Sickle cell disease (SCD), which causes sclerosis and membrane distortion in red blood cells (RBCs), brings about variation in cellular electrical properties ^[38][142]. Liu et al. combined on-chip oxygen control onto a single IFC chip for sickle cell disease diagnosis and monitoring ^[1][110]. They measured the electrical impedance of normal cells and sick cells at three different frequencies under normoxic and hypoxic conditions, respectively. As shown in Figure 14A, normal RBCs and sickle cells were separated clearly according to the measured impedance amplitude and phase value at 156 kHz under the normoxic condition. The results suggested that electrical impedance could serve as a new parameter to diagnose sickle cell disease. Parasite invasion can alter the dielectric properties of RBCs ^[4][39][113,143]. Du et al. demonstrated the discrimination of normal RBCs and P. falciparum-infected RBCs through analyzing the changes in the impedance signal amplitude and phase ^[4][113]. Honrado et al. developed an IFC device integrated with fluorescence interrogation to detect the dielectric properties of RBCs infected by malaria (Figure 14(Bi)) ^[5][114]. As a result, for early-stage infection (6 h), infected cells and normal cells were not distinguishable according to their impedance signals. However, as parasite growth progressed, the membrane capacitance and cytoplasmic conductivity of infected RBCs increased and thus the discrimination between two cell populations gradually became detectable (Figure 14(Bii)).

Holmes et al. discriminated and enumerated CD4 T-cells based on impedance cytometry and immune capture ^[3][40][112,144]. In their study, CD4 T-cells were labeled with small antibody conjugated beads, which changed the electrical properties of target T-cells. Hence, CD4 T-cells could be identified from their corresponding subpopulations based on impedance opacity (|Z_10MHz|/|Z_503kHz|). Recently, Hassan et al. reported an impedance biosensor based on differential immunocapture technology to perform cell counting on CD4 and CD8 T-cells with high accuracy (Figure 14(Ci)) ^[2][41][42][111,145,146]. In this device, antibodies specific to CD4 T-cells were initially adsorbed on a chamber between two conventional IFC modules. As the leukocytes flowed into the chamber, CD4 T-cells were captured and immobilized on the antibodies (Figure 14(Cii)). The cell number of each population can be calculated according to the impedance pulses caused by the passage of cells through the chamber. This protocol can be used to enumerate specific cell types with their corresponding antibodies immobilized in the capture chamber.

Tumor diagnosis is underpinned by determining which cells are malignant in acquired biopsy, leading to the need to accurately distinguish DTCs from normal cells in tissue ^[43][2]. Zhao et al. classified two tumor cell lines (A549 and H1299) based on different cellular membrane capacitance (C_m) and cytoplasm conductivity (σ_p) ^[10][117]. Desai et al. separated lung cancer DTCs (LC-DTCs) from RBCs, peripheral blood mononucleated cells (PBMCs) and normal lung cells based on impedance amplitude ^[11][118]. When LC-DTCs pass through the coplanar electrodes, impedance amplitude signal generates more significant pulse compared to that of normal cells. They also determined five major cancer types (lung, thyroid, breast, ovarian, and kidney cancers) from their corresponding counterpart target cells. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer lacking specific biomarkers. Aimed at this situation, McGrath et al. reported an IFC device to separate single PDAC tumor cells against xenografts ^[13][120]. They found that the phase of impedance signal of six PDAC cell types showed some correlations to specific gene expression, especially the KRAS mutations that led to higher phase variation. T188 and T738 are primary stage tumors with unknown KRAS mutations showing lower impedance phase contrast than other PDAC samples. Zhang et al. developed a microfluidic IFC platform with asymmetrical constriction channel to better detect the dielectric properties and diameters of different types of single tumor cells ^[6][43]. The classification accuracy between two tumor cell lines, A459 and HEP G2 cells, could be significantly improved with the combination of the individual intrinsic bioelectrical markers of membrane capacitance, cytoplasm conductivity and cell diameter. Besides, Ostermann et al. reported that necrotic and viable U937 human lymphoma cells could be clearly discriminated based on the phase of impedance signals by using a commercial IFC device ^[14][121]. Dead and viable cells can be discriminated by impedance signals at high frequency as the imaginary component of cell impedance depends on the membrane integrity of the cell (Figure 25A).

Identification and characterization of CTCs in blood stream is key to monitor the progression of cancer metastasis ^[15][122]. Choi et al. proposed a simple DC impedance microcytometer for identifying CTCs according to the cell volume ^[8][115]. Ren et al. reported an IFC device featuring parallel cyclic deformability channels and coplanar electrodes, to collect both biomechanical and bioelectrical properties for tumor cell analysis ^[12][119]. The deformation and transition time of tumor cells could be obtained from the time points when impedance amplitude changes abruptly (Figure 25B). In clinical application, due to the very small amount of CTCs in a blood sample, it is necessary to pre-enrich CTCs before measurement ^[7][9][52,116]. According to the different membrane capacitance between tumor cells and normal PBMCs, Spencer et al. measured the dielectric properties of MCF-7 cells (a representative of CTCs) at 500 kHz and 2 MHz and distinguished them from leukocytes when mixed in the whole blood ^[7][52]. Compared with optical approaches, electrical impedance measurement shows better performance in separating MCF-7 cells from other blood cells (Figure 25C). Besides, Han et al. reported a microfluidic system integrated with both enrichment and impedance detection units to discriminate CTCs ^[9][116]. In this study, immunomagnetic nanobeads (MNBs) and highly-conductive graphene nanoplates (GNPs) were bonded to the surface of DLD-1 cells (a representative colorectal cancer cell line). Compared with normal blood cells, the impedance signal of DLD-1 cells coated with GNPs shows a phase shift of 100 degrees for identification (Figure 25D), successfully.

Hildebrandt et al. demonstrated that the osteogenic differentiation process of human mesenchymal stem cells (hMSCs) could be monitored by tracking their impedance variation ^[16][123]. Song et al. was the first to propose a dual-micropore microfluidic IFC device to monitor the same differentiation process (Figure 36(Ai)) ^[17][18][124,125]. In this device, when MSCs or osteoblasts passed through micropores, a pulse in impedance amplitude was recorded to determine the proportion of differentiated cells at each stage ^[18][125]. Moreover, a support vector machine (SVM) algorithm was employed in data analysis to reach a classification accuracy of 87%. It is notable that the training data set of SVM included a total number of 1028 impedance signals combining both relative phase at 3 MHz and impedance opacity (|Z_2MHz|/|Z_500kHz|). The optimal SVM-based model was also used to characterize the differentiation process (from MSCs into osteoblasts), in which the proportion of osteoblasts was increasing while that of MSCs was decreasing (Figure 36(Aii)). Zhao et al. reported that the intercellular electrical markers, such as specific membrane capacitance (C_{specific membrane}) and cytoplasm conductivity (σ_cytoplasm) of neural stem cells could be used to evaluate their differentiation processes ^[19][126]. During different stages of differentiation, the distribution difference of C_{specific membrane} differs a lot from that of σ_cytoplasm (Figure 36B). Besides, Xavier et al. developed an IFC device equipped with two pairs of facing electrodes combined with confocal microscopic monitoring of the osteogenic differentiation of skeletal stem cells (SSCs) (Figure 36(Ci)) ^[20][127]. The sample of human bone marrow mononuclear cells (hBMMNCs) extracted from human bone marrow (BM) were cultured in vitro and was injected into a microfluidic chip for impedance detection every two weeks. Changes in the opacity (|Z_2MHz|/|Z_500kHz|) of impedance data could characterize the osteoblast differentiation process of SSCs. (Figure 36(Cii)). In the first two weeks (from BM to P0), the decreased opacity corresponded to the increased cell size and membrane capacitance during SSCs osteogenic differentiation. After P0, there was no significant change of opacity indicating the completion of osteogenic differentiation.

Qualifying pollen or spores, especially their viability and germination capacity, is important for industrial production and plant breeding ^[22][128]. Heidmann et al. measured the viability of pollen samples by using a commercial impedance device ^[22][23][128,129]. In one of these studies, pollen samples were measured before and after heat treatment ^[22][128]. As a result, larger phase of the impedance signal corresponding to viable samples was no longer presented after heat-inactivation, which suggested that heat treatment inactivated the pollen samples and destroyed the integrity of cell membrane. Furthermore, Heidmann et al. predicted the germination rate of tomato pollen population by measuring the amount of viable and dead pollens ^[23][129]. Impe et al. ^[24][130] and Ascari et al. ^[25][131] assessed pollen viability of hazelnut and wheat, and further identified various factors (sugar, H₃BO₃, CaCl₂·2H₂O/Ca(NO₃)₂·2H₂O concentration and pH) affecting pollen viability with the same commercial device. Canonge et al. utilized the IFC device to track and characterize the developmental process of wheat (Triticum aestivum L.) genotype Pavon microspores in gametogenesis and anrogenesis ^[26][132]. According to this study, throughout all sporophytic developmental stages, some of the viable microspores showed a continuous increase in both impedance amplitude and phase. As a result, electrical impedance could serve as a fast and reliable reactivity marker for tracking wheat microspores in androgenesis.

The biological and physiological properties of cell wall unique to plant cells offer the potential to increase phenotyping resolution and identify nonanatomic markers ^[44][147]. Han et al. developed an IFC device to characterize the biophysical properties of two model plant species, herbaceous Arabidopsis thaliana and woody Populus trichocarpa ^[21][60]. In the regeneration process of primary cell wall (PCW), plant cells are gradually covered by the fibrillary network, which becomes thick and interlaced, resulting in the decrease of capacitance of cell membrane and PCW ^[45][148]. Thus, the researchers found that the Arabidopsis cells with regenerated PCW were less deformable and electrically conductive than that without PCW.

IFC devices have been utilized extensively in detection, separation and viability analysis of unicellular microbes, including bacteria ^{[27][28][29][30][31][36][37]}[53,62,133,134,135,140,141], protozoa ^[32][46][136,149] and fungi ^[33][34][35][137,138,139].

Bernabini et al. demonstrated the feasibility to detect bacteria according to cell size in an IFC device ^[29][133]. This device features small cross-sectional area of the flow channel and narrow width of electrodes, since the size of bacteria is usually smaller than that of mammalian cells. Without the measurement of signal phase, E. coli could be identified by volume rather than membrane capacitance, and thus E. coli was indistinguishable from particles in similar size ^[29][133]. To solve this problem, phase metric was introduced and thus the viability and species of bacteria could be determined ^[30][134]. In order to precisely measure the diameter of different bacterium, Choi reported an IFC device with position-adjustable virtual wall ^[28][62]. The movement of virtual wall is modulated by adjusting the flow rate of sample suspension focused by low conductive sheath flow. The cross-sectional area of sample flow could be adjusted to approximate that of bacteria, making the impedance changes caused by the bacteria passage more significant (Figure 47A). Recently, Guler et al. merged the amplitude and phase information of impedance signals to achieve higher size sensitivity and detection throughput of bacteria (Figure 47B) ^[31][135]. Besides, Clausen et al. used two simple IFC devices with coplanar and facing electrodes, respectively, to detect different types of bacteria ^[27][53]. These IFC devices could be used to accurately measure any change in bacteria concentration and distinguish methicillin-sensitive Staphylococcus aureus (MSSA) from E. coli according to the impedance phase signal at 8 MHz (Figure 47C). Moreover, Bertelsen et al. detected and characterized E. coli inactivated by ethanol, heat and autoclaving, respectively ^[36][140]. The population of ethanol-treated bacteria showed a similar amplitude to 1.5-μm polystyrene beads, which was consistent with the hypothesis of membrane disruption. Supported by the experimental data, ethanol treatment caused membrane disruption while heat process did no obvious harm to cell membrane. In detail, the loss of membrane integrity corresponded to changes in impedance signal amplitude and phase (Figure 47D). Notably, Spencer et al. developed a method to optimize the prescription of antibiotic by an impedance-based fast antimicrobial susceptibility test (iFAST) ^[37][141]. By applying microfluidic impedance cytometry with differential electrode configuration, the phenotype response (electrical opacity and electrical diameter) of Klebsiella pneumoniae (K. pneumoniae) to specific antibiotic was accurately analyzed.

Besides, accurate recognition and viability analysis of protozoan pathogens have advanced in parasitic diseases diagnosis of human and livestock ^[46][149]. To this end, Mcgrath et al. detected single protozoan oocysts utilizing a continuous IFC system ^[32][136]. The heat treatment performed on Cryptosporidium parvum (C. parvum) lowered the impedance signal amplitude and phase especially at high frequency representing the internal properties of the oocyst (50 MHz) (Figure 58(Ai)). The difference of viable and inactive populations can be enhanced by increasing the conductivity of medium suspension. In addition, according to the amplitude at 250 kHz and phase at 18.3 MHz, the major human-pathogenic species (C. parvum, Cryptosporidium muris (C. muris) and Giardia Lamblia (G. lamblia)) were discriminated from other parasite species that posed little or no risk to human health (Figure 58(Aii)).

Yeast cells, easily accessible and culturable, have been widely used as an important model organism to study cell growth and division in cell cycle progression ^[47][94]. Xie et al. optimized an IFC device with a constriction channel to detect the size of single budding yeast (S. cerevisiae) cells and calculate the late-budding rates of populations ^[35][139]. As shown in Figure 58B, due to the impact of velocity gradient near the constriction channel, rod particles are aligned with the electric field lines, so that the length of rod particles could be assessed by pulse width of impedance amplitude at 1 MHz. In this way, rod and spherical particles could be clearly discriminated. Moreover, late-budding yeast, namely mother cell with a daughter cell that is nearly mature, can be viewed as a rod-shaped cell, while other yeast is approximately oval. Using the same principle, the shape of target cells as well as the budding stages can be obtained. In another work, Chawla et al. developed a microfluidic platform allowing for long-term culturing and independent monitoring of growth rate of budding yeast (Figure 58(Ci)) ^[33][137]. In this device, multiple cell populations were anchored to pads and their daughter cells were then washed away, flowing through the impedance sensing unit. By analyzing the impedance signal phase at 1.5 MHz, passages of cells through electrodes were recorded. Then by counting the flowing cells in unit time, the growth rate of cell population can be calculated. As shown in Figure 58(Cii), the phase fluctuated drastically as the cell passes through the electrodes, and each phase pulse corresponds to a single cell flowing through the sensing area. This device enabled culturing and monitoring of various groups of budding yeast simultaneously. At the same time, cell populations can be exposed to different medium solution and their growth rates can be calculated indirectly from impedance signal phase. In addition, Opitz et al. focused on monitoring and analyzing of yeast population under different culturing conditions ^[34][138]. In their study, impedance signals at 12 MHz were analyzed to characterize cell viability in a three-day brewing process. On the first day, the high phase indicated that the cell population had high viability and they began to breed by large numbers (Figure 58D). By the end of the third day, the cell population showed lower viability. The cell loss could be ascribed to the depletion of oxygen and the accumulated ethanol.