2. EIS to Detect Suspended or Adherent Single Cells
Different from IFC devices that are commonly used for cell recognition and screening with high throughput, EIS sensing devices are capable of extracting broadband impedance information and tracking dynamic variations of single cells. Recent EIS sensing devices applied in single-cell analysis are summarized in
Table 2. These devices are classified into two categories: one is to determine the optimal frequency at which the impedance of different cell lines or cell states is most sensitive
[48][49] and the other is to continuously monitor the dynamic cell process or cell behavior and phenotypic changes
[50][51][52][53][47][54][45][55][56][57].
Table 2. Applications of EIS measurement for single cells. OT: Observation time. Throughput: Maximum number of single cells that can be simultaneously measured.
EIS sensing technology has been used to investigate the optimal frequency at which the characteristic parameters extracted from EIS signals are most prominent in measuring specific dielectric properties of cells
[48][49]. Park et al. proposed two types of devices to distinguish cancerous from normal human urothelial cell lines (
Figure 6(Ai))
[49]. In one device, single cells were captured at 3D traps by applying negative pressure underneath. Then, the impedance of immobilized single cells was individually measured at frequencies from 5 kHz to 1 MHz. According to the EIS signals in
Figure 6(Aii) plot, 119 kHz was supposed to be the optimal frequency, at which the impedance of two types of cells had the greatest divergence. The real-time impedance of the cell lines was measured at 119 kHz in the other device (an IFC device) to identify cancerous cells. These two devices potentially provide a supplementary platform to detect urothelial cancer of the bladder (UCB). In another study, Tang et al. developed a portable single-cell analytical system combining hydrodynamic traps and EIS measurement to accurately detect the sizes of MCF-7 cells
[48]. Under the hydrodynamic forces, MCF-7 cells could be initially captured at the entrance of the narrow channel and then squeezed into it. Impedance signals were collected from three groups, among which one is the control group of PBS solution without cells, another one is the trapped cells in suspension, and the third one is the squeezed cells (
Figure 6B). According to the sweep-frequency measurement of EIS, the frequency was optimized to 500 kHz, at which, cellular trapping-releasing-squeezing manipulation and cell size could be detected more accurately.
Figure 6. Cell-based assay using EIS sensing devices. (
A) (
i) SEM images of the two devices used to detect cancerous urothelial cells. Left one is an EIS sensing device with a negative pressure trap used to investigate the optimal frequency. Right one is an IFC device to perform high-throughput electrical impedance measurement of normal and cancerous urothelial cells. (
ii) Measurement of the amplitude difference between normal and cancerous urothelial cells in the frequency range of 5 kHz to 1 MHz. Reproduced from
[49] with the permission from Hindawi. (
B) Schematics of a EIS sensing device to measure the amplitude and phase signal of MCF-7 cells under three typical conditions: PBS solution without cells, cell trapped and cell squeezed. Reproduced from
[48] with the permission from Springer. (
C) Using a EIS sensing device with microfluidic traps to distinguish the undifferentiated and differentiated cells by measuring the impedance over the frequency range from 100 kHz to 10 MHz. Reproduced from
[54] with the permission from Elsevier. (
D) (
i) Schematics of an EIS-integrated single-cell culturing device for immobilization and impedance recording of
Schizosaccharomyces pombe (
S. pombe) cells. (
ii) Recorded EIS amplitude and phase signals over the frequency range from 10 kHz to 10 MHz showing the growth and division of single
S. pombe cells. Reproduced from
[47] with the permission from Nature. (
E) Imaginary part of current response for
Arabidopsis mesophyll cells at different status (0 h, 12 h and 24 h after incubation, respectively). Reproduced from
[45] with the permission from Elsevier. (
F) The Bode impedance spectra measured on working electrode before and after cell migration, as well as on reference electrode without cells over the frequency range from 100 Hz to 1 MHz. Reproduced from
[51] with the permission from American Chemical Society. (
G) Recording of |Z|
norm for HeLa cells in the recovery process under different conditions of electroporation.
A,
N,
w and
f stand for pulse amplitude, number, width and frequency, respectively. Reproduced from
[57] with the permission from Nature.
EIS sensing technology has been used to monitor cell behavior and phenotypic changes, including differentiation of stem cells
[53][45][58], cell growth and division
[50][47][59], formation of cell wall
[54], migration of tumor cells
[51][56] and recovery process after electroporation
[57].
In order to characterize the differentiation process of stem cells, Zhou et al. analyzed the impedance data from mouse embryonic stem cells (mESCs) at different time points in a cell differentiation cycle
[54]. In this study, impedance opacity (|Z
1MHz|/|Z
50kHz|) was increasing during the 48-h cell differentiation process, and was significant at above 1 MHz (
Figure 6C). Based on this finding, they observed the metastable transition state, from which stem cells could either differentiate irreversibly or return to pre-differentiation state at 24 h. Zhang et al. proposed a multifunctional microfluidic chip, which featured DEP trapping, electrical stimulation and real-time impedance monitoring of single cells
[53][57][58]. They recorded the real-time impedance changes of two groups of MSCs with (OM group) or without electrical stimulation (OM + ES group)
[53]. The results showed that electrical stimulation could accelerate the response to drug and advance the differentiation of MSCs. Besides, this device provided additional phenotypic indicators that were not available in cell traction force sensor and contributed to multimodal characterization of long-term physiological variations in the cell differentiation process
[58].
Ghenim et al. were the first to monitor the impedance variation in the mitosis of a single mammalian cell
[59]. Zhu et al. presented a microfluidic cell-culturing chip to trap, cultivate and selectively release individual yeast cells
[60]. Then, this device was used to monitor the cell dynamics in a cell cycle of yeast cells (
Figure 6(Di))
[50][47]. As an example, electrodes originally used to generate DEP forces were used to measure the electrical impedance spectrum of rod-shaped
S. pombe cells, which were immobilized in an upright position at the traps
[47]. Cell growth, nuclear division and cytokinesis in a cell cycle were sensitively characterized by EIS signal amplitude at 1 MHz and phase at 5 MHz (
Figure 6(Dii)).
Chen et al. investigated the formation process of primary cell wall of
Arabidopsis mesophyll cells
[45]. The formation of the cell wall reduced the capacitance of entire plant cell and thus led to an increase in the imaginary part of impedance signal
[21]. In support of this hypothesis, they measured the differential current response of
Arabidopsis mesophyll cells at three status of cell wall formation (
Figure 6E).
Cell migration, which serves as the initiation of cancer metastasis, could be recorded by ECIS technology
[61]. Primiceri et al. demonstrated that cell migration could be monitored and automatically analyzed by a EIS biochip
[56]. Nguyen et al. proposed a microfluidic chip with ECIS for monitoring the migration of single cancer cells in 3D matrixes
[51]. In this study, the impedance measurements were performed with a voltage of 10 mV over the frequency range from 100 Hz to 1 MHz and showed the significant decrease of EIS amplitude after cell migration (
Figure 6F). The real-time EIS recording was carried out at 4 kHz and demonstrated that MCF-7 cells were less metastatic than MDA-MB-231 cells. Zhang et al. monitored the recovery processes of HeLa cells after electroporation by using impedance measurement (
Figure 6G)
[57]. HeLa cells were trapped and electroporated with different working modes of center electrodes. Within 5 min after electroporation, normalized amplitude curves were slowly rising corresponded to the reversible EP processes, while those stabilizing at the minimum values indicated the irreversible EP and cell death.