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Microfluidic Screening of Electric Fields for Electroporation.

Garcia PA, Ge Z, Moran JL, Buie CR - Sci Rep (2016)

Bottom Line: The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes.Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes.We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Energy and Microsystems Innovation, Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.

ABSTRACT
Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

No MeSH data available.


Related in: MedlinePlus

Microfluidic device to determine the critical electric field required for bacterial electroporation.(a) Two adjacent microphotographs showing the entire bilaterally converging channel (red-dashed outline) that amplifies the electric field to levels necessary to induce bacterial electroporation. (b) Schematic representation of the magnified constriction region illustrates the increase in green fluorescence due to SYTOX® dye uptake after electroporation with electric fields E ≥ Ecrit. (panel (b) not drawn to scale).
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f1: Microfluidic device to determine the critical electric field required for bacterial electroporation.(a) Two adjacent microphotographs showing the entire bilaterally converging channel (red-dashed outline) that amplifies the electric field to levels necessary to induce bacterial electroporation. (b) Schematic representation of the magnified constriction region illustrates the increase in green fluorescence due to SYTOX® dye uptake after electroporation with electric fields E ≥ Ecrit. (panel (b) not drawn to scale).

Mentions: Complementary to the advances mentioned above, we have developed a microfluidic device to characterize the critical electric field for bacterial electroporation under specific experimental conditions (e.g. pulse duration, buffer conductivity, cell concentration) in a single experiment3. The microfluidic device consists of a bilaterally converging channel to amplify the electric field magnitude to sufficient levels to induce electroporation (Fig. 1). Additionally, the converging shape of the channel produces a spatially linear electric field gradient along its length29. Included in the channel with the cells is SYTOX® Green nucleic acid stain (Life Technologies, Grand Island, NY), a fluorescent dye which shows a >500-fold fluorescence enhancement upon cytoplasmic nucleic acid binding30. The dye cannot penetrate the plasma membrane of living cells, but easily penetrates compromised plasma membranes, such as those induced by electroporation. Thus, the only cells in the channel that fluoresce are those which are exposed to an electric field strength greater than or equal to the critical electroporation threshold for the bacterium under investigation. Coupled with the linear electric field gradient, the dye allows for evaluation of the electric field strength required for electroporation without using discrete steps. Therefore, this microfluidic assay enables precise quantification of the critical electric field for electroporation in a single experiment, which would otherwise require hundreds of discrete experimental trials. Specifically, in our device we test the influence of electric field magnitude and cell type on electroporation. As test cases, we characterize the gram-positive bacteria Corynebacterium glutamicum (ATCC 13032, Manassas, VA, USA) and Mycobacterium smegmatis (ATCC, Manassas, VA, USA), and the gram-negative strain Escherichia coli BL21 (Bioline competent cells BIO-85032, London, UK). C. glutamicum has numerous industrial applications such as the production of enzymes, amino acids, and vitamins31. M. smegmatis is utilized in medical research as a model organism for disease-causing bacteria such as M. tuberculosis32. E. coli BL21 is a commonly used host for high-yield expression of recombinant proteins in biological studies33.


Microfluidic Screening of Electric Fields for Electroporation.

Garcia PA, Ge Z, Moran JL, Buie CR - Sci Rep (2016)

Microfluidic device to determine the critical electric field required for bacterial electroporation.(a) Two adjacent microphotographs showing the entire bilaterally converging channel (red-dashed outline) that amplifies the electric field to levels necessary to induce bacterial electroporation. (b) Schematic representation of the magnified constriction region illustrates the increase in green fluorescence due to SYTOX® dye uptake after electroporation with electric fields E ≥ Ecrit. (panel (b) not drawn to scale).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759816&req=5

f1: Microfluidic device to determine the critical electric field required for bacterial electroporation.(a) Two adjacent microphotographs showing the entire bilaterally converging channel (red-dashed outline) that amplifies the electric field to levels necessary to induce bacterial electroporation. (b) Schematic representation of the magnified constriction region illustrates the increase in green fluorescence due to SYTOX® dye uptake after electroporation with electric fields E ≥ Ecrit. (panel (b) not drawn to scale).
Mentions: Complementary to the advances mentioned above, we have developed a microfluidic device to characterize the critical electric field for bacterial electroporation under specific experimental conditions (e.g. pulse duration, buffer conductivity, cell concentration) in a single experiment3. The microfluidic device consists of a bilaterally converging channel to amplify the electric field magnitude to sufficient levels to induce electroporation (Fig. 1). Additionally, the converging shape of the channel produces a spatially linear electric field gradient along its length29. Included in the channel with the cells is SYTOX® Green nucleic acid stain (Life Technologies, Grand Island, NY), a fluorescent dye which shows a >500-fold fluorescence enhancement upon cytoplasmic nucleic acid binding30. The dye cannot penetrate the plasma membrane of living cells, but easily penetrates compromised plasma membranes, such as those induced by electroporation. Thus, the only cells in the channel that fluoresce are those which are exposed to an electric field strength greater than or equal to the critical electroporation threshold for the bacterium under investigation. Coupled with the linear electric field gradient, the dye allows for evaluation of the electric field strength required for electroporation without using discrete steps. Therefore, this microfluidic assay enables precise quantification of the critical electric field for electroporation in a single experiment, which would otherwise require hundreds of discrete experimental trials. Specifically, in our device we test the influence of electric field magnitude and cell type on electroporation. As test cases, we characterize the gram-positive bacteria Corynebacterium glutamicum (ATCC 13032, Manassas, VA, USA) and Mycobacterium smegmatis (ATCC, Manassas, VA, USA), and the gram-negative strain Escherichia coli BL21 (Bioline competent cells BIO-85032, London, UK). C. glutamicum has numerous industrial applications such as the production of enzymes, amino acids, and vitamins31. M. smegmatis is utilized in medical research as a model organism for disease-causing bacteria such as M. tuberculosis32. E. coli BL21 is a commonly used host for high-yield expression of recombinant proteins in biological studies33.

Bottom Line: The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes.Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes.We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Energy and Microsystems Innovation, Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.

ABSTRACT
Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

No MeSH data available.


Related in: MedlinePlus