In Situ Generation of Fluorescent Copper Nanoclusters Embedded in Monolithic Eggshell Membrane

16 Pages·2017·4.87 MB·English

materials
Article
In Situ Generation of Fluorescent Copper
Nanoclusters Embedded in Monolithic Eggshell
Membrane: Properties and Applications
LuLi,MinHuang,XianhuLiu,DengmingSunandCongyingShao*
CollegeofChemistryandMaterialsScience,HuaibeiNormalUniversity,Huaibei235000,China;
[email protected](L.L.);[email protected](M.H.);[email protected](X.L.);
[email protected](D.S.)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:16August2018;Accepted:30September2018;Published:9October2018
Abstract: Luminescentmetalnanoclustershaveattractedconsiderableresearchattentioninrecent
yearsduetotheiruniquepropertiesandextensiveusageinmanyfields. Threedifferentsynthetic
routesweredevelopedtoinsitugenerateorangeandredemittingcoppernanoclustersembedded
in monolithic eggshell membrane (Cu NCs@ESM) using different reducing reagents including
N H ·H O, NH OH·HCl and Vitamin C at room temperature for the first time. The routes are
2 4 2 2
extremely facile, low-cost and versatile. The obtained Cu NCs@ESM nanocomposites exhibit
excellentphotostabilityandchemicalstability,layingthefoundationforvariouspracticalapplications.
Fluorescentsurfacepatterningwasdemonstratedbasedontheproposedstrategyeasily. Significantly,
theCuNCs@ESMshowsselectivefluorescencequenchingresponsetoHg2+ionsandgoodcatalytic
activityformethyleneblue(MB)reductiondegradationmakingitidealasportablesensingstrip
andrecyclablecatalyst. Theworkprovidesageneralstrategyforthefabricationofothervarious
monolithicnanomaterialswithpotentialapplications.
Keywords: coppernanoclusters;fluorescence;eggshellmembrane;monolithic;applications
1. Introduction
Metalnanoclusters(NCs)consistingofseveraltotensofatomshavedrawnconsiderableresearch
interestduetotheirlowtoxicity,goodbiocompatibility,multifunctionalsurfacechemistry,andexcellent
fluorescencebehaviorssuchasemittingtunability,largeStokesshift,andphotostability[1–4].TheNCs
havebeenwildlyappliedinbioimaging[5–7],sensing[8–10],catalysis[11–14],nanodevices[15–17]and
manyotherfields[18,19]. However,todate,extensiveresearchonfluorescentmetalnanoclustershas
mainlyfocusedonthenoblemetalnanoclusters(especiallyAuandAgNCs). ThestudiesontinyCu
NCsarestilldeficientowingtotheirinherentinstability[17,20].Thelessuseofnoblemetalshasattracted
everincreasingattentionduetotheirscarcenaturalresourcesandhighcost.Researchersturnedtothe
studiesofthefirsttransitionmetalssuchasCu,Fe,Ni,whichcanberegardedasalternativestoAuand
Ag. Copperisfrequentlyusedinindustrybecauseofitshighconductivity,similarpropertiestogold
andsilverandespeciallymuchlowercost.Therefore,thedevelopmentofvarioussyntheticroutesand
extensivepropertiesinvestigationsofCuNCswillbeofgreatpracticalsignificance[21,22].
Recently,manyeffortshaveledtosomeapproachesforCuNCssynthesessuchaspolymerand
biomoleculetemplatedmethods[23–31],electrochemicalreduction[32],sonochemicalsynthesis[33],
microwave-assisted[34–36]andmicroemulsiontechnology[37,38]. However,thoseas-preparedCu
NCsaremostlydispersedinhomogeneoussolution.Agglomeration,precipitationandrecyclingduring
practicalapplicationsarethechiefdrawbacksofsolution-dispersedfluorescentCuNCs. Additionally,
Materials2018,11,1913;doi:10.3390/ma11101913 www.mdpi.com/journal/materials
Materials2018,11,1913 2of16
theseaqueousCuNCsmighthindertheirpotentialapplicationsassolidstatematerialsinchemical
sensingstrips,surfacepatterning,light-emittingdevicesandotherfields. However,todate,thereare
Materials 2018, 11, x FOR PEER REVIEW 2 of 16
onlyfewreportsonthesynthesesofsolidstateCuNCs. Zhangetal. reportedthesynthesisofsolid
fluoresAcedndtitCiounaNllyC, sthienses auqpureaomuso Cleuc uNlCars mhyigdhrto hgienldsevr ithaeeirm ppoltoenytiinalg aappsleicraietisonosf absi lseolaidc isdtadtee mriavtaetriivaless as
pre-geliant cohresm[3ic9a]l. sQenisnignge sttarilp.sp, sruorpfoacsee dpatthteernfliungo,r elisgchet-netmCitutinNg Pdsevteicmesp alnatde dothbeyr pfioelldys(.t hHyomwienvee)r, itno the
date, there are only few reports on the syntheses of solid state Cu NCs. Zhang et al. reported the
agarosehydrogel[40]. Veryrecently, compositepolymerfilmsincorporatingluminescentCuNCs
synthesis of solid fluorescent Cu NCs in supramolecular hydrogels via employing a series of bile acid
werefabricatedinahydrogelbytheprecursorsofpolyvinylpyrrolidoneandpoly(vinylalcohol)by
derivatives as pre-gelators [39]. Qing et al. proposed the fluorescent Cu NPs templated by
Wangetal.[41]. Inthementionedwork,thesolidCuNCswereallgeneratedbasedonpre-formed
poly(thymine) in the agarose hydrogel [40]. Very recently, composite polymer films incorporating
orsimultaneouslypolymerizedhydrogelnetwork. Obviously, sometimespoordispersionofNCs
luminescent Cu NCs were fabricated in a hydrogel by the precursors of polyvinylpyrrolidone and
inhydprooglye(lvwinyolu alldcolheoald) btoy tWhaenpgh eat sael. s[e4g1]r.e Igna tthieo nmmenatikoinnegd iwtdorikffi, tchuel tsotolidf aCbur iNcaCtse wlaerrgee a-lal rgeean,eurantiefdo rm,
photo-sbtaasbelde olun mprien-efoscrmenetdfi olrm ssim. Tuhltuans,eoduesvleyl oppoliynmgesroizmede fhaycdilreogaenld neptrwaoctrikc.a Olsbtvriaotuesgliye, ssotomfeatbimriecsa tpeonoro vel
solidstdaitsepeCrsuioNn Cosf NisCasv ienr hyyudrrgoegnelt wanodulfda slecainda ttoi nthget apshka.se segregation making it difficult to fabricate
Eglagrsghee-allrema,e munbirfoarnme,( EpShMoto),-sgteanbleer allulmyianbeasncednotn feildmisn. dTahiulys, lidfeev,ehlaospibnege nsodmeem foancsilter aatnedd tporabcetiocanle of
natures’strgatieftgsiefso tron faabnroicmataet enroivaellp sroelipda srtaattieo Cnu[ 4N2C].s EisS aM v,earyd uorugebnlet -alnady efarswciantaetirn-ign tsaoslku. blebiomembrane
Eggshell membrane (ESM), generally abandoned in daily life, has been demonstrated to be one
withinterwovenfibrousstructureattachedinsidetheheneggshell,iscomposedofhighlycross-linked
of natureʼs gifts for nanomaterial preparation [42]. ESM, a double-layer water-insoluble
andcystine-abundantproteins,whichmakeESMagoodreducingagentandstabilizerinnanoscience.
biomembrane with interwoven fibrous structure attached inside the hen eggshell, is composed of
Importantly,thethree-dimensionalmonolithicstructurecouldserveasanidealplatform,providing
highly cross-linked and cystine-abundant proteins, which make ESM a good reducing agent and
richsitesandmakingsurethereisuniformloading. Forinstance, previousresearchbyShaoetal.
stabilizer in nanoscience. Importantly, the three-dimensional monolithic structure could serve as an
demonidsteraalt peldattfohramt,E pSrMoviwdinags rainche sfifitecsi eanndt smoalikdinsgt sauterep thlaetrfeo irsm unfioforrmge lnoeardaintign. gFoAr uinsatnandceA, pgreNvCiosus[ 43].
Also,Dreesveiarrechp obryt eSdhatoh eet faol.r dmeamtioonnstoraftAedg thNaPt EsSbMy wESaMs an[4 e4f]fi.cAiengta sinol,idP rsatamtea pnliaktfporrmop foosr egdentehreatfionrgm Aaut ion
of fluoarnedsc eAngt NACusA [g43a].l lAoylson,a Dnoevpia rretpicolretsedim thme ofborilmizaetidonin osf oAligd NEPSsM by[4 E5S].MN [e4v4]e. rAthgealiens, sP,rtaomtahneikb est
ofourpkrnoopwosleedd gthee, tfhoerminatsioitnu osfy fnluthoeressicseonft flAuuoArge saclelonyt CnaunNopCarstiecmlesb eimddmeodbiilnizemd oinn osloitlhidi cEESSMM [4h5a].s n’t
been rNepeovretrethdelyesest,. toA thded biteisotn oaf lolyu,r tkhneowealesdegeo,f thoex iind asittiuo nsyontfhCesuis (oEf 0fl,u0o.r3e4scVen)t, Cinu NcoCms pemarbiesdodnedto int hat
of Agm(Eo0n,o0li.t8h0ic VES)Ma nhdasnA’tu b(eEen0, re1p.5o0rteVd) ymet.a kAedsdirteiolantaelldy, stthued eyasree omf aoixnidaaticohna ollfe Cnug e(E.0, I0n.3t4h Vis), wino rk,
comparison to that of Ag (E0, 0.80 V) and Au (E0, 1.50 V) makes related study remain a challenge. In
we established several facile routes for the in situ generation of fluorescent Cu NCs embedded
this work, we established several facile routes for the in situ generation of fluorescent Cu NCs
in solid state ESM (Cu NCs@ESM), employing different reducing agents at room temperature for
embedded in solid state ESM (Cu NCs@ESM), employing different reducing agents at room
the first time (Scheme 1), and the reaction conditions and properties of obtained Cu NCs@ESM
temperature for the first time (Scheme 1), and the reaction conditions and properties of obtained Cu
nanocoNmCps@oEsiStMes nwaenroecoinmvpeosstiitgesa twederien indveetsatiilg.aTtehde ionr edteictaaill.a Tnhdeoerxeptiecaril manedn teaxlpsetruimdieenstahl asvtuedsiheso hwanvet hat
CuNCsshopwosns tehsast uCnui qNuCess pizoess-easns duncioqmuep soizseit-i oannd-d ceopmepnodseitniotnc-adteapleynsdisenptr coaptaelrytsieiss p[r1o,1p3e,r1ti4e]s. [W1,1it3h,1m4].a ny
attractiWveithfe amtuarneys oatftrtahcetiivne sfietuatugreense roaft etdheC iun NsiCtus -ignecnoerrpatoerda tCedu nNaCnos-cioncmorppoosriatete,din nclaundocinogmpmoosinteo,l ith,
low coisntc,lucdoinnvge mnioennotlittha,i lloorwin cgo,stg, ocoondvsetnaiebnilti ttya,iloeraisnyg, sgtooorda gsetaabinlidty,t reaansysp sotorrta,gaen adndla trrgaensSptoorkt,e asn-sdh ift,
large Stokes-shift, the as-prepared Cu NCs@ESM nanocomposite is expected to exhibit significant
theas-preparedCuNCs@ESMnanocompositeisexpectedtoexhibitsignificantcatalyticandsensing
performcaatnalcyet.icH aenrde isne,ntshinegir paeprfpolrimcaatniocne.s Hinerveiisnu, athleHirg a2p+psltirciaptisonsesn insi nvigsubaals Hedg2o+ nstrflipuso rseesncseinngc ebaqsueedn ocnh ing
fluorescence quenching and catalysis for methylene blue reduction degradation have been
andcatalysisformethylenebluereductiondegradationhavebeendemonstrated.
demonstrated.

SchemSec1h.eSmceh e1m. aStcihcermepartiecs ernetparteisoenntoaftidoinff eorfe ndtifsfyernetnhte tsiycnrtohuettiecs frooruttehse fionrs itthue gienn esritaut iognenoefrafltuioonr eoscf ent
copperflnuaonreosccleunstt ecorsppeemr bneadnodceldusitnerms eomnboelditdheicd eing gmsohneolllimthiecm egbgrsahneell( mCuemNbCrasn@e E(CSuM N).Cs@ESM).
Materials2018,11,1913 3of16
2. MaterialsandMethods
2.1. ChemicalsandMaterials
Coppersulfate(CuSO ,>99%),sodiumhydroxide(NaOH,>96%),sodiumborohydride(NaBH ,
4 4
>98%),hydrazinehydrate(N H ·H O,85%),VitaminC(VC,>99.7%),hydroxylaminehydrochloride
2 4 2
(NH OH·HCl),mercurynitrate(Hg(NO ) ),ethanol(99.7%),andmethyleneblue(MB)werepurchased
2 3 2
fromSinopharmChemicalReagentCo.,Ltd(Shanghai,China). Otherchemicalsareallofanalytical
grades. Allthereagentswereusedasreceivedwithoutfurtherpurification. Deionizedwaterwitha
conductivityof18.2MΩ·cm−1waspurifiedthroughaMilliporewaterpurificationsystemandwas
usedthroughtheexperiment.
FresheggswerepurchasedfromZhenBangsupermarketneartheHuaiBeiNormalUniversity
(Huaibei,China). TheESMswerepeeledofffromthenaturaleggshellsmanuallyandcleanedwith
copiousamountofdeionizedwatercarefully. TheresultantESMswerecutintoproperrectangular
pieces(about1×1.5cm2)andstoredindeionizedwaterbeforeuse.
2.2. Instruments
Fluorescence spectra of Cu NCs@ESMs were recorded with a FP-8300 spectrophotometer
equippedwithsolidsampleholder(Jasco,Tokyo,Japan). Thephotosofallsamplesweretakenwith
aMeiZumobilephone(Zhuhai,China). Thefluorescentphotosunder365nmUVlightweretaken
underaZF-20Dblack-box-styleUVbasedanalyzer(YuHua,GongYi,China). Surfacemorphology
of fluorescent Cu NCs@ESMs were observed under an Olympus IX71 fluorescence microscope
(Tokyo, Japan). The X-ray photoelectron spectra (XPS) were obtained on an ULVAC-PHI X-ray
photoelectron spectrometer (PHI Quantera, Tokyo, Japan) using Al Kα radiation. Quantitative
analyses of the copper contents in Cu NCs@ESMs were performed on Perkin Elmer Opyima
7300ICP-AES(Waltham,MA,USA). TheUV-visabsorptionspectraanddiffusereflectancespectra
(DRS) measurements were carried out using a PGENERAL TU-1901 UV-vis spectrophotometer
(PurkinjeGeneralInstrument,Beijing,China). Thetime-resolvedfluorescencedecaywasinvestigated
by time correlated single photon counting (TCSPC) technique on FLS 920 (Edinburgh Instrument,
Edinburgh,UK).
2.3. SynthesisofCuNCs@ESMUsingN H ·H O
2 4 2
AcleanedpieceofESMplatformwasincubatedinanaqueousofCuSO solution(1mL,50mM)
4
for10min. Then,excessiveCuSO wasremoved,andtheCu2+impregnatedESM(Cu2+/ESM)was
4
transferred into 1 mL N H solution (85%) to initialize the reduction. The process typically took
2 4
approximately5~7hatambienttemperaturetogetstronglyorangeemittingCuNCs@ESM.
2.4. SynthesisofCuNCs@ESMUsingNH OH·HClorVC
2
In a typical process, a piece of ESM was incubated in a solution of CuSO (1 mL, 50 mM) for
4
10min. Afterthat,theobtainedCu2+/ESMwastreatedwithNaOHsolution(1mL,10M)for20min,
andthentransferredinto1mL1MNH OH·HClor2MVCsolution,immediately. Thereactiontook
2
5htogenerateintenseredfluorescentCuNCs@ESMatambienttemperature.
2.5. SurfacePatterningonESMSubstrate
Firstly,“Cu-N-C-s”lettersorthepatternofsmilingfacewerewrittendirectlyonanESMsubstrate
with50mMCuSO solutionas“ink”. Then, theESMwiththeimpregnatedlettersorpatternwas
4
soakedinto10MNaOHsolutionfor20min. Finally,thetreatedESMwastransferredinto2MVC
solutionfor5h,andthefluorescentlettersorpatternwerefabricatedembeddedintheESMsubstrate.
Materials2018,11,1913 4of16
2.6. ResponseofCuNCs@ESMStripstoHg2+Ions
Inatypicalprocedure,theas-preparedmonolithicCuNCs@ESMwascutintosmallpieces. Then,
thesmallpieceswereaddedinto1mLHg(NO ) solutionwithdifferentconcentrationsandincubated
3 2
atambienttemperature. Afterthat,thefluorescencewasobservedunder365nmUVlight. Meanwhile,
theinfluenceofotherinterferenceswasalsoinvestigatedwiththeconcentrationsof500µM.
2.7. CatalyticActivityofCuNCs@ESMforMBReduction
Typically,theas-preparedCuNCs@ESMnanocompositewasbrieflythrowninto1mL30µM
methyleneblue(MB)solutioninthesunlight. ThecatalyticdegradationofMBdyeinthepresenceof
CuNCs@ESMwasmonitoredbytheUV-visspectrometer.
3. ResultsandDiscussion
3.1. SynthesisandCharacterizationofCuNCs@ESMUsingN H ·H O
2 4 2
PreviousresearchdemonstratedtheformationofnobleAuandAgNCsbasedonthereductive
ability of ESM itself in alkaline solution [43,45,46]. What about the in situ synthesis of Cu NCs
employingESMasthesolidstateplatform? Herein,ininitialexperimentstheESMsweretreatedby
thesesimilarwayswithcuriosity[43](FiguresS1–S3). TheresultsshowthecolorofESMsincubated
with Cu2+ rapidly changed from blue to purplish red in alkaline solution, as described in other
literatures[20,47],whichindicatedtheformationofCu-ESMcomplex. However,nofluorescentCu
NCs were further formed, even if the reaction was prolonged to several days or the reaction was
allowedtoproceedatelevatedtemperature(55◦C)employingESMitselfasthereducerinalkaline
solution, whichwasgreatlydifferentfromthatofAu-ESMandAg-ESM.Therefore, newdifferent
routes would be needed to generate fluorescent Cu NCs embedded in monolithic ESM. We then
attemptedtointroduceanexternalreductant,N H ·H O,toinitializethereductionprocess. Typically,
2 4 2
theESMdisplayedabluecoloraftertreatingwithCuSO solutionfor10min,indicatingthatCu2+
4
wasadsorbedontoESMduetothecoordinationbetweenCu2+ andthefunctionalgroups(amines,
amides, and carboxylic groups) of the ESM fibers [47]. Exhilaratingly, after the introduction of
N H ·H O,theabsorbedCu2+couldbereducedtoCu(0)andthusinducedsimultaneousnucleation
2 4 2
andsize-regulatinggrowthoffluorescentCuNCsembeddedinESMplatform. Figure1ashowsthe
obtainedCuNCs@ESMhadayellowishcolorunderroomlightcharacteristicofultrafineCuNCs
andemittedintenseorangefluorescencecenteredat602nmexcitedwith365nmUVlight(Figure1b).
SeparatecontrolexperimentsweresimultaneouslyperformedbytreatingESMswithN H ·H Oand
2 4 2
CuSO ,respectively,andnoorangefluorescencewasobserved. Additionally,theinsitugeneratedCu
4
NCsshowedanexcitationmaximumat317nm(FigureS4).Besides,theexcitationdependenceproperty
ofCuNCsembeddedinESMwasalsoinvestigated,andnoobviousexcitation-dependentbehavior
wasobservedwhentheexcitationwavelengthschangedfrom260to420nm,indicatingthatthesize
distribution of in situ generated Cu NCs is equally uniform (Figure 1c). Fluorescence microscope
images (Figure 1a) show the characteristic interwoven fibrous structure of ESM remained almost
unchangedafterthesurfaceandinsideloadingoforangeemittingCuNCs,whencomparedwiththe
naturalblueemittingESMsubstrate. Itisnoteworthythatthebluefluorescencewasdramatically
suppressedaftertheinsitugenerationofCuNCscomparedwiththeESMitself(insetinFigure1b).
OnepossiblereasonwasthattheoxidationandreductionofsomecomponentsinthenaturalESM
substrateoccurredduringthereactionprocess. Ontheotherhand,theenergytransferbetweenthe
blueemissionfromESMitselfandtheclustersorotherproductswasnotunexpectedwhenexited
withthe365nmUVlight[43]. ThecontentofcopperinmonolithicCuNCs@ESMwasdetermined
to be 1.2% by inductively coupled plasma atomic emission spectroscopy (ICP-AES). XPS analysis
also verified the existence of Cu(0) on the ESM, which showed two strong peaks at 932.2 eV and
952.2 eV in the spectrum, corresponding to the Cu2p and Cu2p of Cu(0), respectively. The
3/2 1/2
absenceofCu2p3/2satellitepeakaround942eVindicatedtheabsenceofCu(II)inthepreparedCu
Materials2018,11,1913 5of16
Materials 2018, 11, x FOR PEER REVIEW 5 of 16
tNheC sp@reEpSaMre(dF iCguu rNe1Cds)@.EASsMw e(Fkignuorwe, 1thde).2 Aps3/ w2be iknndoinwg, etnheer g2py3o/2f bCinud(0in)gis eonnelyrg~y0 o.1f eCVua(0w) aisy ofrnolmy ~t0h.a1t
eoVf Cawu(aI)ys fproecmie tsh.aTth oefr Cefuo(rIe), stpheecvieasle. nTcheersetafotereo,f ththee vCaulenincep srotatteein ofm thater iCxum ino sptrloikteeilny mlieastrbiext wmeoesnt l0ikaenlyd
+li1es[ 1b3e,t2w0]e.en 0 and +1 [13,20].

Figure1.CharacterizationofESMsembeddedwithorwithoutCuNCs.(a)Roomlight(upperpanel)
Figure 1. Characterization of ESMs embedded with or without CuNCs. (a) Room light (upper panel)
and UV light (lower panel) photos of Cu NCs@ESM and controls. Samples from left to right
and UV light (lower panel) photos of Cu NCs@ESM and controls. Samples from left to right
correspond to ESM in H O, ESM in N H ·H O, ESM in CuSO , and Cu NCs@ESM, respectively.
correspond to ESM in H2O2, ESM in N2H24·H42O,2 ESM in CuSO4, an4d Cu NCs@ESM, respectively. The
ThefluorescencemicroscopyimageofESM(middle)andas-obtainedCuNCs@ESM(right)excited
fluorescence microscopy image of ESM (middle) and as-obtained Cu NCs@ESM (right) excited by 365
by365nm;(b)Fluorescenceemissionspectraof1–4samplesexcitedby365nm. Inset:fluorescence
nm; (b) Fluorescence emission spectra of 1–4 samples excited by 365 nm. Inset: fluorescence spectra
spectraofESMitselfbeforeandafterloadingCuNCsinthebluelightregion;(c)Fluorescenceemission
of ESM itself before and after loading Cu NCs in the blue light region; (c) Fluorescence emission
spectraofCuNCs@ESMwithvaryingexcitationwavelengthsfrom260to420nm;(d)XPSspectraof
spectra of Cu NCs@ESM with varying excitation wavelengths from 260 to 420 nm; (d) XPS spectra of
theCuNCs@ESMandESMintheCu2pregion.
the Cu NCs@ESM and ESM in the Cu 2p region.
In the synthetic route, the concentrations of CuSO precursor and N H ·H O reducer
4 2 4 2
In the synthetic route, the concentrations of CuSO4 precursor and N2H4·H2O reducer played
played crucial roles in generating fluorescent Cu NCs. Additional experiments were performed
crucial roles in generating fluorescent Cu NCs. Additional experiments were performed (Figures S5
(FiguresS5andS6) and the results show more than 5 mM Cu2+ and 85% N H ·H O solution
2 4 2
and S6) and the results show more than 5 mM Cu2+ and 85% N2H4·H2O solution (~17 M) were effective
(~17M)wereeffectivetoformstronglyorangeemittingCuNCsembeddedinESMafterthereaction
to form strongly orange emitting Cu NCs embedded in ESM after the reaction duration of 5 h at room
durationof5hatroomtemperature. Interestingly,whenthepreparedmonolithicCuNCs@ESMwas
temperature. Interestingly, when the prepared monolithic Cu NCs@ESM was always immersed in
alwaysimmersedinN H ·H Oreactionsolutionuntilseveraldaysat4◦C,themembranegradually
2 4 2
N2H4·H2O reaction solution until several days at 4 °C, the membrane gradually dissolved due to the
dissolved due to the alkaline hydrolysis and a homogeneous solution with brownish yellow was
alkaline hydrolysis and a homogeneous solution with brownish yellow was obtained, suggesting the
obtained,suggestingtheimmobilizedCuNCswerereleasedintoaqueoussolutionanddispersed
immobilized Cu NCs were released into aqueous solution and dispersed homogeneously. The
homogeneously. Themaximumexcitationandemissionpeakswereobservedat337nmand606nm,
maximum excitation and emission peaks were observed at 337 nm and 606 nm, and the aqueous Cu
andtheaqueousCuNCswasexcitation-independentastheparentCuNCs@ESM(FigureS7).Thelarge
NCs was excitation-independent as the parent Cu NCs@ESM (Figure S7). The large Stokes shift up to
Stokesshiftupto269nmoftheCuNCsmakesitactuallyapplicablewithverylowbackgroundand
269 nm of the Cu NCs makes it actually applicable with very low background and light scattering
lightscatteringinterference,whichisidealforbioimagingandbiosensing.
interference, which is ideal for bioimaging and biosensing.
3.2. SynthesisandCharacterizationofCuNCs@ESMUsingNH OH·HCl
2
3.2. Synthesis and Characterization of Cu NCs@ESM Using NH2OH·HCl
Intheabovecase,thefluorescentCuNCsweresuccessfullyinsitugeneratedinESMbyexternally
In the above case, the fluorescent Cu NCs were successfully in situ generated in ESM by
introducedN H ·H Oasthereducer. Inordertodevelopdiversesyntheticroutes,otherreducing
2 4 2
eaxgteenrntsa,lNlya iBnHtrodanudceNd HN2OHH4·H·H2OCl aws etrheea rlesodutrcieerd. tIon porredpearr teofl dueovreelsocpen dtiCvuerNseC ssy@nEthSeMtice mropultoeysi, nogththere
4 2
rseimduilcainrgsy angthenettisc, rNouaBteH.4H aonwde vNeHr,2vOeHry·HwCela kwlyerree dalesom ittrtiiendg tCou pNreCpsarceo uflludobreesocbetnati nCeud vNiaCsN@aEBSHM
4
employing the similar synthetic route. However, very weakly red emitting Cu NCs could be obtained
reduction. Inparticular,thereactionconditionwasrelativelyharsh,anditseemedverydifficultto
via NaBH4 reduction. In particular, the reaction condition was relatively harsh, and it seemed very
difficult to control a good nucleation and growth of copper clusters due to the strong reducing
Materials2018,11,1913 6of16
Materials 2018, 11, x FOR PEER REVIEW 6 of 16
control a good nucleation and growth of copper clusters due to the strong reducing capacity and
capacity and instability of NaBH4 itself (Figure S8). Meanwhile, no fluorescent CuNCs could be
instability of NaBH4 itself (Figure S8). Meanwhile, no fluorescent CuNCs could be generated by
generated by NH2OH·HCl reduction in the case. One possible reason is that, comparable to NaBH4
NH OH·HClreductioninthecase. Onepossiblereasonisthat,comparabletoNaBH andN H ·H O,
and2 N2H4·H2O, the lower reducibility of NH2OH·HCl was uncapable of convertin4g Cu(II2) to4 Cu2(0)
thelowerreducibilityofNH OH·HClwasuncapableofconvertingCu(II)toCu(0)effectively.
effectively. 2
Intriguingly,itwasfoundthattheCu2+/ESMtreatedsubsequentlywithstrongalkalinesolution
Intriguingly, it was found that the Cu2+/ESM treated subsequently with strong alkaline solution
before throwing into NH OH·HCl could be reduced to obtain red fluorescent Cu NCs effectively.
before throwing into NH22OH·HCl could be reduced to obtain red fluorescent Cu NCs effectively. In
Inthetypicalprocedure,ESMwasbrieflyincubatedinaCuSO solution(1mL,50mM)for10min,
the typical procedure, ESM was briefly incubated in a CuSO4 so4lution (1 mL, 50 mΜ) for 10 min, and
andthentheESMimmobilizedwithCu2+wassoakedinaNaOHsolutionof10Mfor30minduring
then the ESM immobilized with Cu2+ was soaked in a NaOH solution of 10 M for 30 min during which
whichthemembranecolorchangedtopurplishredfromblueindicatingtheformationofESM-Cu
the membrane color changed to purplish red from blue indicating the formation of ESM-Cu complex
complex[20,47],andfinallytransferredinto1MNH OH·HCltoinitiatethereduction. Asthereaction
[20,47], and finally transferred into 1 M NH2OH2·HCl to initiate the reduction. As the reaction
proceededfor1h,thecolorofthemembranechangedfrompurplishredtoyellow,suggestingthe
proceeded for 1 h, the color of the membrane changed from purplish red to yellow, suggesting the
formation of Cu NCs embedded in ESM. Figure 2a shows the images of obtained Cu NCs@ESM
formation of Cu NCs embedded in ESM. Figure 2a shows the images of obtained Cu NCs@ESM by
by NH OH·HCl reduction under alkaline environment. The Cu NCs@ESM emitted obvious red
NH2OH2·HCl reduction under alkaline environment. The Cu NCs@ESM emitted obvious red
fluorescence peaked at 652 nm (Figure 2b), and the maximum excitation was 370 nm (Figure S9).
fluorescence peaked at 652 nm (Figure 2b), and the maximum excitation was 370 nm (Figure S9). The
ThegeneratedCuNCsembeddedinmonolithicESMhaveexcitation-independentproperty(Figure2c)
generated Cu NCs embedded in monolithic ESM have excitation-independent property (Figure 2c)
suggestingtheuniformsizeandnarrowdistributionofCuNCs. XPSanalysisfurtherrevealedthe
suggesting the uniform size and narrow distribution of Cu NCs. XPS analysis further revealed the
existenceofCu(0)embeddedinESM(Figure2d),andthetotalcoppercontentwasdeterminedtobe
existence of Cu(0) embedded in ESM (Figure 2d), and the total copper content was determined to be
2.4%byICP-AES.
2.4% by ICP-AES.

Figure2. PhotosandspectraofCuNCs@ESMbyNH OH·HClreductionunderalkalinecondition.
Figure 2. Photos and spectra of Cu NCs@ESM by NH2O2H·HCl reduction under alkaline condition. (a)
(a)PhotosofCuNCs@ESMunderroomlight(left)and365nmUVlight(middle)alongwiththe
Photos of Cu NCs@ESM under room light (left) and 365 nm UV light (middle) along with the
fluorescencemicroscopyimage(right);(b)FluorescenceemissionspectraofCuNCs@ESMandESM
fluorescence microscopy image (right); (b) Fluorescence emission spectra of Cu NCs@ESM and ESM
excitedby365nm;(c)FluorescencespectraofCuNCs@ESMwithvaryingexcitationwavelengthsfrom
excited by 365 nm; (c) Fluorescence spectra of Cu NCs@ESM with varying excitation wavelengths
300to560nm;(d)XPSspectrumofCuNCs@ESMintheCu2pregion.
from 300 to 560 nm; (d) XPS spectrum of Cu NCs@ESM in the Cu 2p region.
As shown in Figure S10, weakly red emitting Cu NCs were generated after reaction in
As shown in Figure S10, weakly red emitting Cu NCs were generated after reaction in
NH OH·HClsolutionsfor1~6h,whentheCu2+/ESMsweretreatedsubsequentlywith500mM~5M
NNaHO22HOHa·qHuCelo suoslustoiolunsti ofonr 1b~e6f ohr,e wthherno wthine gCuin2+t/oES1MsM weNreH trOeaHte·dH Csul,bsreeqsupeencttliyv ewlyi.th 5E0v0e mnMm~o5r Me,
NaOH aqueous solution before throwing into 1 M NH2O2 H·HCl, respectively. Even more, no
no fluorescence was observed during the whole reaction with a NaOH concentration lower than
fluorescence was observed during the whole reaction with a NaOH concentration lower than 500
500 mM. However, when the concentrations of NaOH solution reached 10~12 M, the visible red
mM. However, when the concentrations of NaOH solution reached 10~12 M, the visible red emitting
emittingCuNCscouldbeinsitugeneratedwithin30min,andthefluorescencestrengthreacheda
Cu NCs could be in situ generated within 30 min, and the fluorescence strength reached a maximum
maximumafter5h. Obviously,intheprocedureemployingNH OH·HClasreductant,thestrong
after 5 h. Obviously, in the procedure employing NH2OH·HCl2 as reductant, the strong alkaline
alkalineconditionwasessentialforthegenerationofredfluorescentCuNCs. First,inacidandneutral
condition was essential for the generation of red fluorescent Cu NCs. First, in acid and neutral
aqueous solution, free carboxyl and amino groups in ESM were partly dissociated into negatively
Materials2018,11,1913 7of16
aqueoussolution, freecarboxylandaminogroupsinESMwerepartlydissociatedintonegatively
chargedspecies,incubatingESMwithCu2+ immediatelyproducedCu2+/ESMassociationbytheir
electrostaticattractionandcoordination. Subsequently,theacidityofsolutionwasadjustedtomore
thanpH12byaddingNaOHandthisresultedinanincreaseinthedegreeofexposureoffreeamino
groupswhichcouldcombinewithCu2+easilytoformastablecomplex.Second,thereducingcapability
oftyrosineresiduescanbegreatlyimprovedbyadjustingthereactionpHabovethepKaofTyr(~10).
Hence,thereducingcapabilityofESMitselfwasnotunexpectedsinceitcontainsTyrresiduesand
possibly other residues with reduction functionality. Third, Kodali et al. reported the presence of
considerableamountofcysteine-richESMproteinswithmultipledisulfidecrosslink[48]. Thedisulfide
bondscouldbecleavedintofree-SHunderalkalineenvironment. Andthe-SHcaneasilycombine
toformstableCu-Sbond,whichcanstabilizethenucleatedclusters. Theinfluencesofotherprocess
parameters,suchasincubationtime,theconcentrationsofCuSO precursorandNH OH·HClwere
4 2
alsoinvestigatedsystematically,whichwereshowninFigureS11–S13.
3.3. SynthesisandCharacterizationofCuNCs@ESMUsingVC
VCisonemildandenvironmentallyfriendlyreagent,whichwasoftenusedinthenanomaterials
preparationinsteadofsomesevereortoxicreductants. Herein,VCwasalsoemployedtogenerate
fluorescentCuNCsembeddedinESM.Excitedly,itwasfoundthatVCcouldinsitureduceCu2+loaded
onESMintofluorescentCuNCsunderalkalinesolution. TheobtainedCuNCs@ESMhadayellowish
color and emitted strong red fluorescence centered at 643 nm under 365 nm UV irradiation with
interwovenfibrousstructure(Figure3a,b).TheCuNCs@ESMhadamaximumexcitationat375nmand
themaximumemissionwavelengthwasalmostinvariablewhentheexcitationwavelengthschanged
from340to570nmindicatingtheuniformsizedistributionofgeneratedNCs(FiguresS14and3c).
Itnoteworthythatdifferentorangeandredfluorescencewereobservedinthedevelopedthreesynthetic
routes, which was confirmed by the excitation and emission spectra of Cu NCs@ESM composites
fabricatedbyemployingdifferentreducingreagents(FiguresS4,S9andS14). Althoughtheexactsize
determinationoftheCuNCsembeddedintheESMwasdifficult,thedifferentsizeofclusterscouldbe
expectedindifferentsyntheticroutesduetothedifferentclusteringprocessofCuatomsunderdifferent
reactionenvironmentandthesize-dependentfluorescencecouldbeachievedbyvaryingthesynthetic
routes [17]. The content of copper in the obtained Cu NCs@ESM was determined to be 1.6% by
ICP-AES.ThegenerationofCu(0)ontheESMplatformwasfurtherdemonstratedbytheCu2P and
3/2
Cu2P peakscenteredat931.6and951.3eV,respectively(Figure3d). Thetime-resolvedfluorescence
1/2
decayoftheobtainedCuNCs@ESMwassimultaneouslyinvestigatedbytimecorrelatedsinglephoton
counting (TCSPC) technique, and the average lifetime of generated Cu NCs was calculated to be
2.84ns(FigureS15). Inthetypicalalkalinecondition,whentheconcentrationsofCuSO precursor
4
wasmorethan10mMandconcentrationsofVCwasmorethan1M,theredfluorescentCuNCswere
generatedinafewminutesandreachedthestrongestfluorescenceabout5h(FiguresS16andS17).
Ithastobenotedthatthestrongalkalineenvironmentplaysakeyroleinthesyntheticroute. Whenthe
ESMwaspre-incubatedin1mLCuSO (50mM)for10min,thentreatedwithNaOHaqueoussolution
4
ofconcentrationslowerthan0.1M,andfinallytransferredto2MVCsolution,nofluorescentCuNCs
couldbegeneratedevenwithaprolongedreaction(FigureS18). Thismaybeduetothereducing
capabilityofESMitselfandVCwasinitiatedeffectivelyinstrongalkalineconditiontogeneratemetal
nucleationandfluorescentclustersasexpected.
To test the effect of copper precursors on the fluorescence of Cu NCs@ESM in the developed
strategies,variouscopperprecursorssuchasCuCl ,Cu(NO ) ,Cu(CH COO) ,CuSO wereemployed
2 3 2 3 2 4
inthethreeroutesreducedbyN H ·H O,NH OH·HClandVC,respectively. Theresultsshowthat
2 4 2 2
brightlyorangeandredemittingCuNCscouldbegeneratednomatterwhatkindoftheabovefour
copper precursors was employed (Figure S19). Interestingly, the ESM platform, even if boiled in
water,wasalmostequallyefficientintheproposedmethodsforgeneratingfluorescentCuNCs@ESM
(FigureS20). Inaddition,UV-VisDRSmeasurementwasconductedtofurtherconfirmthesuccessful
Materials2018,11,1913 8of16
fMoratmeriaatlsi o20n18o, f11C, ux FNOCR sPEoEnR tRhEeVEIESWM substrate[44]. AsshowninFigure4,therewerenocharacter8i osft i1c6
Materials 2018, 11, x FOR PEER REVIEW 8 of 16
surfaceplasmonresonance(SPR)absorptionofCunanoparticles(CuNPs)locatedat~560nminthe
spectra of Cu NCs@ESM fabricated by the three different routes, indicating the in situ generation of
spectraofCuNCs@ESMfabricatedbythethreedifferentroutes,indicatingtheinsitugenerationof
spectra of Cu NCs@ESM fabricated by the three different routes, indicating the in situ generation of
ultra-small Cu NCs rather than the large-sized Cu NPs [31].
ultra-smallCuNCsratherthanthelarge-sizedCuNPs[31].
ultra-small Cu NCs rather than the large-sized Cu NPs [31].

Figure 3. Photos and spectra of Cu NCs@ESM by VC reduction under alkaline condition. (a) Photos
FFiigguurree 33.. PPhhoottooss aanndd ssppeeccttrraa ooff CCuu NNCCss@@EESSMM bbyy VVCC rreedduuccttiioonn uunnddeerr aallkkaalliinnee ccoonnddiittiioonn.. ((aa)) PPhhoottooss
of obtained Cu NCs@ESM under room light (left) and 365 nm UV light (middle) along with the
of obtained Cu NCs@ESM under room light (left) and 365 nm UV light (middle) along with the
of obtained Cu NCs@ESM under room light (left) and 365 nm UV light (middle) along with the
fluorescence microscopy image (right); (b) Fluorescence emission spectra of Cu NCs@ESM and ESM
fluorescencemicroscopyimage(right);(b)FluorescenceemissionspectraofCuNCs@ESMandESM
fluorescence microscopy image (right); (b) Fluorescence emission spectra of Cu NCs@ESM and ESM
excited by 365 nm; (c) Fluorescence spectra of Cu NCs@ESM by varying excitation wavelengths from
excitedby365nm;(c)FluorescencespectraofCuNCs@ESMbyvaryingexcitationwavelengthsfrom
excited by 365 nm; (c) Fluorescence spectra of Cu NCs@ESM by varying excitation wavelengths from
340 to 570 nm; (d) XPS spectrum in the Cu 2p region of the resulting Cu NCs@ESM.
340to570nm;(d)XPSspectrumintheCu2pregionoftheresultingCuNCs@ESM.
340 to 570 nm; (d) XPS spectrum in the Cu 2p region of the resulting Cu NCs@ESM.
2.4 ESM
2.4 E CSuM NCs@ESM reduced by N2H4
C Cuu N NCCss@@EESSMM r reedduucceedd b byy N N2HH42 OH·HCl
C Cuu N NCCss@@EESSMM r reedduucceedd b byy N VHC2OH·HCl
2.0 Cu NCs@ESM reduced by VC
2.0
s 1.6
sb 1.6
bA
A
1.2
1.2
0.8
0.8
200 300 400 500 600 700 800
200 300 400 500 600 700 800
Wavelength(nm)
Wavelength(nm)

FFiigguurree 44.. TThhee UUVV--vviiss DDRRSS ssppeeccttrraa ooff nnaattuurraall EESSMM ((bbllaacckk lliinnee)) aanndd aass--oobbttaaiinneedd CCuu NNCCss@@EESSMM
Fnniaagnnuoorcecoo m4m. ppToohssieitt eeUss Viinn- vsisisti utuD gRgeSne nesreparteaectdetrd ab ybo yNf 2nNHa24t·HuHr4a2·OlH E2reOSdMur ce(tdbioulancc tk(ior enldin (leri)en dea)n,l diNn eHa)s,2-OoNHbHt·aH2inOCelHd r ·eHCduCu clNtiroCends @u(gcEtrSieoMenn
n(lgianrneeoe)c naonlmidnp eVo)Csain tredesd ViunCc tsriioetudn u (gcbetlniuoeenr la(itnbeledu) ,e brlyeins Npee)2,Hcrtei4v·sHepl2eyOc.t irveedlyu.ction (red line), NH2OH·HCl reduction (green
line) and VC reduction (blue line), respectively.
It is noteworthy that the blue fluorescence was also dramatically suppressed after the generation
It is noteworthy that the blue fluorescence was also dramatically suppressed after the generation
of Cu NCs compared with the ESM itself in the NH2OH·HCl and VC reduction routes (Figure S21).
oAf cCcuor NdiCnsg ctoom thpea reexdc iwtaittiho nth sep EecStMra iotsf etlhf ei nf atbhrei cNatHed2O cHom·HpCols aitneds (VFCig urerdesu cSt9io ann dro Su1te4s) , (oFnigeu preo sSs2i1b)l.e
According to the excitation spectra of the fabricated composites (Figures S9 and S14), one possible
reason was that an efficient fluorescence resonance energy transfer (FRET) could happen between the
reason was that an efficient fluorescence resonance energy transfer (FRET) could happen between the
Materials2018,11,1913 9of16
Itisnoteworthythatthebluefluorescencewasalsodramaticallysuppressedafterthegeneration
oMfaCteruialNs 2C01s8c, o11m, xp FaOrRe dPEwERit hREtVhIeEWES MitselfintheNH OH·HClandVCreductionroutes(Figure9S o2f1 1)6.
2
Accordingtotheexcitationspectraofthefabricatedcomposites(FiguresS9andS14),onepossible
ESM protein and Cu NCs, indicating a spatial proximity between the formed Cu NCs and the protein
reasonwasthatanefficientfluorescenceresonanceenergytransfer(FRET)couldhappenbetween
fluorophores inside the ESM, and the excellent stability of the obtained monolithic Cu NCs@ESM
the ESM protein and Cu NCs, indicating a spatial proximity between the formed Cu NCs and
could be expected. On the other hand, the contribution of the oxidation or reduction of some
theproteinfluorophoresinsidetheESM,andtheexcellentstabilityoftheobtainedmonolithicCu
components in the natural ESM substrate during the reaction process was not unexpected. As shown
NCs@ESM could be expected. On the other hand, the contribution of the oxidation or reduction
in Figure S22, the red fluorescence intensity of monolithic Cu NCs@ESM was still strong stored in
ofsomecomponentsinthenaturalESMsubstrateduringthereactionprocesswasnotunexpected.
wet form (dipped in water) for around 15 days and even more than a month in dried form at 4 °C in
AsshowninFigureS22,theredfluorescenceintensityofmonolithicCuNCs@ESMwasstillstrong
the refrigerator. Moreover, photobleaching was not observed even when the product had been
storedinwetform(dippedinwater)foraround15daysandevenmorethanamonthindriedformat
4co◦nCtiinnutohuesrleyf riirgreardaitaotre.dM uonrdeoevr e3r6,5p hnomto UblVea lcihgihntg fowra 1s2n ho t(oFbigsuerrve eSd23ev).e Antwtrhacetnivtheleyp, rthode uflcutohraedscbeenecne
intensity could be maintained without any weakening even under high ionic strength conditions (1
continuouslyirradiatedunder365nmUVlightfor12h(FigureS23). Attractively,thefluorescence
M NaCl solution, Figure S24) for a long period of time. In addition, the Cu NCs@ESM remained very
intensity could be maintained without any weakening even under high ionic strength conditions
stable in the majority of organic solvents while immediate fluorescence quenching occurred in
(1MNaClsolution,FigureS24)foralongperiodoftime. Inaddition,theCuNCs@ESMremained
ethanol as shown in Figure 5a, and the fluorescence intensity decreased continuously with increasing
verystableinthemajorityoforganicsolventswhileimmediatefluorescencequenchingoccurredin
evtohlaunmole afsraschtoiownns ionf Feitghuarneo5l a(,vaonl d%t,h eethflaunoorel:sHce2Onc).e Iitn stehnosuitldy dbeec nreoatseedd tchoant tain luoowu svloyluwmithe finraccrteiaosnin ogf
0.05% could result in an obviously distinguishable response from the background quickly (Figure
volumefractionsofethanol(vol%,ethanol:H O).Itshouldbenotedthatalowvolumefractionof
2
5b). The effective response of Cu NCs@ESM to ethanol content indicates the promise to develop
0.05%couldresultinanobviouslydistinguishableresponsefromthebackgroundquickly(Figure5b).
visual sensing strips for ethanol solution and vapor conveniently based on the monolithic products.
TheeffectiveresponseofCuNCs@ESMtoethanolcontentindicatesthepromisetodevelopvisual
Herein, the fluorescent stability of Cu NCs embedded in ESM substrate was contributed to the
sensingstripsforethanolsolutionandvaporconvenientlybasedonthemonolithicproducts. Herein,
protection of fibrous ESM structure and rich functional groups in the natural ESM acting as
thefluorescentstabilityofCuNCsembeddedinESMsubstratewascontributedtotheprotectionof
nucleation template and capping stabilizer, which avoids the aggregation and oxidation induced
fibrousESMstructureandrichfunctionalgroupsinthenaturalESMactingasnucleationtemplateand
fluorescence quenching. In the presence of ethanol, ESM substrate was denatured mainly due to the
cappingstabilizer,whichavoidstheaggregationandoxidationinducedfluorescencequenching. Inthe
destroy of hydrogen bonds and hydrophobicity of the composed proteins, and a looser structure was
presenceofethanol,ESMsubstratewasdenaturedmainlyduetothedestroyofhydrogenbondsand
expected. Thus, the protection of ESM with Cu NCs was correspondingly weakened and the surface
hydrophobicityofthecomposedproteins,andalooserstructurewasexpected. Thus,theprotectionof
chemical environment of the clusters exhibited a significant change, resulting in easy oxidation of Cu
ESMwithCuNCswascorrespondinglyweakenedandthesurfacechemicalenvironmentoftheclusters
NCs and fluorescence quenching of Cu NCs@ESM composite. In addition, the yellow color of the
exhibitedasignificantchange,resultingineasyoxidationofCuNCsandfluorescencequenchingof
fabricated composite, which was the characteristic color of formed ultralfine Cu NCs, was found to
CuNCs@ESMcomposite. Inaddition,theyellowcolorofthefabricatedcomposite,whichwasthe
fade obviously in the presence of ethanol (Figure S25a). Fascinatingly, the red fluorescence could be
characteristiccolorofformedultralfineCuNCs,wasfoundtofadeobviouslyinthepresenceofethanol
recovered when the quenched composite was replaced in a reducing agent solution such as sodium
(FigureS25a). Fascinatingly,theredfluorescencecouldberecoveredwhenthequenchedcomposite
wboarsorheypdlarciedde i(nNaaBreHd4u),c ianngda tgheen ytesolllouwti ocnolsourc rheaasppsoedairuedm, bwohroichhy sdhroidwee(dN tahBeH pr),oamnidset hoef ytheell orwecycoclleodr
4
ethanol sensing application (Figure S25b).
reappeared,whichshowedthepromiseoftherecycledethanolsensingapplication(FigureS25b).

FFiigguurree 55.. SSoollvveenntt eeffffeeccttss ooff CCuu NNCCss@@EESSMM ggeenneerraatteedd bbyy VVCC rreedduuccttiioonn.. ((aa)) tthhee ssoollvveennttss aarree ((11)) wwaatteerr;;
((22)) aacceettoonnee;; ((33)) ffoorrmmaallddeehhyyddee;; ((44)) mmeetthhaannooll;; ((55)) eetthhyylleennee ggllccyyccooll;; ((66)) iissoopprrooppaannooll;; ((77)) nn--bbuuttyyll aallccoohhooll;;
((88)) etehthyylal caectaettea;te(9; )(c9y)c lcoyhcelxoahneex;a(n1e0;) e(1th0a) neotlh,raensople, crtievsepleyc;t(ibv)eTlyh; e(rbe)s pTohnes eroefspproenpsaer eodf CpureNpCarse@dE SCMu
tNoCetsh@aEnSoMls otolu ettihoannwoli tsholduitfifoerne wntitvho lduimffeerefrnatc vtioolnusm,fer ofrmac0titoon1s,% fruonmd e0r to36 15%n munUdeVr l3ig65h tn.m UV light.
3.4. Surface Patterning on ESM Substrate
Due to the excellent stability and strong fluorescence of the prepared monolithic Cu NCs@ESM
in dried form, it was possible to create fluorescent patterns on the surface of ESM employing the
proposed facile and green route employing VC as the reducing reagent. For example, a red
Materials2018,11,1913 10of16
3.4. SurfacePatterningonESMSubstrate
DuetotheexcellentstabilityandstrongfluorescenceofthepreparedmonolithicCuNCs@ESM
in dried form, it was possible to create fluorescent patterns on the surface of ESM employing
Materials 2018, 11, x FOR PEER REVIEW 10 of 16
the proposed facile and green route employing VC as the reducing reagent. For example, a red
flfluuoorreesscceenntt ssmmiilliinngg fafcaec eanadn d“C“uC-Nu--NC--sC”- sle”ttleertst ewrserwe oebretaoinbetdai bnye dsimbyplsyi mpapinlytinpga itnhteimng onth ae mpieocne oaf
pEiSeMce soufbEstSrMates uwbisthtr aCtueSwOi4t hsoCluutSioOn4 asso l“uintiko”n faosll“oiwnked” fboyl loVwCe idncbuybaVtCionin ucnudbearti oanlkaulnindee rcoanlkdaitliionne
c(Foingduirteio n6)(. FMiguorree 6i)m.pMorotraentilmy,p othrtisa nwtlyo,rkth icsowulodr kbeco uemldpbloeyeemd pfloory e“dhifdodre“nh”i dwderinti”nwg raitnindg aanntdi-
aconutin-ctoeurfnetietirnfegi taipnpglaicpaptiloicnast.i ons.

FFiigguurree 66.. PPhhoottooss ooff tthhee mmoonnoolliitthhiicc CCuu NNCCss@@EESSMM ffaabbrriiccaatteedd bbyy VVCC rreedduuccttiioonn iinn ddrriieedd ffoorrmm uunnddeerr
vviissiibbllee lliigghhtt ((aa)) aanndd 336655 nmnm UUV Vliglhigth (ct)(; cS)u;rSfuacrfea fcleuoflrueoscreenscceen pcaettpearnttienrgn ionfg smofilsinmgi lfiancge (fbac) ean(bd) “aCnud-
“Cu-N-C-s” letters (d) obtained by simply writing the patterning on a piece of ESM with CuSO
N-C-s” letters (d) obtained by simply writing the patterning on a piece of ESM with CuSO4 solution4
solutionas“ink”followedbyNaOHandVCincubation.
as “ink” followed by NaOH and VC incubation.
3.5. MonolithicCuNCs@ESMCompositeasHg2+ResponsiveStrips
3.5. Monolithic Cu NCs@ESM Composite as Hg2+ Responsive Strips
Developing solid state fluorescent materials for sensing purposes (a good example is pH
Developing solid state fluorescent materials for sensing purposes (a good example is pH testing
testing paper) is of great significance with the advantages of reducing liquid handling operations
paper) is of great significance with the advantages of reducing liquid handling operations (such as
(such as accurate volume measurements) and greatly lowered detection costs, which is especially
accurate volume measurements) and greatly lowered detection costs, which is especially useful in
usefulincircumstancesofin-fieldorPOCquantitativeorsemi-quantitativetests. Mercuryisahighly
circumstances of in-field or POC quantitative or semi-quantitative tests. Mercury is a highly toxic
toxicheavymetalelementthatiswidelydistributedintheenvironment. Thereisastrongdemandto
heavy metal element that is widely distributed in the environment. There is a strong demand to
developlow-costandefficienttechniquesforHg2+detection. Asaproof-of-conceptdemonstration,
develop low-cost and efficient techniques for Hg2+ detection. As a proof-of-concept demonstration,
theas-preparedCuNCs@ESMwasusedasvisible“turn-off”stripsensorforHg2+withsatisfactory
the as-prepared Cu NCs@ESM was used as visible “turn-off” strip sensor for Hg2+ with satisfactory
sensitivityandselectivity. AsshowninFigure7b,thefluorescenceofCuNCs@ESMwasquenched
sensitivity and selectivity. As shown in Figure 7b, the fluorescence of Cu NCs@ESM was quenched
completelybydippingthestripinanHg2+solutionfor1h,whilethefluorescenceintensitywasalmost
completely by dipping the strip in an Hg2+ solution for 1 h, while the fluorescence intensity was
unchangedwhenincubatedwithothermetalions(Mn2+, Co2+, Cd2+, Ba2+, Zn2+, Pb2+, Al3+, Fe3+,
almost unchanged when incubated with other metal ions (Mn2+, Co2+, Cd2+, Ba2+, Zn2+, Pb2+, Al3+, Fe3+,
Cu2+, K+, Na+) at the concentration of 500 µM. The current results show that the concentration of
Cu2+, K+, Na+) at the concentration of 500 μM. The current results show that the concentration of Hg2+
Hg2+ aslowto50µMcouldbevisiblyrecognizedbydistinguishablefluorescencequenchingfromthe
as low to 50 μM could be visibly recognized by distinguishable fluorescence quenching from the
backgroundeasily(Figure7a). Thepossiblequenchingmechanismisattributedtothehigh-affinity
mbaectkagllroopuhnidli ceainsitleyr a(cFtiigounrse b7eat)w. Tehene pthoessdib10lec qeunteenrcshoinfgH mg2e+ch(5adn1is0m) a insd atdtr1i0bcuetnedte rtos othfeC hui+gh(-3adf1fi0n)iitny
metallophilic interactions between the d10 centers of Hg2+ (5d10) and d10 centers of Cu+ (3d10) in the Cu
the Cu NCs embedded in ESM. In addition, the formation of certain complexes by coordination
NCs embedded in ESM. In addition, the formation of certain complexes by coordination and
andelectrostaticattractionsbetweenHg2+ andtherichfunctionalgroups(e.g.,hydroxyl,carboxyl,
electrostatic attractions between Hg2+ and the rich functional groups (e.g., hydroxyl, carboxyl, amino
aminoandthiol)intheESMplatformwasnotunexpected. Overall,thesurfacecapping-ligandsand
and thiol) in the ESM platform was not unexpected. Overall, the surface capping-ligands and charge
states of these Cu NCs were greatly changed in the presence of Hg2+ ions mainly due to the
aforementioned interactions, which leads to the effective fluorescence quenching [49].