Accepted Manuscript Tyrosine capped Silver Nanoparticles: a new fluorescent sensor for the quantitative determination of copper(II) and cobalt(II) ions Annalinda Contino, Giuseppe Maccarrone, Massimo Zimbone, Riccardo Reitano, Paolo Musumeci, Lucia Calcagno, Ivan Oliveri PII: DOI: Reference:

S0021-9797(15)30252-6 http://dx.doi.org/10.1016/j.jcis.2015.10.008 YJCIS 20794

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

3 July 2015 29 September 2015 5 October 2015

Please cite this article as: A. Contino, G. Maccarrone, M. Zimbone, R. Reitano, P. Musumeci, L. Calcagno, I. Oliveri, Tyrosine capped Silver Nanoparticles: a new fluorescent sensor for the quantitative determination of copper(II) and cobalt(II) ions, Journal of Colloid and Interface Science (2015), doi: http://dx.doi.org/10.1016/j.jcis.2015.10.008

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Tyrosine capped Silver Nanoparticles: a new fluorescent sensor for the quantitative determination of copper(II) and cobalt(II) ions. Annalinda Continoa,*, Giuseppe Maccarronea, Massimo Zimboneb, Riccardo Reitanoc, Paolo Musumecic, Lucia Calcagnoc and Ivan Oliveria. a

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, 95125 Catania, Italy b CNR-IMM, MATIS via S. Sofia 64, 95123 Catania, Italy c Dipartimento di Fisica e Astronomia, Università degli Studi di Catania, Via S. Sofia 64, 95123 Catania, Italy

Abstract Nanoparticles have been increasingly used as sensors for several organic and inorganic analytes. In this work, we report a study on the synthesis of novel highly fluorescent L-Tyr capped silver nanoparticles (AgNPs) and their use for the determination of metal ions. The AgNPs have been characterized by TEM, UV–Vis and Photoluminescence (PL) spectroscopy and dynamic light scattering (DLS) measurements and used for the quantitative determination of Co(II) and Cu(II) ions. In the L-Tyr capped AgNPs, the α-amino and α-carboxyl groups of the surface-confined amino acid can coordinate the entitled metal ions, giving rise to a decrease of the silver surface plasmon absorption, that is linearly correlated with the metal ions concentrations. The addition of Co(II) and Cu(II) solutions to the L-Tyr AgNPs also induces a paramagnetic quenching of the fluorescence in the PL spectra and the related Stern Volmer plots highlight a linear correlation over the whole concentration range for both metal ions, with a more pronounced effect for the copper(II) ion.

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1. Introduction Several transition elements are important to the chemistry of living systems, the most familiar examples being iron, cobalt, copper, and molybdenum. Though cobalt is understood to be an essential trace element in animal nutrition, the only detailed chemical knowledge of its biochemical action has to do with vitamin B12 and related co-enzymes. On the other hand, copper is found in both plants and animals, and numerous copper-containing proteins have been isolated [1,2]. Due to their essential biological role, disturbances in the homeostasis of these ions cause severe damages and diseases [3], and have been connected with neurodegenerative disorders [4,5]. Furthermore, even if both metals are essential nutrients, they may be harmful if ingested in excessive amounts. Thus an accurate quantitative determination of their concentration in biological samples is increasingly required, with copper(II) also being a valuable marker for the diagnosis of several pathologies as Wilson disease [6]. Traditional methods for the quantitative determination of cobalt(II) and copper(II), such as Atomic Absorption Spectrometry, are highly sensitive and accurate [7]. However these methods are extremely time consuming, while spectrophotometric methods, even though less sensitive, are faster and of easy utilization [7]. Metal nanoparticles (NPs) show size- and shape-dependent optical features, a high surface area to volume ratio, as well as very large extinction coefficients [8], finding many applications as colorimetric sensors for many analytes. These characteristics together with the ease of surface functionalization of gold and silver NPs also with active biomolecules [9] allows chemists to create the desired functionalities for specific applications. Therefore, the unique properties of metal nanoparticles [10,11], in particular of their Surface Plasmons and their interaction with light, have been successfully exploited to detect and accurately determine the concentration of several analytes either organic [12] and inorganic [13-18]. In fact, advantages of using NPs as colorimetric sensors are proven to be a promising approach for simple and cost-effective protocols with high sensitivity and rapid tracking of valuable and toxic metal ions in environmental samples/systems. Furthermore, amplification of light emitted from fluorophores by coupling to metal nanostructures is a promising strategy for significantly improving the detection sensitivity and hence maximizing the potential of fluorescence based technologies in bioapplications [19]. In several papers, gold and silver nanoparticles have been used as fluorescent sensors

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for the determination of metal ions [20-24], showing high sensitivity and wide linear dynamic range. In this paper we report a study on L-tyrosine capped silver nanoparticles (AgNPs), obtained by a new synthetic route that allows to bind the un-oxidized tyrosine, a fluorescent chromophore, on the nanoparticles surface. Although silver colloids are less stable than the gold ones, silver exhibits the highest efficiency of plasmon excitation, and the molar extinction coefficient of AgNPs is approximately 20-fold greater than that of gold nanoparticles (AuNPs) of the same size (6-20 nm), resulting in increased sensitivity [25]. These characteristics and their lower cost compared with AuNPs, have meant that the AgNPs became popular as colorimetric sensors. However, owing to the susceptibility of the silver surface to oxidation, the surface functionalization plays a crucial role in improving the stability and analytical applicability of AgNPs. The choice of L-tyrosine as capping agent for silver nanoparticles was not fortuitous. In fact, functionalization with amino acids, in addition to be widely employed for chiral recognition, with the copper(II) ion playing a pivotal role [26,27], gives rise to very sensitive chemosensors [25]. Furthermore L-Tyr in the oxidized form gives rise to very stable colloids used for metal ions determination [28], as well as for antibacterial agents [29]. The complex capabilities of these new highly fluorescent AgNPs have been exploited for the quantitative

determination

of

copper(II)

and

cobalt(II)

ions

by

UV-Vis

and

photoluminescence spectroscopy.

3

2. Experimental part 2.1. Materials. L-Tyrosine,

was obtained as commercial reagents by Merck and was used as received.

AgNO3 and sodium borohydride (NaBH4) were purchased from Sigma Aldrich. Copper(II) sulphate and cobalt(II) sulphate (Carlo Erba) stock solutions were standardized by EDTA titrations [30]. 2.2. Synthesis of AgNPS. L-Tyrosine

capped AgNPs were synthesized at ice-cold temperature, as pioneered by

Creighton in 1979 [31], employing sodium borohydride (NaBH4) as reducing agent and Ltyrosine as stabilizing agent. The present synthesis was a slight modification of the procedure previously reported for citrate capped AgNPs [32]. Shortly, to 24.6 ml of Milli-Q water placed in an ice bath on a stir plate, a freshly prepared NaBH4 aqueous solution (2.4 ml of 5 x 10−2 M) was added. Subsequently, during continuous stirring, 30.0 ml of a silver nitrate solution (c = 4 × 10−4 M) were dropped, at approximately 1 drop per second. Finally 3.0 ml of a 3 x 10-3 M L-Tyr solution was added as capping agent and the stirring was stopped. 2.3. TEM Analysis. Transmission electron microscopy was performed with a TEM JEOL JEM 2010 F, equipped with the Gatan imaging filter, operating at 200 keV. Samples for TEM were prepared by placing several drops of the colloidal dispersions of AgNPs onto a carbon-coated copper micro-grid (2M STRUMENTI). In order to obtain a good statistical particle size distribution several different areas of the grid were observed and more than 150 Ag particles measured for each sample. The average size diameter estimated by TEM was calculated using the following formula:

, where ni is the number of AgNPs of diameter di and n is

the total number of Ag particles. 2.4. UV-Vis spectroscopic measurements. UV-Vis spectra of the investigated systems were carried out at room temperature, using a diode-array Agilent 8453 spectrophotometer in the 300-700 nm wavelength range. The silver sol was diluted by mixing 1.1 ml of the synthetized colloid with 1.4 ml of water to obtain a final concentration of AgNPs of 1.86 x 10-8 M. The effect of Co2+ and Cu2+ on the UV-Vis spectrum of the L-Tyr-AgNPs was investigated by mixing appropriate amounts of each metal

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ions solutions (0.1 or 1 mM) with 2.0 mL aqueous solution of the diluted sol, reaching a final concentration range of 0.01–0.1 mM of the metal ion. The concentration of the AgNPs solutions was calculated by utilizing the analytical concentration of the silver solution and the diameter values obtained by the TEM measurements and thus estimating the number of silver atoms in a Ag nanoparticle [33]. The species distribution diagrams have been obtained by the simulation and speciation program HYSS [34]. This program allows, provided the equilibria and the concerning stability constants values, as well as the analytical concentration of the components of the investigated solutions, to calculate the concentrations of each species present in solution. In this work the obtained concentrations are plotted as degrees of formation (i.e. Concentration of the species/ analytical concentration) as a function of the pH. 2.5. DLS measurements. The Dynamic Light Scattering (DLS) measurements were carried out by a homemade apparatus as described elsewhere [35]. The analysis of the fluctuations of the scattered light was performed by the intensity auto-correlation function (g2) provided by the hardware correlator operating in single photon counting regime. The field autocorrelation function g1 is computed from the g2 using the Segret relation (g2 = 1+|g1|2). For monodisperse noninteracting particles in Brownian motion, the g1 function is a decreasing exponential with a relaxation rate Γ (Γ=1/ with τ = decay time). In our case, we fit the experimental data with a double exponential. The amplitude relative to the relaxation rates were weighed for the scattering efficiency obtaining a number distribution. Once the main relaxation rate is obtained, it is possible to calculate either the translational diffusion coefficient (Dt) and the hydrodynamic radius (RH) using the following expressions [36]:

=Dtq2

(1)

and RH=kT/(6Dt)

(2)

where q is the scattering vector, defined as q= (4πn/λ)sin(θ/2), being n the refraction index of the solvent, λ the light wavelength, θ the scattering angle,  is the liquid viscosity, k the Boltzmann constant and T the absolute temperature. We used a scattering angle θ =90°. 2.6. Fluorescence measurements. Fluorescence measurements were performed on a Horiba Nanolog spectrofluorometer. The measurements were performed at room temperature with a standard 1 cm cell. The 5

wavelength resolution of both the excitation and the emission slits was set to 5 nm. The intrinsic fluorescence of the L-Tyr AgNPs was excited at 300 nm which is the excitation peak for the tyrosine residue. Analysis of the fluorescence intensity was performed by first subtracting the residual background signal and then integrating over the whole spectral range. The effect of Co2+ and Cu2+ on the emission intensity of the L-Tyr-AgNPs was investigated by mixing appropriate amounts of each metal ion solutions (0.1 mM) with 2 mL of AgNPs aqueous solution, obtaining final concentrations of Co2+ or Cu2+ between 0.2 and 9.0 μM. The mixtures were measured 20 minutes after the preparation.

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3. Results and Discussion 3.1 Synthesis and characterization of L-Tyr capped AgNPs. The silver sol was synthesized at low temperature in order to obtain small particles (5-10 nm) with a narrow distribution range. In fact, it is well known [37] that the procedures derived from the Turkevich method [38] reported for gold, such as Lee–Meisel method [39] in that AgNO3 is used as the metal source and the silver reduction is carried out in a solution of boiling water, produces a broad distribution of particle sizes. Synthesis carried out using Ltyrosine either as reducing and capping agent [40] also require hot temperatures in order that L-Tyr

could reduce the metal ions, giving rise to silver nanoparticles not monodisperse, but

with a bimodal distribution of sizes. In our work, instead, the TEM image, reported in Figures 1, clearly shows that the

L-Tyr

capped AgNPs are highly dispersed with size

distribution consistent with a normal distribution having an average size 6 ± 2 nm. The AgNPs concentration values of the solutions were estimated by the methods reported in Ref [33]. For particles of 6 nm a volume of 113 nm3 was obtained and, considering that each silver atom occupies the volume of a cube with an edge of 0.3 nm, in a 6 nm AgNP there are almost 4700 atoms. Assuming that all the silver ion is reduced, the concentration of Ag in solution is 2.0 x 10-4 M, that corresponds to a concentration of 4.23 x 10-8 M for the AgNPs. For the UV-Vis experiments this colloid was diluted to obtain a final concentration of 1.86 x 10-8 M, as reported in the experimental section. The optical properties of AgNPs have been extensively investigated by UV-vis spectroscopy technique [8,10,41,42] as they exhibit an intense absorption band in this wavelength region. The experimental extinction spectrum of the L-Tyr capped AgNPs in the wavelength range 300-700 nm is reported in Figure 2. This spectrum shows a strong absorption band with a maximum at 400 nm, characteristic of the collective absorption of free conduction band electrons of the nanoparticles, known as Surface Plasmon Resonance (SPR). As reported in the literature [41,42] the SPR peak position, for spherical shaped nanoparticles, depends on their size and on the surrounding medium. Following the approach of Slistan et al [41], for AgNPs in water, it is possible to determine the size from the peak position and from the Full Width at Half Maximum (FWHM) of the band. The experimental extinction spectrum (= 400 nm and FWHM=74 nm) indicates a value of 6 nm for the nanoparticle size, which is consistent with the size distribution (4-8 nm) determined by TEM. DLS measurements were also carried out on the L-Tyr capped AgNPs and the concerning autocorrelation function (g2) is reported in Figure 3. The measured value of the

7

hydrodynamic nanoparticle size was 12 nm, a value larger than that obtained by UV-Vis spectroscopy. This discrepancy can be related both to the molecules adsorbed on the nanoparticle surface (e.g. stabilizers) and to the thickness of the electrical double layer (solvation). In Figure 2, in addition to the UV-Vis spectrum of the

L-Tyr

capped AgNPs recorded

immediately after the preparation, it is also reported the spectrum of the nanoparticles after a period of aging of ca 1 month. The spectrum recorded after a month is superimposable with that obtained immediately after the preparation showing that the L-Tyr Ag colloid is stable for several weeks. The stability of the silver sol, together with the small size of the particles obtained, is a clear evidence that the amino acid is bound on the nanoparticles surface. In fact, if the only capping agent was the BH4- anion, also at ice-cold temperature, larger size nanoparticles would be obtained and the resulting silver sol would be very sensitive to stirring time [33], that it is not the case here reported. Analogously to L-Cys capped AgNPs [32,43], the stability of the L-Tyr sol is due to the pH of the solution. In fact, as reported for AgNPs obtained using Tyr either as reducing and capping agent, for the oxidized tyrosine it is the semiquinone group that binds the silver surface, whereas the aminoacidic residue points towards the bulk of the solution [44] with the carboxylic acid group present as carboxylate anion at the working pH of formation of the sol. In our case it is probably the O- terminal group of the not oxidized tyrosine that interacts with AgNPs, giving rise to a very stable silver sol with very small particles, whereas the amino and the carboxyl groups remain confined on the surface of the nanoparticles. As shown by the species distribution diagram reported in Figure 4, at the pH at which the sol is formed (pH = 9.80) the negatively charged L-Tyr

species has a degree of formation value nearly of 90%, providing the stability of the

silver sol [32].

3.2. Determination of metal ions. 3.2.2 UV-vis results. Analogously to L-Cys capped AgNPs, the amino and the carboxyl groups of the surfaceconfined tyrosine can coordinate the metal ions present in solution. However, owing to the concentration of amino acid, that is higher than in the L-Cys sol, for the L-Tyr nanoparticles, the metal ions bound to the surface confined amino acid could in turn interact with the tyrosine capping another nanoparticle giving rise to aggregation processes triggered exclusively by the metal ions present in solution and allowing their detection at low concentrations [13]. 8

Thus, in order to assess the minimum concentration of metal ion detectable by aggregation processes, the stability of the sol in the presence of cobalt(II) and copper(II) ions was evaluated, as previously described [27,32]. Analogously to L-Cys capped AgNPs that do not show any aggregation even in the presence of 10-4 M of Cu(II) ion, neither copper(II) nor cobalt(II) induce aggregation on the L-Tyr AgNPs. However, upon the addition of either Co(II) and Cu(II) with increasing concentration from 0.2 to 6 μM to the AgNPs, the absorption due to the silver surface plasmon decreases in a little but significant way (Figure 5). In the concerning insets the plots of A400 – A318 vs the concentration of cobalt or copper are reported. The absorbance at 400 nm was subtracted by that at 318 nm in order to minimize the differences in the concentrations due to the experimental errors in the solutions preparation. Both plots show a fairly good linear correlation (r2 = 0.98677 and r2 = 0.99637) between the absorbance and the metal ions concentration values over the entire range. The absorption decrease is probably related to a perturbation effect due to the complexation of the surface bounded tyrosine with the metal ions. L-Tyr AgNPs can be assimilated to a core shell nanoparticles where the core is constituted by silver and the external shell by the Tyr or Tyrmetal complex. Optical properties of core shell nanoparticles are strongly influenced by the properties of the shell. In particular, a reduction of the refractive index of the shell lowers the amplitude of the plasmonic resonance. In other words, the conduction electrons of the nanoparticle, displaced by the incident electromagnetic radiation, gives rise to an induced dipole: the larger the electron displacement induced by the electromagnetic radiation, the larger the induced dipole and consequently the restoring force acting on electrons [8]. The presence of positive charges on the external surface of the particle can reduce the restoring force of the oscillation and, via a lowering of the polarizability, a decrease of the refraction index of the shell. Therefore the complexation of the surface bounded tyrosine with the positively charged metal ions induces a reduction in the refraction index of the shell that, in turn, gives rise to a lower absorbance. The insets of Figure 5 also show that cobalt and copper decrease the absorbance in the same way, as can be inferred by the slopes of the linear correlations and this trend is quite surprising at first sight. In fact, as clearly shown in the literature [45-47], usually differences in the complexation stability constants give rise to different behaviors of the complex species. As specified above, in the L-Tyr-capped AgNPs, the O- terminal group of the amino acid interacts with AgNPs, whereas the α-amino and α-carboxyl groups of the surface confined tyrosine can coordinate the metal ions present in solution, virtually behaving as phenylalanine and, in fact, the stability constants either of protonation and complexation towards cobalt(II) 9

and copper(II) of

L-Tyr

and L-Phe are identical within the experimental error [48]. As

indicated in Figure 6 that reports the species distribution diagrams calculated for the systems Co(II)/L-Tyr and Cu(II)/L-Tyr at the concentrations and at the pH used in our experiments, the two metal ions have a quite different affinity towards the amino acids, with the cobalt complex species that show a maximum degree of formation of 2% as the sum of Co(L-Tyr) and Co(L-Tyr)2 at pH 9.40, whereas the Cu(L-Tyr)2 species reaches a degree of formation of 8% already at pH 8.00. However, the complex species Cu(Tyr)2 forms at the expenses of the zwitterionic amino acid and thus the overall charge distribution on the nanoparticles surface is the same for Co(II) and Cu(II) systems, explaining the observed trend for the two metal ions.

3.2.2 Photoluminescence results. The PL intensity spectra of the L-Tyr and of L-Tyr AgNPs are reported in Figure 7a. As expected, the nanoparticle substrate strongly enhances the fluorescence of the tyrosine, showing a symmetric and narrow fluorescence emission wavelength at 453 nm with a half peak width of 50 nm. In fact, the low quantum yield molecular fluorophore is strongly influenced by the plasmon resonance energy and its scattering efficiency. The fluorescence enhancement by a nanoparticle substrate is optimal when the nanoparticle plasmon resonance is tuned to the emission wavelength of the molecular fluorophore [49]. Upon addition of cobalt (II) (Figure 7b) and copper(II) (Figure 7c) solutions to L-Tyr AgNPs, a remarkably fluorescence quenching – known as the general phenomenon of paramagnetic quenching of fluorescence - is observed [50-53]. In the present case, the quenching can be ascribed to the static effect of the coordination of the metal ions to the aminoacidic group of the tyrosine. Indeed, the change in absorbance with metal concentration shown before points to a modification of the electronic ground state of the tyrosine, an effect not expected for dynamic quenching. Also, as can be seen from the Stern-Volmer plots for the fluorescence quenching of L-Tyr AgNPs by Co2+ and Cu2+ ions reported in Figure 8, the quenching is linear over the whole concentration range indicating a single operating quenching mechanism and thus that the coordination of the metal ions promotes the intersystem crossing (S1→T1 and T1→S0) [54]. However, the two metal ions give rise to different trends (Figure 8). The data clearly shows that copper(II) ion has a more pronounced effect on the

L-Tyr

AgNPs

photoluminescence than the cobalt(II) ion, as confirmed by the values for the constants of static fluorescence quenching obtained by the Stern Volmer plots (KstCo = 0.054 and KstCu = 0.125). This trend is due to the different capability of the two metal ions to accept electrons 10

[54], as well as to their different complexation capability towards the capping amino acid. In fact, copper(II) is more noble than cobalt(II) and thus more prone to accept electrons of the fluorofore favoring the quenching, and, in addition, as shown in Figure 6, the degree of formation of copper(II) complex species is larger than that of the analogous ones of cobalt(II) accounting for the more dramatic quenching effect [55]. The PL experiments show that

L-Tyr

capped AgNPs are pretty sensitive fluorescent

chemosensors for cobalt(II) and copper(II) ions with a linear range of detection from 0.1 μM to 9 μM and a detection limit of 48 ppb and 36 ppb for Co(II) and Cu(II), respectively. Even though traditional methods based on Atomic Spectroscopy are more sensitive, showing LOD of almost 10 ppb, the method here proposed is definitively faster, simpler and exhibits a sensitivity comparable to that (LOD = 40 ppb) of traditional method based on UV-Vis spectrophotometry, that make use of colorimetric reagents.

4.0. Conclusions In this study a new synthetic route for L-Tyr capped AgNPs was developed, obtaining small particles (5-10 nm) with a narrow distribution range. The use of different techniques (TEM, UV–Vis and PL spectroscopy and DLS) and the focused use of solution equilibria allowed us not only to fully characterize novel L-Tyr capped AgNPs but also to smartly exploit them for the quantitative determination of Co(II) and Cu(II) ions in solution. This analytical approach allowed us to develop a fluorescence-based method that shows a sensitivity of about 40 ppb, comparable to that of the traditional methods based on UV-Vis spectroscopy.

Acknowledgements. We thank Salvatore Pannitteri (CNR-IMM, Catania, Italy) for technical assistance and thank Università di Catania (Progetti di Ricerca di Ateneo) for financial support. A special thank to Prof. Santo Di Bella (University of Catania) for helpful discussion. We acknowledge MIUR and European Commission for purpose support through the project PONa3_00136 named BRIT.

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[36] B.J. Berne, R. Pecora, Dynamic light scattering, Wiley, New York, 1976. [37] D. D. Evanoff Jr, G. Chumanov, Synthesis and Optical Properties of Silver Nanoparticles and Arrays, Chem. Phys. Chem., 6 (2005) 1221–1231. [38] J. Turkevich, P. C. Stevenson, J. Hillier, A study of the nucleation and growth processes in the synthesis of colloidal gold, Discuss. Faraday Soc. 11 (1951) 55–75. [39] P. C. Lee, D. Meisel, Adsorption and Surface-Enhanced Raman of Dyes on Silver and Gold Sols, J. Phys. Chem., 86 (1982) 3391-3395. [40] P. R. Selvakannan, A. Swami, D. Srisathiyanarayanan, P. S. Shirude, R. Pasricha, A. B. Mandale, M. Sastry, Synthesis of Aqueous Au Core-Ag Shell Nanoparticles Using Tyrosine as a pH-Dependent Reducing Agent and Assembling Phase-Transferred Silver Nanoparticles at the Air-Water Interface, Langmuir, 20 (2004) 7825-7836. [41] A. Slistan-Grijalva, R. Herrera-Urbina, J.F. Rivas-Silva, M. Ávalos-Borja, F.F. Castillón-Barraza, A. Posada-Amarillas, Classical theoretical characterization of the surface plasmon absorption band for silver spherical nanoparticles suspended in water and ethylene glycol, Physica E, 27 (2005) 104–112. [42] S. K. Ghosh, T. Pal, Interparticle Coupling Effect on the Surface Plasmon Resonance of Gold Nanoparticles:  From Theory to Applications, Chem. Rev., 107 (2007) 4797– 4862. [43] E. Csapó, R. Patakfalvi, V. Hornok, L. Tamás Tóth, Á. Sipos, A. Szalai, M. Csete, I. Dékány, Effect of pH on stability and plasmonic properties of cysteine-functionalized silver nanoparticle dispersion, Colloids Surface B, 98 (2012) 43–49. [44] P. R. Selvakannan, R. Ramanathan, B. J. Plowman, Y. M. Sabri, H. K. Daima, A. P. O’Mullane, V. Bansal, S. K. Bhargava, Probing the effect of charge transfer enhancement in off resonance mode SERS via conjugation of the probe dye between silver nanoparticles and metal substrates, Phys. Chem. Chem. Phys., 15 (2013) 1292012929. [45] V. Cucinotta, A. Giuffrida, G. Maccarrone, M. Messina, A. Puglisi, .E. Rizzarelli, G. Vecchio, Coordination properties of 3-functionalized b-cyclodextrins. Thermodynamic stereoselectivity of copper(II) complexes of the A,B-diamino derivative and its exploitation in LECE, Dalton Trans., (2005) 2731–2736. [46] R. P. Bonomo, E. Conte, G. De Guidi, G. Maccarrone, E. Rizzarelli, G. Vecchio, Characterization and superoxide dismutase activity of copper(II) complexes of 6A,6Xdifunctionalized β-cyclodextrins, J. Chem. Soc., Dalton Trans., (1996) 4351-4355. [47] C. Conato, A. Contino, G. Maccarrone, A. Magrì, M. Remelli, G. Tabbì, Copper(II) complexes with L-lysine and L-ornithine: is the side-chain involved in the coordination? A thermodynamic and spectroscopic study, Thermochim. Acta, 362 (2000) 13-23. [48] A. E. Martell, R. J. Motekaitis, Determination and Use of Stability Constants, VCH Publishers Inc., New York, USA, 1988. [49] F. Tam, G. P. Goodrich, B. R. Johnson, N. J. Halas, “Plasmonic Enhancement of Molecular Fluorescence”, Nano Lett., 7 (2007) 496-501. [50] T. Toyomi Tominaga, H. Imasato, O. Rangel Nascimento, M. Tabak, Interaction of tyrosine and tyrosine dipeptides with Cu2+ ions: a fluorescence study, Anal. Chim. Acta, 315 (1995) 217-224. [51] R. Corradini, A. Dossena, G. Impellizzeri, G. Maccarrone, R. Marchelli, E. Rizzarelli, G. Sartor, G. Vecchio, Chiral Recognition and Separation of Amino Acids by Means of a Copper(II) Complex of Histamine Monofunctionalized β-Cyclodextrin, J. Am. Chem. Soc., 116 (1994) 10267-10274. [52] J. Zhao, D. J. Nelson, Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition, J. Inorg. Biochem., 99 (2005) 383–396. 14

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15

5: Figure

a)

50

b) Frequency %

40

30

20

10

0 2

4

6

8

10

12

Ag diameter [nm]

Figure 1 a) TEM microphotographs and b) related particle size distribution for L-Tyr capped AgNPs.

5: Figure

1.2 L-Tyrosine capped AgNPs L-Tyrosine capped AgNPs after 1 month

1.0

A

0.8 0.6 0.4 0.2 0.0 300

400

500

600

700

 (nm) Figure 2. UV-Vis spectra recorded for L-Tyr capped AgNPs; C°AgNP= 1.86 x 10-8 M.

5: Figure

1.0

g2 ()

0.8 0.6 0.4 0.2 0.0 -4

10

-3

10

-2

10

Time (s)

Figure 3. Correlation function g2 (τ) for the AgNPs L-Tyr capped.

5: Figure

1.0

L-Tyr

0.8 0.6 0.4 0.2 0.0 3

4

5

6

7

8

9

10

11

pH Figure 4. Species distribution diagram for L-Tyr; CTyr = 1.45 x 10-4 M.

5: Figure

1.5 1.08

0

Y = -0.0140x + 1.081 R2 = 0.98677

1.06

A400 - A318

[Co2+]

1.0

A

6 μM

1.04 1.02 1.00 0

0.5

1

2

3

4

5

6

2+

Concentration of Co (M)

0.0 300

400

500

600

700

 (nm)

1.5

1.10

0 A400 - A318

[Cu2+]

1.0

6 μM

1.06 1.04

A

1.02 1.00 0

0.5

0.0 300

Y = -0.0127x + 1.077 R2 = 0.99637

1.08

1

2

3

4

5

6

2+

Concentration of Cu (M)

400

500

600

700

 (nm) Figure 5. a) UV-Vis spectra of L-Tyr AgNPs as a function of various concentrations of (a) Co2+ and (b) Cu2+ ions. In the insets, the plots of A400 – A318 vs the concentration of the concerning metal ion are reported.

5: Figure

1.0 0.8

L-Tyr

0.6 0.4 0.2 0.0 5

6

7

8

9

10

pH Figure 6a. Species distribution diagram for the system Co(II)/L-Tyr; CCo = 6.00 x 10-6 M, CTyr = 1.45 x 10-4 M.

5: Figure

1.0 0.8

L-Tyr

0.6 0.4 0.2 0.0 5

6

7

8

9

10

pH Figure 6b. Species distribution diagram for the system Cu(II)/L-Tyr; CCu = 6.00 x 10-6 M, CTyr = 1.45 x 10-4 M.

5: Figure

Co2+ L-Tyr L-Tyr AgNPs

0.8

a)

0.6

0

b)

[Co2+]

0.8 0.6

9 μM

0.4 0.2 0.0 350

400

450

500

550

600

 (nm)

0.4 0.2 0.0

1.0

400

450

500

550

 (nm)

Cu2+

intensity (a. u.)

Intensity (a. u.)

1.0

Intensity (a. u.)

1.0

0.8

0

c)

[Cu2+]

0.6

9 μM

0.4 0.2 0.0 350

400

450

500

550

 (nm)

Figure 7. a) Luminescence band for L-Tyr and L-Tyr capped AgNPs (λexc = 300 nm). Fluorescence spectral changes for the L-Tyr AgNPs (λex = 300 nm, λem = 453 nm), on addition of b) Co2+ (1.0 – 9.0 μM) and c) Cu2+ (1.0 – 9.0 μM).

5: Figure

2.4 2.2 2.0

Y = 1.0 + 0.125x R2 = 0.9771

I0/I

1.8 Cu2+

1.6

Co2+

1.4 1.2 Y = 1.0 + 0.054x R2 = 0.9997

1.0 0.8 0

2

4

6

8

10

2+

[M ] (M) Figure 8. Stern-Volmer plots for the fluorescence quenching of L-Tyr AgNPs by Cu2+ ion and Co2+ ion.

16