*Review* **Microfluidic Applications of Artificial Cilia: Recent Progress, Demonstration, and Future Perspectives**

**Vignesh Sahadevan 1,† , Bivas Panigrahi 2,† and Chia-Yuan Chen 1,\***

	- <sup>2</sup> Department of Refrigeration, Air Conditioning and Energy Engineering, National Chin-Yi University of Technology, Taichung 411, Taiwan; bivas@ncut.edu.tw

† These authors contributed equally to this work.

**Abstract:** Artificial cilia-based microfluidics is a promising alternative in lab-on-a-chip applications which provides an efficient way to manipulate fluid flow in a microfluidic environment with high precision. Additionally, it can induce favorable local flows toward practical biomedical applications. The endowment of artificial cilia with their anatomy and capabilities such as mixing, pumping, transporting, and sensing lead to advance next-generation applications including precision medicine, digital nanofluidics, and lab-on-chip systems. This review summarizes the importance and significance of the artificial cilia, delineates the recent progress in artificial cilia-based microfluidics toward microfluidic application, and provides future perspectives. The presented knowledge and insights are envisaged to pave the way for innovative advances for the research communities in miniaturization.

**Keywords:** artificial cilia; microfluidics; flow manipulation; biological/medical applications

**Citation:** Sahadevan, V.; Panigrahi, B.; Chen, C.-Y. Microfluidic Applications of Artificial Cilia: Recent Progress, Demonstration, and Future Perspectives. *Micromachines* **2022**, *13*, 735. https://doi.org/ 10.3390/mi13050735

Academic Editor: Kwang-Yong Kim

Received: 23 March 2022 Accepted: 19 April 2022 Published: 3 May 2022

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**Copyright:** © 2022 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/).

## **1. Introduction**

Biological cilia are hair-like microscopic structures found on the outer surfaces of nearly every mammalian cell. These microscopic structures allow the cells to interact and sense their surrounding environment [1]. The length of cilia usually varies between 2–15 µm, and they possess complex internal structures comprised of outer doublet microtubules, central microtubules (Axoneme), and dynein arms, which further determines cilia's function as well as their motion. According to their functions, biological cilia can be broadly classified into two major groups: motile and non-motile, or primary cilia [2]. The cilia could be identified, whether motile or primary, using the arrangements of nine pairs of two microtubules with two central pair-structure known as axonemes. In general, if the cilium is with nine pairs of two microtubules but without two-central pair apparatus, the cilium is widely considered as primary cilium. On the other hand, if the cilium has nine pairs of two peripheral microtubules with two central pair microtubules, the arrangement is considered a motile cilium [3,4]. Motile cilia exhibit spatial, temporal, and even oriental asymmetry in their motion to generate flow around them. In contrast, the non-motile cilia are stationary by nature and act as a sensor. Applications of motile cilia are immense. Motile cilia on the outer membrane paramecia help them propel in the fluid 10 times faster than their body length. Additionally, the cilia on the outer surface of the juvenile starfish allow them to select food by creating vortices around them [5,6]. Non-motile cilia can be found on the kidney tubule, where they sense the direction of urine flow and direct the cells accordingly. The motile cilia exhibit a planar motion and beat in a straight path during the forward stroke and roll back to their original position by moving close to the surface in a tangential manner during the recovery stroke. This spatial asymmetry can generate a substantial flow around the cells [1]. Similarly, cilia on the embryo nodal cell exhibit a tilted conical beating path that produces the fluid flow to determine further left-right symmetry in the body [7,8].

Microfluidics is the science of manipulating the amount of fluid with channels, where at least one dimension is generally in a range of 10~1000 µm [9]. Microfluidics exploits its most apparent characteristic, such as its miniaturized size to efficiently handle a small amount of fluid in a minimal time scale. The flow physics of microfluidics is purely laminar, and the viscous force dominates over the inertial force in the flow regime. Hence, it is quite challenging to manipulate the fluid flow in the microfluidic environment due to the effect of the viscous force. As discussed earlier, nature provides an ingenious way for the microorganisms and eukaryotic cells to manipulate the flow around cells by means of cilia. Taking inspiration from the natural cilia, artificial cilia have now been fabricated in the laboratory environment to manipulate the fluid flow within the microfluidic environment. The applications and usages of these artificial cilia-based microfluidic devices are unparalleled.

In the past two decades, the development of microfluidics and artificial cilia research has increased drastically. The existing reviews on microfluidic based devices focused on any specific topics. For instance, the studies were focused on particular actuation techniques like magnetics [10–14] and light [15], specific applications like mixing [12], robotics [16] and particle manipulation [11,17,18], or fabrication techniques like 3D printing [19]. Few excellent reviews are presented in the field of artificial cilia-based microfluidic devices [20,21]. These reviews have extensively discussed the principles of artificial cilia, fabrication processes, actuation mechanisms, modeling of their motion, etc. Along with these fundamental aspects, this review article has put particular emphasis on describing various biological as well as the industrial applications of these artificial cilia-based microfluidic devices. This will not only bridge the existing knowledge gap in the field of artificial-based microfluidics but will also provide a perspective towards future applications and possible research directions. The arrangement of the article is delineated as follows. First, the importance of the natural cilia dynamics was discussed, followed by the description of the fabrication techniques for artificial cilia and the respective actuation methodologies. In the following sections, dynamic beating behaviors of artificial cilia and their applications in microfluidics were also illustrated. Their roles in contemporary applications were discussed. Finally, in view of the general summary, an outlook on conclusions and future perspectives was offered.

#### **2. From Natural Cilia to Artificial Cilia**

Natural cilia are hair-like structures that perform the operations such as feeding, swimming, transporting, moving, and sensing functions in almost all cell types [22–25]. In motile natural cilia category, their applications in coral reef [22] and the respiratory tract [23] towards particle transportation is exemplary. The natural cilia in the coral surfaces generate metachronal wave motion to create flows using the asymmetric dynamic beating behavior. Due to this flow behavior, the materials such as oxygen and nutrients are transported without the typical wave streams [22]. The natural cilia shielded on the lumen within the human respiratory tract transport the dust and bacteria to remove towards the oropharynx [23]. Natural cilia in the Cactus spines and trachea are well regarded for their directional transporting ability due to their asymmetric motion and anisotropic surface. Beating natural cilia with amorphous sheets and dense tufts can create the propulsion forces which facilitate the locomotion in many species [26–28]. Depending upon the size of the organisms, the functions of the natural cilia vary. For example, including the surface of starfish larvae, many organismic ciliary structures were evolved towards facilitating their feeding. The non-motile natural cilia perform sensing operations and adapt to the ambient atmosphere [23]. They were used for sensing work to defend the predators and sense the food and atmosphere in the Animalia kingdom. The natural cilia in the spider tarsi legs sense air flow and other vibrations. The external vibration makes the natural cilia bend where the electrical impulses create and reach the spiders [23].

Motivated by these biological cilia, congeners such as artificial cilia have been advanced and exploited in microfluidics and micro/nanorobotics to realize performances of propelling, transporting, moving, and mixing [29–35]. Even though the in-depth information and uses of artificial cilia in transportation, moving, and sensing will be seen in the

upcoming chapters, the insights into the evolvement of artificial cilia from natural cilia are explained here by detailing their participation with examples of each significant application. Wang et al. [15] fabricated magnetically actuated cilia using Polydimethylsiloxane (PDMS) and cobalt powder by biomimicking cactus spines and trachea cilia in favor of transportation. The artificial cilia were capable of transporting hydrogel slices directionally using their anisotropic surface and asymmetric motion. The asymmetric stroke of the artificial cilia in the sequential magnetic field drove the hydrogel forward. The asymmetric beating behavior and metachronal wave motion are two intriguing benefits exploited using artificial cilia [16,28,29]. The nonreciprocal or asymmetric beating behavior was achieved by creating the difference in the swept area of forward and recovery strokes of artificial cilia [30]. The nonreciprocal motion was measured by the difference between the swept area of the artificial cilia tip and the swept area of the semicircle. The semicircle was created by the artificial cilia length as the radius. The direction of the particle transportation can be adjusted by changing the levels of forward and recovery strokes [31]. The out-of-phase behavior or the phase difference in the neighboring artificial cilia raised the oscillating waves above the surface of the artificial cilia, known as metachronal wave motion. Another study was reported [34] in favor of transportation, in which the photo-actuated artificial cilia were obtained by mimicking the Paramecium aurelia's complex mechanical functions and surface responsiveness. The light-controllable cilia were fabricated using Diarylethene and are capable of transporting objects, demonstrated to transport the polystyrene bead (PB) of 1 mm in diameter. Recent bioinspired artificial cilia established from natural cilia for transporting ability are reported elsewhere [23,30,31,34,36–40]. Inspired by the starfish surface, researchers designed ciliary bands [29], which can be actuated by ultrasound. The ciliary band was successfully demonstrated for locomotion, trapping polystyrene particles, and transportation of water droplets.

The biomimetic inventions have not ended here—the establishments are also reflected in sensing artificial cilia. For sensing, conceptual-wise, the working principle of the artificial was designed similar to the working principle of natural cilia. Artificial cilia for sensing have two sections. The first section is the cilia part where typical magnetic particles (Carbonyl iron powder (CIP) and, neodymium-iron-boron (NdFeB)), PDMS, glass fibers, and polymer materials were used. The second section is the signal processing section. Graphene nanoplatelets [41], carbon nanotubes (CNTs), SiO2, carbon nanofiber, iron nanowires, and AgNW were employed as the sensing/signal processing part [41–48]. Signals such as the piezoelectric and piezoresistive types were created due to cilia bending and processed through the signal processing units [41]. For instance, in the study [42], the high aspect ratio (HAR) artificial cilia were fabricated by replicating goldfish's neuromasts and arthropod filiform hair. The HAR cilia sensor was comprised of PDMS cilia structure, and graphene nanoplatelet infused microchannel. The change in resistance of graphene nanoplatelet was realized in the presence of external flow or touch over the PDMS cilia sensor.

#### **3. Fabrication Techniques for Artificial Cilia**

#### *3.1. Micro-Molding Fabrication Techniques*

The magnetically actuated artificial cilia fabricated by means of micro-molding techniques required a less complex fabrication process and considered precise by nature. This fabrication technique is one of the template-based fabrication techniques. The micro-mold fabrication process involves four critical steps. The first step is to pattern the mold corresponding to the artificial cilia. The second process introduces the polymer material to the pattern. The third step is arranged for the solidification of the pattern. The final step is separating or peeling off the artificial cilia. Chen and his team [49–55] fabricated a wide range of artificial cilia with universality using micro-molding fabrication processes. For instance, Wu et al. [56] demonstrated a micro-molding fabrication process that involves a series of computerized numerical control (CNC) micromachining processes towards preparing the mould for artificial cilia., PDMS-magnetic composite casting was carried out followed by PDMS casting to create the structure of artificial cilia and its microfluidic environment.

Following the PDMS casting, the sample was kept in the hot plate for curing at 90 ◦C before secluding the artificial cilia [56]. The critical challenge coming up with magnetic artificial cilia is their size, as they are considered relatively oversized than the natural cilia. Recently, the magnetic artificial cilia [57] were showcased of the same size (Radius = 200 nm, Length = 6µm) as their counterpart (i.e., biological cilia). The proposed magnetic artificial cilia were fabricated using a tailored molding process. Figure 1A illustrates the use of micro-molding fabrication process towards the fabrication of multi-segmented magnetic artificial cilia. The fabrication was carried out following processes such as: (i) artificial cilia were patterned in an acrylic sheet, (ii) magnetic and PDMS mixture were poured into the pattern, (iii) the pattern was cured on the hot plate at 85 ◦C for 48 h, (iv) procedures of peeling-off artificial cilia from the acrylic substrate and the following magnetization of the artificial cilia.

## *3.2. Photolithography Fabrication Techniques*

The photolithography fabrication technology was used to fabricate soft patterned thin film on the substrate using light. In general, UV light is a popular alternative to this technique. Still, various lights with different wavelengths, such as X-rays, visible light, and extreme UV rays, were also employed depending on the requirements. In a study [58] where a two-step lithography process was employed to fabricate nickel-iron (Ni-Fe) permalloy-based artificial cilia by the researchers. In the first step, Cu's sacrificial layer was sputtered on the negative photoresist material (NR9 1500Py Futurex), followed by the Ni-Fe layer. The desired thickness of the artificial cilia was defined by the deposition of Ni-Fe. The ciliary structures were formed after removing the photoresist material using acetone. In the second step of lithography, the ciliary structures were pinned on the glass substrate using the Ti anchor to hold the cilia on the substrate. Then, the sacrificial Cu layer was removed by 5% ammonium hydroxide solution.

The photolithography fabrication technique is preferred over some other fabrication techniques for the following reasons. The first reason is that this technique offers high precision. In addition, this method is highly controlled together with high throughput, compared to the bead self-assembly [59,60], which required additional control [58]. The recent discussions and approaches over photolithography processes to fabricate artificial cilia can be found elsewhere [29,44,61,62]. Figure 1B illustrates the photolithography fabrication process depicted fabricating magnetic artificial cilia following processes such as: (i) substrate preparing, (ii) anchor preparing, (iii) cilia body layer preparing, (iv) ciliary shape developing, (v) cilia coating, and (vi) peeling-off.

#### *3.3. 3D/4D/5D Printing Fabrication Techniques*

Previously explained, micro-molding and photolithography fabrication technologies are limited because different molds need to be used for different designs in the micro-molding fabrication process. The photolithography fabrication technology requires photoresist, external light sources, the repeatability of the lithography process, and the cleanroom fabrication setup. In the 3D printing technique eliminates these extensive experiment setups and various sizes of artificial cilia could be made without fabrication complexity. A 3D CAD diagram was used to design the three-dimensional artificial cilium to fabricate it under computer programming [63]. The 3D printing technology for artificial cilia was well illustrated by Liu et al. [64]. Recent discussions and approaches over 3D printing technology (Figure 1C) to fabricate artificial cilia can be found in some recent articles [41,42,46,65].

In 2004, 4D printing technology for multi-material [66] was proposed by by S. Tibbits. In the 4D printing technique, the programmed multi-materials can morph their shape over a period of time, even after they came out of the printer. Recently, 4D printing of artificial cilia was proposed by Tsumori et al. [67]. The printing system printed out the 3D artificial cilia whose anisotropy could be changed simultaneously. Next to 4D printing, 5D-printing was proposed by Tsumori et al. [68] for the magnetic artificial cilia. In this process, three

design parameters (x, y, z) were used to fabricate the artificial cilia's shape. Two more parameters (θ, ψ) were utilized in aligning magnetic chain clusters. Five design parameters (x, y, z, θ, ψ) were optimized simultaneously.

#### *3.4. Facile Bottom-Up Approaches*

The facile bottom-up approach is one of the template-free fabrication methods which is unconstrained and the artificial cilia made by this process is highly flexible by nature. This approach involves simple fabrication and operating steps. Timonen et al. [69] demonstrated the facile bottom-up approach (Figure 1D) to fabricate the magnetic artificial cilia. The magnetic particle such as cobalt was mixed with the solvent toluene and elastomeric poly (styrene-block-isoprene-block-styrene) polymer. Poly (tetrafluoroethylene) (PTFE) was used as the substrate in the study. The suspension was ultrasonicated for 10 s to have the high aspect magnetic artificial cilia. In addition, the bottom-up approach was used to fabricate the minimal model system [70] comprised of microtubule (MT) bundles and molecular motors. The exemplified minimal model system was demonstrated to resemble the beating behaviors of the eukaryotic cilia and flagella by self-assembling the MTs and molecular motors.

## *3.5. Roll-Pulling Approaches*

The roll-pulling approach was showcased by the researchers in the study [71] where the custom-made setup was comprised of aluminum roll, substrate, and the precursor medium. The aluminum roll was surrounded by magnetic pillars fabricated by soft lithography. The aluminum roll and glass substrate were separated in a particular gap and rotated with a constant line speed with the help of a rigid string to eliminate friction. The glass substrate was carried the precursor medium comprised of magnetic particles and PDMS. The precursor medium was then pulled by the pre-molded magnetic pillars during the roll rotation and became filaments of a certain length before breaking. The dimensions of magnetic filaments can be adjusted by the size of micro-pillars and the line speed of the roll and substrate. The proposed fabrication technique has advantages over other fabrication techniques in terms of scaling down the dimension of artificial cilia. For example, the 3D printing technology [42] for PDMS artificial cilia is challenging to fabricate high aspect ratio cilia. It required additional attention to print out PDMS polymer materials for microchannels and biomimetic structures (with less than 150 µm diameter) due to its size limitation.

#### *3.6. Self-Assembly Fabrication Techniques*

Wang et al. [60] demonstrated a new affordable in-situ fabrication process named the self-assembly technique. In this fabrication process, micro-sized beads were self-assembled and constructed to form the artificial cilia and encapsulated with soft polymer coatings. The self-assembly approach for the artificial cilia has been illustrated in Figure 1E [59]. The self-assembly approach was comprised of three electromagnets. The three electromagnets were arranged orthogonally to direct the applied magnetic field. The superparamagnetic particles were assembled to create the artificial cilium due to the controlled magnetic field.

#### *3.7. Field-Effect Spinning Approaches*

High sensing performance can be exploited by developing artificial cilia with identical sensory functions. The field-effect spinning (FES) method was reported in the study [72] for the fabrication of artificial cilia. In the previously reported conventional approach, the fiber filaments were pulled without direction. In contrast to the above-mentioned approach, the FES method was demonstrated to produce the vertical and uniformly sized artificial cilia.

#### *3.8. Dip-Coating Fabrication Techniques*

The dip-coating fabrication technique can be used for the fabrication of large size ciliary structures. High aspect ratio shapes can be fabricated using this approach with the following of four steps. In the first step, to give the shape to the ciliary structure, the metals

like stainless steel pins were used along with the dielectric material. In the second step, the fixture, the dielectric material, and the spaces between the cilia were covered by rubber shims. In the third step, the structure was removed from the downside, and the dielectric material followed the structure of the previously placed fixture immediately. In the fourth step, the downside channels were sealed. The step-by-step fabrication processes along flow charts can be found in the study [73]. *Micromachines* **2022**, *13*, x FOR PEER REVIEW 7 of 36

**Figure 1.** Fabrication techniques for artificial cilia. (**A**) The micro-molding fabrication process demonstrated fabricating multi-segmented magnetic artificial cilia using the following fabrication processes: (i) cilia shape patterning in acrylic sheet, (ii) magnetic and PDMS mixture pouring in the **Figure 1.** Fabrication techniques for artificial cilia. (**A**) The micro-molding fabrication process demonstrated fabricating multi-segmented magnetic artificial cilia using the following fabrication processes: (**i**) cilia shape patterning in acrylic sheet, (**ii**) magnetic and PDMS mixture pouring in the

pattern, (**iii**) cured in the hot surface plate at 85 ◦C for 48 h, and (**iv**) procedures of peeling-off artificial cilia from the acrylic substrate and magnetization. The figure was reproduced with permission from [55], under a Creative Commons BY Non-Commercial No Derivative Works (CC BY-NC-ND 4.0) license, published by Elsevier, 2021. (**B**) Photolithography fabrication process depicted fabricating magnetic artificial cilia using the fabrication following processes: (**i**) substrate preparing, (**ii**) anchor preparing, (**iii**) cilia body layer preparing, (**iv**) ciliary shape developing, (**v**) cilia coating and (**vi**) peeling-off. The figure was reproduced with permission from [74], published by John Wiley and Sons, 2011. (**C**) 3D printing fabrication process illustrated to manufacture magnetic artificial cilia.The 3D printing technique was based on stereolithography. Initially, the resin material was poured, and the UV laser of 355 nm was employed to cure. The cilia body were magnetized using a permanent magnet. The surface of the substrate was set at the level of the layer. The substrate table went down to form the next layer after the curing process of the previous layer, and the process was repeated until the desired shape was achieved. The figure was reproduced with permission from [68], published by the Society of Photopolymer Science and Technology (SPST), 2018. (**D**) The facile bottom-up approach in which elastomeric poly (styrene-block-isoprene-block-styrene), toluene, and magnetic particles were mixed, and the mixture was ultrasonicated. A PTFE dish was placed in the aqueous medium (**left**). The setup was kept in the magnetic field. As a result of the magnetic field, the conical structure was formed by magnetic particles, but the polymer was still in the suspensions (**middle**). Toluene evaporated the suspension when the polymer material covered the empty holes between the magnetic particles and artificial cilia were fabricated (**right**). The figure was reproduced with permission from [69], published by the American Chemical Society, 2010. (**E**) Self-assembly approach in which three magnetic coils were arranged orthogonally to render the magnetic field. The artificial cilium was created by assembling the superparamagnetic particles due to the magnetic field (**right**). The figure was reproduced with permission from [59], published by the National Academy of Sciences, 2010.

## **4. Artificial Cilia Actuation Methodologies**

## *4.1. Optical Actuation Techniques*

Optical actuation is the wireless actuation technology preferred in autonomous wireless microsystems [75]. Optically actuated cilia were established from materials consisting of acrylates or methacrylates and liquid-crystal polymer entailing azobenzene dyes using inkjet printing technology [76]. The visible light had a wavelength of 455–550 nm, and ultraviolet light was used to get the desired bending and complex movements of the artificial cilia in the water. The optical actuation method for the artificial cilia has been illustrated in Figure 2A. The figure illustrates the light actuation process demonstrated by Broer et al. [77]. The artificial cilia were made by the cross-linked polymers functionalized with azobenzene. The polymer was responsive to light and prototyped to transport the particles in the aqueous medium.

#### *4.2. Electrostatic Actuation Techniques*

Electrostatic actuation is done by the force between the conducting electrically charged objects. The electrostatic force used to actuate the artificial cilia can be attractive or repulsive. Den Toonder et al. [78] demonstrated such artificial cilia, which had a length and width of 100 mm, and 20 mm, respectively, and was made of polyimide (PI) and chromium (Cr). The artificial cilia had been electrostatically actuated for microfluidic applications. The rapid ciliary motion of the produced cilia was suitable for delivering the fluid flow over 0.6 mm/s [78]. The recent straightforward method to find the fluid flow velocity can be found elsewhere [79]. The Electrostatic actuation method for the artificial cilia has been illustrated in Figure 2B [80]. The setup comprised one moving electrode in the center, sandwiched by multiple ciliary electrodes deposited on two fixed electrodes on the outside. The electrostatic force was released in the spaces between moving and ciliary electrodes when the applied voltage was exerted between the fixed and moving electrode. Due to the electrostatic force, the moving electrode displaced towards the fixed electrode in parallel as well as perpendicular manner.

#### *4.3. pH Actuation Techniques*

The pH actuation is predominantly used for hydrogel actuators. But the actuation of hydrogel cilia was not limited to pH alone [81]. Hydrogels can be swelled and shrank over 10% of their original volume due to specific stimuli such as temperature (T), light, pH, etc., [82]. It gets attention due to the recent development of microfluidics and MEMS, increasing the need for small devices to work in microscale platforms. Hydrogels are the 3D polymer networks with 99 wt % of water. Changing pH intensity was used to stimulus artificial cilia for hydrogel actuation [21,81]. In the study [83], hydrogel-actuated artificial cilia were fabricated using micropost arrays and microfins. The actuators can be bent and upright straight by treating acids and bases. The soft lithography technique is ideal for manufacturing hydrogel artificial cilia [84]. The schematics of the pH actuation process for hydrogel cilia is seen in Figure 2C.

#### *4.4. Resonance Actuation Techniques*

In the resonance actuation technique, the artificial cilia were actuated under the resonance of the lead– zirconate–titanate (PZT) microstage. The piezoelectric transducer was excited by a signal generator. Photolithography microfabrication technique and deep reactive ion etching (DRIE) were used to fabricate the master mold. PDMS polymer was filled in the mold to manufacture the artificial cilia [85,86]. The resonance-actuated ciliaassisted micromixers can provide an efficient uniform mixing performance than diffusionand-vibration mixtures [86].

## *4.5. Magnetic Actuation*

Many actuation techniques exist to actuate the artificial cilia to harness the desired motions and deliver specific applications. But most actuation techniques have their drawbacks hindering their applications in biological domain. This is the primary reason why the researchers have turned their attention to the magnetic actuation system. For example, the electrostatic actuation system cannot be used for biological fluid because it leads to electrolysis [21,87]. Similarly, water is necessary for the hydrogel actuation of artificial cilia. In the air, water molecules gets absorbed, which leads to a long response time of a few hours even for miniaturized cilia [82,88]. Artificial cilia can be actuated using a permanent magnet [68,89–91] or electromagnet [49,50,92–95]. Both symmetric and asymmetric motions can be obtained from artificial cilia under the influence of the magnetic stimuli [49,58,96–99].

#### 4.5.1. Electromagnetic Actuation

Chen et al. [98] demonstrated the electromagnetic actuation technique for the artificial cilia using the custom-built electromagnetic system with four magnetic coils to achieve real time actuation for various microfluidic applications. The Electromagnetic actuation method for the artificial cilia has been illustrated in Figure 2D [56]. The figure shows the electromagnetic actuation comprised of four solenoidal coils. The complete algorithm to actuate a series of artificial cilia using a single electromagnet can be found elsewhere [95].

#### 4.5.2. Permanent Magnetic Actuation

Rotating a permanent magnet is the alternative method requiring less effort and complexity towards artificial cilia actuation [100]. Hanasoge et al. [58] achieved the asymmetric beating of artificial cilia by rotating the permanent magnet. The study reported that magnetic actuation induced the forward stroke; recovery stroke by releasing the accumulated elastic force. The asymmetric beating was achieved by the interplay of elastic, magnetic, and viscous forces. Figure 2E illustrates permanent magnetic actuation in which the permanent magnet of 0.5 T magnetic field was used to actuate the nano-artificial cilia. The magnetic field was sufficient to generate the deflection up to 7 µm, equivalent to 20◦ of bending angle of artificial cilia.

#### *4.6. Acoustic Actuation Techniques*

The need for simple fabrication and straightforward actuation for the artificial cilia in lab-on-chip applications is in high demand. Orbay et al. [101] fabricated the artificial cilia using initial photolithography and UV polymerization technique which eliminated the complex magnetization process. The artificial cilia were actuated using the piezoelectric transducer (PZT) (81-7BB-27-4L0, Murata Electronics, Japan). The thin epoxy layer was used to connect the PZT to the optical path of the PDMS microchannel. The piezoelectric transducer was driven by the sine waves induced through the function generator. The RF amplifier (25A250A, Amplifier Research, USA) was used to amplify the sine waves. The artificial cilia were tested for the mixing operation where the fluorescein and DI water were used. It was found that the increment of mixing performance and complete mixing can be achieved by the voltage up.

#### *4.7. Electric Stimulation Actuation Techniques*

The electric stimulation actuation technique is practically advantageous for the remote and precise control of the artificial cilia. In this approach, the ciliary structures were made of dielectric material. The dielectric materials were polarized due to the dielectrophoresis in the alternating current (AC) electric field when the dielectric nanoparticles were aligned along the electric field direction, leading to the deformation of the artificial cilia. In the study [102], BaTiO<sup>3</sup> was used as the dielectric nanoparticles along the PDMS cilia body. Dielectric fiber materials were used in a recent study [73]. By converting external physical cues into electric impulses using the piezoelectric or triboelectric effect principles, this actuation technique can also opt for sensors.

#### *4.8. Induced Charge Electro-Osmosis Using AC Electric Field Techniques*

The Induced charge electro-osmosis (ICEO) cilium was fabricated using a basic selforganizing process [103–105] demonstrated by Sugioka and the team. A graphite rod was immersed in the deionized water and intercalated between two Cu electrodes. As shown in Figure 2F, initially, *SW<sup>1</sup>* was turned off. *SW*<sup>2</sup> was turned on by applying DC electric voltage (*Vo*) between two Cu electrodes for duration *tDC* (30–120 s), which led to the formation of Carbon artificial cilia at position *X*o. The fabricated artificial cilia had a fibrous network which was identified using energy-dispersive x-ray spectroscopy (EDS). SW<sup>1</sup> was turned on to actuate the artificial cilia by applying AC electric peak voltage between the graphite rod and the right Cu electrode at time *tAC,* when the non-fixed end of artificial cilia (*X*o) moved with the deflection range *h*. The prototyped artificial cilia were capable of producing asymmetric motions. Using the same ICEO and implementing AC electric field principle, a metachronal wave motion was showcased by actuating three artificial cilia with different lengths [106]. Recently, ICEO actuated artificial cilia were demonstrated to transport the square-shaped polyethylene object [107].

#### *4.9. Pneumatical Actuation Techniques*

The artificial elastomers were actuated using the pressure sources created by electropneumatic actuators. Pneumatically actuated artificial cilia were showcased by Milana and the team [108], where six monolithic PDMS cylinders were offset two times to provide large deflection and swept area. Twelve pressure inputs and pneumatic valves were used to control the six elastomers. Pressure inputs and valves were controlled by the Lab view graphical user interface (GUI). The air inside the elastomer could be diffused with glycerol due to the pressure. The artificial cilia were filled with water to avoid this problem. As a result, the pneumatic signal was converted to hydraulic signals. Figure 2G illustrates the experimental setup of pneumatic actuation. In the same group's previous study [109], the step-by-step fabrication process of two pneumatic artificial cilia was shown. The two independent pneumatic actuators were used to deliver two degrees of freedom and spatial asymmetry. Figure 3F illustrates the pneumatic actuation technique in which two pneumatic actuators are actuated using dedicated pressure sources. Each actuator has an

inflatable cavity body. Similar pneumatic microactuators which were fabricated using the micro-molding processes are reported here [110,111]. Onck et el. [36] established a study where the mixing, pumping, and transport capability of pneumatically actuated artificial cilia were analyzed numerically and experimentally. The study further reported that the antiplectic metachronal wave motion increases the mixing and transportation above and below the ciliary surfaces. *Micromachines* **2022**, *13*, x FOR PEER REVIEW 12 of 36

**Figure 2.** Artificial cilia actuation methodologies**.** (**A**) Optic actuation in which the photoresponsive polymers transported objects by having bending motion as responsive towards the light (left). The **Figure 2.** Artificial cilia actuation methodologies. (**A**) Optic actuation in which the photoresponsive polymers transported objects by having bending motion as responsive towards the light (**left**). The curved lines were the trajectory paths on which the objects were transported, and the dashed lines

were the effective linear distance the objects covered (**right**). The figure was reproduced with permission from [77], published by John Wiley and Sons, 2016. (**B**) The electrostatic actuation setup comprised two fixed electrodes, one moving electrode, and multiple ciliary electrodes (**top**). The electrostatic force was released in the spaces between moving and ciliary electrodes when the applied voltage was exerted in between the fixed and moving electrode. Due to the electrostatic force, the moving electrode moved towards the fixed electrode parallel and perpendicularly. The figure was reproduced with permission from [80], published by the Institute of Electrical Engineers, 2004. (**C**) The schematics of pH actuation process for hydrogel artificial cilia. The hydrogel response towards pH was shown (**left**). The figure was reproduced with permission from [83], published by John Wiley and Sons, 2011. (**D**) The schematics of electromagnetic actuation for artificial cilia. The algorithm was fed into the system to create pulse width modulation through which the artificial cilia were actuated using electromagnet coils. Inset: The beating trajectory of the artificial cilia. The figure was reproduced with permission from [56] under a Creative Commons BY (CC BY) license, published by MDPI, 2017. (**E**) Permanent magnetic actuation in which the permanent magnet of 0.5 T magnetic field was used to actuate the nanorod artificial cilia. The magnetic field was sufficient to generate the deflection up to 7 µm, equivalent to 20◦ of bending angle of artificial cilia. The figure was reproduced with permission from [100], published by the American Chemical Society, 2007. (**F**) ICEO artificial cilia actuation using the electric field (details of the experimental setup were discussed previously in the Induced charge electro-osmosis using AC electric field section). The figure was reproduced with permission from [103] under a Creative Commons BY (CC BY) license, published by AIP, 2020. (**G**) The strategy of pneumatic actuation (details of the experimental setup were discussed previously in the pneumatic actuation section). The figure was reproduced with permission from [108], under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) license, published by the American Association for the Advancement of Science (AAAS), 2020.
