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1Gold nanostars as SERS-active substrates for FT-Raman spectroscopy

Angela Zoppi, Silvana Trigari, Giancarlo Margheri,Maurizio Muniz-Miranda* and Emilia Giorgetti

Surfactant-free and CTAB-stabilized gold nanostars weresynthesized. Their FT-SERS activity was tested. A nanomolarlimit of detection and a Raman enhancement factor of morethan 105 were found.

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Gold1 nanostars as SERS-active substrates for FT-Raman spectroscopy†

Angela Zoppi,ab Silvana Trigari,b Giancarlo Margheri,b Maurizio Muniz-Miranda*c

and Emilia Giorgettib2

Gold nanostars having extinctionmaxima in the near infrared spectral region were synthesized according to

surfactant-free or CTAB-based procedures. The SERS activity of these colloidal suspensions by 1064 nm

excitation was tested with a thiolic derivative of naphtalimide as a ligand molecule. The shielding effect

of the colloid on the observed Raman response was taken into account as well as the influence of

nanoparticle concentration and the addition of chitosan, as stabilizer. A nanomolar limit of detection and

a Raman enhancement factor of more than 105 were found. In particular, an important finding is that

CTAB-free nanostars, even in the more biocompatible chitosan-stabilized form, still behaved as good

SERS substrates in the near infrared where the occurrence of the “optical tissue window” points to their

use in biological systems with minimum fluorescence background.

1. Introduction

Metallic particles, when reduced to the nanoscale, show strongdipolar excitations in the form of localized surface plasmonresonances.1–8 The latter are non-propagating excitations of theconduction electrons, which can lead to a strong enhancementof the electromagnetic eld at the interface between the nano-structure and its environment. This effect was the subject ofextensive research aimed at fundamental studies, as well as atnovel applications in rapidly developing elds, such as nano-biology,9,10 nano-medicine,11,12 photonics,13–15 sensing16 and, inparticular, surface enhanced Raman scattering (SERS).17,18

The resonance frequency of plasmonic oscillations isessentially determined by the dielectric properties of the metaland the surrounding medium, as well as by the particle size andshape. In this respect, branched nanoparticles, otherwiseknown as star-shaped nanoparticles or nanostars (NSs) havereceived great attention for their unusual optical and plasmonicproperties.19–21 The anisotropic shape and, more importantly,the presence of sharp outward branching tips can in fact yieldextraordinary enhancements of the electromagnetic eld due tothe generation of hotspots focused on the sharp tips, that act as“lightening rods”.22,23 This phenomenon can result in a veryhigh SERS activity.24–27

Apart from a recent report demonstrating the good SERSperformance of silver-NSs,28 most published works deal with

gold-NSs, which have fascinated the scientic community sincetheir rst proposal for their broad electromagnetic region ofactivity, from the blue to the near-infrared.29–33 Thanks tomodelling procedures, great advances have also been made inunderstanding the structural parameters that control NSsplasmonic resonances. In particular, the tip morphology,namely its aspect ratio and radius of curvature, was demon-strated to be the main responsible for the extinction in the near-infrared.34–36 The tunability of plasmonic resonances in thisspectral region has received great interest as it is reported to bebenecial for biomedical applications, due to the transparencyof tissues and biological uids. A recent example is representedby gold-NSs as potential photothermic transducers, which, incombination with the low cyto-toxicity of gold, have beenexploited for novel theranostic strategies.37 Among them, SERSis gaining ground as optical diagnostic tool also for in vivobiological applications as a safer alternative to other taggingtechniques typically based on radiotracers.37–39 For this reason,the need of NIR tuned, fully biocompatible SERS active tags, is ademanding task. In spite of the importance of this topic, only acouple of studies report on the employment of NSs in the near-infrared (namely, by excitation at 1064 nm (ref. 40) or at 830 nm(ref. 41)) being most of the research done in the visible spectralregion (i.e. by excitation at 532, 633 and 785 nm excita-tions).26,27,31,33,42–45 Notwithstanding the low intensity of near-infrared Raman response (owing to the l�4 dependence ofRaman scattering intensity), this excitation window is highlydesirable for all those applications requiring removal from theRaman response of any interference of uorescence, due typi-cally to impurities that, even in traces, can add high levels ofshot noise on the Raman bands, making their observation adifficult task, as it oen occurs with biological samples.46

aDepartment of Physics “Enrico Fermi”, University of Pisa, Pisa, ItalybInstitute of Complex Systems CNR, Sesto Fiorentino, ItalycDepartment of Chemistry “Ugo Schiff”, University of Florence, Sesto Fiorentino, Italy.

E-mail: maurizio.muniz@uni.it

† Electronic supplementary information (ESI) available. See DOI:10.1039/c4ra13830a

Cite this: DOI: 10.1039/c4ra13830a

Received 24th November 2014Accepted 23rd December 2014

DOI: 10.1039/c4ra13830a

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In this paper we discuss the potentiality of AuNSs as plat-forms for FT-SERS detection of low analyte concentrations, bylaser excitation at 1064 nm. To this purpose, we used colloidaldispersions having extinctionmaxima at about 930–970 nm andexploited the high affinity of gold towards thiol-terminatedmolecules. In particular, we selected NAFTA8, a N-substituted4-methoxy-1,8-napthalimide derivative (hereaer NF8) bearinga C8 aliphatic chain endowed with a thiol, which has beenalready tested on gold spherical nanoparticles47 and, in itsshorter version with a 6-aliphatic chain, on Au nanostars40 andAu/Ag nanocages.48 The molecular structure of NF8 is reportedin Fig. 1. We tested the FT-SERS efficiency of unprotected andCTAB (cetyltrimethylammonium bromide)-protected AuNSs byvarying the molarity of NF8 from 1.6 � 10�5 M to 1.6 � 10�9 M.The results obtained with 1.6 � 10�6 M NF8 concentration wereused to calculate the effective enhancement values. The inu-ence of a biocompatible stabilizing-agent (namely chitosan) inthe case of unprotected NSs, and of CTAB and colloid concen-trations, in the other one, was considered as well.

2. Experimental2.1. Materials and reagents

Tetrachloroauric(III) acid (HAuCl4), cetyltrimethylammoniumbromide (CTAB), ascorbic acid (AA), silver nitrate (AgNO3),sodium citrate, cyclohexane, acetone, ethanol and chloroformwere purchased from Sigma Aldrich and used as received. Highmolecular weight chitosan (�106 Da) was obtained from HeppeMedical. NF8 was provided by the Institute of Organic Chem-istry with Centre of Phytochemistry BAS, Soa, Bulgaria. Due toits insolubility in pure water, NF8 was dissolved in ethanol orchloroform, while mixtures with NSs were always prepared in

H2O : EtOH 10 : 1 solutions. Ultrapure water (resistivity 18 MU

cm) was used as solvent in all the experiments. All glassware wascleaned with Piranha solution (H2SO4 : H2O2 3 : 1).

2.2. Synthesis of nanostars

Two different kinds of NSs were prepared: one was obtainedaccording to a surfactant-free synthesis procedure (leading tosurfactant-free particles, hereaer named SF-NSs) and anotherone according to a CTAB-based method (leading to CTAB-capped NSs, hereaer named CTAB-NSs). For the sake ofcomparison, the two colloidal solutions were synthesized sothat their main extinction maxima were close to each other andin the near-infrared spectral region, namely at about 960–970nm. These two batches were then used to prepare ten differentsamples (described in Table 1), here named from A to G, andnumbered from 0 to 5 according to the increasing concentrationof the reporter molecule (NF8) probed in the SERSmeasurements.

2.2.1 Surfactant-free nanostars: SF-NSs. Unprotectednanostars were synthesized according to a seed-mediatedprotocol, which employs Au seeds obtained by reduction ofHAuCl4 with citrate. The synthesis procedure was drawn fromref. 36 and the reagents ratios were adapted in order to obtain aplasmon band in the near-infrared spectral region. In partic-ular, gold seeds were prepared by mixing 100 ml of boiling 1mM HAuCl4 with 15 ml of 1% citrate solution. The mixture waslet to react for 150, and then it was cooled and ltered with a 0.22mm nitrocellulose lter. Gold NSs were then prepared in H2O byadding to 10 ml of 0.25 mM HAuCl4, 40 ml of 1 M HCl, 70 ml ofthe Au seeds, 0.1 ml of 3 mM AgNO3 and 0.1 ml of 100 mM AA.The synthesis was performed at room temperature in an amber-brown glass bottle under continuous and strong stirring (about690 rpm). The Au nal concentration was 0.25 mM. This solu-tion (sample A0), which had a negative z-potential (�26mV) andan extinction with a maximum of 0.7 at 960 nm (OP 1 cm), wasused as prepared without any further purication. Since ref. 36does not report any stability check of the surfactant-free NSs, werepeated UV-vis absorption tests one day aer the synthesis andobserved a reduction of the extinction spectrum intensity in 24hours (see Fig. S1, ESI†) that we related to some tendency tosedimentation. Also for this reason, we decided to check theeffect on their long term stability of a biocompatible cappingagent. For this purpose, we added 130 ml of 9 mM chitosan 1%-acetic acid solution to 3 ml of SF-NS. The spectral features weremaintained, apart from a small red-shi of the maximum to 970nm (OP 1 cm) and a �28% reduction of its intensity (sampleB0), while the z-potential changed to +37 mV.

2.2.2 CTAB-protected nanostars: CTAB-NSs. CTAB-protected NSs were synthesized following a protocol based onref. 34, without the use of NaOH. In particular, 7.5 ml of 80 mMCTAB were mixed with 2.64 ml of H2O and 1 ml of 24 mMHAuCl4 solution. Then 320 ml of 100 mM AA were addedtogether with 96 ml of 25 mM AgNO3. Finally, 0.18 ml of cyclo-hexane and 0.26 ml of acetone were added, resulting in a bluishsolution. The Au and CTAB nal concentrations were 2 mM and50 mM, respectively. The reaction was performed at 32 �C in an

Fig. 1 Chemical structure of the ligand molecule: NF8.

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amber-brown glass bottle under continuous and mild stirring(about 560 rpm). The temperature of the bath was controlled bya Velp-digital thermo-regulator coupled to a hot plate. Stirringwas maintained throughout the synthesis procedure; thethermal bath was kept for further 12 hours before the colloidalsuspension was cooled down to room temperature.

Under these conditions, we obtained a colloidal suspensionwith a plasmon maximum at 970 nm and an extinction of 1.4(OP 2 mm). To make a comparison with SF-NSs, we madedilutions. To this aim and also to study the role played by thesurfactant itself, we prepared four different samples: (1)sample C0 was prepared by dilution with a 50 mM CTABsolution (nal concentrations: CAu ¼ 0.2 mM, CCTAB ¼ 50 mM;extinction @ 970 nm ¼ 0.7, OP 1 cm; z-potential +53 mV); (2)sample D0 was obtained by dilution with pure water (nalconcentrations: CAu ¼ 0.2 mM, CCTAB ¼ 5 mM; extinction @970 nm ¼ 0.7, OP 1 cm; z-potential +47 mV); (3) sample E0 wasprepared by purifying with two centrifugation cycles (3000rpm, 24 �C, 100) and dispersing each time the pellet of NSs inpure water. The plasmon band in this case was blue-shied(extinction @ 933 nm ¼ 0.7, OP 1 cm; Z-potential +30 mV);(4) sample F0 was obtained in a similar way, while the nalvolume was adjusted to an extinction maximum of 0.4 at920 nm (OP 1 cm).

To probe the effect of colloid concentration on the FT-SERSresponse, another puried batch was prepared in order toachieve extinction maxz 1 (OP 1 cm) and then diluted down toextinctions near 0.6, 0.4 and 0.2 (G series).

2.3. Characterization

Transmission electron microscope (TEM) images were obtainedwith a Philips CM12 cryo-Gatan UHRST 3500 operating at 100kV. The colloidal solutions were dropped onto carbon-coatedCu grids.

Extinction spectra were obtained at room temperature usinga Cary 5 spectrometer in quartz cuvettes.

z-potential was measured with a Malvern Zetasizer Nanos-eries apparatus.

FT-SERS spectra were recorded using a MultiRaman (Bruker)apparatus operating at 1064 nm and coupled to a liquid-N2

cooled Ge detector. Irradiation power and spectral resolutionwere respectively set at 200 mW and 4 cm�1. A 10 mm glasscuvette was used to record the spectra averaging over 500 scans.For these analyses, 50 ml of differently concentrated NF8 etha-nolic solutions was added to 0.5 ml of the NSs colloids in orderto achieve nal analyte molar concentrations equal to: 1.6 �10�5, 1.6 � 10�6, 1.6 � 10�7, 1.6 � 10�8 and 1.6 � 10�9. Beforerecording the Raman spectra, the samples were homogenizedby sonication for 3000.

Spectral analysis was performed using Origin Pro 8 soware.A commercially available FEM package (COMSOL Multi-

physics) was used to model the nanostars structures byattaching several cones to a sphere until a complete coveragewas obtained. Dimensions of central core and cones were setaccording to the averaged structural features deduced fromTEM images.

2.4. Evaluation of enhancement factors. The experimentalSERS enhancement factors (EFexp) were evaluated according tothe relation:

EFexp ¼ ISERSCsol

IRamanCsurf

(1)

where ISERS and IRaman are respectively the intensities of theligand band in the SERS and bulk spectra, Csurf is the concen-tration of molecules adsorbed on the SERS-active substrate andCsol is the concentration of molecules in the bulk.49 IRaman wasdetermined from a 4.8 � 10�3 M solution of NF8 in chloroformand ISERS was measured from the FT-SERS spectra of 1.6 � 10�6

M NF8 in the colloidal solutions. The calculations were per-formed referring to the Raman band of NF8 at 1400 cm�1.Intensities were measured by curve-tting with Lorentzianfunctions and were normalized with respect to the EtOH bandat 880 cm�1. Csurf was evaluated as

Table 1 Nanostars samples used to measure the FT-SERS spectra of different NF8 concentrations

Batch

no NF8 NF8 concentration (M)

Extinctionmax value

z-potential(mV) 1.6 � 10�9 1.6 � 10�8 1.6 � 10�7 1.6 � 10�63 1.6 � 10�5

SF-NSs 0.7 �26 A0 A3 A4 A50.5 +37 B0 chitosan capped B4

CTAB-NSs Diluted 0.7 +53 C0 CTAB diluted C3 C4 C50.7 +47 D0 H2O diluted D3 D4 D5

Puried 0.7 +30 E0 E1 E2 E3 E40.4 F0 F4

10.60.40.2

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Csurf ¼ 1.6 � 10�6 � Csuper (2)

by measuring the NF8 concentration in the supernatant solu-tion (Csuper), through its absorption peak at 370 nm, once theNSs were all settled down aer a 300-centrifugation at 10 000rpm. Since CTAB concentration inuenced the absorption effi-ciency of NF8 (Fig. S2†), proper reference samples were used toevaluate Csuper. When the absorption band of the supernatantwas not observed, Csurf was taken equal to 1.6 � 10�6 M.

The previous test permits evaluation of the SERS activity ofthe NSs, namely their effective enhancement factor (EFeffective).As a matter of fact, when SERS is performed in colloidalsuspensions, the Raman scattering undergoes the shieldingeffect on the incoming and outgoing radiation provided by thecolloidal nanoparticles. This effect can be taken into account bydening a shielding coefficient, according to the followingrelation:49

EFexp ¼ EFeffectiveKshielding(linc) (3)

The correction factor Kshielding(linc), when calculated inreection mode – which is the case of our FT-SERS congura-tion – is given by:50

KshieldingðlincÞ ¼ 1� e�rn½CextðlincÞþCextðlRamanÞ�h

rn½CextðlincÞ þ CextðlRamanÞ�h

¼ 1� e�ln 10½ExtðlincÞþExtðlRamanÞ�

ln 10½ExtðlincÞ þ ExtðlRamanÞ� (4)

where r is the number of nanoparticles for unit volume, n therefractive index of the solvent, Cext(l) the extinction cross-section, h the pathlength and Ext(l) the experimental extinc-tion of the NF8-NSs solution. In what follows, Kshielding wascalculated from the experimental extinction spectra accordingto the right-term of eqn (4).

3. Results and discussion

We measured the FT-SERS spectra of NF8 mixed with AuNSs,according to the scheme of Table 1. We progressively reducedthe concentration of the reporter molecule in order to estimatethe limit of detection (LOD). We also recorded the extinction

spectra before and aer analyte addition to monitor the colloidsat different stages of the experiments.

3.1. TEM characterization of NSs

The morphology of SF-NSs and CTAB-NSs is compared in Fig. 2,where two representative TEM images are reported. A morebranched aspect can be noticed for SF-NSs, which consist of a�40 nm inner core and many thin outward radiating arms. Onthe contrary, CTAB-NSs have a bigger core (�75 nm) with alower number of spikes with a much lower aspect-ratio. Theaveraged sizes of SF- and CTAB-NSs, evaluated as diameters ofthe circumscribed circles, were found to be 120 nm and �180nm, respectively. The structural features deduced from the

Fig. 2 Representative TEM images of SF-NSs and CTAB-NSs.

Fig. 3 Extinction spectra of (a) SF-NSs, (b) diluted CTAB-NSs and (c)purified CTAB-NSs, before and after the addition of NF8 (finalconcentration: 1.6 � 10�6 M). The position of the excitation wave-length at 1064 nm used for FT-SERS is indicated by the dash-dottedline. The dashed area shows the spectral window containing theRaman bands of NF8 investigated by SERS measurements.

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statistical analysis are reported in the ESI (Fig. S3 and S4†). Theoccurrence of interaction among SF-NSs, which was macro-scopically observed as a tendency to sink, was also evidenced bythe occurrence of small particles aggregates in the TEMmicrographs.

3.2. Extinction spectroscopy

Fig. 3 reports the extinction spectra of the NSs samples beforeand aer the addition of the reporter molecule (nal NF8concentration: 1.6 � 10�6 M).

The spectra of the colloids before functionalization aredominated by amain plasmonic resonance band in the 930–970nm range, that is related to edges and branches of the star-shaped nanoparticles.34 The addition of chitosan to SF-NSs(B0) increased the stability of the colloid for two more weeks.It also caused attenuation of the plasmon resonance (Fig. 3a,pink line), which decreased more than expected from simpledilution, suggesting the occurrence of a NS–polymerinteraction.

On the other hand, dilution of CTAB-NSs with 50 mM CTABsolution (C0) or with pure water (D0) resulted in almost iden-tical extinctions proles (Fig. 3b, cyan and pink lines). It shouldbe noted that, in both cases, the CTAB concentration is farabove the critical micellar concentration (CMCCTAB ¼ 0.9 mM(ref. 51)), leaving the chemical environment felt by the NSspresumably unchanged. In contrast, the purication processinvolving centrifugation and redispersion of the settled NSs ledto a blue shi of the main extinction band (Fig. 3c, E0 and F0,cyan and pink lines). This effect can be associated to materialloss (i.e., loss of the biggest NSs, which mainly contribute to thelong-wavelength portion of the extinction spectra), as well as towashing of excess CTAB. Indeed, the CTAB refractive index issignicantly higher than that of water (nCTAB y 1.4). The CTABde-sorption would decrease the refractive index of the NSsenvironment, causing a blue shi of the extinction spectrum.Both phenomena were likely to occur at every centrifugationstep and should be considered for explaining the spectraldifferences between batches E and F.

Concerning the effect of NF8 addition to the colloidalsuspensions (nal concentration: 1.6 � 10�6 M), a small shi ofthe main resonance is evinced, which was more prominent forpuried samples (Fig. 3c, E4 and F4). For unprotected NSs, achange of the overall shape of the extinction feature was alsoobserved (Fig. 3a, A4), which was less evident for chitosan-capped NSs (Fig. 3a, B4), as a consequence of the protectiveeffect of the polymer. On the whole, samples A4 and B4 gavesimilar features.

The extinction spectra of NF8-colloidal solutions reported inFig. 3 were used to evaluate the Ext(l) term in Kshielding (eqn (4)).

3.3. FT-SERS measurements

The reference Raman spectrum of NF8 is given in Fig. 4. As thesolubility of NF8 in H2O/EtOH 10 : 1 mixture was not sufficientto get a detectable FT-Raman response, the measurement wastaken in chloroform. Fig. 4 shows the characteristic spectralwindow of a 4.8 � 10�3 M solution, aer subtraction of

chloroform spectrum. The typical vibrational bands of NF8closely correspond to those of naphthalimide.52 In particular,themost intense signals at 1400 and 1595 cm�1 (with a shoulderat 1582 cm�1) can be assigned to ring deformationmodes, whilethe band at 1694 cm�1 is associated to C]O stretchingvibration.

Fig. 4 FT-Raman spectrum of NF8 in chloroform (4.8 � 10�3 M) aftersubtraction of the solvent.

Fig. 5 Normal FT-Raman and SERS spectra of surfactant-free NSs.From bottom to top: H2O : EtOH 10 : 1; sample A0 in H2O : EtOH10 : 1; A3 (1.6� 10�7 M); A4 (1.6� 10�6 M); A5 (1.6� 10�5 M); B4 (1.6�10�6 M). The vertical dotted lines indicate the ligand bands. Spectra arevertically translated for a better comparison.

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3.3.1 Effect of ligand concentration. FT-SERS spectra ofNF8 were acquired by mixing the analyte with NSs suspensions,according to the scheme of Table 1. The obtained spectra are

shown in Fig. 5–8, together with the reference spectra of theH2O : EtOH (black line) and NSs : EtOH (cyan line) 10 : 1 solu-tions. In particular, the bands observed in the ethanoliccolloidal suspensions (i.e. NSs : EtOH 10 : 1) are basically thoseof the aqueous solution of ethanol (signals at: 880, 1046, 1086,1279 and 1456 cm�1), with additional weak signals for theCTAB-NSs series at 760, 1303 and 1439 cm�1, which are attrib-uted to the surfactant itself (see Fig. 6–8). For comparison, theFT-Raman spectrum of a 50 mM CTAB solution is also reportedin Fig. 6 (green line). It is interesting to note that the CTABsignal at 760 cm�1, which appears slightly enhanced in thepresence of NSs, is observed even in the puried samples(Fig. 8), indicating the strength of surfactant adsorption to thegold surface. This band is attributed to the CN+ stretchingvibration and its enhancement suggests the positioning of thesurfactant positively-charged head group close to the metallicsurface.53 The presence of CTA+ ions adjacent to the nanostarsurface is also conrmed by the positive z-potential, whichdecreases aer dilution and purication of the colloid as aconsequence of the surfactant desorption (see Table 1).

Aer addition of the ligand, new bands became visible,which closely match those of NF8, already shown in Fig. 4. Inparticular, the absence of any signicant frequency-shibetween the FT-Raman and FT-SERS signals points to excludeany direct interaction of the naphthalimide moiety with themetallic surface and sustains our assumption that NF8 interactswith the NSs only through its terminal-SH functionality. Ingeneral, these bands got weaker by reducing the NF8 molarity

Fig. 6 Normal FT-Raman and SERS spectra of CTAB-protected NSs(CTAB diluted). From bottom to top: H2O : EtOH 10 : 1; CTAB 50 mM;sample C0 in H2O : EtOH 10 : 1; C3 (1.6 � 10�7 M); C4 (1.6 � 10�6 M);C5 (1.6 � 10�5 M). The vertical dotted lines indicate the ligand bands.Spectra are vertically translated for a better comparison.

Fig. 7 Normal FT-Raman and SERS spectra of CTAB-protected NSs(H2O diluted). From bottom to top: H2O : EtOH 10 : 1; sample D0 inH2O : EtOH 10 : 1; D3 (1.6� 10�7 M); D4 (1.6� 10�6 M); D5 (1.6� 10�5

M). The vertical dotted lines indicate the ligand bands. Spectra arevertically translated for a better comparison.

Fig. 8 Normal FT-Raman and SERS spectra of CTAB-protected NSs(purified). From bottom to top: H2O : EtOH 10 : 1; sample E0 in H2-O : EtOH 10 : 1; E1 (1.6 � 10�9 M); E2 (1.6� 10�8 M); E3 (1.6 � 10�7 M);E4 (1.6 � 10�6 M); F4 (1.6 � 10�6 M). The vertical dotted lines indicatethe ligand bands. Spectra are vertically translated for a bettercomparison.

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from 1.6 � 10�5 to 1.6 � 10�9. The trend of the 1400 cm�1

Raman band intensity with the NF8 concentration is reported inthe ESI (Fig. S5†). The minimum analyte concentration still ableto give a detectable Raman signal at 1400 cm�1 was operativelydened as LOD.

For surfactant-free NSs, a 10�7 M LOD was measured (Fig. 5sample A3, dark cyan line). Besides, samples A4 and B4 gave asimilar behaviour (Fig. 5, purple and red lines), reecting thesimilarity of the relative extinction proles, namely the inu-ence of chitosan in the detection of NF8 is negligible.

On the contrary, the hindering action of CTAB appearedevident. A lower LOD (i.e. 10�6 M) was measured for CTAB-NSsdiluted with a CTAB solution (Fig. 6 sample C4, purple line),with respect to the 10�7 M value observed with H2O-dilution(Fig. 7 sample D3, magenta line).

We then puried CTAB-NSs from surfactant excess. Anincrease of the signal-to-noise ratio was rst revealed, which ledto the best LOD, namely 1.6 � 10�9 M (Fig. 8 sample E1, sky-blue line; see also Fig. 9 discussed in the next paragraph).Besides, the combination of the purication process with thedilution of the colloid (sample F4) yielded a further increase ofthe SERS intensity (compare F4 red line and E4 purple line inFig. 8), showing that the colloid concentration plays a criticalrole in the SERS efficiency.

3.3.2 Effect of colloid concentration. As a nal test, weexplored the effect of NSs concentration on the LOD by usingthe samples of the G series. NF8 with different molarities (1.6 �10�8, 1.6 � 10�7, 1.6 � 10�6) was probed on puried CTAB-NSssolutions having extinction maxima of 0.2, 0.4, 0.6, 1.0, while1.6 � 10�9 M NF8 was tested only in the 0.4 extinction colloidalsuspension (refer to Table 1 for the correspondent samplelabels). The resulting FT-SERS spectra are compared in Fig. 9.

The effect of NSs dilution is better visualized by plotting theintensity of the 1400 cm�1 SERS band against the maximumvalue of the colloid extinction. From Fig. 10, a positive effect ofcolloid dilution is clearly deduced only for 1.6 � 10�8 M analyteconcentration (G2 series, blue circles). In this case, the occur-rence of a maximum can be explained as a consequence of thedilution process, which reduces the number of NSs (i.e. theactive substrates), decreases the shielding (which increases thesignal) and increases the number of active sites for binding, dueto the shi to the right of the (CTAB)adsorbed 4 (CTAB)solutionequilibrium. On the contrary, for 1.6 � 10�6 M concentration(G4 series, black circles), the experimental data show a mono-tonic trend, which favours highly concentrated colloids, beingin this case the NSs concentration the main factor inuencingthe measurements.

Fig. 9 SERS spectra taken from purified CTAB-NSs of G series con-taining NF8. From bottom to top: G1 (1.6 � 10�9 M), G2 (1.6 � 10�8 M),G3 (1.6 � 10�7 M) and G4 (1.6 � 10�6 M). NSs plasmonic extinctionmaximum value is reported on the right side. The vertical dotted linesindicate the ligand bands. In each graph, spectra are vertically trans-lated for a better comparison.

Fig. 10 SERS intensity of the 1400 cm�1 band vs. extinction max valueof G-series NSs. NF8 concentrations: 1.6� 10�8 M (blue); 1.6� 10�7 M(red); 1.6 � 10�6 M (black). The intensities were normalized withrespect to the 880 cm�1 band of ethanol.

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In this respect, below an optimal concentration of the ana-lyte, to increase the concentration of the nanoparticles in anattempt to increase the signal may lead to signal attenuation,due to the competition between enhancement and extinctionprocesses.50

3.4. Evaluation of FT-SERS enhancement factors

On the basis of the previous data, we evaluated the experimentaland effective EF values, according to the procedure outlined inparagraph 4 of the Experimental section. The results arereported in Table 2, together with the LODs.

Csurf was determined from the absorption spectra of thesupernatant solutions aer ultracentrifugation of the incubatednanoparticles (see Fig. S6†). The obtained values indicate a 100%conjugation of the Au surface for all the specimens, exceptsamples C and D which contain 50 and 5 mM CTAB, respectively.Under these conditions, in fact, the CTAB critical micellarconcentration is exceeded and a strong interaction of the surfac-tant with NF8 molecules occurs, which, for example, explains thealready mentioned unusual behaviour of NF8 in the absorptionmeasurements (Fig. S6†). As a matter of facts, the dilution andpurication of the CTAB-NS colloids led to a decrease of the z-potential value, from +53 mV (sample C), to +47 mV (sample D)and to +30mV (sample E), suggesting a release of the CTA+ speciesfrom the electric double layer around the particles. These resultsclearly suggest that CTAB molecules prevent the linkage of NF8 tothe nanoparticles surface.

EFexp values can be divided into two groups: approximately104 for samples A, B, C, D, and one order of magnitude higherfor the puried samples E and F. As the shielding factors are allin the 0.45–0.60 range, the EFeffective values are almost doubledwith respect to EFexp and follow the same trend.

On the whole, the data reported in Table 2 point to thefollowing conclusions:

(i) The addition of polymeric chitosan with the aim ofconferring stability to SF-NSs (sample B) does not impair theconjugation of the reporter molecules with the gold surface andmaintains a good SERS efficiency with respect to bare particles(sample A). Chitosan-capped Au-NSs have been recentlydemonstrated to have a good biocompatibility for in vitroexperiments of photothermolysis,55 suggesting this system to betested as a SERS probe as well.

(ii) The interfering role played by CTAB appears evident.Moving from sample C to D to E, for instance, the amount ofadsorbed NF8 grows up approximately from 62%, to 69% to

100%, with a corresponding increase of EF. This is consistentwith the assumption that, above the critical micellar concen-tration, CTAB forms free micellar structures as well as a double-layer covering the nanoparticles surface. These structures areboth believed to capture NF8 molecules, impairing their inter-action with the NSs. As long as CTAB concentration is lowered(through dilution and above all through purication of thecolloid), their competitive effect is reduced, the covering layerbecomes less packed and thus more permeable to NF8 mole-cules, which may now adsorb to the metal surface in a largeramount. In addition, when the colloids are strongly puried(which does not mean that all CTAB is eliminated, as alreadyevinced from SERS spectra) the system sensitivity increases andits LOD improves dramatically (from 10�6 M to 10�9 M).

(iii) According to eqn (1), an increase of Csurf should lead to adecrease of EFexp. However, one order of magnitude increasewas measured when moving from sample C to sample E. It isdue to the increase of the ISERS (see Fig. S5†). We also observed afurther improvement of EFexp when the colloid was puried anddiluted (sample F4), although the surface amount of NF8 didnot vary. An interpretation of this evidence goes beyond thescope of this work. To a rst approximation we hypothesize thatit can be related to a different amount of CTAB interacting withthe surface-conjugated NF8 molecules.

(iv) With respect to bare SF-NSs, puried CTAB-NSs showhigher EFexp and LOD. From a purely morphologic point of view,this is not obvious. As shown by TEM images of Fig. 2, sharpertips and a higher number of branches are present in SF-NSs, ifcompared with CTAB-NSs. These facts suggest the a prioripossibility that SF-NSs give better SERS performances thanCTAB-NSs, mainly due to the more intense lightening rod effectassociated with a smaller apex radius of curvature (3 nm against5 nm).54 Also the higher number of branches, and hence thehigher number of hosted reporters, should contribute, inprinciple, to a higher Raman enhancement. To make acomparison, we used the structural parameters deduced fromTEM images (and reported in Fig. S3 and S4†) to model SF-NSsand CTAB-NSs structures (Fig. S7†). For 1 ml of colloidal solu-tion, we evaluated a tip-surface area of SF-NSs �6 times widerthan that of CTAB-NSs, while the total surface is only 3 timesbroader. Nevertheless, from absorption measurements we didnot evidence any difference between Csurf for SF- and puriedCTAB-NSs cases. Moreover, the improved signal intensityobserved on puried CTAB-NSs (Fig. S5†) strongly suggests theoccurrence of a further parameter affecting the SERS efficiency.

In this respect, it appears important the fact that the colloidalsystems considered for this study are obtained following quitedifferent procedures. In fact, although the experimental condi-tions used for SERS measurements were kept identical and thetwo typologies of colloids were made similar for the intensity oftheir extinction band, the chemical environments felt by thenanoparticles in the two cases are quite different. This was alreadysuggested by the z-potential values which had opposite signs. Forthis reason, the better response provided by puried CTAB-NSs(namely sample E) with respect to bare particles (i.e. sample A)should not be discussed solely in terms of an electromagneticenhancement, but other factors should be invoked. One of these

Table 2 Experimental and effective EF values for a NF8 concentrationequal to 1.6 � 10�6 M. For comparison, also the LODs are reported

Substrate Csurf (M) EFexp Kshielding EFeffective LOD (M)

SF-NSs A 1.6 � 10�6 2.8 � 104 0.46 6.1 � 104 1.6 � 10�7

B 1.6 � 10�6 2.3 � 104 0.45 5.1 � 104

CTAB-NSs C 9.9 � 10�7 2.0 � 104 0.51 3.9 � 104 1.6 � 10�6

D 1.1 � 10�6 2.4 � 104 0.51 4.7 � 104 1.6 � 10�7

E 1.6 � 10�6 1.8 � 105 0.49 3.7 � 105 1.6 � 10�9

F 1.6 � 10�6 3.1 � 105 0.60 5.2 � 105

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is the chemical enhancement, which is reported to provide abouta 10–100 fold increase of the Raman signal intensity.17 Chemicalintensication is achieved by a change in the scattering cross-section of the analyte attached to the metal. In our case, themechanism is reasonably determined by the entity of chemicalinteraction between NF8 and CTAB molecules, at least below aCTAB critical concentration. Along this, also the morphology ofthe particles should be a potential factor affecting the distributionand coverage density of NF8 on the Au surface, with particularregard to the hot-spots. We have le this question opened forfuture investigations.

Although at this point a comparison of our results withanalogous published works on NSs in solution appearscompulsory, it becomes quite puzzling due to the variability ofseveral factors, such as nanoparticle size, morphology,concentration, molecular structure of the test ligand, itsconcentration and incubation time, and so on.

In particular, a critical point in the estimation of EF and itssubsequent comparison with previous published data arisesfrom the evaluation of the number of adsorbed moleculessampled on the SERS-active substrate, i.e. Csurf in eqn (1). Someauthors26,56 considered Csurf equal to the analyte concentration,assuming complete monolayer coverage of the NSs and esti-mating a lower limit for EF. Others31 evaluated EF by modellingthe NSs structure and calculating the surface area. Also in ourcase, an overestimation of Csurf cannot be discarded, due forexample to the presence of small residual spherical goldnanoparticles on which chemisorption may have occurredwithout giving FT-Raman enhancement. Lastly, none of thepublished research works report EFeffective values, but just EFexp,without any evaluation of Kshielding.

With all this in mind, only a coarse comparison can be madewith existing data. The experimental factors acquired from theliterature (measured with SERS excitations at 785, 633 and 532nm)26,31,42,56 roughly range from 2 � 103 to 6 � 105, while theexceptional SERS enhancements found in other reports (see forexample the impressive 1010 of ref. 27) are obtained thanks to acompletely different conguration, that exploits the enhance-ment mechanisms occurring when a metal tip and a metalsubstrate are closely spaced, effect that however rapidlydecreases when the tip distance becomes higher than�10 nm.57

To conclude, despite the inconsistencies among quantica-tion methodologies of the SERS enhancement factor, a fullcompatibility of our experimental results with those present inliterature can be inferred, which places our structures amongthe most efficient SERS substrates ever tested. In particular, theemployment of FT-Raman spectroscopy to probe this typologyof nanostructures, which has not been done before by otherauthors, demonstrates that the nal Raman enhancement (duewhether to electromagnetic or chemical factors) countervailsefficiently the light-scattering efficiency intrinsically penalizedby the near infrared excitation wavelength.

4. Conclusions

In this investigation, we have demonstrated that gold NSshaving plasmon resonances in the NIR range act as efficient FT-

SERS substrates, namely with 1064 nm exciting radiation. Wecompared the behaviour of surfactant-free and CTAB-stabilizedNSs and proved that they give comparable results, although thehighest efficiency was observed with puried CTAB-NSs, forwhich a nanomolar LOD was measured corresponding to ananalyte concentration of 0.6 ng ml�1.

The obtained EFeffective values, in the 4 � 104 O 5 � 105

range, are comparable with the best results reported in litera-ture with analogous structures probed in solution, butmeasured with shorter excitation wavelengths. For CTAB-NSswe also examined the role of particle concentration and foundout that the overall sensitivity could be improved by diluting thecolloid. One of the most interesting ndings of this work isindeed the good efficiency of SF-NSs, which offer alternativeSERS tags much more biocompatibile with respect to CTAB-protected nanoparticles, for which the cellular cytotoxocitydue to desorbed CTAB molecules is ascertained.

Although the overall Raman intensity in the near-infraredspectral region is weaker with respect to the visible case, thegood SERS response shown by our nanostructures appears apromising feature for novel applications based on FT-Ramanspectroscopy. In particular, the possibility of efficientlyexploiting the near-infrared spectral window also with nano-particles capped with biocompatible agents (chitosan in thepresent case), paves the way to the use of these metallic tags inbiological systems, even in vivo models, with minimum uo-rescence background.

Acknowledgements

Funding from PRIN2009 project on “Novel plasmon-basedprocesses and materials for sensor applications” of the ItalianMinistry of Research is acknowledged. The authors also expresstheir thanks to the Institute of Organic Chemistry with Centre ofPhytochemistry BAS, Soa, Bulgaria and, in particular, to Prof I.Timtcheva and Dr G. Dobrikov, for providing the reportermolecule.

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