Gravimetric Viral Diagnostics: QCM Based Biosensors for Early Detection of Viruses

25 Pages·2017·3.2 MB·English

chemosensors
Review
Gravimetric Viral Diagnostics: QCM Based
Biosensors for Early Detection of Viruses
AdeelAfzal1,2,*,AdnanMujahid3,*,RomanaSchirhagl4,SadiaZafarBajwa5,UsmanLatif2
andSaimaFeroz6,7
1 DepartmentofChemistry,CollegeofScience,UniversityofHafrAlBatin,P.O.Box1803,HafrAlBatin31991,
SaudiArabia
2 InterdisciplinaryResearchCentreinBiomedicalMaterials,COMSATSInstituteofInformationTechnology,
1.5KMDefenceRoad,Off.RaiwindRoad,Lahore54000,Pakistan;[email protected]
3 InstituteofChemistry,UniversityofPunjab,Quaid-i-AzamCampus,Lahore54590,Pakistan
4 GroningenUniversity,UniversityMedicalCenterGroningen,AntoniusDeusinglaan1,9713AVGroningen,
TheNetherlands;[email protected]
5 NationalInstituteforBiotechnologyandGeneticEngineering(NIBGE),Faisalabad38000,Pakistan;
[email protected]
6 DepartmentofBiosciences,COMSATSInstituteofInformationTechnology,ParkRoad,ChakShahzad,
Islamabad45550,Pakistan;[email protected]
7 DepartmentofBiology,CollegeofScience,UniversityofHafrAlBatin,FemaleCampus,HafrAlBatin31991,
SaudiArabia
* Correspondence:[email protected](A.A.);[email protected](A.M.);
Tel.:[phone](ext.1675)(A.A.)
AcademicEditor:PeterLieberzeit
Received:18December2016;Accepted:9February2017;Published:13February2017
Abstract: Virusesarepathogenicmicroorganismsthatcaninhabitandreplicateinhumanbodies
causing a number of widespread infectious diseases such as influenza, gastroenteritis, hepatitis,
meningitis,pneumonia,acquiredimmunedeficiencysyndrome(AIDS)etc. Amajorityoftheseviral
diseasesarecontagiousandcanspreadfrominfectedtohealthyhumanbeings. Themostimportant
stepinthetreatmentofthesecontagiousdiseasesandtopreventtheirunwantedspreadistotimely
detectthedisease-causingviruses.Gravimetricviraldiagnosticsbasedonquartzcrystalmicrobalance
(QCM)transducersandnaturalorsyntheticreceptorsareminiaturizedsensingplatformsthatcan
selectivelyrecognizeandquantifyharmfulvirusspecies.Herein,areviewofthelabel-freeQCMvirus
sensorsforclinicaldiagnosticsandpointofcare(POC)applicationsispresentedwithmajoremphasis
onthenatureandperformanceofdifferentreceptorsrangingfromthenaturalorsyntheticantibodies
toselectivemacromolecularmaterialssuchasDNAandaptamers. Aperformancecomparisonof
differentreceptorsisprovidedandtheirlimitationsarediscussed.
Keywords: antibodies;aptamer;epitopeimprinting;molecularlyimprintedpolymers;quartzcrystal
microbalance;virussensor
1. Introduction
Earlydiagnosisofinfectiousdisease-causingagentssuchasvirusesisessentialforclinicaland
pointofcare(POC)applications[1–3]. Sincetheviruseshaveextremelysmallsizeandcaninfectall
livingbeings,i.e.,humans,animals,andplants. Therefore,theirpreciseandaccuratedetectionisof
significantinterest. Virusesusuallyliveinhost’slivingcells,wheretheyreplicate. Thus,theirdetection
isacomplicatedandchallengingtaskduetothecomplexnatureofthemediuminwhichtheyexist.
Sincethediscoveryoffirstvirus, i.e., tobaccomosaicvirus(TMV)in1892, alargenumberoftests
andtoolkitshavebeendevelopedfordifferenttypesofviruses[4,5]. Especially,moderntoolslike
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enzyme-linkedimmunosorbentassay(ELISA)andpolymerizedchainreaction(PCR)amplificationare
highlysensitiveandselectiveprotocolsforvirusrecognition[6,7]. Disposablekitshavingimmobilized
enzymesorDNAoffersimpleandrapidresultsinminimalcost[8,9]. However,suchtestsaremore
suitedforqualitativedetectionofvirusesinremoteareas.
Regardless of various established techniques for the detection and investigation of infectious
viruses,thedevelopmentofsmartbiosensorsanddiagnosticdevicesforquantitativedetermination
ofvirusesismandatory[10–13]. Themajorimpetusintheresearchanddevelopmentofsuchvirus
sensorsistheirpracticalfeaturessuchastheeaseofoperation,simpleandstraightforwarddevice
fabrication,possibilitytointegratesyntheticornaturalantibodies,rapidresponse,highselectivity
andcross-sensitivity, portability, andminiaturizationcapability. Itisduetotheseadvantagesthat
biosensorshavenotonlybeenwidelyemployedforvirusrecognition[14],buthavealsobeenapplied
forthedetectionofmicroorganismsuchaspathogenicbacteria[15,16]andyeast[17–20],livingblood
cells[21–23],anddifferentdiseasesbiomarkers[24,25].
A typical chemical sensor is comprised of natural and-or synthetic receptors coated on a
suitabletransducersuchasoptical,electrochemical,magnetic,gravimetricormass-sensitiveetc.[26].
Amongmanyotherfactors,theselectionofatransductiondevicemainlydependsonthenatureand
physicochemicalpropertiesofthesensitivelayermaterialthatundergoeschangeswhenexposedto
theanalyte. Forinstance,incaseofanopticalsensor,theopticalpropertiesoftheselectivematerial(or
receptorlayer)changeduetoitsinteractionswiththeanalyteofinterest. However,ifthereceptorlayer
isopticallyinactive,alabelingspeciesorindicatorisintroducedthattranslatesthereceptor-analyte
interactionsintoarecognizableopticalsignal[27]. Thus,theinclusionofalabelingmoleculetothe
receptorlayerintroducesthedesiredsensingfeature. Sinceallreceptorsurfacesdonotnecessarily
possesstheoptical,electrochemical,ormagneticcharacteristics,theadditionofalabelingagentcauses
morecomplexityinthereceptor’sbindingmechanismandmayalsoaffectitssensitivity,whichare
obviousdisadvantagesofthelabelingtechniques.
Inthisbargain,acousticormass-sensitivedevicesofferlabel-freedetectionofanalytes,because
mass is a fundamental property of any analyte. Thus, the acoustic wave devices are considered
as universal mass-sensitive or gravimetric transducers. These transducers include: (a) the bulk
acoustic wave devices such as quartz crystal microbalance (QCM) [28,29]; (b) the surface acoustic
wave(SAW)resonators[30,31]; and(c)thesheartransversewave(STW)resonators[32,33], which
arefrequentlystudiedinacombinationwithdifferentreceptormaterialsformolecularrecognition
and biomedical diagnosis. The mass-sensitive biosensors produce direct shift in frequency in the
eventofreceptor-analyteinteractionandanalyterecognition. Amongtheaforementionedacoustic
devices,QCMbasedchemicalandbiosensorshavebeenextensivelystudiedforthedetectionand
quantificationofawiderangeofanalytesfromsmallmoleculesandionstobiologicalmacromolecules
andpathogenicspecies,e.g.,viruses.
Figure1showstheprincipleofaQCMbasedgravimetricsensorforvirusrecognition. AQCM
transducer,ifcoatedwithasuitablyselectivereceptor,iscapableofbindingvirusparticlesaswellas
virusproteins. Inprinciple,QCMishighlysensitivetothechangesinmassloadinganddepending
upon the nature of analyte (e.g., virus particles) these little changes in mass due to selective virus
bindingcanbedetectedeasily. Theselectivereceptorsthereforeplayamajorroleinvirusrecognition
and quantification due to their ability to interact and bind with viruses. Hereby, we present a
comprehensivereviewofthegravimetricormass-sensitiveviraldiagnosticdevicesbasedonQCM
andabroadspectrumofsyntheticandnaturalreceptors.
Thekeyobjectiveofthisworkistodeliveranup-to-dateliteraturereviewandcriticalanalysisof
differentapproachesemployedinthedesignandapplicationofQCMbasedbiosensorsanddiagnostic
devicesfortheexclusiverecognitionofdifferenttypesofviruses. Thesubstanceisdividedintotwo
sectionsinwhichthebasicQCMdevicedesignandassembly,anddifferenttypesofreceptormaterials
withspecialemphasisonthenature,fabricationmethod,andsensorperformancearediscussed. Inthe
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with the benefits and limitations of each receptor. This work also highlights the major achievements
endacomparisonofdifferentreceptorlayersisprovidedalongwiththebenefitsandlimitationsof
and the future research viewpoint.
eachreceptor. Thisworkalsohighlightsthemajorachievementsandthefutureresearchviewpoint.

FigFuigreur1e. 1T.h Tehpe rpinricnicpilpeleo foaf aQ QCCMM-b-baasseedd ggrraavviimmeettrriicc vviirruuss sseennssoorr. .TThhee cchhanangeg eini nmmasass isni nrersepsopnosne steo to
virvuisrubsi nbdinindginwg iwthitthh tehsee sleelcetcivtiever erceecpeptotorri sisd deetteecctteedd aass aa cchhaannggee iinn ffrreeqquueennccyy oof fQQCCMM trtarnasndsudcuecre. r.
2. Q2. uQaurtazrtCz rCyrsytastlaMl Micircorbobalaalnancece::T Trarannssdduucceerr DDeessiiggnn aanndd FFaabbrriiccaattiioonn
ThTishisse cseticotinonp rperseesnetnstsa ab brireieffd deessccrripipttiioonn ooff tthhee pprriinncciippllee,, ddeessigignn, ,aanndd fafbabrirciactaiotino nofo QfCQMCM-ba-bseadse d
gragvraimviemtreitcrictr tarnasndsduucecer.r. DDuurriinngg tthhee lalsats tfefwew dedcaedcaeds,e Qs,CQMC mMeamsueraisnugr tinheg ftrheqeufernecqyu esnhicfyt asnhdif tthaen d
theeleelcetcrotrcohcehmeimcailc aQlCQMC,M i,.ei..,e .E,QEQCMCM [3[43,435,3]5 ]sismimulutaltnaenoeuosulys lycocmopmuptiuntgin gtheth eeleecletrcotrcohcehmeimcailc aalnadn d
frequency changes have been broadly designed for sensing different analytes in the gaseous and
frequencychangeshavebeenbroadlydesignedforsensingdifferentanalytesinthegaseousandliquid
liquid phases. The pioneering work of Sauerbrey [36] on calculating the frequency changes as a
phases. ThepioneeringworkofSauerbrey[36]oncalculatingthefrequencychangesasafunctionof
function of mass adsorbed or deposited on the surface of gravimetric devices has laid the foundation
massadsorbedordepositedonthesurfaceofgravimetricdeviceshaslaidthefoundationfortheir
for their applications in chemical and biosensors.
applicationsinchemicalandbiosensors.
In a typical QCM sensor construction, a thin AT-cut quartz wafer having metal electrodes on
InatypicalQCMsensorconstruction, athinAT-cutquartzwaferhavingmetalelectrodeson
opposite sides is connected with oscillator circuit which drives QCM to resonate at characteristic
opposite sides is connected with oscillator circuit which drives QCM to resonate at characteristic
frequency. A bulk transverse wave is generated that propagates in perpendicular direction of quartz
frequency. Abulktransversewaveisgeneratedthatpropagatesinperpendiculardirectionofquartz
surface. The crystal surface is usually covered with a certain receptor material which binds with
surface. Thecrystalsurfaceisusuallycoveredwithacertainreceptormaterialwhichbindswithtarget
target analyte and thus leads to increase in the rigid mass of crystal. This results a change in
analyteandthusleadstoincreaseintherigidmassofcrystal. Thisresultsachangeinfundamental
fundamental resonance frequency of crystal and may be used to precisely determine the adsorbed
resmonaassn coen fcrreyqsutaeln scuyrfoafcec rtyhsetraelfoarned, rmefaeyrribnegu its eads steonpsorerc siisgenlyald. e terminetheadsorbedmassoncrystal
surfaceQthCeMre,f douree, troe fietsr reixntgreimtaeslys henigsho rsesnigsintiavl.ity towards minor changes in mass, is considered as one
of QthCe Mbe,sdt upelatotfoitrsmesx tfroerm suelcyhh aipgphlsiceantsioitnivs.i tIyf tcoowmabridnsedm winiothr cshuaitnagbeles sinelmecatisvse, imscaotenrsiaidl eorre dreacespotnoer of
thelabyeesrt, pQlaCtfMor mtrasnfsofrosrmucsh ianptop laicna teioxcnesp.tIifocnoamllyb isnmedarwt ittihnys ubiatalabnleces eclaepctaibvlee moaf tdereitaelctoirngre cmeipntiosrculaley er,
QCaMltetrraatinosnfos rimn ssuirnftaocea nmeaxssc,e ip.eti.,o unsaullayllsym ina rtthtei nraynbgael aonf cneg/ccampa2 b[3le7]o. fdetectingminisculealterationsin
surfaceTmhae sss,ein.es.o,ru sruesaplloynisne thoef raa ntgyepoicfanl gQ/CcmM2 [d3e7v].ice, i.e., shift in frequency (Δf), is directly
proTphoerstieonnsaolr tore tshpeo snqsueaorfe aotfy iptsi cfaulnQdaCmMendteavl ircees,oin.ea.t,inshgi fftreinquferenqcuy eanncdy t(h∆ef m),iasssd idreepctolsyitperdo [p3o6r]t, iaosn al
togthiveesnq iuna trhee oEfqiutsatfiuonnd (1a)m beenlotwal: resonatingfrequencyandthemassdeposited[36],asgiveninthe
Equation(1)below: 𝑓2∙∆𝑚
∆∆𝑓f==−−f𝑜o2·∆m/A⁄(cid:112)𝐴ρ√qµρq𝑞μ𝑞 (1)( 1)
whwerheerfeo fios isth tehef ufnudnadmamenentatall reressoonnaanntt ffrreeqquueennccyy ooff QQCCMM, ,Δ∆mm isis ththee chchanagneg eini nmmasass, sA, Ais itsheth e
piepzioeezloeecltercictrailclayllayc taicvteivaer aeareoaf oQf QCMCMin inc mcm2,2,ρ ρqqi iss tthhee ddeennssiittyy ooff qquuaarrttzz ((ii..ee..,, 22..664488 gg//cmcm3)3, )a,nadn dµqµ isq tihset he
shesahreamr omdoudluusluosf oafn anA TA-Tcu-ctuqt uqauratrztzc rcyrystsatall( i(.ie.e.,.,2 2.9.94477 ×× 1100111 1gg/c/mcm·s2·)s. 2).
Chemosensors2017,5,7 4of25
TheEquation(1)ismodifiedformeasurementsinliquidmedium[38,39],i.e.,whenonefaceof
QCMisincontactwithaliquid,theliquidloadingeffectonfrequencychangecanbesummarizedas
giveninEquation(2)below:
(cid:115)
ρ η
∆f = −f3/2 l l (2)
o πρ µ
q q
whereρ isthedensityoftheliquid,andη istheviscosityoftheliquid. Itisimportanttomentionhere
l l
wheneverQCMdevicesarestudiedinliquidphase,theacousticpropertiesofliquidmediumhaveto
betakenintoaccountforsensormeasurements.
Nonetheless,theseequationsshowthatthesensitivityoftheQCMdevicecanbeimprovedby
increasingitsprimaryfrequency,i.e.,ifthefundamentalfrequencyofaQCMisdoubled,itsresponse
wouldbeincreasedbyafactoroffour. However,thiscanonlybeachievedbyreducingthethickness
ofQCMwaferthatwillresultinmechanicallyfragiledevices. Thus,thisparameterpracticallylimits
thefabricationofdevicesbeyondcertainfrequency(f ). Theotherwaytoincreasethesensitivityof
o
QCM devices is to work with overtones, which require special oscillator circuit having band pass
filterforattenuationofharmonicresonances. However,thiswouldalsoleadtoanincreaseinnoise
levelandultimatelymakingsmallimprovementinsignaltonoiseratio. Therefore,anincreaseinthe
fundamentalresonancefrequencyofcrystalisthemoreappropriatemethodoftuningsensitivity.
Typically,QCMhavingfrequencyintherangeof5–20MHzareusedinliquidphasemeasurements.
Although,afewreportssuggesttheuseofQCMwithfrequencyashighas110MHzforliquidphase
operation [40]. This is remarkably high frequency and the authors report that substantially high
detection limit is achieved in the detection of M13-phages in liquid medium, i.e., the sensitivity
improvedbyafactorof200whileusing56MHzQCMascomparedto19MHzQCMdevice[40].
AnotherimportantaspectindeterminingthesensitivityofaQCMdeviceisthenatureanddesign
ofelectrodesonQCMwafers[41,42]. Ifthediameteroftheelectrodeisbigger,theavailablesurfacefor
selectivereceptorlayerintegrationwouldbelarge,whichultimatelyleadstohighersensorresponse.
ForelectrodefabricationonQCMwafer,goldoranyothernoblemetalpastecanbeappliedviascreen
printing. ManyofthecommerciallyavailableQCMhavefusedmetalelectrodesthataffordbetter
supportonQCMsurface. Furthermore,theintegrationofreceptorlayerwithQCMelectrodeshould
befirmandstable,i.e.,thereceptormaterialshouldnotbedeterioratedduringthefabricationand
testingprocesses.
Figure2showsthedesignofasingle,dual,andtetra-electrodeQCMwaferfordetectingviruses.
AmultichannelQCMhavingmorethanoneelectrodesforsensingdifferentanalytesisaninnovative
design[43].Albeitthisuniquetetra-electrode,QCMdesignhasnotbeenimplementedforsimultaneous
recognition of different viruses or virus sub-types, but it could be adopted as a single transducer
platformfordifferentviruses. However,itisimportanttomentionthatthereshouldbeaminimal
distancebetweenthemultipleelectrodestopreventthecross-talkbetweenelectricalsignalsandthereby
reducingsignal-to-noiseratioforimprovedsensitivity. Furthermore,inliquidphasemeasurements,
onesideoftheQCMremainsinairtoavoidexcessivedampingproblems.
These are some general but imperative design and fabrication parameters that regulate the
ultimateQCMdevicesensitivity. Thefollowingstudyemphasizesavarietyofsyntheticreceptors
basedonimprintedpolymers,naturalantibodies,DNAandaptamersforthedetectionofdifferent
virusesincombinationwithQCMdevices.
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Chemosensors 2017, 5, 7 5 of 24

Figure2F.igTuhree d2e.s igThneo fdqeusiagrnt zocfr yqsutaarltmz iccrryosbtaall anmciecr(oQbaClaMnc)et ra(QnCsdMu)c etrrasn:s(dAu)cAersw: o(rAk)i nAg swinogrlkei-negl ectrode
QCMfosrindgelet-eeclteicntrgodaes QpeCcMifi fcorv idreutesc;t(iBng) Aa sdpuecaifli-ce lveicrutrso; d(Be) QAC dMualw-eiltehctrwodoer kQinCgMa wndithr ewfeorrkeinncge aenlde ctrodes
fordetercetfienrgenaces epleecctirfiocdevsi rfours d;eatnecdtin(Cg )aA spteectirfiac- veilreucst;r aondde (QC)C AM teftorar-esliemctruoldtae nQeCoMus fodre stiemctuilotanneoofums ultiple
detection of multiple viruses. The front and back sides of the multichannel tetra-electrode QCM are
viruses. Thefrontandbacksidesofthemultichanneltetra-electrodeQCMareshowninDandE,
shown in D and E, respectively. For sensor measurements, QCM is assembled into a flow cell so that
respectively.Forsensormeasurements,QCMisassembledintoaflowcellsothatitsfrontside(D)is
its front side (D) is exposed to the liquid phase containing the target analyte (e.g., viruses), and its
exposedbtaockt hsiedeli q(Eu) iids epxhpaosseedc toon atiari tnoi nprgevthenet tdaarmgeptinagn aolfy thtee (see.ngs.o,rv siirgunsael.s ),anditsbackside(E)isexposed
toairtopreventdampingofthesensorsignal.
3. Receptors for QCM Virus Sensors
3. ReceptorsAf nourmQbCerM of VaciarduesmSice rnesseoarrsch articles and review papers have been published previously that
emphasize the QCM’s working principle and study their performance in a wide range of complex
Anumberofacademicresearcharticlesandreviewpapershavebeenpublishedpreviouslythat
biochemical processes taking place at the receptor-analyte interface [44–47]. These investigations
emphashizigehltihgehtQ thCe Msu’rsfawceo crhkeimnigstrpyr ainndci mpleechaanndistsict uedveynttsh deuirripnge rthfoe ramttaacnhcmeeinnt oar wbiniddeinrga onf gvearoiofucso mplex
biochemtaircgaelt panroalcyetesss.e Tshtisa kwionrgk ips lparciemaartiltyh feocruesceedp otno rd-iaffneraelnytt teypinest eorff saycneth[e4t4ic– a4n7d]. nTathuerasle reicnevpetosrtsi gations
highlighthtatth deirseuctrlfya icnefluchenecme QisCtrMy adnevdicme seecnhsiatinviistyti acnedv seenletcstidviutyr ifnogr itmhperoavtteadc rhemcoegnnittioorn boif nvdiriunsgeso. Ifnv arious
case of virus sensing, the recognition characteristics are governed by nature of receptor/selective
targetanalytes.Thisworkisprimarilyfocusedondifferenttypesofsyntheticandnaturalreceptorsthat
layer. In the following sections, we shall describe the virus recognition principle, mechanism, and
directlyinfluenceQCMdevicesensitivityandselectivityforimprovedrecognitionofviruses.Incaseof
performance including sensitivity and selectivity of various receptors. The potential receptors for
virussensing,therecognitioncharacteristicsaregovernedbynatureofreceptor/selectivelayer. Inthe
selective sensing of different viruses are divided into four classes based on their nature and
followinvgirusse-cbtiinodninsg, wmeecshhaanlilsmde: s(car) ibSeyntthheetvici raunstibreocdoiegsn bitaisoend porni nmcioplelceu,lmarleyc himanpirsinmte,da npdolypmerefros;r m ance
includin(bg) sneantusirtailv aitnytibaonddiesse; l(ecc) tDivNitAy; o(df)v aaprtiaomuesrrse; caenpd t(oer)s o.tThehre bpioomteancrtoiamlorleecceuplatro rrescfeoprtosres lesucctihv aess ensing
ofdifferpernottevinirsu. sesaredividedintofourclassesbasedontheirnatureandvirus-bindingmechanism:
(a)Syntheticantibodiesbasedonmolecularlyimprintedpolymers;(b)naturalantibodies;(c)DNA;
3.1. Synthetic Antibodies: Molecularly Imprinted Polymers for QCM Virus Sensors
(d)aptamers;and(e)otherbiomacromolecularreceptorssuchasproteins.
Artificially designed polymeric receptors such as molecularly imprinted polymers (MIPs) are
3.1. Synothfteetnic reAfenrtriebdo daise ssy:nMthoelteicc uolra arrlytifIicmiaplr ainnttiebdodPioelsy, mbeecrasufsoer thQeCy McanV miriumsicS ethnes omroslecular recognition
characteristics of naturally occurring receptors or antibodies [48,49]. Among several receptors,
Artsiyfincthiaeltlicy adnetisbiogdnieesd bapsoeldy omn eMriIcPsr ehcaevpe tpolarcsesdu tchhemasselmveos laesc huilgahrllyy eifmficpiernitn rteecdogpnoitiloynm eelermse(nMtsI Ps)are
oftenreffoerr rae wdidaes rsaynngteh oeft bicioo-arnaarlytitfiesc [i5a0l–a5n2]t.i Mboodleiceusl,abr eimcapurisnetinthge isy ac satnramighimtfoircwtahred mwaoyl etoc uinlatrrodreuccoe gnition
the target analyte or a closely related molecule as the template in polymer matrix to develop highly
characteristics of naturally occurring receptors or antibodies [48,49]. Among several receptors,
specific affinity centers [53]. These interaction centers are usually tailored to template size, structure,
syntheticantibodiesbasedonMIPshaveplacedthemselvesashighlyefficientrecognitionelements
and functionality; thus, offering complementary geometrical and chemical fits for the target analyte.
forawiderangeofbio-analytes[50–52]. Molecularimprintingisastraightforwardwaytointroduce
The interactions between such affinity centers and analyte molecules are mainly driven by
thetargneotna-ncoavlyalteento froarcecslo ssuechly ars ehlyadterodgmeno bloencduilneg,a πs-tπh setatcekminpgl, avtaen idnerp Wolayamls eforrmcesa terticx., wtohdicehv aellloowp highly
specificraefvfienrsiitbylec reen-itnecrluss[i5o3n] [.5T4–h5e7s].e interactioncentersareusuallytailoredtotemplatesize,structure,
andfunctionTaheli teya;set houf ssy,notfhfeesriisn, gprcoofomunpdle smenesnittiavirtyy, guenommatecthreicda slealencdtivcithye, mresicisatalnfictes afgoarintshte chtaermgiecatla nalyte.
and thermal degradation, storage stability under ambient conditions are the key features of MIPs,
Theinteractionsbetweensuchaffinitycentersandanalytemoleculesaremainlydrivenbynon-covalent
which make them highly competitive in sensing applications. According to some researchers, the
forces such as hydrogen bonding, π-π stacking, van der Waals forces etc., which allow reversible
selectivity of synthetic antibodies (MIPs) toward specific target molecules is found to be comparable
re-inclutsoio nnat[u5r4a–l a5n7t]i.bodies [58]. The regeneration of these synthetic antibodies after every sensing cycle is
The easeofsynthesis,profoundsensitivity,unmatchedselectivity,resistanceagainstchemicaland
thermaldegradation,storagestabilityunderambientconditionsarethekeyfeaturesofMIPs,which
makethemhighlycompetitiveinsensingapplications. Accordingtosomeresearchers,theselectivity
ofsyntheticantibodies(MIPs)towardspecifictargetmoleculesisfoundtobecomparabletonatural
antibodies[58]. Theregenerationofthesesyntheticantibodiesaftereverysensingcycleisfairlysimple
thatmakesthemviableforseveralroundsofassayswithoutanysignificantlossinsensitivityand-or
Chemosensors2017,5,7 6of25
selectivity. Furthermore,thecostofMIPsforatypicalanalyteisintherangeof0.1–0.5$/mg,whereas
thenaturalantibodiesdependinguponthetargetmoleculesareofferedin100–1000$/mg[59].
TheforemostadvantageofMIPsinchemicaland/biosensingapplicationsisthepossibilityto
easilyintegrateMIPswithdifferenttransducerdevicessuchasQCM[60,61]. Duetotheirexceptional
characteristics,MIPsareconsideredaspotentialcandidateforvirussensingapplicationsandoften
exhibithighrecognitionproficiencyandselectivity[62]. Thereareavarietyofimprintingstrategiesfor
microorganisms’detection[63];however,inthefollowingsub-sections,weshallfocusontheselected
approachesthathavebeentypicallyemployedinQCMbasedviraldiagnostics. Table1showsthe
selectedexamplesofvirus-MIPbasedQCMbiosensorsandtheirdetectionlimits.
Table1.Theselectedexamplesofthemolecularlyimprintedpolymers(MIP)assyntheticreceptorsfor
QCM-basedviraldiagnostics.
Detection
Receptor Template/Target Polymer Reference
Limit
TMV Polyurethane 8ng/mL [64]
5×105virus
PPOV Polyurethane [65]
particles/mL
Soft-lithographically
(surface)imprinted HRV1A,HRV2, Polyurethane 100µg/mL [66]
MIP HRV14
H5N1,H5N3, Poly(acrylamide-co-methacrylic
105
H1N1,H1N3, acid-co-methylmethacrylate-co-N-vinylpyrrolidone) [67]
particles/mL
H6N1 withN,N-(1,2-dihydroxyethylene)bisacrylamide
Denguevirus Poly(acrylicacid-co-acrylamide)withethyleneglycol
Epitopeimprinted NS1protein dimethylacrylate 1µg/L [68]
MIP
HIV-1GP41 Polydopamine 2ng/mL [69]
Plasticantibody Poly(vinylpyrrolidone-co-methacrylicacid)with 2.5×1012virus
replica HRV14 N,N(cid:48)-(1,2-dihydroxyethylene)bisacrylamide particles/mL [70]
3.1.1. Soft-Lithography
Owingtothedelicatenatureofmicroorganisms, soft-lithographyisanappropriatestamping
techniqueforpatterningpolymericmaterials,thusgeneratingselectivebiomimeticsurfaces[71,72].
Itisasurfaceimprintingtechniquethathasbeenextensivelyappliedformacromolecularimprinting
in the last two decades [73,74]. In this case, a suitable monolayer of targeted microorganism or
biomacromoleculeisdepositedastemplateonaninertsubstratetomakethetemplate-stamp,which
is subsequently pressed on the surface of a pre-polymer. The stamp-pressed pre-polymer layer is
allowedtoundergopolymerizationundermoderateconditions,andthenwashedwithmildsolvents
toremovetemplatefromthepolymersurface[75]. Aschematicofthesoft-lithographybasedsurface
imprintingprocedureisshowninFigure3A.Thismethodallowstransferofprecisestructuraldetails
ofbioanalytesontosyntheticpolymersurface. Consequently,thisbiomimeticinterfaceiscapableof
selectivelyrecognizingtargetanalytesthroughnon-covalentchemicalinteractions.
Soft-lithography allows faster mass transfer of bioanalytes with enhanced reversibility.
Furthermore,thepre-polymercanbecoatedonQCMelectrodesbeforesoft-lithographicstamping
procedureforsimpleandeasyintegrationoftheselectivelayerwiththetransducer. Soft-lithographic
imprinting has been extensively studied in combination with QCM devices for different types of
viruses. Forinstance, Dickertetal.[76,77]firstpatternedapre-polymercoatedonQCMelectrode
surface with tobacco mosaic viruses (TMV) using soft-lithography, and the resulting sensor was
reported to be highly sensitivity towards TMV. The device also exhibited reversible sensor signal
indicatingthecompleteremovalofadsorbedspeciesandperfectlayerregenerationforfurtheranalyses.
Inasubsequentwork[64],thesensitivityofQCMdevicescoatedwithsurfaceimprintedpolyurethane
monolayerwassubstantiallyimprovedandtheselectivitywasinvestigatedrevealinghighaffinity
ofstampedpolymersforthetargetedvirus. Thedetectionofparapoxovisvirus(PPOV)isanother
Chemosensors2017,5,7 7of25
Chemosensors 2017, 5, 7 7 of 24
exampleofthesensingplatformusingacombinationofQCMandsoft-lithographicallypatterned
virus (PPOV) is another example of the sensing platform using a combination of QCM and
polymersurfaces[65].
soft-lithographically patterned polymer surfaces [65].
Inalaterstudy[66],thisstrategywasextendedtoimprintdifferentstrainsofhumanrhinovirus
In a later study [66], this strategy was extended to imprint different strains of human rhinovirus
(HRV)asthreeserotypes,i.e.,HRV1A,HRV2,andHRV14,wereusedasthetemplatestoproduce
(HRV) as three serotypes, i.e., HRV 1A, HRV 2, and HRV 14, were used as the templates to produce
therespectivebiomimeticselectivelayersonQCM.Thesensingmeasurementsrevealedthateach
the respective biomimetic selective layers on QCM. The sensing measurements revealed that each
typeofimprintedsurfaceofferedthehighestsensorresponsetoitstemplatedstrainvirusasshownin
type of imprinted surface offered the highest sensor response to its templated strain virus as shown
Figinu rFeig3uBr,et h3Bu,s tphurosv pinrogveinxgc eelxlecneltlesnetl escetlievcittiyv.itSyi.m Siilmarillayr,ltyh, ethseu rsfuarcfaeciem imprpinritnetdedp oploylymmeersrsd deessigignneedd by
sofbty-l itshooftg-lriathpohgyrapprhoyc edpurorceeadrueresu acrcee sssufuclcleyssefmulplylo yemedplionyQedC Min- bQasCeMd-vbiarsaeldd ivaigrnalo sdtiiacsgnfoorstiincsfl ufoenr za
Aivnifrluuesnszuab A-t yvpierus,s is.eu.b,-tHyp5Nes1, ,i.eH.,5 HN53N, 1H, 1HN51N,3H, 1HN1N3,1,a nHd1NH36, Nan1d[ 6H7,67N8]1. [6T7h,7e8]f.a bTrhiec aftaebdricsaetnesdo rs
desmenosnosrtsr adteemdocnosntsriadteedra cbolnessideleercatbivlei tsyelfeocrtisvcirteye fnoirn sgcroefeinniflnug eonfz ianfAluvenirzuas Asu vbi-rtuysp seusba-styepaecsh assu eba-cthy pe
wassubb-etystper ewcoags nbiezsetd rebcyogintsizeodw bnyi mitsp oriwnnte dimspurrinfatecde .suTrhfaecer.e Tphoer treedpodretetedc tdioetnecltiimoni tliwmaits w1a0s5 1v0i5r us
pavrtiirculse sp/amrtiLcl[e6s7/m].LIn [6v7i]e.w Ino vfitehwes oefe txhaemsep elexsa,mitpilseso, bivt iios uosbvthioautss othfta-tl itshooftg-lriathpohgyraapphpyr oaapcphrotaocdhe tsoi gn
sudrfeasciegnM sIuPrsfamcea kMesIPths emmakcaesp athbelemo fcaspelaebcltei voefl ysedleiscttiinvgeluyi sdhiisntigngduififsehrienngt dseifrfoetryepnte sseorfotthyepessa mofe tvhier us,
whsiacmhem vairyubs,e wtehrimche mdaays ibnet rtear-mgreodu apss ienltercat-igvriotyu.p selectivity.

FigFuigruer3e. 3(.A ()AA) Asc shcehmemataitcicr ereppreresseennttaattiioonn ooff ssoofftt--lliitthhooggrraapphhyy: :AA sstatammpp ofo afsassesmemblbedle dvirvuirsu psaprtaicrlteicsl iess is
prepsrseesdseodn toonttho etphree -pproel-ypmoleyrmceora tceodatoend aoQn CaM QeCleMct roeldeec.trAodftee.r Apoftleyrm peorliyzamtieorniz,atthioens,t atmhep sitsarmemp oivs ed
anrdemthoevteedm apnlda tethies wtemasphleadte aiws awyastoheodb taawinayth teo soubrtfaaicne tihme psruinrftaecde siymnptrhienttiecda snytnibthoedtiiecs a;n(Bti)boRdeileast;i v e
sen(Bso) rRreelsaptiovnes eseonfsdorif freersepnotnhsue mofa ndirfhfeinreonvti rhuusm(HanR Vrh)isneorvoitryupse -(iHmRpVri)n steerdotpyopley-mimeprsritnotweda rpdoslydmiffeerrse nt
strtaoiwnsaordfsH dRifVf.eIrtenist esvtriadienns totfh HatReVac. hIt sius be-vtiydpeento tfhHatR eVacish psruebf-etryepnet ioafll yHiRdVe nisti fiperedfebryenthtiealrleys ipdeecnttivifeieMd IP
subrfya cteh.eF riegsupreect3iBvei sMreIpP rosudrufacceed. wFiigtuhrpe e3rBm iisss iroenprfordoumceJden wikiteht aple.r[m66is].siCono pfyrormig hJtenbiyk thete aAl.m [e6r6i]c. an
ChCeompiycrailgShot cbiye ttyh,e2 A00m9.erican Chemical Society, 2009.
3.1.2. Epitope Imprinting
3.1.2. EpitopeImprinting
The living cells, viruses and other microorganisms are sometimes difficult to fit and imprint as
The living cells, viruses and other microorganisms are sometimes difficult to fit and imprint
templates due to their large size and the lack of binding site accessibility [79]. To overcome such
as templates due to their large size and the lack of binding site accessibility [79]. To overcome
challenges in biomacromolecular imprinting, epitope imprinting is introduced as an efficient
suchchallengesinbiomacromolecularimprinting,epitopeimprintingisintroducedasanefficient
macromolecular imprinting method, especially for proteins [80,81]. Epitope imprinting is also a
macromolecular imprinting method, especially for proteins [80,81]. Epitope imprinting is also a
method of choice for imprinting and recognition of viral proteins and different species of viruses.
methodofchoiceforimprintingandrecognitionofviralproteinsanddifferentspeciesofviruses. This
This technique, unlike whole-cell imprinting or soft-lithographic patterning of viruses on polymer
technique,unlikewhole-cellimprintingorsoft-lithographicpatterningofvirusesonpolymersurfaces,
surfaces, introduces a small peptide fragment as the template for imprinting polymers. The resultant
introducesasmallpeptidefragmentasthetemplateforimprintingpolymers. Theresultantimprinted
imprinted material interacts with target protein through its epitope, i.e., a small part of the antigen.
materialinteractswithtargetproteinthroughitsepitope,i.e.,asmallpartoftheantigen. Aschematic
A schematic of the epitope imprinting process in shown in Figure 4A.
oftheeTphiteo pneatiumraplr inantitnibgopdryo-caenstsigienns hboinwdninign Finigspuirreed4A e.pitope imprinting approach is useful in
proTdhuecninagtu arratlifaicnitailb oredcye-patnotrisg feonr bbiinodainnaglyitnessp rierecdogenpiittioopn.e Aimlbperiti nitt ihnagsa bpeperno aecxhteinssuivseelfyu lsitnudpireodd ufocri ng
artpifiroctieailnr reecceopgtnoirtisonfo [r59b,i8o2a],n saolmytee srerseecaorcghneirtsio anls.o Ainlvbeesittigitatheads itbs eaepnpleicxatteinonsisv ienl ydisatgundoiseids offo vrirpursoetse in
recaongdn ivtiiroanl p[5r9o,t8e2in],ss. oTmhee sreelseecatriochne orfs palespotiidnev efrsatiggmateendt iatss taepmpplilcaattei oannsdi nitsd sieaqguneonsicse oafrev iirmuspeosrtaanndt tvoi ral
proactehiinevs.e Timheprsoevleecdt iorencoogfnpiteiponti daes ftrhaeg msuernfatcae sgtreomuppsl aoten atnhed eitpsitsoepqeu eyniecled asrpeecimifipc osrttrauncttutroala cahnide ve
Chemosensors2017,5,7 8of25
impCrohevmeodsenrsoercs o20g1n7,i 5ti, o7 n asthesurfacegroupsontheepitopeyieldspecificstructuralandfu8n ocf t2i4o nal
memoryinpolymers. Thesessurfacegroupsaresuitedforepitopeimprintingduetotheiraccessibility
andffuunnccttioionnaal lmiteymthorayt ilnea pdoslytmoethrse. Tsehleescetsiv seurrfeaccoeg gnroituiopns aorfet saurgiteedte fdors pepeictioepse. imprinting due to their
accessibility and functionality that leads to the selective recognition of targeted species.
Taiandcoworkers[68,83]adoptedtheepitopeimprintingapproachforQCM-basedserological
Tai and coworkers [68,83] adopted the epitope imprinting approach for QCM-based serological
assay of dengue virus, i.e., mosquito-borne virus, infections. They used acrylic polymer system
assay of dengue virus, i.e., mosquito-borne virus, infections. They used acrylic polymer system for
for epitope imprinting of 15-mer peptide that is known as the linear epitope of dengue virus NS1
epitope imprinting of 15-mer peptide that is known as the linear epitope of dengue virus NS1
protein [68]. The resulting epitope-imprinted polymer (EIP) QCM sensor achieved the direct and
protein [68]. The resulting epitope-imprinted polymer (EIP) QCM sensor achieved the direct and
quantitativedetectionoftheNS1protein,asshowninFigure4B.Furthermore,theEIP-QCMsensors
quantitative detection of the NS1 protein, as shown in Figure 4B. Furthermore, the EIP-QCM sensors
exhibitedagoodcorrelationwithenzyme-linkedimmunosorbentassay(ELISA)indicatingreliable
exhibited a good correlation with enzyme-linked immunosorbent assay (ELISA) indicating reliable
detedcetitoecntioofn doef ndgenugeuvei vruirsu.sT. Thhees seennssoorr aallssoo ccoorrrercetcltyl yidiednetnifiteifide sdamsapmlesp floers tfhoer pthreesepnrcees oenr acebsoerncaeb osfe nce
ofvivriurusss pspeecciieess.. TThhee EEIIPP sseennssoorr ccooaatitninggs srermemaianiende dstasbtaleb lfeorf oar paeprieordi oodf oonfeo nmeomntoh natnhda ncodulcdo ubled be
regerneegreanteerdatfiedv efivtiem tiems.esT. hTehea naanlaylysissist itmimee ooff oonnee ssaammppllee wwaas s202–03–03 0mmini nwiwthi tthhet hdeetdeecttieocnt iloimnilti mofi t of
1–101µ–1g0/ µLg./SLu. cShucdhe dveicveicseasr aereu suesfeufulli ninc clilinniiccaall ddiiaaggnnoossiiss ooff ddeenngguue evivruirsu isnfinecfteicotnios nass tahsetyh peryevpernevt tehnet the
useoufsem oof nmoocnloonclaolnaanl tainbtoibdoiedsie[s8 [48,48,58]5.].

FiguFriegu4.re( A4.) A(As) cAh esmchaetmicarteipc rreespernetsaetnitoantioonf tohfe tehpei teoppiteoipme pimrinptriinntginpgr opcreodcuedreu;r(eB; )(BT)h Tehfere fqrueqeunecnycsyh ifts
asasfhuinftcst iaosn ao ffutnhcetioenp iotof pthee- imeppitroipnet-eidmpproilnytmede pr’osly(EmIePr)’sc o(EmIPp)l ecoxmatpiolenxwatiiothn dweinthg udeenvgiurue svNiruSs1 NpSro1 tein.
ThepErIoPt-ecino.a tTehde QECIPM-cocahtiepd isQcCaMpa bchleipt oisb icnadpaNblSe1 tpo rboitnedin NinS1th perroatneigne ionf t5hen gr/anmgLe toof 550 nµgg//mmL Ltow ith
100µ50L μingj/emctLi ownitohf 1t0h0e μuLn pinujercifitieodn oNf Sth1ep urontpeuinrifsioedlu NtiSo1n .pFroigteuinre s4oBlutisiorne.p Friogduurec e4Bd iws irtehprpoedrumciesds iwonithfr om
Taieptearlm.[i6ss8i]o.nC foropmyr Tigahi ettb ayl. t[h6e8]A. CmoepryirciagnhtC bhye tmheic AalmSeorciciaenty C,2h0e0m5i.cal Society, 2005.
Chen and coworkers [69] used a synthetic peptide, i.e., 35 amino acid residue as template to
Chen and coworkers [69] used a synthetic peptide, i.e., 35 amino acid residue as template to
developed epitope-imprinted polydopamine coated on QCM for human immunodeficiency virus
developed epitope-imprinted polydopamine coated on QCM for human immunodeficiency virus
(HIV) type-1 detection. The selection of epitope was made by similar amino acid residues of HIV-1
(HIVg)lytcyopper-o1tedine te4c1t, ioi.ne.., Tahmeinseol eacctiido nnoufmebpeirtso p5e79w–6a1s3.m Tahdee bdyevsicime ielxahriabmiteidn ohaigchid sreenssiidtiuveitsy oafnHdI V-1
glycsoeplerocttieviinty4 1f,oir. eH.,IaVm-1in golyaccoidprnouteminb e(rGsP54719)– 6w1i3th. Tthhee deesvtiimceateexdh idbeitteedctihoing hlismeint siotfi v2it ynagn/mdLs.e lTehctei vity
forHhiIgVh-p1oginlyt coofp trhoist eaipnp(rGoPac4h1 )isw itisth apthpeliceasbtiimlitayt etod rdeaelt escatmiopnlelsim, withoerfe2 tnhge /smenLso.rT ehxehhibiigtsh preocionvteoryf this
apprvoaaluchesi osfi t8s6.a5p%p–l9ic4a.1b%il iotfy HtoIVr-e1a GlsPa4m1 ipnl essp,ikwehde hruemthaen suerninseo.r Tehxehseib fiitnsdriencgosv aerrey invtearluesetsinogf i8n6 v.5ie%w– o9f4 .1%
ofHtIhVe-ir1 pGotPe4n1tiainl fosrp cikliendicahl utemstas nofu tarringeet. vTirhuessees afinndd-oinr gvsiraalr epriontteeinres swtiinthgouint uvsiienwg loabfetlhinegir inpdoitceantotria. lfor
clinicaltestsoftargetvirusesand-orviralproteinswithoutusinglabelingindicator.
3.1.3. Plastic Antibody Replica
3.1.3. PlaDstiicckeArnt tainbdo dcyowRoerpkleicrsa [70,86] employed an innovative strategy to produce plastic antibody
replica for virus sensing via two-step soft-lithography technique. Firstly, they used natural
Dickert and coworkers [70,86] employed an innovative strategy to produce plastic antibody
antibodies for a specific target as the template in synthetic polymer system, which could be
replicaforvirussensingviatwo-stepsoft-lithographytechnique. Firstly,theyusednaturalantibodies
Chemosensors2017,5,7 9of25
Chemosensors 2017, 5, 7 9 of 24
for a specific target as the template in synthetic polymer system, which could be precipitated in
precipitated in appropriate solvent to produce nanoparticles. Then, natural antibody-imprinted
appropriatesolventtoproducenanoparticles. Then,naturalantibody-imprintednanoparticleswere
nanoparticles were used as the master stamp on the surface of pre-polymer already coated on QCM
used as the master stamp on the surface of pre-polymer already coated on QCM electrode. Thus,
electrode. Thus, two-step soft-lithography leads to the formation of precisely patterned polymer
two-stepsoft-lithographyleadstotheformationofpreciselypatternedpolymerinterfacethatperfectly
interface that perfectly mimics natural antibodies used as template.
mimicsnaturalantibodiesusedastemplate.
The authors compared QCM sensor results of natural antibodies and their plastic antibody
TheauthorscomparedQCMsensorresultsofnaturalantibodiesandtheirplasticantibodyreplica
replica for HRV (the template and target analyte) and foot-and-mouth disease virus (FMDV; the
forHRV(thetemplateandtargetanalyte)andfoot-and-mouthdiseasevirus(FMDV;theinterferent),
interferent), as shown in Figure 5. Astonishingly, plastic antibody replica exhibited higher
asshowninFigure5. Astonishingly,plasticantibodyreplicaexhibitedhigherresponse,i.e.,sixtimes
response, i.e., six times higher, as compared to natural receptors. It was suggested that high
higher,ascomparedtonaturalreceptors. Itwassuggestedthathighsensitivityofplasticantibody
sensitivity of plastic antibody replica might be attributed to the higher surface area of
repMlicIPa-mnaingohptabretiacltetrsi. bFuuterdthteormthoereh, igthhee rsseunrsfoarc ereasrpeaonosfeM oIfP -bnoatnh opnaatrutircalle sa.nFdu rstyhnetrhmetoirce ,rethceepsteonrss or
restpoownasredos fFbMoDthVn awtausr anleagnlidgisbylen tshheotwicinregc evpertyo rlsotwo wcraorsdss-sFeMnsDitiVvitwy aasnnde egxlicgeilblelnets hseolwecitnivgitvy.e rTyhliso w
croasps-psreonascithi vpitryovainddese xance allletenrtnsaetlievcet iavnitdy .iTnnhoisvaaptipvreo wacahy ptroo pvrideceissealny aplatettrenrant isvyenatnhedtiicn nreocveapttiovres wthaayt to
preacries elpylapsatitct ercnopsiyens thoef tincarteucreapl toarnstitbhoadtiaerse apnlads titchacto pciaens obfen autsuerda liann tmibiocdroieosrgaanndistmhast’ cdaentebcetiuonse d
inmanidcr boiooragsasnayis.m s’detectionandbioassay.

FigFuirgeur5e. 5A. Ac ocmompaprairsiosonno offt hthees esennssoorr rreessppoonnssee ooff nnaattuurraall aannttiibbooddieies saanndd ththeieri rsysnytnhtehteict icrerpelipclai ca
prepared via imprinting for human rhinovirus (HRV) and foot-and-mouth disease virus (FMDV)
prepared via imprinting for human rhinovirus (HRV) and foot-and-mouth disease virus (FMDV)
recognition. HRV is the template as well as the target analyte, while FMDV is used for
recognition.HRVisthetemplateaswellasthetargetanalyte,whileFMDVisusedforcross-sensitivity
cross-sensitivity measurements. Reproduced with permission from Schirhagl et al. [70]. Copyright
measurements. ReproducedwithpermissionfromSchirhagletal.[70]. CopyrightbyWiley-VCH
by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009.
VerlagGmbH&Co.KGaA,Weinheim,Germany,2009.
3.2. Natural Antibodies for QCM Virus Sensors
3.2. NaturalAntibodiesforQCMVirusSensors
This section reviews strategies for fast and reliable detection of viruses with the help of natural
Thissectionreviewsstrategiesforfastandreliabledetectionofviruseswiththehelpofnatural
antibody (NAb)-based QCM biosensors. NAb-QCM biosensors for viral recognition have versatile
antibody(NAb)-basedQCMbiosensors. NAb-QCMbiosensorsforviralrecognitionhaveversatile
advantageous features compared to other sensing devices. They are eminently suitable for the rapid
advdaiangtnagoseios uosf vfeirautuserse swcitohm hpigahr esdentsoitiovtihtye ransden sspiencgifidcietvy.i cNesa.turTahl eayntaibroedeiems ianreen ptlryotseuinista pbrloedfuocredth e
rapbiyd tdheia igmnmosuisneo sfyvstierums etos pwriotthecht itghhe bsoendsyi tbiyv iitdyenantidfyisnpge acnifidc inteyu.trNalaiztuinrga lpaanthtoibgoednsie [s87a]r.e Npartoutreailn s
proadnuticbeoddibeys tfhuenicmtiomnu anse tshyes tesemlectotivper orteeccetptthoersb oind ygbrayviidmeentrtiicfy vinirgala nddiangenuotsrtaicl izdienvgicpeas thanodge nofsfe[8r 7].
Nastuitera-slpaenctiifbico adfifeinsiftuyn wctiitohn eiatshethr evisrealle schtievlle prreocteepintosr osri nwigtrha tvhiem pertoritceivnisr arelldeiaasgendo bsyti tchdee vviircuessesa nwdhoenff er
sitet-hsepye ecnifitecra tfhfien hitoystw biothdye.i tIhne fravcti,r athles haenltligpernoitce ainmsionro waciitdhst ohcecpurro atse ipnastcrheelesa asnedd fbuyntchtieonv iarsu spersotweihne n
thebyinednitnegr tshiteesh oons tthbeo dviyr.uIsn cfoaactt. ,theantigenicaminoacidsoccuraspatchesandfunctionasprotein
bindingTshiete ascocnumthuelavtiirouns ocfo abti.oanalyte (the targeted virus) on the surface of QCM biosensor coated
wiTthh eNaAcbc uremsuulltast iino nthoef rebsioonaannatl yfrteeq(uthenectya rsgheiftte tdhavti irsu us)seodn atsh tehes usernfascoer soifgnQaClsM. Thbeio sseennssitoivrictyo aotfe d
witthheN NAAbbr-eQsCulMts biniotsheensroers odneapnentdfrse qonu etnhec ytyspheif otft haanttiibsoudsye dusaesd tahneds eitns soorriesnigtantaiolsn. oTnh ethsee enlseicttirvoidtye of
surface. Although considerable data has been documented on NAb-based methods for the detection
theNAb-QCMbiosensordependsonthetypeofantibodyusedanditsorientationontheelectrode
of pathogenic viruses by conventional techniques [88,89], relatively little work has been done on
surface. AlthoughconsiderabledatahasbeendocumentedonNAb-basedmethodsforthedetection
specifically utilizing NAb-QCM biosensors as viral diagnostics. Nonetheless, NAb-QCM based
of pathogenic viruses by conventional techniques [88,89], relatively little work has been done on
detection systems have been developed to detect harmful virus species causing fatal diseases to
specifically utilizing NAb-QCM biosensors as viral diagnostics. Nonetheless, NAb-QCM based
Chemosensors2017,5,7 10of25
detectionsystemshavebeendevelopedtodetectharmfulvirusspeciescausingfataldiseasestohuman
beings,plants,andanimalsinthelasttwodecades[90–92]. Table2showstheselectedexamplesof
NAb-QCMbiosensorsforviruses.
Table 2. The selected examples of the natural antibodies (NAb) as receptors for QCM-based
viraldiagnostics.
Receptor Target Fabrication/ImmobilizationMethod DetectionLimit Reference
Anti-H5attachedtonanobeadsimmobilized
Anti-H5NAb AIVH5N1 0.128HAU [93]
on16-mercaptohexadecanoicacidmonolayer
3-mercaptopropanoicacidand
Anti-MCMVNAb MCMV 11-mercaptoundecanoicacid(10:1ratio) 250ng/mL [94]
crosslinkedwithanti-MCMV
Monoclonalanti-CIV ProLinker™Bimmobilizedanti-CIV
CIVH3N2 14nM [95]
NPantibody monoclonalantibody
Secondaryantibodieslinkedthrough
Secondaryantibodies HepBV 2ng/mL [96]
carboxylatedhyper-branchedpolymer
For instance, Li et al. [93] fabricated a QCM immunosensor using magnetic nanobeads and
polyclonalanti-H5antibodiesforthedetectionofavianinfluenzavirus(AIV)H5N1inagricultural,
food,environmental,andclinicalsamples. Thesurfaceantigenhemagglutinin(HA)wasdeposited
onQCMthroughself-assembledmonolayerof16-mercaptohexadecanoicacid. ThetargetAIH5N1
viruseswerethencapturedbytheimmobilizedanti-H5antibodiesattachedtomagneticnanobeads.
Theadditionofmagneticnanobeadscoatedwithanti-H5resultsinamplificationofbindingreaction
betweenantibodyandvirusantigens. Thisanti-H5coatedNAb-QCMimmunosensorexhibitedgood
sensitivity(limitofdetection: 0.128HAU[97])duetonanobeadsamplification. Itwasalsonoticedthat
signalamplificationwasmoresignificantatlowervirusconcentrationthatcouldbefavorableforearly
stagescreeningofH5N1virus.
Thissetupwasalsousedtoquantifyvirusesfromchickentrachealswabsamples. Authorsdidnot
observeanysignificantinterferencewithAIVsubtypesH3N2,H2N2,andH4N8[93]. Itisimperative
tomentionherethatbyincreasingthenumberofactivebindingsitesatsensorinterfacialcoatings,
amplified mass loading can be achieved by QCM based immunosensors. In this perspective, the
fabricationofnanomaterialswithNAbcouldleadtoenhancedmassdepositiononelectrodesurface
thus,amplifyingsensorresponse. Thishasalsobeendemonstratedinareport[98],whereauthors
developedAunanoparticlesfunctionalizedwithantibodiestoamplifytherecognitionprocess. Ithas
beenshownthatAumodifiedreceptorsincreasethedetectionlimittothreeordersofmagnitudehigher
ascomparedtodirectQCMsensingwithoutamplification.
Huangetal.[94]reportedaNAb-QCMbiosensorfortheselectiverecognitionofmaizechlorotic
mottle virus (MCMV). They mixed 3-mercaptopropanoic acid and 11-mercaptoundecanoic acid
(10:1 ratio) to prepare a self-assembled monolayer on QCM gold electrodes. Then, anti-MCMV
antibody was used as the cross-linking agent for specific recognition of MCMV. The MCMV was
cultivatedincorn,andtheinfectedtissueswerecollectedafter14daysfortests. Figure6Ashowsthe
surfacemodificationofQCMelectrodestofabricateNAb-QCMbiosensorforMCMVdetection.
Theanti-MCMVcoatedNAb-QCMbiosensorsuccessfullydetectedMCMVintheconcentration
rangeof250ng/mLto10µg/mL.Thedetectionlimitwasreportedtobe250ng/mL,whichwasvery
closetothatachievedbyconventionalELISAmethod. Thedevicewasalsofoundtobe45foldmore
selectivetowardsMCMVascomparedtoothervirusessuchasmaizedwarfmosaicvirus(MDMV),
sugarcanemosaicvirus(SCMV),andwheatstreakmosaicvirus(WSMV)atthesameconcentration.
Furthermore, the NAb-QCM biosensor was not only capable to recognize MCMV in a mixture of
MCMV,MDMV,WSMV,andSCMV,butalsodistinguishedbetweenhealthyandinfectedcornleaf
sampleswithhighaccuracy[94],asshowninFigure6B.