*5.5. Detection of Viruses*

The key to the use of impedance biosensors for virus sensing is the immobilization of antibodies, where the antibody must be immobilized on the surface of the electrode first. However, the impedance signal will be altered due to the binding of both antibodies and antigens.

Pathogenic avian influenza viruses have been detected by impedance sensors [112]. The polyclonal antibody was immobilized on the surface of a gold microelectrode by protein A. The antibody–antigen binding reaction in the same research could be amplified by red blood cells, and the proposed impedance immunosensor could be completed in as little as 2 h. A portable impedance biosensor for avian influenza virus H5N2 was developed by Wang et al. [113]. Magnetic nanobeads and interdigitated array microelectrodes were integrated on a microfluidic chip, where the magnetic nanobeads captured avian influenza virus subtype-specific antibodies. The entire detection time was less than 1 h, which was greatly reduced compared with traditional avian influenza virus detection. The experimental results indicate that the sensitivity of the impedance biosensor is comparable to that of real-time reverse transcriptase PCR. Jacob Lum designed an impedance biosensor, mainly for avian influenza virus subtype H5N1 [55]. For this purpose, 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 avian influenza virus H5N1 to generate an impedance signal. Chicken red blood cells were used as biomarkers attached to interdigital microelectrodes. The results showed that the impedance signal can be increased by more than 100%. For the detection of human immunodeficiency virus, Shafiee et al. designed a microfluidic sensing chip to measure the impedance spectrum of virus nanolysates [114]. Two gold electrodes were designed on both sides of the microchannel cavity. The lysis of the virus results in the release of the ions and charged molecules of the virus into a non-ionic solution. That research demonstrated that impedance spectroscopy provides a convenient and rapid tool for the detection of multiple pathogens. Singh et al. developed an electrical impedance sensing chip to detect influenza H1N1 virus, as shown in Figure 8a [115,116], for which three microelectrodes were fabricated on a glass substrate. The authors used reduced graphene oxide and monoclonal antibodies as electrode modifications.

**Figure 8.** Glass-based impedance biosensors for detecting other organisms and chemicals. (**a**) An electrical impedance sensing chip was developed to detect influenza H1N1 virus. Reproduced with permission from [115]. Copyright Scientific Reports 2017. (**b**) Impedance sensors were developed for DNA hybridization. Reproduced with permission from [117]. Copyright Sensors and Actuators B: Chemical 2011. (**c**) Single-stranded DNA probes were functionalized onto electrodes. Complementary DNA hybridization was then induced using electrochemical impedance spectroscopy. Reproduced with permission from [118]. Copyright Biosensors and Bioelectronics 2012. (**d**) Galectin-1 protein is a biomarker of bladder cancer. An impedance immunosensor was developed to detect Galectin-1 protein in urine. The dielectrophoretic force was used to capture Galectin-1 antibody to improve the sensitivity of the sensor. Reproduced with permission from [119]. Copyright Biosensors and Bioelectronics 2016.

#### *5.6. Detection of Other Analytes and Chemicals*

Berdat et al. used an impedance sensor based on an interdigitated microelectrode array to sense DNA [116], for which 5 μm wide microelectrodes were fabricated using a lift-off process method. The complementary probe was first immobilized on the electrode and hybridized to the target ssDNA. Finally, impedance sensors were used to detect the pathogen *Salmonella choleraesuis* in dairy products. Javanmard et al. developed a set of impedance sensors for DNA hybridization, as shown in Figure 8b [117]. Oligonucleotide probes were immobilized on the surface of the microchannel, and target DNA strands were immobilized on the surface of polystyrene beads. Contact between the probe and the target DNA strand results in the hybridization of the DNA, leading to the capture of the polystyrene beads on the surface of the microchannel. An impedance chip for sensing DNA hybridization was developed by Hadar et al., as shown in Figure 8c [118]. Single-stranded DNA probes were functionalized onto electrodes, and complementary DNA hybridization was then induced using electrochemical impedance spectroscopy. DC-biased AC electroosmotic vortex was utilized to design a label-free electrochemical impedance spectroscopy (EIS)-based DNA biosensing chip [119]. Based on the electro-osmotic vortex, 20-base target DNA fragments were hybridized to achieve 90% within 141 s. The ultrasensitive detection limit was 0.5 aM. Another study indicated that the electric field was manipulated by alternating current (AC) electrokinetics to improve hybridization efficiency and reduce hybridization time [120]. Thus, the chip was realized for faster and more efficient detection.

Galectin-1 protein is a biomarker of bladder cancer. An impedance immunosensor for detecting bladder cancer in urine was developed, as shown in Figure 8d [121]. Before measuring the impedance signal, the authors used dielectrophoretic force to capture nanoprobes (Gal-1 antibody) on the surface of the microelectrode, in order to improve the sensitivity of the sensor. Alsabbagh et al. designed a microfluidic impedance biosensor for the detection of myocardial infarction proteins by electrochemical impedance spectroscopy [122]; in particular, Troponin I, which is a biomarker for the diagnosis of myocardial infarction, was targeted. Self-assembled thiolated oligonucleotides tested on gold electrodes were found to perform better, as they improved the performance of the impedance signal. Fluorescence analysis and electrochemical impedance spectroscopy were integrated to measure aggregated C-reactive proteins [123]. The circular array of electrodes was designed to create electrokinetic flow for C-reactive protein aggregation. Interdigitated microelectrode arrays were modified by the self-assembled monolayers of mercaptocaproic acid for detecting the arthritis anti-CCP-ab biomarker [124]. The experimental results showed that the sensor response increased linearly with the stepwise increase of the biomarker concentration. A polyaniline (PANI)/MoS2-modified screen-printed electrode was detected for anti-cyclic citrullinated peptide [125]. Among them, the polymerized PANI-Au nanomatrix was utilized to entrap the aCCP antibodies for amplification of the higher signal. A peptide-based electrochemical sensor was used to detect autoantibodies for the diagnosis of rheumatoid arthritis [126]. The developed peptides were modified on the gold surface of the working electrode by a self-assembled monolayer method. Subsequently, the sensor was clinically demonstrated to have better sensitivity using 10 clinically validated samples from rheumatoid arthritis patients and 5 healthy control samples.

Chiriaco et al. designed a microfluidic electrochemical impedance biosensor to detect cholera toxin [127] with sensitivity less than 10 pM. Liu et al. developed a biosensor for the rapid screening of toxic substances in drinking water [128]. Cells are damaged due to toxins in water, resulting in decreased impedance and an increase in resonance frequency. Impedance and mass sensing measurements can help to improve sensor accuracy. An impedance biosensor was used to detect the cytotoxicity of tamoxifen in cervical cancer cells [129]. The experimental results showed that the dose of tamoxifen resulted in a significant reduction in the number of HeLa cells. The same article demonstrated that impedance biosensors can be used for the evaluation of novel drugs and cytotoxicity.

A microfluidic impedance sensor was designed for pesticide detection in vegetables [130]. Anti-chlorpyrifos monoclonal antibody was immobilized on an interdigitated electrode array. The capture of chlorpyrifos produced a change in impedance. Zeng et al. integrated magnetic focusing into impedance microsensors for oil monitoring [131]. The highly focused magnetic field was derived from two electromagnetic coils and eight silicon steel tips, where the silicon steel tips greatly improved the sensitivity of the sensor.

#### *5.7. Conclusions of the Glass Chips*

Microfluidic impedance sensor glass chips have two great development directions: electrochemical impedance spectroscopy and electrical impedance flow cytometry. The difference between the two lies in the state of the measured object. For example, in an electrical impedance flow cytometer, as shown in Figure 6, the cells are stationary. The cells may be attached to the bottom of a microgroove or a microchamber, where the culture medium is quiescent. On the other hand, cells may be trapped by the microstructures and focused by the dielectrophoretic force, thus, fixing them in one position. In an electrical impedance flow cytometer, the cells are in a dynamic state, as shown in Figure 7. For the impedance sensing of other organisms, the main technology relies on the modification of electrodes and the combination of antigens and antibodies.

#### **6. Paper-Based Impedance Biosensors**

Lei et al. combined the electrical impedance measurement technique with the hydrophilic properties of a paper base into a system for recording and analysis [132]. Through impedance analysis, a trend proportional to cell proliferation was observed. A paperbased electrochemical impedance DNA sensor for tuberculosis detection was developed by Teengam et al., as shown in Figure 9a [133]. Carbon graphene inks were printed as working and counter electrodes, while reference electrodes and conductive pads were screen-printed with silver/silver chloride ink. Pyrrolidine peptide nucleic acid (acpcPNA) was immobilized on cellulose paper, and changes in impedance were induced in the presence of *Mycobacterium tuberculosis*. Rengaraj et al. developed paper-based electrodes for the impedance detection of bacteria in water, as shown in Figure 9b [134]. The paper-based electrodes were made of carbon on hydrophobic paper formed by screen printing. The cellulose was cross-linked before use in order to enhance the strength of the paper substrate and the electrical properties of the screen-printed electrodes. The same article was the first to combine the hydrophobicity of paper substrates with the electrochemical functionalization of electrodes. A paper-based microfluidic impedance chip was developed to sense alpha-fetoprotein in human serum using peptide-modified plastic paper [135]. The sensor included two layers, where the upper layer was cellulose chromatography paper, and the lower layer was plastic paper. Among them, the sensing electrodes were printed with Ag-20 wt % graphene. The limit value of alpha-fetoprotein in PBS detected by the sensor was 1 ng/mL. Lei et al. used hydrogel to encapsulate cells and then cultured them on top of paper substrates [136]. The resulting analysis could more accurately distinguish the impedance differences between two cells and under the action of a drug. Some studies have used electrochemical impedance spectroscopy to detect miRNA-34a, which is a biomarker of cancer and Alzheimer's disease [137]. PAMAM dendrimers were modified on the surface of screen-printed electrodes. The results indicated that the difference between miRNA-34a and miRNA-15 or miRNA-660 could be distinguished through electrochemical impedance spectroscopy. Cardiac troponin I is a biomarker for the early diagnosis of acute myocardial infarction. A paper-based impedance immunosensor was developed to detect cardiac troponin I [138], for which multi-walled carbon nanotubes were immobilized on carbon ink electrodes. Then, cardiac troponin I antibody was immobilized on multi-walled carbon nanotubes. Finally, cardiac troponin I was captured by the antibody, affecting the impedance signal. Li et al. designed paper-based electrochemical impedance spectroscopy to detect coronavirus (COVID-19), as shown in Figure 9c [139]. The carbon ink was first printed on paper, and a layer of zinc oxide nanowires was grown on the carbon ink. Then, probes and blocking molecules were immobilized on its surface. Finally, the target molecules were captured for impedance sensing. The results showed that the enhanced ZnO nanowires can improve impedance sensitivity. Electrochemical impedance spectroscopy was used to analyze artificial sweat using hand-painted electrodes [140]. Electrodes were drawn on the opposite side of the paper in order to reduce the double-layer capacitance. The silver electrode pattern paper chip had stronger impedance stability than the graphite electrode paper chip. Research and development of paper-based electrochemical impedance spectroscopy

was conducted for the detection of microRNA 155 [141], for which gold nanoparticles changed the properties of paper-based electrodes. S. Karuppiah developed a paper-based impedance biosensor for monitoring bacteria in water [142]. The electrodes of the biosensor were screen-printed with graphene (G) and then surface-modified with graphene oxide (GO). The lectin concanavalin A was then immobilized on the modified electrode described above. A paper-based impedance sensor was used to sense miRNA-155 and miRNA-21 for the early diagnosis of lung cancer, as shown in Figure 9d [143]. The authors used MoS2 crystals and MoS2 nanosheets to modify the paper-based electrodes. Paper-based electrochemical impedance spectroscopy was used to detect foodborne pathogens (Listeria) [144]. Tungsten disulfide nanostructures were used as paper-based electrodes for impedance sensors.

**Figure 9.** Paper-based impedance biosensors. (**a**) A paper-based electrochemical impedance DNA sensor was developed for tuberculosis detection. Reproduced with permission from [133]. Copyright Analytica Chimica Acta 2018. (**b**) Paper-based electrodes for impedance sensors were used to detect the bacteria in water. Reproduced with permission from [134]. Copyright Sensors and Actuators B: Chemical 2018. (**c**) A paper-based electrochemical impedance spectroscopy was designed to detect coronavirus (COVID-19). Reproduced with permission from [139]. Copyright Biosensors and Bioelectronics 2021. (**d**) Paper-based impedance sensor was used to sense miRNA-155 and miRNA-21 for early diagnosis of lung cancer. Reproduced with permission from [143]. Copyright Talanta 2022.

Paper-based chips have great advantages due to their low material cost. Microelectrodes with small line widths on paper-based chips and antibodies combined onto the microelectrodes will be key technologies for the development of paper-based chips.

#### **7. Stretchable Biosensors**

Furniturewalla et al. designed a microfluidic impedance cytometer on a flexible circuit board in the form of a portable wristband [145]. Lock-in amplification, a microfluidic biosensor, a microcontroller, and a Bluetooth module are integrated into the wristband. Flexible and stretchable biosensors for skin physiological parameter monitoring have been developed [146], where screen printing was used to fabricate sensing electrodes in flexible and stretchable conductive materials originally intended for epidermal tattooing. A retractable body biosensor for sensing the biomarker cortisol in sweat was published, as shown in Figure 10a [147]. A pullable body impedance biosensor was designed at the bottom layer and attached to the skin, following which microfluidic microvalves and microchannels were applied to this wearable patch. A stretchable microfluidic immunobiosensor patch was used for sensing neuropeptide Y in human sweat [148]. Conductive microfibers that can be stretched help to improve the sensitivity of the biosensor patches. Sensors attached to the skin can detect biomarker concentrations in human sweat at levels as low as fm. A wearable microfluidic impedance immunosensor for sweat cortisol detection was designed, as shown in Figure 10b [149], where microfluidic channels and chambers were integrated into the wearable patch, and Ti3C2Tx MXene nanosheets were incorporated in the porous structure of graphene. The wearable microfluidic impedance immunosensor could detect cortisol down to 88 pM.

**Figure 10.** Stretchable impedance biosensors. (**a**) All-polymer electrochemical microfluidic biosensors were used for sensing the biomarker cortisol in sweat. The pullable body impedance biosensor was designed at the bottom layer and attached to the skin. Reproduced with permission from [147]. Copyright Biosensors and Bioelectronics 2020. (**b**) A wearable microfluidic impedance immunosensor was designed for sweat cortisol detection. Microfluidic channels and chambers were integrated into the wearable patch. Reproduced with permission from [149]. Copyright Sensors and Actuators B: Chemical 2021.

Stretchable chips for microfluidic impedance sensors comprise a novel research direction. At present, impedance sensing signals are mostly measured on the skin. The stretchability and bendability of such materials are key features, especially the flexibility of the electrode materials. In this line, conductive hydrogels may be a helpful technology.

#### **8. Conclusions**

Impedance biosensors integrated with microfluidic technology are powerful tools for understanding electrical information in microscopic and sub-microscopic organisms. The integrated sensors have the key characteristics of improved sensitivity, reduced reagent consumption, short analysis time, reduced instrument size, and simple operation. In this paper, the developed microfluidic impedance sensors were classified into six categories: silicon chip-, PCB chip-, polymer chip-, glass chip-, paper chip-, and stretchable chip-based. No matter which type of chip is to be developed, the microfabrication of microelectrodes and the bonding of chemicals are key technologies.

Silicon-based chips are made by the MEMS process developed by Taiwan Semiconductor Manufacturing Co., Ltd., which can become the commercialization direction of electrical impedance biosensors. The development of PCB chips was limited by the line width of the electrodes, which makes it impossible to improve sensitivity. Therefore, PCB chips are more suitable for production or commercial designs with a fast sensing speed and low sensitivity requirements. Polymer materials are suitable for mass production. Therefore, polymer chips can be one of the options for commercialization. Microfluidic impedance sensor glass chips have two great development directions: electrochemical impedance spectroscopy and electrical impedance flow cytometry. The difference between the two lies in the state of the measured object. In an electrical impedance flow cytometer, the cells are stationary. In an electrical impedance flow cytometer, the cells are in a dynamic state. For the impedance sensing of other organisms, the main technology relies on the modification of electrodes and the combination of antigens and antibodies.

Since the development of microfluidic impedance sensors, the development of the principle has mostly stagnated since 2014, especially electrical impedance flow cytometers. Therefore, with regard to the development of sensing applications, specific antigen– antibody binding may not easily to become a break-point in relevant research. Instead, paper-based chips and stretchable chips are expected to become the focus of future development related to microfluidic impedance sensors. As the COVID-19 virus has spread globally in recent years, this variable virus needs to be sensed by electrical impedance biosensors.

**Author Contributions:** Writing—original draft preparation, Y.-S.C. and C.-H.H.; writing—review and editing, P.-C.P., J.S. and K.F.L.; funding acquisition, J.S. and K.F.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Science and Technology Council, Taiwan (Project No. MOST111-2221-E-182-006-MY3) and the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (Project No. 2022R1A2C4001652).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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