## 4 Discussion  
In this work, we have investigated the feasibility of dielectrophoresis to be induced in a conventional electroporation cuvette and the possibilities to affect the efficiency of transfection as an alternative to the HV/LV methodology (which relies on electrophoresis). It was shown that depending on the order of application (PEF + DEP or DEP + PEF) the transfection efficiency can be either improved or inhibited, which is an excellent and anticipated result. Considering the positive results, the electric field gradient and spatial distribution can be further improved by the introduction of an additional array of needle electrodes in the cuvette. With an increase of the number of needle electrodes, the next step could be using the 2- or 5-mm gap cuvettes, which would increase the volume and potentially allow even large numbers of cells to be processed. Based on our study, it can be concluded that the field gradient factor \(\
abla\,\rvert\,E\,\rvert^{2}\) of \(10^{14}\) is sufficient to induce detectable changes in the electrotransfer of DNA. Increasing it further may improve the processing time, however, we speculate that it is unlikely to change the efficacy of the treatment (refer to Figure 5B), while improvement of the spatial \(E\) field distribution should be the priority. Moreover, within the framework of our study, the DEP force and spatial distribution of the cells inside the cuvette were not simulated. The reason behind this is the inability for experimental confirmation of such simulations in the mL range cuvette. After electroporation the parameters such as cell size, conductivity, and extracellular medium conductivity dynamically change due to the biophysics of the electropermeabilization process. It implies that the Clausius-Mossotti factor becomes unpredictable, i.e., the range of applicable values due to electroporation enable both pDEP and nDEP to be equally plausible. Therefore, in order to perform spatial cell simulation, a microfluidic chip has to be developed for tuning of the simulation parameters. In our study, DEP characterization was limited to the use of \(\
abla\,\rvert\,E\,\rvert^{2}\), which is a common practice since it does not depend on cell radius or Clausius-Mossotti factor and thus, no significant approximations are required. Furthermore, we employed square wave DEP, which involves high frequency harmonics in the signal. The effect of the harmonics of a square waveform contributes to a generation of a stronger DEP force (due to the RMS value) and additional DEP forces at its corresponding frequencies [28]. The application of square waves is easier and cheaper in terms of engineering, it is also advantageous in the context of electroporation since many commercial bipolar electroporators can be easily adapted for DEP-assisted electrotransfection. Moreover, we have used a low conductivity buffer (0.1 S/m) to perform the experiments. However, the polarizability factor or the real part of the Clausius-Mossotti factor will be altered in buffers of different conductivities. The reason to use a low conductivity buffer was to maximize the DEP forces index, which for CHO cells increase with the decrease of conductivity [31]. [PERSON] et al. have also shown that in mediums with conductivity close to 0.1 S/m the real part of Re [\(p\)] for CHO cells is positive with a peaking DEP force index in the frequency of \(10^{6}\)-\(10^{7}\)[31]. Our results in part support the data, i.e., during the PEF + DEP protocols the highest transfection efficiency was reached when (1 MHz) the DEP3 sequence was used. Considering the available knowledge [29; 31], the application of square waves, low conductivity buffer, and DEP frequencies including MHz pulsing allows maximizing the DEP force. Such an approach increased the number of transfected cells 1.4-fold (when compared to PEF only), which is competitive even against the HV/LV methodology [14; 33].