Microcontact-BSA imprinted capacitive biosensor for real-time, sensitive and selective detection of BSA

Microcontact-BSA

imprinted

capacitive

biosensor

for

real-time,

sensitive

and

selective

detection

of

BSA

Gizem

Ertürk

1

,

Dmitriy

Berillo,

Martin

Hedström

2

,

Bo

Mattiasson

2,

*

DepartmentofBiotechnology,LundUniversity,Lund,Sweden

ARTICLE INFO Articlehistory: Received10April2014

Receivedinrevisedform19June2014 Accepted19June2014

Availableonline25June2014 Keywords:

Microcontactimprinting Capacitivebiosensor BSAdetection

ABSTRACT

Ananalyticalmethodispresented,combiningnovelmicrocontactimprintingtechniqueandcapacitive

biosensortechnologyforthedetectionofBSA.Glasscoverslipswereusedforpreparationofprotein

stamps.Themicrocontact-BSAimprintedgoldelectrodeswerepreparedinthepresenceofmethacrylic

acid (MAA)and poly-ethyleneglycol dimethacrylate(PEGDMA)asthecross-linkerbybringing the

proteinstampandthegoldelectrodeintocontactunderUV-polymerization.Real-timeBSAdetection

studieswereperformedintheconcentrationrangeof1.010 20

–1.010 8_{Mwithalimitofdetection}

(LOD)of1.010 19_{M.Cross-reactivitytowardsHSAandIgGwere5and3%,respectively.Theelectrodes}

wereusedfor>70assaysduring2monthsandretainedtheirbindingpropertiesduringallthattime.The

NIP (non-imprinted) electrode was used as a reference. The microcontact imprinting technology

combinedwiththebiosensorapplicationsisapromisingtechnologyforfutureapplications.

ã2014PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/3.0/).

1.Introduction

Molecularimprinting is the technology of creating arti_ficial recognitionsitescomplimentaryinbothformandfunctiontothe “template” molecule [1–4]. Molecularly imprinted polymers (MIPs)areformedbythepolymerizationofafunctionalmonomer aroundthe moleculartemplate in the presenceof cross-linker. MIPs have been used in solid-phase extractions, analytical separations,catalysis,drugdeliverysystemsandasa biorecogni-tionelementinbiosensors[5–11].MIPtechnologyissuccessfully usedfortherecognitionoflowmolecularweighttemplates,but there are still some difficulties in the design of MIPs for macromoleculartemplateslikeproteins[12,13].Duetothis,many researchers have focused on imprinting the template protein directlyontoasubstrate,thuscreatingasubstratesurface,which will berecognized by the target protein[14–16]. Microcontact imprintingisthesurface coatingtechnique usedforemploying recognitioncavitiesforlargemoleculesandassemblies [17–20]. Thegeneralprocedureofthemethoddependsonthe polymeriza-tion between two surfaces – a protein stamp and a polymer support.Inthefirststep,theproteinstampisformedbyadsorption ofthetemplateproteinontothepre-cleanedglasssurface.Then,

theproteinstampisbroughtintocontactwiththesecondsurface, monomer-coated substrate. By this way, thin polymer film is formedonthesupportviaUV polymerization.Asthelast step, templateproteinisremovedfromthesurfaceandspecificprotein recognitionsitesareformedonlyatthesurfaceoftheimprinted support[14_–19].Thismethodhassomeadvantageslikereducing activitylossoftheimprintedmoleculeandinadditionthemethod requires very small amount of template molecule for the imprintingprocess[21,22].Microcontactimprintingmethodhas beenusedforvariousproteins[23–27].

BSA(bovine serumalbumin)isa proteinwiththemolecular weightof66.5kDaandithasmanyusesinbiomedicalapplications andenzymaticreactions.Itisusedtopreventadhesionofenzymes duringapplications[28].Itisagenerallyusedproteinreagentin proteinassays,likeBradfordassay,tomeasuretheconcentrationof aproteininsolution.Furthermore,BSAhasastructuralhomology with HSA (human serum albumin) [29]. Due to this, BSA is frequentlystudiedasamodelproteininsteadofHSA.Moreover, BSAis acommonlyusedtargettoanalyzewhendesigningnew immunochemicalassays.

Determination of micro-quantities of BSA is possible with methodslikeradioimmunoassay(RIA)orenzyme-linked immu-nosorbentassay(ELISA)[30].Therearealsosomedeterminations likeFT-IRspectroscopy,polarographicandfluorimetric measure-ments usedfor BSA detection[28,31,32].Someof the methods requirea labelledreagentlikearadioisotope orenzymelabeled antibody/antigen[30].Someofthemarereallyexpensiveandneed time-consuming, complex procedures. Low selectivity and

* Correspondingauthor.

E-mailaddress:[email protected](B.Mattiasson).

1

DepartmentofBiology,HacettepeUniversity,Ankara,Turkey.

2

CapSenzeHB,MediconVillage,S-22381Lund,Sweden.

http://dx.doi.org/10.1016/j.btre.2014.06.006

2215-017X/ã2014PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

ContentslistsavailableatScienceDirect

Biotechnology

Reports

sensitivityistheanotherdrawbackofthesemethods[33].Direct, label-free,fastandsensitivemeasurementofvariousanalyteswith biosensors has attracted considerable interest [34]. Highly sensitivebiosensorconceptsmakeitpossibletoassay biomacro-molecules at concentrations below the limit of detection of conventionalmethods[35].Capacitivebiosensorsarethe electro-chemicalsensorsthatmeasurechangesinthedielectricproperties whenananalyteinteractswitha biorecognitionelementonthe sensor surface, causing a decrease in the capacitance [36–41]. Capacitivebiosensorshavebeenusedforthedetectionofvarious analyteslikeantigens,antibodies,proteinsandheavymetalions [42_–47].Thesetypesofbiosensorshavealotofadvantageslike inherent rapidity, high sensitivity, simplicity, low cost, easy manipulationandreal-timemeasurementwithoutlabeling.

In the study reported here, a capacitive biosensor with an automated-flowinjectionsystemwasusedforBSAdetection.BSA is most commonly used model proteinin the macromolecular imprintingstudies.However,toourknowledge,thisis thefirst microcontact-BSAimprintingstudyforthedetectionofBSAwith the capacitive biosensor. Microcontact imprinting method was applied for the imprinting of BSA onto the pre-modified gold electrodesurface.Aftermodificationofthegoldelectrodesurface withpoly-tyramineandacryloylchloride,theproteinstampwas broughttogetherwithamixtureofmonomerandcross-linkerin contactwiththeelectrode.Thus,themicrocontactBSAimprints wereintroducedtotheelectrodesurfaceviaUV-polymerization. AfteroptimizationoftheconditionssuchaspH,ionicstrengthand buffertype,BSAdetectionstudieswereperformedusingstandard solutions of BSA at different concentrations. In between each injection,regenerationoftheBSAsensorwasperformedusinga low-pHbuffer[48]. Selectivityof the developedBSA-imprinted electrodewastestedtogetherwithotherproteins(HSA,IgG)and selectivityasaresultofimprintingefficiencywasindicatedwith thecomparisonoftheresultsobtainedfromNIP(non-imprinted) electrode.

2.Materialsandmethods 2.1.Materials

Bovine serum albumin (BSA), tyramine (99%,

HOC6H4CH2CH2NH2) and human serum albumin (HSA) were

obtained from Sigma (Steinheim, Germany). Acryloyl chloride and 1-dodecanethiolwereobtainedfrom Aldrich(Deisenhofen, Germany). Glutaraldehyde 50% (w/v), triethylamine, 3-amino-propyl-triethoxysilane (APTES) and

a

-

a

'-azoisobutyronitrile (AIBN)werepurchasedfromFluka(Buchs,Switzerland).Human gammaglobulin(humanIgG)waspurchasedfromOctapharmaAB (Stockholm,Sweden).Glassmicroscopecoverslips(2440mm) (Menzel-Glaser) were used as the base for protein stamp in microcontact imprinting. All other chemicals used were of analyticalgrade.Allbufferswerepreparedwithwaterprocessed usingareverseosmosisstepwithaMilli-QsystemfromMillipore (Bedford,MA,USA).Priortouse,allbufferswere_filteredthrougha Milliporefilter(poresize0.22

m

m)anddegassedfor1h. 2.2.Preparationofthemicrocontact-bovineserumalbumin(BSA) imprintedelectrodes

The microcontact-BSA imprinted capacitive electrodes were preparedinthreesteps:

(a) Preparationoftheglasscoverslips(proteinstamps): Glasscoverslips(2440mm)wereusedforthepreparationof proteinstampsinthisprocedure.Inthefirststep,coverslipswere

cleaned in 10mL of 1M HCl, de-ionized water,1M NaOH, de-ionizedwaterandethanol,respectivelyinultrasoniccleanerfor 10minineachstep.Aftercleaning,thecoverslipsweredriedwith nitrogengas.

The cleanedcover slips were immersed in 10% (v/v) APTES (3-amino-propyl-triethoxysilane) in ethanol at room tempera-ture for 1h to introduce amino groups on the surface. Subsequently,theelectrodeswererinsedwithethanoltoremove anyunboundAPTESmolecules.For theactivationoftheamino groups on the APTES modified cover slip surface, they were immersed in 5% (v/v) glutaraldehyde (GA) solution in 10mM phosphatebuffer(pH7.4)atroomtemperaturefor2h.Then,the coverslipswererinsedwithphosphatebuffertoremoveexcess GAanddriedwithnitrogengas.Inthelaststep,thecoverslips wereimmersedin0.1mg/mLBSAsolution(inphosphatebuffer, 10mM,pH7.4)at4Cfor24hfortheimmobilizationofBSAonto thesurface.Finally,thecoverslipswerewashedwithphosphate bufferandthen,driedwithnitrogengas.Theywerekeptat4Cin aclosedPetridishuntiluse.

(b)Preparationofthecapacitivegoldelectrodes:

Inthefirststep,goldelectrodeswerewashedwithethanol, de-ionizedwater,acetone,de-ionizedwaterandpiranhasolution(3:1, H2SO4:H2O2,v/v),respectivelyfor10minineachstepinultrasonic

cleaner.Then,theelectrodeswereplasmacleaned(Mod.PDC-3XG, Harrick,NY)for30min.Priortomodificationofthesurfaceofthe electrodes,electropolymerizationoftyraminewasperformedby cyclicvoltammetry(CV)inethanolicsolutionof10mMtyramine withasetpotentialrangeof0–1.5V(vs.Ag/AgCl)andascanrateof 50mVs 1for15scans,asdescribedbefore[45].Bythisway, poly-tyraminewasdepositedontheelectrode,andfreeprimaryamino groupswereintroducedonthesurfaceoftheelectrode.Thecoated electrodeswererinsedwithwateranddriedwithnitrogengas.

In the second step, the electrodes were immersed in a solutioncontaining30mMacryloylchlorideand30mM triethyl-amine(intoluene)overnight,at roomtemperature.Hence, the reactionoftheacryloylchloridewiththeaminogroupsonthe surfaceof theelectrodegeneratedamidegroups.Aftermodi fi-cation,theelectrodewasrinsedwithdistilled wateranddried withnitrogengas.

Cyclic voltammetry (CV) is used to evaluate the degree of insulationoftheelectrodesurfaceaftereachstep.

(c)Microcontact imprinting of BSA onto the capacitive gold electrode:

ThemonomersolutioncontainingMAA(methacrylicacid)and PEGDMA(Poly ethylene glycoldimethacrylate) (1mM: 1.5mM) waspreparedandtheinitiator(AIBN)wasaddedtothissolution. Monomer solution (1.5

m

L) was pipetted onto the electrode surface.Then,theproteinstamp(theglasscoverslip)wasbrought intocontactwiththismonomersolution.Thepolymerizationwas initiatedunderUVlight(365nm,400W)andcontinuedfor15min. Afterpolymerization,thecoverslipwasremovedandanytemplate protein(BSA)thatgotstuckontheelectrodesurfacewaseluted away(Fig.1).Thiselution/washingwas doneasa securitystep since theprint proteinwas immobilized totheglass plateand wouldinprinciplestayonthatplateandthusberemovedwhen theplatewastakenaway.Finally,theelectrodewasimmersedin 1-dodecanethiol(10mMinethanol)for20mininordertocover barepartsofthegoldsurface.Whennotinuse,electrodeswere keptat4CinaclosedPetridishfilledwithnitrogengas.

Non-imprinted(NIP)electrodeswerepreparedwiththesame procedurewithoutimmobilizationofthetemplateprotein,BSA, ontotheglasscoverslips.

2.3.RealtimeBSAdetectionwiththemicrocontact-BSAimprinted electrode

2.3.1.Capacitivemeasurement:

The capacitive measurements were performed with the automated flow-injection system, as described by Erlandsson et al. [43]. The BSA imprinted electrode was inserted in the electrochemicalflowcellandconnectedtotheplatinumauxiliary and reference electrodes. The capacitance measurement was performed via current pulse method, as described previously. The capacitancewas calculated as a function of time from the resultingpotentialprofile(Fig.2).

Priortoanalysis,aregenerationsolution(25mMglycine–HCl, pH2.5)wasinjectedtothesystemtocleanthesurface,anditwas repeatedbetweeneachanalyteinjections.Afterastablebaseline wasrecorded,thestandardsolutionsofdifferentconcentrationsof BSA (1.010 20_–1.010 8_{M) were injected sequentially. The}

bindingofthetargetprotein(BSA)totheimprintedcavitiesonthe surfaceoftheelectroderesultedina decreaseoftheregistered capacitance and the change was calculated automatically by CapSenzeSmartSoftware(CSS).Inalloftheanalysis,theflowrate was100

m

L/minandtheinjectedsamplevolumewas250

m

L.

Theeffectsoftype(phosphateandTris–HClbuffers,10mM), pH(6.0–8.0)andionicstrengthoftherunningbuffertotheBSA detectionwere evaluated bymonitoring the changeof capaci-tance signal at the same standard concentration of BSA (1.010 10_M).

2.3.2.Selectivityofthemicrocontact-BSAimprintedelectrode InordertoshowtheselectivityoftheBSAimprintedelectrode, the responsesof thecapacitive system against the competitive proteinsHSAandIgGweremonitored.Theproteinsolutionswere appliedinsingularmannerandalso,mixedsolutionsofHSA,IgG and BSA were studied in competitive manner. The protein concentration was 1.010 10_{M for each protein during the}

analysis.Samplesofsolutionsoftheindividualproteinswerealso analyzedusingNIP-electrodes.

2.3.3.Reproducibility

BSA wasdetected repeatedly, usingthe assaycycle; equili-bration-injection-regeneration,for70times.Thereproducibility of the assay was evaluated by monitoring the change in capacitanceatthesameconcentrationofstandardBSAsolution, 1.010 10M.

Fig.1.Schematicrepresentationofthemicrocontact-BSAimprintedcapacitivebiosensor.(A)Preparationoftheglasscoverslips(proteinstamps),(B)preparationofthe capacitivegoldelectrodes,(C)microcontactimprintingofBSAontothegoldelectrodesurfaceviaUV-polymerization,(D)removaloftemplateprotein(BSA)fromthe electrodesurface.

3.Resultsanddiscussion

3.1.Electrochemicalcharacterizationofthebiosensorsurface Properinsulationoftheelectrodesurfaceisanimportantstep inthecapacitivebiosensorassay[40–47].Cyclicvoltammetry(CV) isthegenerallyusedmethodinthepresenceofapermeableredox coupletoevaluatethedegreeofinsulationoftheelectrodesurface. As shown in Fig. 3, the degree of insulation increased after modificationoftheelectrodesurfacewithtyramineandacryloyl chloride. The density of the surface after each step increased, compared to that of the bare surface. Finally, treatment with 1-dodecanethiolreducedtheredoxcurrentssubstantiallyandthe surfacewascompletelyblocked.Thecyclicvoltammetryresults

showthatthesurfaceoftheelectrodeisinsulatedwellanditcan beusedinthesubsequentcapacitivemeasurements.

3.2.Optimizationofthecapacitivebiosensor

TheBSAimprintedelectrodewasplacedintheelectrochemical flowcell andit was connectedtotheautomatedflow-injection system.

The operating conditions of the capacitive system were optimizedfortype,pHandionicstrengthoftherunningbuffer.

Fortheinfluenceoftypeofbuffer;10mMphosphateand10mM Tris–HCl; were tested. The pH of the buffer solution was investigated in therange of 6.0–8.0. StandardBSA solutions of 1.0_10 10_{Mwerepreparedineachofthesebuffersandinjected}

Fig.3.Cyclicvoltammogramsrecordedinasolutionof100mMKClcontaining100mMK3[Fe(CN)6]using:bareelectrode(red-a),goldelectrodeafterelectropolymerization

oftyramine(green-b),goldelectrodeaftersurfacemodificationwithacryloylchloride(black-c),aftertreatmentwith1-dodecanethiol(blue-d).Thescanrangewasfrom 0.3 to0.8V(vs.Ag/AgCl),atascanrateof0.1Vs 1

.

Fig.2. Schematicdiagramshowingthecapacitiveimmunosensorwithanautomatedflowinjectionsystem.

intothesystem.Therewasnosignificantcapacitancedifference betweenthesebuffersinthestudiedBSAconcentration(Fig.4(A)). However,phosphatebufferatpH7.4gaveamorestablebaseline and thus, the capacitance change was more clear. Due to the results,10mMphosphatebuffer(pH7.4)wasusedastherunning bufferinthesubsequentstudies.

The effect of ionic strength of the running buffer was also investigated for the optimization of the conditions. The effect ofionicstrengthwasstudiedbyaddingdifferentconcentrationsof NaCl to the running buffer for the standard BSA solution of 1.010 10_{M.AsshowninFig.4(B),thechangeinthecapacitance}

decreasedwiththeincreasingionicstrengthofthemedium.Thus, maximumcapacitancechangewasobservedintherunningbuffer whichdidnotcontainanysalt.

3.3.Capacitivemeasurements-realtimeBSAdetection

AfteroptimizationofBSAdetectionconditions,real-timeBSA detectionstudies fromaqueousBSA solutionswerecarried out withtheautomatedflow-injectioncapacitivesystemasdescribed inSection3.2.

TheBSAimprintedelectrodewasplacedintheelectrochemical flowcelland it wasconnectedtotheautomatedflow injection system.

Therunningbufferwascontinuouslypassedthroughtheflow system by the pump at a flow rate of 100

m

L/min. Standard solutions of BSA in the concentration range of 1.010 20_–

1.010 8M were prepared in the same running buffer and sequentiallyinjectedintothesystem.Phosphatebuffer(10mM,pH 7.4)wasusedasrunningbuffer.Eachsolutionwasinjectedfor3 timesthroughtheflowsystem.Afterinjectionandequilibration

periods,intotal15min,regenerationbufferwasinjectedduring 2.5minbeforerunningbufferwasusedforreconditioninguntilthe baseline signal was achieved. The decrease in capacitance increasedwiththeincreasingconcentrationsofBSA,asexpected (Fig.5(A)).Inordertoobtainareliableanalyticalsignal,anaverage ofthelastfivecapacitancereadingswascalculated.Thegraphwas obtainedbyplottingthecapacitancechange( pFcm 2_)versusthe

logarithmofBSAmolarconcentration(Fig.5(B)).Analmostlinear relationship was obtainedbetween1.010 18_{and 1.010} 8_M

and the limit of detection (LOD) was determined to be1.010 19M,based onIUPACguidelines.Duetotheresults, thecapacitancechangeasafunctionoflogconcentrationofthe analyte inthe studiedconcentrationrangewas linear withthe regressionequationofy=52.27x+1805.2(R2_{=0.9477).Whennot}

inuse,theelectrodeswerestoredat4_{CinaclosedPetridish.} 3.4.SelectivityoftheBSAimprintedelectrodeagainstcompeting proteins

InordertotesttheselectivityoftheBSAimprintedelectrode, HSA and IgG were selected as competing proteins. For this purpose,theinteractionsbetweentheaqueoussolutionsofBSA, HSAandIgGmoleculesandpre-mixedproteinsolutionshaving BSA/HSA,BSA/IgG,BSA/HSA/IgGandtheBSAimprintedelectrode were also investigated. As seen from Fig. 6(A), the change in capacitance wasvery low for the standard HSA (1.010 10_M,

10mMphosphatebuffer,pH7.4)andIgGsolutions(1.010 10_M,

Fig.4. (A)EffectoftypeandpHoftherunningbufferonBSAdetection(BSA concentration: 1.01010

M; flow rate: 100mL/min; sample volume: 250mL; regenerationbuffer: 25mMglycine–HCl,pH:2.5; T:25_{C). (B)Effectofionic}

strengthoftherunningbufferonBSAdetection(BSAconcentration:1.010 10

M; flowrate:100mL/min;samplevolume:250mL;runningbuffer:10mMphosphate, pH:7.4;regenerationbuffer:25mMglycine–HCl,pH:2.5;T:25C).

Fig.5.(A)CapacitanceoftheBSAimprintedelectrodevs.BSAconcentrations(M) under optimum conditions (Flow rate: 100mL/min; sample volume: 250mL; runningbuffer:10mMphosphate,pH:7.4;regenerationbuffer;T=25C).Att=0 thetargetBSAanalytewasinjectedtotheflowcellcontainingthegoldtransducer containingthemicrocontactimprintingsurface.Priortoeachinjection,thesurface wasregeneratedusing25mMglycine–HCl,pH2.5(notshowninthisgraph).(B) Capacitancechangevs.logarithmofBSAconcentrationsforthemicrocontact-BSA imprintedelectrodeunderoptimumconditions(Flowrate:100mL/min;sample volume:250mL;runningbuffer:10mMphosphate,pH:7.4;regenerationbuffer: 25mMglycine–HCl,pH:2.5;T:25C).

10mM phosphate buffer, pH 7.4) compared to that from the standardBSAsolution(1.010 10M,10mMphosphatebuffer,pH 7.4).Asa secondconfirmation oftheselectivity,thepre-mixed proteinsolutionsofBSA(1.010 10_{M)/HSA(1.010} 10_M),BSA

(1.010 10M)/IgG (1.010 10M) and BSA (1.010 10M)/HSA (1.010 10_{M)/IgG(1.010} 10_{M)wereinjectedintothe}

capaci-tivesystem.Pre-mixedproteinsolutionscausedalower capaci-tance changecompared tothe singularstandard BSA solution. Thisdifferencecouldbestemmedfromthecompetitiveeffectsof HSAandIgGproteins.However,itcouldbeclearlyobservedthat, theBSAimprintedelectrodeshowedhighaffinityforthetemplate protein (BSA) and the electrode could detect BSA in singular mannerand also under competitive conditions.The calculated selectivity coefficients are summarized in Table 1. Due tothe results,the BSA imprinted capacitive electrode exhibited good selectivity for the template protein, BSA, compared to other proteinswithcross-reactivitiesof5and3%againstHSAandIgG, respectively.

3.5.RealtimeBSAdetectionwiththeNIP(non-imprinted)electrode RealtimeBSAdetectionwasalsoperformedwith NIP-electro-des. Standard BSA solutions in the concentration range of 1.010 20_–1.010 6_{M were prepared in the running buffer}

(10mMphosphate,pH7.4)andtheanalyseswereidenticaltothat withtheimprintedelectrodes.Nochangeinthecapacitancecould be observed for the lower BSA concentrations. The limit of detection (LOD) was determined to be 1.010 10M, based on IUPACrecommendations.

To evaluate the analytical efficiency of the imprinting procedure, standard BSA (1.010 10M), HSA (1.010 10M) andIgG(1.010 10_{M)solutionswereinjectedtothecapacitive}

systeminaserialmanner(Fig.6(B)).Itwasobservedthat,there wasnosignificantdifferenceinthecapacitancechangewiththe changingproteinsfor theNIP electrode.Thechange in capaci-tancewasalmostinthesamevalueforallthree.Thecalculated selectivitycoefficientsforNIPelectrodewere1.07and0.376for BSA,comparedtoHSAandIgG,respectively(Table1).Therewasa big difference in the selectivity coefficients of NIP and BSA imprintedelectrode.Theseresultsindicatethat,theimprintingof theproteinontotheelectrodesurfacegeneratescavitieshighly specific for the template protein. In addition, the imprinting efficiencyvalueswerecalculatedandtheresultsaresummarized

in Table 1. The enhanced selectivity coefficients of the BSA

imprintedcapacitivesensoraccordingtocompetingproteinsare approximately 21 and 85 for BSA against HSA and IgG, respectively.

3.6.Reproducibility

The BSA imprinted electrodes were evaluated in terms of reproducibilitybymonitoringthecapacitancechange( pFcm 2₎

atthesameconcentrationofstandardBSAsolution(1.010 10M) for 70 times.After injection and equilibrationperiods, in total 15min,regeneration bufferwas injected during 2.5min before running buffer was used for reconditioning until the original baselinesignalwasachieved.ThecapacitanceoftheBSAimprinted sensorversusthenumberofinjectionsisshowninFig.7.TheBSA detection activity of the sensor retained about the same level during the injections. Microcontact imprinting method has an advantage of reducing activity loss of the imprinted molecule duringtheapplication[21,22].Inthisstudy, thesameelectrode was used in whole experiments and triplicate injections were madeforeachanalysis.TheresultsshowthattheBSAimprinted electrodecanbereusedforBSAdetectionwithgood reproducibili-tywithoutanysignificantlossintheactivity.Atotalof80assays during a period of 2 months were carried out on the same electrode,stillwithretainedperformance.

Table1

Selectivitycoefficientsofmicrocontact-BSAimprintedelectrodeandnon-imprinted(NIP)electrode[BSA:bovineserumalbumin,HSA:humanserumalbumin,IgG:human gammaglobulin,D:capacitancechangeforthemicrocontact-BSAimprintedandNIPelectrode,k:selectivitycoefficientforBSAversuscompetingproteins,k’:relative selectivitycoefficientformicrocontact-BSAimprintedelectrodeversusNIPelectrode].

Capacitancechange,D Capacitancechange,D Selectivitycoefficient,k Selectivitycoefficient,k Relativeselectivitycoefficient,k’

Protein Imprinted Non-imprited(NIP) Imprinted Non-imprinted(NIP)

BSA 987 32 – – –

HSA 44 30 22.43 1.07 20.96

IgG 31 85 31.84 0.376 84.68

Fig.6.(A)Selectivityofmicrocontact-BSAimprintedcapacitivebiosensoragainst competitorproteins;HSA(humanserumalbumin)andIgG(immunoglobulinG)in singularandcompetitivemanner(proteinconcentration:1.01010_M;flowrate:

100mL/min;samplevolume:250mL;runningbuffer:10mMphosphate,pH:7.4; regenerationbuffer:25mMglycine–HCl,pH:2.5;T:25_{C).(B)Imprintingefficiency}

ofthemicrocontact-BSAimprintedelectrodevs.NIP(non-imprinted)electrode (protein concentration: 1.010 10

M;flow rate: 100mL/min; sample volume: 250mL;runningbuffer:10mMphosphate,pH:7.4;regenerationbuffer:25mM glycine–HCl,pH:2.5;T:25_C).

4.Conclusion

Thisstudywas carriedouttoevaluate thepossibilitytouse microcontactimprintingof proteinmoleculeson electrodesfor capacitivebiosensor measurements.Asmodel target,theacidic BSAproteinwaschosen.WiththeacidicfunctionalmonomerMAA chosen in the study, it could beexpected that somerepulsion mightoccurwhichcouldreducethesurfaceaffinityinthebinding step. However, since the electrode should be utilized for repetitivelyanalyticalcycles,thissystemwaschosentofacilitate regeneration (complex dissociation) of the surface rather than optimizingbindingstrength.Infact,bothselectivityandstability provedtobeatanacceptablelevel.

Thisisapromisingmethodthatcanbeutilizedforthecreationof biorecognitionimprintsexhibitinghighselectivityandoperational stabilityforthetargetusingthebiosensortechnology.Inthefuture, thecapacitivebiosensortechnology combinedwith themicrocontact imprintingmethodcanbeusedinvariousapplications,includingthe diagnosis of diseaseswhere real-time,rapid,highly selectiveand very sensitivedetectionofaknownbiomarkerisrequired.

Acknowledgements

GEwassupportedbyafellowshipfromHacettepeUniversity, Turkey.The support fromProf. Adil Denizli and Prof. M.Aşkın Tümer,bothatHacettepeUniversity,isgratefullyacknowledged.

The project was also supported by the Swedish Research Council and the instrument used for analysiswas a loan from CapSenzeHB,Lund,Sweden.

References

[1]R.Arshady,K.Mosbach,Synthesisofsubstrate-selectivepolymersby host-guestpolymerization,Macromol.Chem.182(1981)687–692.

[2]G.Wulff,A.Sarhan,K.Zabrocki,Enzyme-analoguebuiltpolymersandtheiruse fortheresolutionofracemates,TetrahedronLett.14(1973)4329–4332. [3]G.Wulff,Enzyme-likecatalysisbymolecularlyimprintedpolymers,Chem.

Rev.102(2002)1–27.

[4]K.Haupt,Molecularlyimprintedpolymers,thenextgeneration,Anal.Chem.1 (2003)376–383.

[5]L.I.Andersson,Molecularimprinting:developmentsandapplicationsinthe analyticalchemistryfield,J.Chromatogr.BBiomed.Sci.Appl.745(2000)3–13. [6]C.Alexander,L.Davidson,W.Hayes,Imprintedpolymers:artificialmolecular recognitionmaterialswithapplicationsinsynthesisandcatalysis,Tetrahedron 59(2003)2025–2057.

[7]X. Li, S.M. Husson, Two-dimensional molecular imprinting approach to produceopticalbiosensorrecognitionelements,Langmuir22(2006)9658– 9663.

[8]G.Ertürk,L.Uzun,M.A.Tümer,R.Say,A.Denizli,FabfragmentsimprintedSPR biosensor for real-time human immunoglobulin G detection, Biosens. Bioelectron.28(2011)97–104.

[9]K.Haupt,K.Mosbach, Molecularly imprintedpolymers andtheir usein biomimeticsensors,Chem.Rev.100(2000)2495–2504.

[10]K.Haupt,Molecularlyimprintedpolymersinanalyticalchemistry,Analyst126 (2001)747–756.

[11]L.Uzun,R.Say,S.Ünal,A.Denizli,Productionofsurfaceplasmonresonance basedassaykitforhepatitisdiagnosis,Biosens.Bioelectron.24(2009)2878– 2884.

[12]D.R.Kryscio,N.A.Peppas,Surfaceimprintedthinpolymerfilmsystemswith selectiverecognitionforbovineserumalbumin,Anal.Chim.Acta718(2012) 109–115.

[13]N.Bergmann, N.A.Peppas,Molecularlyimprintedpolymerswithspecific recognitionformacromoleculesandproteins,Prog.Polym.Sci.33(2008)271– 288.

[14]H.D. Inerowicz, S. Howell, F.E. Regnier, R. Reifenberger, Multiprotein immunoassayarraysfabricated bymicrocontact imprinting,Langmuir18 (2002)5263–5268.

[15]Y.W.Chen,J.Rick,T.C.Chou,Asystematicapproachtoformingmicro-contact imprintsofcreatinekinase,Org.Biomol.Chem.7(2009)488–494. [16]P.C. Chou, C-reactive protein thin-film molecularly imprinted polymers

formedusingamicro-contactapproach,Anal.Chim.Acta542(2005)20–25. [17]P.C.Liao,Y.C.Tyan,C.Y.Wang,J.F.Hsu,T.C.Chou,H.Y.Lin,Assessingthebinding selectivityofmolecularlyimprintedpolymerartificialantibodiesbymass spectrometry-basedprofilingsystem,J.Biomed.Mater.Res.A91A(2009)597– 604.

[18]H.Y.Lin,C.Y.Hsu,J.L.Thomas,S.E.Wang,H.C.Chen,T.C.Chou,Themicrocontact imprintingofproteins:theeffectofcross-linkingmonomersforlysozyme, ribonucleaseAandmyoglobin,Biosens.Bioelectron.22(2006)534–543. [19]J.O.Foley,E.Fu,L.J.Gamble,P.Yager,Microcontactprintedantibodiesongold

surfaces:function,uniformityandsiliconecontamination,Langmuir24(2008) 3628–3635.

[20]L.Chen,S.Xu,J.Li,Recentadvancesinmolecularimprintingtechnology: currentstatus,challengesandhighlightedapplications,Chem.Soc.Rev.40 (2011)2922–2942.

[21]G.Şener,E.Özgür,A.Y.Rad,L.Uzun,R.Say,A.Denizli,Rapidreal-timedetection ofprocalcitoninusingamicrocontactimprintedsurfaceplasmonresonance biosensor,Analyst138(2013)6422–6428.

[22]B. Osman,L. Uzun, N.Beşirli,A.Denizli,Microcontactimprintedsurface plasmonresonancesensorformyoglobindetection,Mat.Sci.Eng.C33(2013) 3609–3614.

[23]H.Y. Lin, J. Rick, T.C. Chou, Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer formed using a micro-contact imprintingmethod,Biosens.Bioelectron.22(2007)3293–3301.

[24]C.Y.Hsu,H.Y.Lin,J.L.Thomas,T.C.Chou,Synthesisofandrecognitionby ribonucleaseAimprintedpolymers,Nanotechnology17(2006)77–83. [25]P.J.Yoo,S.J.Choi,J.H.Kim,D.Suh,S.J.Baek,T.W.Kim,H.H.Lee,Unconventional

patterningwithamodulus-tunablemold:fromimprintingtomicrocontact imprinting,Chem.Mater.16(2004)5000–5005.

[26]A.P. Quist, E. Pavlovic, S. Oscarsson, Recent advances in microcontact imprinting,Anal.BionalChem.381(2005)591–600.

[27]H.W.Chien,W.B.Tsai,Fabricationoftunablemicropatternedsubstratesforcell patterningviamicrocontactprintingofpolydopaminewithpoly(ethylene imine)-graftedcopolymers,ActaBiomater.8(2012)3678–3686.

[28]Y.Jinghua,W.Fuwei,Z.Congcong,Y.Mei,Z.Xiaona,W.Shaowei,Molecularly imprinted polymeric microspheres for determination of bovine serum albumin based on flow injection chemiluminescence sensor, Biosens. Bioelectron.26(2010)632–637.

[29]J.S. Mandeville,H.A.Tajmir-Riahi, Complexes ofdendrimers with bovine serumalbumin,Biomacromolecules11(2010)465–472.

[30]T.-Y.Lin,C.-H.Hu,T.-C.Chou,Determinationofalbuminconcentrationby MIP-QCMsensor,Biosens.Bioelectron.20(2004)75–81.

[31]D.B.Luo,J.G.Lan,C.Zhou,C.X.Luo,PolarographicbehaviourofCo(II)-BSAor HSAcomplexinthepresenceofaguanidinemodifier,Anal.Chem.75(2003) 6346–6350.

[32]A.Wittemann,M.Ballauf,Secondarystructureanalysisofproteinsembedded insphericalpolyelectrolytebrushesbyFT-IRspectroscopy,Anal.Chem.76 (2004)2813–2819.

[33]C.Berggren,B.Bjarnason,G.Johansson,Capacitivebiosensors,Electroanalysis 13(2001)173–180.

[34]B.Mattiasson,K.Teeparuksapun, M.Hedström,Immunochemical binding assaysfordetectionandquantificationoftraceimpuritiesinbiotechnological production,TrendsBiotechnol.28(2009)20–27.

[35]P.Bataillard,F.Gardies,N.Jaffrezic-Renault,C.Martelet,Directdetectionof immunospeciesbycapacitancemeasuremets,Anal.Chem.60(1998)2374– 2379.

[36]W.Limbut,M.Hedström,P.Thavarungkul,P.Kanatharana, B.Mattiasson, Capacitivebiosensorfordetectionofendotoxin,Anal.Bioanal.Chem.389 (2007)517–525.

[37]M.Stelzle,E.Sackmann,Sensitivedetectionofproteinadsorptiontosupported lipid bilayers by frequency-dependent capacitance measurements and microelectrophoresis,Biochim.Biophys.Acta981(1989)135–142. [38]V.Billard,C.Martelet,P.Binder,J.Therasse,Toxindetectionusingcapacitance

measurementsonimmunospeciesgraftedontoasemiconductorsubstrate, Anal.Chim.Acta249(1991)367–372.

Fig.7.Reproducibilityofthemicrocontact-BSAimprintedcapacitivebiosensor (BSAconcentration:1.010 10

M;flowrate:100mL/min;samplevolume:250mL; runningbuffer:10mMphosphate,pH:7.4;regenerationbuffer:25mMglycine– HCl,pH:2.5).

[39]M.Labib,M.Hedström,M.Amin,B.Mattiasson,Anovelcompetitivecapacitive glucose biosensor based on concanavalin A-labeled nanogold colloids assembled on a polytyramine-modified gold electrode, Anal. Chim.Acta 659(2010)194–200.

[40]M.Labib,M.Hedström,M.Amin,B.Mattiasson,Acapacitiveimmunosensorfor detectionofcholeratoxin,Anal.Chim.Acta634(2009)255–261.

[41]M.Hedström,I.Y.Galaev,B.Mattiasson,Continuousmeasurementsofabinding reactionusingacapacitivebiosensor,Biosens.Bioelectron.21(2005)41–48. [42]M.Labib,M.Hedström,M.Amin,B.Mattiasson,Amultipurposecapacitive

biosensor for assay and quality control of human immunoglobulin G, Biotechnol.Bioeng.104(2009)312–320.

[43]D. Erlandsson,K.Teeparuksapun,B. Mattiasson,M.Hedström,Automated flow-injectionimmunosensorbased oncurrentpulsecapacitive measure-ments,Sens.Actuat.B190(2014)295–304.

[44]S.Basheer,D.Samyn,M.Hedström,M.S.Thakur,B.L.Persson,B.Mattiasson,A membrane protein based biosensor: use of a phosphate-H+

symporter

membrane protein (Pho84) in the sensing of phosphate ions, Biosens. Bioelectron.27(2011)58–63.

[45]K. Teeparuksapun, M. Hedström, E.Y. Wong, S. Tang, I.K. Hewlett, B. Mattiasson, Ultrasensitive detection of HIV-1 p24 antigen using nano-functionalizedsurfacesinacapacitiveimmunosensor,Anal.Chem.82(2010) 8406–8411.

[46]K. Teeparuksapun, M. Hedström, P. Kanatharana, P. Thavarungkul, B. Mattiasson,Capacitiveimmunosensorfordetectionofhostcellproteins,J. Biotechnol.157(2012)207–213.

[47]M.Labib,HedströmM,AminM,MattiassonB,Acapacitiveimmunosensorfor detectionofstaphylococcalenterotoxinB,Anal.Bioanal.Chem.393(2009) 1539–1544.

[48]S.Loyprasert,M.Hedström,P.Kanatharana,P.Thavarungkul,B.Mattiasson, Sub-attomolar detection of cholera toxin using a label-free capacitive biosensor,Biosens.Bioelectron.27(2010)41–48.