## 3 Results  
The application of a voltage potential across a distance induces an electric field inside the cuvette. In the case of DEP, it is advantageous to have an electric field as high as possible to induce the highest force. However, the electric field amplitude should be below the electroporation threshold to prevent any effects of DEP on permeabilization and/or viability of cells. Therefore, the FEM analysis was performed to characterize the possible effects of DEP pulsing sequence. The spatial distribution of electric field inside the cuvette when 20 V of potential is applied is shown in Figure 3.  
Figure 3: Spatial distribution of electric field inside the electroporation cuvette with the center needle electrode, where (**A**) side-view; (**B**) top-view. Figure 2: The mesh (**A**) and the photograph of the prototype (**B**) of the dielectrophoresis-assisted electroporation cuvette. It can be seen that the induced electric field is non-homogeneous. Most of the volume is subjected to the sub-threshold electric field (<0.6 kV/cm). The higher electric field is generated only in close proximity with the electrode. In order to generate a significant DEP force, the gradient of electric field should be maximized. A value for the factor of around 10\({}^{13}\) V\({}^{2}\)/m\({}^{3}\) is required to produce a detectable DEP force [32]. The \(\
abla\,|\,E\,|\,^{2}\) factor inside the proposed cuvette was estimated and the results are presented in Figure 4.  
It can be seen that the peak value exceeds 8 \(\times\) 10\({}^{14}\), which should be sufficient to induce dielectrophoretic motion. The gradient can be further improved by the introduction of additional needle electrodes inside the cuvette. However, within the scope of this paper, it was important to show the proof of concept and the capability to control transfection efficiency via DEP sequence in a traditional cuvette. Finally, the proposed cuvette was tested experimentally in transfection experiments. The electroporation sequences were applied separately (without DEP) and with various parameters DEP. The results are summarized in Figure 5.  
Figure 4: Spatial dependence of \(\
abla\,|\,E\,|\,^{2}\) inside the cuvette. As it can be seen in Figure 5A, the PEF only treatment resulted in a 27% \(\pm\) 5% transfection efficiency. The value was used as a reference for comparison of the DEP-assisted treatments and is marked as a dashed line in the graphs (PEF only). The DEP only independently on the frequency did not induce detectable transfection. The combination of DEP sequences with electroporation showed significant effects on the treatment outcome. Depending on the order of application (PEF + DEP or DEP + PEF) the efficacy of the treatment was either improved or inhibited, e.g., when 1 MHz DEP3 sequence was applied after PEF, the number of transfected cells increased 1.4-fold (from 27% to 37%). If the same DEP3 sequence was applied before PEF, the treatment efficacy was inhibited 1.3-fold (from 27% to 20%). Similar tendencies were observed for 1 and 100 kHz sequences. During the PEF + DEP protocol, the frequency response was statistically significant (\(p\) < 0.05) between DEP1 and DEP3 (1 kHz and 1 MHz, respectively). When the DEP + PEF methodology was employed, the DEP2 and DEP3 (100 kHz and 1 MHz) sequences showed statistically significant differences versus each other. Furthermore, the effects of exposure time during the DEP application step were studied and the results are summarized in Figure 5B. It can be seen that independently on the time of the exposure (1, 3 or 5 min) the differences between DEP-assisted protocols are not significant (\(p\) > 0.05) or lost within the standard deviation of experimental data. It implies that 1 min is sufficient to produce a saturated response. Finally, considering that 1 MHz DEP (DEP3) is the highest intensity treatment (due to high repetition of pulses), the viability of the cells after PEF + DEP3 treatments was compared.