Microchip Electroporation

Tao Geng and Chang Lu
Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, USA.

Electroporation has been widely used to facilitate delivery of exogenous molecules such as nucleic acids and drugs into cells or tissues. It occurs when an external electric field is applied to cells and the transmembrane potential (ΔψE) exceeds a critical threshold 1,2. The transmembrane potential (ΔψE) can be described using the following equation:

ΔψE=0.75 g(λ)aE cosθ(1)

where g(λ) is a function of the membrane and buffer conductivities, a is the diameter of the cell, E is the field intensity and θ is the angle between the normal to the membrane surface and the field direction. If the field intensity/duration is not too high/too long, nanoscale pores created in the plasma membrane can reseal after removal of the field and cells remain viable. Classical electroporation is typically conducted by exerting short electric pulses of defined duration and intensity to a cuvette with embedded electrodes inside to diminish the excessive heating and damage to cells due to the electric current. A pulse generator such as special capacitor discharge equipment is required to generate the high voltage pulses. By tuning the electric parameters (e.g. field intensity, field duration and pulse pattern), electroporation efficiency and cell viability can be optimized.

Due to its physical nature, electroporation can be easily miniaturized on a microfluidic platform 3-19. Electroporation microchips are typically designed and fabricated with standard microfabrication technology such as soft lithography, and a variety of microelectrodes can be incorporated into the chips to generate the field necessary for electroporation. The required voltage is dramatically decreased by reducing the distance between microscale electrodes 3,9,20 or creating physical constraints with subcellular dimensions 6,10,21. Although pulsed voltage is used in the vast majority of the reports, a constant dc voltage can also be applied to the channels with alternating wide and narrow sections for electroporation 22-24. 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 intensity in the wide sections that does not affect membrane integrity. Cells experience 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.

Based on Eqn. 1, ΔψE is highest at the poles of a cell (i.e., θ → 0) where the surface normal is aligned with the field direction. When a cell remains static during the application of the electric field (this is typically the case for conventional electroporation), permeabilization of the membrane and gene delivery occur mostly at the poles 25,26. This limitation was overcome in recent flow-through electroporation work by creating channel geometry and flow conditions which gave rise to complex cell migrations 27. The entire surface area of cells was exposed to the electric field for permeabilization during the course of the flow. Such practice dramatically improved the uptake without substantial impact on the cell viability.

Irreversible electroporation can also be applied to either kill tumor cells 19 or produce release of intracellular materials for analysis 7,15,28-30. In these cases, excessive electroporation leads to cell death and the electrophoretic force enables rapid release of intracellular molecules.

Microchip electroporation also allows obtaining real-time information about the electroporation process at single cell level while studying a population of cells with high throughput to establish the distribution 5,6,10,13,14,16. In principle, both the temporal resolution and the throughput in these methods can be significantly improved by applying a fast imaging digital camera. Since electrical stress may also cause dynamic responses from proteins in the cytoskeleton and introduce complex modulations to the biochemical and biophysical properties of cells, this tool will also allow studies of the time courses of these events 14.

  1. Weaver, J.C. & Chizmadzhev, Y.A. Theory of electroporation: A review. Bioelectrochem Bioener 41, 135-160 (1996).
  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. Lee, S.W. & Tai, Y.C. A micro cell lysis device. Sens Actuators A: Phys 73, 74-79 (1999).
  4. Lin, Y.-C., Jen, C.-M., Huang, M.-Y., Wu, C.-Y. & Lin, X.-Z. Electroporation microchips for continuous gene transfection. Sens Actuators B: Chem 79, 137-143 (2001).
  5. Huang, Y. & Rubinsky, B. Microfabricated electroporation chip for single cell membrane permeabilization. Sens Actuators A: Phys 89, 242-249 (2001).
  6. Davalos, R., Huang, Y. & Rubinsky, B. Electroporation: Bio-electrochemical mass transfer at the nano scale. Nanoscale and Microscale Thermophysical Engineering, 4, 147-159 (2000).
  7. McClain, M.A., et al. Microfluidic devices for the high-throughput chemical analysis of cells. Anal Chem 75, 5646-5655 (2003).
  8. 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).
  9. Lu, H., Schmidt, M.A. & Jensen, K.F. A microfluidic electroporation device for cell lysis. Lab Chip 5, 23-29 (2005).
  10. Khine, M., Lau, A., Ionescu-Zanetti, C., Seo, J. & Lee, L.P. A single cell electroporation chip. Lab Chip 5, 38-43 (2005).
  11. Fei, Z., et al. Gene transfection of mammalian cells using membrane sandwich electroporation. Anal Chem 79, 5719-5722 (2007).
  12. 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).
  13. 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).
  14. 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).
  15. Marc, P.J., Sims, C.E., Bachman, M., Li, G.P. & Allbritton, N.L. Fast-lysis cell traps for chemical cytometry. Lab Chip 8, 710-716 (2008).
  16. Valley, J.K., et al. Parallel single-cell light-induced electroporation and dielectrophoretic manipulation. Lab Chip 9, 1714-1720 (2009).
  17. 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).
  18. Ziv, R., Steinhardt, Y., Pelled, G., Gazit, D. & Rubinsky, B. Micro-electroporation of mesenchymal stem cells with alternating electrical current pulses. Biomed Microdevices 11, 95-101 (2009).
  19. Neal, R.E., 2nd & Davalos, R.V. The feasibility of irreversible electroporation for the treatment of breast cancer and other heterogeneous systems. Ann Biomed Eng 37, 2615-2625 (2009).
  20. Lin, Y.-C. & Huang, M.-Y. Electroporation microchips for in vitro gene transfection. J Micromech Microeng, 542 (2001).
  21. Munce, N.R., Li, J., Herman, P.R. & Lilge, L. Microfabricated system for parallel single-cell capillary electrophoresis. Anal Chem 76, 4983-4989 (2004).
  22. Wang, H.Y. & Lu, C. Electroporation of mammalian cells in a microfluidic channel with geometric variation. Anal Chem 78, 5158-5164 (2006).
  23. 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).
  24. Geng, T., et al. Flow-through electroporation based on constant voltage for large-volume transfection of cells. J Control Release 144, 91-100 (2010).
  25. Tekle, E., Astumian, R.D. & Chock, P.B. Selective and asymmetric molecular transport across electroporated cell membranes. Proc Natl Acad Sci U S A 91, 11512-11516 (1994).
  26. Golzio, M., Teissie, J. & Rols, M.P. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U.S.A. 99, 1292-1297 (2002).
  27. Wang, J., Zhan, Y., Ugaz, V.M. & Lu, C. Vortex-assisted DNA delivery. Lab Chip 10, 2057-2061 (2010).
  28. Wang, J., et al. Detection of kinase translocation using microfluidic electroporative flow cytometry. Anal Chem 80, 1087-1093 (2008).
  29. Bao, N., Wang, J. & Lu, C. Microfluidic electroporation for selective release of intracellular molecules at the single-cell level. Electrophoresis 29, 2939-2944 (2008).
  30. Wang, J., Fei, B., Zhan, Y.H., Geahlen, R.L. & Lu, C. Kinetics of NF-kappa B nucleocytoplasmic transport probed by single-cell screening without imaging. Lab Chip 10, 2911-2916 (2010).