Microchip Electroporation

Tao Geng and Chang Lu*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
*Department of Chemical Engineering, Virginia Tech, Blacksburg, VA 24061, USA.

Electroporation, also termed as electropermeabilization, is a robust physical method for breaking cell membranes to either release intracellular components out of cells or introduce exogenous molecules into cells.1-3 When an electric field is applied to a cell, cell membrane charges like a capacitor, as it has several orders of magnitude higher conductivity than cytoplasm and extracellular medium. Thus, a potential difference is formed across the intact cell membrane within an extremely short charging time (on the scale of microseconds). As the transmembrane potential (ΔψE) exceeds a critical threshold (typically 0.2 to 1 V irrespective of cell types), electroporation occurs with nanoscale eletropores created within the plasma membrane via the localized structural rearrangements of lipid molecules.1,2 Assuming that the cell is a sphere and the membrane charging time is much shorter than the electric field duration, the ΔψE induced by an external electric field can be described using the following equation2,4:

ΔψE=0.75 g(λ)aE cosθ(1)

where g(λ) is a function of the conductivities of cytoplasm, cell membrane and extracellular buffer, a is the cell diameter, E is the electric field intensity, and θ is the angle between the direction of the electric field E and the normal from the cell center to a point on the membrane surface.

Depending on the degree of the conformational changes in the membrane structure, electroporation can be reversible or irreversible. If the electric pulse amplitude/duration is not too high/long, the pores are transient and can rapidly reseal after the removal of the field, ensuring that the cells remain viable. Reversible electroporation has been widely applied to the delivery of diverse foreign molecules into cells, including small-molecule fluorescent dyes, drugs, oligonucleotides, DNA, RNA, peptides, proteins, and nanoparticles etc.5-8 When the electric pulses are highly intensive, excessive electroporation leads to the generation of permanent pores and cell death, followed by rapid release of intracellular molecules due to electrophoretic forces. Irreversible electroporation has been used to selectively kill target cells for cell enrichment9,10, inactivation of microorganisms in food products11, and release intracellular materials (e.g., DNA, RNA, enzymes, and other cytoplasmic proteins) for cell analysis or the production of therapeutic proteins12-14.

Conventional electroporation is implemented by exerting short electric pulses of defined duration and intensity to a cuvette with electrodes fabricated out of aluminum, stainless steel, platinum or graphite, and arranged in a plate-to-plate manner.15 A pulse generator such as special capacitor discharge equipment is required to generate high-voltage pulses. By tuning the electric parameters (e.g., electric pulse amplitude, duration, frequency and shape), electroporation efficiency and cell viability (specially for molecular delivery) can be optimized.16 Although traditional electroporation systems have been widely used, they require high voltage input and suffer from adverse environmental conditions such as electric field distortion, local pH variation, metal ion dissolution and excess heat generation, leading to low electroporation efficiency.

Due to its physical nature, electroporation can be easily miniaturized on microfluidic platforms. Microfluidic electroporation systems overcome many drawbacks of bench-scale electroporators owing to its unique characteristics of miniaturization and integration.17-19 First, microfluidic electroporation devices are fabricated with standard microfabrication techniques, and hence a wide spectrum of microelectrodes can be incorporated into the microchips to generate the field necessary for electroporation. By shrinking the distance between microelectrodes to a few tens of micrometers20-22 or creating physical constraints with subcellular dimensions23-25, the required voltage is dramatically decreased to a few volts. Second, the electroporation microchips provide relatively more uniform electric field distribution, favorable fluidic and chemical environment, and rapid heat dissipation in small-volume microstructures. Third, individual cells could be manipulated on chips to probe single-cell dynamics and identify cell heterogeneity. The miniaturization of the systems also makes them very suitable for assays involving rare cell populations and expensive reagents due to the substantially reduced sample consumption. Fourth, the flow-through format of the microfluidic electroporation also allows the continuous processing of large cell populations with high throughput to obtain statistically meaningful results at the single-cell level or produce in a large volume.23,25-29 Fifth, the utilization of transparent materials (e.g., polydimethylsiloxane and glass) for microchips allows in situ observation and real-time monitoring of electroporation process using fluorescent probes, which facilitates the exploration of electroporation mechanism. Finally, microfluidic electroporation can be integrated with other analytical processes such as dielectrophoresis, electrophoresis, electroosmosis and hybridization to implement a total analysis analytical system, which is especially critical to of intracellular content analysis.30-44 Based on the strategies used to facilitate electroporation at the microscale, microfluidic electroporation methods are divided into five categories: (1) electrode incorporation and configuration, (2) channel geometry variation and constriction structures, (3) hydrodynamics-enhanced electroporation, (4) compartmentalized electroporation, and (5) other miscellaneous methods. Figure 1 demonstrates representative microfluidic electroporation methods.

Figure 1. Schematic of microfluidic electroporation methods. (a) Electrode incorporation and configuration: interdigitated 2D planar electrodes incorporated in a microchamber; (b) Channel geometry variation and constriction structures: a fluidic channel comprising a number of alternating wide and narrow sections where cells undergo electroporation while flowing through the narrow section; (c) Hydrodynamics-enhanced electroporation: hydrodynamic focusing for single-cell electroporation; (d) Compartmentalized electroporation: single-cell electroporation within a microfluidic droplet surrounded by oil.

The most straightforward way to implement electroporation at the microscale is constructing microelectrode structures inside simple microchannels or microchambers. The design of the microelectrodes is therefore of crucial importance for the electroporation process, because the geometry defines the electric field distribution and uniformity and hence greatly affects the electroporation efficiency. The electrodes utilized in microfluidic electroporation can be generally sorted into four groups: (1) parallel plate electrodes, (2) coplanar electrodes, (3) 3D electrodes, and (4) wire electrodes. Parallel plate electrodes that mimic the architecture of commercial electroporation systems is one of the simplest arrangements.45-54 In such systems, the microchannel is sandwiched between two substrates coated with various electrodes such as gold, aluminum, stainless steel and indium tin oxide, and therefore the electric field is uniformly distributed in between the space of the two electrodes. The metal electrodes could also be modified with high-aspect-ratio nanoscale structures to locally focus electric field intensity to the surface of the cell membranes.48,49,51,55 Cell suspension can be continuously flowed through the channel, but cells cannot be visualized in real-time when metal electrodes are involved. Microelectrodes are also often arranged in coplanar configuration due to the convenience in the microfabrication and visualization. However, the electric field distribution is more complex in such architecture, and hence a variety of electrode geometries have been proposed to obtain optimal electric fields for electroporation. The microelectrode geometries include parallel strip electrodes56-62, interdigitated electrodes in rectangular33-35,63-70, castellated37,44, circular38-40, curved71 or saw-tooth32,42,43 formats, and circular/square electrode arrays30,31,72-77. Combined with microchannel or microchamber structures, the systems could process either suspended or adherent cells. However, 2D planar electrodes suffer from non-uniform electric field distribution and high possibility of decaying away from the metal layers due to water electrolysis. Therefore, 3D electrodes including vertical sidewall electrodes36,78-84, electrode arrays in the form of pillars85,86, needles87,88 or nails89,90, and “liquid electrodes”91-94 have been reported to address these challenges in spite of the complexity in manufacturing. Although microelectrodes offers diverse forms of electric fields on-chip, it is still beneficial to use macroscale wire electrodes by simply inserting them in the open reservoirs connected to the microchannel network. Platinum wires and Ag/AgCl wires are two most commonly used electrodes because of their chemical stability.9,95-122

Constriction segments or structures in microchannels or microchambers can generate high localized electric fields, and thus electroporation occurs when cells of interest are flow through or positioned within such constricted regions. Due to the high electrical resistance of the small constriction regions, cells experience high field intensity in such configurations even when the overall input voltage is low. A simple fluidic channel comprising a number of alternating wide and narrow sections has been created for flow-through electroporation based on a constant voltage.9,103-106,108,109,112-114,116-120 Appropriate combination of the voltage and the channel geometry yields high field intensity in the narrow sections that is beyond the electroporation threshold and low field intensity in the wide sections that does not affect membrane integrity. Cells undergo electroporation while flowing through the narrow section(s), and the duration of electroporation is determined by the residence time(s) in the narrow section(s). Having multiple narrow sections in the channel renders flowing cells subject to field variations equivalent to multiple pulses. Alternatively, the constriction segments of a channel could also be constructed by reducing the depth of a segment of the channel.123,124 Moreover, microscale holes and channels with subcellular dimensions have been used for the entrapment and electroporation of single or multiple cells.27,125-136 More recently, nanoscale structures such as nanopores, nanostraws, and nanochannels have been implemented to provide even smaller constriction features and offer higher precision and efficiency for electroporation.137-144

Microfluidics creates favorable hydrodynamic conditions for cell manipulation in fluidic networks, which has been harnessed to enhance the performance of microfluidic electroporation.115,119,145-149 For instance, a spiral-shaped electroporation channel enabled the system to generate permeabilization over the whole cell membrane surface, thereby substantially enhancing the electroporation efficiency without compromising the cell viability.115 Also, hydrodynamic focusing was incorporated to facilitate single cell electroporation.119,145

Confining cells into tiny reaction volumes offers a number of advantages for electroporation including increased contact between the cell and the delivered molecule as well as potential for single cell/molecule screening and analysis. Microfluidics provides an ideal platform for the spatiotemporally regulated electroporation processes in the form of either droplets60,150-154 or microwell arrays31,59,69,71,155-157. These formats are especially suitable for single-cell electroporation.

Other than the abovementioned technologies, miscellaneous electroporation methods have been developed on chips based on microvalves107, salt bridges158 and light29,159,160.

In conclusion, the fusion of microfluidics with electroporation has created a myriad of versatile tools for analyzing intracellular contents and delivering foreign molecules into cells at microscale. Microfluidic electroporation allows more precise control over the process and more flexible integration with other tools than their bench-scale counterpart. Additionally, microfluidic platform provides unique opportunities for incorporating hydrodynamic effects into electroporation-based processes, and these effects often produce results that are otherwise not achievable. These exciting breakthroughs will benefit biotechnology and life science in broad realm including cell therapy, electrochemotherapy, large-scale functional screening of genome, and cell biology studies.


  1. 1. Weaver, J.C. & Chizmadzhev, Y.A. Theory of electroporation: A review. Bioelectrochem. Bioenerg. 41, 135-160 (1996).
  2. 2. Teissie, J., Golzio, M. & Rols, M.P. Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge. Biochim. Biophys. Acta 1724, 270-280 (2005).
  3. 3. Geng, T. & Lu, C. Microfluidic electroporation for cellular analysis and delivery. Lab Chip 13, 3803-3821 (2013).
  4. 4. Ho, S.Y. & Mittal, G.S. Electroporation of cell membranes: a review. Crit. Rev. Biotechnol. 16, 349-362 (1996).
  5. 5. Neumann, E., Schaefer-Ridder, M., Wang, Y. & Hofschneider, P.H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841-845 (1982).
  6. 6. Rols, M.P., et al. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat. Biotechnol. 16, 168-171 (1998).
  7. 7. Gothelf, A., Mir, L.M. & Gehl, J. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat. Rev. 29, 371-387 (2003).
  8. 8. Andre, F. & Mir, L.M. DNA electrotransfer: its principles and an updated review of its therapeutic applications. Gene Ther. 11 Suppl 1, S33-42 (2004).
  9. 9. Bao, N., Le, T.T., Cheng, J.X. & Lu, C. Microfluidic electroporation of tumor and blood cells: observation of nucleus expansion and implications on selective analysis and purging of circulating tumor cells. Integr. Biol. 2, 113-120 (2010).
  10. 10. Eppich, H.M., et al. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nat. Biotechnol. 18, 882-887 (2000).
  11. 11. Jeyamkondan, S., Jayas, D.S. & Holley, R.A. Pulsed electric field processing of foods: a review. J. Food Prot. 62, 1088-1096 (1999).
  12. 12. Vitzthum, F., et al. Amplifiable DNA from gram-negative and gram-positive bacteria by a low strength pulsed electric field method. Nucleic Acids Res. 28, E37 (2000).
  13. 13. Ganeva, V., Galutzov, B. & Teissie, J. High yield electroextraction of proteins from yeast by a flow process. Anal. Biochem. 315, 77-84 (2003).
  14. 14. Oshima, T. & Sato, M. Bacterial sterilization and intracellular protein release by a pulsed electric field. Adv. Biochem. Eng. Biotechnol. 90, 113-133 (2004).
  15. 15. Potter, H. Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem. 174, 361-373 (1988).
  16. 16. Canatella, P.J., Karr, J.F., Petros, J.A. & Prausnitz, M.R. Quantitative study of electroporation-mediated molecular uptake and cell viability. Biophys. J. 80, 755-764 (2001).
  17. 17. Fox, M.B., et al. Electroporation of cells in microfluidic devices: a review. Anal. Bioanal. Chem. 385, 474-485 (2006).
  18. 18. Lee, W.G., Demirci, U. & Khademhosseini, A. Microscale electroporation: challenges and perspectives for clinical applications. Integr. Biol. 1, 242-251 (2009).
  19. 19. Movahed, S. & Li, D. Microfluidics cell electroporation. Microfluid. Nanofluid. 10, 703-734 (2011).
  20. 20. Lee, S.W. & Tai, Y.C. A micro cell lysis device. Sens. Actuators A 73, 74-79 (1999).
  21. 21. Lin, Y.-C. & Huang, M.-Y. Electroporation microchips for in vitro gene transfection. J. Micromech. Microeng. 542 (2001).
  22. 22. Lu, H., Schmidt, M.A. & Jensen, K.F. A microfluidic electroporation device for cell lysis. Lab Chip 5, 23-29 (2005).
  23. 23. Huang, Y. & Rubinsky, B. Flow-through micro-electroporation chip for high efficiency single-cell genetic manipulation. Sens. Actuators A 104, 205-212 (2003).
  24. 24. Munce, N.R., Li, J., Herman, P.R. & Lilge, L. Microfabricated system for parallel single-cell capillary electrophoresis. Anal. Chem. 76, 4983-4989 (2004).
  25. 25. Khine, M., Lau, A., Ionescu-Zanetti, C., Seo, J. & Lee, L.P. A single cell electroporation chip. Lab Chip 5, 38-43 (2005).
  26. 26. Huang, Y. & Rubinsky, B. Microfabricated electroporation chip for single cell membrane permeabilization. Sens. Actuators A 89, 242-249 (2001).
  27. 27. Khine, M., Ionescu-Zanetti, C., Blatz, A., Wang, L.P. & Lee, L.P. Single-cell electroporation arrays with real-time monitoring and feedback control. Lab Chip 7, 457-462 (2007).
  28. 28. Valero, A., et al. Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab Chip 8, 62-67 (2008).
  29. 29. Valley, J.K., et al. Parallel single-cell light-induced electroporation and dielectrophoretic manipulation. Lab Chip 9, 1714-1720 (2009).
  30. 30. Cheng, J., et al. Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nat. Biotechnol. 16, 541-546 (1998).
  31. 31. Xu, Y., Yao, H., Wang, L., Xing, W. & Cheng, J. The construction of an individually addressable cell array for selective patterning and electroporation. Lab Chip 11, 2417-2423 (2011).
  32. 32. Lee, S.-W. & Tai, Y.-C. A micro cell lysis device. Sens. Actuators A 73, 74-79 (1999).
  33. 33. Suehiro, J., Shutou, M., Hatano, T. & Hara, M. High sensitive detection of biological cells using dielectrophoretic impedance measurement method combined with electropermeabilization. Sens. Actuators B 96, 144-151 (2003).
  34. 34. Suehiro, J., Hatano, T., Shutou, M. & Hara, M. Improvement of electric pulse shape for electropermeabilization-assisted dielectrophoretic impedance measurement for high sensitive bacteria detection. Sens. Actuators B 109, 209-215 (2005).
  35. 35. Suehiro, J., Ohtsubo, A., Hatano, T. & Hara, M. Selective detection of bacteria by a dielectrophoretic impedance measurement method using an antibody-immobilized electrode chip. Sens. Actuators B 119, 319-326 (2006).
  36. 36. Lu, H., Schmidt, M.A. & Jensen, K.F. A microfluidic electroporation device for cell lysis. Lab Chip 5, 23-29 (2005).
  37. 37. Ramadan, Q., et al. Simultaneous cell lysis and bead trapping in a continuous flow microfluidic device. Sens. Actuators B 113, 944-955 (2006).
  38. 38. de la Rosa, C. & Kaler, K.V. Electro-disruption of Escherichia coli bacterial cells on a microfabricated chip. Conf. Proc. IEEE Eng. Med. Biol. Soc. 1, 4096-4099 (2006).
  39. 39. de la Rosa, C., Prakash, R., Tilley, P.A., Fox, J.D. & Kaler, K.V. Integrated microfluidic systems for sample preparation and detection of respiratory pathogen Bordetella pertussis. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2007, 6303-6306 (2007).
  40. 40. de la Rosa, C., Tilley, P.A., Fox, J.D. & Kaler, K.V. Microfluidic device for dielectrophoresis manipulation and electrodisruption of respiratory pathogen Bordetella pertussis. IEEE Trans. Biomed. Eng. 55, 2426-2432 (2008).
  41. 41. MacQueen, L.A., Buschmann, M.D. & Wertheimer, M.R. Gene delivery by electroporation after dielectrophoretic positioning of cells in a non-uniform electric field. Bioelectrochemistry 72, 141-148 (2008).
  42. 42. Sedgwick, H., Caron, F., Monaghan, P.B., Kolch, W. & Cooper, J.M. Lab-on-a-chip technologies for proteomic analysis from isolated cells. J. R. Soc. Interface 5 Suppl 2, S123-130 (2008).
  43. 43. Nakayama, T., Namura, M., Tabata, K.V., Noji, H. & Yokokawa, R. Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition. Lab Chip 9, 3567-3573 (2009).
  44. 44. Bahi, M.M., Tsaloglou, M.N., Mowlem, M. & Morgan, H. Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification. J. R. Soc. Interface 8, 601-608 (2011).
  45. 45. Lin, Y.-C., Jen, C.-M., Huang, M.-Y., Wu, C.-Y. & Lin, X.-Z. Electroporation microchips for continuous gene transfection. Sens. Actuators, B 79, 137-143 (2001).
  46. 46. Gong, X., et al. Wax-bonding 3D microfluidic chips. Lab Chip 10, 2622-2627 (2010).
  47. 47. Choi, Y., et al. A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells. Biomed. Microdevices 12, 855-863 (2010).
  48. 48. Shahini, M. & Yeow, J.T. Carbon nanotubes for voltage reduction and throughput enhancement of electrical cell lysis on a lab-on-a-chip. Nanotechnology 22, 325705 (2011).
  49. 49. Shahini, M. & Yeow, J.T. Cell electroporation by CNT-featured microfluidic chip. Lab Chip (2013).
  50. 50. Shahini, M., van Wijngaarden, F. & Yeow, J.T. Fabrication of electro-microfluidic channel for single cell electroporation. Biomed. Microdevices (2013).
  51. 51. Miyano, N., et al. Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy. Lab Chip 8, 1104-1109 (2008).
  52. 52. Yamauchi, F., Kato, K. & Iwata, H. Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer. Langmuir 21, 8360-8367 (2005).
  53. 53. Fujimoto, H., Kato, K. & Iwata, H. Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency. Anal. Bioanal. Chem. 397, 571-578 (2010).
  54. 54. Huang, K.S., Lin, Y.C., Su, K.C. & Chen, H.Y. An electroporation microchip system for the transfection of zebrafish embryos using quantum dots and GFP genes for evaluation. Biomed. Microdevices 9, 761-768 (2007).
  55. 55. Jokilaakso, N., et al. Ultra-localized single cell electroporation using silicon nanowires. Lab Chip 13, 336-339 (2013).
  56. 56. Lin, Y.-C. & Huang, M.-Y. Electroporation microchips for in vitro gene transfection. J. Micromech. Microeng., 542 (2001).
  57. 57. He, H., Chang, D.C. & Lee, Y.K. Micro pulsed radio-frequency electroporation chips. Bioelectrochemistry 68, 89-97 (2006).
  58. 58. Cheng, W., Klauke, N., Sedgwick, H., Smith, G.L. & Cooper, J.M. Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform. Lab Chip 6, 1424-1431 (2006).
  59. 59. Cheng, W., Klauke, N., Smith, G. & Cooper, J.M. Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes. Electrophoresis 31, 1405-1413 (2010).
  60. 60. Zhan, Y., Wang, J., Bao, N. & Lu, C. Electroporation of cells in microfluidic droplets. Anal. Chem. 81, 2027-2031 (2009).
  61. 61. Zhan, Y., et al. Release of intracellular proteins by electroporation with preserved cell viability. Anal. Chem. 84, 8102-8105 (2012).
  62. 62. Geng, T., Bao, N., Sriranganathanw, N., Li, L. & Lu, C. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform. Anal. Chem. 84, 9632-9639 (2012).
  63. 63. Lin, Y.C., Li, M., Fan, C.S. & Wu, L.W. A microchip for electroporation of primary endothelial cells. Sens. Actuators A 108, 12-19 (2003).
  64. 64. Lin, Y.C., Li, M. & Wu, C.C. Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement. Lab Chip 4, 104-108 (2004).
  65. 65. Jen, C.-P., Wu, W.-M., Li, M. & Lin, Y.-C. Site-specific enhancement of gene transfection utilizing an attracting electric field for DNA plasmids on the electroporation microchip. J. Microelectromech. Syst. 13, 947-955 (2004).
  66. 66. Li, M., Lin, Y.C. & Su, K.C. Using magnetic nanoparticles to enhance gene transfection on magneto-electroporation microchips. in Progress on Advanced Manufacture for Micro/Nano Technology 2005, Pt 1 and 2, Vol. 505-507 (eds. Jywe, W., et al.) 661-666 (2006).
  67. 67. Huang, K.S., Lin, Y.C., Su, C.C. & Fang, C.S. Enhancement of an electroporation system for gene delivery using electrophoresis with a planar electrode. Lab Chip 7, 86-92 (2007).
  68. 68. Yang, S.-C., Huang, K.-S., Chen, H.-Y. & Lin, Y.-C. Determination of optimum gene transfection conditions using the Taguchi method for an electroporation microchip. Sens. Actuators B 132, 551-557 (2008).
  69. 69. Kim, S.H., Yamamoto, T., Fourmy, D. & Fujii, T. Electroactive microwell arrays for highly efficient single-cell trapping and analysis. Small 7, 3239-3247 (2011).
  70. 70. Adamo, A., Arione, A., Sharei, A. & Jensen, K.F. Flow-through comb electroporation device for delivery of macromolecules. Anal. Chem. 85, 1637-1641 (2013).
  71. 71. Huang, H., et al. An efficient and high-throughput electroporation microchip applicable for siRNA delivery. Lab Chip 11, 163-172 (2011).
  72. 72. Choi, Y.S., Kim, H.B., Kwon, G.S. & Park, J.K. On-chip testing device for electrochemotherapeutic effects on human breast cells. Biomed. Microdevices 11, 151-159 (2009).
  73. 73. Jain, T. & Muthuswamy, J. Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells. Biosens. Bioelectron. 22,, 863-870 (2007).
  74. 74. Jain, T. & Muthuswamy, J. Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture. Lab Chip 7, 1004-1011 (2007).
  75. 75. Jain, T. & Muthuswamy, J. Microelectrode array (MEA) platform for targeted neuronal transfection and recording. IEEE Trans. Biomed. Eng. 55, 827-832 (2008).
  76. 76. Vassanelli, S., et al. Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray. N. Biotechnol. 25, 55-67 (2008).
  77. 77. Odorizzi, L., et al. An integrated platform for in vitro single-site cell electroporation: Controlled delivery and electrodes functionalization. Sens. Actuators B 170, 182-188 (2012).
  78. 78. Wang, S., Zhang, X., Wang, W. & Lee, L.J. Semicontinuous flow electroporation chip for high-throughput transfection on mammalian cells. Anal. Chem. 81, 4414-4421 (2009).
  79. 79. Wang, S., Zhang, X., Yu, B., Lee, R.J. & Lee, L.J. Targeted nanoparticles enhanced flow electroporation of antisense oligonucleotides in leukemia cells. Biosens. Bioelectron. 26, 778-783 (2010).
  80. 80. Dalmay, C., et al. A microfluidic biochip for the nanoporation of living cells. Biosens. Bioelectron. 26, 4649-4655 (2011).
  81. 81. He, H., Chang, D.C. & Lee, Y.K. Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells. Bioelectrochemistry 70, 363-368 (2007).
  82. 82. He, H., Chang, D.C. & Lee, Y.K. Nonlinear current response of micro electroporation and resealing dynamics for human cancer cells. Bioelectrochemistry 72, 161-168 (2008).
  83. 83. Chang, W.C. & Sretavan, D.W. Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls. Biosens. Bioelectron. 24, 3600-3607 (2009).
  84. 84. Homhuan, S., Zhang, B., Sheu, F.S., Bettiol, A.A. & Watt, F. Single-cell electroporation using proton beam fabricated biochips. Biomed. Microdevices 14, 533-540 (2012).
  85. 85. Lu, K.Y., et al. Three dimensional electrode array for cell lysis via electroporation. Biosens. Bioelectron. 22, 568-574 (2006).
  86. 86. Xie, C., Lin, Z., Hanson, L., Cui, Y. & Cui, B. Intracellular recording of action potentials by nanopillar electroporation. Nat. Nanotechnol. 7, 185-190 (2012).
  87. 87. Wilke, N., Hibert, C., O’Brien, J. & Morrissey, A. Silicon microneedle electrode array with temperature monitoring for electroporation. Sens. Actuators A 123–124, 319-325 (2005).
  88. 88. Koester, P.J., Tautorat, C., Beikirch, H., Gimsa, J. & Baumann, W. Recording electric potentials from single adherent cells with 3D microelectrode arrays after local electroporation. Biosens. Bioelectron. 26, 1731-1735 (2010).
  89. 89. Braeken, D., et al. Local electrical stimulation of single adherent cells using three-dimensional electrode arrays with small interelectrode distances. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2009, 2756-2759 (2009).
  90. 90. Braeken, D., et al. Single-cell stimulation and electroporation using a novel 0.18 micro CMOS chip with subcellular-sized electrodes. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2010, 6473-6476 (2010).
  91. 91. Demierre, N., et al. Characterization and optimization of liquid electrodes for lateral dielectrophoresis. Lab Chip 7, 355-365 (2007).
  92. 92. Shafiee, H., Caldwell, J.L., Sano, M.B. & Davalos, R.V. Contactless dielectrophoresis: a new technique for cell manipulation. Biomed. Microdevices 11, 997-1006 (2009).
  93. 93. Mernier, G., Hasenkamp, W., Piacentini, N. & Renaud, P. Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis. Sens. Actuators B 170, 2-6 (2012).
  94. 94. Shah, D., Steffen, M. & Lilge, L. Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis. Biomicrofluidics 6, 14111-1411110 (2012).
  95. 95. Shin, Y.S., et al. Electrotransfection of mammalian cells using microchannel-type electroporation chip. Anal. Chem. 76, 7045-7052 (2004).
  96. 96. Kim, J.A., et al. A multi-channel electroporation microchip for gene transfection in mammalian cells. Biosens. Bioelectron. 22, 3273-3277 (2007).
  97. 97. McClain, M.A., et al. Microfluidic devices for the high-throughput chemical analysis of cells. Anal. Chem. 75, 5646-5655 (2003).
  98. 98. Hargis, A.D., Alarie, J.P. & Ramsey, J.M. Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis. Electrophoresis 32, 3172-3179 (2011).
  99. 99. Mellors, J.S., Jorabchi, K., Smith, L.M. & Ramsey, J.M. Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. Anal. Chem. 82, 967-973 (2010).
  100. 100. Gao, J., Yin, X.F. & Fang, Z.L. Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip. Lab Chip 4, 47-52 (2004).
  101. 101. Xia, F., Jin, W., Yin, X. & Fang, Z. Single-cell analysis by electrochemical detection with a microfluidic device. J. Chromatogr. A 1063, 227-233 (2005).
  102. 102. Ling, Y.Y., Yin, X.F. & Fang, Z.L. Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis. Electrophoresis 26, 4759-4766 (2005).
  103. 103. Wang, H.Y. & Lu, C. Electroporation of mammalian cells in a microfluidic channel with geometric variation. Anal. Chem. 78, 5158-5164 (2006).
  104. 104. Wang, H.Y., Bhunia, A.K. & Lu, C. A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage. Biosens. Bioelectron. 22, 582-588 (2006).
  105. 105. Wang, H.Y. & Lu, C. High-throughput and real-time study of single cell electroporation using microfluidics: effects of medium osmolarity. Biotechnol. Bioeng. 95, 1116-1125 (2006).
  106. 106. Wang, H.Y. & Lu, C. Microfluidic chemical cytometry based on modulation of local field strength. Chem. Commun. 3528-3530 (2006).
  107. 107. Wang, J., Stine, M.J. & Lu, C. Microfluidic cell electroporation using a mechanical valve. Anal. Chem. 79, 9584-9587 (2007).
  108. 108. Wang, H.Y. & Lu, C. Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply. Biotechnol. Bioeng. 100, 579-586 (2008).
  109. 109. Wang, J., et al. Detection of kinase translocation using microfluidic electroporative flow cytometry. Anal. Chem. 80, 1087-1093 (2008).
  110. 110. Bao, N., Jagadeesan, B., Bhunia, A.K., Yao, Y. & Lu, C. Quantification of bacterial cells based on autofluorescence on a microfluidic platform. J. Chromatogr. A 1181, 153-158 (2008).
  111. 111. Bao, N. & Lu, C. A microfluidic device for physical trapping and electrical lysis of bacterial cells. Appl. Phys. Lett. 92, 214103 (2008).
  112. 112. Bao, N., Wang, J. & Lu, C. Microfluidic electroporation for selective release of intracellular molecules at the single-cell level. Electrophoresis 29, 2939-2944 (2008).
  113. 113. Bao, N., Zhan, Y. & Lu, C. Microfluidic electroporative flow cytometry for studying single-cell biomechanics. Anal. Chem. 80, 7714-7719 (2008).
  114. 114. Wang, J., Fei, B., Zhan, Y., Geahlen, R.L. & Lu, C. Kinetics of NF-kappaB nucleocytoplasmic transport probed by single-cell screening without imaging. Lab Chip 10, 2911-2916 (2010).
  115. 115. Wang, J., Zhan, Y., Ugaz, V.M. & Lu, C. Vortex-assisted DNA delivery. Lab Chip 10, 2057-2061 (2010).
  116. 116. Zhan, Y., Martin, V.A., Geahlen, R.L. & Lu, C. One-step extraction of subcellular proteins from eukaryotic cells. Lab Chip 10, 2046-2048 (2010).
  117. 117. Geng, T., et al. Flow-through electroporation based on constant voltage for large-volume transfection of cells. J. Control. Release 144, 91-100 (2010).
  118. 118. Geng, T., Zhan, Y., Wang, J. & Lu, C. Transfection of cells using flow-through electroporation based on constant voltage. Nat. Protoc. 6, 1192-1208 (2011).
  119. 119. Bao, N., et al. Single-cell electrical lysis of erythrocytes detects deficiencies in the cytoskeletal protein network. Lab Chip 11, 3053-3056 (2011).
  120. 120. Zhan, Y., et al. Low-frequency ac electroporation shows strong frequency dependence and yields comparable transfection results to dc electroporation. J. Control. Release 160, 570-576 (2012).
  121. 121. Ma, S., et al. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria. Integr. Biol. 6, 973-978 (2014).
  122. 122. Sun, C., Cao, Z., Wu, M. & Lu, C. Intracellular tracking of single native molecules with electroporation-delivered quantum dots. Anal. Chem. 86,, 11403-11409 (2014).
  123. 123. Fox, M., Esveld, E., Luttge, R. & Boom, R. A new pulsed electric field microreactor: comparison between the laboratory and microtechnology scale. Lab Chip 5, 943-948 (2005).
  124. 124. Fox, M.B., Esveld, D.C., Mastwijk, H. & Boom, R.M. Inactivation of L. plantarum in a PEF microreactor: The effect of pulse width and temperature on the inactivation. Innov. Food Sci. Emerg. Technol. 9, 101-108 (2008).
  125. 125. Huang, Y. & Rubinsky, B. Micro-electroporation: improving the efficiency and understanding of electrical permeabilization of cells. Biomed. Microdevices 2, 145-150 (1999).
  126. 126. Huang, Y. & Rubinsky, B. Microfabricated electroporation chip for single cell membrane permeabilization. Sens. Actuators A 89, 242-249 (2001).
  127. 127. Huang, Y., Sekhon, N.S., Borninski, J., Chen, N. & Rubinsky, B. Instantaneous, quantitative single-cell viability assessment by electrical evaluation of cell membrane integrity with microfabricated devices. Sens. Actuators A 105, 31-39 (2003).
  128. 128. Huang, Y. & Rubinsky, B. Flow-through micro-electroporation chip for high efficiency single-cell genetic manipulation. Sens. Actuators A 104, 205-212 (2003).
  129. 129. Diaz-Rivera, R.E. & Rubinsky, B. Electrical and thermal characterization of nanochannels between a cell and a silicon based micro-pore. Biomed. Microdevices 8, 25-34 (2006).
  130. 130. Kurosawa, O., et al. Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli. Meas. Sci. Technol. 17, 3127 (2006).
  131. 131. Suzuki, T., et al. High throughput cell electroporation array fabricated by single-mask inclined UV lithography exposure and oxygen plasma etching. Conf. Proc. IEEE Solid-State Sensors, Actuators and Microsystems 2007. 687-690 (2007).
  132. 132. Lee, E.S., et al. Microfluidic electroporation of robust 10-microm vesicles for manipulation of picoliter volumes. Bioelectrochemistry 69, 117-125 (2006).
  133. 133. Robinson, D.B., Lee, E.S., Iqbal, Z., Rognlien, J.L. & Davalos, R.V. Reinforced vesicles withstand rigors of microfluidic electroporation. Sens. Actuators B 125, 337-342 (2007).
  134. 134. Khine, M., Lau, A., Ionescu-Zanetti, C., Seo, J. & Lee, L.P. A single cell electroporation chip. Lab Chip 5, 38-43 (2005).
  135. 135. Ionescu-Zanetti, C., Blatz, A. & Khine, M. Electrophoresis-assisted single-cell electroporation for efficient intracellular delivery. Biomed. Microdevices 10, 113-116 (2008).
  136. 136. Valero, A., et al. Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab Chip 8, 62-67 (2008).
  137. 137. Fei, Z., et al. Gene transfection of mammalian cells using membrane sandwich electroporation. Anal. Chem. 79, 5719-5722 (2007).
  138. 138. Fei, Z., et al. Micronozzle array enhanced sandwich electroporation of embryonic stem cells. Anal. Chem. 82, 353-358 (2010).
  139. 139. Boukany, P.E., et al. Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat. Nanotechnol. 6, 747-754 (2011).
  140. 140. Gao, K., et al. Design of a microchannel-nanochannel-microchannel array based nanoelectroporation system for precise gene transfection. Small 10, 1015-1023 (2014).
  141. 141. Ishibashi, T., Takoh, K., Kaji, H., Abe, T. & Nishizawa, M. A porous membrane-based culture substrate for localized in situ electroporation of adherent mammalian cells. Sens. Actuators B 128, 5-11 (2007).
  142. 142. Nelson, E.M., Kurz, V., Shim, J., Timp, W. & Timp, G. Using a nanopore for single molecule detection and single cell transfection. Analyst 137, 3020-3027 (2012).
  143. 143. Xie, X., et al. Nanostraw-electroporation system for highly efficient intracellular delivery and transfection. ACS Nano 7, 4351-4358 (2013).
  144. 144. Kang, W., et al. Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons. Lab Chip 14, 4486-4495 (2014).
  145. 145. Zhu, T., et al. Electroporation based on hydrodynamic focusing of microfluidics with low dc voltage. Biomed. Microdevices 12, 35-40 (2010).
  146. 146. Wei, Z., et al. A laminar flow electroporation system for efficient DNA and siRNA delivery. Anal. Chem. 83, 5881-5887 (2011).
  147. 147. Selmeczi, D., Hansen, T.S., Met, O., Svane, I.M. & Larsen, N.B. Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip. Biomed. Microdevices 13, 383-392 (2011).
  148. 148. Yun, H. & Hur, S.C. Sequential multi-molecule delivery using vortex-assisted electroporation. Lab Chip 13, 2764-2772 (2013).
  149. 149. Vickers, D.A., Ouyang, M., Choi, C.H. & Hur, S.C. Direct drug cocktail analyses using microscale vortex-assisted electroporation. Anal. Chem. 86, 10099-10105 (2014).
  150. 150. Luo, C., et al. Picoliter-volume aqueous droplets in oil: electrochemical detection and yeast cell electroporation. Electrophoresis 27, 1977-1983 (2006).
  151. 151. Qu, B., Eu, Y.J., Jeong, W.J. & Kim, D.P. Droplet electroporation in microfluidics for efficient cell transformation with or without cell wall removal. Lab Chip 12, 4483-4488 (2012).
  152. 152. Xiao, K., et al. Electroporation of micro-droplet encapsulated HeLa cells in oil phase. Electrophoresis 31, 3175-3180 (2010).
  153. 153. Im do, J., et al. Digital microfluidic approach for efficient electroporation with high productivity: transgene expression of microalgae without cell wall removal. Anal. Chem. 87,, 6592-6599 (2015).
  154. 154. Shih, S.C., et al. A versatile microfluidic device for automating synthetic biology. ACS Synth. Biol. (2015). DOI: 10.1021/acssynbio.5b00062.
  155. 155. Jain, T., McBride, R., Head, S. & Saez, E. Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays. Lab Chip 9, 3557-3566 (2009).
  156. 156. Jain, T., Papas, A., Jadhav, A., McBride, R. & Saez, E. In situ electroporation of surface-bound siRNAs in microwell arrays. Lab Chip 12, 939-947 (2012).
  157. 157. Wang, J., Yang, S.-C., Wang, C., Wu, Q. & Wang, Z. A DEP-assisted single-cell electroporation chip with low operation voltage. Conf. Proc. IEEE Sensors 2010, 2097-2100 (2010).
  158. 158. Kim, S.K., Kim, J.H., Kim, K.P. & Chung, T.D. Continuous low-voltage dc electroporation on a microfluidic chip with polyelectrolytic salt bridges. Anal. Chem. 79, 7761-7766 (2007).
  159. 159. Wang, C.H., et al. Dielectrophoretically-assisted electroporation using light-activated virtual microelectrodes for multiple DNA transfection. Lab Chip 14, 592-601 (2014).
  160. 160. Witte, C., et al. Spatially selecting a single cell for lysis using light-induced electric fields. Small 0, 3026-3031 (2014).