*5.1. Detection of Bacteria*

Ruan et al. used electrochemical impedance spectroscopy to detect *E. coli* O157:H7 [50]. An anti-*E. coli* O157:H7 antibody was immobilized on the surface of an indium tin oxide (ITO) electrode. The binding of the antibody to the antigen changed the impedance signal. The limit of the sensor to detect bacteria was as low as 6 × <sup>10</sup><sup>3</sup> cells/mL. Yang et al. used an impedance sensor to detect Salmonella typhimurium and observed impedance changes during bacterial growth [51]. The material of the interdigitated electrode array was ITO. Four frequencies (10 Hz, 100 Hz, 1 kHz, and 10 kHz) were used to record impedance growth curves in the experiment. The impedance changes only when the bacterial count reaches 105–106 CFU/mL. Experimental data indicated that the greatest impedance change was observed at 10 Hz. [Fe(CN)6]3−/4<sup>−</sup> was used as a redox probe [52], which increases the electron transfer resistance on the antibody-immobilized microelectrode surface. Impedance immunosensors for the detection of Listeria were developed [53], for which TiO2 nanowires were immobilized on gold electrodes, and monoclonal antibodies were immobilized on the nanowires. Listeria was then specifically captured using the antibodies. The impedance immunosensors sense impedance changes induced by the nanowire–antibody–bacteria system. Tan et al. designed a microfluidic impedance immunosensor for the detection of *Escherichia coli* and *Staphylococcus aureus* [54]; in particular, specific antibodies were immobilized on alumina nanoporous membranes, and bacteria were captured on the nanoporous membranes by antibodies. In a 2 h rapid assay, the sensitivity was as high as 102 CFU/mL. J. Lum designed an impedance biosensor, mainly for avian influenza virus subtype H5N1, as shown in Figure 4a [55]. Immunomagnetic nanoparticles and interdigitated microelectrodes were designed on a microfluidic chip. The polyclonal antibody was immobilized on the surface of the microelectrode, which binds to the avian influenza virus H5N1 to generate an impedance signal. Chicken red blood cells were used as biomarkers attached to the interdigital microelectrodes. The results showed that the impedance signal was increased by more than 100%. An impedance biosensor with focusing and sensing electrodes was designed to detect *E. coli* O157:H7 [56]. The sensitivity lower limit of detection for impedance sensor measurements was 3 × <sup>10</sup><sup>2</sup> CFU/mL. By focusing on p-DEP, the measurement sensitivity can be increased by 2.9 to 4.5 times. Couniot et al. integrated a CMOS process to design an impedance sensor for bacterial detection in urine, as shown in Figure 4b [57], where the detection of impedance spectroscopy was mainly directed at *Staphylococcus epidermidis*. Pyrex wafers were used as substrates for the impedance sensors. Liu et al. developed impedance-based biosensors that can be used to simultaneously detect Salmonella Serotypes B, D, and E by relying on three sensing IDE arrays, as shown in Figure 4c [58]. The sensor was designed with focusing electrodes to generate dielectrophoretic force, where the focusing force can increase the sensitivity by 4–4.5 times, and the detection limit is as low as 8 cells/mL. Other results indicated that the sensor could also distinguish between dead and live cells. Dastider et al. designed a microfluidic impedance sensor that can sense low concentrations of *E. coli* O157:H7 [59]. Upstream of the microchannel, the authors designed interdigitated focusing electrodes, in which the electrodes were arranged in a 45-degree-inclined manner. Positive DEP forces were used to focus cells in the center of the microchannel. Then, downstream of the microchannel, three sets of interdigital electrode arrays (IDEAs) were designed to sense impedance. An *E. coli* antibody was functionalized on the sensing electrode, which captured *E. coli* and resulted in a change in impedance. Experiments have shown that the microfluidic impedance sensor can detect coliform bacteria at a concentration of 39 CFU/mL. A microfluidic impedance sensor was used to detect Salmonella Serotypes B and D in food, as shown in Figure 4d [60]. There are two sensing areas in the chip, and the sensing electrodes are composed of interdigitated electrodes. The shocks were coated with antibodies against Salmonella. The experimental results demonstrate that the impedance sensor can detect Salmonella as low as 300 cells/mL.

**Figure 4.** Glass-based impedance biosensors for detecting bacteria. (**a**) A polyclonal antibody was immobilized on the surface of a microelectrode and bound to avian influenza virus H5N1 to generate an impedance signal. Reproduced with permission from [55]. Copyright Biosensors and Bioelectronics 2012. (**b**) Pyrex wafers were used as substrates for impedance sensors. Reproduced with permission from [57]. Copyright Biosensors and Bioelectronics 2015. (**c**) An impedance-based biosensors were used to simultaneously detect Salmonella Serotypes B, D, and E. Reproduced with permission from [58]. Copyright Scientific Reports 2018. (**d**) There were two sensing areas in the chip, and the sensing electrodes were composed of interdigitated electrodes. Reproduced with permission from [60]. Copyright PLoS ONE 2019.

Many of the keys aspects in the design of impedance biosensors used to sense bacteria are related to improving the sensitivity. As the combination of antigen and antibodies can lead to changes in the impedance signal, many studies on antibodies immobilized on electrodes have been carried out.

## *5.2. Detection of Blood Samples*

The manufacturing of electrical impedance measurement systems became possible with microfabrication technology. For example, wet etching was used to obtain microchannels on a coverslip [2], and gold electrodes were plated on both sides of the microchannel. The authors scanned the electric impedance spectroscopy of ionic salt solutions, air, and deionized (DI) water in the frequency range from 100 Hz to 2 MHz. Their experimental results showed the efficacy of electrical impedance spectroscopy for human polymorphonuclear leukocytes and teleost fish red blood cells. Mishra et al. used three microelectrodes on a chip to sense the impedance of human CD4(+) cells in blood, as shown in Figure 5a [61]. The reference electrode, working electrode, and counter electrode were microfabricated on glass wafers. As the protein adsorbed onto the microelectrode surface, the detected impedance value increased even more. The impedance also increased with the number of captured cells. Kuttel et al. used impedance spectroscopy to detect red blood cells infected with *Babesia bovis* [62]. The change in impedance was mainly due to the presence of the parasite in the cell changing the impedance value of the original red blood cells, white blood cells, or platelets. Therefore, infected cells could be easily and quickly distinguished from healthy cells. In areas without good infrastructure or in very remote areas, the use of impedance spectroscopy to detect parasites in whole blood samples can greatly reduce the time of diagnosis for medical personnel. Holmes et al. developed a high-speed microfluidic single-cell impedance cytometer using dual frequency for whole blood analysis [63], which is mainly used for the impedance measurement and identification of T lymphocytes, monocytes, and neutrophils. The experiments showed that, at low frequencies, T lymphocytes and neutrophils can be distinguished. The cells were conjugated with fluorescently labeled antibodies, allowing the system to analyze fluorescence and impedance simultaneously. Han et al. developed a microfluidic chip that integrates red blood cell lysis with a microfluidic impedance cytometer, as shown in Figure 5b [64]. Their laboratory developed a buffer that not only lyses red blood cells but also increases the identification of monocytes and neutrophils. The method for multi-step cell lysis described in this paper is of great help for the microfluidic system, in terms of whole blood analysis. Lei et al. designed an electrical impedance to monitor the blood coagulation process in a microfluidic chip, which can obtain impedance results consistent with clinical reports at different temperatures and blood cell counts, as shown in Figure 5c [65]. This device provides a new analytical method for the sensitive and real-time monitoring of coagulation in whole blood samples. Song et al. developed a microfluidic impedance flow cytometer to identify undifferentiated and differentiated mouse embryonic stem cells [66], where two micropores and three electrodes were designed in the chip. The experimental results indicated that undifferentiated stem cells and polystyrene spheres could be distinguished at any frequency, while undifferentiated and differentiated stem cells require higher frequency and opacity to be distinguished. Du et al. designed an electrical impedance flow cytometer targeting red blood cells infected with *Plasmodium falciparum* [67]. The physiological and electrical properties of erythrocytes were altered 48 h after *P. falciparum* infection. In addition to the cytometer, the authors incorporated new offset parameters to make it easier to distinguish infected erythrocytes from uninfected erythrocytes. Spencer et al. used a microfluidic impedance cytometer for the detection of a representative circulating tumor cell (the MCF7 tumor cell line) [68]. The red blood cells were removed by lysis, and the buffer did not affect the dielectric properties of the MCF7 cells. Through impedance analysis, MCF7 cells were shown to have a larger size and membrane capacitance. The experimental results indicated that 100 MCF7 cells could be detected in 1 mL of whole blood. The average recovery rate was as high as 92%. Liu et al. developed an electrical impedance microflow cytometer that can control oxygen flow for the analysis of sickle blood cells, as shown in Figure 5d [69]. The two-layer microfluidic channel was separated by a 150 μm thick PDMS film. The upper layer is a serpentine gas channel that controls oxygen, while the lower layer is a microchannel through which sickle cells and red blood cells flow. Ti/Au electrodes were designed to measure the impedance of sickle cells in the lower microchannel. Under normoxic conditions, the authors distinguished between normal and sickle cells using impedance signals measured at intermediate frequencies. The same research demonstrated that impedance signals can be obtained without the need for hemolysis. Their experimental results also proved that the impedance signal of the microflow cytometer can be used as an indicator of red blood cell disease and sickle cell disease.

**Figure 5.** Glass-based impedance biosensors for detecting blood. (**a**) Three microelectrodes were used to sense the impedance of human CD4(+) cells in blood. Reproduced with permission from [61]. Copyright Biosensors and Bioelectronics 2005. (**b**) A microfluidic impedance cytometer was for red blood cell lysis. Reproduced with permission from [64]. Copyright Analytical Chemistry 2011. (**c**) A microfluidic chip was designed to detect electrical impedance for monitoring the blood coagulation process. Reproduced with permission from [65]. Copyright PLoS ONE 2013. (**d**) An electrical impedance microflow cytometer with controlled oxygen flow for the analysis of sickle blood cells. Reproduced with permission from [69]. Copyright Sensors and Actuators B: Chemical 2018.

#### *5.3. Static Cell Analyzed by Electrical Impedance Spectroscopy*

A microfluidic impedance sensor was designed to measure the cell migration of cancer cells in a three-dimensional extracellular matrix [70]. A total of 16 sets of sensing electrode arrays and cell grabbing arrays were designed in the microchannel. Under continuous monitoring, the migration of MDA-MB-231 cells allowed for a rapid change in impedance amplitude (of about 10 Ω/s). Liu et al. designed a microfluidic chip with embedded measurement electrodes to monitor the cell migration process using impedance measurement technology [71]. Cells were measured and recorded in the microfluidic channel as they pass through multiple parallel electrodes. This method enables the accurate and objective recording of cell migration activity and the calculation of migration rates among different stimulating drugs. Huang et al. designed a microchannel filled with Matrigel to quantify cell migration velocity as an assay tool, as shown in Figure 6a [72]. The successful measurement of cells suspended in a 3D environment and the induction of cell migration by stimulatory factors were used to record the migration speed of cells. The measurement sensitivity was better than that of a traditional trans-well assay.

**Figure 6.** Static cell analyzed with electrical impedance spectroscopy. (**a**) A microchannel was designed filled with Matrigel to quantify cell migration velocity. Reproduced with permission from [72]. Copyright Analytica Chimica Acta 2020. (**b**) This chip was designed to induce angiogenesis to extend into the microchannel. Reproduced with permission from [73]. Copyright Sensors and Actuators B: Chemical 2022. (**c**) The relationship between the electrochemical impedance spectroscopy and withstand voltage was established with four different cells (HeLa, A549, MCF-7, and MDA-MB-231). Reproduced with permission from [74]. Copyright Sensors and Actuators B: Chemical 2012. (**d**) Because of the constriction of the microchannel, tumor cells, thus, are elongated for sensing impedance. Reproduced with permission from [75]. Copyright Biosensors and Bioelectronics 2014. (**e**) A microfluidic impedance sensing chip with droplets and microelectrode arrays was used to monitor the osteogenic differentiation of bone marrow mesenchymal stem cells. Reproduced with permission from [76]. Copyright Biosensors and Bioelectronics 2019. (**f**) Single stem cells were captured in a 20 μm chamber by dielectrophoresis. Reproduced with permission from [77]. Copyright Talanta 2021.

Lei et al. developed a perfusion three-dimensional (3D) cell culture microfluidic chip combined with real-time and non-invasive impedance monitoring [73]. This device can simulate complex 3D biological microenvironments to culture cells and monitor the impedance changes under different concentrations of drug stimulation through impedance measurements. The impedance results are analyzed to determine the cell proliferation and chemosensitivity of 3D cell cultures. Lei et al. designed an impedance measurement device for cell colonies cultured on hydrogels [74–76]. Huang et al. constructed a 3D biological barrier using Matrigel and induced angiogenesis to extend into the microchannel, as shown in Figure 6b [77]. The angiogenesis process could be monitored by label-free impedance, using electrodes at the bottom of the microchannel. The device can also successfully quantify the time and distance of angiogenesis, thereby providing a reliable and quantitative method for the assay of angiogenesis.

Bieberich et al. developed electrical cell impedance spectroscopy to monitor the impedance response of PC12 and embryonic stem cells forming synapses [78]. Jang et al. published a study combining a cell capture method with microfabricated impedance spectroscopy [79], in which three micropillars were designed in the center of the microchannel to capture single HeLa cells. Cho et al. detailed the integrated microfluidic capture of singlecell technology with electrical impedance spectroscopy [80]. Hildebrandt et al. developed electrochemical impedance spectroscopy to distinguish the osteogenic differentiation of human mesenchymal stem cells [81]. In the application of cellular impedance to infectious parasites, Houssin et al. designed an electrochemical impedance spectroscopy approach to detect the presence of oocysts [82]. Dalmay et al. developed impedance spectroscopy to distinguish cancer stem cells and U87 glial cells (differentiated cells) [83]. The impedance spectrum designed by Bagnaninchi et al. can instantly monitor adipose stem cell (ADSC) differentiation [84]. Hong et al. established the relationship between electrochemical impedance spectroscopy and the withstand voltage of four different cells (HeLa, A549, MCF-7, and MDA-MB-231), as shown in Figure 6c [85]. Under a strong electric field, the cytoplasmic resistance decreases due to the opening of ion channels. The experimental results showed that different cells have not only different impedance spectra but also different withstand voltages. Chen et al. designed a microfluidic chip for capturing single cells and measuring impedance values [86]. Zhao et al. proposed to convert the measured impedance values into membrane capacitance (C-specific membrane) and cytoplasmic conductivity (σ cytoplasm), as shown in Figure 6d [87]. Due to the constriction of the microchannel, tumor cells become elongated. The experimental results demonstrated that tumor cells can be distinguished using two parameters: C-specific membrane and sigma cytoplasm. Ruan et al. integrated dielectrophoretic force and impedance sensors to detect lung circulating tumor cells [88]. Fan et al. designed a microfluidic impedance sensing chip with droplets and microelectrode arrays to monitor the osteogenic differentiation of bone marrow mesenchymal stem cells, as shown in Figure 6e [89]; the authors also proposed a model of cellular droplets. Lei et al. captured single stem cells in a 20 μm chamber by dielectrophoresis, as shown in Figure 6f [90]; the cells were measured in the impedance spectrum range of 2–20 kHz.

#### *5.4. Dynamic Cell Analyzed by Microfluidic Impedance Cytometry*

The development of microfluidic impedance flow cytometry is important for cell analysis. In this subsection, microfluidic impedance flow cytometry is divided into two parts for discussion: one is the principle of microfluidic impedance flow cytometry, and the other includes the sensing applications of microfluidic impedance flow cytometry.

Sun et al. designed two microfluidic impedance cytometers with parallel facing electrodes and coplanar electrodes [91]. For impedance measurements, parallel facing electrode designs are more sensitive than coplanar electrode designs. Holmes et al. used a microfabricated flow cytometer for the discrimination of micron-sized polymer beads [92]. Fluorescently labeled proteins were immobilized on the beads, which can be used to analyze the immune response. Negative dielectrophoretic force was used to focus the

polymer beads by the focusing electrodes. An electrical impedance flow cytometer for the high-speed analysis of particles was developed [93]. The impedance signal of polystyrene beads could be obtained in as little as 1 ms. Compared with microchannels fabricated by soft lithography, Kummrow et al. used ultraprecision milling technology to design 3D microchannels with horizontal and vertical focusing capabilities [94]. Fiber optics, mirrors, and electrodes were integrated into a flow cytometer for blood cells. Spencer et al. conducted a study on how the position of particles in a microchannel affects impedance measurements [95]. Impedance is related to the position of the particle in the vertical direction. A flow cytometer was designed by Barat et al. in order to measure both the optical and electrical properties of particles [96]. Daniel Spencer integrated optical fibers and waveguides into an impedance flow cytometer to measure electrical impedance (electrical volume and opacity), fluorescence, and large-angle side scatter without the need for particle focusing [97]. Haandbæk et al. published an article on microfabricated flow cytometry at high frequencies [98]. The experimental results indicated the ability at high frequencies to distinguish not only wild-type yeast and mutant strains but also opacity values at frequencies above 50 MHz.

David et al. used impedance flow cytometry to measure the viability and membrane potential of *Bacillus megaterium* cells [99]. A microfluidic impedance cytometer was designed for platelet analysis by Evander et al. [100], where the focusing electrodes allow for secondary focusing of the sample through the dielectrophoretic force. Lin et al. used a microfluidic impedance cytometer to detect quantified protein biomarkers [101]. For whole blood analysis, Simon et al. developed a microfabricated AC impedance cytometer with multi-frequency AC impedance and side scatter analysis capabilities [102]. Xie et al. proposed the concept of using a microfabricated impedance cytometer to detect electronic biomarkers [103]. By changing the dielectric properties of the particle, the authors designed a nanoelectronic barcode particle as an electronic biomarker. McGrath et al. used a microfluidic impedance flow cytometer to distinguish the oocysts of protozoan parasites, as shown in Figure 7a [104]. This chip can distinguish between *Cryptosporidium parvum*, *Cryptosporidium muris*, and *Giardia lamblia* within minutes. A microfluidic impedance cytometer integrating inertial focusing and liquid electrodes was developed for the high-throughput measurement of human breast tumor cells and leukocytes, as shown in Figure 7b [105]. The purpose of inertial focusing is to reduce cell adhesion and ensure that single cells pass through the sensing area. An interesting study involved the use of two microneedles, placed on either side of a microchannel to sense impedance values [106]. For clinical analysis and judgment, Sun et al. used multi-frequency impedance spectroscopy and machine learning to rapidly distinguish the survival of cancer cells under the action of anti-matriptase-conjugated drugs [107]. Sui et al. developed an impedance flow cytometer to detect spheroid green algae cells (Picochlorum SE3) at different salt concentrations, as shown in Figure 7c [108]. Mahesh et al. published a study on the observed "double-peak" characteristics of individual cells with high sensitivity to changes in cell membrane capacitance [109]. This phenomenon has limitations: it operates at the lower frequencies (400–800 kHz) of the beta-dispersion mechanism, and the microelectrodes must be coplanar and paired. The authors pointed out that changes in cell size and membrane capacitance can be resolved using a single frequency. A microfluidic impedance cytometer was used for the analysis of antigen-specific T lymphocytes, as shown in Figure 7d [110]. The experimental results demonstrated that differences in impedance can be observed among dead, healthy, and activated lymphocytes. Caselli et al. used artificial intelligence methods to decipher signals from microfluidic impedance cytometers [111]. The authors demonstrated two advances: (i) the use of a neural network to determine the dielectric properties of single cells in raw impedance data streams and (ii) resolving the impedance signatures of coincident cells. The results demonstrated that the neural network could increase the signal processing capability of the microfluidic impedance cytometer.

**Figure 7.** Dynamic cell analyzed by microfluidic impedance cytometry. (**a**) A model of the impedance flow cytometer was established to distinguish Cryptosporidium parvum, Cryptosporidium muris, and Giardia lamblia in minutes. Reproduced with permission from [104]. Copyright Scientific Reports 2017. (**b**) A microfluidic impedance cytometer integrating inertial focusing and liquid electrodes was developed. Reproduced with permission from [105]. Copyright Analytical Chemistry 2017. (**c**) An impedance flow cytometer was developed to detect spheroid green algae cells (Picochlorum SE3) at different salt concentrations. Reproduced with permission from [108]. Copyright Scientific Reports 2020. (**d**) A microfluidic impedance cytometer was used for the analysis of antigen-specific T lymphocytes. Reproduced with permission from [110]. Copyright Sensors and Actuators B: Chemical 2021.
