A smartphone based surface plasmon resonance imaging (SPRi) platform for on-site biodetection

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ContentslistsavailableatScienceDirect

Sensors

and

Actuators

B:

Chemical

j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b

A

smartphone

based

surface

plasmon

resonance

imaging

(SPRi)

platform

for

on-site

biodetection

Hasan

Guner

a

_,

_Erol

_Ozgur

a

_,

_Guzin

_Kokturk

b

_,

_Mehmet

_Celik

c

_,

_Elif

_Esen

b

_,

_Ahmet

_E.

_Topal

a

_,

Sencer

Ayas

d

_,

_Yildiz

_Uludag

b

_,

_Caglar

_Elbuken

a

_,

_Aykutlu

_Dana

a,∗

a_UNAM,_Institute_of_Materials_Science_and_{Nanotechnology,}_Bilkent_University,_Ankara,_06800,_Turkey

b_{UEKAE-BILGEM,}_The_Scientiﬁc_and_{Technological}_Research_Council_of_Turkey_(TUBITAK),_Gebze,_Kocaeli,_41470,_Turkey

c_Department_of_Computer_Engineering,_Middle_East_Technical_University,_Ankara,_06800,_Turkey

d_Canary_Center_at_Stanford_for_Cancer_Early_Detection,_Department_of_Radiology,_Stanford_School_of_Medicine,_Palo_Alto,_CA_94304,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2July2016

Receivedinrevisedform5August2016

Accepted10August2016

Availableonline10August2016

Keywords: Plasmonics

Surfaceplasmonresonanceimaging

Opticaldiscs

Mobilesensing

Biosensors

Opticalsensor

a

b

s

t

r

a

c

t

Wedemonstrateasurfaceplasmonresonanceimagingplatformintegratedwithasmartphonetobe usedintheﬁeldwithhigh-throughputbiodetection.InexpensiveanddisposableSPRsubstratesare pro-ducedbymetalcoatingofcommercialBlu-raydiscs.Acompactimagingapparatusisfabricatedusinga 3DprinterwhichallowstakingSPRmeasurementsfrommorethan20.000individualpixels.Real-time bulkrefractiveindexchangemeasurementsyieldnoiseequivalentrefractiveindexchangesaslowas 4.12×10−5RIUwhichiscomparablewiththedetectionperformanceofcommercialinstruments.Asa demonstrationofabiologicalassay,wehaveshowncaptureofmouseIgGantibodiesbyimmobilized layerofrabbitanti-mouse(RAM)IgGantibodywithnanomolarlevellimitofdetection.Ourapproach inminiaturizationofSPRbiosensinginacost-effectivemannercouldenablerealizationofportableSPR measurementsystemsandkitsforpoint-of-careapplications.

1. Introduction

Thewide useofmobilephonesallacrosstheworld created signiﬁcantopportunitiesforhealthcareapplicationsusingmobile devices. Improvement of healthcare services requires democ-ratization of the services with higher quality and lower cost. Early diagnosis, close monitoring,patient comfort are some of theconcerns that healthcare providers are strivingto improve on.Thedevelopmentoflab-on-a-chip platformsin thelast two decades brought severalexamples of novel platforms that can beused for rapid diagnosisof widespreaddiseases. Portability, shortturn-around-time,cost-efﬁciencyandconnectivityaresome ofthecriticalassetsthatsuccessfuldevicesshouldpossess.The advancementsinsuchareasenabledtheuseoflab-on-a-chip sys-temsason-siteorpoint-of-caresystemsnotonlyforremoteor resource-limitedsettings,butalsoforhome-monitoringofelderly populationatdevelopedcountries.

Oneofthemainbottlenecksintransformingthelab-on-a-chip systemsintopoint-of-carediagnosticdevicesistherequirementto miniaturizeandcombineseveraloff-chipcomponents.The

mar-∗ Correspondingauthor.

E-mailaddress:aykutlu@unam.bilkent.edu.tr(A.Dana).

riageofthelab-on-a-chipsystemswithmobilephoneswasthe tippingpointthatyieldedaplethoraofintegratedscreeningand diagnosticdevices.MobilephonesprovidepowerfulCPUs,touch screen displays,advanced connectivityfeatures as wellas high pixel-count,sensitivecamerasandintegratedlightsources. There-fore,theuseofmobilephonesforapplicationsrequiringoptical detectionisaninterestingandrapidlydevelopingfieldofresearch. Forinstance,immunodiagnosticassays,lateralflowassays, micro-scopicimaging,flowcytometry,colorimetricdetection,photonic crystalandsurfaceplasmonresonance(SPR)basedbiosensinghave beendemonstratedusingmobilephoneplatforms[1–12].Inthis study,tothebestofourknowledgewepresentthefirstsurface plasmonresonanceimagingonasmartphone.

SPR biosensing is a popular method for quantitative anal-ysis and characterization of biomolecular interactions [13–15]. SPRprovideslabel-freeandreal-timedetectionofbindingevents withhighsensitivity.Surfaceplasmonsareelectromagneticwaves propagating along and evanescently decaying away from the metal/dielectric interface. This field confinement around the boundarymakesplasmonresonancecouplingconditionextremely sensitivetothelocalrefractiveindexchangescausedbyspecific adsorptionoftargetanalytesontothemolecularprobesresidingon themetalsurface.Recently,somestudieshavedemonstratedSPR sensingusingsmartphone. Preechaburanaetal.reported

angle-http://dx.doi.org/10.1016/j.snb.2016.08.061

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572 H.Guneretal./SensorsandActuatorsB239(2017)571–577

resolvedSPRchemicaldetectionusinganSPRcouplerattachedon thesmartphonescreenutilizingtheilluminationfromthescreen [8].Inordertodevelopamorepracticalsystem,Liuetal.[9]and Bremeretal.[10]showedtheuseofopticalﬁbersforSPR detec-tiononcellphones.Bothofthesesystemsusedtheback-sideLED andcameraofthecellphoneasthelightsourceandthesensor, respectively.Rocheetal.demonstratedlocalizedSPRsensingon cellphonewithincreasedsensitivityusinggoldnanoparticlesand nanorods[11].Duttaetal.reportedlocalizedSPR(LSPR)sensing oncellphoneforbiomoleculardetectionandmeasuringsize vari-ationofmetalnanoparticles[12].Allofthesestudies,demonstrate single-spotor1DspatiallyresolvedSPRsensing,whichlimitstheir applicabilityforhigh-throughputandmultiplexeddetection.Inthis article,wedemonstrateSPRimagingor2DSPRsensingonacell phone,unveilingthepotentialofmulti-analytedetectionaswell asimplementationofarray-basedadvancedbiochemicalanalysis usingalow-cost,integratedplatform(SeeSupplementarymaterial TableS1fordetailedcomparisonwithpreviousstudies).

SPRimagingissuperiortosingle-spotSPRsinceitcanbeusedfor detectionofmultipleanalytesinasinglesampleforpanelassays. Thisleadstosigniﬁcantbeneﬁtsintermsofcostandmeasurement time.Similarly,themultiplesensingpointsonthesensorcanbe usedfordetectionoftheanalyteatseveralsampledilutionswhich iscriticalforserialdilutionassays.Also,image-based bioanalyti-caldetectionhelpstheoperatortoviewtheresultsatonceand interpretthem moreeasily.Additionally, image-based2D sens-ingcanprovidereplicatedmeasurementsonthesamesensorchip togetherwithcontrolsthatleadstohigherreliability,precisionand on-chipself-calibration. On-chipcontrolandself-calibrationare especiallycriticalforpoint-of-caresensingapplicationsthat suf-ferfromhigherrorratesduetovaryingoperatingconditionsand thewide rangeof userlevels.We believethedemonstrationof suchadvancedbiochemicaltechniquesusingmobilephonesand cost-effectivesensorswillleadtoa paradigmshiftintheglobal healthcaremarket.Implementationofadvanceddetection appli-cationsoncontinuouslyimprovingfeature-richmobilephoneswill pavethewaytowardshighlysensitivediagnosistoolsreachingto thepeoplefromallsocio-economiclevels.

Here, we present surface plasmon resonance imaging on a smartphone.We have developedvery low-costgratingcoupled SPRsensor chipsusingoff-the-shelfopticalstorage discs. Addi-tionally,wedesignedacompactopticalsystem,usinga3D-printed apparatusthathoststheLEDsource,collimator,bandpassﬁlter, linearpolarizer,beamsplitterplateandanexternalimaginglens whichcanbeeasilyattachedtothesmartphone.Weemployeda silver/gold(Ag/Au)bilayerstructurecoatedontheperiodic cor-rugationsofBlu-raydiscsinordertoperformplasmonresonance imaging at thecentral regionof visible spectrum (␭r∼500nm)

undernormalincidenceilluminationinaqueousenvironment.This allowedtheoptimaluseoftheCMOSsensorofthesmartphone whilemaintaininghighsensitivity,chemicalstabilityandbiological affinity[16–20].Amicrofluidicchannelisplacedonthe bimetal-liclayerforcontrolledplumbingoftheliquids.TheuseofBlu-ray discsandstandardmetaldepositiontechniquestogetherwiththe low-costmicrofluidicchannelresultedinsignificantcost-reduction whichcanallowthesystemtobeusedforapplicationsrequiring disposableSPRsensors.

2. Materialsandmethods

2.1. Smartphoneattachmentforsurfaceplasmonresonance imaging

Anopticalattachmentwasdevelopedwhich converts smart-phone into a real-time surface plasmon resonance imaging

platformbasedonintensityinterrogationmechanism.ASamsung I8552Galaxy Win wasused asthe smartphone. Theprototype accessorywasfabricatedoutofpolylacticacid(PLA)filamentusing a 3D printer (MakerBot Replicator 2). Optical configuration of theimagingplatformisschematicallyillustratedinFig.1a.Light emittingfrom a 520nm LEDsource iscoupledto a multimode fiberopticcable (actingas a spatialfilter) and collimatedby a fiberopticcollimator.Abandpassinterferencefilter(␭c=520nm,

␭FWHM=10nm)isusedtonarrowthespectralbandwidthof

illu-mination.Collimatedbeamoflightbecomestransversemagnetic (TM)polarizedpassingthroughalinearpolarizingfiltersheetand isdirectedontothesensorsurfaceatnormalincidenceby reflect-ingfromabeamsplitterplate.Lightreflectingoffthesensorsurface passesthroughthebeamsplitterplateandisfocusedonthe smart-phone’scamerasensorbyanexternalplasticimaginglens(focal length=8mm).Imagingspotcoversapproximately160pixelsin diameterwhichcorrespondstomorethan20.000individual pix-els,althoughthecameraiscapableofvideorecordingat720×480 pixelsresolution.Imagingresolutioniscalculatedas12␮m/pixel. OnlygreenchannelofRGBimageisanalyzedsinceithasthehighest spectralresponsivityattheoperationwavelength.Theattachment measures143_×75_×44mm3 _and _weighs₂₁₅_g_including₂ _AAA

batteries(Fig.1b).

An Android application software was developed to analyze imagedata,reportintensitychangesandestimateanalyte concen-trationinthefielduse(Fig.1c).Whentheappisinitiated,zoomed inviewoftheimagingspotisdisplayedcontinuouslyatthe back-ground.First,regionofinterests(ROI)onthesensorsurfaceare definedbytappingmenubuttonandenteringROIparametersin thesettingsmenu.Toestimateanalyteconcentrationsfrom inten-sitychanges,predeterminedcalibrationlineparametersaresetin slope,interceptandunitfields.Timeaveragingfeaturecanbe acti-vatedbyenteringthenumberofconsecutiveframestobeaveraged. “Reference”,“Dark”and“Baseline”buttonsareusedtotakeand storereference,darkandbaselineintensityvalues,respectively,as describedinSubsection2.3.Averagedintensitychangesand esti-matedanalyteconcentrationsforeachROIsegmentaredisplayed separatelyontherightsideofthescreenwhenthe“Start record-ing”boxischecked.Uncheckingthe“Startrecording”itemstops recordingandsavesmeasurementdatainatextfile.Screen cap-turesfromapplicationsoftwarearepresentedinSupplementary materialFig.S1.

2.2. DesignandfabricationofSPRisensorchips

Opticalstoragediscscanbeexploitedasgratingcoupled sur-faceplasmonresonancesensorsbymetalcoating,thankstothe periodic corrugations [21,22]. Depending on thedisc type and refractive index of the surrounding medium, surface plasmons canbe excitedatwavelengthsranging fromultravioletto near infraredspectral regions.SPR substratesproduced fromBlu-ray discs (BD)(gratingperiod =320nm, grating depthd=20nm) exhibitplasmonresonanceswithinthevisiblespectrumwhen illu-minatedatnormalincidenceinaqueousmedium,asindicatedby thereflectancespectracalculations(Fig.S2). Numerical calcula-tionsareperformedusingacommercialsoftware(PCGrate)which employsmodifiedintegralmethod(MIM)[23].PCGrateallows cal-culationof diffraction efficienciesof multilayered 1-D gratings. Calculationmodeshouldbeswitchedfromnormaltoresonance modefor SPR coupler simulations. Polarization of the incident beamissetasnon-polarizedinordertoexaminethereflectance spectraforbothtransverseelectric(TE)andtransversemagnetic (TM)polarizedilluminations.SurfaceprofileofthegratingonBDis definedassine-trapezoidalusingbuilt-ingeometricaltools. Com-plexrefractiveindices of gold andsilver are extrapolatedfrom previouslytakenexperimentaldata[24].Accordingtothe

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simu-Fig.1. Surfaceplasmonresonanceimagingplatformintegratedwithasmartphone.(a)Schematicillustrationand(b)photographoftheimagingapparatus.(c)Custom

developedsmartphoneapplicationforreal-timeandon-sitemonitoringofmultiplesensingspots.

lationresults,silvercoatedBDexhibitssharpplasmonresonance around500nmwavelength,whereasgoldcoatedBDdoesnot dis-play surfaceplasmon resonant behaviorat normal incidenceof excitationduetoitsintrinsicabsorptioncharacteristicsatthis por-tionofthespectrum.However,goldhassuperiorpropertiesover silverintermsofchemicalstabilityandadequacyforfurthersurface functionalizationchemistryforbiosensingapplications.Inorderto overcomethisdichotomy,wecameupwithahybridsolutionin whichthick(>80nm)silvercoatingatthebottomactsasa plas-monexcitationlayerandthin(<10nm)goldfilmontopfunctions asa surfaceforthesubsequentsurfacemodification,aswellas extendingtheshelflifeofthesensorchip.ThethicknessesofAg andAulayersareoptimizedusingnumericalsimulations.Silver layerthicknessabove80nm isfoundtoyieldalmost sameSPR reflectancespectrawithsemi-infinitesilverlayerconfigurationas showninFig.S3.Asthethicknessofthetopgoldlayerisincreased, ontheotherhand,plasmonresonancecurvedegradesandloses itssharpnessasshowninFig.S4.Thus,goldcoatingthicknessis keptas2nminordertosatisfybothsensitivityandsurface chem-istryrequirements.Apartfromthepreviousstudieswhichusethe Ag/Aubimetallicstructuretoincreasesensitivity[16–20],weused thisconfigurationtoobtainaresponsebelow520nmwhichisnot possiblewiththecommonlyusedsinglegoldlayeredcouplers.

Blu-raydiscs(BD)areconvertedtoplasmonicsurfacesby peel-ingoffthetransparentthinprotectivecoatingontopofthediscwith tweezeraftercuttinganotchonthesideofthedisc,followedby metallizationusingthermalvacuumevaporation(VaksisThermal Evaporator).First,athingermaniumlayerisdepositedtoactasan adhesionlayeraswellastoreducethesurfaceroughnessof sub-sequentsilvercoating[25,26].Germanium(3nm),silver(80nm) andgold(2nm)metallayersaredepositedat0.4 ˚A/s,0.6–1.0 ˚A/s and0.2 ˚A/sgrowthrates,respectively,under0.005mTorrchamber pressure.SchematicillustrationandSEMimageofaAg/Aubilayer coatedBDstructureareshowninFig.2.

SensitivityperformanceofabimetallicBDbasedSPRsensoris investigatedusingnumericalsimulations.Thespectralbulk refrac-tiveindexsensitivity,S_␭BCiscalculatedas316nm/RIU.Theratioof thechangeinresonancewavelengthtothechangeinthethickness oflayerformedbytheadsorptionofmoleculesontothesensor surfacegivesthespectralsurfacecoatingsensitivity,S_␭SC,andit iscalculatedas0.7nm/nmforacoatingmaterialwithrefractive index,n=1.45.

Blu-raydiscs asdescribedherealloweasyreplicationof our workby otherswithoutneeding anyinfrastructure investment. Moreover,sensor surfacescanbeproducedat lowercostsona largescalefollowingthemanufacturingprocessesofBlu-raydiscs. Infactroll-to-rollprintingofgratingsisdemonstrated[27],which canbeusedasanalternativetoBlu-raydiscs.Itisimportantto notethatplanarsensorsurfacesthatarefabricatedusinganyofthe processesmentionedabovearecompletelycompatiblewithour opticalreadoutconﬁguration.

Alow-costmicroﬂuidicchannelisfabricatedusinganextremely simple,yetveryeffectivemethodbasedonlasercuttingofa3mm thicktransparentacrylicplateandadoublesidedadhesivetape(3M 468MPAdhesiveTransferTape)(Fig.S5).Bothchannelgeometry andholesforinletandoutletaredeﬁnedbyahighpowerCO2laser

cuttingsystem(EpilogZing24).Channelgeometryisdefinedon thedoublesidedtapeat8Wlaserpowerand100%scanningspeed. Channelframe,inlet andoutletholesaredefinedontheacrylic plateat30W laserpowerand15%scanningspeed.First,double sidedtapeisbondontotheacrylicplate.Then,acrylicplateand BDchiparebondtogethertightlybyapplyingmechanicalpressure forhalfaminute.Schematicillustrationofaflowcellintegrated SPRisensorchipisshowninFig.2.Tygontubings(Cole-Palmer) areconnectedtotheinletandoutletoftheflowcell.Epoxy adhe-sive(BisonEpoxy5Minutes)isusedtocompletelysealthetubings attheinletandoutletconnections.Thevolumeofaflowcellis8

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Fig.2.GratingcoupledSPRisensorchip.(a)SchematicoftheSPRichipassemblyfabricatedbyintegratingabimetallicBlu-raydiscchipandadisposableﬂuidicchannel.(b)

Cross-sectionschematicviewand(c)SEMimageofabimetallicBDchip.Groovedepth,dis20nm,andpitchwidth,is320nm.FilmthicknessesofAgandAulayersare

80nmand2nm,respectively.

microliters(16×5x0.1mm3_)._An_external_peristaltic_pump_is_used

forcontrolledplumbingoftheliquidsthroughthechannel. 2.3. Measurementprotocol

Inatypicalmeasurement,anSPRisensorchipispluggedintothe sampleholderunit.Next,LEDlightsourceisturnedonand polariz-ingfilterisadjustedtotransverseelectric(TE)polarizationwhere electricfieldofthetransmittedlightisparalleltothegratinglines ofthesensorsurface.Inletportoftheflowcellisconnectedtothe pumpandoutletportisconnectedtoawastetube.Flowcellisfilled withtherunningbuffersolution.Orientationofthesamplewith respecttotheincidentlightbeamisfinelytunedbyadjustingset screwsbehindthesampleholdercoveruntiltheilluminationspot displayedonthetouchscreenhasacircularshapeandgetslocated atthecenterofthescreen.Havingthesampleproperlyaligned, surfaceimageisrecordedunderTEpolarizedilluminationtobe takenasnormalizationreference.Thenthelightsourceisturned offandscreenimageiscapturedasdarkreading.Fortherestofthe measurement,thelightsourceisturnedonandpolarizingfilter isadjustedtoTMpolarization.Beforeperformingtheassay proto-col,surfaceimageisrecordedtoestablishbaselinereadingwhile thesampleisinitsbarestateandsurroundedbybuffersolution. Intensitychangeateachpixeliscalculatedbytakingthedifference betweenactualandbaselineintensities.Timeandareaaveraging optionsareprovidedintheapplicationsoftwaretoobtainenhanced signalduringthecourseofthemeasurement.Fromsurface activa-tiontothecaptureofanalytemolecules,thewholeassayprotocol canbemonitoredinreal-time.

2.4. Experimentalsetupforwavelengthinterrogationofplasmon resonance

Reflectancespectraofsurfaceplasmonresonancesexcitedon thesensorchipsareprobedinreal-timeusinganormalincidence SPRspectroscopysetup(Fig.S6).Whitelightemittedfromahigh powerbroadbandLEDsourcedrivenbyastableDCcurrentsource (Keithley2400SourceMeter)iscoupledtoamultimodefiberoptic cableandcollimatedbyacollimationlensattheoutputofthefiber. Collimatedbeamoflightpassesthroughanadjustablelinear polar-izer,anadjustableiristonarrowdownthebeamdiameter,anda non-polarizingbeamsplittercube.Polarizedandnarrowedbeam isreflectedoffthesensorsurface,directedbythebeamsplitter towardsthespectrometerfiberopticcomponents,collectedbya fibercouplinglensandanalyzedbyaspectrometer(OceanOptics Maya2000Pro).Eachreflectancespectrummeasurementistaken at20msofintegrationtime.Thespectralpositionoftheresonance dipisprobedusingcentroidalgorithm[28].Resonancewavelength shiftnoiseofaspectrogramaveragedover75spectraiscalculated as1.5pm.Takingtheexperimentalspectralbulkrefractiveindex

sensitivity,S_␭BEas356nm/RIUforBDSPRsensors,theminimum detectablebulkrefractiveindexchange,␴nBEisfoundas4.2×10−6

RIUforthewavelengthinterrogationsetup. 2.5. Optimizationoftheilluminationwavelength

Theilluminationwavelength(␭i)oftheimagingplatformwhich

yieldsthehighestintensitysensitivitywasidentiﬁedbymeasuring reﬂectancespectraforvaryingrefractiveindicesofthe surround-ingliquidmedium(Fig.S7).Liquidsolutionswithrefractiveindices rangingfrom1.335to1.365werepreparedbydissolvingglycerol (n=1.474)indeionizedwater(n=1.335)atvaryingconcentrations. Thehighestbulkrefractiveindexsensitivitywasfoundtobeabout 800%/RIUatthewavelengthof520nmforTDKBD-Rsubstrate. Bothresonancewavelengthshiftandintensitychange(␭i=520nm)

showlineardependenceontherefractiveindexchangewithinthe 1.335<nd<1.365range.

3. Resultsanddiscussion

3.1. MicroarrayimagingofAg/Aubimetallicsensingspots

WehaveperformedSPRimagingofAg/Aubimetallicmicrospot arrayunderbulkdielectricmediawithchangingrefractiveindices. Thebimetallicmicroarraystructurewasfabricatedusing conven-tionalopticallithographyprocessesinadditiontometaldeposition steps.Thediameterofeachspotis110␮mandthespacingbetween adjacentspotsis 130␮m.First, wehaverecorded videoof illu-minatedsensorsurfaceat30frames/sduringthesuccessiveﬂow ofglycerolsolutionswithincreasingrefractiveindices.Each200 consecutivevideoframeswereaveragedinordertoenhancethe signal-to-noise ratio. As the refractive index of the surround-ingmediumgetshigher,plasmonresonancecurveshiftstowards longerwavelengthsresultingindarkerimaging spotsunderTM polarizedillumination.Fig.3ashowsgreenchannelviewofan aver-agedRGBimageofamicrospotsensorarrayin20%v/vglycerol solutionenvironment (nd=1.3596).SPRidifferenceimageswere

generated bytaking TE imagesasnormalizationreference, and TMimagesinpurewater mediumasbackground(Fig.3b).Line profileofarowofmicroarrayrevealstheuniformityofthe reflec-tivitychangeacrossthespots(Fig.3c).Averagereflectivitychange ofspotsweremeasuredateachglycerolconcentrationlevelfrom 1%to25%v/v(SeeFig.S8fortimeresolvedresponsesof individ-ualsensingspotsduringfluidexposures).Theglycerolcalibration curvefor4×4spotslocatedwithintherectangularframeshownin Fig.3aexhibitshighlinearity(R2₌_0.9951)_within_a_dynamic_range

of22200×10−6RIUbetweennd=1.3354and1.3576(Fig.3d).The

errorbarsshowninFig.3dshowtwicethestandarddeviation(2SD) ofthemeasuredvaluedemonstratingthelowintra-sensor variabil-itybetween16spots.Theslopeofthecalibrationcurvewhichgives

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Fig.3. Microarrayimagingofsensingspotsunderbulkdielectricmediawithdifferentrefractiveindices.(a)GreenchannelviewofanRGBimageofamicroarrayofAg/Au

bimetallicsensingspotsunder20%v/vglycerolsolutionenvironment(Refractiveindexofglycerolsolution,nd:1.3596).(b)3DrepresentationofSPRidifferenceimage.

Baselineimageistakenunderpurewatermedium(Refractiveindexofpurewater,nd:1.335).(c)Lineproﬁleofarowofmicrospots.(d)Percentagereﬂectivitychangewith

respecttotherefractiveindexofbulkmediumatvaryingconcentrationsofglycerolsolutionsfrom1%to25%v/v.Bulkrefractiveindexsensitivityobtainedfromlinear

calibrationcurveis485%/RIU.Errorbarsdenote2SDofmeasurementsfrom16spotsshowninpart(a),demonstratingintra-sensorvariability.(Forinterpretationofthe

referencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

theexperimentalreﬂectivitybulkrefractiveindexsensitivity,SRBE

wasfoundas485%/RIU.Intensitynoiseofasinglepixelinterms ofpercentagereﬂectivity,␴Rwasfoundas0.2%,whereas

averag-ingarectangularareaof10-by-10pixels(120×120␮m2₎_reduces

thereﬂectivity noise downto 0.02% level. Thus, theminimum detectablebulkrefractiveindexchange,␴novera10-by-10pixels

areaiscalculatedas4.12×10−5RIUforthesmartphoneplatform. ThelowcostAg/AubimetallicBDSPRstructureexhibitsrelatively low refractive index sensitivity as compared to the previously reportedSPRgrating,prismandwaveguidecouplers[13,29–32]. However,sincethenoiselevelisalsoreducedbyareaaveraging, overallresolutionofthesystemiscomparablewiththepreviously demonstratedstudies.

3.2. Real-timemonitoringofbovineserumalbumin(BSA) adsorption

Wehavetakentime-resolvedintensitychangemeasurements inresponsetotheproteinadsorptiononthesensorsurface.Bovine serumalbumin(BSA) isknowntoadsorbontothegoldsurface forming a 4–7nm thick monolayer [33]. 1mg/mL (15␮M) BSA (Sigma Aldrich) dissolved in 10mM phosphate buffer solution (PBS,pH=7.4)wasinjectedthroughtheﬂowcellfor5minafter athoroughPBSwash.Followingtheformationofself-assembled monolayeronthegoldcoating, thesensorsurface wascleaned byPBS washagain.SPRi differenceimageof arbitrarilydeﬁned 4-by-4rectangularregionofinterestsaftertheBSAadsorptionis

showninFig.4a.Averagereflectivitychangeof16ROIsovertime isasshowninFig.4bandthesteady-statereflectivitydifference is foundas4.37±0.53%.Resonancewavelength shiftcausedby theBSAadsorptionwasmeasuredusingSPRwavelength interro-gationsetuprepeatingthesameBSAprotocol.Theresonancedip shiftsfrom503.8nmto507nmasshowninFig.4c.Reflectivityat 520nmdecreasesby6.44%from52.46%to46.02%,inagreement withimagingresults.

Wehaveperformedkineticanalysisofthereactionbasedona simpleinteractionmodelexplainedintheSupplementarymaterial. Theassociationrate,kaoftheBSA-Aucomplexformationis

calcu-latedas1150M−1s−1,withtheassumptionthatthedissociation rate,kdisnegligiblysmallascanbededucedfromthesensorgram

curveaftersecondPBSinjection.

3.3. Demonstrationofabiodetectionassay

Asanexemplarybiodetection experiment,captureofmouse IgG antibodyby immobilized layerof rabbit anti-mouse(RAM) IgGantibodyprotocolwasimplementedasdirectassay.First, sur-face of a thin gold ﬁlm coated BD chip wascleaned by argon plasmatreatment.Sensorsurfacewascoatedwithself-assembled monolayer(SAM)of 11-mercaptoundecanoicacid(11-MUDA)by immersingthechipin2mMethanolsolutionof mercaptounde-canoicacidovernightfollowedbyrinsingwithethanolandwater, and drying in the fume hood. Then, BD chip was integrated withaﬂow cell.Ethanol(70%)and PBSsolutionswereinjected

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Fig.4. Real-timemonitoringofBSAadsorptionontothegoldsurface.(a)Arbitrarilydeﬁned4-by-4rectangularregionofinterests(96×96␮m2_each)_on_entirely_Ag/Au_bilayer

coatedsensorspotimage(top)andbackgroundcorrectedSPRidifferenceimageofROIs(bottom)takenafterBSAadsorption.(b)Sensorgramshowingaveragereﬂectivity

changeofROIspotsduringtheimplementationofBSAprotocol.TheinsetschematicillustratesbindingofBSAmoleculesonthegratingsurface.Errorbarsdenote2SDof

measurementsfrom16spots.(c)ReﬂectancespectratakenbeforeandafterBSAadsorptionusingthenormalincidenceSPRspectroscopysetup(Fig.S6).

Fig.5.NanomolarleveldetectionofcaptureofmouseIgGbyimmobilizedlayerofRAMIgG(a)SpectralsensorgramshowingtheimmobilizationstepsofRAMIgGtakenby

usingthenormalincidenceSPRspectroscopysetup(Fig.S6).(b)Dose-responsecurveforthecaptureofmouseIgGtakenbyusingSPRimagingplatform.Theinsetschematics

illustrateimmobilizationofRAMIgG(bluecolored)onthesensorsurfaceandselectivebindingofmouseIgG(redcolored)withRAMIgG,respectively.Errorbarsdenote2SD

ofmeasurementsfrom3spots.(Forinterpretationofthereferencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

onto the surface at 100␮L/min flow rate for further cleaning. Surfaceofthe11-MUDA SAMlayerwasactivatedbythe expo-sureof400mM1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC)and100mMN-hydroxysuccinimide(NHS)mixture(1:1)at 100␮L/minflowratefor4min.Bothreagentswerepreparedin distilledwaterandmixturewaspreparedjustbeforeuse.Diluted RAMIgG(50␮g/mL)in0.1mMsodiumacetatebuffersolutionwas injectedontotheactivatedsensorsurfaceat50␮L/minflowrate for3min.RAMIgGantibodieswereimmobilizedontheactivated surfacebyaminecouplingchemistry.FollowingtheRAMFc immo-bilization,non-reactedNHSesterswerecappedbytheexposure of1Methanolaminesolutionat100␮L/minflowratefor4minto preventnon-specificanalytebindings.Sensorsurfacewascleaned byPBSwashfor5minaftereachstep.Thewholeimmobilization processfromsurfaceactivationtoblockingstepwasprobedin real-timeusingthewavelengthinterrogationsetup(Fig.5a).

SPRchipimmobilizedwithRAMIgGistakenoutthewavelength interrogationsetupandpluggedintothesmartphoneattachment. MouseIgG solutionsat concentrationsrangingfrom1.33nMto 830nMwereinjectedsuccessivelyat50␮L/minﬂowratefor5min. Intensitychangesofindividualpixelsatthreedistinctlocationson thesensorsurfaceisshowninFig.5b.Dose-responsecurvereveals

thatnanomolarleveldetectionofantibodyanalyteisachievable withina dynamic range froma few nanomolarstomicromolar concentration.

4. Conclusion

In this work, we demonstrated the useof a smartphone as ahand-heldsurface plasmonresonanceimagingbiosensor with highsensitivity.Developinganattachableimagingaccessoryfrom inexpensiveopticalcomponentsand3Dprintedparts,andusing easy-to-implementproceduresforfabricatingminiaturizedsensor chipsintegratedwithﬂowcellsfromextremelycheapsubstrates likeopticalstoragediscs,weofferapromisingdetectionplatform thatenablesbiosensingintheﬁeldwithhighlevelofportabilityand affordablecost.Thecapabilitytoperformparallelassaysinashort amountoftimeallowstheuseoftheinstrumentforpoint-of-care applicationswheremonitoringofmultipleparametersisdesirable.

Acknowledgement

ThisworkwassupportedbytheStatePlanningAgencyofthe TurkishRepublicProjectUNAM.

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AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.snb.2016.08.061.

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Biographies

HasanGunerisaPh.D.studentattheUNAMMaterialsScienceandNanotechnology

ProgramatBilkentUniversity.Hisresearchinterestsareintheﬁeldofdesignand

implementationofplasmonresonancesensingsystems.

ErolOzgurisapost-doctoralresearchassociateattheUNAMMaterialsScienceand

NanotechnologyProgramatBilkentUniversity.Hecurrentlyworksonlargescale

productionandvariousapplicationsofphotonicandplasmonicbiosensors.

GuzinKokturkisaresearcherattheUEKAE−BILGEM−TheScientiﬁcand

Techno-logicalResearchCouncilofTurkey(TUBITAK).SheworksinBioelectronicsDevices

andSystemsGroup.Herresearchinterestsaredevelopmentofbiosensordevices

andbiologicalassaysfordifferentbiosensorapplicationareas.

MehmetCelikisaPh.D.studentattheDepartmentofComputerEngineeringat

MiddleEastTechnicalUniversity.Hisresearchinterestsareintheﬁeldofcomputer

visionandmachinelearning.

ElifEsenisresearcherattheUEKAE−BILGEM−TheScientiﬁcandTechnological

ResearchCouncilofTurkey(TUBITAK).SheisaPh.D.studentatMolecular

Biol-ogy,GeneticsandBiotechnolgyprogramattheIstanbulTechnicalUniversityand

continuestoworkintheﬁeldsofbiosensordeviceandsensorchipdevelopment.

AhmetEminTopalisaPh.D.studentattheUNAMMaterialsScienceand

Nano-technologyProgramatBilkentUniversity.Hisresearchinterestsareintheﬁeldof

colorimetricbiomolecularsensingandimagingusingplasmonresonances.

SencerAyasisapost-doctoralfellowattheCanaryCenteratStanfordforCancer

EarlyDetection,DepartmentofRadiology,StanfordSchoolofMedicine.Hisresearch

isonthedesignandrealizationofplasmonicnanostructuresandmetasurfaces,with

applicationstoimagingandspectroscopy.

YildizUludagistheHeadofComputationalBiologyAndSecurityApplicationsUnit

attheUEKAE−BILGEM−TheScientiﬁcandTechnologicalResearchCouncilof

Turkey(TUBITAK).ShereceivedherPh.D.fromCranﬁeldUniversity(UK)and

con-tinuestoworkintheﬁeldsofbiosensordeviceandsensorchipdevelopment.

CaglarElbukenisAssistantProfessoratBilkentUniversity,National

Nanotech-nology Research Center.His researchinterests include lab-on-a-chipdevices,

microdroplet-basedmicroﬂuidicsystemsandsensingtechnologiesforportable

applications.

AykutluDanaisAssociateProfessoratBilkentUniversity,NationalNanotechnology

ResearchCenter.HereceivedhisPh.D.fromStanfordUniversityandcontinuesto