Fluorescence Probing of Thiol-Functionalized Gold Nanoparticles: Is ...

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Fluorescence Probing of Thiol-Functionalized Gold Nanoparticles: Is Alkylthiol Coating of a Nanoparticle as Hydrophobic as Expected? Alexander Kyrychenko,*,†,‡ Galina V. Karpushina,† Denis Svechkarev,† Dmitry Kolodezny,† Sergey I. Bogatyrenko,† Alexander P. Kryshtal,† and Andrey O. Doroshenko†,‡ †

V. N. Karazin Kharkiv National University, 4 Svobody Square, Kharkiv 61022, Ukraine Ukrainian-American Laboratory of Computational Chemistry, Kharkiv, Ukraine and Jackson, Mississippi, United States



S Supporting Information *

ABSTRACT: Understanding the interaction of fluorescent dyes with monolayer-protected gold nanoparticles (AuNPs) is of fundamental importance in designing new fluorescent nanomaterials. Among a variety of molecular sensors and reporters, fluorescent probes based on a 3-hydroxychromone (3HC) skeleton appear to be very promising. They exhibit the phenomenon of dual band emission, resulting from excitedstate intramolecular proton transfer (ESIPT), known to be highly sensitive to a nature of microenvironment surrounding a fluorophore. In this study, dodecanethiol-protected gold nanoparticles were synthesized, and, owing to the transmission electron micrograph imaging, their average diameter was found to be ∼1.4 nm. Fluorescence titrations of the 3HC ESIPT probes with AuNPs in toluene solutions demonstrate significant changes in the intensity ratio of their normal and tautomeric emission bands, suggesting that the probe molecules become noncovalently bound to a dodecanethiol layer of AuNPs. Despite expected fluorescence quenching induced by close proximity to the metal surface, no fluorescence lifetime decrease was observed, indicating that a bound-fluorophore is shielded from a nanoparticle core. Further spectral analysis revealed that the ratiometric fluorescence changes could be interpreted as a consequence of intermolecular hydrogen bonding between a probe and residual ethanol molecules, trapped into the dodecanethiol shell of AuNPs during the synthesis. Evidences for residual traces of ethanol in the ligand shell of the nanoparticles were also observed in NMR spectra, suggesting that alkylthiol-coated gold nanoparticles may not be as hydrophobic as one could expect. To elucidate structural features of dodecanethiol-stabilized gold nanoparticles at the supramolecular level, a molecular dynamics (MD) model of AuNP was developed. The model was based on the all-atom CHARMM27 force field parameters and parametrized according to available experimental data of the synthesized AuNPs. Our MD simulations show that in bulk toluene the 3HC probe molecule becomes weakly bound to a dodecanethiol monolayer, so that a fluorophore favors residence in an outer shell of AuNP. In addition, MD simulations of transfer of AuNP from bulk ethanol to toluene demonstrate that a small population of ethanol molecules are able to penetrate deeply into the dodecanethiol layer and may indeed be trapped into the ligand shell of alkylthiolfunctionalized gold nanoparticles. The results of our fluorescence experiments and molecular dynamics simulation suggest that 3hydroxychromones can be used as a noncovalent fluorescent labeling agent for alkylthiol-stabilized noble metal nanoparticles.



fully utilized for fluorescence sensing of mercury(II) ions in aqueous solution.9 A selective chemodosimeter for mercury ions based on the mercury-promoted intramolecular cyclic guanylation of thiourea connected with 1,8-naphthalimide has been suggested as well.10 In addition, a thiourea based receptor composed of linked acridinedione functionalized gold nanoparticles has been proposed for selective recognition of fluoride ions in solution using both steady-state and time-resolved fluorescence techniques.11 Due to the rapid development of the computational tools, molecular dynamics (MD) simulations become widely applied for modeling nanoparticles composed of different materials and,

INTRODUCTION Dye-functionalized gold nanoparticles possess a lot of interesting properties that make them useful for many chemical and biological applications.1 Though similar functions can also be characteristic to colloidal nanoparticles of a different nature, such as fluorescent quantum dots, there are several features unique to gold particles. Gold nanoparticles of controllable size can be easily synthesized.2 They are colloidally stable and can be conjugated with different fluorescent labels in a straightforward way enabling emission color tuning.3,4 That is why dyefunctionalized AuNPs have been widely applied to resolving a diversity of experimental tasks: They are very attractive contrast agents, as they can be applied in fluorescent labeling,5 confocal microscopy imaging,6 and various analytic detection methods with a large variety of fluorescent techniques.7,8 For example, rhodamine-6G-functionalized gold nanoparticles were success© 2012 American Chemical Society

Received: June 20, 2012 Revised: September 12, 2012 Published: September 12, 2012 21059

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thus, have contributed significantly to our understanding of structure and mechanism of their formation.12 MD simulations of alkanethiol-stabilized spherical gold nanoparticles have shown that the monolayer organization depends strongly on the temperature, molecular chain length, and nanoparticle core size.13 Multiscale modeling of gold nanoclusters and thiolstabilized nanoparticles has attracted significant interest covering a range from all-atom models to coarse-grained approaches. The engineering of new electronic materials for nanotechnology often requires fabrication of self-assembled arrays of passivated metal, oxides, and semiconductor nanoparticles. Since assembling and aggregation of AuNPs into a superlattice lead to a dramatic change in their optical properties, therefore the aggregation behavior of monolayer-protected AuNPs has intensively been considered.14 3-Hydroxychromone (3HC) dyes offer unique features for ratiometric fluorescence sensing of a large number of parameters for a microenvironment in the vicinity of a fluorophore. The origin for their environment-sensitive behavior is believed to be an excited-state intramolecular proton transfer (ESIPT) reaction, leading from a normal form to phototautomer, as shown schematically in Figure 1a.

Article

MATERIALS AND METHODS

Preparation of Gold Nanoparticles. Synthesis and purification of 2-[4-(5-phenyloxazol-2-yl)phenyl]-3-hydroxychromone (1) and 2-(benzimidazol-2-yl)-3-hydroxychromone (2) (Figure 1b) was described elsewhere.22−24 The dodecanethiol-functionalized AuNPs were prepared by the following method:25,26 Solution of tetraoctylammonium bromide (0.3 g) in 30 mL of toluene was mixed with solution of 0.11 g of HAuCl4·3H2O in water (25 mL). The mixture was stirred for 10 min at room temperature, and then the decolorized water layer was moved off. Dodecanethiol (0.28 g) in 10 mL of toluene was added then, and the mixture was stirred for another 10 min. After this a water solution of NaBH4 (0.011 g in 10 mL, ∼10 molar excess) was added in small portions during 40 min, and the reaction mixture was left overnight at permanent stirring; then the organic phase was collected, and the solvent was evaporated in waterjet pump vacuo. The resulted solid was first washed with deionized water, after this it was washed with ethyl alcohol at ultrasound sonification; the ethyl alcohol washing was repeated 5 times to remove the excess of unbound thiol (in the following cycles: dispersion in ethanol − ultrasound sonification − centrifugation − elutriation − drying in air). After this the thiol-coated nanoparticles were finally dried out in air at room temperature during a week. The synthesized AuNPs were stable in solid state and in solutions in the nonpolar solvents. The prepared in a such way AuNPs powder was stored at 4−5 °C. Spectroscopy. The electronic absorption and fluorescence spectra were recorded using HITACHI U-3210 spectrophotometer and Hitachi F-4010 spectrofluorimeter, respectively. Fluorescence decays were measured using a homemade timeresolved fluorescence spectrometer, utilizing a standard scheme of time-correlated single-photon counting, constructed from commercial building blocks purchased from PicoQuant GmbH (Berlin, Germany: TimeHarp 200 and pulsed UV LEDs driven by PDL 800-B device). Fluorescence emission was selected by using a MDR-12 monochromator (LOMO, Saint Petersburg, Russia) and detected using a Hamamatsu H5783P PMT (Hamamatsu, Japan) photomultiplier. Samples were excited at 375 nm by a subnanosecond pulsed LED PLS 370 (PicoQuant GmbH, Berlin, Germany) operating with a repetition rate of 10 MHz. Transmission electron micrograph (TEM) images were measured by PEM-125K transmitting electron microscope with the accelerating voltage 100 kV (production of JSC Selmi, Ukraine). Samples for microscopic investigations were prepared by evaporation of AuNPs dilute hexane solution (of spectrophotometric concentration) on the surface of carbon films of approximate thickness near 100 nm. The TEM images were registered with a CCD video camera and treated mathematically on the desktop computer. The 1H and 13C NMR spectra were recorded on a Bruker 500 spectrometer (Bruker BioSpin GmbH, Germany) in CDCl3 at 500 and 125 MHz, respectively. Concentrations of gold nanoparticles in toluene solution were calculated from their molar absorbance coefficient (ε, dm 3 ·mol −1·cm −1 ), which was determined according to Lambert−Beer law (eq 1, in which A is the AuNPs absorbance at wavelength λi, l is the path length, and CAuNPs is the AuNPs concentration, M).

Figure 1. a) Scheme of the excited state intramolecular proton transfer (ESIPT) in 3-hydroxychromones. b) Molecular structure of 2-[4-(5phenyloxazol-2-yl)phenyl]-3-hydroxychromone (1) and 2-(benzimidazol-2-yl)-3-hydroxychromone (2).

Therefore, a number of 3HC derivatives have been employed to probe changes of medium polarity,15 viscosity,16 and pH17 as well as water admixtures and metal ion content.18 In the past decade, 3HC derivatives have been also applied to study membrane potential and noncovalent interactions with model biological systems such as lipid bilayers and vesicles19,20 as well as single- and double-stranded DNA.21 This paper is devoted to the study of noncovalent binding of two fluorescent dyes of 3HC series (Figure 1b) to alkylthiolcoated gold nanoparticles in nonpolar solvents. The fluorescence response observed upon titration of 3HC probes by dodecanethiol-coated gold nanoparticles revealed, however, unexpectedly high hydrogen bonding ability of the investigated hydrophobic nanocomposites. This feature was explained by possible penetration of alcohol molecules into the AuNPs thiol coating upon synthesis and purification of the latter. Fluorescence measurements, 1H and 13C NMR data, and molecular dynamics modeling were applied for supporting of this hypothesis.

ε = A × l × CAuNPs 21060

(1)

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in particle edges determination of ±1 pixel on each side, which gives ±0.48 nm taking into account the TEM image scale of 0.24 nm per pixel. In addition, the particle size distribution was also approximated using Gaussian function as shown in Figure 2.

The molar absorbance of AuNPs in toluene was found to be 245000 and 149000 at 400 and 500 nm, respectively. Molecular Dynamics Modeling and Simulation Setup. A molecular dynamics (MD) model of a dodecanethiolfunctionalized gold nanoparticle was based on the all-atom CHARMM27 force field for proteins and lipids, recently ported to the GROMACS package.27,28 The force field parameters of bonding interactions of gold-attached dodecanethiol were developed based on mapping of DFT (B3PW91 with the LanL2DZ basis set for Au and cc-pVDZ for the other elements) optimized geometry of all-atom gold-dodecanethiol (Au−S− (CH2)11−CH3) into harmonic-type potentials which describe bond stretching and angle bending and keeping bond lengths and angles at their equilibrium values. Finally, the topology file of gold-dodecanethiol was built in the CHARMM27 format using the SwissParam force-field generation tool.29 The nonbonded parameters for gold−gold interactions were developed by reproducing experimental gold−gold nearestneighbor distances of ∼0.288 nm found in the face-centered cubic crystal structure.30 An initial configuration of a gold nanoparticle was built as described previously.14 The bond length and angle parameters for 1 were optimized by DFT calculations at the B3LYP/cc-pVDZ level and adopted for the GROMACS force field format. Dihedral rotation around the C−C bond connecting the chromone and the diphenyloxazole moiety was modeled using the periodic RyckaertBellemans dihedral potential.31 Partial charges needed for Coulomb interactions were derived from the B3LYP/cc-pVDZ electron densities by fitting the electrostatic potential to point (ESP) charges. All covalent bonds in gold-dodecanethiol and the dye 1 were constrained to their equilibrium values by using the P-LINCS algorithm.32 The repulsion and dispersion terms of nonbonded interactions were computed using the LennardJones potential energy function.31 Electrostatics was treated with a particle-mesh Ewald (PME) model,33 using a short-range cutoff of 1.0 nm, and van der Waals interactions switched off between 0.8 to 1.0 nm. Temperature was maintained using the thermostat of Bussi et al.34 Periodic boundary conditions were applied as well as isotropic pressure-coupling to a ParrinelloRahman barostat35 with a coupling constant of 1 ps. MD simulations were carried out at the constant number of particles, constant pressure, P = 1 atm, and the constant temperature, T = 298 K (NPT ensemble). Simulations were run using an integration time step of 1 fs with neighbor list updates every 10 fs. The MD simulations were carried out using the GROMACS set of programs, version 4.5.5.31 Molecular graphics and visualization were performed using the VMD 1.8.7 software package.36

Figure 2. The size distribution diagram for AuNPs based on the statistical analysis of transmission electron micrograph (TEM) images. According to the TEM imaging statistics, the mean diameter of AuNP was found to be 1.39 ± 0.48 nm, which is in a good agreement with the value of 1.29 ± 0.57 nm estimated alternatively from the Gaussian fitting of the size distribution diagram.

The analysis of the Gaussian fit parameters gave us the particle mean size of 1.29 ± 0.57 nm, which is in a good agreement with the size value of 1.39 ± 0.48 nm resulting from the statistical processing. To estimate the composition of the gold core of AuNPs, the mean number of gold atoms per one particle was calculated using eq 2,37 where NA is the Avogadro number (6.022 × 1023), ρ is the density of gold (19.3 g/cm3), D is the diameter of the AuNPs in centimeters, and M stands for the atomic mass of gold (197 g/mol)

NA πρD3 (2) 6M According to eq 2, the gold nanoparticles with the average diameter of 1.39 ± 0.48 nm should be composed of 85 ± 25 Au atoms. Fluorescence Probing of AuNP. Among a variety of fluorescence probes, those revealing an ESIPT reaction have significant advantage for fluorescence sensing of local structure and solvation dynamics because they can offer both traditional and ratiometric detection principles. In the case of the traditional detection, conventional spectral properties such as position of emission maximum, quantum yield, polarization, or lifetime of a sensing fluorophore changing in response to variations in polarity or viscosity of an environment could serve as an analytical signal. The wavelength-ratiometric method offers, in addition to all the above fluorescence signals, an analytical response estimated from the ratio of two emission intensities measured at two wavelengths, corresponding to those of a normal form (FN) and tautomer (FT) (Figure 1a). Fluorescence spectra of two 3-hydroxychromone derivatives 1 and 2 in toluene are characterized by dual band emission attributed to the normal and tautomeric forms.24 Since the ratio of the integral fluorescence intensities of the two bands depends neither on fluorescent molecule concentration nor on excitation light intensity, a ratiometric signal may, therefore, be n=



RESULTS AND DISCUSSION Structure of Dodecanethiol-Stabilized AuNP. To study the size and structure of the synthesized nanoparticles, the transmission electron micrograph (TEM) imaging was applied. To estimate the spatial dimensions of AuNP, the TEM image characterized with the best particles distribution (the absence of close-packed aggregates and second layer fragments) was selected and analyzed. The image was divided into 6 sectors, so that within each sector no less than 150 nanoparticle sizes in pixels were measured to ensure that the result is statistically representative. The sampling of 1009 particles was further used for statistical analysis, according to which the mean particle diameter was found to be 1.39 ± 0.48 nm. The relatively high statistic vagueness can be primarily explained by an uncertainty 21061

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Figure 3. Fluorescence titration of 1 by AuNPs in toluene: Changes in fluorescence (a) and absorption (b) spectra of 1 observed upon the titration with AuNPs. The arrows indicate the changes of signal intensity which accompany the increase in the concentrations of AuNPs. The concentration of 1 was constant and equal to 3 × 10−7 M, whereas the concentration of AuNPs was varied: 0 M, 7.1 × 10−7 M, 1.3 × 10−6 M, 2.2 × 10−6 M, 3.1 × 10−6 M, 4.3 × 10−6 M, and 9.3 × 10−6 M, respectively. c) The fluorescence spectra normalized to the maximum of the band F2 (see parts a−b for more details). d) The fluorescence decays measured at 420 nm (F1) and 565 nm (F2), respectively.

ratiometric signal changes suggest the existence of noncovalent interactions between the fluorophore molecules and AuNPs, because in the absence of these interactions we could expect the trivial linear decrease in the dual emission intensity keeping the F1/F2 ratio constant. It should also be noted that in the presence of AuNPs, there are no significant spectral shifts in the emissions bands positions, λ(F1) = 420 nm and λ(F2) = 555 nm, respectively. Upon the addition of AuNPs, fluorescence decays of 1 measured at the two wavelengths corresponding to the F1 and F2 bands maxima revealed, however, no changes, as demonstrated in Figure 3d. These findings indicate that there is no nanoparticle-induced fluorescence quenching of the bound fluorophore, which is often observed for other systems.38 Figure 4 shows results of the fluorescence titration of 2 by AuNPs in toluene. Fluorescence experiments with the probe 2 revealed the spectral trends which are similar in many aspects to those observed for 1. The absolute fluorescence intensity of both the F1 and F2 bands was gradually decreased upon adding AuNPs as demonstrated in Figure 4a. However, this behavior could also be explained by the strong absorption of AuNPs appearing at the excitation wavelength and overlapping with the spectral range of the two fluorescence bands of 2 (not shown). The fluorescence spectra, normalized to the intensity of the long-wavelength band F2, also demonstrate ratiometric signal changes as seen in Figure 4b. Therefore, such spectral behavior also provides strong evidence for the binding of the probe 2 to AuNPs. It is interesting that no decrease of fluorescence lifetimes was again observed upon the probe-to-nanoparticle binding, as follows from Figure 4c. The changes in the relative intensities of the short- and longwavelength bands in the fluorescence spectra of 1 and 2 are also consistent with the formation of intermolecular hydrogen

measured in the presence of high concentrations of AuNPs. In the presence of AuNPs we can expect two types of the spectral behavior of the probes: In the first case, if interactions between a probe and nanoparticles are weak, we would not observe any ratiometric changes in the probe fluorescence response, so that the intensity of the both emission bands should decrease proportionally. In the second case, if the probe became bound to the surface of AuNPs, despite an overall decrease in the fluorescence signal, the normal-to-tautomer band ratio should be changed. The latter indicates that the probe became transferred from the bulk solution to the alkylthiol coating of AuNPs. Figure 3a shows the changes in fluorescence spectra of 1 observed in toluene solution in the presence of varying concentrations of AuNPs. As can be seen, the absolute fluorescence signal of 1 was gradually decreased upon adding AuNPs. The decrease in the overall emission of 1 could, however, be explained by the phenomenon of the first-order inner filter effect (absorption of the excitation light by AuNPs). The second-order inner-filter effect (reabsorption of the probe fluorescence by AuNPs) could not be excluded as well, because of the strong growth of the absorption of AuNPs, which takes place in the 3HCs emission spectral range as shown in Figure 3b. It is interesting to note that the decrease of the intensity of the both fluorescence bands was accompanied also with changes in their ratio. As a result, the relative intensity of the F1 band increases gradually with respect to that of the normalized intensity of the F2 one as shown in Figure 3c. This change of the ratio F1/F2 could not be a consequence of the probe fluorescence reabsorption by AuNPs, because the F2 band should be less affected owing of the smaller absorption of the nanoparticles in the long-wavelength range. The observed 21062

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Figure 5. A F1/F2 intensity ratio of the two fluorescence bands versus the concentration of the dodecanethiol ligand plotted for the dyes 1 (a) and 2 (b). To determine the partition coefficient Kp, the ratio data points were fitted to eq 3 (solid red line).

noncovalent binding to micelles, lipid vesicles, or living cells.42 The binding between a dye and AuNPs should be driven by van der Waals and electrostatic interactions that arise from the collective properties of coating ligands covalently bound to the nanoparticle core. The apparent partition coefficient Kp can be determined from the binding curves shown in Figure 5. The coefficient Kp was determined by fitting the ratio Ri of the two fluorescence bands F1 and F2 of the probe to eq 3

Figure 4. a) Fluorescence titration of 2 by AuNPs in toluene. The concentration of 2 was equal to 1 × 10−7 M, and the concentration of AuNPs was varied: 0 M, 8.4 × 10−8 M, 3.1 × 10−7 M, 6.6 × 10−7 M, 9.7 × 10−7 M, 1.6 × 10−6 M, 2.2 × 10−6 M, 3.1 × 10−6 M, 4.2 × 10−6 M, 5.5 × 10−6 M, 7.1 × 10−6 M, and 9.6 × 10−6 M, respectively. b) The fluorescence spectra normalized to the maximum of the band F2. c) The fluorescence decays measured at 420 and 525 nm. The decays are shown with the same color-coding as indicated in parts a-b. Both decays were acquired after pulse excitation by a LED diode emitting at 375 nm.

R i = R 0 + (R max − R 0)

K p[Ligand] [Toluene] + K p[Ligand]

(3)

here R0  F1/F2 bands intensity ratio in the absence of AuNPs, Rmax = the maximal ratio at complete partitioning, [Ligand] = the molar concentration of dodecanethiol ligand in solution, [Toluene] = the molar concentration of bulk toluene (10.9 M), and Kp = the mole fraction partition coefficient.43 In our experiments, Kp was found to be (6.2 ± 1.1) × 104 and (7.0 ± 0.5) × 104 for 1 and 2, respectively. NMR Spectra. The NMR spectra of a freshly prepared sample of AuNPs measured in CDCl3 revealed the presence of trace amounts of residual ethanol (1H: 3.29 ppm broadened quartet, this signal was absent in the neat solvent spectrum; the alcohol contents could be estimated as ∼5−7%mol with respect to the integrated intensity of thiol bound to the gold core high field signal; 13C: 59.34 ppm) and definite quantity of thiol unbound to gold as well (1H: triplet at 2.60 ppm and quintet at 1.60 ppm, nearly the same relative molar amount as ethanol; 13 C: 39.24 ppm). Broad signals observed at 1.1−1.4 ppm and 0.75−0.78 ppm in the proton NMR spectrum correspond to thiol residues covalently linked to the gold core of AuNPs. We do not expect the presence of water molecules in the nanoparticles thiol coating because of the high hydrophobicity of the latter. In any case the water traces would be masked by

bonds of 3-hydroxychromones with protic solvents.39−41 The carbonyl group in 3HCs contains an unshared electron pair capable of interaction with hydrogen-bond donor solvents such as alcohols and water. This hydrogen bonding can lead to the appearance of small changes in absorption and more prominent ones in the fluorescence spectra.41 It has been shown that the ratio of the two emission bands in 3HCs could be used as a measure of the ground state distribution between its intermolecular hydrogen-bonded and non-hydrogen-bonded species.40 To analyze association of the probes 1 and 2 with AuNPs, the ratio F1/F2 is plotted as a function of the concentration of AuNPs as shown in Figure 5. Despite different absolute values of the ratio for probes 1 and 2, both plots revealed the increase in the above ratio which reaches some plateau seen at the high concentrations of AuNPs. This type of a hyperbolic-like curve is typical for the saturation behavior, and it was often observed in titration experiments when fluorescent probes underwent 21063

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Figure 6. An all-atom molecular dynamics model of the dodecanethiol-coated AuNP: a-b) Gold-nanoparticle building scheme: Several initial configurations of AuNPs were built, in which a gold core, composed of ligand-free gold atoms was surrounded by gold-attached dodecanethiol residues. In these configurations a total number of the free (metallic) and ligand attached gold atoms was kept to be 85. c-d) Stable structures of AuNP obtained for the ratio of free-gold and gold-dodecanethiol varied from 47:38 to 35:50. e) The mass density distribution of all gold atoms in the two boundary configurations of AuNP (a ratio of free gold to dodecanethiol-attached gold 47:38 (red solid) and 35:50 (blue dotted)) estimated by MD simulations. The distribution of the gold atoms was averaged along the three spatial axes x, y, and z. Average diameter of the gold core was found to be 1.4 ± 0.4 nm.

In our model, a total number of ligand-free and ligand-attached gold atoms were kept to be about 85, which corresponds to the experimental value estimated for AuNPs with the diameter of 1.4 nm. After constructing a symmetrical initial configuration of AuNP, in which dodecanethiol molecules are spherically located around the gold core, MD simulations of the annealing were employed to ensure a fully sampled distribution of dodecanethiol residues on the core surface of AuNP. Finally, the pre-equilibrated structure of AuNP was placed into a cubic simulation box and solvated by ∼1000 toluene molecules. It should be noted that the Au−Au distance within the gold core are found to be 2.9 ± 0.1 Å which corresponds with the face centered cubic (fcc) packing in the bulk metallic gold.30 To find stable structures, several initial configurations of AuNP were constructed in which a ratio of free-to-bound gold atoms was varied from 60:25 to 25:60. After 20-ns long MD equilibrations, the stable nanoparticle structures were observed for the ratio ranging from 47:38 to 35:50 as shown in Figures 6c and 6d, respectively. To test the agreement between the experimental size of AuNPs estimated from the TEM image analysis and the gold core diameter obtained in our MD simulations, we calculated the mass density distribution of all the gold atoms for the two boundary MD-modeled

the 1.48 ppm signal caused by residual humidity of the deuterated chloroform used for the spectra measurements. Interestingly, that aged nanoparticles, which NMR spectra were measured after a year of their preparation demonstrate no other signals except 1.1−1.3 ppm and 0.75−0.85 ppm in the 1H and 32.04, 29−30.5 (broaden), 22.75 and 14.13 ppm in the 13C NMR spectra. Probably, the trace amount of unbound thiol still present in the organic coating of gold nanoparticles just after their synthesis diffuses to the metallic core and binds covalently to it during the continuous storage. The outlined experiments reveal the possibility for the presence of definite amounts of proton donor impurities in the thiol coating of gold nanoparticles, which could not be removed at the conventional room temperature drying in air during several weeks or even months after their synthesis. Molecular Dynamics Simulations of AuNP. To study interactions between a 3HC dye and a nanoparticle at the supramolecular level, we developed an all-atom molecular dynamics model of dodecanethiol-stabilized gold nanoparticles. The model was based on the all-atom CHARMM27 force field parameter data set.27 To build an initial configuration of AuNP, a free-gold core was surrounded by gold-attached dodecanethiol molecules as shown schematically in Figures 6a and 6b. 21064

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Figure 7. MD simulations of noncovalent binding of the dye 1 to AuNP in toluene solution: Snapshots of 1 bound AuNP composed of Au:AuDodecanethiol with the ratio 35:50 are shown for front, side, and top views, respectively. The dye 1 is drawn using van der Waals representation. For clarity, toluene solvent molecules are not shown.

environment of the dodecanethiol chains, the bound dye was shielded from a gold nanoparticle metallic core. These findings can also explain the small changes in the fluorescence decays of 1 and 2 observed upon the binding of these dyes to AuNPs as shown in Figures 3d and 4c. To verify our hypothesis that the synthesized alkylthiolcoated AuNPs may still contain some residual amounts of ethanol molecules, which could be trapped into the hydrophobic shell during the nanoparticle preparation (upon washing-off the thiol excess), we have performed MD simulations mimicking the final steps of the synthesis of AuNPs. To simulate interaction and trapping of ethanol molecules into the nanoparticle thiol shell we first performed MD simulations of AuNP solvated in bulk ethanol. AuNP was composed of metallic crystalline gold and gold-dodecanethiol with the ratio of 35:50. Second, the equilibrium structure of AuNP in ethanol was used to prepare a two phases system composed of ethanol and toluene, as shown in Figure 8(lef t). Finally, slow transfer of AuNP from polar ethanol solvent to nonpolar toluene was simulated using an MD umbrella sampling. During the sampling, AuNP was slowly transferred from ethanol to toluene under action of the “umbrella” sampling potential: a harmonic potential of 100 kJ mol−1 nm−2 was applied along the vertical axes z and the pulling rate was 0.02 nm/ps. After 10 ns of the MD simulation sampling, which should reproduce the transfer of AuNP from ethanol to toluene, some ethanol molecules could indeed be trapped into a dodecanethiol shell of the gold nanoparticle, as can be seen from Figure 8(right). These ethanol molecules, bound to AuNP, were also transferred to the nonpolar toluene phase, so that they can, in principle, remain trapped in the nanoparticle after further synthetic procedures. Depending on the pulling rate, about 20−30 ethanol molecules could be trapped, so that an ethanol:dodecanethiol ratio is about ∼0.5:1. Some of these trapped ethanol molecules would be removed at further roomtemperature drying, while a definite amount of them will remain noncovalently bound in the solid AuNPs powder sample and, therefore, they might be the reason for the detected unexpected anomalously high H-bonding ability of the latter.

configurations of AuNP, which are opposite in terms of a ratio of free gold to dodecanethiol-attached gold atoms and varied from 47:38 to 35:50. Taking into account a spherical shape of a nanoparticle, the mass distribution of the gold atoms was also averaged along three spatial axes. Figure 6e shows that the calculated mass density profiles were very similar for the nanoparticles of two different compositions 47:38 to 35:50, so that for both AuNPs types the average diameter of the gold core agreed with the experimentally found value ∼1.4 ± 0.4 nm. To study noncovalent binding and favorable localization of the dye 1 on AuNP, we applied unconstrained MD simulations based on free distribution of the probe molecule between bulk toluene and AuNP. First, we placed one molecule of the probe 1 in bulk solution at the vicinity of a nanoparticle surface. To achieve better MD statistics, several initial configurations were constructed in which the probe was placed at random locations and orientations with respect to the nanoparticle. To remove high energy contacts, these systems were equilibrated at NPT conditions for 2 ns. After the initial equilibration period, binding interactions between the probe molecule and AuNP were studied using a series of unconstrained MD runs with a total simulation time of 50 ns. These independent MD runs, started from different initial locations of the probe with respect to a nanoparticle surface, showed that finally the probe molecule become bound to AuNP. We have found that the probe molecule favors to move from bulk solution toward the nanoparticle interface in the majority of the MD runs. When the probe became bound to AuNP, it tends to be buried deeper into the hydrophobic region of a dodecanethiol shell of the nanoparticle. However, we also observed that, after the initial binding of the probe to AuNP, it was able to leave the nanoparticle and diffuse back to bulk toluene in about 20% of the MD runs. Therefore, the binding mode of 1 to thiol-coated AuNP could be characterized as “weak binding”. Figure 7 shows MD snapshots of the stable configurations of dye 1 bound to AuNP shown at different viewing points. As can be seen from Figure 7, the probe molecule becomes bound to the outer interfacial region of the nanoparticle, so that the probe orients itself with a long molecular axis lying roughly parallel to the AuNP surface. The analysis of the bound geometries shows that both the chromone and diphenyloxazole moieties favor binding to the nanoparticle, so that no preferable residence of these moieties in the dodecanethiol shell was detected. We also found that, due to the dense and crowded



SUMMARY AND CONCLUSIONS Fluorescence labeling of gold nanoparticles (AuNPs) has broad perspectives for their biological and biomedical applications. One difficulty in designing new nanomaterials coated with dye21065

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fluorescence decays of the bound form of 1 and 2, measured at the emission peaks maxima of the normal and tautomer bands, have been revealed. The latter indicates that there is no nanoparticle-induced fluorescence quenching of the fluorophore bound to the nanoparticle organic coating. To study the dye binding at the supramolecular level, we have combined our experimental fluorescence study with a series of classical MD simulations of the dye 1 binding to a dodecanethiol-protected gold nanoparticle. First, we have developed an all-atom MD model of AuNP and a dye molecule using available parameters for the CHARMM27 force field.28 Second, we simulated the free passive diffusion and distribution of the probe 1 between bulk toluene solution and a ligandprotected nanoparticle. We have found that the probe molecule favors the residence in the outer interfacial region of the nanoparticle. The unexpectedly high hydrogen bonding ability of the thiolcoated gold nanoparticle surface observed in our titration experiments was interpreted as a result of ethyl alcohol molecules trapped into the thiol shell upon the preparation of AuNPs. The NMR experiments and MD modeling revealed the high probability for such phenomenon. Thus, freshly synthesized thiol coated gold nanoparticles could not be considered as strongly hydrophobic environment, as expected according to the conventional insights of their structure. Finally, the results obtained in this work point out that the noncovalent binding of fluorescent dyes of the 3-hydroxychromone series to a ligand-protected nanoparticle opens up possibilities for their easy and express fluorescence labeling. On the other hand, the lack of the strong fluorescence quenching of organic dye molecules in the outer shells of thiol coating gives the promise for their use for covalent chemical bonding to AuNPs.

Figure 8. Molecular dynamics simulations of transfer of the dodecanethiol coated AuNP from ethanol to toluene modeled by using an umbrella pulling potential. The MD cell was filled by bulk ethanol and toluene solvents molecules placed at upper and lower parts of a simulation cell, respectively. AuNP was first solvated in ethanol, and it then was slowly moved to toluene under action of the umbrella sampling potential (red arrow). Lef t: The initial configuration of the system (t = 0 ns) in which AuNP was placed in ethanol. Toluene molecules were not shown for clarity. Right: The final configuration of the system in which AuNP was transferred from ethanol to toluene. It should be noticed that, after the transfer, some ethanol molecules (magenta) were trapped into a dodecanethiol shell of a nanoparticle.

functionalized ligands is the lack of reliable spectroscopic and imaging techniques for in situ investigation of interactions between a dye and a nanoparticle. The utility and effectiveness of these composites are predicated by the design of stabilizing ligands and by the separation distance within the dye-tonanoparticle core architecture. This is why environmentsensitive fluorescence dyes are a powerful tool for studying the structure and morphology of AuNPs as well as ligand packing of their coating layer.4 In this work, we have synthesized small dodecanethiolprotected gold nanoparticles of 1.39 ± 0.48 nm diameter. To elucidate the possibility for noncovalent labeling of a nanoparticle by fluorescent dyes, we have investigated the emission properties of the two dyes, 2-[4-(5-phenyloxazol-2yl)phenyl]-3-hydroxychromone (1) and 2-(benzimidazol-2-yl)3-hydroxychromone (2) (Figure 1b), at the presence of alkylthiol-coated gold nanoparticles using steady-state and time-resolved emission spectroscopy. Our experiments based on the steady-state fluorescence titration of the dyes by the nanoparticles have shown the changes in ratiometric spectral response occurring upon intensity redistribution of the two emission bands F1 and F2 assigned to those of the normal and tautomer forms of 1 and 2. The absolute intensity of the both fluorescence band in toluene solution was decreased in the presence of AuNPs with the accompanying relative increase in the intensity of the short-wavelength band with respect to the long-wavelength one. These changes could not be explained by the trivial second order inner-filter effect (reabsorption of dye emission by the nanoparticles) and, therefore, should be interpreted in the terms of the noncovalent binding of the dyes to the AuNP surface demonstrating, however, its definite proton donor ability. In addition, no changes in the



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AUTHOR INFORMATION

S Supporting Information *

The molecular dynamics topology file of gold-dodecanethiol and PDB and GROMACS coordinate files for equilibrated dodecanethiol-coated gold nanoparticles in toluene. This material is available free of charge via the Internet at http:// pubs.acs.org. Corresponding Author

*Phone: (+38)-057-707-5335. E-mail: alexander.v. [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS A.K., G.V.K., D.S., D.K., and A.O.D. acknowledge financial support by the Swiss National Science Foundation − SCOPES 2009-2012 Program (Project No. IZ7320_27864/1). The authors express their gratitude to Mr. A. Pinto and Prof. E. Vauthey (Université de Genève, Genève, Switzerland) for help with the NMR measurements.



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