Electrical Lysis as a Tool for Cellular Analysis and Identification

Max Vogel

Abstract

Cellular lysis is often an essential sample preparation step in isolating cellular components for biochemical analysis. In this study, the principles of electrical cellular lysis were reviewed and compared to other methods of cellular lysis (mechanical, chemical, thermal, etc.). After putting electrical lysis in context with other methods, the parameters affecting cellular lysis were examined. Electrode geometry and material, cell concentration, media conductivity, throughput, and other parameters were considered in the review of electrical lysis. Using ANSYS electronics, several electrode arrays were designed based on the findings of the study on electrical lysis. Using ANSYS, simulations were conducted to determine the optimal voltage, electrode geometry, and excitation arrangement for creating an electric field capable of lysing cells to isolate cellular components for biochemical analysis and cellular identification. A preliminary determination of an ideal electrode array was determined based on ANSYS simulations.

Introduction

Sample preparation is an essential part in diagnostic assays. Using cellular components such as proteins or DNA, pathogens can be rapidly identified and treated using treatment plans tailored to the specific pathogen. Within this context, sample preparation allows for the isolation and/or enrichment of specific cells or compounds of interest to facilitate their biochemical analysis. Although other cellular components like organelles can be isolated using lysis techniques, DNA and protein are the most useful for biochemical analysis and cell identification [1]. After lysis, the lysates might require to be further processed in order to purify a target from cell debris and other byproducts of lysis.

Implementing lysis on a microscale presents numerous advantages in terms of diagnostic capacity. Microfluidic channels have a much higher surface area to volume ratio than macroscale operations, leading to an increased ability to detect a cellular component of interest once it has been isolated from a cell. 

Almost every cell is surrounded by a phospholipid bilayer that partitions the cytosol from the external environment. This selectively permeable barrier keeps cellular components like organelles and proteins contained, and allows the cell to control what enters and exits from the external environment [2]. The goal of cellular lysis in the context of sample preparation for a diagnostic assay is to break down this membrane in order to isolate intracellular components like DNA and proteins. A multitude of different methods can be used to lyse cells. Some of the most common methods are mechanical (use of physical force), thermal, chemical, laser, and electrical lysis [1]. Each method attempts to exploit a chemical property of the cell membrane in order to rupture the cell. Based on a review of a number of factors including time for lysis to occur, notable costs associated with each method, additional reagents required to achieve lysis, throughput, lytic efficiency, and impact of the method on cellular components, electrical lysis was determined to be the most effective method for cellular lysis. 
Application of electrical lysis on a microfluidic platform is often achieved by creating an electric field using electrodes. Most cells lyse when the applied electric field reaches a threshold voltage of 1000 V/cm [3]. By changing the distance between electrodes, electrode geometry, and the arrangement of excitations the magnitude of the electric field throughout the channel can be manipulated.

Materials and Methods

ANSYS Electronics 2020R1 was used to design electrodes and run simulations to generate theoretical electric field gradients. Twelve simulations were performed for each electrode geometry. In the first set of six simulations, positive and negative excitations were assigned to the electrodes by row. In the second set of six simulations, positive and negative excitations alternated between adjacent electrodes. Each excitation condition was simulated with applied voltages of 10V, 20V, 30V, 40V, 50V, and 60V. 

Figures

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FIGURE 1: Pore formation in cell membranes from the application of an electric field. When applied, the field can drastically increase the conductivity of the membrane to the point where the membrane becomes a conductor rather than an insulator. Essentially, the application of the electric field causes a dielectric breakdown of the membrane that creates pores allowing charged compounds to flow freely between the cell’s interior and the surrounding environment [4,5].

Results

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Figure 2: 40um circles

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Figure 3: 60um circles

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Figure 4: 80um circles

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Figure 5: 40um squares

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Figure 6: 60um squares

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Figure 7: 80um squares

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Figure 8: 40um triangles

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Figure 9: 80um triangles

Conclusions

  • Based on ANSYS simulations, circle electrodes were selected as the ideal geometry for electrical lysis
  • Smaller distances between electrodes allowed for threshold electric fields to be achieved at lower voltages
  • Alternating positive and negative excitations created an electric field that covered a greater area of the channel

Future Direction

  • More electrode geometries, distances, materials, and voltages will be tested 
  • Simulations will be run at different media conductivities to approximate the compatibility of the device with biological samples

References

  1. Bao N., Lu C. (2008) Microfluidics-Based Lysis of Bacteria and Spores for Detection and Analysis. In: Zourob M., Elwary S., Turner A. (eds) Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York, NY. https://doi.org/10.1007/978-0-387-75113-9_30
  2. Rye, Connie, et al. “3.4 The Cell Membrane.” Concepts of Biology 1st Canadian Edition, BCcampus, 1 May 2019, opentextbc.ca/biology/chapter/3-4-the-cell-membrane/.
  3. Wang, Hsiang-Yu, et al. “A Microfluidic Flow-through Device for High Throughput Electrical Lysis of Bacterial Cells Based on Continuous Dc Voltage.” Biosensors and Bioelectronics, vol. 22, no. 5, 2006, pp. 582–588., doi:10.1016/j.bios.2006.01.032.
  4. Morshed, Bashir, et al. “Electrical Lysis: Dynamics Revisited and Advances in On-Chip Operation.” Critical Reviews in Biomedical Engineering, vol. 41, no. 1, Mar. 2013, pp. 37–50., doi:10.1615/critrevbiomedeng.2013006378.
  5. Tsong T.Y. (1989) Electroporation of Cell Membranes. In: Neumann E., Sowers A.E., Jordan C.A. (eds) Electroporation and Electrofusion in Cell Biology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-2528-2_9