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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

Fluorescent images for detection of boundary between electroporated and non-electroporated bacteria.Fluorescent images (a) before and (b) after delivering a 1.8-kV exponentially decaying (t = 1.0 ms; τ = 5.0 ms) pulse in 0.01× phosphate buffered saline (PBS) buffer (PBS diluted 100 times in DI water) and 5 μM SYTOX® Green nucleic acid stain to C. glutamicum bacteria (scale bar = 200 μm).
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f3: Fluorescent images for detection of boundary between electroporated and non-electroporated bacteria.Fluorescent images (a) before and (b) after delivering a 1.8-kV exponentially decaying (t = 1.0 ms; τ = 5.0 ms) pulse in 0.01× phosphate buffered saline (PBS) buffer (PBS diluted 100 times in DI water) and 5 μM SYTOX® Green nucleic acid stain to C. glutamicum bacteria (scale bar = 200 μm).

Mentions: The critical electric field is quantified by analyzing fluorescent images captured before and after electric pulsing. Figure 3 displays fluorescent images of C. glutamicum before and after a truncated (t = 1.0 ms) 1.8-kV exponentially decaying pulse (with decay constant τ = 5 ms) was delivered. Prior to pulse delivery, some background fluorescence was detected (Fig. 3a). The background fluorescence is proportional to the number of dead or already-compromised cells in the channel. Figure 3b shows fluorescence detected 100 ms after pulse delivery, in which the fluorescence is qualitatively enhanced compared to Fig. 3a. The representative panels provide the raw data used during image processing to correlate the location of fluorescence enhancement with the simulated electric field distribution (Fig. 2). The fluorescent images demonstrate that electroporation can be induced and detected in our microfluidic device, sampling a continuum of electric field strengths in a single experiment.


Microfluidic Screening of Electric Fields for Electroporation.

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

Fluorescent images for detection of boundary between electroporated and non-electroporated bacteria.Fluorescent images (a) before and (b) after delivering a 1.8-kV exponentially decaying (t = 1.0 ms; τ = 5.0 ms) pulse in 0.01× phosphate buffered saline (PBS) buffer (PBS diluted 100 times in DI water) and 5 μM SYTOX® Green nucleic acid stain to C. glutamicum bacteria (scale bar = 200 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Fluorescent images for detection of boundary between electroporated and non-electroporated bacteria.Fluorescent images (a) before and (b) after delivering a 1.8-kV exponentially decaying (t = 1.0 ms; τ = 5.0 ms) pulse in 0.01× phosphate buffered saline (PBS) buffer (PBS diluted 100 times in DI water) and 5 μM SYTOX® Green nucleic acid stain to C. glutamicum bacteria (scale bar = 200 μm).
Mentions: The critical electric field is quantified by analyzing fluorescent images captured before and after electric pulsing. Figure 3 displays fluorescent images of C. glutamicum before and after a truncated (t = 1.0 ms) 1.8-kV exponentially decaying pulse (with decay constant τ = 5 ms) was delivered. Prior to pulse delivery, some background fluorescence was detected (Fig. 3a). The background fluorescence is proportional to the number of dead or already-compromised cells in the channel. Figure 3b shows fluorescence detected 100 ms after pulse delivery, in which the fluorescence is qualitatively enhanced compared to Fig. 3a. The representative panels provide the raw data used during image processing to correlate the location of fluorescence enhancement with the simulated electric field distribution (Fig. 2). The fluorescent images demonstrate that electroporation can be induced and detected in our microfluidic device, sampling a continuum of electric field strengths in a single experiment.

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