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Spectrochimica Acta Part A 85 (2012) 111– 119
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy
j ourna l ho me page: www.elsev ier .com/ locate /saa
urface-enhanced Raman scattering study of riboflavin on borohydride-reducedilver colloids: Dependence of concentration, halide anions and pH values
angfang Liua, Huaimin Gua,∗, Yue Linb, Yajing Qia, Xiao Donga, Junxiang Gaoa, Tiantian Caia
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, ChinaSchool of Educational Science, South China Normal University, Guangzhou 510631, China
r t i c l e i n f o
rticle history:eceived 10 July 2011eceived in revised form8 September 2011ccepted 21 September 2011
eywords:
a b s t r a c t
The influences of concentration, halide anions and pH on the surface-enhanced Raman scattering (SERS)of riboflavin adsorbed on borohydride-reduced silver colloids were studied. The optimum concentrationfor the SERS of riboflavin is 10−6 mol/L while the SERS enhancement varies for different modes. The addi-tion of 0.2 mol/L halide (NaCl, NaBr, and NaI) aqueous solutions, leads to a general decrease of the SERSintensity and a change of spectral profile of riboflavin excited at 514.5 nm. Riboflavin interacts with thesilver surface possibly through the C O and N–H modes of the uracil ring. The SERS spectra of riboflavinwere recorded in the 3.4–11.6 pH range. By analyzing several SERS marker bands, the protonated, depro-
iboflavinurface-enhanced Raman scatteringpectroscopyilver colloidsoncentrationalide anionsH
tonated or the coexistence of both molecular species adsorbed on the colloidal silver particles was proved.
© 2011 Elsevier B.V. All rights reserved.
. Introduction
Surface-enhanced Raman scattering (SERS) spectroscopy is aeliable high-resolution analytical technique for probing the naturef the chemisorption or physisorption process of target moleculesn a metal surface [1,2]. A combination of fluorescence quenchingnd Raman intensity enhancement provided by the silver sur-ace allows the acquisition of spectra at sub-micromolar levels3]. Indeed, recent advances have made SERS a versatile techniqueor diverse applications not only in analytical sciences but also iniomedicine, artwork conservation, nanotechnology, environmen-al science and archaeological.
Generally, the observed SERS signal enhancement is currentlyypothesized to be caused by a combination of three resonanceenominators: (i) the surface plasmon resonance in the metalanoparticle, (ii) the metal–molecule charge-transfer resonance athe Fermi energy, and (iii) resonances within the molecule itself4]. These three components are often treated as independentlyontributions to the overall effect. However, each resonance has a
lightly different effect on the resulting Raman spectrum, and it isecessary to invoke one or more of these resonances to completelyescribe a particular SERS experiment [4].∗ Corresponding author. Tel.: +86 20 85216972; fax: +86 20 85216052.E-mail address: [email protected] (H. Gu).
386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2011.09.043
For maximum sensitivity, SERS requires controlled aggregationof the substrates used. It has been shown that the SERS signalsare not only dependent on the size of the nano metal particles[5] but also on the pH [6,7], concentration of analytes [8,9] andthe presence of anions (particularly Cl−) [10,11]. A comprehensivesummary of chloride anions activation in SERS has been providedby Otto et al. [12]. Interestingly, different kinds of substrates canbe used to obtain SERS signals of analytes, such as silver and goldparticles [13], roughened electrodes [1] and nanoshell colloids [14].
Riboflavin, also known as vitamin B2 (Fig. 1), is a water-solublecompound that is essential for cell growth since it acts as a pre-cursor to flavoenzyme co-enzymes, flavin mononucleotide (FMN)and flavin adenosine dinucleotide (FAD) [15]. As a drug, it is oftenapplied in treating some clinic diseases such as tongue inflam-mation and anal conjunctivitis [16]. Riboflavin can also inducephotooxidation damage of cell-matrix compounds in the skin andeye, which would cause inflammation, accelerate aging and muta-tion [17]. In addition, the greenish-yellow fluorescence emittedfrom riboflavin is an ideal probe for conformational dynamics. Yanget al. successfully applied it to study protein dynamics withoutperturbation in 2003 [18].
Raman and SERS of riboflavin have been studied by several
groups. Lee et al. reported the SERS spectra of several active speciesof flavins [19]. In addition, Kim and Carey reported a compara-tive study of the Raman spectra of riboflavin in H2O and D2O [20].Studies of riboflavin on Ag nanoparticle-coated glass capillary [21],
112 F. Liu et al. / Spectrochimica Acta
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Fig. 1. Chemical structure of riboflavin.
ough silver films [22] and in silver colloids [23] have also beeneported. However, all of these studies were carried out withoutonsidering the influence of the aqueous solution environment onhe SERS of riboflavin. The interactions aimed at in this paper wereot or not well studied.
Considering the enormous biological importance of riboflavin,he present work is to study the influence of concentration, halidenions and pH on the adsorption of riboflavin on silver nanopar-icles, using the Normal Raman (NR) and SERS spectroscopyechnique. The results highlight the remarkable dependence of thedsorption mechanism of riboflavin on the experimental condi-ions. The experimental Raman bands are studied from the changesn integrated intensity, peak position and spectral profile with par-icular attention focus on (i) the influence of concentration on theERS spectra of riboflavin, (ii) the influence of halide anions on theurface enhancement of riboflavin, (iii) the protonated, deproto-ated molecular species and adsorption orientations of riboflavint different pH values, (iv) the interaction of adsorbates with sil-er colloids, and (v) the enhancement mechanisms, particularly thehemical enhancement effect of riboflavin on silver surface.
. Experimental
.1. Chemicals
The experimental materials of riboflavin, silver nitrate (AgNO3),odium borohydride (NaBH4), sodium chloride (NaCl), sodium bro-ide (NaBr), sodium iodide (NaI), sodium hydroxide (NaOH) and
cetic acid (CH3COOH) were of analytical grade. The riboflavin had certified purity of 98.5% and was used without any further purifi-ation. Doubly deionized water was used for all experiments.
All glassware was washed with nitric acid followed by gentlecrubbing with a soap solution. A silver colloid, used as a SERSubstrate, was prepared by the chemical reduction of sodium boro-ydride with silver nitrate according to the standard procedure
roposed by Creighton et al. [24]. Briefly, silver nitrate powdersith 8.5 mg were dissolved into 50 ml doubly distilled water, andhen rapidly added dropwise to 150 ml of ice-cold aqueous solutionf 2 × 10−3 mol/L sodium borohydride with vigorous stirring using
Part A 85 (2012) 111– 119
a magnetic stirrer for 1 h. The resulting yellowish silver colloid wasfound to be stable over several days at room temperature. The cor-rectness of the preparation of the solution was verified by observedthe absorption maximum of an active silver sol at 423 nm (data notshown), consistent with those reports in the literature.
Samples for SERS measurement were prepared as follows: thedesired concentration of riboflavin in fresh silver colloid (usedwithin a few days after preparation) was attained by mixing a spe-cific volume of stock solution with doubly deionized water in anappropriate volume. The volume proportion of silver sol:riboflavinsolution was 9:1. For studying the halide anions effect, 0.2 mol/Lhalide (NaCl, NaBr, and NaI) aqueous solutions were added todoubly deionized water and tested with 1 × 10−6 mol/L aqueoussolution of riboflavin in silver colloids. The volume proportionof silver sol:Na(X) solution:riboflavin was 4:1:1. The final con-centration of halide anions used in the measurements was about6.6 × 10−2 mol/L. The concentration of riboflavin solutions usedfor SERS measurement at different pH values was 1 × 10−7 mol/L.Dilute acetic acid and sodium hydroxide solutions were used toadjust the pH of the silver sol in the 3.4–11.6 pH range. The vol-ume ratio of silver colloid:acetic acid/sodium hydroxide:riboflavinsolution was kept at 2:2:1.
All the samples were left to incubate for 30 min at 25 ◦C after thefinal addition (riboflavin or anion) before the SERS measurement. Ineach SERS measurement, a droplet (0.02 mL) of incubated solutionwas evenly dropped on a clean glass slide. All measurements weremade on the same day.
2.2. Instrumentation
Raman spectra were acquired using a triple grating Ramanmicro-spectrometer (Acton Spectro @2300i, Princeton Acton, USA)equipped with a argon ion laser (� = 514.5 nm) and a liquid nitrogencooled coupled charge device (CCD) detector (Pixis 256, PrincetonActon, USA). The Raman system was equipped with a BX-41 Olym-pus microscope and 45× Objective. Spectra were recorded from100 cm−1 to 1900 cm−1 with a spectral resolution of 2 cm−1. Foreach Raman scan, the acquisition time was 10 s and the laser powerfocused on the sample was about 5 mW. Normal Raman spectrum ofanhydrous riboflavin powder was recorded by an i-Raman® Ramanspectrometer, the power at the sample was about 10 mW (785 nmlaser), and the acquisition time was 30 s. The absorption spectrumwas recorded by a Perkin Elmer Lambda 35 UV–Vis spectrometerwith a scan speed 480 nm/min.
3. Results and discussion
3.1. Normal Raman spectrum of riboflavin
As seen from the chemical structure of riboflavin (Fig. 1), thismolecule contains a tricyclic ring system which is composed of abenzene ring (I), a pyrazine ring (II) and a uracil ring (III). Riboflavinfurther possesses a carbon chain on the nitrogen atom of thepyrazine ring in which four of the hydrogen positive ions arereplaced by hydroxyl groups.
Fig. 2 presents the Normal Raman spectrum of riboflavin pow-der, using laser excitation wavelength of 785 nm. It is difficult tomeasure normal Raman spectrum of riboflavin in aqueous solu-tion by the conventional Raman spectrum technique because thestrong fluorescence disturbs the Raman signal. Prominent vibra-tional bands were observed at 294, 740, 1153, 1178, 1222, 1344,
1398, 1461, 1534 and 1576 cm in Fig. 2. Vibrational mode assign-ments were made by consulting the earlier literature [23]. Themost intense band at 1344 cm−1 in the Normal Raman spec-trum is attributed to the C–N stretching and the C–C stretching
F. Liu et al. / Spectrochimica Acta Part A 85 (2012) 111– 119 113
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Fig. 2. Normal Raman spectroscopy of riboflavin powder.
ibration, which represents the characteristic frequency of theyrazine ring of the molecule. The modes have prevailing contribu-ions from the C–C stretching vibrations of the benzene ring of theiboflavin molecule appear as strong bands at 1461 and 1620 cm−1,espectively. The 1534 and 1576 cm−1 bands have significant con-ributions from the C–N stretching, while the 1398 cm−1 bandas prevailing contribution from the C–C stretching. These obser-ations indicate the relationship of vibrational modes betweendjacent rings. Sharp and intense bands at around 1178 and222 cm−1 appear in the normal Raman spectrum of the molecule
n the solid state. These bands are ascribed to the C–N stretchingode of the uracil ring and the C–CH3 stretching mode of the ben-
ene ring. The band at 1287 cm−1 has a dominate contribution fromhe C O bending vibration while the 519 cm−1 band has salientontribution from the C–C O bending mode, both of them emanaterom the uracil ring. The ring breathing mode appears at 740 cm−1
hile the pyrazine ring bending mode locates at 672 cm−1.
.2. Concentration dependence of SERS of riboflavin
Many intermolecular interactions such as caging effect,otional narrowing and diffusion dynamics may be obtained from
he concentration dependence of the vibrational bands [25–28].nalysis of parameters such as line width, peak position and inten-ity provides information about the static and dynamic processesaking place in a system while the band shape analysis of SERSands may add further additional information about the dynamicalrocess of the system [28]. In view of the above facts, experi-ental measurements of the Raman bands of riboflavin in silver
olloid at different concentrations were carried out to study theoncentration-dependent enhancement in the SERS process.
SERS spectra of riboflavin in nanocolloidal solutions at con-entrations from 1 × 10−9 mol/L to 1 × 10−4 mol/L are presentedn Fig. 3. It is found that the vibrational bands in normal Ramanpectrum are richer than that in SERS spectra. The strong bandst 740 and 1461 cm−1 in the normal Raman spectrum appear veryeak in the SERS spectra. Even the strong bands at 294, 1178 and
222 cm−1 in the normal Raman spectrum disappear in the SERSpectra of riboflavin. These phenomena may be due to the waterffect in the SERS measurement as well as the rising of the back-
round of inelastic scattering and the intense scattering of silverurface to the laser light.Prominent vibrational bands were observed at 550, 625, 841,085, 1157, 1345, 1405, 1524 and 1627 cm−1 in Fig. 3. It is evident
Fig. 3. Dependence of the SERS spectra on the concentration of riboflavin. The exci-tation wavelength is 514.5 nm and the laser power at the sample is 5 mW.
that the enhancement of all vibrational bands of riboflavin is sen-sitive to the concentration variations. At very low concentrations,the SERS enhancement observed using the silver colloid is too weakto be detected even for the most intense band at 1345 cm−1 (datanot shown). It was also reported previously that at very low con-centrations the SERS amplification was missing, due to a criticalconcentration that was necessary to start the aggregate forma-tion [29,30]. The minimum concentration of riboflavin required toaggregate the colloid to a required extent was found to be about3.2 × 10−10 mol/L. Below this value, the SERS may be only possi-ble if some aggregating agent is added to the colloid. It is seenfrom Fig. 3 that the optimum SERS spectrum of riboflavin appearsat the concentration of 1 × 10−6 mol/L. The integrated intensity ofthe SERS spectrum reduces with the increase of the concentrationfrom 10−6 mol/L. This saturation effect is assumed as a result of pre-cipitation of metallic silver [30], as well as a saturation caused bycomplete coverage of the SERS substrate [31].
The SERS enhancement is not sufficient when the concentration
of riboflavin is too high or too low. The concentration variation ofriboflavin in solution is probably caused by a different coverage ofriboflavin on the metal surface. To explain this surface coverageeffect, the existence of two different groups of riboflavin molecules
114 F. Liu et al. / Spectrochimica Acta
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bending mode below 200 cm−1.
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ig. 4. Variation of integrated intensity of different bands (w.r.t. the 550 cm−1 band)ith molar concentration.
hould be considered: (1) those molecules standing in the firstayer, which correspond to those molecules that strongly interact
ith the metal, adopting a more rigid position in a predominantormal orientation with respect to the surface and (2) moleculeslaced in layers superimposed to the first one, which may adoptore variable orientations.While at higher concentration of riboflavin, the silver surface is
overed by the riboflavin multilayer, thus reducing the contribu-ion of the first layer to the overall SERS spectrum. Decreasing theoncentration of riboflavin produces more contributions of the firstayer to the SERS spectrum. Therefore, the pronounced SERS with
aximum intensity appears only at the monomer coverage, whereoth charge transfer and electromagnetic enhancement contribu-ion reach maxima.
With further decreasing the concentration, there are lessiboflavin molecules adsorbed on silver surface and thus theub-monolayer is exposed. The absence of sufficient scatteringolecules results in weak SERS intensity. Therefore, in order to
btain maximum SERS signals, the concentration of riboflavineeds to reach an appropriate value. This result is consistent withhe earlier work of S. Sanchez-Cortes et al. [32] who researchedhe effects of the concentration ratio, chloride and pH on SERS ofytosine and its methyl derivatives.
Following the discussion of the concentration dependence ofhe SERS integrated intensity, in order to obtain more informa-ion about the influence of concentration on the SERS spectra ofiboflavin, the enhancement of different vibrational modes underach molar concentration is studied. In this section, six promi-ent Raman bands are chosen for the analysis. The intensitiesf these six bands are plotted with respect to the Raman band
≈550 cm−1) at each molar concentration (see Fig. 4). This methodas also adopted by Mishra et al. [28]. When taking the 550 cm−1and as an internal standard, a basic requirement, although still
able 1aman peak positions at different molar concentrations of riboflavin.
Conc. (mol/L) Peak position (cm−1)
≈550 cm−1 band ≈841 cm−1 band ≈1085 cm−1 ban
10−4 550.71 839.60 1091.86
10−5 559.30 845.80 1087.86
10−6 555.00 841.67 1085.85
10−7 550.71 841.67 1085.86
10−8 550.71 839.60 1085.86
10−9 550.71 835.46 1089.86
Part A 85 (2012) 111– 119
needs further studying, is that this mode should remain constantwith the changes of the concentration. Also, the absolute inten-sity is not a reliable parameter. The Raman activity of the 550 cm−1
band increases moderately from 10−9 mol/L to 10−6 mol/L from theexperimental data. Without taking the relative intensity, nothingcan be regard as an enhancement. However, the intensity comparedwith the 550 cm−1 mode gives a rough qualitative estimation aboutthe enhancement. We only want to ensure that enhancement ispresent in some bands. It is seen from Fig. 4 that the SERS enhance-ment is not the same for all six modes. The enhancements increaseconsistently up to 10−6 mol/L concentration for the 1345, 1524and 1627 cm−1 bands, and then decrease with further increasingthe concentration. On the other hand, the intensities reach max-ima at 10−8 mol/L concentration for the 841 and 1085 cm−1 bands,and then decrease with further increasing the concentration. Theenhancement is supported on the whole by the Raman activity andthe ratio of this activity for different vibrational modes varies underdifferent molar concentrations.
The peak positions of the above six Raman bands at differ-ent concentrations are presented in Table 1. The first band at1524 cm−1 involves significant amounts of C–N stretching bend-ing of the pyrazine ring and uracil ring. This mode is red-shifted by17 cm−1 experimentally in the riboflavin + silver complex with thedecrease of the adsorbate concentration. The next band at 845 cm−1
with concentration of 10−5 mol/L is obtained at 835 cm−1 with con-centration of 10−9 mol/L. The red shift in this mode is 10 cm−1.The C–C O bending mode of uracil ring appears experimentallyat 559 cm−1 with concentration of 10−5 mol/L while this mode isobserved at 550 cm−1 with adsorbate concentration of 10−9 mol/L.The red shift of this mode is 9 cm−1. The most intense band at1345 cm−1 has a shift of 4 cm−1 while the C–C stretching modeof benzene ring at about 1627 cm−1 has a red shift of 8 cm−1 bydecreasing the concentration from 10−4 mol/L to 10−5 mol/L. Theshifts observed in the above-mentioned bands can actually beattributed to the different contributions to the overall SERS spec-trum from several adsorbed forms whose relative importance mayvary with the adsorbate concentration. These discussions indicatethat the SERS spectra of riboflavin are of both concentration-dependent and mode-specific nature.
3.3. Halide anions-dependent enhancement
Fig. 5 displays the SERS spectra of riboflavin in the presence ofhalide anions. It is evident that all three halide anions, Cl−, Br−, andI−, affect the enhancement of Raman signals of riboflavin consider-ably. The presence of the halide anions induces the following maineffects: (i) a decrease of SERS intensity varying with different halideanions; (ii) a change of spectral profile, which is different for eachkind of halide anion; (iii) a shift of vibrational band, which is not thesame for all modes; (iv) a significant increase of the Ag-adsorbate
The addition of a relatively low amount of 0.2 mol/L halide (NaCl,NaBr, and NaI) aqueous solutions, leads to a general decrease ofthe SERS intensity when exciting at 514.5 nm. Particularly, in the
d ≈1345 cm−1 band ≈1524 cm−1 band ≈1627 cm−1 band
1345.45 1534.26 1631.421341.59 1528.81 1623.991347.38 1524.81 1629.561347.38 1515.81 1627.711347.38 1522.58 1629.561347.38 1517.10 1627.71
F. Liu et al. / Spectrochimica Acta Part A 85 (2012) 111– 119 115
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ig. 5. SERS spectra of riboflavin in silver colloids for the 10−6 mol/L concenration:a) riboflavin, (b) riboflavin + Cl− , (c) riboflavin + Br− , and (d) riboflavin + I− .
resence of Br− (Fig. 5c) and I− (Fig. 5d), the Raman signals fromiboflavin become very weak, especially for the most intense bandt about 1347 cm−1. This suggests that in the presence of Br− and I−,iboflavin molecules are prevented to adsorb on the colloidal silverurface. This phenomenon was also reported by several authorsnd was explained as a consequence of a competitive adsorptionetween the halide anions and the target analytes [33].
The interaction strength of halide anions with silver particlesncreases in the order of Cl− < Br− < I− ions [34]. To study what hap-ens in the presence of riboflavin, the SERS spectra of riboflavin inhe presence of Cl− + Br−, Cl− + I−, Br− + I− and Cl− + Br− + I− ionsere measured, respectively (see Fig. 6). As can be seen from
he spectra region 100–400 cm−1 in Fig. 6, in the coexistence ofiboflavin, Cl− and Br− ions, there appear only the Ag–Br− stretch-ng mode (153 cm−1) and the riboflavin modes (321 and 361 cm−1),
hereas in the coexistence of riboflavin, Cl−, Br− and I− ions, onlyhe Ag–I− (146 cm−1) stretching mode is observed. These resultsndicate that the interaction strength order of halide ions with silveroes not change even when riboflavin is present.
The band positions and relative intensities of the SERS spectraf riboflavin in the presence of Cl−, Br− and I− as well as the normalaman spectrum are listed in Table 2. A comparison of the SERSith the normal Raman spectrum of riboflavin shows that (i) the
and at 343 cm−1 due to C–C bending vibrations of the carbon chainhifts by 13–26 cm−1 to lower wavenumbers in the SERS spectra,hile this mode is too weak to be detected in the presence of Br−
−
nd I ions; (ii) similar band shifts are also observed for the C–Ntretching and C–C stretching vibrations of the uracil ring at about094 cm−1; (iii) the bands at about 1461 and 1620 cm−1, whichan be assigned to the C–C stretching mode of benzene ring, shiftFig. 6. SERS spectra of 10−6 mol/L riboflavin in silver colloids in the presence of: (a)Cl− + Br− , (b) Cl− + I− , (c) Br− + I− , and (d) Cl− + Br− + I− .
1–14 cm−1 to the lower wavenumbers for 1461 cm−1 and 3–7 cm−1
to higher wavenumbers for 1620 cm−1; (iv) the elimination of theband appearing at 687 cm−1 which is assigned to the pyrazine ringbending mode; (v) I− ions result in the largest band shifts for theC–C O bending vibrations at about 519 cm−1 and C–N stretchingmode of pyrazine ring at about 1344 cm−1.
In summary, halide anions cause the C–C bending vibrations ofthe carbon chain, the C–N stretching and C–C stretching modesof the uracil ring to shift obviously to lower wavenumbers whilethey cause the C–C O bending vibrations and C–N stretching modeof pyrazine ring to shift to higher wavenumbers. The upshift inwavenumbers indicates strengthening of the corresponding bond.
A complete understanding of halide anions effect on the SERSintensity is still not achieved at present, since there are adsorbateswhose SERS signals are positively enhanced by the halide anions,while others are negatively influenced by them [32]. This differencecan be attributed to the nature of the adsorbate and the double con-tribution of the halide anions to both the EM and CT mechanisms,thus making analysing the effect of halide anions difficult. The SERS
intensity quenching observed at low concentration when excitingat 514.5 nm may be attributed to a surface-coverage restrictionimposed by the co-adsorption of the halide anions effectively
116 F. Liu et al. / Spectrochimica Acta Part A 85 (2012) 111– 119
Table 2Frequencies and assignments of normal Raman and SERS bands of riboflavin.
NRS (cm−1) SERS (cm−1) Assignments
Cl− Br− I−
158 m 158 vs 151 vs � (Ag–Halide anions)216 m 214 vw 216 m � (N–C, Ag–N)III
294 m343 w 330 w 317 w ı (C–C)n n = 5
406 vw443 vw 451 vw 446 vw 446 vw
500 w519 m 550 m 537 s 531 vw 559 m ı (C–C O)595 m 623 s 619 m 619 vw 608 vw672 m 687 vw ı (Ring II)740 s 743 m 741 w Ring breathing
784 m 764 vw 768 vw804 vw 810 vw 808 m 806 vw 812 vw842 vw 841 m 837 m 856 w ı (C8–H, C11–H)921 vw 913 w 915 w 921 vw
1019 vw 1005 w 1005 m 1003 w1058 w 1031 m 1029 w
1094 vw 1085 s 1089 m 1093 m � (C2–N3, C5–C4, C5–C14)1117 vw1153 m 1157 s 1159 m1178 s 1179 m 1177 vw � (C2–N1, C2–N3, C4–N3, C9–CH3,
C4–C5, C5–C14)1222 s 1204 vw � (C4–N3, C10–CH3)
1287 vw 1285 m 1263 m 1296 w 1275 vw ı (C2 O, C4 O, N3-H), � (C14-N1)1312 vw ı (N–H), � (C12-N13)
1344 vs 1347 vs 1349 vs 1355 m 1357 w � (C14–N13, C7–C12)1398 m 1405 s 1405 w � (N1–C2, C7–C8, C10–C11, C7–C12)1461 m 1458 w 1460 w 1447 m � (C8–C9, C10–C11, C11–C12)1496 w ı (CH3)1534 m 1524 s 1536 m 1539 vw 1530 vw � (C5–N6, C14–N1)1576 m 1571 m 1579 m 1586 w 1588 w � (C5–N6, C14–N13, C14–N1, C5–C14)
1627
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1620 w 1627 vs 1627 vs
bbreviations: vw, very weak; w, weak; m, medium; s, strong; vs, very strong; �, str
ompeting with riboflavin for adsorption sites on the metal sur-ace. When the concentration of halide aqueous solutions in silverolloids is too high (0.2 mol/L), superfluous halide anions may formn absorption layer so that riboflavin molecules cannot be adsorbedn silver surface directly.
The effect of the halide anions on the spectral profile is anothernteresting fact that should be analyzed to obtain more informationbout the influence of Cl−, Br− and I− ions. Comparing Fig. 5b withig. 3, it is a noteworthy fact that some of the changes inducedy Cl− on the SERS of riboflavin are similar to those observedhen lowering down the adsorbate concentration: (i) down shift
f C O bending bands of uracil ring (from 1285 to 1263 cm−1);ii) up shift of the band at 1157 cm−1; (iii) a new band appears atbout 158 cm−1. The above results suggest that the effect of Cl− ions also connected to a surface-coverage variation, since the SERSf riboflavin at low concentration resembles that of the SERS ofiboflavin in the presence of Cl− ions. Also, the changes in adsorp-ion of riboflavin on the silver surface may lead to the same result.n addition, these phenomena are assumed to be associated withhe aggregation of silver sol in the absence of Cl− [33]. Due to theggregation effect, large average radii of new particles lead to aecrease of the available surface and a resultant increase of thedsorbate/surface ratio. Therefore, the effect of Cl− is similar to thatbserved when lowering the adsorbate concentration.
In fact, the SERS spectrum of riboflavin obtained after addingalide anions is close to that of a multilayer surface-coverage, sincehe changes induced by the halide anions are stronger than thoseaused by reducing the concentration. In this case, the decrease
f SERS intensity with adding halide anions could account forhe strong contribution of the CT mechanism, where absorbedolecule always show weak SERS intensity at dense surface cov-rage [33].
vs 1623 m � (C7–C8, C9–C10, C10–C11, C7–C12)
g; ı, bending.
It may be deduced from the increase of the above adsor-bate/surface ratio in the presence of halide anions that the negativeeffect of surface concentration induced by the co-adsorption ofhalide anions, which competes with riboflavin for the adsorptionon the silver surface, is compensated by the multilayer surfacecoverage and the aggregation of silver sol created by the halideanions. The intensity, frequency and shape of the bands may alsochange due to the adsorbate reorientation. However, not onlybands shift but also the new bands are obtained in our experi-mental results, which may only be induced by the reorientation ofmolecules. The shift of most modes has been interpreted in termsof an edge-on adsorption of the adsorbate [33]. The SERS signalsinduced by the chemi-adsorption are intensified with lowering ofthe adsorbate concentration; while the multilayer adsorption con-figuration reduce the effective contribution of first layer moleculesto the overall SERS signals at higher concentration. This effect isin contrast to the observation of a reorientation from face-on toedge-on, in going from low to high surface-coverage [33]. There-fore, when first-layer adsorption occurs at low surface-coverage,riboflavin may strongly interact with the surface by adopting amore rigid position and a predominant near-flat orientation; whileat dense surface-coverage riboflavin may orient in layers super-imposed to the first, adopting more random orientations. In thesecases, riboflavin molecules may be considered to be more ‘free’,than when lying on the metal surface, this is in agreement with theSERS spectra.
The effect of riboflavin is also noted in the <300 cm−1 region. Theexistence of a weak band attributed to silver–adsorbate stretching
vibration at ≈216 cm−1 in the absence of halide anions, demon-strates the adsorption of riboflavin on the metal, which may beproduced by the electron transition between riboflavin and sil-ver surface. When Cl− appears, this mode increases, indicating the
a Acta Part A 85 (2012) 111– 119 117
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Fig. 7. SERS spectra of riboflavin recorded at different pH values as indicated in thefigure for the 1 × 10−7 mol/L concentration. The excitation wavelength is 514.5 nmand the laser power at the sample is 5 mW.
F. Liu et al. / Spectrochimic
ossibility of the increasing of CT interaction between adsorbatend metal. However, this band is quenched when Br− and I− appear,ndicating the existence of adsorption competition between halidenions and riboflavin. A very intense band is observed at about58 cm−1 which is attributed to the (Ag–Cl−/Br−/I−) stretchingibration (see Fig. 5). It demonstrates the adsorption of the halidenions on the metal. The changes in intensity and profile shape ofhe SERS spectra suggest different adsorption behavior and surface-overage of riboflavin on silver surface. In addition, the adsorptionrientation of riboflavin on the silver surface varies due to differentalide anions. In the presence of Cl−, the riboflavin molecules aressumed to adsorb in a predominant tilted orientation to the silverurface. While in the presence of Br− or I−, the riboflavin moleculesdsorb in a less tilted, closer to perpendicular orientation with theespect of the silver surface.
Based on the vibrational features of the SERS of riboflavin, its possible to find the groups through which riboflavin interacts
ith the colloidal silver surface. The obvious different Raman bandsetween Fig. 2 and Fig. 5a indicate that the riboflavin has a strong
nteraction with silver surface. As can be seen from Table 2, theelative intensity and band position of the C O bending vibrationalode at 1287 cm−1 are obviously changed by the comparison of the
ormal Raman and SERS spectra. This indicates that the riboflavinolecule is adsorbed on the silver surface through the of the uracil
ing, it may explain at least partially the large shifts of the bandositions for the C–C O bending, the C–N bending and C–C bend-
ng of uracil ring. If halide anions are supposed to interact with theolecule through its C O and N–H modes of uracil ring, it may
lso result in the changes of the above modes. In this case, the–C stretching vibrations of the benzene ring and the C–C bend-
ng vibrations of the carbon chain should be less affected becausehey locate far from the uracil ring. However, the obvious changesf these two modes can be observed. This may be also due tohe changes of the adsorption orientation of riboflavin on the sil-er surface resulted from the interaction between halide anionsnd riboflavin molecules. It is possible that both electromagneticnd charge transfer mechanisms are involved in the interactionetween riboflavin and the silver colloids, while the contributionf the charge transfer mechanism to SERS may be dominant.
.4. SERS of riboflavin at different pH values
The physical processes and chemical reactions involved in bio-ogical processes are often very sensitive to the concentration ofhe hydrogen or hydroxyl ions of the medium. SERS spectroscopy
onitors the molecules adsorbed on a metal surface without givingnformation about the bulk solution and represents a versatile toolor recording the spectrum in the pH domain with low solubility,ue to its potential of detection on the micro-molar level [7]. Thenhancement of a particular band is maximum at a particular pHf the colloidal solution while the same pH does not produce theame enhancement for all bands.
The SERS spectra of riboflavin in the pH interval 3.4–11.6 areresented in Fig. 7. The pH of the silver colloid was adjusted by theilute acetic acid and sodium hydroxide solutions. Small aliquots of
× 10−7 mol/L riboflavin solution were added to the correspondingH-adjusted colloid up to a final concentration of 5 × 10−8 mol/L.
The SERS spectral table can be divided into three parts. Firstly,t pH 3.4–4.5, as it was expected, the spectra reveal the presencef the protonated species adsorbed on the silver surface; secondly,n the 6.5–10.6 pH range, both molecular species, protonated andeprotonated (neutral) coexist on the silver surface; and thirdly, at
H 11.6 only deprotonated molecules are adsorbed onto the silverurface.Marker bands of the adsorbed protonated molecular species inhe SERS spectra at pH 3.4–4.5 are the most intense ones at 1460,
118 F. Liu et al. / Spectrochimica Acta
Fig. 8. Logarithm (log10) intensity (peak area) of SERS vibrational band at 1345 cm−1
of riboflavin molecules using a concentration of 1 × 10−7 mol/L, prepared from solu-t
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of riboflavin with silver surface were investigated. The Ag atoms areattached to the riboflavin molecule quite possibly through the C O
ions by increasing pH of the dye. Points represent averages of three measurements.
406, 1349, 1314, 1155 and 621 cm−1. The assignments of theseands are shown in Table 2.
The shape of the SERS spectrum at pH 6.5 is similar to that ofhe SERS spectrum at pH 4.5, indicating the presence of mainlyrotonated riboflavin molecular species adsorbed on the silver sur-ace. Nevertheless, compared with the SERS spectrum at pH 4.5,hanges in band position and intensity of the SERS spectrum at pH.5 show the coexistence of a few deprotonated riboflavin molec-lar species adsorbed onto the silver surface. On examining thehange of the spectra from pH 4.5 to 6.5, the characteristic band forhe protonated riboflavin molecular species at 1406 cm−1 is blue-hifted to 1408 cm−1 and is further shifted in the SERS spectra atigher pH values, whereas the other bands at 1460, 1349, 1314,155 and 621 cm−1 decrease in intensity. The bands at 1460, 1314nd 621 cm−1 disappear totally in the SERS spectrum at pH 11.6.
The pKa value of riboflavin is 10.2 [35]. The coexistencef deprotonated molecular species with the protonated onesdsorbed already on the silver surface at pH 6.5 is not sur-rising. It was reported by several authors that the adsorptionn metal surfaces of deprotonated molecular species at pH val-es lower by two or more units than the pKa value of theolecule [7,36–38].The presence of the protonated and deprotonated riboflavin
pecies adsorbed on the silver surface is more evident from pH.8 to 10.6. This is revealed by the presence in the spectra of theands attributed to both molecular species. The contributions ofhe protonated molecular form are the bands at 1460, 1314, 1155nd 621 cm−1, while the contributions of the deprotonated molec-lar species are mainly the band at 1537 and 1324 cm−1. With
ncreasing of pH from 7.8 to 10.6, the relative intensities of theand at 1537 and 1324 cm−1, due to the deprotonated riboflavinolecules, slightly increase, and the relative intensities of the
eaks at 1460, 1314, 1155 and 621 cm−1, given by the protonatedolecules decrease. Consequently, an increase of the number of
eprotonated riboflavin molecules adsorbed on the silver surfaces observed, whereas the number of protonated species decreases.n addition, at pH 10.6, a new peak appears at 1206 cm−1, which isttributed to the deprotonated riboflavin molecules, becomes veryntense at higher pH values.
At pH 11.6, the lack of the characteristic bands of the protonated
iboflavin molecular species and the enhancement of the intenseands at 1536, 1324 and 1206 cm−1 demonstrate the adsorptionPart A 85 (2012) 111– 119
exclusively of deprotonated riboflavin molecules on the silver sur-face.
In order to verify the above conclusions, intensities of vibra-tional modes at different pH values are studied. The most intensesurface enhanced Raman band at 1345 cm−1 is used to assess signalenhancement because this mode is related to both the protonatedand deprotonated molecular species of riboflavin. The enhance-ment is determined by plotting the logarithm (log10) of intensityof this SERS band versus pH. Fig. 8 shows the approximate pH pro-file from this set of SERS spectra. The maximum intensity at acidicpH values is found to be at pH 4.5 while the maximum intensityat alkaline pH values appears at approximately pH 11.6. The abovetwo maxima observed in the pH profile suggest the presence oftwo species of riboflavin molecules: protonated and deprotonated.From Fig. 8 it is found that the band intensity increases significantlyfrom pH 3 to pH 4.5, indicates the presence of protonated molec-ular species. Beginning with pH 4.5, the number of protonatedmolecules exponentially decreases while the number of deproto-nated molecules is not strength enough to stop the intensity fromreducing. With further increase of the pH values, the deprotonatedriboflavin molecules are dominant on the silver surface, thus theintensity of the mode increases slightly. The obviously enhance-ment of the intensity observed from pH 10.6 to pH 11.6 revealsthat only deprotonated molecules are adsorbed on the silver surfaceunder the strong basicity condition.
As mentioned above, riboflavin interacts with the silver surfaceriboflavin through the C O and N–H modes of the uracil ring. Fol-lowing the variations of the ring-breathing mode of riboflavin atabout 741 cm−1 in the SERS spectra (Fig. 7), this vibration is lessenhanced in the 3.4–4.5 pH range. The protonated molecules areassumed to adsorb in a predominant tilted orientation of the uracilring to the silver surface only through the N–H mode of the uracilring.
In the 6.5–8.3 pH range, when both molecular species coex-ist on the silver surface, the ring-breathing mode becomes moreenhanced; consequently, the riboflavin molecules are adsorbed ina less tilted, closer to perpendicular orientation of the uracil ringwith the respect of the silver surface.
As shown in Fig. 7, at pH 10.6 and 11.6, the ring-breathing modeis less enhanced; therefore, a flat orientation of the uracil ring withrespect of the surface is suggested.
4. Conclusions
In this paper, SERS of riboflavin adsorbed on silver surface underdifferent aqueous solution environment have been studied. Therelative intensity of SERS of riboflavin is significantly varied withdifferent concentrations of adsorbate while a maximum enhance-ment is achieved at 10−6 mol/L. The SERS enhancement of riboflavinis both concentration-dependent and mode-specific. SERS spectraof riboflavin on the silver surface suggest the existence of a first-layer, which changes the adsorption behavior in the presence ofhalide anions. The changes of SERS intensity and the spectral profilein the presence of halide anions are governed by the CT mechanismand co-adsorption of anions and molecules. SERS of riboflavin aredominated by the first-layer molecules at low surface-coverage,with the molecular plane parallel to the silver surface. In contrast,at higher halide anions concentration (0.2 mol/L), the SERS spectraare attributed to the adsorbates being in predominant tilted orien-tation (Cl−) or near perpendicular (Br− or I−) to the silver surface.The interaction of riboflavin with halide anions also the interaction
and N–H modes of the uracil ring. SERS spectra of riboflavin wererecorded in the 3.4–11.6 pH range, demonstrating the existence
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f protonated, deprotonated or the coexistence of both molecularpecies adsorbed on the colloidal silver particles.
cknowledgement
The authors are grateful for the support of this research by theational Natural Science Foundation of China (60678050).
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