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Single cytidine u

State Key Laboratory of Bioelectronics, So

China. E-mail: xuewang@seu.edu.cn

† Electronic supplementary informationpreparation of Au NCs, photophysical punder different pH and reaction time, a10.1039/c4nr02180k

‡ These authors contributed equally.

Cite this: Nanoscale, 2014, 6, 10355

Received 22nd April 2014Accepted 27th June 2014

DOI: 10.1039/c4nr02180k

www.rsc.org/nanoscale

This journal is © The Royal Society of C

nits-templated syntheses of multi-colored water-soluble Au nanoclusters†

Hui Jiang,‡ Yuanyuan Zhang‡ and Xuemei Wang*

Ultra-small metallic nanoparticles, or so-called “nanoclusters” (NCs), have attracted considerable interest

due to their unique optical properties that are different from both larger nanoparticles and single atoms.

To prepare high-quality NCs, the stabilizing agent plays an essential role. In this work, we have revealed

and validated that cytidine and its nucleotides (cytidine 50-monophosphate or cytidine 50-triphosphate)can act as efficient stabilizers for syntheses of multicolored Au NCs. Interestingly, Au NCs with blue,

green and yellow fluorescence emissions are simultaneously obtained using various pH environments or

reaction times. The transmission electron microscopy verifies that the size of Au NCs ranges from 1.5 to

3 nm. The X-ray photoelectron spectroscopy confirms that only Au (0) species are present in NCs.

Generally, the facile preparation of multicolored Au NCs that are stabilized by cytidine units provides

access to promising candidates for multiple biolabeling applications.

Introduction

The ultra-small metallic nanoparticles (NPs) have attractedconsiderable interest in the recent decade.1,2 Nowadays, todistinguish these particles from those traditional NPs (such ascolloidal gold or silver particles), NPs with a size of smaller than2 nm are dened using a new term: nanoclusters (NCs).3

Theoretical investigations have demonstrated that the contin-uous density of states splits into discrete energy levels when theparticle size approaches the Fermi wavelength of electrons, thusexhibiting signicantly different optical, electronic and chem-ical properties from either larger NPs or single atoms. Forexample, the well-known surface plasmon resonance (SPR)effect by Au NPs is diminished completely when their size isreduced to 2 nm, while uorescence (FL) emission usuallyemerges at this size. These unique properties offer greatpossibilities in exploring their potential applications insensing,4–8 catalysis,9 bioimaging,10–13 and therapy.13 The widelyreported metallic NCs include but are not limited to: Au,14–17

Ag,18,19 Cu,20 Pt,21 and Au–Ag alloy NCs.22 Among these, Au andAg NCs have attracted more attention. Although Ag NCs havehigher FL quantum yields, they may cause undesired toxicity inbiological systems due to the gradual release of low-molecular-weight silver species.23 In comparison, Au NCs, which is the

utheast University, Nanjing 210096, PR

(ESI) available: The feed amount forroperties of Au NCs, the FL spectrand XPS results are included. See DOI:

hemistry 2014

focus of this work, are generally biocompatible and suitable forlong-term applications.10

To prepare stable, multi-colored, and water-soluble NCs forbiomedical purposes, the stabilizing agent or pre-formedtemplate plays an essential role. An ideal stabilizer shouldprecisely control the growth rate of NCs and overcome theirtendency to aggregate into larger NPs spontaneously as toreduce their surface energy.3 Recently, DNA oligomers havebeen shown success as stabilizers due to their exiblesequences and versatile complexation with metallic cores, aspioneered by Dickson's group and investigated by severalgroups.24–26 Liu and co-workers have recently validated that AuNCs can be easily prepared even when the DNA oligomers areshortened to a single base (i.e., adenosine and itsnucleotides).27

Herein, we prepared Au NCs templated by another seriesof single-base contained nucleoside/nucleotide, namely,cytidine (Cyt), cytidine 50-monophosphate (CMP) or cytidine50-triphosphate (CTP) at room temperature. Interestingly, AuNCs with blue, green and yellow FL emissions are simulta-neously obtained in various pH environments or usingdifferent reaction times (Scheme 1), while only blue-emittedAu NCs are prepared by using adenosine and its nucleo-tides.27 The transmission electron microscopy and X-rayphotoelectron spectroscopy indicate that the three kindsof Au NCs show size-dependent emissions and the blue-emitted Au NCs are intermediates for the preparationof green or yellow-emitted NCs. Generally, the simultaneousfacile preparation of multi-colored Au NCs provides accessto promising candidates for multiple biolabelingapplications.

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Scheme 1 Illustration of the room-temperature syntheses of multi-colored Au NCs stabilized by cytidine and cytidine nucleotides.

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Materials and methodsReagents and instruments

Cytosine (C4H5N3O), Cyt (C9H13N3O5), CMP (C9H12N3O8PNa2),and CTP (C9H16N3O14P3Na2) were purchased from SigmaAldrich (St. Louis, MO, USA). All other reagents were of analyt-ical grade from Sinoreagent, China. A citrate buffer at a series ofpH values was prepared bymixing different molar ratios of citricacid and trisodium citrate.

The UV-vis and uorescence (FL) spectra were recorded on aBiomate 3S spectrometer (Thermo Co. Ltd., US) and aRF-5301PC uoremeter (Shimadzu, Tokyo, Japan), respectively.The transmission electron microscopy (TEM) and high-resolu-tion TEM images were obtained on a JEM-2100 TEM (JEOL,Japan) at an applied accelerating voltage of 200 kV. The X-rayphotoelectron spectroscopic data were recorded on a PHI 5000VersaProbe X-ray photoelectron spectrometer (XPS) with an AlKa ¼ 280.00 eV excitation source. The mass spectroscopy (MS)was recorded on a LCQ Fleet electrospray ionization (ESI†) massspectrometer (Thermo, USA).

Preparation and separation of Cyt/Cyt nucleotides stabilizedAu NCs

Au(III) precursors are prepared using a mixture of HAuCl4 (nalconcentration 1.0 mM) and Cyt (CMP or CTP) at a molar ratio of1 : 1 or 1 : 2. The reduction of Au(III) precursors is initiated bythe injection of citrate of various pHs (nal concentration50 mM) to the mixture. The FL spectra for the reaction solutionare recorded aer different reduction times (i.e., the timeinterval from the injection until measurement). All experiments

10356 | Nanoscale, 2014, 6, 10355–10362

are performed at room temperature unless otherwise indicated.The typical luminescent Au species, i.e., blue-emitted Au–Cyt(b-AuCyt) or Au–CMP (b-Au–CMP), green-emitted Au–Cyt (g-Au–Cyt), and yellow-emitted Au–CMP (y-Au–CMP) are preparedaccording to the feed amount in Table S1.†

The synthesized Au uorescent species can be separatedfrom the reaction solution by ethanol precipitation and ultra-ltration. As is well known, uorescent Au NCs, smaller poly-meric gold species, and larger gold structures can formsimultaneously when using citrate as a reducing agent. Thelarge-sized NPs can be rst precipitated and removed by theaddition of a small amount of absolute ethanol (volume ofethanol to sample at �0.1 : 1). Then, more ethanol is graduallyadded to the ltrate. The solution turns turbid when the volumeratio of ethanol to ltrate reaches 1 : 1 or higher. The precipi-tates are collected aer ultracentrifugation at 8000 rpm for 10min. These crude samples can be re-dispersed in doublydistilled water and further puried by using a Nanosep ultra-lter (Pall, US) with a molecular weight cut-off (MWCO) of10 kDa. The components with MW higher than 10 kDa are thenprecipitated with ethanol. The precipitates are dried in vacuum.The obtained solid samples can be stored at �20 �C for days tomonths. These luminescent products can be well dispersed in asmall amount of doubly distilled water for TEM and MScharacterization.

Results and discussionInteraction of cytosine and its derivatives with HAuCl4

The interaction between nucleotides and metals is well-knownas both DNA and RNA are ideal scaffolds for metal NPs

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deposition, which is a process with potential application in 1Dnanoelectronics.28 Our group has theoretically evaluated thebinding capacity between adenine (or thymine) and Au clustersby quantum chemical calculations.29

In this work, we attempt to use the complexes formed by Auand cytidine/cytidine nucleotides as precursors for thesynthesis of uorescent Au NCs. The Au(III)–Cyt complexation isinitially evidenced by an indirect method. First, a mixture ofAuCl4

� (with a cumulative stability constant of �4.0 � 105) andAg+ is maintained clear for several hours (Fig. S1, ESI†). WhenCyt and AuCl4

� were premixed, the sequent addition of Ag+

solution generated insoluble AgCl immediately. This indicatesthe replacement of Cl� by Cyt and the strong Au(III)–Cytcomplexation.

To quantify the interaction between the cytosine unit andHAuCl4, we investigated the UV titration processes of thesecompounds by HAuCl4. Although in a previous work CTP-coatedAu NPs were prepared via ligand exchange on the citrate-stabi-lized Au NPs and it suggested the Au–cytosine interaction,30 thepossible stoichiometric relationship remains unclear. Fig. 1Arepresents a typical titration process. The characteristic UVabsorption peak at �276 nm from Cyt (1 mL, 40 mM) graduallyincreases aer the addition of 2–40 mL of 2mMHAuCl4. AnotherUV peak emerges at �220 nm upon titration of HAuCl4 due tothe ligand-to-metal charge transfer in AuCl4

�.31 The relation-ship between the change in the peak absorbance (DAbs276nm)and the concentration of HAuCl4 is shown in Fig. 1B (solidsquare). Considering that HAuCl4 also contributes to theabsorbance at approximately 276 nm, the increase caused byHAuCl4 should be subtracted according to a standard calibra-tion curve at 276 nm (curve not shown). Thus, the “real” changein absorbance tends be stable at HAuCl4 of �40 mM (hollowtriangle, Fig. 1B), which is exactly the same as the concentrationof Cyt, indicating that the complexation occurs at a molar ratio

Fig. 1 (A) Titration of AuCl4� (0–80 mM, interval: 4 mM) to 40 mMCytmoni

peak absorbance at�276 nm is located between 0.1 and 1.0, i.e., the lineaapparent increment (solid square) and subtracted increment (hollow triamaximum DAbs276nm appears with the addition of 40 mM AuCl4

�, indica

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of 1 : 1. The same molar ratio is obtained for Au–CMP (Fig. S2,ESI†) and Au–cytosine (Fig. S3, ESI†), while a molar ratio of 1 : 2is found for Au–CTP (Fig. S4, ESI†).

In summary, the UV titration results conrm that all thesecytosine-based molecules show a stoichiometric interactionwith Au(III) with a ratio of either 1 : 1 or 1 : 2. The possiblecomplexation mechanisms between cytosine and metal havebeen investigated. Theoretical calculations suggests the pres-ence of Ag–N3 (cytosine) bonds in silver nanoclusters.32 Ramaninvestigations also indicate strong bonding between Au(III) andN3/O2 (cytosine).33,34 These stable precursors provide the keybasis for the subsequent formation of uorescent Au species.

Fast generation of blue-emitted Au species

Citrate is a classical reducing agent for the preparation ofcolloidal Au NPs from HAuCl4. Herein, we used citrates atvarious pHs to reduce Au–Cyt precursors. Interestingly, the Au–Cyt mixture at a molar ratio of 1 : 2 shows signicant blue FLemission shortly aer the injection of citrate at pH 4–6 (Fig. 2A).Upon reduction for 1 h, the optimized FL excitation/emissionwavelength is 360 nm/455 nm for pH 4, 380 nm/470 nm for pH5 and 375 nm/500 nm for pH 6, respectively, with an intensity (I)order of IpH5 > IpH4 > IpH6. Similar blue FL emission (peak at�460 nm) is also observed by the reduction of Au–CMPprecursors at a molar ratio of 1 : 2 at pH 4–6 (Fig. 2B). Furtherinvestigation reveals the indispensable role of citrate. As shownin Fig. S5A, ESI,† no FL is observed for CMP itself (curve a) andAu–CMP precursors (curve b), while signicant FL appears aer5 min incubation with citrate (curve c), indicating that thereduction of Au(III) is essential for the generation of blue-emitted uorescent species.

Comparatively, Au–CTP and Au–cytosine are ineffectiveprecursors. Although similar FL around 460 nm is observed bythe reduction of Au–CTP precursor (molar ratio of 1 : 2), the

tored by UV spectra. The Cyt concentration of 40 mMcan ensure that itsr detection range for UV-vis absorptionmethod. (B) The relationship ofngle) in peak absorbance at 276 nm and concentration of AuCl4

�. Theting a “saturated” complexation ratio of 1 : 1.

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Fig. 2 (A) FL excitation (a, c and e) and emission (b, d and f) spectra for Au–Cyt (molar ratio of 1 : 2) after reduction by citrate at pH 4 (a and b), 5 (cand d), or 6 (e and f) for 60min. Inset: the photographs for a sample prepared at pH 5 under exposure to sunlight (left) and illumination at 365 nm(right); (B) FL emission spectra for Au–CMP (molar ratio of 1 : 2) after reduction by citrate at pH 3–7 (a–e) for 60 min (Excitation at 360 nm for pH4–7 and 350 nm for pH 3); (C and D) Comparison of FL peak intensity by using Au–Cyt (C) or Au–CMP (D) at a molar ratio of 1 : 1 (a, c and e) or1 : 2 (b, d and f) at pH 4 (a and b), 5 (c and d) and 6 (e and f), respectively. All data are recorded exactly at a reduction time of 15, 30 and 60min witha same excitation wavelength at 360 nm. Excitation/emission slit width: 3/3 nm.

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intensity is weak and only 1/50–1/100 that of the Au–Cyt or Au–CMP system (Fig. S5B, ESI†). As for Au–cytosine, the FL is almostnegligible aer the reduction (Fig. S6, ESI†). This result isconsistent with that of adenine,27 suggesting that the deoxyri-bose residue is important for the construction of active uo-rescent species.

The dependence of FL emission intensity upon reductiontime was investigated using Au–Cyt and Au–CMP precursors(Fig. 2C and D and S7). To facilitate the comparison, the FLexcitation wavelength is xed at 360 nm for all groups.Surprisingly, the FL decays very rapidly for Au–Cyt and Au–CMPat a molar ratio of 1 : 1 (i.e., at the “saturated” complexationratio) and may nearly disappear aer reduction for only 1 h(solid symbols, Fig. 2C and D). The rate of decline is relativelyslower for a molar ratio of 1 : 2 (hollow symbols, Fig. 2C and D).Thus, it can be concluded that these obtained uorescent Auspecies, which are denoted as b-Au–CMP or b-Au–Cyt, areunstable due to the complicated reduction steps. The presenceof excess ligands (i.e., Cyt or CMP) may favor their occupation of

10358 | Nanoscale, 2014, 6, 10355–10362

more complexation sites with Au cores, which stabilize theintermediates and reduce further attack by the citrate. Accord-ing to this viewpoint, the yield of blue-emitted Au uorescentspecies will be improved by using Au–ligand precursors at amolar ratio of 1 : 2. Therefore, the average FL levels for Au–Cytor Au–CMP at a ratio of 1 : 2 are generally higher than those at aratio of 1 : 1. On the other hand, the optimal pH for preparationof the samples with a remarkable blue FL emission is 4–6, whichdiffers from pH 3 reported for poly-C30 stabilized Au NCs.35 Thisresult indicates that the deprotonated state of cytosine (pKa 4.2)is now critical for formation of uorescent species when usingonly a single cytidine unit.

It should be noted that b-Au–CMP or b-Au–Cyt can beprecipitated and collected as white solids by the addition of anequal volume of ethanol to the reaction mixture. Since thereduction processes are terminated by this step, the FL spectraof Au species show little changes aer storage at �20 �C for afew days.

This journal is © The Royal Society of Chemistry 2014

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Generation of green or yellow-emitted Au species aer long-term reduction

We further extended the FL monitoring of mixtures to 1–7 h atpH 3–7. The use of different pH conditions results in changes inboth the FL wavelength and intensity. Aer reduction of Au–Cytprecursors at a molar ratio 1 : 1 for �90 min, the peak wave-length of FL emission stays at �462 nm at pH 3 and 4 (Fig. S8Aand B, ESI†), but is red-shied by �22 nm (to �484 nm) at pH 5(Fig. S8C, ESI†) and �38 nm (to �500 nm) at pH 6–7 (Fig. S8Dand E, ESI†). The FL intensity slowly increases with increasingtime for Au–Cyt mixture at pH 6 and 7 (curve d and e, Fig. S8F,ESI†). This signicant change indicates the generation of newAu species aer a long-term reduction by citrate. The Au–Cytmixture turns green aer a reduction at pH 5–7 for 7 h,accompanied by the appearance of a well-dened absorptionpeak at ��370 nm (curve c–e, Fig. S9, ESI†). For a colorlessmixture prepared at pH 3 and 4, this peak is non-distinguish-able (curve a and b, Fig. S9, ESI†). More interestingly, themixture even shows signicant green FL emission at 505 nmaer a reduction at pH 6 or 7 (Fig. 3A and inset). The peakwavelength is maintained at 505 nm, indicating that these Auspecies are very stable at room temperature.

Therefore, Au–Cyt precursors that are rapidly reduced tounstable b-Au–Cyt by citrate and b-Au–Cyt can be transformedinto stable, green-emitted Au uorescent species (g-Au–Cyt)using a long-term reduction. Note that the formation of thesespecies is also pH-dependent: it is preferable to synthesizeb-Au–Cyt species at pH 4–6, while g-Au–Cyt species should beprepared at pH 6–7.

Other uorescent species may also be produced by using theAu–CMP precursors aer a long-term reduction by citrate. ForAu–CMP (molar ratio of Au : CMP¼ 1 : 1), the FL emission peakwavelength at �460 nm is rather stable at pH 3 with a gradualincrease in peak intensity during a 24 h reduction (Fig. S10A,

Fig. 3 (A) FL emission spectra of Au–Cyt (molar ratio of 1 : 1) after 1 daynm. Inset shows the green-emitted Au species (reduction at pH 7) underspectra for Au–CMP species (molar ratio of 1 : 2) obtained by the reductithan 10 kDa. Excitation/emission slit width: 5/5 nm. Inset shows the yelloat 365 nm (right).

This journal is © The Royal Society of Chemistry 2014

ESI†). This peak wavelength is red-shied quickly to�473 nm atpH 4 (Fig. S10B, ESI†) and to �485 nm at pH 5–7 in 1.5 h(Fig. S10C–E, ESI†), respectively. It is noteworthy that a shoulderpeak at�550 nm emerges aer 4 h reduction at pH 4 (Fig. S10B,ESI†). This shoulder peak arises continuously and is evencomparable with the main peak (at �473 nm) aer a reductionfor 24 h. Some yellow-emitting Au species (y-Au–CMP) with MW> 10 kDa can be obtained aer ultraltration (Fig. 3B inset). TheFL emission spectrum for y-Au–CMP (Fig. 3B) is relativelysymmetric with a single peak at 562 nm (curve b), while thespecies with MW < 10 kDa shows a weak, dual emission at�470 nm and �550 nm (curve d), respectively.

Similar FL changes were observed for Au–CTP (molar ratio ofAu : CTP ¼ 1 : 2). No signicant shis in the FL emission peak(�460 nm) are observed during a 24 h reduction at pH 3 and 4(Fig. S11A and B, ESI†). The peak shis signicantly to�487 nmaer reduction at pH 5–7 for �2 h (Fig. S11C–E, ESI†). Ashoulder peak at approximately 550 nm also appears aer along-term reduction at pH 5–7, suggesting the presence ofspecies similar to y-Au–CMP. However, unlike y-Au–CMP, theuorescent Au–CTP components exhibit a MW lower than10 kDa (curve b, Fig. S12, ESI†). Therefore, it is reasonable thatthis shoulder peak, corresponding to the components with aMW larger than 10 kDa, is much weaker than that of y-Au–CMP.

Although the uorescent Au species can be prepared at roomtemperature, it is of no doubt that external heat can signi-cantly accelerate the growth of Au species. Aer only 1 hreduction by citrate (pH 6.0) at 80 �C, signicant FL emission at505 nm, 485 nm and 487 nm was observed by using Au–Cyt, Au–CMP, and Au–CTP precursors, respectively (Fig. S13, ESI†). Notethat the metal–ligand molar ratio also affects the FL. For Au–CMP and Au–CTP, both the FL excitation and emission inten-sities at a molar ratio of 1 : 2 are �4-fold higher than that at a

reduction by citrate at pH 3–7 (a–e). Excitation/emission slit width: 5/5illumination at 365 nm. (B) FL excitation (a and c) and emission (b and d)on of citrate pH at 4.0 with a MW of higher (a and b) or lower (c and d)w-emitted Au species under exposure to sunlight (left) and illumination

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molar ratio of 1 : 1. However, Au–Cyt at a molar ratio of 1 : 1shows an even stronger FL.

The up-conversion uorescent properties of Au species is ofgreat interest due to their promising applications in bioimagingwith low background and high contrast.36 Upon excitation at680 nm, the FL emission of Au–Cyt remains at 505 nm, but itsintensity is only �1/8 of that excited at 350 nm (curve e,Fig. S13A, ESI†). This intensity ratio is comparable to that of Au–Ag alloy NCs.37 Thus, the as-prepared Au species may provideaccess to potential bio-label candidates in up-conversion uo-rescent bioimaging.

Fig. 5 The TEM images (A, C and E) and size-distribution histograms(B, D and F) of b-Au–CMP (A and B), g-Au–Cyt (C and D), and y-Au–CMP (E and F) NCs. A inset: the high resolution TEM for b-Au–CMPNCs. To obtain larger NCs (MW > 10 kDa) for TEM imaging, all sampleswere treated with an ultrafiltration device (MWCO 10 kDa) to removepossible polymeric species and other small molecules.

Characterization of the luminescent Au species

The above luminescent Au species are precipitated and washedrepeatedly with absolute ethanol. The samples are then re-sus-pended in deionized water and further puried by ultraltra-tion (MWCO 10 kDa). Similar UV-vis and uorescent spectra areobserved for these puried samples (Fig. S14†) compared withthe corresponding species generated in reaction buffers. In allcases, the excitation wavelengths show little effect on theemission wavelengths. The quantum yields range from 0.4% to3.3% (Table S2†) by using quinine sulfate as a standard. Theseluminescent Au species mainly show uorescent lifetimes of theorder of nanoseconds (Fig. 4 and Table S2†): for example,6.28 ns (62%)/15.55 ns (23%)/0.82 ns (15%) for b-AuCMP. Thesedata are comparable to <10 ns reported for poly(amidoamine)dendrimers encapsulated Au NCs, indicating that the uores-cence may come from the Au species themselves.38

The morphology of b-Au–CMP (Fig. 5A), g-Au–Cyt (Fig. 5C),and y-Au–CMP (Fig. 5E) is characterized by TEM. The resultsshow that all products are ultrasmall particles with a size of1.54 � 0.46 nm (b-Au–CMP), 2.30 � 0.72 nm (g-Au–Cyt), and2.56 � 0.76 nm (y-Au–CMP) in diameter, based on the sizestatistics of 290, 323 and 273 particles (Fig. 5B, D and F),respectively. No Au SPR absorption at �520 nm is observed forall these particles, corresponding to a features of NCs instead oflarger NPs. The high-resolution TEM (Fig. 5A inset) reveals

Fig. 4 Fluorescent lifetimes of b-Au–CMP (black), g-Au–Cyt (red),and y-Au–CMP (green). The excitation wavelength is 370 nm for b-Au–CMP and 452 nm for g-Au–Cyt and y-Au–CMP, respectively.

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typical structural lattices with an interplanar spacing of about0.24 nm, which corresponds to the (111) planes of face-centeredcubic Au, conrming the formation of crystalline Au.

These results clearly show the size effect, i.e., dependence ofFL on the size of Au NCs. The larger size corresponds to the red-shied FL emission, similar to those known for other lumi-nescent nanostructures such as CdTe quantum dots.39 It is well-known that the size-dependent FL is not clearly observed whenDNA oligomers are used as templates, mainly due to the greatvariety in the number and sequences of bases, and their relatedsecondary structures.40 In our case, although the stabilizers(Cyt and CMP) are different ligands, they both contain singlenucleotides from a monomer base, which can largely preventthe creation of complicated environments around the Au coreby exible DNA wrapping styles. The possible factors that affectFL locations are generally limited to their size. Thus, it ispossible to prepare size-dependent multi-colored Au NCs onlyby tuning the reaction time or buffer pH.

We also use XPS technique to determine the valence of Au.The spectrum for g-Au–Cyt shows binding energy (BE) peaks ofAu 4f, C 1s, N 1s and O 1s, corresponding to the binding of Cytto Au atoms (curve a, Fig. S15A, ESI†). According to the cali-bration by C1s at 284.6 eV, the Au 4f7/2 and 4f5/2 doublet givesrise to peaks at 83.8 eV and 87.5 eV, respectively (Fig. S15B,ESI†). The spin–orbital splitting of 3.7 eV and the peak area ratio(Au 4f7/2 : Au 4f5/2) of 4 : 3 both validate that only Au (0) ispresent.41 For b-Au–CMP (curve b, Fig. S15A, ESI†), similar BEpeak positions are observed for the Au 4f7/2 and 4f5/2 doublet. It

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Fig. 6 The ESI-MS curve for y-Au–CMP (molar ratio of 1 : 2).

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is interesting that no Au (I) species are observed here, which arewidely present in glutathione (GSH)-stabilized Au NCs,42 indi-cating the complete reduction of Au (III) by citrate. In addition,since bulk Au (0) atoms have Au 4f7/2 BE of 84.0 eV,43 theobserved negative BE shi (�0.2 V) for Au 4f7/2 suggests theenhanced electron density around Au (0) due to the ligand (Cytor CMP) to metal (Au) charge transfer.

Themass spectra of the Au NCsmay provide some additionalinformation. For example, y-Au–CMP is mainly comprised ofAu cores and ligands with different ratios (Fig. 6). Generally,the highly abundant components include the species of[AuCMP]� (m/z 518.42), [Au3CMP4]

3� (m/z 625.75), [Au3CMP4]2�

(m/z 939.08), [Au2CMP2]� (m/z 1036.92), [Au4CMP5]

3� (m/z798.83), [Au4CMP5]

2� (m/z 1198.67), [Au2CMP5]3� (m/z 667.33)

and [Au5CMP5]2� (m/z 1295.58). The high abundance for

[Au3CMP4]3� and [Au3CMP4]

2� among all peaks indicates thatAu and CMP are prone to form complexes at a ratio of 3 : 4.

Conclusions

In summary, we reported the syntheses of multi-colored uo-rescent Au NCs stabilized by cytidine and its nucleotides atdifferent pH values and reaction times. Three typical NCs with asize in the range of 1.5–3 nm show size-dependent uorescentemission at �470 nm, �505 nm and �550 nm, respectively.Only Au (0) species are present in these NCs, which is quitedifferent from those reported for well-known GSH-stabilized AuNCs. The facile preparation of multi-colored Au NCs offersaccess to promising candidates for various biolabelingapplications.

Acknowledgements

This work is supported by the National Basic Research Programof China (2010CB732404), the National Natural Science Foun-dation of China (81325011, 21175020, and 20905012), theNational high-tech R&D program of China (2012AA022703), and

This journal is © The Royal Society of Chemistry 2014

the open research fund from State Key Laboratory of AnalyticalChemistry for Life Science (SKLACLS1212), Nanjing University.

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