外文文献分子印迹聚合物接枝的石墨颗粒糊电极制成的血液肝素传感器.doc

外文文献分子印迹聚合物接枝的石墨颗粒糊电极制成的血液肝素传感器 Sensors andActuators B259 (2018)455462Contents listsavailable atScienceDirectSensors andActuators B:Chemicaljo urnal homepage:.elsevier./locate/snbBlood heparinsensor madefrom apaste electrode of graphiteparticlesgrafted withmolecularly imprinted polymerYasuo Yoshimi?,Yuto Yagisawa,Rina Yamaguchi,Maki SekiDepartment of AppliedChemistry,Shibaura Instituteof Technology,3-7-5Toyosu,Koto-ku,Tokyo135-8548,Japana rt i c le i n foArticle history:Received23SeptemberxxReceived inrevised form12DecemberxxAepted13DecemberxxAvailable online14DecemberxxKeywords:Molecularly imprinted polymer(MIP)HeparinGraphite paste electrodeVoltammetrya bs tr ac tAreal-time heparinmonitor couldbe used to optimizethe dosageof heparinduring extracorporealcirculationprocedures.This reportdescribes thedevelopment of a graphite-paste(GP)electrode withmolecularlyimprinted polymer(MIP)grafted ontoit.Heparin-imprinted poly(methacryloxyethyltri-ammonium chloride-co-acrylamide-co-methylenebisacrylamide)was grafteddirectly ontographiteparticles.The grafted particles werethoroughly mixedwith oil to fabricatethe MIP-GP electrode.Tradi-tional cyclic voltammetry wasperformed with the electrodein physiological saline orbovine whole bloodcontaining5mM ferrocyanideand08units/mL heparin.The current intensity increasedwith heparinconcentration,due toexpansion of the effective surface arearesulting fromheparin-promoted mobilityof the oilin the MIP-GP electrode.No signif i cantdifference wasfound in the sensitivity of the current tounfractionated heparin among the electrodesfabricated because of the electrode homogenizationresult-ing fromthorough mixing of the MIP-grafted particlesand oil.(A previousMIP-grafted indiumtin oxideelectrodeexhibited lowersensitivity in blood thanin saline.)Only60s wereneeded tostabilize the cur-rent.The current at the MIP-GP electrode was also sensitive tolow-molecular-weight heparin in blood,but insensitive to chondroitin sulfate C(CSC),which isa heparin analog.The non-imprinted polymer(NIP)-grafted electrode was insensitive to heparin.Thus,the MIP-GP electrode,which operatedthrougha newheparin-sensing mechanism,is anexcellent candidatefor application as adisposable sensortomonitor heparinlevels in blood.?xxThe Author(s).Published byElsevier B.V.This isan openaess articleunder theCC BYlicense(creativemons/licenses/by/4.0/).1.IntroductionIn extracorporeal therapy(e.g.,hemodialysis,cardiopulmonarybypass),blood contactsartif i cial materialsin the blood vessels,creating arisk ofclotting.The clotscan plugtubes used for extra-corporeal perfusion,or blockblood capillaries.Clotting isgenerallyprevented by administering anticoagulants,among whichheparinis themost widelyused injectableanticoagulant andis metabolizedrapidlyby thehuman body.Controlling anticoagulationduring amedicalprocedure isvery importantbecause a heparin overdosecouldinduce bleeding,while anunderdose couldcause clotting.Theadministered doseof heparin and itsantidote,protamine sulfate,are usuallydetermined bymeasuring the“activated clottingtime”(ACT).However,the ACTof heparin added toblood duringextra-corporealtherapyis approximately600s1,which istoo longtobe ofpractical usein surgery.In addition,ACT isinsensitive tolow-molecular-weight heparin(LMWH)2,which producesa lower?Corresponding author.E-mail address:yosimisic.shibaura-it.ac.jp(Y.Yoshimi).risk ofbleeding thanconventional unfractionatedheparin(UFH)3.Thus,a sensorthat coulddetect heparin,including LMWH,inreal timeis neededto monitorheparin dosage.A newsensor has been developedfor thequantif i cation ofhep-arin based onaheparin-specif ic molecularlyimprinted polymer(MIP),which aresynthetic polymersthat containspecif ic bindingsitesformed byimprinting thestructure of the targetmolecule,referred to as the template,during the polymerization process.Thisheparin sensorwas developed by graftingthe MIPlayer with theheparin templateonto the surface ofa fl at indiumtin oxide(ITO)electrode4,5.The oxidativecurrent of hexacyanoferrate(II)(orferrocyanide)at the MIP-grafted ITO(MIP-ITO)electrodewassensi-tiveto LMWH aswell asUFH in whole blood5.The sensorcurrentcould bestabilized inless than30s,indicating that the electrodeispractical forapplication as a real-time heparinsensor.Sensorsfor thediagnosis ofwhole blood samples areusually disposableforhygienic reasonsand thereforemust exhibithigh reproducibilitybecausethe sensorcan beusedforonly onesample.However,largevariations insensitivity existedamongthefabricated electrodes,soactual use of the MIP-ITO electrodeasadisposable sensorwas verydiff i cult.This waslikely due to diffi cultyin ensuringuniform radicals:/doi/10.1016/j.snb.xx.12.0840925-4005/?xxThe Author(s).Published byElsevier B.V.This isan openaess articleunder theCC BYlicense(creativemons/licenses/by/4.0/).456Y.Yoshimi et al./Sensors andActuators B259 (2018)455462graft polymerizationat the surface of the electrode.Thus,the MIPlayertends to be heterogeneous,and reproducibility of the sensi-tivity of the graftedelectrode isvery poor.In addition,sensitivitytoward heparininblood was alsopoor due to inhibitionby plasmaproteins.In the present study,a newmethod toimprove thequality ofMIP-grafted electrodes was developedby using a fl uidized bedforgraft polymerization,which enableduniform chemical reactions6,and acarbon pastingmethod thatallowed massproduction ofthe electrodes withhigh homogeneity7.First,the graphitecarbonparticles werecoated with a radicalpolymerization photo-initiator,and thiswas followed by radicalpolymerization tograft the MIPonto thefluidizedinitiator-coated graphite.Finally,the graftedgraphite was mixedthoroughly withoiltofabricate homogenouscarbonpaste electrodes.The heparin-sensitivity and the repro-ducibilityof the outputcurrent at the MIP-grafted graphitepaste(MIP-GP)electrode wereevaluated in whole bloodand in saline.The sensingmechanism of the MIP-GP wasexamined using thesensitivity of the contact angle ofsaline on the MIP-GP electrodetothe presenceof the template.2.Materials andmethods2.1.ChemicalsSpherical graphite particles8mm indiameter(SG-BH8)weredonated byIto GraphiteCo.,Ltd.(Kuwana,Japan).Ethanol,N,N-dimethylformamide(DMF),sulfuric acid,hydrochloric acid,and acetic acid were purchased fromKanto ChemicalCo.,Ltd.(Tokyo,Japan).Zinc chloride,sodium diethyldithiocarbamate,formaldehyde,sodium saltof UFH(180units/mg;from porcineintestinalmucosa),acrylamide AAm,N,N-methylenebisacrylamide(MBAA),potassium hexacyanoferrate(II)(or ferrocyanide)trihy-drate,potassium nitrate,and sodiumchondroitin C(from sharkcartilage)werepurchasedfrom WakoPure ChemicalIndustry Co.Ltd.(Osaka,Japan).(2-(Methacryloxy)ethyl)trimethyl ammoniumchlorideacrylamide(METMAC)was purchasedfrom Sigma-AldrichInc.(St.Louis,USA).Dalteparin sodium(LMWH)was purchasedasFragmin?from Pfi zerInc.(New York,USA).Bovine whole blood waspurchasedfrom ameat processingpany(Tokyo ShibauraZokiKK,Tokyo).2.2.Introduction ofradical photopolymerizationinitiator on thegraphite surfaceThediethyldithiocarbamate methylene group,which isa radi-cal photopolymerizationinitiator,was introducedonto the surfaceof graphite particles viachloromethylation usinga proceduredescribedby Samuelsetal.8,as shown in Fig.1.A three-neck,200-mL,round-bottom flask wascharged first with5gof the graphite,then0.25g(1.9mmol)of zinc chloride,and finally with100mL of1:1(v:v)concentrated hydrochloricacid and aceticacid.After -plete dissolution of thezinhloride,the graphitesuspension wascooledin an ice bath and bubbled with argongas forone hour.Next,the argonstream wasstopped and10mL offormaldehydeaqueous solution(37%)was added to the suspension.The suspen-sion wasplaced in anicebathand bubbled withhydrogen chloridegasproduced bydropwise addition of concentratedhydrochloricacid intoconcentrated sulfuricacid inanother flask for4h.Thehydrogen chloridegas at the outletwas absorbedbyasaturatedaqueous solutionof sodiumhydrogen carbonate.When bubblingceased,thesuspension was stirredovernight atroom temperature.The treated graphitewascollected bysuction filtration,washedwith500mL ofdistilled water,and dried under vacuum overnight.The driedparticles weresuspended ina0.12M ethanolicsolu-tionofsodium diethyldithiocarbamatefor24h tointroduce thediethyldithiocarbamatemethylenegroupused asan initiatorofradical polymerization.Progress of the reactionwas confi rmedbythe turbidityof thesupernatant dueto NaClcrystals producedbythe reaction.The initiator-coated graphitewas washed with dis-tilled waterfollowedbymethanol,and thendried undervacuum.The driedinitiator-coated graphitewas packedinabrown bottlecoveredin aluminumfoil and stored ina refrigerator.2.3.Graft polymerizationof the graphiteparticlesurfaceThe METMACwas recrystallizedin acetonebefore use.The UFHsodium(0.16g),METMAC(0.87g),and AAm(1.00g)were dis-solved in6mL ofdistilled water,the MBAA(1.00g)was dissolvedin18mL ofDMF,and thetwo solutionswere mixedtogether.Theinitiator-coated graphite(0.7g)was suspendedin thebinedsolution,and thesuspensionwasplaced ina quartzcrystal testtube(26.5mm innerdiameter)andbubbledvigorously for20minwith nitrogen gas to remove dissolvedoxygen.While beingbub-bledwithnitrogengasand stirredwith amagic stirrer,thesuspension wasirradiated withlight froma xenonlamp(LC-5,Hamamatsu Photonics,Co.Ltd.,Hamamatsu,Japan)guided byanoptical fiber.The treatedparticles werecollected bysuction fil-tration andwashed with1:3(v:v)water andDMF,followed bywashingwith1M aq.sodium chloridetoremovethe heparinfromthe grafted layer.The isolatedparticles werewashedwithdistilledwater,driedundervacuumovernight,andstoredinadesiator.The particleswere observedusinga scanning electrodemicroscope(SEM)(JSM-6010LV,Jeol,Akishima,Japan).A non-imprinted poly-mer(NIP)was alsoprepared usingthe sameprocedure describedabove,but withoutthe UFHtemplate.2.4.Preparation of MIP-GP electrodeThegrafted particles(7mg)and3mg siliconeoil(KF-96-300CS,Shin-Etsu ChemicalCo.,Ltd.,Tokyo)were mixedand groundinto apasteinapolytetraf luoroethylene mortarand apestle for20min.The graphitepaste waspacked into the tipofahematocrit capil-lary(Hirschmann Laboger?te GmbH&Co.KG,Eberstadt,Germany)with aninner area of1mm2.The particleswere packedto alengthof approximately1mm usinga glassrod.The tipof thepackedgraphite paste was polishedusing paraffinpaper(Hakuai Co.,Ltd.Tokyo),andacopper(diameter0.8mm)wire wasinserted into thegraphite pastethrough theother endof thecapillary tomaintainthe electricalconnection.2.5.Electrochemical sensingSamplescontaining08unit/mL heparinand5mM potassiumhexacyanoferrate(II)(or ferrocyanide)in physiologicalsaline or inbovine wholeblood wereaddedto an electrochemicalcell VC-4(BAS Inc.,Tokyo).The preparedcarbon-paste electrode,platinumwire,and Ag/AgCl electrode(RE-1B,BAS Inc.)were insertedas theworking,counter,and referenceelectrodes,respectively.The elec-trodes wereconnected toan electrochemicalanalyzer HZ-5000(Hokuto Denko,Tokyo).Traditional cyclicvoltammetry wasper-formed with ascanrate of0.2V/s.Scanning wascontinued untilthe current stabilized.The relationbetween the peak anodiur-rent ofhexacyanoferrate(II)and the heparin concentrationwasdetermined.2.6.Measurement ofcontact angleThe MIP-GP orNIP-GP waspacked intoa1cm1cm squareholeina100-?m-thick poly(ethyleneglycol terephthalate)(PET)film thatwas pastedonto aglass plateon whicha conductiveY.Yoshimi etal./Sensors andActuators B259 (2018)455462457Fig.1.Scheme showingimmobilization of the photo-initiator ontothegraphitesurface.Fig.2.SEM imageof(A)non-treated and(B)MIP-grafted graphiteparticles.ITO layerwas deposited(IN-100,Furuuchi ChemicalCo.,Tokyo,Japan).The packedpastewassmoothed witha spatulato obtaina flatplate electrode.The contact angle of the physiologicalsalinedroplet containing0or10unit/mL UFH(or CSC)on thepasteelec-trodewas measured usingan OCA15EC instrument(DataPhysicsInstruments GmbH,Filderstadt,Germany).The advancingcontactangle wasmeasured3min after20?L ofsaline wasdropped on thepaste electrode.The receding contact anglewasmeasured1minafter10?L ofsaline wasremoved fromthe droplet.3.Results3.1.Morphology of grafted particlesAparison of the SEMimages of the non-treated graphiteparticlesand MIP-grafted particlesis shown in Fig.2.Although thenon-treated graphiteparticles hada smoothsurface,the surfaceof the graftedparticles wasrough and the particleswere par-tially coagulated.The imagealso indicatesthat graftpolymerizationon particlescovered with the photo-initiator(diethyldithiocar-bamidomethylene)proceeded suessfully.However,the shapeof the treatedparticles wasinhomogeneous,especially that ofthe coagulatedparticles,which suggeststhat homogenousgraftpolymerization remainsdiffi cult evenwhen usinga fluidized bedreaction.3.2.Effect ofgraftedlayeronthe redox reactionCyclic voltammograms of5mM hexacyanoferrate(II)at theMIP-grafted graphitepaste(MIP-GP)electrode andat the graphitepaste electrodeprepared withnon-treatedgraphiteusingthe sameprocedure(non-treated GP)are shownin Fig.3.Both the anodic andcathodic currents wereenhanced by the MIP grafting.The peaksofboth currents at the MIP-GP were more than5times greater thanthat at the non-treated GP.The differencebetween thepeak poten-tials of the cathodiurrent and anodic current at the MIP-GP was110120mV,while that at the non-treated GP was approximately250mV,which indicatesthat theelectrochemical reactionofhex-acyanoferrate(II)at bothelectrodeswasquasi-reversible9,butthat thereaction at the MIP-GP wascloser toa reversibleelectro-chemicalreaction(difference inpeak potential of58mV)than thatat the non-treated GP.The peak current andpeak potentialresultsdemonstrate that the activityof theGP electrodewas enhanceddramaticallyby polymergrafting.However,thepeak current at458Y.Yoshimi etal./Sensors andActuators B259 (2018)455462Fig.3.Cyclicvoltammogramof5mM potassiumhexacyanoferrate(II)at theMIP-grafted GP electrode(solid lineand brokenline correspondtoaheparin con-centration of0and8units/mL,respectively)and non-treated GP electrode(dottedline:0unit/mL heparin).the5mM hexacyanoferrateMIP-GP wasgreaterthan the calcu-lated peakcurrent of1.5mA/cm2at thesame hexacyanoferrate(II)concentration andpotential scanrate(0.2V/s)with thereversiblereaction(electron transferwas veryrapid paredto diffusionofthe redoxspecies9).Thus,the redoxcurrent wasunlikely tohavebeen increased by the grafted polymerdue onlyto electrodeacti-vation.Instead,the polymergrafting likelyincreased the effectivesurface area of thegraphite-paste electrode.These resultsare incontrastto thoseof previousstudies thatreported that the redoxcurrentsof hexacyanoferratesat the ITO electrodewere decreasedby MIPgraftingbut that the differencebetween the redox peakcur-rents was increased,indicating that MIP onITO limiteddiffusionof hexacyanoferrate(II)and(III)ions andelectron transferfromthe ionstotheITO electrode4,10.These resultsdemonstrate thatthe contribution of the graftedlayer totheredoxreaction at thegraphite surfacewas pletelydifferent fromthatof theITOelec-trode.The addition of UFH increased boththe anodic and cathodiurrentsat the MIP-GP electrode,as shownin Fig.3.The depen-dences ofthe anodic and cathodic peak potentialsare shown inFig.4.The anodic peak potentialshifted towardpositive poten-tials butthe cathodicone shiftedtoward negativepotentials withincreasingheparin concentration.The reasonfor thisbehavior isprobablythe decreasein reversibilityoftheelectrochemical reac-tionofhexacyanoferrate(II)and(III)9.The resultsindicate thatthe heparin decelerateselectron transferbetween thehexacyano-ferrates and the MIP-GP surface,or decreasestheelectrodeactivityof the MIP-GP.Noheless,theanodicand cathodiurrents wereincreased by the heparin,suggesting that the effectivearea of theMIP-GP isincreasedby the heparin.3.3.Sensitivity of UFH in whole bloodObtaininga stablecurrent requiredonly about60s aordingtocyclic voltammetrywiththe MIP-GP electrodeeither inblood orsaline.The relationbetween thestabilized peakanodic current ofhexacyanoferrate(II)at the MIP graftedelectrode and UFH concen-tration in saline andin thebovine wholeblood areshownin Fig.5.The coeffi cientof variation(standard deviation/average)was lessthan4%ofthe current ofthe threeMIP-GP electrodes,as showninFig.5(A)for bothsaline andblood.The highreproducibility wasdueto homogenizationofthe MIP-graphite during pasting ratherthanto graftpolymerization usingafluidized bed.The currentincreasedwith the UFH concentration.The UFHsensitivity ofthe currentinwhole bloodwas approximately90%of that in saline.In contrast,the coeffi cientof variationwas2040%ofthe current forthecon-ventional MIP-grafted ITO electrodes5,as shownin Fig.5(B).Inaddition,the currentdecreased uponan increasein UFHin samplefl uidsatthe MIP-ITO.The sensitivity ofthecurrent atthe MIP-ITOto UFHin wholebloodwasless thanhalf ofthatin saline.Theseresults indicate that thereproducibility andstability ofthesen-sitivity inwholeblood atthe MIP-GP issuperior tothose attheMIP-ITO.The MIP-graftedparticleswere mixedduringpastingwithoil,which producedelectrodes thatweremorehomogeneous thanthoseprepared byconventional methodsof graftingMIP ontoaf lat ITOsurface,where it is difficultto ensurehomogenous radicalpolymerization.The relationbetween thecurrent atthe non-imprinted polymergraftedGP(NIP-GP)and theUFH concentrationis shownin Fig.6.The currentwas alsolarger thanthatatthenon-treated GPelec-trode,but was insensitive toheparin.These resultsindicate that theNIP layeralso enhancedthe oxidativecurrent ofhexacyanoferrate(II)and that the imprinted cavity isessential tofurther enhance-mentofthecurrentby heparin.Fig.4.Relation between(A)anodicand(B)cathodicpeakpotentialofcyclicvoltammetryof5mM ferrocyanideatthe MIP-GP electrodeand UFHconcentration inphysiologicalsaline.(n=5).Y.Yoshimi etal./Sensors andActuators B259 (2018)455462459Fig.5.Relation betweenanodic peakcurrent of5mM hexacyanoferrate(II)atthe(A)newly developed MIP-GP electrodeand(B)conventional M