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ElectronicsupplementaryinformationtoSynthesis and functionalization of monodisperse near-ultraviolet and visibleexcitable multifunctional Eu3+:Bi3+:REVO4 nanophosphors for bioimaging andbiosensingapplicationsAlbertoEscudero,1,2*CarolinaCarrillo-Carrión,1,3MikhailV.Zyuzin,1SumairaAshraf,1RaimoHartmann,1NuriaO.Núñez,2ManuelOcaña,2andWolfgangJ.Parak.11. AG Biophotonik, Fachbereich Physik, Philipps-UniversitätMarburg. Renthof 7. D-35037,Marburg,Germany.2. Instituto de Ciencia de Materiales de Sevilla. CSIC – Universidad de Sevilla. C.AméricoVespucio49.E-41092,Seville,Spain.3.CICbiomaGUNE.PaseoMiramón182.E-20009,SanSebastian,Spain.*Correspondingauthor:alberto.escudero@csic.esIndex
1. Synthesisofthenanoparticles2. Physicochemicalcharacterizationofthenanoparticles3. Colloidalstabilitystudies4. Opticalproperties5. Imagingset-up
5.1 Fluorescencemicroscopy5.2 Confocallaser-scanningmicroscopy
6. Nanoparticleuptakebycellsandimaging7. DissolutionatacidicpH8. Flowcytometrystudies
Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2016
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1.SynthesisofthenanoparticlesEuropiumandbismuthdopedYVO4andGdVO4nanoparticlesweresynthesisedfromappropriate RE precursors and sodium orthovanadate in ethylene glycol – watermixtures,asdescribedintheexperimentalsectionofthepaper.TableS1:Experimentalconditionsforthesynthesisofallpreparedsamples.
Sample [Eu3+](M) [Bi3+](M) [Y3+](M) [VO43-](M) PAA(mg/mL)
Bi0Y 0.0016 0 0.0184 0.1 0Bi5Y 0.0016 0.001 0.0174 0.1 0Bi10Y 0.0016 0.002 0.0164 0.1 0Bi15Y 0.0016 0.003 0.0154 0.1 0Bi20Y 0.0016 0.004 0.0144 0.1 0
Bi0YPAA2 0.0016 0 0.0184 0.1 2Bi5YPAA2 0.0016 0.001 0.0174 0.1 2Bi10YPAA2 0.0016 0.002 0.0164 0.1 2Bi15YPAA2 0.0016 0.003 0.0154 0.1 2Bi20YPAA2 0.0016 0.004 0.0144 0.1 2Bi20YPAA5 0.0016 0.004 0.0144 0.1 5Bi20YPAA8 0.0016 0.004 0.0144 0.1 8
[Eu3+](M) [Bi3+](M) [Gd3+](M) [VO43-](M) PAA
(mg/mL)Bi0Gd 0.0016 0 0.0184 0.1 0Bi5Gd 0.0016 0.001 0.0174 0.1 0Bi10Gd 0.0016 0.002 0.0164 0.1 0Bi15Gd 0.0016 0.003 0.0154 0.1 0Bi20Gd 0.0016 0.004 0.0144 0.1 0Bi0GdPAA 0.0016 0 0.0184 0.1 2Bi5GdPAA 0.0016 0.001 0.0174 0.1 2Bi10GdPAA 0.0016 0.002 0.0164 0.1 2Bi15GdPAA 0.0016 0.003 0.0154 0.1 2Bi20GdPAA 0.0016 0.004 0.0144 0.1 2
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2.PhysicochemicalcharacterizationofthenanoparticlesTable S2: Morphological and colloidal characterization data of all the synthesizedsamples.DLS(numberdistribution)andζ-potentialmeasurementswererecordedinwateratpH7.Errorsrefertostandarddeviationsorincaseoftheunitcellvolumetoabsoluteerrors.N.r.=notrecorded.
Sample Particlesize(TEM)(nm)
Hydrodynamicdiameter(nm)
Crystallitesize(nm)
ζ-potential(mV)
Unitcellvolume(Å3)
Bi0Y N.r. 142(9) N.r. N.r. N.r.Bi5Y N.r. 139(7) N.r. N.r. N.r.Bi10Y N.r. 148(4) N.r. N.r. N.r.Bi15Y N.r. 137(5) N.r. N.r. N.r.Bi20Y 107(14) 138(7) N.r. -19(1) N.r.
Bi0YPAA2 88(14) 82(3) 62 -37(2) 319.24(3)Bi5YPAA2 N.r. 94(4) 77 N.r. 321.01(4)Bi10YPAA2 N.r. 98(3) 79 N.r. 322.55(4)Bi15YPAA2 N.r. 110(4) 77 N.r. 324.03(2)Bi20YPAA2 93(7) 108(10) 79 -42(3) 325.58(2)Bi20YPAA5 N.r. 79(4) N.r. -37(2) N.r.Bi20YPAA8 51(5) 51(2) 57 -39(2) 325.61(3)Bi0Gd N.r. 47(2) N.r. N.r. N.r.Bi5Gd N.r. 52(1) N.r. N.r. N.r.Bi10Gd N.r. 57(4) N.r. N.r. N.r.Bi15Gd N.r. 58(3) N.r. N.r. N.r.Bi20Gd 51(15) 58(4) N.r. -25(1) N.r.Bi0GdPAA 39(7) 40(2) 53 N.r. 330.32(4)Bi5GdPAA N.r. 41(3) 54 N.r. 331.65(3)Bi10GdPAA N.r. 45(2) 56 N.r. 332.79(5)Bi15GdPAA N.r. 46(1) 54 N.r. 333.74(2)Bi20GdPAA 40(8) 41(2) 65 -41(2) 335.37(2)
Table S3: ICP-determined europium, bismuth, yttrium, gadolinium, and vanadiumcontentofselectedsamples.RE=YorGd.
Sample Eu/(Eu+Bi+RE)(%) Bi/(Eu+Bi+RE)(%) V/(Eu+Bi+RE)
Bi20YPAA2 7.96 20.6 1.17Bi20YPAA8 7.91 20.4 1.05Bi20GdPAA 8.07 19.7 1.15Bi20Y 8.04 19.9 1.03Bi20Gd 7.88 20.0 1.06
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TableS4:Meanhydrodynamicdiameter(nm)offreshlypreparedsuspensionsofPAA-functionalizedandLbL-functionalizedEu,Bi-dopedYVO4andGdVO4NPsobtainedbyDLS in water and in different buffer media of biological interest. C.a = aproximateresults.
Bi20YPAA2 Bi20GdPAA Bi20Y@PAH@PAA@PAH
Bi20Gd@PAH@PAA@PAH
Water 108 41 157 84MESpH6.5 109 48 164 125PBSpH7.4 125 81 c.a.700 c.a.1250Cellmedium >1100 >1900 c.a.1400 c.a.1450Serum-
supplementedcellmedium
182 113 c.a.250 c.a.530
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Figure S1: TEM micrographs of the Bi20Y (A), Bi0YPAA2 (B), Bi20Gd (C), andBi0GdPAA(D)samples.
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FigureS2:Thermogravimetry(TG)analysisofselectedNPsfunctionalisedwithPAA.ThecurvescorrespondingtothesamplessynthesizedintheabsenceofPAA(Bi20YandBi20Gd)arealsoincluded.
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FigureS3:Energy-dispersiveX-ray(EDX)spectraofsingleparticlesofBi20YPAA2(A)andBi20GdPAA(B)samples.
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FigureS4:UnitcellvolumeoftheEu-andBi-dopedYVO4(A)andGdVO4(B)samplessythesized inpresenceofPAA (2mg/mL)with increasing thebismuth content.Theerrorbarsaresmallerthanthesymbolsize.
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3.Colloidalstabilitystudies
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FigureS5:EvolutionofthehydrodynamicdiameterobtainedfromDLS(dinamiclightscattering) analyses of the YVO4-based nanoparticles (A to E; blue triangles =Bi20YPAA2,greentriangles=Bi20Y,redcircles=Bi20Y@PAH@PAA@PAHsamples),andGdVO4-basednanoparticles(FtoJ;bluetriangles=Bi20GdPAA,greentriangles=Bi20Gd, red circles = Bi20Gd@PAH@PAA@PAH samples) samples inwater (pH 7),MES (N-Morpholino)ethanesulfonic acidhydrate)50mMatpH6.5, PBS (phosphatebuffered saline) at pH 7.4, serum-supplemented cell medium, and serum-free cellmedium.
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FigureS6:HydrodynamicdiameterobtainedbyDLSoftheBi20Ysample(A),andafterthedepositionofonePAHlayer(B),oneextraPAAlayer(C)andoneextraPAHlayer(D).RedlinescorrespondtoanalysescarriedoutinMESbuffer50mMatpH6.5andblacklinescorrespondtomilli-QwateratpH7.
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Figure S7: Hydrodynamic diameter obtained by DLS of the Bi20Y sample (A) andBi20Gdsample(B) inwateratpH7(black lines),MES50mMatpH6.5(red lines),PBS at pH 7.4 (blue lines), serum-containing cell medium (green lines), and cellmedium(purplelines).
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4.Opticalproperties
FigureS8:(A)Excitationspectra(emissionrecordedatλem=622nm)oftheEu–Bi-doped YVO4 samples functionalized with PAA (2 mg/mL) with increasing the Bicontent.a=Bi0YPAA2,b=Bi5YPAA2,c=Bi10YPAA2,d=Bi15YPAA2,e=Bi20YPAA2.(B) Excitation spectra (λem = 622 nm) of the Eu – Bi- doped GdVO4 samplesfunctionalizedwithPAA(2mg/mL)withincreasingtheBicontent.a=Bi0GdPAA,b=Bi5GdPAA, c = Bi10GdPAA, d = Bi15GdPAA, e = Bi20GdPAA. (C) Evolution of theabsorption maximum of the Eu-Bi-YVO4 samples functionalized with PAA withincreasing the Bi content. (D) Evolution of the absorption maximum of the Eu-Bi-GdVO4 samples functionalized with PAA with increasing the Bi content. The linesrepresentingthefitsareonlyintentedasguidesfortheeye.
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Figure S9: Evolution of the emission intensity (λem = 622 nm) of Bi20YPAA2 NPsunder continuous UV irradiation in a in a wide-field fluorescence microscope(Axiovert200M,Zeiss,Germany).TheparticleswereinmobilisedwithFluoromount–G(SouthernBiotech).TheUVirradiationwasstoppedfromminutes31to44.
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FigureS10:Emissionspectra(λex=355nm)ofaqueoussuspensions(0.05mg/mL)oftheBi20GdPAA(blue),Bi20YPAA2(black)andBi20YPAA8(red)samples.
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5.Imagingset-up 5.1FluorescencemicroscopyImagingwas performedwith awide-field fluorescencemicroscope (Axiovert 200M,Zeiss,Germany).APlanApochromat63x/1.4OILDIC∞/0.17oilobjectivewasusedwhileimagingthestainedfixedsamples(cellswithluminescentparticlesinside).Theimmersionoilwas ImmersolTM518F.The tripodwasequippedwithamercuryarclampofthetypeNHBO103andaCCDcameraofthetypeAxiocamHRc.A340±24nmexcitation filter and a longpass (>600nm) emission filterwereused to detect thenanoparticles.Automaticimagingwasperformedand9to16imagespersamplewerecaptured.Theredchannelimageswerecollectedwith3000msofexposuretime.Themicromanagersoftware(basedonimageJ)wasused.Theimagesweredeconvolutedandoptimizedafteracquisition.FiltersetswereobtainedfromAHFAnalysentechnik.FilternameExcitation(nm)Emission(nm)BeamSplittersExciter/Emitter1-Chroma31001FITC480535505DCLPD48030x/D53540m2-ZeissFilterSet49G365BP445-450FT395G365/445503-AHFCy5BP620/30BP700/40BST660LPXRET620/60x,ET700/75m4-PBFI/SBFI(Alexared)LP340600LP400DCLPD340-26/600LPwhereDCLP=DichroicLongPassBP=bandpassLP=longpass
5.2Confocallaser-scanningmicroscopy
ThenanoparticleuptakebyHeLacellswasliveimagedusingaconfocallaser-scanningfluorescencemicroscope(CLSM510Meta,Zeiss)equippedwithaportableincubator(Pecon,Germany) tomaintain theµ-ibidiplates(Ibidi#80826,1cm2/well)at37°Cwith5%CO2.ImageserieswereacquiredusingaPlan-Apochromat63x/1.40OilDICM27 objective. The nanoparticleswere excited at 405 nm (with a diode laser, 20%
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power) and their fluorescence was recorded between 604 and 754 nm. Untreatedcells(i.e.cellsnotincubatedwithnanoparticles)wereusedascontrol.6.Nanoparticleuptakebycellsandimaging
FigureS11:Fluorescence imagesofHeLacellsnot incubatedwithnanoparticles (i.e.controlexperiment).(A)Redchannel,nanoparticles;(B)bluechannel,cellnuclei;(E)greenchannel,cellmembranes;(D)transmissionimage,and(E)mergedimage.
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Figure S12: Fluorescence images of HeLa cells incubated with Bi20GdPAAnanoparticlesfor24h.(A)Redchannel,nanoparticles(B)yellowchannel,lysosomes;(C)bluechannel,cellnuclei;(D)greenchannel,cellmembranes;(F)mergedimage.
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Figure S13: Fluorescence images of HeLa cells incubated withBi20Y@PAH@PAA@PAHLbL-functionalizednanoparticlesfor24h.(A)Redchannel,nanoparticles(B)yellowchannel, lysosomes; (C)bluechannel,cellnuclei; (D)greenchannel,cellmembranes;and(E)mergedimage.
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Figure S14: Fluorescence images of HeLa cells incubated withBi20Gd@PAH@PAA@PAHLbL-functionalizednanoparticlesfor24h.(A)Redchannel,nanoparticles(B)yellowchannel, lysosomes; (C)bluechannel,cellnuclei; (D)greenchannel,cellmembranes;(F)mergedimage.
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FigureS15:ConfocalmicroscopyimagesoflivingHeLacellswithoutnanoparticles(AtoC);andHeLacellsincubatedfor24hourswithBi20YPAA8(DtoF),andBi20GdPAA(GtoI)nanoparticles.Leftcolumn:transmissionimages,centralcolumn:fluorescenceimages;rightcolumns:mergedimages.Thescalebarsrepresent20µm.
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7.DissolutionatacidicpH
FigureS16:(A)ImageofsuspensionsoftheBi20YPAA2(left)andBi20GdPAA(right)samples(initialconcentration=0.5mg/mL)inMES50mMbufferatpH3.5.(B):SamesuspensionsinMES50mMbufferatpH3.5after21daysofaging.
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8.Flowcytometrystudies
Figure S17: Flow cytometry controlmeasurements of untreated cells (A), cellswithDAPI(B),anddeadcellswithDAPI(C).1,and22DdensityplotofA.untreatedcells,B.cellswithDAPI,C.deadcellswithDAPI.Column1showstheforward–areascatteringversussidescattering;Column2showstheforward-widthscatteringversusforward-areascattering;Column3showstheDAPIintensityhistogram.
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