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Rhodamine B-Coated Gold Nanoparticles as Effective“Turn-On” Fluorescent Sensors for Detection of Zinc IIIons in WaterDaniela Simona Tira a , Monica Focsan a , Sorin Ulinici b , Dana Maniu a & Simion Astilean aa Nanobiophotonics Center, Institute of Interdisciplinary Research in Bio-NanoSciences andFaculty of Physics, Babes-Bolyai University, Cluj-Napoca, Romaniab S.C. ICPE, Bistrita, RomaniaAccepted author version posted online: 10 Apr 2013.

To cite this article: Spectroscopy Letters (2013): Rhodamine B-Coated Gold Nanoparticles as Effective “Turn-On” FluorescentSensors for Detection of Zinc II Ions in Water, Spectroscopy Letters: An International Journal for Rapid Communication, DOI:10.1080/00387010.2013.782557

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1

Rhodamine B-coated Gold Nanoparticles as Effective “Turn-on” Fluorescent

Sensors for Detection of Zinc II Ions in Water

Daniela Simona Tira1, Monica Focsan1, Sorin Ulinici2, Dana Maniu1, Simion Astilean1,

1Nanobiophotonics Center, Institute of Interdisciplinary Research in Bio-NanoSciences and Faculty of Physics, Babes-Bolyai University, Cluj-Napoca, Romania 2S.C. ICPE,

Bistrita, Romania

Prof. dr. Simion Astilean, Babes-Bolyai University, Institute of Interdisciplinary Research in BioNanoSciences and Faculty of Physics, Nanobiophotonics Center,

Treboniu Laurian 42, 400271, Cluj-Napoca, Romania Tel.: +40 264 454554/+40 264 405300; Fax: +40 (264) 591906; E-mail: simion.astilean@phys.ubbcluj.ro

Abstract We design a Fluorescence Resonance Energy Transfer system consisting of fluorophore Rhodamine B and gold nanoparticles for sensing of zinc ions in aqueous solution. The electrostatic attraction between positively charged N-atoms in Rhodamine molecules and negatively charged citrate corona of gold nanoparticles leads to substantial fluorescence quenching. However, the quenching is switched off in the presence of zinc ions ions and therefore the system can be used as effective “turn-on” fluorescence sensor. UV-Vis absorption, fluorescence spectroscopy and transmission electron microscopy were used for sensor evaluation. The approach of “turn-on” fluorescence proves a real potential for sensing metallic ions in water. KEYWORDS: Gold nanoparticles, Zn2+ ions, fluorescence, FRET

1. INTRODUCTION

The detection of metallic ions in aquatic ecosystems, drinking waters and biological

systems is a problem of major interest due to the serious health problems that such

species can cause to living organisms. The fluorescent methods implemented for

detection of metal ions require a combination between an organic molecule whose

mission is to attach to the target compound (metal ion) and a fluorescence probe which

provides the fluorescence signal. The recognition event is translated into a change in

intensity of fluorescence signal, fluorescence lifetime, or a shift of fluorescence

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wavelength. In principle, fluorescent chemosensors satisfy these requirements and could

be particularly appropriate for detection of various metallic ions [1,2]. However the

approach of fluorescent chemosensors based exclusively on organic compounds has

several limits, including complicated synthetic procedures, poor water solubility and loss

of activity caused by fluorophore photobleaching. Gold nanoparticles (GNPs) could

represent promising alternatives to various organic molecules used as scaffold units in

fabrication of fluorescent chemosensors. This is because GNPs exhibit localized surface

plasmon resonance (LSPR) of large extinction coefficient, high stability under

illumination and “super quenching” properties against nearby fluorophores - which allow

their integration in so-called “turn-on” fluorescence sensors [3]. So far several

spectroscopic techniques based on GNPs have been demonstrated for detection of metal

ions, ranging from the exploitation of LSPR [4–7] and nonlinear optical properties [7] to

surface-enhanced Raman scattering (SERS) [8,9] and fluorescence spectroscopy [10,11]. For

example, Lin et al. reported several functional GNPs based sensors that allow the

sensitive and selective detection of metal ions through colorimetric and fluorescence

changes [12]. Wang et al. reported a sensitive GNPs-amplified SPR sensor for the

detection of mercury ions in aqueous media [13]. Xu et al. revealed that the association of

dye molecules onto the surface of metallic nanoparticles leads to formation of organic-

inorganic hybrid moieties which have numerous applications in light energy conversion,

cell imaging or detection of molecules of different substances [14]. Makarova et al. [15] and

Templeton et al. [16] modified the surface of GNPs using fluorescein isothiocyanate,

demonstrating that dye molecules chemisorbed on the gold nanoparticles surface do not

induce any aggregation.

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In this work, we report a sensitive GNPs-based nanosensor for the detection of zinc

(Zn2+) ions in water. The concept is based on the substantial fluorescence quenching of

Rhodamine B (RhB) molecules after dye adsorption onto GNPs surface. If target are Zn2+

ions in solution, as a result of interaction between GNPs and Zn2+ ions, the RhB

molecules are desorbed from the metal surface and the fluorescence recovered. The

analysis of fluorescence intensity changes provides valuable information on the

interactions between GNP, fluorescent probe and target compound. Finally we

demonstrate that the system RhB-coated GNPs can be used as effective “turn-on”

fluorescence sensor for precise dosing of Zn2+ ions in water.

2. EXPERIMENTAL SECTION

2.1. Chemicals

Tetrachloroauric acid (HAuCl4 · 4H2O, 99.99%), sodium citrate (C6H5Na3O7), and zinc

acetate ((C2H3O2)2Zn) were obtained from Aldrich. Rhodamine B (C28H31CIN2O3) was

purchased from Sigma. All reagents used through the experiments were of analytical

grade and used without any purification. All glassware and magnetic stir bars were

cleaned in aqua regia solution (HCl:NO3 3:1) and then rinsed with deionized water.

Ultrapure water (resistivity of 18 MΩ cm) was used for preparation of colloids and all the

other samples.

2.2. Synthesis Of Gold Nanoparticles

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Spherical GNPs were synthesized according to Turkevich-Frens method by

aqueous reduction of HAuCl4 with sodium citrate where the citrate ions act as both

reducing agent and a capping agent [17,18]. Briefly, 500 ml of 10-3 M HAuCl4 was brought

to boil on a heat plate, with vigorous stirring. 10 ml of 38.8 mM sodium citrate was added

all at once and the stirring and boiling continued for 15 min. The solution was then

removed from heat and kept stirring another 15 min. After the colloid got cold the

volume was adjusted to 500 ml by adding deionized water. Before being mixed with RhB

solution, as prepared colloidal GNPs were separated from the reactive solution by

centrifugation (6000 rpm, 10 minutes) and resuspended in ultra pure water. The molar

concentration of GNPs was determined from measured optical absorbance by the means

of Beer-Lambert’s law. The transmission electron microscopy (TEM) examination was

performed by drop-drying the colloidal solution on carbon-coated copper grids at room

temperature.

2.3. Samples Preparation

Stock solutions of RhB (5·10-4 M) and zinc acetate (25·10-3 M) were prepared in ultrapure

water of 18 MΩ cm resistivity. To analyze the fluorescence quenching of RhB as

function of GNPs concentration, various volumes (0.2, 0.4, 0.6, 0.8, 1 ml) of GNPs stock

solution (0.3 10-8 M) were added to RhB solution to a final concentration of 1.87·10-6 M.

A similar procedure was used to analyze the fluorescence recovery of RhB as function of

increasing concentrations of zinc acetate in RhB-coated GNPs solution.

2.4. Instruments

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UV-Vis absorption measurements were carried out using a Jasco V-670

spectrophotometer. Fluorescence spectra were collected using a Jasco LP-6500

spectrofluorimeter. All fluorescence measurements were recorded in the right angle cell

geometry (10 mm cell), under the same excitation conditions, specifically bandwidths of

1 nm in excitation and 3 nm in emission and excitation wavelength set at 532 nm. TEM

images were obtained with a JOEL model JEM 1010 microscope.

3. RESULTS AND DISCUSSION

3.1. Spectroscopic Characterization Of The Interaction Between Rhb Molecules And

Gold Nanoparticles

Rhodamine B was chosen here as fluorescencent dye due to its suitable photophysical

properties in water, including a high fluorescence quantum yield (0.71), high extinction

coefficient (>75,000 cm−1 M−1) and good photo-stability [19,20]. In neutral solution RhB is

a zwitterion due to the deprotonation of the carboxylic group at the benzene ring [21] and

therefore would tend to interact with negatively charged GNPs. The UV-Vis absorption

spectrum of RhB molecules in solution (see Fig. 1A, curve a) presents a strong absorption

band at 556 nm, characteristic to the π-π* electronic transition of monomers, and a weak

band at 520 nm assigned to the dimeric states of RhB molecules. It is known that in

aqueous solutions, the monomers are in equilibrium with dimers and the molar fraction of

the dimers varies with RhB concentration [21,22]. In our case for a concentration of about

10-6 M, the feature of absorption spectrum suggests that most of RhB molecules should

exist as monomers.

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The colloidal solution of citrate-capped GNPs exhibits a well-resolved extinction band at

521 nm (Fig. 1A curve b) assignable to LSPR of individual spherical nanoparticles of

18±2 nm diameter, as given by TEM analysis (see representative TEM pictures in Fig.

1B). The molar concentration of GNPs in solution (cGNP= 0.3 10-8 M) was calculated by

using Beer-Lambert’s law from measured absorbance of 0.8 and molar extinction

coefficient of ε521 = 2.6 108 cm-1 M-1 given in literature [22]. It is well known that the

spectral position and shape of LSPR band of GNPs is strongly dependent on

nanoparticles size and shape as well as local surrounding medium and inter-particles

distance [23]. For instance, upon adding 1 ml RhB solution of 10-5 M concentration to

same volume of colloidal solution (1ml), a new plasmonic absorption band at about 639

nm is observed (Fig. 1A curve c). This new emerging band together with the drastic color

change of the mixture solution – from original red to violet blue (see insert in Fig. 1) -

represents the hallmark of electromagnetic coupling of nanoparticles with formation of

small aggregates (Fig. 1A, curve c). The main plasmonic band decreases and shifts

slightly to red as result of dielectric changes around nanoparticles [24]. The induced

aggregation of citrate-capped GNPs is further confirmed by TEM analysis. The TEM

pictures recorded before (Fig. 1B) and after (Fig. 1C) addition of RhB molecules clearly

prove the partial aggregation of GNPs in the form of linear and branched chains, as result

of dye adsorption. We can admit that RhB molecules are adsorbed onto the GNPs surface

due to the electrostatic attraction between positively charged N-atoms in RhB molecules

and negatively charged citrate corona of GNPs. More specifically, it was established that

RhB and citrate molecules of the gold NPs shell can form a hydrogen bond between

carboxyphenyl group of RhB and one of the carboxyl groups of citric acid, while another

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carboxylate group of citric acid interacts with the N+ center of the pyronine group of

RhB, forming a scissors-like structure [25]. A spherical GNP of 18 nm in diameter

exhibiting a surface area of approx. 1017 nm2 can accommodate at least 1000 molecules

of RhB on its surface, considering an uniform distribution of molecules with a molecular

surface “print” at about 1 nm2. Within this assumption, the minimum number of RhB

molecules necessary to coat all GNPs in a specified sample can be estimated. As for

example for coating a number of Nnanoparticles = NAvogadro·c =6.023·1023·10-8= 6·1015

nanoparticles/L a number of about nRhB= 6·1018 RhB molecules /L are necessary, which

is consistent with the molar concentrations used in this investigation (cRhB = nRhB/NAvogadro

= 6·1018/6.023·1023 = 0.996·10-5≈ 1·10-5 M).

The likely reason for the formation of nanoparticle chains should be the nonuniform

distribution of RhB molecules on the surface of gold nanoparticles, which makes that

bare area on the surface of nanoparticles can be attracted against area covered by the RhB

from other nanoparticles. Previous studies have demonstrated the possibility of GNPs

coverage by a dye shell as result of increasing number of RhB molecules in solution [25].

On the contrary to the process of aggregation observed above, large GNPs aggregates

with no particular form were previously observed with eosin molecules [26].

Previous studies demonstrated also that electroactive molecules within dye solutions of

high concentrations can associate onto the solid substrate and their relative orientation

determine two different types of dimeric structures. Parallel arrangements determine the

appearance of H-aggregates in the form of vertical stacks. This type of aggregates yields

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a blue shift of the absorption band of the dye. The other type of dye association, named J-

aggregates, have a “head-to-tail” arrangement and exhibit a red shift of the absorption

band relative to the monomer band [27]. Comparing the extinction spectrum (spectrum c in

Fig 1) of the mixed solutions with the extinction spectrum of free RhB (spectrum a) the

absorption band of the dye is blue-shifted by 3-4 nm. The adsorption of RhB molecules

from solution leads to a concentration of molecules onto the surfaces of GNPs and this

local concentration can exceed the equivalent solution concentration for formation of

dimers and aggregates. Therefore, although RhB concentration in solution is relatively

small, we can not exclude the formation of some patches of H-aggregates onto the

surface of GNPs.

Next we study the fluorescence of RhB molecules in the presence of GNPs. The

fluorescence intensity of RhB is strongly reduced upon the addition of GNPs, as seen in

Fig. 2. The electrostatic interaction between RhB and the citric acid corona of

nanoparticles as well as the formation of hydrogen bonds does not suppose an important

change in the electronic transition moment of xanthene group of RhB molecules.

Therefore we exclude any important effect in the reduction of fluorescence quantum yield

as direct result of molecule-molecule interaction (RhB - citric acid corona). On the other

hand, although RhB concentration in solution is relatively small, the accumulation of dye

on the surface of GNPs with formation of RhB dimers and aggregates can not be totally

excluded. Therefore it is conceivable that molecules in dimers and aggregates could

contribute to partial decrease of fluorescence signal. However, the main effect on

quantum yield decrease should be correlated to Fluorescence Resonance Energy Transfer

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(FRET). This is because for a constant concentration of RhB molecules in solution the

quenching increases drastically with the amount of GNPs, as illustrated in Fig.2.

Additionally, it is important to note that RhB molecules show a strong fluorescence

emission band centered at 577 nm which exhibit good overlapping with the absorbance

band of aggregated GNPs – a pre-requisite condition for an efficient resonance energy

transfer. Indeed, it is expected that the excited-state energy from the fluorophores to be

transferred to the nanoparticle through a non-radiative process, named resonance energy

transfer (RET) or FRET. The FRET can occur only when there is a sufficient overlap

between the absorption band of acceptor (here GNPs) and the fluorescence band of donor

(here RhB molecules) and the molecule are in close vicinity, as the efficiency of FRET

depends on the inverse sixth power of the distance of separation between the donor and

acceptor. This FRET occurs via an induced-dipole-dipole interaction when the dye

molecule exchanges energy with the GNP of similar resonance wavelength without

requiring of photon emission [28]. By plotting the ration (F0/F) vs. GNPs concentration,

where F0 and F represent the fluorescence intensity of RhB solution in the absence and

presence of GNPs, respectively, a sloping curve was obtained (see inset in Fig. 3),

achieving thus some information about the quenching mechanism of RhB fluorescence.

The obtained plot in our case is not linear, indicating that the RhB molecules exhibit a

faster quenching than resulted from a pure static mechanism [28,29]. We should exclude the

possibility of dynamic collisional quenching mechanism because of permanent and strong

interaction existing between molecules and nanoparticles.

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The deviation from linearity can be modeled by the extended model of classical Stern-

Volmer equation presented bellow [30], where K represents the binding equilibrium

constant, Qtot is the total concentration of active sites on the nanoparticle quenchers and

β is the dye fluorescence intensity factor (β = F/cRhB):

+

+=

βKF

KQFF tot

110

The upward deviation from the standard linear behavior can be explained by the

contributions of several factors, among them the fact that a fraction of RhB molecules are

captured in between nanoparticles as result of aggregation. Therefore, it is conceivable

that the rate of nonradiative decay of molecules located at the interstice between

nanoparticles should be higher that of molecules adsorbed directly onto the surface of

individual nanoparticles. Moreover, the spectra of RhB and GNPs aggregates overlap

completely which in turn increases the energy transfer efficiency in the system. This

could explain the accelerated static quenching as function of GNPs concentration and the

existence of non-Forster resonance energy transfer mechanism from RhB molecules to

GNPs. We admit also that the direct energy transfer (without emitted photons) can occur

simultaneously with effects like inner filter effect and reabsorbing effect. However FRET

is the only effect which involves a true quenching mechanism by increasing the non-

radiative rate of de – excitation and induce modification of fluorescence lifetime without

emission of photons. The re-absorption of emitted photons does not impact on the excited

state of emitter molecules (i.e. fluorescence lifetime) but on the intensity of collected

signal. It is conceivable that a number of molecules can accumulation on the surface of

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GNPs with formation of dimers and aggregates which could contribute to a decrease of

fluorescence signal (chemical quenching). To correct for the formation of dimers in

solution, although RhB concentration is relatively small, all spectra presented in Figs 2

and 3 were corrected by normalization to the spectrum of free RhB solution (reference

spectrum).

Assuming the value of quantum efficiency of fluorescence for free RhB in water (i.e

φ0=0.71) [19,20], we also calculate the quantum efficiency of RhB fluorescence in the

presence of GNPs as follows below, where F0 and Fq are the fluorescence intensities of

RhB molecules in the absence and in the presence of GNPs respectively, k is the

correction factor for chemical quenching (formation of dimers) on the surface of GNPs

due to high local concentration of the dye:

( )q q 0 0φ F / F k φ=

The calculated values for φq using different concentrations of RhB were between 0.011

and 0.045, that confirms the high quenching effectiveness of the fluorescence of RhB by

GNPs.

3.2. Turn-On Fluorescence For Sensing Zn2+ Ions Based On Rhb-Coated Gnps System

Next, we evaluate the sensitivity in detection of Zn2+ by “turning-on” the fluorescence

emission of RhB molecules in RhB-coated GNPs hybrid system.

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As mentioned above, while the free RhB molecules are highly fluorescent in solution, the

fluorescence emission of RhB adsorbed onto GNPs surface is quenched via energy

transfer. However, as we can see in Fig. 4, in the presence of Zn2+ ions, the fluorescence

intensity of RhB molecules recovers gradually with the increase of added Zn2+ ions. This

means that RhB molecules are released from the GNPs’ surface, restoring their

fluorescence and proving the successful demonstration of a “turn-on” fluorescence sensor

for the detection of Zn2+ ions. The detachment of RhB molecules from the surface of gold

nanoparticles is triggered by a stronger affinity of Zn2+ cations toward gold surface and

citrate corona, although the mechanism of detachment is not clear.

The release of RhB molecules from the surface of GNPs after mixing RhB-coated GNPs

with zinc acetate was studied by UV-vis spectroscopy and TEM analysis. Firstly, the

extinction spectrum indicates a better spectral separation of the “fingerprint” band of free

RhB molecules at 556 nm from the red-shifted plasmonic band corresponding to GNPs

aggregate (see Fig 4, spectrum b). With increasing concentration of zinc ions added to

solution, the LSPR bands increases also in intensity and features larger and lager

aggregates.

The TEM image in Fig 4 shows GNPs aggregates which exhibit size and form

particularly different relative to aggregates existing in solution before adding Zn+2 ions.

In fact the addition of metallic ions modifies the ionic strength of colloidal solution and

this induces accelerated aggregation as result of dramatic change in charge surface of

GNPs after chemical displacement of RhB.

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Finally, it is important to point out that the normalized fluorescent signal recovery [(Fon-

Foff)/F0] follows a simple linear dependence as function of Zn2+ ions ions concentration

(see inset in Fig. 3). The calibration equation y(x) = 0.0097 + 0.02x can be used to

quantify Zn2+ ions ions concentration as low as 0.05 mg / L. The limit of detection was

calculated by considering a ratio of 3 between the minimum spectrometer signal which

can be measured and the noisy level (S/N = 3). The concentration of zinc in drinking

water ranges from 0.01 to 0.1 mg/L which fits rather well with the sensitivity of detection

in the above experiments [31]. However, higher concentrations of Zn2+ ions could occur

accidentally in drinking waters as result of water treatment and galvanized pipes and

tanks used in distribution systems. The beverages may also contain high levels of zinc

ions if stored in metal containers coated with zinc to resist rust. US Environmental

Protection Agency (EPA) has stated that drinking water should contain no more than 5

mg/L (or 5 ppm) because of taste [31]. This means that the RhB-coated GNPs system

studied here can be used as reliable probe for quantification the level of concentrations of

Zn ions in various water sources.

4. CONCLUSIONS

In summary, a simple and rapid method based on “turn-on” fluorescence has been

demonstrated for real time detection of Zn2+. The fluorescence recovery shows that Zn2+

can be detected rapidly within a low detection limit. The FRET platforms based on

similar conjugated systems can be extended to other analytes as different metals exhibit

different stability constants in water. The analysis we performed here will facilitates the

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development of similar detection systems toward discrimination between metallic ions

and their detection in real samples collected from rivers or tap water.

ACKNOWLEDGEMENTS

This work was supported by CNCSIS–UEFISCSU, project number

PNII_ID_PCCE_312/2008

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Figure. 1 (A). UV-Vis absorption spectra of RhB solution (a), GNPs solution (b), and 1:1

mixture between RhB and GNPs solutions (c). Inset shows the photographic images of

RhB solution before and after mixing with GNPs. (B). TEM image of GNPs; (C). TEM

image of RhB-coated GNPs.

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Figure. 2. Evolution of normalized fluorescence spectra of RhB solution as function of

increasing amounts of GNPs: 200 µl (b), 400 µl (c), 600 µl (d), 800 µl (e), and 1 ml (f).

Spectrum (a) represents the fluorescence of RhB solution free of GNPs. The inset shows

the relationship between (F0/F) ratio and GNPs concentration. The spectra were

normalized to fluorescence spectrum of free RhB solution (spectrum a). Excitation

wavelength: 532 nm.

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Figure. 3. Recovery of fluorescence emission in RhB-coated GNPs system as function of

Zn2+ ions concentration: 1mM (b), 5mM (c), 12.5mM (d) and 25 mM (e). The inset

shows the linear plot of fluorescence ratio (Fon-Foff)/F0 as a function of Zn2+ ions

concentration, where F0 is the fluorescence intensity of pure RhB solution (reference

spectrum (f)), Foff is the “turn-off” fluorescence intensity corresponding to RhB-coated

GNPs solution as recorded before adding Zn2+ ions (spectrum (a)) and Fon is the current

“turn-on” fluorescence intensity of RhB-coated GNPs solution as measured at different

concentrations of Zn2+ ions (spectra (b), (c), (d), and (e)). Excitation wavelength: 532

nm.

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Figure. 4. UV-Vis extinction spectra recorded before (curve a) and after (curve b) RhB

molecules displacement from the surface of GNPs. The reference spectra of pure Zn

acetate and RhB solutions are given in curve (c) and curve (d), respectively. Inset shows

a representative TEM picture of GNPs aggregates produced after RhB displacement.

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