monodispersesodu.tw

REVIEWMicrofluidicsinmacro-biomoleculesanalysis:macroinsideinananoworldIulianaOita&HadewychHalewyck&BertThys&BartRombaut&YvanVanderHeyden&DebbyMangelingsReceived:27February2010/Revised:13May2010/Accepted:18May2010/Publishedonline:13June2010#Springer-Verlag2010AbstractUseofmicrofluidicdevicesinthelifesciencesandmedicinehascreatedthepossibilityofperforminginvestigationsatthemolecularlevel.
Moreover,micro-fluidicdevicesarealsopartofthetechnologicalframeworkthathasenabledanewtypeofscientificinformationtoberevealed,i.
e.
thatbasedonintensivescreeningofcompletesetsofgeneandproteinsequences.
Adeeperbioanalyticalperspectivemayprovidequantitativeandqualitativetools,enablingstudyofvariousdiseasesand,eventually,mayoffersupportforthedevelopmentofaccurateandreliablemethodsforclinicalassessment.
Thiswouldopenthewaytomolecule-baseddiagnostics,i.
e.
establishaccuratediagnosisanddiseaseprognosisbasedonidentificationand/orquantificationofbiomacromolecules,forexampleproteinsornucleicacids.
Finally,thedevelopmentofdisposableandportabledevicesformolecule-baseddiag-nosiswouldprovidetheperfecttranslationofthesciencebehindlife-scienceresearchintopracticalapplicationsdedicatedtopatientsandhealthpractitioners.
Thisreviewprovidesananalyticalperspectiveoftheimpactofmicro-fluidicsonthedetectionandcharacterizationofbio-macromoleculesinvolvedinpathologicalprocesses.
Themainfeaturesofmolecule-baseddiagnosticsandthespecificrequirementsforthediagnosticdevicesaredis-cussed.
Further,thetechniquescurrentlyusedfortestingbio-macromoleculesforpotentialdiagnosticpurposesareidentified,emphasizingthenewestdevelopments.
Subse-quently,thechallengesofthistypeofapplicationandthestatusofcommerciallyavailabledevicesarehighlighted,andfuturetrendsarenoted.
KeywordsMicrofluidics.
Diagnostics.
Proteins.
Nucleicacids.
PCR.
VirusIntroductionThetechnologicaladvancesofthelastcenturyenabledscientificclarificationofseveralmajormysteriesoflife,i.
e.
allorganismsaremadeofcells,whicharechemicalsystemscomposedmainlyofcomplexcarbonchainsandsharingthesameinformationsystem[1].
Adeeperinsightintothecells,onthenanometrescale,highlightsthemaincompo-nents:nucleicacids—thesourceofgeneticinformation—andproteins—themainexecutivemolecules.
Elucidationofthewayinwhichsub-cellularcomponentsworktogethertoformfunctionalcellsandorganismswillenablecompleteunderstandingofcellularprocessesandthewaythecellrespondstotheenvironment[1].
Eventually,thedecipher-ingofcellularprocesseswillhelpustounderstanddiseasemechanisms,andtofindtheproperwaytodiagnoseandcurethem.
Fastassessmentofbio-macromoleculessuchasproteins,peptides,over/underexpressionofgenemarkers,andgenemutationscanbeextremelyvaluableinformationfordiagnosisandprognosisofseveralpathologies[2,3].
Forexample,thepresenceofvariouscancersanddiseasesisI.
Oita:Y.
VanderHeyden:D.
Mangelings(*)DepartmentofAnalyticalChemistryandPharmaceuticalTechnology,CenterforPharmaceuticalResearch(CePhaR),VrijeUniversiteitBrussel-VUB,Laarbeeklaan103,Brussels1090,Belgiume-mail:debby.
mangelings@vub.
ac.
beH.
Halewyck:B.
Thys:B.
RombautDepartmentofPharmaceuticalBiotechnology&MolecularBiology,CenterforPharmaceuticalResearch(CePhaR),VrijeUniversiteitBrussel-VUB,Laarbeeklaan103,Brussels1090,BelgiumAnalBioanalChem(2010)398:239–264DOI10.
1007/s00216-010-3857-7sometimeslinkedtoabnormalconcentrationsofspecificproteins[4].
Also,investigationofnucleicacidsequences,especiallyforidentificationofageneticmutation,isapracticalapproachusedtoidentifyorconfirmdifferentpathologies[4,5].
Ininfectiousdiseases,themaincauseofmortalityindevelopingcountriesandoneofthemajorcausesindevelopedcountries[6],thepathogenicsourcebecomesevenmoretraceableandisperfectcandidateformolecule-baseddiagnostics.
Inallthesesituations,complexsamples,availableinsmallamounts,havetobeprocessedrapidly,preferablynearthepatient'sbedside,andaclinicallysignificantresponsehastobeobtained.
Inthiscase,themedicalresponsecanbeadjustedmorerapidlytothepatient'sreaction,enablingapersonalizedmedicalapproach.
Foryears,consistenteffortshavebeenmadetodevelopanalyticalapplicationsenablingfast,accurate,precise,andreproducibleinsightsintotheworldofmacro-biomolecules.
Pandora'sboxinthebioscienceswasopenedaroundthe1990s,whenadvancesinincrementaltechnology,theminiaturizationboom,andprogressinengineeringemergedinmicrofluidicdevices[7].
Microfluidicscanberegardedasaframework,anenablingtechnology[8]relatedtofluidflowinginchannelsofmicroornano-size,offeringthepossibilityofdevelopingproductswithbetterperformanceandadditionalfeatures(Fig.
1).
Thedawnofexpectationsstartedwiththeconceptofthemicro-totalanalyticalsystem(μTAS)[9].
μTASwassupposedtoperformautomaticsampling,sampletransport,anynecessarychemicalreac-tions,anddetectiononasingle,miniaturized,platform[9].
Theconceptwasdeclaredthestate-of-artstrategy,becauseofitsamazingadvantages,i.
e.
fasterseparations,shortertransporttime,lowersampleandreagentconsumptionandthepossibilityofmulti-componenttestingunderthesameconditions[9].
SincetheconceptofμTASwasannounced,therewasanexplosiveinterestonthetopic,asshownbythehugeamountofpaperspublishedsofar(7745accordinglytoScopusdatabase,December2009),ofwhichalmostafifthisrelatedtothelifesciencesandmedicalapplications(Fig.
2).
Over70%ofallpapersontheuseofmicrofluidicdevicesinthelifesciencesandmedicinearetargetedonthecharacterizationofbiologicalsystemsatthemolecularlevel(Fig.
2).
Theuseofmicrofluidicswasperceivedasapossiblenewdimensioninbioanalysis.
Microfluidicdevice-basedapplicationsshiftedtheperspectivefromthegeneraltothecellularandsub-cellularlevelandenabledthemeasurementofmolecularvariation,thedynamicsofseconds-longprocesses,andbio-macromolecularmotion[10].
Valuablebio-moleculeswereisolated,characterized,quantified,andusedtoexploreinteractionswithothermolecules.
Oncetheeuphoriageneratedbythispromisingnewsolutionforoldunsolvedproblemsbecametransformedintoachaseforapplicationdevelopment,alonglistofpracticalproblemswasrevealed.
Becauseacompletelynewplatformwastobedeveloped,choiceofthematerialandthedesignofthedevicewerethefirstproblemstooccur.
Further,applicationsdevelopmentrevealedthatthetech-nologyrequiredforproductionoffullyintegrateddeviceswasnotyetmastered.
Also,solutionswereneededfortheextremelysensitiveandminiaturizeddetectionsystems,whilethecomplexityofsamplesemphasizedtheneedforsampleclean-uporenrichmentofanalytesofinterest.
Sometechnicalproblemshavealreadybeenanswered,oratleastbetterunderstood,whileseveralarestillopenchallengesforthetechnologyavailable.
Thisreviewtriestogiveanoverview,fromananalyticalperspective,oftheimpactofmicrofluidicsonthedetectionandcharacterizationofthebio-macromoleculesinvolvedinpathologicalprocesses,focussingespeciallyonthosewithahighpotentialtobedevelopedasdiagnosticdevicesforinfectiousdiseasesandcancer.
Giventheimportanceofthefield,inwhichvolumeofliteraturedoubleseveryfouryears,manuscriptsmainlypublishedduringthelastthreeyearsarediscussed.
Wewillfirstreviewthemainrequirementsfordevelop-ingdiagnosticallyrelevantapplications.
Further,on-chipsampletreatment,on-chipPCR,separation,andimmunoaf-finitytechniques,anddetectionschemeswillbeidentified.
Subsequently,thechallengesofthistypeofapplicationandthestatusofcommerciallyavailabledeviceswillbediscussed.
Wewillconcludewithfutureopportunitiesoftheresearch.
Throughoutthereview,examplesofthenewestresearch,promisingapproaches,andopportunitieswillbeemphasized.
Manuscriptsreportingtheuseofmicrofluidicsformicro-vascularnetworkchips,theisolationofbiomacromoleculesonchips,andapplicationsrelatedtocellsotherthanpathogensarebeyondthescopeofthisreview.
High-densitymicroarrays,althoughasourceofclinicallyvaluableinformation,arealsonotincludedbecauseofdisadvan-tagessuchascomplexity,highcost,lackofrobustness,anddifficultyofinterpretation.
MainrequirementsfordevelopingdiagnosticrelevantapplicationsAnimpressiveamountofresearchhasfocusedondevelopingapplicationsthatcanhelpmedicalpractitionersachievefasterandmoreaccuratediagnosis,reliablyassessdiseaseprogno-sis,ormonitortreatment,onasolidquantitativebasis[11].
Thefinalobjectivewouldbethedevelopmentofportableautomaticdevicesabletoprovidefastlaboratorygraderesultswithouttheneedforspecialreagents.
Thedevicesshouldbeabletoprovideresultsnotonlyforpatientbedside240I.
Oitaetal.
usebutalsoinmajorpublichealththreatssuchaspandemicsriskandsuspicionsofbiowarfareagentsuse[12].
TheapplicationofμTASconceptwouldfitperfectlytosuchanapplication—asampleofbodilyfluid,forexampleblood,urine,ornasalsecretion,iscollectedandisintroducedintotheworkstationwhereminimaltreatmentisapplied(e.
g.
abloodsampleisdilutedwithEDTAtopreventclotting)andseparationisperformedifnecessary(e.
g.
plasmaisseparatedfromwholebloodorcellsarelysed).
Furthermore,multipleanalytesarethencapturedatthereceptorsite.
Afterwashingandintroductionofsecondaryreagents,analytelevelsarereadusingtheworkstationreadout[13].
Duringthedevelopmentofmolecularminiaturizeddiagnostics,bioanalysisshouldprovidetwosolutions:1.
extractionoftheanalyteofinterestfromthesample,and2.
conversionofanalytepropertiesintoareadablesignal.
Findingthesesolutionswouldbeequivalenttotranslationofthescienceintopracticalapplicationsdedicatedtomassconsumersandhealthpractitioners.
Thetechnologyavailabletodayhasenabledthedevelop-mentofanumberofbiosensorsforcancerbiomarkersanalysis,whereasmostofthemulti-arraysensorchipsfortestingatornearthepatientbedsidearestillindevelopmentorresearchstage[3].
Unfortunately,theuseofproteinsfordiagnostictestsislimitedbycurrentdetectionmethods,whichareonlysensitiveenoughwhenthediseaseissignificantlyadvancedandproteinconcentrationshavealreadyreachedcriticalthresholds[4].
Thedevelopmentofmicrofluidicdevicesenablingdifferentialproteomicprofilinganddetectionofapanelofseveralproteinshasthepotentialtorevolutionizethebiomedicalresearchandtoincreasethesensitivityoftests[14].
Viraldetectionisanotherfieldwheretheuseofmicrofluidicdeviceshasthepotentialtoimprovedetectionlimits,simplifyprocedures,andreducethetimeneededforconfirmationofaviralinfection[15].
Currenttechniquesusedtostudyand/oridentifymacro-biomoleculesfordiagnosticrelevantapplicationsSeveralmacro-biomoleculesareroutinelytestedfordiag-nosticpurposesusinganumberofclassicalmolecularbiologytools,forexampleslabgelelectrophoresisorenzyme-linkedimmunosorbentassay(ELISA).
Usually,themethodsinvolvemultiplemanualsteps,withseveralcriticalstepsandlongincubationtimes,anduselargevolumesofbuffersandexpensivereagentsavailableinminuteamounts(antibodies).
Foramorepreciseassess-ment,blottingcanbeperformedbytransferringthebandsfromtheslabgelelectropherogramtoanitrocelluloseorabcdefFig.
1Severalcommerciallyavailablemicrofluidics-baseddevicesusedforbio-analyticalpurposesa.
DynaflowSystemforion-channeldrugdiscovery(Cellectricon,Mlndal,Sweden);b.
LC–MSmicrofluidics-basedchip(Agilent,SantaClara,CA,USA);c.
Nano-titerplates(microfluidicChipShop,Jena,Germany);d.
15-cyclescontinuous-flowpolymerasechainreaction(PCR)chip(microfluidicChipShop);e.
96-sampleSentrixArrayMatrix(top)andthemulti-sampleSentixBeadChips(Illumina,SanDiego,CA,USA);f.
Disposablechipforgenerationofpicoliter-volumedroplets.
EachdropletisfurtherusedasaPCRreactor(RainDanceTechnologies,Lexington,MA,USA)Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld241Nylonmembraneusingahighelectricfield.
Themembranecontainingthetransferredbandsisthenincubatedwithfunctionalizedantibodiesforspecificproteins.
Detectionisgenerallyachievedusingfunctionalizedantibodies,eitherradioactivelyorfluorescentlylabelledorcovalentlyboundtoanenzymetheactivityofwhichcanbeeasilymeasured.
Nucleicacidshaveahugeadvantageoverproteins,becauseuseofpolymerasechainreaction(PCR)enablessignalincreasesthroughtarget-basedamplificationandreliabledetectionofjustafewcopiesofnucleotidesequences[2,16].
Forproteins,enzyme-linkedimmuno-sorbentassay(ELISA)isthemostusedtechnique.
Limitsofdetection(LOD)forELISAareinthepicomolarrange,butlargevolumesareneededandthetechniqueisrathercomplicatedinvolvingtargetcapturingbyanantibodyandsandwichingwithasecondantibody,whichisalsoresponsibleforsignalgeneration[2].
Withinthiscontext,applicationsdevelopedusingthemicrofluidicdeviceswouldbringportability,highersensi-tivity,costreduction,shorteranalysistime,andlesslaboratoryspaceconsumption,overcomingmostoftheinconveniencesoftheclassicaltoolsofmolecularbiology[17].
Inmicrofluidics,thereductioninsizeresultsinahighsurfacearea-to-volumeratioandsurfaceeffectsbecomeextremelyprominent.
ThephysicsunderlyingmicrofluidicdeviceshasbeenexcellentlydescribedbySquiresandQuake[18].
Thepaperemphasizesthevarietyofphenom-enaandthemannerinwhichtheyhadbeenexploitedtodevelopmicrofluidicdevices.
Theinitialpromiseofmicrofluidics—thedevelopmentofaμTASthatwouldintegrateallanalyticaloperationsonasingleplatform[9]—isonlypartiallyfulfilledatthemoment.
Thenumberofmanuscriptsreportingthedevel-opmentofacompleteapplication,integratingsampletreatmentisequaltoorbelowthenumberofmanuscriptsreportingpartialapplications,forexampletheanalysisofanalreadyprocessedsampleorthedevelopmentofamethodofsamplepreparation.
Inthiscontexttheintegrationofthepre-separationsampleprocessingiscurrentlytheweakestlink[16,19].
ExpectationshavebecomemorematureandrealisticandthesolutionhasbeenfoundbychangingtheperceptionofμTAS:theomnipotentchipisnowseenasamicrofluidicsplatform,composedofasetofcombinablebuildingblocks,whereeachblockperformsasingleoperation[17].
AlmostalldevicesreportedintheliteratureChemicalsciencesEnvironmentalsciencesOthersHealthsciencesDevicemanufacturinandtheoreticalaspectsBiochemistryGeneticsandMolecularBiologyMedicineNeuroscienceMultidisciplinaryImmunologyandMicrobiologyPharmacologyToxicologyandPharmaceuticsHealthProfessions0501001502002503003504001992199419961998200020022004200620082010YearNumberofpapersabFig.
2Characterizationofthescientificliteratureavailableonmicrofluidicdevices.
a.
Yearlydynamicsofthenumberofpublishedpapersb.
Domainsofinterestformicrofluidicdevicesresearch242I.
Oitaetal.
canbefurtherintegratedinsuchamicrofluidicsplatform.
Thisapproachenablestheimplementationofbio-analyticalassayinabetter,foreseeableandlessriskymanner[17].
Thedevelopmentofsingle-unitblocksavoidsconflictsbetweenthetechnicalrequirementsforvariousoperations;forexample,insampletreatmenthighthroughputisthemaindemandwhereasforanalyticalseparationshighresolutionismostimportant[16].
Thetransportofsamplesandreagentsinmicrofluidicchannelsisperformedbyroutineapproachesforseparationscience,forexamplehighpressure,vacuum,orelectricalfield.
Thelastispreferredformostapplications,eitheruniform,asinelectrophoreticseparations,ornon-uniform,asindielectrophoresisinwhichaforceisexertedonadielectricparticlesubjectedtoanon-uniformelectricfield.
Dielectrophoresisismostlyusedforanalytessuchasdielectricparticles,withouttheneedforthemtobecharged.
Theselectivityofdielectrophoresiscanbeeasilytunedbyalteringthefieldfrequency.
Otherresearchersaretryingtousethemagneticfieldforcontrolledtransportofparamag-neticparticles[20].
Analternativelab-on-a-chiptechnologyisdroplettechnology,alsocalleddigitalmicrofluidics.
Inthiscase,theflowisnotcontinuousbutasdiscretedroplets.
Thedevicesoperatesimilarlytobench-topequipment,onlywithasignificantvolumereductiontothenLrangeandmoreautomation.
Generationandmanipulationofdropletsareperformedinaccordancewiththreemainprinciples—electrowetting,dielectrophoresis,andimmiscible-fluidflows.
Detailedcharacterizationofdifferentmicrofluidicplatformsandrecommendationsforselectionofthemostappropriateapproachbasedonapplicationcanbefoundelsewhere[17].
Foranalyticalpurposes,twomainapproachesareusedforseparationoftheanalyteofinterest,fromthesamples—affinity-basedseparationandcapture,basedforexampleantigen–antibodyreactions,andphysicochemical-basedseparations,forexamplecapillaryelectrophoresis(CE)orliquidchromatography(LC).
SamplingandsampletreatmentSamplingandevenminimalsamplepreparation,forexampleseparatingplasmafromwholeblood,areper-formedoff-microfluidicdevice.
Themaininterestsinusingmicrofluidicdevicesforsampletreatmentarerelatedtotheisolationand/orconcentrationofanalytesortheremovalofinterferingcomponents.
Thereforemethodsinvolvingcon-tinuousphaseseparationsshouldbefavoured,becausetheyhavenoneedforcarefulsampleloading[16].
However,theprocessingoflargersamplevolumes(sometimesevenhundredsofmicroliters)isstillaproblem.
Antigen–antibodyandstreptavidin–biotinaffinityarethefavouriteapproachesforisolatingtargetanalytes.
Theisolationsareperformedwithinamagneticfieldusingmagneticbeadsorinelectricalfieldsusingpolystyrenebeads(Table1).
Sometimes,thesuccessfulisolationdescribedintheliteratureismoreaproofofconceptshownforstandardsolutionsandnotyettestedonrealclinicalsamples(Table1).
Atrialforalternativewaystoavoidtheuseofantibodies[31]hasbeenreported,butinsomecasestheuseofimmobilizedantibodiescanresultinextremelysensitivedevices[32–35].
Averyinterestingexample,withanintendeduseforsinglenucleotidepolymorphismdiagnosis,ispresentedinFig.
3.
Dielectrophoresis[36],solid-phaseextraction(SPE)[21,37–41]orsize-exclusionbasedseparationswithnanopores[42]andmicrofabricatedplasticmembranes[43]areotherpossiblemethodsreportedfortargetisolation.
Isolationandconcentrationisparticularlyimportantforproteins,usuallypresentincomplexmixtures(forinstanceinserumthereareover10000differenttypes)andinhighdynamicranges(theproteinofinterestispresentatpicomolarlevels,whilelessinterestingproteinscanbepresentat30–50gL1concentrations).
Fortheparticularcaseofnucleicacidisolation,commonlyusedprocedureswithcommerciallyavailablekitsarecumbersome,andincludeasubstantialnumberofmanualsteps.
Forexample,somepurificationkitsrequireapproximatelytenpipettingsteps,threemechanicalmixingsteps,sixcentrifugationsteps,andtentube-transfersteps[44].
Thecommonautomationapproachesforthesetestsarebenchtop-dependent,andthereforenotsuitedtoemergingbedsideapplicationsthatrequirecompact,automated,androbustoperationalsettings[19].
Still,severalsuccessfulattemptstoautomatenucleicacidsextractionusingmicrofluidicdeviceshavebeenreported.
AplasticchiphasbeenusedforviralRNAextractionfromalysateofmammaliancellsinfectedwithinfluenzaA(H1N1)virus[40].
TheRNAisolationwasachievedbyμSPE,byreversiblebindingofthenucleicacidstosilicaparticlestrappedinaporouspolymermonolith[40].
Theprocedureisextremelysimpleandonlyrequires10min.
Asolid-phasemethodtoisolatePCR-amplifiablegenomicDNAfromchemicallylysedbloodcellshasalsobeenused[37].
Inthiscase,aporoussiliconmatrixintegratedinthebiochipwasusedtoperformtheDNAextractionin20min[37].
Bloodwasinitiallyregardedasthemaintargetforthedevelopmentofdiagnostictests.
Moreandmoreapplicationsfocusondevelopmentoftestsforlessinvasiveandcomplexbodyfluids,forexampleurine,saliva,ornasalsecretions.
However,wholebloodandplasmasamplesremainaconstanttargetforsample-preparationdevicesusingDNAextraction[37,45,46],pathogencapturing[47],proteindepletion[38,48],orplasmaseparationfromwholeblood[49].
Microfluidicsmayalsooffersolutionsformanipulationofsamplesrankedashighlybio-hazardous.
AninterestingMicrofluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld243Table1Selectedon-chipsampletreatmentusingconjugatedbeadsforaffinity-basedisolationofthetargetanalyteNo.
TargetanalyteTestedsampleCapturingtechniqueMicrofluidicdeviceProcessedsamplevolumeTotaltime(isolation+separation)Ref.
1DenguevirusesClinicalserumAntibody-conjugatedsuperparamagneticbeadMicrofluidicsystemwiththreeintegratedfunctionaldevicesforpumping,mixing,andseparation25μL10min[21]2WestNilevirusSerafrominfectedchickenMicroarrayofprobemoleculesimmobilizedonasemipermeablemembranefollowedbyextractionfromthemembraneusingfunctionalizedmagneticbeadsElectrophoreticflowcell20μLmin12–3min[22]3Denguevirusserotype2andenterovirus(EV)71ClinicalserumAntibody-conjugatedsuperparamagneticbeadPDMSchipintegratingthesamplepurification/enrichmentandRT-PCRdiagnosis20–100μLmin160min[23]4CholeratoxinsubunitB(CTB)SolutionAntibody-conjugatedsuperparamagneticbeadPDMSchipintegratingthesamplepurification/enrichmentanddetection100μL60min[24]5PeptidesdisplayedonE.
colicellsAlibraryofE.
colicellsStreptavidin-functionalizedpolystyrenemicrospheresContinuous-flowmicrofluidicsortingdevice>108cellsh1[25]6gDNAfromleukocytesHumanwholebloodsampleAntibody-conjugatedsuperparamagneticbeadPDMSchipintegratingtheleukocytespurification,DNAextractionandfastanalysisofgeneticgene200μL20min[26]7Alpha-fetoprotein(AFP)Spikedserum(0.
1μgmL1)Streptavidin-functionalizedpolystyrenemicrospheresPDMS-glasshybridimmunoassaymicrochip100μLmin155min[27]8AFP,CEA,andPSAantigenSolution,10ngmL1Streptavidin-functionalizedpolystyrenemicrospheresMultiplexelectro-immunosensingsystemPDMS-glasshybrid90μL55min[28]9Alpha-fetoprotein(AFP)Solution1–1,000ngmL1Antibody-conjugatedsuperparamagneticbeadPMMAchips5μL20min[29]10Antibodiesassociatedwithaninfectionbythedenguevirus(immunoglobulinG(IgG)andimmunoglobulinM(IgM)SerumVirus-conjugatedsuperparamagneticbeadIntegratedchipiscomposedofthreepolydimethylsiloxanelayersandoneglasslayer100μL30min[30]11Pathogen-specificDNAWholebloodspikedwithHepatitisBvirus(HBV)andE.
coliAntibody-conjugatedsuperparamagneticbeadCentrifugalmicrofluidicsonapolymerbasedCDplatform100μL12min[46]244I.
Oitaetal.
applicationconsistsinthemanipulationanddisruptionofbacterialcellsandvirusesinaclosedsystemandwithminimuminterventionfromtheoperator[37,39,47,50–52].
Dielectrophoresis(DEP)hasbeenusedformanipu-lationanddisruptionofcellsofBordellapertussiss,abacterialrespiratorypathogen[36],andforcaptureandlysisofVacciniavirusparticles[51].
Thedisintegrationofthevirusouterlayerwasprovedbyrevealingthedamagedandexposedtubulesnetworksbyscanningelectronmicroscopy(SEM)[51].
Rapidisolationandcountingofhumanimmunodeficien-cyvirus(HIV)fromonly10μLunprocessedwholebloodhasbeenperformedonamicrofluidicplatform[47].
Thechipsurfacewascoatedwithanti-gp120antibodiestocaptureHIVbybindingtothegp120-glycoproteinonthesurfaceofHIVenvelope.
Theresearchgroupthatdevel-opedtheon-chipisolationofHIVintendstodeveloparapid(<10min),handheld,low-cost,anddisposablemicro-fluidicHIVmonitoringplatformforrapid,bedside,clinicalHIVmonitoring.
AnotherresearchgroupreportedcaptureofHIVonamicrochipbasedonCD4+T-lymphocyteaffinity[53].
PCRonachipThepolymerasechainreaction(PCR)ispossiblythemostusedtoolinmolecularbiology[1].
ItamplifiesspecificDNAsequences(target)inthepresenceofapairofprimersthathybridizewiththeflankingsequencesofthetarget,thefourdeoxyribonucleosidetriphosphates(dNTPs),andaheat-stableDNApolymerase[54].
Theamplificationisperformedincyclesofthreesteps:strandseparation,hybridizationofprimersandDNAsynthesis.
Eachstepisisothermalandrequiresaspecifictemperature,i.
e.
95°C,54°C,and72°C,respectively.
Theamplificationiscarriedoutrepetitivelyjustbychangingthetemperatureofthereactionmixture[54].
ForRNAamplification,eitherareverse-transcriptase(RT)PCRapproachoranucleicacidsequence-basedamplification(NASBA)procedurecanbeused.
NASBAisatranscription-basedRNAamplificationKeyboardinterfaceDNAextraction/PCRchamberSpiralmicrocoils/micro-heatersCircularmicrocoilsarraySampleloading/mixingchamberMembrane-typemicromixerLeukocytepurificationchamberTwo-waycircularmicropumpWastechamberPneumaticmicrovalveMicrotemperaturesensorDigitaldisplayASICdigitalcontrollerMicrofluidicchipabFig.
3aAphotographofas-sembledmagnetic-bead-basedmicrofluidicsystemperformingon-chipsinglenucleotidepoly-morphismgenotypingassociat-edwithgeneticdiseases.
gDNAisextractedfromleukocytesintheDNAextraction/PCRchamber.
bAhand-heldsystemincludingamicrofluidicchip,anASICcontroller,acontrolcir-cuitboard,andEMVshasbeendeveloped.
(Reproduced,withpermission,fromRef.
[26])Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld245system,whichismoresensitive,"userfriendly",andfasterthanPCR[55].
Asprovedalsobyclinicalstandards,PCRisaninvaluabletoolfornucleicacidsanalysis,especiallyinviraldiseasesandcancerdiagnosis[56].
Analysisofheterogeneousnucleicacidmixturesisasourceofimportantclinicalinformation,especiallyinassessmentofantiretroviralresistantmutationsorearlycancerdetection[57].
Individually,genotypicinvestigationofminorityviralpopulationsmightbeasourceofvaluableinformationsuchasthetendencytodevelopdrugresistance.
IntegrationofPCRonmicrofluidicplatformsreducescostbyreducingreagentvolumestotensofnanolitres.
Italsoreducescomplexitybyintegrationofseveralassaystepsintoasingledevice,andreducesthetimesrequiredforthermocycling[58].
InthecurrentmodeofoperationofPCR,sampleprocessing,amplification,andanalysisofthePCRmixturesarestand-aloneoperations.
TheminiaturizationofPCRhasthepotentialtoidentifythemissinglinkthatintegratessampleprocessingwithdownstreamPCRandanalysisofPCRmixtures[56].
Inthismode,samplemanipulation,withpotentialeffectsonthemeasurement,canbetightlyregulatedandaccountedfor.
Moreover,avoidingconventionalextractionandmanip-ulationlimitssampleloss.
Theintegrationoftheseoperationswouldbebeneficialforcharacterizationofcancersatthemolecularlevel,enablingmeaningfulquantitativeassessmentofcancerpathogenesisanddevel-opmentofmoreeffectivetherapy[56].
Miniaturizationalsobringsotherpotentialbenefits,forexamplemulti-paralleltreatmentofadefinednumbersofcells,singlecell-basedanalysis,orevensinglegeneanalysis[56].
Thefeasibilityofrapid,single-moleculeamplificationofnucleicacidswasestablishedusingamicrofluidicsystemdevelopedtoenablerapidPCRanalysisofindividualDNAmoleculeswithprecisetemperaturecontrol[57].
PCRwasperformedonheterogeneoussamplescontainingsyntheticCYP2D6.
6wild-typeandmutanttemplates,onaquartzchipdesignedtoenableadequatemixingbyBrowniandiffusionaftereachstageofreagentdispensing.
Sampleswereloadedfromamicrotitreplateontothemicrochipthroughanintegratedcapillary,whichminimizesthecontaminationrisk.
ThechipdesignallowedeightparallelPCRreactions.
Thermocyclingwasperformedbyheatingwithnineembeddedresistiveheatersbuiltfromplatinumtracersandcoolingbyrecirculatingwaterunderneaththechip.
Thesystemhadanimpressivedetectionset-upcomprisingtwodifferentlasersforexcitation,i.
e.
a488nmopticallypumpedsolid-statelaserandthe633-nmlineofanHeNelaser,aseriesofdichroicmirrorsandbandpassfilters,andthreecharged-coupleddevice(CCD)cameras,eachcollectinglightofadifferentwavelength,i.
e.
515,550,and685nm.
ThermocouplesandopticalDNAmeltanalysiswereusedtodemonstratethechip'sabilitytorapidlythermocycle.
Whenthedesiredtempera-turewas68.
5°C,sevenoutoftheeightchannelsmanagedaccuratecontrolwithina1°range.
Theefficiencyofamplificationwasprovedbymeasuringthefluorescenceemissionresultingfromamplificationofa1:1mixtureofwildandmutanttemplatesofCYP2D6.
6usingtwoTaqmanprobes.
Theprocedureisfast,reproducibleandabletoamplifyaheterogeneoussamplecontainingtwotemplateswithoutmixingtheamplificationproductsbetweentem-plates.
Useoftheproposedmicrofluidicsetupovercomesthehighreagentcostandcumbersomereactionassemblyrequirementsofplate-basedlimitingdilutionPCRmethodsandthelowthroughputandmanualhandlingofcurrentmicrochipmethods.
Almost96templateswereanalyzedin25minbyuseof1μLPCRreactionvolume.
ComparedwithdigitalPCRthisoperationwasperformedalmostfivetimesfasterandusingaPCRreactionvolumeafactorof1400lower[57].
Smallchannel-to-channeldifferencesinamplificationwerenoticed,butthesearepotentiallyreduciblebyrefinementoftheheatingprocedure.
Whentransfer-messenger(tm)RNApurification,nucleicacidsequence-basedamplification(NASBA),andreal-timedetectionwereintegratedonamicrofluidicdeviceforthefirsttime(Fig.
4),thechipwasabletoidentifycrudeE.
colibacteriallysatesinlessthan30min[59].
Celllysiswasperformedoff-chip,usingacommerciallyavailabledevice.
tmRNA,from100lysedE.
colicells,waspurifiedbySPEusingsilicabeadsimmobilizedonthechipsurface.
Devicecloggingbydebris,orbubbleformationbecauseofpassageofairwereavoidedbyimmobilizationofsilicabeadsinathinlayerthatleavesenoughfreespacewithinthechannel.
RNAwaselutedin5-μLfractionsbyflowofdeionizedwaterthroughthesilicabedchamber.
Theamplificationusedcustom-designedhigh-selectivityprimersandreal-timedetectionwasperformedat530nmusingmolecularbeaconprobes(oligonucleotideswithanFAMfluorophoreatthe5′endandaBHQ1quencheratthe3′end)[59].
AninfraredtemperaturecontrolsystemhasbeendescribedforcompletelycontactlesstemperaturecontrolPCRinmicro-fluidicchipsinafluidicchanneltoosmalltoenableconventionaltemperaturecontrolusingthermocouple-basedsensing[60].
ThesystemcomprisedanIRpyrometersensingthesurfacetemperatureaboveaPCRchamber.
ThedesignofthesystemensuredrapidequilibrationbetweenthePCRsolutionandthechambersurface[60].
Fornon-contacttemperaturecontrol,thesurfacetemperaturerelativetothatofthePCRsolutiontemperaturewascalibratedusingtheboilingpointofwaterandanazeotropewithinthechip.
SuccessfulPCRofafragmentofaBacillusanthracisgenewasperformedbyuseofthedescribedsystem[60].
Forthefirsttime,reversetranscriptionPCRhasbeenperformedonsingle-copyviralRNAinmonodisperse246I.
Oitaetal.
isolatedpico-dropletreactorsusingafused-silicachipwithhydrophobiccoating.
Thedevicewascoupledwithanoffchipvalvingsystemforgenerationofmono-dispersedropletswith70nLvolume[61].
RNAwasisolatedinmonodispersepicoliterdropletsemulsifiedinoil.
Ineachdroplet,real-timereversetranscriptionPCRwithfluores-cencedetectionofamplificationwasperformed.
Afterapproximately23amplificationcycles,RNAfrom0.
05–47plaqueformingunits(pfu)/dropletwasdetectedbyreal-timefluorescence[61].
TheuseofmicrodroplettechnologylimitstheinteractionbetweenthemicrofluidicsurfaceandPCRsample/reagents;thisisresponsibleforPCRinhibitionandcarry-overcontamination.
Severalothermanuscripts(Table2)havereportedfullyintegrateddeviceswithimpressivelowestamplifiedcon-centration,butarealμTASisstillinitsresearchphase.
SeparationtechniquesOnchipcapillaryelectrophoresis(CE)isthemostusedseparationtechnique.
Detectionisfrequentlyachievedbyfluorescence,althoughelectrochemicalmethodsinvolvingamperometricdetection[73]andcontactlessconductivitydetection[74]havealsobeenreported.
Separationsareper-formedonhome-made,hybridglass-polydimethylsiloxane(PDMS)[65,73]chipsorpoly(methylmethacrylate)(PMMA)chips[74–77].
Afewapplicationsusecommer-ciallyavailabledevices[75,78,79].
CEonchiphasbeenusedtoquantifyPCRproducts[65,80],oxidativestressbiomarkersinurine[73],hepaticcancerbiomarkersinspikedserum[77,81,82],thrombin—amarkerforvarioushaemostasis-relateddiseasesandcon-ditions—indilutedplasma[76],viruses,forexamplehumanrhinovirus(HRV)orswineinfluenzavirus[75,78,83],inflammatorybiomarkers[84],andK-ras,theoncogeneformutationscloselyassociatedwithcolorectalcancer[79](Table3).
Theseparationmodesinvolvedfree-solutionelectrophoresis[73,75,77–79,84],capillarygelelectropho-resis(CGE)[65,73–75,80,82,86,87],oraffinitycapillaryelectrophoresis[42,77,78,81,88].
CGEhasbeenperformedtoresolveandinvestigatetheabundanceofproteinsincomplexsamplesinordertoidentifyvirusesandbacteriophages[89],todetectthepresenceoffood-bornepathogenicbacteriaindecayedfoodsamples[80],ortoanalyseofRNA–RNAinteractions[90],asalternativestocellcultureorPCR.
Severalmanuscriptsreporttheuseofon-chiptransientisotachophoresis(ITP)toachievepreconcentrationofanalytes[82,85,88].
UsingITP,humanserumalbumin(HSA)anditsimmunocomplexwithamonoclonalantibodywerepreconcentrated800-fold[85]and2000-fold[88]onlineonstandardcross-channelPMMAmicro-chips.
Anotherinterestingapplicationusesanintegratednanoporousmembranetoperformsimultaneousconcen-trationanddetectionoftheinactivatedswineinfluenzavirus.
Detectionwasachievedbycouplingwithafluorescentlabelledantibody.
Thefluorescentantibodycomplexwaselectrophoreticallyseparatedfromtheun-boundantibodyin6minbyuseoflessthan50μLclinicalsample[83].
IntactproteinseparationsfromE.
colicelllysatehavebeenperformedonatwo-dimensionalmicrofluidicsystemwithtenchannelscombiningisoelectricfocusing(IEF)andsodiumdodecylsulfate(SDS)polyacrylamidegelelectro-phoresis(PAGE)[86].
Proteinprofilingwasusedforbacterialidentificationandcharacterizationusingamicro-chipseparationplatform[87].
Themethodispotentiallyuniversallyapplicable,especiallyforbiothreatagents,andovercomesthedisadvantagesofPCR,i.
e.
highcostandtheneedforspecialworkingprocedurestoavoidcontamina-tion.
Themethodincludesfoursteps—thebacteriaorsporesareharvestedandlysed,andtheconstituentproteinsaresolubilized,labelledwithafluorescenttag,andanalyzedusingchipgelelectrophoresis(CGE).
Theproteinfingerprintsfromthemodelorganismswereusedforbacterialidentificationorcharacterization.
ChamberforfutureapplicationRPCRNAPurificationSilicaBeadChamberInputNASBAChamber(NC)NASBAPortWasteOutputabFig.
4IntegratedmicrofluidicdeviceforRNApurificationandreal-timeNASBA.
aPhoto-graphofthedevice.
Eachchipcanperformtwoseparatereac-tionswiththesamereagents,butdifferentsamples,toincorporatecontrols.
bSingle-devicearchi-tectureshowingthedistinctfunctionalmicrofluidicmodules:RNApurificationchamber(RPC)andreal-timeNASBAchamber.
(Reproduced,withpermission,fromRef.
[59])Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld247Table2Selectedon-chipPCRapplicationsObjectiveChiparchitectureAmplifiedsequenceLowestconcentrationsuccessfullyamplified.
DetectionoftheamplifiedsequenceRef.
MicrocirculatingPCRchipThreebio-reactorswithsuction-typemembraneandthreemicrovalvesoperatingatthreedifferenttemperatures150basepairsassociatedwiththehepatitisCvirus102copiesμL1OfflineafterextractionfromopenreactionchambersafterfinishingthePCRprocedure[62]ConcurrentelectrochemicaldetectionElevenparallelchannels489-bpgenefragmentOn-line,electrochemical,square-wavevol-tammetry[63]ExtractionofgenomicDNAanddetectionofsinglenucleotidepolymorphismThreemajormodulesforrapidpurification,DNAextractionandfastanalysisofgeneticgeneGenomicDNAfromleukocytes33.
26±2.
5ngDNAμL1Off-line,opticalevaluationinUV[64]IntegrationofPCRandCEonasingleplatformTri-layeredglass-PDMSwithintegratedpneumatically-actuatedvalvesandpumpsforfluidhan-dling,athin-filmresistiveelementthatactssimultaneouslyasaheaterandatemperaturesensor,andchannelsforcapillaryelectropho-resis(CE)On-line,alaserdiodeandachargedcoupleddevice(CCD)camera[65]Detectionofα-thalassemia-1dele-tionusingsalivasamplesDNAextractionchamber,sampleloadingchamber,wastecollectionchamber,PCRreactionchambersgDNAextractedfromsaliva12.
00pgμL1Off-line,fluorescence,byanexternalopticaldetectionmodule[66]DevicearchitectureforelectrochemicalpatterninganddetectionofmultipleDNAsequencesAmpliconsdiagnosticofhuman(H1N1)andavian(H5N1)influenza400nmolL1On-lineintegratedelectrochemicalarray[67]Unsealedreactorsforreal-timeisothermalhelicase-dependentamplificationAnarrayof4unsealedreactorsforreal-timehelicase-dependentamplificationBNI-1fragmentofSARScDNAOn-linefluorescence,CCDcameraandimageanalysis[68]QuantitativePCRsystemforDNAamplificationanddetectionTwomicromodulesforthermalandmicrofluidiccontrolwiththreeserpentine-shapemicro-pumps350and150-bpdetectiongenesassociatedwithtwoviruses,specificallyhepatitisBvirus(HBV)andhepatitisCvirus(HCV),10copiesμL1On-linefluorescence[69]FungalpathogenicnucleicaciddetectionMicrofluidicmicroarrayassemblydeviceonaCD-likeglasschipBotrytiscinereaandDidymellabryoniae0.
5nmolL1On-line,confocallaserfluorescentscannerfollowedbyimageanalysis[70]SinglenucleotidepolymorphismgenotypingofPCRampliconsfromwholebloodThinfilmtransistorphotosensorintegratingamicrofluidicchannel,aDNAchipplatform,andaphotodetectorBiotinylatedtargetDNA0.
5nmolL1On-linechemiluminescencephotodetector[71]Poly(methylmethacrylate)continuous-flowPCRmicrofluidicchipChiponthePMMAsubstratewith20parallelchannelsDNAtemplatewitha990-basepairfragmentofPseudomonasOff-line[72]248I.
Oitaetal.
Table3SelectedCEapplicationsonachipAnalyteDetectionPerformanceRef.
RT-PCRmixtureobtainedafteramplificationofa234-basepairRNAisolatedfromamultiplemielomacancerlineLaser-induced-fluorescenceSufficientsensitivityevenwithdramaticreductionininstrumentcostandcomplexity;separationresolutioncomparablewithabenchtop,commerciallyavailablesystem;signal-to-noiseratio32.
3;LOD0.
1ngμL1[65]8-Hydroxydeoxyguanosine(8-OH-dG)DNAadductinurine(oxidativestressbiomarker)Electrochemically(amperometricdetection)andviascanningelectronmicroscope(SEM)imagingLOD20attomoles;rangefrom100nmolL1–150μmolL1);separationefficienciesofapprox.
120,000–170,000platesm1[73]DNAContactlessconductivitymeasurementHighsignal-to-noiseratio;labelfreedetection[74]Humanrhinovirusserotype2(HRV2)FluorescenceAnalysistime10s;purityassessmentoffractionscollectedfromsize-exclusionchromatographypurificationofthelabellingmixtureandmonitoringaffinitycomplexformation[75]Thrombinlevelsinplasmadilutedto10%(v/v)FluorescenceLessthan1min;run-to-runandchip-to-chipreproducibility(RSD)ofmigrationtimes<10%;LOD540nmolL1[76]AFPfluorescentlylabelledAFPinspikedserumsamplesFluorescenceQuantitativeassay;eitherthemethodofstandardadditionoracalibrationcurve;AFPatngmL1levelsin10μLhumanseruminafewtensofminutes[77]K-rasoncogeneformutationshighlyassociatedwithcolorectalcancerFluorescenceAnalysistime11minusingtheCAEsystemand85sforPMMAmicrochips[79]α-Fetoprotein(AFP)fromspikedserumsamplesLaser-induced-fluorescence(LIF)Totalassaytime<10min;LOD0.
1ngmL1;CV<2%;quantitationrangefrom24to922ngmL1;goodcorrelationoftestresultsfor68patientserumsampleswithacommerciallyavailablereferencemethod[81]InactivatedswineinfluenzaFluorescenceMicrochip-basedconcentration;separatethevirus/fluorescentantibodycomplexfromtheunboundantibodyelectrophoretically;totalassaytime6min;<50μLsample[83]Humanserumalbumin(HSA)anditsimmunocomplexwithamonoclonalantibodyFluorescence800-foldsignalenhancement;LOD7.
5pmolL1;analysistime25s[85]Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld249MicrochipLCcoupledwithMShasbeenreportedforidentificationofautoantigens[91],definingaproteomesignatureforinvasiveductalbreastcarcinoma[92],andbiomarkerscreeningapplicationsinMCF-7breastcancercellularextracts[14].
Amethodveryapplicableinaclinicalenvironmentwasalsodeveloped[92].
ImmunoaffinitytechniquesAffinity-basedmethodsexploitthespecificbindingofbiomoleculesinordertoisolateandcharacterizetheminthepresenceofthousandsofothercompounds[93].
Themainactorsareantibodies,butaptamers,forexamplecell-surfacereceptorsandoligonucleotides,arealsovaluablealternatives[94].
Low-molecular-massaptamershavesev-eraladvantagesoverantibodies—fastertissuepenetration,longershelf-life,sustainingreversibledenaturation,lowertoxicity,thepossibilityofbeingproducedagainsttargetssuchasmembraneproteins,anduseofhighlyautomatedtechnology[95].
Awidearrayofclinicaldiagnostictestsemploysimmunoassaysandimmunoblottingapproaches[96].
Performingimmunoassaysonmicrofluidicdeviceshastheadvantagesofhighthroughput,shortanalysistime,smallvolumeandhighsensitivity,andfulfilsmostoftheimportantcriteriaforclinicaldiagnoses[97].
Themainadvantagesofmicrofluidicsasenablingtechnologyhasbeenclearlyprovedbyseveralapplications[77,98,99]withsimilarorimprovedperformancecomparedwithELISAbutmuchsimplerandfasterandwithlowervolumes.
FuturedevelopmentswillincreaseevenmoretheadvantagesoverELISA,creatingfullyintegrateddevices[100].
Analysisofinflammatorybiomarkers,C-reactivepro-tein(CRP),prostate-specificantigen(PSA),argininevasopressin(AVP),alpha-fetoprotein(AFP)cancercells,andinfectionmarkersarejustsomeoftheclinicallyimportantapplicationscurrentlyimplementedonmicro-fluidicimmunoassaychips.
Achiphasbeenusedforrapidisolationandquantificationofinflammatorybio-markersinmicrodissectedareasofaskinbiopsy.
Thebiomarkerswereisolatedbyimmunoaffinitycapturewithintheextractionportofthechipbyuseofapanelof12antibodiesimmobilizedonadisposableglassfibredisk[84].
Aspecificaptamer,i.
e.
anoligonucleotide,hasbeenusedforhighlyselectivecaptureandenrichmentofargininevasopressin(AVP)andpossiblediagnosisofimmunologi-calshockorcongestiveheartfailurebasedonAVPquantification[101].
TraceamountsofAVPwereenriched,elutedisocraticallyusingamicrofluidicplatform,anddetectedlabel-freebycoupledmatrix-assistedlaserdesorp-tion/ionizationmassspectrometry(MALDI-MS).
Theaptamer–analytebindingwasthermallydisruptableen-ablingeasydeviceregeneration[101].
Affinity-basedisolationhasenabledthefirststepsfromthebenchtoptotheclinicinthedevelopmentofaptamericbiosensorsforcancercells.
ProstatetumourcellsweresuccessfullyisolatedandidentifiedonaPMMAmicrochip[32].
Theproceduremanagedtodiscriminaterarecirculat-ingprostatetumourcellsresidentinaperipheralbloodmatrixwithoutstaining,usingantibodiesandaptamersforprostate-specificmembraneantigen(PSMA)immobilizedonthesurfaceofacapturebedfixedwithinthechip[32].
Similarly,theefficacyofon-chiprecognitionandcaptureofbreastcancercellsusinganantibody-basedmicrofluidicdevicehasbeenreported[34].
TheprocedureusedabiochipetchedontoPDMSwiththeinnersurfaceofthemicrochannelscoatedwithepithelialmembraneantigen(EMA)andepithelialgrowthfactorreceptor(EGFR)[34].
Applyingthesameprinciplebutfromareversedperspective,virus-boundmagneticbeadcomplexeshavebeenusedforrapidserologicalanalysisofantibodiesassociatedwithaninfectionbytheDenguevirus[30].
Denguevirusinfectionwasconfirmedbyuseofamicro-fluidicsystemthatintegratedone-waymicropumps,afour-membrane-typemicromixer,two-waymicropumps,andanon-chipmicrocoilarray[30].
Detectionisachievedusingfluorescence-labelledsecondaryantibodies.
Theprocedurewasperformedautomaticallyonasinglechipwithin30min,whichisafactorofeightfasterthanthetraditionalmethod.
Also,theLOD(21pg)wasreducedbyafactorofapproximately38comparedwiththetraditionalmethod[30].
C-reactiveprotein(CRP),ageneralinflammationandcardiovasculardiseaseriskassessmentmarker,hasbeendetectedinaone-stepsandwichimmunoassayusingafluorescencemicroscope[35].
Samplecollectionandimmunoreactionwereintegratedonamicrofluidicchip.
CRPwasdetectedfrom5μLhumanserumatconcen-trationsof10ngmL1inlessthan3min,andafter13minforconcentrationsbelow1ngmL1[35].
AnothermanuscriptreportedCRPassayinasimulatedserummatrixbyon-chipimmunoaffinitychromatography[102].
CRPwasfluorescentlylabelledinaone-stepreactionandinjecteddirectlyintotheimmunoaffinitycapillarycontain-ingmonoclonalanti-CRPattachedtoa5.
0-μmstreptavidin-coatedsilicabead.
Thelimitofdetectionwas57.
2ngmL1andchromatographicruntimeswerelessthan10min[102].
Animportantstepintheminiaturizationandintegrationoflaboratoryoperationsinself-containeddeviceswasmadebydesigningamicrofluidicchipforcombinatoriallibraryscreening(Fig.
5)[25].
Thechipwasusedtomapacombinatorialpeptidelibraryofpossibleepitopesforanti-T7tagantibodyandanti-FLAGtagantibody.
Thepeptides250I.
Oitaetal.
weredisplayedonE.
colicellsasinsertionswithinanexternalloopoftheoutermembraneproteinOmpX.
Thebindingpeptideswereselectedinseveralsteps(Fig.
5).
Inthefirststep,thelibrarywasincubatedoff-chipwiththetargetbiotinylatedantibodies.
Cellswiththebindingpeptideswerecapturedonstreptavidin-functionalized5.
6-μmpolysty-renemicrospheres.
Inthesecondstep,adisposablechipwasusedforserialdieletrophoresisactivatedcellsortingtoseparatecellswithbindingpeptidesfromcellswithnon-bindingpeptides.
Thesortingwasperformedonthebasisofsize,incontinuous-flow,withasortingspeedofmorethan108cellsh1.
Theantibody-bindingtargetcellscapturedonmicrosphereswerefunnelleddielectrophoreti-callybecauseoftheirdifferentpolarization[25].
Inthethirdstep,thecollectedbeadswithattachedcellsweregrownovernightandthesortinginthesecondstepwasrepeated.
TheDNAofthesortedcellswassequencedautomaticallytodeterminethesequenceofthebindingpeptides.
Celllibraryscreeningusingmicrofluidicsortingwascomparablewithacombinationofconventionalcellsorting-methods,i.
e.
oneroundofsequentialmagneticselectionandtworoundsoffluorescenceactivatedcellsorting.
Themicrofluidicsortingchip[25]hadincreasedtoleranceofflowdisturbancesandmicrobubblesandmorerobustpurityperformanceusinghighcellconcentrationsattheinlet.
Theauthorsalsomentionapossibilityofincreasedthroughputbyoptimizationofchiparchitecture,usingparallelchannelsfabricatedonasinglechip.
Thetechniquecouldbefurtherdevelopedtomonitorpoly-clonalsignaturesofserumantibodiesfordiseaseprofiling,forotheraffinity-basedscreeningusingsubstrate-functionalizedbeads,andforautomatedreagentgenera-tion,whereinligandsforagivenproteinorcelltypecouldbediscoveredusingself-regeneratinglibraries.
Prostate-specificantigen(PSA)hasbeenrapidlyandsensitivelyquantifiedinhumanserumsamplesusinganimmunosensorcoupledtoaglassycarbonelectrode(GCE)modifiedwithmultiwallcarbonnanotubes(MWCNT)(CNT–GCE)integratedwithmicrofluidicsystems[103].
PSAwascapturedimmunologicallywiththeimmobilizedanti-tPSAandhorseradishperoxidase(HRP)enzyme-labelledsecondantibodiesspecifictoPSA.
Thedetectionreliesonbackelectrochemicalreductionof4-tert-butylca-techolcatalyzedbyHRPinthepresenceofhydrogenperoxide.
TheprocedurehadanLODof0.
08μgL1whenelectrochemicaldetectionwasused[103].
Anewapproachtoimmunoblottingcombineson-chipintegrationofpolyacrylamidegelelectrophoresis(PAGE)Fig.
5Schematicdepictionofantibodyfingerprintingusingamicrofluidicsortingdevice,cas-setteB(nottoscale).
Left:Micrographofthesortingdeviceinoperationshowingthefirstsortingstage.
Right:Micrographofthesecondstageatthecol-lectionpoint.
(Reproduced,withpermission,fromRef.
[25])Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld251withsubsequentin-situimmunoblotting[98].
Themanu-scriptreports"handsfree"electrophoretictransferofresolvedspeciestoablottingmembraneasadirected,efficientmethodforproteinidentificationwithoutaneedforpressure-drivenflowandvalving[98].
alpha-ActininandPSAwereidentifiedandquantifiedfrommulti-proteinsamplesinthe101–105nmolL1rangewithLODsof0.
05pgand1.
8pg,respectively.
Moreover,detectionsensitivitywasenhancedapproximatelyfivefoldbytargetproteinenrichmentontheblottingmembrane[98].
Aninterestingapplicationofaffinity-basedmethodsisthedevelopmentofencodedmicrobeads.
Thesearesmartmicrostructureswithbothmolecularrecognitionabilityandbuild-incodesforrapidmicrobeadsidentification[104].
Thecodescanbeoptical,electronic,graphical,orphysicalsignaturescreatingcombinationssimilartotheblackandwhitestripesoftraditionalbarcodes[105,106].
Themostpopularapproachusescombinationsofquantumdots(QDs)togeneratefluorescentcodes.
QDsareinorganicnano-crystalswithfluorescentpropertieswhichdependonboththeircompositionandsize[106].
Theyhaveseveralamazingproperties,forexamplehighfluorescenceyield,remarkablestability,andextremelynarrowemission,enablingmanynon-overlappingcolourstobeusedsimul-taneously[106].
Moreover,theuseoftheencodedmicrobe-adscanaddmultiplexingcapabilitytotheassaybyenablingmulti-analyteanalysis[97].
DetectionschemesOpticaldetectionschemesarestillthefavouritechoiceformeasurementsinmicrofluidicsystems[107–109].
Therearetwomajorapproaches—couplingofthemacro-scaleopticalinfrastructureas"off-chipapproach"orintegrationofmicro-opticalfunctionsontomicrofluidicdevicesas"on-chipapproach"[110].
Severalsolutionsforintegration,forexampleplanarwaveguides,couplingschemestotheoutsideworld,evanescent-wavebaseddetectors,andopticalfluidicsintegrationproblems,andperspectivesandlimitationsofthesedifferentsolutionshavebeendiscussedindetail[107].
Irrespectiveofthedetectionapproach,theLODsachievedareprofoundlyaffectedbysensorsizeandshape,becauseofanalytetransportlimitationsratherthansignaltransductionlimitations[110].
Productionofinexpensive,sensitive,andportableopticaldetectionsystemsis,therefore,currentlyofmajorimportanceinthemanufactureofcommercialdevicesforportablediagnosticdevices[108].
Laser-inducedfluorescence(LIF)isthemostpopulardetectiontechniqueinmicrochip-basedapplications.
Be-causethenumberoffluorescentanalytesisratherlimited,aderivatizationstepwithafluorescentdyeisusuallyinvolved[42,51,65,89,111].
UnlikeUVdetection,analternativeto"diodearraydetectors"enablingsimultaneousdataacquisitionatmultiplewavelengthsisnotthataccessibleforfluorescentdetection.
Fluorescentdetectorsarebulkyandexpensive.
Insomecases,theabilitytocombinetheavailabledetectiontechnologywiththemostappropriatelabelisarealchallenge,butalsothelabellingitselfisamajoranalyticalburden.
Inmanycasesthedetectionreliesonmeasurementoffluorescenceintensity,performedbycapturingthesignalwithaCCDcameracoupledtoafluorescencemicroscopeandfollowedbygraphicalanalysisofthecapturedimages.
Forinstance,CCDcamerashavebeenusedtomonitor,capture,andspecificallyisolatevirusesandbacteria[112,113],tocontrolDNAhybridization[114],totypeStaphylococcusaureusstrains[115],toquantifyproteinsafterseparation[98],ortoscreenandidentifynewserologicalbiomarkersforinflammatoryboweldisease[62].
Asfluorescentprobes,fluorescein'sderivativesarestillextremelypopular[89,116–119],butthecyaninefamily(Cy)dyes[75,120,121]andAlexaFluortypedyes[31,33,120,122,123]arealsoused.
Inmostimmunoassays,thedetectionproblemissolvedbyusingfluorescentlabelledantibodies.
Theuseoffluorophoreswithhigherintensityyield,forexampleQDs,hasenabledvisualisationofHIVaftercaptureonamicrofluidicdeviceusingonlyastandard10*fluorescencemicroscope[47].
Similarly,theuseofQDprobesresultedin30-foldsignalamplification,whichimpliedareductioninobservedlimitsofdetectionbynearlytwoordersofmagnitude[124].
AcombinationoftwodyeswithdifferentcolourshasbeenusedtostudyVacciniavirusinfection[51].
Abluefluorescentcell-permeableDNAcounterstaindyeandagreen-fluorescentcell-permeablelipophilicdyewereused[51].
Evolvingtomorecomplexhardware,atwo-colourdetectionsystemwasincorporatedforthefirsttimeintoaCE–LIFchiptomeasuresimultaneouslyfluorescencefromreferencestandards(650nm)andtheanalyte(450nm)inthesample[89].
Thisapproachenabledlocationofstandardpeakswithoutinterferencefromsampleorbackgroundpeaks[89].
Fluorescencedetectionhastheadvantageofsensitivity,butthederivatizationstepsrequiredareamajordrawback.
Alternativesenablinglabel-freedetectionare,therefore,alwaysofinterest.
Whenanimmediateanswerfromanunprocessedsampleisneeded,fluorescencedetectionisoflimitedvalue.
Tocombinetheadvantagesoffluorescencewithlabel-freedetection,thegeneforgreenfluorescentproteinhasbeenincorporatedintotheVesicularstomatitisvirus(VSV)genometomonitortheinfectiononachip[125].
Anotherapproachenabledrapiddetectionofbacteriabymonitoringoff-chipbioluminescence[126].
Adenosinetriphosphate(ATP)extractedfrombacterialcellswas252I.
Oitaetal.
treatedwithluciferintoinducethebioluminescencereactionoffirefly,luciferin-ATP[126].
Surfaceplasmonresonance(SPR)tendstobethemethodofchoiceforlabel-freeopticaldetectionasanalternativetofluorescence.
Thetechniquedetectsvariationsinrefractiveindexbyobservingchangesinanoptimumplasmoncouplingangleorwavelengthwhenbindingoccursatmetal–dielectricinterfaces.
Themaindisadvan-tageofthistechniqueisthenon-specificmeasureformassaccumulation,thusanychangeduetonon-specificallyloadedmoleculescannotbedifferentiatedfromthetarget[127].
IncaseofsurfaceenhancedRamanspectroscopy(SERS),theabsorptionandscatteringofpropertiesofmetallicnanoparticlesenabletheiruseasenhancedchro-mophoresinmolecularlabelling[128].
Inthisway,thedisadvantagesofSPRareovercome.
SERSapplicationsformicrofluidicdevicesaredescribedindetailelsewhere[127].
ThetechniquehasbeenusedtoidentifyDenguevirusserotype2onchipatlevelshigherthan30pmolL1andwithexcellentspecificityagainstotherserotypes[129].
Thecombinationofliquidsandopticsinthesamephysicalvolumeisonewaytoenrichthefunctionalityofthesensors[123].
Qβphageshavebeendetectedandmonitoredonanoptofluidicchipusinganti-resonantreflectingopticalwaveguides[123].
Time-dependentfluo-rescencecorrelationspectroscopydatawereusedtocalculatediffusioncoefficients,flowvelocities,andcon-centrationsofviruses.
Thedevicecanalsobeusedasaninexpensiveandportablesensorcapableofdiscriminatingbetweenvirusesofdifferentsizes.
Thetechniqueissensitivetopicomolarconcentrationsandcanbeusedtodetectanddistinguishfluorescentobjectsinthesizerangeofviruses,e.
g.
phagesof26nm[123].
Electrochemicaldetectionisanalternativewithgrowingpopularity.
Itissensitive,compatiblewithawidearrayofbiochemicalreactions,andeasilyminiaturized.
Therearethreepossibilities:voltammetric,conductometric,andpoten-tiometricdetection.
Thisdetectionapproachisnotaffectedbyscalereductionandhasanexcellentperformanceevenwhenmicrometer-sizeelectrodesareemployed.
Anoverviewoftheavailableapproachesisgivenelsewhere[130].
Severalinterestingapplicationsusingelectrochemicaldetection,SERS,andSPRarelistedinTable4.
Massspectrometry(MS)enablesdetailedqualitativeandquantitativeassessmentofcellularbiomarkerswithhighsensitivityandreliability.
Thefabricationofmassspectrom-etersandinterfacesbetweenmicrofluidicplatformsandMSdetectorsonmicroandnanoscalesiscurrentlyoneofthemostinvestigatedtopics[140–142].
Thereductioninscalebringsadvantagessuchasimprovedprocesscontrolandautomation,shorteranalysistimes(minutesorseconds),reducedsampleconsumption,amenabilitytomultiplexingandhigh-throughputprocessing,andloweranalysiscosts[9,140–142].
Unfortunately,MSrequiresmulti-stepsamplepre-treatmentprocedures,sometimesincludingliquidchromato-graphic(LC)separations[14].
TwomainMSstrategiesarecurrentlysuccessfullyimplementedforquantitativeproteo-mics,namelylabel-freeandstableisotopelabelling.
Thesestrategiesaredescribedindetailelsewhere[140–142].
AnalysisofacomplexcellularextractonafullyintegratedmicrofluidicsystemusingMSdetectionhasbeenreported[14].
Proteinsfrombreastcancercellularextractsweretrypticallydigested,cleanedfromsaltsandlabelledwithanisobarictagforrelativeandabsolutequantitation(iTRAQ)reagents.
Bovineproteinswerealsoaddedtothesampleasstandards.
TheseparationwasperformedusingaglassmicrochipLC–MS,designedin-houseandenclosingfourdistinctivefunctionalelementsincludingapump,asamplingvalve,aseparationchannel,andanelectrosprayionization(ESI)interface.
MobilephasepropulsionthroughtheLCchannelandESIinterfacewasachievedbyEOFpumping.
Thepumpingunitconsistedoftwoarraysof200microchannels,eachof2cmlengthand1.
5–1.
8μmdepth,connectedinparallel.
Thelargenumberofchannelsensuredsufficientflowrateandthesmallsizeresultedinsufficienthydraulicresistancetopressurizeaback-flowleakage.
TheEOFgeneratedinthemultichannelspumpensuredmobilephasepropulsion.
Thevalvinghadasimilardesign.
Separationwasperformedinachannelpackedwith5μmZorbaxC18particles.
Sampleinjectionwasperformedelectrokineticallyandagradientwasgeneratedtoperformtheseparation.
Quantitativeanalysisofanentireproteinextracthasbeenperformedwithoutanysamplepre-fractionationanddifferentialproteinexpressionanalysisinMCF-7cellsculturedinthepresenceofβ-oestradiolandtamoxifen[14].
Thechipenabledreliableidentificationof40–50proteinsand,inanotherexperiment,wasabletoidentifyfiveproteinsofseveralpreviouslyreportedhumanputativecancerbiomarkersthatwereupordownregulated[14].
ChallengesandtrendsResearchersdevelopingapplicationsortechniquesusefulformoleculardiagnosisemployingmicrofluidicsarefacingchallengesatthreelevels—thedevicelevel,thesamplelevel,andtheapplicationlevel.
ChallengesatthedevicelevelThedeviceitselfisasourceofchallengesresultingfromdesign,fabricationandoperation.
Severalcommercialsolutionsareavailable,buthome-madeadapteddevicesareverypopularanditisimpressivetoseethatthecreativityofresearchershasnoboundaries.
IntheacademicMicrofluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld253world,lackoffundssometimesresultsingoodbrainactivity.
However,inseveralcases,theenthusiasmofresearchersleadstocomplicatedsolutions,whicharenottestedforreproducibilityandenlargethegapbetweenacademiaandindustry.
Thedevicesarefabricatedinvariousways,andfabricationisasignificantpartofmicrofluidics-relatedmanuscripts.
Artificialpolymers,PDMS[33,36,51,113,114,125,143–145],PMMA[47,77,80,132]orcyclicpolyolefin[40,99]arethematerialsofchoiceformassproductionandmosthome-madeapplications.
Polymershavea"JekyllandHyde"characterincomparisonwithothermaterialclasses[146].
Theyhaveseveraladvantages,i.
e.
arebiocompatibleandUV–visibletransparent,andtheiruseenablesrelativelyinexpensivefabricationofcomplexmicroandnanostructures,withhighreproducibility.
More-over,hugenumbersofmaterialsandmethodsareavailableformicrostructurefabrication.
Selectionoftheappropriatematerialandmicrofabricationmethodforagivenapplica-tionis,therefore,extremelydifficult,especiallyforscien-tistsunfamiliarwithpolymerchemistry.
Thisexplainswhythescientificliteratureisdominatedbyafewfavouritematerialsonly[146].
Polymericmaterialscanalsobeasourceofchallenges,especiallythosecontainingpolyimide,whichisincompat-iblewithaggressivestepsthatmightoccurduringmanu-facture[147].
Amodularapproachhasbeenusedformanufacturinganelectricalbiosensorcomposedofsingle-strandedmodifiedDNAprobeusedtoperformsimulta-neousmonitoringanddifferentiationofDNAsequencerepresentativesofPCRampliconsderivedfromhuman(H1N1)andavian(H5N1)influenza.
Initially,theelectrodeandchambersubstrateswereindividuallyprocessed;thetwosubstrateswerethenbondedtocompletetheprocessingofthedevice.
Themodularapproachwasasolutiontotherelativelyharshconditionsoccurringasaresultoftheuseofsulfuricacidforinsitucleaningandpreparationoftheelectrodes[147].
Thedesignofthedeviceshouldbekeptassimpleaspossibletoenablemassproduction,butsometimesitisproblematictocontroltheaccuratedeliveryofmanyreagentssimultaneously,toensureadequatemixing,andtoavoidcontamination.
Someresearchershavemanagedtofindsimplesolutions,forexampleintegrationofseparatereservoirsforsampleandreferencesolutions[77],hencereducingthenumberofwashingstepsandtherisksofcontamination.
Asimplesolutionhasbeendescribedinwhichmagneticbeadsusedthroughouttheapplicationwereeffectivelyretainedbyplacingamagnetonasideofthechip[126].
Althoughtechnologicaladvancesinrecentdecadeshaveenabledreadyaccesstomicro-components,fabricationonamicrometerscale(typicalsurfaceareasformicro-electromechanicalsystemsare100–10.
000μm2[120])withorwithouttheuseofbiologicalreagentsisachallengethatcannotbeneglected.
Whensurfacesarefunctionalizedwithbiologicalmacromolecules,supplementaryaspectssuchasbiomoleculestabilityandcompatibilityalsobecomecriti-Table4ApplicationsemployingadetectionmethodotherthanfluorescenceAnalyteLODDetectionRef.
α-Fetoprotein(AFP),hepato-cellularcarcinomabiomarker0.
1ngElectrochemical[52]Thyroglobulin,cancerbiomarker1pgmL1Surfaceplasmonresonance[31]8-Hydroxydeoxyguanosine(8-OH-dG)DNAadduct,biomarkerforoxidativestress20attomolesElectrochemical[73]Breastcarcinomamarkers<7fmolμL1NanoscaleMS[92]Prostate-specificantigen(PSA)0.
08μgL1Electrochemical[103]NucleicacidsequencesassociatedwithDenguevirusserotype230picomolarOn-chipsurfaceenhancedRamanspectroscopy(SERS)[129]Urinaryproteins(lysozyme,albumin)0.
1ppmElectrochemical[131]TracelevelofAFP1pgmL1Electrochemical[132]Alanineaminotransferase(ALT)oraspartateaminotransferase(AST)inhumanserum,liverdiseasebiomarker0.
145μAU1LforALT0.
463μAU1forASTElectrochemical[133]Tumourmarkers<0.
5μgL1Electrochemical[134]K-rasoncogene20picomolarSERS[135]Insulinandalbumin0.
9ngL1SERS[136]PSASinglemoleculeSERS[137]AFP2ngmL1Resonantmicrocantilever[138]Hepatitisbsurfaceantigen(hbsag)<10pgμL1Surfaceacousticwave[139]254I.
Oitaetal.
cal.
Awiderangeofbiomoleculeshavebeenimmobilizedontheinnersurfacesofdevices,e.
g.
anti-Micoplasmapneomoniaeantibodies[148],anti-gp120antibodies[47],anti-AFPantibodies[77],bacterialcells[149],DNA[114,147],H1N1probe[147],andHsp60[113].
Amicrospottingtoolhasbeenreportedforsequentialdepositionofbiomoleculesatthesamelocationonanactivesurface[120].
Thetechniqueenablespropercoatingofthesensingsurfacewithbioactivelayersandparalleldepositionofthreedifferentbiomoleculesinasinglerun[120].
Anothertechnique,the"layer-by-layer"(LbL)technique[150],enablespolyelectrolytecoatingstobeappliedtothesurfaceofdigitallyencodedmicrocarriers.
Thecoatingofmicro-carrierswithantibodies,andtheuseofthecoatedmicro-carriersascapturingagents,havebeenreported[150].
Microfluidicdevicesarecharacterizedbylargesurfacearea-to-volumeratios.
Largecapillaryforcesarehencegenerated,whichmaydrivefluidsintounwantedareasofthedevice,riskingcontaminationofotherfluidstreams.
Withinthechannels,themixingofliquidscanonlybeperformedbydiffusion,becauseofthelaminarflows.
Toperformadequatemixing,longchannelsareneeded.
Anewmembrane-typemicromixerhasbeendescribed[112].
Mixingwasachievedbyinjectingcompressedaircon-trolledbyanelectromagneticvalve(EMV).
Themicro-mixerwasusedintheprocessofincubationoftheviralsamplesandthemagneticbeads[112].
Otherapproachesformixingliquidsincludetheuseofcentrifugalforceandcapillaryaction[114],andmagneticforce[151].
Realsampleanalysisinvolvesthecompleteintegrationofsamplepreparation,andanalyteseparationanddetection[19].
Severalmanuscriptshavereportedinte-grateddetectiondevices,forexampleaminiaturelaser-inducedfluorescencedetectionmodule[89],aprototypeofanintegratedfluorescencedetectionsystem,andanopticalfibrelightguideonalaminate-basedmultichannelchip[117],andevenanewdesignofacontrollablemicro-lensstructurecapableofenhancinganLIFdetectionsystem[116].
ChallengesatthesamplelevelBioanalyticalsamplesarewellknownfortheircomplexity,i.
e.
oftenalargenumberofdifferentmoleculesispresentoverwiderangesofconcentrationindifferentmatrices.
Moreover,availablesamplevolumesareusuallylow,i.
e.
inthemicrolitrerange.
Also,biologicalcolloids,forexamplemacromolecularsolutionsandviralorbacterialsuspen-sions,areknownforwidedistributionsofcharges,sizes,andshapes,whichcanbeaffectedalsobytheexperimentalconditions.
Comparedwiththese,small-moleculespeciesareahomogenouspopulationcomposedofvirtuallyidenticalentities.
Thedistributionofanalytepropertieswillgenerateadistributionofelectrophoreticmobilities,forexample,whencapillaryelectrophoresisisusedassepara-tiontechnique.
Thisisoneofthecausesofthebroadandoftenirregularpeakshapessometimesobtainedforsamplesofbiologicalorigin[152].
Occasionally,thenon-uniformityofanalytepropertiesrequiresalternativesolutions,forexampleuseofanasymmetricelectricalfieldforelectrophoreticseparations.
Dielectrophoresiswasthesolutionfoundforseparationofcellsorparticles,whenthelargesizevariationsturnedintoaseparationasset[36,51,113,144].
Wallinteractionsareanothercommonproblemforsamplesofbiologicalorigin,especiallyforprotein-containingsamples.
BSA-FITChasbeenusedasmodelproteintodemonstratehowproteinmoleculesareadsorbedanddistributedontheinnerwallsurfaceofthePMMAmicrochannel[132].
Thesurfaceofthechannelswasinitiallycoatedwithpolyethyleneimine(PEI)containingabundantaminogroupstocovalentlyimmobilizeAFPmonoclonalantibody.
BSA–FITCbindingwithinthechannelswasthenstudiedtoenableoptimizationofthesystemtoreducenon-specificbinding,andquantificationofAFPwasachievedwithLODdownto1pgmL1.
TheutilityofthechiptodetectAFPfromhealthyhumanserumwasdemonstrated[132].
Gonzalesetal.
[153]studiedtheadsorptionofmajorpolymerasechainreaction(PCR)mixturecomponentsonthecapillarychannelwall.
NoneofthepolymericmaterialsorflowvelocitiestestedwasfoundtoaffectsubsequentPCRamplification.
PCRinhibitionoccurredonlyafterexposureofthemixturetotubinglengthsof3morwhenthesamplevolumewasreduced.
IndividualtestingofPCRproductsrevealedsignificantDNAadsorptionandanevengreateradsorptionofthefluorescentdyeused.
ThesefindingsimplythatadsorptionofreactioncomponentsbywallsurfacesisresponsibleforinhibitionofPCRinpolymerictubing.
Thesephenomenaincreasesubstantiallywithincreasingtubinglengthsorwithsamplevolumereduction,butnotwithcontacttimesortypicalflowvelocitiesfordynamicPCRamplification[153].
TheseresultsindicatetheneedforcarefulconsiderationofchemicalcompatibilitybetweenpolymericcapillariesandDNAdyeswhenquantitativemicrofluidicdevicesaredeveloped[153].
ChallengesattheapplicationlevelDevelopmentofapplicationsisusuallyfocusedonmain-tainingthestabilityofsamplesandreagents,preventingfalse-positiveresultsbecauseofnon-specificbinding,increasingprecision,andreducingtheLOD.
Toavoidnon-specificbinding,moresensitiveandselectiveapproachesareusedforthedesignofdevices.
Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld255Molecular-imprintedpolymers(MIP),forinstance,areartificialrecognitionmaterialsdesignedtointeractnon-covalentlywiththeanalyte.
Across-linkedpolymercontaininghighlyselectiverecognitionsitescreatedbymeansofsoft-lithographyhasbeenusedforspecificrecognitionofviruses[154,155].
TheshapeandsurfacechemistryoftheMIPfacilitatedhighlyspecificinteractionwiththevirus,andnon-specificinteractionswerethuseliminated[154,155].
Insomecases,theapplicationsaremorethanroutineanalysessuchasseparation,identification,orassay—forinstance,adeviceusedforcellgrowthandon-chipinfectivityassayofswineinfluenzavirus[125].
TrendsThehugenumberofresearchprojectsonthedevelopmentofportablediagnosticsisanobjectivemeansofquantifyingthegreatexpectationsofmicrofluidicsinthemoleculardiagnosisfield.
Paperisincreasinglyregardedasapromisingsupportforinexpensive,portable,fullydisposable,andeasytousedevicesforcomplicatedmoleculardiagnostics—aseasytointerpretasthehome-usedpregnancytest.
Photolithographycanbeusedtobuildselectivelyhydrophobicbarriersinthefilterpaper,enablingthehydrophilicpaperchannelstocontrolthetransportofaqueoussolutionsbycapillaryforceswithouttheneedforexternalpumping[156].
Severalotherresearchgroupsfocusedonthecalibrationofpaper-basedmicrofluidicdevices[157],onthetechniquesusedtogeneratehydrophilicchannelsinfilterpaper[158],ontheapplica-tionofelectrochemicaldetection[159],oronthedevelopmentofahand-heldopticalcolorimeter[160].
Useofmicrofluidicdevicesgivesdeeperinsightintothelifesciencesandmedicinebecauseitenables,forinstance,studyoftheproteinsinasinglecell[161],quantificationofmultipleproteinsinasinglesample[33,162],identificationofasinglenucleotidepolymorphism[135],ordetectionbelowthelimitsofclassicalmethods,forexamplethe"bio-barcode"assay[163–165].
The"bio-barcode"assay(Fig.
6)usestwotypesoffunction-alizedparticles:1.
magneticparticlesfunctionalizedwithrecognitionelements,i.
e.
monoclonalantibodiesorahapten-modifiedoligonucleotide;and2.
goldnanoparticlesfunctionalizedwithasecondrecog-nitionelementandabifunctionaloligonucleotidebar-codeDNA[2].
Theanalyteofinterest,thetarget,usuallyaprotein,isinitiallycapturedandenrichedbythemagneticmicropar-ticle(Fig.
6).
Further,thetargetissandwichedbetweenmagneticmicroparticleandthegoldnanoparticles.
Thesandwichesareeasilyseparatedfromthesampleinamagneticfield.
Inafurtherstep,theDNAofthebarcodeisreleasedbyheating.
Signalamplificationisachievedbecauseforeachtargetrecognitionthousandsofbar-codesarereleased.
HalfofthereleasedDNAisdetectedbyuseofascanometricassaybasedontheaffinityofthereleasedDNAforacomplementary"universal"scanometricgoldnanoparticleDNAprobe.
TheotherpartiscomplementarytothechipimmobilizedDNA,responsibleforsortingandbindingbarcodescomplementarytothetargetsequence.
Theconcepthasunparalleledsensitivityfortargetdetec-tion,i.
e.
itcanbebetweenoneandsixordersofmagnitudemoresensitivethanconventionalELISA.
Byuseofthismethod,PSAwasmeasuredintheserumofpatientsafterradicalprostatectomy,evenincaseswhentheavailableimmunoassayswerenotabletodetectit[166].
Atthecrossroadoffourmajorscientificfields,i.
e.
biology,physics,chemistry,andmedicalscience,biosen-sorsareoneofthemoststudiedsubjectsofrecentyears.
Althoughsignificantprogresshasbeenachieved,andanalysisatthesingle-moleculelevel[5]orbasedonsingle-cellcomposition[161,167]ispossible,viablesolutionsforreal-time,bedsidediagnosticdevicesarestillawaited.
Recentprogressintechnologyopensthewaytowardmassproductionofbiosensorsandbedsidedevices.
Theuseofpolymericmaterialsforfabricationofmicrofluidicsystemswillsimplifymanufacturingprocesses[168].
Newtransducingandbiocompatibleinterfacesareexpectedtobedevelopedbasedoncompositeswhichintegratenano-particles,carbonnanotubes(CNTs),andnanoengineered"smart"polymers[168].
Thedifficultyinproducinglow-cost,sensitive,andportableopticaldetectionsystemslimitsthenumberofthecommercialdevicesforbedsidediag-nostics,butseveraltechnologicalsolutionsforbiomolecule-compatibleoptofluidicintegrationhavealreadybeendescribed[169].
Linder[170]hasdescribedtwotypesoftechnologyexpectedtoextendtheuseofmicrofluidicdevicesoutsideclinicallaboratories,i.
e.
amplificationchemistrythatresultsintheaccumulationofanopaquematerialatthesurfaceofreactionsite,andasolutionforlong-termstorageofmultiplereagentsandforthesequentialdeliveryofthereagentstothereactionsiteinsideamicrofluidicdevice.
Innovativetechniquesinvolvingnanoengineeredmaterialshavebeenusedtodeveloppotentiallylessexpensivedetectionsystemswithoutalignmentrequirements,withoutminiaturizationdisadvantages,andwhicharemorereadilyadaptedforbedsideuse[108].
Thecombinationofprogressintechnologyandthelifesciencesbringsusonestepclosertomicrofluidicdeviceswithhighersensitivity,andim-256I.
Oitaetal.
provedbiocompatibilityandstabilityoftheimmobilizedmolecules,whichmakesthemfeasiblebiosensorsandbedsidedevices.
Theuseofproteinbiomarkersfordevelopmentofmicro-electricalsensors,andtheunderlyingtechnicalconcepts,havebeendescribed[171].
Similarly,emergingopticalandmicrofluidictechnologysuitableforbedsidegeneticanal-ysissystems[169]andDNAbiosensors[172]havebeenreviewed.
SomeearlystagecommercialproductsbasedonelectrochemicalDNAbiosensorsintegratedinanalyticalmicrofluidicdeviceshavealsobeenreported[172].
Thereiscurrentlyadiscrepancybetweenthemolec-ularbaseddiagnostictestsapprovedbyregulatoryagenciessuchastheFDA(Table5)withinthelastthreeyears,andtheliteratureandtheavailabletechnology.
Theapprovedtestsarerathertraditional,andmostlybasedonimmunoassays.
SeveraltestsusePCRforconfirmationofviralinfection,cancerdiagnosis,andforprognosisordiagnosisofgeneticdiseases,forexamplecysticfibrosisinnew-borns(Table5).
Still,comparedwiththenumberofpublishedpapers(hundredsoreventhousands)FDA-cleareddiagnosticdevicesinvolvingmicrofluidicsarerestrictedmostlytoimmunochromatographicassaystoconfirmseveralvirusesandbacteria,andseveralotherdevicesintendedformeasurementofcholesterollevelinbloodcontrolorforglycaemiccontrolinpeoplewithdiabetes.
Alltheothertestsmakeuseofhighlyspecializedreagentsandequipment.
Thewholesituationisevenmorecontradictoryifweconsiderthatmorethanhundredcompaniesareproducingandcommercializingminiatur-izedanalyticaldevices[173].
AccordingtoastudypublishedbyAnalyticalChemistry[8],microfluidicsisnowatthe"slopeofenlightenment"stageoftheGartnerhypecyclemodelofthelifecycleoftechnology(Fig.
7a).
Iftheyearlychangeinpaperspublishedonmicrofluidics(Fig.
7b)isconsidered,itisclearthatmajoreventsinscienceandtechnologyalsoactedastriggersformicrofluidics.
Thetechnologywasredefined,anewcyclestarted,morepowerfulapplicationsweredefined,anddeeperinsightwasobtained.
ConcludingremarksMicrofluidicsarecurrentlyamongthemostfashionableresearchtopics.
Muchoftheresearchexploitstheadvan-tagesofmicrofluidicstosolvecurrentproblemsinbioanalysis,e.
g.
smallvolumes,limitedstability,andhighcost.
Anoverwhelmingnumberofmanuscriptsisdevotedtotryingtoextendresearchfindingsintoroutineclinicaluse.
However,onlyafewdiagnosticdevicesareavailablecommercially,andtheincreaseinthenumberofregistereddevicesisnotfollowingthenumberofpublishedpapersorFig.
6BiobarcodeassaydevelopedforquantificationofPSAinpatientserum.
SchematicrepresentationofthePSAAu–NPprobes(A)andthePSAbio-barcodeassay(B).
Fordetails,seetext(reproducedwithpermissionfromRef.
[166])Microfluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld257Table5MoleculardiagnosticsFDAapprovedwithinthelastthreeyears(source:https://www.
accessdata.
fda.
gov/scripts/cdrh/devicesatfda/index.
cfm,accessed02.
2010)ProductNameMarketingauthorisationholderApprovaldateApplicationrangeWorkingprincipleInstrumentation1VIDASfPSArtAssayBiomerieux10/8/09Assessmentofthechancethatthemanhasprostatecancerbasedonmeasurementoffreeprostate-specificantigen(fPSA)TwostepenzymeimmunoassaysandwichmethodwithafinalfluorescencedetectionReady-to-usereagentstrips(10wells/strip)tobeusedwithbenchtopchemistryanalyzers2ArchitectCoreAbbottLaboratories04/10/09Detectsantibodies(antiHBc)associatedwiththehepatitisBvirus(HBV)coreantigeninserumorplasmaTwo-stepimmunoassaywithchemiluminescencedetectionBenchtopanalyzer3CervistaHPVThirdWaveTechnologies03/12/09IdentifyDNAfromhumanpapillomavirus(HPV)types16and18incervicalsamplesDNAextractedfromcervicalsamplesisamplifiedinathermalcycler.
AsecondisothermalreactionwillgenerateafluorescentsignalBenchtopPCR4CobasTaqManRocheMolecularSystems10/03/10QuantifytheamountofhepatitisCviralRNAinapatient'sbloodtohelpthephysicianstodetermineanindividual'sresponsetotreatmentNucleicacid(RNA)isseparatedfromthecellsinthebloodsample.
SeparatedRNAisamplifiedandtheamountofHCVRNAinthepatient'sbloodismeasuredonthebasisoftheamountoflightproducedBenchtopqPCR5Tspot.
TBOxfordImmunotec07/30/08Detecttheimmuneresponseofthymuscells(Tcells)foundinanindividual'swhitebloodcellsthatarestimulatedbyproteinsproducedbythebacteriathatcausestuberculosisCombinationofatwostepenzymeimmunoassaysandwichmethodwithafinalvisualdetectionBenchtop-wells6Spot-LightHER2CISHInvitrogen07/01/08MeasurethenumberofcopiesofHer-2geneonchromosome17inbreastcancercellstoassessifthepatientiseligiblefortreatmentwiththecancerdrugHerceptin(Trastuzumab).
On-slidebindingofHer-2genewithamatchingdigoxigenin-taggedDNAprobe.
Afluorescent(FITC)taggedantibodytodigox-igeninfollowedbyahorseradishperoxidaseconjugatedantibodytoFITCandDABfurtherrevealstheprobes.
Fluorescencemicroscopy7xTAGrespiratoryviralpanel(RVP)LuminexMolecularDiagnostics01/03/08IdentifiesnucleicacidsofmultiplerespiratoryvirusesinnasopharyngealswabspecimensfromindividualssuspectedofrespiratorytractinfectionsMultiplexdetectionofviralnucleicacidsbasedonbeads;selectiveisolationofviralRNAfollowedbyPCRMultiplesteps,benchtopPCRequipment8DakoTOP2AFISHpharmDxDakoDenmark01/11/08Assessmentoftheriskofpost-surgicalrecurrenceofbreastcancerandlong-termsurvivalbasedonthemeasurementofthenumberofcopiesoftheTOP2A(Topoisomerase2alpha)geneonchromosome17inbreastcancercellsOnslidebindingofHer-2genewithamatchingaredfluorescent-taggedDNAprobebindstomatchingDNAoftheTOP2Ageneandagreenfluorescent-taggedDNAprobebindstothematchingcentralportionofchro-mosome17incellsontheslideFluorescencemicroscopy9GeneSearchBLNTestVeridexLLC07/16/07Rapiddetectionofmetastaseslargerthan0.
2mminlymphnodestissueremovedfrombiopsiesofbreastcancerpatientsChemicalamplificationoftwogeneproductsabundantinbreasttissueandscarceinlymphnodecells.
FluorescencedetectionBenchtopPCR10MesomarkFujirebioDiagnostics01/24/07Assessmentofmetastasesforararecanceroftheinternalbodylining(mesothelioma)Colorimetricreactionforspecificproteinfragmentsreleasedbycellsofmalignantmesotheliomaintoapatient'sbloodBenchtopanalyzer258I.
Oitaetal.
fundingofbiomarkeranalysisonmicrofluidicplatforms.
Therearetwodifferentviewsontheevolutionofthesedevices:theoptimisticviewisthatmicrofluidicswillbecomeaintegralpartofthefutureofbioanalysis;thepessimisticviewisthatmicrofluidicswillremainanichepreoccupationforresearchwithoutanypracticalconsequences[8].
Althougha"killerapplication"hasnotyetbeende-veloped[8],smallstepstowardsitaretakeneveryday.
Thebeginningiscomplete,andapplications,forexampleTable5(continued)ProductNameMarketingauthorisationholderApprovaldateApplicationrangeWorkingprincipleInstrumentation11MammaPrintAgendiaLaboratory2009Assessapatient'sriskofdistantbreastcancermetastasisMicroarray-basedgeneexpressionanalysisofRNAextractedfrombreasttumourtissueBenchtop–bioanalyzer12AVantageA/H5N1flutestArborVita04/01/09RapiddiagnostictestfortheH5N1influenzaAviralsubtype(avianorbirdflu)RapidproteomicstestforthespecificdetectionofH5N1Portableb1996–cloningofDollythesheep2004–firstcarbonnanotubetransistor%increaseofthenumberofpapers2002200014020092007200520042003200119991998199719961995199401001201601992199419961998200020022004200620082010Year60802040200820062001–decodingofhumangenomeaFig.
7aGartnerhypecyclemodel(reproducedwithpermis-sion).
bEvolutionofpercentagechangeinthenumberofpaperspublishedyearlyonmicrofluidicsMicrofluidicsinmacro-biomoleculesanalysis:macroinsideinananoworld259MammaPrint(Table5),thatusesamicrofluidicplatformtogiveaprognosisofthefutureevolutionofbreastcancer,willincreasinglybecomepartofourlife.
Technically,applicationswithinthelifesciencesareincreasinglytryingtoresembletheworlddepictedin'70s–'80ssciencefictionmovies:deviceswerealwayssmall(handheld),incrediblysleek,andprovidedalltheinformationnecessaryatamoment'snotice[173].
Useofmicrofluidicsdevicesinlifescienceandmedicinehaverevealednewperspectivesandenableddeeperinvestigation,buttheyhavestilltofindtheirwaytomoregeneralizedandcommercializedapplications.
AcknowledgementsThisworkwassupportedbyaHorizontaleOnderzoeksactie(HOA)oftheVrijeUniversiteitBrusselandaresearchgrant(G.
0051.
08)oftheFWO-Vlaanderen.
DebbyMangelingsisapostdoctoralfellowoftheResearchFoundationFlanders(FWO).
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