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Method for Electric Parametric Characterization and Optimization of Electroporation on a Chip Mengxi Wu,∥,† Deyao Zhao,‡,† Zewen Wei,§ Wenfeng Zhong,∥ Hao Yan,∥ Xiaoxia Wang,‡ Zicai Liang,*,‡ and Zhihong Li*,∥ ∥

National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Institute of Microelectronics, Peking University, Beijing 100871, China ‡ Institute of Molecular Medicine, Peking University, Beijing 100871, China § National Center for Nanoscience and Technology, Beijing 100190, China S Supporting Information *

ABSTRACT: We have developed a rapid method to optimize the electric parameters of cell electroporation. In our design, a pair of ring-dot formatted electrodes was used to generate a radial distribution of electric field from the center to the periphery. Varied electric field intensity was acquired in different annulus when an electric pulse was applied. Cells were cultured on the microchips for adherent cell electroporation and in situ observation. The electroporation parameters of electric field intensity were explored and evaluated in terms of cell viability and transfection efficiency. The optimization was performed in consideration of both cell viability, which was investigated to decrease as electric field increases, and the transfection rate, which normally increases at stronger electric field. The electroporation characteristics HEK-293A and Hela cells were investigated, and the optimum parameters were obtained. Verified by a commercial electroporation system as well as self-made microchips endowed the optimization with wider meaning. At last, as applications, we acquired the optimal electroporation pulse intensity of Neuro-2A cells and a type of primary cell (human umbilical vein endothelial cell, HUVEC) by one time electroporation using the proposed method.

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hand, irreversible breakdown of cell membrane (cell lysis) is also strongly dependent on the electric field.17 Many researchers have investigated the transfection efficiency and cell viability under various electric conditions and came up with the optimization by using commercial electroporation systems,18,19 microchips,11,20,21and other proposed systems.22,23 While optimization remains to be hard work as electroporation differs greatly among cell types even different batches of the same cell type. Optimization is achieved mainly by exhaustivity of electric parameters in most studies. Recently, J. A. Kim et al.24 proposed an easy optimization method of electrical parameters for electroporation by using a multichannel electroporation chip formed by PDMS. Moreover, M. J. Kim et al.25 proposed a simpler device, consisting of a single channel with multiple electric field zones. However, the situation of electroporation in the constrained microchannels is different from the one on an electroporation chip or in a cuvette, so is the following characterization procedure. In addition, only five different electric field zones can be achieved in one experiment. On the other hand, microchips without using microchannels are also suggested; for example, T. Jain and J. Muthuswamy26 developed a chip using a microelectrode array to generate

lectroporation has been widely studied and used as an effective transfection method in gene delivery, molecular therapies, and other biology engineering fields. The transport of DNA across cell membranes is enhanced greatly when cells are exposed to a proper electric field.1 The dramatic permeabilization is believed as a result of the reversible structure breakdown of cell membrane and transient hydrophilic nanopores induced by electric field.2 Once the primary hydrophilic pathways are formed, the expansion in population and location is engendered rapidly by the interaction of both external membrane charging and internal membrane discharging.3 Through these pores, foreign macromolecules such as genes, drugs, antibodies, and other reagents can be introduced into cells predominantly by electrical drift.4 It is generally believed that the efficiency of electroporation is greatly influenced by several electrical parameters, such as electric pulse amplitude, duration, and number of pulses.5−11 Moreover, several studies indicate the orientation of the electric field of great importance especially for ellipsoidal complex cells.12,13 Among these parameters, pulse amplitude, which determines the electric field intensity applied to cells, plays the most important role in the formation of pores. In most cases, electroporation is characterized as a threshold phenomenon, which means the rearrangement of the molecular structure of the membrane occurs and transient pores form only when the transmembrane voltage induced by electric field exceeds a certain threshold (typically 200 mV−1 V).4,14−16 On the other © 2013 American Chemical Society

Received: January 2, 2013 Accepted: April 2, 2013 Published: April 2, 2013 4483

dx.doi.org/10.1021/ac400017x | Anal. Chem. 2013, 85, 4483−4491

Analytical Chemistry

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localized varied electric fields. However, the scale of electrode array is still limited, and each pixel of the array is too small and contains too few cells for reliable statistics. Olofsson et al.27 developed a purpose-made capillary electrode to generate a high electric field close to the interface by field focusing effects. The radially varied electric field was used to guarantee that a part of the cells was electroporated. Equipped with a computer-controlled scanning system, the capillary electrode was scanned over a cell culture to acquire specific patterns of electroporated cells.28 Although it is an excellent method to generate a focused electric field pattern, their method is not suitable for electroporation parameter optimization and characterization. In their system, the electric field is focused, which means that the electrical field distributes with a steep slope and the distribution is sensitive to the gap between the capillary electrode and dielectric surface. While for parameter characterization and optimization applications, the distribution of the electrical field should be with fixed and smooth gradients, which are important for reliable statistics. In this study, we propose a rapid and effective optimization method by using a ring-dot formatted microchip. A pair of electrodes consists of a ring electrode and a dot electrode in the center in order to generate radially distributed electrical field. A fixed and smooth gradient of electric field is acquired when electric pulses are applied. Therefore, adherent cells on the same microchip are exposed to varied electric field intensity under one pulse voltage. Besides, in situ observation can be easily realized using the proposed microchip according to our electroporation procedures. The transfection rate and cell viability under varied electric field are evaluated by a combined staining method. The optimum value of electric field intensity is obtained according to the statistics. By using this method, characterization and optimization of HEK-293A, Hela, Neuro2A, and HUVEC cells have been performed. The results demonstrate that the cell viability and the transfection rate exhibit obvious different dependence on electric field intensity among these four types of cells. The annular-interdigitated electroporation microchip, which was proved efficient for cell electroporation in our previous work,29 has been utilized to verify the conclusion. The optimal parameters are proved also effective for commercial electroporation systems, such as Eppendorf Multiporator electroporation system.

compared to pulse duration (usually between several microseconds and milliseconds). Therefore eq 1 can be reduced as Um = 1.5rcellE cos θ

(3)

for most conditions to describe the transmembrane potential. The transmembrane potential which plays an important role on the transfection efficiency and cell viability is determined by external electric field. According to eq 3, it is clear that primary destabilization of a cell membrane occurs only at the areas of the cell membrane facing the electrodes where θ is close to 0 or π, which has been observed in experiments.32 Generally, the electric field intensity affects the cell in two ways: 1) to trigger electroporation when it is larger than a critical value for the membrane destabilization to appear and 2) to enlarge the geometry of the part of the cell membrane that is permeabilized, when it keeps increasings.33 However, the referenced electric field intensity for either effective electroporation or destructive damages to cell membrane varied in different publications.4,14−16 The optimized condition of electric field intensity for electroporation still needs to be achieved by experimental statistics.

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MATERIALS AND METHODS Design and Fabrication of Microchips. The detail design of a ring-dot formatted electroporation microchip and the corresponding simulation of electric field distribution are illustrated in Figure 1. The microchip consisted of a glass

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THEORETICAL BASIS When a spherical cell with a nonconductive membrane is exposed to an external electric field, the transmembrane potential Um is described as the equation below30 Um = 1.5rcellE cos θ(1 − exp(−t /τm))

Figure 1. (A): The photo of a single ring-dot formatted microchip and the schematic view of manufacture process. (B): The corresponding simulation of electric field distribution and the close-up cross section with z axis exaggerated. (C): The distribution curve of electric field intensity along the radius of ring.

(1)

where rcell is the radius of cell, E is the external electric field intensity, θ is the angle between the position where Um is measured and the direction of electric field, and τm is the charging time of the cell membrane. The charging time of the cell membrane can be described as31 τm = rcellCm(1/σi + 1/(2σe))

substrate and a pair of microelectrodes on the surface. More specifically, a 4-in. Pyrex7740 glass wafer was used as substrate, since it has high resistivity and transparency. A chrome layer with 30 nm thickness was sputtered on the substrate as adhesive material. Gold was used as electrode because of high biocompatibility as well as high conductivity and good chemical stability. The total thickness of chrome/gold layer was 330 nm. Then electrodes were patterned by photolithography and wet etching. The radius of dot electrode was 800 μm. The inner and

(2)

where Cm is the membrane capacitance, and σi and σe are intracellular conductivity and conductivity of the external medium, respectively. For most mammalian cells, the charging time is quite short (about several hundreds of nanoseconds31) 4484



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mm cuvette, 150 μL of the mixture was added, and then pulses were applied. Immediately after electroporation, the electroporated mixture was transferred to a 96-well plate (20 μL each well), and then 200 μL of cell culture medium was added to each well. Cells, Plasmids, and Staining. HEK-293A, Hela, Neuro2A, and HUVEC cells were used as target cells. The first three types of cells were originally purchased form the Chinese Academy of Medical Science and then were cultured by ourselves in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin (Life Technologies, Gibco). We are grateful to Dr. Jincai Luo from the Institute of Molecular Medicine of Peking University for providing HUVECs. HUVECs were maintained in M199 culture medium supplemented with 20% FBS (Hyclone), antibiotics, 5 ng/mL FGF (Sigma-Aldrich), and 80 mg/mL heparin (Sigma-Aldrich). All the cells were cultured in a 5% CO2 high-humidity atmosphere at 37 °C. Transfection efficacy of plasmid DNA was determined by using pEGFP-C3 plasmid encoding an enhanced green fluorescent protein (Clontech). Purifications of plasmids were performed using an EndoFree Plasmid Maxi Kit (Qiagen, German). To evaluate the transfection rate and the survival rate, a combined staining method was used before observation. PI (Propidium iodide) and Hoechst (bisBenzimide H 33342) staining dye (both purchased from Sigma-Aldrich) were added. Cells whose membranes were not intact were identified by PI exclusion staining, while Hoechst staining was used to identify the total cells remained. Image Processing and Analysis. The fluorescent images were processed and analyzed by using Image-Pro Plus (Media Cybernetics Inc., USA) image analysis software. To acquire the performance of the whole area, the visual field was scanned, and then images were tiled according to the position. In the statistics process, the area of a ring-dot formatted microchip was divided into several radial annulus rings. The radius of considered rings increased in a step of 100 μm. Fluorescent spots of GFP, PI, and Hoechst, which meant transfected cells, necrotic cells, and total cells, respectively, were collected and counted. The image processing drew a salutary lesson from the method proposed by R. J. Blatt et al.34 For each transfection condition, efficiency was evaluated by the number of GFPexpressing cells, while the viability of electroporation was obtained by comparing the number of living cells stained by Hoechst dye and excluded by PI staining between treated and control samples. The survival rate and the transfection rate were obtained as the equations below:

outer radius of ring electrode was 2800 and 3100 μm, respectively. At last, the patterned wafer was incised to microchips whose sizes were 15 mm × 15 mm. The entire manufacture was a standard MEMS machining process. The pair of electrodes consisted of a ring electrode and a dot electrode in the center of the ring. The radius of central dot electrode and the spacing between two electrodes were designed as 800 μm and 2 mm, respectively. By using the ring-dot formatted electrodes, a specific distribution of electric field was acquired. As shown in Figure 1B, a radial gradual electric field was generated in the area between ring-electrode and central dot-electrode because of varied curvatures of different ring annulus. The electrical simulation was performed using COMSOL Multiphysics ver. 3.5a (COMSOL Inc.). A buffer layer with 80 μm thickness whose conductivity was 3.5 mS/cm was considered in simulation. The close-up cross section view with z axis exaggerated demonstrated annular cylindrical distribution of electric field with good longitudinal uniformity throughout the buffer layer. Figure 1C shows radial distribution of electrical field, with a gradient from about 2.5 × 105 V/m to 0.7 × 105 V/m when 250 V voltage is applied. Abrupt changes near 0.8 mm and 2.8 mm are caused by the edge effects of electrodes. Experimental Procedures. Figure 2 shows the schematic view of adherent cell electroporation procedures. Cultured cells

Figure 2. Procedures of adherent cell electroporation.

were harvested and resuspended to a density of 4 × 105 cells/ mL in culture medium. For each chip, 200 μL of the mixture was loaded (that means 0.8 × 105 cells per chip) and cultured for twenty-four hours in an incubator for cells to adhere. Before electric pulses were applied, the adherent cells were washed by elctroporation buffer (25 mM KCl, 0.3 mM KH2PO4, 0.85 mM K2HPO4, 36 mM myo-inositol, pH 7.2, conductivity 3.5 mS/cm at 25 °C) two times. Then 10 μL of the plasmid-buffer mixture was dropped on a chip (with pEGFP-C3 plasmid added to a final concentration of 20 μg/mL). Pulses were generated by a BTX ECM830 stimulator (BTX, USA). Immediately after electroporation, 200 μL of cell culture medium was added onto each microchip. Twenty-four hours later, the transfection results were observed under a fluorescence microscopy (Olympus). To verify the optimal electric conditions, a commercial Eppendorf Multiporator (Eppendorf, Germany) was used. Cultured cells were also harvested and resuspended to a density of 3 × 106 cells/mL in electroporation buffer (with pEGFP-C3 plasmid added to a final concentration of 20 μg/mL). For a 2

survival rate = (total cell number − necrotic cell number) /(number of control group) × 100%

transfection rate = (transfected cell number) /(total cell number − necrotic cell number) × 100%

The experiment results were indicated using the survival rate and the transfection rate in two aspects together. The survival rate was clear to evaluate the damage and toxicity of electroporation; on the other hand, the transfection rate was present at the level of transfection efficiency among the survived cells. 4485



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Figure 3. Images of HEK-293A cells after electroporation (Pulse amplitude: 250 V, duration: 100 μs, pulse number: 3). (A): Fluorescent images of transfection cells. (B): DIC, bright field image; PI, fluorescent image of necrotic cells determined by PI staining; Merge, merged images of bright field, GFP and PI; Hoechst, fluorescent images of total cells remained after electroporation by Hoechst staining.

Figure 4. Statistics of HEK-293A cells electroporation efficiency. The relationships between the survival rate and the transfection rate and intensity of electric field (left). The variation trend of transfection cell amount with the intensity of electric field (right). The survival rate is determined by comparing the number of live cells between treated versus nontreated samples, and the transfection rate is acquired by comparing number of transfected live cells and number of total live cells.

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Electric Parameters. It is proposed that optimum electrical conditions cannot be determined by one parameter but by the combinations of various parameters, such as electric field, pulse duration, and number. To determine the effect of electric field intensity to cell electroporation, in all the followed experiments the duration and number of pulses were fixed as 100 μs and 3, respectively, which were proved optimum values for a various types of cells in our other work (as shown in Supplementary Figure 1). Besides, several publications19,35 indicate the same parameters for a wider range. More efficiently, fine-tuning of electric parameters could be carried out after optimization of electric field intensity.

RESULTS AND DISCUSSION Plasmid Electroporation of HEK-293A Cells. Electric pulses with the amplitude of 250 V were applied, and twentyfour hours after electroporation, the performance was observed and evaluated, as illustrated in Figure 3. Cells in the peripheral annulus were less successfully transfected than the interior annulus, and considerable successfully transfected cells present a green ring due to the radial gradual electric field distribution (as shown in Figure 3A). On the other hand, interior annulus was exposed to a stronger electric field, which led to serious cell mortality and destructive lysis. According to DIC and Hoechst fluorescent view, an obvious dead ring appeared around the dot electrode since cells were destructively damaged by electric pulses and broken up with no remains after twenty-four hours. 4486



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Figure 5. Images of HEK-293A cells after electroporation. (A): Bright field and green fluorescent images of cells after electroporation using Eppendorf cuvettes. (B): Bright field and fluorescent images after adherent electroporation using annular-interdigitated formatted microchips. (C): Images of suspension electroporation under optimal condition using annular-interdigitated formatted microchips.

commercial Eppendorf electroporation system were used to validate the electroporation characteristics and the optimization conditions of HEK-293A. The results of electroporation using an Eppendorf cuvette are shown in Figure 5A. When 120 V voltage (for a 2 mm cuvette, the electric field intensity was 6 × 104 V/m) was applied, cells were slightly damaged, but only a small part of total cells are transfected. When 240 V (about 1.2 × 105 V/m) were applied, the amount of successfully transfected cells increased obviously. Cells were seriously damaged under 340 V (about 1.7 × 105 V/ m). The transfection rate under optimum electroporation conditions using a commercial cuvette was about 70%, while the survival rate was around 30%. The performances of microchips have been proved to be better than traditional electroporation devices since the electrodes can be designed refined and smart to eliminate many potential adverse effects associated with macroscale environments.36 An annular-interdigitated formatted microchip was proposed in our previous study for its excellent performance. Hence high cell viability and a high transfection rate were acquired (Figure 5B). When 30 V voltage (6 × 104 V/m) was applied, the survival rate and the transfection rate were 73% and 29%, respectively. When 60 V (about 1.2 × 105 V/m, the optimum electric field intensity) was applied, the amount of successfully transfected cells reached a peak value, more precisely, the survival rate was 53% and the transfection rate was as high as 95%. Cells were seriously damaged, and the survival rate was 22% under 84 V (about 1.7 × 105 V/m), though the transfection rate could be as high as 91%.

Varied voltages were also applied in our experiment, and consistent results were observed under all conditions (shown in Supplementary Figure 2). Electroporation Characteristics of HEK-293A Cells. The transfected ring and dead ring were owing to the specific distribution of electric field produced by a ring-dot formatted microchip. Thus the transfection efficiency and cell mortality both varied with the intensity of electric field. To identify the electroporation characteristics dependence on electric field, quantitative analysis was carried out, illustrated in Figure 4. Briefly, cell lysis played a more dominant role when electric field becomes stronger. Over 50% of the total cells were damaged and broken up when the electric field intensity was around 1 × 105 V/m. On the other hand, the transfection rate increased with electric field intensity increasing first, but saturated with a value of 65%, when electric field intensity was higher than 1.3 × 105 V/m. Considering both the survival rate and the transfection rate, the amount of successfully transfected cells reached the peak value when electric field intensity is about 1.2 × 105 V/m. The lower electric field was not effective for enough transfection, while cell lysis reduced the cell viability when the electric field exceeded this intensity. Therefore the optimized intensity was around 1.2 × 105 V/m in order to realize the most transfection cell. The statistics also indicated the electric field intensity for HEK-293A electroporation should not be stronger than 1.4 × 105 V/m. Verify the Optimal Conditions of HEK-293A Cells. With the guidance indicated by ring-dot formatted microchips, selfmade annular-interdigitated formatted microchips and a 4487



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Figure 6. (A): Images of Hela cells after electroporation (pulse amplitude: 300 V, duration: 100 μs, pulse number: 3). (B): Statistics of Hela cells electroporation efficiency. The survival rate is determined by comparing the number of live cells between treated versus nontreated samples, and the transfection rate is acquired by comparing number of transfected live cells and number of total live cells. (C): Optimal adherent electroporation results using annular-interdigitated formatted microchips (pulse amplitude: 75 V, duration: 100 μs, pulse number: 3).

Figure 6C shows the transfection results under an optimum electric field, which means the amplitude of electric pulses is 75 V for an annular-interdigitated formatted chip. Optimization of Neuro-2A and HUVEC Cells. In this study, we proposed a rapid optimization method of electroporation parameters. As application, we used the method to find the optimal electric field intensity of Neuro-2A and primary cell (human umbilical vein endothelial cell, HUVEC). The optimized electrical field was determined by one time electroporation. The results are shown in Figure 7 and Figure 8, respectively. Neuro-2A cells were applied with 150 V in a ring-dot formatted microchip electroporation as they were less tolerant to electric field. Pulse duration and pulse number were fixed as 100 μs and three. Bright field and fluorescent field images are shown as Figure 7A. Cells located nearer to the central dot electrode were damaged more seriously, thus fewer cells survived, while successfully transfected cells which were GFP-expressing were observed more near to the center of a ring-dot formatted microchip. The survival rate and the transfection rate were evaluated (illustrated in Figure 7B). The survival rate decreased from 50% to about 15% as the intensity of electric field increased from 4 × 104 V/m to over 1.1 × 105 V/m. The transfection rate experienced an increase from 5% to about 20% and decreased after electric field exceeded 1 × 105 V/m. According to the statistics, we chose 8 × 104−9 × 104 V/m as the optimum spectrum of electric field, and optimized electroporation was done using an annulus-interdigitated formatted microchip under 45 V electric pulses (the

The optimum electroporation condition was found to be applicable to suspension electroporation which was more convenient for biology studies and applications. High transfection efficiency (the transfection rate was more than 90%) was acquired (Figure 5C) under the optimum condition, that was 60 V for annular-interdigitated formatted microchips. Electroporation of Hela Cells. To test the feasibility of the ring-dot formatted microchip optimization method, Hela cells were thus electroporated and studied. Transfection results and the analysis are shown in Figure 6. Figure 6A shows the transfection of Hela using a ring-dot formatted microchip. Hela cells were found to be less sensitive to electric field. Electric pulses with the amplitude of 300 V were applied. Similar with HEK-293A electroporation results, a dead ring was observed obviously around the dot electrode, and peripheral cells were less successfully transfected than interior ones. The survival rate and the transfection rate were evaluated by the same method of HEK-293A electroporation. Figure 6B indicates the statistics of the survival rate and the transfection rate. In general, Hela was more electrically stable than HEK293A. When the electric field increased to as strong as 1 × 105 V/m, the survival rate still kept more than 60% yet (for HEK293A the survival rate fell to a level at about 50%). A small part of the cells, representing about 15%, could survive even under an extreme condition that electric field intensity was as high as 2 × 105 V/m. The transfection rate increased from about 10% to 80%. The amount of successfully transfected cells reached the peak value when electric field intensity was about 1.5 × 105 V/m, which was regarded as an optimum electric condition. 4488



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Figure 7. (A): Images of Neuro-2A cells after electroporation (pulse amplitude: 150 V, duration: 100 μs, pulse number: 3). (B): Statistics of Hela cells electroporation efficiency. Optimal (C) suspension and (D) adherent electroporation results using annular-interdigitated formatted microchips (pulse amplitude: 45 V, duration: 100 μs, pulse number: 3).

Figure 8. (A): Images of HUVEC cells after electroporation (pulse amplitude: 350 V, duration: 100 μs, pulse number: 3). (B): Statistics of Hela cells electroporation efficiency. (C): Optimal suspension electroporation results using annular-interdigitated formatted microchips (pulse amplitude: 80 V, duration: 100 μs, pulse number: 3).

corresponding intensity of electric field was about 9 × 104 V/ m), shown as Figure 7C and Figure 7D. For suspension electroporation, the cell-buffer mixture was loaded on each chip to an excess density in consideration of the loss of quantity induced by the damage of electroporation. HUVEC cells were found to need a high voltage for electroporation. We used 350 V for a ring-dot formatted microchip to determine the optimal electric field. The results are shown as Figure 8A. However, as high voltage was applied, cells were obviously damaged especially near to the dot

electrode. According to the statistics (shown in Figure 8B), the survival rate experienced a dramatic fall from about 50% at 1 × 105 V/m to below 20% at 1.2 × 105 V/m, followed by a gentle decline to about 5% as the electric field intensity rose to about 2.8 × 105 V/m. On the other hand, the transfection rate soared to about 30% at 1.2 × 105 V/m and rose slowly till about 2.2 × 105 V/m. Rapid fluctuation happened over that point mainly because cell mortality reduced the sample size of statistics. In consideration of both the survival rate and the transfection rate, we chose 1.5 × 105−2 × 105 V/m as the optimal spectrum of 4489



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electric field. As the survival rate within the optimal spectrum was at a quite low level (about 10% or even below), the original amount of cells on each chip should be excessive (about 1 × 105 cells per chip) in order to acquire enough of an amount of survival and transfected cells. The optimum electroporation was done using annulus-interdigitated formatted microchips under 80 V electric pulses (the corresponding intensity of electric field was about 1.6 × 105 V/m), as shown in Figure 8C.

ACKNOWLEDGMENTS We are grateful to Dr. Jincai Luo from Institute of Molecular Medicine of Peking University for providing HUVECs. This work was supported by the National Natural Science Foundation of China (Grant No. 61176111).

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1 shows the dependence of transfection efficiency on electric pulse duration and number. The data indicate the optimal duration and number of electric pulses are 100 μs and three, respectively. Supplementary Figure 2 shows the images of HEK-293A cells after ring-dot electroporation treatment when electric pulses applied are 200 V, 250 V, and 300 V. Consistent results were observed under varied conditions. On the other hand, transfection ring and dead ring extended as the electric field increases. Supplementary Figure 3 shows the comparisons of the electroporation characteristics of HEK-293A, Hela, Neuro-2A, and HUVEC cells, evaluated by the survival rate, the transfection rate, and transfected cell density. Different types of cell exhibited varied characteristics. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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CONCLUSIONS Despite a number of methods have been established to optimization of the conditions of cell electroporation, most of the methods are based on exhaustivity, which means parallel experiments are carried out under various selected conditions. As a consequence, finding the optimal electric parameters is costly and time-consuming. In this study, we proposed a rapid method for the optimization of electroporation parameters by using a ring-dot formatted microchip. This device provided continuously varied electric field so that varied electroporation conditions can be achieved on one electroporation chip. Therefore, the optimized electric field could be determined by one time electroporation. By this method, the electroporation conditions of Neuro-2A and HUVEC were acquired, which were of significant meanings especially the latter one. We also acquired the electroporation characterization of HEK-293A, Hela, Neuro-2A, and HUVEC cell lines. The survival rate and the transfection rate were evaluated, and the dependence on the electric field intensity was presented. According to these data, different cell lines exhibited discrepant characteristics (shown in Supplementary Figure 3). Guidance and prediction could be obtained in accordance with these experimental results.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Z. Liang) and [email protected] (Z. Li). Author Contributions †

These authors contributed equally to the work.

Notes

The authors declare no competing financial interest. 4490



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