micromachines

Review DEP-on-a-Chip: Dielectrophoresis Applied to Microfluidic Platforms

Haoqing Zhang 1, Honglong Chang 1,* and Pavel Neuzil 1,2,3,*

1 Ministry of Education Key Laboratory of Micro/Nano Systems for Aerospace, School of Mechanical Engineering, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China; [email protected] 2 Central European Institute of Technology, Brno University of Technology, Brno 61300, Czech Republic 3 Department of Microelectronics, Faculty of Electrical Engineering, Brno University of Technology, Technická 3058/10, Brno 61600, Czech Republic * Correspondence: [email protected] (H.C.); [email protected] (P.N.)



Received: 17 May 2019; Accepted: 19 June 2019; Published: 24 June 2019


Abstract: Dielectric particles in a non-uniform electric field are subject to a force caused by a phenomenon called dielectrophoresis (DEP). DEP is a commonly used technique in microfluidics for particle or cell separation. In comparison with other separation methods, DEP has the unique advantage of being label-free, fast, and accurate. It has been widely applied in microfluidics for bio-molecular diagnostics and medical and polymer research. This review introduces the basic theory of DEP, its advantages compared with other separation methods, and its applications in recent years, in particular, focusing on the different electrode types integrated into microfluidic chips, fabrication techniques, and operation principles.

Keywords: dielectrophoresis; microfluidics; two-dimensional electrodes; parallel electrodes; interdigitated electrode; castellated electrodes; three-dimensional electrodes

1. Introduction The establishment of lab-on-a-chip (LOC) technology made it possible to gradually integrate large and costly bench-top laboratory instruments into palm-sized devices containing chips in a scale of a few centimeters or less, without sacrificing their performance [1]. LOC-based devices have been used in molecular biology [2], clinical diagnostics [3], and point-of-care systems [4]. The fabrication of microfluidic chips requires a demanding technological process to integrate a variety of structures and components to perform different processes, such as sample pretreatment [5], sample delivery [6], particle manipulation [7], on-chip reaction [8], and result detection and analysis [9]. Particle separation [10], a subcategory of particle manipulation, is a crucial process affecting detection precision. Many techniques have so far been used for this, such as filtration [11,12], centrifugation [13], the use of magnetic [12,14] and acoustic force [15,16], chromatography [17,18], electrophoresis [19,20], and dielectrophoresis (DEP) [21,22]. Compared to other methods, DEP is becoming one of the most promising separation techniques for micro- and nano-scale systems because of its low running cost, ease of integration into microfluidics, speed, efficiency, sensitivity, and selectivity. Finally, it is also a label-free method [23], making sample processing very simple. Here, we present the theory of DEP, a comparison between DEP and other separation methods, and an explanation of its applications in microfluidic chips. We review the advantages and disadvantages of major systems, their impact, and potential, in particular, focusing on different electrode types integrated into microfluidic chips with structures, fabrication techniques, and operation principles. The brief review will be of interest to researchers in DEP microfluidics.

Micromachines 2019, 10, 423; doi:10.3390/mi10060423 www.mdpi.com/journal/micromachines Micromachines 2019, 10, 423 2 of 22

2. Microfluidic Separation Techniques Particle separation is a technique that extracts or obtains target particles from a solution or suspension or isolates them from each other based on differences in the particles’ physical and or chemical properties or differences between the properties of the particles and the carrier fluid. It can also be considered as a concentration or purification process for target chemicals or particles. Filtration [24–26] is a commonly used and effective separation method based on particle size differences. A porous medium with a specific pore size forms a filter, allowing particles smaller than the pore size, as well as the carrier fluid, to pass through the filter, where oversized particles are collected. However, it is challenging to apply filtration to microfluidic chips once the porous medium becomes blocked during the filtration process, thus gradually decreasing the throughput unless a more complex ultrafiltration system is used [27]. These disadvantages or complexities hinder the wide employment of this technique for LOC systems. The centrifugation method [28–32] is used to separate particles based on specific mass while being subjected to centrifugal force. However, it requires the application of an additional tool to generate the force. Magnetic force separation [33–36] mainly depends on the magnetic susceptibility of the particles. It is possible to bind magnetic beads with the particles of interest, but this increases the system’s complexity. Acoustic tweezers [37–40] are a separation method based on sound waves. Particles can be induced to move toward acoustic pressure nodes or pressure antinodes in a standing acoustic field by an acoustic-radiation force. The movements are dependent on the particles’ density, and precise movement can be obtained by controlling the position of these nodes. However, the precision and throughput limit the application of acoustic tweezers for LOC systems. Chromatography [41–44] is a powerful separation technique widely applied in microfluidic platforms, where the mobile phase carries the target particles and passes through a stationary phase. Different particles in the mixture travel at different speeds, resulting in their separation. However, the chromatography in microfluidic chips require complex structures, including valves, pumps, and stationary phases, subsequently increasing its fabrication complexity. The requirement of sample labeling and especially sample loss due to unspecific adhesion to the large surface area of the stationary phase prevents its wide application. Electrophoresis [45–47] is a technique to separate charged particles in the suspension based on a uniform electric field and is widely applied in the study of deoxyribonucleic acids (DNA), ribonucleic acids, and proteins. However, it is only available for charged particles, limiting its potential application. In comparison with other separation methods, DEP [48–50] has the unique advantage of being label-free, fast, and accurate, which is why it has been widely applied in microfluidics for bio-molecular diagnostics, and medical and polymer research.

3. Dielectrophoresis DEP is a method used to separate suspended particles from the suspension or carrier fluid by generating polarization forces in a non-uniform electric field (NUEF) [51]. Converse to electrophoresis, the particles in DEP do not carry electrical charges, but they must be polarizable. These particles that are exposed to an electric field and become polarized forming electric dipoles; the resulting force on these dipoles is called DEP force (FDEP). FDEP is 0 N (Figure1A) when the particle is in a uniform electric field, while in NUEF, the force is either positive (pDEP) or negative (nDEP) making the particle move in a certain direction depending on its polarization (Figure1B) [51]. The amplitude of FDEP of a spherical particle in the suspension follows Equations (1) and (2) [52].

3 2 F = 2πεmε r Re[CM( f )] E (1) DEP 0 ext ∇ RMS Micromachines 2019, 10, 423 3 of 22

where εm is the medium relative permittivity, ε0 is the vacuum permittivity, rext is a radius of the Micromachines 2019, 10, x 3 of 22 spherical particle, ERMS is the root-mean-square value of the applied electric field, and CM(f ) is the Clausius–Mossotti factor. where εm is the medium relative permittivity, ε0 is the vacuum permittivity, rext is a radius of the εp∗ εm∗ spherical particle, ERMS is the root‐meanCM‐square( f ) = value −of the applied electric field, and CM(f) is the (2) ε + 2ε Clausius–Mossotti factor. p∗ m∗ ∗ ∗ where εp∗ and εm∗ are defined as: 𝜀 𝜀 𝐶𝑀𝑓 ∗ ∗ (2) 𝜀 2𝜀 σp σm ∗ ∗ ε∗ = εpε0 j , ε∗ = εmε0 j (3) where 𝜀 and 𝜀 are defined pas: − π f m − π f 𝜎 ∗ ∗ 𝜎 𝜀 𝜀𝜀 𝑗 , 𝜀 𝜀𝜀 𝑗 (3) where εp is the relative permittivity of a particle,𝜋𝑓σp is the electric𝜋𝑓 conductivity of a particle, σm is the electric conductivity of the medium, and f is the frequency of the electric field. where εp is the relative permittivity of a particle, σp is the electric conductivity of a particle, σm is the Theelectric NUEF conductivity is powered of the by medium, the direct and current f is the (DC) frequency or alternating of the electric current field. (AC) and is generated by geometryThe of insulating NUEF is powered posts in combinationby the direct current with channel (DC) or tapering, alternating etc., current in the (AC) microfluidic and is generated channel [10] also knownby geometry as AC /DC-iDEPof insulating [53 ].posts The in microfluidic combination chips with based channel on DC-iDEP tapering, haveetc., in a simple the microfluidic structure with no gaschannel bubble [10] generation also known during as AC/DC particle‐iDEP separation. [53]. The However, microfluidic the chips power based loss inon the DC DC‐iDEP electrical have a field resultssimple in Joule structure heat generation,with no gas bubble excessively generation increasing during theparticle temperature separation. inside However, the device. the power Compared loss to this,in the DC AC-DEP electrical is morefield results widely in used Joule because heat generation, the Joule excessively heat generation increasing is greatlythe temperature suppressed. The deviceinside the selectivity device. Compared then depends to this, on the the AC AC‐DEP frequency is more as widely well asused the because field strength; the Joule thus,heat the generation is greatly suppressed. The device selectivity then depends on the AC frequency as well as parameters of both should be optimized. the field strength; thus, the parameters of both should be optimized.

FigureFigure 1. Diagram 1. Diagram of the of the positive positive dielectrophoresis dielectrophoresis (pDEP)(pDEP) principle. principle. Reproduced Reproduced with with permission permission fromfrom [51], [51],© 2011 © 2011 Elsevier. Elsevier. (A ()A The) The particle particle is is symmetricallysymmetrically polarized polarized in ina uniform a uniform electrical electrical field, field, resulting in net DEP force (FDEP) with zero amplitude. (B) The particle is asymmetrically polarized in resulting in net DEP force (FDEP) with zero amplitude. (B) The particle is asymmetrically polarized in the non‐uniform electric field (NUEF) and the net FDEP, resulting in moving the particle into an area the non-uniform electric field (NUEF) and the net F , resulting in moving the particle into an area with the maximum amplitude of the electric field. DEP with the maximum amplitude of the electric field. 4. Dielectrophoresis (DEP)‐on‐a‐Chip 4. Dielectrophoresis (DEP)-on-a-Chip A variety of DEP microfluidic devices have been developed for use in bioparticle separation. AThe variety gradient of DEPof the microfluidic electric field devicesis an important have been factor developed that affects for the use DEP in bioparticle performance. separation. The The gradientelectrodes’ of thegeometry electric mainly field is anproduces important the factorNUEF thatin the affects microfluidic the DEP performance.chip, so the shape The electrodes’ and geometrydistribution mainly of produces the electrodes the NUEFare vital in to the the microfluidicNUEF amplitude. chip, Here so the we shapediscuss and different distribution electrodes of the electrodesapplied are in vital microfluidics to the NUEF approaches, amplitude. such Here as DEP we discussperformed diff byerent external electrodes electrodes applied and in 2D microfluidics and 3D approaches,electrodes. such as DEP performed by external electrodes and 2D and 3D electrodes.

4.1. External4.1. External Electrodes Electrodes ResearchersResearchers have have developed developed an an iDEP iDEP microfluidic microfluidic chip chip made made of of polydimethylsiloxane polydimethylsiloxane (PDMS) (PDMS) coveredcovered with with a glass a glass lid. lid. TheThe device’s device’s performance performance was wasevaluated evaluated in a hydrodynamic in a hydrodynamic study of blood study of alteration [54]. The chip structure consisted of an inlet and an outlet port, and a ≈ 1 cm long, ≈ 50 μm blood alteration [54]. The chip structure consisted of an inlet and an outlet port, and a 1 cm long, deep main channel with 38 dead‐end branches distributed evenly on both sides of the main ≈channel 50 µm deep main channel with 38 dead-end branches distributed evenly on both sides of the main ≈ Micromachines 2019, 10, 423 4 of 22

Micromachines 2019, 10, x 4 of 22 channel (Figure2A). The width and depth of the branches were 200 µm and 50 µm, respectively. (Figure 2A). The width and depth of the branches were ≈ 200 μ≈m and ≈ 50 μ≈m, respectively. An An electric field was introduced into the device by inserting two Pt electrodes, one into the inlet electric field was introduced into the device by inserting two Pt electrodes, one into the inlet and one and one into the outlet. A sample of blood with a volume of 2 µL was injected into the main into the outlet. A sample of blood with a volume of ≈ 2 μL was injected≈ into the main channel and channel and pumped through it using internal capillary forces. The branches hydro-dynamically pumped through it using internal capillary forces. The branches hydro‐dynamically trapped the red trappedblood thecells red (RBCs). blood cellsThen, (RBCs). the DC Then, power the supply DC power was supply activated was to activated establish to establishthe DC‐DEP the DC-DEPforce, force,preventing preventing more more RBCs RBCs from from being being trapped trapped inside inside the thedead dead-end‐end channels channels near near the theinlet inlet port. port. Additionally,Additionally, the the electro-osmotic electro‐osmotic flow flow generated generated by by the the DC DC electric electric field field removed removed RBCs RBCs from from the the cross junctionscross junctions formed formed at the channels at the channels and branches. and branches. As a result, As a result, the plasma the plasma was separatedwas separated from from the blood,the formingblood, a forming plasma areaa plasma without area RBCs. without This RBCs. method This obtained method aobtained plasma puritya plasma of 99%.purity The of iDEP99%. deviceThe performediDEP device the plasmaperformed separation the plasma without separation diluting without the blood diluting samples. the blood This devicesamples. was This used device with was lower voltageused with in comparison lower voltage to other in comparison iDEP devices to other [55 ].iDEP devices [55].

FigureFigure 2. 2.DEP DEP devices devices with with external external electrodes.electrodes. ( (AA) )Schematic Schematic of of iDEP iDEP chips chips achieving achieving plasma plasma separationseparation without without diluting diluting the bloodthe blood samples. samples. Reproduced Reproduced with with permission permission from [from54], © [54],2015 © Springer 2015 Nature.Springer (B) SchematicNature. (B of) Schematic chip used forof chip heterogeneous used for emulsionheterogeneous droplets emulsion separation. droplets Reproduced separation. with permissionReproduced from with [56 permission], © 2017 Elsevier. from [56], © 2017 Elsevier.

AA simple simple PDMS PDMS microfluidic microfluidic chipchip waswas fabricatedfabricated using using soft soft lithography lithography to to perform perform the the wall-inducedwall‐induced DEP DEP [56 [56]] (Figure (Figure2B). 2B). This This device device was usedwas toused separate to separate oil droplets oil droplets from Janus from particles, Janus havingparticles, two having or more two distinct or more physical distinct properties physical onproperties the surface on the covered surface with covered oil phase. with oil The phase. suspension The containingsuspension the containing particles ofthe interest particles was of moving interest from was themoving sample from inlet the to sample the main inlet channel, to the andmain the buchannel,ffer from and the the sheath buffer fluid from inlet the sheath pushed fluid the inlet particles pushed to the move particles along to the move wall along of the the main wall channel.of the Themain length channel. and width The length of the and sample width inlet of the and sample sheath inlet fluid and inlet sheath were fluid5 inlet mm were and ≈ 5150 mmµ andm, ≈ 1150 mm, ≈ ≈ ≈ andμm, ≈ 2501 mm,µm, respectively.and ≈ 250 μm, The respectively. sample inlet The was sample next inlet to a was confinement next to a confinement chamber of chamber6 mm of6 ≈ mm.6 mm≈ × 6 mm. The length of both outlets was ≈ 9 mm, and their widths were ≈ 120 μm ≈and ≈ 180× μm, The length of both outlets was 9 mm, and their widths were 120 µm and 180 µm, respectively. respectively. The depth of the≈ confinement chamber was ≈ 80≈ μm, allowing≈ only droplets of a The depth of the confinement chamber was 80 µm, allowing only droplets of a diameter of < 80 µm diameter of < 80 μm to pass through the channel.≈ The main channel, with a length and depth of ≈ 1 to pass through the channel. The main channel, with a length and depth of 1 mm and 250 µm, mm and ≈ 250 μm, respectively, connected the inlets and outlets. The channel tapering≈ generated≈ the respectively, connected the inlets and outlets. The channel tapering generated the NUEF after four NUEF after four Pt electrodes were inserted into the inlets and outlets. The deionized water first Ptwetted electrodes the werechannel, inserted and then into thethe inletsJanus anddroplets outlets. covered The deionized by oil droplets water were first wettedinjected the into channel, the andconfinement then the Janus chamber droplets and pushed covered close by oil to dropletsthe main were channel injected by the into fluid the from confinement the sheath chamber fluid inlet. and pushedThe DC close power to the supply main channelwas then by applied the fluid to from the thechannel, sheath and fluid the inlet. Janus The and DC oil power droplets supply were was thengradually applied separated to the channel, from each and other the Janus with and the oilhelp droplets of the F wereDEP and gradually moved into separated the different from each outlets. other withThe the separation help of the processFDEP wasand dependent moved into on the the di amplitudefferent outlets. of the The applied separation voltage process and droplet was size. dependent The onfabrication the amplitude process of the was applied rather voltagesimple because and droplet the electrodes size. The fabricationwere inserted process into the was chip rather and simple not becausemonolithically the electrodes integrated were into inserted the device. into the chip and not monolithically integrated into the device.

4.2.4.2. Two-Dimensional Two‐Dimensional Electrodes Electrodes Thin-filmThin‐film 2D 2D electrodes electrodes based based on a on simple a simple fabrication fabrication process process with di withfferent different shapes wereshapes developed were to meetdeveloped the requirements to meet the ofrequirements integration of and integration to increase and DEP to separationincrease DEP effi separationciency within efficiency the microfluidic within chips.the microfluidic Here, we introduce chips. Here, the most we commonlyintroduce the used most electrode commonly configurations, used electrode parallel configurations, [52,57–60] and

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Micromachines 2019, 10, x 5 of 22 castellated electrodes [61–64], and briefly present other such configurations, such as quadrupole electrodesparallel [[52,57–60]65,66]. and castellated electrodes [61–64], and briefly present other such configurations, such as quadrupole electrodes [65,66]. 4.2.1. Parallel and Interdigitated Electrodes 4.2.1. Parallel and Interdigitated Electrodes The electrodes from both parallel and interdigitated electrode arrays are typically rectangular. The electrodesThe electrodes have identical from both widths parallel and gapsand interdigitated resulting in a electrode constant electricarrays are field typically between rectangular. them. Thus, theThe system electrodes based onhave them identical has a highwidths separation and gaps e resultingfficiency ofin particlesa constant of electric interest. field between them. Thus,A microfluidic the system based device on was them proposed has a high to separateseparation green efficiency fluorescent of particles protein of interest. labeled MDA-MB-231, A microfluidic device was proposed to separate green fluorescent protein labeled and other types of cancer cells from healthy ones [52]. The microfluidic chip consisted of two parts MDA‐MB‐231, and other types of cancer cells from healthy ones [52]. The microfluidic chip consisted (Figure3A). First, the electrode part was made using a glass wafer as a substrate, which was sputtered of two parts (Figure 3A). First, the electrode part was made using a glass wafer as a substrate, which and coated with a sandwich layer of Cr/Au. This sandwich metal layer was then patterned using a wet was sputtered and coated with a sandwich layer of Cr/Au. This sandwich metal layer was then etching process to form a planar interdigitated electrode array. This consisted of 20 pairs of electrodes patterned using a wet etching process to form a planar interdigitated electrode array. This consisted with widths and gaps of 39 µm and 36 µm, respectively, protruding into the channel by 2 µm of 20 pairs of electrodes≈ with widths and≈ gaps of ≈ 39 μm and ≈ 36 μm, respectively, protruding≈ into to 3 µm, providing powerful NUEF. Next, the microfluidic structure was formed using PDMS and ≈the channel by ≈ 2 μm to ≈ 3 μm, providing powerful NUEF. Next, the microfluidic structure was a soft lithography technique. The width of the channel was 50 µm and it was enlarged to 250 µm. formed using PDMS and a soft lithography technique. The ≈width of the channel was ≈ 50 μm≈ and it Finally,was enlarged these two to parts, ≈ 250 μ them. electrodes Finally, these and two microfluidic parts, the structure, electrodes were and microfluidic bonded together, structure, assisted were by an oxygenbonded plasma.together, Theassisted sample by an was oxygen pumped plasma. into The the channel,sample was and pumped the NUEF into was the formedchannel, by and the the AC powerNUEF supply was formed with a setby amplitudethe AC power of 15 supply V peak with to peaka set amplitude (VPP) at a setof 15 frequency V peak to of peak 40 kHz, (VPP) obtaining at a set a separationfrequency accuracyof 40 kHz, and obtaining purity a of separation100% and accuracy81%, and respectively. purity of ≈ 100% and ≈ 81%, respectively. ≈ ≈

FigureFigure 3. 2D3. 2D electrode electrode microfluidic microfluidic chips chips with with DEP. DEP. (A )( SchematicA) Schematic of theof the device device to separateto separate cancer cancer cells

fromcells blood from cells blood by cellsFDEP bygenerated FDEP generated by parallel by parallel electrodes. electrodes. Reproduced Reproduced with with permission permission from from [52 ], © 2016[52], John © 2016 Wiley John and Wiley Sons. and (B) Sons. The device (B) The combined device combined with induced-charge with induced electro-osmosis‐charge electro (ICEO)‐osmosis for particle(ICEO) focusing for particle and DEP focusing achieving and DEP simpler achieving chip structure simpler chip to perform structure particle to perform focusing particle and separation.focusing Reproducedand separation. with permission Reproduced from with [60 permission], © 2017 Americanfrom [60], Chemical © 2017 American Society. ( CChemical) Hybrid Society. DEP-inertial (C) deviceHybrid with DEP top‐ sheathinertial flow device to improve with top the sheath separation flow to e ffiimproveciency. the Reproduced separation with efficiency. permission Reproduced from [64 ], © 2018with Elsevier. permission from [64], © 2018 Elsevier.

A microfluidicA microfluidic chip chip was fabricatedwas fabricated to combine to combine particles particles and DEP and to performDEP to continuousperform continuous separation of yeastseparation cells fromof yeast silica cells particles from silica [60] by particles ICEO, using[60] by a microscopeICEO, using glass a microscope slide as the glass device slide substrate. as the Thedevice glass wassubstrate. coated The with glass a thin was layer coated of with indium a thin tin layer oxide of (ITO) indium and tin subsequently oxide (ITO) and patterned, subsequently forming patterned, forming parallel electrodes. The channels, one inlet and four outlets, were made in the parallel electrodes. The channels, one inlet and four outlets, were made in the PDMS (Figure3B) and PDMS (Figure 3B) and bonded to the glass substrate using an oxygen plasma surface‐activation bonded to the glass substrate using an oxygen plasma surface-activation method. The microfluidic method. The microfluidic chip had three regions: focusing, transition, and separation. First, the chip had three regions: focusing, transition, and separation. First, the suspension, consisting of silica suspension, consisting of silica particles, yeast cells, and carrier fluid, was injected into the chip, and particles, yeast cells, and carrier fluid, was injected into the chip, and the ICEO focused the particles the ICEO focused the particles in the focusing region. The side branches, with optimized size in the in thetransition focusing region, region. ensured The sidethe particle branches, stream with flowed optimized into the size separation in the transition region in region,a straight ensured line. The the particle stream flowed into the separation region in a straight line. The separation efficiency was separation efficiency was > 96% with the AC power supply at a set value of 225 VPP at 1 MHz. This > 96%general with‐purpose the AC DEP power microfluidic supply at chip a set has value great ofpotential 225 VPP in atmolecular 1 MHz. diagnostics This general-purpose in LOC systems DEP

Micromachines 2019, 10, 423 6 of 22 microfluidicMicromachines chip2019, 10 has, x great potential in molecular diagnostics in LOC systems because it exhibited6 of 22 high throughput. The integration of the ICEO module simplified the chip structure, as there is no because it exhibited high throughput. The integration of the ICEO module simplified the chip needstructure, for a complicated as there is no flow need rate for control a complicated module andflow narrow rate control microfluidic module channel and narrow to focus microfluidic the particle. Itchannel provided to anfocus alternative the particle. method It provided to focus an the alternative particles, method resulting to focus in an the integrated particles, device. resulting in an integratedA microfluidic device. platform with a hybrid DEP-inertial system was developed to separate particles [64] (Figure3C). The channel, which was 200 µm wide and 40 µm deep, consisted of 15 symmetric A microfluidic platform with a hybrid≈ DEP‐inertial system≈ was developed to separate particles U-shaped segments with a length of 700 µm and was patterned in PDMS using soft lithography. [64] (Figure 3C). The channel, which≈ was ≈ 200 μm wide and ≈ 40 μm deep, consisted of 15 Thesymmetric inlet and U outlet‐shaped holes segments were also with punched a length in of the ≈ 700 PDMS. μm Aand lift-o wasff techniquepatterned patternedin PDMS using the thin-film soft Tilithography. (50 nm)/Pt (150 The nm) inlet electrodes and outlet on holes a glass were slide also in a punched zigzag distribution. in the PDMS. The A gap lift‐ andoff widthtechnique of the electrodespatterned were the thin both‐film 20Ti (50µm. nm)/Pt The bonding (150 nm) betweenelectrodes PDMS on a glass and slide the glassin a zigzag slide distribution. was performed The by ≈ oxygengap and plasma. width Aof suspensionthe electrodes flow were of both polystyrene ≈ 20 μm. beads The bonding with diameters between ofPDMS5 µ andm and the glass13 µslidem was ≈ ≈ injectedwas performed from the by inlet, oxygen and plasma. the sheath A suspension flow from flow the top of polystyrene pushed the beads particles with along diameters the bottom of ≈ 5 μ ofm the channel,and ≈ 13 keeping μm was theminjected in from the NUEF. the inlet, The and field the strengthsheath flow of thefrom AC the power top pushed supply the was particles optimized along to the27 V bottomat of1 MHz,the channel, obtaining keeping a separation them in ethefficiency NUEF. of The13 fieldµm strength and 5 µofm the particles AC power of supply100% and ≈ PP ≈ ≈ ≈ ≈ was96%, optimized respectively. to ≈ The27 V variedPP at ≈ particle1 MHz, size obtaining separations a separation were achieved efficiency inside of ≈ 13 this μ chipm and by ≈ adjusting5 μm ≈ theparticles voltage of without ≈ 100% needingand ≈ 96%, a redesignrespectively. of the The chip varied layout. particle The size implementation separations were of the achieved top sheath inside flow improvedthis chip theby adjusting separation the e ffivoltageciency without as the particles needing a were redesign forced of tothe the chip bottom layout. of The the implementation channel and were of the top sheath flow improved the separation efficiency as the particles were forced to the bottom not influenced by the electric field decay along the vertical direction. of the channel and were not influenced by the electric field decay along the vertical direction. An isomotive DEP microfluidic device was developed to separate living and dead yeast cells An isomotive DEP microfluidic device was developed to separate living and dead yeast cells (Figure4)[ 67]. The device consisted of three sections: three inlets, a separation section, and two (Figure 4) [67]. The device consisted of three sections: three inlets, a separation section, and two outlets. The electrodes for DEP were made of a metal sandwich of Ti/Pt/Au with a thickness of 50 nm, outlets. The electrodes for DEP were made of a metal sandwich of Ti/Pt/Au with a thickness of≈ ≈ 50 50 nm, and 300 nm, respectively, on a glass substrate. The electrode width and gap between them ≈ nm, ≈ 50 nm,≈ and ≈ 300 nm, respectively, on a glass substrate. The electrode width and gap between were both 10 µm. The fluidic channels were patterned on top of the electrodes using SU-8 photoresist them were≈ both ≈ 10 μm. The fluidic channels were patterned on top of the electrodes using SU‐8 with a thickness of 100 µm and then sealed by a glass coverslip. The length and width of the main photoresist with a ≈thickness of ≈ 100 μm and then sealed by a glass coverslip. The length and width channel were 600 µm and 100 µm, respectively. The side of the coverslip facing the chamber of the main channel≈ were ≈ 600≈ μm and ≈ 100 μm, respectively. The side of the coverslip facing the waschamber coated was with coated ITO, with serving ITO, as serving a ground as a electrode.ground electrode. The electrode The electrode biasing biasing network network was formedwas usingformed the using resistor the array resistor next array to the next main to channelthe main and channel was connectedand was connected to the electrodes to the electrodes to perform to the isomotiveperform the DEP. isomotive DEP.

FigureFigure 4. 4.An An isomotive isomotive DEP DEP microfluidicmicrofluidic device with with parallel parallel electrodes. electrodes. (A ()A The) The fabrication fabrication process process ofof the the device. device. ( B(B)) DiagramDiagram ofof samplesample process sequence sequence and and photograph photograph of ofthe the device device with with details details showingshowing the the main main channel channel forfor DEP. Reproduced with with permission permission from from [67], [67 ©], ©20072007 Royal Royal Society Society of of ChemistryChemistry (RSC). (RSC).

Micromachines 2019, 10, 423 7 of 22 Micromachines 2019, 10, x 7 of 22

The suspension containing yeast cells was pumped pumped into into the the main main channel channel from from the the middle middle inlet, inlet, and thethe cells cells in in suspension suspension were were focused focused to move to move in a streamline in a streamline with the with help the of sheath help of flow sheath generated flow generatedby the buff erby from the buffer the other from two the side other channels. two side The channels. living and The dead living yeast and cells dead were yeast separated cells inwere the separatedDEP section in underthe DEP a non-uniform section under isomotive a non‐uniform electric isomotive field. The electric device performancefield. The device was performance evaluated by was evaluated by applying AC power supply with a set value of 14 VPP at different frequencies. applying AC power supply with a set value of 14 VPP at different frequencies. A microfluidic microfluidic device with interdigitated electrodes was fabricated to separate living and dead Listeria cells (Figure 55A)A) [[68].68]. The The Cr/Pt Cr/Pt with with a a thickness of of ≈ 1010 nm nm and and 100 100 nm, nm, respectively, respectively, were ≈ sputtered using a magnetron system on a glass substrate, and then then the the interdigitated interdigitated electrodes were patterned usingusing thethe photolithography photolithography method method with with subsequent subsequent etching. etching. The The gap gap and and the the width width of the of theelectrodes electrodes were were both both15 ≈ µ15m. μ Then,m. Then, a rectangular a rectangular chamber chamber was constructedwas constructed of silicon of silicon rubber rubber with ≈ witha thickness a thickness of 0.3of ≈ mm,0.3 mm, and aand20 a ≈ µ20L suspension μL suspension containing containing the Listeriathe Listeriacells cells was was pipetted pipetted into into the ≈ ≈ thechamber. chamber. A glass A glass coverslip coverslip was was then then used used to seal to seal the chamberthe chamber after after loading loading the suspension.the suspension. The The AC AC power supply was applied with a set value of 1 VPP from 10 kHz to 15 MHz, and the cell power supply was applied with a set value of 1 VPP from 10 kHz to 15 MHz, and the cell separation separationperformance performance was monitored was throughmonitored a charge-coupled through a charge device‐coupled camera. device The camera. separation The e ffiseparationciency of efficiencyliving and of dead livingListeria andcells dead was Listeria> 90% cells at a setwas frequency > 90% at of a 50set kHz. frequency This system of 50 combined kHz. This antibody system combinedrecognition antibody and DEP recognition and thus improved and DEP theand selectivity thus improved of capturing the selectivity bacteria. of capturing bacteria.

Figure 5. Devices with interdigitated electrodes. ( (AA)) Schematic Schematic of of the the system with simple particle manipulation and and top top view view of of electrodes. electrodes. Reproduced Reproduced with with permission permission from from [68], [68 ©], 2002© 2002 Elsevier. Elsevier. (B) Drawing(B) Drawing of the of the chip chip combining combining antibody antibody recognition recognition with with DEP, DEP, thus thus improving improving the the selectively selectively of capturing bacteria. Reproduced Reproduced with permission from [69], [69], © 20062006 RSC. RSC.

A DEP microfluidicmicrofluidic device with interdigitated electrodes was developed to concentrate and selectively capture capture ListeriaListeria cellscells bonding bonding with with antibodies antibodies (Figure (Figure 55B)B) from otherother types of cellscells [[69].69]. The interdigitated electrodes were made of a Ti/Pt sandwich with a thickness of 20 nm and 80 nm, The interdigitated electrodes were made of a Ti/Pt sandwich with a thickness≈ of ≈ 20 nm and≈ ≈ 80 nm,respectively, respectively, by a by sputtering a sputtering and aand lift-o a liftff process‐off process on an on oxidized an oxidized silicon silicon substrate. substrate. The widthThe width and gap of the electrodes were 23 µm and 17 µm, respectively. The electrodes were then covered and gap of the electrodes were≈ ≈ 23 μm and≈ ≈ 17 μm, respectively. The electrodes were then covered with a SiO layer with a thickness of 300 nm using plasma-enhanced chemical-vapor deposition, with a SiO2 layer with a thickness of ≈ ≈ 300 nm using plasma‐enhanced chemical‐vapor deposition, preventing electro-osmoticelectro‐osmotic currents currents near near the electrodes the electrodes via the formation via the offormation a bimetallic of electrochemical a bimetallic electrochemicalcell. The PDMS patternedcell. The PDMS with fluidic patterned structure with wasfluidic bonded structure to the was substrate bonded using to the an substrate oxygen plasma using enhancement process at 200 W for 10 s. The length, width, and depth of the channel were an oxygen plasma enhancement≈ process≈ at ≈ 200 W for ≈ 10 s. The length, width, and depth of the 3000 µm, 260 µm, and 16 µm, respectively. The tubes were inserted into the inlet and outlet to channel≈ were≈ ≈ 3000 μm, ≈ ≈260 μm, and ≈ 16 μm, respectively. The tubes were inserted into the inlet anddeliver outlet the to suspension deliver the into suspension the channel. into The the AC channel. power The supply AC waspower applied supply with was a setapplied value with of 20 a V setPP valueat 1 MHz of 20 to V capturePP at 1 MHz the Listeria to capturecells bonding the Listeria with cells antibodies bonding from with the antibodies suspension. from The the result suspension. showed 1 Thethat result> 90% showedListeria thatcells > bonding 90% Listeria with cells antibodies bonding were with collectedantibodies with were a flowcollected rate with of 0.2a flowµL minrate −of, ≈ · ≈ and0.2 the μL∙ increasedmin−1, and flow the increased rate decreased flow therate capture decreased effi ciency.the capture efficiency.

4.2.2. Castellated Castellated Electrodes Electrodes The castellated electrodeselectrodes generated generated an an electrical electrical field field gradient, gradient, forming forming a maximum a maximum value value at the at theedges edges of the of the microelectrodes microelectrodes and and a minimum a minimum value value at the at centerthe center between between two two electrodes. electrodes. A microfluidic device that combined an insulator with 2D electrodes was developed to focus and separate particles (Figure 6A,B) [70]. The electrodes were designed in a parallel configuration with a total number of 30 electrodes having 15 electrodes on each side of the main channel. The

Micromachines 2019, 10, 423 8 of 22

MicromachinesA microfluidic 2019, 10, x device that combined an insulator with 2D electrodes was developed to focus8 of and 22 separate particles (Figure6A,B) [ 70]. The electrodes were designed in a parallel configuration with a total metalnumber sandwich of 30 electrodes of Ti/Pt having was patterned 15 electrodes using on a each lift‐ sideoff technique of the main on channel. a Pyrex The glass metal substrate. sandwich The of fluidicTi/Pt was structure patterned was using made a lift-o in ffa techniqueSU‐8 layer on with a Pyrex a thickness glass substrate. of ≈ 20 The μ fluidicm, forming structure a castellated was made structurein a SU-8 layer(Figure with 6A). a thickness A PDMS ofcover20 withµm, formingaccess holes a castellated was mounted structure on (Figuretop of the6A). structure A PDMS to cover seal ≈ it.with Two access opposite holes wasNUEFs mounted were ongenerated top of the in structure the first to part seal of it. Twothe main opposite channel NUEFs by were applying generated two differentin the first VPP part amplitudes of the main on channel each side by applyingof the channel. two di ffTheseerent VtwoPP amplitudesNUEFs allowed on each the side dielectric of the particleschannel. Thesein the twomain NUEFs channel allowed to reach the dielectrican equilibrium, particles resulting in the main in particles channel to being reach confined an equilibrium, to the centerresulting of the in particleschannel. beingThe equilibrium confined to depended the center on of the the voltage channel. amplitude The equilibrium and the frequencies depended on of the powervoltage supply. amplitude Then, and the the third frequencies AC power of thesupply power was supply. applied Then, to perform the third the AC particle power separation supply was in theapplied second to performpart of the the main particle channel. separation The suspension, in the second containing part of thepolystyrene main channel. beads Thewith suspension, a diameter ofcontaining ≈ 5 μm and polystyrene yeast cells, beads was used with to a diameter evaluate the of chip’s5 µm performance. and yeast cells, This was microfluidic used to evaluate chip, with the ≈ thischip’s combination performance. of castellated This microfluidic electrodes chip, and with an thisinsulator, combination provides of a castellated good alternative electrodes option and for an cellinsulator, separation provides by DEP. a good The alternative combination option of forinsulator cell separation sidewalls by and DEP. castellated The combination electrode of achieved insulator accuratesidewalls particle and castellated focusing electrode and continuous achieved separation. accurate particle focusing and continuous separation.

Figure 6. Devices with castellated electrodes. ( (AA)) Schematic Schematic of of an an insulator insulator and 2D electrode device toto focus and separate particles by applying different different frequencies and amplitudes of an AC power supply [[70].70]. ((BB)) 3D3D drawing drawing and and scanning scanning electronic electronic microscopy microscopy image image of the of device.the device. Reproduced Reproduced with withpermission permission from [from70], © [70],2008 © Elsevier. 2008 Elsevier. (C) Schematic (C) Schematic of the combined of the combined interdigitated interdigitated and castellated and castellatedelectrode device. electrode It was device. used It to was quantitatively used to quantitatively evaluate the evaluate particle andthe particle cell separation. and cell Reproduced separation. Reproducedwith permission with from permission [71], © from2003 Elsevier.[71], © 2003 (D) SchematicElsevier. (D of) aSchematic device based of a on device screen-printing based on screenfabrication.‐printing Reproduced fabrication. with Reproduced permission with from permission [63], © 2015 from Elsevier. [63], © 2015 Elsevier. A microfluidic device, combining interdigitated and castellated electrodes, was developed A microfluidic device, combining interdigitated and castellated electrodes, was developed to to quantitatively assess the particle and cell separation (Figure6C) [ 71]. The Pt electrodes were quantitatively assess the particle and cell separation (Figure 6C) [71]. The Pt electrodes were patterned to form both an interdigitated electrode system as well as castellated topology on a glass patterned to form both an interdigitated electrode system as well as castellated topology on a glass substrate. The height, width, and gap of the interdigitated electrodes and castellated electrodes substrate. The height, width, and gap of the interdigitated electrodes and castellated electrodes were were 100 nm 10 µm 10 µm and 100 nm 20 µm 20 µm, respectively. The fluidic chamber, ≈ 100 ≈nm × 10 μ×m × 10 μ×m and ≈ 100 nm≈ × 20 μm× × 20 μm,× respectively. The fluidic chamber, with a with a length, width, and height of 10 mm, 4 mm, and 30 µm, respectively, was made of length, width, and height of ≈ 10 mm,≈ ≈ 4 mm, and≈ ≈ 30 μm, respectively,≈ was made of poly(methyl poly(methyl methacrylate) (PMMA), and then the PMMA was bonded to a glass substrate on top of the methacrylate) (PMMA), and then the PMMA was bonded to a glass substrate on top of the electrodes. The suspension of polystyrene beads with a diameter of 500 nm, 2 µm, and 6 µm, and electrodes. The suspension of polystyrene beads with a diameter ≈of ≈ 500 nm,≈ ≈ 2 μm, and≈ ≈ 6 μm, RBCs were used to quantitatively evaluate the system’s performance. The suspension was pumped and RBCs were used to quantitatively evaluate the system’s performance. The suspension was into the chamber, and an AC power supply was applied with a set value ranging from 15 V to 20 V pumped into the chamber, and an AC power supply was applied with a set value rangingPP from 15PP and from 1 MHz to 15 MHz. VPP to 20 VPP and from 1 MHz to 15 MHz. An AC-DEP chip was developed with a low-cost screen-printing thick-film technique (Figure6D). An AC‐DEP chip was developed with a low‐cost screen‐printing thick‐film technique (Figure A glass slide was used as a substrate, and a woven mesh and electrodes first patterned a commercial 6D). A glass slide was used as a substrate, and a woven mesh and electrodes first patterned a carbon paste, and contact wires were printed on its surface as using screen printing [63]. Three types of commercial carbon paste, and contact wires were printed on its surface as using screen printing [63]. electrodes, 10 µm thick, were printed on the glass: offset castellated electrodes, non-offset castellated Three types≈ of electrodes, ≈ 10 μm thick, were printed on the glass: offset castellated electrodes, electrodes, and interdigitated line electrodes. Once the carbon paste hardened, the ultraviolet (UV) non‐offset castellated electrodes, and interdigitated line electrodes. Once the carbon paste hardened, the ultraviolet (UV) curable dielectric paste was then printed and cured using UV light to form microfluidic channels with a depth of ≈ 50 μm, and the contact wires were separated from the fluid. Finally, the PDMS layer with inlet and outlet ports was bonded on top of the channels, assisted by an oxygen plasma surface‐activation method. The device’s performance and efficiency in capturing

Micromachines 2019, 10, 423 9 of 22 curable dielectric paste was then printed and cured using UV light to form microfluidic channels Micromachineswith a depth 2019 of, 10, x50 µm, and the contact wires were separated from the fluid. Finally, the PDMS9 of 22 ≈ layer with inlet and outlet ports was bonded on top of the channels, assisted by an oxygen plasma yeastsurface-activation cells from the method. suspension The device’swere optimized performance using and an eACfficiency electric in capturingfield of different yeast cells frequencies from the andsuspension strengths. were The optimized result indicated using an that AC electricthe voltage field amplitude, of different the frequencies frequency and of strengths. AC power The supply, result theindicated carrier that fluid the conductivity, voltage amplitude, and the the flow frequency rate significantly of AC power affected supply, the the capture carrier fluidrate. The conductivity, capture rateand was the flow > 95% rate after significantly applying the affected AC power the capture supply rate. with The set capture values rateof 10 was VPP> at95% 100 afterkHz. applying This simple the fabrication technique has great potential to be applied in mass production. AC power supply with set values of 10 VPP at 100 kHz. This simple fabrication technique has great potential to be applied in mass production. 4.2.3. Other Types of Electrodes 4.2.3.In Other addition Types to the of Electrodes main types of electrode shapes, many different electrodes with specific shapes were Indesigned addition to to provide the main better types and of more electrode stable shapes, NUEF, many such diasff annularerent electrodes [72,73], oblique with specific [74–76], shapes and curvedwere designed electrodes to provide[77]. The better characteristics and more of stable these NUEF, electrodes such have as annular been reviewed [72,73], oblique elsewhere [74– [51]76], and and arecurved not the electrodes subject [of77 this]. The study; characteristics here, we only of these introduce electrodes the quadrupole have been reviewed and trapezoid elsewhere electrodes. [51] and are notThe the performance subject of this of quadrupole study; here, electrodes we only introduce was first the simulated, quadrupole designed, and trapezoid and fabricated electrodes. based on a Thepolynomial performance with of the quadrupole assumption electrodes that it wasobeyed first Laplace’s simulated, equation designed, [66] and to fabricated generate based a well on‐ defineda polynomial NUEF with that the separates assumption yeast that cells. it obeyedA similar Laplace’s electrode equation type was [66] then to generate used to a pattern well- defined actin usingNUEF DEP that on separates a myosin yeast substrate cells. A along similar electric electrode field type lines. was then used to pattern actin using DEP on a myosinA microfluidic substrate along platform electric was field proposed lines. to separate bacteria from whole blood via DEP generatedA microfluidic by a quadrupole platform microelectrode was proposed embedded to separate at bacteria the bottom from wholeof a small blood well via in DEP the generated chip [65] (Figureby a quadrupole 7). The blood microelectrode was pumped embedded into the atdevice the bottom and first of desalinated a small well by in theinline chip dialysis [65] (Figure without7). membrane.The blood was Then, pumped the target into bacteria the device were and separated first desalinated and concentrated by inline using dialysis DEP. without This membrane.obtained a concentrationThen, the target efficiency bacteria wereof ≈ separated79% and ≈ and78% concentrated of Escherichia using coli DEP. and This Staphylococcus obtained a concentration aureus at a processingefficiency ofrate of79% ≈ 0.6 and mL∙h78%−1. The of resultsEscherichia were coli verifiedand Staphylococcus by performing aureus a polymeraseat a processing chain reaction, rate of ≈ ≈ showing0.6 mL thath 1. bacterial The results 16S wereribosomal verified DNA by levels performing were enriched a polymerase ≈ 307‐ chainfold in reaction, comparison showing with thatthe ≈ · − original.bacterial 16S ribosomal DNA levels were enriched 307-fold in comparison with the original. ≈

FigureFigure 7. AA device device with with quadrupole quadrupole electrodes. electrodes. (A) The(A) principleThe principle of the of device. the device. (B) Schematic (B) Schematic showing showingred blood red cells blood (RBC) cells separation (RBC) separation by quadrupole by quadrupole electrode configuration. electrode configuration. Reproduced Reproduced with permission with permissionfrom [65], © from2017 [65], RSC. © 2017 RSC.

AA trapezoidaltrapezoidal electrode electrode array array (TEA) (TEA) based based microfluidic microfluidic chip was chip developed was developed to separate to polystyrene separate polystyrenebeads of diff beadserent diametersof different (Figure diameters8)[ 78 (Figure]. The gold 8) [78]. TEA The was gold patterned TEA was on a patterned Pyrex glass on substrate a Pyrex glassusing substrate a photolithography using a photolithography method. The widths method. of twoThe baseswidths in of a two single bases trapezoidal in a single electrode trapezoidal were electrode were ≈ 120 μm and ≈ 20 μm, respectively, and their height was ≈ 60 μm. The microfluidic structure was patterned in PDMS using a soft lithography method. The width and depth of the microfluidic channel were ≈ 50 μm and ≈ 30 μm, respectively. The fluidic structure was aligned to

Micromachines 2019, 10, 423 10 of 22

120 µm and 20 µm, respectively, and their height was 60 µm. The microfluidic structure was ≈ ≈ ≈ patternedMicromachines in 2019 PDMS, 10, x using a soft lithography method. The width and depth of the microfluidic10 of channel 22 were 50 µm and 30 µm, respectively. The fluidic structure was aligned to the TEA, and then the the TEA,≈ and then ≈the PDMS with a patterned structure was bonded to the glass substrate using an PDMSoxygen with plasma a patterned method. structure was bonded to the glass substrate using an oxygen plasma method.

Figure 8. A trapezoidal electrode array (TEA) microfluidic chip. Reproduced with permission from [78], Figure 8. A trapezoidal electrode array (TEA) microfluidic chip. Reproduced with permission from ©[78],2005 © RSC. 2005 (ARSC.) Schematic (A) Schematic of the of chip the for chip particle for particle separation. separation. (B) Schematic (B) Schematic of the of chipthe chip for particle for fractionation.particle fractionation. (C) Image (C) of Image the TEA of the and TEA the and device. the device.

TheThe systemsystem had three three sections: sections: focus, focus, separation, separation, and andcollection. collection. In the Infocus the section, focus section,100 100trapezoidal trapezoidal electrodes electrodes forced forced the theflow flow that that contained contained polystyrene polystyrene beads beads to move to move in a in streamline a streamline alongalong one one side side ofof thethe channel. In In the the separation separation section, section, a TEA a TEA with with 20 20electrodes electrodes performed performed the thebead bead separationseparation based based on on the the beads’ beads’ diameter, diameter, achieving achieving diff erentdifferent dielectrophoretic dielectrophoretic velocities. velocities. The streamsThe ofstreams the two of separated the two separated beads were beads split were into split two into outlets two outlets and collected and collected in the in collecting the collecting section. section. TheThe polystyrene polystyrene beads with with diameters diameters of of ≈ 6 μ6 mµm and and ≈ 15 μ15mµ mwere were used used to test to test the theseparation separation ≈ ≈ effiefficiencyciency by by applying applying anan AC power supply supply with with a a set set value value of of 8 V 8PP V PPat 50at 50kHz. kHz. The The effect eff ofect the of flow the flow raterate was was investigated investigated to evaluate the the separation separation performance. performance. The The results results showed showed that that the thepurities purities of of beadsbeads with with diameters diameters ofof ≈ 66 μµmm and and ≈ 1515 μµmm were were 96.8 96.8 ± 0.6%0.6% and and 99.5 99.5 ± 0.5%,0.5%, respectively, respectively, at a atflow a flow ≈ ≈ ± ± raterate of of ≈ 1010 mLmL∙hh−1.. In In comparison comparison with with other other DEP DEP devices, devices, the the TEA TEA system system gently gently processed processed the the ≈ · − samplesample to to prevent prevent cellcell damage from from long long-term‐term exposure exposure in in the the electric electric field. field. TheThe integrated integrated circuit circuit (IC) (IC) technique technique was was used used to to fabricate fabricate the the electrode electrode array array to to manipulate manipulate yeast cellsyeast and cells mammalian and mammalian cells (Figure cells (Figure9)[ 79]. 9) The [79]. IC The electrodes IC electrodes array array with awith static a static random random access access memory elementmemory was element designed was and fabricateddesigned usingand a standardfabricated complementary using a standard metal-oxide-semiconductor complementary metal‐oxide‐semiconductor (CMOS) process. The array, with a size of ≈ 1.4 mm × 2.8 mm, consisted (CMOS) process. The array, with a size of 1.4 mm 2.8 mm, consisted of 32,768 individual pixels, of 32,768 individual pixels, and each pixel≈ was ≈ 11× μm × 11 μm. The microfluidic channel was formed in a double‐sided adhesive film placed on the IC electrode array and then sealed by a

Micromachines 2019, 10, 423 11 of 22 andMicromachines each pixel 2019 was, 10, x11 µm 11 µm. The microfluidic channel was formed in a double-sided11 adhesive of 22 ≈ × film placed on the IC electrode array and then sealed by a coverslip with holes connected to the fluid coverslip with holes connected to the fluid input and output tubes. The entire setup was placed on a input and output tubes. The entire setup was placed on a Cu block serving as a cooler. The DEP was Cu block serving as a cooler. The DEP was performed in a set value of 5 VPP at 1.8 MHz to test the cell performedmanipulation, in a set resulting value of in 5 cell VPP movementat 1.8 MHz at to a testspeed the of cell ≈ 30 manipulation, μm s−1. Additionally, resulting this in cell setup movement could at a speed of 30 µm s 1. Additionally, this setup could split the water from oil in a volume of pL. split the water≈ from oil− in a volume of pL. This IC microfluidic system has good potential to be Thiswidely IC microfluidic used because system the electrode has good array potential can be to mass be widely produced used using because a standard the electrode CMOS fabrication array can be massprocess. produced using a standard CMOS fabrication process.

FigureFigure 9. 9.An An integrated integrated circuit circuit (IC) (IC) DEP DEP microfluidicmicrofluidic system. Reproduced Reproduced with with permission permission from from [79], [79 ], © 2008© 2008 RSC. RSC. (A ()A Schematic) Schematic of of the the device. device. ( (BB)) PhotographPhotograph of the the system. system. (C (C) )The The schematic schematic of ofthe the IC IC electrodes.electrodes. (D (D) Image) Image of of the the IC IC electrodes. electrodes.

4.3.4.3. Three-Dimensional Three‐Dimensional Electrodes Electrodes

WeWe discussed discussed the the DEP DEP microfluidic microfluidic chips chips with with a variety a variety of 2D of electrodes. 2D electrodes. However, However,FDEP isFDEP mainly is availablemainly atavailable the bottom at the of bottom the microfluidic of the microfluidic channels nearchannels the thin-filmnear the thin electrodes.‐film electrodes. There is aThere significant is a significant decrease of FDEP in a vertical direction [64], which is inversely proportional to the distance decrease of FDEP in a vertical direction [64], which is inversely proportional to the distance from the electrodesfrom the generating electrodes the generating electric field. the electric The polarized field. The particles polarized that flow particles at a certain that flow distance at a abovecertain the electrodesdistance mightabove the not electrodes be dielectrophoretically might not be dielectrophoretically trapped leading to trapped the development leading to the of development a 3D electrode systemof a to3D perform electrode a more system powerful to perform NUEF a through more powerful the entire NUEF depth through of the channels. the entire This depth also providedof the channels. This also provided a gradient in the velocity generated by the hydrodynamic force in the a gradient in the velocity generated by the hydrodynamic force in the device, improving the separation device, improving the separation performance of the microfluidic devices [80–90]. performance of the microfluidic devices [80–90]. 4.3.1. Metal 3D Electrodes 4.3.1. Metal 3D Electrodes The thin‐film electrodes patterned on the side wall of the main channel could be used as 3D The thin-film electrodes patterned on the side wall of the main channel could be used as 3D electrodes (Figure 10) [28]. Compared to the planar electrodes at the bottom of the channel, the electrodes (Figure 10)[28]. Compared to the planar electrodes at the bottom of the channel, the electrodes electrodes patterned on both side walls provided a much stronger NUEF and suppressed the field patterned on both side walls provided a much stronger NUEF and suppressed the field strength strength decay along the channel’s height, resulting in cell focusing and separation [70]. The kidney decay along the channel’s height, resulting in cell focusing and separation [70]. The kidney cells

Micromachines 2019, 10, 423 12 of 22 Micromachines 2019, 10, x 12 of 22 cellsHEK293 HEK293/nerve/nerve cells cells N115 N115 and kidneyand kidney cell HEK293 cell HEK293/polystyrene/polystyrene beads beads were were used toused verify to verify the chip’s the chip’sperformance performance by applying by applying an AC an power AC power supply supply with optimized with optimized voltage voltage and frequency. and frequency.

FigureFigure 10. 10. AA vertical vertical thin thin-film‐film device device and and the the 2D 2D thin thin-film‐film electrodes electrodes were were used used as as 3D 3D electrodes. electrodes. ReproducedReproduced with with permission permission from from [28], [28 ],© ©20092009 John John Wiley Wiley and and Sons. Sons. (A) Schematic (A) Schematic of the of device. the device. (B) The(B) Thedrawing drawing of the of cell the separation cell separation in the in separation the separation zone. zone.

A microfluidic device made by PDMS molding, combining acoustophoresis and DEP methods, A microfluidic device made by PDMS molding, combining acoustophoresis and DEP methods, was proposed, designed, fabricated, and tested to wash and separate uncoated and half-coated latex was proposed, designed, fabricated, and tested to wash and separate uncoated and half‐coated latex particles with aluminum with a mean diameter of 5 µm (Figure 11A,B) [80]. The brass mold particles with aluminum with a mean diameter of ≈ 5≈ μm (Figure 11A,B) [80]. The brass mold was was machined, making microfluidic channels with a depth and length of 200 µm and 61 mm, machined, making microfluidic channels with a depth and length of ≈ 200≈ μm and ≈ ≈61 mm, respectively. Two piezoelectric ceramic transducer slides made of lead-zirconate-titanate (PZT) and respectively. Two piezoelectric ceramic transducer slides made of lead‐zirconate‐titanate (PZT) and metal electrodes were placed next to each other and tightened at both sides of the channels on the brass metal electrodes were placed next to each other and tightened at both sides of the channels on the mold. The liquid PDMS precursor was then poured into the mold and cured. Finally, the cured PDMS, brass mold. The liquid PDMS precursor was then poured into the mold and cured. Finally, the cured patterned with structures attached to PZTs and electrodes, was peeled off from the mold and bonded to PDMS, patterned with structures attached to PZTs and electrodes, was peeled off from the mold and a glass slide using an oxygen plasma surface-activation process. The chip consisted of two main units, bonded to a glass slide using an oxygen plasma surface‐activation process. The chip consisted of two one for washing and one for separation. The suspension containing target particles, with contents main units, one for washing and one for separation. The suspension containing target particles, with of 1 107 particles mL 1, and the buffer were pumped into the channel from two different inlets. contents≈ × of ≈ 1 × 107 particles· − ∙mL−1, and the buffer were pumped into the channel from two different The standing waves generated by the PZTs in the washing section of the chip caused the particles to inlets. The standing waves generated by the PZTs in the washing section of the chip caused the move from the carrier into the buffer solution with low electrical conductivity, effectively suppressing particles to move from the carrier into the buffer solution with low electrical conductivity, effectively the amplitude of the Joule heating effect. Subsequently, the solution reached the 3D separation unit of suppressing the amplitude of the Joule heating effect. Subsequently, the solution reached the 3D the chip, where the FDEP separated the uncoated and half-coated particles using an AC power supply separation unit of the chip, where the FDEP separated the uncoated and half‐coated particles using an with set values of 16 VPP at 3 MHz. Integrating the washing and separation units provided a valuable AC power supply with set values of 16 VPP at 3 MHz. Integrating the washing and separation units system for cell separation in clinical diagnostics. However, the fabrication of the 3D electrodes is provided a valuable system for cell separation in clinical diagnostics. However, the fabrication of the a challenging process. Additionally, the process of tightening the screws into the brass mold is another 3D electrodes is a challenging process. Additionally, the process of tightening the screws into the challenge because there is a high chance of liquid leakage. brass mold is another challenge because there is a high chance of liquid leakage.

Micromachines 2019, 10, 423 13 of 22 Micromachines 2019, 10, x 13 of 22

FigureFigure 11. DevicesDevices with with 3D 3D electrodes. ( (AA)) Photograph Photograph of of the the device device combined combined with with piezoelectric ceramicceramic transducertransducer slides slides made made of lead-zirconate-titanateof lead‐zirconate‐titanate (PZT) (PZT) slides slides and metal and electrodes. metal electrodes. (B) Principle (B) Principleof the device. of the The device. PZT slides The werePZT slides used to were focus used the particles,to focus andthe theparticles, particles and were the separated particles inwere the separatedfollowing in NUEF the following area. Reproduced NUEF area. with Reproduced permission fromwith [80permission], © 2016 Americanfrom [80], Institute© 2016 American of Physics Institute(AIP) Publishing. of Physics (C )(AIP) Schematic Publishing. and simulation (C) Schematic of the device and simulation with a carbon of electrode the device array. with Reproduced a carbon electrodewith permission array. Reproduced from [81], © with2013 permission John Wiley from and Sons.[81], © 2013 John Wiley and Sons. 4.3.2. Carbon 3D Electrodes 4.3.2. Carbon 3D Electrodes Apart from the metal electrodes, electrodes made of carbon were also used in DEP microfluidic Apart from the metal electrodes, electrodes made of carbon were also used in DEP microfluidic devices as carbon is more electrochemically stable than metal, resulting in the application of a higher devices as carbon is more electrochemically stable than metal, resulting in the application of a higher voltage without electrolysis of the solution. The cost of the material and process was also lower than voltage without electrolysis of the solution. The cost of the material and process was also lower than for metal electrodes as carbon electrodes were formed directly from the photoresist. These advantages for metal electrodes as carbon electrodes were formed directly from the photoresist. These are of interest to researchers developing carbon electrodes to perform DEP in microfluidics. advantages are of interest to researchers developing carbon electrodes to perform DEP in A 3D carbon electrode system was proposed to concentrate λ-DNA (Figure 11C) [81]. The carbon microfluidics. electrodes were made by carbonization of SU-8 photosensitive material. The fused silica substrate A 3D carbon electrode system was proposed to concentrate λ‐DNA (Figure 11C) [81]. The was coated with SU-8, then the SU-8 was patterned, exposed to a temperature of 900 C, and carbon electrodes were made by carbonization of SU‐8 photosensitive material. The≈ fused◦ silica carbonized. These carbon electrode pillars had a height and diameter of 95 µm and 58 µm, substrate was coated with SU‐8, then the SU‐8 was patterned, exposed to a temperature≈ of ≈ ≈900 °C, respectively. They were then connected to the carbon pads by dispensing indium. The microfluidic and carbonized. These carbon electrode pillars had a height and diameter of ≈ 95 μm and ≈ 58 μm, channels, with a depth and width of 100 µm and 2 mm, respectively, were made on a stack of respectively. They were then connected≈ to the carbon≈ pads by dispensing indium. The microfluidic pressure-sensitive double-sided adhesive tape with a thickness of 100 µm and then covered by a layer channels, with a depth and width of ≈ 100 μm and ≈ 2 mm, respectively,≈ were made on a stack of of polycarbonate (PC). pressure‐sensitive double‐sided adhesive tape with a thickness of ≈ 100 μm and then covered by a Finally, both substrates, fused silica with carbon electrodes and the stack of PC layers, were adhered layer of polycarbonate (PC). to each other and sealed using a rolling press. A suspension containing λ-DNA was pumped into the Finally, both substrates, fused silica with carbon electrodes and the stack of PC layers, were channel with a flow rate of 2 µL min 1. The AC power supply amplitude was set to 16 V with adhered to each other and≈ sealed· using− a rolling press. A suspension containing λ‐DNAPP was different frequencies to characterize the system’s performance. The result showed that λ-DNA was pumped into the channel with a flow rate of ≈ 2 μL∙min−1. The AC power supply amplitude was set trapped by pDEP or nDEP at set frequencies between either 10 kHz or 50 kHz for pDEP and either to 16 VPP with different frequencies to characterize the system’s performance. The result showed that 75 kHz or 250 kHz for nDEP. λ‐DNA was trapped by pDEP or nDEP at set frequencies between either 10 kHz or 50 kHz for pDEP A compact disc (CD) microfluidic system with 3D carbon electrodes was developed to separate and either 75 kHz or 250 kHz for nDEP. latex particles, with a diameter of 8 µm, from yeast cells (Figure 12)[91]. The microfluidic system A compact disc (CD) microfluidic≈ system with 3D carbon electrodes was developed to separate consisted of four sections: a programmable motorized spin stand combined with an optical detection latex particles, with a diameter of ≈ 8 μm, from yeast cells (Figure 12) [91]. The microfluidic system system, a function generator to apply the AC power supply, a CD platform to hold the DEP chips when consisted of four sections: a programmable motorized spin stand combined with an optical detection spinning, and carbon-DEP chips. The formation of carbon electrodes was described in [81]. The SU-8 system, a function generator to apply the AC power supply, a CD platform to hold the DEP chips was first patterned on the substrate, and then the pyrolysis was conducted to carbonize the SU-8, when spinning, and carbon‐DEP chips. The formation of carbon electrodes was described in [81]. The SU‐8 was first patterned on the substrate, and then the pyrolysis was conducted to carbonize the SU‐8, forming the 3D electrodes at a height of ≈ 40 μm and ≈ 70 μm, respectively. All the electrodes

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Micromachines 2019, 10, x 14 of 22 forming the 3D electrodes at a height of 40 µm and 70 µm, respectively. All the electrodes were ≈ ≈ connectedwere connected to the electricto the electric leads. A leads. thin SU-8A thin layer SU was‐8 layer patterned was patterned around the around 3D electrodes the 3D toelectrodes protect the to connectingprotect the connecting leads from contactleads from with contact the suspension. with the suspension.

Figure 12.12. Compact disc (CD) microfluidicmicrofluidic device withwith 3D3D carboncarbon electrodes.electrodes. Reproduced with permission fromfrom [ 91[91],], © ©2010 2010 RSC. RSC. (A) Schematic(A) Schematic of the of device. the device. (B) The ( imageB) The of image the carbon of the electrodes carbon andelectrodes the schematic and the ofschematic the chip. of the chip.

The fluidic fluidic structure with with two two chambers chambers and and two two channels channels was was patterned patterned in in PC PC and and sealed sealed by by a a double-sided pressure-sensitive adhesive. The width and height of the main channel were 600 µm double‐sided pressure‐sensitive adhesive. The width and height of the main channel were ≈ ≈ 600 μm and 100 µm, respectively. The width and height of the channel for suspension retrieval were 1 µm and ≈ ≈ 100 μm, respectively. The width and height of the channel for suspension retrieval were≈ ≈ 1 and 100 µm, respectively. The loaded suspension in the inlet chamber was delivered into the μm and≈ ≈ 100 μm, respectively. The loaded suspension in the inlet chamber was delivered into the main channel, and and its its flow flow rate rate was was controlled controlled by by a a centrifugal centrifugal force force caused caused by by rotating rotating the the CD CD at at ≈ 800 revolutions per minute. The AC power supply with a set value of 20 V at 200 kHz was applied ≈800 revolutions per minute. The AC power supply with a set value of 20 VPP at 200 kHz was applied to separate the yeast cells from latex particles at a flow rate of 35 mL min 1. to separate the yeast cells from latex particles at a flow rate of ≈ ≈ 35 mL·∙min−1. 4.3.3. Polymer 3D Electrodes 4.3.3. Polymer 3D Electrodes The mixture of silver platelet PDMS was another alternative to fabricate the electrode. Different The mixture of silver platelet PDMS was another alternative to fabricate the electrode. from the fabrication of a metal electrode, it removed the sputtering and photolithography applied in Different from the fabrication of a metal electrode, it removed the sputtering and photolithography metal film electrodes, simplifying the fabrication process and device structure. applied in metal film electrodes, simplifying the fabrication process and device structure. The 3D electrodes were developed based on PDMS (Figure 13)[89]. The ITO leadouts were first The 3D electrodes were developed based on PDMS (Figure 13) [89]. The ITO leadouts were first patterned on a glass substrate using a photolithography method. A second photolithography was patterned on a glass substrate using a photolithography method. A second photolithography was then applied to form the 3D electrode mold. Ag-PDMS 3D electrodes made of silver platelets with then applied to form the 3D electrode mold. Ag‐PDMS 3D electrodes made of silver platelets with a a diameter of 1 µm and PDMS precursor mixture with a ratio of 85% were poured on the top of diameter of ≈ 1≈ μm and PDMS precursor mixture with a ratio of 85% were poured on the top of the the ITO leadouts and the PDMS precursor was subsequently cross-linked. The width and gap of the ITO leadouts and the PDMS precursor was subsequently cross‐linked. The width and gap of the electrodes were both 200 µm. The PDMS layer patterned with inlets, outlets, and a channel made electrodes were both ≈ ≈ 200 μm. The PDMS layer patterned with inlets, outlets, and a channel made by soft lithography was aligned to the electrodes and bonded to the glass substrate using an oxygen by soft lithography was aligned to the electrodes and bonded to the glass substrate using an oxygen plasma activation method. The diameters of the two inlets and outlets were 6 mm and 5 mm, plasma activation method. The diameters of the two inlets and outlets were ≈ ≈ 6 mm and ≈ ≈ 5 mm, respectively. The width of the channels connected to inlet A and inlet B was 100 µm and 200 µm, respectively. The width of the channels connected to inlet A and inlet B was ≈ ≈ 100 μm and ≈ ≈200 μm, respectively. The width of the main channel was 200 µm. There were three vaulted obstacles along respectively. The width of the main channel was ≈ ≈ 200 μm. There were three vaulted obstacles along the main channel with a pitch and radius of 400 µm and 100 µm, respectively. the main channel with a pitch and radius of ≈ ≈ 400 μm and ≈ ≈100 μm, respectively.

Micromachines 2019, 10, 423 15 of 22 Micromachines 2019, 10, x 15 of 22

Figure 13. A 3D Ag-Ag‐ polydimethylsiloxanepolydimethylsiloxane (PDMS) electrode microfluidicmicrofluidic device.device. Reproduced with permission fromfrom [ 89[89],], © ©2015 2015 John John Wiley Wiley and and Sons. Sons. (A ()A Schematic) Schematic of theof the device. device. (B )( TheB) The drawing drawing of the of separation.the separation. (C) The(C) photoThe photo of the of device. the device. (D) The (D dimension) The dimension of the structure.of the structure. (E) The ( fabricationE) The fabrication process ofprocess the device. of the device.

The suspensionsuspension containing containing yeast yeast cells cells and gold-coatedand gold‐coated polystyrene polystyrene microspheres microspheres with a diameter with a ofdiameter25 µm of was ≈ 25 pumped μm was intopumped the channel into the connected channel connected to inlet A. to The inlet suspension A. The suspension was moving was along moving the ≈ upperalong the side upper wall ofside the wall main of channelthe main by channel the dynamic by the focusingdynamic flowfocusing generated flow generated by the bu byffer the from buffer the channelfrom the connected channel connected to inlet B. to The inlet AC B. power The AC supply power was supply applied was with applied different with amplitudesdifferent amplitudes of VPP at 1of MHz VPP at to 1 investigate MHz to investigate the separation the separation efficiency atefficiency different at flow different rates. flow The resultsrates. The showed results that showed> 90% yeastthat > cells 90% andyeast cells100% and gold-coated ≈ 100% gold polystyrene‐coated polystyrene microspheres microspheres were separated were by separated applying by voltages applying of ≈ 1 voltages18.75 V of ≈ and18.75 12.5VPP and V ≈ at12.5 1 MHz VPP at with 1 MHz a flow with rate a offlow300 rateµ ofm ≈ s 300. μm∙s−1. ≈ PP ≈ PP ≈ · − A 3D3D Ag-PDMS Ag‐PDMS electrode electrode microfluidic microfluidic device device was was developed developed to separate to separate microalgae microalgae cells in cells ballast in waterballast (Figure water (Figure 14)[90]. 14) The [90]. electrode The electrode fabrication fabrication process process was described was described in [89]. in The [89]. length The length and width and ofwidth the mainof the channel main channel were were2000 ≈ µ2000m and μm and100 ≈ µ100m, respectively. μm, respectively. One sideOne ofside the of main the main channel channel was ≈ ≈ designedwas designed as a triangular as a triangular shape shape to increase to increase the inhomogeneity the inhomogeneity of the electric of the fieldelectric and field improve and theimproveFDEP. Thethe F suspensionDEP. The suspension containing containing cells of interest cells of and interest the bu andffer the were buffer pumped were intopumped the channel into the from channel two inletsfrom two forcing inlets the forcing cells to the move cells along to move the triangular along the sidetriangular of the mainside of channel. the main Two channel. types ofTwo microalgae types of cells,microalgaePlatymonas cells,and PlatymonasClosterium and, and Closterium polystyrene, and particles polystyrene were particles used to test were the used chip’s to performance test the chip’s by applyingperformance an optimized by applying AC poweran optimized supply withAC power a value supply of 10 with V ata 30value MHz, of ≈ obtaining10 VPP aat separation 30 MHz, ≈ PP eobtainingfficiency ofa separation> 90%. efficiency of > 90%.

Micromachines 2019, 10, 423 16 of 22 Micromachines 2019, 10, x 16 of 22

Figure 14. AA 3D 3D Ag Ag-PDMS‐PDMS electrode electrode microfluidic microfluidic device. device. Reproduced Reproduced with with permission permission from from [90], [90 ©], ©20192019 John John Wiley Wiley and and Sons. Sons. (A (A) )Schematic Schematic of of the the system. system. (B (B) )The The image image of of the device. ( (C)) The schematic in the separation area and the dimension of thethe electrodes.electrodes.

4.3.4. Silicon 3D Electrodes 4.3.4. Silicon 3D Electrodes The developments in IC and micro-electro-mechanical-system fabrication technology have also The developments in IC and micro‐electro‐mechanical‐system fabrication technology have also been used for DEP, allowing the chip structure to be made from single crystal silicon. As a result, been used for DEP, allowing the chip structure to be made from single crystal silicon. As a result, the the chip fabrication and structure were simplified as using electrically conductive silicon formed both, chip fabrication and structure were simplified as using electrically conductive silicon formed both, the microfluidic channel walls also serving as 3D electrodes, obtaining a potential for mass production. the microfluidic channel walls also serving as 3D electrodes, obtaining a potential for mass A square-shaped pillar structure together with the channels was proposed and fabricated from production. silicon (Figure 15A) [82] using a deep reactive ion etching (DRIE) process and was then sandwiched in A square‐shaped pillar structure together with the channels was proposed and fabricated from silicon with two glass wafers by anodic bonding. Living/dead yeast cells were used to test the device’s silicon (Figure 15A) [82] using a deep reactive ion etching (DRIE) process and was then sandwiched performance by applying the AC power supply with a gradually increasing amplitude from 0 V to in silicon with two glass wafers by anodic bonding. Living/dead yeast cells were used to testPP the 25 V at a frequency range from 20 kHz to 100 kHz. device’sPP performance by applying the AC power supply with a gradually increasing amplitude from The electrode pillars could also be designed in a non-uniform height to separate living and dead 0 VPP to 25 VPP at a frequency range from 20 kHz to 100 kHz. yeast cells (Figure 15B) [83]. The device consisted of two glass wafers sandwiching a patterned silicon wafer forming the 3D electrodes as well as the separation chamber. The glass wafer was anodically bonded to the silicon wafer, and then the DRIE was performed to etch the silicon, forming two sets of pillars of different heights, either 100 µm or 2.5 µm, as well as the chamber. These two structures ≈ ≈ were then bonded together, forming the asymmetric electrode structures. The device’s performance was tested using two populations of cells, dead and live yeast, one exhibiting the pDEP and the other exhibiting nDEP, using an AC power supply with a set amplitude of 20 VPP at 20 kHz. Another DEP microfluidic device with symmetric silicon electrodes was proposed to perform a sequential DEP field flow cell separation (Figure 15C) [84]. The chip fabrication process was similar to the previous one shown in Figure 15B. The only difference was that the silicon electrodes were symmetrical. This electrode geometry, compared to the coplanar electrodes, suppressed Joule heat, and increased the separation efficiency. The suspension was first pumped into the channel, and the particles were then trapped in different locations by performing the DEP. One population of particles

Micromachines 2019, 10, 423 17 of 22

(group A) was trapped in the center of the channel, which was the weakest position of the electric field, while the other population (group B) was trapped near the electrode field. Group A was then flushed out of the channel by increasing the flow rate. The electric field was then removed, and group B was subsequently flushed out. This array structure increased separation efficiency. Living/dead yeast cells wereMicromachines used to 2019 verify, 10, x the chip’s performance. 17 of 22

FigureFigure 15.15. MicrofluidicMicrofluidic devicesdevices withwith siliconsilicon 3D3D electrodes.electrodes. (A)) SchematicSchematic ofof thethe devicedevice withwith siliconsilicon electrodeelectrode pillars of a a uniform uniform height. height. Reproduced Reproduced with with permission permission from from [82], [82 ©], 2008© 2008 Elsevier. Elsevier. (B) (SchematicB) Schematic of ofthe the device device with with silicon silicon electrode electrode pillars pillars of of a a non non-uniform‐uniform height. Reproduced withwith permissionpermission fromfrom [[83],83], ©© 20092009 AIPAIP Publishing. Publishing. ((CC)) SchematicSchematic ofof thethe devicedevice withwith symmetricalsymmetrical siliconsilicon electrodeselectrodes [[84].84]. ((DD)) SchematicSchematic ofof thethe devicedevice withwith bulkbulk siliconsilicon electrodeelectrode [[85].85]. Silicon can also be used as a bulk electrode without arrays, simplifying the chip structure and The electrode pillars could also be designed in a non‐uniform height to separate living and dead fabrication process (Figure 15D) [85]. The simulation showed that there was no volume without a NUEF, yeast cells (Figure 15B) [83]. The device consisted of two glass wafers sandwiching a patterned suggesting good separation efficiency. The fabricated chip consisted of three parts: heavily doped silicon wafer forming the 3D electrodes as well as the separation chamber. The glass wafer was silicon patterned with corrugated channels forming the DEP, sandwiched by two glasses mechanically anodically bonded to the silicon wafer, and then the DRIE was performed to etch the silicon, forming connected by two subsequent anodic bonding processes. The device’s performance was again verified two sets of pillars of different heights, either ≈ 100 μm or ≈ 2.5 μm, as well as the chamber. These two by separating dead from living yeast cells using an AC power supply with a set voltage amplitude of structures were then bonded together, forming the asymmetric electrode structures. The device’s 25 V . performancePP was tested using two populations of cells, dead and live yeast, one exhibiting the pDEP 5.and Conclusions the other exhibiting nDEP, using an AC power supply with a set amplitude of 20 VPP at 20 kHz. Another DEP microfluidic device with symmetric silicon electrodes was proposed to perform a sequentialDEP is DEP a particle field separationflow cell separation technique (Figure based on 15C) particle [84]. polarization The chip fabrication in a NUEF. process It has been was provensimilar toto bethe a previous viable tool one for shown LOC in in the Figure application 15B. The of medicine, only difference molecular was diagnostics, that the silicon and electrodes any other fields were relatedsymmetrical. to particle This separationelectrode geometry, because of compared its selectivity to the and coplanar accuracy. electrodes, It is also suppressed a label-free Joule method. heat, Inand this increased review, wethe describedseparation DEP efficiency. theory The and suspension compared it was to other first pumped separation into techniques the channel, to show and the its particles were then trapped in different locations by performing the DEP. One population of particles (group A) was trapped in the center of the channel, which was the weakest position of the electric field, while the other population (group B) was trapped near the electrode field. Group A was then flushed out of the channel by increasing the flow rate. The electric field was then removed, and group B was subsequently flushed out. This array structure increased separation efficiency. Living/dead yeast cells were used to verify the chip’s performance.

Micromachines 2019, 10, 423 18 of 22 advantages for microfluidic devices. We also introduced the recent developments in DEP microfluidic devices, classified by electrode type. For each system, we explained the chip structure, fabrication process, electrode type, and operation principle. These microfluidic devices have the potential to be widely used in laboratories and diagnostic centers. However, the wider employment of DEP would probably require higher levels of system integration, including sample pretreatment, post DEP processing, as well as product analysis. In addition, the DEP itself should be designed in a more efficient way of using 3D or semi-3D operations. That could be achieved either by designing the 3D electrode geometry or making the DEP area shallow and suppressing the low efficiency space to promote the commercialization and wide utilization of DEP.

Acknowledgments: This work was supported by the Foreign Experts Program of P.R. China (W099109). We also acknowledge the support of CEITEC Nano Research Infrastructure (ID LM2015041, MEYS CR, 2016–2019), CEITEC Brno University of Technology, and Grant Agency of the Czech Republic under the contract GA16-11140S. Conflicts of Interest: The authors declare no conflict of interest.

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