mic omachines
Re iew
DEP-on-a-Chip: Dielec opho esis Applied o
Mic o luidic Pla o ms
Haoqing Zhang 1, Honglong Chang 1,* and Pa el Neuzil 1,2,3,*
1Minis y o Educa ion Key Labo a o y o Mic o/Nano Sys ems o Ae ospace, School o Mechanical
Enginee ing, No hwes e n Poly echnical Uni e si y, 127 Wes Youyi Road, Xi’an 710072, China;
[email p o ec ed]
2Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no 61300, Czech Republic
3
Depa men o Mic oelec onics, Facul y o Elec ical Enginee ing, B no Uni e si y o Technology, Technick
á
3058/10, B no 61600, Czech Republic
*Co espondence: [email p o ec ed] (H.C.); [email p o ec ed] (P.N.)
Recei ed: 17 May 2019; Accep ed: 19 June 2019; Published: 24 June 2019


Abs ac :
Dielec ic pa icles in a non-uni o m elec ic ield a e subjec o a o ce caused by
a phenomenon called dielec opho esis (DEP). DEP is a commonly used echnique in mic o luidics
o pa icle o cell sepa a ion. In compa ison wi h o he sepa a ion me hods, DEP has he unique
ad an age o being label- ee, as , and accu a e. I has been widely applied in mic o luidics o
bio-molecula diagnos ics and medical and polyme esea ch. This e iew in oduces he basic heo y
o DEP, i s ad an ages compa ed wi h o he sepa a ion me hods, and i s applica ions in ecen yea s,
in pa icula , ocusing on he di e en elec ode ypes in eg a ed in o mic o luidic chips, ab ica ion
echniques, and ope a ion p inciples.
Keywo ds:
dielec opho esis; mic o luidics; wo-dimensional elec odes; pa allel elec odes;
in e digi a ed elec ode; cas ella ed elec odes; h ee-dimensional elec odes
1. In oduc ion
The es ablishmen o lab-on-a-chip (LOC) echnology made i possible o g adually in eg a e la ge
and cos ly bench- op labo a o y ins umen s in o palm-sized de ices con aining chips in a scale o
a ew cen ime e s o less, wi hou sac i icing hei pe o mance [
1
]. LOC-based de ices ha e been
used in molecula biology [2], clinical diagnos ics [3], and poin -o -ca e sys ems [4].
The ab ica ion o mic o luidic chips equi es a demanding echnological p ocess o in eg a e
a a ie y o s uc u es and componen s o pe o m di e en p ocesses, such as sample p e ea men [
5
],
sample deli e y [
6
], pa icle manipula ion [
7
], on-chip eac ion [
8
], and esul de ec ion and analysis [
9
].
Pa icle sepa a ion [
10
], a subca ego y o pa icle manipula ion, is a c ucial p ocess a ec ing de ec ion
p ecision. Many echniques ha e so a been used o his, such as il a ion [
11
,
12
], cen i uga ion [
13
],
he use o magne ic [
12
,
14
] and acous ic o ce [
15
,
16
], ch oma og aphy [
17
,
18
], elec opho esis [
19
,
20
],
and dielec opho esis (DEP) [
21
,
22
]. Compa ed o o he me hods, DEP is becoming one o he mos
p omising sepa a ion echniques o mic o- and nano-scale sys ems because o i s low unning cos ,
ease o in eg a ion in o mic o luidics, speed, e iciency, sensi i i y, and selec i i y. Finally, i is also
a label- ee me hod [23], making sample p ocessing e y simple.
He e, we p esen he heo y o DEP, a compa ison be ween DEP and o he sepa a ion me hods,
and an explana ion o i s applica ions in mic o luidic chips. We e iew he ad an ages and
disad an ages o majo sys ems, hei impac , and po en ial, in pa icula , ocusing on di e en
elec ode ypes in eg a ed in o mic o luidic chips wi h s uc u es, ab ica ion echniques, and ope a ion
p inciples. The b ie e iew will be o in e es o esea che s in DEP mic o luidics.
Mic omachines 2019,10, 423; doi:10.3390/mi10060423 www.mdpi.com/jou nal/mic omachines
Mic omachines 2019,10, 423 2 o 22
2. Mic o luidic Sepa a ion Techniques
Pa icle sepa a ion is a echnique ha ex ac s o ob ains a ge pa icles om a solu ion o
suspension o isola es hem om each o he based on di e ences in he pa icles’ physical and o
chemical p ope ies o di e ences be ween he p ope ies o he pa icles and he ca ie luid. I can
also be conside ed as a concen a ion o pu i ica ion p ocess o a ge chemicals o pa icles.
Fil a ion [
24
–
26
] is a commonly used and e ec i e sepa a ion me hod based on pa icle size
di e ences. A po ous medium wi h a speci ic po e size o ms a il e , allowing pa icles smalle han he
po e size, as well as he ca ie luid, o pass h ough he il e , whe e o e sized pa icles a e collec ed.
Howe e , i is challenging o apply il a ion o mic o luidic chips once he po ous medium becomes
blocked du ing he il a ion p ocess, hus g adually dec easing he h oughpu unless a mo e complex
ul a il a ion sys em is used [
27
]. These disad an ages o complexi ies hinde he wide employmen
o his echnique o LOC sys ems.
The cen i uga ion me hod [
28
–
32
] is used o sepa a e pa icles based on speci ic mass while being
subjec ed o cen i ugal o ce. Howe e , i equi es he applica ion o an addi ional ool o gene a e
he o ce.
Magne ic o ce sepa a ion [
33
–
36
] mainly depends on he magne ic suscep ibili y o he
pa icles. I is possible o bind magne ic beads wi h he pa icles o in e es , bu his inc eases
he sys em’s complexi y.
Acous ic weeze s [
37
–
40
] a e a sepa a ion me hod based on sound wa es. Pa icles can be
induced o mo e owa d acous ic p essu e nodes o p essu e an inodes in a s anding acous ic ield
by an acous ic- adia ion o ce. The mo emen s a e dependen on he pa icles’ densi y, and p ecise
mo emen can be ob ained by con olling he posi ion o hese nodes. Howe e , he p ecision and
h oughpu limi he applica ion o acous ic weeze s o LOC sys ems.
Ch oma og aphy [
41
–
44
] is a powe ul sepa a ion echnique widely applied in mic o luidic
pla o ms, whe e he mobile phase ca ies he a ge pa icles and passes h ough a s a iona y phase.
Di e en pa icles in he mix u e a el a di e en speeds, esul ing in hei sepa a ion. Howe e ,
he ch oma og aphy in mic o luidic chips equi e complex s uc u es, including al es, pumps,
and s a iona y phases, subsequen ly inc easing i s ab ica ion complexi y. The equi emen o sample
labeling and especially sample loss due o unspeci ic adhesion o he la ge su ace a ea o he s a iona y
phase p e en s i s wide applica ion.
Elec opho esis [
45
–
47
] is a echnique o sepa a e cha ged pa icles in he suspension based on
a uni o m elec ic ield and is widely applied in he s udy o deoxy ibonucleic acids (DNA), ibonucleic
acids, and p o eins. Howe e , i is only a ailable o cha ged pa icles, limi ing i s po en ial applica ion.
In compa ison wi h o he sepa a ion me hods, DEP [
48
–
50
] has he unique ad an age o being
label- ee, as , and accu a e, which is why i has been widely applied in mic o luidics o bio-molecula
diagnos ics, and medical and polyme esea ch.
3. Dielec opho esis
DEP is a me hod used o sepa a e suspended pa icles om he suspension o ca ie luid by
gene a ing pola iza ion o ces in a non-uni o m elec ic ield (NUEF) [
51
]. Con e se o elec opho esis,
he pa icles in DEP do no ca y elec ical cha ges, bu hey mus be pola izable. These pa icles ha
a e exposed o an elec ic ield and become pola ized o ming elec ic dipoles; he esul ing o ce on
hese dipoles is called DEP o ce (F
DEP
). F
DEP
is 0 N (Figu e 1A) when he pa icle is in a uni o m
elec ic ield, while in NUEF, he o ce is ei he posi i e (pDEP) o nega i e (nDEP) making he pa icle
mo e in a ce ain di ec ion depending on i s pola iza ion (Figu e 1B) [51].
The ampli ude o F
DEP
o a sphe ical pa icle in he suspension ollows Equa ions (1) and (2) [
52
].
FDEP =2πεmε0 3
ex Re[CM( )]∇E2
RMS (1)
Mic omachines 2019,10, 423 3 o 22
whe e
εm
is he medium ela i e pe mi i i y,
ε0
is he acuum pe mi i i y,
ex
is a adius o he
sphe ical pa icle, E
RMS
is he oo -mean-squa e alue o he applied elec ic ield, and CM( ) is he
Clausius–Mosso i ac o .
CM( )=
ε∗
p−ε∗
m
ε∗
p+2ε∗
m
(2)
whe e ε∗
pand ε∗
ma e de ined as:
ε∗
p=εpε0−j
σp
π ,ε∗
m=εmε0−jσm
π (3)
whe e εpis he ela i e pe mi i i y o a pa icle, σpis he elec ic conduc i i y o a pa icle, σmis he
elec ic conduc i i y o he medium, and is he equency o he elec ic ield.
The NUEF is powe ed by he di ec cu en (DC) o al e na ing cu en (AC) and is gene a ed by
geome y o insula ing pos s in combina ion wi h channel ape ing, e c., in he mic o luidic channel [
10
]
also known as AC/DC-iDEP [
53
]. The mic o luidic chips based on DC-iDEP ha e a simple s uc u e wi h
no gas bubble gene a ion du ing pa icle sepa a ion. Howe e , he powe loss in he DC elec ical ield
esul s in Joule hea gene a ion, excessi ely inc easing he empe a u e inside he de ice. Compa ed
o his, he AC-DEP is mo e widely used because he Joule hea gene a ion is g ea ly supp essed.
The de ice selec i i y hen depends on he AC equency as well as he ield s eng h; hus, he
pa ame e s o bo h should be op imized.
Mic omachines2019,10,x3o 22
whe eε
m
is hemedium ela i epe mi i i y,ε
0
is he acuumpe mi i i y,
ex
isa adiuso  he
sphe icalpa icle,E
RMS
is he oo ‐mean‐squa e alueo  heappliedelec ic ield,andCM( )is he
Clausius–Mosso i ac o .
𝐶𝑀󰇛
𝑓
󰇜𝜀
∗𝜀

∗
𝜀
∗2𝜀

∗(2)
whe e𝜀
∗ and𝜀
∗a ede inedas:
𝜀
∗𝜀
𝜀
𝑗
𝜎
𝜋𝑓 , 𝜀
∗𝜀
𝜀
𝑗
𝜎
𝜋𝑓(3)
whe eε
p
is he ela i epe mi i i yo apa icle,σ
p
is heelec icconduc i i yo apa icle,σ
m
is he
elec icconduc i i yo  hemedium,and is he equencyo  heelec ic ield.
TheNUEFispowe edby hedi ec cu en (DC)o al e na ingcu en (AC)andisgene a ed
bygeome yo insula ingpos sincombina ionwi hchannel ape ing,e c.,in hemic o luidic
channel[10]alsoknownasAC/DC‐iDEP[53].Themic o luidicchipsbasedonDC‐iDEPha ea
simples uc u ewi hnogasbubblegene a iondu ingpa iclesepa a ion.Howe e , hepowe loss
in heDCelec ical ield esul sinJoulehea gene a ion,excessi elyinc easing he empe a u e
inside hede ice.Compa ed o his, heAC‐DEPismo ewidelyusedbecause heJoulehea 
gene a ionisg ea lysupp essed.Thede iceselec i i y hendependson heAC equencyaswellas
he ields eng h; hus, hepa ame e so bo hshouldbeop imized.

Figu e1.Diag amo  heposi i edielec opho esis(pDEP)p inciple.Rep oducedwi hpe mission
om[51],©2011Else ie .(A)Thepa icleissymme icallypola izedinauni o melec ical ield,
esul inginne DEP o ce(F
DEP
)wi hze oampli ude.(B)Thepa icleisasymme icallypola izedin
henon‐uni o melec ic ield(NUEF)and hene F
DEP
, esul inginmo ing hepa iclein oana ea
wi h hemaximumampli udeo  heelec ic ield.
4.Dielec opho esis(DEP)‐on‐a‐Chip
A a ie yo DEPmic o luidicde icesha ebeende eloped o useinbiopa iclesepa a ion.
Theg adien o  heelec ic ieldisanimpo an  ac o  ha a ec s heDEPpe o mance.The
elec odes’geome ymainlyp oduces heNUEFin hemic o luidicchip,so heshapeand
dis ibu iono  heelec odesa e i al o heNUEFampli ude.He ewediscussdi e en elec odes
appliedinmic o luidicsapp oaches,suchasDEPpe o medbyex e nalelec odesand2Dand3D
elec odes.

4.1.Ex e nalElec odes
Resea che sha ede elopedaniDEPmic o luidicchipmadeo polydime hylsiloxane(PDMS)
co e edwi haglasslid.Thede ice’spe o mancewase alua edinahyd odynamics udyo blood
al e a ion[54].Thechips uc u econsis edo aninle andanou le po ,anda≈1cmlong,≈50μm
deepmainchannelwi h38dead‐endb anchesdis ibu ede enlyonbo hsideso  hemainchannel
Figu e 1.
Diag am o he posi i e dielec opho esis (pDEP) p inciple. Rep oduced wi h pe mission
om [
51
],
©
2011 Else ie . (
A
) The pa icle is symme ically pola ized in a uni o m elec ical ield,
esul ing in ne DEP o ce (F
DEP
) wi h ze o ampli ude. (
B
) The pa icle is asymme ically pola ized in
he non-uni o m elec ic ield (NUEF) and he ne F
DEP
, esul ing in mo ing he pa icle in o an a ea
wi h he maximum ampli ude o he elec ic ield.
4. Dielec opho esis (DEP)-on-a-Chip
A a ie y o DEP mic o luidic de ices ha e been de eloped o use in biopa icle sepa a ion.
The g adien o he elec ic ield is an impo an ac o ha a ec s he DEP pe o mance. The elec odes’
geome y mainly p oduces he NUEF in he mic o luidic chip, so he shape and dis ibu ion o he
elec odes a e i al o he NUEF ampli ude. He e we discuss di e en elec odes applied in mic o luidics
app oaches, such as DEP pe o med by ex e nal elec odes and 2D and 3D elec odes.
4.1. Ex e nal Elec odes
Resea che s ha e de eloped an iDEP mic o luidic chip made o polydime hylsiloxane (PDMS)
co e ed wi h a glass lid. The de ice’s pe o mance was e alua ed in a hyd odynamic s udy o
blood al e a ion [
54
]. The chip s uc u e consis ed o an inle and an ou le po , and
a≈1 cm long
,
≈50 µm
deep main channel wi h 38 dead-end b anches dis ibu ed e enly on bo h sides o he main
Mic omachines 2019,10, 423 4 o 22
channel (Figu e 2A). The wid h and dep h o he b anches we e
≈
200
µ
m and
≈
50
µ
m, espec i ely.
An elec ic ield was in oduced in o he de ice by inse ing wo P elec odes, one in o he inle
and one in o he ou le . A sample o blood wi h a olume o
≈
2
µ
L was injec ed in o he main
channel and pumped h ough i using in e nal capilla y o ces. The b anches hyd o-dynamically
apped he ed blood cells (RBCs). Then, he DC powe supply was ac i a ed o es ablish he DC-DEP
o ce, p e en ing mo e RBCs om being apped inside he dead-end channels nea he inle po .
Addi ionally, he elec o-osmo ic low gene a ed by he DC elec ic ield emo ed RBCs om he c oss
junc ions o med a he channels and b anches. As a esul , he plasma was sepa a ed om he blood,
o ming a plasma a ea wi hou RBCs. This me hod ob ained a plasma pu i y o 99%. The iDEP de ice
pe o med he plasma sepa a ion wi hou dilu ing he blood samples. This de ice was used wi h lowe
ol age in compa ison o o he iDEP de ices [55].
Mic omachines2019,10,x4o 22
(Figu e2A).Thewid handdep ho  heb ancheswe e≈200μmand≈50μm, espec i ely.An
elec ic ieldwasin oducedin o hede icebyinse ing woP elec odes,onein o heinle andone
in o heou le .Asampleo bloodwi ha olumeo ≈2μLwasinjec edin o hemainchanneland
pumped h oughi usingin e nalcapilla y o ces.Theb ancheshyd o‐dynamically apped he ed
bloodcells(RBCs).Then, heDCpowe supplywasac i a ed oes ablish heDC‐DEP o ce,
p e en ingmo eRBCs ombeing appedinside hedead‐endchannelsnea  heinle po .
Addi ionally, heelec o‐osmo ic lowgene a edby heDCelec ic ield emo edRBCs om he
c ossjunc ions o meda  hechannelsandb anches.Asa esul , heplasmawassepa a ed om he
blood, o mingaplasmaa eawi hou RBCs.Thisme hodob ainedaplasmapu i yo 99%.The
iDEPde icepe o med heplasmasepa a ionwi hou dilu ing hebloodsamples.Thisde icewas
usedwi hlowe  ol ageincompa ison oo he iDEPde ices[55].

Figu e2.DEPde iceswi hex e nalelec odes.(A)Schema ico iDEPchipsachie ingplasma
sepa a ionwi hou dilu ing hebloodsamples.Rep oducedwi hpe mission om[54],©2015
Sp inge Na u e.(B)Schema ico chipused o he e ogeneousemulsiond ople ssepa a ion.
Rep oducedwi hpe mission om[56],©2017Else ie .
AsimplePDMSmic o luidicchipwas ab ica edusingso li hog aphy ope o m he
wall‐inducedDEP[56](Figu e2B).Thisde icewasused osepa a eoild ople s omJanus
pa icles,ha ing woo mo edis inc physicalp ope ieson hesu aceco e edwi hoilphase.The
suspensioncon aining hepa icleso in e es wasmo ing om hesampleinle  o hemain
channel,and hebu e  om heshea h luidinle pushed hepa icles omo ealong hewallo  he
mainchannel.Theleng handwid ho  hesampleinle andshea h luidinle we e≈5mmand≈150
μm,≈1mm,and≈250μm, espec i ely.Thesampleinle wasnex  oacon inemen chambe o ≈6
mm×6mm.Theleng ho bo hou le swas≈9mm,and hei wid hswe e≈120μmand≈180μm,
espec i ely.Thedep ho  hecon inemen chambe was≈80μm,allowingonlyd ople so a
diame e o <80μm opass h ough hechannel.Themainchannel,wi haleng handdep ho ≈1
mmand≈250μm, espec i ely,connec ed heinle sandou le s.Thechannel ape inggene a ed he
NUEFa e  ou P elec odeswe einse edin o heinle sandou le s.Thedeionizedwa e  i s 
we ed hechannel,and hen heJanusd ople sco e edbyoild ople swe einjec edin o he
con inemen chambe andpushedclose o hemainchannelby he luid om heshea h luidinle .
TheDCpowe supplywas henapplied o hechannel,and heJanusandoild ople swe e
g aduallysepa a ed omeacho he wi h hehelpo  heFDEPandmo edin o hedi e en ou le s.
Thesepa a ionp ocesswasdependen on heampli udeo  heapplied ol ageandd ople size.The
ab ica ionp ocesswas a he simplebecause heelec odeswe einse edin o hechipandno 
monoli hicallyin eg a edin o hede ice.
4.2.Two‐DimensionalElec odes
Thin‐ ilm2Delec odesbasedonasimple ab ica ionp ocesswi hdi e en shapeswe e
de eloped omee  he equi emen so in eg a ionand oinc easeDEPsepa a ione iciencywi hin
hemic o luidicchips.He e,wein oduce hemos commonlyusedelec odecon igu a ions,
Figu e 2.
DEP de ices wi h ex e nal elec odes. (
A
) Schema ic o iDEP chips achie ing plasma
sepa a ion wi hou dilu ing he blood samples. Rep oduced wi h pe mission om [
54
],
©
2015 Sp inge
Na u e. (
B
) Schema ic o chip used o he e ogeneous emulsion d ople s sepa a ion. Rep oduced wi h
pe mission om [56], ©2017 Else ie .
A simple PDMS mic o luidic chip was ab ica ed using so li hog aphy o pe o m he
wall-induced DEP [
56
] (Figu e 2B). This de ice was used o sepa a e oil d ople s om Janus pa icles,
ha ing wo o mo e dis inc physical p ope ies on he su ace co e ed wi h oil phase. The suspension
con aining he pa icles o in e es was mo ing om he sample inle o he main channel, and he
bu e om he shea h luid inle pushed he pa icles o mo e along he wall o he main channel.
The leng h and wid h o he sample inle and shea h luid inle we e
≈
5 mm and
≈
150
µ
m,
≈
1 mm,
and
≈
250
µ
m, espec i ely. The sample inle was nex o a con inemen chambe o
≈
6 mm
×
6 mm.
The leng h o bo h ou le s was
≈
9 mm, and hei wid hs we e
≈
120
µ
m and
≈
180
µ
m, espec i ely.
The dep h o he con inemen chambe was
≈
80
µ
m, allowing only d ople s o a diame e o <80
µ
m
o pass h ough he channel. The main channel, wi h a leng h and dep h o
≈
1 mm and
≈
250
µ
m,
espec i ely, connec ed he inle s and ou le s. The channel ape ing gene a ed he NUEF a e ou
P elec odes we e inse ed in o he inle s and ou le s. The deionized wa e i s we ed he channel,
and hen he Janus d ople s co e ed by oil d ople s we e injec ed in o he con inemen chambe and
pushed close o he main channel by he luid om he shea h luid inle . The DC powe supply was
hen applied o he channel, and he Janus and oil d ople s we e g adually sepa a ed om each o he
wi h he help o he F
DEP
and mo ed in o he di e en ou le s. The sepa a ion p ocess was dependen
on he ampli ude o he applied ol age and d ople size. The ab ica ion p ocess was a he simple
because he elec odes we e inse ed in o he chip and no monoli hically in eg a ed in o he de ice.
4.2. Two-Dimensional Elec odes
Thin- ilm 2D elec odes based on a simple ab ica ion p ocess wi h di e en shapes we e de eloped
o mee he equi emen s o in eg a ion and o inc ease DEP sepa a ion e iciency wi hin he mic o luidic
chips. He e, we in oduce he mos commonly used elec ode con igu a ions, pa allel [
52
,
57
–
60
] and
Mic omachines 2019,10, 423 5 o 22
cas ella ed elec odes [
61
–
64
], and b ie ly p esen o he such con igu a ions, such as quad upole
elec odes [65,66].
4.2.1. Pa allel and In e digi a ed Elec odes
The elec odes om bo h pa allel and in e digi a ed elec ode a ays a e ypically ec angula .
The elec odes ha e iden ical wid hs and gaps esul ing in a cons an elec ic ield be ween hem. Thus,
he sys em based on hem has a high sepa a ion e iciency o pa icles o in e es .
A mic o luidic de ice was p oposed o sepa a e g een luo escen p o ein labeled MDA-MB-231,
and o he ypes o cance cells om heal hy ones [
52
]. The mic o luidic chip consis ed o wo pa s
(Figu e 3A). Fi s , he elec ode pa was made using a glass wa e as a subs a e, which was spu e ed
and coa ed wi h a sandwich laye o C /Au. This sandwich me al laye was hen pa e ned using a we
e ching p ocess o o m a plana in e digi a ed elec ode a ay. This consis ed o 20 pai s o elec odes
wi h wid hs and gaps o
≈
39
µ
m and
≈
36
µ
m, espec i ely, p o uding in o he channel by
≈
2
µ
m
o
≈
3
µ
m, p o iding powe ul NUEF. Nex , he mic o luidic s uc u e was o med using PDMS and
a so li hog aphy echnique. The wid h o he channel was
≈
50
µ
m and i was enla ged o
≈
250
µ
m.
Finally, hese wo pa s, he elec odes and mic o luidic s uc u e, we e bonded oge he , assis ed by
an oxygen plasma. The sample was pumped in o he channel, and he NUEF was o med by he AC
powe supply wi h a se ampli ude o 15 V peak o peak (V
PP
) a a se equency o 40 kHz, ob aining
a sepa a ion accu acy and pu i y o ≈100% and ≈81%, espec i ely.
Mic omachines2019,10,x5o 22
pa allel[52,57–60]andcas ella edelec odes[61–64],andb ie lyp esen o he suchcon igu a ions,
suchasquad upoleelec odes[65,66].
4.2.1.Pa allelandIn e digi a edElec odes
Theelec odes ombo hpa allelandin e digi a edelec odea aysa e ypically ec angula .
Theelec odesha eiden icalwid hsandgaps esul inginacons an elec ic ieldbe ween hem.
Thus, hesys embasedon hemhasahighsepa a ione iciencyo pa icleso in e es .
Amic o luidicde icewasp oposed osepa a eg een luo escen p o einlabeled
MDA‐MB‐231,ando he  ypeso cance cells omheal hyones[52].Themic o luidicchipconsis ed
o  wopa s(Figu e3A).Fi s , heelec odepa wasmadeusingaglasswa e asasubs a e,which
wasspu e edandcoa edwi hasandwichlaye o C /Au.Thissandwichme allaye was hen
pa e nedusingawe e chingp ocess o o maplana in e digi a edelec odea ay.Thisconsis ed
o 20pai so elec odeswi hwid hsandgapso ≈39μmand≈36μm, espec i ely,p o udingin o
hechannelby≈2μm o≈3μm,p o idingpowe ulNUEF.Nex , hemic o luidics uc u ewas
o medusingPDMSandaso li hog aphy echnique.Thewid ho  hechannelwas≈50μmandi 
wasenla ged o≈250μm.Finally, hese wopa s, heelec odesandmic o luidics uc u e,we e
bonded oge he ,assis edbyanoxygenplasma.Thesamplewaspumpedin o hechannel,and he
NUEFwas o medby heACpowe supplywi hase ampli udeo 15Vpeak opeak(VPP)a ase 
equencyo 40kHz,ob ainingasepa a ionaccu acyandpu i yo ≈100%and≈81%, espec i ely.

Figu e3.2Delec odemic o luidicchipswi hDEP.(A)Schema ico  hede ice osepa a ecance 
cells ombloodcellsbyFDEPgene a edbypa allelelec odes.Rep oducedwi hpe mission om
[52],©2016JohnWileyandSons.(B)Thede icecombinedwi hinduced‐cha geelec o‐osmosis
(ICEO) o pa icle ocusingandDEPachie ingsimple chips uc u e ope o mpa icle ocusing
andsepa a ion.Rep oducedwi hpe mission om[60],©2017Ame icanChemicalSocie y.(C)
Hyb idDEP‐ine ialde icewi h opshea h low oimp o e hesepa a ione iciency.Rep oduced
wi hpe mission om[64],©2018Else ie .
Amic o luidicchipwas ab ica ed ocombinepa iclesandDEP ope o mcon inuous
sepa a iono yeas cells omsilicapa icles[60]byICEO,usingamic oscopeglassslideas he
de icesubs a e.Theglasswascoa edwi ha hinlaye o indium inoxide(ITO)andsubsequen ly
pa e ned, o mingpa allelelec odes.Thechannels,oneinle and ou ou le s,we emadein he
PDMS(Figu e3B)andbonded o heglasssubs a eusinganoxygenplasmasu ace‐ac i a ion
me hod.Themic o luidicchiphad h ee egions: ocusing, ansi ion,andsepa a ion.Fi s , he
suspension,consis ingo silicapa icles,yeas cells,andca ie  luid,wasinjec edin o hechip,and
heICEO ocused hepa iclesin he ocusing egion.Thesideb anches,wi hop imizedsizein he
ansi ion egion,ensu ed hepa icles eam lowedin o hesepa a ion egioninas aigh line.The
sepa a ione iciencywas>96%wi h heACpowe supplya ase  alueo 225VPPa 1MHz.This
gene al‐pu poseDEPmic o luidicchiphasg ea po en ialinmolecula diagnos icsinLOCsys ems
Figu e 3.
2D elec ode mic o luidic chips wi h DEP. (
A
) Schema ic o he de ice o sepa a e cance cells
om blood cells by F
DEP
gene a ed by pa allel elec odes. Rep oduced wi h pe mission om [
52
],
©
2016 John Wiley and Sons. (
B
) The de ice combined wi h induced-cha ge elec o-osmosis (ICEO) o
pa icle ocusing and DEP achie ing simple chip s uc u e o pe o m pa icle ocusing and sepa a ion.
Rep oduced wi h pe mission om [
60
],
©
2017 Ame ican Chemical Socie y. (
C
) Hyb id DEP-ine ial
de ice wi h op shea h low o imp o e he sepa a ion e iciency. Rep oduced wi h pe mission om [
64
],
©2018 Else ie .
A mic o luidic chip was ab ica ed o combine pa icles and DEP o pe o m con inuous sepa a ion
o yeas cells om silica pa icles [
60
] by ICEO, using a mic oscope glass slide as he de ice subs a e.
The glass was coa ed wi h a hin laye o indium in oxide (ITO) and subsequen ly pa e ned, o ming
pa allel elec odes. The channels, one inle and ou ou le s, we e made in he PDMS (Figu e 3B) and
bonded o he glass subs a e using an oxygen plasma su ace-ac i a ion me hod. The mic o luidic
chip had h ee egions: ocusing, ansi ion, and sepa a ion. Fi s , he suspension, consis ing o silica
pa icles, yeas cells, and ca ie luid, was injec ed in o he chip, and he ICEO ocused he pa icles
in he ocusing egion. The side b anches, wi h op imized size in he ansi ion egion, ensu ed he
pa icle s eam lowed in o he sepa a ion egion in a s aigh line. The sepa a ion e iciency was
>96% wi h he AC powe supply a a se alue o 225 V
PP
a 1 MHz. This gene al-pu pose DEP

Mic omachines 2019,10, 423 6 o 22
mic o luidic chip has g ea po en ial in molecula diagnos ics in LOC sys ems because i exhibi ed
high h oughpu . The in eg a ion o he ICEO module simpli ied he chip s uc u e, as he e is no
need o a complica ed low a e con ol module and na ow mic o luidic channel o ocus he pa icle.
I p o ided an al e na i e me hod o ocus he pa icles, esul ing in an in eg a ed de ice.
A mic o luidic pla o m wi h a hyb id DEP-ine ial sys em was de eloped o sepa a e pa icles [
64
]
(Figu e 3C). The channel, which was
≈
200
µ
m wide and
≈
40
µ
m deep, consis ed o 15 symme ic
U-shaped segmen s wi h a leng h o
≈
700
µ
m and was pa e ned in PDMS using so li hog aphy.
The inle and ou le holes we e also punched in he PDMS. A li -o echnique pa e ned he hin- ilm
Ti (50 nm)/P (150 nm) elec odes on a glass slide in a zigzag dis ibu ion. The gap and wid h o he
elec odes we e bo h
≈
20
µ
m. The bonding be ween PDMS and he glass slide was pe o med by
oxygen plasma. A suspension low o polys y ene beads wi h diame e s o
≈
5
µ
m and
≈
13
µ
m was
injec ed om he inle , and he shea h low om he op pushed he pa icles along he bo om o he
channel, keeping hem in he NUEF. The ield s eng h o he AC powe supply was op imized o
≈27 VPP
a
≈
1 MHz, ob aining a sepa a ion e iciency o
≈
13
µ
m and
≈
5
µ
m pa icles o
≈
100% and
≈
96%, espec i ely. The a ied pa icle size sepa a ions we e achie ed inside his chip by adjus ing
he ol age wi hou needing a edesign o he chip layou . The implemen a ion o he op shea h low
imp o ed he sepa a ion e iciency as he pa icles we e o ced o he bo om o he channel and we e
no in luenced by he elec ic ield decay along he e ical di ec ion.
An isomo i e DEP mic o luidic de ice was de eloped o sepa a e li ing and dead yeas cells
(Figu e 4) [
67
]. The de ice consis ed o h ee sec ions: h ee inle s, a sepa a ion sec ion, and wo
ou le s. The elec odes o DEP we e made o a me al sandwich o Ti/P /Au wi h a hickness o
≈50 nm,
≈50 nm
,
and ≈300 nm
, espec i ely, on a glass subs a e. The elec ode wid h and gap be ween hem
we e bo h
≈
10
µ
m. The luidic channels we e pa e ned on op o he elec odes using SU-8 pho o esis
wi h a hickness o
≈
100
µ
m and hen sealed by a glass co e slip. The leng h and wid h o he main
channel we e
≈
600
µ
m and
≈
100
µ
m, espec i ely. The side o he co e slip acing he chambe
was coa ed wi h ITO, se ing as a g ound elec ode. The elec ode biasing ne wo k was o med
using he esis o a ay nex o he main channel and was connec ed o he elec odes o pe o m he
isomo i e DEP.
Mic omachines2019,10,x6o 22
becausei exhibi edhigh h oughpu .Thein eg a iono  heICEOmodulesimpli ied hechip
s uc u e,as he eisnoneed o acomplica ed low a econ olmoduleandna owmic o luidic
channel o ocus hepa icle.I p o idedanal e na i eme hod o ocus hepa icles, esul inginan
in eg a edde ice.
Amic o luidicpla o mwi hahyb idDEP‐ine ialsys emwasde eloped osepa a epa icles
[64](Figu e3C).Thechannel,whichwas≈200μmwideand≈40μmdeep,consis edo 15
symme icU‐shapedsegmen swi haleng ho ≈700μmandwaspa e nedinPDMSusingso 
li hog aphy.Theinle andou le holeswe ealsopunchedin hePDMS.Ali ‐o  echnique
pa e ned he hin‐ ilmTi(50nm)/P (150nm)elec odesonaglassslideinazigzagdis ibu ion.The
gapandwid ho  heelec odeswe ebo h≈20μm.Thebondingbe weenPDMSand heglassslide
waspe o medbyoxygenplasma.Asuspension lowo polys y enebeadswi hdiame e so ≈5μm
and≈13μmwasinjec ed om heinle ,and heshea h low om he oppushed hepa iclesalong
hebo omo  hechannel,keeping hemin heNUEF.The ields eng ho  heACpowe supply
wasop imized o≈27VPPa ≈1MHz,ob ainingasepa a ione iciencyo ≈13μmand≈5μm
pa icleso ≈100%and≈96%, espec i ely.The a iedpa iclesizesepa a ionswe eachie edinside
hischipbyadjus ing he ol agewi hou needinga edesigno  hechiplayou .Theimplemen a ion
o  he opshea h lowimp o ed hesepa a ione iciencyas hepa icleswe e o ced o hebo om
o  hechannelandwe eno in luencedby heelec ic ielddecayalong he e icaldi ec ion.
Anisomo i eDEPmic o luidicde icewasde eloped osepa a eli inganddeadyeas cells
(Figu e4)[67].Thede iceconsis edo  h eesec ions: h eeinle s,asepa a ionsec ion,and wo
ou le s.Theelec odes o DEPwe emadeo ame alsandwicho Ti/P /Auwi ha hicknesso ≈50
nm,≈50nm,and≈300nm, espec i ely,onaglasssubs a e.Theelec odewid handgapbe ween
hemwe ebo h≈10μm.The luidicchannelswe epa e nedon opo  heelec odesusingSU‐8
pho o esis wi ha hicknesso ≈100μmand hensealedbyaglassco e slip.Theleng handwid h
o  hemainchannelwe e≈600μmand≈100μm, espec i ely.Thesideo  heco e slip acing he
chambe wascoa edwi hITO,se ingasag oundelec ode.Theelec odebiasingne wo kwas
o medusing he esis o a aynex  o hemainchannelandwasconnec ed o heelec odes o
pe o m heisomo i eDEP.

Figu e4.Anisomo i eDEPmic o luidicde icewi hpa allelelec odes.(A)The ab ica ionp ocess
o  hede ice.(B)Diag amo samplep ocesssequenceandpho og apho  hede icewi hde ails
showing hemainchannel o DEP.Rep oducedwi hpe mission om[67],©2007RoyalSocie yo 
Chemis y(RSC).
Figu e 4.
An isomo i e DEP mic o luidic de ice wi h pa allel elec odes. (
A
) The ab ica ion p ocess
o he de ice. (
B
) Diag am o sample p ocess sequence and pho og aph o he de ice wi h de ails
showing he main channel o DEP. Rep oduced wi h pe mission om [
67
],
©
2007 Royal Socie y o
Chemis y (RSC).
Mic omachines 2019,10, 423 7 o 22
The suspension con aining yeas cells was pumped in o he main channel om he middle inle ,
and he cells in suspension we e ocused o mo e in a s eamline wi h he help o shea h low gene a ed
by he bu e om he o he wo side channels. The li ing and dead yeas cells we e sepa a ed in he
DEP sec ion unde a non-uni o m isomo i e elec ic ield. The de ice pe o mance was e alua ed by
applying AC powe supply wi h a se alue o 14 VPP a di e en equencies.
A mic o luidic de ice wi h in e digi a ed elec odes was ab ica ed o sepa a e li ing and dead
Lis e ia cells (Figu e 5A) [
68
]. The C /P wi h a hickness o
≈
10 nm and 100 nm, espec i ely, we e
spu e ed using a magne on sys em on a glass subs a e, and hen he in e digi a ed elec odes we e
pa e ned using he pho oli hog aphy me hod wi h subsequen e ching. The gap and he wid h o he
elec odes we e bo h
≈
15
µ
m. Then, a ec angula chambe was cons uc ed o silicon ubbe wi h
a hickness o
≈
0.3 mm, and a
≈
20
µ
L suspension con aining he Lis e ia cells was pipe ed in o he
chambe . A glass co e slip was hen used o seal he chambe a e loading he suspension. The AC
powe supply was applied wi h a se alue o 1 V
PP
om 10 kHz o 15 MHz, and he cell sepa a ion
pe o mance was moni o ed h ough a cha ge-coupled de ice came a. The sepa a ion e iciency o
li ing and dead Lis e ia cells was >90% a a se equency o 50 kHz. This sys em combined an ibody
ecogni ion and DEP and hus imp o ed he selec i i y o cap u ing bac e ia.
Mic omachines2019,10,x7o 22
Thesuspensioncon ainingyeas cellswaspumpedin o hemainchannel om hemiddleinle ,
and hecellsinsuspensionwe e ocused omo einas eamlinewi h hehelpo shea h low
gene a edby hebu e  om heo he  wosidechannels.Theli inganddeadyeas cellswe e
sepa a edin heDEPsec ionunde anon‐uni o misomo i eelec ic ield.Thede icepe o mance
wase alua edbyapplyingACpowe supplywi hase  alueo 14VPPa di e en  equencies.
Amic o luidicde icewi hin e digi a edelec odeswas ab ica ed osepa a eli inganddead
Lis e iacells(Figu e5A)[68].TheC /P wi ha hicknesso ≈10nmand100nm, espec i ely,we e
spu e edusingamagne onsys emonaglasssubs a e,and hen hein e digi a edelec odeswe e
pa e nedusing hepho oli hog aphyme hodwi hsubsequen e ching.Thegapand hewid ho 
heelec odeswe ebo h≈15μm.Then,a ec angula chambe wascons uc edo silicon ubbe 
wi ha hicknesso ≈0.3mm,anda≈20μLsuspensioncon aining heLis e iacellswaspipe edin o
hechambe .Aglassco e slipwas henused oseal hechambe a e loading hesuspension.The
ACpowe supplywasappliedwi hase  alueo 1VPP om10kHz o15MHz,and hecell
sepa a ionpe o mancewasmoni o ed h oughacha ge‐coupledde icecame a.Thesepa a ion
e iciencyo li inganddeadLis e iacellswas>90%a ase  equencyo 50kHz.Thissys em
combinedan ibody ecogni ionandDEPand husimp o ed heselec i i yo cap u ingbac e ia.

Figu e5.De iceswi hin e digi a edelec odes.(A)Schema ico  hesys emwi hsimplepa icle
manipula ionand op iewo elec odes.Rep oducedwi hpe mission om[68],©2002Else ie .(B)
D awingo  hechipcombiningan ibody ecogni ionwi hDEP, husimp o ing heselec i elyo 
cap u ingbac e ia.Rep oducedwi hpe mission om[69],©2006RSC.
ADEPmic o luidicde icewi hin e digi a edelec odeswasde eloped oconcen a eand
selec i elycap u eLis e iacellsbondingwi han ibodies(Figu e5B) omo he  ypeso cells[69].
Thein e digi a edelec odeswe emadeo aTi/P sandwichwi ha hicknesso ≈20nmand≈80
nm, espec i ely,byaspu e ingandali ‐o p ocessonanoxidizedsiliconsubs a e.Thewid h
andgapo  heelec odeswe e≈23μmand≈17μm, espec i ely.Theelec odeswe e henco e ed
wi haSiO2laye wi ha hicknesso ≈300nmusingplasma‐enhancedchemical‐ apo deposi ion,
p e en ingelec o‐osmo iccu en snea  heelec odes ia he o ma iono abime allic
elec ochemicalcell.ThePDMSpa e nedwi h luidics uc u ewasbonded o hesubs a eusing
anoxygenplasmaenhancemen p ocessa ≈200W o ≈10s.Theleng h,wid h,anddep ho  he
channelwe e≈3000μm,≈260μm,and≈16μm, espec i ely.The ubeswe einse edin o heinle 
andou le  odeli e  hesuspensionin o hechannel.TheACpowe supplywasappliedwi hase 
alueo 20VPPa 1MHz ocap u e heLis e iacellsbondingwi han ibodies om hesuspension.
The esul showed ha >90%Lis e iacellsbondingwi han ibodieswe ecollec edwi ha low a eo 
≈0.2μL∙min−1,and heinc eased low a edec eased hecap u ee iciency.
4.2.2.Cas ella edElec odes
Thecas ella edelec odesgene a edanelec ical ieldg adien , o mingamaximum aluea 
heedgeso  hemic oelec odesandaminimum aluea  hecen e be ween woelec odes.
Amic o luidicde ice ha combinedaninsula o wi h2Delec odeswasde eloped o ocus
andsepa a epa icles(Figu e6A,B)[70].Theelec odeswe edesignedinapa allelcon igu a ion
wi ha o alnumbe o 30elec odesha ing15elec odesoneachsideo  hemainchannel.The
Figu e 5.
De ices wi h in e digi a ed elec odes. (
A
) Schema ic o he sys em wi h simple pa icle
manipula ion and op iew o elec odes. Rep oduced wi h pe mission om [
68
],
©
2002 Else ie .
(
B
) D awing o he chip combining an ibody ecogni ion wi h DEP, hus imp o ing he selec i ely o
cap u ing bac e ia. Rep oduced wi h pe mission om [69], ©2006 RSC.
A DEP mic o luidic de ice wi h in e digi a ed elec odes was de eloped o concen a e and
selec i ely cap u e Lis e ia cells bonding wi h an ibodies (Figu e 5B) om o he ypes o cells [
69
].
The in e digi a ed elec odes we e made o a Ti/P sandwich wi h a hickness o
≈20 nm
and
≈80 nm
,
espec i ely, by a spu e ing and a li -o p ocess on an oxidized silicon subs a e. The wid h and
gap o he elec odes we e
≈
23
µ
m and
≈
17
µ
m, espec i ely. The elec odes we e hen co e ed
wi h a SiO
2
laye wi h a hickness o
≈
300 nm using plasma-enhanced chemical- apo deposi ion,
p e en ing elec o-osmo ic cu en s nea he elec odes ia he o ma ion o a bime allic elec ochemical
cell. The PDMS pa e ned wi h luidic s uc u e was bonded o he subs a e using an oxygen plasma
enhancemen p ocess a
≈
200 W o
≈
10 s. The leng h, wid h, and dep h o he channel we e
≈3000 µm
,
≈260 µm
,
and ≈16 µm
, espec i ely. The ubes we e inse ed in o he inle and ou le o
deli e he suspension in o he channel. The AC powe supply was applied wi h a se alue o 20 V
PP
a 1 MHz o cap u e he Lis e ia cells bonding wi h an ibodies om he suspension. The esul showed
ha >90% Lis e ia cells bonding wi h an ibodies we e collec ed wi h a low a e o
≈
0.2
µ
L
·
min
−1
,
and he inc eased low a e dec eased he cap u e e iciency.
4.2.2. Cas ella ed Elec odes
The cas ella ed elec odes gene a ed an elec ical ield g adien , o ming a maximum alue a he
edges o he mic oelec odes and a minimum alue a he cen e be ween wo elec odes.
Mic omachines 2019,10, 423 8 o 22
A mic o luidic de ice ha combined an insula o wi h 2D elec odes was de eloped o ocus and
sepa a e pa icles (Figu e 6A,B) [
70
]. The elec odes we e designed in a pa allel con igu a ion wi h a o al
numbe o 30 elec odes ha ing 15 elec odes on each side o he main channel. The me al sandwich o
Ti/P was pa e ned using a li -o echnique on a Py ex glass subs a e. The luidic s uc u e was made
in a SU-8 laye wi h a hickness o
≈
20
µ
m, o ming a cas ella ed s uc u e (Figu e 6A). A PDMS co e
wi h access holes was moun ed on op o he s uc u e o seal i . Two opposi e NUEFs we e gene a ed
in he i s pa o he main channel by applying wo di e en V
PP
ampli udes on each side o he
channel. These wo NUEFs allowed he dielec ic pa icles in he main channel o each an equilib ium,
esul ing in pa icles being con ined o he cen e o he channel. The equilib ium depended on he
ol age ampli ude and he equencies o he powe supply. Then, he hi d AC powe supply was
applied o pe o m he pa icle sepa a ion in he second pa o he main channel. The suspension,
con aining polys y ene beads wi h a diame e o
≈
5
µ
m and yeas cells, was used o e alua e he
chip’s pe o mance. This mic o luidic chip, wi h his combina ion o cas ella ed elec odes and an
insula o , p o ides a good al e na i e op ion o cell sepa a ion by DEP. The combina ion o insula o
sidewalls and cas ella ed elec ode achie ed accu a e pa icle ocusing and con inuous sepa a ion.
Mic omachines2019,10,x8o 22
me alsandwicho Ti/P waspa e nedusingali ‐o  echniqueonaPy exglasssubs a e.The
luidics uc u ewasmadeinaSU‐8laye wi ha hicknesso ≈20μm, o mingacas ella ed
s uc u e(Figu e6A).APDMSco e wi haccessholeswasmoun edon opo  hes uc u e oseal
i .Twoopposi eNUEFswe egene a edin he i s pa o  hemainchannelbyapplying wo
di e en VPPampli udesoneachsideo  hechannel.These woNUEFsallowed hedielec ic
pa iclesin hemainchannel o eachanequilib ium, esul inginpa iclesbeingcon ined o he
cen e o  hechannel.Theequilib iumdependedon he ol ageampli udeand he equencieso  he
powe supply.Then, he hi dACpowe supplywasapplied ope o m hepa iclesepa a ionin
hesecondpa o  hemainchannel.Thesuspension,con ainingpolys y enebeadswi hadiame e 
o ≈5μmandyeas cells,wasused oe alua e hechip’spe o mance.Thismic o luidicchip,wi h
hiscombina iono cas ella edelec odesandaninsula o ,p o idesagoodal e na i eop ion o 
cellsepa a ionbyDEP.Thecombina iono insula o sidewallsandcas ella edelec odeachie ed
accu a epa icle ocusingandcon inuoussepa a ion.

Figu e6.De iceswi hcas ella edelec odes.(A)Schema ico aninsula o and2Delec odede ice
o ocusandsepa a epa iclesbyapplyingdi e en  equenciesandampli udeso anACpowe 
supply[70].(B)3Dd awingandscanningelec onicmic oscopyimageo  hede ice.Rep oduced
wi hpe mission om[70],©2008Else ie .(C)Schema ico  hecombinedin e digi a edand
cas ella edelec odede ice.I wasused oquan i a i elye alua e hepa icleandcellsepa a ion.
Rep oducedwi hpe mission om[71],©2003Else ie .(D)Schema ico ade icebasedon
sc een‐p in ing ab ica ion.Rep oducedwi hpe mission om[63],©2015Else ie .
Amic o luidicde ice,combiningin e digi a edandcas ella edelec odes,wasde eloped o
quan i a i elyassess hepa icleandcellsepa a ion(Figu e6C)[71].TheP elec odeswe e
pa e ned o o mbo hanin e digi a edelec odesys emaswellascas ella ed opologyonaglass
subs a e.Theheigh ,wid h,andgapo  hein e digi a edelec odesandcas ella edelec odeswe e
≈100nm×10μm×10μmand≈100nm×20μm×20μm, espec i ely.The luidicchambe ,wi ha
leng h,wid h,andheigh o ≈10mm,≈4mm,and≈30μm, espec i ely,wasmadeo poly(me hyl
me hac yla e)(PMMA),and hen hePMMAwasbonded oaglasssubs a eon opo  he
elec odes.Thesuspensiono polys y enebeadswi hadiame e o ≈500nm,≈2μm,and≈6μm,
andRBCswe eused oquan i a i elye alua e hesys em’spe o mance.Thesuspensionwas
pumpedin o hechambe ,andanACpowe supplywasappliedwi hase  alue anging om15
VPP o20VPPand om1MHz o15MHz.
AnAC‐DEPchipwasde elopedwi halow‐cos sc een‐p in ing hick‐ ilm echnique(Figu e
6D).Aglassslidewasusedasasubs a e,andawo enmeshandelec odes i s pa e neda
comme cialca bonpas e,andcon ac wi eswe ep in edoni ssu aceasusingsc eenp in ing[63].
Th ee ypeso elec odes,≈10μm hick,we ep in edon heglass:o se cas ella edelec odes,
non‐o se cas ella edelec odes,andin e digi a edlineelec odes.Once heca bonpas eha dened,
heul a iole (UV)cu abledielec icpas ewas henp in edandcu edusingUVligh  o o m
mic o luidicchannelswi hadep ho ≈50μm,and hecon ac wi eswe esepa a ed om he luid.
Finally, hePDMSlaye wi hinle andou le po swasbondedon opo  hechannels,assis edbyan
oxygenplasmasu ace‐ac i a ionme hod.Thede ice’spe o manceande iciencyincap u ing
Figu e 6.
De ices wi h cas ella ed elec odes. (
A
) Schema ic o an insula o and 2D elec ode de ice
o ocus and sepa a e pa icles by applying di e en equencies and ampli udes o an AC powe
supply [
70
]. (
B
) 3D d awing and scanning elec onic mic oscopy image o he de ice. Rep oduced wi h
pe mission om [
70
],
©
2008 Else ie . (
C
) Schema ic o he combined in e digi a ed and cas ella ed
elec ode de ice. I was used o quan i a i ely e alua e he pa icle and cell sepa a ion. Rep oduced
wi h pe mission om [
71
],
©
2003 Else ie . (
D
) Schema ic o a de ice based on sc een-p in ing
ab ica ion. Rep oduced wi h pe mission om [63], ©2015 Else ie .
A mic o luidic de ice, combining in e digi a ed and cas ella ed elec odes, was de eloped
o quan i a i ely assess he pa icle and cell sepa a ion (Figu e 6C) [
71
]. The P elec odes we e
pa e ned o o m bo h an in e digi a ed elec ode sys em as well as cas ella ed opology on a glass
subs a e. The heigh , wid h, and gap o he in e digi a ed elec odes and cas ella ed elec odes
we e
≈100 nm ×10 µm×10 µm
and
≈100 nm ×20 µm×20 µm
, espec i ely. The luidic chambe ,
wi h a leng h, wid h, and heigh o
≈
10 mm,
≈
4 mm, and
≈
30
µ
m, espec i ely, was made o
poly(me hyl me hac yla e) (PMMA), and hen he PMMA was bonded o a glass subs a e on op o he
elec odes. The suspension o polys y ene beads wi h a diame e o
≈
500 nm,
≈2µm
,
and ≈6µm
, and
RBCs we e used o quan i a i ely e alua e he sys em’s pe o mance. The suspension was pumped
in o he chambe , and an AC powe supply was applied wi h a se alue anging om 15 V
PP
o 20 V
PP
and om 1 MHz o 15 MHz.
An AC-DEP chip was de eloped wi h a low-cos sc een-p in ing hick- ilm echnique (Figu e 6D).
A glass slide was used as a subs a e, and a wo en mesh and elec odes i s pa e ned a comme cial
ca bon pas e, and con ac wi es we e p in ed on i s su ace as using sc een p in ing [
63
]. Th ee ypes o
elec odes,
≈
10
µ
m hick, we e p in ed on he glass: o se cas ella ed elec odes, non-o se cas ella ed
elec odes, and in e digi a ed line elec odes. Once he ca bon pas e ha dened, he ul a iole (UV)
Mic omachines 2019,10, 423 9 o 22
cu able dielec ic pas e was hen p in ed and cu ed using UV ligh o o m mic o luidic channels
wi h a dep h o
≈
50
µ
m, and he con ac wi es we e sepa a ed om he luid. Finally, he PDMS
laye wi h inle and ou le po s was bonded on op o he channels, assis ed by an oxygen plasma
su ace-ac i a ion me hod. The de ice’s pe o mance and e iciency in cap u ing yeas cells om he
suspension we e op imized using an AC elec ic ield o di e en equencies and s eng hs. The esul
indica ed ha he ol age ampli ude, he equency o AC powe supply, he ca ie luid conduc i i y,
and he low a e signi ican ly a ec ed he cap u e a e. The cap u e a e was >95% a e applying he
AC powe supply wi h se alues o 10 V
PP
a 100 kHz. This simple ab ica ion echnique has g ea
po en ial o be applied in mass p oduc ion.
4.2.3. O he Types o Elec odes
In addi ion o he main ypes o elec ode shapes, many di e en elec odes wi h speci ic shapes
we e designed o p o ide be e and mo e s able NUEF, such as annula [
72
,
73
], oblique [
74
–
76
], and
cu ed elec odes [
77
]. The cha ac e is ics o hese elec odes ha e been e iewed elsewhe e [
51
] and
a e no he subjec o his s udy; he e, we only in oduce he quad upole and apezoid elec odes.
The pe o mance o quad upole elec odes was i s simula ed, designed, and ab ica ed based on
a polynomial wi h he assump ion ha i obeyed Laplace’s equa ion [
66
] o gene a e a well- de ined
NUEF ha sepa a es yeas cells. A simila elec ode ype was hen used o pa e n ac in using DEP on
a myosin subs a e along elec ic ield lines.
A mic o luidic pla o m was p oposed o sepa a e bac e ia om whole blood ia DEP gene a ed
by a quad upole mic oelec ode embedded a he bo om o a small well in he chip [
65
] (Figu e 7).
The blood was pumped in o he de ice and i s desalina ed by inline dialysis wi hou memb ane.
Then, he a ge bac e ia we e sepa a ed and concen a ed using DEP. This ob ained a concen a ion
e iciency o
≈
79% and
≈
78% o Esche ichia coli and S aphylococcus au eus a a p ocessing a e o
≈0.6 mL·h−1
. The esul s we e e i ied by pe o ming a polyme ase chain eac ion, showing ha
bac e ial 16S ibosomal DNA le els we e en iched ≈307- old in compa ison wi h he o iginal.
Mic omachines2019,10,x9o 22
yeas cells om hesuspensionwe eop imizedusinganACelec ic ieldo di e en  equencies
ands eng hs.The esul indica ed ha  he ol ageampli ude, he equencyo ACpowe supply,
heca ie  luidconduc i i y,and he low a esigni ican lya ec ed hecap u e a e.Thecap u e
a ewas>95%a e applying heACpowe supplywi hse  alueso 10VPPa 100kHz.Thissimple
ab ica ion echniquehasg ea po en ial obeappliedinmassp oduc ion.
4.2.3.O he Typeso Elec odes
Inaddi ion o hemain ypeso elec odeshapes,manydi e en elec odeswi hspeci icshapes
we edesigned op o idebe e andmo es ableNUEF,suchasannula [72,73],oblique[74–76],and
cu edelec odes[77].Thecha ac e is icso  heseelec odesha ebeen e iewedelsewhe e[51]and
a eno  hesubjec o  hiss udy;he e,weonlyin oduce hequad upoleand apezoidelec odes.
Thepe o manceo quad upoleelec odeswas i s simula ed,designed,and ab ica edbased
onapolynomialwi h heassump ion ha i obeyedLaplace’sequa ion[66] ogene a eawell‐
de inedNUEF ha sepa a esyeas cells.Asimila elec ode ypewas henused opa e nac in
usingDEPonamyosinsubs a ealongelec ic ieldlines.
Amic o luidicpla o mwasp oposed osepa a ebac e ia omwholeblood iaDEP
gene a edbyaquad upolemic oelec odeembeddeda  hebo omo asmallwellin hechip[65]
(Figu e7).Thebloodwaspumpedin o hede iceand i s desalina edbyinlinedialysiswi hou 
memb ane.Then, he a ge bac e iawe esepa a edandconcen a edusingDEP.Thisob aineda
concen a ione iciencyo ≈79%and≈78%o Esche ichiacoliandS aphylococcusau eusa a
p ocessing a eo ≈0.6mL∙h−1.The esul swe e e i iedbype o mingapolyme asechain eac ion,
showing ha bac e ial16S ibosomalDNAle elswe een iched≈307‐ oldincompa isonwi h he
o iginal.

Figu e7.Ade icewi hquad upoleelec odes.(A)Thep incipleo  hede ice.(B)Schema ic
showing edbloodcells(RBC)sepa a ionbyquad upoleelec odecon igu a ion.Rep oducedwi h
pe mission om[65],©2017RSC.
A apezoidalelec odea ay(TEA)basedmic o luidicchipwasde eloped osepa a e
polys y enebeadso di e en diame e s(Figu e8)[78].ThegoldTEAwaspa e nedonaPy ex
glasssubs a eusingapho oli hog aphyme hod.Thewid hso  wobasesinasingle apezoidal
elec odewe e≈120μmand≈20μm, espec i ely,and hei heigh was≈60μm.Themic o luidic
s uc u ewaspa e nedinPDMSusingaso li hog aphyme hod.Thewid handdep ho  he
mic o luidicchannelwe e≈50μmand≈30μm, espec i ely.The luidics uc u ewasaligned o
Figu e 7.
A de ice wi h quad upole elec odes. (
A
) The p inciple o he de ice. (
B
) Schema ic showing
ed blood cells (RBC) sepa a ion by quad upole elec ode con igu a ion. Rep oduced wi h pe mission
om [65], ©2017 RSC.
A apezoidal elec ode a ay (TEA) based mic o luidic chip was de eloped o sepa a e polys y ene
beads o di e en diame e s (Figu e 8) [
78
]. The gold TEA was pa e ned on a Py ex glass subs a e
using a pho oli hog aphy me hod. The wid hs o wo bases in a single apezoidal elec ode we e
Mic omachines 2019,10, 423 16 o 22
Mic omachines2019,10,x16o 22

Figu e14.A3DAg‐PDMSelec odemic o luidicde ice.Rep oducedwi hpe mission om[90],©
2019JohnWileyandSons.(A)Schema ico  hesys em.(B)Theimageo  hede ice.(C)The
schema icin hesepa a iona eaand hedimensiono  heelec odes.
4.3.4.Silicon3DElec odes
Thede elopmen sinICandmic o‐elec o‐mechanical‐sys em ab ica ion echnologyha ealso
beenused o DEP,allowing hechips uc u e obemade omsinglec ys alsilicon.Asa esul , he
chip ab ica ionands uc u ewe esimpli iedasusingelec icallyconduc i esilicon o medbo h,
hemic o luidicchannelwallsalsose ingas3Delec odes,ob ainingapo en ial o mass
p oduc ion.
Asqua e‐shapedpilla s uc u e oge he wi h hechannelswasp oposedand ab ica ed om
silicon(Figu e15A)[82]usingadeep eac i eione ching(DRIE)p ocessandwas hensandwiched
insiliconwi h woglasswa e sbyanodicbonding.Li ing/deadyeas cellswe eused o es  he
de ice’spe o mancebyapplying heACpowe supplywi hag aduallyinc easingampli ude om
0VPP o25VPPa a equency ange om20kHz o100kHz.
Figu e 14.
A 3D Ag-PDMS elec ode mic o luidic de ice. Rep oduced wi h pe mission om [
90
],
©
2019 John Wiley and Sons. (
A
) Schema ic o he sys em. (
B
) The image o he de ice. (
C
) The
schema ic in he sepa a ion a ea and he dimension o he elec odes.
4.3.4. Silicon 3D Elec odes
The de elopmen s in IC and mic o-elec o-mechanical-sys em ab ica ion echnology ha e also
been used o DEP, allowing he chip s uc u e o be made om single c ys al silicon. As a esul ,
he chip ab ica ion and s uc u e we e simpli ied as using elec ically conduc i e silicon o med bo h,
he mic o luidic channel walls also se ing as 3D elec odes, ob aining a po en ial o mass p oduc ion.
A squa e-shaped pilla s uc u e oge he wi h he channels was p oposed and ab ica ed om
silicon (Figu e 15A) [
82
] using a deep eac i e ion e ching (DRIE) p ocess and was hen sandwiched in
silicon wi h wo glass wa e s by anodic bonding. Li ing/dead yeas cells we e used o es he de ice’s
pe o mance by applying he AC powe supply wi h a g adually inc easing ampli ude om 0 VPP o
25 VPP a a equency ange om 20 kHz o 100 kHz.
The elec ode pilla s could also be designed in a non-uni o m heigh o sepa a e li ing and dead
yeas cells (Figu e 15B) [
83
]. The de ice consis ed o wo glass wa e s sandwiching a pa e ned silicon
wa e o ming he 3D elec odes as well as he sepa a ion chambe . The glass wa e was anodically
bonded o he silicon wa e , and hen he DRIE was pe o med o e ch he silicon, o ming wo se s o
pilla s o di e en heigh s, ei he ≈100 µm o ≈2.5 µm, as well as he chambe . These wo s uc u es
we e hen bonded oge he , o ming he asymme ic elec ode s uc u es. The de ice’s pe o mance
was es ed using wo popula ions o cells, dead and li e yeas , one exhibi ing he pDEP and he o he
exhibi ing nDEP, using an AC powe supply wi h a se ampli ude o 20 VPP a 20 kHz.
Ano he DEP mic o luidic de ice wi h symme ic silicon elec odes was p oposed o pe o m
a sequen ial DEP ield low cell sepa a ion (Figu e 15C) [
84
]. The chip ab ica ion p ocess was simila
o he p e ious one shown in Figu e 15B. The only di e ence was ha he silicon elec odes we e
symme ical. This elec ode geome y, compa ed o he coplana elec odes, supp essed Joule hea ,
and inc eased he sepa a ion e iciency. The suspension was i s pumped in o he channel, and he
pa icles we e hen apped in di e en loca ions by pe o ming he DEP. One popula ion o pa icles

Mic omachines 2019,10, 423 17 o 22
(g oup A) was apped in he cen e o he channel, which was he weakes posi ion o he elec ic ield,
while he o he popula ion (g oup B) was apped nea he elec ode ield. G oup A was hen lushed
ou o he channel by inc easing he low a e. The elec ic ield was hen emo ed, and g oup B was
subsequen ly lushed ou . This a ay s uc u e inc eased sepa a ion e iciency. Li ing/dead yeas cells
we e used o e i y he chip’s pe o mance.
Mic omachines2019,10,x17o 22

Figu e15.Mic o luidicde iceswi hsilicon3Delec odes.(A)Schema ico  hede icewi hsilicon
elec odepilla so auni o mheigh .Rep oducedwi hpe mission om[82],©2008Else ie .(B)
Schema ico  hede icewi hsiliconelec odepilla so anon‐uni o mheigh .Rep oducedwi h
pe mission om[83],©2009AIPPublishing.(C)Schema ico  hede icewi hsymme icalsilicon
elec odes[84].(D)Schema ico  hede icewi hbulksiliconelec ode[85].
Theelec odepilla scouldalsobedesignedinanon‐uni o mheigh  osepa a eli inganddead
yeas cells(Figu e15B)[83].Thede iceconsis edo  woglasswa e ssandwichingapa e ned
siliconwa e  o ming he3Delec odesaswellas hesepa a ionchambe .Theglasswa e was
anodicallybonded o hesiliconwa e ,and hen heDRIEwaspe o med oe ch hesilicon, o ming
wose so pilla so di e en heigh s,ei he ≈100μmo ≈2.5μm,aswellas hechambe .These wo
s uc u eswe e henbonded oge he , o ming heasymme icelec odes uc u es.Thede ice’s
pe o mancewas es edusing wopopula ionso cells,deadandli eyeas ,oneexhibi ing hepDEP
and heo he exhibi ingnDEP,usinganACpowe supplywi hase ampli udeo 20VPPa 20kHz.
Ano he DEPmic o luidicde icewi hsymme icsiliconelec odeswasp oposed ope o ma
sequen ialDEP ield lowcellsepa a ion(Figu e15C)[84].Thechip ab ica ionp ocesswassimila 
o hep e iousoneshowninFigu e15B.Theonlydi e encewas ha  hesiliconelec odeswe e
symme ical.Thiselec odegeome y,compa ed o hecoplana elec odes,supp essedJoulehea ,
andinc eased hesepa a ione iciency.Thesuspensionwas i s pumpedin o hechannel,and he
pa icleswe e hen appedindi e en loca ionsbype o ming heDEP.Onepopula iono 
pa icles(g oupA)was appedin hecen e o  hechannel,whichwas heweakes posi iono  he
elec ic ield,while heo he popula ion(g oupB)was appednea  heelec ode ield.G oupA
was hen lushedou o  hechannelbyinc easing he low a e.Theelec ic ieldwas hen emo ed,
andg oupBwassubsequen ly lushedou .Thisa ays uc u einc easedsepa a ione iciency.
Li ing/deadyeas cellswe eused o e i y hechip’spe o mance.
Figu e 15.
Mic o luidic de ices wi h silicon 3D elec odes. (
A
) Schema ic o he de ice wi h silicon
elec ode pilla s o a uni o m heigh . Rep oduced wi h pe mission om [
82
],
©
2008 Else ie .
(
B
) Schema ic o he de ice wi h silicon elec ode pilla s o a non-uni o m heigh . Rep oduced wi h
pe mission om [
83
],
©
2009 AIP Publishing. (
C
) Schema ic o he de ice wi h symme ical silicon
elec odes [84]. (D) Schema ic o he de ice wi h bulk silicon elec ode [85].
Silicon can also be used as a bulk elec ode wi hou a ays, simpli ying he chip s uc u e and
ab ica ion p ocess (Figu e 15D) [
85
]. The simula ion showed ha he e was no olume wi hou a NUEF,
sugges ing good sepa a ion e iciency. The ab ica ed chip consis ed o h ee pa s: hea ily doped
silicon pa e ned wi h co uga ed channels o ming he DEP, sandwiched by wo glasses mechanically
connec ed by wo subsequen anodic bonding p ocesses. The de ice’s pe o mance was again e i ied
by sepa a ing dead om li ing yeas cells using an AC powe supply wi h a se ol age ampli ude o
25 VPP.
5. Conclusions
DEP is a pa icle sepa a ion echnique based on pa icle pola iza ion in a NUEF. I has been p o en
o be a iable ool o LOC in he applica ion o medicine, molecula diagnos ics, and any o he ields
ela ed o pa icle sepa a ion because o i s selec i i y and accu acy. I is also a label- ee me hod.
In his e iew, we desc ibed DEP heo y and compa ed i o o he sepa a ion echniques o show i s
Mic omachines 2019,10, 423 18 o 22
ad an ages o mic o luidic de ices. We also in oduced he ecen de elopmen s in DEP mic o luidic
de ices, classi ied by elec ode ype. Fo each sys em, we explained he chip s uc u e, ab ica ion
p ocess, elec ode ype, and ope a ion p inciple. These mic o luidic de ices ha e he po en ial o be
widely used in labo a o ies and diagnos ic cen e s.
Howe e , he wide employmen o DEP would p obably equi e highe le els o sys em
in eg a ion, including sample p e ea men , pos DEP p ocessing, as well as p oduc analysis.
In addi ion, he DEP i sel should be designed in a mo e e icien way o using 3D o semi-3D
ope a ions. Tha could be achie ed ei he by designing he 3D elec ode geome y o making he DEP
a ea shallow and supp essing he low e iciency space o p omo e he comme cializa ion and wide
u iliza ion o DEP.
Acknowledgmen s:
This wo k was suppo ed by he Fo eign Expe s P og am o P.R. China (W099109). We also
acknowledge he suppo o CEITEC Nano Resea ch In as uc u e (ID LM2015041, MEYS CR, 2016–2019), CEITEC
B no Uni e si y o Technology, and G an Agency o he Czech Republic unde he con ac GA16-11140S.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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