ContentslistsavailableatScienceDirect
Sensors
and
Actuators
B:
Chemical
j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b
Non-enzymatic
assay
for
glucose
by
using
immobilized
whole-cells
of
E.
coli
containing
glucose
binding
protein
fused
to
fluorescent
proteins
Ana
Charneca
a,
Amin
Karmali
a,∗,
Manuela
Vieira
baChemicalEngineeringandBiotechnologyResearchCenterandDepartmentalAreaofChemicalEngineeringofInstitutoSuperiordeEngenhariadeLisboa,
R.ConselheiroEmídioNavarro,1,1959-007Lisboa,Portugal
bElectronicsTelecommunication&ComputerDepartmentofInstitutoSuperiordeEngenhariadeLisboa,R.ConselheiroEmídioNavarro,1,
1959-007Lisboa,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received23June2014
Receivedinrevisedform5June2015 Accepted5June2015
Availableonline20June2015 Keywords:
Fluorescentindicatorproteins Onionmembranes
Geneticallyencodednanosensor3.2mM Highthroughputglucoseassay
Immobilizationon96-wellmicrotiterplates FRET
a
b
s
t
r
a
c
t
Glucosemonitoringinvivoisacrucialissueforgainingnewunderstandingofdiabetes.Glucosebinding protein(GBP)fusedtotwofluorescentindicatorproteins(FLIP)wasusedinthepresentstudysuchas FLIP-glu-3.2mM.RecombinantEscherichiacoliwhole-cellscontaininggeneticallyencodednanosensors aswellascell-freeextractswereimmobilizedeitheroninnerepidermisofonionbulbscaleoron 96-wellmicrotiterplatesinthepresenceofglutaraldehyde.GlucosemonitoringwascarriedoutbyFörster ResonanceEnergyTransfer(FRET)analysisduethecyanoandyellowfluorescentproteins(ECFPand EYFP)immobilizedinboththesesupports.
TherecoveryoftheseimmobilizedFLIPnanosensorscomparedwiththefreewhole-cellsandcell-free extractwasintherangeof50–90%.Moreover,thedatarevealedthattheseFLIPnanosensorscanbe immobilizedinsuchsolidsupportswithretentionoftheirbiologicalactivity.Glucoseassaywasdevised byFRETanalysisbyusingthesenanosensorsinrealsampleswhichdetectedglucoseinthelinearrange of0–24mMwithalimitofdetectionof0.11mMglucose.Ontheotherhand,storageandoperational stabilitystudiesrevealedthattheyareverystableandcanbere-usedseveraltimes(i.e.atleast20times) withoutanysignificantlossofFRETsignal.Toauthor’sknowledge,thisisthefirstreportontheuseof suchimmobilizationsupportsforwhole-cellsandcell-freeextractcontainingFLIPnanosensorforglucose assay.Ontheotherhand,thisisanovelandcheaphighthroughputmethodforglucoseassay.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Glucoseisanimportantphysiologicalanalyteinvolvedinmajor catabolic pathwayssuch as glycolysis and oxidative phosphor-ylation. Therefore, the maintenance and regulation of glucose concentrationarecriticalissuesforproperphysiologicalfunction [1].Continuousbloodglucosemonitoringisakeyissueof mod-erndiabetestreatment,particularlyintype1diabetesaswellas insulin-dependenttype2diabetes[2].Thesearchfortheideal glu-cosesensorhasbeena long-timegoalofmany researchersand asaresult,manyglucosesensorshavebeendevelopedinthelast few decades[3].Although many glucose sensing systems have foundinvivo applications,theneedfora reliable,specific, sen-sitiveandstableglucosesensorishighlyrequired[4].Thereare many parameters that affect thedevelopment of an optimized glucosesensorsuchasselectivity,linearrange,biocompatibility,
∗ Correspondingauthor.Tel.:+351218317052;fax:+351218317267. E-mailaddress:akarmali@deq.isel.ipl.pt(A.Karmali).
responsetime,reproducibilityandreversibilityofsignal[5]. More-over,theseenzymeelectrodesarebasedonachemicalconversion [6]withaconsumptionofglucoseandO2andproductionof
hydro-genperoxideandd-gluconicacidinvivo.Apromisingalternative to electrochemistry has been investigated by several research groupswhichisfluorescence-basedglucosesensing[2,7,8].This isapowerfulmethodsuitableforfast,sensitive,reagentless,and non-invasivedetectionofneutralanalytessuchasglucose[7–9].
Glucosebindingprotein(GBP)hasbeenusedforglucosesensing by severalresearchworkers [8]. Maturegenetically engineered GBPwasfusedtoenhancedyellowfluorescentprotein(EYFP)and enhanced cyanofluorescent protein (ECFP)containing histidine affinitytagforglucosesensingin plants[10].Thelevelsof glu-cosewerequantifiedbyFluorescenceResonanceEnergyTransfer (FRET)measurementswhich allowthedeterminationofratioof emissionintensityofEYFP/ECFP[11].Althoughseveralresearch workershaveusedthesegeneticallyencodednanosensorsfor glu-coseassay,theywereusedinasolubleandpurifiedform[10].Inthe lastdecade,thereisanincreasinginterestonwhole-cellbiosensors [12–14]sincemicrobialcellscanbegeneticallymanipulatedtobe http://dx.doi.org/10.1016/j.snb.2015.06.037
usedaswhole-cellbiosensorsforawiderangeofbiomoleculesin ordertooptimizethesensitivity,selectivityandrobustness.Onthe otherhand,thenatureofimmobilizationsupportsusedandthe highthroughputassaymethodarealsocriticalissuesforagood glucosebiosensor.
Therefore, the present work involves the immobilization of recombinantwhole-cells andcell-free extractcontaining genet-ically encoded biosensors on onion membranes and 96-well microtiterplates.Thesenanosensorswillbeusedforglucoseassay eitherinfreeorimmobilizedforms.
2. Materialsandmethods
2.1. Chemicals
Glutaraldehyde, Coomassie Blue dye, 96-well tissue culture microtiterplates(polystyrene),imidazole,ampicillin,glucose oxi-daseandperoxidasewerepurchasedfromSigma–Aldrich(St.Louis, MO, USA). LB medium components were supplied by HiMedia Laboratories(Mumbai,India).Onionswereobtainedfromalocal supermarket.Allotherchemicalsusedwereofanalyticalgrade. 2.1.1. Recombinantplasmidscontaininggeneticallyencoded glucosenanosensors
Recombinant plasmids containing FLIP-glu- 3.2mM was obtainedfromAddgene,USA.Thisnanosensorhadadissociation constant(Kd)of3.2mMforglucose[15].
2.2. Methods
2.2.1. Maintenanceandgrowthconditionsofrecombinant Escherichiacolistrains
RecombinantplasmidcontainingFLIP-glu-3.2mMwasusedto transformE.colistrainsandtherecombinantstrainsweregrownin solidLuriaBertani(LB)mediumcontaining100g/mlampicillinof culturemediumat37◦Cfor24hinordertoobtainsinglecolonies. 2.2.2. Productionoffluorescentindicatorprotein(FLIP)
nanosensors
TheproductionandextractionofFLIPnanosensorsfrom recom-binantE.coliwascarriedoutasdescribedpreviouslywithmajor modifications[10].Briefly,singlecoloniesofrecombinantE.coli containingFLIP-glu-3.2mMweregrowninLBmediumcontaining 100g/mlampicillininthedarkat21◦C,150rpmfor2–3days.The cellswereharvestedbycentrifugationat10,000rpmfor5minat 4◦C,washedwithsalineandcentrifugedagainatsamespeed.The supernatantwasdiscardedandthepelletwasstoredat−20◦C.
Thecells werethawed,resuspendedin 2volumesof 20mM Tris–HClbufferpH8.0containing1mMbenzamidineandsonicated at4◦Cfor30s,forthreetimesandcentrifugedat19,000rpmfor1h. Thesupernatantwasrecoveredandusedasthecell-freeextract whichwasasourceofFLIPnanosensors.
2.2.3. ImmobilizationofFLIPglucosenanosensors
Asfarasimmobilizationoninnerepidermisofonionbulbscale isconcerned,itwascarriedoutaspublishedpreviouslywithmajor modifications[16].Preliminaryexperimentswerecarriedoutin ordertooptimizeseveralparameterssuchastheamountof whole-cells,cell-freeextract,concentrationofglutaraldehydeanddrying timeonthesupport.Briefly,circularmembraneoftheepidermis werecut(5–10mmdiameter)and suitablealiquotsof recombi-nantE.colicellsandcell-freeextractsin20mMTris–HClbufferpH 8.0containing1mMbenzamidineweretransferredtothese mem-branes,driedfor1hatroomtemperature.Asuitableamountof glutaraldehyde(8l)wasaddedtothemembraneandincubated atroomtemperaturefor45min.Subsequently,membraneswere
washedseveraltimeswith20mMTris–HClbufferpH8.0 contain-ing1mMbenzamidineandstoredinthesamebufferat4◦C.Asfar asfluorescencemeasurementsareconcerned,thesemembranes weretransferredtoa96-wellmicrotiterplatecontaining200l of50mMphosphatebufferpH7.0containing1mMbenzamidine perwellandreadingswerecarriedoutinamicrotiterplatereader. Thesemembraneswerestoredat4◦Cinthesamebuffersystem andtheywerere-usedseveraltimes.Regardingthe immobiliza-tionofwhole-cellsandcell-freeextractsontissueculture96-well microtiterplates,suitablealiquotsofthesebiosensorswere trans-ferredtotheseplatesanddriedovernightatroomtemperature. Subsequently,appropriateconcentrationofglutaraldehyde(20l) wasaddedtothewellsandthesameprocedurewasfollowedas describedabove.Thesemicrotiterplateswerestoredat4◦Cinthe samebuffersystemandtheywerere-usedseveraltimes.
2.2.4. Spectrameasurementoffreeandimmobilizedglucose nanosensors
Fluorescent measurements were carried out eitheron spec-trofluorimeter(JASCOFP-8300inaquartzcuvette)oronFluorstar Optimamicrotiterplatereader(excitation:433nmandemission: 485nmand528nm)in100lof50mMphosphatebufferpH7.0 containing1mMbenzamidine per wellasdescribed previously withmajormodifications[10].Briefly,measurementswerecarried outintheSpectrofluorimeter(Jasco)and spectrawereobtained in50mMphosphatebufferpH7.0containing1mMbenzamidine (100l)asthebackground.Increasingconcentrationsofglucose (0.01,0.1,1.0e10mM)inthesamebuffersystemwereaddedto thesolubleformsofcell-freeextractorwhole-cells(100l) con-tainingtheglucosenanosensor.Thesameprocedurewascarried outfortheimmobilizedglucosenanosensoroncircularmembrane andon96-wellmicrotiterplatesandspectraweremeasuredeither inthewavelengthrangeof400–600nm(Spectrofluorimeter)orin microtiterplatereader(excitation:433nmandemission:485nm and528nm).
2.2.5. Glucoseassay
FRETanalysiswascalculated astheratioofthefluorescence intensityat528nmdividedbythefluorescenceintensityat485nm. Spectrameasurementswerecarriedoutofthefreeformofboth nanosensorsinthepresenceandabsenceofglucoseastheligand. Thepresence ofglucose altersthefluorescenceintensityof the yellow(530nm)andcyano(485nm)ofFLIPnanosensors.The spec-trameasurementswerecarriedoutbyusingfreeandimmobilized formsofbothnanosensorsinthepresenceandabsenceofglucose astheligand.Acalibrationcurveforglucosewascarriedoutby usingFRETmeasurementsinthelinearrangeof0–24mMglucose asdescribedinSection2.2.4.Forcomparativepurposes,glucose wasassayedinseveralsamplesofhumanserabyusingthepresent FRETmethodandaconventionalcolorimetricassaymethodbased onglucoseoxidaseandperoxidase[6].
2.2.6. StabilityofimmobilizedFLIPglucosenanosensors
Immobilized FLIPglucosenanosensors (microtiterplatesand onion membranes) were used to investigate their storage and operationalstabilities.Therefore,fluorescencemeasurementswere carried out inthe presence andabsence of glucosein order to determinetheirstabilityasafunctionoftimebymeasurementsof glucoseassayinmicrotiterplatereader.Thesenanosensorswere storedin50mMphosphatebufferpH7.0containing1mM benza-midineat4◦C.
2.2.7. Selectivityofgeneticallyencodednanosensors
Theselectivityofthesenanosensorswasinvestigatedbyusing severalcarbohydratesasligandsinordertostudytheireffectonthe
ratioofEYFP/ECFPcomparedwiththeglucosebyusingdifferent ligandconcentrations.
2.2.8. Proteinassay
Proteinconcentrationofcell-freeextractswerecarriedoutby coomassiebluedyebindingmethodbyusingBSAastheprotein standardasmentionedpreviouslywithminormodifications[17].
3. Resultsanddiscussion
3.1. Productionofgeneticallyencodedglucosenanosensors
Genetically-encodedFRETnanosensors have been developed formeasuringthedynamic changes in concentrationofseveral metabolitesofbiologicalinterest[10].ThepresentFRETsensor con-tainsGBPwhichwasfusedtoafluorescentpairwithoverlapping emissionandexcitationspectra(i.e.ECFPandEYFP).Thebinding ofthemetaboliteofinterestinducesaconformationalchangethat affectstherelativedistanceand/ororientationbetweenthetwo flu-orescentproteinswhichcancauseeitheranincreaseoradecrease inFRETefficiency.TheseFLIPnanosensorsweredetectedinLB cul-turemediumbymeasurementoffluorescencespectrumbetween 400and600nm(Fig.1A)sincetherearetwofluorescencepeaks atabout485and530nm.Moreover,thefluorescencespectrumof cell-freeextractofFLIP-glu-3.2mMnanosensorwasobtainedin thepresenceandabsenceofglucosewhichrevealedadecreasein fluorescenceofEYFPandanincreaseofECFPasshowninFig.1B. 3.2. ImmobilizationofFLIPglucosenanosensors
The96-welltissue culturemicrotiterplatesand onion mem-branes were selected as immobilization supports since they
exhibitedthebestresultsasfarasrecoveryandretentionof bio-logicalactivityareconcerned.Thecellwallofinnerepidermiscells containsseveralbiologicalmacromoleculessuchas polygalactur-onicacid,hemicelluloses,proteinsandlignin[18].Therefore,onion membranesexhibitabiocompatiblemicroenvironmentandstable supportforimmobilizationofwhole-cellsandcell-freeextract.
Theimmobilizationofthesenanosensorsonboththesesupports resultedinrecoveryoffluorescenceintherangeof50–90%which wasrepresentedasthepercentageoftheratioEYFP/ECFPcompared withthefreeformofthenanosensor(Figs.2and3).
Asfarasglutaraldehydeisconcerned,severalconcentrations weretestedand itwasfoundthatlowerconcentrations of glu-taraldehydeexhibited higher recoveryof fluorescenceof either theimmobilizedwhole-cellsorcell-freeextractonbothsupports (Fig.2).Theeffectoftheamountofwhole-cellsandcell-freeextract onfluorescencerecoveryataconstantconcentrationof glutaralde-hyde(i.e.2%,v/v)wasalsoinvestigatedwhichrevealedthatlower amountsofnanosensorexhibitedhigherrecoveryoffluorescenceof eithertheimmobilizedwhole-cellsorcell-freeextractonboth sup-ports(Fig.3).Therefore,thesedataindicatethattherate-limiting stepseemstobethelowcapacityoftheonionmembranesaswell asthewellsofthemicrotiterplates[16,18].
3.3. Glucoseassay
Glucoseassaywascarriedoutbyusingfreeandimmobilized genetically encoded nanosensor in the range of 0–100mM as shown inFig. 4.Asexpected, theratioof EYFP/ECFP decreased astheglucose concentrationincreasedfor thefree formofthe nanosensorforwhole-cellsandcell-freeextract(Fig.4A).The cell-freeextractpresentedhigherFRETvaluesthanwhole-cellsbecause it has a higher amount of soluble protein compared withthe
0 200 400 600 800 1000 1200 1400 450 500 550 600 650 700 Fluor escence (A UF) wavelenght (nm) FLIPglu- 3.2mM LB medium 0 200 400 600 800 1000 1200 450 500 550 600 AUF λλ (nm) 1 m M glucose No glucose
A
B
0 20 40 60 80 100 0 5 10 15 20 Rec ov er y ( % ) Glutaraldehyde (%, v/v) whole cells cell-free extract 0 20 40 60 80 100 120 0 5 10 15 20 Rec ov er y ( % ) Glutaraldehyde (%, v/v) whole-cells
A
B
Fig.2. Effectofglutaraldehydeconcentrationontherecoveryofglucosenanosensor immobilizedonbothsupports.Aconstantamountofwhole-cells(1.9and0.5g) andcell-freeextract(2.9and29.0g)ofFLIP-glu-3.2mMwereusedand glu-taraldehydeconcentrationwasvariedforonionmembraneandtissuecultureplate, respectively.TherecoveryrepresentspercentageoftheratioEYFP/ECFPinthe pres-enceofglucosecomparedwiththefreeformofthebiosensor.(A)Innerepidermis ofonionbulbscale;(B)96-welltissueculturemicrotiterplate.
whole-cells(Fig.4A).Moreover,thenanosensorincell-freeextract andwhole-cellsmaybeindifferentconformationswhichhasbeen alsomentionedbyotherresearchworkersintheliterature[12,19]. However,asfarastheimmobilizedcell-freeextractand whole-cells on onion membrane are concerned,the data revealed an increaseintheratioasafunctionofglucoseconcentrationwhich suggestthatthereisanincreaseinFRETefficiency(Fig.4B). Sim-ilarresultswerealsoobtainedwithimmobilizednanosensorsin 96-wellmicrotiterplates(datanotshown).Thisresultwas unex-pectedbut it maybeexplained by thefact that thecovalently immobilizednanosensorisboundtotheimmobilizationsupport insuchawaythatbothfluorescentproteins(i.e.EYFPandECFP) maybelocatedfurtherapart.Subsequently,thebindingofglucose toGBPinducesa conformationalchangewhichmayapparently bring closer both these two fluorescent proteins and therefore thereisanincreaseinFRETefficiency.Severalresearchworkers have reported that FRET is apparently a very complex process whenfreeand immobilizedwhole-cellsorcell-freeextractsare involved [19–22].Regardingthedifferencesin FRETefficiencies betweenthefreeandimmobilizednanosensors,therearea num-beroffactorsaffectingimmobilizedandsolubleproteinssuchas
Fig.3.Effectoftheamountofwhole-cellsandcell-freeextractontherecoveryof glucosenanosensorimmobilizedonbothsupports.Increasingamountsof whole-cellsandcell-freeextractofFLIP-glu-3.2mMwereusedataconstantconcentration ofglutaraldehyde(2%).TherecoveryrepresentspercentageoftheratioEYFP/ECFP inthepresenceofglucosecomparedwiththefreeformofthebiosensor.(A)96-well tissueculturemicrotiterplate;(B)innerepidermisofonionbulbscale.
restrictedmobilityonimmobilization,chemicalmodificationdue totheimmobilizationmethodused,natureofthe microenviron-mentanddiffusionlimitation[20].Thereareseveralreportsinthe literaturethathave mentioneddifferentfluorescentbehaviorin theFRETpairbetweenfreeandimmobilizednanosensorsdueto somefactorsmentionedabove.Apparently,thisdifferencemaybe duetothereducedflexibilityof thelinkerdomainbetweenthe fluorescentproteinsbecauseoftheattachmenttosolidsurface. Thisissueemphasizestheimportancetoretainflexibilityofboth thefusionproteinsindevisingFRETpairsforimmobilizationto solidsurfaces[19,21].Ingeneral,FRETtheoryassumesthatina FRETcoupleonlyasingledonorandasingleacceptorarepresent withveryweakcoupling.However,incell-freeextractsor whole-cells, it is generally unknown if a single or multiple acceptors arepresentwhichmarkedlycomplicatesthecalculationofFRET efficiency.Moreover,otherproblemsmustbeevaluatedand con-trolledsuchascross-talkbetweenFRETpartners,effectofpHon theirmicroenvironmentanddifferenceinstoichiometricratiosof donorandacceptorbiomolecules[23,24].However,this immobi-lizednanosensorwassuccessfully usedtoassayglucosein real samplesofhumansera(Table1).Acalibrationcurveforglucose assayhasbeenpresentedinFig.4Cbyusingimmobilizedcell-free extractononionmembrane.Alimitofdetection(LOD)of0.11mM Table1
ComparativeanalysisofglucoseassaybythepresentFRETmethod(i.e.immobilized whole-cellsinonionmembrane)andconventionalcolorimetricmethodbyusing threedifferenthumanserasamples.
Serumsamples PresentFRET
method(mM) Colorimetric method(mM) Humanserum1 3.1±0.15 3.3±0.11 Humanserum2 5.9±0.32 6.1±0.28 Humanserum3 8.4±0.43 8.7±0.51
Fig.4. EffectofglucoseconcentrationontheratioofEYFP/ECFPoffreeand immo-bilizedFLIP-glu-3.2mMnanosensor.(A)Freewhole-cellsandcell-freeextract;(B) immobilizedcell-freeextractandwhole-cellsononionmembraneand(C) cali-brationcurveforglucoseassaybyusingimmobilizedcell-freeextractononion membrane.
glucosewasobtainedaccordingtoIUPACandICH[25].However,
upconversionluminescencenanosensorshavebeenusedtoassay forglucosewitha LODof64nMwhich ismuchlowerthanthe valuepresentedinthiswork[26].Acomparativestudywascarried outbyusing awell-establishedconventional colorimetricassay forglucosewithglucoseoxidaseandperoxidasewhichrevealed thattheresultsareslightlylowerwiththeFRETmethodcompared thecolorimetricmethod(Table1).Theseresultsmaybeexplained bythefactthatsomeinterferingsubstancesinhumanserummay affecttheFRETmethod.However,thisdifferenceisnotsignificant sinceverysimilarresultswereobtainedforbothmethods(Table1).
Fig.5.Stabilityofwhole-cellsandcell-freeextractcontainingFLIP-glu-3.2mM nanosensorimmobilizedonbothsupports.Thestability/operationalstability repre-sentsthepercentageoftheratioEYFP/ECFPinthepresenceofglucoseasafunction oftime:(A)innerepidermisofonionbulbscale;(B)96-welltissueculturemicrotiter plateand(C)Operationalstabilityoftheimmobilizednanosensorononion mem-braneasafunctionofNo.ofcyclesforglucoseassay.
Similarresultswereobtainedbyusingimmobilizedwhole-cells andcell-freeextracton96-wellmicrotiterplates(datanotshown). 3.4. StabilityofimmobilizedFLIPglucosenanosensors
The data presented in Fig.5 revealed that the immobilized nanosensors exhibited a very high storage stability at 4◦C of over 6 and 7 months for onion membrane and tissue culture microtiterplate,respectively(Fig.5AandB).Ontheotherhand, theoperationalstabilityoftheseimmobilizednanosensorswasalso investigatedbyassayingforglucoseasafunctionofthenumberof cycleswhichrevealedthattheycouldbeusedatleast20cycles withoutanysignificantlossofbiological activityasfarasFRET signalisconcerned(Fig.5C).
2 2.2 2.4 2.6 2.8 100 10 1 0 Ratio [Carbohydrate] mM Glucose Galactose Fructose Xylose Arabinose Sacarose
Fig.6. SelectivityofFLIP-glu-3.2mMnanosensor.Differentconcentrationsof sev-eralsugarswereusedtoinvestigatetheireffectontheratiobyusingfreewhole-cells.
3.5. Selectivityofgeneticallyencodednanosensors
The selectivity of FLIP-glu-3.2mM nanosensor was investi-gatedbyusingseveralmonoanddisaccharidesontheratiowhich revealedthatgalactoseexhibitedaverysmallcross-reactivitywith thenanosensorcomparedwiththeglucose(Fig.6).Thisresultis inagreementwithpublishedreports ontheselectivityofthese geneticallyencodednanosensors[10].
4. Conclusions
RecombinantE.coliwhole-cellsandcell-freeextract contain-inggeneticallyencodednanosensorswereimmobilizedononion membraneandtissueculturemicrotiterplateswithhighrecovery ofbiologicalactivity.Thismethodwasusedtoquantifyglucosein realhumanserasamplesandtheresultsareinagreementwith awell-establishedcolorimetricassaymethod.Thisassaymethod isbasedon96-wellmicrotiterplatewhichcanbeusedasahigh throughputassaymethodforglucosesinceitischeap,rapidand manysamplescanbeassayedona singleplatform.Thestorage andoperationalstabilityofthesenanosensorsarehighand there-foretheycanbere-usedseveraltimes.Toauthorknowledge,thisis thefirstreportontheuseofimmobilizedwhole-cellsandcell-free extract containing genetically encoded nanosensors for glucose assaybyusingthesetwonovelsupports.
Acknowledgements
WewouldliketothankFundac¸ãoparaaCiênciaeaTecnologia/ MCTES (Portugal) for financial support (PTDC/EEA-ELC/11854/ 2009;Pest2012-2014forUnit702).
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Biographies
AnaCharnecacompletedherBachelordegreeinChemicalandBiological Engineer-ingin2012atInstitutoSuperiordeEngenhariadeLisboa(ISEL)inPortugaland sincethenshehasbeeninvolvedinresearchactivitiesasaresearchassistantina biosensorresearchprojectforglucose.Shehasseveralpublicationsininternational journalsaswellasininternationalscientificconferences.
AminKarmaligothisPh.D.attheageof26fromKing’sCollegeLondon-U.K.,and postdoctoratefromGulbenkianInstituteofScienceinPortugal.HegothisD.Sc. (aggregation)in2001atÉvoraUniversity.HeisafullProfessorinBioengineeringand HeadofBiotechnologyandChemicalEngineeringResearchCenterofISELinLisbon, hasseveralresearchprojectsfundedbyEuropeanandNationalResearchCouncil, hassupervised15M.Sc.and7Ph.D.thesis(completed),publishedmorethan70 papersand4patentsinreputedjournalsandisareviewerofseveralinternational journalsandeditorialmemberofsomejournals.
ManuelaVieirawasborninLisbon,Portugal.In1986,shereceivedtheMastersof Scienceinsolidstatephysics-microelectronicandin1993thePh.D.in semicon-ductormaterialsbothfromtheNewUniversityofLisbon.Sheisfullprofessorin electronicsintheDepartmentofElectronicsTelecommunicationandComputers (ISEL,Portugal)andtheheadofagroupinappliedresearchinmicroelectronic opto-electronicandsensors(GIAMOS).Shehasseveralscientificpapersand20yearsof experienceinthefieldofthinfilmsanddevices,herresearchactivitieshavebeen mainlyrelatedtothedevelopmentofopticalsensors.
