Paper-Based Sensors: Emerging Themes and Applications

22 Pages·2017·3.12 MB·English

sensors
Review
Paper-Based Sensors: Emerging Themes
and Applications
AmritaTribhuwanSingh1,2,DarlinLantigua2,3,AkhilMeka1,2,ShainleeTaing1,2,
ManjotPandher2,3andGuldenCamci-Unal2,*
1 DepartmentofBiologicalSciences,UniversityofMassachusettsLowell,OneUniversityAvenue,Lowell,
MA01854,USA;[email protected](A.T.S.);[email protected](A.M.);
[email protected](S.T.)
2 DepartmentofChemicalEngineering,UniversityofMassachusettsLowell,OneUniversityAvenue,Lowell,
MA01854,USA;[email protected](D.L.);[email protected](M.P.)
3 BiomedicalEngineeringandBiotechnologyProgram,UniversityofMassachusettsLowell,
OneUniversityAvenue,Lowell,MA01854,USA
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:10July2018;Accepted:23August2018;Published:28August2018
Abstract: Paper is a versatile, flexible, porous, and eco-friendly substrate that is utilized in the
fabricationoflow-costdevicesandbiosensorsforrapiddetectionofanalytesofinterest. Paper-based
sensorsprovideaffordableplatformsforsimple,accurate,andrapiddetectionofdiseases,inaddition
tomonitoringfoodquality,environmentalandsunexposure,anddetectionofpathogens.Paper-based
devicesprovideaninexpensivetechnologyforfabricationofsimpleandportablediagnosticsystems
that can be immensely useful in resource-limited settings, such as in developing countries or
austereenvironments,wherefully-equippedfacilitiesandhighlytrainedmedicalstaffareabsent.
In this work, we present the different types of paper that are currently utilized in fabrication of
paper-based sensors, and common fabrication techniques ranging from wax printing to origami-
and kirigami-based approaches. In addition, we present different detection techniques that are
employedinpaper-basedsensorssuchascolorimetric,electrochemical,andfluorescencedetection,
chemiluminescence,andelectrochemiluminescence,aswellastheirapplicationsincludingdisease
diagnostics,cellcultures,monitoringsunexposure,andanalysisofenvironmentalreagentsincluding
pollutants. Furthermore,mainadvantagesanddisadvantagesofdifferenttypesofpaperandfuture
trendsforpaper-basedsensorsarediscussed.
Keywords: papermicrofluidics;point-of-carediagnostics;biosensors;low-costplatforms
1. Introduction
Rapid,simple,accurate,andlow-costdetectionofanalytessuchasbiomarkersandenvironmental
reagents is an important need in biochemical research. For example, methods currently utilized
in immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA)) for the detection of
biomarkersinbodilyfluids(e.g.,blood,urine,serum)providesensitiveandreliableresults. Similarly,
spectrophotometricmethodsandconventionalELISAusedtoquantitativelyanalyzeenvironmental
reagents(e.g.,heavymetalionsinindustrialwastewater)alsoyieldconsistentandaccurateresults.
However,thesophisticatedandlongprocedures,requirementofhighlytrainedpersonneltoexecute
theexperimentsandevaluatetheresults,andtheneedforlargeamountsofreagentsandsamples
prevent the use of these analytical tools in resource-poor environments [1]. Therefore, there is an
unmetneedfordevelopmentofsimpleandlow-costdetectionsystemstoprovideunskilleduserswith
theabilitytoeasilydetectanalytesandevaluateresultsinresource-limitedsettings. Applicationsof
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Sensors2018,18,2838 2of22
paper-basedbiosensorsincludediagnosisofdiseases,monitoringhealthconditions,environmental
agentsorsunexposure,detectionofpathogens(e.g.,bacteria,fungi)[2],andcontrollingwaterand
foodsafetyandquality[3–5].
Paper-based microfluidic approaches enable the fabrication of low-cost, simple, flexible, and
portable diagnostic platforms [6]. This simple technology uses paper as a substrate to create
microfluidicchannelsbypatterninghydrophobicmaterialsonhydrophilicpaper[7].Totestabiological
substance(e.g.,blood,urine,saliva,sweat,tear)oranenvironmentalreagent(e.g.,heavymetalion,
hydrogensulfidegas)thatcontainsananalyteofinterest,thesampleisappliedtothedeviceandwicked
toadetectionzonebycapillaryactionwithouttheneedtouseanexternalpump[8,9].Analytedetection
fromthesampleisfacilitatedbyachemicalreactionwhichinducesachangeincolor,electrochemical
propertiesandlightabsorptionoremission. Amongthesemethods,themostfrequentlyuseddetection
approachisbasedoncolorimetricchange.Inthismethod,resultscanbesimplyevaluatedbyformation
ofacolorproductgeneratedbyligand-analytebinding(e.g.,antibody-antigen)whichcanbequantified
usinglow-costbenchtopscanners,single-lensreflexcameras,orcellphones[10]. Themainadvantages
of paper include (i) high surface to volume ratio; (ii) adsorption properties; (iii) capillary action;
(iv) compatibility with biological samples; (v) chemical functional groups for immobilization of
proteins and antibodies; and (vi) straightforward sterilization [11,12]. Paper also allows for easy
disposalviaincineration. Furthermore,theabilitytostoreandtransportreagentswithinthepaper
matrixeliminatestheneedforuserstohandlechemicalsolutions. Inaddition,paperislightweight
andaccessibleglobally[13]. Finally,paper-basedmicrofluidicdevicescanbeeasilyfabricatedata
low-costusingpracticalfabricationtechniquessuchaswaxprinting[14].
Paper-baseddipstickassays,lateralflow,andverticalflowimmunoassaysareroutinelyperformed
forrapiddetectionoftargetanalytes[15]. Simpledipsticktechniqueswereinitiallyutilizedtoquantify
theglucoseinurinein1956[16]. Althoughthedipsticktechniquewasconvenientandeasytointerpret,
itsmaindrawbacksincludedlonganalysistimesandinaccuracy. Therefore,in1980sseverallateral
flowdevicesweredevelopedtoachievebetterperformanceparameters. Thesedevicesweremainly
used for pregnancy tests [17]. Over time, lateral flow assays became standard platforms for other
applicationsincludingscreeningbloodcoagulation[18],detectionofpesticidesinbeverageandfood
sample[19]anddetectionofpathogens(e.g.,Pseudomonasaeruginosa,Staphylococcusaureus)[20]. Inthe
lateralflowconfiguration,fluidisdrawntowardsasampledetectionzonehorizontally,whereasfluid
flowisdirectedverticallyintheverticalflowimmunoassays. Althoughthedirectionoffluidflow
isthemostapparentdifferencebetweenthesetwoassayformats, verticalflowprovidesdetection
morerapidly(asshortas5min)withahigherdetectionsensitivityinnanogramspermL.Moreover,
Hook’seffect(aphenomenonthatiscausedbythepresenceofexcessamountofantibodiespreventing
agglutination),whichcanleadtoinaccurateresultsinlateralflowassays,isalsoeliminatedinthe
verticalflowconfiguration[21]. Thus,morerecentstudiesuseverticalflowassaysfordetectionof
analytes.Forexample,Rivasetal.fabricatedapaper-basedvertical-flowdevicetoanalyzemicroarrays
ofDNAfordetectionofN.meningitidis[22].
Despite the promising applications of paper-based devices and sensors, there are some
limitations regarding sensitivity, accuracy, and detection of multiple analytes at the same time.
Recently, paper-based sensors have been fabricated via patterning to address these drawbacks.
Inadditiontothecolorimetrictechniques,otherdetectionapproachessuchaschemiluminescence,
electrochemiluminescence,fluorescence,andelectrochemistryhavebeenemployedformonitoring
analytes. These analytical approaches have been implemented in paper-based microfluidic
platforms; however, these techniques also exhibit disadvantages associated with cost, simplicity,
andsensitivity[23].
Inthisreview,wepresentdifferenttypesofpaperusedinthefabricationofbiosensorsandthe
currentfabricationtechniquesrangingfromwaxprintingtoorigami-andkirigami-inspiredapproaches
(Figure1)whichpresentuniqueadvantagesindetectionofanalytes.Inaddition,weintroducedifferent
detectionmechanismsusedbypaper-basedsensorsandtheirapplicationsinvariousfieldsranging
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frommedicaldiagnosticstoanalysisofenvironmentalpollutants,sensinganalytesincellcultures,
andformonitoringsunexposure. Lastly,weconcludebydiscussingthepotentialfutureapplications
Sensors 2018, 18, x FOR PEER REVIEW 3 of 23
of paper-based sensors. Overall, we systematically cover the most recent developments and new
applicationsofpaper-basedsensors.Theexamplesincludedinthispaperdemonstratethatinexpensive
developments and new applications of paper-based sensors. The examples included in this paper
anddpeomrotanbslteraptea ptehra-tb ainseexdpseennssivoers ahnadv epothrteaabblei liptyapteorm-boasneidto rseannsdordse hteacvtea nthaely atebsilaittyl otow mcoonnciteonrt raantido ns
(updteotefcMt a)n.aTlyhteesse atd leovwic ceosnacerenturasteiofunls (nuopt toon flMy)i. nThfueslely deevqiuceips paered urseesfeual rncoht oinnslyti tiun tfiuolnlys ebquutipaplseod in
resoruesrecaer-clihm initsetdituetniovnirso bnumt eanlstos .in resource-limited environments.

Figure 1. Overview of the analytical capabilities that are employed in paper-based sensors. Paper-
Figure 1. Overview of the analytical capabilities that are employed in paper-based sensors.
based assays with strategies for fluid and analyte handling, sample processing and analysis, and
Paper-basedassayswithstrategiesforfluidandanalytehandling,sampleprocessingandanalysis,
quantification. Reproduced with permission from [3].
andquantification.Reproducedwithpermissionfrom[3].
2. Paper Types
2. PaperTypes
There are different types of paper employed in paper-based sensors depending on the
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metbhroandda cnhdrotmheataopgrpalpichayt ipoanpoerf dthuee tsoe intss osur.pTerhioerm woicsktinexgt aebnisliitvye [l2y4–u2s7e]d. Tmhias tpeariratilcuislaWr thyaptem oaf npabprearn d
chrohmasa mtoegdriaupmh yrepteanpteiornd aunedt ofloitws sruaptee roiworinwg itcok iintsg thabiciklintyes[s2 (41–8207 μ].mT)h aisndp aprotirceu sliazre t(y1p1e μomf)p. aOptehrerh as
medtyiupmes roeft penaptieorn saunchd aflso twher Watehaotwmianng ftiolteirts ptahpicekr nNeos.s 4(,1 w80asµ ums)edan dduep otore itssi zlaerg(1e1r µpomre). sOizteh oerf 2ty0p–2e5s of
papμemrs auncdh haisgthheer Wretheanttmiona nrafitelt e[2r8p].a MpeorreN roe.ce4n,twlya, sfiultseer dpadpueer thoasit bseleanrg uesrepdo inre psaizpeero-bfa2s0e–d2 s5eµnmsorasn d
[24–26], which is also manufactured by Whatman (Maidstone, United Kingdom). This type of paper
higherretentionrate[28]. Morerecently,filterpaperhasbeenusedinpaper-basedsensors[24–26],
has been used for its relatively uniform thickness and wicking properties as well as superior
whichisalsomanufacturedbyWhatman(Maidstone,UnitedKingdom). Thistypeofpaperhasbeen
adsorption and retention of reagents compared to the similar types of paper [25].
usedforitsrelativelyuniformthicknessandwickingpropertiesaswellassuperioradsorptionand
Because different physical and/or chemical characteristics may be required for biosensors, other
retentionofreagentscomparedtothesimilartypesofpaper[25].
categories of paper have been explored depending on the target application. Nitrocellulose
Becausedifferentphysicaland/orchemicalcharacteristicsmayberequiredforbiosensors,other
membranes have been used due to their chemical functional groups that enable covalent
categoriesofpaperhavebeenexploreddependingonthetargetapplication.Nitrocellulosemembranes
immobilization of biomolecules. Nitrocellulose allows for charge-charge interactions, weak hydrogen
have been used due to their chemical functional groups that enable covalent immobilization of
bonds, and van der Waals interactions with protein-based substrates [27,29]. As a result of their high
biomproolteecinu-lbeisn.dNinigtr oacbeilliltuielso,s ethaelsleo wmsemfobrrcahnaersg aer-ec hcaormgmeionntleyr aucsteiodn isn, wELeIaSkAh aynddro ggoelnd bnoanndops,aartnicdlev-an
derbWasaeadl sasinsateyrsa [c3t0i]o.n Tshwesiet hmpemrobterainn-ebsa hsaevdes aulsbos tbreaetnes u[s2e7d, 2b9e]c.aAussea threeysu plrteovfetnhte diriffhuisgihonp arontde liena-cbhinindgi ng
abiloitfi seasm, tphleess ethmroeumghb rtahne emseamrebrcaonme,m wohniclhy aullsoewdsi nforE aL hISigAhearn ddeggroeled onf arentoenptaiortnic alne-db sausbesdeqausseanytlsy [a3 0].
Thelsoenmgeerm rebarcatnioens thimavee wailtshoinb etheen suesnesdorb [e2c8a]u. Nseittrhoecyelplurleovseen mtedmiffbursainoens aanred slmeaocohtihn agnodf hsaavmep al eusntifhorromu gh
thepmoerem sbizrea noef ,0w.4h5 iμchma. lTlhowesse fmoermabhriagnheesr cdaeng bree emoofdriefiteedn tvioian waanxd psruinbtsienqgu tehnatt liys afollolonwgeedr rbeya chteioatnintgim e
with[2in8],t hheowseenvseorr w[2a8x] .pNenietrtoracteilolnu ltohsreoumgehm thber annitersoacerellusmlosoeo mtheamnbdrahnaev eis asluonwi fworhmenp coormespiazreedo fto0 .t4h5aµt m.
of the filter paper.
In addition to these paper types, bioactive paper has also been used in biosensors. In the
bioactive paper, the paper matrix is modified with biomolecules. Without modification, only cationic
Sensors2018,18,2838 4of22
Thesemembranescanbemodifiedviawaxprintingthatisfollowedbyheating[28],howeverwax
penetrationthroughthenitrocellulosemembraneisslowwhencomparedtothatofthefilterpaper.
Inadditiontothesepapertypes,bioactivepaperhasalsobeenusedinbiosensors. Inthebioactive
paper,thepapermatrixismodifiedwithbiomolecules. Withoutmodification,onlycationicmolecules
adsorbontothewetcellulosefibersthatmakeuppaper. Inaddition, whileproteinsareadsorbed
on cellulose, the rate and extent of this process has not been effective when compared to other
hydrophilic surface [31]; thus, the cellulose fibers must be modified in a way that allows them to
absorb biomolecules. This can be done first by activating the surface of the paper (e.g., aldehyde,
amide)andthencovalentlyconjugatingbiomolecules. Furthermore,abioactivepaper-baseddevice
wasproducedviapiezoelectricinkjetprintingofenzymeswithinbiocompatiblesol-gelsilicalayers,
whichweresupportedbypaper[32]. Ithasalsobeenshownthatthegraftcopolymerizationofglycidal
methacrylateontocellulosefilterpaperhasallowedfortheimmobilizationofbiomolecules,which
allowedfordotELISAandgoldnanoparticlebasedtests[33].
Glossypaperhasalsobeenstudiedasaviableoptionforpaper-basedsensors. Glossypaperis
composedofcellulosefibersandinorganicfillersthatareblendedintothepapermatrix. Arenaetal.
usedglossypaperinordertodevelopaflexiblepaper-basedsensingdevicefordetectionofethanol[34].
Thisspecifictypeofpaperisusedinlieuoffilterpaperasitiseasiertoalterthesurfacepropertiesof
theglossypaper.
Different types of common paper have also been successfully used in the production of
paper-basedsensors. Conventionalprintingpaperwasselectedforuseinawearabledevice,intended
to be foldable and flexible [35]. The paper was subject to wax printing and then carbon black ink
wasspreadalongthesurfaceofthepaper. Inaddition,papertowelhasbeenutilizedasasurfacefor
printingofcarbonblack-modifiedelectrodes[36]. Papertowelhasalsobeenusedaswickinglayerfor
abiosensorproducedfromfilterpaper[37]. Papertowelischeaperthanfilterpaperandpossessesa
highporosity,whichmakesitaviablematerialforanalysisofawiderangeofanalytes.
3. FabricationandPrinting
Thefabricationmethodthatisusedtoproducethebiosensorscanimpactthesimplicityandtheir
applications. Therearenumerousapproachesavailablethatinvolvechemicalmodificationorphysical
deposition onto the paper, both of which alter the material characteristics of the cellulose matrix.
Theapproachesthatwillbecoveredinthisreviewarewaxprinting,photolithography,inkjetprinting,
lasercutting,polydimethyl-siloxane(PDMS),hotembossing,hydrophobicsilanization,andtheuseof
origami-andkirigami-basedapproaches. Severaladvantagesanddisadvantagesofeachprocedure
arelistedinTable1.
Table1.Advantagesanddisadvantagesofthemainfabricationtechniquesforpaper-basedsensors.
Procedure Advantages Disadvantages
WaxPrinting Low-cost,easyfabrication,shortfabricationtime Lowresolution,unstableuponheating
Expensiveandsophisticatedequipment,
Photolithography Highresolution,suitableforlarge-scaleproduction
unstabilityagainstbendingorfolding
Efficient,reducedcross-contamination,
InkjetPrinting Expensivebio-inkprinter
rapidfabrication,highresolution
LaserCutting Simple,inexpensive Specializedequipment
Lowresolution,sophisticated
PDMS Low-cost,flexible
equipmentforfabricationofmolds
HotEmbossing Shortfabricationtime,efficient Specializedequipment
HydrophobicSilanization Low-cost,rapidfabrication Limitationwithsimpledesigns
OrigamiandKirigami Intricateandinnovativedesigns,simplefabrication
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3.1. WaxPrinting
Waxprintingcanbeusedtocreatemicrofluidicchannelsforpaper-basedsensors. Thisprocedure
iswidelyusedduetoitssimplicity,non-toxicity,andlowcostcomparedtotheothertechniques. Inone
study,waxprintingwasperformedforfabricationofpaper-basedmicrofluidicdevicesduetothelow
costofwaxprinters[38]. Thewaxpatternsaredirectlyprintedonthepaper,whicharethenusedto
controltheflowofthefluidthatincludesthesamplesandthereagents. Dungchaietal. waxpatterned
chromatographypaperandsubsequentlymeltedthewaxusingahotplateinthreedimensions(3D)
ofthepaperinordertoformhydrophobicbarriers. Namwongetal. usedahomehairdryertomelt
thewaxintothepaper[39]. Inanotherstudy,waxmicrostructureswereprintedonanitrocellulose
membrane,andthenbakedtoenabletheprintedwaxtopenetratethewaxandcreatehydrophobic
channelsinthedevice[29]. Thefabricationprocesstookonly10min. However,theuseofwaxprinting
isnotsuitableforhighresolutionprintingandthepatternsarenotstableathightemperaturesasthe
waxeasilymelts.
3.2. Photolithography
Photolithographycanbeusedtofabricatepaper-basedsensorswithhighresolution, andisa
suitableapproachforlarge-scaleproduction. Paper-basedmicrofluidicdevicesthatarecapableof
detectingmultiplebiomarkersweregeneratedusingphotolithography[40]. First,photoresistwas
pouredontothepaper,evenlydistributed,andthenpaperwasbaked. Afterwards,thepaperwas
covered with a patterned transparent film that was generated with a laser printer and irradiated
withUV.Followingbaking, theunpolymerizedphotoresistwasremovedusingacetone, andafter
dryingthepaperwasexposedtoairplasmatocreatehydrophilicareas. Thisprocedurerequiresthe
use of expensive processing equipment and involves long and complex steps. In another study,
photolithography was utilized to fabricate patterned sensors without the use of air plasma for
generation of hydrophilic channels [41]. The group first fabricated the microfluidic channels in a
1:1mixtureofanacryloxy-terminatedsiloxanepolymerandanacrylatebasedpolymer. Thesolution
wasthendegassedinanultrasonicbathandslowlypipettedontopaper. Acetonewasusedtoremove
the uncrosslinked polymer. This procedure took only 3 min to fabricate the sensor. Ouyang et al.
fabricatedanelectrochromatographicmicrofluidicpaper-basedanalyticaldevice(µPAD)fordetection
ofmultiplemetalcomplexesbyusingphotolithography[42]. Chromatographypaperwastreated
withathinnedphotoresistanddriedfor1min. Aphotomaskandthetreatedpaperwascombined
throughexposureofUVlight. Thecombinedpaperwasthenwashedwithacetone. Cathodeswere
incorporated into the device to allow detection of specifically targeted metal analytes. However,
sensorsproducedusingphotolithographycanbesubjecttodamagewhenbendedorfolded.
3.3. Polydimethyl-Siloxane(PDMS)Printing
Printing a solution of PDMS on paper, due to its hydrophobic nature, offers a solution to the
inflexibilityofsensorsthatarefabricatedbyphotolithography. Alow-costmethodwasdevelopedfor
fabricatingflexiblesensorsviaPDMSpatterningonpaper[43]. PDMS-patternedpaperallowsforthe
sensortobebendedorfoldedwithoutdamagingthemicrofluidicchannels. Inordertofabricatesensor
patternswiththismethod,PDMSalongwithhexanesina3:1(PDMS:hexanes)ratio,wasloadedinto
aplotterandprintedontothepaper. PDMShasmanyadvantagesoverphotolithography: itcosts
lessthanSU-8photoresist,doesnotrequireUVlighttocrosslink,curesrapidly,andrequiresnopost
printingsteps. However,theplottermustbemodifiedwithcustomprintercartridgesinordertoload
thePDMS,whichyieldsalowresolutionduringprinting.GervaisandDelamarchecapturedantibodies
bypatterningPDMSsubstratesusingstencilsunderahumiditychambertopreventevaporationof
reagents[44]. Thedevicewaswashedwithpurifiedwater,PBSsolution,ethanol,andO plasmaand
2
driedunderastreamofN . Shangguanetal. fabricatedaPDMSonpaperdevicebyintegratingtwo
2
layersofPDMS—achannellayerandapatternedlayer[45]. Thebaseprecursorandthecuringagent
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ina10:1(w/w)ratiowereusedtopreparePDMS.PDMSwasthenpouredoverastenciltogenerate
patterns. Thepatternedlayerandchannellayerwerecombinedtoproducemicrofluidicchannels.
ThePDMSpolymerwascuredbyheating.
3.4. InkjetPrinting
Inkjetprintinghasallowedresearcherstostreamlinethefabricationprocessbyonlyrelyingon
theuseofinkjetprintingequipmentwithouttheneedforadditionalinstrumentation. Inkjetprinting
becamepopularduetoitsnon-contactoperationandreducedcross-contaminationbetweensamples
andreagents. Typically,commerciallyavailableprintersareusedwithorwithoutmodificationtoprint
biomoleculesforsensors. Forexample,aCanoninkjetprinterandinkcartridgesweremodifiedfor
fabricationofpaper-basedmicrofluidicdevices[46]. Inanotherwork,athermalinkjetprintingmethod
wasusedtoprintproteinsandenzymesonpaperchips. Similarly,Gasparetal. usedinkjetprintingto
createsilverinterdigitatedelectrodesonthesurfaceofpapersubstrates[47]. Koetal. fabricatedan
electrochemicalsensorbysimplyutilizingcarbonnanotubesasconductiveinkandprintedelectrodes
usingacommercialofficeinkjetprinter[48]. Theuseofinkjetprintinghasallowedgroupstorapidly
fabricatedevices,however,eitheranexpensivebio-inkprinteroramodifiedinkjetprinterisnecessary.
3.5. LaserCutting
Laser cutting is used as a simple and inexpensive way to produce patterns on paper-based
devices. Chitnis et al. used a CO laser cutter to create hydrophilic patterns on paper, which was
2
modified with a hydrophobic coating [49]. This group was able to convert hydrophobic regions
that are present on the surface and in the interior of the paper into hydrophilic regions by laser
cutting. Thisprocessresultedingenerationofcarbonylandhydroxylgroupsatthepapersurface.
Itisimportanttooptimizethecuttingparameterssothatthelasercutterdoesnotcutthroughthe
papercompletely. Spicar-Mihalicetal. alsousedacommercialCO lasertoeasilycutthroughbacked
2
andunbackednitrocellulosemembrane,glassfibers,andMylar-basedsubstrates[50]. Thelaserwas
also used to pattern chromatography paper. Mahmud et al. reported that laser cutting produces
small-scalechannelsinµPADs,allowingasmallamountofsampletoflowthrough[51]. Theyused
differenttypesofpaperincludingnitrocellulose,filter,andchromatographypaper,andanadhesive
film to pair the aluminum foil and paper together. A CO laser was used to produce barriers by
2
strippingthehydrophilicmaterialinthepaper. Althoughlasercuttingisasimplemethod,itrequires
theuseofspecializedequipmentsuchaslasercutterorengraver,2Dgraphicsproductionsoftware,
anddouble-sidedadhesiveforassemblyofthepaperdevices.
3.6. HotEmbossing
Theuseofhotembossingforfabricationofpaper-basedsensorsyieldshydrophilicchannelsthat
arehollow,whichallowsforspontaneouscapillaryflow. Thismethodisefficient,astheroll-to-roll
processofhotembossingallowsforhighthroughputduetolowcycletimes. Amicrofluidicdevice
was fabricated by embossing PowerCoat® HD paper (Boulogne-Billancourt, Paris, France) coated
withalayerofrubberforwater-resistanceandpoly(vinylalcohol)forhydrophilicity[52]. Embossing
wasperformedwithanEitreNanoImprintLithographydevice, inwhichtheembossingchamber
washeatedup,andpressurewasappliedtothedevicefor60s. Thechamberwasthencooledand
the pressure was set back to atmospheric levels. The device was able to detect glucose in 12 min.
Kimetal.introducedtwomethodsinproductionofµPADsbyslightlyorcompletelyinfusingparafilm
tothedevice,inwhichresultedinlaminatedpaper(slightlyinfused)orinfusedpaper(completely
infused)[53]. Thermallypressingparafilmintothepapercausedittobondtogether. Thelaminated
paperwasformedbyhot-pressingat45◦Cfor3min,whiletheinfusedpaperwaspressedforlonger
atahighertemperature. PatternswerethenconstructedbyirradiationusingaCO laserengraver.
2
Yuetal.hadasimilardesignutilizingparafilmforpaper-baseddevices[54].Althoughtheirtechniques
differedbybonding,parafilmandthepaperwerepressedbetweenaluminumfoilsandglassslides,
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whereitwasthenheatedinanovenat120◦Cfor2–5min.Afterward,thedevicewasirradiatedbyaUV
lamp. Althoughtheuseofhotembossingcanbeefficient,itrequirestheuseofspecializedequipment.
3.7. HydrophobicSilanization
Silanization is the process of covering the surface of a substrate with alkoxysilane molecules.
Yang et al. have shown that silanizing paper with trichloromethylsilane enables the formation of
hydrophobicsubstrates[55]. Theprocessissimple, low-cost, andrapidlyexecuted. Afilterpaper
sheet was silanized by soaking it in 2.0% trimethoxy(octadecyl)silane and then heating it for an
hour. Afterwards, the hydrophobic paper was aligned onto a paper mask, printed with a readily
availablelaserprinter. Themaskwassaturatedwithsodiumhydroxide(NaOH),turningmasked
regionshydrophilicviawetetching. Usingthisdevice,aglucoseassayforartificialurinewascarried
out,whichprovidedpromisingdetectionresults. Analternativeglucoseassaywasdevelopedusing
trimethoxyoctadecylsilane(TMOS)asahydrophobicreagentinsteadofsilanizingthefilterpaper[56].
ThepapersubstratewassoakedinTOSsolutioninn-hexanefor10sanddriedfor1minafterwards.
Microchannelswereprintedontothehydrophilicsubstrateusingamask. Bothpaperandmaskwere
compressed between two glass slides for 5 min and was readily available for use. This process is
cost-effectiveasitdidnotinvolveexpensiveequipmentandcanbeimmediatelyfabricatedwithin
7min.
3.8. OrigamiandKirigami
Theuseoforigami(foldingofpaper)[57]andkirigami(cuttingofpaper)[26]techniquesduring
Sensors 2018, 18, x FOR PEER REVIEW 9 of 23
thefabricationofmicrofluidicdeviceshasgivenresearchersnewopportunitiesforfabricatingtheir
devices(Fig4u. Dreet2e)c.tiTonh eTepcrhinnicqiupelse soforigamiwereusedtocreateauniquedeviceinwhichtwozonesare
separatedbyaDcrifefearseent[ t5e7ch].niOqunees szuochn easi scotlhoreimdeetrtiec catsisoanys,z cohnemeialunmdintehsecenocteh, eerleicstrtohcheeemnilzuymmineesicmenmce,o bilization
zone. Inthieslewctororckh,etmhiecarle, asneda rflcuhoerersscednecsei bganseedd teachpnaipqueers- bcaans ebed eamnpalolyyetidc ianl pdaepveri-cbeasteode sleencstorros ctoh deemteicct allydetect
the presence of an analyte of interest [60]. These techniques have been extensively used since they
glucoseusinganorigami-inspireddevice[58].
provide highly sensitive results rapidly compared to traditional techniques such as the ELISA.

Figure 2. Construction of paper-based devices using origami and kirigami. (a) Quilling and kirigami
Figure2.Cpornoscetsrsuecs taioren doefpipctaepd.e (rb-b) aSscheedmdateicv ifcoer sthues cinregatioorni goaf m2Di aanndd 3kDir isgenasmoris. u(asi)ngQ ouriilglainmgi aanndd kirigami
processesarkeiridgeampiic. t(ce)d P.h(obto)gSrcaphhesm shaotiwc 2foDr stehnesocr rbeeafotiroe nanodf a2fDtera tnhed in3tDrodsuecntsioonr sofu 0s.1in0 gMo PrBigS asmoluitaionnd kirigami.
(c)Photogrcaopnhtasinsinhgo w0.0210D Mse cnosoomrabsseief obrreillaianndt balfutee. rStohluetiionnt rios dinutrcotdiouncedo fa0t .p1o0inMt oPf BreSds oarlruotwio. n(dc) ontaining
Photographs show 3D sensor before and after the introduction of 0.010 M coomassie brilliant blue,
0.010Mcoomassiebrilliantblue.Solutionisintroducedatpointofredarrow.(d)Photographsshow
0.010 M rhodamine B, 0.010 M methyl orange and 0.010 M bromocresol green. Solutions are
3Dsensorbinetfroodreucaedn dat apfotienrts tohf ereidn tarrorodwus.c Sticoalne boafrs0 .r0ep1r0esMentc 1o0o mmma. sRseiperbodruilcleida nwtitbhl pueer,m0is.0si1o0n fMromrh odamine
B,0.010M[m26e].t hylorangeand0.010Mbromocresolgreen.Solutionsareintroducedatpointsofred
arrows.Scalebarsrepresent10mm.Reproducedwithpermissionfrom[26].
4.1. Colorimetric Techniques
Colorimetric assays include methods utilized to detect the presence and concentration of an
analyte by evaluating the color formation or color change via i) direct imaging using a single-lens
reflex (SLR) camera, mobile devices, or low-cost desktop scanners in combination with software such
as MATLAB for quantification [25], or ii) traditional spectrophotometers by measuring the
absorbance of the sample at specific wavelengths. This detection technique is the most commonly
used one as it provides accurate results at a lower cost. Color formation or color change can be
induced by using enzymes, ELISA-based immunoassays, or gold nanoparticles (Figure 3).
Sensors2018,18,2838 8of22
Electrodes in the detection zone were drawn by a pencil and an electron transfer mediator
(ferrocenecarboxylic acid) was incorporated to catalytically oxidize glucose. The immobilization
zone contained a circle of hydrophobic barriers. The two zones were combined by folding along
thecrease allowingtheelectrodestomeet withthereactionmixture. Linetal. producedahighly
stretchablepaper-basedphotodetectorarraybyprintingZnOnanowiresandcarbonelectrodeson
printingpaper[59]. ThiswasachievedbytheMiuraorigamifoldingtechnique. Oneofthebenefitsof
theorigami-basedmethodistheabilitytoreusethedevicewithoutdegradation. Theseapproachesare
favorableduetotheirsimplicity,reliability,andaffordability. Inadditiontoorigami,kirigamiwasalso
employedforfabricationof2Dand3Dverticalflowpaper-baseddevices[26]. Specifically,kiragami
wasusedtoachieveduplicatepatternsusingonlyoneseriesofcuts,inwhichpaperwasfolded,cut,
andthenunfolded. Themainfabricationtechniquesforpaper-basedsensorsisprovidedinTable2.
Table2.Mainfabricationtechniquesforpaper-basedsensors.
Fabrication Detection
Procedure RecognitionElement AnalyteDetected Reference
Technique Method
Waxprinting Meltedwax Electrochemical Glucoseoxidase Glucose [38]
Ascorbicacidand
Meltedwax Colorimetric Iron [38]
1,10-phenanthroline
Limitingreagentof
Meltedwax Colorimetric Phenolphthalein [39]
acid-basereaction
Meltedwax Colorimetric Anti-HumanIgG HumanIgG [29]
Oxidaseenzymesthat Glucose,lactate,
Photolithography Hydrophobicphotoresist Colorimetric [40]
produceH2O2 uricacid
Glucoseoxidase,
Hydrophobicphotoresist Colorimetric potassiumiodide,trehalose, Glucose [41]
horseradishperoxide
Hydrophobicphotoresist Colorimetric Glycine,sodiumnitroprusside Acetoacetate [41]
Sulfanilamide,citricacid,and
Hydrophobicphotoresist Colorimetric Nitrite [41]
n-(1-napthyl)ethylenedi-amine
Glucoseoxidase,potassium
PDMS HydrophobicPDMS Colorimetric Glucose [43]
iodide,horseradishperoxide
HydrophobicPDMS Colorimetric BromothymolBlue pH [43]
HydrophobicPDMS Colorimetric Tetrabromophenolblue Proteins [43]
Tetrabromophenolblueand Totalserumprotein,
HydrophobicPDMS Fluorescence NBTalkalinePhosphatase alkalinephosphatase, [45]
ColorDevelopmentKit aspartateaminotransferase
Detectionantibodiesand C-reactiveprotein
HydrophobicPDMS Colorimetric [44]
captureantibodies (fromhumanserum)
Inkjetprinting Printedbiomolecules Colorimetric 3,3(cid:48)-diaminobenzidine Horseradishperoxidase [46]
Printedconductiveelectrodes Electrochemical Changeincapacitance Humidity [47]
Hydrophilicpatternsprinted Catalyticactionof Luminol-based
Lasercutting Chemiluminescence [49]
usingCO2lasercutter ironinhemoglobin hemoglobin
Embossedpaperwithlayerof Glucoseoxidase,horseradish
Hotembossing Colorimetric Glucose [52]
rubberandpoly(vinyl)alcohol peroxidase,ando-Dianisidine
Cholinesteraseand
Parafilmandpaper
Colorimetric dithiodipropionic Methomyl [54]
heatedinanoven
nitrobenzeneacid
Uricase,horseradish
Parafilmandpaper
Colorimetric peroxidase, Uricacid [54]
heatedinanoven
tetra-methylbenzidine
Hydrophobic Selectivewetetchingof Glucoseoxidase,potassium
Colorimetric Glucose [56]
silanization hydrophobicfilterpaper iodide,horseradishperoxide
Printedelectrodes
Origami Electrochemical Glucoseoxidase Glucose [57]
followedbybaking
4. DetectionTechniques
Differenttechniquessuchascolorimetricassays,chemiluminescence,electrochemiluminescence,
electrochemical,andfluorescencebasedtechniquescanbeemployedinpaper-basedsensorstodetect
thepresenceofananalyteofinterest[60]. Thesetechniqueshavebeenextensivelyusedsincethey
providehighlysensitiveresultsrapidlycomparedtotraditionaltechniquessuchastheELISA.
Sensors2018,18,2838 9of22
4.1. ColorimetricTechniques
Colorimetric assays include methods utilized to detect the presence and concentration of an
analytebyevaluatingthecolorformationorcolorchangeviai)directimagingusingasingle-lens
reflex(SLR)camera,mobiledevices,orlow-costdesktopscannersincombinationwithsoftwaresuch
asMATLABforquantification[25],orii)traditionalspectrophotometersbymeasuringtheabsorbance
ofthesampleatspecificwavelengths. Thisdetectiontechniqueisthemostcommonlyusedoneasit
providesaccurateresultsatalowercost. Colorformationorcolorchangecanbeinducedbyusing
enzymes,ELISA-basedimmunoassays,orgoldnanoparticles(Figure3).
Sensors 2018, 18, x FOR PEER REVIEW 10 of 23

Figure 3. The mechanism of TB diagnosis using a paper-based sensor. If extracted DNA sequences
Figure3. ThemechanismofTBdiagnosisusingapaper-basedsensor. IfextractedDNAsequences
contain IS6110 sequences (specific to Mycobacterium tuberculosis complex), detection oligonucleotide
containIS6110sequences(specifictoMycobacteriumtuberculosiscomplex),detectionoligonucleotide
sequences will hybridize with the sequences. If IS6110 sequences are not present, the color of the
sequenceswillhybridizewiththesequences. IfIS6110sequencesarenotpresent, thecolorofthe
mixture does not change, and stays red after hybridization with DNA. The color change can be
miqxutuarnetifdieode sthnrooutgchh tahneg aen,aalynsdis sotfa ay psircetudrea tfatekrenh vyibar simdiazrattpihoonnwe. iRthepDroNduAc.eTd hweitcho ploerrmcihsasinogne frcoamn be
qua[6n1t]i.fi ed through the analysis of a picture taken via smartphone. Reproduced with permission
from[61].
4.1.1. Enzymatic Techniques
4.1.1. EnzymaticTechniques
Enzymatic detection is achieved by a reaction between an enzyme and a specific substrate
forEmniznygm ana teicnzdyemteec-stiuobnstrisatae cchoimevpeledx,b wyhaichre parcotidouncebse at wcoeleonr cahnanegnez [y4m,6,e12a,1n6d,17a,5s8p]e. cMifiacrtisnuebz settr ate
foraml.i ndgeteacnteedn zpyrmoteei-nsus basntdra gteluccoomsep ilne xa,rwtifhiciicahl uprriondeu scaemspalceos lionr ac hpaanpgeer d[4e,v6i,c1e2,, 1w6h,1ic7h,5 w8]a.sM faabrrtiincaetzede tal.
detveicat epdhoptorolittheiongsraapnhdy g[6lu2]c.o Tsheeiyn saturtdiifiecdi athleu roixniedastaiomnp olfe sgliuncoasep atop egrludcoenviicc ea,ciwd haincdh fworamsaftaiobnri coaf ted
viahpydhrootgoelinth poegroraxpidhey in[6 t2h]e. pTrheeseynscteu odfi ethde tghleucooxsied oaxtiiodnasoe fegnlzuycmoes.e Htoydgrlougceonn ipcearocxididaen wdafso rremduactieodn of
hydbyro hgoernsepreardoisxhid peeirnoxthideapser e(sHeRnPce) ooxfitdhiezignlgu cioodseidoex tiod aiosdeienne,z ywmhiec.hH ryesdurlotegde ninp tehreo xfoidrmewataiosnr eodf uac ed
bybhroowrsne rcaodliosrh. Spiemrouxltiadnaesoeus(Hly,R pPr)ootexiindsi zwinerge ioddetiedcetetdo uiosidnign ete,twrah-bicrohmreospuhletnedol ibnluthe e(TfoBrPm), awtihoincho f a
broewlecntrcoosltoatri.caSlilmy iunlttearnacetoeuds wlyi,thp rportoetieninssw toe rcehadnegtee cthteedir ucoslionrg frtoemtra y-eblrloowm otop bhleuneo. Ilnb alunoeth(TeBr rPe)p,owrth, ich
eledctertoecsttiaotnic aolfl yacitnivteer aincgterdedwieintths porfo atneitnibsiototiccsh oafn gβe-lathcteaimrcs owloars fsrtoumdiyede llboyw a tcoobmlupeet.iItinvea neontzhyemrarteipc ort,
detaescstaiyo nusoinfga cntiitvroecienfignr e[6d3i]e. nAt scoolfoar nchtiabniogtei cfrsoomf yβe-lllaocwta tmo srewd ainsdsitcuadteided prbeyseancceo omf paenttiitbiivoeticesn. z ymatic
assayusingnitrocefin[63]. Acolorchangefromyellowtoredindicatedpresenceofantibiotics.
4.1.2. ELISA-based Immunoassays
An immunoassay demonstrates the interaction between an antigen and an antibody that is
specific to a particular antigen. Conventional ELISA requires large volume of reagents, long
procedures, and tedious washing steps. The paper-based ELISA approach is advantageous due to the
superior adsorbent properties of paper, significantly smaller volume (1–5 μL) of sample requirement,
and rapid blocking step [64]. In one study, rabbit IgG was immobilized on paper and blocked using
bovine serum albumin (BSA). Alkaline phosphatase (ALP)-conjugated IgG antibody was then added
to bind the rabbit IgG [65]. Yellow colored substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium (BCIP/NBT) was placed on the sample zone to form a conjugate which subsequently
produced a purple precipitate. A desktop scanner was used to scan the colorimetric result, which was
then quantified using the NIH ImageJ software (Bethesda, MD, USA).
Sensors2018,18,2838 10of22
4.1.2. ELISA-basedImmunoassays
Animmunoassaydemonstratestheinteractionbetweenanantigenandanantibodythatisspecific
to a particular antigen. Conventional ELISA requires large volume of reagents, long procedures,
andtediouswashingsteps. Thepaper-basedELISAapproachisadvantageousduetothesuperior
adsorbentpropertiesofpaper,significantlysmallervolume(1–5µL)ofsamplerequirement,andrapid
blockingstep[64]. Inonestudy,rabbitIgGwasimmobilizedonpaperandblockedusingbovineserum
albumin(BSA).Alkalinephosphatase(ALP)-conjugatedIgGantibodywasthenaddedtobindthe
rabbitIgG[65]. Yellowcoloredsubstrate5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium
(BCIP/NBT) was placed on the sample zone to form a conjugate which subsequently produced a
purple precipitate. A desktop scanner was used to scan the colorimetric result, which was then
quantifiedusingtheNIHImageJsoftware(Bethesda,MD,USA).
4.1.3. GoldNanoparticles
Gold nanoparticles (GNPs) are typically used to label secondary antibodies as detection
componentsinimmunoassays. Acolorformationtakesplaceduetoaggregationofgoldnanoparticles
basedontheirspecificinteractionswiththeanalyte(e.g.,antigen)ofinterest. Imagingsoftwarecanbe
usedtoquantifythecolorinformationinwhichthepixelvaluescorrelatewithanalyteconcentrations.
In a research study by Tsai et al. tuberculosis was diagnosed using gold nanoparticle-tagged
antibodies [66]. The changes in the aggregation properties of gold nanoparticles were quantified
using the surface plasmon resonance (SPR) effect. This technique utilizes plasmons, which are a
quantum wave produced by a large number of electrons when disturbed from their equilibrium.
Thistechniquewasutilizedtostudytheeffectofasingle-strandedDNAhybridizedwithatarget
causingaggregation. Thecolorimetricchangewasquantifiedusingamobiledeviceandyieldeda
detectionlimitof1.95×10−2ng/mL.GNPsensorshavevariousmeritssuchasbiologicalcompatibility
andhighsurfaceareaforaffinityreactions. However,GNPsmightleakfromthepapermembranes
uponwashing. Toaddressthisissue,Lietal. usedchitosantostabilizetheGNPs[67]. Thepaper-based
sensorwascoatedwithTiO toindicatethatGNPswereleachingout. Ferreiraetal. demonstrated
2
theuseofsilvernanoparticles(AgNPs)forquantificationofascorbicacid(AA)usingpaper-based
sensors[68]. Ascorbicacidwasdetectedbyacolorchangefromlightyellowtogreyuponreacting
withAgNPsthatwereimpregnatedinthedevice. Thecolorchangewasimagedbyadesktopscanner
oracustommadescannerthatmeasuresthetransmittanceoflight,andcolorintensitywasquantified
inthetestregions.
4.2. Chemiluminescence
Chemiluminescenceinvolvesthedetectionoflightgeneratedfromachemicalreaction,when
reactive intermediate molecules generate an emission upon returning to ground state from their
excited state. Chemiluminescent detection of uric acid was performed via an enzymatic reaction
thatproducedhydrogenperoxideasaby-productwhiledecomposingthesubstrate[69]. Hydrogen
peroxidethenreactedwithrhodaminederivatives,whichwereaddedtothemicrofluidicchannelsof
thesensor. Luminescencewasobservedafterinjectingluminolandiodophenolusingacomputerized
RFL-200detector(Xi’anYimaOpto-ElectricalTechnologyCo.,Ltd.,Xi’an,Shaanxi,China),producing
asignalwiththepeakheightrepresentingitsconcentration. Chemiluminescenceisconsideredone
ofthesimplestdetectiontechniquesbecauseitdoesnotrelyheavilyonsophisticatedinstruments.
In addition, chemiluminescence is one of the most precise and sensitive detection technique [70].
However,itrequiresahighconcentrationofenzymes,whichmightblocktheporousstructureofpaper
andcanpossiblyreducetheoverallefficiencyofdetection.