On-chip electroporation and impedance spectroscopy of single-cells

Abstract We present an integrated microplatform for handling, electroporation and label free analysis of single mammalian cells in suspension. Accurate axial positioning of a cell flowing through a channel is accomplished through microfluidic control and flow direction reversal; electroporation is achieved by applying 2 kV cm −1 at 50 kHz through on-chip microelectrodes; label-free detection of changes in the cell undergoing electroporation is performed by means of electric impedance spectroscopy (EIS). Shuttling the cell back and forth allows either (a) assessment of a single cell at multiple points of time to evaluate dynamic processes or (b) increased quality of EIS results by averaging subsequent passages of the same cell through the measurement region. Electrical parameters are extracted from the measurement by fitting the impedance magnitude spectrum to an equivalent-circuit model of the microchannel and by using a three-shell model for the cell. The fitting procedure is shown to be robust and suggests cell swelling, exchange of intra- and extracellular liquids, and change of the cell membrane and nuclear envelope capacitance. Cell swelling was in agreement with bright-field micrographs, while the exchange of intra- and extracellular liquids has also been observed via established fluorescent markers. The quantification of cellular changes as well as of the changes in cellular dielectric properties induced by electroporation enables a better understanding and control of the cell electroporation process.

[1]  Vasan Venugopalan,et al.  Investigation of laser-induced cell lysis using time-resolved imaging , 2004 .

[2]  Richard D. Rabbitt,et al.  Electric impedance spectroscopy using microchannels with integrated metal electrodes , 1999 .

[3]  Peter R. C. Gascoyne,et al.  Correlations between the dielectric properties and exterior morphology of cells revealed by dielectrophoretic field‐flow fractionation , 2013, Electrophoresis.

[4]  T. Mak,et al.  Cell lysis inside the capillary facilitated by transverse diffusion of laminar flow profiles (TDLFP) , 2006, Analytical and bioanalytical chemistry.

[5]  M. Flentje,et al.  Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status , 2014, PloS one.

[6]  Damijan Miklavčič,et al.  Electroporation in Biological Cell and Tissue: An Overview , 2009 .

[7]  Luke P. Lee,et al.  Single-cell electroporation arrays with real-time monitoring and feedback control. , 2007, Lab on a chip.

[8]  R. M. Boom,et al.  Inactivation of L. plantarum in a PEF microreactor The effect of pulse width and temperature on the inactivation , 2008 .

[9]  Chang Lu,et al.  A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage. , 2006, Biosensors & bioelectronics.

[10]  H. Morgan,et al.  Microfluidic Impedance Cytometry: Measuring Single Cells at High Speed , 2010 .

[11]  Nayoun Won,et al.  In vivo imaging of cancer cells with electroporation of quantum dots and multispectral imaging , 2010 .

[12]  T. Tsong,et al.  Formation and resealing of pores of controlled sizes in human erythrocyte membrane , 1977, Nature.

[13]  M. Rols,et al.  Direct visualization at the single-cell level of electrically mediated gene delivery , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Chang Lu,et al.  Electroporation of mammalian cells in a microfluidic channel with geometric variation. , 2006, Analytical chemistry.

[15]  Paul Theodor Pyl,et al.  The Genomic and Transcriptomic Landscape of a HeLa Cell Line , 2013, G3: Genes, Genomes, Genetics.

[16]  S. Gawad,et al.  Impedance spectroscopy flow cytometry: On‐chip label‐free cell differentiation , 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[17]  W. Jin,et al.  Determination of different forms of human interferon‐γ in single natural killer cells by capillary electrophoresis with on‐capillary immunoreaction and laser‐induced fluorescence detection , 2004, Electrophoresis.

[18]  Robert Meissner,et al.  Distinguishing drug-induced minor morphological changes from major cellular damage via label-free impedimetric toxicity screening. , 2011, Lab on a chip.

[19]  Yu-Cheng Lin,et al.  Electroporation microchips for continuous gene transfection , 2001 .

[20]  J Teissié,et al.  An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. , 1993, Biophysical journal.

[21]  Mark Bachman,et al.  Fast electrical lysis of cells for capillary electrophoresis. , 2003, Analytical chemistry.

[22]  Hsan-Yin Hsu,et al.  Parallel single-cell light-induced electroporation and dielectrophoretic manipulation. , 2009, Lab on a chip.

[23]  Christian H. Reccius,et al.  Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry. , 2009, Lab on a chip.

[24]  Dino Di Carlo,et al.  On-chip cell lysis by local hydroxide generation. , 2005, Lab on a chip.

[25]  Guillaume Mernier,et al.  Multiple-frequency Impedance Measurements in Continuous Flow for the Evaluation of Electrical Lysis of Yeast Cells , 2010 .

[26]  A. T. Esser,et al.  Membrane electroporation: The absolute rate equation and nanosecond time scale pore creation. , 2006, Physical review. E, Statistical, nonlinear, and soft matter physics.

[27]  A. Irimajiri,et al.  A dielectric theory of "multi-stratified shell" model with its application to a lymphoma cell. , 1979, Journal of theoretical biology.

[28]  Dino Di Carlo,et al.  Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation. , 2003, Lab on a chip.

[29]  Yi-Kuen Lee,et al.  Nonlinear current response of micro electroporation and resealing dynamics for human cancer cells. , 2008, Bioelectrochemistry.

[30]  J. Cooper,et al.  Lab-on-a-chip technologies for proteomic analysis from isolated cells , 2008, Journal of The Royal Society Interface.

[31]  Urban Seger,et al.  Dielectric spectroscopy in a micromachined flow cytometer: theoretical and practical considerations. , 2004, Lab on a chip.

[32]  D. Holmes,et al.  Single cell impedance cytometry for identification and counting of CD4 T-cells in human blood using impedance labels. , 2010, Analytical chemistry.

[33]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[34]  Peter Wiktor,et al.  Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy. , 2011, Nature chemistry.

[35]  W. J. Dower,et al.  High efficiency transformation of E. coli by high voltage electroporation , 1988, Nucleic Acids Res..

[36]  P. Leder,et al.  Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[37]  P. Eriksson,et al.  Selective Introduction of Antisense Oligonucleotides into Single Adult CNS Progenitor Cells Using Electroporation Demonstrates the Requirement of STAT3 Activation for CNTF-Induced Gliogenesis , 2001, Molecular and Cellular Neuroscience.

[38]  Dong-Chul Han,et al.  Electrotransfection of mammalian cells using microchannel-type electroporation chip. , 2004, Analytical chemistry.

[39]  Koji Asami,et al.  Characterization of heterogeneous systems by dielectric spectroscopy , 2002 .

[40]  J. Yeow,et al.  Cell electroporation by CNT-featured microfluidic chip. , 2013, Lab on a chip.

[41]  T. Reese,et al.  Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. , 1990, Biophysical journal.

[42]  Boris Rubinsky,et al.  Micro-Electroporation: Improving the Efficiency and Understanding of Electrical Permeabilization of Cells , 1999 .

[43]  J. Vienken,et al.  Rotation of cells in an alternating electric field theory and experimental proof , 2005, The Journal of Membrane Biology.

[44]  Stephen R Quake,et al.  Whole-genome molecular haplotyping of single cells , 2011, Nature Biotechnology.

[45]  U. Zimmermann,et al.  Particles in a homogeneous electrical field: A model for the electrical breakdown of living cells in a coulter counter , 1979 .

[46]  Y. Feldman,et al.  Time domain dielectric spectroscopy study of human cells. II. Normal and malignant white blood cells. , 1999, Biochimica et biophysica acta.

[47]  A. Steinbach,et al.  Surviving High-Intensity Field Pulses: Strategies for Improving Robustness and Performance of Electrotransfection and Electrofusion , 2005, The Journal of Membrane Biology.

[48]  Shengnian Wang,et al.  Micro-/nanofluidics based cell electroporation. , 2013, Biomicrofluidics.

[49]  Saeid Movahed,et al.  Microfluidics cell electroporation , 2011 .

[50]  R. Jaenisch,et al.  Microfluidic Control of Cell Pairing and Fusion , 2009, Nature Methods.

[51]  S. Gawad,et al.  Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing. , 2001, Lab on a chip.