Skip to main content
Log in

Measurement of local electric field in microdevices for low-voltage electroporation of adherent cells

  • Technical Paper
  • Published:
Microsystem Technologies Aims and scope Submit manuscript

Abstract

In this study, we present the measurement of the local electric field in a microdevice designed for electroporation of adherent cells. The microdevice mainly consists of a coverslip that has a transparent conductive layer and an insulating layer. The insulating layer has small cylindrical holes that focus the field lines to reduce the voltage required for electroporation. We estimated the local electric field at the cells by analyzing the ionic current based on a simple equivalent circuit model and investigated the correlation between the field strength and the efficiency of electroporation. We prepared various designs with matrices of electrodes with diameters ranging from 5 to 10 μm and center-to-center distances between adjacent electrodes ranging from 20 to 75 μm to perform systematic and statistical investigations. Furthermore, we discussed the efficiency of the electrode design in terms of the degree of field focusing, the applicability of optical observations, and the probability of positioning cells on the electrodes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D’Errico-Grigioni A, Ferri E, Baccarani M, Lemoli RM (2006) Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Stem cells 24(2):454–461. doi:10.1634/stemcells.2005-0198

    Article  Google Scholar 

  • Boukany PE, Morss A, Liao WC, Henslee B, Jung H, Zhang X, Yu B, Wang X, Wu Y, Li L, Gao K, Hu X, Zhao X, Hemminger O, Lu W, Lafyatis GP, Lee LJ (2011) Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat Nanotechnol 6(11):747–754. doi:10.1038/nnano.2011.164

    Article  Google Scholar 

  • Chuang YJ, Tseng FG, Lin WK (2002) Reduction of diffraction effect of UV exposure on SU-8 negative thick photoresist by air gap elimination. Microsyst Technol 8(4):308–313. doi:10.1007/s00542-002-0176-8

    Article  Google Scholar 

  • Di Carlo D, Ionescu-Zanetti C, Zhang Y, Hung P, Lee LP (2005) On-chip cell lysis by local hydroxide generation. Lab Chip 5(2):171–178

    Article  Google Scholar 

  • Fox M, Esveld D, Valero A, Luttge R, Mastwijk H, Bartels P, van den Berg A, Boom R (2006) Electroporation of cells in microfluidic devices: a review. Anal Bioanal Chem 385(3):474–485. doi:10.1007/s00216-006-0327-3

    Article  Google Scholar 

  • Hakamada K, Shintaku H, Nagata T, Fujimoto H, Kawano S, Miyake J (2013) Development of a microfabricated device for low-voltage electropermeabilization of adherent cells. J Biosci Bioeng 115(3):314–319. doi:10.1016/j.jbiosc.2012.10.005

    Article  Google Scholar 

  • He H, Chang DC, Lee Y-K (2007) Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells. Bioelectrochemistry 70(2):363–368. doi:10.1016/j.bioelechem.2006.05.008

    Article  Google Scholar 

  • Huang Y, Rubinsky B (2001) Microfabricated electroporation chip for single cell membrane permeabilization. Sens Actuators A 89(3):242–249. doi:10.1016/s0924-4247(00)00557-4

    Article  Google Scholar 

  • Huang Y, Rubinsky B (2003) Flow-through micro-electroporation chip for high efficiency single-cell genetic manipulation. Sens Actuators A 104(3):205–212. doi:10.1016/s0924-4247(03)00050-5

    Article  Google Scholar 

  • Huang K-S, Lin Y-C, Su C–C, Fang C-S (2007) Enhancement of an electroporation system for gene delivery using electrophoresis with a planar electrode. Lab Chip 7(1):86–92

    Article  Google Scholar 

  • Huang H, Wei Z, Huang Y, Zhao D, Zheng L, Cai T, Wu M, Wang W, Ding X, Zhou Z, Du Q, Li Z, Liang Z (2011) An efficient and high-throughput electroporation microchip applicable for siRNA delivery. Lab Chip 11(1):163–172

    Article  Google Scholar 

  • Jain T, Muthuswamy J (2007) Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture. Lab Chip 7(8):1004–1011

    Article  Google Scholar 

  • Jain T, McBride R, Head S, Saez E (2009) Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays. Lab Chip 9(24):3557–3566

    Article  Google Scholar 

  • Jimbo Y, Kasai N, Torimitsu K, Tateno T, Robinson HPC (2003) A system for MEA-based multisite stimulation. IEEE Trans Biomed Eng 50(2):241–248

    Article  Google Scholar 

  • Khine M, Lau A, Ionescu-Zanetti C, Seo J, Lee LP (2005) A single cell electroporation chip. Lab Chip 5(1):38–43

    Article  Google Scholar 

  • Kurosawa O, Oana H, Matsuoka S, Noma A, Kotera H, Washizu M (2006) Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli. Meas Sci Technol 17(12):3127–3133. doi:10.1088/0957-0233/17/12/S02

    Article  Google Scholar 

  • Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, Forster M, Stocking C, Wahlers A, Frank O, Ostertag W, Kuhlcke K, Eckert HG, Fehse B, Baum C (2002) Murine leukemia induced by retroviral gene marking. Science 296(5567):497. doi:10.1126/science.1068893

    Article  Google Scholar 

  • Lin Y-C, Li M, Wu C–C (2004) Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement. Lab Chip 4(2):104–108

    Article  Google Scholar 

  • Marelli M, Divitini G, Collini C, Ravagnan L, Corbelli G, Ghisleri C, Gianfelice A, Lenardi C, Milani P, Lorenzelli L (2011) Flexible and biocompatible microelectrode arrays fabricated by supersonic cluster beam deposition on. J Micromech Microeng 21(4):045013

    Article  Google Scholar 

  • Marie R, Schmid S, Johansson A, Ejsing L, Nordström M, Häfliger D, Christensen CBV, Boisen A, Dufva M (2006) Immobilisation of DNA to polymerised SU-8 photoresist. Biosens Bioelectron 21(7):1327–1332. doi:10.1016/j.bios.2005.03.004

    Article  Google Scholar 

  • Miyano N, Inoue Y, Teramura Y, Fujii K, Tsumori F, Iwata H, Kotera H (2008) Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy. Lab Chip 8(7):1104–1109

    Article  Google Scholar 

  • Neumann E, Schaeferridder M, Wang Y, Hofschneider PH (1982) Gene-transfer into mouse lyoma cells by electroporation in high electric-fields. EMBO J 1(7):841–845

    Google Scholar 

  • Onuki-Nagasaki R, Nagasaki A, Hakamada K, Uyeda TQP, Fujita S, Miyake M, Miyake J (2008) On-chip screening method for cell migration genes based on a transfection microarray. Lab Chip 8(9):1502–1506

    Article  Google Scholar 

  • Rols MP, Teissie J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 75(3):1415–1423. doi:10.1016/S0006-3495(98)74060-3

    Article  Google Scholar 

  • Shintaku H, Azuma S, Kawano S (2009) Measurements of electric field and electrokinetic phenomena using two kinds of tracer particles with different mobilities. J Fluid Sci Technol 4(3):687–698

    Article  Google Scholar 

  • Singh AV, Lenardi C, Gailite L, Gianfelice A, Milani P (2009) A simple lift-off-based patterning method for micro- and nanostructuring of functional substrates for cell culture. J Micromech Microeng 19(11):115028

    Article  Google Scholar 

  • Stroh T, Erben U, Kuhl AA, Zeitz M, Siegmund B (2010) Combined pulse electroporation–a novel strategy for highly efficient transfection of human and mouse cells. PLoS One 5(3):e9488. doi:10.1371/journal.pone.0009488

    Article  Google Scholar 

  • Techaumnat B, Washizu M (2007) Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells. J Phys D Appl Phys 40(6):1831–1837. doi:10.1088/0022-3727/40/6/036

    Article  Google Scholar 

  • Valero A, Post JN, van Nieuwkasteele JW, Ter Braak PM, Kruijer W, van den Berg A (2008) Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab Chip 8(1):62–67

    Article  Google Scholar 

  • Valley JK, Neale S, Hsu H-Y, Ohta AT, Jamshidi A, Wu MC (2009) Parallel single-cell light-induced electroporation and dielectrophoretic manipulation. Lab Chip 9(12):1714–1720

    Article  Google Scholar 

  • Vernier PT, Levine ZA, Wu YH, Joubert V, Ziegler MJ, Mir LM, Tieleman DP (2009) Electroporating fields target oxidatively damaged areas in the cell membrane. PLoS One 4(11):e7966. doi:10.1371/journal.pone.0007966

    Article  Google Scholar 

  • Wang H-Y, Lu C (2006) Electroporation of mammalian cells in a microfluidic channel with geometric variation. Anal Chem 78(14):5158–5164. doi:10.1021/ac060733n

    Article  Google Scholar 

  • Wang M, Orwar O, Olofsson J, Weber S (2010) Single-cell electroporation. Anal Bioanal Chem 397(8):3235–3248. doi:10.1007/s00216-010-3744-2

    Article  Google Scholar 

  • Weaver JC (1993) Electroporation: a general phenomenon for manipulating cells and tissues. J Cell Biochem 51(4):426–435

    MathSciNet  Google Scholar 

  • Winterbourne DJ, Thomas S, Hermon-Taylor J, Hussain I, Johnstone AP (1988) Electric shock-mediated transfection of cells. Characterization and optimization of electrical parameters. Biochem J 251(2):427–434

    Google Scholar 

  • Xu Y, Yao H, Wang L, Xing W, Cheng J (2011) The construction of an individually addressable cell array for selective patterning and electroporation. Lab Chip 11(14):2417–2423

    Article  Google Scholar 

  • Zimmermann U (1986) Electrical breakdown, electropermeabilization and electrofusion. In: Reviews of physiology, biochemistry and pharmacology, vol 105. Springer, Heidelberg, pp 175–256. doi:10.1007/BFb0034499

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Hirofumi Shintaku or Satoyuki Kawano.

Appendix

Appendix

1.1 Effect of Au surface electrode

The electrical impedance of the microdevice was measured by an LCR meter (ZM2353, NF Corp, Japan) at 1.0 kHz with amplitude of 100 mV to assess the effect of the Au surface electrodes. The measurement for each well was conducted using phosphate-buffered saline and the same Pt wire electrode used for cell electroporation, whereas cells were not cultured in wells. Figure 9 shows the relation between the impedance and the total electrode area S of a well. It shows that the impedance with Au surface electrodes (indicated by Au) is lower than that without Au surface electrodes (indicated by ITO). In the case of ITO, the dependence of the impedance on S is not clear since there is a large variation. On the other hand, in the case of Au, the impedance decreases with increasing S and there is a relatively small variation in the measured values as shown in an inset of Fig. 9a. These results suggest that the Au surface electrodes reduce the interfacial impedance and they are expected to have superior uniform electrical properties to ITO electrodes.

Fig. 9
figure 9

Comparison of electrical characteristics of ITO and Au surface electrodes: a the impedance of a well in the microdevice against the total electrode area. Measurements were performed at 1.0 kHz and 100 mV. b Measured current signal when a pulsed voltage was applied using a well with 8-μm diameter electrodes and a center-to-center distance of 50 μm. The applied voltage has a rectangular waveform with amplitude of 0.5 V and a period of 1.0 ms. The current obtained Au surface electrodes decays slower than that obtained without Au

We also measured transient currents under the application of a pulsed voltage, which was used for electroporation. The data indicated by ITO and Au in Fig. 9b were obtained using the same electrode design of d = 8 μm and w = 50 μm. The applied voltage (which is qualitatively indicated by the broken line in Fig. 9b) increases to 0.5 V at t = 0 ms, is switched to the opposite polarization of −0.5 V at t = 0.5 ms, and is changed to 0 V at t = 1.0 ms. The current exhibits peaks at t = 0 and 1.0 ms and a trough at t = 0.5 ms, after which it decays with time. Comparison of the results for Au and ITO reveals that they have similar currents at the peaks and the trough, whereas their decay characteristics clearly differ. The current for Au has a longer tail than that for ITO, as clearly shown in the inset of Fig. 9b. Since the membrane potential is related to the charge accumulated on the cell membrane (Techaumnat and Washizu 2007), which is related to the current integrated over time, the different decay characteristics for Au and ITO are practically significant. In addition, He et al. (2007) reported that the long pulse duration reduces the critical electric field for electroporation. Thus, the above results indicate that Au surface electrodes are advantageous for low-voltage electroporation. In addition, ITO is easy to be damaged by electrolysis compared to Au. Therefore, all experiments were performed using electroporation plates with Au surface electrodes.

As mentioned above, there is no significant difference in the current values for Au and ITO at t = 0.0, 0.5, and t = 1.0 ms. This is consistent with the fact that R hole is simply determined by the geometry of the holes, as expressed by Eq. (2), but it is independent of the electrode material. On the other hand, Au and ITO clearly have different current decay characteristics after the peaks and trough (see Fig. 9b), indicating that C EDL is material dependent. For reference, the capacitance per unit area c EDL was extracted from the impedance data. The Au surface electrode was found to have an approximately three times larger c EDL (~120 mF/m2) than ITO (~40 mF/m2). That is, the different current decay characteristics of Au and ITO are caused by the different electrical properties of the EDL.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shintaku, H., Hakamada, K., Fujimoto, H. et al. Measurement of local electric field in microdevices for low-voltage electroporation of adherent cells. Microsyst Technol 20, 303–313 (2014). https://doi.org/10.1007/s00542-013-1797-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00542-013-1797-9

Keywords

Navigation