DTIC ADA534282: Optimization of Antibody-Conjugated Magnetic Nanoparticles for Target Preconcentration and Immunoassays

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AnalyticalBiochemistryxxx(2010)xxx–xxx
ContentslistsavailableatScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Optimization of antibody-conjugated magnetic nanoparticles for target
preconcentration and immunoassays
Joshua E. Smitha,b,⇑, Kim E. Sapsfordc,d,1, Weihong Tana, Frances S. Liglerd,⇑
aCenterforResearchattheBio/NanoInterface,DepartmentofChemistryandShandsCancerCenter,UFGeneticsInstituteandMcKnightBrainInstitute,
UniversityofFlorida,Gainesville,FL32611,USA
bDepartmentofChemistryandPhysics,ArmstrongAtlanticStateUniversity,Savannah,GA31419,USA
cGeorgeMasonUniversity,Manassas,VA20110,USA
dCenterforBio/MolecularScienceandEngineering,NavalResearchLaboratory,Washington,DC20375,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Biosensorsbasedonantibodyrecognitionhaveawiderangeofmonitoringapplicationsthatapplytoclin-
Received24August2010 ical,environmental,homelandsecurity,andfoodproblems.Inanefforttoimprovethelimitofdetection
Receivedinrevisedform12October2010 oftheNavalResearchLaboratory(NRL)ArrayBiosensor,magneticnanoparticles(MNPs)weredesigned
Accepted7November2010
and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently
Availableonlinexxxx
labeledprotein,AlexaFluor647–chickenIgG(Alexa647–chickIgG).Antibody-labeledMNPs(Alexa647–
chick–MNPs)wereusedtopreconcentratethetargetviamagneticseparationandasthetracertodem-
Keywords:
onstratebindingtoslidesmodifiedwithanti-chickenIgGasacaptureagent.Afulloptimizationstudyof
Immunoassay
theantibody-modifiedMNPsandtheiruseinthebiosensorwasperformed.Thisinvestigationlookedat
Magneticnanoparticles
theAlexa647–chick–MNPcomposition,MNPsurfacemodifications,targetpreconcentrationconditions,
Totalinternalreflectionfluorescence
ArrayBiosensor andtheeffectthatmagneticextractionhasontheAlexa647–chick–MNPbindingwiththearraysurface.
Proteinmicroarrays TheresultsdemonstratetheimpactofmagneticextractionusingtheMNPslabeledwithfluorescentpro-
teins both for target preconcentration and for subsequent integration into immunoassays performed
underflowconditionsforenhancedsignalgeneration.
(cid:2)2010ElsevierInc.Allrightsreserved.
Biosensorsareunderdevelopmentfortargetscreeninginclin- ies have demonstrated high binding affinities with extraordinary
ical, environmental, water, and food samples [1–4]. An essential specificity for target molecules even in complex sample matrices
component of these systems is the recognition elements, often and with low target concentrations [5]. The Array Biosensor
antibodies,forselectiveidentificationoftargetanalytes.Antibod- developed at the Naval Research Laboratory (NRL),2 which
typically performs multiplexed immunoassays, has been used
successfully for the detection of a variety of proteins, molecules,
⇑
Correspondingauthors.Fax:[phone](J.E.Smith),[phone] viruses, and bacteria in complex sample matrices [6,7]. The two-
(F.S.Ligler).
dimensional nature of the sensing surface facilitates simultaneous
E-mail addresses: [email protected] (J.E. Smith), frances.ligler@
analysisofmultiplesamplesformultipleanalytes. Theimmunoas-
nrl.navy.mil(F.S.Ligler).
1 Presentaddress:DivisionofBiology,OfficeofScienceandEngineering,Centerfor saysdevelopedtodatearerapid(15–25min)andautomated,with
DevicesandRadiologicalHealth,USFoodandDrugAdministration,SilverSpring,MD little or no sample pretreatment prior to analysis [8]. Limits of
20993,USA. detection(LOD)obtainedwiththeNRLArrayBiosensorarecompa-
2 Abbreviations used: NRL, Naval Research Laboratory; LOD, limit of detection;
rable to other rapid biosensor technologies and enzyme-linked
ELISA,enzyme-linkedimmunosorbentassay;PCR,polymerasechainreaction;MP,
immunosorbent assays (ELISAs). However, the NRL system falls
magnetic particle; HSA, human serum albumin; MNP, magnetic nanoparticle;
MATEFF, magnetically assisted transport evanescent field fluoroimmunoassay; short of the LODs desired for some targets, particularly bacterial
Alexa647–chick–MNPs, MNPs functionalized with fluorescently labeled target species, compared with those obtained by the more time-
chickenIgGwithAlexaFluor647;MTS,3-mercaptopropyltrimethoxysilane;GMBS, consuming and complex ‘‘gold standard’’ methodologies such as
N-(c-maleimidobutyryloxy)succinimideester;TEOS,tetraethylorthosilicate;Mes,2-
cellcultureandpolymerasechainreaction(PCR).Toovercomethis
(N-morpholino) ethane sulfonic acid; PDMS, poly(dimethyl) siloxane; carboxyl–
silane,carboxyethylsilanetriolsodiumsalt;EDC,1-ethyl-3-[3-dimethylaminopropyl] limitation, one approach would be to include a target preconcen-
carbodiimidehydrochloride;NHS,N-hydroxysuccinimide;Rb–anti-chickIgG,rabbit tration step prior to the immunoassay. However, to keep the
anti-chickenIgY; PBS,phosphate-buffered saline; UV–Vis,ultraviolet–visible; PEG, detection method practical, any sample treatment steps must be
polyethyleneglycol;BSA,bovineserumalbumin;PBST,PBS+0.05%Tween;PBSCD,
simple to perform, add minimal time to the analysis, and improve
PBS/0.1%casein/0.05%deoxycholicacid;CCD,charge-coupleddevice;DOC,deoxy-
the overall assay results.
cholicacid.
0003-2697/$-seefrontmatter(cid:2)2010ElsevierInc.Allrightsreserved.
doi:10.1016/j.ab.2010.11.005
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
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1. REPORT DATE 3. DATES COVERED
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Optimization of antibody-conjugated magnetic nanoparticles for target
5b. GRANT NUMBER
preconcentration and immunoassays
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Naval Research Laboratory,Center for Bio/Molecular Science and REPORT NUMBER
Engineering,Washigton,DC,20375
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13. SUPPLEMENTARY NOTES
14. ABSTRACT
Biosensors based on antibody recognition have a wide range of monitoring applications that apply to
clinical environmental, homeland security, and food problems. In an effort to improve the limit of detection
of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed
and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently
labeled protein, AlexaFluor647?chicken IgG (Alexa647?chick IgG). Antibody-labeled MNPs (Alexa647?
chick?MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to
demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study
of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at
the Alexa647?chick?MNP composition, MNP surface modifications, target preconcentration conditions
and the effect that magnetic extraction has on the Alexa647?chick?MNP binding with the array surface.
The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent
proteins both for target preconcentration and for subsequent integration into immunoassays performed
under flow conditions for enhanced signal generation.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 9
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Prescribed by ANSI Std Z39-18
2 Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx
Immunomagnetic separation (IMS) is one preconcentration surface is subject to shear and subsequent dissociation from the
technique that is commonly used prior to detection for sample surface in the flow conditions normally used in such immunoas-
preparation and cleanup. Magnetic particles (MPs) are becoming says[32].Onewaytoaddressthisproblemwouldbetodecrease
increasinglypopularforautomatedseparations[9,10].Thesemag- thesizeoftheMPsused.Nano-sizedmagnetiteparticlesenveloped
netic materials are easily manipulated using magnetic fields and in lipid membranes, produced by bacteria, have been used in a
areremovedfromsolutionsin amatterofminutes.Withsurface number of studies [33]. Modifiable iron oxide-based MNPs have
modification, MPs have been labeled with a variety of biological alsobeensynthesizedwithawell-definedsizeandshape[34,35].
moleculesthathavetheabilitytoscavengefortargetsofinterest MNPsarebeingusedinELISAs[15–17],lateralflowimmunoassays
and separate them from complex biological media, potentially [36],andamagneticforcemicroscopybioassayforbiotin–strepta-
improving the LOD of subsequent analysis techniques. Commer- vidin[37]andIgGdetection[38].AlthoughMorozovandMorozova
ciallyavailableMPsaretypically0.5to2lmindiameterandcome [31,32] have studied micron-sized MPs interacting with protein
with a variety of chemically active surfaces that can be used to microarrays,todatetherearelimitedstudiesthatusenano-sized
functionalizetheparticlewiththedesiredcaptureagent,offering MPs to facilitate both target concentration and signaling events
alargesurfaceareafortargetcapture. forimmunoassays[36–38].
CommonformatsforquantificationoftargetscollectedbyMPs Inthisstudy,fluorescentlytaggedantibodiesattachedtoMNPs
aretypicallyindependentoftheparticlesthemselves.Suchmeth- were employed in a simple target preconcentration step. The
ods include culture, flow cytometry analysis [11], PCR coupled extracted target–antibody–MNPs were introduced directly to the
with hybridization [12], electrochemical measurements [13,14], ArrayBiosensorunderflowconditionstoinitiatesignaltransduc-
andELISAs[15–17].Whenfluorescencespeciesareadded,quanti- tion,andtheeffectofthetargetpreconcentrationandnanoparti-
ficationoftheresultingfluorescentimmunomagnetic–targetcom- cle-based fluorescence signal generation was evaluated. This
plex is normally achieved using devices such as a spectrometer methodwasusedtoimprovetheoverallLODoftheArrayBiosen-
[18,19], a flow cytometer [11,20], or a fluorescence microscope sor(Scheme1). Unlike thepreviouslymentionedMATEFFs,these
[21,22]. Increasingly, researchers are using the properties of the MPs are not used simply to localize the target to the evanescent
MPs themselves to determine the presence of the bound target fieldsensingsurfacebutrathertosimultaneouslyperformbotha
[23,24]withdevicessuchasgiantmagnetoresistive(GMR)sensors target concentration and a signaling function on the microarray.
[25,26],thesuperconductingquantuminterferencedevice(SQUID) For optimization purposes, a simple direct binding assay was
[27], and the magnetic permeability-based assay [28]. Interest- investigated. MNPs were functionalized with the fluorescently
ingly, Colombo and coworkers [29] recently used the proton T labeled target chicken IgG with AlexaFluor647 (Alexa647–chick–
2
relaxation time of water molecules surrounding human serum MNPs).Thesensorsurfacewaspatternedwithrabbit–anti-chicken
albumin(HSA)-modifiedmagneticnanoparticles(MNPs)asasen- IgG.Theassaywasusedtoevaluatethesurfacecompositionofthe
sorforanti-HSAdetection. modifiedMNPspreparedunderavarietyofconjugationconditions,
Advancesinmicrofluidicsandintegratedtechnologieshavere- theextractiontimeforpreconcentrationexperiments,thesample
sultedin theuseofMPscoupledwithplanarsurfaces[15,16,24– concentration achieved by magnetic extraction, and the effect
26]. Wellman and Sepaniak [30] demonstrated that magnetic of magnetic extraction on the MNP on particle aggregation and
beadsfunctionalizedwithafluorescenceantibodycomplexcould binding. The final signals were produced as the result of the
be transported, using an external magnetic field, into the region bindingoftheMNPstotheslidesurface.
ofanevanescentfieldfordetection,atechniquereferredtoasmag-
netically assisted transport evanescent field fluoroimmunoassay Materialsandmethods
(MATEFF). Morozov and Morozova [31] investigated a number of
methodsforinteractingantibody-labeledMPswithproteinmicro- Materials
arrays, including a magnetic brushing technique, magnetic scan-
ning, and a push/pull method that used a magnet below the Unlessotherwisespecified,chemicalswereofreagentgradeand
substrate to concentrate the beads on the surface and a second used as received. All chemicals, including 3-mercaptopropyl
magnetabovethesubstratetoremoveweaklyboundornonspecif- trimethoxysilane(MTS),N-(c-maleimidobutyryloxy)succinimide
icallyboundMPs[31].Theyrecentlyextendedtheirstudiestolook ester(GMBS),tetraethylorthosilicate(TEOS),and2-(N-morpholino)
more closely at force differentiation and shear stress under flow ethanesulfonicacid(Mes),werepurchasedfromSigma–Aldrich(St.
[32].ThesestudiesusecomplicatedschemestofacilitateMPinter- Louis, MO, USA) unless otherwise noted. Poly(dimethyl) siloxane
action with the surface, in large part due to the relatively large (PDMS),usedfor makingthe assay flowcells, was obtained from
sizes of the commercial MPs used. The binding of large antigen– NusilSiliconeTechnology(Carpinteria,CA,USA).Borosilicateglass
antibody–MPcomplexes to an antibodyimmobilizedon a sensor slidesfromDaigger(VernonHills,IL,USA)wereusedinallofthe
Remove supernatant
Dilute sample Concentrated sample Biosensor Magnetic Nanoparticles (MNPs)
Chicken IgG
Fluorescent Tag (Alexa647)
Y Chicken IgGAntibody
magnetic suspend analysis
extraction
m
ag
n
et
YYYYYYYY
Scheme1. Magneticextraction,sampleconcentration,andanalysis.TheMNPsmodifiedwithfluorescentlytaggedantibodyareextractedfromthelargevolumesample,the
extractedMNPsareresuspendedin0.2mlofbuffer,andanalysisisperformedusingtheArrayBiosensorwiththeMNPsfunctioningasthetracer.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx 3
assays described. Carboxyethylsilanetriol sodium salt (carboxyl– activatedMNPs.Thesolutionwasincubatedfor2hwithvortexing
silane)waspurchasedfromGelest(Morrisville,PA,USA).1-Ethyl- every 15 to 30min. The MNPs were magnetically extracted and
3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), washed three times with 500-ll aliquots of 10mM PBS buffer.
N-hydroxysuccinimide (NHS), and NeutrAvidin were purchased After the third wash, the Alexa647–chick–MNP complex was
fromPierceBiotechnology(Rockford,IL,USA).Ammoniumhydrox- resuspended in 500ll of 30mM hydroxylamine with 1% bovine
ide was obtained from Fisher. The biotin-SP-conjugated rabbit serumalbumin(BSA)in10mMPBSbuffer(pH7.4)andincubated
anti-chicken IgY (Rb–anti-chick IgG) and chicken IgY (chick IgG) for30min.Finally,theAlexa647–chick–MNPswerewashedthree
were purchased from Jackson ImmunoResearch (West Grove, PA, times and resuspended in 500-ll aliquots of 10mM PBS with
USA).NotethatIgYistheoriginaldesignationfortheIgG-likeprotein 0.05% Tween 20 and 0.1% BSA at pH 7.4. The final concentration
foundinbothserumandeggyolk;therefore,IgGisusedthroughout oftheMNPswas2mg/ml,andthesampleswerestoredat4(cid:3)Cun-
this article. Fluorescent labeling of the chicken IgG was achieved tilused.Foruse,theAlexa647–chick–MNPsweredilutedinbuffer
using succinimide ester-functionalized Alexa647 purchased from forfinalconcentrationsrangingfrom0.01to0.4mg/ml.
MolecularProbes(Eugene,OR,USA).
Slidepreparation,MNPextraction,andimmunoassay
MNPsynthesis
Microscopeslides,usedaswaveguides,werecleanedbyimmer-
IronoxideMNPsweresynthesizedbycoprecipitatingironsalts. sionin10%(w/v)KOHin2-propanolfor30minatroomtempera-
Usingamechanicalstirrer,a155-mlsolutionofammoniahydrox- ture, followed by rinsing with 18mX Milli-Q water and drying
ide (2.5%) and iron chloride was mixed at 350rpm for 10min as with a nitrogen stream. The slides were immediately immersed
describedpreviously[39].Theironsaltsolutionwasferricchloride in a toluene solution containing 2% MTS for 1h under nitrogen.
hexahydrate (0.5M), ferrous chloride tetrahydrate (0.25M), and The silanized slides were then rinsed with toluene, dried with
HCl(0.33M)toafinalvolumeof100ml.TheMNPsgeneratedwere nitrogen, and immediately immersed in 1mM GMBS in absolute
washedwith3-mlaliquotsofwaterthreetimesandwithethanol ethanol for 30min at room temperature. The slides were rinsed
once. The MNPs were dispersed in a 3-ml ethanol solution that with water and incubated in 25lg/ml NeutrAvidin in PBS over-
containedapproximately1.2%ammoniumhydroxideatafinalcon- night at 4(cid:3)C before being washed in PBS (pH 7.4). Slides were
centrationofapproximately7.5mg/ml. either used immediately for patterning or stored in PBS at 4(cid:3)C
TheMNPswerecoatedwithsilicabyadding200llofTEOS.The until required. Patterning of the biotinylated Rb–anti-chick IgG
hydrolysisprocesswasconductedwhilesonicatingfor90min.An- (10lg/ml) in PBS+0.05% Tween (PBST) was carried out using a
otheraliquotofTEOS(10ll)wasadded,andsonicationwascon- 6-channelpatterningPDMSflowcellclampedontotheNeutrAvi-
tinued for an additional 90min. The sample was again washed din-functionalizedslidesurfaceandinjectingthebiotinylatedcap-
with3-mlaliquotsofethanolthreetimes.An80-llaliquotofthe tureantibodyinto4or5ofthechannels[6–8].Biotinylatedgoat
carboxyl–silane as a sodium salt was added to 1ml of 10mg/ml anti-mouse IgG (10lg/ml in PBST) was introduced into the
silica-coatedMNPs in 10mMphosphate-buffered saline (PBS, pH remainingchannelsforuseasanegativecontrol.Theslideswere
7.4) and continuously mixed for 4h. Finally, the particles were then incubated overnight at 4(cid:3)C. After the channels were rinsed
washedthreetimeswith10mMPBSandstoredatroomtempera- with1mlofPBST,theslidewasremovedfromthePDMSpattern-
tureuntilused. ing template and placed in PBS blocking solution containing 1%
casein. After approximately 1h, the slides were rinsed with
DyelabelingofchickIgG 18mX Milli-Q water and assembled in a 6- or 12-channel assay
PDMS flow cell, with the flow channels orientated perpendicular
AlexaFluorlabelingofthechickIgGpriortoattachmenttothe tothestripesofimmobilizedbiotinylatedantibodies.Eachchannel
MNPs was carried out according to the procedure of Anderson washooked uptoan ISMATECmultichannelpump(Cole–Parmer
andNerurkar[40].Labeledantibodieswereseparatedfromunin- Instruments,VernonHills,IL,USA)atoneend(outlet),andsyringe
corporated dye using size exclusion chromatography (BioGel barrels(1ml)werethenattachedattheoppositeend(inlet),ready
P10).Protein-to-dyeratiosweredeterminedusingultraviolet–vis- fortheimmunoassay.
ible(UV–Vis)spectroscopy. The Alexa647–chick–MNPs were first sonicated and vortexed
briefly to suspend the sample. The Alexa647–chick–MNPs were
OptimizedMNPlabelingwithAlexa647–chickIgG directly diluted in 1ml of the PBS/0.1% casein/0.05% deoxycholic
acid(PBSCD)buffer(pH7.4)fordirectimmunoassaysorprepared
Theoverallgoalofthisstudywastooptimizeproteinimmobi- for extraction studies. A typical extraction procedure involved
lization onto the surface of the MNPs with respect to its subse- diluting 200ll of the stock Alexa647–chick–MNPs in 10ml of
quentimagingontheNRLArrayBiosensor.Thissectiondescribes PBSCD. The samples were prepared for the assay as described in
theoptimizedconditionsforthebestMNPsforthedualuseofcon- detailinScheme2(seedescriptionbelow).Thesamplesthatwere
centratingtheanalyteandasthetracerforthearray.Theinvesti- treatedbypathsAandBwerenotconcentrated,andthesamples
gation described here was the research involved to arrive at this thatfollowpathCwereconcentrated5-foldpriortointroduction
optimized system for the described purpose of using MNPs for intotheArrayBiosensor.
theimprovementoftheLODfortheNRLArrayBiosensor.Anum- The MNPs labeled with Alexa647–chick IgG, prepared as de-
berofparameterswereinvestigated,ashighlightedinResultsand scribedaboveandsonicatedfor5min,wereappliedtoeachchan-
discussion. The following protocol represents the final optimized nel(0.8ml) ataflowrate of0.1ml/min.Thechannelswerethen
procedure only and not all of the prerequisite investigations re- washed with 1ml of PBSCD at 0.25ml/min. The PDMS flow cell
quired in arriving at this optimized procedure. A 250-ll solution wasremoved,andtheslidewaswashedwith18mXMilli-Qwater,
of4mg/mlcarboxyl-modifiedMNPswaswashedthreetimeswith driedwithnitrogen,andimagedontheArrayBiosensor.
250-llaliquotsof0.5mMMesbuffer(pH5.0).Modificationofthe
chicken IgG was carried out by adding 50ll of a 20-mg/ml EDC Immunoassayarrayimagingandanalysis
solution to the washed particles and incubated for 15min. Next,
100lg of Alexa647–chick IgG with a 1:5 molar equivalent of an The slides were imaged using a Peltier-cooled charge-coupled
amine–PEG (polyethylene glycol, 5000Da) was added to the device (CCD) camera as described previously [6–8]. Briefly,
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
4 Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx
Diluted Sample Ref magneticextractionhasontheMNParraybindingeventwasalso
explored. For these studies, the optimization, demonstration of
1-1 mlno extraction Path:A sample concentration, and operation of the array with the MNP
tracers were performed using a direct binding assay. The assay
used Alexa647–chick–MNPs and slides patterned with Rb–anti-
Ex chickIgGslidesurfaces.
For the initial investigations with micron-sized MPs (see
1-1 mlextracted Resuspendin 1 ml Supplementary Fig. 1 in supplementary material), the samples
Path:B
mm
agag werenot extractedprior to introducingthe samplesto the Array
nn
etet Biosensor. The microparticles were internally labeled with
Alexa647 and functionalized with chicken IgG [41]. In these
Conc
studies, MPs modified with chicken IgG in various buffer condi-
Resuspendin 0.2 ml Path:C tions, surfactants, and blocking proteins were investigated. The
mm MPsdemonstratedsuccessfulbindingunderstaticconditionswith
5-1 mlsamples extracted agag
netnet buffercontainingcasein(1%)anddeoxycholicacid(DOC,0.05%)to
5-1 mlsamples minimizenonspecificparticlearraysurfacebinding.However,the
direct binding assay performed poorly under flow conditions,
Scheme 2. Sample extraction and concentration of the MNPs. Three sample resulting in low signal intensities. This poor assay performance
classificationsexist:RefobtainedfollowingpathA,ExobtainedfollowingpathB,
was probably due to the large diameter of the magnetic beads
andConcobtainedfollowingpathC.TheRefsampleisasampletakendirectlyfrom
thedilutedsamplewithnomagneticextractionperformed(noconcentration).Both andtheshearforceatthesurfaceunderflowconditions.
the Ex and Conc samples are magnetically extracted. However, after magnetic Toaddressthesizeissue,smallernano-sizedMNPssynthesized
extraction, the supernatant is removed from the Ex sample and the sample is in-housewereinvestigated.IronoxidecoreMNPs,coatedwithsil-
suspended in the volume of 1ml (resulting in no concentration). For the Conc
ica, were functionalized with carboxyl–silane, followed by the
sample,thesampleissuspendedinasmallervolumeof0.2mlafterextractionand
removalofthesupernatant.Thiscauseda5timesconcentrationoftheextracted attachmentofchickenIgGviaEDCcouplingchemistry.Transmis-
MNPs. sion electron microscope (TEM) and scanning tunneling micro-
scope (STM) images suggest that the nanoparticles are spherical,
silica-coated MNPs with sizes from 30 to 120nm, although the
evanescentwaveexcitationofthesurface-boundfluorescentspe-
majority exhibited an approximately 65-nm diameter (Supple-
cieswasachievedusinga635-nm,12-mWdiodelaser(LaserMax,
mentary Fig. 2). To generate a signal in the evanescent field of
Rochester,NY,USA).Lightwaslaunchedintooneendoftheslideat
the NRL Array Biosensor, the chicken IgG attached to the MNPs
an appropriate angle through a 1-cm focal length lens equipped
wasalsolabeledwithAlexa647dye.Typicalratioswerekeptbe-
with a line generator to generate evanescent wave excitation.
tween 2 and 4 dye molecules per chicken IgG, as determined by
The fluorescence emission was monitored at right angles to the
UV–Vis spectroscopy, to ensure that free lysines were available
planar surface. A two-dimensional graded index of refraction
forcouplingtotheMNPsurface.
(GRIN)lensarray(NipponSheetGlass,Somerset,NJ,USA)wasused
A number of factors that could affect the MNP performance
toimagethefluorescentpatternontothePeltier-cooledCCDcam-
were investigated, particularly when these silica-based materials
era(SpectraSource,Teleris,WestlakeVillage,CA,USA)[6–8].Long-
areusedwithasurfacesuchastheglassslides.Asastartingpoint,
pass(Schott0G-0665,SchottGlass,Duryea,PA,USA)andbandpass theEDC-activatedMNPswereinitiallyexposedto350lgofpuri-
(CorionS40-670-S,Franklin,MA,USA)filtersweremountedonthe fiedAlexa647–chickIgG.TheAlexa647–chickIgGMNPswerethen
devicescaffoldingtoeliminateexcitationandscatteredlightprior diluted 50, 20, or 5ll in 1ml of the running buffer PBSCD (PBS/
toCCDimaging. 0.1%casein/0.1%DOC)forassaystudies.ACCDimageoftheseini-
DatawereacquiredintheformofdigitalimagefilesinFlexible tialassaysisshowninFig.1.Thefirstfourlanesofthearraywere
Image Transport System (FITS) format. To analyze the images, a modifiedwithrabbit–anti-chickenforthetargetcapture,andthe
custom software application was written in LabWindows/CVI fifth lane was modified with goat-anti-mouse to serve as the
(NationalInstruments).Theprogramcreatesamaskconsistingof control.
data squares (enclosing the areas where the capture antibody is Strongsignalswerefoundintheregionsoftheslidefunctional-
patterned) and background rectangles that are located on either ized with Rb–anti-chick IgG. The signal intensity was concentra-
side of each data square. The average background value is sub- tion dependent. No signal was observed in the control lane,
tractedfromtheaveragedatasquarevalue,andthenetintensity demonstrating the specificity of the interaction. More important,
valueiscalculatedandimportedintoaMicrosoftExcelfilefordata these initial experiments illustrate that these antigen-coated
analysis. MNPs, unlike their larger counterparts (see Supplementary
Fig. 1), bind to the surface under flow conditions. However, the
Resultsanddiscussion nonuniform/speckled fluorescence signal observed in Fig. 1 sug-
geststhataggregationoftheMNPswasproblematic.Aggregation
MPsonthearraybiosensor
Bt-Rb-anti-chick C
Inanefforttoimprovethisbiosensor’sLODwhilemaintaininga
rapidanalysistime,theconceptofimmunomagneticconcentration
50 µl MNPs/ 1ml
ofthe targetcoupledwithsimultaneousfluorescentlabelingwas
investigated (see Scheme 1). In general, magnetic extraction was 20 µl MNPs/ 1ml
performedbyusingapermanentmagnet,thesupernatantwasre- 5 µl MNPs/ 1ml
moved,andtheextractedsamplewassuspendedin0.2mlofbuf-
fer. This concentrated sample was then introduced to the Array
Fig.1. Assay under flow conditions using the Alexa647–chick–MNPs at varying
Biosensorunderflowconditions,andthearraywasanalyzedusing
concentrationsataflowrateof0.1ml/min.Thefirstfourlanesofthearraywere
aCCDcamera.Thismethodwasemployedwhileoperatingthebio-
modifiedwithbiotinylated(Bt)rabbit–anti-chickenIgG,andthefifthlanewasthe
sensor with the MNPs as the tracer. In addition, the effect that controllaneofthearray(labeledC)andwasmodifiedwithgoat–anti-mouseIgG.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx 5
causesthenanoparticlestobehavelikelargerparticlesratherthan surfacebydecreasingthecarboxylconcentrationdidnotdecrease
monodispersednanoparticles,anditwaspossiblethatevenlarger thespecklednatureofthefluorescencesignalobservedintheCCD
aggregatesweredissociatedfromthesurfacebysheerforces.This images(datanotshown).Thissuggestedthataggregationwasstill
investigation demonstrated that the nano-sized particles showed anissueforthesesamples.Thespecklednatureofthefluorescence
promiseasatracerunderflowconditionsfortheArrayBiosensor. signalcausedamuchgreaterstandarddeviationoftheintensities
However, further experimentation to optimize the quality of the withinandbetweenspots.ThisspecklednaturecausedpoorLOD
MNPsactingasthetracerwasrequired. results from the assay and concomitant reproducibility concerns.
Therefore, the 100% carboxyl-treated MNPs were used for the
OptimizationofMNPsastracerforthearray remainder of these optimization studies because they produced
thegreatestamountofabsolutefluorescencesignal.
ToimprovetheperformanceoftheseMNPswiththebiosensor Simultaneously,theimpactoftheassaybufferandtheamount
onthe arrayslide, thisaggregationissue was examined.The fea- of protein used in the MNP immobilization reaction on particle–
turesstudiedtolimitaggregationincludethecompositionofcar- particleaggregationwereevaluatedtoreducenonspecificinterac-
boxyl groups present in the MNP modification procedure, the tions of the particles with the array surface and to promote
assay buffer conditions, and the amount of Alexa647–chick IgG selective binding of the Alexa647–chick–MNPs with the array
usedbytheMNPsandtheEDCattachmentmethodintheprotein surface (Supplementary Fig. 3). The buffer affects nonspecific
modificationreaction.Theseparameterswereinvestigatedbothto bindingofthenanoparticlesduetoelectrostaticandphysicalinter-
improve the compatibility of the particles with the array surface actions with the array surface as well as with other particles in
andtopreventparticle–particleinteractions. solution.SupplementaryFigs.3Aand3BshowMNPsreactedwith
Theproportionofcarboxylsilanepresentinthesurfacemodifi- 350lgofAlexa647–chickIgGwithPBS/0.1%casein/0.1%Tween20
cationreactionfortheMNPswasvariedtochangethedensityof andPBS/0.1%casein/0.1%DOC,respectively.Thefirstfourlanesof
charged groups and protein binding sites on the surface. The sil- the array were modified with rabbit–anti-chicken for the target
ica-coatedMNPsurfacewasexposedtosilanesolutionscontaining capture,andthefifthlanewasmodifiedwithgoat–anti-mouseto
100%,75%,50%,or25%carboxylgroups,withtheremainderofthe serveasthecontrol.Changingthesurfactantinthebufferhadlittle
solution consisting of silane containing an EDC-unreactive phos- effectoneliminatingthespeckledfluorescencesignalobservedin
phonategroup.Thesesilaneswereselectedbecausetheymaintain theCCDimages.
anegativecharge,andanegativelychargedsurfaceisnecessaryto Similarly,theamountofproteinintheimmobilizationreaction
reducetheaggregationobservedby thesesilica-basednanoparti- was optimized to reduce nonspecific particle–particle solution
cles. The carboxyl percentages represent the percentages of car- interaction due to electrostatic and physical interactions. When
boxylgroupspresentinthesolutionexposedtotheMNPsanddo the amount of Alexa647–chick IgG was reduced from 350 to
notnecessarilyequatetothepercentagesofcarboxylgroupspres- 100lg(cf.SupplementaryFigs.3Aand3C)andbothsampleswere
entonthefinalMNPsurface.Theamountofproteinimmobilized analyzed using the PBS/casein/DOC buffer, the resulting fluores-
to the MNPs was either 350 or 100lg of Alexa647–chick IgG in cencesignalwasmuchmoreuniforminintensitywithintheindi-
the presence of EDC-activated MNPs. The Alexa647–chick–MNPs vidual data points. This result suggests that less aggregationwas
in0.8ml(150llin1mlPBS/0.1%casein/0.05%DOC)werepassed observedwiththelowerconcentrationofAlexa647–chickusedin
over an antibody-functionalized surface at a flow rate of 0.1ml/ the protein attachment procedure. Other buffer types were used
min.Theresultingbargraph(Fig.2A)showstherelativeintensity with the 100lg Alexa647–chick IgG prepared samples (data not
of the captured Alexa647–chick–MNPs. The black and gray bars shown), but PBS/casein/DOC provided the highest quality perfor-
represent the MNPs fabricated using either 350 or 100lg of manceoftheAlexa647–chick–MNPsonthearray.
Alexa647–chick IgG, respectively. For the 100-lg samples, only ThenextstepintheoptimizationoftheMNPsforthearrayin-
100 and 25% carboxyl were used; these two amounts confirmed volvestheproteinattachmentreactionprocedure.Heretheeffect
thetrendbasedonamorecompleterangetestedusingMNPsmod- of the EDC reaction conditions used to attach 100lg of
ifiedwith350lgofantigen.Theconcentrationofcarboxylgroups Alexa647–chick IgG on the surface of 100% carboxyl MNPs was
ontheparticlesurfacewasmoreimportantforenhancingtheuse investigated.Inthestudiesmentionedabove,theEDCwasnotre-
oftheparticlesasatracerthantheconcentrationofproteinusedin moved from the MNP solution prior to the addition of the
thecouplingreaction.Higherconcentrationsofcarboxylgroupsin- Alexa647–chick IgG (EDC method I). Because the protein chicken
creasedtheamountofproteinbound. IgGcontainsbothcarboxylandaminegroups,itwaspossiblethat
However, in the case of the MNPs exposed to 350lg of the multilayersofAlexa647–chickIgGcouldformonthesurfaceofthe
Alexa647–chick IgG, decreasing the protein concentration on the MNPs.Multilayerscouldencourageincreasedaggregation,causing
A B
120 120
y 100 y 100
sit sit
en 80 en 80
nt nt
e i 60 e i 60
v v
ati 40 ati 40
el el
R 20 R 20
0 0
100 75 50 25 EDC I EDC IIEDC IIIEDC IVEDC V
% Carboxyl in reaction EDC method
Fig.2. OptimizationoftheMNPsurfacetreatment.(A)EffectofthepercentagecarboxylcompositionusedintheMNPmodificationreactiononthefluorescenceintensity
observedonthearray.TheblackbarsrepresenttheproteinattachmentreactiontotheMNPswith350lgofAlexa647–chickIgG,andthegraybarsrepresenttheprotein
reactionproceduretotheMNPswith100lgofAlexa647–chickIgG.(B)EffectoftheEDCmethod(refertoTable1)usedtoattach100lgofAlexa647–chickIgGonthesurface
of100%carboxylMNPs.TheresultingbargraphshowstherelativeintensityoftheMNPscapturedbythearraysurfaceforthreeseparateslides.Slide1(black)andslide2
(lightgray)involvedPBSwashesfortheMNPs,whereasslide3(gray)usedMeswashes.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
6 Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx
Table1 and PBS/0.1% casein/0.05% DOC was used as the buffer in the
EDCprotocolsinvestigatedforMNPmodificationwithAlexa647–chickIgG. immunocaptureexperiments.
EDCexposure EDCMNPactivationprocedurepriortoadditionof
method Alexa647–chickIgG MNPextraction
EDCI EDC/resuspendinIgGsolution
EDCII EDC/magneticextraction/suspendinIgGsolution The next step was to investigate the extraction/concentration
EDCIII EDC/magneticextraction/PBSorMeswash/magnetic procedures, outlined in Scheme 2, of the Alexa647–chick–MNPs
extraction/suspendinIgGsolution
todeterminehowtheMNPsperforminadirectassayformatand
EDCIV EDC+NHS/magneticextraction/PBSwash/magnetic
tocharacterize theimpactofthetargetconcentration processon
extraction/suspendinIgGsolution
EDCV EDC(5(cid:2))/magneticextraction/Meswash/magnetic theassay.Forthisinvestigation,threedistinctsamplepreparation
extraction/suspendinIgGsolution protocolswereused.Tobegin,200lloftheAlexa647–chick–MNPs
wasdilutedin10mlofPBSCD.PathAwassimplya1-mlaliquotof
theoriginaldilutedsamplewithnoextraction/concentrationpro-
cedureperformedonthesample.Thissamplewasusedastheref-
nonuniformarrayspots,nonreproduciblestandarddeviations,and erence(Ref)forcomparisonpurposes.PathBprovidedacontrolfor
greaterstandarddeviationsinthemeasuredfluorescentintensity theextractionprocesswithoutconcentration;a1-mlaliquotofthe
signals.ToinvestigatetheeffectoftheEDCmethodontheamount originaldilutedsamplewasconcentratedusingamagnetandsus-
ofAlexa647–chickIgGattachedtothesurfaceoftheMNPs,fivedif- pendedinafresh1-mlaliquotofthePBSCDrunbuffer.Thissample
ferentprocedureswereperformed.Theseproceduresaresumma- waslabeledextracted(Ex).PathCwasa1-mlaliquotoftheorigi-
rizedinTable1. nal diluted sample that underwent the extraction procedure and
The general approach used 100ll of the prepared Alexa647– wasthenconcentratedina0.2-mlaliquotofthePBSCDrunbuffer,
chick–MNPs and diluted the sample to 1ml of PBSCD. Then resultinginaneffectivesampleconcentrationoffivetimesthatof
0.8ml of this solution was passed over an Rb–anti-chick IgG- theoriginaldilutedsample.Thissamplewaslabeledconcentrated
functionalized surface at a flow rate of 0.1ml/min. The resulting (Conc).PathCwasperformedatotaloffivetimestoobtaina1-ml
bar graph (Fig. 2B) shows the relative intensity of the Alexa647– sampletobeusedforcharacterization.
chick–MNPspreparedusingEDCmethodsItoVandcapturedby Tobeefficient,theextractiontimerequiredtorecoverthebulk
the rabbit–anti-chick IgG-functionalized surface. The net intensi- of the MNPs from the large volume samples was studied. For
tiesobtainedfromtheCCDimageswerenormalizedtotheEDCI extraction, an Eppendorf magnet was used to extract the
procedure, which produced the brightest fluorescence intensity Alexa647–chick–MNPs. The Eppendorf magnet system can hold
ontheslidesurface.Slides1and2usedaPBSwashforEDCmethod up to 6 (cid:2) 1.5-ml Eppendorf tubes. For the extraction study, four
III,wherethesebarsareblackandlightgray,respectively.Forslide 1-ml aliquots of the diluted sample were placed in the magnet
3(darkgraybars),thisdatasetusedEDCmethodIIIwithMesbuf- apparatus, and after 3, 7, 15, or 30min, the sample supernatant
ferasthewashsolution.Thewashproceduresattemptedtoreduce wascollected.Thiswasachievedbyremovaloftheliquidportion
theopportunityformultilayersofproteinstoformabovetheMNP of the sample from the Alexa647–chick–MNPs, and the particle
surfaceand,therefore,reduceaggregation. samplesweresuspendedin0.5mlofPBSCD.Thisresultedinacon-
Asexpected,theEDCIprocedureresultedinthebrightestsig- centrationfactorof2.TheextractedAlexa647–chick–MNPsanda
nals obtained from the Rb–anti-chick IgG-functionalized regions portionofthepreextractedsample(both0.5ml)werepassedover
becauseofagreaterchanceformultilayerformation.Thesesam- anantibody-patternedsurfaceataflowrateof0.1ml/min.Relative
pleshadthegreaternumberoffluorophore-labeledproteinsand, signalintensityversusextractiontime,takenfromtheCCDimage
therefore, more signaling molecules present on the MNPs. The (data not shown), suggested that 15min was optimal for the
EDCIIprocedureincludedremovaloftheEDCpriortotheaddition extractionprocedure.Thesedataalsodemonstratedthat,although
ofAlexa647–chickIgGandresultedinasignificantdecreaseinthe the Alexa647–chick–MNPs were concentrated by a factor of 2 as
overallintensityoftheMNPs.Thisprocedurereducedthechance determined by UV–Vis spectroscopy, this did not translate to an
for multilayerformationand, therefore,decreasedthe numberof increased intensity from the CCD array biosensor image for the
signalingmoleculesontheMNPsurface.Afurtherreductioninsig- concentrated samples. In fact, the fluorescence appeared to be
nalwasobservedintheEDCIIIprocedure,whereaPBSwashstep slightlylowerthanthatfortheoriginalpreextractedsample.Son-
wasincludedpriortoIgGexposure.TheEDCreactiveintermediate icationoftheconcentratedAlexa647–chick–MNPsfor5minversus
was found to be more stable using a wash step at the lower pH thepreviouslyused1-minsonicationtimegaveasignalintensity
(Mes[pH5.0–5.5]vs.PBS[pH7.4])andproducedahigherrelative improvementuptoafactorofapproximately2.Thissuggeststhat
intensityfortheMeswashedsamplesinFig.2B.Allvariationsof the drop in signal that was observed when the samples were
theEDCIItoVpreparationconditionswerenottestedunlessthe extracted was likely due to aggregation. Aggregation of the
initialtrialseitherreducedaggregationorproducedhighersignals Alexa647–chick–MNPs led to the larger particle effect (described
thantheEDCImethod. previously),whichessentiallyindicatedthatlargerparticleswere
NeithertheadditionofNHS(EDCIV,lightgraybar),whichisre- not as effectively captured by the array surface under the shear
portedtostabilizethereactiveEDCintermediate,noranexcessof forceoftheflowconditions(seeSupplementaryFig.1).Therefore,
EDC(EDCV,darkgraybar)seemedtoincreasetheintensityofthe these results indicate that magnetic extraction can have a detri-
signal obtained relative to the EDC III procedure. UV–Vis absor- mentaleffectontheperformanceoftheMNPsasatracerinimmu-
bance of the MNPs in suspension also confirmed that more IgG noassays. This phenomenon was further investigated to improve
was removed from the reaction solution when EDC I versus EDC theperformanceofextractedparticlesonthearraysystem.
IIIwasused(datanotshown).AlthoughEDCmethodIprobablyre- A number of extraction experiments using the optimized
sults in multilayers of Alexa647–chick IgG on the surface of the Alexa647–chick–MNPs coupled with a 5-min sonication prior to
MNPs, it also produces the brightest fluorescence signals when use on the immunoassay were performed. Alexa647–chick IgG
theMNPsarecapturedonthesurfaceofthebiosensor.Therefore, (100lg) was attached to the surface of 100% carboxyl-modified
EDC method I along with addition of 100lg Alexa647–chick IgG MNPs using the EDC I method (no wash). The Alexa647–chick–
to the EDC-activated MNPs that were made in the presence of MNPs(200ll)weredilutedto10mlinPBSCDasdescribedabove
100% carboxyl–silane, was used as the optimized MNP protocol, forScheme2usingthe15-minextractiontimewhereappropriate.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx 7
Table2 C Bt-Rb-anti-chick
AveragesolutionandsurfacemeasurementcharacterizationoftheAlexa647–chick–
MNPsamplespre-andpostextractiondisplayedasConc/Refratio(seeScheme2).
Ref
Extraction
Chick–MNPsample Solution Solution Surface carried out day
Ex
absorbance fluorescence fluorescence before the assay
(400nm) emission(670nm) Conc
MNP 3.0 2.3 1.2 Ref
MNPovernight 2.9 2.1 1.6 Extraction
5A:1PPEGA 3.0 1.6 0.9 Ex carried out day
1A:1PPEGA 4.0 2.4 1.3 of the assay
1A:5PPEGA 4.5 2.6 1.5 Conc
1A:10PPEGA 4.3 1.9 0.6
5A:1PPEGB 2.8 1.4 0.6 Fig.3. Effectoftimeofanalysisontheassayresults.Thesampleswerepreparedfor
1A:1PPEGB 3.6 2.4 1.0 theassayasfollows:Ref,Ex,andConc(refertoScheme2).Thisprocedurewas
1A:5PPEGB 3.7 2.0 0.9 performedeitherthedaybeforetheassaywasusedandthesampleswerestoredin
PBSat4(cid:3)C(overnight)oronthedayoftheassay.Thesamplesweresonicatedfor
5minpriortouse.TheresultingCCDimageisshown.C,control;Bt,biotinylated.
For the Ref and Conc samples, these samples were sonicated for
5min,and50llofthesesampleswasdilutedin950llof18mX
the surface under the flow conditions of the assay. This may ex-
Milli-Qwater.ThiswasdonesothatUV–Visandfluorescencespec-
plainthedecreaseinsignalfromthesurface.Itwasfoundthatif
troscopy measurements could be obtained. Table 2 summarizes
extractedsampleswereallowedtositovernightintherefrigerator
the relative ratio of the Conc versus the Ref samples for solution
beforeperformingtheimmunoassay,asignificantincrease(nearly
and surface characterization of the NRL Array Biosensor for an
double)inthefluorescentsignalobtainedfromtheCCDimageof
average of five separately prepared batches of these Alexa647–
the surface was observed (Fig. 3 and Table 2, MNPs overnight).
chick–MNPs. The concern was that the MNPs were being lost
These data suggested that if MNP aggregation was the cause of
throughtheextractionprocedure.Therefore,UV–Visspectroscopy
theselowersignalintensities,theaggregationwasatleastpartially
wasusedtodeterminewhethertheMNPsthemselveswerebeing
reversible,andfurtherinvestigationtoovercomethisproblemwas
removedfromsolution.
performed.
The solution UV–Vis values obtained at 400nm demonstrated
thatanincreaseintheconcentrationoftheMNPsinthesolution
OptimizedMNPsforextractionandimprovedarrayperformance
was achieved. Likewise, the solution fluorescence at 650nm
Alexa647–chick–MNPs and the intensity taken from the CCD
The main issue with extracting the samples and leaving the
imagesfromtheRb–anti-chickIgG-functionalizedregionsfollow-
MNPsovernightpriortoassayistheincompatibilityforrapidanal-
ing the immunoassay were obtained. As shown in Table 2, both
ysistime.Therefore,wedecidedtoinvestigatetheadditionofPEG
thesolutionabsorbanceand,toalesserextent,thesolutionfluo-
to the surface of the Alexa647–chick–MNPs to see whether this
rescence showed an increase in intensity following extraction.
TheabsorbanceresultsuggeststhattheMNPsampleswereeffec-
tively collected through the magnetic extraction procedure but A C Bt-Rb-anti-chick
thattheextractionprocedurehasanadverseeffectonthefluores-
cence signaling. In addition, the observed concentration did not 100 ng/ml Alexa647-chick IgG
translatetoasignificantincreaseinCCDsurfacefluorescencesig- 1A:5P PEG A
nal generated from the immunoassay-captured Alexa647–chick–
1A:5P PEG A PBSTB
MNPs.ThisislikelyaresultofMNPaggregationandlargerparti-
clesthatmightnotbeaseffectivelycapturedbythesurfaceunder 1A:1P PEG A
theflowconditionsoftheassay.Inaddition,thisobservationwas 5A:1P PEG A
alsonotedforsamplespreparedusingtheEDCIIImethod.There-
1A:1P PEG A EDC III
fore, the potential for multilayers of chick IgG on the surface of
theMNPscausingtheseobservationscanbeeffectivelyruledout.
B
Atthispoint,theMNPshavebeendemonstratedtofunctionwell B 3300000000
eitherasthetracerfortheArrayBiosensororastheconcentration
agent,butwhenusingtheMNPsforbothfunctionssimultaneously, 2255000000
further investigation into the effect that magnetic extraction has yy
ontheMNParraybindingeventneedstobeperformed. nsitnsit 2200000000
tahteedTlhofewronemrextththapenhieamxsempeiuncntteohdaissisniacnyrv-eecasastpeigtiuanrteiCodCnDAwlfleauxsoatr6oe4s7dc–eetcnehcriemcksii–ngMenaNwlPhgseentwheaers-r et InteNet inte 11110505000000000000
N
aresultoftheextractionortheconcentrationoftheMNPs.Anex- 55000000
tra control sample was added to the immunoassay called the Ex
sample(seeScheme2,pathB).Althoughtheabsorbancemeasure- 00
55AA::11PP 11AA::11PP 11AA::55PP 11AA::11PP wWaAsShH
ments suggested that the concentrations of the Alexa647–chick–
CChhicickk--IIggGG ((AA))/:PPEEGG ( P(P) r)a Rtioatio
MNPsintheRefandExsolutionswerethesame(Scheme2,paths
AandB,respectively),boththesolutionfluorescenceandsurface
Fig.4. EffectofmodificationofMNPswithAlexa647–chickIgGandPEGmolecules.
intensitytakenfromtheCCDimagesuggestadropinthefluores- Alexa647–chick IgG in the presence of either PEG A (5000MW) or PEG B
cencefortheAlexa647–chick–MNPsfollowingextraction(seethe (10,000MW)atdifferentmoleratioswasattachedtotheMNPsusingtheEDCI
lowerhalfofFig.3).Thissuggestedthattheextractionprocessit- method(nowash)ortheEDCIIImethod(Meswash).(A)TheresultingCCDimage
forthedifferentMNPsmodifiedwithAlexa647–chickIgG/PEGA.Bt,biotinylated.
selfwasaffectingthefluorescencefromtheMNPs.Aggregationof
(B)BargraphplottingthenetintensitiestakenfromtheCCDimageforthedifferent
theAlexa647–chick–MNPsmayproduceparticlestoolargetore-
Alexa647–chick IgG MNPs modified with either PEG A (black) or PEG B (gray)
main bound to the surface during the shear force that occurs at molecules.
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assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
8 Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx
would help to prevent the aggregation. PEG is known to prevent chance of losing the target analyte. Furthermore, little time was
nonspecificinteractionsbyincreasingthehydrophiliccharacteris- added to the overall assay protocol, and the target was concen-
ticsofthenanoparticles[42].Inthiscase,thePEGwasemployedto trated prior to performing the analysis. The next step will be to
reducetheparticle–particleinteractionsandthenonspecificinter- demonstrate sandwich assays where the analyte is pulled out of
actions occurring between the functionalized nanoparticles and solutionbytheMNPscoatedwithfluorescentantibodiesandcon-
thearraysurface.Carboxyl-activatedMNPs,activatedusingeither centratedpriortobiosensoranalysis.
theEDCI(nowash)orEDCIII(Meswash)protocol,weresimulta-
neously exposed to Alexa647–chick IgG (100lg) and either Acknowledgments
amine–PEGA(5000MW)oramine–PEGB(10,000MW)atdiffer-
ent mole ratios. Alexa647–chick–PEG–MNPs were then diluted The work was supported by NRL 6.2 work unit 62123 and by
50llin1mlofPBS/0.1%casein/0.05%DOC.Thesamples(0.8ml) NSF/NIRT Grant EF-0304569. The views expressed are those of
werepassedover anantibody-patternedsurfaceata flowrateof the authors and do not represent those of the U.S. Navy, U.S.
0.1ml/min. The resulting CCD image for the different Alexa647– DepartmentofDefense,orU.S.Government.
chickIgG/PEGA-modifiedMNPsisshowninFig.4A(PEGBnotdis-
playedinimage).Thebargraph(Fig.4B)plotsthenetintensities
AppendixA.Supplementarydata
takenfromtheCCDimageforthedifferentAlexa647–chick–MNPs
modifiedwitheitherPEGA(black)orPEGB(gray)molecules.As
Supplementarydataassociatedwiththisarticlecanbefound,in
illustrated in Fig. 4B, the smaller PEG A-modified Alexa647–
theonlineversion,atdoi:10.1016/j.ab.2010.11.005.
chick–MNPs produce slightly stronger fluorescent signals from
theCCDimagethandoesthecorrespondingratioofPEGB-modi-
fied Alexa647–chick–MNPs. For both PEG molecules (A and B), References
the fluorescence intensity increased slightly with increasing PEG
[1] D.V. Lim, J.M. Simpson, E.A. Kearns, M.F. Kramer, Current and developing
ratioscomparedwiththeExsamples.
technologies for monitoring agents of bioterrorism and biowarfare, Clin.
Data from the extraction experiments are summarized in Microbiol.Rev.18(2005)583–607.
Table2forboththesolutionandsurfacecharacterization.Therel- [2] S.Song,H.Xu,C.Fan,Potentialdiagnosticapplicationsofbiosensors:current
ative intensity ratio determined from the intensity of the Conc andfuturedirections,Int.J.Nanomed.1(2006)433–440.
[3] L.M.Eubanks,T.J.Dickerson,K.D.Janda,Technologicaladvancementsforthe
sample divided by the intensity of the Ref sample is presented. detectionofandprotectionagainstbiologicalandchemicalwarfareagents,
The ratios are compared in the UV–Vis spectroscopy at 400nm, Chem.Soc.Rev.36(2007)458–470.
in the solution fluorescence at 670nm, and the average fluores- [4] C.Blasco,Y.Picó,Prospectsforcombiningchemicalandbiologicalmethodsfor
integratedenvironmentalassessment,TrendsAnal.Chem.28(2009)745–757.
cence intensities determined from the CCD array image. The
[5] P.J.Conroy,S.Hearty,P.Leonard,R.J.O’Kennedy,Antibodyproduction,design,
extractionexperimentswithAlexa647–chick–MNPsmodifiedwith anduseforbiosensor-basedapplications,Semin.CellDev.Biol.20(2009)10–
the smaller of the amine–PEG molecules (PEG A, 5000MW) 26.
[6] C.R.Taitt,L.C.Shriver-Lake,M.M.Ngundi,F.S.Ligler,Arraybiosensorfortoxin
showed promising initial data. The Alexa647–chick–MNPs modi-
detection:continuedadvances,Sensors8(2008)8361–8377.
fiedwithPEGA,ataratioof1:5chickIgG/PEGA,providedasimilar [7] K.E Sapsford, C.R Taitt, F.S Ligler, Planar waveguides for fluorescence
enhancementinCCDfluorescentsignalinlesstimethantheregu- biosensors, in: F.S. Ligler, C.R. Taitt (Eds.), Optical Biosensors: Today and
Tomorrow,Elsevier,Amsterdam,Netherlands,2008,pp.139–184.
larAlexa647–chick–MNPsextractedandleftovernight.Therefore,
[8] F.S.Ligler,K.E.Sapsford,J.P.Golden,L.C.Shriver-Lake,C.R.Taitt,M.A.Dyer,S.
using PEG A in a commodification procedure with 100lg of Barone,C.J.Myatt,Thearraybiosensor:portable,automatedsystems,Anal.Sci.
Alexa647–chick IgGonthepreviouslydescribedoptimizedMNPs 23(2007)5–10.
[9] M.A.M.Gijs,Magneticbeadhandlingon-chip:newopportunitiesforanalytical
improved immunoassay performance with the shortest time
applications,MicrofluidicsNanofluidics1(2004)22–40.
betweenextractionandanalysis. [10] M.Magnani,L.Galluzzi,I.J.Bruce,Theuseofmagneticnanoparticlesinthe
developmentofnewmoleculardetectionsystems,J.Nanosci.Nanotechnol.6
Conclusion (2006)2302–2311.
[11] S.Yitzhaki,E.Zahavy,C.Oron,M.Fisher,A.Keysary,ConcentrationofBacillus
sporesbyusingsilicamagneticparticles,Anal.Chem.78(2006)6670–6673.
ThisextensivestudyhasdemonstratedtheuseofMNPsastrac- [12] C.Metzger-Boddien,D.Khaschabi,M.Schönbauer,S.Boddien,T.Schlederer,J.
ersforimmunoassaysperformedonabiosensorsurfaceandchar- Kehle, Automated high-throughput immunomagnetic separation–PCR for
detection of Mycobacterium avium subsp. paratuberculosis in bovine milk,
acterized the effect that extraction of the MNPs has on the
Int.J.FoodMicrobiol.110(2006)201–208.
performanceoftheseMNPs.TheuseofMNPsforthesimultaneous [13] H.Kuramitz,Magneticmicrobead-basedelectrochemicalimmunoassays,Anal.
functionoftargetpreconcentrationandsignaltransductioninbio- Bioanal.Chem.394(2009)61–69.
[14] D. Tang, B. Su, J. Tang, J. Ren, G. Chen, Nanoparticle-based sandwich
sensorassaysperformedunderflowconditionswasdemonstrated
electrochemical immunoassay for carbohydrate antigen 125 with signal
using a direct binding assay format. The optimal conditions for enhancement using enzyme-coated nanometer-sized enzyme-doped silica
synthesizingtheMNPsweredeterminedbyexploringthesurface beads,Anal.Chem.82(2010)1527–1534.
[15] M.Tudorache,I.A.Zdrojewska,J.Emnéus,Evaluationofprogesteronecontent
composition, antibody functionalization procedures, and various
insalivausingmagneticparticle-basedimmunosupportedliquidmembrane
blocking buffers. In addition to investigating the MNP synthesis, assay(m-ISLMA),Biosens.Bioelectron.22(2006)241–246.
the best extractiontime and methodfor introducingtheconcen- [16] M.Tudoracher,A.Tencaliec,C.Bala,Magneticbeads-basedimmunoassayasa
sensitivealternativeforatrazineanalysis,Talanta77(2008)839–843.
trated MNPs to the biosensor were ascertained. We determined
[17] G.Pappert,M.Rieger,R.Niessner,M.Seidel,Immunomagneticnanoparticle-
that magnetic separation of the MNPs had an adverse effect on basedsandwichchemiluminescence–ELISAfortheenrichmentandquantifi-
thesemodifiedparticles,andfurtherstudyintonanoparticlesur- cationofE.coli,Microchim.Acta168(2010)1–8.
facetreatmentwasperformedbyaddingPEGpolymersalongwith [18] H.Yu,Useofanimmunomagneticseparation–fluorescentimmunoassay(IMS–
FIA)forrapidandhighthroughputanalysisofenvironmentalwatersamples,
theantibodyduringtheimmobilizationstep. Anal.Chim.Acta376(1998)77–81.
Previous experiments using micron-sized MPs in conjunction [19] J.Wang,Q.Wang,L.Ren,X.Wang,Z.Wan,W.Liu,L.Li,H.Zhao,M.Li,D.Tong,
withsensingunderflowconditionswerefoundtobeproblematic. J.Xu,Carboxylatedmagneticmicrobead-assistedfluoroimmunoassayforearly
biomarkersofacutemyocardialinfarction,ColloidsSurf.B72(2009)112–120.
The fabrication of nano-sized MNPs was essential to reduce the
[20] K.Hibi,A.Abe,E.Ohashi,K.Mitsubayashi,H.Ushio,T.Hayashi,H.Ren,H.Endo,
shearing effect of the fluid flow on the surface-bound particles. Combination of immunomagnetic separation with flow cytometry for
Including the MNPs in the assay instead of removing the bound detectionofListeriamonocytogenes,Anal.Chim.Acta573-574(2006)158–163.
[21] L.S.L. Yu, J. Uknalis, S.-I. Tu, Immunomagnetic separation methods for the
targetorrequiringtheadditionofasecondaryfluorescentspecies
isolation of Campylobacter jejuni from ground poultry meats, J. Immunol.
minimized the number of steps in the assay and reduced the Methods256(2001)11–18.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005
Optimizationofantibody-conjugatedMNPs/J.E.Smithetal./Anal.Biochem.xxx(2010)xxx–xxx 9
[22] J.Steingroewer,H.Knaus,T.Bley,E.Boschke,Arapidmethodforthepre- [33] Y.Amemiya,T.Tanaka,B.Yozaand,T.Matsunaga,Noveldetectionsystemfor
enrichment and detection of Salmonella typhimurium by immunomagnetic biomolecules using nano-sized bacterial magnetic particles and magnetic
separationandsubsequentfluorescencemicroscopicaltechniques,Eng.Life forcemicroscopy,J.Biotechnol.120(2005)308–314.
Sci.5(2005)267–272. [34] S.Santra,R.Tapec,N.Theodoropoulou,J.Dobson,A.Hebard,W.Tan,Synthesis
[23] Y.Lalatonne,F.Benyettou,D.Bonnin,N.Lièvre,P.Monod,M. Lecouvey,P. and characterization of silica-coated iron oxide nanoparticles in micro-
Weinmann, L. Motte, Characterization of magnetic labels for bioassays, J. emulsion:theeffectofnonionicsurfactants,Langmuir17(2001)2900–2906.
Magn.Magn.Mater.321(2009)1653–1657. [35] Y.Lu,Y.Yin,B.T.Mayers,Y.Xia,Modifyingthesurfacepropertiesofsuperpara-
[24] T.Aytur,J.Foley,M.Anwar,B.Boser,E.Harris,P.R.Beatty,Anovelmagnetic magneticironoxidenanoparticlesthroughasol-gelapproach,NanoLett.2
beadbioassayplatformusingamicrochip-basedsensorforinfectiousdisease (2002)183–186.
diagnosis,J.Immunol.Methods314(2006)21–29. [36] K. Taton, D. Johnson, P. Guire, E. Lange, M. Tondra, Lateral flow immuno-
[25] R.L. Edelstein, C.R. Tamanaha, P.E. Sheehan, M.M. Miller, D.R. Baselt, L.J. assay using magnetoresistive sensors, J. Magn. Magn. Mater. 321 (2009)
Whitman,R.J.Colton,TheBARCbiosensorappliedtothedetectionofbiological 1679–1682.
warfareagents,Biosens.Bioelectron.14(2000)805–813. [37] T.Osaka,T.Matsunaga,T.Nakanishi,A.Arakaki,D.Niwa,H.Iida,Synthesisof
[26] J.Schotter,A.Shoshi,H.Brueckl,Developmentofamagneticlab-on-a-chipfor magnetic nanoparticles and their application to bioassays, Anal. Bioanal.
point-of-caresepsisdiagnosis,J.Magn.Magn.Mater.321(2009)1671–1675. Chem.384(2006)593–600.
[27] H.E. Horng, S.Y. Yang, Y.W. Huang, W.Q. Jiang, C.-Y. Hong, H.C. Yang, [38] X.Hong,Y.Liu,J.Li,W.Guo,Y.Bai,Detectionofmagnetic-labeledantibody
NanomagneticparticlesforSQUID-basedmagneticallylabeledimmunoassay, specific recognition events by combined atomic and magnetic force
IEEETrans.Appl.Supercond.15(2005)669–671. microscopy,J.Magn.Magn.Mater.321(2009)2607–2611.
[28] F.Ibraimi,K.Kriz,H.Merin,D.Kriz,Magneticpermeabilitybaseddiagnostic [39] J.K.Herr,J.E.Smith,C.D.Medley,D.Shangguan,W.Tan,Aptamer-conjugated
testforthedeterminationofthecanineC-reactiveproteinconcentrationin nanoparticlesforselectivecollectionanddetectionofcancercells,Anal.Chem.
undilutedwholeblood,J.Magn.Magn.Mater.321(2009)1632–1634. 78(2006)2918–2924.
[29] M.Colombo,S.Ronchi,D.Monti,F.Corsi,E.Trabucchi,D.Prosperi,Femtomolar [40] G.P. Anderson, N.L. Nerurkar, Improved fluoroimmunoassays using the dye
detection of autoantibodies by magnetic relaxation nanosensors, Anal. AlexaFluor647withtheRAPTOR,afiberopticbiosensor,J.Immunol.Methods
Biochem.392(2009)96–102. 271(2002)17–24.
[30] A.D. Wellman, M.J. Sepaniak, Multiplexed, waveguide approach to [41] G.U.Lee,S.Metzger,M.Natesan,C.Yanavich,Y.F.Dufrêne,Implementation
magneticallyassistedtransportevanescentfieldfluoroassays,Anal.Chem.79 of force differentiation in the immunoassay, Anal. Biochem. 287 (2000)
(2007)6622–6628. 261–271.
[31] V.N. Morozov, T.Y. Morozova, Active bead-linked immunoassay on protein [42] L. Wang, C. Lofton, M. Popp, W. Tan, Using luminescent nanoparticles as
microarrays,Anal.Chim.Acta564(2006)40–52. stainingprobesforAffymetrixGeneChips,Bioconjug.Chem.18(2007)610–
[32] T.Y. Morozova, V.N. Morozov, Force differentiation in recognition of cross- 613.
reactiveantigensbymagneticbeads,Anal.Biochem.374(2008)263–271.
Pleasecitethisarticleinpressas:J.E.Smithetal.,Optimizationofantibody-conjugatedmagneticnanoparticlesfortargetpreconcentrationandimmuno-
assays,Anal.Biochem.(2010),doi:10.1016/j.ab.2010.11.005