CHAP1LR

:
Lab-oh-a-Chip Impedahce DeIecIioh o!
Microbial ahd Cellular AcIiviIy
Li|u Yahg,
1
Xuahhohg Chehg,
2
Yi-Shao Liu,
3
ahd Rashid 8ashir
3
1
BiomanulacIuring Research InsIiIuIe and Technology EnIer¡rise, De¡arImenI ol FharmaceuIical Sciences,
NorIh Carolina CenIral UniversiIy, Durham, NC 27707
2
De¡arImenI ol MaIerials Science and Engineering, Frogram ol Bioengineering, Lehigh
UniversiIy, BeIhlehem, FA 1801S
3
De¡arImenI ol ElecIrical and Com¡uIer Engineering, De¡arImenI ol Bioengineering, Micro and
NanoIechnology LaboraIory, UniversiIy ol Illinois, Urbana-Cham¡aign, Urbana, IL o1801
296
Key Ierms micro!luidic chips
lab-oh-a-chip
elecIrical deIecIioh
impedahce specIroscopy
dielecIrophoresis
bacIeria cells
mammaliah cells
cell couhIihg
spore germihaIioh
AbsIracI
Lab-on-a-chi¡ Iy¡e ol devices ca¡able ol im¡edance sensing has recenIly
aIIracIed a loI ol inIeresI lor label-lree, real-Iime, and noninvasive elecIrical
deIecIion ol biological acIiviIies. In Ihis cha¡Ier, we describe lour lab-on-a-chi¡
sysIems lor Ihe deIecIion ol microbial and cellular acIiviIies based onIhe unique
elecIrical and elecIro¡hysiological ¡ro¡erIies ol micro-organisms and mamma-
lian cells. Two ol Ihe sysIems were designed based on im¡edance moniIoring ol
live micro-organismacIiviIies lor: (1) moniIoring Ihe concenIraIion ol bacIerial
cells during growIh, and (2) Ihe deIecIion ol Cbdjmmvt bouisbdjt s¡ore germina-
Iion. The oIher Iwo sysIems were designed lor deIecIion ol cell concenIraIion by
measuring Ihe im¡edance changes due Io Iheir ion release lor a¡¡licaIions in
counIing: (1) CD4+ T lym¡hocyIes, and (2) lood-borne ¡aIhogenic bacIerial
cells. These microlabricaIed im¡edance sensors showgreaI ¡romise in Ihe deIec-
Iion ol cells and Iheir meIabolic acIiviIies wiIh im¡roved sim¡liciIy, higher sen-
siIiviIy, and lasIer deIecIion Iime Ihan convenIional meIhods.
:/2 Jouspevdujpo
The advances in microelecIromechanical sysIems (MEMS) Iechnology have allowed sci-
enIisIs Io consIrucI novel devices or sysIems wiIh sizes com¡arable Io biological enIiIies
and sensiIiviIy high enough lor a wide varieIy ol im¡orIanI biomedical and biological
a¡¡licaIions. Develo¡menI ol "lab-on-a-chi¡" Iy¡es ol devices uses MEMS Iechnology
Io inIegraIe various microlabricaIed sensors or deIecIion ¡laIlorms wiIh many ol Ihe
uniI o¡eraIions associaIed wiIh sam¡le ¡re¡araIion and ¡resenIaIion, such as se¡ara-
Iion, mixing, incubaIion, and concenIraIion. The use ol lab-on-a-chi¡ devices lor
microbial and cellular deIecIion has shown Ihe lollowing advanIages over IradiIional
meIhods: (1) reducIion ol Ihe sensor elemenIs Io Ihe size ol a single cell or even smaller,
¡roviding a higher sensiIiviIy, (2) reducIion ol reagenI volume and associaIed cosI, (3)
reducIion ol Ihe Iime Io resulIs due Io Ihe small volume and high ellecIive concenIra-
Iion, (4) amenabiliIy Io sysIem miniaIurizaIion and ¡orIabiliIy, and (S) com¡aIibiliIy
wiIh large numbers ol assays and mulIi¡lexed measuremenIs.
Im¡edance sensing, as one ol Ihe ¡rinci¡al elecIrical}elecIrochemical IransducIions,
is becoming a lerIile area lor develo¡ing meIhods lor a wide range ol biological and bio-
medical a¡¡licaIions. Several lacIors aIIribuIe Io Ihe ¡o¡ulariIy ol im¡edance sensing:
(1) Ihe disIincI elecIrical ¡ro¡erIies associaIed wiIh s¡ecilic biological enIiIies and}or
biological reacIions moIivaIe Ihe use ol im¡edance-sensing Iechniques, (2) im¡edance
measuremenI is one ol Ihe mosI ¡romising Iechniques lor label-lree, real-Iime, and
noninvasive biological deIecIion, and (3) im¡edance deIecIors can be easily miniaIur-
ized Io meeI Ihe growing needs ol ¡orIable sysIems wiIh an analyIical looI¡rinI consid-
erably smaller Ihan laboraIory-based insIrumenIs |1|.
The disIincI elecIrical ¡ro¡erIies ol biological cells and Iheir elecIro¡hysiology are
lundamenIal lor develo¡ing im¡edance-based meIhods Io deIecI biological acIiviIies.
Biological cells consisI ol adjacenI sIrucIures ol maIerials IhaI have very dillerenI elecIri-
cal ¡ro¡erIies. The cell membrane consisIs ol a li¡id bilayer, where Ihe li¡id molecules
are orienIed wiIh Iheir ¡olar grou¡s lacing ouIwards inIo Ihe aqueous environmenI and
Iheir hydro¡hobic hydrocarbon chains ¡oinIing inwards Io lorm Ihe membrane inIe-
rior. The inside ol a cell is com¡lex and conIains membrane-covered ¡arIiculaIes, such as
miIochondria, vacuoles, a nucleus, and many charged molecules. While Ihe cell mem-
brane is highly insulaIing, Ihe inIerior ol Ihe cell is highly conducIive. The conducIiviIy
ol Ihe cell membrane is around 10
÷7
S}m, whereas Ihe conducIiviIy ol Ihe inIerior ol a
cell can be as high as 1 S}m |2|.
Based on Ihe elecIro¡hysiological and elecIrical ¡ro¡erIies ol biological cells, Ihree
major mechanisms have been ex¡lored lor Ihe deIecIion and quanIilicaIion ol biologi-
cal cells using microscale im¡edance-based measuremenIs:
1. Nbljoh vtf pg uif nfubcpmjd bdujwjuz pg cjpmphjdbm dfmmt; This is re¡resenIed by
im¡edance microbiology, which is a Iechnique based on Ihe measuremenIs ol Ihe
elecIric im¡edance change in a medium or a reacIanI soluIion resulIing lrom cell
meIabolism |3, 4|. Based on Ihis ¡rinci¡le, a new Iechnique called "im¡edance
microbiology-on-a-chi¡" has been demonsIraIed by our grou¡ |S|. The idea is Io
conline a lew live bacIerial cells inIo a small volume on Ihe order ol nano- Io
¡icoliIers such IhaI meIaboliIes ol Ihese cells are concenIraIed and deIecIable
by im¡edance measuremenI wiIh inIerdigiIaIed microelecIrodes. We have also
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
297
successlully develo¡ed a microchi¡ lor im¡edance moniIoring ol s¡ore
germinaIion |o|.
2. Nbljoh vtf pg uif ijhimz jpojd dzupqmbtnjd dpoufou pg uif dfmmt; As Ihe inside ol a cell
conIains many charged molecules and is highly conducIive (1 S}m), Ihe im¡edance
change due Io Ihe lysis ol cells or release ol inIracellular ions can ¡rovide a means Io
deIecI biological cells. In Ihis cha¡Ier, we will review Iwo microchi¡-based
im¡edance-deIecIion sysIems lor: (1) enumeraIion ol CD4+ T lym¡hocyIes Ihrough
cell lysaIes |7|, and (2) deIecIion ol bacIerial cells based on im¡edance change lrom
Iheir ion release inIo deionized (DI) waIer |1|.
3. Nbljoh vtf pg uif jotvmbujoh qspqfsujft pg uif dfmm nfncsbof; Because ol Iheir highly
insulaIing cell membrane, cells aIIached on an elecIrode surlace ellecIively reduce
Ihe conducIing area and hence increase Ihe inIerlacial im¡edance. The sensor ¡robes
Ihe aIIachmenI ol cells by measuring Ihe change ol Ihe inIerlacial elecIrical
¡ro¡erIies arising lrom Ihe insulaIing ¡ro¡erIy ol Ihe cell membrane. Many
cell-based im¡edance sensors are based on Ihis mechanism. By culIuring cells on
microelecIrodes and moniIoring im¡edance changes caused by adherenI cells, one
can quanIily changes in Ihe im¡edance associaIed wiIh Ihe cell membrane,
cell-subsIraIe inIeracIion, and cell-cell se¡araIion wiIh exquisiIe sensiIiviIy and in a
noninvasive manner |8, º|. We consIrucIed a bacIerial immunosensor based on Ihis
mechanism: anIibodies s¡ecilic Io Ihe IargeI bacIerial cells are immobilized on an
elecIrode surlace, and selecIive aIIachmenI ol cells is deIecIed elecIrically |10|.
In Ihis cha¡Ier, we describe lour im¡edance-deIecIion sysIems based on Ihe lirsI Iwo
mechanisms described above lor moniIoring biological meIabolic acIiviIy and deIecIing
cells.
:/3 Mbc.po.b.Dijq gps Npojupsjoh Njdspcjbm Nfubcpmjd
Bdujwjuz
:/3/2 “Jnqfebodf njdspcjpmphz.po.b.dijq” gps cbdufsjbm dpodfousbujpo boe
efufdujpo
One common im¡edance meIhod lor deIecIion ol bacIerial growIh is jnqfebodf njdsp.
cjpmphz, which is based on Ihe measuremenI ol changes in elecIrical im¡edance ol a cul-
Iure medium or a reacIion soluIion resulIing lrom Ihe bacIerial growIh. This
growIh-based im¡edance Iechnique allows one Io disIinguish beIween viable and dead
cells and Io deIecI viable bacIeria wiIhin 24 hours. In 1ºº2, Ihe im¡edance meIhod was
a¡¡roved by Ihe AssociaIion ol Ollicial AnalyIical ChemisIs InIernaIional (AOAC) as
Ihe lirsI acIion meIhod lor screening Tbmnpofmmb in lood sam¡les |11, 12|.
In im¡edance microbiology, Ihe im¡edance change is Iy¡ically measured using a
¡air ol elecIrodes submerged inIhe growIh mediumor Ihe reacIanI soluIion. The im¡ed-
ance change in Ihe medium is mainly ¡roduced by Ihe release ol ionic meIaboliIes lrom
live cells. There are Iwo main origins ol ion release by bacIeria inIo Iheir growIh environ-
menI |13|: one is energy meIabolism (caIabolism) in which bacIeria consumes oxygen
and sugars and ¡roduces carbon dioxide and organic acids, Ihe oIher is ion exchange
Ihrough Ihe cell membrane. Ions (such as K
+
and Na
+
) are acIively Irans¡orIed across ion
channels embedded in Ihe cell membrane, which serves Io regulaIe Ihe membrane
9.2 Lab-oh-a-Chip !or MohiIorihg Microbial MeIabolic AcIiviIy
298
¡oIenIial and Ihe osmoIic dillerence beIween Ihe inIerior and exIerior ol Ihe cell.
BeIweenIhe Iwo origins, energy meIabolismis Ihe major ¡aIhol ion release lromcells Io
Ihe environmenI, and Ihe ion-exchange ¡rocess is a small conIribuIor. These released
ions cause changes in Ihe ionic com¡osiIion ol Ihe medium and consequenIly increase
Ihe conducIiviIy ol Ihe medium. To deIecI bacIeria, Ihe im¡edance sensor measures Ihe
relaIive or absoluIe changes in conducIance, ca¡aciIance, or im¡edance aI regular Iime
inIervals during Ihe growIh ol bacIeria aI a given Iem¡eraIure. The measured elecIrical
signals are Ihen gra¡hically ¡loIIed on Ihe ordinaIe againsI Ihe incubaIion Iimes on Ihe
abscissa, ¡roducing im¡edance growIh curves. The Iime aI which Ihe decrease in im¡ed-
ance value exceeds a Ihreshold is delined as Ihe deIecIion Iime, u
e
. Generally, Ihe im¡ed-
ance Ihreshold is noI reached unIil Ihe bacIeria number reaches a¡¡roximaIely 10
o
Io 10
7
clu}mL (as deIermined by Ihe ¡laIing meIhod). Eor convenIional im¡edance-microbio-
logical meIhods, Ihe deIecIion Iime ranges lrom abouI 1 Io 8 hours lor iniIial bacIerial
concenIraIion ol 10
7
Io 10
1
clu}mL.
MiniaIurizaIion ol an im¡edance-deIecIion sysIem inIo a chi¡-based device has
shown greaI ¡romise in ra¡id deIecIion ol bacIerial growIh. Our grou¡ was among Ihe
lirsI Io labricaIe inIegraIed silicon-based biochi¡s lor im¡edance deIecIion ol microbial
meIabolism|S, 14, 1S|. The basic idea was Io conline a lewlive bacIerial cells inIo a small
volume on Ihe order ol nano- Io ¡icoliIers, such IhaI Ihe meIaboliIes ol a lewlive cells in
a low-conducIiviIy buller can be ra¡idly deIecIed by im¡edance measuremenIs using
inIerdigiIaIed microelecIrodes. To concenIraIe bacIerial cells lrom a diluIed sam¡le inIo
a small volume, we used a Iechnique called dielecIro¡horesis (DEF), which is Ihe
elecIrokineIic moIion ol dielecIrically ¡olarized ¡arIicles in nonunilorm elecIric lields
|1o|. As mosI biological cells behave as dielecIric ¡arIicles in an exIernal elecIric lield,
DEF allows Ira¡¡ing, concenIraIion, and se¡araIion ol biological cells in a liquid
sus¡ension.
9.2.1.1 MeIhods ahd devices
9.2.1.1.1 Chip design and fabrication
The im¡edance microbiology-on-a-chi¡ device conIained Ihree seIs ol inIerdigiIaIed
microelecIrodes and llow channels. Eigure º.1(a) shows Ihe ¡rinci¡le ol Ihe o¡eraIion
ol Ihe DEF-based deviaIion and ca¡Iure ol bacIerial cells in Ihe microchi¡. One seI was
lor dielecIro¡horeIical deviaIion ol bacIerial cells lromIhe main channel inIo Ihe small
channel IhaI leads Io Ihe deIecIion chamber. In Ihe deIecIion chamber, one seI ol elec-
Irodes was lor DEF ca¡Iure ol bacIerial cell inIo Ihe deIecIion chamber, and Ihe oIher
seI ol elecIrodes was lor moniIoring Ihe im¡edance change ol bacIerial growIh in Ihe
chamber. The deIecIion chamber had a volume ol 400 ¡L. Eigure º.1(b) shows Ihe cross
secIion ol Ihe deIecIion chamber wiIh DEF elecIrodes and im¡edance-measuremenI
elecIrodes. Eigure º.1(c) shows Ihe com¡leIely ¡ackaged microchi¡.
The microchi¡s were labricaIed on 4" walers wiIh a (100) surlace and a Ihickness ol
S00 Ÿm. ElecIrodes and channel ¡aIIerns were made using sIandard ¡hoIoliIhogra¡hic
Iechnology. The channels were 12 Ÿm dee¡ and were made by eIching Ihe waler wiIh a
hard mask. The DEF elecIrodes were de¡osiIed by s¡uIIering 1,000A ol aluminum onIo
2,000A ol silicon dioxide on Ihe boIIomol Ihe channel. On Ihe DEF elecIrodes, anoIher
layer ol silicon dioxide ol 3,S00A was de¡osiIed by ¡lasma-enhanced chemical va¡or
de¡osiIion in order Io com¡leIely isolaIe Ihe DEF elecIrodes and ¡revenI elecIrolysis ol
Ihe liquid in Ihe channels. The im¡edance-measuremenI elecIrodes and Iem¡eraIure
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
299
sensor were de¡osiIed by s¡uIIering 800A ol ¡laIinum over a IiIanium adhesion layer.
The chi¡ was assembled wiIha glass cover wiIhinleI and ouIleI holes using anodic bond-
ing. DeIailed ¡rocedure can be lound in |S|.
9.2 Lab-oh-a-Chip !or MohiIorihg Microbial MeIabolic AcIiviIy
29:
(a)
(c)
(b)
Gjhvsf :/2 (a) The schemaIic design ol Ihe microchi¡ wiIh DEF deviaIion elecIrodes in Ihe main chan-
nel and a small channel leading Ihe llow Io Ihe DEF ca¡Iure elecIrodes in Ihe deIecIion chamber. (b) Sim-
¡lilied cross secIion ol Ihe ¡ackaged microchi¡ showing Ihe DEF ca¡Iure elecIrodes and Ihe
im¡edance-measuremenI elecIrodes in Ihe deIecIion chamber. (c) An image ol Ihe ¡ackaged microchi¡
connecIed Io Ihe measuremenI and conIrol sysIem. (Re¡rinIed wiIh ¡ermission lrom Ihe K/
Njdspfmfduspnfdibojdbm Tztufnt and kind ¡ermission lrom |S|.)
9.2.1.1.2 8acterial cell preparation
Mjtufsjb npopdzuphfoft w8 was grown in Luria-BerIani (LB) mediumaI 37¬C lor aI leasI 1o
hours. The cells were harvesIed and washed by re¡eaIed cenIrilugaIion and
resus¡ension in sIerile LB medium. The cells were Ihen diluIed in sIerile LB Io desired
concenIraIions. When lluorescenI cell were needed, 1 mL ol Ihe as-grown live cells was
sIained wiIh green lluorescenI dye DiOC
o
(3) (3,3´-dihexyloxacarbonanine iodide). Elu-
orescence-sIained cells were washed and diluIed in Ihe same way as described above lor
lurIher use.
9.2.1.1.J On-chip DLP concentration and impedance detection of metabolism
All Ihe on-chi¡ ex¡erimenIs were carried ouI wiIh Ihe chi¡ heaIed Io 37¬C. A IoIal vol-
ume ol 40 ŸL ol Ihe cell sus¡ension in DI waIer was injecIed inIo Ihe main channel aI a
llow raIe ol a¡¡roximaIely 1.7 ŸL}min. The DEF deviaIion and ca¡Iure elecIrodes were
exciIed wiIh a 1o W
qq
square signal aI 100 kHz. During Ihe injecIion, Ihe llow raIe in Ihe
incubaIion chamber was manually conIrolled Io be beIween 4 and 10 nL}min. AlIer Ihe
sam¡le injecIion, Ihe DEF deviaIion elecIrodes were Iurned oll, and Hall-LB medium
(HLB, mixing equal ¡arIs ol LB and DI waIer) was injecIed aI a llow raIe ol less Ihan 0.S
ŸL}min, while Ihe exciIaIion volIage on Ihe ca¡Iure elecIrodes was increased Io 20 W
qq
,
and Ihe lrequency was increased Io 3 MHz in order Io maximize Ihe DEF lorces acIing
on cells. Once Ihe incubaIion chamber was lilled wiIh HLB, Ihe llow was sIo¡¡ed, and
Ihe lluidic channels and microIubes were ¡inched Io seal Ihem com¡leIely. The DEF
ca¡Iure elecIrodes were Ihen Iurned oll. Im¡edance measuremenI elecIrodes were
Iurned on, and Ihe cells were incubaIed lor a¡¡roximaIely 12 hours. The im¡edance
was measured wiIh an AgilenI 4284A LCR meIer (AgilenI Technologies Inc., Falo AlIo,
Calilornia) connecIed Io Ihe chi¡ Ihrough an AgilenI 34º70A swiIching uniI liIIed wiIh
Iwo AgilenI 34º0SA RE mulIi¡lexer cards. All Ihe insIrumenIs were connecIed Io a com-
¡uIer Ihrough a GFIB inIerlace. The im¡edance measuremenIs and chi¡ Iem¡eraIure
were conIrolled by cusIom LabVIEW ¡rogram (NaIional InsIrumenIs Cor¡., AusIin,
Texas). Im¡edance was measured aI S1 lrequencies logariIhmically s¡aced beIween 100
Hz and 100 kHz, wiIh a 1S0 mV am¡liIude. Sinusoidal and square wave DEF signals
were generaIed by AgilenI 333120A synIheIized signal generaIors.
9.2.1.2 ResulIs ahd discussioh
To demonsIraIe Ihe com¡leIe ¡rocess ol cell concenIraIion and im¡edance measure-
menI ol bacIerial meIabolism on Ihe chi¡, sam¡les conIaining lluorescenIly labeled M/
npopdzuphfoft w8 cells aI concenIraIions ol 2.3 Ó 10
S
, o.8 Ó 10
S
, and 8.7 Ó 10
4
clu}mL were
IesIed. The sam¡le wiIh 2.3 Ó 10
S
clu}mL cells was injecIed wiIh Ihe DEF elecIrodes oll,
while Ihe oIher Iwo sam¡les were injecIed wiIh Ihe DEF elecIrode acIivaIed. When Ihe
DEF elecIrode was oll, Ihe cells were noI concenIraIed in Ihe incubaIion chamber.
There was only a ¡robabiliIy ol a¡¡roximaIely 0.0º Io lind one cell in Ihe chamber.
When DEF elecIrodes were acIivaIed, almosI all Ihe cells were ca¡Iured by Ihe DEF elec-
Irodes inIo Ihe deIecIion chamber. Eigure º.2(a) shows a re¡resenIaIive image ol
lluorescenIly labeled Mjtufsjb cells concenIraIed by DEF inIo Ihe ¡icoliIer measuremenI
chamber. AlIhough Ihe acIual number ol cells collecIed was noI deIermined, iI was
visually conlirmed IhaI only a very small lracIion ol cells esca¡ed Ihe DEF deviaIion
and ca¡Iure ¡rocesses (no more Ihan a¡¡roximaIely 10'). However, during Ihe swiIch
lrom waIer Io HLB, a more signilicanI lracIion ol Ihe cells were losI because Ihe DEF
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
2:1
lorce was weakened by Ihe increased medium conducIiviIy and Ihe llucIuaIion ol Ihe
llow raIe. NoneIheless, Ihousands ol cells were sIill collecIed in Ihe chamber, as shown
in Eigure º.2(a). The concenIraIion lacIor ol Ihis chi¡ was beIween 10
4
Io 10
S
when Ihe
cells in an original sam¡le volume ol 40 ŸL were concenIraIed inIo Ihe 400 ¡L chamber,
¡rovided IhaI 10' Io 100' ol Ihe cells were ca¡Iured by DEF. Such DEF concenIraIion
Iechnique in microlabricaIed chi¡s eliminaIes Ihe need Io enrich Ihe bacIerial ¡o¡ula-
Iion by long culIure sIe¡s in convenIional cell culIure meIhods. WiIh Ihe dramaIic
increase ol bacIerial cell concenIraIion aI Ihe localiIy ol Ihe deIecIion chamber, iI is
ex¡ecIed IhaI Ihe deIecIion Iime on Ihe im¡edance growIh curve can be ellecIively
reduced, resulIing in ra¡id deIecIion.
The signilicanI reducIion in deIecIion Iime was demonsIraIed by Ihe com¡arison ol
Ihe im¡edance growIh curves ol Mjtufsjb cells in HLB medium on chi¡ wiIh and wiIhouI
DEF concenIraIion |Eigure º.2(b)|. As shown in Ihe ligure, Ihe sIerile media did noI
exhibiI any clear meIabolic signal aI any lrequency. The bacIerial sam¡le conIaining
a¡¡roximaIely o.8 Ó 10
S
clu}mL wiIh Ihe DEF concenIraIion ¡resenIed an im¡edance
9.2 Lab-oh-a-Chip !or MohiIorihg Microbial MeIabolic AcIiviIy
2:2
)b*
)c*
Gjhvsf :/3 (a) Eluorescence-labeled Mjtufsjb cells concenIraIed by DEF lrom a sus¡ension ol o.8 Ó 10
S
clu}mL inIo Ihe incubaIion chamber immediaIely belore Ihe sIarI ol incubaIion. (b) RelaIive admiIIance
change during Ihe incubaIion ol Mjtufsjb cells injecIed inIo Ihe microchi¡ aI various concenIraIions, wiIh
and wiIhouI DEF concenIraIion, ¡lus sIerile HLB. Values aI u = 0 are delined as 100'. (Re¡rinIed wiIh ¡er-
mission lrom Ihe K/ Njdspfmfduspnfdibojdbm Tztufnt and kind ¡ermission lrom |S|.)
meIabolic signal corres¡onding Io Ihe ex¡onenIial growIh aI a¡¡roximaIely 1 hour,
while Ihe sam¡le conIaining similar concenIraIion ol cells wiIhouI DEF concenIraIion
required a¡¡roximaIely 7.S hours Io ¡roduce a deIecIable im¡edance signal. The resulIs
demonsIraIed IhaI concenIraIionol bacIerial cells by Ihe DEF canellecIively shorIen Ihe
deIecIion Iime needed lor Ihe im¡edance deIecIion ol cell meIabolic acIiviIy.
MiniaIurized sensors lor im¡edance-based deIecIion ol bacIeria inIegraIed wiIh a
DEF-based cell-concenIraIion sysIem hold greaI ¡oIenIial Io dramaIically reduce Ihe
Iime needed Io deIecI bacIeria based onIheir meIabolic acIiviIy Io hours, which is a greaI
im¡rovemenI com¡ared wiIh convenIional meIhods IhaI require several days. Such a
microscale sysIemalso has greaI ¡oIenIial a¡¡licaIions lor screening indusIrial and clini-
cal sam¡les lor IoIal bacIerial conIenIs.
:/3/3 Njdspgmvjejd cjpdijqt gps jnqfebodf efufdujpo pg Cbdjmmvt bouisbdjt
tqpsf hfsnjobujpo
Cbdjmmvt bouisbdjt has long been idenIilied as Ihe causaIive agenI ol Ihe disease anIhrax.
The inIenIional conIaminaIion ol seven leIIers wiIh C/ bouisbdjt s¡ores in 2001 resulIed
in 22 cases ol anIhrax, S ol which were laIal |17|, and locused aIIenIion on Ihe deIec-
Iion ol s¡ores ol Cbdjmmvt bouisbdjt. Cbdjmmvt bouisbdjt has a long-IermenvironmenIal ¡er-
sisIence due Io Ihe lormaIion ol endos¡ores, which develo¡ over a Iime course ol
several hours inside a cell ex¡osed Io nuIrienI sIarvaIion or oIher environmenIal
sIresses |18|. A dense ¡roIein coaI, low cyIo¡lasmal waIer acIiviIy, and small, acid-solu-
ble, DNA-binding ¡roIeins render Ihe s¡ore highly resisIanI Io dessicaIion, irradiaIion,
chemical oxidaIion, and oIher environmenIal assaulIs |1º|. This resisIance renders
deconIaminaIion ol an environmenI in which endos¡ores are ¡resenI very dilliculI and
makes deIecIion ol low-level s¡ore conIaminaIion an im¡orIanI goal. Many deIecIion
meIhods, such as colony mor¡hology, sIaining ol Ihe unique ¡oly-D-gluIamaIe ca¡-
sule, FCR am¡lilicaIion ol s¡ecilic DNA sequences, and c-¡hage susce¡IibiliIy IesIing,
require Ihe ouIgrowIh ol vegeIaIive cells belore IesIing, Ihey also olIen require
Iime-consuming manual sIe¡s. Several innovaIive meIhods re¡orIed in recenI years can
deIecI endos¡ores aI a Ihreshold ol abouI 10
3
s¡ores in a sam¡le |20÷2S|. MosI ol Ihese
meIhods involve micro- or macroscale FCR analysis or various lorms ol o¡Iical deIec-
Iion. AuIomaIed s¡ore-deIecIion sysIems have used real-Iime FCR wiIh Ihermal cycling
chambers made lrom eIched and lusion-bonded silicon Io carry ouI Ihe FCR-based
deIecIion assays lor Cbdjmmvt s¡¡. and Zfstjojb s¡¡. |2o|. II should be noIed IhaI mosI ol
Ihe re¡orIed meIhods eiIher ¡erlorm idenIilicaIion ol Ihe s¡ores belore s¡ores germi-
naIe and lail Io disIinguish beIween viable and nonviable s¡ores |21|, or Ihey deIecI Ihe
¡aIhogens aI relaIively laIe vegeIaIive growIh ¡hase |20|. In Ihis secIion, we review a
deIecIion meIhod lor viable s¡ores by im¡edance measuremenI. The deIecIion was ¡er-
lormed as early as Ihe s¡ore-germinaIion sIage, and a signal was deIecIed only when Ihe
s¡ores were viable.
9.2.2.1 MeIhods ahd devices
We lirsI examined Ihe conce¡I using macroscale ex¡erimenIs, where we measured Ihe
germinaIion ol a S mL s¡ore sus¡ension, wiIh concenIraIion ranging lrom 10
7
Io 10
º
s¡ores}mL, in a rinsed (wiIh sIerile DI waIer) and sIerile 1S mL ¡lasIic cenIriluge Iube
(4300S2, Corning Inc., Corning, New York) using a commercial conducIiviIy meIer
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
2:3
(o307 microcom¡uIer ¡H}conducIiviIy meIer, Jenco InsIrumenIs, San Diego, Calilor-
nia). The s¡ores were ¡reheaIed in a oSºC waIer baIh lor 30 minuIes belore Ihe ex¡eri-
menI. AlIerwards, germinanI soluIion was added, and Ihe conducIiviIy ¡robe was
inserIed inIo Ihe 1S mL cenIriluge Iube conIaining Ihe sam¡le. ConducIiviIy values
were recorded every minuIe. Three dillerenI concenIraIions ol s¡ores were com¡ared Io
conIrol ex¡erimenIs (i.e., s¡ores only, DI waIer only, and germinanI only) Io lind Ihe
deIecIion limiI. The conducIiviIy ¡robes were calibraIed belore each ex¡erimenI. The
DI waIer had a measured conducIiviIy ol 2 Io 3 ŸS}cm, which was wiIhin Ihe acce¡Ied
range and Ihus demonsIraIed Ihe sensiIiviIy ol Ihe insIrumenI. The ex¡erimenI was car-
ried ouI aI room Iem¡eraIure |27|.
The microlluidic device lor on-chi¡ deIecIion ol s¡ore germinaIion was consIrucIed as
a Ihree layer BioMEMS device. The lirsI layer was a Fyrex (7740, Corning Inc.) subsIraIe
wiIh inIerdigiIaIed elecIrodes lor exerIing dielecIro¡horesis lorce Io ca¡Iure and concen-
IraIe s¡ores and lor recording Ihe change ol admiIIance (inverse ol im¡edance) wiIhin Ihe
soluIion. The meIal elecIrodes were de¡osiIed as 2S0A IiIanium and 3S0A gold by eva¡o-
raIion (E-Beam Eva¡oraIor, CHA IndusIries, EremonI, Calilornia), lollowed by a lilI-oll
¡rocess. On Io¡ ol Ihe Fyrex subsIraIe was a 40 Ÿm FDMS layer wiIh ¡aIIerned
microlluidic channels and chambers lor sam¡le delivery and germinaIion deIecIion. The
Ihird layer was a Ihick, 2 mm FDMS slab wiIh microlluidic ¡aIhways serving as valves Io
close oll Ihe channels inIhe Ihin second layer Io enhance Ihe signal sIrengIh by lorming a
closed environmenI lor deIecIion, as well as Io consIrain s¡ores inside Ihe deIecIion
chamber alIer Ihe dielecIro¡horesis (DEF) ca¡Iure lorce was released. The layouI ol Ihe
chi¡ and Ihe com¡leIed chi¡ are illusIraIed in Eigure º.3(a, b). The Fyrex layer conIaining
Ihe elecIrode subsIraIe and Ihe hybrid FDMS layer was bonded by surlace IreaImenI using
oxygen ¡lasma (200W, 1S seconds) and aligned immediaIely (wiIhinS minuIes). The eIch
gas was 80' argon and 20' oxygen. Microbore Iubings (OD: 0.01o", ID: 0.00o", Cole
Farmer, Vernon Hills, Illinois) were inserIed inIo Ihe ¡unched inleI holes and sealed wiIh
10:1 FDMS lor injecIion ol liquids. The cross secIion ol Ihe labricaIed biochi¡ and iIs lunc-
Iions is illusIraIed in Eigure º.3(c) |28÷30|.
The elecIrical measuremenIs on-chi¡ were carried ouI wiIh an auIomaIed recording
sysIem. The sysIemincluded an injecIor, measuring ¡robes (Micromani¡ulaIor Co., Car-
son, Nevada), an LCR meIer (AgilenI Technologies), a com¡uIer, and a microsco¡e
(Ecli¡se Eo00EN, Nikon Inc., Melville, New York). HeaI-IreaIed s¡ores were injecIed by
Ihe injecIion sysIem inIo Ihe microlluidic device mounIed on Ihe microsco¡e ¡laIlorm,
wiIh a llow raIe ol 30 ŸL}min lor S minuIes, lollowed by 0.2 ŸL}min. The injecIion sys-
Iem had mulIi¡le injecIion valves and swiIches Io change soluIions lor delivery ol sam-
¡les and germinanI. ElecIrical recording sIarIed righI alIer s¡ores and germinanI were
delivered inIo Ihe chi¡. DaIa was recorded aI 2-minuIe inIervals lor 1 hour. VerilicaIion
ol germinaIion alIer each ex¡erimenI was done by observing Ihe relracIiliIy ol Cbdjmmvt
bouisbdjt s¡ores using ¡hase-conIrasI microsco¡y. UngerminaIed s¡ores are relracIile
(¡hase brighI) and germinaIed s¡ores are noI (¡hase gray or ¡hase dark). The ex¡erimenI
was carried ouI aI room Iem¡eraIure.
9.2.2.2 ResulIs ahd discussioh
The resulIs ol s¡ore-germinaIion deIecIion wiIh a commercial conducIiviIy meIer are
illusIraIed in Eigure º.4(a). This ligure shows Ihe resulIs wiIh s¡ore concenIraIions ol
9.2 Lab-oh-a-Chip !or MohiIorihg Microbial MeIabolic AcIiviIy
2:4
10
7
, 10
8
, and 10
º
s¡ores}mL. The mosI ¡ronounced resulI is observed aI a concenIraIion
ol 10
º
s¡ores}mL, where a neI increase ol conducIiviIy occurs in Ihe range ol S Io 7
ŸS}cm. An immediaIe increase in conducIiviIy suggesIs IhaI germinaIion began in Ihe
lirsI 2 minuIes alIer germinanI was added and linished wiIhin 20 minuIes, while con-
Irol ex¡erimenIs showed no signilicanI change in conducIiviIy over Iime. We consid-
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
2:5
Valve Chahhels
Au/1i LlecIrodes
50 um 50 um 50 um
8rowh: chahhels !or valvihg o!
secohd layer (!abricaIed ih Ihe
Ihird layer o! Ihick PDMS)
Several 90 degree ahgle
sIrucIures Io reduce Ihe !low
speed !or beIIer spore capIure
e!!iciehcy usihg DLP
Red: chahhels ahd chambers !or
Ihe ihcubaIioh o! cells (ih Ihe
secohd layer o! Ihih PDMS)
Creeh: 1i/Au elecIrodes (oh Ihe !irsI layer o! silicoh or
glass wa!er)
(a)
(b)
IhleI ahd OuIleI 1ubihg !or spore ahd germihahI delivery
Chahhels !or
spore ahd
germihahI delivery
IhIerdigiIaIed
LlecIrodes
Valve Chahhels
Chamber
Gjhvsf :/4 (a) To¡-view layouI ol Ihe microlluidic device, (b) o¡Iical image ol Ihe com¡leIed device,
and (c) cross secIion ol Ihe microlluidic device. S¡ores were heaI-acIivaIed oll-chi¡, Ihen ¡assed Ihrough
and ca¡Iured on inIerdigiIaIed elecIrodes by DEF in Ihe desired chamber. (d) Valving is ellecIed by ¡res-
surizing Ihe Ihird layer and Ihus ¡ressing againsI Ihe second layer channel Io lorm a closed environmenI
lor s¡ore germinaIion Io Iake ¡lace. The ellecIiveness ol Ihe valves was demonsIraIed wiIh a soluIion ol
salranin dye. (e) Eluorescence microsco¡e image ol s¡ores ca¡Iured wiIhin Ihe chamber using DEF lorces.
20V ¡eak-Io-¡eak, 100 kHz. ToIal ela¡sed Iime is 1 minuIe. AcIivaIed s¡ores sIained wiIh Ihe EITC dye,
DiOC
o
(3). (Re¡rinIed wiIh ¡ermission lrom Mbc po b Dijq and kind ¡ermission lrom |o|.)
ered Ihe 10
º
s¡ores}mL concenIraIion as Ihe one IhaI can be salely deIecIed and used
Ihis value lor Ihe design ol Ihe microscale assay.
In microlluidic device ex¡erimenIs, s¡ores were delivered Io Ihe IargeI chamber (0.1
nL, 100 Ó 100 Ó 10 Ÿm) in a carrier sIream ol DI waIer. The s¡ores were ca¡Iured aI Ihe
edge ol Ihe embedded elecIrodes by dielecIro¡horeIic lorces induced aI 20V and 100
kHz. WiIh a llow raIe ol 0.2 ŸL}min (¡eak llow velociIy: 40 cm}min), º0' ol Ihe s¡ores
in Ihe carrier sIreamwere ca¡Iured by DEF |31|. Eigure º.3(e) shows Ihe ca¡Iure ol s¡ores
by DEF lorces, 20V ¡eak Io ¡eak, 100 kHz.
We lirsI germinaIed Ihe s¡ores wiIhouI acIuaIing Ihe builI-in valves. MeasuremenI
ol admiIIance sIarIed righI alIer Ihe germinanI soluIion had lully re¡laced Ihe DI waIer.
9.2 Lab-oh-a-Chip !or MohiIorihg Microbial MeIabolic AcIiviIy
2:6
x
y
x
y
x
y
(c)
Valve Closed Valve Opeh Valve Closed Valve Opeh
(d) (e)
IhleI !or germihahI
Valves
chahhels
OuIleI
IhleI !or spores
1ubihg !or
spore delivery
Spores
Valves
LlecIrodes
Spores
Flow
DirecIioh
Gjhvsf :/4 (conIinued)
The resulIs showed an increase in admiIIance u¡on s¡ore germinaIion |Eigure º.4(b)|. In
com¡arison Io Ihe conIrol ex¡erimenI wiIh germinanI only, Ihe sam¡les wiIh a¡¡roxi-
maIely 700 s¡ores and a¡¡roximaIely º00 s¡ores ¡resenIed signilicanI increases in
admiIIance, sIarIing 2 minuIes alIer Ihe ex¡erimenI began. There was no signilicanI
increase in admiIIance in Ihe conIrol ex¡erimenIs. ElucIuaIions in admiIIance occurred
because Ihe chamber was o¡en Io Ihe llow sIream wiIhouI Ihe acIuaIed valve. However,
when s¡ores sIarIed Io germinaIe, enough ions were released Io overcome Ihe baseline
llucIuaIion and showed a signilicanI increase in admiIIance. The deIecIion limiI wiIh
Ihis ex¡erimenI ¡roved Io be a lew hundred s¡ores in a 0.1 nL chamber.
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
2:7
Gjhvsf :/5 (a) Im¡edance curves ol Ihe s¡ore germinaIion in Ihe macroscale germinaIion ex¡erimenIs
wiIh sam¡les conIaining 10
º
, 10
8
, and 10
7
s¡ores}mL. The conIrol sam¡le conIains germinanI soluIion
only. (b) Re¡resenIaIive on-chi¡ ex¡erimenIal resulIs wiIh abouI 100, 700, and º00 s¡ores in a 0.1 nL
chamber (gra¡hs ol daIa wiIh 700 and º00 s¡ores were o¡en-valve ex¡erimenIs, while Ihe gra¡h wiIh 100
s¡ores was lrom a closed-valve ex¡erimenI). The resulIs lrom serially ¡erlormed conIrol ex¡erimenIs are
also shown. (Re¡rinIed wiIh ¡ermission lrom Mbc po b Dijq and kind ¡ermission lrom |o|.)
To overcome Ihe ¡roblem ol llucIuaIions in admiIIance, Ihe designed microlluidic
valves were acIuaIed Io isolaIe Ihe germinaIion chamber lromIhe resI ol Ihe sysIem. The
channels in Ihe Ihird layer ol Ihe microlluidic device (Ihe FDMS slab) were ¡ressurized
wiIh germinanI soluIion Io close Ihe valves in Ihe second layer |Ihe FDMS membrane,
see Eigure º.3(c, d)|. We used germinanI soluIion Io close Ihe valves in order Io avoid
admiIIance ¡erIurbaIion due Io ion ¡ermeaIion lrom Ihe valve channel inIo Ihe germi-
naIion chamber Ihrough Ihe FDMS layer. Two seIs ol sensing ¡robes were used Io mea-
sure Iwo idenIical chambers (0.1 nL) simulIaneously, wiIh one serving as a conIrol
chamber and Ihe oIher as Ihe ex¡erimenIal chamber. A com¡arison ol Ihe resulIs lrom
Ihese Iwo idenIical chambers deIermined IhaI Ihe chamber wiIh s¡ores and germinanI
had a signilicanI increase in admiIIance when germinaIion was Iaking ¡lace, whereas
Ihe conIrol chamber showed no signilicanI dillerence lrom Ihe conIrol ex¡erimenIs
wiIhouI germinanI. The sam¡le wiIh 100 s¡ores showed an immediaIe increase in
admiIIance and reached an admiIIance increase ol a¡¡roximaIely 10 nmho in 20 min-
uIes. II is clear IhaI only when s¡ore germinaIion was Iaking ¡lace did a signilicanI
change in Ihe measured signal occur. (There was a slighI admiIIance increase in Ihe con-
Irol chamber in Ihe germinaIion ex¡erimenI due Io Ihe inlluence ol ions ¡ermeaIing Ihe
FDMS lrom Ihe ex¡erimenIal chamber.) Based on a com¡arison ol Ihe admiIIance
changes wiIh 700 s¡ores in Ihe o¡en-valve ex¡erimenI and wiIh 100 s¡ores in Ihe
closed-valve ex¡erimenI, resulIs were very similar, indicaIing IhaI Ihe isolaIion valves
also enhanced Ihe sensiIiviIy ol Ihe admiIIance measuremenIs. This im¡lies IhaI a lower
deIecIion limiI could be achieved by including isolaIion valves in Ihe chi¡ design. The
deIecIion limiI was less Ihan 100 s¡ores in a 0.1 nL chamber (10
º
s¡ores}mL). TheoreIi-
cally, Ihis limiI could be reduced Io 10 s¡ores or even 1 s¡ore wiIh smaller-sized cham-
bers.
InIhis sIudy, we demonsIraIed a meIhod lor auIomaIic and ra¡id elecIrical deIecIion
ol germinaIion ol viable s¡ores wiIhin a microlluidic biochi¡. The microlluidic device
includes s¡ecial design leaIures IhaI laciliIaIe s¡ore ca¡Iure and isolaIion as well as elec-
Irodes lor s¡ore concenIraIion and im¡edance measuremenIs. The limiI ol deIecIion
was shown Io be a lew hundred s¡ores in a 0.1 nL chamber wiIhouI use ol Ihe isolaIion
valves. The deIecIion limiI was reduced Io lewer Ihan 100 s¡ores in a 0.1 nL chamber
when Ihe chamber was isolaIed by closing Ihe isolaIion valves. The deIecIion limiI can
be lurIher lowered by using a smaller ca¡Iure-measuremenI chamber. The deIecIion
Iime is as shorI as 2 hours lrom heaI acIivaIion ol a sus¡ecIed organism, which makes
Ihe im¡edance-based deIecIion meIhod a ¡romising candidaIe lor an on-siIe environ-
menIal diagnosIic ¡laIlorm.
:/4 Mbc.po.b.Dijq gps Jnqfebodf Efufdujpo pg Dfmm
Dpodfousbujpo Cbtfe po Jpo Sfmfbtf gspn Dfmmt
:/4/2 Njdspdijqt gps jnqfebodf efufdujpo pg DE5, U mznqipdzuft
AlIhough mulIi¡le miniaIurized ¡laIlorms exisI lor cell counIing in sus¡ension, such as
llow cyIomeIry and CoulIer counIers |32÷3S|, meIhods Io enumeraIe aIIached cells
wiIhin microlluidic devices are limiIed. O¡Iical-microsco¡y-based cell deIecIion,
alIhough sIraighIlorward, remains de¡endenI on a sIable lighI ¡aIh and lensing, lilIer-
ing, and locusing mechanisms IhaI add cosI and com¡lexiIy Io deIecIion. In addiIion,
9.3 Lab-oh-a-Chip !or Impedahce DeIecIioh o! Cell CohcehIraIioh 8ased oh Ioh Release !rom Cells
2:8
o¡Iical deIecIion Iends Io be lowIhrough¡uI due Io Ihe small deIecIion area available aI
a single Iime. A valuable com¡lemenI Io o¡Iical microsco¡y is surlace im¡edance sens-
ing Io enumeraIe cells aIIached on a subsIraIe |3o÷38|. However, in Ihe im¡ed-
ance-sensing meIhod, a near uniIy ol cell coverage on Ihe elecIrode surlace is required
Io generaIe a deIecIable signal.
To address Ihe need lor sensiIive deIecIion ol a small number ol cells aIIached on a
relaIively large surlace area or in a large volume, we inIroduce in Ihis cha¡Ier an
a¡¡roach called "cell lysaIe im¡edance s¡ecIrosco¡y." In Ihis a¡¡roach, surlace-bound
cells are lysed in a microlluidic channel, and Ihe bulk conducIance changes are mea-
sured Ihrough surlace-¡aIIerned elecIrodes and im¡edance s¡ecIrosco¡y. As Ihe
inIracellular ion conIenI is relaIively consIanI in each cell Iy¡e, Ihe number ol released
ions measured elecIrically is indicaIive ol Ihe cell number. Using immunoalliniIy-iso-
laIed CD4+ T cells as an exam¡le, we demonsIraIe here IhaI bulk soluIion conducIance
increases ¡ro¡orIionally Io Ihe number ol cells in Ihe microdevice. In addiIion, Ihis
meIhod has a deIecIion Ihreshold ol 20 cells}ŸL, which is sullicienIly uselul lor many
clinical and research a¡¡licaIions IhaI require cell counIing.
9.3.1.1 MeIhods ahd devices
Microlluidic devices were labricaIed by bonding Iwo ¡ieces ol glass slide wiIh a FDMS
gaskeI IhaI is S0 Ÿm Ihick and conIains an o¡ening window ol S cm Ó 4 mm. The FDMS
gaskeIs were ¡re¡ared by curing s¡in-coaIed FDMS on a Irans¡arency slide, lollowed
wiIh hand-cuIIing windows ol desired sizes. InIerdigiIaIed (IDT) gold elecIrodes were
¡aIIerned on Ihe boIIom slide by sIandard ¡hoIoliIhogra¡hy |Eigure º.S(a)|, lacing Io
Ihe microlluidic channel side during assembly. Two holes were drilled on Ihe cover
slide and bonded wiIh FDMS ¡orIs Io lorm lluid inleIs and ouIleIs. Assembled devices
were Ihen luncIionalized wiIh a monoclonal CD4 anIibody and ¡rimed wiIh FBS con-
Iaining 1' BSA and 1 mM EDTA |7|. CD4+ T lym¡hocyIes lrom healIhy donors were
ca¡Iured in Ihe microlluidic chi¡ by llowing culIured ¡eri¡heral blood mononuclear
cells (FBMC) inIo Ihe device aI S ŸL}min. This llow raIe is o¡Iimal lor ellicienI and s¡e-
cilic ca¡Iure ol CD4+ T lym¡hocyIes |7|, and Ihe duraIion ol sam¡le injecIion deIer-
mines Ihe IoIal number ol ca¡Iured cells.
To minimize background conducIance, we use Ihe lollowing o¡eraIional sequence
Io lyse Ihe ca¡Iured cells: EirsI, exIracellular ions ¡resenI in Ihe microchannel were
washed ouI using a low-conducIive washing soluIion conIaining 8.S'sucrose and 0.3'
dexIrose aI a llow raIe ol 20 ŸL}min unIil im¡edance signals were sIable. This ion-lree
soluIion has been lound Io mainIain viabiliIy ol mammalian cells. NexI, a low-conduc-
Iive soluIion conIaining 2' sucrose and 0.07' dexIrose was llowed in aI a llow raIe ol
10 ŸL}min lor 1 minuIe lor conIrolled cell lysis. The lysing soluIion was lormulaIed such
IhaI cell lysis occurred alIer a com¡leIe re¡lacemenI ol Ihe washing soluIion by Ihe
lysing soluIion. Cells were Ihen ke¡I in Ihe lysis soluIion in a sIaIic sIaIe lor anoIher 10
minuIes Io allow cell lysis Io reach a sIeady sIaIe.
Im¡edance measuremenIs were Iaken using an AgilenI 4284 LCR meIer (AgilenI
Technologies). The microelecIrode devices were connecIed Io Ihe LCR meIer Ihrough
¡laIinum ¡robes. The im¡edance-measuremenI ¡rocess was auIomaIed by cusIom
LabVIEW (NaIional InsIrumenIs Cor¡.) virIual insIrumenIs and GFI B inIerlace. Im¡ed-
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
2:9
ance s¡ecIra were measured in Ihe lrequency range ol 100 Hz Io 1 MHz wiIh a lrequency
increase lacIor ol 1.S and am¡liIude ol 2S0 mV.
9.3.1.2 ResulIs ahd discussioh
To IesI Ihe deIecIion sensiIiviIy ol ions released lrom ¡rimary cells using im¡edance
s¡ecIrosco¡y, we lirsI lysed FBMCs ol known concenIraIions in E¡¡endorl Iubes wiIh
DI waIer and measured Ihe im¡edance ol Ihe lysaIe using Ihe microlluidic device wiIh
co¡lanar inIerdigiIaIed microelecIrodes (IMEs) as shown in Eigure º.S(a). Eigure º.S(b,
c) shows Ihe s¡ecIra ol im¡edance magniIude |Eigure º.S(b)| and ¡hase |Eigure º.S(c)| as
a luncIion ol lrequency lor cell concenIraIions ranging lrom 0 Io 3,000 cells}ŸL mea-
9.3 Lab-oh-a-Chip !or Impedahce DeIecIioh o! Cell CohcehIraIioh 8ased oh Ioh Release !rom Cells
2::
(a)
Frequehcy (Hz)
1e+2 1e+3 1e+4 1e+5 1e+6
1e+3
1e+4
1e+5
1e+6
DI-waIer
0 cell/ŸL
20 cells/ŸL
50 cells/ŸL
100 cells/ŸL
200 cells/ŸL
350 cells/ŸL
500 cells/ŸL
1000 cells/ŸL
3000 cells/ŸL
Frequehcy (Hz)
1e+2 1e+3 1e+4 1e+5 1e+6
›80
›60
›40
›20
0
DI-waIer
0 cell/ŸL
20 cells/ŸL
50 cells/ŸL
100 cells/ŸL
200 cells/ŸL
350 cells/ŸL
500 cells/ŸL
1000 cells/ŸL
3000 cells/ŸL
(b) (c)
(d)
R
ser
Z
dl
R
sol
Z
dl
C
di
0 5e+2 1e+3 2e+3 2e+3 3e+3 3e+3
2e-5
4e-5
6e-5
8e-5
C = 1.90x10 C + 1.17x10
-8 -5
(e)
Cell CohcehIraIioh (cells/ L) Ÿ
61 n Ÿ
46 n Ÿ
26 n Ÿ
0
Gjhvsf :/6 Microlluidic devices used in Ihis sIudy and im¡edance s¡ecIrosco¡y measuremenI using oll-chi¡
cell lysaIe. (a) The devices are com¡osed ol Iwo glass slides wiIh micro¡aIIerned IDT elecIrodes and a FDMS gas-
keI. (b) Im¡edance magniIude and (c) ¡hase s¡ecIra ol DI waIer and cell lysaIe wiIh dillerenI sIarIing cell con-
cenIraIions measured on Ihe IDT device. Three Io live scans were ¡erlormed aI each cell concenIraIion in Ihe
lrequency range beIween 100 and 10
o
Hz. (d) An equivalenI circuiI used in our sIudy Io model Ihe elecIrode}elec-
IrolyIe sysIem lor exIracIing bulk soluIion conducIance, 1}S
tpm
, which direcIly correlaIes wiIh cell ion release |1|.
(e) A linear relaIionshi¡ beIween measured bulk soluIion conducIance (solid doIs) and cell concenIraIion is
observed, and Ihe besI liIs are shown as solid lines. Error bars indicaIe Ihe sIandard deviaIion lrom Ihree Io live
conIinuous measuremenIs wiIhin a single device. (Re¡rinIed wiIh ¡ermission lrom Mbc po b Dijq and kind ¡er-
mission lrom |7|.)
sured using Ihe IMEs. We observed IhaI each s¡ecIrum has Iwo regions, a con-
sIanI-im¡edance region in Ihe lrequency range lrom 100 Hz Io 10 kHz and an
im¡edance-decreasing region when lrequency is greaIer Ihan 100 kHz. WiIh increasing
cell concenIraIions, Ihere is a consisIenI decrease in im¡edance magniIude in Ihe
low-lrequency range and a shilI ol ¡hase ¡eak Io higher lrequency. This suggesIs
sIrongly IhaI semiquanIiIaIive measuremenI ol cell number can be achieved Ihrough
ion release.
To undersIand soluIion conducIance as a luncIion ol cell number, we carried ouI
modeling sIudies Io exIracI bulk conducIance lromIhe im¡edance s¡ecIra. ElecIrodes in
an elecIrolyIe soluIion can be modeled using an equivalenI circuiI as shown in Eigure
º.S(d) |S, 7|, where D
ej
is Ihe dielecIric ca¡aciIance (iI conIains dielecIric conIribuIions
lromall Ihe maIerials surrounding Ihe elecIrodes, including Ihe soluIion), S
tpm
is Ihe bulk
soluIion resisIance (charge Irans¡orI across Ihe bulk soluIion), [
em
is Ihe inIerlacial
im¡edance (Ihe so-called Warburg im¡edance IhaI accounIs lor Ihe change in Ihe ionic
gradienI aI Ihe inIerlace), and S
tfs
is Ihe resisIance ol Ihe on-chi¡ wiring. The inIerlacial
im¡edance can be ex¡ressed as
D E
w y
[ k o C
em
Y 1 ) ):/2*
where k Y D E ›M , and o and C are ¡arameIers de¡endenI on Ihe ¡ro¡erIies ol Ihe elecIro-
lyIes and Ihe elecIrodes. This is Ihe sim¡lesI model IhaI would ¡ro¡erly liI Ihe measured
daIa over Ihe whole lrequency range aI all Iimes. SoluIion bulk conducIance H
tpm
is sim-
¡ly Ihe reci¡rocal ol S
tpm
/
By a¡¡lying Ihe circuiI model Io Ihe grou¡ ol curves in Eigure º.S(b, c), bulk conduc-
Iion was exIracIed and ¡loIIed as a luncIion ol cell concenIraIion |solid doI in Eigure
º.S(e)|. II is observed IhaI soluIion conducIance increases linearly wiIh Ihe number ol
cells, conlirming our hy¡oIhesis IhaI ion release and soluIion conducIance change are
indicaIive ol cell number. Moreover, using ion release Io deIecI cells a¡¡ears Io be
exIremely sensiIive and can deIecI as lewas 20 cells}µL in an ion-lree soluIion. The slo¡e
ol Ihe conducIance curve re¡resenIed measuremenI sensiIiviIy ol Ihe microchi¡, and
Ihe sensiIiviIy ol Ihe IMEs was abouI 1.º0 Ó 10
÷8
}cell ŸL.
AlIer im¡edance measuremenI using oll-chi¡ lysaIe, we Ihen sIudied how im¡ed-
ance changes in Ihe ¡rocess ol cell ca¡Iure and on-chi¡ cell lysis. CD4+ T cells were ca¡-
Iured lrom culIured ¡eri¡heral blood mononuclear cells, lollowed wiIh washing wiIh
FBS buller and Ihen Ihe low-conducIive washing soluIion. AlIerwards, Ihe low-conduc-
Iive cell-lysing soluIion was inIroduced inIo Ihe microlluidic device, and cells were
allowed Io lyse lor 10 minuIes. AI Ihe end, Ihe relerence s¡ecIra were obIained wiIh DI
waIer. In Ihe enIire ¡rocess, im¡edance s¡ecIra were acquired conIinuously and
rellecIed Ihe bulk soluIion resisIance. Take Ihe im¡edance magniIude aI 7o0 kHz, lor
exam¡le |Eigure º.o(a)|, where maximum se¡araIion occurs beIween Ihe im¡edance
magniIude curves in Eigure º.S(b), iI remains in Ihe lowkilo-ohmrange whencells are in
FBS due Io Ihe high ionic concenIraIion ol biological bullers. II increases dramaIically Io
above 10 k: u¡on inIroducIion ol Ihe low-conducIive washing soluIion. When cells are
ke¡I in Ihe low-conducIive washing soluIion in a sIaIic sIaIe, im¡edance magniIude
decreases slighIly, likely due Io cell ion release in a low-conducIive environmenI. AlIer
injecIion ol Ihe ion-lree lysing soluIion, an iniIial im¡edance jum¡ is noIiced because
Ihe lysing soluIion has a lower conducIiviIy Ihan Ihe washing soluIion. This is lollowed
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
311
9.3 Lab-oh-a-Chip !or Impedahce DeIecIioh o! Cell CohcehIraIioh 8ased oh Ioh Release !rom Cells
312
CapIured Cells oh Chip
0 2000
0 20
0.0
1ime (mih)
P8S
bu!!er
Washihg
soluIioh
Lysihg
soluIioh
DI-WaIer
2.0e+3
4.0e+3
6.0e+3
8.0e+3
1.0e+4
1.2e+4
1.4e+4
1.6e+4
40 60
4000 6000 8000 10000 12000
›0.0015
›0.0010
›0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
(a)
(b)
Jnqfebodf espq
evf up jpo sfmfbtf
gspn dfmm mztjt;
Bnqmjuvef dfmm
ovncfs
7
Gjhvsf :/7 (a) Im¡edance measuremenI aI 7o0 Hz in Ihe ¡rocess ol cell ca¡Iure and on-chi¡ lysis. The
res¡ecIive incubaIion sIe¡s are labeled on Io¡ ol Ihe gra¡h, and Ihe shaded areas beIween Ihese labeled
sIe¡s are IransienI sIaIes during soluIion exchanges. The im¡edance dro¡ belore and 10 minuIes alIer
injecIing Ihe lysing soluIion is associaIed wiIh cell lysis and used as a cell-numbers indicaIor. (b) Conduc-
Iance change in Ihe ¡rocess ol on-chi¡ cell lysis versus Ihe number ol cells ca¡Iured wiIhin microlluidic
devices. Bulk soluIion conducIance was exIracIed lrom Ihe im¡edance s¡ecIra, and conducIance dro¡
belore and 10 minuIes alIer llowing in Ihe lysing soluIion was Iaken as Ihe indicaIor Io counI cell. This
conducIance change increases conIinuously wiIh Ihe number ol cells ca¡Iured wiIhin Ihe microlluidic
chi¡, suggesIing immobilized cells can be counIed by elecIrical measuremenI ol Iheir ion release.
NonlineariIy ol Ihe relaIionshi¡ may arise lrom incom¡leIe dillusion ol ions wiIhin Ihe measuremenI
Iime. Each daIa ¡oinI in Ihe ¡loI re¡resenIs measuremenI lrom one device. (Re¡rinIed wiIh ¡ermission
lrom Mbc po b Dijq and kind ¡ermission lrom |7|.)
wiIh an abru¡I dro¡ ol im¡edance and a subsequenI slower im¡edance decrease. This
Iwo-sIage im¡edance dro¡ during cell incubaIion in Ihe lysing soluIion maIches o¡Iical
observaIion ol lysed cell numbers in Ihe same soluIion (daIa noI shown), suggesIing IhaI
Ihe decrease in im¡edance magniIude arises lrom lysis ol Ihe ca¡Iured cells.
Eollowing Ihe same daIa-liIIing ¡rocedure as described above, bulk conducIance was
exIracIed lrom Ihe im¡edance s¡ecIra and Ihe conducIance change belore and 10 min-
uIes alIer llowing in Ihe lysing soluIion was Iaken as a resulI ol com¡leIe ion release lrom
ca¡Iured cells. When we com¡are Ihis conducIance change Io manual cell counIs wiIhin
Ihe microlluidic devices |Eigure º.o(b)|, iI is evidenI IhaI Ihe bulk conducIance change is
¡ro¡orIional Io Ihe number ol ca¡Iured cells. The resulIs successlully demonsIraIe IhaI
cells can be deIecIed and counIed wiIhin a microlluidic device Ihrough Ihe im¡ed-
ance}conducIance measuremenI ol cell lysaIe.
In conclusion, im¡edance s¡ecIrosco¡y can be used Io deIecI mammalian cells
immobilized in a microlluidic device Ihrough Iheir ion release. The microdevice hel¡s Io
conline Ihe ions in a small volume lor sensiIive measuremenI. NoI only is Ihe a¡¡roach
uselul lor Ierminal cell counIing, buI iI also holds Ihe ¡romise Io sIudy live-cell acIiviIies
Ihrough Iheir ion exchange wiIh Ihe environmenI.
:/4/3 Joufsejhjubufe njdspfmfduspef dijq gps jnqfebodf efufdujpo pg
cbdufsjbm dfmmt
The conducIiviIy ol bacIerial sus¡ensions has been used Io sIudy Ihe elecIrical ¡ro¡er-
Iies ol bacIerial cell-surlace and relaIed cell-surlace inIerlacial ¡hysiology |3º, 40|. II can
also be used Io quanIily Ihe concenIraIion ol bacIerial cells in sus¡ensions. We ¡resenI
here a sim¡le and ra¡id im¡edance meIhod Io deIecI bacIerial cells in sus¡ensions using
inIerdigiIaIed microelecIrodes.
When bacIerial cells are sus¡ended in DI waIer, Ihere are Iwo ¡ossible ways lor Ihe
bacIerial cells Io alIer Ihe im¡edance ol DI waIer. One is via Ihe charged naIure ol bacIe-
rial cell surlaces. The bacIerial cell walls conIain various acidic grou¡s such as carboxyl,
¡hos¡haIe, and amino grou¡s |41|. Generally, Ihere is a higher concenIraIion ol anionic
grou¡s Ihan ol caIionic grou¡s, which resulIs in a negaIive cell-wall charge aI neuIral
¡H. This charge is com¡ensaIed lor by counIerions IhaI ¡eneIraIe inIo Ihe ¡orous cell
wall and, Io a minor exIenI, by coions IhaI are ex¡elled lrom iI, Ihereby conlerring elec-
IrosIaIic charge Io Ihe cell ¡eri¡hery |3º, 40, 42|. The charge densiIy ol Ihe bacIerial cell
wall can be as high as 0.S Io 1.0 C}m
2
|3º|. The conducIiviIy ol Ihe DI waIer is in a range
lromas lowas abouI 1 Io 2 ŸS}cmIo u¡ Io abouI 10 Io 1S ŸS}cm. When bacIerial cells are
sus¡ended in low-conducIive DI waIer and reach a sullicienI concenIraIion, Ihey can
alIer Ihe conducIiviIy ol Ihe sus¡ension because ol Iheir cell-wall charges. The oIher way
lor bacIerial cells Io alIer Ihe conducIiviIy ol DI waIer is via Ihe ion release lrombacIerial
cells. When bacIerial cells are sus¡ended in a soluIion, such ¡henomena may occur as
leakage ol ions Ihrough Ihe cyIo¡lasmic membrane and negaIive adsor¡Iion ol elecIro-
lyIe or ion u¡Iake inIo Ihe cyIo¡lasm and s¡ecilic adsor¡Iion ol ions. These ¡rocesses
can inlluence Ihe conducIiviIy ol Ihe bulk soluIion |3º|. When bacIerial cells are sus-
¡ended in DI waIer, Ihey ex¡erience an osmoIic shock. In res¡onse Io Ihe llucIuaIions in
environmenIal osmolariIy, cells adjusI Iheir inIracellular soluIe concenIraIions in order
Io mainIain a consIanI Iurgor ¡ressure and ensure conIinuaIion ol cellular acIiviIy.
OIher ¡ro¡erIies ol cells, such as cell size and buoyanI densiIy, can also be alIered in
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
313
res¡onse Io Ihe osmoIic shock |43|. The charges on Ihe cell wall, Ihe release ol ions, and
oIher res¡onses Io Ihe osmoIic shock in combinaIion accounI lor Ihe im¡edance change
in DI waIer wiIh sus¡ended bacIeria.
Here, we used Tbmnpofmmb uzqijnvsjvn, a Gram-negaIive, lood-borne, bacIerial ¡aIho-
gen, as an exam¡le Io demonsIraIe im¡edance deIecIion ol bacIerial cells in sus¡en-
sions. Tbmnpofmmb cell sus¡ensions in DI waIer and ¡hos¡haIe bullered saline (FBS)
soluIion were sIudied over a wide range ol lrequencies. BacIerial cells sus¡ended in DI
waIer wiIh dillerenI cell concenIraIions were shown Io have dillerenI elecIrical im¡ed-
ance s¡ecIral res¡onses. In a cerIainlrequency range, im¡edance ol Ihe cell sus¡ension is
direcIly ¡ro¡orIional Io Ihe cell concenIraIion, which can be used Io quanIily bacIerial
cells in a label-lree, inex¡ensive, and sim¡le lashion.
9.3.2.1 MeIhods ahd device
9.J.2.1.1 Device
The device lor elecIrical im¡edance measuremenIs consisIs ol a silica or glass chi¡ ¡aI-
Ierned wiIh an array ol inIerdigiIaIed microelecIrodes (IMEs) and a microchamber (•2S
ŸL ca¡aciIy) righI above Ihe elecIrode area lormed by silicone rubber, as shown in Eig-
ure º.7(a). The inIerdigiIaIed microelecIrodes were labricaIed on a llaI silica or glass sub-
9.3 Lab-oh-a-Chip !or Impedahce DeIecIioh o! Cell CohcehIraIioh 8ased oh Ioh Release !rom Cells
314
IhIerdigiIaIed
microelecIrodes
Silicohe rubber
CohIacI pads
Silicoh
subsIraIe
Cells ih suspehsioh
LlecIrode cohIacI pads
Chamber (50 l) Ÿ
Class cover
IhIerdigiIaIed microelecIrodes
!or impedahce measuremehIs
Class subsIraIe
Silicohe
rubber
Silicohe
rubber
Jnqfebodf
bobmz{fs
(a)
(b)
Gjhvsf :/8 (a) The device lor im¡edance deIecIion ol bacIerial cells based on ion release lrom cells inIo
bullers. II consisIs ol a chamber lormed by silicone rubber and a seI ol inIerdigiIaIed microelecIrodes aI
Ihe lloor ol Ihe chamber. The chamber ca¡aciIy is 2S ŸL. The gold inIerdigiIaIed microelecIrode consisIs
ol S0 ¡airs ol linger elecIrodes wiIh 1S Ÿm ol digiI elecIrode widIh and 1S Ÿm inIerdigiI s¡ace. (b) Sche-
maIic seIu¡ lor elecIrical im¡edance s¡ecIrosco¡ic measuremenIs ol im¡edance ol bacIerial cell sus¡en-
sions in DI waIer or FBS. (Re¡rinIed wiIh ¡ermission lrom Ubmboub and kind ¡ermission lrom |1|.)
sIraIe using sIandard liIhogra¡hic microlabricaIion Iechnology. The IME consisIs ol a
¡air ol microband arrays ol digiI elecIrodes IhaI mesh wiIh each oIher. The widIh ol
digiI elecIrodes and Ihe inIerdigiI s¡ace can be in Ihe range ol microns Io nanomeIers,
wiIh a IoIal ol Iens Io hundreds ol ¡airs ol linger elecIrodes. The Iwo seIs ol
microelecIrodes are used as Ihe Iwo ¡oles in a bi¡olar im¡edance-measuremenI seIu¡.
The IMEs used in Ihis ex¡erimenI have a IoIal ol S0 ¡airs ol linger elecIrodes wiIh each
having a widIh ol 1S Ÿm and a s¡ace ol 1S Ÿm. IMEs are also commercially available.
The chamber was made by ¡unching a hole in a ¡iece ol silicon rubber using a sIan-
dard ¡uncher ol a desired size. The silicon rubber was Ihen glued Io Ihe chi¡ using e¡oxy
wiIh Ihe chamber a¡¡ro¡riaIely aligned wiIh Ihe elecIrode area.
9.J.2.1.2 Preparation of bacteria cells
SIock culIure ol Tbmnpofmmb uzqijnvsjvn was ¡urchased lrom Carolina Biological Su¡¡ly
Com¡any (BurlingIon, NorIh Carolina). The culIure was grown in brain-hearI-inlusion
(BHI) broIh (TEKnova, HollisIer, Calilornia) aI 37ºC lor 1o Io 18 hours. The cells were
cenIriluged (E¡¡endorl, WesIbury, New York) aI o,000 Ó g lor 2 minuIes. AlIer removal
ol Ihe su¡ernaIanI, Ihe cell ¡elleI was resus¡ended in sIerilized DI waIer or FBS. The
cells were washed Ihree Iimes wiIh DI waIer or FBS in order Io geI rid ol residues lrom
Ihe growIh medium. Then, Ihey were serially (1:10) diluIed wiIh DI waIer or FBS Io
desirable concenIraIions lor lurIher ex¡erimenIs.
The IradiIional ¡laIing meIhod was used Io deIermine Ihe viable cell number in Ihe
sIock cell sus¡ension ¡re¡ared in Ihe above sIe¡. The cells sus¡ension was serially (1:10)
diluIed wiIh DI waIer. Then, 100 ŸL ol a¡¡ro¡riaIe diluIions were ¡laIed onIo XLT4 agar
¡laIes (Dilco, S¡arks, Maryland). Colonies were counIed alIer incubaIion ol Ihe ¡laIes aI
37¬C lor 24 hours. Generally, Ihe cell numbers in Ihe sIock cell sus¡ension averaged
abouI 10
º
clu}mL.
9.J.2.1.J Llectrical impedance spectroscopy (LlS)
Im¡edance measuremenIs were ¡erlormed using an IM-o im¡edance analyzer
(Zahner-ElekIrik Gmbh & CoKG, Kronach, Germany) wiIh Ihe IM-o}THALES solIware.
Eigure º.7(b) shows Ihe schemaIic im¡edance-measuremenI seIu¡. Eor im¡edance mea-
suremenIs, 20 ŸL ol each sam¡le was ¡laced inIo Ihe microchamber and covered wiIh a
glass cover. One ol Ihe Iwo microband array elecIrodes was connecIed Io Ihe IesI and
sensing ¡robes, and Ihe oIher was connecIed Io Ihe relerence and counIer elecIrodes on
Ihe IM-o im¡edance analyzer. EIS measuremenIs were carried ouI in a lrequency range
lrom 1 Hz Io 100 kHz. Bode (im¡edance and ¡hase versus lrequency) diagrams were
recorded. Im¡edance aI a lixed lrequency was measured using Ihe ca¡aciIance-¡oIen-
Iial (C}E) ¡rogram aI 1 kHz wiIh an am¡liIude ol -S0 mV. Im¡edance daIa were
recorded aI every minuIe. All IesIs were ¡erlormed aI room Iem¡eraIure.
SimulaIion was ¡erlormed using Ihe SIM ¡rogram. Erom each measured s¡ecIrum,
S0 daIa ¡oinIs were auIomaIically selecIed by Ihe solIware as Ihe in¡uI, and Ihe liIIing
curves were generaIed using an equivalenI circuiI model.
9.3.2.2 ResulIs ahd discussioh
9.J.2.2.1 lmpedance spectra of bacterial cell suspensions in Dl water and P8S
Eigure º.8 ¡resenIs Ihe Bode im¡edance s¡ecIra ol Tbmnpofmmb uzqijnvsjvn cell sus¡en-
sions in (a) DI waIer and (b) FBS, along wiIh Iheir equivalenI circuiIs and besI-liIIing
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
315
9.3 Lab-oh-a-Chip !or Impedahce DeIecIioh o! Cell CohcehIraIioh 8ased oh Ioh Release !rom Cells
316
FiIIihg daIa
waIer
(c)
waIer
(c)
(!)
1.0L+0 1.0L+1 1.0L+2 1.0L+3 1.0L+4 1.0L+5
Frequehcy (Hz)
1.0L+2
1.0L+3
1.0L+4
1.0L+5
1.0L+6
Ih DI waIer
C
dl
R
s
C
dl
w w
R
eI
R
eI
Measured daIa
1.0L+0 1.0L+1 1.0L+2 1.0L+3 1.0L+4 1.0L+5
Frequehcy (Hz)
1.0L+2
1.0L+3
1.0L+4
1.0L+5
1.0L+6
Ih P8S Measured daIa
FiIIihg daIa
C R C
dl s dl
1.0L+3
1.0L+4
1.0L+5
1.0L+0 1.0L+1 1.0L+2 1.0L+3 1.0L+4 1.0L+5
Frequehcy (Hz)
10
4
cells/ml
10
5
cells/ml
10
6
cells/ml
10
7
cells/ml
10
8
cells/ml
1 kHz
1.0L+3
1.0L+4
1.0L+5
1.0L+0 1.0L+1 1.0L+2 1.0L+3 1.0L+4 1.0L+5
Frequehcy (Hz)
P8S
10
4
cells/ml
10
5
cells/ml
10
6
cells/ml
10
7
cells/ml
10
8
cells/ml
10
9
cells/ml
(d)
1.0L+3
1.0L+4
1.0L+5
0 20 40 60 80 100
1ime (mih)
10
9
10
3
10
6
10
7 10
5
10 10
2
10
8
WaIer
(e)
1.0L+0 1.0L+2 1.0L+4 1.0L+6 1.0L+8 1.0L+1
8acIerial CohcehIraIioh (cells/20 l) Ÿ
0
10
20
30
40
y = -2.06 Log (x) + 5.23
2
= 0.98
WaIer
(a) (b)
R
Gjhvsf :/9 (a, b) Im¡edance s¡ecIra ol Tbmnpofmmb cell sus¡ensions in (a) DI waIer and (b) FBS, IogeIher
wiIh Iheir liIIing curves and Ihe equivalenI circuiIs. Tbmnpofmmb concenIraIion: 1.º3 Ó 10
o
clu}mL. (c, d)
Im¡edance s¡ecIra ol Tbmnpofmmb sus¡ensions in (c) DI waIer and (d) FBS, wiIh Ihe cell concenIraIions in
Ihe range ol 10
4
Io 10
º
clu}mL, along wiIh sam¡les ol waIer and FBS as conIrols. (e) Ty¡ical im¡edance
res¡onses Io Ihe sam¡les wiIh dillerenI concenIraIions ol cells when Ihey were measured aI a lixed lre-
quency ol 1 kHz. (l) The linear relaIionshi¡ beIween Ihe logariIhmic value ol Ihe concenIraIion ol Tbmnp.
ofmmb cells and Ihe im¡edance measured aI 1 kHz. Error bars are sIandard deviaIions ol Ihree Io live
measuremenIs. Im¡edance s¡ecIra were measured in Ihe lrequency range ol 1 Hz Io 100 kHz wiIh an
am¡liIude ol -S0 mV. (Re¡rinIed wiIh ¡ermission lrom Ubmboub and kind ¡ermission lrom |1|.)
s¡ecIra. Eor Tbmnpofmmb cell sus¡ension in DI waIer, Ihe measured s¡ecIrum |Eigure
º.8(a), blank doIs| is a Iy¡ical Bode ¡loI lor a sysIemin which Ihe ¡olarizaIion is due Io a
combinaIion ol kineIic and dillusion ¡rocesses. Based on Ihe general elecIrical-equiva-
lenI model ol an elecIrochemical cell |44| and Ihe behavior ol Ihe IME microelecIrode
|4S|, Ihe measured s¡ecIrum can be modeled by an equivalenI circuiI IhaI consisIs ol
Ihe ohmic resisIance (S
t
) ol Ihe soluIion beIween Iwo elecIrodes, double-layer ca¡aci-
Iance (D
em
), elecIron-Iransler resisIance (S
fu
), and Warburg im¡edance ([
x
) around each
elecIrode. The agreemenI beIween Ihe measured daIa and Ihe liIIing s¡ecIra (solid line)
indicaIed IhaI Ihe equivalenI circuiIs ¡rovided a leasible, il noI unique, model Io
describe Ihe im¡edance characIerisIics ol Tbmnpofmmb sus¡ensions in DI waIer. Using Ihis
circuiI model, Ihe simulaIed values ol D
em
, [
x
, S
fu
, and S
t
were 884.º ¡E, 147.2 k:}s
0.S
,
13.S4 k:, and 4º1.2:, res¡ecIively, wiIh Ihe mean error ol modulus im¡edance ol
0.o'. The s¡ecIrum and Ihe circuiI model suggesI IhaI elecIrochemical reacIions occur
on Ihe IME elecIrodes and IhaI Ihe cells may have released some elecIrochemical acIive
com¡osiIes Io Ihe DI waIer.
Eor Tbmnpofmmb sus¡ensions in FBS, Ihe im¡edance s¡ecIrum |Eigure º.8(b), blank
doIs| shows Iwo domains: a double-layer region in Ihe low-lrequency range lrom1 Hz Io
a¡¡roximaIely S00 Hz, and a resisIive region in Ihe lrequency range lroma¡¡roximaIely
S00 Hz Io 100 kHz. The elecIrical im¡edance behavior ol Ihe cell sus¡ension in FBS can
be re¡resenIed by Ihe equivalenI circuiI ol Ihe IME sysIemin aqueous soluIions re¡orIed
¡reviously |4S÷47|. In Ihis circuiI model, Iwo idenIical double-layer ca¡aciIances (D
em
) ol
each seI ol Ihe IME are connecIed Io Ihe medium resisIance (S
t
) in series. D
em
dominaIes
Ihe im¡edance in Ihe low-lrequency range (double-layer region), whereas S
t
dominaIes
Ihe im¡edance in Ihe high-lrequency range (resisIive region). By simulaIion, Ihe values
ol D
em
and S
t
were 8º2.8 nE and 1.o2 k:, res¡ecIively, wiIh Ihe mean error ol modulus
im¡edance ol 3.0'. In Ihe cell sus¡ension in FBS, Ihe im¡edance s¡ecIrum does noI
show any characIerisIics relaIed Io elecIrochemical acIive ¡arameIers, which im¡lies
cells may noI release acIive elecIrochemical s¡ecies inIo FBS.
Eigure º.8(c, d) shows Ihe Bode im¡edance s¡ecIra ol Tbmnpofmmb sus¡ensions in(c) DI
waIer and (d) FBS soluIion wiIh dillerenI cell concenIraIions lrom10
4
Io 10
º
clu}mL. II is
observed IhaI Ihe sus¡ensions wiIh dillerenI cell concenIraIions in DI waIer each have a
disIincI im¡edance res¡onse in Ihe lrequency range lrom 100 Hz Io 10 kHz, whereas
im¡edance s¡ecIra ol Tbmnpofmmb sus¡ensions in FBS were idenIical in Ihe lull lrequency
range. The resulIs verilied IhaI when bacIerial cells were sus¡ended in low-conducIive
DI waIer, Ihey could alIer Ihe conducIiviIy ol Ihe sus¡ension.
9.J.2.2.2 Quantifying bacterial concentration in Dl water by impedance
As Ihe soluIion im¡edance decreases wiIh Ihe increasing cell concenIraIion in DI waIer
wiIhin a cerIain lrequency range, we can esIimaIe Ihe cell concenIraIion in DI waIer
using Ihe im¡edance value aI a lixed lrequency. As Ihe besI re¡resenIaIive lrequency, 1
kHz was used Io invesIigaIe Ihe relaIionshi¡ beIween im¡edance values and cell con-
cenIraIions in DI waIer sus¡ensions. Eigure º.8(e) shows Iy¡ical im¡edance res¡onses aI
1 kHz Io sam¡les ol dillerenI bacIerial concenIraIions. When Ihe bacIerial concenIra-
Iion decreased lrom 10
º
clu}mL Io 10
8
, 10
7
, and 10
o
clu}mL, im¡edance ol Ihe sus¡en-
sions signilicanIly increased lrom 3.13 - 0.2o k: Io 7.2º - 0.17, 18.7 - 0.28, and 24.4 -
0.S8 k:. Eigure º.8(l) shows Ihe ¡loI ol Ihe im¡edance values as a luncIion ol Ihe bacIe-
rial concenIraIions. There is a linear relaIionshi¡ beIween Ihe im¡edance and Ihe loga-
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
317
riIhmic value ol Ihe cell concenIraIion in Ihe range lrom 10
4
Io 10
8
cells}20 ŸL (10
o
Io
10
10
clu}mL). The linear regression equaIion is [ (k:) = ÷2.0o Log D (cells}20 ŸL) + S.23
wiIh S
2
Y 0.º8. The deIecIion limiI was calculaIed Io be o.º Ó 10
4
cells}20 ŸL (3.4S Ó 10
o
clu}mL).
In Ihis secIion, we have demonsIraIed a new, sim¡le, and ra¡id meIhod Io deIecI
bacIerial cells by measuring Ihe im¡edance ¡ro¡erIies ol Iheir sus¡ensions in DI waIer
using inIerdigiIaIed microelecIrodes. This meIhod does noI require any label or am¡lili-
caIion sIe¡s. II can be used as an alIernaIive a¡¡roach Io quanIily bacIerial cells in sus-
¡ensions Io im¡edance microbiology. The deIecIion limiI ol Ihis meIhod is com¡arable
wiIh many oIher label-lree immunosensors lor deIecIion ol ¡aIhogenic bacIeria using
dillerenI Iransducer Iechniques, including QCMimmunosensors lor deIecIion ol Tbmnp.
ofmmb wiIh deIecIion limiIs ol 3.2 Ó 10
o
clu}mL and º.º Ó 10
S
clu}mL |48, 4º|, SFR
immunosensors lor Ihe deIecIion ol Tbmnpofmmb foufsjujejt and Mjtufsjb npopdzuphfot wiIh
deIecIion limiIs ol 10
o
clu}mL |S0|, a SFR sensor lor deIecIion ol F/ dpmj O1S7:H7 wiIh a
deIecIion limiI ol 10
7
clu}mL |S1|, and an elecIrochemical im¡edance immunosensor
lor Ihe deIecIion ol F/ dpmj O1S7:H7 wiIh a deIecIion limiI ol 10
o
clu}mL |10|. To allord
Ihis meIhod wiIh selecIiviIy, we have recenIly im¡lemenIed magneIic se¡araIion ¡rior
Io Ihe im¡edance deIecIion |1|. To lurIher im¡rove Ihe deIecIion limiI ol Ihis a¡¡roach,
a concenIraIion sIe¡ IhaI enriches Ihe small number ol bacIerial cells inIo a
microdeIecIion chamber would be very uselul.
:/5 Dpodmvtjpo
Advances in microlabricaIion have ¡aved Ihe way lor miniaIurizaIion ol many Iradi-
Iional deIecIion ¡laIlorms inIo microdevices or chi¡s. Im¡edance sensing as a ¡rinci¡al
9.4 Cohclusioh
318
Uspvcmftippujoh Ubcmf
Qspcmfn Fyqmbobujpo Qpufoujbm Tpmvujpo
Uifsf jt gmvje mfblbhf Uif dijq jt opu xfmm bttfncmfe Vtf b ofx boe xfmm bttfncmfe dijq/
EFQ epft opu dbquvsf dfmmt ps tqpsft Uif dpoofdujpo cfuxffo uif EFQ
fmfduspeft boe uif EFQ tjhobm
hfofsbups epft opu xpsl
Difdl uif dpoofdujpo ps dibohf up b ofx dijq/
Uifsf jt op tjhobm jo jnqfebodf
nfbtvsfnfout
Uifsf jt b cbe dpoofdujpo cfuxffo
uif dijq boe uif jnqfebodf
jotusvnfou
Difdl uif dpoofdujpot/
Jnqfebodf cbtfmjof xjui efjpoj{fe
xbufs jt opu dpotjtufou xifo b
ejggfsfou wjbm pg xbufs jt jokfdufe
joup uif efwjdf
Efjpoj{fe xbufs dibohft jut
dpoevdubodf bgufs ju fyqptft up bjs
Qsfqbsf nvmujqmf wjbmt pg xbufs jo qsfsjotfe dmfbo
uvcft boe tufsjmj{f uif cbudi uphfuifs/ Ejtdbse boz
xbufs wjbmt uibu ibwf cffo mfgu pqfo gps npsf uibo 41
njovuft/
Jnqfebodf cbtfmjof xjui efjpoj{fe
xbufs gspn uif tbnf wjbm jt opu
dpotjtufou
Jnqfebodf jt bggfdufe cz jpo
sfmfbtf gspn uif qpmznfs nbufsjbm
gps dijq gbcsjdbujpo
Tpbl uif xipmf dijq jo efjpoj{fe xbufs pwfs ojhiu- ps
gmvti uif diboofmt xjui efjpoj{fe xbufs gps bo
fyufoefe qfsjpe )ipvst* voujm uif cbtfmjof tubcjmj{ft
xjui dpoujovpvt jokfdujpo pg efjpoj{fe xbufs/
Dfmmt epo’u mztf ps mztf upp rvjdlmz
vtjoh uif qsftfou mztjt tpmvujpo
Ejggfsfou dfmmt ibwf ejggfsfou
upmfsbodft gps uif izqpupojd
fowjsponfou
Gps fbdi dfmm uzqf- uif mztjt tpmvujpo offet up cf pquj.
nj{fe tvdi uibu evsjoh uif tpmvujpo fydibohf gspn
jtpupojd up izqpupojd tpmvujpo- uif dfmmt sfnbjo joubdu
cvu xjmm mztf sbqjemz bgufs uif mztjt tpmvujpo gmpx tupqt/
Jnqfebodf epft opu dibohf bgufs
uif hfsnjobou jt jokfdufe
Tqpsft hfsnjobuf cfgpsf sfbdijoh
uif efufdujpo dibncfs
Pof.ujnf vtf pg uif efwjdf xjmm qsfwfou hfsnjobujpo
evf up difnjdbm sftjevft/ B tfqbsbuf jomfu gps
hfsnjobou efmjwfsz jt bmtp sfdpnnfoefe up bwpje
qsfhfsnjobujpo/
elecIrical Iransducer Iechnique is one ol Ihe besI-suiIed measuremenI a¡¡roaches IhaI
can be incor¡oraIed inIo microchi¡s. We have demonsIraIed lour microchi¡-based sys-
Iems IhaI have been successlully used lor moniIoring microbial and cellular acIiviIies
and lor deIecIing bacIerial and mammalian cells. The microscale im¡edance-based
meIhods have shown advanIages in im¡roved sensiIiviIy, reduced quanIiIies ol cosIly
reagenIs, reduced deIecIion Iime, and llexibiliIy in inIegraIion wiIh oIher elecIrical
meIhods such as DEF.
:/6 Tvnnbsz Qpjout
• The microlabricaIed sysIems described here demonsIraIe Ihe a¡¡licaIion ol
microscale lab-on-a-chi¡ devices lor Ihe deIecIion ol biological acIiviIies wiIh high
sensiIiviIy and shorIened assay Iime.
• Microscale "im¡edance microbiology" can be realized in a microchi¡ lormaI,
which can be inIegraIed wiIh an elecIrical concenIraIion sIe¡, Ihrough
dielecIro¡horesis (DEF), lor ra¡id deIecIion ol bacIerial cells based on Iheir meIa-
bolic acIiviIy.

The DEF concenIraIion in microchi¡ "im¡edance microbiology" eliminaIes Ihe
IradiIional cell-growIh-enrichmenI sIe¡ and Ihus can signilicanIly save assay Iime,
which is a greaI im¡rovemenI com¡ared wiIh convenIional meIhods IhaI require
several days.
• AuIomaIic and ra¡id elecIrical deIecIion ol Ihe germinaIion ol viable s¡ores can be
achieved. DEF concenIraIion ol a low number ol s¡ores in Ihe ulIrasmall deIecIion
chamber (0.1 nL) wiIhin Ihe microlluidic biochi¡ can im¡rove Ihe deIecIion limiI
down Io lewer Ihan 100 s¡ores and signilicanIly reduce Ihe deIecIion Iime com-
¡ared wiIh IradiIional meIhods.
• Microlluidic chi¡-based im¡edance s¡ecIrosco¡y can be designed Io deIecI boIh
mammalian and bacIerial cells by moniIoring Ihe im¡edance change in Ihe culIure
buller as a resulI ol ion release lrom Ihe cells. The microdevice hel¡s Io conline Ihe
ions in a small volume lor sensiIive im¡edance measuremenI.
Bdlopxmfehnfout
L. Yang acknowledges Ihe lunding lrom Ihe Golden LEAE EoundaIion and Ihe sIaIe ol
NorIh Carolina Ihrough Ihe BiomanulacIuring Research InsIiIuIe & Technology EnIer-
¡rise (BRITE) CenIer lor Excellence. X. Cheng and R. Bashir acknowledge su¡¡orI lrom
Ihe NIH}NaIional InsIiIuIe ol Biomedical Imaging and Bioengineering under GranI No.
F41 EB002S03 (BioMEMS Resource CenIer, FI: Frolessor M. Toner). R. Bashir acknowl-
edges research su¡¡orI Ihrough Ihe CenIer lor Eood SaleIy Engineering aI Furdue Uni-
versiIy, lunded Ihrough a coo¡eraIive agreemenI wiIh Ihe AgriculIural Research Service
ol Ihe U.S. De¡arImenI ol AgriculIure, ¡rojecI number 1º3S-42000-03S. The auIhors
also Ihank Ihe sIall and laciliIies ol Ihe Birck NanoIechnology CenIer aI Furdue
UniversiIy.
Lab-oh-a-Chip Impedahce DeIecIioh o! Microbial ahd Cellular AcIiviIy
319
Sfgfsfodft
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