An

MMJI

a

ARRAA

KTGSF

1

mdctrtastes

ycaiuhn

0h

Synthetic Metals 185– 186 (2013) 61– 65

Contents lists available at ScienceDirect

Synthetic Metals

journa l h om epage: www.elsev ier .com/ locate /synmet

simple method to synthesize fluorescent modified goldanoparticles using tryptamine as the reducing and capping agent

arcos Alberto de Carvalho, Patrícia Fernanda Andrade, Fabiana Cristina Andrade Corbi,aria do Carmo Gonc alves, André Luiz Barboza Formiga, Italo Odone Mazali,

uliano Alves Bonacin ∗, Pedro Paulo Corbinstitute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil

r t i c l e i n f o

rticle history:eceived 27 May 2013eceived in revised form 12 August 2013ccepted 10 September 2013vailable online 30 October 2013

a b s t r a c t

A simple method to synthesize fluorescent modified gold nanoparticles using tryptamine as the reducingand capping agent is described. The presented method produces gold nanoparticles with 36.65 ± 5.30 nmaverage size. The modified gold nanoparticles were characterized by elemental and thermal analyses,dynamic light scattering, transmission electron microscopy, zeta potential, X-ray powder diffractionand spectroscopic techniques, such as electronic spectroscopy in ultraviolet–visible and fluorescence

15

eywords:ryptamineold nanoparticlesolid state 15N NMRluorescence

excitation–emission. In addition, modified gold nanoparticles were analyzed by solid state N nuclearmagnetic resonance spectroscopy, which confirmed the coordination of tryptamine on the gold nanopar-ticles surface. A prominent characteristic observed is the fluorescence of tryptamine which was notquenched after the coordination to gold nanoparticles. The results presented in this paper confirm themodification of gold nanoparticles by tryptamine and suggest potential use of such nanoparticles aslabeling dye in biological systems.

. Introduction

Gold nanoparticles (AuNP) have been used for a long time,ainly due to their optical properties for staining glass [1]. The

iversity of properties of AuNP depends on the size, shape and theapping agents. Such parameters can be controlled during the syn-hesis and the preparation of AuNP commonly involves chemicaleduction of gold salts in aqueous, organic, or mixed solvent sys-ems. Furthermore, capping agents such as carboxylates, aminesnd thiol groups are necessary to promote stabilization over theurface of nanoparticles [2–5]. AuNP surface is extremely reac-ive and, under certain conditions, the nanoparticles may aggregateither by replacing the capping agent or by variations in the ionictrength [6].

The focus on studies of gold nanoparticles has changed in the lastears and the attentions are directed to the application in biologi-al systems [7,8]. They can be functionalized by bioactive moleculesnd be employed in drug delivery systems for cancer treatment orn photodynamic therapy [9,10]. Sperling et al. have proposed the

ses of AuNP in four conceptual applications: labeling, delivering,eating and sensing [11]. Labeling of cells is essential for early diag-osis of certain types of diseases. Once the problem is identified, the

∗ Corresponding author. Tel.: +55 19 3521 3103; fax: +55 19 3521 3023.E-mail address: jbonacin@iqm.unicamp.br (J.A. Bonacin).

379-6779/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.synthmet.2013.09.038

© 2013 Elsevier B.V. All rights reserved.

medical treatment can be started. This makes it possible to providea higher success rate in healing, minimize the consequences andgenerate a lower treatment cost.

Gold nanoparticles are very interesting contrast agents consid-ering they can be visualized by a large number of techniques,in which those based on the interaction between nanoparticlesand scatter visible light could be highlighted. The use of opticaltechniques in biological imaging provides great potential for under-standing chemical process in cells, and is essential for an earlydiagnosis in some diseases. Nevertheless, the success of obtain-ing optical images depends on how sensitive and stable the opticallabels are [12].

Organic fluorescent dyes are widely used for optical labeling, butthey present a limitation caused by self-photodegradation. In thisaspect, gold nanoparticles are immensely more interesting sincethey present controlled chemical inertia, photostability and arebiocompatible [12,13].

Some classes of biological molecules may be used as reducingagent in the synthesis of metallic nanoparticles by the presence ofamine functional group, and the main advantage is produce non-toxic by-products [14]. Among the known biological molecules,triptamine draws attention due to its structure, reactivity and func-

tionality.

Tryptamine (TRA) or [3-(2-aminoethyl)indole] is an importantbiomolecule widely studied in medicinal chemistry, which acts onseveral molecular receptors in the central nervous system, and

62 M.A. de Carvalho et al. / Synthetic Metals 185– 186 (2013) 61– 65

pataicm

ttiqbb

maidmMar

2

2

9(bPisfmicw5oioCpyfDppauU

0

Fig. 1. Biosynthesis of tryptamine from tryptophan [17].

articipates on the regulation of several biological processes. Inddition, TRA contains a flexible ethyl amino side chain bondedo an indolic ring, being produced from tryptophan decarboxyl-tion by the action of decarboxylase enzyme (Fig. 1). TRA structure,n particular its indolic ring, may be part of the structure of moreomplex compounds, e.g. serotonin, lysergic acid diethylamide andelatonin [15–17].Another interesting molecule used to produce metallic nanopar-

icles is the tryptophan. Iosin et al. have presented a smart methodo synthesize gold nanoparticles by using tryptophan as the reduc-ng and stabilizing agent [18]. Although simple and efficient, theuenching of the tryptophan fluorescence was observed. Thisehavior does not permit the use of modified gold nanoparticlesy tryptophan as a fluorescent probe.

This article presents a simple method to synthesize fluorescentodified gold nanoparticles by tryptamine (Au-TRA), wherein TRA

cts as the reducing and capping agent. The Au-TRA were character-zed by electronic spectroscopy, transmission electron microscopy,ynamic light scattering analysis, powder X-ray diffraction, ele-ental and thermal analyses and fluorescence spectroscopy.oreover, the characterization of the modified nanoparticles was

lso performed by using the solid-state 15N{1H} nuclear magneticesonance.

. Experimental

.1. Materials and methods

Tryptamine (98%) and potassium tetrachloroaurate(III) (KAuCl4,8%) were purchased from Sigma–Aldrich Laboratories. Ethanol99.5%) was obtained from J.T. Baker. Elemental analyses for car-on, hydrogen and nitrogen were performed using a CHNS/Oerkin Elmer 2400 Analyzer. The UV–vis spectrum was obtainedn a Cary 50 Proble Varian/Agilent spectrophotometer, using theupernatant of the Au-TRA nanoparticles centrifuged at 4000 rpmor 10 min, in aqueous solution, at 25 ◦C. Fluorescence measure-

ents of the Au-TRA nanoparticles in aqueous solution and TRAn ethanol were carried out using a Cary Eclipse (Varian) fluores-ence spectrophotometer. The spectrofluorimeter was equippedith 1.0 cm quartz cells, and the widths of the two slits were

nm in excitation and 10 nm in emission, respectively. Morphol-gy and particle size of the Au-TRA nanoparticles were investigatedn a Carl Zeiss CEM902 transmission electron microscope (TEM),perated at an acceleration voltage of 80 kV, and equipped with aastaing-Henry energy filter spectrometer within the column and aroscan slow scan CCD camera. Scanning electron microscopy anal-ses were performed in an FEI Inspect F50 – High Resolution SEMrom the Brazilian Nanotechnology National Laboratory (LNNano).rops of an Au-TRA suspension were deposited on carbon coatedarlodion film supported in a copper grid and dried at room tem-

erature. Measurements of apparent hydrodynamic particle sizend zeta potential of the Au-TRA nanoparticles were performedsing a Zetasizer Nano ZS (Malvern Instruments, Worcestershire,K) using the supernatant of the Au-TRA nanoparticles centrifuged

Fig. 2. X-ray powder diffractogram of crystalline Au-TRA.

at 4000 rpm for 10 min, in aqueous solution, also at 25 ◦C (see Sup-plementary Material). The 15N{1H} nuclear magnetic resonancespectra were recorded in solid state for TRA and Au-TRA on aBruker Avance 300 MHz spectrometer, using the combination ofcross-polarization, proton decoupling and magic angle spinning(CP/MAS) of 10 kHz.

Thermogravimetric and differential thermal analyses (TG/DTA)of a solid sample of the Au-TRA nanoparticles were performed on aSimultaneous TGA/DTA SEIKO EXSTAR 6000 thermoanalyzer, usingthe following conditions: synthetic air, flow rate of 50 cm3 min−1

and heating rate of 5 ◦C min−1, from 25 ◦C to 1000 ◦C (see Supple-mentary Material). The Au-TRA nanoparticles were analyzed on aShimadzu XRD-6000 diffractometer (CuK� radiation, � = 1.5406 A)with a graphite monochromator and room temperature. The sam-ple was scanned over the 2� range from 5◦ to 50◦.

2.2. Synthesis of the Au-TRA nanoparticles

The Au-TRA nanoparticles were synthesized by adding anethanolic solution of TRA (5.0 × 10−4 mol) to an ethanolic solutionof KAuCl4 (5.0 × 10−4 mol) under constant stirring at room temper-ature for 40 min. After this period, 25 cm3 of distilled water wereadded and the system was left to stand for 80 min. A purple solidwas obtained and collected by filtration, washed with cold waterand dried under vacuum. The mass of solid obtained was of 0.050 g.Elemental analysis led to the following results (%): C 22.7; H 2.22;N 4.87%. The C/H and C/N proportions found for Au-TRA are closeto those observed for the free tryptamine, which is an indicativeof the maintenance of the integrity of the TRA molecule. TG/DTAanalyses are presented as Supplementary Material.

2.3. Results and discussions

Several methods to prepare metallic nanoparticles using dif-ferent reducing agents are described in the literature. Goldnanoparticles (AuNP), for example, have been prepared by citratereduction of HAuCl4 at high temperatures or by reduction of goldsalts using borohydride [19–21]. We have used an ethanol/watermixture to avoid the production of undesirable residues whichcould later prejudice the use of the AuNP for biological application.The reduction of KAuCl4 had occurred through the transferenceof electrons from the amine group of TRA to Au3+, leading to the

formation of Au [22].

The X-ray power diffraction (XRD) technique was applied toinvestigate the crystallinity of the Au-TRA nanoparticles. The XRDpattern exhibits two peaks, as can be seen in Fig. 2, one at 38.2◦

M.A. de Carvalho et al. / Synthetic Metals 185– 186 (2013) 61– 65 63

ap[aoaoM

iCcitfotrfcacst

Fig. 3. Solid-state 15N{1H} (CP/MAS) of TRA and Au-TRA.

nd another at 44.5◦, which are assigned to (1 1 1) and (2 0 0)lanes of gold in the face-centered-cubic (fcc) crystalline structure23]. It is worth noting that gold nanoparticles prepared using themino acid tryptophan as reducing/stabilizing agent were previ-usly described, and presented an intense diffraction peak at 38.2◦,ttributed to the (1 1 1) plane [18]. Scanning electron microscopyf the sample used for XRD analysis is presented in Supplementaryaterial.Tryptamine and the Au-TRA nanoparticles were also character-

zed by solid state 15N NMR spectroscopic measurements (SSNMR).onsidering the 15N SSNMR spectra shown in Fig. 3, it is possible toonfirm that one of the coordination sites of the ligand (TRA) to golds the nitrogen atom of the NH2 group. In the free TRA spectrum,he nitrogen atom of the NH2 group is observed at 26.8 ppm whileor the Au-TRA nanoparticles, this signal is shifted downfield, beingbserved at 44.9 ppm. The �ı (ıAu-TRA–ıTRA) of 18.1 ppm indicateshe coordination of the TRA to Au through the NH2. The observedesults are also compared to those published by Abbehausen et al.or a gold complex with 2-mercaptothiazoline (Au-MTZ), whereoordination of MTZ to gold through the N atom was confirmed by

�ı (ıAu-MTZ–ıMTZ) of 15.3 ppm in the 15N NMR spectra [24]. Con-erning the nitrogen atom of the indolic ring, it was also observed ahift of 5.8 ppm upon coordination. The observed data may indicatehat this group does not participate directly in the coordination of

Fig. 4. UV–vis spectrum of the Au-TRA nanoparticles in water.

Fig. 5. Transmission electron microscopy image of Au-TRA and the histogram ofparticle diameter distribution.

TRA to gold, but is somewhat affected upon coordination of TRAto gold. It is important to emphasize that we cannot exclude dif-ferences in the intermolecular forces in the solid state when freeTRA molecule and the Au-TRA nanoparticles are compared, whichcan also lead to small differences in the 15N NMR spectra. The15N NMR data also indicate the presence of polymorphism in theTRA molecules when coordinated to gold nanoparticles. This effectis probably due to the change in the conformation of the indolicring, leading to the appearance of two signals at 143.3 ppm and at141.3 ppm for the indolic nitrogen. Gaigalas et al. [25] have demon-strated that TRA can be bound to a polycrystalline gold surface usingthe DTSP protein cross-linking, known as dithiobis(succinimidylpropionate), which suggests that the indole ring of the immobilizedtryptamine does not interact directly with the gold surface.

The dynamic light scattering analysis (DLS) of the Au-TRAnanoparticles has shown an average particle size of 46 nm, and pos-itive surface charges with average values of +18.8 mV in the zetapotential (see Electronic Supplementary Material).

The UV–vis electronic spectrum of the Au-TRA nanoparticlesexhibits two bands centered at 535 nm and at 260 nm, as can beobserved in Fig. 4. The band at 535 nm corresponds to character-istic red color ascribed to the optical interaction with the surface

64 M.A. de Carvalho et al. / Synthetic Metals 185– 186 (2013) 61– 65

of (—

pbt

husAmabb

tae

aptsobHitSsAimbal

icaarttofsw

Fig. 6. Fluorescence spectra of excitation and emission

lasmons, as accounted for by the Mie theory [26,27], while theroad band with its maximum at 260 nm is attributed to a � → �*ransition of indolic ring of the TRA.

Transmission electron microscopy (TEM) studies of Au-TRAave shown a relatively narrow sized diameter distribution of non-niform shaped particles (some spherical, some hexagonal andome rectangular), as presented in Fig. 5. The mean values for theu-TRA nanoparticle diameter and the standard deviation, esti-ated from the histogram (Fig. 5), are 36.65 ± 5.30 nm. A grayer

rea, in comparison to the surrounding area without particles, cane observed around and between the particles and is suggested aseing the TRA layer.

Fluorescence spectra are presented in Fig. 6, and show thathe excitation and emission wavelengths of the TRA are 302 nmnd 352 nm, respectively, while the wavelengths of excitation andmissions of Au-TRA were 276 nm and 356 nm, respectively [28].

TRP is a zwitterionic molecule in neutral aqueous solution (NH3+

nd COO− groups in the same structure) and this suggests that theositive charge from protonated amine group can interact elec-rostatically with the negative charge from gold nanoparticles’surfaces as described in Ref. [18]. Immediately after adsorptionf TRP on AuNP’s surface, its fluorescence is drastically quenchedy transfer of fluorescence resonance energy from TRP to AuNP.owever, TRA molecule maintains the fluorescence after the mod-

fication of AuNP. The fluorescence is not quenched by TRA, becausehere is an effective coordination of TRA to gold by amine group.o, the spectroscopy features of TRA are transferred to the hybridystem, what reinforces the hypothesis of bonding between theuNP and TRA through the amine nitrogen, and not through the

ndole ring as corroborated by NMR spectroscopy data. Further-ore, changes in the fluorescence intensities could be explained

y the coupling of TRA in the nanoparticle’s surface and addition-lly by the zeta potential presented by Au-TRA, as suggested byiterature [29].

Based on spectra analysis it is possible to infer that two states arenvolved in the spectroscopy behavior of the system. TRA moleculean be excited to the second state (state 2) and then it can induce

radiationless decay to the emissive state (state 1), as observed in schematic Jablonsky diagram in Fig. 6. According to presentedesults, it can be observed a considerable influence of AuNP inhe excited state of TRA (state 2). It is observed a blue shift tohe excitation of TRA chemically bound on AuNP surface. On the

ther hand, there is a small difference in the emission spectra fromree TRA and AuNP-TRA. Probably, excitation process from groundtate to state 2 is accompanied by a change in the dipole moment,hat should not occur in state 1. This can be inferred from the

) TRA, ( ) Au-TRA and qualitative Jablonsky diagram.

absorption UV–vis spectrum for free TRA which shows only a transi-tion at 260 nm. The same transition is responsible for the excitationspectrum. Zeta potential about +30 mV was registered for Au-TRA,as presents in Supplementary Material. This means that Au-TRA’ssurface can influence the state 2 which shows the most promi-nent change in the dipole moment (charge separation) [29,30].This could explain the changes in the excitation spectrum after thecoordination on nanoparticle’s surface and the slight enhancementin the intensity of the spectra since state 2 is more susceptibleto the surface’s charge, what is not seen in the case of emissionspectrum.

3. Conclusion

A simple method to produce modified gold nanoparticles bytryptamine as the reducing and capping agent in water at roomtemperature was developed. The solid state 15N NMR spectroscopicmeasurements led us to propose the coordination of TRA to Authrough the nitrogen atom of the NH2 group, and transmissionelectron microscopy studies of Au-TRA have shown a relatively nar-row sized diameter distribution (36.65 ± 5.30 nm) of non-uniformshaped particles (some spherical, some hexagonal and some rect-angular).

Unlike the results reported in the literature wherein tryptophanfluorescence is quenched by gold nanoparticles, the tryptamineretains its fluorescence even after coordination on gold surfaces.Due to this interesting characteristic observed by Au-TRA, futurestudies are necessary in order to evaluate the application ofsuch nanoparticles as fluorescent labeling or carriers of specificbiomolecules.

Acknowledgments

This study was supported by grants from the Brazilian Agen-cies FAPESP (Fundac ão de Amparo à Pesquisa do Estado de SãoPaulo – Proc. 2012/08230-2), CAPES, CNPq (Conselho Nacionalde Desenvolvimento Científico e Tecnológico, Brazil) and LNNano(Laboratório Nacional de Nanotecnologia, Campinas-SP).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.synthmet.2013.09.038.

tic Me

R

[

[

[[[[[

[[[

[

[

[

[[

[[[

3356.

M.A. de Carvalho et al. / Synthe

eferences

[1] M. Quinten, Optical Properties of Nanoparticles System, Wiley-VCH Verlag &Co. KGaA, Weinheim, Germany, 2011, pp. 1–8.

[2] M.-C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293.[3] P. Zhao, N. Li, D. Astruc, Coord. Chem. Rev. 257 (2013) 638.[4] A.H. Faraji, P. Wip, Bioorg. Med. Chem. 17 (2009) 2950.[5] J.A. Dahl, B.L.S. Maddux, J.E. Hutchison, Chem. Rev. 107 (2007) 2228.[6] T. Kim, K. Lee, M.-S. Gong, S.-W. Joo, Langmuir 21 (2005) 9624.[7] P.F. Jiao, H.Y. Zhou, L.X. Chen, B. Yan, Curr. Med. Chem. 18 (2011) 2086.[8] X. Cao, Y. Ye, S. Liu, Anal. Biochem. 417 (2011) 1.[9] Y. Cheng, A.C. Samia, J.D. Meyers, I. Panagopoulos, B. Fei, C. Burda, J. Am. Chem.

Soc. 130 (2008) 10643.10] E.C. Dreaden, A.M. Alkilany, X. Huang, C.J. Murphy, M.A. El-Sayed, Chem. Soc.

Rev. 41 (2012) 2740.11] R.A. Sperling, P.R. Gil, F. Zhang, M. Zanella, W.J. Parak, Chem. Soc. Rev. 37 (2008)

1896.

12] H. He, C. Xie, J. Ren, Anal. Chem. 80 (2008) 5951.13] X. Huang, P.K. Jain, I.H. El-Sayed, M.A. El-Sayed, Nanomedicine 2 (2007) 681.14] D.V. Leff, L. Brandt, J.R. Heath, Langmuir 12 (1996) 4723.15] R.E. Kantor, S.D. Dudlettes, A.T. Shulgin, Biol. Psychiatry 15 (1980) 349.16] S.A. Barker, J.A. Monti, S.T. Christian, Int. Rev. Neurobiol. 22 (1981) 83.

[

[[

tals 185– 186 (2013) 61– 65 65

17] R.S.G. Jones, Prog. Neurobiol. 19 (1982) 117.18] M. Iosin, P. Baldeck, S. Astilean, J. Nanopart. Res. 12 (2010) 2843.19] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, J. Chem. Soc. Chem.

Commun. (7) (1994) 801.20] A. Kumar, S. Mandal, P.R. Selvakannan, R. Pasricha, A.B. Mandale, M. Sastry,

Langmuir 19 (2003) 6277.21] J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, J. Phys. Chem. B

110 (2006) 15700.22] N. Wangoo, K.K. Bhasin, S.K. Mehta, C.R. Suri, J. Colloid Interface Sci. 323 (2008)

247.23] Powder Diffraction Database (1994) File 04-0784 (JCPDS-ICDD).24] C. Abbehausen, J.F. Castro, M.B.M. Spera, T.A. Heinrich, C.M. Costa-Neto, W.R.

Lustri, A.L.B. Formiga, P.P. Corbi, Polyhedron 30 (2011) 2354.25] A.K. Gaigalas, V. Reipa, G. Niaura, J. Colloid Interface Sci. 203 (1998) 299.26] G. Mie, Ann. Phys. 25 (1908) 377.27] S.H. Toma, J.A. Bonacin, K. Araki, H.E. Toma, Eur. J. Inorg. Chem. 2007 (21) (2007)

28] M. Schmitt, K. Feng, M. Böhm, K. Kleinermanns, J. Chem. Phys. 125 (2006)144303.

29] E.M. Goldys, M.A. Sobhan, Adv. Funct. Mater. 22 (2012) 1906.30] A.G. Sazo, D.M. Rayner, J. Am. Chem. Soc. 102 (1980) 554.