Electroporation is a widely used nonviral technology for transfection of genes and drugs [1,2,3,4]. Under the application of a strong electric field, supraphysiological membrane potential is induced across the cell membrane to cause the formation of nanopores and thus change cell permeability . When the applied field strength is sufficiently high, cell membranes are permanently perforated, leading to cell inactivation. The electroporation technique has numerous applications, such as the delivery of exogenous reagents like genes, drugs, and nanoparticles or the extraction of intracellular components like proteins, nucleic acids, etc. [6,7]. It also plays an important role in recent breakthroughs such as gene editing (CRISPR-Cas9) [8,9] and cell reprogramming (induced neurons) . Moreover, this method is used in the beverage, wine, and dairy industries for disinfecting liquid products [11,12,13,14] and shows great potential in the field of water treatment as well [15,16,17]. However, to maintain the high electric field strength required for electroporation, electric voltages up to 105 volts are usually employed, which increases the issues concerning operational safety.
Recently, various kinds of microfluidics devices have been reported in the study of electroporation under controlled experimental conditions [6,7]. Early devices implemented 2D electrodes, including rectangular , interdigitated , and saw tooth electrodes  and other shapes [21,22,23] for generating high electric field strength. However, the electric field distribution is nonuniform because of the decay of field strength away from the electrode surface. To resolve this issue, devices involving 3D electrodes were developed, and the electroporation efficiency was significantly improved [24,25]. Although local high field strength can be developed using different electrode designs and configurations, problems such as electrolysis and corrosion of electrodes were unavoidable. As a consequence, insulator-based microfluidic devices were developed where insulating structures (e.g., micropillars) with geometric variations are used [26,27,28,29,30,31]. The use of insulating structures not only enhances local electric field strength but also minimizes the adverse effects observed in conducting electrode-based devices. Insulator-based dielectrophoresis (iDEP) devices have been demonstrated manipulating cells, particles, and proteins [32,33,34]. In our previously reported work, an iDEP device involving insulating PDMS micropillars was developed to inactivate microorganisms under various electric field and flow conditions .
In this paper, we present electroporation-based bacterial inactivation operating on an enhanced electric field induced by insulating microbeads. Electric voltage was applied to a pair of mesh electrodes inside a chamber that consists of densely packed microbeads. The use of microbeads can enhance local electric field strength at lower applied voltages that are sufficient to electroporate bacteria. The inactivation performance is tested for two types of bacteria: Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) under various electric field conditions. Both of the bacteria are abundantly found in raw water and are widely used as indicator organisms in the water treatment process [36,37]. Figure 1a shows a schematic of our proposed device, with its working principle illustrated in Figure 1b.
In the device shown in Figure 1a, the motion of the fluid is governed by the continuity equation
and the steady-state momentum equation
, where p is the pressure and μ is the dynamic viscosity of the liquid.
denotes the electrical body force with ρe being the net charge density and E the strength of the applied electric field. However, the electrical body force terms can be ignored based on an assumption of a thin electric double layer (EDL); hence, a slip boundary condition can be imposed , and such slip velocity can be expressed as 
where ueo is the electroosmotic mobility of the fluid and is dependent on the surface potential ζs and permittivity εm and viscosity μ of the liquid . This mobility is given by the Smoluchowski equation as
Similarly, the electric potential distribution outside the EDL region is governed by the Laplace equation
, and the electric field strength can be obtained as
. In the presence of electric field, cells experience electrophoresis due to their electrostatic charges. In addition, the formation of nonuniform electric field generated inside the region of the packed beads results in the cell dielectrophoresis effect. Therefore, considering the combined effect of electroosmotic flow, electrophoresis, and dielectrophoresis, the cell velocity can be estimated as 
where u is the velocity of the fluid, up is the electrophoretic mobility of the cell of radius r, and c is a correction factor. Since the conductivity of the working fluid is much lower than that of cells, cells experience a negative DEP effect (refer to Supplementary Information S1). In most iDEP devices, the Joule heating effect can play a significant role as
, where Q is the volumetric Joule heating related to the electrical conductivity of the medium (σ) and the electric field strength (E).
Additionally, cells undergo the electroporation effect due to the charges induced across the cell membrane. These induced charges give rise to a voltage, termed the transmembrane potential (TMP), which is given by Schwan’s equation as 
where f is the shape factor and θ is the angle between the line of electric field and the line joining the center of the cell to the point of interest. Further, the shape factor is given by 
where r is the radius and l is the length of the cell. For rod-shaped cells such as E. coli, l >> 2r. Thus, f = 1. According to the theory of electroporation, when the transmembrane potential reaches the critical value of 0.2–1 V , nanopores are formed within the cell membrane, thereby allowing exchanges of ions, drugs, molecules, genes, etc. If the applied field strength is very high, the pores do not reseal, and the cell membrane is damaged permanently. Two major theories can be found in the literature, and they are the theory of electromechanical compression  and the pore energy model . Schwan’s equation remains valid as long as the conductivity of the cell membrane is several orders higher than the conductivity of the suspending medium . Successful electroporations have been demonstrated with various buffer conductivities ranging from deionized (DI) water to saline solution (i.e., 1.6 S/m) without affecting the viability of the cell [17,47]. However, the use of a highly conductive buffer can generate a significant amount of heat, which can affect the electroporation process.
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