Gd

SN4

a

ARRAA

KGGBS

1

nshdAitretdiiplpip“

0h

Colloids and Surfaces B: Biointerfaces 103 (2013) 475– 481

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

elatin–nanogold bioconjugates as effective plasmonic platforms for SERSetection and tagging

orina Suarasan, Monica Focsan, Dana Maniu, Simion Astilean ∗

anobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in Bio-Nano-Sciences and Faculty of Physics, Babes-Bolyai University, M. Kogalniceanu 1,00084 Cluj-Napoca, Romania

r t i c l e i n f o

rticle history:eceived 25 September 2012eceived in revised form 25 October 2012ccepted 27 October 2012vailable online 10 November 2012

eywords:old nanoparticles

a b s t r a c t

It is well known that standard citrate-reduced gold nanoparticles (AuNPs) are unstable at high ionicstrength solution, which limits their applications in the biomedical field. In this work we present anenvironmentally friendly approach for the stabilization of citrate-reduced AuNPs in aqueous solution.Specifically, the stability of the AuNPs against salt-induced aggregation was greatly improved in thepresence of gelatin biopolymer and stabilization of individual or small assemblies of nanoparticles canbe controlled by the amount of gelatin. Furthermore, the gelatin–nanogold bioconjugates were demon-strated to be operational as highly sensitive surface-enhanced Raman scattering (SERS) active substrate

elatinioconjugatesERS

for the detection of Rose Bengal fluorophore in solution at very low concentration. The results suggest thatsuch bioconjugates can be successfully employed not only for detection of analytes, but more interestinglyfor building SERS-active tags in view of imaging purpose. The stabilization of bioconjugates was analyzedby localized surface plasmon resonance spectroscopy (LSPR), transmission electron microscopy (TEM),dynamic light scattering (DLS) and zeta-potential, and the chemical interaction of gelatin with AuNPs

r tran

was inferred from Fourie

. Introduction

In the last decade noble-metal nanoparticles, especially goldanoparticles (AuNPs), have received great interest in biomedicalcience due to their unique optical and chemical properties whichave promoted several relevant applications in biosensing [1],rug delivery [2], diagnostic [3] and imaging [4]. The key feature ofuNPs optical properties is expressed by a strong absorption band

n the visible region related to collective oscillation of free elec-rons under light excitation, known as localized surface plasmonesonance (LSPR). Since plasmon excitation induces electric-fieldnhancement around nanoparticles, surface enhanced Raman scat-ering (SERS) is the most specific application of AuNPs devoted toetection of (bio)molecules at extremely low concentrations in var-

ous biomedical environments [5,6]. Beyond the traditional interestn fabrication of highly sensitive SERS substrates for analytical pur-ose, an increasing importance is given nowadays to spectroscopic

abels called SERS-active tags which are designed for imaging pur-ose, as example for tracking the presence of labeled nanoparticles

n cells [7]. Such SERS-active tags are nano-constructs made oflasmonic nanoparticles which are deliberately decorated withreporter molecules” and enveloped in a shell of biocompatible

∗ Corresponding author. Tel.: +40 264 454554x115; fax: +40 264 591906.E-mail address: simion.astilean@phys.ubbcluj.ro (S. Astilean).

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2012.10.046

sform infrared spectroscopy (FT-IR).© 2012 Elsevier B.V. All rights reserved.

material [8]. For any class of fabricated SERS substrates, the mainchallenge is to provide good colloidal stability, biocompatibilityand chemical affinity toward specific biomolecules, cells or tissues[9]. As it is well known, due to their negatively charged surfaceprovided by the sodium citrate used in the classical synthesis,AuNPs are unstable at high ionic strength aqueous solution andstart to aggregate in biological media, greatly limiting their appli-cations. Nevertheless the long term stability of AuNPs under theextreme/physiological conditions can be improved by conjugationwith various proteins or biopolymers [10,11]. In this case bio-conjugation not only ensures the stabilization of the system but,more significantly, the biopolymer layer provides a biocompatiblenanoparticle surface, an essential requirement for applying suchSERS-active tags in different intracellular imaging studies.

Considering the affinity of AuNPs to sulfur atoms, the biopoly-mers that posses functional groups such as SH facilitate theformation of such bioconjugates. Gelatin is a natural, biocom-patible biopolymer derived from collagen denaturation througha hydrolysis process and – as a result – contains the same 18amino acids in varying amounts [12]. It has a unique advantagefrom the other biopolymer due to its electrical nature which canbe easily adapted via methods and conditions of collagen pro-

cessing [13]. Specifically, during extraction from collagen, gelatincan be modified trough an alkaline or acid collagen denaturationtreatment to yield a positive or negative charged gelatin [14]. Inaddition, thanks to its relatively abundant content of functional

4 ces B:

gaatb[alaacem

fAsnopsc4wa[gibsm(Ituo

2

2

9BCSpgw

2

mTaa

bsogp

i–

76 S. Suarasan et al. / Colloids and Surfa

roups (i.e. SH, NH2 and COOH), gelatin is easy to cross-linknd therefore it can be successfully used as a stabilizing/coatinggent for nanoparticles. Indeed, gelatin was successfully used inhe synthesis of silver–gold bimetallic nanoparticles [15] or car-oxylic single-walled carbon nanotubes–AuNPs nanocomposites16] and, more recently, as surface capping agent for stabilizationnd in situ synthesis of size controlled-AuNPs [17]. Due to its excel-ent biocompatibility, gelatin biopolymer was widely employed for

variety of biomedical applications, including bio-mimetic miner-lization [18] or pharmaceutical carriers [19]. Moreover, as gelatinan be easily cross-linked with various molecular biomarkers, it isxpected that this biopolymer can be an ideal candidate for imple-enting SERS active tags.However, no previous investigations have been reported on the

ormation of bioconjugates between gelatin and pre-synthesizeduNPs (referred here as gelatin@AuNPs) aiming to analyze theirtability under simulated biological conditions and demonstrateew bio-oriented functionalities. In this study, apart from a thor-ugh characterization of bioconjugation process, we highlight aromising application of gelatin@AuNPs as biocompatible SERSubstrates for both analytical and imaging purpose. As proof of con-ept we encoded gelatin@AuNPs substrates with Rose Bengal (RB,,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein), a fluorophorehich presents some relevant applications in biology and medicine

nd specifically in photodynamic therapy as photosensitizing agent20]. RB molecule is a hydrophilic fluorophore with a carboxylroup which is negatively charged at physiological pH, allow-ng the formation of stable RB-gelatin@AuNPs SERS complex. Theioconjugates were characterized using a variety of specific mea-urements, including LSPR spectroscopy, transmission electronicroscopy (TEM), dynamic light scattering (DLS), zeta-potential

�-potential) measurements and Fourier transform infrared (FT-R) spectroscopy. In view of future intracellular spectral studies,he colloidal stability of gelatin@AuNPs bioconjugates was checkednder simulated biological conditions at physiological temperaturef 37 ◦C.

. Materials and methods

.1. Materials

Hydrogen tetrachloroaurate-(III) trihydrate (HAuCl4·3H2O,9.99%), gelatin (Type A) from porcine skin and Roseengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,20H2Cl4I4Na2O5) were purchased from Sigma–Aldrich (Germany).odium chloride (NaCl) and sodium citrate (C6H5Na3O7) wereurchased from Merck (Germany). All chemicals were of analyticalrade, and all aqueous solutions were prepared with ultrapureater (resistivity ∼18 M�).

.2. Bioconjugation procedures

Spherical AuNPs were synthesized according to the Turkevichethod by chemical reduction of HAuCl4 with sodium citrate [21].

he as-prepared AuNPs in solution exhibit a plasmonic resonancet 520 nm which is reliable with an average diameter of 18 ± 2 nms provided by TEM measurements.

The pure gelatin stock solution (10 mg/mL) was freshly preparedy dissolving gelatin powder in ultrapure water, under continuoustirring for 10 min at 50 ◦C to ensure the complete solubilizationf the powder. From the stock solution, different concentrations ofelatin solutions (0.03, 0.05, 0.07, 0.1, 0.3, 1, 5 and 10 mg/mL) were

repared.

The formation of gelatin@AuNPs bioconjugates was realizedn one step process simply by mixing 100 �L of gelatin solution

at different concentration values – with 1 mL of colloidal gold

Biointerfaces 103 (2013) 475– 481

solution. Subsequently, we denoted as 0.03gelatin@AuNPs and10gelatin@AuNPs the bioconjugates prepared in the presence ofgelatin concentration of 0.03 and 10 mg/mL respectively. The sam-ples were centrifuged for 10 min at 12,000 rpm to remove the freegelatin and then were redispersed in ultrapure water.

The stability of the nanoparticles was investigated under (i)addition of 220 �L of 1 M NaCl aqueous solution at room tem-perature and (ii) at temperature of 37 ◦C, in order to mimic thephysiological conditions, in view of their future inset in biologi-cal applications or intracellular studies. LSPR spectroscopy, particlesize analysis by DLS and �-potential measurements were employedas versatile methods for investigation of bioconjugates.

For SERS measurements, two set of bioconjugates were selected:so called “individual” 10gelatin@AuNPs, exhibiting the plasmonicband at 525 nm for excitation with 532 nm laser line and “inter-connected” 0.03gelatin@AuNPs with plasmonic band at 622 nm forexcitation with 632 nm laser line. The two sets of bioconjugateswere incubated for 2 h at room temperature with RB at concentra-tion of 3.84 × 10−6 M in final solution.

2.3. Characterization methods

For LSPR measurements the optical absorbance spectra of theprepared solutions were recorded at room temperature using aJasco V-670 UV–Vis–NIR spectrophotometer with a band width of2 nm and 1 nm spectral resolution. The stability of the bioconju-gates at physiological temperature of 37 ◦C was monitored usingthe same spectrophotometer with a Peltier single cell holder unit.

TEM images were obtained with a JEOL JEM 1010 transmissionelectron microscope. For TEM measurements the samples wereprepared by placing a drop of gelatin@AuNPs colloidal dispersiononto carbon-coated copper grids and dried at room temperature.Uranyl acetate dihydrate was used as the negative contrast agent.

The particle size distribution and �-potential of the colloidalsolutions, before and after bioconjugation, were measured usinga particle analyzer (Nano ZS90 Zetasizer, Malvern Instruments)equipped with a He–Ne laser (633 nm, 5 mW). The numbers ofmeasurements were optimized automatically by the software. Eachcolloidal sample was measured three times and the mean value hasbeen reported.

Infrared absorption measurements were recorded with a BrukerEquinox 55 Fourier transform infrared (FT-IR) spectrometer. Eachspectrum was measured in attenuated total reflectance (ATR) modewith 60 scans and 2 cm−1 resolution. For FT-IR measurements inATR mode the films of gelatin and gelatin@AuNPs were preparedby dropping colloidal dispersion onto glass slides and dried at roomtemperature.

The SERS measurements were recorded in aqueous solutionusing a Confocal Raman microscope (CRM 200 from WiTec)equipped with a 20× (N.A. 0.4) microscope objective and a spec-trometer (UHTS 300) equipped with CCD detector operating at−60 ◦C. The spectra were recorded using for excitation eithera 632.8 nm He–Ne laser with a power of 1.5 mW or a 532 nmfrequency-doubled YAG laser with a power of 3.7 mW. The typicalexposure time for each SERS measurement was 20 s.

3. Results and discussion

3.1. Characterization of gelatin@AuNPs bioconjugates

The position and shape of LSPR band can be used to transduce

changes of polarizability in the vicinity of individual nanoparticleas result of their surface modification and electromagnetic coupling[22]. Therefore, we analyze such spectral modifications to infer thebinding of gelatin layer on the surface of citrate-synthesized AuNPs

S. Suarasan et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 475– 481 477

Fig. 1. (A) Reference spectrum (curve a) and time evolution of 0.03gelatin@AuNPs spectra recorded at (curve b) 10 min, (curve c) 2 h, (curve d) 4 h, (curve e) 24 h, (curve f)2 me ev(

aFpasoe

Fpd

6 h, (curve g) 29 h and (dashed line) 48 h; (B) reference spectrum (curve a) and ticurve d) 15 min and (dashed line) 1 h.

nd conclude about the stability of gelatin@AuNPs bioconjugates.irstly, we compare the spectra of two representative samples pre-ared at low (Fig. 1A) and high (Fig. 1B) gelatin concentrations

nd therefore referred as 0.03gelatin@AuNPs and 10gelatin@AuNPsamples (see Section 2). We use for reference the LSPR bandf citrate-coated AuNPs (Fig. 1A curve a) located at 520 nm andxhibiting a bandwidth of 64 nm, parameters which are consistent

ig. 2. (A) DLS analysis of AuNPs (curve a), individual 10gelatin@AuNPs (curve b) and

otential data for AuNPs (curve a), gelatin induced interconnected 0.03gelatin@AuNPs (ciameter and �-potential dependence of gelatin concentration.

olution of 10gelatin@AuNPs spectra recorded at (curve b) 5 min, (curve c) 10 min,

with standard plasmon resonance of spherical nanoparticles of18 ± 2 nm diameter as provided by TEM measurements and hydro-dynamic diameter of 25 nm with a standard deviation of ±3.1, cal-

culated for n = 3 measurements (polydispersity index 0.256), givenby DLS data (Fig. 2A, curve a). A higher hydrodynamic diameterthan the geometrical diameter measured by TEM is not surprisingas DLS gives information about the effective hydrodynamic volume

gelatin induced interconnected 0.03gelatin@AuNPs (curve c), respectively; (B) �-urve b) and individual 10gelatin@AuNPs (curve c). Insets show the gelatin@AuNPs

478 S. Suarasan et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 475– 481

corded

oa−

dJttrlooa(nAan

pwmdFcctiaDn

ast1Titntgt(stm

Fig. 3. (A) LSPR spectra of 10gelatin@AuNPs and (B) 0.03gelatin@AuNPs re

f nanoparticles in solution. The presence of citrate anions layert the surface of reference AuNPs is confirmed by �-potential of31.5 ± 1.8 mV (�-deviation = 4.02) (Fig. 2B, curve a).

Fig. 1A shows a selection of LSPR spectra recorded successivelyuring the stabilization process of 0.03gelatin@AuNPs sample.

ust after mixing AuNPs with gelatin solution, we notice thathe intensity of standard plasmonic band of individual nanopar-icles (∼520 nm) starts to decrease steadily and shift slightly toed while a new broad plasmonic band emerges at longer wave-ength (∼622 nm). The new emerging band reveals a phenomenonf aggregation by which small aggregates are formed in solutionn the expense of free AuNPs. The aggregation process is limitednd ceases after 2 days. A stable spectrum can be finally recordedFig. 1A, dashed curve) reflecting a composite plasmonic reso-ance originated from a mixture of small aggregates and individualuNPs covered by gelatin. This is a typical example of controlledggregation process conducting to formation of small ensembles ofanoparticles, as already noticed by Han et al. [23].

In fact, upon the addition of low amount of gelatin, we sus-ect that the surface of nanoparticles covered with citrate ionsill be only partially neutralized by the presence of the biopoly-er (see spectroscopic details of molecular binding later). The

ecrease of total negative surface charge (�-potential data inig. 2B) reduces the interparticles repelling force and promotesloser approach between nanoparticles, causing a controlled pro-ess of aggregation. Indeed, when gelatin is not enough to inducehe homogeneous neutralization of entire nanoparticle, anisotropicnteractions between nanoparticles could take place conducting toggregates of particular geometry. The TEM pictures in Fig. 1C and

show representative examples of arrangements (short chains andecklace rings).

Similarly, we analyze the modifications in LSPR spectra recordedt different time intervals during the stabilization process of secondample, referred as 10gelatin@AuNPs (Fig. 1B). The first observa-ion is that the stabilization process is fast, lasting no more than5 min, opposite to about 2 days in the case of 0.03gelatin@AuNPs.he only modification is a red shift of 5 nm (see spectra and insetn Fig. 1B) from 520 nm to 525 nm with an almost no modifica-ion in the band shape, clearly indicating the behavior of individualanoparticles in solution. The red shift is due to the increase ofhe refractive index at the surface of nanoparticles as result ofelatin layer deposition. Corroborating evidences from TEM pic-ure (Fig. 1E), DLS (Fig. 2A, curve b) and �-potential measurements

Fig. 2B, curve c) confirm the presence of gelatin layer onto AuNPsurface. In particular, DLS measurements indicate an increase ofhe hydrodynamic diameter from 25 ± 3.1 nm to 65 ± 5 nm with a

odification of �-potential from −31.5 ± 1.8 mV to +6.44 ± 2.4 mV.

before (solid lines) and after (dashed lines) addition of 1 M NaCl solution.

The positive potential of bioconjugates is explained by the presenceof NH3

+ groups in gelatin layer (see details later).As mentioned before, citrate-reduced AuNPs are subject of

aggregation when transferred in cells culture media or biologi-cal fluids, which could have a negative impact on their biologicalapplications. However, the biopolymer conjugation is expectedto avoid such aggregation process of nanoparticles by the mech-anisms of electrostatic or steric repulsion. Here we investigatethe stability of the as-prepared colloidal bioconjugates against astrong aggregation agent for various ionic strengths induced byadding aliquots from a solution of 1 M NaCl. While the additionof salt solution has no significant effect on the plasmonic spec-trum of 10gelatin@AuNPs sample (Fig. 3A), the addition of thesame amount of salt solution in 0.03gelatin@AuNPs sample leadsto broadening and red-shifting of the spectra assignable to smallaggregates (Fig. 3B). Moreover, the LSPR band of 10gelatin@AuNPsremains unchanged in solution even at physiological temperatureof 37 ◦C, demonstrating the possibility of use 10gelatin@AuNPs bio-conjugates as individual nanoparticles in biological media. In thecase of 0.03gelatin@AuNPs sample, the balance between individualand associated nanoparticles changes after adding salt solution bydecreasing the number of individual nanoparticles, as expected. It isworth mentioning that heating/cooling cycles from 20 ◦C to 60 ◦Cdid not disrupt the as-formed chain networks (spectral data notshown here) which is positive for applications as SERS substrates(see details later).

We have investigated the evolution of LSPR spectra for sev-eral examples of samples prepared with gelatin concentrationsranging from 0.03 to 10 mg/mL, as described in Section 2. All sam-ples exhibit stable LSPR spectra (Fig. 4) after shorter or longerincubation time, consistent with mixtures of isolated and smallaggregates those ratios can be controlled by gelatin concentration.The corresponding DLS measurements (inset in Fig. 2A) providesadditional information, confirming the presence of aggregatesof hydrodynamic diameter as large as 200–250 nm at very lowgelatin concentration (0.03 mg/mL), decreasing to 106 ± 26.4 nmand finally to 65 ± 5 nm (n = 3) as gelatin concentration increases(10 mg/mL). Accordingly, the surface charge of bioconjugates varieswith gelatin concentrations in the range 0.03–0.3 mg/mL untilit reaches a steady-state positive value of +6.44 ± 2.4 mV for10 mg/mL (inset in Fig. 2B).

3.2. FT-IR analysis of gelatin–AuNPs interaction

The polymeric chain of gelatin (Fig. 5) exhibits both amine,carboxyl and amide functional groups, which – depending onthe pH – makes the polymer positively or negatively charged

S. Suarasan et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 475– 481 479

Fig. 4. LSPR spectra of reference sample (curve a) and gelatin@AuNPs bioconju-g0f

[cct

gCtsNi(m

i1aA1cstwhc�

(gb

s

bioconjugates (Fig. 7, spectrum a) and, for reference, we collected anormal Raman spectrum from a solution of much higher concentra-tion (10−2 M) under 633 nm laser excitation line (Fig. 7, spectrum b).Table 1 summarizes the spectral positions and the assignments of

Table 1Raman and SERS bands of free RB and RB-gelatin@AuNPs complex presented in Fig. 7.

RB (Raman) RB-gelatin@AuNPs (SERS) Assignments

614 vs 605 vs �(C I) + ı(CCO)765 vw 783 vw �(C Cl)1013 m 1003 m �(C OH)

1166 vw 1163 s �(C O) + �(C C) + ı(C H)1271 s 1268 m ı(CCC) ring + ı(C H)

ates formed in presence of different gelatin concentrations: 0.03 mg/mL (curve b),.05 mg/mL (curve c), 0.07 mg/mL (curve d), 0.1 mg/mL (curve e), 0.3 mg/mL (curve), 1 mg/ml (curve g), 5 mg/mL (curve h) and 10 mg/mL (curve i), respectively.

14]. As gelatin has an isoelectric point (pI) around 7–9 [24] wean conclude that in our case, at physiological pH, the positivelyharged gelatin peptide chains adsorb electrostatically on nega-ively charged citrate-capped AuNPs.

The details of molecular structure and specific interaction ofelatin with AuNPs is further characterized by FT-IR spectroscopy.urve a from Fig. 6 – a typical FT-IR spectrum of pure gelatin – showshe characteristic bands due to the vibrations of the amide groupuch as C O stretching vibrations from 1632 cm−1 (amide I band),

H bending vibrations at 1528 cm−1 (amide II band), C N stretch-ng vibrations coupled with in-plane N H bending at 1236 cm−1

amide III band). The bands from 1400 to 1500 cm−1 region areainly due to the COO− symmetrical stretching vibrations [25].It is well known that the amide I band is sensitive to changes

n the gelatin chain conformation [26]. The wave number range660–1650 cm−1 was known as �-helical and 1640–1620 cm−1

s �-antiparallel sheets [27]. In our case, after conjugation withuNPs (Fig. 6, curve b), the amide I band shift from 1632 cm−1 to650 cm−1, suggesting that the secondary structure of gelatin washanged from �-antiparallel sheets to �-helix. The �-antiparallelheets secondary structure of gelatin is confirmed by the posi-ion of amide III band at 1236 cm−1. According to Fu et al. [28],hich successfully used amide III region for determination of �-elix and �-sheets secondary structure, the 1330–1295 cm−1 rangeorresponds to �-helix structure and 1250–1220 cm−1 indicates a-antiparallel sheets structure.

Moreover, the amide III band observed in the case of pure gelatinmainly due to C N stretching vibration), disappears in the case of

elatin bind to the surface of AuNPs. This behavior indicates thelocking of the C N stretching vibration due to the coordination ofNH2 functional group from amide group to the gold nanoparticles

urface. In addition, the intensities of characteristic amide I, amide II

Fig. 5. Gelatin primary structures: (a) car

Fig. 6. FT-IR spectra of gelatin (curve a) and 10gelatin@AuNPs bioconjugates (curveb).

and carboxylic bands of gelatin decrease after the conjugation withAuNPs. These results suggest that the amide and carboxylic groupsillustrated in Fig. 5, can be especially active sites for the coordina-tion with surface of AuNPs, clearly indicating that the nanoparticleswere indeed conjugated with the biopolymer.

3.3. Gelatin@AuNPs bioconjugates as SERS active substrates

In this study we demonstrate the versatility of gelatin@AuNPsbioconjugates to operate as SERS substrates able to detect analytesin solution or, alternatively, to provide biocompatible platformsfor spectroscopic labeling with SERS and/or fluorescence tags. Tothis end, two examples of gelatin@AuNPs bioconjugates (individ-ual and aggregated) were tested as SERS substrates in solutionof 3.6 × 10−6 M RB concentration under laser excitation lines at532 nm and 633 nm. No Raman signal could be recorded from solu-tion of identical RB concentration (3.6 × 10−6 M) prepared without

1297 s 1290 s ı(CCC) ring + ı(C H)1339 m 1327 vs �s(C O)1488 vs 1481 vs �as(C C) ring

1547 vw 1541 vw �(C C) ring

boxylic group and (b) amide group.

480 S. Suarasan et al. / Colloids and Surfaces B:

Fig. 7. Reference Raman spectrum of free RB of 3.6 × 10−6 M (spectrum a) and10−2 M (spectrum b) in solution recorded at 633 nm; SERS spectra of RB-individualgr

m[

blsb(ras(mtsoiorlvestwTaottpRcioimS

palSr

[1] S.K. Dondapati, T.K. Sau, C. Hrelescu, T.A. Klar, F.D. Stefani, J. Feldmann, ACS

elatin@AuNPs (spectrum c); RB-interconnected gelatin@AuNPs (spectrum d)ecorded at 633 nm and RB-individual gelatin@AuNPs (spectrum e) recorded at 532.

ain normal Raman bands according to reported data in literature29–32].

The SERS measurements reveal the presence of RB molecules inioconjugates by assuming local field enhancement at the molecu-

ar location as result of using for excitation laser lines at optimizedpectral positions relative to LSPR and molecular absorptionands, respectively. For RB-incubated individual gelatin@AuNPsRB-10gelatin@AuNPs), the 532 nm laser line is simultaneouslyesonant with LSPR band of AuNPs at 525 nm and the electronicbsorption band of RB at 548 nm, enabling to record in this case aurface-enhanced resonance Raman scattering (SERRS) spectrumFig. 7, spectrum e) [31]. In principle, the SERRS is the result of

ultiplying two resonance-enhancement effects, the first one dueo the excitation of plasmonic substrate, providing SERS, while theecond one is due to the laser excitation of electronic transitionf the molecule, providing a resonant Raman scattering (RRS). Its difficult in our case to discriminate the relative contributionsf two mechanisms. Nevertheless the RRS contribution is clearlyevealed (Fig. 7, spectrum e) by the occurrence of two strong bandsocated at 1619 cm−1 and 1496 cm−1 assignable to molecular ringibrations (Table 1) which represent the hallmarks of chromophorexcitation. Under typical SERRS conditions the fluorescence emis-ion of molecules should be quenched by nonradiative losses inhe metal. However, this behavior is not observable in our casehere the SERRS bands occur on top of fluorescence background.

o explain this result, we admit that part of RB molecules arettached to polymer not direct to metal surface to prevent flu-rescence quenching, but not too far from metal to compromisehe SERS activity. It is noteworthy that RB molecules are nega-ively charged which means that molecules interact directly withositive sites in polymer chain conducting to formation of stableB-gelatin@AuNPs SERS complex. In the case of aggregated bio-onjugates (RB-0.03gelatin@AuNPs) the main LSPR band at 622 nms located far from the excitation laser line at 532 nm. As resultnly small fraction of individual bioconjugates existing in solutionn presence of aggregated bioconjugates are SERS-active, which

akes that the recorded fluorescence completely overlaps theERRS signal of RB reporter molecule (spectra not shown here).

Next we analyze the SERS spectra recorded from the two sam-les (0.03gelatin@AuNPs and 10gelatin@AuNPs) under excitationt 633 nm laser line. As the electronic absorption band of RB is

ocated at 548 nm no resonant Raman effects can contribute toERS. In the case of 0.03gelatin@AuNPs sample the laser line isesonant with LSPR band of small aggregates at 633 nm which

Biointerfaces 103 (2013) 475– 481

allows recording a very good SERS signal (Fig. 7, spectrum d). Weassume that a significant contribution of SERS originates from thenumber of hot-spots occurring between small gaps induced byaggregation [33]. On the contrary, in the case of 10gelatin@AuNPssample, the laser line is out of resonance of bioconjugates whichmakes the SERS signal extremely weak (Fig. 7, spectrum c).

According to the surface selection rules for Raman scattering,it will be preferentially enhanced those vibrations of the adsorbedmolecules for which polarizability variation is perpendicular to themetal surface [34]. However the relative intensities of vibrationalbands measured in SERRS (Fig. 7, spectrum e) and SERS (Fig. 7, spec-trum d) could be different because the surface selection rules whichcontrol the enhancement of vibrations in SERS are not identicallyoperable in the case of electronic transitions. As regard to SERSspectra, we note the presence of 605 and 1327 cm−1 bands assignedto C I stretching and C O symmetric stretching, respectively the1481 and 1606 cm−1 bands attributed to C C symmetric and asym-metric stretching from aromatic rings. The presence of these twolast bands in SERS spectra evidenced that the RB’s quinoidal struc-ture (Fig. 7, spectrum e) is preserved. On the other hand, theC O symmetric vibration at 1339 cm−1 undergoes a clear increasein intensity and shift by about 10 cm−1, indicating that the RBmolecule binds gelatin@AuNPs through the C O bond [29]. Takingthis into account, we assume that the RB molecules are perpendic-ularly oriented at the gold surface being bound through C O− and

COO− groups.In conclusion we demonstrate that gelatin biopolymer is not

only an excellent coating for pre-synthesized AuNPs but enablesplasmonic nanoparticles to be further exploited as imaging SERS orfluorescent labels.

4. Conclusions

In this paper, we have successfully demonstrated the bio-conjugation of citrate-stabilized AuNPs with gelatin biopolymerin aqueous solution. Below a critical concentration of gelatin(0.3 mg/mL), a process of controlled aggregation takes place con-ducting to formation of small chains or rings of bioconjugatesassemblies. Single-particle bioconjugates formed at higher con-centration of gelatin (10 mg/mL) present good stability againstsimulated biological conditions (e.g. solutions of various ionicstrengths, physiological temperature of 37 ◦C). We have finallydemonstrated the functionality of gelatin@AuNPs bioconjugatesas highly effective plasmonic platform for SERS detection of RBfluorophore. Thanks to their ability to adsorb and incorporatetarget analytes from solution, gelatin@AuNPs bioconjugates openthe possibility to be further exploited in multiplex detection ofbiomolecules via SERS measurements or, alternatively, to operateas SERS-active tags in biomedical imaging.

Acknowledgments

This work was supported by CNCSIS–UEFISCSU, project num-ber PNII ID PCCE 312/2008. M. Focsan gratefully acknowledgesthe financial support from the Sectoral Operational Programmefor Human Resources Development 2007–2013, co-financed bythe European Social Fund, under the project number POSDRU89/1.5/S/60189 with the title “Postdoctoral Programs for Sustain-able Development in a Knowledge Based Society”.

References

Nano 4 (2010) 6318.[2] L. Rastogi, A.J. Kora, J. Arunachalam, Mater. Sci. Eng. C: Mater. Biol. Appl. 32

(2012) 1571.[3] M. Larguinho, P.V. Baptista, J. Proteomics 75 (2012) 2811.

ces B:

[

[

[[

[[[[

[

[

[[[[[[[

[

[

[[

S. Suarasan et al. / Colloids and Surfa

[4] A.A. Sousa, J.T. Morgan, P.H. Brown, A. Adams, M.P.S. Jayasekara, G. Zhang, C.J.Ackerson, M.J. Kruhlak, R.D. Leapman, Small 8 (2012) 2277.

[5] J. Kneipp, B. Wittig, H. Bohr, K. Kneipp, Theor. Chem. Acc. 125 (2010) 319.[6] J. Kneipp, H. Kneipp, K. Kneipp, Chem. Soc. Rev. 37 (2008) 1052.[7] L. Jiang, J. Qian, F. Cai, S. He, Anal. Bioanal. Chem. 400 (2011) 2793.[8] S. Boca, D. Rugina, A. Pintea, L. Barbu-Tudoran, S. Astilean, Nanotechnology 22

(2011) 055702.[9] S.H. De Paoli Lacerda, J.J. Park, C. Meuse, D. Pristinski, M.L. Becker, A. Karim, J.F.

Douglas, ACS Nano 4 (2010) 365.10] M. Iosin, V. Canpean, S. Astilean, J. Photochem. Photobiol. A: Chem. 217 (2011)

395.11] T. Simon, S. Boca, S. Astilean, Colloid. Surf. B: Biointerface 97 (2012) 77,

http://dx.doi.org/10.1016/j.colsurfb.2012.03.037.12] V.H. Segtnan, T. Isaksson, Food Hydrocolloid. 18 (2004) 1.13] Y. Tabata, in: P.X. Ma, J. Elisseeff (Eds.), Scaffolding in Tissue Engineering, Taylor,

Francis Group, USA, 2006 (chapter 4).14] S. Young, M. Wong, Y. Tabata, A.G. Mikos, J. Control. Release 109 (2005) 256.15] Y. Liu, X. Liu, X. Wang, Nanoscale Res. Lett. 6 (2011) 22.

16] J.-J. Zhang, M.-M. Gu, T.-T. Zheng, J.-J. Zhu, Anal. Chem. 81 (2009) 6641.17] M.P. Neupane, S.J. Lee, I.S. Park, M.H. Lee, T.S. Bae, Y. Kuboki, M. Uo, F. Watari,

J. Nanopart. Res. 13 (2011) 491.18] H. Tlatlik, P. Simon, A. Kawska, D. Zahn, R. Kniep, Angew. Chem.: Int. Ed. Engl.

45 (2006) 1905.

[[

[[

Biointerfaces 103 (2013) 475– 481 481

19] M.R.C. Marques, E. Cole, D. Kruep, V. Gray, D. Murachanian, W.E. Brown, G.I.Giancaspro, Pharmacopeial Forum 35 (2009) 1029.

20] A. Uppal, B. Jain, P.K. Gupta, K. Das, Photochem. Photobiol. 87 (2011) 1146.21] J. Turkevich, P.C. Stevenson, H. Hillier, Discuss. Faraday Soc. 11 (1951) 55.22] T. Sannomiya, J. Vörös, Trends Biotechnol. 29 (2011) 343.23] X. Han, J. Goebl, Z. Lu, Y. Yin, Langmuir 27 (2011) 5282.24] M. Nahar, D. Mishra, V. Dubey, N.K. Jain, Nanomedicine 4 (2008) 252.25] S. Tang, Y. Li, J. Colloid Interface Sci. 360 (2011) 71.26] H. Torii, M. Tasumi, in: H.H. Mantsch, D. Chapman (Eds.), Theoretical Analyses

of the Amide I Infrared Bands of Globular Proteins. Infrared Spectroscopy ofBiomolecules, Wiley-Liss Inc., New York, 1996 (chapter 1).

27] D.M. Hashim, Y.B. Che Man, R. Norakasha, M. Shuhaimi, Y. Salmah, Z.A. Sya-hariza, Food Chem. 118 (2010) 856.

28] F.-N. Fu, D.B. DeOliveira, W.R. Trumble, H.K. Sarkar, B.R. Singh, Appl. Spectrosc.48 (1994) 1432.

29] H.R. Zhu, L. Parker, Chem. Phys. Lett. 162 (1989) 424.30] V.A. Narayanan, D.L. Stokes, T. Vo-Dinh, J. Raman Spectrosc. 25 (1994)

415.

31] A.M. Gabudean, M. Focsan, S. Astilean, J. Phys. Chem. C 116 (2012) 12240.32] W. Maciejewska, K. Polewski, M. Grundwald-Wyspiadska, Carbohydr. Res. 246

(1993) 253.33] M. Potara, M. Baia, C. Farcau, S. Astilean, Nanotechnology 23 (2012) 055501.34] D. Sajan, V.B. Jothy, T. Kuruvilla, I.H. Joe, J. Chem. Sci. 122 (2010) 511.