, requiring electroporation occurring only at the contact area of paired cells while avoiding excessive cell electroporation at other places of the cell membranes. As the electric field will decrease due to the presence of the cells, a higher 0 was applied (i.e. 0 = 10V) in the electro-fusion process. The distributions of the electric field and TMP for electro-fusion of a pair of cells with equal radii were shown in Fig. 5. The same as the case of a single cell, the electric field near the planar microelectrode is the highest in the planar structure. In the micro-cavity structure,
440
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Fig. 7. TMP as a function of the length of the side wall microelectrode.
again the electric field intensity near the side wall of the microcavity is higher than that near the pole of P1 . The steady-state TMP at P1 is always higher than that at P2 in the co-planar structure. When the TMP at P2 reaches a sufficiently high value (∼1.2 V), a large number of pores formed in the membrane near P2 . While the TMP at P1 reaches ∼1.8 V, almost reaching the TMP required for the irreversible electroporation (i.e., ∼2 V), irreversible electroporation may occur in the area of P1 . Therefore, the co-planar structure is not a preferable condition for cell fusion. On the other hand, the microcavity structure with b = 0 has changed the distribution of TMP. TMP at the contact point of the paired cells (i.e., P2 ) reaches ∼1.1 V, and TMP near the microelectrode (P1 ) is also ∼1.1 V. Under this condition, the TMP at both P1 and P2 are quite close, and reversible electroporation occurs at both the contact point (P2 ) as well as P1 . Since TMP at P1 is below the critical value for irreversible electroporation (i.e., ∼2 V) cell will not rupture near the position of P1 . Since TMP at P3 is much lower than that at P1 and P2 , and no electroporation occurs at the pole of P3 . In the case of a chain of cells with more than 2 cells, the TMP at the contact points of three cells (Fig. 6) show that only two cells will be fused, and the cell away from the electrode will not be fused with the other two due to very low TMP at the corresponding contact point P3 . Note that the micro-cavity structure with b=0 shown in Fig. 5 is not the optimal structure since the TMP at P1 is still slightly higher than that at P2 , and reversible electroporation still occurs at the non-contacting area of P1 . To find an optimal microelectrode structure, we investigate the dependence of TMP on the length of microelectrode on the side walls of the micro-cavity, as shown in Fig. 7. As b increases, TMP at P1 dramatically decreases. When b is relatively low (i.e., b ≤ 10 m), the TMPs at P2 and P3 have small variation with an increase in b. When b exceeds 10 m, further increase in b yields a decrease in TMP at P2 and an increase in the TMP at P3 . To have reversible electroporation at the contact area of P2 , one needs to have higher TMP at P2 than those at P1 and P3 . Fig. 7 shows that the micro-cavity structure with microelectrodes of b = 5 m, b = 7.5 m and b = 10 m meets the requirements for cell electro-fusion. Fig. 8 compares the pore density along cell membranes at 40 s in the co-planar microelectrode array (Fig. 8A) and in the microcavity structure with the sidewall electrode b = 0 (Fig. 8B) and b = 7.5 m (Fig. 8C). For the co-planar microelectrode structure, the highest pore density occurs at the pole P1 near the microelectrode in addition to relatively high pore density at the contact point of the paired cells, P2 . Since TMP at P1 is about 1.8 V, the induced electroporation may be irreversible, yielding cell rupture near P1 . For the micro-cavity structure with b = 0, although the pore density at P1 is
Fig. 8. Pore density along cell membranes for two paired cells in the discrete coplanar microelectrode array (A), and in the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
still the highest, the pore density at P2 is also increased. In addition, the TMPs at P1 and P2 are around 1.1 V, and the formed pores at P1 and P2 are reversible, and cell rupture will be avoided. For the case of b = 7.5 m, the highest pore density appears at the contact point of P2 , and very low pore density occurs at the point P1 . In addition, the TMP at P2 is close to 1.1 V, thus the electroporation at the contact point of the paired cells is reversible for cell fusion, and the cell would not rupture in the area near P1 . In the case of a chain with more than 2 cells, the distributions of the pore density of 3 cells as shown in Fig. 9 also show that multiple cell fusion could be avoided in the micro-cavity structure. To validate the above theoretical predictions, we conducted the cell electro-fusion experiments with the microfluidic devices which have the co-planar and micro-cavity microelectrode structures. Fig. 10 depicts the sequential fluorescence images of a pair of cells during the fusion step. In the device with co-planar microelectrode, fluorescence leaks out from the cell’s membrane near the electrode, and then diffuses into the liquid medium. The fluorescence intensity inside the cell away from the microelectrode remains almost the same, implying that no rupture occurs for that cell. In the device with micro-cavity electrode of b = 0, (a)–(e) in Fig. 10B show that fluorescence is confined inside the two paired
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
441
Fig. 10. Cell electroporation during cell electro-fusion using the discrete co-planar microelectrode array (A) and the micro-cavity structure (B). The cells were exposed to a high-voltage electroporation pulse (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses: 5).
Fig. 9. Pore density along cell membrane in the discrete co-planar microelectrode array (A) and the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
cells without leaking into the fluid medium. When time is larger than 21 s (f–h in Fig. 10B), the fluorescence intensity inside the cell in contact with the microelectrode inside the cavity decreases, and this is because its fluorescence is transferred through the formed pores near P2 into the other cell. Cell rupture did not occur in the process since the fluorescence intensity inside the fluid medium outside the paired cells does not significantly increase. Obviously, the fluorescence intensity inside the fluid medium in Fig. 10A is much higher than that in Fig. 10B, and the enhanced fluorescence intensity in the fluid medium around the paired cells arises from the fluorescent dye leaked from the ruptured cell in contact with the microelectrode. The experimental results qualitatively agree with the aforementioned theoretical predictions. 4. Conclusions A systematical model for controllable cell electroporation has been developed and validated to illustrate the effect of the microelectrode configuration on the electroporation process. Based on the distribution of the formed nanopore density predicted from the validated model, one could modify and optimize the design of the device for controllable electroporation prior to experimental test. The design tool would be useful for the electroporation applications such as cell electrofusion, lysis, and gene transfection etc. The following results are obtained from both the theoretical predictions and the experimental observations:
(1) In the device with co-planar micro-electrodes, the TMP at the point P1 in the vicinity of the microelectrode is always about 50% higher than that at the contact point of the paired cells (P2 ), and cell rupture occurs near P1 during electroporation/electrofusion. (2) The micro-cavity structure improves the TMP distribution, and can create a desirable TMP distribution by varying the length of the side wall micro-electrode to minimize the risk of membrane rupture occurring at the microelectrodes and also avoid the possibility of multiple cells fusion. (3) With the micro-cavity structure, reversible electroporation appears in the contact area of P2 in a chain of two paired cells, yielding cell-electrofusion. (4) In the micro-cavity structure, multiple cell fusion will not occur since the TMP at the contact area other than P2 is relatively low. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81371691, 31571005, 81501617), the Fundamental Research Funds for the Central Universities (No. 106112016CDJZR238807), the Visiting Scholar Foundation of Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education (No. CQKLBST-2014-005). References [1] J.C. Weaver, Y.A. Chizmadzhev, Theory of electroporation: a review, Bioelectrochem. Bioenerg. 41 (1996) 135–160. [2] S. Movahed, D.Q. Li, Microfluidics cell electroporation, Microfluid. Nanofluid. 10 (2011) 703–734. [3] R. Shirakashi, V.L. Sukhorukov, R. Reuss, A. Schulz, U. Zimmermann, Effects of a pulse electric field on electrofusion of Giant Unilamellar Vesicle (GUV)-jurkat cell: (measurement of fusion ratio and electric field analysis of pulsed GUV-jurkat cell), J. Therm. Sci. Technol. 7 (2012) 589–602. [4] U. Zimmermann, J. Vienken, G. Pilwat, W.M. Arnold, Electro-fusion of cells: principles and potential for the future, Ciba Found. Symp 103 (1984) 60–85.
442
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
[5] A. Biragyn, K. Tani, M.C. Grimm, S. Weeks, L.W. Kwak, Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity, Nat. Biotechnol. 17 (1999) 253–258. [6] J.M. Melancon, T.P. Foster, K.G. Kousoulas, Genetic analysis of the herpes simplex virus type 1 UL20 protein domains involved in cytoplasmic virion envelopment and virus-induced cell fusion, J. Virol. 78 (2004) 7329–7343. [7] V.L. Sukhorukov, R. Reuss, J.M. Endter, S. Fehrmann, A. Katsen-Globa, P. Geßner, et al., A biophysical approach to the optimisation of dendritic-tumour cell electrofusion, Biochem. Biophys. Res. Commun. 346 (2006) 829–839. [8] R.J. Orentas, D. Schauer, Q. Bin, B.D. Johnson, Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine, Cell. Immunol. 213 (2001) 4–13. [9] R.A. Miller, F.H. Ruddle, Pluripotent teratocarcinoma-thymus somatic cell hybrids, Cell 9 (1976) 45–55. [10] A. Szczerbakowa, U. Maciejewska, P. Pawłowski, J.S. Skierski, B. Wielgat, Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum, Acta Physiol. Plant. 23 (2001) 169–179. [11] Y. Okada, The fusion of Ehrlich’s tumor cells caused by HVJ virus in vitro, Biken J. 1 (1958) 103–110. [12] U. Zimmermann, J. Vienken, Electric field-induced cell-to-cell fusion, J. Membr. Biol. 67 (1982) 165–182. [13] U. Karsten, P. Stolley, I. Walther, G. Papsdorf, S. Weber, K. Conrad, et al., Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas, Hybridoma 7 (1988) 627–633. [14] J. Yang, L.P. Zhao, Z.Q. Yin, N. Hu, J. Chen, T.Y. Li, et al., Chip-based cell electrofusion, Adv. Eng. Mater. 12 (2010) B398–B405. [15] C.T.S. Ching, T.P. Sun, W.T. Huang, S.H. Huang, C.S. Hsiao, K.M. Chang, A circuit design of a low-cost, portable and programmable electroporation device for biomedical applications, Sens. Actuators B: Chem. 166–167 (2012) 292–300. [16] W. Kang, J.P. Giraldo-Vela, S.S.P. Nathamgari, T. McGuire, R.L. McNaughton, J.A. Kessler, et al., Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons, Lab Chip 14 (2014) 4486–4495. [17] S.C. Buergel, C. Escobedo, N. Haandbaek, A. Hierlemann, On-chip electroporation and impedance spectroscopy of single-cells, Sens. Actuators B: Chem 210 (2015) 82–90. [18] L. Odorizzi, C. Ress, C. Collini, E. Morganti, L. Lorenzelli, N. Coppede, et al., An integrated platform for in vitro single-site cell electroporation: controlled delivery and electrodes functionalization, Sens. Actuators B: Chem. 170 (2012) 182–188. [19] J. Wang, C. Lu, Microfluidic cell fusion under continuous direct current voltage, Appl. Phys. Lett. 89 (2006) 234102. [20] A.M. Skelley, O. Kirak, H. Suh, R. Jaenisch, J. Voldman, Microfluidic control of cell pairing and fusion, Nat. Methods 6 (2009) 147–152. [21] E.W.M. Kemna, F. Wolbers, I. Vermes, A. van den Berg, On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation, Electrophoresis 32 (2011) 3138–3146. [22] N. Hu, J. Yang, S.W. Joo, A.N. Banerjee, S. Qian, Cell electrofusion in microfluidic devices: a review, Sens. Actuators B: Chem. 178 (2013) 63–85. [23] B. Techaumnat, M. Washizu, Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells, J. Phys. D: Appl. Phys. 40 (2007) 1831–1837. [24] L. Rems, M. Uˇsaj, M. Kanduˇser, M. Reberˇsek, D. Miklavˇciˇc, G. Pucihar, Cell electrofusion using nanosecond electric pulses, Sci. Rep. 3 (2013) 3382. [25] N. Hu, J. Yang, Z.Q. Yin, Y. Ai, S. Qian, I.B. Svir, et al., A high-throughput dielectrophoresis-based cell electrofusion microfluidic device, Electrophoresis 32 (2011) 2488–2495. [26] N. Hu, J. Yang, W.S. Hou, X.L. Zheng, Y. Cao, J. Yang, et al., SOI-based cell electrofusion chip, Chem. J. Chin. U 30 (2009) 42–45. [27] Y. Cao, J. Yang, Z.Q. Yin, H.Y. Luo, M. Yang, N. Hu, et al., Study of high-throughput cell electrofusion in a microelectrode-array chip, Microfluid. Nanofluid. 5 (2008) 669–675. [28] N. Hu, J. Yang, S. Qian, S.W. Joo, X. Zheng, A cell electrofusion microfluidic device integrated with 3D thin-film microelectrode arrays, Biomicrofluidics 5 (2011) 034121. [29] N. Hu, J. Yang, S. Qian, X. Zhang, S.W. Joo, X. Zheng, A cell electrofusion microfluidic chip using discrete coplanar vertical sidewall microelectrodes, Electrophoresis 33 (2012) 1980–1986.
[30] G. Mernier, W. Hasenkamp, N. Piacentini, P. Renaud, Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis, Sens. Actuators B: Chem. 170 (2012) 2–6. [31] Y.C. Chung, Y.S. Chen, S.H. Lin, Enhancement for gene transfection of low-descent-velocity bacteria using magnetic attraction in electroporation chip, Sens. Actuators B: Chem. 213 (2015) 261–267. [32] G. Pucihar, D. Miklavcic, T. Kotnik, A time-Dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells, IEEE Trans. Biomed. Eng. 56 (2009) 1491–1501. [33] K.A. DeBruin, W. Krassowska, Modeling electroporation in a single cell. I. Effects of field strength and rest potential, Biophys. J. 77 (1999) 1213–1224. [34] S.A. Freeman, M.A. Wang, J.C. Weaver, Theory of electroporation of planar bilayer membranes: predictions of the aqueous area, change in capacitance, and pore-pore separation, Biophys. J. 67 (1994) 42–56. [35] T.R. Gowrishankar, A.T. Esser, Z. Vasilkoski, K.C. Smith, J.C. Weaver, Microdosimetry for conventional and supra-electroporation in cells with organelles, Biochem. Biophys. Res. Commun. 341 (2006) 1266–1276. [36] T. Kotnik, D. Miklavˇciˇc, T. Slivnik, Time course of transmembrane voltage induced by time-varying electric fields – a method for theoretical analysis and its application, Bioelectrochem. Bioenerg. 45 (1998) 3–16. [37] N. Hu, X. Zhang, J. Yang, S.W. Joo, S. Qian, A cell electrofusion microfluidic chip with micro-cavity microelectrode array, Microfluid. Nanofluid. 15 (2013) 151–160. [38] H. Pauly, H.P. Schwan, Impendance of a suspension of ball-shaped particles with a shell; a model for the dielectric behavior of cell suspensions and protein solutions, Zeitschrift für Naturforschung, Teil B: Chemie. Biochemie. Biophysik. Biologie. 14 B (1959) 125–131.
Biographies Xiaoling Zhang received her Ph.D. in Biomedical Engineering from Chongqing University in 2015. She is a postdoctoral fellow in Key Laboratory of Optoelectronic Technology and System, Ministry of Education, and a researcher in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. Her current research interests include bio-MEMS and microfluidics. Ning Hu is an associate professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from Chongqing University in 2010. His research focuses on bio-MEMS, biomicrofluidics, lab-on-a-chip technology, and cell electrofusion. Xi Chen is a Ph.D. student in Bioengineering College, Chongqing University. He obtained his master’s degree in Biomedical Engineering from Chongqing University in 2015. His research focuses on magnetoelastic sensors and microfluidics. Ting Fan defended her BS in Biomedical Engineering at Chongqing University in 2016. Her research focuses on lipid vesicles, microfluidics. Zhenyu Wang is a faculty in College of Biomedical Engineering of Chongqing Medical University, She obtained her Ph.D. from Chongqing University in 2013. Her research focuses on lipid vesicles, microfluidics, bio-MEMS, and ultrasound. Xiaolin Zheng is a professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from Chongqing University in 1995. His research focuses on bio-MEMS, biomedical devices. Jun Yang is a professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from City University of Hong Kong in 2004. His research focuses on bio-MEMS, microfluidics, and biosensors. Shizhi Qian received his Ph.D. in Mechanics and Applied Mechanics from University of Pennsylvania in 2004. He is an associate professor in Department of Mechanical and Aerospace Engineering at the Old Dominion University. His current research interests include electrokinetics, and microfluidics and nanofluidics.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Controllable cell electroporation using microcavity electrodes Xiaoling Zhang a,b , Ning Hu a,∗ , Xi Chen a , Ting Fan a , Zhenyu Wang c , Xiaolin Zheng a , Jun Yang a,∗ , Shizhi Qian d,∗ a
Key Laboratory of Biorheological Science and Technology, Ministry of Education, Chongqing University, Chongqing, 400030, PR China Key Laboratory for Optoelectronic Technology and Systems, Ministry of Education, Chongqing University, Chongqing, 400030, PR China College of Biomedical Engineering, Chongqing Medical University, Chongqing, 400016, PR China d Institute of Micro/Nanotechnology, Old Dominion University, Norfolk, VA, 23529, USA b c
a r t i c l e
i n f o
Article history: Received 21 June 2016 Received in revised form 23 August 2016 Accepted 30 August 2016 Available online 31 August 2016 Keywords: Electro-fusion Transmembrane potential Microelectrode Microcavity Myoblast cell
a b s t r a c t Cell electro-fusion includes four steps, cell alignment, cell electroporation, reconstruction of cytomembrane and cytoplasm exchange. The cell alignment and electroporation steps are highly related to the intensity and distribution of the electric field, which depend on the applied voltage as well as the microelectrode structure. The microelectrode structures were first evaluated based on the numerical analysis of the electric field and the transmembrane potential induced on biological cells when the cell electroporation and electro-fusion were performed based on different designs of microelectrodes. Microelectrodes in a micro-cavity geometry can induce electroporation around the contact area of the paired cells for high-yield electro-fusion. Microfluidic chips with co-planar microelectrodes and microelectrodes within micro-cavities have been fabricated and tested for electro-fusion of Myoblast cells, and the experimental results confirmed the numerical analysis. © 2016 Published by Elsevier B.V.
1. Introduction When cells are subject to an external electric field, a voltage difference is induced across the cell membrane, which is termed as the transmembrane potential (TMP). If TMP exceeds the membrane breakdown voltage, which is about 1 V for most cells, nanopores are created on the cell membrane, which is called electroporation or electropermeabilization [1,2]. The created nanopores can be reversible or irreversible, decided by the duration and the intensity of the external electric field. For the irreversible electroporation, the TMP required is around twice as high as the reported reversible electroporation potential [3]. One of the most important applications of the cell reversible electroporation is cell electro-fusion, which uses a high-strength electric field to induce cell alignment/pairing, membrane permeabilization, and fusion of paired cells to form hybrid cells [4]. Since the formed hybrid cells contain the genetic materials from parent cells, cell electro-fusion provides a feasible technology and approach to understand and study genetics [5,6], immunology [7,8], hybridization, and crossbreeding [9,10]. Compared with the virus- [11] and PEG-induced cell
∗ Corresponding authors. E-mail addresses: [email protected] (N. Hu), [email protected] (J. Yang), [email protected] (S. Qian). http://dx.doi.org/10.1016/j.snb.2016.08.172 0925-4005/© 2016 Published by Elsevier B.V.
fusion techniques, electro-fusion attracts high attention due to its non-toxicity, wide adaptability to cell types, easy implementation and high reproducibility [4,12,13]. In addition, the electro-fusion efficiency is much higher than the other methods. Generally, the electro-fusion process can be divided into four sequential steps: cell alignment (pairing), reversible electroporation on cell membrane, reconstruction of cytomembrane, and cytoplasm exchange between two cells [4]. Cell alignment and reversible electroporation are the most important two steps, which highly depend on the local electric field intensity as well as its gradient. Typically, cell alignment is achieved under a low-amplitude (around 100–300 V cm−1 ) and high-frequency (around 1–3 MHz) sinusoidal alternating current (AC) field, while a series of short-duration (around 10–50 s) and high-strength (around 1–10 kV cm−1 ) electric pulses is applied to induce reversible electroporation [14]. For a successful cell electro-fusion, the reversible electroporation should take place only at the contact area of the paired cells, which requires that the TMP distribution should be well controlled to induce electroporation at the contact area. The TMP distribution is controlled by the electric field around the cell, which depends on the geometry of the microelectrodes as well as the microfluidic channel. In the last decade, although many kinds of microelectrode/microfluidic structures have been developed for cell electroporation [15–18] and electro-fusion [19–22], there are few studies focused on the controllable electroporation.
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Techaumnat and Washizu [23] conducted a numerical analysis of the TMP on biological cells with the effects of an orifice plate in electroporation and electro-fusion. Electric field is constricted at the orifice, where the magnitude and localization of the TMP can be controlled, and yields high electro-fusion efficiency. Rems [24] reported that nanosecond pulses can induce selective electroporation at the contact areas of paired cells, regardless of the cells’ sizes. In the nanosecond range, cell membranes are still in the charging phase, and electric field at the contact area reaches the highest value, yielding selective contact electroporation. Previously we developed and tested continuous protruding microelectrode array [25–27] for cell electrofusion. As the most important process of cell electrofusion, the location and intensity of reversible electroporation are highly dependent on the electric field distribution around the cells. To obtain desirable cell alignment and reversible electroporation efficiency, two kinds of discrete microelectrode arrays, 3D thin film microelectrode [28] and discrete co-planer vertical sidewall microelectrode [29], have been developed to optimize the electric field distribution. Although the cell electrofusion efficiency is improved by this structure, we found that irreversible electroporation occurring at the contact point between discrete microelectrodes and their adjacent cells. Therefore, the existing microelectrode structures still need to be optimized to achieve controllable electroporation. In addition to cell electrofusion, electroporation has been widely used in other applications such as cell lysis [30] and gene transfection [31]. Depending on the application, the desirable position for cell electroporation is different. In addition, most previous studies related to cell electroporation focused on experimental investigation using different structures of microelectrodes and/or microchannel, and there is no appropriate design tool for the design and optimization of the device for achieving selective cell electroporation at the desirable position. This study develops a mathematical model for the electroporation process, and is further
435
Fig. 1. Configuration for the discrete co-planar microelectrode array (A) and the micro-cavity structure (B).
validated by experimental results. Based on the distribution of the formed nanopore density predicted from the validated model, one can easily modify and optimize the design for controlling the position of cell electroporation, which would improve the efficiency of the electroporation applications.
Fig. 2. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array with one cell, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with one cell, (C) The absolute value of TMP of P1 and P2 for the case of single cell electroporation in the discrete co-planar microelectrode and in the micro-cavity structure.
436
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Table 1 Values of constants and parameters used in the simulations. Parameter
Value
Microchannel size (H × W × L) Microelectrode length (l) Cavity size (Lca × Wca ) Cell radius (a) Medium conductivity (out ) Medium permittivity (εout ) Cytoplasmic conductivity (in ) Cytoplasmic permittivity (εin ) Cell membrane thickness (dm ) Cell membrane conductivity (m0 ) Cell membrane permittivity (εm ) 0 creation rate coefficient (˛) equilibrium pore density when = 0 (N0 ) pore creation rate (q) characteristic voltage of electroporation (VEP ) pore radius (rp ) conductivity of solution inside the pore (p ) relative entrance length of pores (n) energy barrier within pore (w0 ) Faraday’s constant (F) gas constant (R) absolute temperature (T)
30 m × 80 m × 60 m 20 m 20 m × 20 m 7.5 m 0.012 S m−1 80 0.3 S m−1 70 5 nm 5e-7 S m−1 4.5 8 V for single cell electroporation 10 V for cell electro-fusion 1e9 m−2 s−1 1.5e9 m−2 2.46 258 mV 0.76e-9 0.16 S m−1 0.15 2.65 9.65e4 Cmol−1 8.314 J K−1 mol−1 295 K
Reference
2. Materials and methods
Fig. 1 shows schematic diagrams of the microchannels with a pair of discrete co-planar microelectrode (A) and microelectrodes within the micro-cavity structure (B), respectively. The purple faces in Fig. 1 denoted the microelectrodes, which are part of the vertical side walls of the microchannel. In the co-planar structure, the planar electrodes of length l are on the same plane of the corresponding vertical side walls. In the 2nd design with micro-cavities, the size of which are Lca × Wca and determined based on the size of the cells to be fused in the experiments. Microelectrodes can be fabricated along one side wall of the micro-cavity (i.e., b = 0) as well as part of the other two side walls with the length of b. The entire vertical side walls of the micro-cavity will become the microelectrode when b = Wca . We consider spherical cells of radius a suspended in a buffer solution with conductivity out , which fills the microchannel of height H, width W and length L. To investigate the electric field distribution and TMP, we assume a potential 0 is imposed on one electrode and the opposite electrode is ground. For the case of single cell electroporation, only one cell was fixed in the vicinity of a microelectrode. For electroporation in cell electrofusion, two cells were aligned as cell-cell pair and fixed near the microelectrode. We also considered a chain of three cells to investigate the case of electro-fusion of multiple cells. The position of the pole at the microelectrode was denoted by P1 , and the first and second contact point of the paired three cells are denoted as P2 and P3 , respectively. Between the paired cells, we inserted a small separation of 0.001 m so that cells are not completely contiguous. The electric potential inside (in ) and outside (out ) the cell can be described by the following electrostatic equations [32]:
∂ (∇ ˚in ) ∂t
−∇ (out ∇ out ) − ε0 εout ∇
[7] [33] [33] [33] [33] [33] [32] [33] [33]
The required voltage for electroporation/electro-fusion (0 ) was applied via the embedded discrete electrodes. Thus, the boundary condition on electrodes was assumed as
2.1. Mathematical model
−∇ (in ∇ in ) − ε0 εin ∇
Eppendorf Co. [35] [36] [7] [33]
= 0,
∂ (∇ ˚out ) ∂t
(1)
= 0 or0.
(3)
Electrical insulation boundary condition was imposed on the rest walls of the microchannel. Since the cell membrane is very thin, it was not physically included in the model. To take into account the thin membrane, a current density flowing through the membrane was used [33]: J (t) =
m (t) (˚in − out ) ε0 εm ∂ (˚in − out ) + . dm dm ∂t
(4)
The first and the second terms on the right-hand side of Eq. (4) represent the conductive and the capacitive components of the electric current through the membrane, respectively. Here, J (t) is the current density; εm and dm are the relative permittivity and the thickness of the membrane, respectively; m is the conductivity of the membrane; in and out are, respectively, the potentials at the cytoplasm/membrane and membrane/medium interfaces. Note that the membrane conductivity varies during the electroporation process, and is modeled by m (t) = m0 + N (t) p rp2 A.
(5)
In the above, m0 is the membrane conductivity without electroporation; rp is the formed pore radius; p is the conductivity of solution inside the pore; N is the pore density governed by following equation [32,33] with an initial condition of N = N0 : 2 dN (t) = ˛e(˚(t)/VEP ) dt
1−
N (t) −q(˚(t)/VEP )2 e N0
,
where N0 is the pore density in the non-electroporated membrane; the parameters ˛, q, and VEP describe the characteristics of the electroporation process; and the induced transmembrane potential (TMP = ) is defined as (t) ≡ in (t) − out (t)
= 0.
(2)
Here, ε0 is dielectric permittivity of the vacuum; εi and i are, respectively, the relative dielectric permittivity and conductivity of the cytoplasm (i = in) and the medium (i = out).
(6)
(7)
The parameter A in Eq. (5) is given by [33] e vm − 1 . A= v e m (w0 ew0 −nvm − nvm ) / (w0 − nvm ) − (w0 ew0 +nvm + nvm ) / (w0 + nvm )
(8)
In the above, vm = F/RT with F, R, and T being the Faraday’s constant, the gas constant, and absolute temperature of the suspension,
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
437
respectively; n is the relative entrance length of pores; and w0 is the energy barrier within pore. In this current study, we assume that all pores have equal and time-independent radii, which is about 0.8 nm [32,34]. The timedependent Eqs. (1) and (2) are simultaneously solved subject to the boundary conditions, Eqs. (3) and (4) in which m is calculated from Eqs. (5)–(8), with the commercial finite element package, COMSOL, using the values of parameters listed in Table 1. 2.2. Cell culture and cell preparation In our experiments, Myoblast cells (Third Military Medical University, Chongqing, China), which were labelled by green fluorescence (pAdTrack-GFP), were used to monitor the process of electroporation. Myoblast cells were cultured in F10 supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin. They were incubated at 37 ◦ C in humidified atmosphere containing 5% CO2 and 95% air. Before the experiment, cells were trypsinized by using EDTA (0.25/0.02%; Sigma) and collected in a small tube. After collecting the cells, the electro-fusion medium (Osmolaity: 90 ± 10 mOsmol/kg, conductivity: 0.012 S/m, Eppendorf Co., electro-fusion buffer for eukaryotic cell, German) was used to wash the cells three times and suspend them as a concentration of 1.0 × 106 (5.0 × 106 ) cells/mL for single cell electroporation (cell electro-fusion). 2.3. Experiments protocol The microfluidic chips, which integrated discrete co-planar microelectrodes and micro-cavity microelectrodes respectively, are designed and fabricated on SOI wafer [29,37]. The discrete microelectrodes and micro-cavity microelectrodes are designed based on the optimized microelectrode structures from the numerical simulation of the electric field inside the microchannel. To fabricate the side wall electrodes with a certain length of b, an insulator made by SiO2 -Ployisilicon-SiO2 with length of Lca -b is fabricated first based on the method described by the previous work [29,37]. Then the highly doped silicon is etched by the inductively coupled plasma technique to form the designed micro-cavity electrode. In addition, these fabricated microfluidic chips are packaged to connect the external power generator for experiments. Before experiments, the microfluidic chips were rinsed by the electro-fusion buffer for 10 s. A droplet of the suspending solution was pipetted into the microfluidic chips. And then AC electric pulses (Vp-p : 2–3 V, frequency: 1 MHz, duration: 30 s) were applied to the microelectrodes. Due to the positive DEP under the considered conditions, cells are attracted to the microelectrodes. A little higher voltage of DC pulse signal (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses number: 5) was applied to induce cell irreversible electroporation. The fluorescence of the dye in the cell leaked out after the cell was electroporated, which was monitored with a CCD camera (Motic 3000, China) through an Olympus BX 61 microscope (Olympus Optical Co. Ltd., Japan). 3. Results and discussion 3.1. Single cell electroporation For cells suspended in an infinite domain and exposed to a uniform electric field of Ee , if the nanopores are not created, TMP () is given by the Schuwan equation [38]: = 1.5Ee a cos ,
(9)
where a is the radius of the cell, and is the angle between the direction of electric field Ee and the outward normal from the center of the cell to the site on the cell membrane. TMP is symmetrical
Fig. 3. Pore density along cell membrane for a single cell in the discrete co-planar microelectrode array (A) and the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
on both sides of the cell, and reaches its maximum value at the poles ( = 0 and 180◦ ). For the cell confined in a microchannel as shown in Fig. 1, the distribution of TMP is highly dependent on the spatial distribution of the electric field, which is decided by the structure of the microelectrode and microchannel. Fig. 2 depicts the spatial distribution of the electric field strength in the microchannel with discrete co-planar microelectrode array (Fig. 2A) and the micro-cavity microelectrode with b = 0 (Fig. 2B) in the presence of one cell in the vicinity of the electrode. For the case of co-planar microelectrode, the electric field strength near the microelectrode (i.e., the pole P1 ) is the highest, and decreases with increasing the distance from the microelectrode. In contrast, the electric field is concentrated within the micro-cavity, however, the electric field at the poles of the cell, P1 and P2 , is not the highest. The electric field in the area between the cell and the side walls of the microcavity is the highest. Since the distance between the two opposite microelectrodes in the micro-cavity is larger than that of co-planar case, under the same voltage 0 , the maximum magnitude of the
438
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Fig. 4. Single cell electroporation using the discrete co-planar microelectrode array (A) and the micro-cavity structure (B). The cells were exposed to a high-voltage electroporation pulse (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses: 5).
Fig. 5. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array with two cells, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with two cells, (C) The absolute value of TMP of P1 and P2 for the case of the cell electro-fusion in the discrete co-planar microelectrode and in the micro-cavity structure.
electric field in the microchannel with co-planar microelectrodes is higher than that with the micro-cavity microelectrodes. Due to the same reason, the steady-state TMP shown in Fig. 2C in micro-cavity structure is lower than that in co-planar structure at the same position. Fig. 2C shows that the TMP at P1 located in the vicinity of the microelectrode is always higher than that at P2 in the discrete coplanar microelectrode array. In the micro-cavity structure, the TMP
at P1 is a little higher than that at P2 at the beginning, and is significantly lower than that at P2 when it reaches the steady state. If we also extend the microelectrode to cover part of the side walls inside the micro-cavity, the steady-state TMP at P1 is considerably lower than that at P2 , as shown by the results for b = 7.5 m in Fig. 2C. The results suggest that we can achieve the highest TMP at P2 , where cell fusion occurs, and relatively low TMP at P1 to avoid cell rupture
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
439
Fig. 6. (A) Spatial distribution of the electric field strength in the discrete co-planar microelectrode array microfluidic channel with three cells, (B) Spatial distribution of the electric field strength in the micro-cavity microfluidic channel with three cells, (C) The absolute value of TMP at P1 and P2 for the case of three cells in the discrete co-planar microelectrode (no symbols) and in the micro-cavity structure (with symbols).
occurring there by adjusting the length of the microelectrodes on the side wall of the micro-cavity. When TMP exceeds the threshold voltage, many nanopores are induced along cell membrane and cell electroporation occurs. Fig. 3 shows the pore density of one cell induced along the cell membrane at 40 s in the discrete co-planar microelectrode array (Fig. 3A) and in the micro-cavity structure with b = 0 (Fig. 3B) and b = 7.5 m (Fig. 3C). The purple lines in Fig. 3 denoted as the microelectrode and the black ones denoted as the insulting material. Membrane areas with the pore density ranging from 1/10 Max to Max were marked with color legend and Max represents the corresponding maximum pore density. For the discrete co-planar microelectrode array, the highest pore density occurs near the microelectrode, and very limited pores formed at the other pole, P2 , since the TMP at P1 is much higher than that at P2 , as shown in Fig. 2C. For the microcavity structure with b = 0, the highest pore density also occurs near P1 , but there were also a large number of pores formed near P2 . In contrast, when b = 7.5 m, the highest pore density occurs near P2 instead of P1 . These pore density results are consistent with the results of the TMP shown in Fig. 2C. Because the distance between two electrodes in the micro-cavity structure is longer than that in the discrete co-planar structure, the intensity of the electric field is lower, yielding lower TMP and accordingly lower pore density, as shown in Figs.2C and 3. To validate the above theoretical predictions, we conducted experiments on single cell electroporation in the microchannels with discrete co-planar microelectrode array and the micro-cavity microelectrode with b = 0. Fig. 4 depicts a time-sequential fluorescence images, which show single cell was electroporated and finally ruptured in the discrete co-planar microelectrode array (Fig. 4A) and the micro-cavity microelectrode (Fig. 4B). Fig. 4A shows that cell electroporation and rupture occur near the microelectrode in the co-planar microelectrode structure. Cytoplasm leaked into the microchannel from the formed nanopores. Because cells carried
green fluorescence, it can be seen that cytoplasm diffused in fluid medium within the microchannel. When the leakage of cytoplasm reached to a certain extent, the cells dissolved away. This is consistent with our previous simulation results shown in Figs.2C and 3. Fig. 4B demonstrates that cell rupture occurs near the pole, P2 , instead of near P1 , which is consistent with the theoretical results shown in Figs.2C and 3. Therefore, one can use different microelectrode structures to control the electroporation positions, which will have three main applications: cell lysis (releasing the subcellular contents of the cells), cell transfection (inserting DNA or other molecule into the cells), and cell electro-fusion (paired cells merged into a hybrid cell). For cell lysis, it would be easier to collect the subcellular contents released from the cells away from the microelectrode, and one can control the electroporation occurring near the pole of P2 . For cell transfection, DNA or other molecule could be first concentrated near the microelectrode, and electroporation occurring near the microelectrode will improve the transfection efficiency. For successful cell fusion, the membrane breakdown should take place only near the contact area of the paired cells (i.e., near the pole of P2 ) and avoid cell rupture at other parts of the cell membrane. 3.2. Electroporation in cell electro-fusion In this section we will control the electroporation for the application of cell electro-fusion, requiring electroporation occurring only at the contact area of paired cells while avoiding excessive cell electroporation at other places of the cell membranes. As the electric field will decrease due to the presence of the cells, a higher 0 was applied (i.e. 0 = 10V) in the electro-fusion process. The distributions of the electric field and TMP for electro-fusion of a pair of cells with equal radii were shown in Fig. 5. The same as the case of a single cell, the electric field near the planar microelectrode is the highest in the planar structure. In the micro-cavity structure,
440
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
Fig. 7. TMP as a function of the length of the side wall microelectrode.
again the electric field intensity near the side wall of the microcavity is higher than that near the pole of P1 . The steady-state TMP at P1 is always higher than that at P2 in the co-planar structure. When the TMP at P2 reaches a sufficiently high value (∼1.2 V), a large number of pores formed in the membrane near P2 . While the TMP at P1 reaches ∼1.8 V, almost reaching the TMP required for the irreversible electroporation (i.e., ∼2 V), irreversible electroporation may occur in the area of P1 . Therefore, the co-planar structure is not a preferable condition for cell fusion. On the other hand, the microcavity structure with b = 0 has changed the distribution of TMP. TMP at the contact point of the paired cells (i.e., P2 ) reaches ∼1.1 V, and TMP near the microelectrode (P1 ) is also ∼1.1 V. Under this condition, the TMP at both P1 and P2 are quite close, and reversible electroporation occurs at both the contact point (P2 ) as well as P1 . Since TMP at P1 is below the critical value for irreversible electroporation (i.e., ∼2 V) cell will not rupture near the position of P1 . Since TMP at P3 is much lower than that at P1 and P2 , and no electroporation occurs at the pole of P3 . In the case of a chain of cells with more than 2 cells, the TMP at the contact points of three cells (Fig. 6) show that only two cells will be fused, and the cell away from the electrode will not be fused with the other two due to very low TMP at the corresponding contact point P3 . Note that the micro-cavity structure with b=0 shown in Fig. 5 is not the optimal structure since the TMP at P1 is still slightly higher than that at P2 , and reversible electroporation still occurs at the non-contacting area of P1 . To find an optimal microelectrode structure, we investigate the dependence of TMP on the length of microelectrode on the side walls of the micro-cavity, as shown in Fig. 7. As b increases, TMP at P1 dramatically decreases. When b is relatively low (i.e., b ≤ 10 m), the TMPs at P2 and P3 have small variation with an increase in b. When b exceeds 10 m, further increase in b yields a decrease in TMP at P2 and an increase in the TMP at P3 . To have reversible electroporation at the contact area of P2 , one needs to have higher TMP at P2 than those at P1 and P3 . Fig. 7 shows that the micro-cavity structure with microelectrodes of b = 5 m, b = 7.5 m and b = 10 m meets the requirements for cell electro-fusion. Fig. 8 compares the pore density along cell membranes at 40 s in the co-planar microelectrode array (Fig. 8A) and in the microcavity structure with the sidewall electrode b = 0 (Fig. 8B) and b = 7.5 m (Fig. 8C). For the co-planar microelectrode structure, the highest pore density occurs at the pole P1 near the microelectrode in addition to relatively high pore density at the contact point of the paired cells, P2 . Since TMP at P1 is about 1.8 V, the induced electroporation may be irreversible, yielding cell rupture near P1 . For the micro-cavity structure with b = 0, although the pore density at P1 is
Fig. 8. Pore density along cell membranes for two paired cells in the discrete coplanar microelectrode array (A), and in the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
still the highest, the pore density at P2 is also increased. In addition, the TMPs at P1 and P2 are around 1.1 V, and the formed pores at P1 and P2 are reversible, and cell rupture will be avoided. For the case of b = 7.5 m, the highest pore density appears at the contact point of P2 , and very low pore density occurs at the point P1 . In addition, the TMP at P2 is close to 1.1 V, thus the electroporation at the contact point of the paired cells is reversible for cell fusion, and the cell would not rupture in the area near P1 . In the case of a chain with more than 2 cells, the distributions of the pore density of 3 cells as shown in Fig. 9 also show that multiple cell fusion could be avoided in the micro-cavity structure. To validate the above theoretical predictions, we conducted the cell electro-fusion experiments with the microfluidic devices which have the co-planar and micro-cavity microelectrode structures. Fig. 10 depicts the sequential fluorescence images of a pair of cells during the fusion step. In the device with co-planar microelectrode, fluorescence leaks out from the cell’s membrane near the electrode, and then diffuses into the liquid medium. The fluorescence intensity inside the cell away from the microelectrode remains almost the same, implying that no rupture occurs for that cell. In the device with micro-cavity electrode of b = 0, (a)–(e) in Fig. 10B show that fluorescence is confined inside the two paired
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
441
Fig. 10. Cell electroporation during cell electro-fusion using the discrete co-planar microelectrode array (A) and the micro-cavity structure (B). The cells were exposed to a high-voltage electroporation pulse (amplitude: 14 V, duration: 40 s, interval of two pulses: 1 s, pulses: 5).
Fig. 9. Pore density along cell membrane in the discrete co-planar microelectrode array (A) and the micro-cavity structure with b = 0 (B) and b = 7.5 m (C).
cells without leaking into the fluid medium. When time is larger than 21 s (f–h in Fig. 10B), the fluorescence intensity inside the cell in contact with the microelectrode inside the cavity decreases, and this is because its fluorescence is transferred through the formed pores near P2 into the other cell. Cell rupture did not occur in the process since the fluorescence intensity inside the fluid medium outside the paired cells does not significantly increase. Obviously, the fluorescence intensity inside the fluid medium in Fig. 10A is much higher than that in Fig. 10B, and the enhanced fluorescence intensity in the fluid medium around the paired cells arises from the fluorescent dye leaked from the ruptured cell in contact with the microelectrode. The experimental results qualitatively agree with the aforementioned theoretical predictions. 4. Conclusions A systematical model for controllable cell electroporation has been developed and validated to illustrate the effect of the microelectrode configuration on the electroporation process. Based on the distribution of the formed nanopore density predicted from the validated model, one could modify and optimize the design of the device for controllable electroporation prior to experimental test. The design tool would be useful for the electroporation applications such as cell electrofusion, lysis, and gene transfection etc. The following results are obtained from both the theoretical predictions and the experimental observations:
(1) In the device with co-planar micro-electrodes, the TMP at the point P1 in the vicinity of the microelectrode is always about 50% higher than that at the contact point of the paired cells (P2 ), and cell rupture occurs near P1 during electroporation/electrofusion. (2) The micro-cavity structure improves the TMP distribution, and can create a desirable TMP distribution by varying the length of the side wall micro-electrode to minimize the risk of membrane rupture occurring at the microelectrodes and also avoid the possibility of multiple cells fusion. (3) With the micro-cavity structure, reversible electroporation appears in the contact area of P2 in a chain of two paired cells, yielding cell-electrofusion. (4) In the micro-cavity structure, multiple cell fusion will not occur since the TMP at the contact area other than P2 is relatively low. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81371691, 31571005, 81501617), the Fundamental Research Funds for the Central Universities (No. 106112016CDJZR238807), the Visiting Scholar Foundation of Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education (No. CQKLBST-2014-005). References [1] J.C. Weaver, Y.A. Chizmadzhev, Theory of electroporation: a review, Bioelectrochem. Bioenerg. 41 (1996) 135–160. [2] S. Movahed, D.Q. Li, Microfluidics cell electroporation, Microfluid. Nanofluid. 10 (2011) 703–734. [3] R. Shirakashi, V.L. Sukhorukov, R. Reuss, A. Schulz, U. Zimmermann, Effects of a pulse electric field on electrofusion of Giant Unilamellar Vesicle (GUV)-jurkat cell: (measurement of fusion ratio and electric field analysis of pulsed GUV-jurkat cell), J. Therm. Sci. Technol. 7 (2012) 589–602. [4] U. Zimmermann, J. Vienken, G. Pilwat, W.M. Arnold, Electro-fusion of cells: principles and potential for the future, Ciba Found. Symp 103 (1984) 60–85.
442
X. Zhang et al. / Sensors and Actuators B 240 (2017) 434–442
[5] A. Biragyn, K. Tani, M.C. Grimm, S. Weeks, L.W. Kwak, Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity, Nat. Biotechnol. 17 (1999) 253–258. [6] J.M. Melancon, T.P. Foster, K.G. Kousoulas, Genetic analysis of the herpes simplex virus type 1 UL20 protein domains involved in cytoplasmic virion envelopment and virus-induced cell fusion, J. Virol. 78 (2004) 7329–7343. [7] V.L. Sukhorukov, R. Reuss, J.M. Endter, S. Fehrmann, A. Katsen-Globa, P. Geßner, et al., A biophysical approach to the optimisation of dendritic-tumour cell electrofusion, Biochem. Biophys. Res. Commun. 346 (2006) 829–839. [8] R.J. Orentas, D. Schauer, Q. Bin, B.D. Johnson, Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine, Cell. Immunol. 213 (2001) 4–13. [9] R.A. Miller, F.H. Ruddle, Pluripotent teratocarcinoma-thymus somatic cell hybrids, Cell 9 (1976) 45–55. [10] A. Szczerbakowa, U. Maciejewska, P. Pawłowski, J.S. Skierski, B. Wielgat, Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum, Acta Physiol. Plant. 23 (2001) 169–179. [11] Y. Okada, The fusion of Ehrlich’s tumor cells caused by HVJ virus in vitro, Biken J. 1 (1958) 103–110. [12] U. Zimmermann, J. Vienken, Electric field-induced cell-to-cell fusion, J. Membr. Biol. 67 (1982) 165–182. [13] U. Karsten, P. Stolley, I. Walther, G. Papsdorf, S. Weber, K. Conrad, et al., Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas, Hybridoma 7 (1988) 627–633. [14] J. Yang, L.P. Zhao, Z.Q. Yin, N. Hu, J. Chen, T.Y. Li, et al., Chip-based cell electrofusion, Adv. Eng. Mater. 12 (2010) B398–B405. [15] C.T.S. Ching, T.P. Sun, W.T. Huang, S.H. Huang, C.S. Hsiao, K.M. Chang, A circuit design of a low-cost, portable and programmable electroporation device for biomedical applications, Sens. Actuators B: Chem. 166–167 (2012) 292–300. [16] W. Kang, J.P. Giraldo-Vela, S.S.P. Nathamgari, T. McGuire, R.L. McNaughton, J.A. Kessler, et al., Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons, Lab Chip 14 (2014) 4486–4495. [17] S.C. Buergel, C. Escobedo, N. Haandbaek, A. Hierlemann, On-chip electroporation and impedance spectroscopy of single-cells, Sens. Actuators B: Chem 210 (2015) 82–90. [18] L. Odorizzi, C. Ress, C. Collini, E. Morganti, L. Lorenzelli, N. Coppede, et al., An integrated platform for in vitro single-site cell electroporation: controlled delivery and electrodes functionalization, Sens. Actuators B: Chem. 170 (2012) 182–188. [19] J. Wang, C. Lu, Microfluidic cell fusion under continuous direct current voltage, Appl. Phys. Lett. 89 (2006) 234102. [20] A.M. Skelley, O. Kirak, H. Suh, R. Jaenisch, J. Voldman, Microfluidic control of cell pairing and fusion, Nat. Methods 6 (2009) 147–152. [21] E.W.M. Kemna, F. Wolbers, I. Vermes, A. van den Berg, On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation, Electrophoresis 32 (2011) 3138–3146. [22] N. Hu, J. Yang, S.W. Joo, A.N. Banerjee, S. Qian, Cell electrofusion in microfluidic devices: a review, Sens. Actuators B: Chem. 178 (2013) 63–85. [23] B. Techaumnat, M. Washizu, Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells, J. Phys. D: Appl. Phys. 40 (2007) 1831–1837. [24] L. Rems, M. Uˇsaj, M. Kanduˇser, M. Reberˇsek, D. Miklavˇciˇc, G. Pucihar, Cell electrofusion using nanosecond electric pulses, Sci. Rep. 3 (2013) 3382. [25] N. Hu, J. Yang, Z.Q. Yin, Y. Ai, S. Qian, I.B. Svir, et al., A high-throughput dielectrophoresis-based cell electrofusion microfluidic device, Electrophoresis 32 (2011) 2488–2495. [26] N. Hu, J. Yang, W.S. Hou, X.L. Zheng, Y. Cao, J. Yang, et al., SOI-based cell electrofusion chip, Chem. J. Chin. U 30 (2009) 42–45. [27] Y. Cao, J. Yang, Z.Q. Yin, H.Y. Luo, M. Yang, N. Hu, et al., Study of high-throughput cell electrofusion in a microelectrode-array chip, Microfluid. Nanofluid. 5 (2008) 669–675. [28] N. Hu, J. Yang, S. Qian, S.W. Joo, X. Zheng, A cell electrofusion microfluidic device integrated with 3D thin-film microelectrode arrays, Biomicrofluidics 5 (2011) 034121. [29] N. Hu, J. Yang, S. Qian, X. Zhang, S.W. Joo, X. Zheng, A cell electrofusion microfluidic chip using discrete coplanar vertical sidewall microelectrodes, Electrophoresis 33 (2012) 1980–1986.
[30] G. Mernier, W. Hasenkamp, N. Piacentini, P. Renaud, Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis, Sens. Actuators B: Chem. 170 (2012) 2–6. [31] Y.C. Chung, Y.S. Chen, S.H. Lin, Enhancement for gene transfection of low-descent-velocity bacteria using magnetic attraction in electroporation chip, Sens. Actuators B: Chem. 213 (2015) 261–267. [32] G. Pucihar, D. Miklavcic, T. Kotnik, A time-Dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells, IEEE Trans. Biomed. Eng. 56 (2009) 1491–1501. [33] K.A. DeBruin, W. Krassowska, Modeling electroporation in a single cell. I. Effects of field strength and rest potential, Biophys. J. 77 (1999) 1213–1224. [34] S.A. Freeman, M.A. Wang, J.C. Weaver, Theory of electroporation of planar bilayer membranes: predictions of the aqueous area, change in capacitance, and pore-pore separation, Biophys. J. 67 (1994) 42–56. [35] T.R. Gowrishankar, A.T. Esser, Z. Vasilkoski, K.C. Smith, J.C. Weaver, Microdosimetry for conventional and supra-electroporation in cells with organelles, Biochem. Biophys. Res. Commun. 341 (2006) 1266–1276. [36] T. Kotnik, D. Miklavˇciˇc, T. Slivnik, Time course of transmembrane voltage induced by time-varying electric fields – a method for theoretical analysis and its application, Bioelectrochem. Bioenerg. 45 (1998) 3–16. [37] N. Hu, X. Zhang, J. Yang, S.W. Joo, S. Qian, A cell electrofusion microfluidic chip with micro-cavity microelectrode array, Microfluid. Nanofluid. 15 (2013) 151–160. [38] H. Pauly, H.P. Schwan, Impendance of a suspension of ball-shaped particles with a shell; a model for the dielectric behavior of cell suspensions and protein solutions, Zeitschrift für Naturforschung, Teil B: Chemie. Biochemie. Biophysik. Biologie. 14 B (1959) 125–131.
Biographies Xiaoling Zhang received her Ph.D. in Biomedical Engineering from Chongqing University in 2015. She is a postdoctoral fellow in Key Laboratory of Optoelectronic Technology and System, Ministry of Education, and a researcher in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. Her current research interests include bio-MEMS and microfluidics. Ning Hu is an associate professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from Chongqing University in 2010. His research focuses on bio-MEMS, biomicrofluidics, lab-on-a-chip technology, and cell electrofusion. Xi Chen is a Ph.D. student in Bioengineering College, Chongqing University. He obtained his master’s degree in Biomedical Engineering from Chongqing University in 2015. His research focuses on magnetoelastic sensors and microfluidics. Ting Fan defended her BS in Biomedical Engineering at Chongqing University in 2016. Her research focuses on lipid vesicles, microfluidics. Zhenyu Wang is a faculty in College of Biomedical Engineering of Chongqing Medical University, She obtained her Ph.D. from Chongqing University in 2013. Her research focuses on lipid vesicles, microfluidics, bio-MEMS, and ultrasound. Xiaolin Zheng is a professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from Chongqing University in 1995. His research focuses on bio-MEMS, biomedical devices. Jun Yang is a professor in Key Laboratory of Biorheological Science and Technology, Ministry of Education, in Chongqing University. He obtained his Ph.D. from City University of Hong Kong in 2004. His research focuses on bio-MEMS, microfluidics, and biosensors. Shizhi Qian received his Ph.D. in Mechanics and Applied Mechanics from University of Pennsylvania in 2004. He is an associate professor in Department of Mechanical and Aerospace Engineering at the Old Dominion University. His current research interests include electrokinetics, and microfluidics and nanofluidics.