## 3 Results and Discussion
### Membrane Failure Due to High Field Strength  
Membrane failure was also observed when subjecting the erythrocyte suspension to a higher voltage excitation, 7 \(V_{rms}\) (Figure 6a). Mechanical strain, \(\varepsilon\) = (\(d-d_{0}\))/\(d_{0}\) was averaged from six different measurements. The value of \(\varepsilon\) increased within the first second and gradually decreased in an exponential function within the duration of loading. After the electrical field was removed, erythrocytes  
Figure 5: Creep and recovery test during cyclic loading conditions. Error bar represents the standard deviation.  
recovered but never returned to their initial states (Figure 6c). Eventually, erythrocytes membranes showed multiple thorny projections similar to a typical echinocyte. The dimple area disappeared, and an apparent reduction in cell volume was observed. This failure mode was different from the membrane damage observed in the fatigue test, where only one thorny projection was formed in the fraction of cells under the same type of square waveforms.  
Another interesting observation was that a small number of erythrocytes under 7 \(V_{rms}\) excitations turned into ghost cells, indicating the release of hemoglobin from erythrocytes. Figure 5a shows the time lapse of a representative cell under electrodeformation for 4.5 s. The cell responded to the electric excitation within 0.5 s and was fully stretched, retracted rapidly, and gradually became transparent, observed under the bright-field microscopy through a band pass filter of 414 \(\pm\) 46 nm, which is near the peak of the hemoglobin absorption spectra. The field pulse was long enough; it is likely that the electrical breakdown of erythrocyte membranes occurred at such a high electric field strength, where the maximum value of the carrier sinusoidal waveform is up to 5 kV/m. Although the field strength utilized here was below the critical field strength required for the breakdown of membranes, 20-40 kV/m [35; 36], it is likely that the electromechanical coupling exerted high tensile stress that contributed to the lysis of cellular plasma membranes and opened up pathways for hemoglobin release.  
The effects of the induced release of hemoglobin were also recognized from the smaller deformation of erythrocyte membranes under a constant high electric field strength. As the electrical conductivity of erythrocyte interior is a function of hemoglobin concentration, its electrical conductivity is expected to decrease with the loss of hemoglobin. This process affects the Clausius-Mossotti factor, \(Re(f_{CM})\), which is proportional to the magnitude of the DEP force exerted on cell membranes (Equation (1)). The value of \(Re(f_{CM})\) decreases from 2.1 to 0.7 according to the ellipsoid model, and 0.87 to 0.48 according to the spherical model [21], when the interior conductivity of erythrocyte reduces from 0.31 to 0.07 S/m (Figure 7b,c). Correspondingly, when the interior conductivity of erythrocyte reduces to a same level as the surrounding medium, 0.018 S/m, the value of \(Re(f_{CM})\) further reduces to \(-\)0.015 and \(-\)0.004 based on the ellipsoidal and spherical models, respectively, both indicating a negative DEP. These mathematical predictions were confirmed by experimental observations in that the transparent ghost erythrocytes were slowly repelled away from the higher field strength.  
Figure 6: Erythrocyte response to high field strength excitations. (**a**) Induced membrane failure test was conducted by subjecting erythrocyte suspension to a constant high electric field strength for a duration of 22 s and removed after that. \(V_{rms}\) value is 7 V. (**b**) Such a load caused a strain in erythrocyte membranes as a function of time. It decreased gradually in an exponential function (\(n\) = 6). Error bar represents the standard deviation. (**c**) After the loading condition was removed, cells recovered with permanent damages in the membranes, similar to the echinocytes with small volume and thorny projections at multiple places.