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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.:+966-13-720-3426(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 Chemosensors2017,5,7;doi:10.3390/chemosensors5010007 www.mdpi.com/journal/chemosensors Chemosensors2017,5,7 2of25 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 Chemosensors2017,5,7 3of25 Chemosensors 2017, 5, 7 3 of 24 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. Chemosensors2017,5,7 5of25 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.

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Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen,. The Netherlands; [email protected]. 5 Keywords: antibodies; aptamer; epitope imprinting; molecularly imprinted polymers; quartz crystal microbalance; virus sensor. 1. Introduction.
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Gravimetric Viral Diagnostics: QCM Based Biosensors for Early Detection of Viruses PDF

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

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.:+966-13-720-3426(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 Chemosensors2017,5,7;doi:10.3390/chemosensors5010007 www.mdpi.com/journal/chemosensors Chemosensors2017,5,7 2of25 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 Chemosensors2017,5,7 3of25 Chemosensors 2017, 5, 7 3 of 24 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. Chemosensors2017,5,7 5of25 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.

Description:
Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen,. The Netherlands; [email protected]. 5 Keywords: antibodies; aptamer; epitope imprinting; molecularly imprinted polymers; quartz crystal microbalance; virus sensor. 1. Introduction.
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