Microfluidic Electroporation Assay with Linear Electric Field Gradient

Figure 1 shows the microfluidic device used to determine the critical electric field from a population of cells based on their response to pulsed electric field exposure. Upon application of a voltage along the device, the electric field within the microfluidic device exhibits a linear gradient, allowing cells to experience location-dependent electric field strengths within the channel. The cells are introduced with the nucleic acid stain SYTOX® Green into the channel. SYTOX® permeates the cell membranes and fluorescence is enhanced >500-fold in the cells that experience E ≥ E crit , where E crit is the critical electric field strength to cause electroporation of the cell. The location of the transition between electroporated and non-electroporated cells can be determined from fluorescent images and enables facile determination of the critical electric field threshold.

The electric field gradient is a function of the applied voltage and the channel geometry. Figure 2 shows the channel with a linear electric field gradient along the 3-mm constriction length. Figure 2a,b shows the electric field distribution within the channel. Additionally, by varying the applied voltage one can broaden or compress the range of electric fields applied to the cells (Fig. 2c). The converging geometry in the device results in a linear electric field gradient, as can be seen in Fig. 2c. Due to the symmetrical geometry of the channel, two E crit measurements are possible from a single experiment. The bilaterally converging geometries can be tuned to have steeper or shallower electric field gradients by modifying the constriction ratio and adjusting the channel length.

In reversible (transient) electroporation, maintaining high cell viability is a pre-requisite for protein expression. The goal of the microfluidic assay is to determine the critical electric field for the onset of electroporation. Thus, we determine the location of the transition between non-electroporated and electroporated cells, which occurs at significantly lower electric fields than irreversible electroporation (i.e., cell death). In order to maintain high cell viability, we designed the microfluidic device to achieve maximum electric field strengths of approximately 15 kV/cm at an applied voltage of 2.5 kV. This ensured that the electric field range evaluated was below the 15 kV/cm experimental limit in which E. coli viability is compromised after exposure to a 1.0 ms pulsed electric field34. Exposure of cells to stronger electric fields, longer pulse duration, or even a larger number of pulses will have a negative impact on cell viability34,35. Therefore, the present assay was limited to a single 1.0-ms electric pulse to establish the lowest electric field required for electroporation while preserving high cell viability.

Representative Fluorescent Images for Electroporation Assay

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.

Image Processing Methodology

Although the increase in fluorescence due to dye uptake by electroporated cells is often easy to locate visibly, the precise location of the onset of fluorescence can be more accurately quantified with image analysis. Figure 4a shows the summed fluorescence intensity (defined as the sum of the intensities in the shaded pixels) as a function of position along the channel. The red “Before” data is taken from the last image before the pulse is applied (the same image shown in Fig. 3a) and the blue “After” data is captured 200 ms after the pulse. In most cases, this slight delay allows the cells to fully uptake the dye and reach their steady-state post-pulse fluorescence intensity. In some cases dye uptake requires more time than 200 ms; in these cases the “After” data is captured 1 s after the pulse. These exceptions are indicated with an asterisk in Table 1.

Table 1 Estimates of critical electric field [kV/cm] magnitude (±ΔE crit ) for electroporation of C. glutamicum, M. smegmatis, and E. coli BL21 exposed to a single exponentially decaying (t = 1.0 ms; τ = 5.0 ms) pulse in 0.01× PBS buffer with 5 μM SYTOX® Green nucleic acid stain. Full size table

The critical electric field is defined as the electric field magnitude at the location of the onset of electroporation-induced fluorescence enhancement. To quantitatively estimate this location, we use the two-sample Kolmogorov-Smirnov (KS) test, a well-established statistical procedure for determining whether or not two data sets are drawn from the same underlying probability distribution36. The KS test considers the null hypothesis that two discrete datasets are drawn from the same (unknown) continuous probability distribution against a user-specified alternative hypothesis. The standard two-sided KS test considers the alternative hypothesis that the distribution functions of the two datasets are unequal, without regard to which is larger or smaller. However, in this work we are specifically interested in finding the regions of the channel where the post-pulse fluorescence intensity exceeds the pre-pulse intensity. Therefore, we use a one-sided KS test which evaluates the null hypothesis defined above (that both datasets are drawn from the same distribution) against the alternative hypothesis that the cumulative distribution function underlying the pre-pulse dataset is larger than that of the post-pulse dataset (as opposed to the two being simply unequal). Defined this way, the KS test will reject the null hypothesis in favor of the alternative hypothesis in regions of the channel where the post-pulse intensity significantly exceeds the pre-pulse intensity.

Consider two sets of fluorescence intensity values, I before and I after , which respectively represent the sets of intensity values before and after the pulse for a given sub-region R of the channel. From these datasets one can construct empirical cumulative distribution functions (CDFs), S before and S after . Then let F before and F after be the corresponding true (but unknown) population cumulative distribution functions for I before and I after . Both F before and F after are normalized, so that both . The null hypothesis H 0 to be tested is that the underlying distribution functions are identical, i.e. that both datasets are drawn from the same distribution:

where x is the distance along the channel constriction and R is the sub-region of the channel under consideration. In the regions of the channel where the electric field is insufficient to induce electroporation, there should be a negligible difference between the pre- and post-pulse intensity data. In these regions, the KS test should fail to reject the null hypothesis (i.e., in these cases we would expect the KS test to conclude that the two datasets are indeed drawn from the same distribution).

However, as the region R moves toward areas of increasing electric field strength, eventually the post-pulse data will deviate from the pre-pulse data due to electroporation-induced fluorescence enhancement in the post-pulse data. If enough of this data is included in R, the null hypothesis will be rejected in favor of the one-sided alternative hypothesis H 1 :

We note that, somewhat counterintuitively, if , this implies that a significant portion of the data points in the pre-pulse data (captured before the pulse) have a lower intensity. Thus, we should expect that H 1 will be favored for regions R where the post-pulse fluorescence is significantly enhanced due to electroporation.

Here, we perform the KS test sequentially for a 51-pixel stencil (which constitutes the region R) that begins at one end of the channel and moves horizontally along the channel, one pixel at a time. At each stencil location, the KS test is performed for the pre-pulse and post-pulse intensity values (shown in Fig. 4a) for the 51 adjacent pixels and a value H is recorded at the pixel occupying the center of the stencil (see Fig. 4b). The binary parameter H is defined as follows:

In words, we expect that H = 1 in regions where electroporation has occurred and H = 0 in regions where electroporation has not occurred.

Finally, to estimate the critical electric field, for each dataset we examine the locations where H = 1 and among these points, find the location where the electric field is minimized. This location is taken as the location of the onset of electroporation and the electric field at this location (calculated by linear interpolation of the simulation data displayed in Fig. 2c) is taken as the minimum electric field for electroporation for the given experimental trial.

Figure 4b shows the KS test result H as a function of position for an example case of C. glutamicum exposed to a 1.8-kV 1-ms pulse. For this particular case, the critical electric field for electroporation is estimated as 4.07 kV/cm. Note that here the KS test is able to identify two transition zones, one on each end of the channel. This enables one to exploit the bilateral symmetry of the channel geometry to obtain two estimates of E crit in one experiment. The value 4.07 kV/cm is the average of the KS-test-determined values on each end. Table 1 shows quantitative estimates of the critical electric field for electroporation of C. glutamicum, M. smegmatis and E. coli BL21 using the method described above. Note that all of the data displayed in Table 1 consists of a single estimate for each experiment; in these cases, the entire channel was not within the microscope’s field of view and so the KS test was only able to detect one transition.

Critical Electric Field Threshold for Electroporation