Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry

12 Pages·2015·1.71 MB·English

RESEARCHARTICLE
Detection of Cytomegalovirus Antibodies
Using a Biosensor Based on Imaging
Ellipsometry
HongliuSun1☯,CaiQi2☯,YuNiu3,TengfeiKang3,YongxinWei4,GangJin3,XianzhiDong5,
ChunhuaWang1,WeiZhu6*
1 SchoolofPharmaceuticalSciences,BinzhouMedicalUniversity,Yantai,China,2 InstituteofEquipment
Technology,ChineseAcademyofInspectionandQuarantine,#3,Beijing,China,3 InstituteofMechanics,
ChineseAcademyofSciences,#15,Beijing,China,4 FoodLaboratory,BeijingInspectionandQuarantine
a11111
TestingCenter,#6,Beijing,China,5 InstituteofBiophysics,ChineseAcademyofSciences,#15,Beijing,
China,6 InstituteofRadiationMedicine,ShandongAcademyofMedicalSciences,#18877,Jinan,China
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
OPENACCESS
Citation:SunH,QiC,NiuY,KangT,WeiY,JinG,et
al.(2015)DetectionofCytomegalovirusAntibodies
UsingaBiosensorBasedonImagingEllipsometry. Background
PLoSONE10(8):e0136253.doi:10.1371/journal.
pone.0136253 Cytomegalovirus(CMV)isthemostcommoninfectiouscauseofmentaldisabilityinnew-
bornsindevelopedcountries.Thereisanurgentneedtoestablishanearlydetectionand
Editor:GotthardKunze,IPK,GERMANY
high-throughputscreeningmethodforCMVinfectionusingportabledetectiondevices.
Received:April3,2015
Accepted:August3,2015
Methods
Published:August21,2015
AnantibodyanalysismethodisreportedforthedetectionandidentificationofCMVantibod-
Copyright:©2015Sunetal.Thisisanopenaccess
iesinserumusingabiosensorbasedonhighspatialresolutionimagingellipsometry(BIE).
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits CMVantigen(CMV-3A)wasimmobilizedonsiliconwafersandusedtocaptureCMVanti-
unrestricteduse,distribution,andreproductioninany bodiesinserum.AnantibodyagainsthumanimmunoglobulinG(anti-IgG)wasusedtocon-
medium,providedtheoriginalauthorandsourceare
firmtheIgGantibodyagainstCMVcapturedbytheCMV-3A.
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. Results
Funding:Theauthors’laboratoryworkissupported OurresultsshowthatthisassayisrapidandspecificfortheidentificationofIgGantibody
bygrantsfromtheYantaiScienceandTechnology againstCMV.Further,patientserumwasquantitativelyassessedusingthestandardcurve
DevelopmentFund(2012128),theYouthBackbone
method,andthequantitativeresultswereinagreementwiththeenzyme-linkedimmunosor-
TeacherFundofBinzhouMedicalUniversity,the
NationalNaturalScienceFund(81202516),the bentassay.TheCMVantibodydetectionsensitivityofBIEreached0.01IU/mL.
ShandongProvinceNaturalScienceFund
(ZR2013HL039),Scienceandtechnology
developmentplanofShandongAcademyofMedical Conclusions
Sciences(2014-08),theSpecialFundforAgro-
ThisnovelbiosensormaybeavaluablediagnostictoolforanalysisofIgGantibodyagainst
scientificResearchinthePublicInterest
CMVduringCMVinfectionscreening.
(201303045),theNationalKeyScientificInstrument
andEquipmentDevelopmentProjectofChina
PLOSONE|DOI:10.1371/journal.pone.0136253 August21,2015 1/12
CytomegalovirusDetectionUsingBiosensors
(2012YQ090197),theNationalBasicResearch Introduction
Program(Project973)ofChina(2009CB320300),the
CMVisthemostcommoninfectiouscauseofmentaldisabilityinnewbornsindeveloped
NationalBasicResearchProgramofChina
(2009CB320302),theNationalHighTechnology countries[1].DetectionofCMVantibodiesiseffectiveforsystematicscreeningforCMVinfec-
ResearchDevelopmentProgramofChina tion[2].Forinstance,theCMVimmunoglobulinG(IgG)avidityassaycanhelptodistinguish
(2008AA02Z419),theInternationalScience& primaryfromnon-primaryhumanCMVinfections[3–5].Thekeyimmunemethodsusedfor
TechnologyCooperationProgramofChina
CMVantibodydetectionare:enzyme-linkedimmunosorbentassay(ELISA)[6],Elecsys[7],
(2012DFG31880),theProjectsofSino-PortugalS&T
Cooperation2010–2015,theNationalNatural electrochemiluminescenceimmunoassay(ECLIA)[8],immunofluorescenceassay(IFA)[9],
ScienceFoundationofChina(21305147)andthe flowcytometry(FCM)[10]andimmunoblots[11].Additionally,newimmunemethodsfor
InstrumentDevelopingProjectoftheChinese CMVantibodydetectionhavebeendeveloped,suchasthechemiluminesentmicroparticle
AcademyofSciences(KJCX2-YW-M04,KJCX2-YW- immunoassay(CMIA)[12]andproteinmicroarrays[13,14].However,thesemethodssuffer
M03).
frominherentlimitations,suchaslengthoftestingtime,theneedsforexpensiveequipment,
CompetingInterests:Theauthorshavedeclared specialistskills,lowsensitivity,andcomplicatedsamplepreparationprocesses.Forexample,
thatnocompetinginterestsexist. conventionalELISAisstillthemaindiagnostictestforCMV,andcommercialCMVELISA
Kitsareavailable.AlthoughELISAisoftenusedasacomparisonmethodforCMVantibody
detection[13],obviousshortcomingsincludetheneedoftracerlabel,platewashing,theindi-
rectformatofdetection,andthelengthoftimenecessaryfortesting.Thus,arapid,simple,
direct,andhigh-throughputmethodforCMVantibodydetectionisurgentlyneeded.
Thefirstbiosensorbasedonimagingellipsometry(BIE)wasdevelopedin1995[15,16].
Comparedtothemethodsabove,theadvantagesofBIEareevident,e.g.,high-throughputmul-
tiplexedanalysisandquantitative,label-freerapidtesting.PreviousapplicationsofBIEmainly
focusonthebiomedicalfields[17,18],suchashigh-throughputdiseasediagnosisofhepatitisB
virus(HBV)marker[19,20],thedetectionofavianinfluenzavirus(AIV)[21]andantibodies
againstsevereacuterespiratorysyndrome(SARS)detection[22].Thus,thebiosensortechnol-
ogyoffersimportanttoolsfordiseasediagnosis.Assuch,newapplicationshaverecentlybeen
developed,includingthosefortheanalysisoftheinteractionbetweentropomyosinallergens
andantibodies[23],andtheinteractionbetweensolubleN-ethylmaleimide-sensitivefactor
attachmentreceptor(SNARE)proteins[24].Todate,however,BIEhasnotbeenappliedfor
thedetectionofCMVantibody,particularlyfortheidentificationoftheCMVIgGandIgM
antibodies.
ThepurposeofthisstudywastodetectantibodiesagainstCMV-3Ainpatientserumusing
aBIEmicroarraywithCMV-3A,aswellastospecificallyidentifycapturedIgGantibody
againstCMV.AntibodiesagainstCMVqualitativelyweredetectedusingBIE,andthen,goat
antibodyagainsthumanIgG(anti-IgG)wasaddedtotheareawiththecapturedCMVanti-
bodytoconfirmIgGantibodyagainstCMV.Astandardcurverepresentingdifferentconcen-
trationgradientswasalsoestablishedforthequantitativedetectionofCMVantibodies.As
such,theconcentrationofCMVantibodyinserumwasquantitativelydetectedbyBIEand
thencomparedusingELISAs.
MaterialsandMethods
Materialsandreagents
SiliconwaferswerepurchasedfromtheGeneralResearchInstituteforNonferrousMetals
(China).N-hy-droxysuccinimide(NHS)and1-(3-Dimethyla-minopropyl)-3-ethylcarbodii-
midehydrochloride(EDC),Tween-20,bovineserumalbumin(BSA),IgG,andBlockingBuffer
(10×,B6429)werepurchasedbySigma.Anti-IgGandagoatantibodyagainsthumanIgM
(anti-IgM)werepurchasedfromBeijingBoShengBio-technologyCo.Ltd.CMV-3Awaspur-
chasedfromGalaxyBio.TheCMV-3AwasafusionofthreesegmentsofPp150,Gp52and
Pp65,whichhavestrongantigenicityepitopes.Fusionofthemulti-epitopebothenhancesthe
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CytomegalovirusDetectionUsingBiosensors
sensitivity/specificityandreducesfalsenegatives.Thus,CMV-3Awasusedastheligandinthe
BIEassay.PurifiedCMVantibody(Pp65,C-Term,Rabbit,1mg/mL)waspurchasedfrom
Antibodies-online.com.PatientserumsampleswerepurchasedfromQiLuHospitalofShan-
dongUniversityandclinicalinformationislistedinTableAinS2File.
TORCHELISAKitsusedtoanalyzeserumsampleswerepurchasedfromMEDSONInc.
Thedetectionprocessandanalysiswereexecutedaccordingtothemanufacturer’sinstructions.
MicroplateswerecoatedwithnativeCMVantigens,highlypurifiedbysucrosegradientcentri-
fugationandinactivated.Thesolidphasewasfirsttreatedwiththedilutedsample,andIgG
moleculestoCMVwerethencaptured,ifpresent,bytheantigens.Afterwashingoutallofthe
othersamplecomponents,boundanti-CMVIgGmoleculesweredetectedbytheadditionof
specificpolyclonalanti-H-IgGantibodieslabelledwithperoxidase(HRP)inthesecondincuba-
tion.Theenzymecapturedonthesolidphase,actingonthesubstrate/chromogenmixture,
generatesanopticalsignalthatisproportionaltotheamountofanti-CMVIgGantibodies
presentinthesample.Acalibrationcurve,calibratedagainstthefirstW.H.Ointernational
standard,makespossibleaquantitativedeterminationoftheIgGantibodyinthepatient.
NEsolutionwaspreparedwithNHS(0.05M)andEDC(0.2M)indeionizedwater(18.3
MOcm)fromaMilli-Qplussystem(Millipore,Bedford,MA).Phosphate-bufferedsaline
(PBS)waspreparedwith140mMNaCl,2.7mMKCl,10mMNa HPO ,and1.8mMKH PO
2 4 2 4
(pH7.3)indeionizedwater.PBSTbufferwaspreparedwith1%Tween-20inPBS.CMV-3A
(0.1mg/mL)waspreparedwithPBST.BSA(10mg/mL)waspreparedwithPBST.Theblocking
reagentwasa1:1(vol:vol)mixtureof10mg/mLBSAand10×BlockingBuffer.PurifiedCMV
antibody,IgG,anti-IgG,andanti-IgMweredilutedtoaconcentrationof0.1mg/mLwith
PBST.SerumsamplesweredilutedasindicatedwithPBSTforqualitativeorquantitative
detection.
BIEprinciple
TheBIEcombineshighspatialresolutionimagingellipsometry(FigAinS1File)withamicro-
fluidicsystem(FigBinS1File)toanalyzebiomolecularinteraction[25].
Themicrofluidicsystemisusedforsurfacepatterningandarrayproduction,aswellasfor
solutiondelivery,ligandimmobilizationandtargetcapture[26].Themicrofluidicsystemhas
fourmainparts:asampleplate,multi-cellarray,micro-channelsandpumps.Amulti-cellarray
wasformedwhenapolydimethyl-siloxane(PDMS)patternmoldcontactedthesurfaceofasili-
consubstrate,eachcellhavinganinletandanoutletforsolutionpassage.Thephysicalsizeof
eachcellwas~1.5×1×0.5mm3.Theinletmicro-channelswereplacedintosampleplate,and
theoutletmicro-channelswereconnectedwithpumps(ISM939,ISMATEC,Switzerland.
www.ismatec.com)offeringnegativepressure.Thesimplechanneljunctionscanbeusedin
serialorparallelformatstosimultaneouslyanalyzesingleormultiplesamples.
Imagingellipsometryisadisplaytechniqueforultrathinfilmandsurfacecharacterization
[27],whichisusedtoreadandanalyzetheproteinarraysmadebymicrofluidicsystems.The
incidentwaveofpolarizedlightirradiatesthesampleasaprobebeamandistherebymodified
resultinginareflectiveortransmissionbeamhavingtheabilitytocarrysampleinformation,
suchasproteinlayerthickness.Whenimagingellipsometryisusedtodetectlayerthickness,
thereflectionintensityisrepresentedingrayscale,andthevariationinlayerthicknessleadsto
changesinthegrayscalevalue.Iftherefractiveindexisinvariant,thegrayscalevalueisdirectly
proportionaltothethicknessoftheproteinlayerwithintherangeof0~30nmlayerthickness,
i.e.,I=kd,whereIisthelightintensityanddisthelayerthickness[28].Undertheseconditions
kisaconstantandcanbedeterminedfromaproteinlayerwithknowngrayscalevaluesand
knownthickness[29].Thereisalsoarelationshipbetweensurfaceconcentrationandfilm
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CytomegalovirusDetectionUsingBiosensors
thickness:surfaceconcentration(μg/cm2)(cid:1)K×d,whereK=0.12[27].Thus,thegrayscale
valuedirectlyreflectslayerthicknessandsurfaceconcentration.Thehigherthegrayscalevalue,
thethickerthelayerandthehigherthesurfaceconcentration.
Surfacemodification
Siliconwaferswerecutinto20×10mm2rectanglesandrinsedwithdeionizedwater.After
soakinginpiranhasolution(30%H O :98%H SO =1:3,vol/vol)for30mintoincreasethe
2 2 2 4
numberofsilanolgroupsonthewafersurface,andrinsingwithdeionizedwaterandethanol,
thewafersweresoakedinamixtureof3-aminopropyltriethoxy-silane(APTES)andabsolute
ethanol(APTES:absoluteethanol=1:10,vol/vol)andincubatedfor2hwithgentleagitation.
ThereactionofAPTESwiththesurfacesilanolgroupsresultedincovalentimmobilizationof-
O-Si(OH) -(CH ) -NH ,formingalayerofdenselypackedaminogroupsonthesurface.After
2 2 3 2
rinsingthreetimeswithabsoluteethanol,thewaferswereincubatedinasaturatedsolutionof
succinicanhydrideinethanolfor(cid:3)3hwithgentleagitation.TheCH CH COOCO-groupof
2 2
succinicanhydridereactedwiththe-NH of-O-Si(OH) -(CH ) -NH groupimmobilizedon
2 2 2 3 2
thesurface,generatingcarboxylgroups(-(CH ) NH-CO-(CH ) -COOH).Onceprepared,the
2 3 2 2
waferswerestoredinethanol.
CMVdetectionprocedure
Theabove-modifiedsiliconwaferswereusedasthesubstrate.Apieceofsiliconwaferwas
placedonthemicrofluidicmoldofthemicrofluidicsystemsothatsurfacesofthewaferswere
patternedtoformsmall,regularcellsinanarrayformat.Then,thecarboxylgroupswereacti-
vatedbypumping10μLofNEintoeachcellataflowrateof5μL/minandpassedthroughthe
surfaceofthewafer.InthepresenceofNHS,EDCtransferscarboxylgroupstotheSulfo-NHS
ester,whichreactswiththeaminogroupsofproteinstoimmobilizetheproteinsonsurface.
Next,theligandwasimmobilized:CMV-3Aasligandorprobewaspumpedintoeachcell
(10μLpercellat1μL/min).Third,blockingoccurredbypumpingtheblockingreagentinto
eachcell(50μLpercellat1μL/min)andpassingitthrougheachligandarea.Thus,asensing
arraysurfacewithCMV-3AwasformedtocatchCMVantibodies.Fourth,targetswere
detected.PBSTwasusedasblankcontrol(50μLpercellat1μL/min)andpurifiedCMVanti-
bodyasapositivecontrol(50μLpercellat1μL/min)bypumpingthemintoseveralofthe
individualcells.Simultaneously,patientserumsampleswerealsopumpedintoremainingcells
(50μLpercellat1μL/min).TheCMVantibodiesintheserumwerecapturedwhentheyinter-
actedwiththeCMV-3Aonthesensingsurface.AllcellswererinsedwithPBST(20μLpercell
at20μL/min)betweeneverytwoconsecutiveoperationsteps.Finally,themicroarraywafers
wereremovedfromthemicrofluidicsystem.Afterbeingrinsedwithdeionizedwateranddried
withnitrogen,thewaferswereanalyzedusingimagingellipsometry.IftheCMVantibodiesin
thesolutionorseruminteractedwiththeCMV-3Aonthesurfaceandformedacomplex,the
layerinthatareabecamethicker.Theexperimentalresultswererecordedasimagesingray-
scale,andbindingofCMVantibodiesresultedinasignificantincreaseingrayscalevalue.
QualitativedetectionofCMVantibodiesandpatientserum
TodetectthespecificityofantibodiesagainstCMV,CMV-3Aasligandwasimmobilizedon
twocolumns(Fig1).PBSTbufferwasaddedasblankcontroltotwoareasonthefirstrow.
Simultaneously,purifiedCMVantibody(0.1mg/mL)wasaddedaspositivecontroltotwo
areasonthesecondrow.NormalserumwithoutCMVantibodieswasaddedasnegativecon-
troltotwoareasonthethirdrow.Threepatientserumsamples(No.956,933,and978;see
TableAinS2Fileforsampleinformation)wereanalyzedonthefollowingrows,respectively.
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Fig1.SpecificityandqualitativedetectionofCMVantibodiesusingBIE.(A)Grayscaleimagesof
differentCMVantibodiessamples;and(B)3-DgrayscaledistributionmapofdifferentCMVantibodysamples
inimageA.Inthefirststep,CMV-3Awasimmobilizedastheligandontwocolumns.Inthesecondstep,
PBSTbufferwasaddedasablankcontroltotwoareasonthefirstrow.Simultaneously,purifiedCMV
antibodywasaddedasapositivecontroltotwoareasonthesecondrow.NormalserumwithoutCMV
antibodieswasaddedasanegativecontroltotwoareasonthethirdrow.AccordingtoELISAdata,No.956
hadahighCMVantibodyconcentration,andNo.933and978hadmediumCMVantibodyconcentrations.To
observequalitativedetectionofCMVantibodiesinserum,sampleswithhigherconcentrationswerechosen.
doi:10.1371/journal.pone.0136253.g001
TheincreaseinlightreflectiondensityateacharearevealedthatCMVantibodiesinthesam-
plesinteractedwiththeCMV-3Aimmobilizedonthechip.
WefurtheranalyzedthetypesofCMVantibodiescapturedbytheCMV-3A(Fig2).Inthe
firststep,IgGwasimmobilizedasligandonthefirstandsecondcolumns.Simultaneously,
CMV-3Awasimmobilizedonthethird,fourth,fifth,andsixthcolumns.Inthesecondstep,
sampleNo.948wasaddedtothethirdandfourthcolumns,andsampleNo.940wasaddedto
thefifthandsixthcolumns.PBSTbufferwasaddedasblankcontroltoremainingareas.Inthe
thirdstep,anti-IgGandanti-IgMwereaddedtothirdandfourthrowstoidentifyandconfirm
Fig2.IdentificationofIgGantibodyagainstCMVbyBIE.(A)GrayscaleimagesofIgGantibody
identificationinserum;and(B)grayscalevalueandP-valueoftheIgGantibodyidentification.“*”indicates
significantchangesingrayscalevalues.Inthefirststep,IgGwasimmobilizedasligandonthefirstand
secondcolumns.CMV-3Awasimmobilizedonthethird,fourth,fifth,andsixthcolumns.Inthesecondstep,
PBSTbufferwasaddedasblankcontroltothecorrespondingareasintheimage.SampleNo.948was
addedtothethirdandfourthcolumns,andsampleNo.940wasaddedtothefifthandsixthcolumns.Inthe
thirdstep,PBSTbufferwasaddedasblankcontroltothefirstareasineverycolumn.Anti-IgGandanti-IgM
wereaddedtothethirdandfourthrows,respectively.
doi:10.1371/journal.pone.0136253.g002
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iftheantibodycapturedbytheligandwasIgGorIgM.PBSTbufferwasaddedasblankcontrol
toremainingareas.
Calibrationcurveandquantitativedetectionofclinicalserumsamples
Toestablishacalibrationcurve,aserumsample(No.942,21.8IU/ml,seeTableAinS2File)
wasused,andfivelevelsofserialdilutioncontaining0.011,0.043,0.170,0.681,and2.725IU/
mLofCMVantibodywerepreparedinPBST.CMV-3Awasimmobilizedasligandontwocol-
umns(Fig3).Afterblocking,PBSTbuffer,CMVantibodyandnormalserumwithoutCMV
antibodywereaddedasblank,positive,andnegativecontrols(respectively)tothefirst,second,
andthirdrows,respectively.The0.011-,0.043-,0.170-,0.681-and2.725-IU/mLsampleswere
addedfromthefourthtotheeighthrows,respectively.ConcentrationplottedontheX-axis
andthevariationofthegrayscalevaluecomparedtoblankcontrolwasplottedontheY-axisto
generatethecalibrationcurve(Fig3).
Afteracquiringthecalibrationcurve,theconcentrationsofantibodiesinsamplesof
unknownconcentrationcouldbedeterminedonthecurveaccordingtotheirgrayscalevalues.
Measurementsofeverysamplewererepeatedintwoareasononechip(Fig4).Commercial
ELISACMVantibodykitswereusedascontrolsaccordingtothemanufacturer’sinstructions.
Statisticalanalyses
Forstatisticalanalyses,thecorrespondingP-valueswerecalculatedwithsinglefactoranalysis
ofvarianceinMicrosoftOfficeExcelaccordingtothequantityofareasinimagesandpatients’
serumsamplesinTableAinS2File[30].Inqualitativeandquantitativedetectionexperiments,
significantchangesingrayscalevaluedetectionareascomparedtotheblankcontrolareasand
thedetectionareasweredeemedpositivesignalsifP-valuewas<0.05.IfthePwas(cid:3)0.05,the
detectionareasweredeemednegativesignals.IncomparisonoftheELISAandBIEdata,the
resultsofthetwomethodsweredeemedinagreementifPwas(cid:3)0.05.IfPwas<0.05,thetwo
methodsweredeemedindisagreement.Thecorrelationcoefficient(r-value)ofBIEandELISA
wascalculatedwithanalysisofcorrelationcoefficientinMicrosoftOfficeExcel(TableBinS2
File).
Results
Specificityconfirmationandqualitativedetection
Comparedtoblankcontrolsareas,thepurifiedCMVantibodyandpatientserumsample
detectionareashadmarkedlythickerfilms,withtheaveragegrayscalevaluedisplayingsignifi-
cantincreases,whilenegativecontrolareasdidnot(Fig1).Themeangrayscalevalueofblank
controlswasmeasuredat117.45±0.92,andthevalueofthepurifiedCMVantibodywasmea-
suredat176.8±3.39.Thus,themeangrayscalevalueofpurifiedCMVantibodyminusthe
meangrayscalevalueofblankcontrolwas59.4(P=0.002).Thevalueofthenegativecontrol
was125.9±3.18.Themeangrayscalevalueofthenegativecontrolminusthemeangrayscale
valueoftheblankcontrolwas8.4(P=0.07).Thisindicatedthattherewerespecificinteractions
betweenthepurifiedCMVantibodyandtheCMV-3A.Therefore,CMVantibodiesinpatient
samplescouldbecapturedbytheCMV-3Aimmobilizedonthesubstrate.
Indeed,theaveragegrayscalevalueoftheserumsamplesanalyzedsignificantlyincreased
relativetothecontrols(Fig1).Themeanserumvaluewas253.58±0.49,andthemeangray-
scalevalueofserumminusthemeangrayscalevalueoftheblankcontrolwas136.1
(P=2.8×10−5),indicatingthatCMVantibodieswereabundantintheserumsamples.Thiswas
consistentwiththeELISAresults(TableAinS2File).
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Fig3.CalibrationcurveandsensitivityofBIEfordetectingCMVantibodies.(A)GrayscaleimagesofdifferentCMVantibodyconcentrationlevels
detected;(B)3-DgrayscaledistributionmapofdifferentconcentrationsofCMVantibodiesdetected;and(C)standardcurveofCMV-3Aasligandtodetect
fiveserialdilutionsofcontainingCMVantibodies(0.011,0.043,0.170,0.681,and2.725IU/mL).Inthefirststep,CMV-3Awasimmobilizedastheligandon
twocolumns.Inthesecondstep,PBSTbufferwasaddedasablankcontroltotwoareasonthefirstrow.Simultaneously,purifiedCMVantibodywasadded
asapositivecontroltotwoareasonthesecondrow.Negativeserumwasaddedasanegativecontroltotwoareasonthethirdrow.Theserialdilutionsof
CMVantibodieswereaddedasanalyticalsamplesonthefollowingrows.Thesameconcentrationwasmeasuredintwoduplicateareas.
doi:10.1371/journal.pone.0136253.g003
IdentificationofIgGantibodyagainstCMVbyBIE
WhenpurifiedhumanIgGwasusedastheligand,theaveragegrayscalevalueoftheanti-IgG
detectionareassignificantlyincreased,whiletheanti-IgMdetectionareasdidnot(lefttwocol-
umnsinFig2).Themeangrayscalevalueoftheanti-IgGminusthemeangrayscalevalueof
theblankcontrolwas96.2(P=0.003).Themeanvalueoftheanti-IgMminusthemeangray-
scalevalueoftheblankcontrolwas8.15(P=0.29).Thesedataareindicativeofthespecific
interactionsbetweentheanti-IgGandthehumanIgGimmobilizedonthechip,whichcould
beusedasreferencestodeterminewhethertheantibodiesinsamplesareIgG.
ComparedtotheCMVantibodyareas,theanti-IgGdetectionareasdisplayedsignificant
increases,whiletheanti-IgMdetectionareasdidnot(Fig2).Forexample,thevalueofsample
948was156.2±2.6,whilethevalueoftheanti-IgGwas189.6±4.4,foradifferenceof33.4
(P=0.02).However,thevalueoftheanti-IgMwas154.25±0.25,andthedifferencewasnot
obvious(P=0.53).ThisindicatedthatIgGCMVantibodieswerecapturedbythechip.For
sampleNo.940detection,thevalueoftheanti-IgGwas237.7±11,andtheincreasewas77.3
(P=0.01).TheresultalsoindicatedthatthecontentofIgGinsamplesNo.948and940was
different.
BIEsensitivityandcalibrationcurve
Fiveconcentrationgradientsofaserumsample(No.942,21.8IU/mL,seeTableAinS2File)
measuredinseriallydilutedsampleswereusedtodeterminethesensitivityoftheBIEassay
(Fig3).WefoundthatthechangeinsignalintensitywasconsistentwiththeincreaseinCMV
antibodyconcentration.TheCMVantibodydetectionsensitivitylevelsreached0.01IU/mL.
Thegrayscalevaluesoftheblankcontrolwere124.7±0.5,andthegrayscalevaluesofthedilu-
tionsaslowas0.01IU/mLwere132.3±0.1,i.e.,~6.1%greaterthanthatofthecontrols
(P=0.03).Repeatingthesetests>10times,ourresultsdemonstratedthatthesensitivityofthe
assayreached0.01IU/mLorless.Eachvariationingrayscalevaluewaslinkedtoacorrespond-
ingconcentrationoftheCMVantibodyovertherangeof0.011–2.725IU/mL,andthe
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calibrationcurveisshowninFig3.Theconcentrationsofunknownsamplescouldbedeter-
minedaccordingtocorrespondinggrayscalevalueonthecalibrationcurve.
QuantitativedetectionofCMVantibodiesinclinicalserum
41CMVpatients(TableAinS2File)withquantitativeresultsbyELISAweretestedwithBIE
(Fig4).Fordifferentserumsamples,thechangesinBIEsignalintensityweredifferent.The
comparisonofresultsbetweenthetwomethodsisshowninFig5.Singlefactoranalysisand
correlationcoefficientanalysisrevealedthattheresultswereinagreementbetweenELISAand
BIE(F=1.38<F =3.96;P=0.24>0.05,r=0.7)(TableBinS2File).
0.05
Discussion
High-throughputdetectionissuitableformassCMVscreeninginwomenofchildbearingage.
Whencoupledwithmicrofluidictechnologies,BIEcangreatlyimprovethethroughputfor
CMVdetectionononechip.Themicroarraysdevelopedherehavemultiplecellsimmobilized
withdifferentligandsfordifferentCMVantibodysubtypes.Imagingellipsometryisalsosuit-
abletohigh-throughputanalysis.Itcanbeusedtovisualizethevariationinsignalfromall
unitsofthemicroarraywithhighspatialresolution.Patientsampleanalysisresultsfromone
microarraycansimultaneouslybeidentified,andthedatacanbeobtainedinseveralseconds
usinganimagingellipsometer.Presently,ourmethodcanprovidesimultaneous48reaction
areas.Withitsenhancedthroughputandlowercost,BIEmaybeusedinclinicalmassCMV
screeningsforhealthybirthsinthefuture.
AntibodydetectioniswidelyavailablefortheclinicaldiagnosisofCMVinfection.However,
theidentificationofantibodytypesmayhelptodeterminethecourseofdisease,andBIEmay
beusedasaclinicalprimaryscreeningtoolforCMVpatients.IgMisproducedinlarge
amountsearlyininfection(reachingapeakinthefirstmonth),andisfollowedbyIgGproduc-
tion[7,31].LevelsofIgMdeclineinthemonthsfollowingtheonsetofinfection,whereasIgG
levelspersistfortherestofthepatient’slife[32,33].DeterminingIgGaviditycanprovideaddi-
tionalguidanceoninfectionstatus,andlowavidityIgGisinitiallypresentbutincreasesover
time[34].ApositiveresultforIgMcombinedwithlow-to-moderateIgGsuggestsavidityapri-
maryCMVinfectionwithinthepast3to4months[35].ThevisualizedBIEimage(Fig2)
couldreflecttypesviadifferenceinbrightness,sothistechnologycanachieverapidscreening.
Todate,ourworkissimplyademonstrationforIgGtypeantibodydetectionwithBIE.IgM
andotherantibodiesneededtoscreenforspecificantigencanbeincorporatedinthefuturefor
furtherpracticalapplications.
Presently,mostmethodsmentiontherelativesensitivity(%)ofdetectionforCMVantibod-
ies[8,36],butlittleinprovidedaboutabsolutesensitivity.Comparedothermethods,theabso-
lutesensitivityofBIEcanreach0.01IU/mL,andithasgoodresolutionintherangeof0.1–1.0
IU/mLonthecalibrationcurveforquantitativedetection.Thereferencerangefordetectionof
CMVantibodieshaspreviouslybeenpublished.Forexample,Elecsys(RocheDiagnostics)
immunoassayshaveanequivocalrange(0.5–1.0IU/mL)ofCMVIgG[12].Inourexperiment,
ELISAasacontrolmethodhadareferencerangeof0.4–0.6IU/mL.Therefore,thesensitivity
ofourBIEbiosensorisbelowthereferencerangeandhasalreadyreachedclinicalstandards.
However,thereisasubstantialdiscordancebetweentheELISAandtheBIEinsomepatients
(i.e.,940,956,964,984,and980).Samplesfromthesepatientsdisplayedstrongersignalusing
ELISAthanBIE.InELISAs,anti-CMVIgGmoleculesaredetectedbytheadditionofpoly-
clonalspecificanti-H-IgGantibodieslabelledwithhorseradishperoxidase(HRP).Incontrast,
theBIEbiosensordirectlyidentifiedthevariationofthesurfaceanti-CMVIgGconcentration
aftercapturingtargetwithoutasecondarylabel.Thus,thislabel-freemethodmayavoidsome
PLOSONE|DOI:10.1371/journal.pone.0136253 August21,2015 8/12
CytomegalovirusDetectionUsingBiosensors
Fig4.DetectionofCMVantibodiesinpatientserumusingBIE.Inthefirststep,CMV-3Awasimmobilizedastheligandonsixcolumns.Inthesecond
step,PBSTbufferwasaddedasablankcontroltosixareasonthefirstrow.Simultaneously,purifiedCMVantibodywasaddedasapositivecontroltothe
lasttwoareasonthesecondrow.Patientserumsampleswereaddedasanalyticalsamplesonthefollowingareas,respectively.Thesameserumsample
wasmeasuredintwoduplicateareas(No.940,959,938nosample,P15-9,andPBSTcontrolareunderlined).
doi:10.1371/journal.pone.0136253.g004
Fig5.ComparisonofBIEwithELISA.P15-1andP15-2wereusedashealthycontrols.ConcentrationofCMVantibody(0.5IU/mL)locatedinthenormal
referencerange(0.4–0.6IU/mL).Thecorrelationcoefficient(r-value)andP-valuewerecalculated.
doi:10.1371/journal.pone.0136253.g005
PLOSONE|DOI:10.1371/journal.pone.0136253 August21,2015 9/12
CytomegalovirusDetectionUsingBiosensors
interferencefactor.Inaddition,thegrayscaleimagesoffereduniformareas,helpingtoavoid
falsepositivesignals.Whenthecorrelationcoefficient(r-value)andP-valuewerecalculated
afterexcludingthenumberofseveremutations(940,956,964,and984),r-value=0.93and
P=0.8thestatisticalresultsshowedmoreagreementbetweenELISAandBIE.
Inconclusion,wehavedevelopedalabel-freeandmultiplexscreeningCMVIgGbiosensor
method.Thehigh-throughputdetectionissuitableformassCMVscreeningofwomenof
childbearingage.Further,ourresultsdemonstratethatthesensitivityofBIEhasalready
reachedclinicaldiagnosestandardsforanti-CMVIgG.Thus,onthebasisofanti-CMVIgG
detection,BIEcanbeusedforqualitativeandquantitativedetectionofmoretypesofCMV
antibodiesusingasimpleandfastprocedure.
SupportingInformation
S1File.ContainsFigA.Imagingellipsometry.(a)Imagingprinciple[23];and(b)Laboratory
prototype.FigB.Microfluidicsystem.(a)Internalstructure;and(b)Laboratoryprototype.
(DOCX)
S2File.ContainsTableA.ClinicalinformationofCMVpatientsfromQiLuHospitalofShan-
dongUniversity.TableB.StatisticalanalysisprocessofcomparisonbetweentheELISAand
BIEdata.
(DOCX)
Acknowledgments
WethankQiLuHospitalofShandongUniversityforclinicalCMVpatientsamples.
AuthorContributions
Conceivedanddesignedtheexperiments:WZGJ.Performedtheexperiments:HLSCQYN.
Analyzedthedata:TFKYGWXZD.Contributedreagents/materials/analysistools:HLSWZ.
Wrotethepaper:CQ.Collectedthedata:HLSWZCHW.
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