Paper-based Diagnostic Devices

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Schultz, Noxon, Sisley, Martinez. In Portable Biosensors _{This paper is a postprint of a paper submitted}

and Point-of-Care Systems; 2017 _{to and accepted for publication in Portable}

Biosensors and Point-of-Care Systems and is subject to Institution of Engineering and Technology Copyright. The copy of record is available at the IET Digital Library.

Paper-based

diagnostic

devices

Spencer

A. Schultz

1

_,

_Isabelle

_C.

_Noxon

1

_,

_Tyler

_A.

_Sisley

1

and

Andres

W. Martinez

1

2.1 Introduction

Paper, broadly defined as any porous hydrophilic membrane, has served as a

platformfordiagnosticassaysformillennia.Oneoftheearliestrecordedexamples

of a paper-based assay comes from c. 77 A.D. when Pliny the Elder, a Roman

naturalist, describeda qualitative testfor detecting the presence of adulterants in

verdigrisusingpapyrusimpregnatedwithanextractofgallnuts[1,2].Startingwith

simplequalitativecolorimetricassaysandmovingallthewaytothecurrentfieldof

paperfluidics,paperoffersseveraluniqueadvantagesasaplatformforconductinga

widevarietyof diagnosticassaysaswellasforcollecting,storingandtransporting

samples.Thischapterwillprovideanoverviewofexistingdiagnosticdevicesmade

primarilyoutofpaperandthenfocusonpaper-basedmicrofluidicdevices,thenext

generationofpaper-baseddiagnosticdevicesthatpromisestoextendtheuseofpaper

asamaterialforfabricatingdiagnosticdeviceswellintothefuture.

Paperisdefinedformallyasaflexiblesheetmadefrompressedcellulosicfibers

(Figure2.1)[3].TheprocessofmakingpaperwasdevelopedinChinaduringtheHan

Dynasty(206B.C.to220A.D.)[4].WrittenrecordscreditTsaiLun,anofficialinthe

HanCourt,withdeveloping themodernformofpaperas wellas thepapermaking

processin105A.D.[5].Althoughtheequipmentusedformakingpaperhasevolved

overtime,theessentialstepsofthepapermakingprocesshaveremainedunchanged:

first,adilutesuspensionofcellulosicfibersispreparedinwater;then,thefibersare

depositedontoascreenformingamat;and,finally,thematoffibersispressedand

driedtoproduceasheet ofpaper[5].Thecellulosicfibersusedtomakepapercan

comefrommanysources,butmostdiagnosticdevicesaremadewithpaperproduced

fromcottonfibersmadeprimarilyofcellulose(Figure2.2(a))[4].Manyothertypes

of porous hydrophilicmembranes have been developed over time and have been

incorporatedintodiagnosticdevices[3].Amongthese,porousmembranesmadefrom

nitrocelluloseareusedmostcommonlybecauseoftheirhighprotein-bindingaffinity

1_Department_of_Chemistry_and_{Biochemistry,}_California_Polytechnic_State_University,_San_Luis_Obispo,

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AN: 1579526 ; Kintzios, Spiridon E..; Portable Biosensors and Point-of-Care Systems

100 µm

Figure2.1 SEMmicrograph(400)ofthesurfaceofWhatmanNo.1 chromatographypaper

HO

OH

OH _OH

OH

O

O O O O

O

O O

O

O2N

O₂N (a)

O2N _O 2N

NO2

O

n

n O

O HO

(b)

Figure2.2 Chemicalstructuresofcellulose(a)andnitrocellulose(b)

(Figure2.2(b))[3].Anenormousdiversityofpaperswithvaryingchemical

compo-sition,thickness,andporesizeareavailable,andeachhasuniquecharacteristicsthat

couldbeharnessedforspecificapplicationsinpaper-baseddiagnosticdevices.

Paperhasmanyinherentcharacteristicsthatmakeitattractiveasaplatformfor

diagnostic assays.Paper isinexpensive andwidely available,so scientists across

the globe andthroughouthistoryhave beenable toworkwithpaper anddevelop

teststhatarecompatiblewithpaper[2,4,6].Paperistypicallywhiteandprovidesan

excellent background for assays that produce a colored product or a change in

color. Another attractive characteristic of paper is that it has a large

surface-to-volumeratio, soasmallvolumeof fluid(1mL)cantypicallyproduceaspoton

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largesurface-to-volume ratioof paperalso allowsforreagentsandsamples tobe

driedandstoredonpaper-baseddevices.Papercanevenbemodifiedchemicallyin

ordertocovalentlybondreagentstothefibers[7].Paper canalsoserveasthe

sta-tionaryphaseforchromatographicseparationsofanalytes[8].Paperisdisposable—

one simple method for disposing paper-based tests is through incineration. Paper

wicks fluid bycapillaryaction, so fluids will movethrough apaper-based device

withoutanyexternalsourcesofpower.Andfinally,asmentionedpreviously,paper

canbeproducedfromawidevarietyoffibersandwithawidevarietyof

character-istics(e.g.,poresize,wickingspeed,proteinbindingability),whichcanbeharnessed

for specific applications and can even be combined in a single device to enable

particularcapabilities.

2.2 Current

paper-based

diagnostic

devices

Oneofthebest-knownexamplesofapaper-basedtestislitmuspaper,whichisused

todeterminewhetherasolutionisacidicorbasic[7].Scientificreportsdatingbackto

thenineteenthcenturydescribetheuseoflitmuspaperforanalyzingsamples,anditis

still sold commercially and used routinely by professional scientists and students

[6,9].Inadditiontolitmuspaper,ahugevarietyofothercolorimetrictestshavebeen

developedtodetectthepresenceofanalytesonpaper,butmostofthesetestshavenot

beendevelopedintocommercialproducts[2,10–12].Currently,therearetwoprimary

formsofcommerciallyavailablepaper-baseddiagnosticdevices:dipstickassaysand

lateral-flowassays(LFAs)[3].Athirdformofpaper-baseddevicesknownas

paper-basedarraysareusedprimarilytocollect,store,andtransportsamplesbutarealso

usedinacademicresearchtoconductassays.

2.2.1 Dipstick

devices

Dipstickassaysareoneofthefirstexamplesofpaper-baseddiagnosticdevicesfor

clinicaluse[4].Dipstickdevicescompriseoneormoresmalltestpadsofpaperthat

havebeen impregnatedwithreagentsfor specificcolorimetricassaysandthatare

adhered to aplastic strip, which serves as a handle for the device (Figure 2.3).

Dipstickassaysareperformedbyquicklyimmersingthedipstickintothesampleto

be tested and then observing the colorsthat are produced on the test pads. The

colors produced on the test pads can often be compared to a color-coded chart

providedwiththedipstickinordertomakeasemiquantitativedeterminationofthe

concentrationoftheanalyteinthesample[3].

The first pH dipstick test strips were patented and commercialized in the

1920s[4].ThesedevicescanbepurchasedtodeterminethepHofasolutionwithin

agiven range withvaryingamountsof sensitivity, bothof whichare determined

bythenumberoftestpadsonthedipsticksaswellasthespecificindicatorsusedon

thetestpads [13].The firstdipstickassaysforclinical useweredevelopedinthe

1950sand wereintroduced commercially inthe 1960s [3]. Theseinitial dipstick

assays were developed to quantify the concentration of glucose in urine by

harnessing enzymatically catalyzed reactions involving glucose oxidase and

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Figure2.3 Dipstickdeviceforanalysisofwater[18]

test pads to detect or analyze glucose, bilirubin, ketones, nitrite, urobilinogen,

protein,blood,leukocytes,pH,andspecificgravity[3,15,16].Thesetestsallowfor

the diagnosis of a variety of conditions and diseases including kidney disease,

urinary tract infections, carbohydrate metabolism disorders, and liver disease

[4,15,16].Formoresophisticatedurinalysistests,thedipstickscanbeanalyzedbya

detector thatanalyzesthe colorof thetestpads inordertoimprovethe accuracy

andprecisionofthetests[3,17].Anothercommonapplicationfordipsticktestsis

for theanalysis ofwater samples.Dipstickdevicesfortestingdrinkingwaterand

swimming poolscanbeusedtoquicklydeterminepH,totalhardness, total

chlor-ine,totalbromine,freechlorine,totalalkalinity,andcyanuricacidlevels[18].

Themainadvantagesofdipstickdevicesarethattheyareinexpensive,easyto

use and provide immediate results. Dipstick tests are also simple to design and

manufacture.Thedisadvantagesof dipstickassaysarethattheyaretypically

qua-litativeorsemiquantitative,theyoftenhavelimitedsensitivityandselectivity,and

theycanonlybedevelopedforanalytesthatcanreacttoproduceacolorchangeorbe

linked to a reaction that produces a color change [6]. Dipstick assays often also

requirearelativelylarge volumeofsamplein ordertowetthe testpads.For these

reasons,dipstickdevicesaremostpracticalfortestingrelativelysimplesamples,such

as urineand water, thatareabundant andcontain relativelyhighconcentrationsof

analytes,suchasglucoseorchlorine.Inordertoovercomesomeofthelimitations

withdipstickassays,LFAsweredevelopedtoexpandtherangeofanalytesthatcould

bedetectedaswellasthesensitivityandselectivityoftheassays.

2.2.2 Lateral-flow

devices

Asthename suggests,lateral-flow devicesrelyonthecapillarywicking offluids

across aporousmembraneinordertodetectananalyte[19].Lateral-flowdevices

aremadetypicallyofseveraldifferenttypesofpaperthataretreatedwithdifferent

reagents andheld in contact with each other to create a single fluidic path that

wicksasamplefromasampleporttoanabsorbentpad,whichservesasa

waste-collection zone (Figure 2.4) [20]. In a typical LFA, a few drops of sample are

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Sample port

Sample pad

Test line Control line

Housing

Absorbent pad Conjugate pad

Membrane

Figure2.4 Schematicdiagramofalateral-flowdevice.Reproduced by permissionofEMDMilliporeCorporation. 2002,2008EMD MilliporeCorporation

whichitinteractswithalabelingreagent—typicallyanantibodyboundtoacolored

particle—that binds specifically to the analyte. The labeled analyte then wicks

across amembrane containing acapture reagent—typically a secondantibody—

thatisimmobilizedonthedeviceinanarrowtestlineandthatcapturesselectively

the labeled analyte. Thisprocess generatesa colored lineor band on the device

whentheanalyteispresent.Alltheothercomponentsofthesampleaswellasany

excesslabelingreagentcontinuetowickalongthedeviceintothewastezone.Most

commercialdevicesalsoincludeacontrollinecontainingasecondcapturereagent

onthemembranethatbindsonlytothelabelingreagentandservestoconfirmthat

thecapturereagentsareworkingproperly[3].

LFAsare widelyused inlaboratories,hospitals, andinthehome wherethey

candetectconditionssuch as pregnancy,disease, druguse,contamination,

infec-tion,andorganmalfunction[3,21–23]. ThefirstLFAsweredevelopedinthe late

1960sandhavebecomeauser-friendly,robust,affordable,point-of-care platform

fordiagnostics[19].Thefirst commercialLFAsweredevelopedtodetecthuman

chorionicgonadotropin(hCG);ahighlevelof thisbiomoleculeisapositive

indi-cator for pregnancy [24]. This test was fast (1–3 min), simple to use even for

untrainedusers,required littleuser input,used smallsample sizes, andwas

sub-sequently made commercial by1988[24].The hCG testisnowused globally to

detectpregnancyandcanbepurchasedforlessthanUS$1[25].

ThethreemainfacetsofLFAsarethedeviceorplatform,samplepreparation,

andtheimmunochemicalreaction.TheLFAplatformiscomposedofasamplepad,

afiberglass conjugate padfor storageof the labeled reagent, amembrane thatis

typicallynitrocellulose, anabsorbent waste pad, andoften asmallplastic holder

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brittleandflammable,itisthemembraneusedinmostLFAsbecauseitisporous,

wicks fluids by capillary action, and its surface has a high affinity for binding

proteins[4,23].Thisbindingcharacteristicisessentialforthedetectionofanalytes,

asLFAsrelyonselectivebindingandretentionofthelabeledanalyte.Fabrication

of commercial LFAs begins with treatment of the different components. The

sample padis pretreated,the fiberglass conjugate padisloaded withthe labeled

reagent,andthemembraneisprintedwithtestandcontrollinesofcapturereagents

andthenblockedfornonspecificadsorption[20].Eachcomponentisdried,andthe

deviceisassembled,cutintostrips,andplacedintocartridgesforpackaging[26].

The hCG urinetest does not require much sample preparation,but there are

many otherLFAsthatdetectbiological componentsfrom bloodplasma orserum

that dorequire treatment. Often samplepreparation includes collection of blood,

treatmentwithanticoagulants,separationofbloodcellsfromplasmaorserum,and

dilution of the sample [19]. Thesesteps are important, but are cumbersomeand

timeconsuming—especiallyatthepointofcareinwhichexpensiveequipmentfor

sample treatmentisdifficulttoobtainornotavailable[27].

The immunochemical reaction is arguably the most important component of

LFAs. There are two main types: immunoassays that rely on antibody–antigen

interactions, and nucleic acid-basedassays that relyon nucleicacid aptamers and

target moleculebindingor nucleicacidhybridization[19].These immunochemical

reactions rely heavily on the sensitive and specific interactions that consequently

determinethesensitivityandselectivityoftheassayitself[19].Thecomponentofthe

immunochemical reactionthatallowsforvisualdetectionisthecolorimetricprobe,

whichisconjugatedtoanantibody,protein,orDNAsequencetomakethelabeling

reagent.Theprobesaremostcommonlylatexbeads,goldnanoparticles,fluorescent

tags, quantumdots,andenzymes.The typeof colorimetricprobe isdeterminedby

several factors including the type of sample, the analyte, pH, and device storage

conditions.Immunochemicalreagentscanbesynthesizedagainstamyriadofproteins

and biomolecules. The heterogeneity of immunochemical combinations is part of

whatmakesLFAssuchawidelystudiedandversatileplatform[19].

ThemainadvantagesofLFAs,aswithmostotherpaper-baseddevices,arethat

theyareinexpensiveandeasytouse—the usertypicallyonlyhastoapply afew

dropsofsampletothedeviceandthenreadtheresultingcoloredlines,unlessmore

sophisticatedsamplepreparationisrequired[3].WhatsetsLFAsapartfromother

paper-baseddevices, likedipsticks,isthatbyharnessing the wickingcapabilities

of paper, LFAs perform what is effectively a chromatographic separation of the

analytefromtheothercomponentsofthesampleaswellasaconcentrationofthe

analytealongthetestline.Boththeseactions leadtohighlysensitiveandspecific

assays.Somemoresophisticated LFAs includeasecondstepinwhichan

ampli-ficationreagentisaddedtothedeviceinordertoenhancethecolorofthetestline

andfurtherimprovethesensitivityofthedevice[3].AnotheradvantageofLFAsis

thatas theyrely onimmunochemical reactionstodetectanalytes, thesamebasic

devicedesigncanbeusedtodetectahugerangeofanalytesbysimplychangingthe

labeling reagent and the capture reagent [19].On their own,LFAs are typically

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withelectronicreadersthatanalyzetheintensityofcolorthatdevelops atthe test

lineorrelyonfluorescentlabelsinordertomake quantitativedeterminations[3].

2.2.2.1 Vertical-flowdevices

Vertical-flowdevicesrely onthesameanalyte-detectionprinciplesaslateral-flow

devicesandcanbeusedtoconductthesametypesofassays,but,insteadoflining

up the different components of the device horizontally as in LFAs, the various

componentsarestackedvertically.Theadvantageofstackingthelayersvertically

isthat the pathlengthfor the sample towick throughthe device issignificantly

reduced;however,theseassaystypicallyrequiremultiplestepsbytheuserinorder

tobeperformedcorrectly[3].

2.2.3 Paper-based

arrays

Paper-basedarraysareusedprimarilyforthecollection,storage,andtransportation

of samples. Paper-based arrays typically comprise a card marked with multiple

circularzonesthatcanbefilledwithliquidsamples.Thezonesareoftenpretreated

withreagents toimprove the stability ofthe analytes or todisinfectthe samples.

Samples,suchasblood,canbespottedonthearrayanddried,andthenthe array

canbe shippedtoacentral laboratorywherethe samples canbeeluted fromthe

arrayandanalyzed [28].Thisprocess allowsfor large numbersof samplestobe

collectedandstoredinaconvenientformatthatdoesnotrequirerefrigeration[29].

The sample collection can also be performed in the field with minimal

instru-mentation[29].

Onecommonapplicationforpaper-basedarraysisindriedbloodspot(DBS)

testing [28].This techniquewasdevelopedintheearly1900sbutgained

wide-spread use in the1960s and1970s when RobertGuthrie used DBSto develop

atechniqueforthesystematicscreeningofnewbornsformetabolicdiseases[30].

NowknownastheGuthrietest,newbornsaroundtheworldaretestedforaseries

of metabolic disorders and diseases by collecting their blood from a heal

prickon a DBScard[28].In fact, DBScan be used todetect awide range of

analytesanddiseasesincludingHIV,dengue,hepatitis,andsmallmoleculedrugs

[28,29,31].

Analternativeapplicationofpaper-basedarraysisforthesynthesisoflibrariesof

shortpeptidesorsmallmoleculesviaatechniqueknownasspotsynthesis[32–35].

Onceprepared,thesearrayscanbeusedtoconductawiderangeofassaysincluding

bindingassays,enzymeassays,andstudieswithlivingorganisms[36].

2.3 Paper-based

microfluidic

devices

In2007,theWhitesidesgroupatHarvardUniversitypublishedanarticledescribing

thefirstexampleofwhatwouldbecomeknownaspaper-basedmicrofluidic

devi-cesormicrofluidicpaper-basedanalytical devices(microPADs)(Figure2.5)[37].

Havingcontributedasubstantialbodyof worktothefield ofconventional

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Figure2.5 Apaper-basedmicrofluidicdevicefordetectingglucose(leftzones) andprotein(rightzones)

Whitesides was looking to develop a new class of devices that married the

advantagesoflateral-flowanddipstickdevices,suchascapillarywickingandlow

cost, with the advantages of conventional microfluidic devices, such as low

volumesofsample,multiplexing,andcontrolledsequentialsteps.Theultimategoal

oftheWhitesidesgroupwastodevelopanewclassofultra-low-costpoint-of-care

diagnostic devices thatcould bedeployed toresource-limit settings andimprove

accesstohealthcaretopopulationsaroundtheglobe.

Paper was a natural choice as a material for fabricating this new class of

devices forallthesamereasonsmentioned inthe introduction.Themain

innova-tion describedintheinitialpublicationfromtheWhitesidesgroupwasthe

devel-opmentofamethodtopatternpaperintoanetworkofhydrophilicchannelsandtest

zonesboundedbyhydrophobicbarriers[37,40].Thechannelscouldwicksamples

and distributethem into the testzones whereassays wouldtakeplace. Although

examples of patterning paper usingwaxes had been described previously [2,41],

whatsettheworkfromtheWhitesidesgroupapartwasthefocusonthe

develop-ment of anew class of lowcost, verysimple to useportable diagnostic devices

[37,42].Thisworkcatalyzedasurgeofinterestinthedevelopmentofpaper-based

assaysandresultedintheestablishmentofthenewfieldofpaperfluidics[3,6,12].

2.3.1 Fabrication

of

paper-based

microfluidic

devices

Fabrication ofpaper-basedmicrofluidic devicesinvolvesthepatterningofapiece

of paper using hydrophobic inks to define hydrophilic channels and test zones

boundedbyhydrophobicbarriers[12].Severalmethodsfor patterningpaperhave

been developed since 2007, each with their own set of advantages and

dis-advantages [6,12].Althoughthe methodsare verydiverse interms of equipment

and inks, they can be classified broadly into two categories: indirect and direct

patterning.Inindirectpatterning,thefirststepoftheprocessinvolvescoatingthe

entirepieceofpaperwithahydrophobicinkorreagentsuchasawax,apolymer,or

aresin.In thesecondstep,thehydrophobicreagent isetchedawayselectivelyto

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Indirect patterning Direct patterning Paper

Hydrophobic barrier

Hydrophilic channel

Figure2.6 Cross-sectionalschematicdiagramofindirectanddirectpatterning ofpaper

producehydrophilic channels.In directpatterning,thehydrophobicinkisprinted

directlyonthepaperinthedesiredpattern(Figure2.6).

Thefirstpublishedmethodforfabricatingpaper-basedmicrofluidicdeviceswas

anexampleofindirectpatterningandwasanadaptedformofphotolithography[37].

In this approach, thepaper is firstimpregnated with SU-8 photoresist,anegative

photoresist,anddried.ThepaperisthenexposedtoUVlightthroughaphotomaskin

order tocross-linkthe exposedportionsof the photoresist.Finally,theunexposed

portions of the photoresist are dissolved and removed from the paper using a

solventsuch as acetoneto yieldthe desiredpattern of channelsandtest zonesin

thepaper[37,40,42].Otherphotopolymers,inadditiontoSU-8,havealsoshownto

beusefulforpatterningpaperviaindirectpatterning[3].

Indirectpatterningwasalsoachievedusinganinkjetprinter[43].Theprocess

involvesfirstcoatingapieceofpaperwithpolystyrenetomakeithydrophobic.The

polystyreneisthenselectivelyremovedfromspecificareasofthepaperbyprinting

toluene ontothe paper usinganinkjet printer inthe desiredpattern. The toluene

washesthepolystyreneoutofthepaperandreturnsthewashedpapertoitsoriginal

hydrophilicstate.

A third example of indirect patterning using plasma treatment involves

impregnatingasheetofpaper withalkyl ketenedimer,areagent usedcommonly

forsizingpapertomake itmorehydrophobic[44].Thereagentisthenselectively

etched awaybyplacingthe paper betweentwometal stencilsandtreatingit ina

vacuum plasma reactor. The areas of the paper that are exposed to the plasma,

through the metal stencils, are made hydrophilic andthe areasof paper thatare

protectedbythestencilremainhydrophobic.

In direct patterning, hydrophobic agents are applied onto the paper in the

desired pattern. Hydrophobic agents are applied typically through conventional

printingtechnologies,suchasinkjetprinting,screenprinting,flexographicprinting,

andstamping,butcouldevenbeappliedbyhand.Eachtechniquehasitsownsetof

advantagesanddisadvantagesintermsofcost,resolution,throughput,and

techni-caldifficulty.Oneofthemostpopularmethodsofdirectpatterningiswaxprinting

[45–47]. In wax printing, asolid-ink printer is used toprint wax directly onthe

surfaceofpaperinanydesiredpattern.Theprintedpaperisthenheatedtoreflow

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completed inlessthan5minfor anentiresheetof paper,andfeaturesasmallas

1 mm can be patterned reliably [46].The equipment required for wax printing,

namelythesolid-inkprinterandtheheater(e.g.,oven,hotplate,thermallaminator,

heatgun,andiron)canbepurchasedforlessthanUS$1,000,andtheprocessdoes

notrequireacleanroomorafumehood,sowaxprintingcanbeperformedbyusers

with limited resources or limited experience with fabrication [12]. Another

advantage of waxprinting isthat thereis virtually noproduction-cost penaltyfor

makingsmallbatchesofdevicesatatime,whichmakesitwellsuitedfor

laboratory-scaleprototyping.

In comparing indirect and direct patterning methods, indirect patterning

methodsaregenerallymoretimeconsumingastheyinvolveatleasttwosteps,but

oftenseveralmore.Indirectpatterningmethodsalsoconsumelargerquantitiesof

reagents and solvents as the entire piece of paper is treated and made

hydro-phobic. Direct patterning methodsare generallyfaster, less expensive, andless

technically challenging than indirect methods. One advantage of indirect

patterning,however,isthat ittends toallowforhigherresolutionpatterns tobe

produced. Specifically in the case of photolithography, the resolution of the

patternis controlled by the resolution of the exposure to UV light, so smaller

featurescanbeproducedusingthismethodcomparedtowaxprinting[12].Inone

instance,directandindirectmethodsofpatterningwerecombinedtopatternpaper

withTeflonin orderto generatemicroPADsthat werecompatible withorganic

solvents[48].

A third method of preparing microPADs is via shaping or cutting. In this

approachthe desired network of channels andtest zones iscut out of asheet of

paper,eithermanuallyorusingalasercutterorcuttingplotter[49,50].Thismethod

issimpletoperformanddoesnotrequireanyreagents.Thedevicesfabricatedvia

cuttingusuallyhavetobeencasedinplastictoprotectthepaper,providestructural

supporttothepaper,andfacilitatemanipulationofthedevices[12].

Once the patterning processis complete, devicescan be furthermodified to

addadditionalcapabilities.Onecommonpost-patterningstepistoencasedevices

inplasticusingeithertape,thermallaminationsheetsortoner[49,51,52].Another

optionistoaddadditionalfeaturestothedevicesuchaselectrodes.Electrodesare

typicallyprinted ontothe devices after they are patterned usingconductive inks

[53–57].Devicescanalsobemodifiedtocontrolthewickinginthedeviceeitherby

incorporating valves [58], adding reagents or shunts that slow down wicking

[59,60], or adding additional layers of paper or plastic to speed up wicking

[52,57,61].Finally,multiplelayersofpatternedpapercanbestackedontopofeach

other to produce three-dimensional (3D) microPADs (Figure 2.7) [62–65]. The

layersofpaper inthe3D devicecan eitherbe bondedtoeach otherpermanently

usingpermanentadhesivesortapes,ortheycanbeheldtogethertemporarilyusing

amanifoldorremovableadhesivesortapes.

Tocompletethefabricationprocess,reagentsfortheassaymustbeappliedto

the device.Typically reagentsare deposited onto microPADsfrom solution, and

the solutions are then allowed to dry leaving the reagent behind on the device.

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1 cm Cellulose powder Channel 0.2 min 2 min 4 min

1 mm Tape Channel

Figure2.7 Topviewandcross-sectionalviewofathree-dimensional microPADassembledfromtwolayersofpatternedpaperand alayerofdouble-sidedtape.ReproducedbypermissionofThe NationalAcademyofSciencesfromReference62. 2008 NationalAcademyofSciences,USA

deposited using an inkjet printer or liquid-dispensing robot. More recently, a

methodforsoliddepositionofreagentsusingcustom-madepencilswasdescribed,

whichledtoimproved shelflifeofthereagentsonthedevices[66].

2.3.2 Applications

of

paper-based

microfluidic

devices

Initial research on paper-based microfluidic devices focused primarily on the

development of methods of fabricating devices. The first devices consisted

gen-erallyof asampleinletthatdirectedthe sampleinto onemorechannels andthen

into multipletestzones inwhichreagentsfor colorimetric assays weredeposited

(Figure2.5).Theseinitialdevicesrequired smallvolumesoffluid (20mL)and

could performmultiple assayssimultaneously fromasinglesample-addition step

[37,42].Asworkwithpaper-devicesexpanded,newapplicationsanduniqueassays

were developed, many of which were enabled by the unique capabilities of

microPADs.Thissection will highlightfourareasofapplications for microPADs

thatshowpromiseforfuturediagnosticdevices.

Colorimetric assays, assays that result in a color change in response to the

presenceofanalyte,remainoneofthemostcommontypesofassaysconductedon

microPADs [3]. Awide range of colorimetric assays have developedfor

micro-PADsincludingindicator-basedassays,enzymaticassays,sorbent-basedassays,as

wellas combinationsas theseapproaches such asenzyme-linkedimmunosorbent

assays (ELISAs) [3].These assays have beenshown tobe usefulfor detecting a

widerangeof analytesincludingions,smallmolecules,proteins,andDNA.

Con-current with the development of colorimetric assays on microPADs, several

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Hydrophobic

Glucose 6 mm

CE

WE Lactate

5 mm RE Hydrophilic _{5 mm}

(a) Uric acid

5 mm (b)

Figure2.8 Schematicofanelectrochemicalpaper-basedmicrofluidicdevice includingthreeelectrodes(a),andimageofanactualdevicefor detectingglucose,lactate,anduricacid(b).Adaptedwithpermission fromReference53. 2009AmericanChemicalSociety

developed. One promising approach is theuse of digital image colorimetry,in

whichadigitalimageoftheresultsofacolorimetricassayisobtained,andthen

thepixelintensityofthecolorismeasured[67,68].Theimagescouldbeobtained

using a scanner, a digital camera or, most interestingly in the context of

portablepoint-of-carediagnosticassays,acameraphone[42].Theresultscanbe

calibrated througheither externalcalibrationorstandard additionandcan

typi-callyproduce resultswithrelativeerrorsandrelative standarddeviationswithin

tenpercent[68].

As an alternative to colorimetric assays, paper-based assays that rely on

electrochemical detection offer many advantages, especially when it comes to

performing quantitative tests [53,55,56]. Electrochemical paper-based assays

have been demonstrated for a wide range of analytes with high accuracy and

precision, and with the abilityto detect analytes at femtomolar concentrations

[6,69,70].Theelectrodesforelectrochemicalassaysaretypicallyprinteddirectly

on themicroPADs(Figure 2.8),and anexternalreader is usedtypicallyto

per-formtheassay[6].

A third type of paper-based assays that eliminate the need for sophisticated

external readersarechronometricassays,inwhichthesignalfromtheassayisthe

time that it takes the sample to wick across a channel in a device (Figure 2.9)

[64,71–74].TheseassaysrelyontheuniquepropertiesofmicroPADstowickfluids

alongchannelsinpaperaswellastheabilitytopatternreagentsinmultiplelayers

ofpaperandthenassembletheselayersintoasingle3Ddevice.Inthesedevices,a

reagent that creates and impermeable or semi permeable barrier is added to a

channelinthedevice.Thepresenceoftheanalytetriggersachemicalreactionthat

resultsinthedegradationofthebarrier.So,whentheanalyteispresent,thesample

wicks acrossthe channelmorequickly thanwhen the analyte isabsent.

Chrono-metricassaysstandoutfortheirsimplicity,sensitivity,andlowlimitsofdetection,

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Add sample

Wait for the left Measure the time to region to turn green appearance of color in the right region relative to the left

(a)

Start the measurement when layer 1 turns green

Add sample

Stop the measurement when layer 1 turns green

layers

1 2 3 4 5

Assay region Control region

Laminate Hydrophilic paper

Substrate and immobilized GOx Hydrophobic paper

Phase-switching reagent (2) Buffer containing MgCI2 (b) Green dye Enzyme substrate (1)

Figure2.9 Achronometricpaper-basedassay(a)andacross-sectionalschematic ofthelayersandcomponentsmakingupthedevice(b).Adaptedwith permissionfromReference74. 2013AmericanChemicalSociety

Inadditiontodevelopingspecifictypesofquantitativeassays,thepaperfluidics

communityhas devotedconsiderable efforttoward developingdevicescapableof

performing multiple sequential stepsin order to automatically perform multistep

assays,such as ELISAs or enzyme-inhibition assays.For example, a device that

performs a typical lateral-flow immunoassay and then automatically adds an

amplification reagent to enhance the intensity of the test line on the device and

improvethe limit of detectionof the assay was developedby designinga device

with multiple fluid inlets leading to a single-test zone (Figure 2.10) [50,75,76].

Alternatively,asimilartypeofassaywasachievedbycarefullydesigningaseriesof

channelsthatwouldwickfluidfromasinglesampleinletanddeliverittoatestzone

atdifferenttimes[77].Thedevelopmentoffluidicdiodesprovidedathirdoptionfor

achievingautomatedmultistepassaysonmicroPADs [78,79].Finally,paper-based

enzyme-inhibitionteststhatrequireincubationofanenzymewithasamplefollowed

byintroductionoftheenzymesubstratewereautomatedbyusingerodiblepolymeric

bridges,whichallowedfortheintroductionofsingle-usetimedshut-offvalvesinto

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Rehydration of dry reagents

Gold

Paper enhancement

network reagents

Buffer

Conjugate Wicking

pad

15 s

Source pads

Arrival of yellow

Original signal Unamplified Amplified signal Amplified Enlarged Gold particle –

Test

control

particle antibody conjugate

Antigen

Sample

capture antibody 4 Min Water

8 min 30 min

(b) Control

indicates

Arrival of red _{0 ng/mL} ₅ ₁₀ ₂₀ ₄₀

card failure

40000

11 Min

30000

Amplified

Unamplified

10000

0

0 10 20 30 40 50

18 Min Concentration (ng/mL)

(a) (c)

Figure2.10 Multisteppaper-basedassay.(a)Imagesofthedeviceperforming multiplesequentialdeliveryofreagentsusingdyesassample reagents.(b)ImageofactualdevicefordetectingMalaria. (c)Resultsofthetestshowingtheimprovedsignalobtainedusing themultistepdevice(amplified).Adaptedwithpermissionfrom Reference76. 2012AmericanChemicalSociety

2.4 Conclusions

Paperoffersseveraladvantagesasaplatformforconductingsimple,point-of-care

diagnosticassays.Current,commerciallyavailablepaper-baseddiagnosticdevices,

suchasdipstickassaysandLFAs,willcontinuetobeusedwidelyduetotheirlow

cost and simplicity. A newgeneration of paper-based devices is currently being

developed and promises to extend those applications of paper for point-of-care

diagnostics.

Signal

20000

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Theconceptofmakingdiagnosticdevicesoutof paperisbeingadoptedbya

growingcommunityofresearchers.MicroPADswerefirstdevelopedasdiagnostic

devices for usein developingcountries butare now being developedtodetect a

wide rangeof analytes andcould beused tomonitorair,soil, andwater quality;

theycouldbeusedasdiagnosticdevicesforanimalsandplants;theycouldbeused

inhomehealthcaretodiagnose diseaseormonitordruglevels; andtheycouldbe

usedbythemilitaryandfirst-responderstoassessaperson’shealthstatusordetect

toxins, biohazards, or explosives. MicroPADs also have many potential

applica-tionsinbasicresearch.

Oneofthegreataspectsofpaper-basedmicrofluidic devicesisthatthereisa

verylowbarriertoentry,bothintermsofthecostofequipmentandthetechnical

expertisethatisrequiredtofabricatedevices.Apairofscissorsandapapertowelis

allthatisreallyneededtomakesimplepaper-baseddevices.So,muchlike

open-sourcesoftware,welookforwardtoseeingcontributionstothisfieldfromallkinds

ofscientistsfromallovertheworldandseeingwherewecangowiththissimple

yet-powerfultechnology.

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