Operando investigation of the synergistic effect of electric field treatment and copper for bacteria inactivation

Abstract

As the overuse of chemicals in our disinfection processes becomes an ever-growing concern, alternative approaches to reduce and replace the usage of chemicals is warranted. Electric field treatment has shown promising potential to have synergistic effects with standard chemical-based methods as they both target the cell membrane specifically. In this study, we use a lab-on-a-chip device to understand, observe, and quantify the synergistic effect between electric field treatment and copper inactivation. Observations in situ, and at a single cell level, ensure us that the combined approach has an enhancement effect leading more bacteria to be weakened by electric field treatment and susceptible to inactivation by copper ion permeation. The synergistic effects of electric field treatment and copper can be visually concluded here, enabling the further study of this technology to optimally develop, mature, and scale for its various applications in the future.

Fig. 1. The lab-on-a-chip (LOAC) device and its application in this study.

Examples of images and data analysis are presented for the EFT-Cu condition with 500 ns pulse width, 500 µs period, 40k pulses, 80 V applied voltage, and a 0.5 mg/L Cu concentration. a A digital photo shows the LOAC device in size respect to a hand and vertical image to show the gold electrode layer. The zoomed in image shows an individual channel of the chip captured by the microscope. The curved electrode design is easily shown in respect to key parameters (w, w₀, and x, further explained in Supplementary Note 2). This one channel shows the scale, and the red dashed portion represents the area selected for image and data analysis consistent for all experiments. b The fabrication flow for both the top and cross-sectional view. The dashed line visible in the top view represents the cross-sectional view shown. c A COMSOL Multiphysics simulation of the electric field strength produced across an individual channel. The colored strength key is shown for reference to this example condition. d The plotted representation of the simulated linear electric field strength established by the curved electrode. e The individual differential interference contrast (DIC) channel image zoomed in with a yellow rectangle to highlight the immobilized cells that are well attached and closely packed together. f The individual fluorescent channel image effectively stained with the propidium iodide (PI) dye, and a zoomed in portion with a yellow rectangle to show a high electric field strength region where cells are inactivated and appear bright red. g The overall inactivation percentage over the x-axis and electric field strength as translated from the COMSOL Multiphysics simulation. The inactivation here refers to positive PI-staining further explained in the methods section. In all our results presented, the entire region is analyzed and averaged with the duplicate values on the second half of the x-axis. The dashed blue lines are presented to guide the reader through a typical analysis process. The error bars indicate 95% confidence intervals from the mean values based on 30 replicate channels. (For any references to colors in the figures, the reader is referred to the online/web version of this article).

Fig. 2. Microbial inactivation of combined electric field treatment and copper (EFT-Cu).

The overall inactivation percentage over the electric field strength for five varied Cu concentrations 0-2 mg/L are presented when the pulse width is 500 ns (a), 1 μs (b), and 2 μs (c). The overall inactivation here refers to positive PI-staining further explained in the methods section. The error bars indicating 95% confidence intervals from the mean values for inactivation efficiency in Fig. 2a–c are shown in Supplementary Figs. 2–4 in the SI. The red dashed line represents the lethal electroporation threshold (LET) where overall inactivation reaches 50%. (d-f) The overall inactivation percentage over increasing Cu concentration for increasing values of electric field strength and their corresponding linear fit lines for pulse widths 500 ns (d), 1 μs (e), and 2 μs (f). The slope values for linear fit lines shown in panels d–f plotted for pulse widths 500 ns (g), 1 μs (h), and 2 μs (i). There is no statistical analysis provided for Fig. 2d–i as they are data analyzed and obtained from the data points, trends, and relationships shown in Fig. 2a–c and not pulled from the total replicate data.

Fig. 3. Microscopy images of the LOAC device under fluorescent channel and quantified inactivation percentage taken every 30 min over the course of 3 h.

Cu-only using no EFT + 2 mg/L Cu (a), EFT-only using EFT + 0 mg/L Cu (b), and EFT-Cu where EFT + 2 mg/L Cu (c) are all shown. The pulse width for EFT is operated under 500 ns. Images for 1 min into the experiment are also presented for each condition to show cell damage immediately following any pulse application. The overall inactivation here refers to positive PI-staining further explained in Supplementary Note 5. Due to time and processing constraints, there are only 5 replicate channels for each experiment collected and analyzed in the time series results presented (compared to the usual capacity of 30). Only one representative channel for each experiment is shown. The zoomed in portions are shown for 1 and 180 min for each condition, and areas of significance are highlighted by yellow dashed circles to guide the readers. d The overall inactivation percentage over time in minutes for Cu-only, EFT-only, and EFT-Cu. The dashed line refers to the calculated theoretical additive. Individual plots are shown for specific electric field strengths appearing in increasing order from left to right and top down. The error bars represent 95% confidence intervals from the mean values for the 5 total replicates collected and analyzed for each of these and all additional plots with electric field strengths 29-39 kV/cm. (For any references to colors in the figures the readers are referred to the online/web version of this article).

Fig. 4. Microscopy images of the LOAC device under fluorescent and DIC channels where the red indicates PI fluorescence and diffusion of the dye into the individual cells and the relative time points where each image’s time stamp is shown underneath.

Cu-only using no EFT + 2 mg/L Cu (a), EFT-only using EFT + 0 mg/L Cu (b), and EFT-Cu where EFT + 2 mg/L Cu (c) are all shown. Conditions for Cu-only and EFT-Cu show both the DIC and fluorescent channels, while the EFT-only condition shows only the fluorescent channel due to time and processing constraints. Short videos for each condition are available in Supplementary Movies 1–6. The pulse width for EFT is operated under 2 μs. (d–f) The relative PI intensity of fluorescence as measured by the microscope normalized over time in seconds is also shown for Cu-only (d), EFT-only (e), and EFT-Cu (f) where importance is placed on when the cells reach 100% saturation of the dye intensity. (For any references to colors in the figures the reader is referred to the online/web version of this article).