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Advances in paper-based point-of-care diagnostics
BiosensorsandBioelectronics54(ContentslistsavailableatScienceDirectBiosensorsandBioelectronicsjournalhomepage:/locate/biosAdvancesinpaper-basedpoint-of-carediagnosticsJieHua,b,1,ShuQiWangc,1,LinWanga,b,FeiLib,d,BelindaPingguan-Murphye,TianJianLub,n,FengXua,b,nnTheKeyLaboratoryofBiomedicalInformationEngineeringofMinistryofEducation,SchoolofLifeScienceandTechnology,Xi'anJiaotongUniversity,Xi'an710049,P.R.ChinabBioinspiredEngineeringandBiomechanicsCenter(BEBC),Xi'anJiaotongUniversity,Xi'an710049,P.R.ChinacBrighamandWomen'sHospital,HarvardMedicalSchool,Boston02139,MA,USAdDepartmentofChemistry,SchoolofScience,Xi'anJiaotongUniversity,Xi'an710049,P.R.ChinaeDepartmentofBiomedicalEngineering,FacultyofEngineering,UniversityofMalaya,KualaLumpur50603,MalaysiaaarticleinfoArticlehistory:Received25July2013Receivedinrevisedform30October2013Accepted31October2013Availableonline19November2013Keywords:Micro?uidicsPoint-of-care(POC)Paper-baseddiagnosticsabstractAdvanceddiagnostictechnologies,suchaspolymerasechainreaction(PCR)andenzyme-linkedimmunosorbentassay(ELISA),havebeenwidelyusedinwell-equippedlaboratories.However,theyarenotaffordableoraccessibleinresource-limitedsettingsduetothelackofbasicinfrastructureand/ortrainedoperators.Paper-baseddiagnostictechnologiesareaffordable,user-friendly,rapid,robust,andscalableformanufacturing,thusholdinggreatpotentialtodeliverpoint-of-care(POC)diagnosticstoresource-limitedsettings.Inthisreview,wepresenttheworkingprinciplesandreactionmechanismofpaper-baseddiagnostics,includingdipstickassays,lateral?owassays(LFAs),andmicro?uidicpaper-basedanalyticaldevices(μPADs),aswellastheselectionofsubstratesandfabricationmethods.Further,wereporttheadvancesinimprovingdetectionsensitivity,quanti?cationreadout,proceduresimpli?ca-tionandmulti-functionalizationofpaper-baseddiagnostics,anddiscussthedisadvantagesofpaper-baseddiagnostics.Weenvisionthatminiaturizedandintegratedpaper-baseddiagnosticdeviceswiththesample-in-answer-outcapabilitywillmeetthediverserequirementsfordiagnosisandtreatmentmonitoringatthePOC.&2013ElsevierB.V.Allrightsreserved.Contents1.2.Introduction........................................................................................................586Substrates,fabricationmethodsandworkingprinciplesofpaper-baseddiagnosticdevices.........................................5862.1.Papersubstratesandalternatives.................................................................................5862.2.Dipstickassays................................................................................................5882.3.Lateral?owassays(LFAs).......................................................................................5882.4.Micro?uidicpaper-basedanalyticaldevices(μPADs)..................................................................588Reactionmechanismofpaper-baseddiagnostics...........................................................................5893.1.Chemicalreaction:colorchange..................................................................................5893.2.Biologicalreaction.............................................................................................5903.2.1.AntigenCantibodybinding................................................................................5903.2.2.Nucleicacidhybridization................................................................................5903.2.3.Functionalnucleicacid-basedreaction......................................................................5903.3.Electrochemicalreaction........................................................................................590Advancesindevelopmentofpaper-baseddiagnostics.......................................................................5904.1.Sensitivity...................................................................................................5904.2.Quanti?cation................................................................................................5914.3.Simpli?cation.................................................................................................5933.4.Correspondingauthor.Correspondingauthorat:TheKeyLaboratoryofBiomedicalInformationEngineeringofMinistryofEducation,SchoolofLifeScienceandTechnology,Xi'anJiaotongUniversity,Xi'an710049,P.R.China.E-mailaddresses:tjlu@mail.(T.J.Lu),fengxu@mail.(F.Xu).1Theauthorscontributedequallytothiswork.nnn/$-seefrontmatter&2013ElsevierB.V.Allrightsreserved.
http://dx.doi.org/10.1016/j.bios.586J.Huetal./BiosensorsandBioelectronics54(4.4.Multi-functionalization.........................................................................................5.Disadvantagesofpaper-baseddiagnostics................................................................................6.Conclusionandfutureperspectives.....................................................................................Acknowledgments.......................................................................................................References.............................................................................................................5961.IntroductionAnnually,infectiousdiseases,includingacquiredimmunode?-ciencysyndrome(AIDS),tuberculosis(TB)andmalaria,causeapproximately15milliondeaths,accountingforabout25%ofdeathsworldwide(BissonnetteandBergeron,2010;Morensetal.,2004).Morethan95%ofthedeathsoccurindevelopingcountriesduetothelackofcost-effectivemedicalinterventions(Leeetal.,2010;Yageretal.,2006).Diagnosticsarecommonlyusedtodiagnosethecauseofsymptomsinpatients,tomonitortheef?cacyoftreatment,andtoscreenforpotentialdiseasesinasymptomaticbuthigh-riskpopula-tion(HayBurgessetal.,2006).Assuch,diagnosticsareofimportanceinthehealthcaresystemandhaveacriticalimpactondecision-makingclinicallyandepidemiologically.Forexample,malariacausesonechilddeathinevery45sandnearlyonemillioninfantdeathsperyearinAfrica,despitethefactthatthisdiseaseisbothpreventableandcurable.Sinceimmediatetreatmentisrequiredupontheappearanceofsymptoms,rapidandspeci?cdiagnosisofmalariawouldbecriticalforthetreatmentandprevention(Mukhopadhyay,2010).However,thediagnosisisoftenbasedontheclinicalexperi-encewithoutanylaboratoryevidenceinresource-limitedsettings.Ifarapidtestkitisaffordable,simple-to-operate,andaccuratetodiagnosemalaria,itcanbealifesaver.Althoughadvanceddiagnostictechnologies,suchaspolymerasechainreaction(PCR)andenzyme-linkedimmunosorbentassay(ELISA),havealreadybeenimplementedindevelopedcountries,theycannotbewidelyusedindevelopingcountriesbecauseoflimitedavailabilityoflaboratoryinfrastructure,skilledpersonneland?nancialsupports(HayBurgessetal.,2006;Mabeyetal.,2004;Martinezetal.,2010b;Yageretal.,).Comparedwithstandardlaboratorytesting,point-of-care(POC)diagnosticsarerapid,simpleandinexpensive,andthushavegreataccessibilitytoresource-limitedsettings(Hartetal.,2011;Tabak,2007).Therefore,POCdiagnosticsareessentialtoinitiateandscaleupon-sitemedicalcareforthepreventionandcontrolofinfectiousdiseases(Haucketal.,2010;Leeetal.,2010;Wangetal.,b).Micro?uidictechnologieshavebeenwidelyemployedindevel-opingPOCdiagnosticstoaddressglobalhealthissuesbecauseoftheirevidentadvantages(HawkinsandWeigl,2010).Forexample,micro-?uidicdevices,coupledwithdifferentfunctionalunits(e.g.,pumps,valvesandreactors)andintegratedintoaminiaturizedanalyticalsystem(Chinetal.,2007;HawkinsandWeigl,2010),canmanipulatesmallvolumesof?uids(Whitesides,2006).Therefore,micro?uidicdevicessigni?cantlyreducetheconsumptionofsamplesandreagents,thecomplexityofoperationprocedures,andthelengthofassaytimewithoutcompromisingspeci?cityandsensitivity(Chinetal.,2007;Laksanasopinetal.,2009;Whitesides,2006).Ontheotherhand,mostmicro?uidicdevices,whicharemadeofglass,silicon,andpolymerslikepoly(dimethylsiloxane)(PDMS)andpoly(methylmethacrylate)(PMMA)(Martinezetal.,2010b;Nilghazetal.,2012),requirebothcomplexfabricationprocessesandexternalinstruments,therebymakingthemunsuitableforPOCtesting(Nilghazetal.,2012).Incontrast,paper-basedmicro?uidicsholdgreatpotentialtodeliverPOCdiagnosticstodevelopingcountriesintermsofbeingaffordable,sensitive,speci?c,user-friendly,rapidandrobust,equipment-free,anddeliverabletoend-users(ASSURED)(HawkinsandWeigl,2010;Leeetal.,2010;Martinezetal.,2010b).Thedetailedcomparisonbetweenchip-basedandpaper-baseddevicesislistedinTable1.Althoughtherearesomerecentreviewsonpaper-baseddiag-nostics(ParoloandMerko?i,2013;Yetisenetal.,2013),weherefocusonthesubstratesandmethodstodesignandfabricatepaper-basedmicro?uidics,theworkingprinciplesandreactionmechan-ismofpaper-baseddiagnostics,andmostimportantly,thelatestadvancesinimprovingapplicationsofpaper-baseddiagnosticsatthePOC.Wealsodiscussthechallengesassociatedwithpaper-baseddiagnosticsforimprovingtheirperformanceandaccessibility.2.Substrates,fabricationmethodsandworkingprinciplesofpaper-baseddiagnosticdevicesPaperfabricationisoneofthemostimportanttechnologiesinhumanhistory(Grifantini,2009),whichwasborninthe2ndcenturyADinChina(Rooz,2010).Currently,paper(includingmembrane)basedmaterialshavebeenutilizedforbiochemicalanalyses,includingdipstickassays,lateral?owassays(LFAs),andmicro?uidicpaper-basedanalyticaldevices(μPADs)(ParoloandMerko?i,2013).Dipsticksrefertourineteststripsatthebeginning.TheyweredevelopedbyaParisianchemist,JulesMaunmené,in1850andmarketedbyanEnglishphysiologist,GeorgeOliver,in1883.Subsequently,pHteststripswerepatentedandcommercia-lizedinthe1920s(FosterandGruntfest,1937).Till1956,the?rstlatexagglutinationassaydevelopedbyPlotzandSingerlaidthetechnicalbasisofLFAs(WongandTse,2009).Sincethen,thebasicprincipleofLFAshasbeenfurtherre?nedandwidelyusedforrapiddetectionofinfectiousdiseases.Morerecently,Whitesidesetal.introducedpaper-basedmicro?uidicstofabricatelowcostandsimpleμPADs,whichwerehighlightedasoneof10emergingtechnologiesin2009byTechnologyReview(Grifantini,2009).Thesubstratesandfabricationmethodsofbasicdipsticks,conven-tionalLFAsandemergingμPADs,andtheirworkingprinciplesareintroducedasfollows.2.1.PapersubstratesandalternativesNowadays,avarietyofpapermaterialshavebeendevelopedandtwomainkindshavebeenwidelyutilizedtofabricatediagnosticdevicesforPOCtesting(Ballerinietal.,2012;Yetisenetal.,2013).Oneiscellulose?berbasedmaterials,suchas?lterTable1Thecomparisonbetweenchip-parisonChip-baseddevicePaper-baseddeviceMaterialGlass,silicon,polymer,etc.PaperandmembraneManufactureChannelfabricationHydrophilicchannelsandsurfacemodi?cationandhydrophobicbarriersDrivingforcePumpCapillaryforceandevaporationResultanalysisReaderReaderorvisualdetection
J.Huetal./BiosensorsandBioelectronics54(587Fig.1.Threekindsofpaper-baseddiagnosticplatforms:dipsticks(ACB),lateral?owteststrips(C),andmicro?uidicpaper-basedanalyticaldevices(μPADs,DCF).(A)DiagnosticpHteststrips().(B)Urineteststrips().(C)Atypicalschematicviewofalateral?owteststrip,includingcomplexandsandwichformats(Millipore,2009).(D)Atypicaltwo-dimensional(2D)μPAD(Martinezetal.,2010b).(E)Atypicalthree-dimensional(3D)μPAD(Martinezetal.,2008b).(F)Atypicalmicro?uidicpaper-basedelectrochemicaldevice(μPED)(Nieetal.,2010b).588J.Huetal./BiosensorsandBioelectronics54(paperandchromatographypaper,whicharethemajorsubstratesofdipsticksandμPADs(Pelton,2009).Theotherisnitrocellulosemembrane,whichisthekeymaterialforLFAs(Millipore,2009;WongandTse,2009).Cellulose,whichisalinearchainmacro-moleculecomposedofhundredsofglucoseunits(O'Sullivan,1997),is?brous,hydrophilic,biodegradable,andinsolubleinwaterandmostorganicsolvents.Nitrocelluloseisproducedbypartialnitrationofcellulose.Nitrationstrengthenstheporouspropertyofcelluloseandchangescellulosefromhydrophilictohydrophobic.Bothcellulose?bersandnitrocellulosemembranesareporousmaterials.Porosity,togetherwithsurfacechemistryandopticalpropertiesofthesematerials,iscriticalforthepreparationofpaper-baseddiagnosticdevices(Pelton,2009).Surfacechemistryhasanimpactonmoleculeorparticleimmobi-lization,non-speci?cadsorption,andcolorexpressionindiagnos-ticassays.Porosityandthesurfacechemistrytogetherdeterminewetpropertiesofthesematerials,andthusaffectthebehaviorof?uidon/inthedevice.Moreover,theopticalpropertiesofthesematerialscanin?uencetheaccuracyofcolorimetricor?uorescentreadouts(Pelton,2009).Forexample,manycommercialpapermaterialsareaddedwith?uorescentmolecules(i.e.,opticalbrighteningagents)inordertomakethemappearwhite.How-ever,suchmodi?cationcanleadtoahighbackgroundin?uorescence-baseddiagnosticassays.Tothisend,polymericadditivesareselectivelyaddedinthepapermakingprocesstoadjustthepropertiesofthesematerials.Forexample,somerewettingagents(e.g.,surfactants)havebeenaddedtofabricatenitrocellulosemembranes(WongandTse,2009).Inaddition,someotherpapersubstratesandalternativeshavebeenexploredtodeveloppaper-baseddiagnosticdevices.Glavanetal.(2013)silanizatedcardstockpaperwitha?uorinatedalkyl-trichlorosilanetoobtainomniphobicRFpaperandthenutilizedthepapertofabricatepressure-drivenopen-channelmicro?uidicdevices.SimilartoPDMS-basedmicro?uidicsystems,thesedevicesexhibitcomparableperformanceinthecontrolof?uid?ow.Inaddition,thesedevicesareinexpensive,lightweight,anddisposable.Inanotherstudy,Hanetal.(2013b)employedasoftpaper/polymercompositetofabricaterecon?gurablethree-dimensionalmicro?uidicdevicesonbenchtop.Additionally,somealternativematerials,suchas?exible?lm(Fockeetal.,2010),cottonyarn(Safaviehetal.,2011),cottoncloth(Nilghazetal.,2012)andvegetableparchment(Yanetal.,2012),wereusedasamatrixtofabricatemicro?uidicdiagnosticdevices.2.2.DipstickassaysDipsticks,suchaspHteststripsandurineteststrips,aresimpletodesign,easytomanufactureandconvenienttouse.Generally,pHteststripsaremanufacturedbysoakingapieceof?lterpaperintoamixtureofacidCalkaliindicatorswithacertainconcentra-tionratio.Afterdried,thepaperisimpregnatedwithdetectionregents.Whenanunknownsampleisdispensedonthepaper,thedetectingregentsreactwiththeanalyte(Ht)anddevelopacolor.Byreferringtoastandardindicatorcard,thepHvalueofthesolutioncanbeindicatedandthustheconcentrationofHtissemi-quanti?ed.Further,pHteststrips(Fig.1A)havebeenusedtomeasurethelevelofpHinurineorsalivatomonitordietintakeandtoobtainthegeneralhealthinformationofhumanbody(YoungandYoung,2010).Colorindicatorscanbeaddedtodetectleadacetate,potassiumiodide,etc.()aswell.Similarly,urineteststrips(Fig.1B)havebeendesignedtodetectmetabolicproductsinurine,whichhavebecomebasicdiagnostictoolstoindicatepathologicalchanges.Forinstance,urinarymetabolicproducts(e.g.,protein,glucose,andsalt)frompatientswithnephriticordiabeticdiseasescanbedetectedusingastandardurineteststrip.2.3.Lateral?owassays(LFAs)Lateral?owteststripsaretypicallycomposedofanitrocellulosemembrane,samplepad,conjugatepad,wickingorabsorbentpadandbackingpad(Millipore,2009)(Fig.1C).Theabsorbentpadprovidesadrivingforcebasedoncapillaryeffect,andthebackingpadprovidesacertainmechanicalsupporttothedevice.NitrocellulosemembraneisthemostpopularandimportantmaterialinLFAsbecauseitprovidesaplatformforbothreactionanddetectionduringtheassay(Millipore,2009).Capturingmolecules,e.g.,antibodies,canbedepositedonthenitrocellulosemembranestoformtestandcontrollinesbyelectro-staticinteraction,hydrogenbondsand/orhydrophobicforces(Millipore,2009).Eachtwoadjacentcomponentsoverlapwitheachotherbyasmallpartinordertocoordinatethe?uid?ow.Whentheassayisperformed,asmallvolumeofsampleisappliedontothesamplepad.Thesamplepadispretreatedwithabuffer(e.g.,toadjustpH)toimprovetheperformanceoforcompatibilitywiththeothercomponentsusedintheassay(WongandTse,2009).Themixturemigratesalongthepadandthencarriesconjugatedparticles,whicharepreloadedontotheconjugatepad,alongthenitrocellulosemembrane.VariousparticleshavebeenemployedinLFAsfortheiruniqueoptical,electronic,and/orstructuralproperties,suchasgoldnanoparticles(AuNPs)(DanielandAstruc,2004)andupconversionnanoparticles(UCNPs)(Linetal.,2012).Therearetwoformats,i.e.,sandwichandcompetitive(orinhibition)formats,forLFAs(WongandTse,2009).Inthesandwichformat,theconjugatedparticlesreactwiththeanalyteofinterest(ifexists)toformparticleCanalytecomplexes,andthenthecomplexescontinuetomigratealongthe?uid?ow.Thecomplexesarecapturedatthetestlineviatheinteractionbetweenanalytesandcorrespondingcapturingmolecules.Theobsessiveconjugatedparticles,whicharefreeofanalytes,canexceedthetestline.Theseparticlesarethencapturedbyanothertypeofcapturingmoleculesandthusformacontrolline.Inthecompe-titiveformat,theconjugatedparticlescanreactwithcapturingmoleculesdepositedatbothtestandcontrollines.Assuch,theanalytecompetesforthebindingsiteswiththecapturingmole-culesatthetestline,leadingtonon-aggregationofconjugatedparticlesatthetestline.Intheabsenceofanalyte,conjugatedparticlescanbecapturedatbothtestandcontrollines.Bothsandwichandcompetitiveformatassayscanbeusedforqualita-tiveandquantitativedetectionofproteinsandnucleicacidsinpreviousstudies(WongandTse,2009).Ingeneral,sandwichformatassaysareutilizedforananalytewithmultipleantigenepitopes,whilecompetitiveformatassaysaredesignedtodetectananalytewithasingleantigenepitope(WongandTse,.Micro?uidicpaper-basedanalyticaldevices(μPADs)Paperishydrophilicandporous,thusprovidinganaturalplatformforfabricatingmicro?uidicchannels.Micro?uidicpaper-basedanaly-ticaldevices(μPADs)arepioneeredbyWhitesidesetal.atHarvardUniversityin2007(MaoandHuang,2012).Theydevelopedbothtwo-dimensional(2D)(Fig.1D)andthree-dimensional(3D)(Fig.1E)μPADsbypatterningpaperwithavarietyofassaydesigns(Nilghazetal.,2012),inwhichμPADsaremainlybasedoncapillaryforcetodriveaqueous?uidmovement(Martinezetal.,2010b).2DμPADsaremadebypatterningphysicalorchemicalhydrophobicboundariestoformmicrochannelsonpaper(CassanoandFan,2013;Grifantini,2009).Recently,variousapproaches,includingcutting,photolithography,plotting,inkjetetching,plasmaetching,waxprinting,etc.,havebeenproposedtocreatechannelsandbarriersinpaper(Martinezetal.,2010b).Thedimensionsoftheresultingchannelstogetherwiththecharacteristicsofpaperandambientconditions(temperatureandhumidity)canaffectthewickingrateof?uid(Martinezetal.,2010b).ThereagentsrequiredforbiochemicalreactionscanbeimmobilizedJ.Huetal./BiosensorsandBioelectronics54(589:GC.skheuc.;nodec.aimspc)set)02gey-ocef11niil.)blkersat3a00icgese1d22((aw.w.t0.w..lb2lewwy-(aatdcc.wwwtnlawneenae//a///t/gkdrc:::eppgnenoatmpteutelflpetthoHthoRCPtchrphne/Ubr4os8bdanrtiaome)gilLnanoTinzLkom/AcM/l()iitUMoawcIe14mMmL/TS,tCAnfUAdeA0//0maDN.143(1N20455Lpet,.ga.geuj,tnnoeecgAs,adgNaGnaDtngipenieItz2aeme1tlyriialdBa-msobspatSi,n2x2GVgcubnImlHHOlCAaaChHraafGRTrs.,eesattanrteysasresdctserebftineetretatepmsedeanpeondsanipoiatiomtrrgrcctoepatmteeeenpraupplscteusilpaacgenscaituoilmmrspepfdeserlArTeualaapattmtcedrSrelxHsseetEeoeupoepIeeaovpcatHtNaolaLpBpiapLpPELpotrrntaioynpsitdlcaaanoaeobciim:TrnittdnoSicnoirAloimiaafatctekaeCaahedltrnnzcdnaincixeoioaaCpeitccrccenaubelssiaAraPerReAerNyherrpeiforstlmasntraslclistisnanacnneotnacioiomeoihigiithtwcmoteclcccomehaoaoaii?aMCeeerBerBrlanirentaallanoa:Ts;LrseeArirv1aebdepclyiatbcoistuhyrplioavrndaycyrdnheeelpsiaaaccpb?iemdr-deerpoheeancpnrpudaertooptcmihcaldmipii.tseachfuDipo?naAmsoeysPnleyarrmstrμocuiinbahgemnpotyedtimhpa:nal:1acisyesmatsrkirurnemksaDDsAaLCtoctrD-VAboa....AI1.2345atFFaSPhcSPHLμ;,;nstsiysypaetaossrsrstaewoaokplcaa?DDwdaiiplAtrAonsraaP?operPteepμμglitidataractDLrifMliDFs23enotaoilysrr:oamykasrcsaAhc2moistsaFfLtsasDnemaluapAAabliPDFPsLμmuTaAhonpaperwithdifferentpatterns(e.g.,four-leafclover)byhanddispensingorinkjetprinting(Martinezetal.,2010b).Functionalchemicalorbiologicalmoleculescanbeimmobilizedonpaperbyphysicalabsorption,chemicalcoupling,andcarrier-mediated(e.g.,goldparticles)deposition(Pelton,2009;SicardandBrennan,2013).Whenthereagentsaredried,thepaper-baseddevicescanbeusedforbiochemicalanalyses.3DμPADsareproducedbystackinglayersofpatternedpaperinsuchawaythatchannelsinadjacentlayersofpaperconnectwitheachother(Martinezetal.,2008b).Both2Dand3DμPADscanserveasasubstratefor?lteringsamples,performingchromatographicseparations,andtakingbiochemicalreactions(Martinezetal.,2010b).Comparedwith2DμPADs,3DμPADshaveseveralpotentialadvantagesduetotheircapabilitytoincorporatecomplexnetworksofchannels,thusprovidingmultiplefunctional-ities(Martinezetal.,2010b).Micro?uidicpaper-basedelectrochemicaldevices(μPEDs)areonecategoryofμPADswithelectrodes.Inprinciple,thereporteddigitalresultsofμPEDsareelectrochemicalsignalsinsteadofcolorimetricsignalscommonlyusedintypicalμPADs.Theelectro-desusedinμPEDsarepreparedfromconductinginks(carbonormetal)byscreen-printing(Nieetal.,2010b),inkjet-printing(Kit-Ananetal.,2012),orpencil-drawing(Dossietal.,2013)onpaperorplastic.Atypicalelectrode-basedsensingunitcomprisesofthreeelectrodes,includingaworkingelectrode(WE),acounterelectrode(CE)andareferenceelectrode(RE)(Fig.1F)(Nieetal.,2010b).Theworkingelectrodeisbiologicallyorchemicallymod-i?edinordertoachievesensitiveandspeci?csensingfunction.Thespeci?cityofμPEDsissimilarlyachievedbythemethodsusedinpaper-basedcolorimetricassays,suchasantibodyCantigenreaction,nucleicacidhybridizationreaction,orenzymaticreaction(seeSection3).Differentfromcolorimetricassays,thesereactionsusedinμPEDsaremainlyredoxreactions,eitherdirectorindirect.Toensuresuf?cientsensitivityoftheelectrochemicalreactions,materialssuchasAuNPs,carbonnanotubesand/orgraphemenanosheets,areemployedtomodifytheworkingelectrode(Luetal.,2012;Wangetal.,2012a;Yanetal.,2012).3.Reactionmechanismofpaper-baseddiagnosticsVariouspaper-baseddiagnosticshavebeendesignedtodetectpHvalue,urinemetabolites,bloodglucose,liverfunction,hor-mones,infectiousagents,etc.Accordingtothereactionmechanism,thesetestscanbecategorizedintochemical,biologicalandelectro-chemicalreactions(Table2).3.1.Chemicalreaction:colorchangeMostchemicalreactionswithcolorchangecanbeachievedonpaper,suchasacidCalkalireaction,precipitationreaction,redoxreactionandenzymaticreaction,etc.Thesereactionsgenerallyinvolveaone-stepprocedure.Takinglitmusteststripsasanexample,litmusisapurplewater-solublemixturewithcolorchangewhenthepHvaluerangesfrom4.5to8.3.Thesolutionoflitmuschangestoredunderacidicconditionsandtoblueunderalkalineconditions.Therefore,litmusredandblueteststripsareproducedforthedetectionofalkalineandacidconditions,respectively.pHteststripscanbedispensedwithseveralcom-poundstoexhibitdifferentcolorchangesinresponsetodifferentpHvalues.Semi-quantitativedetectionofHtconcentrationsofsolutionscanthenbeachievedbygradingthepHvaluesofsolutionsfrom1to14.Inaddition,moresensitivepHteststripswithprecisionof0.1and0.01havebeendeveloped().ThesepHteststripshaveanarrowtestingpHrangeandtheycanbecombinedtoquantitativelyindicatepHvaluerangingfrom1to14.590J.Huetal./BiosensorsandBioelectronics54(3.2.Biologicalreaction3.2.1.AntigenCantibodybindingAntigenCantibodybindingbasedimmunoassaysdetecteitherantigenorantibodypresentinaclinicalsample.Homepregnancyteststripshavebeenoneofthemostsuccessfuldiagnosticpaper-basedimmunoassayssofar.Itmeasuresahormone,humanchorionicgonadotropin(hCG),inurinefrompregnantwomen.hCGisaheterodimericglycoproteinwithαandβsubunits.αsubunitisidenticaltothatofsomeotherhormones(e.g.,luteiniz-inghormone),whileβsubunitisuniquetohCG.Homepregnancyteststripsjustmakeuseofβsubunitandcontainthreekindsofantibodies,i.e.,anti-hCGantibody,monoclonalantibody(MAb)andimmunoglobinG(IgG).Anti-hCGantibody,conjugatedwithcoloredparticles,canspeci?callyrecognizeandbondwithhCGinthesample.MAbandIgGcanbondtohCGandanti-hCGantibody,respectively,thusformingthetestandcontrollines.Sincethereactionsbetweenthesemoleculescannotbeobservedbythenakedeye,signalmoleculesneedbeemployedtoindicatewhetherthereactionsoccurornot.Thisideahasbeenusedtomeasuretumormarkers,e.g.,primaryhepaticcarcinoma(Yangetal.,2011),andtodiagnoseinfectiousdiseases,e.g.,AIDS(VandenBerketal.,2003).Additionally,ELISAhasalsobeenrealizedonpaper-baseddevices(Apiluxetal.,2013;Chengetal.,2010;Liuetal.,2011b).3.2.2.NucleicacidhybridizationComparedwithantigen/antibody(protein)testing,nucleicacidtesting(NAT)ismoresuitableforearlydetectionofgeneticandinfectiousdiseases(CrawandBalachandran,2012;Yuetal.,2012).Typically,aNATassayrequirestwotypesofoligonucleotideprobes,i.e.,detectorprobeandcaptureprobe.Detectorandcaptureprobesarebothcomplementarywithtargetnucleicacidsequence,whilethedetectorprobeisusedtocombinewithatagtomakethereactionvisibleormeasurable.Thetagcanbecoloredparticles(e.g.,AuNPs)forcolorimetricassays(Huetal.,2013),orelectroactivemolecules(e.g.,thionine)forelectrochemicalmeasurements(Luetal.,2012).Sincegeneticandinfectiousdiseasescanbedeter-minedbyafragmentofgene-speci?cnucleicacids,NATbasedstripspromiseagreatpotentialforrapidandreliablediagnosis.3.2.3.Functionalnucleicacid-basedreactionFunctionalnucleicacids,includingDNAzymes,aptamersandaptazymes,arenucleicacidswhichhavefunctionsnotlimitedtonucleicacidhybridization(Liuetal.,2009).MostDNAzymespossessenzymeactivityinthepresenceofspeci?cions,andthismechanismcanbeutilizedforsensinggivenmetalionsbymeasuringtheenzymaticactivity.Similartoantibodies,aptamersareessentiallynucleicacidmoleculesthatcanspeci?callybindawiderangeofproteins(LiuandLu,2006).AcombinationofDNAzymesandaptamersresultsinaptazymes.TheyhavebeenemployedforcolorimetricsensingmetalionsandbiochemicalmoleculesinLFAs(Liuetal.,2006;Mazumdaretal.,2010).Themechanismoftheirsignaldetectionreliesontargetanalyte-inducedcleavageofnanoparticleconjugatesordisassemblyofnanoparticleaggregates,whichiscapturedatthetestlineforcolordevelopmentonlateral?owteststrips.3.3.ElectrochemicalreactionElectrochemicaldetectioncanbeachievedonthebasisofbothredoxreactionsandnon-redoxreactions(Hanetal.,2013a).Redoxreactionsareinvolvedinelectronstransferbetweenmoleculesorparticles(e.g.,enzymeandnanoparticles),whilenon-redoxreac-tionsarerelatedwiththechangesofelectricalproperties,suchasimpedance,resistance,conductance,andpotential(Hanetal.,2013a).Sinceelectrochemicaldetectionpossessesfeaturessuchashighsensitivityandselectivity,lowcost,andportability,ithasbeenextensivelyappliedinvariousassays,thusprovidinganalternativedetectionschemeforpaper-baseddiagnostics(Dungchaietal.,2009;Peietal.,2013).Themostsuccessfulexampleofelectrochemicaldetectionisthebloodglucosemeterandteststripfordiabeticpatientsthatconstituteabout5%oftheworld'spopulation(HellerandFeldman,2008).Forexample,atotalofabout6billionelectrochemicalstripswereproducedin2007,whichsurpassedtheamountofanyotherassays(HellerandFeldman,2008).Theglucosemeterisinessentialanamperometer,anditmeasuresthequantityofanelectroactivespeciesasaresultoftheoxidationofglucosebyreagentsstoredintheteststrips(Nieetal.,2010a).Brie?y,theteststripisimpregnatedwithglucoseoxidaseandothercomponents(e.g.,ferrocyanide).Whenadropofbloodisadded,glucoseoxidasecatalyzestheoxidationofglucose,andtheglucosemeterquanti?estheelectronsgeneratedbytheoxidationandcorrelatesthemtothelevelofglucoseinblood(Nieetal.,2010a).MoredetailedinformationabouttheprincipleanddevelopmentofglucosemeterscanbefoundinChemicalReviews(HellerandFeldman,2008;Wang,2008).More-over,arecentreviewhasdiscussedthecapabilitiesandlimitationsofcurrentμPEDs(Maxwelletal.,2013).Besidestheabovementionedreactions,therearechemilumi-nescent(Geetal.,2012;Wangetal.,2012b),electrochemilumi-nescent(Yanetal.,2013)andphotoelectrochemicalreactions(Wangetal.,2013a)usedforpaper-baseddiagnostics.Thesereactionscanberegardedasanintegrationoftwoormoreofchemical,biological,and/orelectrochemicalreactions.4.Advancesindevelopmentofpaper-baseddiagnosticsForaqualitativedetection,colorimetricchangesinpaper-baseddiagnosticassayscanbevisualizedbythenakedeyetoyieldayes/noanswer.Incontrast,imaginganalysisbyahandheldreaderoracellphone(Martinezetal.,2008a),orvisualestimationbythenakedeye(bycomparingthecolorchangetoapredeterminedscorechart)(Dinevaetal.,2005)isusedtoreportquantitativereadouts.However,bothqualitativeandquantitativestrategiessufferfromlowsensitiv-ityandpooraccuracy.Additionally,theperformanceofpaper-baseddiagnosticscanbeenhancedbyreducingcomplexityandincreasingfunctionalitysuchastheintegrationofsamplepretreatmentandresultanalysis.Here,wepresentthestate-of-the-artadvancesinimprovingpaper-baseddiagnosticsforPOCapplications.4.1.SensitivityForanumberofanalytes,conventionalpaper-baseddiagnosticsdonothaveenoughsensitivityforclinicalapplications.Forexample,incurrentLFAs,variousstrategieshavebeenwidelyemployedtoincreasetheirsensitivity,suchasenzyme-based(Heetal.,2011;Paroloetal.,2013a)andmetalions(goldorsilver)-based(Rohrmanetal.,2012)signalenhancement.Enzyme-ormetalion-basedenhancementstrategieshavetwostepstoful?llitsreaction,i.e.,?rstlydetectionandthenampli?cation.Thesetwostrategiescanimprovethedetectionlimituptohundredsoffolds.Liuetal.improvedthesensitivityinLFAsbyadoptinghorseradishperoxidase(HRP)toenhancethecolorintensityinthepresenceofHRPsubstrate,3-amino-9-ethyl-carbazole(AEC),whichloweredthedetectionlimitfrom0.5nMto50pM(Maoetal.,2009).TheyfurtheroptimizedtheconjugationofHRPandthiolatedDNAtoAuNPsbyadjustingtheirimmobilizationsequenceandaddingsodiumdodecylsulfate(SDS),whichloweredthedetectionlimitby1000times(Fig.2A)(Heetal.,2011).Ontheotherhand,Paroloetal.(2013a)testedthreedifferentHRPsubstrates,i.e.,AEC,3,3′-diaminobenzidineJ.Huetal./BiosensorsandBioelectronics54(591Fig.2.Generalmethodstoimprovesensitivityinlateral?owassays(LFAs).(A)Enzyme-basedsignalenhancement(Heetal.,2011).(B)Gold-orsilver-basedsignalenhancement(Rohrmanetal.,2012).(C)Dualgoldnanoparticle(AuNP)conjugate-basedsignalampli?cation(Choietal.,2010).(D)Oligonucleotide-linkedAuNPaggregates-basedsignalenhancement(Huetal.,2013).(E)Thermalcontrast(Qinetal.,2012).(F)Architecturemodi?cation(Paroloetal.,2013b).tetrahydrochloride(DAB)and3,3′,5,5′-tetramethylbenzidine(TMB),andfoundthatTMBwasmoresuitablethantheothertwo.Rohrmanetal.(2012)presentedastrategytoimprovethedetectionofHIVRNAusinggoldandsilverenhancement(Fig.2B).Theunderlyingmechanismmaybeduetothefactthatmetallicions(goldorsilver)arereducedanddepositedonthesurfaceofAuNPs,thusincreasingthesizeandopticalextinction(Rohrmanetal.,2012).Recently,coloredparticleconjugationoraggregationhasbeenemployedtoimprovethesensitivityinLFAswithintegrationofdetectionandampli?cationprocedures,i.e.,ampli?cationanddetec-tionareperformedatthesametime(Choietal.,2010;Liuetal.,2011a;Tangetal.,2009).Thisstrategycanimprovethedetectionlimitfromseveralfoldstoseveral10-fold.Choietal.(2010)devel-opedamethodforsignalampli?cationbyusingtwokindsofAuNPconjugates,inwhichoneAuNPconjugatewasusedtodevelopcolorandtheothertoenhancethecolordevelopment(Fig.2C).ByoptimizingthecombinationoftwoAuNPs,thedetectionlimitoftroponin?wasaslowas0.01ng/mL,whichwas100-foldmoresensitivethanthatintheconventionalLFAs.Huetal.(2013)successfullydevelopedimprovednucleicacidLFAsbyusingoligonucleotide-linkedAuNPaggregates,whichholdgreatpotentialtodetectabroadrangeofnucleicacids.Itscorrespondingdetectionlimitwasaslowas0.1nMwithalmost3-foldsignalampli?cation(Fig.2D).Inaddition,Qinetal.(2012)improvedthesensitivityofLFAsto36-foldbyusingthermalcontrast(Fig.2E).Theunderlyingmechanismisthatmetalnanoparticlesgenerateheatinthepresenceofopticalstimulation(GovorovandRichardson,2007).Uponstimu-lation,thesurfaceplasmonatthemetalCdielectricinterfaceofmetalnanoparticlestransfersfromanexcitedstatetoagroundstate,thusreleasingheat(Qinetal.,2012).Bymodifyingthedimensionofteststrips,Paroloetal.(2013b)improvedthesensitivityby8-fold(Fig.2F).Accordingly,thevolumeofsampleandtheamountofreagentsdepositedonteststripswereenlarged,thusincreasingtheamountoftargetClabelcomplexesatthetestline,whichwascon?rmedbyusingmathematicalsimulations.4.2.Quanti?cationClinicaldiagnosisoftenrequiresquantitativemeasurementsofproteins,nucleicacids,andotherbiomarkers.However,visualcolorimetricmeasurementsbythenakedeyeisnotsuf?cientforthequantitativepurposeduetothevariationinvisualperceptionofcoloramongend-usersandunderdifferentlightingconditions(Dungchaietal.,2009).Inordertoachievequantitativeanalysisinpaper-baseddiagnostics,camerasorscannersareusedtorecordthecolorintensityduetoitsrelationshipwiththeamount
of592J.Huetal./BiosensorsandBioelectronics54(Fig.3.Typicalmethodstoachievequantitativedetectionofanalytes.Optics-basedanalysis:(A)scanner-basedanalysis(Glynouetal.,2003),(B)reader-basedanalysis(Maoetal.,2009),and(C)mobilephone-basedanalysis(Mudanyalietal.,2012).(D)Electrochemistry-basedanalysis:glucosemeter-oranalogousreader-basedanalysis(Nieetal.,2010a).(E)Nakedeyes-basedanalysis(Zhangetal.,2012).analyte.Adesktopscannerorcommerciallyavailableportablestripreader(e.g.,DT1030)canbeusedtoobtaintheimagesofassayresults,andtheintensityoftestzonescanbeanalyzedbyimage-processing(Fig.3AandB)(Glynouetal.,2003;Maoetal.,2009).Inaddition,ahand-heldopticalcolorimeterwasdevelopedforquantifyingcolorimetricassaysinμPADsbytheanalysisofthetransmissionoflightthroughpaper(Ellerbeeetal.,2009).Bondetal.(2013)investigatedtheuseofspectrophotometricmeasure-mentofbloodspottedonchromatographypaperasalowcost(o$0.01/test)alternativetotheHemoCuemethod(arecom-mendedstandard-of-careinresource-limitedsettings).Thankstotherapidincreaseinthecoverageandusageofmobilephones(especiallysmartphone)worldwide,mobilephonespromisepotentialtoolsindiagnosticsandtelemedicine(Martinezetal.,2008a).Martinezetal.(2008a)usedacameraphoneasaprobabledetectorfordigitizingtheresultsofpaper-basedcolorimetricassays,whichwascomparabletodesktopscanner,portablescanneranddigitalcamerabasedquanti?cation.Additionally,cameraphonecantransfertheassayresultsfromanon-siteoperatortoanoff-sitelaboratoryforresultinterpretationbyatrainedmedicalprofessional.Further,Ozcanetal.developedacompactandlightdigitalrapid-diagnostic-test(RDT)readerplatformandmanuallyattachedittotheexistingcameraunitofamobilephone(Fig.3C)(Mudanyalietal.,2012).Itwasdemonstratedthatthemobilephone-basedRDTreaderworkedwellwithvariousLFAsfordetectionofmalaria,TBandHIV,thusprovidingreal-timedetection/quanti?cationof
theJ.Huetal./BiosensorsandBioelectronics54(593targetanalytes.ArecentreviewhasdetailedtheuseofmobilephonebasedtechniquesforPOCdiagnostics(Zhuetal.,2013).Electrochemistry-baseddetectionhasalsobeenusedforquanti-?cationinpaper-basedmicro?uidicassays.Dungchaietal.(2009)usedphotolithographytomakeaμPADandthenemployedscreen-printingtechnologytoprepareelectrodesonthedevice.Theyfurtherdemonstratedthedevicewiththeabilitytodetermineglucose,lactateanduricacidinbiologicalsamplesbasedonoxidaseenzymereaction.Whitesidesetal.coupledsimpleelectrochemicalμPADswithacommercialglucosemetertorapidlyquantifytheamountofcompoundsrelevanttohumanhealth(e.g.,glucose,cholesterol,lactateandalcohol)inbloodorurine(Fig.3D)(Nieetal.,2010a).Yuetal.usedcollapsibleμPADandscreen-printedelectrodes(SPEs)tocreatesimple,lowcost,disposaldevicesfordetectionofDNAwithanexcellentanalyticalperformance(Luetal.,2012).Thedetectionlimitfortargetnucleicacidwasaslowas0.2aM.Ontheotherhand,abarcodedetectionstrategywasdevelopedtosemi-quantifytheconcentrationofanalytebythenakedeye,inwhichtheconcentrationsofanalyteswereexpressedasthenumberoftestlines(Fig.3E)(ChoandPaek,2001;Fangetal.,2011;Fungetal.,2009;Zhangetal.,.Simpli?cationEffortshavebeenmadetosimplifythefabricationprocedureanddetectionprotocolofpaper-baseddiagnostics.Forexample,tosimplythefabricationprocedure,Phillipsetal.recentlypublishedasimplemethodforassembling3DμPADsusingsprayadhesivetopermanentlybondmultiplelayersofpaper,thusavoidingtediousalignmentandassemblysteps(Lewisetal.,2012).Further,Martinezetal.presentedanewmethodforfabricating3DμPADsusingtonerasathermaladhesivetobondmultiplelayersofpatternedpaper(Schillingetal.,2013).Thefabricationprocessisrapid,involvesminimalequipment(alaserprinterandalaminator),andcanproducecomplexchannelnetworkswithdimensionsdownto1mm.Onetheotherhand,CassanoandFan(2013)reportedaμPADfabricationmethodbysimplecraft-cuttingandlamination.Theconstructeddevicesusingthismethodhavebeenexploitedforsimultaneousdetectionofbovineserumalbumin(BSA)andglucoseinsyntheticurinewithcolorimetricassays.LiuandCrooks(2011)reportedamethodbasedontheprinciplesoforigami(paperfolding)toprepare3DμPADs.Therequiredreagentsweredepositedonasinglesheetof?atpaperusingphotolithography,andthepaperwasthenfoldedbyhandtoassemble3DμPAD,whichseemspromisingforlowcostandsimpleapplicationsatthePOC(Fig.4A).Tosimplifythedetectionprotocol,Khanetal.(2010)developedadiagnosticpaperwiththreearmstreatedwithdifferentantibodysolutions(A,BandD)forinstantaneousABObloodtyping.Martinezetal.developedafullyenclosedμPAD,whichcouldpreventembeddedmicrochannelsfromcontaminationandavoidevaporation(Schillingetal.,2012).Chengetal.(2010)developedapaper-basedELISA(P-ELISA)whichrequiresalessvolumeofsampleandreagents,simplerequipment,andlessturnaroundtimethanconventionalELISA.Liuetal.(2011b)developedaportable3DμPADforperformingELISA(Fig.4B).Allthereagentsrequiredforanalysiswere?rststoredinadryformwithinthedeviceandthendissolvedinabufferanddeliveredtothetestzones.Thitwasmovedsequentiallytodesiredpointstocompleteaspeci?creaction
requiredFig.4.Representativemethodstosimplythefabricationprocedureanddetectionprotocolofpaper-baseddiagnostics.(A)A3DμPADsfabricatedbyorigami(paperfolding)(LiuandCrooks,2011).(B)Adisposable3DμPADforenzyme-linkedimmunosorbentassay(ELISA)(Liuetal.,2011b).(C)2Dpapernetwork(2DPN)formatassay(Fuetal.,2012).(D)Aself-poweredμPADforon-chip?uorescenceassay(Thometal.,2013).594J.Huetal./BiosensorsandBioelectronics54(inELISA.Viathemovementoftheteststrip,theELISAprocedurewassigni?cantlysimpli?ed.Recently,Apiluxetal.(2013)reportedapaper-baseddeviceforautomatingthemulti-stepprocedureofsandwich-typeELISA.TheirmainstrategyistouseseparatechannelstoperformdifferentstepsusedinELISA.Ontheotherhand,Yageretal.developed2Dpapernetworks(2DPNs),whichexperimentallyandtheoreticallydemonstratedthecapabilityofpaper-baseddevicestocarryoutmulti-stepchemicalproceduresthatarehardtoperformincurrentLFAs(Fuetal.,b;Kauffmanetal.,2010;Lutzetal.,2011).Further,Yageretal.performedgold-basedsignalampli?cationinthe2DPNs(Fuetal.,a,2012).Sincethedistancesbetweeneachinletandthedetectionpadweredifferent,thearrivaltimeofsolutionsintheinletswasdifferent,enablingsequentialreactionandsignalenhancementbyaddingthereagentssimultaneouslyondifferentinlets(Fuetal.,2010a).Thisassaywasfurthersimpli?edbyusinga2DPNcardtostorethegoldenhancementsolution(Fuetal.,2011a)ordryreagents(Fuetal.,2012)(Fig.4C).Fornucleicacidtesting,Govindarajanetal.(2012)reportedalowcostμPADforPOCextractionofbacterialDNAfromrawviscoussamplesusingmicro?uidicorigami.Asdemonstrated,Escherichiacoliwithabacterialloadaslowas33CFUmLà1wasreliablyextractedfrompigmucin(simulatingsputum)andsubsequentlydetected.Additionally,electroniccomponents,suchaspowersourcesandcapacitors,playasigni?cantroleinpaper-baseddiagnosticdevices.Researchershavemadeeffortstointegratepaper-baseddiagnosticdeviceswithpowersourcesandcapacitors.Forexample,Phillipsetal.developedaμPADwithmultiplegalvaniccells(alsotermedas?uidicbatteries)integrateddirectlyintothemicro?uidicchannels(Thometal.,2012).These?uidicbatteriesprovidedpowerforon-chipdevices(e.g.,aUVLED)andmadeanon-chip?orescenceassaypossible(Fig.4D).SuchastrategycouldmakeμPADsindependentofexternallypoweredreaders.Theysubsequentlyreportedtwogeneraldesignsforthese?uidicbatteriesviaconnectioninseriesand/orinparalleltoprovidepredictableandtunablesourcesofpower(desiredcurrentandtime)foron-chipassays(Thometal.,2013).LiuandCrooks(2012)developedabattery-poweredelectro-chemicalsensingplatformwithanelectrochromicdisplayforPOCdiagnostics.Theyemployedanintegratedmetal/airbatterythatpoweredboththeelectrochemicalsensorandelectrochromicread-out.Zhangetal.(2013)developeda3DorigamiμPADwithastable,environment-friendlyandnoblemetal-free(Ag/Agt)primarybattery(C|FeCl3|NaCl|AlCl3|Al)fordrivingluminal(Ru(bpy)32t)electrochemiluminescencesystemtodetectglucose.Yuetal.developedanovelmicro?uidicphotoelectrochemicalpaper-basedanalyticalplatform,whichintegratedaninternalche-miluminescentlightsource,apapersupercapacitor,andanexternaldigitalmulti-meter(DMM)(Geetal.,2013;Wangetal.,c).Thepapersupercapacitorwasformedviaascreen-printedcarbonworkingelectrodeandcounterelectrode.Itcouldcollectandstorethephotocurrentsgeneratedfromthepapersamplezoneunderaninternalchemiluminescentlightsourcefor1min.Oncetheswitchwasturnedoff,thesupercapacitorinstantaneouslyreleasedthestoredelectricalenergythroughtheDMMand
producedFig.5.Multifunctionalpaper-baseddiagnosticswithmultipledetectionorsamplepretreatment.(A)SimultaneousvisualdetectionofmultipleviralampliconsbasedonaLFA(Dinevaetal.,2005).(B)AprogrammableμPADforurinalysis(Novaketal.,2013).(C)Amultiplexedtransaminaseteststrip(Pollocketal.,2012).J.Huetal./BiosensorsandBioelectronics54(595anabout13-fold-ampli?edandDMM-detectablecurrent,whichwasmoresensitivethanthedirectphotocurrentmeasurement.4.4.Multi-functionalizationOneapproachformulti-functionalizationistosimultaneouslydetectmultipleanalytes.Dinevaetal.(2005)developedaLFAwiththreetestlinesforvisualdetectionandidenti?cationofmultiplenucleicacidamplicons,i.e.,hepatitisBvirus(HBV)DNA,hepatitisCvirus(HCV)RNA,andhumanimmunode?ciencyvirustype1(HIV-1)RNAatthesametime(Fig.5A).Inaddition,theyovercamethelowsensitivityofLFAsbyusingdetectorprobeslabeledwithmultiplecoloredparticles,paredwithLFAs,μPADshaveinherentadvantagesinmultipledetectionsduetotheir2Dor3Dstructures.Forexample,Martinezetal.(2010a)developedaprogrammable(post-fabrication)3DmPADsformultiplepatternsof?uid?ow.Further,theyusedthedeviceforurinalysis,withwhichtheend-usercouldchoosetorunasingleormultiplecolorimetricassay(s)forthedetectionofglucose,proteins,ketonesandnitrite(Fig.5B)(Novaketal.,2013).Anotherapproachformulti-functionalizationistointegratesamplepretreatment,separation,reactionanddetection.Yangetal.(2012)developedaμPADthatseparatedbloodplasmafromwholebloodusingthemeshworkofpaper?berstoentangleagglutinatedredbloodcellsandthenperformedacolorimetricassayforplasmaglucose.Further,theydevelopedasimple,rapid,lowcostPOCdiagnostictestforsicklecelldiseasesbyrunningthehemoglobinsolubilityassayinpaper.Duetotheentanglementeffectofthemeshworkofpaper?bers,thepolymerizedhemoglo-binwaspreventedfromdiffusingthroughthepaper,whilethesolublehemoglobinwasfreetowickthroughthepaper(Yangetal.,2013).Themigrationdistanceofsolublehemoglobinfromthecenterofthebloodstainandcorrespondingnormalizedcolorintensityshowedsigni?cantdifferencesbetweennormal,sicklecelltraitandsicklecelldiseasebloodsamples.Whitesidesetal.developeda3DμPADfordetectionoftwoenzymaticmarkersofliverfunction(alkalinephosphatase,ALP,andaspartateamino-transferase,AST)andtotalserumprotein(Vellaetal.,2012).Thedevicecanperformtheentireprocedureofsamplepreparationandqualitativedetection.Amobilephonewasthenusedtodigitizethecolorintensityandtosendtheresulttomedicalprofessionalforoff-siteanalysis,thusgivingaquantitativereadout.Basedonthisstrategy,manyotherbiomarkers(e.g.,alanineaminotransferase,ALT)couldbemeasuredaswell.Further,theydemonstratedamajorprogresstomakethedeviceforrapid,semi-quantitativemeasure-mentofASTandALTfroma?ngerstickwhole-bloodspecimenforlowcost,POCliverfunctiontesting(Fig.5C)(Pollocketal.,2012).Withawellclinicalapplicability,thedevicehasbeenoptimizedforvisualreadoutinASTandALTrangescomparabletocurrentcutoffsusedclinicalmanagementdecisionsforHIVandTBtreatmentmonitoring(Pollocketal.,2012).5.Disadvantagesofpaper-baseddiagnosticsPaper-basedanalyticaldevicesprovidecost-effectivesolutionsforPOCdiagnostics.Theyprovideend-userswithanidealpre-liminaryscreeningtoolforhealthcare.Suchdevicesaresimple,inexpensiveanduseful(Whitesides,2013).However,paper-baseddiagnosticsneedtobefurtherimprovedintermsofclinicalperformance.Asreported,existingpaper-basedanalyticaldevicesshowvaryingspeci?cityandsensitivity(Pikeetal.,2013),whichmaycausefalse-negativeandfalse-positiveresults.Homepreg-nancytestsmayyieldfalse-negativeresultsandleadtounsafesex,contraceptivenon-adherence,andahigherrateofsexuallytransmittedinfectionsandpregnancy(RahmanandBerenson,2013).Ontheotherhand,false-positivehomepregnancytestingresultscanresultinsigni?cantpatientanxietyandunnecessaryinterventions(Nakhaletal.,2012).POCglucosemetersalsohavevaryinglevelsofclinicalperformanceandaccuracy(Watkinsonetal.,2012).Usinginappropriateglucosemetersmayoverestimateglucoselevels,whichleadstoinappropriateinsulindoseadjust-ment(Pereraetal.,2011).OraQuickAt-HomeHIVtest,aFDA-provedPOCHIVtest,hasa92%sensitivityanda99.98%speci?city(Arnold,2012).Intheory,itcancauseonefalsenegativeinevery12HIV-infectedindividuals.Paper-baseddiagnosticdevicesarecriticizedforvaryingsensi-tivitiesandspeci?cities,whichmaybemainlyduetothefollowingfourreasons.First,varyingdetectionmethodsanddiversesub-stratescanbecoupledindifferentcombinationstopreparetestingdevices.Thedetectionmethodsvaryfromopticaltoelectroche-micalsensing.Foragivendetectionmethod,arangeofsubstratescanbeassessedtoreachanoptimalsensitivityandspeci?city,aswellastoreducecost.Forexample,arangeofHi-FlowPlusnitrocellulosemembranes(fromHF075toHF240,Millipore)withdifferent?owspeedscanbeusedinLFAs,dependingontherequirementsforsamplevolume,assaytime,speci?cityandsensi-tivity(Millipore,2013).Ingeneral,thequickerthecapillary?owspeedis,thehigherthespeci?cityis,andthenthelowerthesensitivityis(Millipore,2009).BycomparingmembranesHF240withHF180,Maoetal.(2009)foundthatthesignalofthetestlineonHF240wassigni?cantlyhigherthanthatonHF180.Second,thereexistsvariationinreportingresultswiththenakedeyeamongend-users,especiallyforcolorimetricdetection-basedLFAs.Thesubjec-tivejudgmentfromoperatorsanddifferencesintheilluminationsettingcanleadtocontroversialreadouts,especiallywhenthedetectionsignalisclosetothreshold.Third,therobustnessoftestingdevicescangreatlyaffecttheperformances.Theusedreagentssuchasenzymes,antibodiesorantigensshouldwithstandharshenvir-onmentalconditionsduringstorage,shippingandtesting(ThenandGarnier,2013).Temperatureandhumidity,whichaffectthemigra-tionspeedofliquidandtherecognitionbetweenmolecules,inadditiontothelong-termstabilityofreagents,canleadtovaryingsignals.Fourth,batch-to-batchvariationisanotherwell-knownchallengewhichneedstobeaddressedtoensurereproducibilityforPOCtesting(Abeetal.,2010;Lietal.,2011).Therefore,thesensitivityandspeci?cityofpaper-baseddiagnosticsneedtobefurtherimprovedforwideapplicationsinclinicalorhomesettings.6.ConclusionandfutureperspectivesPaper-basedmicro?uidicdeviceshavebeenwidelyemployedtodevelopPOCdiagnosticsduetotheirlowcostandeasyscale-upinmanufacturing.Withtheinterfacingbetweenmaterialsscienceandbiomedicalengineering,paper-baseddiagnosticsarebecomingsim-pler,moresensitive,moreaccurate,andmulti-functional.Whencombiningpaperdiagnosticswithmobilephonebasedopticaldetection,telemedicineplaysanimportantroleinimprovinghealth-careservicesinresource-limitedsettings(Wangetal.,2011).How-ever,thepotentialofpaper-baseddiagnosticswillnotbemaximizeduntilotheraspectsofPOCdiagnosticsareachievedonpaper,suchassamplepretreatment(Govindarajanetal.,2012),plasmaseparation(Carvalhaletal.,2010),nucleicacidisolationandampli?cation(CrawandBalachandran,2012).Theseaspectsareofimportanceforpaper-basedNAT,whicharesuperiortoconventionalimmunoassaysintermsofspeci?cityandsensitivity.Webelievethattheoreticalsimulationandoptimizationcanmakeagreatcontributiontothedevelopmentofpaper-baseddiagnostics(Fridleyetal.,2013).Wealsoenvisionthatthefullyintegratedpaper-basedNATwiththe596J.Huetal./BiosensorsandBioelectronics54(capabilityofachievingsample-in-answer-outcapabilitywillbeabigstepforwardinimprovingpatientcareatthePOC.AcknowledgmentsThisworkwas?nanciallysupportedbytheNational111ProjectofChina(B06024),theMajorInternationalJointResearchProgramofChina(),theKey(KeyGrant)ProjectofChineseMinistryofEducation(313045),SouthWisdomValleyInnovativeResearchTeamProgram,InternationalScience&TechnologyCoop-erationProgramofChina(2013DFG02930),NationalInstrumenta-tionProgram(No.),theFundamentalResearchFundsfortheCentralUniversities,andChinaPostdoctoralScienceFoundationfundedproject().FLgratefullyacknowl-edgedthefundingfromtheScienceandTechnologyResearchandDevelopmentProgramsupportedbyShaanxiProvinceofChina().BP-MreceivedfundingfromtheMinistryofHigherEducation(MOHE),GovernmentofMalaysiaunderthehighimpactresearch(UM.C/HIR/MOHE/ENG/44).FXwasalsopartiallysup-portedbytheChinaYoung1000-TalentProgramandProgramforNewCenturyExcellentTalentsinUniversity(NCET-12-0437).ReferencesAbe,K.,Kotera,K.,Suzuki,K.,Citterio,D.,2010.Anal.Bioanal.Chem.398,885C893.Apilux,A.,Ukita,Y.,Chikae,M.,Chailapakul,O.,Takamura,Y.,2013.LabChip13,126C135.Arnold,C.,2012.Lancet380,.Ballerini,D.R.,Li,X.,Shen,W.,2012.Micro?uid.Nano?uid.13,769C787.Bissonnette,L.,Bergeron,M.G.,2010.Clin.Microbiol.Infect.16,.Bond,M.M.,Elguea,C.,Yan,J.,Pawlowski,M.,Williams,J.,Wahed,A.,Oden,M.,Tkaczyk,T.S.,Richards-Kortum,R.,2013.LabChip13,.Carvalhal,R.F.,SimaoKfouri,M.,deOliveiraPiazetta,M.H.,Gobbi,A.L.,Kubota,L.T.,2010.Anal.Chem.82,.Cassano,C.,Fan,Z.H.,2013.Micro?uid.Nano?uid.15,173C181.Cheng,C.M.,Martinez,A.W.,Gong,J.,Mace,C.R.,Phillips,S.T.,Carrilho,E.,Mirica,K.A.,Whitesides,G.M.,2010.Angew.Chem.Int.Ed.49,.Chin,C.D.,Linder,V.,Sia,S.K.,2007.LabChip7,41C57.Cho,J.H.,Paek,S.H.,2001.Biotechnol.Bioeng.75,725C732.Choi,D.H.,Lee,S.K.,Oh,Y.K.,Bae,B.W.,Lee,S.D.,Kim,S.,Shin,Y.B.,Kim,M.G.,2010.Biosens.Bioelectron.25,.Craw,P.,Balachandran,W.,2012.LabChip12,.Daniel,M.C.,Astruc,D.,2004.Chem.Rev.104,293C346.Dineva,M.A.,Candotti,D.,Fletcher-Brown,F.,Allain,J.P.,Lee,H.,2005.J.Clin.Microbiol.43,.Dossi,N.,Toniolo,R.,Pizzariello,A.,Impellizzieri,F.,Piccin,E.,Bontempelli,G.,2013.Electrophoresis34,.Dungchai,W.,Chailapakul,O.,Henry,C.S.,2009.Anal.Chem.81,.Ellerbee,A.K.,Phillips,S.T.,Siegel,A.C.,Mirica,K.A.,Martinez,A.W.,Striehl,P.,Jain,N.,Prentiss,M.,Whitesides,G.M.,2009.Anal.Chem.81,.Fang,C.,Chen,Z.,Li,L.,Xia,J.,2011.J.Pharm.Biomed.Anal.56,.Focke,M.,Kosse,D.,Müller,C.,Reinecke,H.,Zengerle,R.,vonStetten,F.,2010.LabChip10,.Foster,L.S.,Gruntfest,I.J.,1937.J.Chem.Educ.14,274.Fridley,G.E.,Holstein,C.A.,Oza,S.B.,Yager,P.,2013.MRSBull.38,326C330.Fu,E.,Kauffman,P.,Lutz,B.,Yager,P.,2010a.Sens.ActuatorsB:Chem.149,325C328.Fu,E.,Liang,T.,Houghtaling,J.,Ramachandran,S.,Ramsey,S.A.,Lutz,B.,Yager,P.,2011a.Anal.Chem.83,.Fu,E.,Liang,T.,Spicar-Mihalic,P.,Houghtaling,J.,Ramachandran,S.,Yager,P.,2012.Anal.Chem.84,.Fu,E.,Lutz,B.,Kauffman,P.,Yager,P.,2010b.LabChip10,918C920.Fu,E.,Ramsey,S.A.,Kauffman,P.,Lutz,B.,Yager,P.,2011b.Micro?uid.Nano?uid.10,29C35.Fung,K.-K.,Chan,C.P.-Y.,Ren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