Electroporator – Principle, Types, Protocol, Applications

Electroporator is a specialized electronic pulse generator that is used to deliver controlled high-voltage electrical pulses to biological cells. It is used in electroporation process for temporary opening of the cell membrane.

It acts on the lipid bilayer membrane of the cell and makes small aqueous pores for short time. Through these pores, different macromolecules like DNA, RNA, proteins, and therapeutic drugs can enter or come out from the cell.

Modern electroporator can control voltage, pulse duration, and number of pulses. This helps in proper molecule delivery and also maintains cell survival. It usually gives exponential decay wave for bacteria and yeast and square wave for mammalian cells and tissues.

The machine is mainly made up of high-voltage power supply, capacitor bank, and high-speed semiconductor switch. These parts store and release electrical energy into the biological sample.

What is Electroporation?

Electroporation is a biophysical technique that is used to introduce foreign molecules inside the living cell by using short high-voltage electrical pulses. It is also known as electropermeabilization.

In this process, the applied electric field acts on the lipid bilayer membrane of the cell. It causes temporary disturbance in the membrane and forms very small pores called nanopores.

Through these temporary pores, molecules like DNA, RNA, proteins, antibodies, and drugs can pass into the cell. After the electrical pulse is stopped, the pores close naturally and the cell membrane reseals again.

This technique is used in biotechnology, gene therapy, vaccine development, and drug delivery. It helps in delivery of materials into cell without permanent damage of the cell.

Principle of Electroporation

Electroporation is based on application of short high voltage electric pulse to the cell membrane. It increases the permeability of the cell membrane for short time.

When electric field is applied, the membrane potential of the cell increases. After reaching a critical value, the phospholipid bilayer become unstable.

The lipid molecules of the membrane are rearranged. Small water filled pores are formed in the membrane. These pores are called hydrophilic pores or nanopores.

Through these pores, DNA, RNA, proteins, and drugs can pass into or out of the cell. These molecules normally cannot pass through the intact cell membrane.

After removal of electric pulse, the membrane again return to normal state. The pores are closed and the entered molecules remain inside the cell.

Types of Electroporation

The following are the types of electroporation–

1. Reversible electroporation
   In this type temporary pores are formed in the cell membrane and again closed. The cell remain alive after molecule entry.
2. Irreversible electroporation (IRE)
   In this type electric field causes permanent membrane damage. Cell death takes place and it is used in tissue and cardiac ablation.
3. Non-thermal irreversible electroporation (N-TIRE)
   It is a tumor ablation technique. It destroy targeted tissue by repeated electric bursts without heat damage.
4. High-frequency irreversible electroporation (H-FIRE)
   It uses high-frequency bipolar electric bursts. It destroy tumor cells without causing strong muscle contraction.
5. Bulk electroporation (BEP)
   In this method uniform electric field is applied to many suspended cells at same time. It is generally done inside a cuvette.
6. Single-cell electroporation (SCEP)
   In this method single cell is isolated and electric field is applied. Microfluidic devices are used in this process.
7. Localized single-cell membrane electroporation (LSCMEP)
   In this technique electric field is focused on small region of single cell membrane. It helps in targeted delivery with less cell damage.
8. Flow-through electroporation
   In this method cell suspension passes through a treatment chamber. Large volume of cells are electroporated continuously.
9. Micro-electroporation and nanotransfection
   In this method nanochannels or micro-electrodes are used. Molecules are delivered in controlled and less invasive way.
10. Nucleofection
    It is a special form of electroporation. It is used to deliver molecules like CRISPR components directly into nucleus.
11. Electrochemotherapy (ECT)
    It is the clinical use of reversible electroporation. It helps chemotherapeutic and cytotoxic drugs to enter tumor cells.
12. Gene electrotransfer (EGT)
    It is the use of electroporation for transfer of DNA into cells. It is used in gene therapy and DNA vaccines.

Parts of Electroporator

The following are the main parts of electroporator–

1. High-voltage power supply
   It is used to produce and supply electrical power. It charges the system up to a fixed voltage.
2. Capacitor bank
   It is used for storage of electrical energy. The stored energy is released into the sample during electroporation.
3. High-speed switching mechanism
   It is used to release the stored energy as electric pulse. It contains solid-state electronic parts like transistors or silicon-controlled rectifiers (SCRs).
4. Electrode interface
   It is the physical connection between machine and biological sample. It delivers the electrical pulse directly to the sample.
5. Cuvettes
   These are small plastic chambers with parallel metal plates. These plates are generally made of aluminium, stainless steel or platinum and are used for cell suspension.
6. Specialized electrodes
   These are electrodes used for special samples. Needle arrays, tweezers, and plate electrodes are used for in vivo, in ovo, or tissue samples.
7. Modular extensions
   These are interchangeable modules present in some electroporators. Capacitance extender (CE) module is used for mammalian cells and pulse controller (PC) module is used for high-voltage microbial electroporation.

Steps for performing electroporation – electroporation protocol

The following are the steps of electroporation protocol–

1. The cells of interest are cultured up to required growth phase. Then cells are harvested by centrifugation and washed properly to remove salts and ions.
2. The washed cells are resuspended in cold low-conductance electroporation buffer. This helps to prevent electrical arcing during pulse.
3. The prepared cells are mixed with purified DNA, RNA, proteins, or drugs. This mixing is generally done in a tube kept on ice.
4. The cell and molecule mixture is transferred into pre-chilled electroporation cuvette. The cuvette is tapped gently to remove air bubbles and outside of cuvette is dried.
5. The cuvette is placed inside the electroporator. Required voltage, capacitance and pulse duration are set according to the cell type.
6. The machine is activated and high-voltage electric pulse is delivered to the cell mixture. Temporary pores are formed in the cell membrane.
7. Immediately after pulse, recovery medium or broth is added into the cuvette. The mixture is gently mixed and transferred into a new tube.
8. The cells are kept for short recovery period. During this time, the temporary pores in the cell membrane are closed again.
9. The recovered cells are transferred into suitable growth medium or spread on selective agar plate. Then plates are incubated under proper condition.
10. The successfully transfected cells survive and grow. Colonies are formed on the plate when selectable genetic marker is used.

How to Prepare Cells for Electroporation?

The following are the steps for preparation of cells for electroporation–

1. The cells are grown up to proper density and healthy condition. Mammalian cells are generally used in actively dividing stage and bacterial or yeast cells are grown up to mid-log phase.
2. The cells are collected from the growth medium. This is generally done by centrifugation and then the supernatant is removed.
3. The collected cells are washed properly to remove salts from the growth medium. Excess salt increases conductivity and may cause electrical arcing during electroporation.
4. Bacterial cells are washed several times with ice-cold low-conductance solution. 10% glycerol or 0.5 M sucrose is commonly used for washing.
5. Yeast cells are washed with sterile water and osmotic stabilizer like 1 M cold sorbitol. Sometimes lithium acetate (LiAc) and dithiothreitol (DTT) are used before electroporation to weaken the thick cell wall.
6. Mammalian cells are washed carefully and antibiotics are removed from the medium. Antibiotics should not be present because electric pulse may allow them to enter into the cell and cause toxicity.
7. The washed cells are resuspended in suitable electroporation buffer. The buffer should be low-conductance and suitable for the particular cell type.
8. Bacterial cells are suspended in high-resistance buffer like 10% glycerol at high cell density. Generally 1×10¹¹ to 1×10¹² cells/ml are used.
9. Mammalian cells are suspended in specialized low-conductance electroporation buffer. Generally 5 to 10 million cells/ml are used.
10. Cells and cuvettes are kept cold before electroporation depending on the cell type. Pre-chilling is commonly used for bacteria and yeast and it also reduce heat damage in some mammalian cells.
11. The target molecule like DNA, RNA, or protein is prepared in sterile nuclease-free water. Salt containing buffer is avoided because it may cause arcing.
12. The target molecule is mixed gently with prepared cells just before electroporation. Then the mixture is placed into electroporation cuvette for application of electric pulse.

Applications of Electroporator

The following are the applications of electroporator–

• It is used for delivery of foreign DNA, RNA, plasmids and small molecules into mammalian, bacterial, yeast and plant cells.
• It is used in genetic engineering, recombinant protein production and gene expression studies.
• It is used in gene therapy for delivery of therapeutic genes into target cells.
• It is used for treatment study of genetic and acquired diseases like cancer, muscular dystrophy and cystic fibrosis.
• It is used to deliver CRISPR-Cas9 components into difficult cells like stem cells and primary immune cells.
• It is used in genome editing for precise genetic modification of cells.
• It is used in cancer treatment by irreversible electroporation techniques like N-TIRE and H-FIRE.
• It is used in electrochemotherapy for increasing delivery of cytotoxic anti-cancer drugs into tumor cells.
• It is used in vaccine development by increasing delivery and uptake of DNA vaccines.
• It is used in cell therapy for ex vivo genetic manipulation of immune cells like CAR-T cells and NK cells.
• It is used in cell fusion. In hybridoma technology, it helps in fusion of B lymphocytes with myeloma cells for production of monoclonal antibodies.
• It is used in biomanufacturing for development of stable cell lines.
• It is used for large scale production of therapeutic proteins, monoclonal antibodies and viral vectors.
• It is used in food preservation by Pulsed Electric Field (PEF) treatment.
• It is used for non-thermal pasteurization and sterilization of water, hospital wastewater and liquid foods.
• It is used for pathogen inactivation by destroying pathogenic microorganisms without affecting natural vitamins, colour and flavour.
• It is used for extraction of biomolecules from microbial and plant tissue.
• It increases yield of extracted juices, sugars and other valuable compounds.
• It is used in cardiac ablation for treatment of arrhythmias and heart rhythm irregularities.

Advantages of Electroporator (Electroporation machine)

The following are the advantages of electroporator–

• It is a fast and simple method. Large number of cells can be transfected within short time.
• It gives high efficiency of molecule delivery. The success rate is high when proper electric parameters are used.
• It can be used for different cell types. Bacteria, yeast, plant, insect and mammalian cells can be treated by this method.
• It is useful for difficult cells also. Primary cells and stem cells can be transfected by electroporation.
• It can deliver different types of molecules. Small molecules, DNA, RNA, proteins and large plasmids can be introduced into cells.
• It is a non-viral physical method. Viral vectors are not required in this technique.
• It does not need chemical carrier. So toxicity due to chemical transfection reagent is avoided.
• It reduces risk of immunogenicity and permanent genetic integration which are related with viral delivery.
• It gives reproducible result. Modern electroporator can control voltage, pulse length and other electric parameters.
• It allows direct entry of molecules into cytoplasm or nucleus. The molecules do not need to depend on endocytosis pathway.
• It needs less complex preparation steps. The membrane barrier is crossed directly by temporary pore formation.

Disadvantages of Electroporator (Electroporation machine)

The following are the disadvantages of electroporator–

• It may cause cell damage. High-voltage electric pulse can damage the cell membrane permanently.
• Cell death may occur if the voltage, pulse duration and buffer condition are not properly optimized.
• It needs specialized equipment. Electroporator and electroporation cuvette are required for this process.
• The machine and consumables are costly. So it is not always suitable for small laboratory use.
• It needs optimization for every new cell type. Field strength, pulse time and buffer conductivity should be adjusted properly.
• It may not deliver very large DNA fragments or highly complex molecular structures efficiently.
• Metal toxicity may occur. Electric pulse can oxidize metal plates of cuvette and release toxic metal ions into the sample.
• Transfection efficiency may be variable. Some cells take up molecules better than other cells in the same population.
• Electrical arcing may occur when high salt buffer or high conductivity buffer is used. This damages the sample and reduces cell viability.
• In in vivo or clinical use, electric pulse may stimulate nerve and muscle cells. This causes pain and involuntary muscle contraction.
• Anesthesia or paralytic agents may be needed in some clinical applications.
• Cells are very fragile just after electric pulse. Careful handling is required during recovery period to prevent cell death.

Precautions for Electroporator

The following are the precautions of electroporator–

• Cells should be washed properly before electroporation. All salts from the growth medium should be removed because salts can cause electrical arcing.
• Low-conductance and high-resistance electroporation buffer should be used. High salt buffer should be avoided.
• DNA, RNA, or ligation mixture should be diluted properly in sterile nuclease-free water. Generally it is diluted at least 1:5.
• Buffers containing Tris or EDTA should be avoided. These buffers may cause arcing and reduce transfection efficiency.
• The outside surface of electroporation cuvette should be dried before placing it into the machine. Moisture or condensation should not remain outside the cuvette.
• Antibiotics should not be added in electroporation medium during pulse. Penicillin or Streptomycin may enter the cell through temporary pores and cause cell death.
• Antibiotics should be added only after recovery period. Generally it is added after 4 to 6 hours of electroporation.
• Cells and cuvettes should be kept cold when required. Pre-chilling and post-chilling on ice helps to reduce heat damage.
• Electroporation cuvettes should not be reused. Reuse can cause salt build-up, cross contamination and reduced transformation efficiency.
• Plastic cuvettes or multi-well plates should not be autoclaved. The plastic material may melt in autoclave.
• The electroporator should not be opened for internal repair. It contains dangerous high-voltage electronic parts.
• The machine should be cleaned with damp cloth only. Harsh detergents, organic solvents and spilling of liquid inside the station should be avoided.

References

1. Asymmetric waveforms decrease lethal thresholds in high frequency irreversible electroporation therapies. (n.d.). ResearchGate.
2. Electroporation-based applications in biotechnology. (n.d.). ResearchGate.
3. In vivo electroporation for gene therapy. (n.d.). ResearchGate.
4. A model for determining nsPEF effects on cellular targets and apoptosis. (n.d.). ResearchGate.
5. A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection. (n.d.). PMC.
6. An introduction to electroporation – A tool for transfection and competent cell generation. (n.d.).
7. Laboratory of Biocybernetics. (n.d.). Applications in food and biotechnology. University of Ljubljana.
8. Ng, M. M. (2019). Design your own electroporation protocol episode 1 – Converting between square and exponential decay waves. BTX Online Blog.
9. Synthego. (n.d.). CRISPR transfection protocols guide: How to select the best method.
10. Thermo Fisher Scientific. (n.d.). CRISPR transfection.
11. Kotnik, T., Kramar, P., Pucihar, G., Miklavčič, D., & Tarek, M. (n.d.). Cell membrane electroporation—Part 1: The phenomenon. IEEE Electrical Insulation Magazine.
12. Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques. (n.d.). PMC.
13. Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line. (n.d.). PMC.
14. Comparison of different electroporation parameters on transfection efficiency of sheep testicular cells. (n.d.). PMC.
15. Control of the electroporation efficiency of nanosecond pulses by swinging the electric field vector direction. (n.d.). PMC.
16. ATUM. (n.d.). Culture and induction protocol to screen for protein expression in Saccharomyces cerevisiae.
17. DNA vaccine VGX-3100 with electroporation induces regression of cervical intraepithelial neoplasia 2/3 and clears HPV infection with robust T cell responses. (n.d.). PMC.
18. Ng, M. M. (2019). Design your own electroporation protocol episode 1 – Converting between square and exponential decay waves. BTX Online.
19. Ng, M. M. (2019). Design your own electroporation protocol episode 7 – Buffer considerations. BTX Online.
20. Does electroporation really efficient for knocking down despite the decreased siRNA concentration compared to lipofection. (n.d.). ResearchGate.
21. Does the shape of the electric pulse matter in electroporation? (n.d.). PMC – NIH.
22. Ng, M. M. (2019). Exclusive compendium: Optimizing an electroporation protocol. Harvard Bioscience.
23. Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture. (n.d.). PMC.
24. Electrochemotherapy (ECT) and irreversible electroporation (IRE) -advanced techniques for treating deep-seated tumors based on electroporation. (n.d.). PMC.
25. Thermo Fisher Scientific. (n.d.). Electrocompetent cells.
26. BEX CO., LTD. (n.d.). Electrode for in vivo electroporation.
27. Bulldog-Bio. (n.d.). Electrodes for in vivo, in ovo, in utero, ex vivo electroporation.
28. Wikipedia contributors. (2026). Electroporation. In Wikipedia, The Free Encyclopedia.
29. MaxCyte. (n.d.). Electroporation applications.
30. Thermo Fisher Scientific. (n.d.). Electroporation cuvettes.
31. BTX Online. (n.d.). Electroporation education.
32. BTX Online. (n.d.). Electroporation FAQ (Frequently asked questions).
33. New England Biolabs. (n.d.). Electroporation protocol (C2986).
34. Thermo Fisher Scientific. (n.d.). Electroporation support—Getting started.
35. Electroporation and cell killing by milli- to nanosecond pulses and avoiding neuromuscular stimulation in cancer ablation. (n.d.). ODU Digital Commons.
36. Hoover, A., Jin, M., Amador, S., Michels, C., Cram, J., Segura, M., & Paglia, M. (n.d.). Electroporation and lipid nanoparticles as delivery methods for targeted CRISPR/Cas9 gene editing in primary human T-cells. ElevateBio.
37. Electroporation of mammalian cells by nanosecond electric field oscillations and it’s inhibition by the electric field reversal. (n.d.). CORE.
38. Bio-Rad. (n.d.). Electroporation.
39. Electroporation-mediated gene delivery. (n.d.). PMC – NIH.
40. Tamang, S. (2023). Electroporation: Definition, principle, steps, uses. Microbe Notes.
41. NanoEntek. (2025). Electroporation: From principle to applications. NanoEntek Blog.
42. Electroporation: an arsenal of application. (n.d.). PMC.
43. Electroporator systems and electroporation technology: A comprehensive analysis of biophysical principles, instrumentation, and multidisciplinary applications. (n.d.).
44. Kumar, T. (2025). Exploring the benefits of electroporation in NK cell transfection. Cell & Gene.
45. Thermo Fisher Scientific. (n.d.). Factors influencing transfection efficiency.
46. Full article: High transfection efficiency and cell viability of immune cells with nanomaterials-based transfection reagent. (n.d.). Taylor & Francis.
47. Thermo Fisher Scientific. (n.d.). General CRISPR RNP transfection guidelines.
48. BTX Online. (n.d.). General optimization guide for electroporation.
49. AI Labs: Microfluidics. (n.d.). How electroporation for gene editing can revolutionize biotechnology [Video]. YouTube.
50. In ovo electroporation of plasmid DNA and morpholinos into specific tissues during early embryogenesis. (n.d.). PMC.
51. Irreversible electroporation: Background, theory, and review of recent developments in clinical oncology. (n.d.). PMC.
52. Biocompare. (n.d.). Key considerations for electroporation optimization. Bench Tips.
53. Microfluidic electroporation for cellular analysis and delivery. (n.d.). PMC – NIH.
54. Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses. (n.d.). PMC.
55. Ruiz-Fernández, A. R., Campos, L., Gutierrez-Maldonado, S. E., Núñez, G., Villanelo, F., & Perez-Acle, T. (2022). Nanosecond pulsed electric field (nsPEF): Opening the biotechnological Pandora’s box. International Journal of Molecular Sciences, 23(11), 6158.
56. Nanosecond pulsed electric fields (nsPEFs) for precision intracellular oncotherapy: Recent advances and emerging directions. (n.d.). PMC.
57. Nanosecond pulsed electric fields (nsPEFs) for precision intracellular oncotherapy: Recent advances and emerging directions. (n.d.). PubMed.
58. Nepa Gene Co., Ltd. (n.d.). Needle electrodes/other electrodes | Product.
59. BTX Online. (n.d.). Non invasive electrodes – In vivo electroporation – Research area.
60. Thermo Fisher Scientific. (n.d.). Overview of transfection methods.
61. Lowe-Power, T. (n.d.). Preparation of electrocompetent cells | protocols. Lowe-Power Lab.
62. BTX Online. (n.d.). Protocol 0906 ElectroSquarePorator™.
63. Pulse parameters and thresholds for (ir)reversible electroporation on hepatocellular carcinoma cells in vitro. (n.d.). PMC.
64. Recent advancements in electroporation technologies: From bench to clinic. (n.d.). PMC.
65. Santra, T. S., & Tseng, F. G. (2013). Recent trends on micro/nanofluidic single cell electroporation. Micromachines, 4(3), 333-356.
66. Reversible and irreversible effects of electroporation on contractility and calcium homeostasis in isolated cardiac ventricular myocytes. (n.d.). Circulation: Arrhythmia and Electrophysiology – American Heart Association Journals.
67. Eppendorf. (2001). Saccharomyces cerevisiae. Transformation Protocol No. 4308 915.531.
68. Safety and immunogenicity of VGX-3100 formulations in a healthy young adult population. (n.d.).
69. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial. (n.d.). PMC.
70. Thermo Fisher Scientific. (n.d.). Six considerations for competent cell selection.
71. The advantages of microfluidic technology in achieving precise single-cell genomics. (n.d.).
72. Ziya, D. (2023). The electroporation technique: Principles, applications, and advancements. Journal of Biomolecular Research & Therapeutics, 12(9), 336.
73. The impact of non-electrical factors on electrical gene transfer. (n.d.). PMC – NIH.
74. Transfection by electroporation. (n.d.). PMC – NIH.
75. Nepa Gene Co., Ltd. (n.d.). Tweezer electrodes | Product.
76. Thompson, J. R. (1999). Transformation of Saccharomyces cerevisiae by electroporation (U.S. Patent No. 5,879,891A). U.S. Patent and Trademark Office.
77. iGEM. (n.d.). Yeast electroporation.