*Review* **A Review on Microfluidics-Based Impedance Biosensors**

**Yu-Shih Chen 1, Chun-Hao Huang 1, Ping-Ching Pai 2, Jungmok Seo 1,3,\* and Kin Fong Lei 1,2,3,\***

	- **\*** Correspondence: jungmok.seo@yonsei.ac.kr (J.S.); kflei@mail.cgu.edu.tw (K.F.L.)

**Abstract:** Electrical impedance biosensors are powerful and continuously being developed for various biological sensing applications. In this line, the sensitivity of impedance biosensors embedded with microfluidic technologies, such as sheath flow focusing, dielectrophoretic focusing, and interdigitated electrode arrays, can still be greatly improved. In particular, reagent consumption reduction and analysis time-shortening features can highly increase the analytical capabilities of such biosensors. Moreover, the reliability and efficiency of analyses are benefited by microfluidics-enabled automation. Through the use of mature microfluidic technology, complicated biological processes can be shrunk and integrated into a single microfluidic system (e.g., lab-on-a-chip or micro-total analysis systems). By incorporating electrical impedance biosensors, hand-held and bench-top microfluidic systems can be easily developed and operated by personnel without professional training. Furthermore, the impedance spectrum provides broad information regarding cell size, membrane capacitance, cytoplasmic conductivity, and cytoplasmic permittivity without the need for fluorescent labeling, magnetic modifications, or other cellular treatments. In this review article, a comprehensive summary of microfluidics-based impedance biosensors is presented. The structure of this article is based on the different substrate material categorizations. Moreover, the development trend of microfluidics-based impedance biosensors is discussed, along with difficulties and challenges that may be encountered in the future.

**Keywords:** microfluidic; impedance biosensor; electrical impedance flow cytometer; electrochemical impedance spectroscopy

## **1. Introduction**

Biosensors are mainly used to measure or perceive signals from biological responses. Electrical biosensors can be generally classified into potential, current, and impedance sensors [1]. H. E. Ayliffe pioneered the measurement of single-cell impedance in a microchannel in 1999 [2]. Biological substances can be detected using a pair of microelectrodes with a gap of several μm, in a microchannel 10 μm in width. This narrow microchannel allowed for a more accurate impedance measurement of human polymorphonuclear leukocytes and teleost fish red blood cells. Subsequently, the electrical and equivalent circuit models of single cells were established [3,4].

As early as 1984, Giaever et al. designed a device on a Petri dish that could monitor impedance in order to investigate the density and cell migration of fibroblasts [5]. The impedance signal was obtained by gold electrodes 130 μm in width, which were deposited by a metal evaporator using a mask. Fibroblasts were attached to the gold electrodes, and their cellular response could be represented by the impedance measured across the electrodes [6]. The measured impedance values could differentiate normal fibroblasts from transformed cells. When fibronectin and gelatin were coated on the gold electrodes, the fibroblasts showed a better response. Based on the results of electrical signal monitoring, cell movement was observed. Later, Giaever published an article proposing that the phenomenon of cell movement is called micromotion [7], following which the authors

**Citation:** Chen, Y.-S.; Huang, C.-H.; Pai, P.-C.; Seo, J.; Lei, K.F. A Review on Microfluidics-Based Impedance Biosensors. *Biosensors* **2023**, *13*, 83. https://doi.org/10.3390/ bios13010083

Received: 21 November 2022 Revised: 20 December 2022 Accepted: 28 December 2022 Published: 3 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

put forward a mathematical model establishing the theory of cell movement analysis. Experimentally, small fluctuations in impedance electrical signals were measured as direct evidence of cell movement. Unlike cells observed by light microscopy, impedance biosensors are designed to continuously track the vertical movement of detected cells [8]. The range of cell motion can be as small as 1 nm. The impedance value and fluctuation of each type of cell differs and, so, may be used as a cell fingerprint. Regarding electrical impedance spectroscopy, Grossi et al. carried out a series of literature reviews [9]. The impedance spectrum is usually matched with an equivalent circuit model, and the impedance spectrum obtained by each test object can be expressed as the electrical fingerprint of the test object. Sun et al. used electrochemical impedance spectroscopy to explain the dielectric properties of individual polyelectrolyte microcapsules with different shell thicknesses [10]. The authors built a complete equivalent circuit model of a single solid spherical particle in suspension, and the resistance of the shell and the capacitance of the inner core were used to determine the permittivity and conductivity of individual capsule shells. The study indicated that the conductivity of the six- and nine-layer microcapsule shells could be estimated as 28 ± 6 and 3.3 ± 1.7 mS m<sup>−</sup>1, respectively.

On the other hand, the first microchannel-based flow cytometer was designed by Kamentsky et al. in 1965 [11], with a cell throughput of 500 cells per second. Initially, optical detection was used, using wavelengths of 253 nm and 410 nm. Later on, S. Gawad developed an impedance spectroscopy flow cytometer using Pt as an electrode, which could reduce the electrode impedance and allow the measurement frequency to be as low as 10 kHz [12]. By using a three-electrode design, the impedance value when the cells pass through can be easily measured. The peak impedance value is related to the cell size, and the electrode spacing and the time difference between two impedance signals could also be used to determine the velocity of the measured particle. Gawad et al. established a model to discriminate cell size, membrane capacitance, and cytoplasmic conductivity using a miniature cytometer [13]. Moreover, Cheung et al. obtained amplitude, opacity, and phase information using a microfluidic impedance cytometer, which can be used to distinguish different cells [14], where the measured amplitude can determine the cell size. Opacity was used to distinguish polystyrene beads from red blood cells (RBCs), while phase information was used to distinguish between RBCs and ghosts. RBCs and RBCs fixed in glutaraldehyde could also be distinguished by opacity. The definition of opacity was published in an article by R. A. Hoffman [15]; in particular, it is the ratio of the high-frequency impedance to low-frequency impedance of a particle. Some scholars have studied the development of fluidic impedance cytometers for micro-single cells [16].

Impedance biosensors integrated with microfluidic technology can be categorized into two major technologies: electrical impedance flow cytometry and electrochemical impedance spectroscopy. In the design of a microchannel, an electrical impedance flow cytometer can measure dynamic objects using impedance technology. On the other hand, electrochemical impedance spectroscopy is usually combined with microcavity structures. Static objects can be measured using impedance measurement techniques. In this review article, microfluidics-based impedance biosensors are comprehensively summarized and classified according to the different substrate materials. The development trend, difficulties, and challenges associated with microfluidics-based impedance biosensors, which are expected to be encountered in the future, are also discussed.

#### **2. Silicon-Based Impedance Biosensors**

Silicon-based impedance biosensors are only possible with the fabrication of the electrodes by micromachining. Therefore, the development of silicon impedance biosensors began earlier; however, a number of articles have also been published recently, due to relevant special processes.

An impedance biosensor with a nanometer-wide interdigitated electrode array was developed [17], with electrode widths and spacings of 250–500 nm microfabricated using deep UV lithography. The same article also verified the binding of biomolecular structures to nanoscale electrode surfaces. The impedance signal results indicate that the immobilization of glucose oxidase on the electrode can be monitored. A silicon-based microfabricated biochip was designed to measure the electrical impedance spectrum [18], which could measure the conductance change in a 30 nl volume of bacterial suspension and showed the viability of the bacteria. The same research demonstrated electrical impedance values for the live micro-organism *Listeria innocuous*. By-products after bacterial metabolism have also been shown to change the electrical impedance value; for example, Radke et al. developed an impedance biosensor for bacterial detection using immobilized antibodies. The interdigitated electrode arrays were designed on silicon-based biosensors [19]. *Escherichia coli*-specific antibodies were immobilized on the electrodes, and impedance changes due to bacteria immobilized on the interdigitated gold electrodes were observed. Impedance signals at low frequencies showed that bacteria bound to the sensor electrode surface within 5 min. The rate of binding was the most pronounced in the first 20 min but slowed down significantly after 35 min. At high frequencies, impedance does not change over time. Test concentrations can be as low as 10 CFU/mL of bacterial suspension. The same research team used an impedance biosensor with immobilized antibodies and interdigitated electrode arrays to detect pathogenic *Escherichia coli* O157:H7 and Salmonella infants [20]. It mainly detects bacteria in food or water. P-type (100) silicon wafers were used in that article. Coplanar impedance sensors were designed on glass and fabricated by photolithography [21]. They verified that the spacing of the set of coplanar electrodes is a more important parameter than the electrode area. As the spacing of the electrode design increases, the impedance value increases accordingly. An impedance sensor was used to monitor drug-affected spheroids in a microcavity [22]. Silicon wafers were microfabricated into microcavities through wet anisotropic etching. The impedance sensor was designed with 15 microcavities, and 4 electrodes in each cavity were used to sense the impedance of the spheroids. OLN93 cell spheroids were most loosely organized and peaked at around 180 kHz, while Bro cell spheroids had a more compact structure and showed a peak at 100 kHz. They also observed the impedance of the spheroids 8 h after drug administration. Impedance increased with forskolin, camptothecin, and staurosporine and decreased with doxorubicin and tamoxifen. Single-cell impedance and optical sensing were integrated into a single chip for real-time viability assessment [23]. Single-cell capture microwell chips were obtained by etching silicon wafers with KOH. The induction chip is composed of two cavities (upper and lower). The adhesion changes of RAW264.7 macrophages can be assessed through the impedance value of the sensing chip. In an interdigitated electrode array, the gap between the electrodes is an important factor to improve the sensitivity of the biosensor [24]. Three-dimensional interdigitated electrode arrays were fabricated to sense the impedance signals of proteins. C-reactive protein-specific antibodies were immobilized on the electrode surface. The results showed that electrochemical impedance spectroscopy can be used to monitor the concentration of C-reactive protein in human serum. An impedance biosensor that can sense the concentration of picesterol was developed [25]. The electrodes on the silicon wafer were designed with interdigitated electrodes, where the distance between two electrodes was 15 μm. Cortisol-specific monoclonal antibodies were immobilized on the surface of the microelectrodes. Cortisol binding to antibodies can be signaled by changing the impedance values. The experimental results showed that the impedance biosensor can accurately detect cortisol in the range of 1 pM to 10 nM in saliva. An electrical impedance biosensor was constructed through a CMOS-process, using high-density sensing electrodes for the detection of breast tumor cells (MCF-7) [26]. A total of 96 × 96 gold microelectrodes were designed with a sensing area of 3.5 mm × 3.5 mm. The impedance signal is read out by an integrated circuit fabricated with 0.18 μm CMOS technology. The results showed that the increase in impedance was associated with cell binding to the electrode surface. Ma et al. designed an impedance biosensor that can detect suspended DNA, as shown in Figure 1a [27], where the sensing electrodes are fabricated using 0.35 μm CMOS technology. They proposed that the impedance of a solution is highly dependent on the concentration. Moreover, the impedance value of the sample

solution is also highly correlated with the length of the DNA fragment. In other words, the biological samples obtained after PCR can be tested using the biosensors designed in that article. Lisandro Cunci developed electrochemical impedance biosensors that can detect telomerase in cancer cells [28]. The flexible heater and temperature sensor were designed together in the biosensor. Single-stranded DNA probes were immobilized on the surface of the interdigitated gold electrode array. Jurkat cells were tested for telomerase and showed a 14-fold increase in electrical resistance. The sensitivity of electrochemical impedance spectroscopy biosensors was enhanced using electric field focusing of magnetic beads in microwells [29], where the antibody is immobilized on the surface of the magnetic beads. Microwells are fabricated on silicon wafers using high aspect ratio SU-8 microstructures. In the same article, prostate-specific antigen in a PBS buffer and human plasma were used to validate the argument that focused magnetic beads can improve the sensitivity. The experimental results showed that prostate-specific antigens at low concentrations (i.e., tens or hundreds of fg/mL) could be detected. Pursey et al. integrated surface plasmon resonance and electrochemical impedance spectroscopy into a microfluidic chip, targeting bladder cancer-associated DNA sequences, as shown in Figure 1b [30]. Gold electrodes are microfabricated on the silicon layer, and 20 sensors on the same wafer simultaneously detect three different DNA markers for bladder cancer. Signals were measured within a short period of 20 min. Impedance biosensors that can monitor bacteria have also been developed. In one study, 216 three-dimensional interdigitated electrodes of 3 μm width and with 3 μm gap were microfabricated on a silicon wafer [31]. To improve the sensitivity, the three-dimensional electrodes were separated by an insulating layer. In addition to sensing the impedance signals of bacteria, the three-dimensional electrode can also concentrate bacteria. An impedance sensor fabricated in microgrooves was developed, where silicon substrates were microfabricated to create the microgrooves, as shown in Figure 1c [32]. Two gold electrodes were microfabricated in the microgrooves, which can be used to sense the impedance value of three-dimensional cancer cells, and changes in impedance can reflect the proliferation and apoptosis of three-dimensional cancer cells. Impedance sensors can also identify cancer cells that are affected by drugs. A biosensor combining impedance and photoelectrochemical analysis of cancer cells was designed, as shown in Figure 1d [33], where monocrystalline silicon was used as a substrate for the biosensor. The serrated interdigitated electrodes not only can focus cells but also sense impedance signals. They identified four different types of cancer cells: esophageal cancer (CE81T cell), esophageal cancer (OE21 cell), lung adenocarcinoma (A549 cell), and bladder cancer (TSGH-8301 cell).

The development of silicon chips for microfluidic impedance sensors depends on the development of the microelectromechanical system. Many silicon-based microfabrication processes and metal microelectrode processes are derived from microelectromechanical system microfabrication. Therefore, this is discussed in the first part of this article. Due to the special process requirements, several articles have focused on silicon-based microfluidic impedance sensors. In recent years, the COVID-19 virus has spread throughout the world, and this multi-mutated virus needs to be sensed through the use of biosensors. The MEMS process developed by Taiwan's Taiwan Semiconductor Manufacturing Company may provide the direction for such a technology to be commercialized.

**Figure 1.** The silicon-based impedance biosensors. (**a**) A low-cost 0.35 mm CMOS technology by TSMC (Taiwan) was used to fabricate the micro-array chip that sensed DNA characterization. Reproduced with permission from [27]. Copyright Scientific Reports 2013. (**b**) Integrated surface plasmon resonance and electrochemical impedance spectroscopy in a microfluidic chip [30]. Reproduced with permission from [30]. Copyright Sensors and Actuators B: Chemical 2017. (**c**) Silicon substrates were microfabricated to create microgrooves. Reproduced with permission from [32]. Copyright Microsystems and Nanoengineering 2020. (**d**) A biosensor combining impedance and photoelectrochemical was designed. Reproduced with permission from [33]. Copyright Biosensors 2022.

#### **3. Printed Circuit Board (PCB)-Based Impedance Biosensors**

Printed circuit boards (PCBs) are flat plates that were originally used to make circuits, which are very commercialized. Their electrode width and electrode spacing are not very small; therefore, the sensitivity limit of the sensor is not low either.

Electrochemical impedance spectroscopy biosensors consist of PCBs with gold-coated electrodes, which are mainly used to detect plant pathogens [34], where the antibody is first bound to the surface of the electrochemical sensor. For papaya ring spot virus, the biosensor was shown to be capable of detecting papaya ring spot virus coat protein with high sensitivity. A chip using a microfluidic impedance sensing system to detect the transgenic protein Cry1Ab was designed, as shown in Figure 2a [35]. Gold electrodes were printed on commercial printed circuit boards, with the spacing between two printed electrodes being 250 μm. The impedance signal at the optimal test frequency (358.3 Hz) presented a good linear relationship with the concentration of the transgenic protein Cry1Ab in the range of 0–0.2 nM. Clinically, the degree of red blood cell agglutination is divided into five grades by visual inspection, which is routinely conducted in hospitals [36]. An electrical impedance blood-sensing chip was designed by Chang et al. to distinguish the degree of blood agglutination. The interdigitated electrode array was designed on a PCB. ZnO nanowires were synthesized on the surface of the electrode array in order to improve the sensitivity of impedance measurement. An electrical impedance sensor was used to detect the degree of fibrosis in liver tissue, as shown in Figure 2b [37], where liver tissue was detected by a pair of gold electrodes on the PCB. The experimental results indicate that the maximum resistance difference between healthy and fibrotic tissue was about 2 kΩ at day 8.

**Figure 2.** The PCB-based impedance biosensors. (**a**) Gold electrodes were printed on commercial printed circuit boards. Reproduced with permission from [35]. Copyright Scientific Reports 2013. (**b**) An electrical impedance sensor was used to detect the degree of fibrosis in liver tissue. Reproduced with permission from [37]. Copyright Biosensors 2022.

The development of PCB chips is limited by the electrode line width in traditional PCB processes. Microelectrodes that are not tiny enough will lead to an inability to improve the sensitivity. Therefore, PCB chips are more suitable for the production or commercial design where the induction is fast and the sensitivity requirements are not high.

#### **4. Polymer-Based Impedance Biosensors**

In addition to glass, the most commonly used substrates in the field of microfluidics are polymer chips. Different from the stretchable chips mentioned in later chapters, the polymer chips mentioned in this chapter are formed of hard and inflexible materials.

A study considering electrochemical impedance spectroscopy on polymer substrates was developed [38], in which the authors designed a nanoscale interdigitated electric shock array, in which the electrode width was only 200 nm and the electrode spacing was 500 nm. Gold nanometer interdigitated electrode arrays were patterned on cyclic olefin copolymer substrates. Experiments have demonstrated selective iDEP capture and impedance detection on polystyrene microspheres and *Bacillus subtilis* spores [39]. The authors used oxides to passivate the sensing electrode of the sensor in order to avoid the metal and electrolyte having adverse effects on the electrode surface. Cyclic olefin copolymer was used as the substrate of the sensor. Studies have used all-polymer electrochemical microfluidic biosensors for electrochemical impedance spectroscopy, as shown in Figure 3a [40], where polymer materials from Topas Corporation were used as substrates and conductive polymer bilayers were used as electrode materials. Electrochemical impedance spectroscopy was able to detect ampicillin in a concentration range from 100 pM to 1 μM and kanamycin A from 10 nM to 1 mM. Figure 3b shows how Pires et al. combined an impedance sensor and a current sensor to detect biofilms in water [41]. Two microfluidic chambers were designed on a cyclic olefin copolymer substrate with four impedance sensors and three current sensors in each cavity. A conductive polymer (PEDOT:TsO) was fabricated as an interdigitated electrode array for impedance biosensors [42], where the cyclic olefin copolymer produced by TOPAS was used as the base material for the conductive polymer electrodes. A microfluidic impedance sensor was used to detect the food additive clenbuterol hydrochloride [43], where the electrodes were patterned on poly (ethylene terephthalate) films. Polyaniline@graphene oxide nanocomposites were used to functionalize the sensing electrodes, and the microfluidic impedance sensors could detect down to 0.12 ppb. Sharif et al. integrated a microfluidic system and a magnetic separation procedure

to develop a novel impedance sensor for the detection of various foodborne pathogens [44]. Their results showed that the impedance sensor was effective for the detection of various foodborne pathogens, including *Escherichia coli* (*E. coli* O157:H7), *Vibrio parahaemolyticus* (*V. parahaemolyticus*), *Staphylococcus aureus* (*S. aureus*), and *Listeria monocytogenes* (*L. monocytogenes*). Polymethyl methacrylate (PMMA) was used as the substrate. D-dimer is a biomarker in the blood that can be used to diagnose deep vein thrombosis and pulmonary embolism [45]. Lakey et al. designed a polymer microfluidic impedance sensor for the detection of D-dimer, where interdigitated electrode arrays were patterned on polyethylene naphthalate (PEN) substrates. Ma et al. developed an electrochemical impedance spectroscopy approach for the detection of endotoxins [46]. The electrodes were screen-printed on a polyethylene terephthalate (PET) substrate, which contained carbon for the working and auxiliary electrodes and Ag/AgCl for the reference electrode. The sensitivity of the impedance biosensors could be as low as 500 pg mL<sup>−</sup>1, and the total measurement time was only half an hour. Niaraki et al. used graphene microelectrodes to monitor neuronal growth and detachment after death, as shown in Figure 3c [47]. Kapton Polyimide (PI) was used as a substrate for patterned graphene electrodes. The microelectrodes fabricated by this research team feature a wrinkled surface morphology, which allows for a fast response time to be achieved. Chmayssem et al. integrated a cell culture chamber with electrochemical impedance spectroscopy, as shown in Figure 3d [48]. The researchers used a screen-printing technique to fabricate the microelectrodes on polyethylene terephthalate (PET) sheets. The electrode material was selected, and Ag/AgCl was used as the low interface impedance electrode on the PET sheet. On the top layer of electrodes was carbon-biocompatible ink rich in IrOx particles. Hantschke et al. integrated electrophoretic and electrical impedance sensors for point-of-care (POC) diagnostics [49], where the electrodes and microchannels were fabricated on polymethylmethacrylate (PMMA) substrates.

**Figure 3.** The polymer-based impedance biosensors. (**a**) All-polymer electrochemical microfluidic

biosensors are used. Reproduced with permission from [40]. Copyright Biosensors and Bioelectronics 2013. (**b**) Two microfluidic chambers were designed on a cyclic olefin copolymer substrate. Reproduced with permission from [41]. Copyright Biosensors and Bioelectronics 2013. (**c**) Kapton Polyimide (PI) was used as a substrate. Reproduced with permission from [47]. Copyright Biosensors and Bioelectronics 2022. (**d**) Microelectrodes were microfabricated on polyethylene terephthalate (PET) sheets. Reproduced with permission from [48]. Copyright Biosensors 2022.

The key to the use of polymer chips in microfluidic impedance biosensors is the combination of microelectrodes and polymer substrates. The sensitivity of the biosensors were determined by the size of the microelectrodes. Polymer chips are used similarly to glass chips, with both being hard and inelastic materials. On the other hand, polymer materials are suitable for mass production. Therefore, polymer chips can be one of the options for commercialization.

#### **5. Glass-Based Impedance Biosensors**

Microfluidic chips with glass as the substrate are very common. There have been some articles on materials that can replace glass, such as ITO glass, Pyrex glass, and SiO2 glass. Therefore, the classification in this chapter is based on what the biosensors on glass can monitor. In this line, the subsections are classified regarding the detection of bacteria, blood, cells, DNA, proteins, toxins, and viruses.
