Delayed Fluorescence of Dyes Sensitized by Eu3+ ... - ACS Publications

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Delayed Fluorescence of Dyes Sensitized by Eu3+ Chelate Nanoparticles Leonid Yu. Mironov,*,† Peter S. Parfenov,† Anna V. Shurukhina,‡ Yaroslav I. Lebedev,† and Anastasiya A. Metlenko† †

ITMO University, 49 Kronverkskiy Prospekt, Saint-Petersburg, 197101 Russia Laboratory “Photoactive Nanocomposite Materials”, Saint-Petersburg State University, Ulyanovskaya Street 1, Peterhof, Saint-Petersburg, 198504 Russia

‡

S Supporting Information *

ABSTRACT: Dye-doped nanoparticles of Eu3+ chelate complexes with naphtoyltrifluoroacetone and 1,10-phenanthroline were synthesized using two different reprecipitation techniques. The nanoparticles were characterized by atomic force microscopy, absorption spectroscopy, steady-state, and timeresolved fluorescence spectroscopy. Fluorescent spectroscopy of chelate nanoparticles doped with Oxazine 170 and Nile blue dyes indicates that a single dye molecule efficiently quenches luminescence of more than a hundred chelates. The Eu3+ chelates have a significantly longer luminescence lifetime than organic dyes, which leads to the appearance of delayed dyes fluorescence with microsecond lifetimes. The fluorescence brightness of dye-doped chelate nanoparticles was determined to be 50 times higher than that of a single Rhodamine 6G molecule. The combination of high fluorescence brightness and long fluorescence lifetime of the dye-doped chelate nanoparticles is promising for time-gated applications.

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chelates were incorporated into silica12,13 and polymeric NPs14 to enhance photostability and luminescence brightness. Silica has no absorption in the near-UV region, and it plays the role of an inert container that takes many chelates into its structure providing more brightness per particle. The same functionality is usually provided by a polymeric host that incorporates Ln3+ chelates. Recently, Eu3+ chelates were incorporated into NPs of conjugated polymers.15,16 Conjugated polymers efficiently absorb UV light and act as energy donors for Eu3+ chelates providing better luminescent brightness per particle compared to silica NPs. Moreover, NPs consisting of only Eu3+ chelates were prepared by the reprecipitation technique.17−19 Analogous to time-resolved immunoassays, the long lifetime of Eu3+ luminescence is utilized in timeresolved fluorescence imaging to overcome the autofluorescence of cells. The choice of Ln3+ ions with visible luminescence is restricted in the case of organic complexes. Only Eu3+ and Tb3+ ions have sufficient energy gaps to minimize quenching by high-frequency vibrations of organic ligands, although the luminescence quantum yield was still lowered by adjacent water molecules.20,21 The possibility of two-color imaging with two-photon excited Eu3+ and Tb3+ chelates was demonstrated.22

INTRODUCTION Fluorescent nanoparticles (NPs) are widely used as fluorescent labels and probes for biomedical applications.1 Significant efforts are directed toward the development of both organic2 and inorganic3 NPs. Although the composition and optical properties of different NPs vary greatly, researchers usually aim to design brightly fluorescent NPs that can provide high signalto-noise ratio. In this paper, we have studied NPs composed of Eu3+ chelates and doped with fluorescent dyes. Ln3+ chelates are well-known luminescent materials that possess several useful properties such as sharp luminescence peaks, long luminescence lifetime, and strong near-UV absorption.4 The first observation of the Ln3+ luminescence excited by the energy transfer from organic ligands was reported in 1942 by S.I. Weissman.5 He studied Ln3+complexes with organic ligands and observed characteristic luminescence of Eu3+, Sm3+, and Tb3+ under excitation via absorption bands of ligands. Possible applications of highly luminescent Ln3+ chelates have attracted much attention toward the design and characterization of new chelate compounds. Ln3+ chelates were applied in time-resolved immunoassays.6,7 These analyses utilize long luminescence lifetimes of Ln3+ to achieve higher signal-to-noise ratio. Significant efforts were put to shift the excitation region of Eu3+ chelates toward the visible spectrum8,9 as the visible light excitation is less harmful for biological objects and provides lower autofluorescence of cells and tissues. Different hybrid materials containing Ln3+ chelates were synthesized to provide additional functionality.10,11 Eu3+ © XXXX American Chemical Society

Received: April 18, 2017 Revised: July 22, 2017

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DOI: 10.1021/acs.jpcc.7b03648 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

Figure 1. (a) Chemical structure of the Eu(NTA)3phen chelate, Ox-170, and NB. (b, c) Representative AFM image of M1 chelate NPs with particle height histogram. The particle height histogram is based on a series of AFM measurements.

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In this paper, we study colloidal NPs composed of Eu3+ chelates and doped with fluorescent dyes. The possibility of using Ln3+ chelates as energy donors for organic dyes has been shown both for Eu3+ and Tb3+ chelates.23,24 Polymeric NPs doped with Eu3+ chelates have been used as energy donors for dyes in immunoassays.25 Förster resonance energy transfer (FRET) between Eu3+ chelates and dyes shifts the fluorescence spectra of NPs toward longer wavelengths and opens the way to tune the fluorescent properties of NPs. Dye-doped chelate NPs composed of the Eu3+ chelate complexes with methoxybenzoyltrifluoroacetone, pivaloyltrifluoroacetone, and naphtoyltrifluoroacetone (NTA) have been investigated previously.26 The formation of Eu3+ chelates followed by their agglomeration in NPs occurred directly in the water solution of europium nitrate after the addition of organic ligands. The spontaneous formation of chelate complexes yields inhomogeneous chelates of unknown composition. In this study, a new method of dye-doped chelate NP synthesis, based on the reprecipitation of presynthesized chelates, has been introduced and compared with the previously mentioned one. The impact of the NP synthesis method on their optical properties was studied. We have chosen NTA as the chelating ligand since the chelates of Eu3+ with NTA and 1,10-phenanthroline are hydrophobic and easily form chelate NPs in the aqueous media. Time-resolved assays and imaging with organic dyes are virtually impossible, since organic dyes usually have nanosecond fluorescence lifetimes, unlike the Ln3+ ions; although the situation is different in the case of energy transfer between an Ln3+ chelate and a dye. We have shown the presence of delayed dye fluorescence in the microsecond time region caused by the energy transfer from Eu3+ chelates. This fact opens a way to produce new fluorescent labels for time-gated bioassays.

EXPERIMENTAL SECTION Materials. The β-diketone NTA (99%) and 1,10-phenanthroline (phen, 99%) were purchased from Sigma-Aldrich. Oxazine 170 (Ox-170, 95%) and Nile blue (NB, 95%) perchlorates were purchased from Sigma-Aldrich. Eu3+ nitrate hexahydrate (99%) was produced by Novosibirsk rare metals plant. Triton X-100 (TX-100) was produced by Merck (>95%). The solvents isopropyl alcohol (99%), ethyl alcohol (99%), and acetonitrile (99%) were purchased from Vekton (St.Petersburg, Russia). Acetonitrile was distilled before usage. All other chemicals were used without purification. Synthesis of Eu3+ Chelates. NTA (0.3 mM), phen (0.1 mM), and sodium methoxide (0.3 mM) were dissolved in 5 mL of hot ethyl alcohol. The solution was placed under stirring followed by the addition of 1 mL of Eu3+ nitrate (0,5 mM) solution in alcohol. The ratio of NTA:phen:Eu3+ in the resulting solution was equal to 3:1:1. The addition of Eu3+ nitrate results in the formation of a white residue that was filtered, washed with alcohol, and dried in a desiccator. Preparation of Nanoparticles. Chelate nanoparticles can be produced via reprecipitation of hydrophobic chelates into the water environment. We used two different synthesis methods to produce colloidal solutions of dye-doped chelate nanoparticles. In the first method (M1), 100 μL of an isopropyl alcohol solution of NTA (3 × 10−3 M), and phen (10−3 M) were injected into 10 mL of Eu3+ nitrate water solution (10−5 M) containing different amount of dyes (0−200 nM). The formation of chelates followed by their agglomeration in NPs occurred directly in a water solution under sonication. The nonionic surfactant TX-100 (0.24 mM) was added after the sonication to stabilize the NP solution. In the second method (M2), 200 μL of an acetonitrile solution of the presynthesized Eu(NTA)3phen chelates (5 × 10−4 M) were added to a water solution of dyes (10 mL). The B


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Figure 2. (a, c) Time dependent absorption spectra of NP solutions prepared using M1 and M2, respectively, without TX-100. (b, d) Time dependent absorption spectra of NP solutions prepared using M1 and M2, respectively, with TX-100.

Earlier it was shown that chelate NPs can be doped not only with neutral dyes but also with cationic and anionic dyes,30 although at high concentrations some part of the dyes remains in solution. Ox-170 and NB dyes were chosen as dopant molecules for their high quantum yield of fluorescence and significant spectral overlap of their absorption with the Eu3+ luminescence. The fluorescence spectrum of the dyes in the NPs is slightly shifted compared to the fluorescence spectrum of the dyes in a water solution. The dye fluorescence in NPs can be excited by visible light using dye absorption and by the UV using FRET between chelates and dyes. The position of the fluorescence spectra should coincide if no significant amount of dye remains in the solution (Figure S1 of the Supporting Information, SI). Through the comparison of dye fluorescence excited directly and by the FRET, we found the highest concentration at which no significant amount of dye remains in the solution equal to 200 nM (∼2 mol %). The drop of an NP solution was placed onto a mica substrate to perform AFM measurements. A representative AFM image of undoped chelate NPs is shown in Figure 1b. A particle height analysis of M1 NPs based on a series of AFM images shows that 90% of NPs have diameters less than 30 nm, however, a few larger particles with sizes up to 100 nm were observed (Figure 1c). We did not observe significant differences between size distributions of M1 and M2 NPs, the particle height histogram of M2 NPs is provided in the SI (Figure S2). Assuming a single chelate has a size of approximately 1 nm, it is estimated that one particle with the size of 10 nm consists of roughly 1000 chelates, corresponding to a peak molar extinction coefficient of 4 × 107 M−1 cm−1. The Eu3+ chelate complexes with NTA have high absorption in the near UV range with the maxima at the 340 nm. There is no significant difference in the absorption spectra of NPs produced without the addition of surfactant by M1 and M2, although the change of the absorption over time varies greatly. The chelates produced by M1 tend to aggregate over a period

acetonitrile solution of Eu(NTA)3phen was heated before the reprecipitation in order to dissolve the necessary concentration of the complexes. Some part of the solid presynthesized chelates did not dissolve and remained in the solution. The agglomeration of chelates was conducted under the sonication and TX-100 (0.24 mM) was also used to stabilize the NPs. This is the first demonstration of dye-doped chelate NP synthesis from the presynthesized chelates. Characterization. The size distribution was measured by atomic force microscopy (AFM). A drop of NP solution was placed onto a mica substrate, and the surface topology was imaged with a Solver PRO-M AFM in the tapping mode with NSG01 cantilever (tip radius 10 nm). To minimize the NP aggregation on the mica surface the stock solution of NPs was diluted 2-fold with distilled water. UV−vis spectra were collected using a PerkinElmer LAMBDA 650 spectrophotometer with 1 cm quartz cells. Fluorescence and delayed fluorescence spectra were obtained with a PerkinElmer LS50B fluorescence spectrometer. The lifetimes of the Eu3+ luminescence and delayed dyes fluorescence were measured using a Cary Eclipse fluorescence spectrophotometer. Quantum yields of NPs fluorescence were measured with a Hamamatsu C9920 system of absolute quantum yield measurements, the accuracy of quantum yield measurements is ±0.02.

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RESULTS AND DISCUSSION Size and Stability of Nanoparticles. The reprecipitation of hydrophobic molecules from a water-miscible solvent to water is an easy and widely used method of NPs synthesis. It was used to produce dye-doped polymeric NPs,27 conjugated polymer NPs,28 NPs composed of small organic molecules,29 and chelate NPs. Different conditions of the chelates reprecipitation have led to variations of NPs sizes.18 Figure 1a represents the chemical structures of the chelates and the dyes used in this study. Usually, the hydrophobic neutral dyes are used to dope NPs composed of hydrophobic compounds. C


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wavelength (370 nm) was negligible by comparison with the chelate absorption. Since NB and Ox-170 dye absorption significantly overlaps with the luminescence of Eu3+ ions, the existence of energy transfer via Förster mechanism can be concluded. Mixed alcohol solutions of Eu3+ chelates and fluorescent dyes with the same concentrations were prepared for a comparison. The absence of Eu3+ luminescence quenching and dyes sensitized fluorescence indicated that chelates aggregation in the water environment is crucial for efficient energy transfer. Dye molecules can be incorporated inside chelate NPs or attached to the surface of NPs. The addition of dye molecules after the formation of chelate NPs leads to the appearance of sensitized dyes fluorescence, but the efficiency of Eu3+ quenching was significantly lower than for dye-doped NPs produced by M1 or M2. Fluorescence spectra of chelate NPs produced by M1 and M2 with different dopant concentrations are shown in Figure 4 (left side). The luminescence of Eu3+ ions gradually decreased with the increase of dye concentration for all studied NP compositions, while fluorescence of NB and Ox-170 reached maxima at dye molar fractions of 0.005−0.01 and then decreased for NPs produced by M1. However, the fluorescence of NB and Ox-170 in NPs produced by M2 does not show such a significant decrease at high dye concentration. The formation of dimers causes the concentration quenching of the dye fluorescence, as oxazine dyes are known to form Haggregates.32 However, there is also another reason for the sensitized fluorescence decrease. The amount of chelate complexes was maintained constant with an increase of doping dye concentrations, therefore, at some point dye molecules start to compete for the energy absorbed by the chelates. In the absence of a dye dimerization, this competition slows the growth of sensitized dyes fluorescence and completely stops it after the saturation is reached. It can be seen that the efficiency of Eu3+ luminescence quenching is lower in the NPs produced by M2 that corresponds to the less efficient energy transfer between Eu3+ ions and oxazine dyes. As the single oxazine dye quenches fewer chelates in the NPs produced by M2 than in the NPs produced by M1, it is clear that the competition between dye molecules for the energy absorbed by chelates is weaker in M2 NPs. Therefore, in the NPs produced by M2 aggregation induced quenching of dyes fluorescence occurs together with the involvement of new chelates into the energy transfer process that partially compensates for the negative effect of concentration quenching. The quenching of Eu3+ luminescence was modeled by using the Stern−Volmer relationship, which is expressed as follows:

of time with the appearance of nonstructured absorption in the longer wavelength region (Figure 2a), within the same period of time chelates produced by M2 show only a reduction in the intensity of the absorption without changes in the form of spectra (Figure 2b). To prevent rapid chelate aggregation, the TX-100 surfactant was used. The effect of TX-100 on the stability of chelate NP absorption was tested with the three different concentrations of the surfactant: 0.12 mM, 0.24 mM, and 0.48 mM. The TX-100 concentration of 0.12 mM was found to be insufficient to stabilize NPs and prevent fast changes in the absorption and fluorescence of NP solutions. Although the highest used TX-100 concentration of 0.48 mM provides excellent stability of chelate absorption, it leads to a significant reduction of the sensitized dye fluorescence caused by partial destruction of NPs. As seen from Figure 2(c, d), 0.24 mM of TX-100 stabilizes the absorption of NPs produced by M1 and M2 over the same period of time the changes of absorption were observed without detergent; moreover, this concentration of TX-100 does not affect the fluorescent properties of the dye-doped chelate NPs. The addition of the dyes does not change the absorption of NPs (Figure S3). Optical Properties of Nanoparticles. The luminescence of Eu3+ ions is highly sensitive to the symmetry of the environment.31 The intensity of 5D0−7F1 transition is generally independent of the environment as it has a magnetic dipole nature. The 5D0−7F2 transition is often referred to as hypersensitive transition since its intensity changes significantly with the symmetry change of the Eu3+ site. The luminescence spectra of undoped chelate NPs produced by M1 and M2 are shown in Figure 3. The intensities of 5D0−7F1 transition for

Figure 3. Luminescence spectra of NPs solutions prepared using M1 (blue) and M2 (red).

I0/I = 1 + KSV[Q]

(1) 3+

where I0 and I are luminescence intensities of Eu in the undoped and doped NPs, respectively, KSV is the Stern− Volmer quenching constant, and [Q] is the concentration of the dye. The Stern−Volmer relationship implies a linear relation of quenching efficiency and quencher concentration, however, positive deviations were observed in the case of high quenching efficiencies. This phenomenon was called “amplified quenching” or “superquenching” and was observed in different nanoaggregate systems: conjugated polymer NP33 and small organic molecules NPs.29,34 As seen from Figure 4 (right side), in the case of chelate NPs produced by M1 with NB or Ox-170 as a dopant, the I0/I relations significantly deviate from the linearity at the high concentrations of the dyes, while those of NPs produced by M2 remain linear. To obtain KSV, the experimental data on chelate luminescence quenching were

NPs produced by different methods are normalized to be equal. The intensity of the 5D0−7F2 transition, which is twice as low for M2 NPs, indicates that presynthesized Eu3+ chelates have more symmetrical structure than those produced directly in the water solution by M1. Since the synthesis of M1 chelates involves the water solution of Eu3+ nitrate it is possible that some −OH groups remain bonded to the Eu3+ ions and lead to the appearance of defect sites of different symmetry. The presynthesized Eu3+ chelates also have longer luminescence lifetime compared to the chelates produced by M1. The quenching of Eu3+ luminescence accompanied by decreasing luminescence lifetimes was observed in the dyedoped NPs. At the same time, new bands corresponding to the dye fluorescence appeared in the emission spectra of NPs despite the fact that the dye absorption at the excitation D


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Figure 4. (Left) Concentration-dependent fluorescence spectra of dye-doped chelate NPs under 370 nm excitation. The concentration of dyes is expressed in molar fraction. (Right) Luminescence quenching of Eu3+ versus molar fraction of the dyes. The scattered squares are experimental data, the solid lines represent the linear fit of the luminescence quenching.

linearly fit, and KSV were determined from the slope of a fit. Since the concentration of dyes is expressed as the molecular fraction, obtained KSV corresponds to the number of chelate complexes quenched by a single dye molecule. The Förster theory of energy transfer states that the rate of the energy transfer process is proportional to the overlap integral of a donor fluorescence spectrum and the molar extinction spectrum of an acceptor. Although the overlap of the Eu3+ luminescence spectrum and absorption spectra of the dyes is different for NB and Ox-170, we observed no correlation between the quenching efficiency and the overlap factor; in fact, Ox-170 has a higher value of the overlap integral but shows less quenching efficiency than NB. The values of the Förster

distances were calculated to be 5.2 and 5.4 nm for the chelates produced by M1 as energy donors and Ox-170 and NB as energy acceptors, respectively. The luminescence spectrum of chelates produced by M2 differs from that of M1 chelates (Figure 2); also, the M2 chelates possess a lower luminescence quantum yield that leads to lower but comparable Förster distance values of 4.8 and 4.9 nm for Ox-170 and NB, respectively. Since the differences of Eu3+ luminescence quenching between NPs produced by M1 and M2 cannot be adequately explained in terms of the energy transfer theory, the most probable reason is the different ability of NPs to uptake dye molecules in its structure. E


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Dye molecules have fluorescence lifetimes in the region of nanoseconds, but in the case of dye fluorescence sensitization by the Ln3+ chelates, delayed fluorescence of dyes is observed. Figure 5(a, d) represents the delayed fluorescence spectra of NPs produced by M2 and doped with 0.5 mol % of Ox-170 or NB. The delay time was changed from 20 to 100 μs, and the gate time was 10 μs for all measurements. Both the luminescence of Eu3+ and the fluorescence of dyes are presented in the spectra, although the delayed fluorescence of dyes decays faster than Eu3+ luminescence. The reason is that energy transfer from Eu3+ ions to dyes shortens the lifetime of Eu3+ luminescence, therefore, the delayed dye fluorescence has the lifetime of quenched Eu3+ ions. However, the Eu3+ luminescence originates also from the Eu3+ ions that have no adjacent molecules of Ox-170 or NB. The NPs produced by M1 show a similar behavior of NB and Ox-170 delayed fluorescence. We measured decay kinetics at 613 and 670 nm to test the influence of the doping dyes concentration on lifetimes of the Eu3+ luminescence and the delayed dye fluorescence. Eu3+ ions have no luminescence at 670 nm, therefore, only delayed fluorescence of dyes contributes to the signal at 670 nm. As the change of Eu3+ luminescence lifetime is almost the same for NPs doped with NB or Ox-170, we herein discuss only NPs doped with Ox-170. Lifetimes of 136 and 183 μs were found for Eu3+ luminescence in the undoped NPs produced by M1 and M2, respectively. The reprecipitation of presynthesized chelates yields the formation of NPs having monoexponential decay of Eu3+ luminescence, unlike the NPs produced by M1 that clearly have several components of Eu3+ luminescence decay. The increase of a molar fraction of dyes in the NPs led to the gradual decrease of Eu3+ luminescence lifetime, which was caused by the FRET, and appearance of nonexponential decay kinetics Figure 5(b, c). The delayed fluorescence of dyes has a lifetime of quenched Eu3+ ions acting as energy donors. Since every single dye molecule accepts energy from many adjacent chelates, it is obvious that decay of delayed fluorescence cannot be monoexponential. At the same time, the increase of dye concentration lowers the delayed fluorescence lifetime as more chelates are quenched by the dyes (Figure 5(e, f)).

The brightness of NPs fluorescence is often defined as ε · Φ, where ε is the peak molar extinction coefficient and Φ is a quantum yield of NPs fluorescence. To evaluate the brightness of dyed-doped chelate NPs, absolute quantum yields of NPs solutions were obtained with an integrating sphere (Table 1). Table 1. Absolute Quantum Yields of NPs Fluorescence under 370 nm Excitationa undoped NPs NB/0.005 NB/0.01 Ox-170/0.005 Ox-170/0.01

M1

M2

0.14 0.1 0.09 0.14 0.12

0.08 0.07 0.08 0.09 0.08

a

The concentration of dyes is expressed in molar fraction; the quantum yield values of dye-doped NPs represent the dyes emission.

Fluorescence quantum yields of 7−14% were determined for chelate NPs doped with different amount of NB or Ox-170. The calculated fluorescence brightness of dyed-doped chelate NPs is 50 times higher than that of a single Rhodamine 6G molecule, assuming that a single NP has a molar extinction coefficient of 4 · 107 M−1 cm−1 for 1000 of chelates per NP. Delayed Fluorescence of Dyes. The long luminescence lifetime of Ln3+ chelates is widely used in biomedical research to separate a useful luminescence signal from the cells autofluorescence. The choice of appropriate Ln3+ ion with visible luminescence for time-resolved measurements is restricted to Eu3+, Tb3+, Sm3+, and Dy3+ chelates.6 The quantum yields of the luminescence of Sm3+ and Dy3+ ions coordinated with organic ligands are low as the energy gap of the ions does not allow one to sufficiently suppress nonradiative deactivation. Furthermore, to sensitize Tb3+ luminescence, it is necessary to use ligands with a higher triplet energy level than that used in Eu3+ chelates, as the 5D4 Tb3+ energy level is higher than the 5D0 Eu3+ energy level. The absorption of Tb3+ chelates is usually shifted toward the shorter wavelength region by comparison with the absorption of Eu3+ chelates.

Figure 5. (a, d) Delayed fluorescence spectra of M2 NPs doped with 0.5 mol % of Ox-170 and NB. (b, c) Normalized luminescence lifetime decay curves of M1 and M2 NPs doped with different molar fraction of Ox-170 at 613 nm. (e, f) Normalized delayed fluorescence lifetime decay curves of M1 and M2 NPs doped with different molar fractions of Ox-170 at 670 nm. F


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(4) Bünzli, J.-C. G. On the Design of Highly Luminescent Lanthanide Complexes. Coord. Chem. Rev. 2015, 293−294, 19−47. (5) Weissman, S. I. Intramolecular Energy Transfer. The Fluorescence of Complexes of Europium. J. Chem. Phys. 1942, 10, 214−217. (6) Xu, Y. Y.; Hemmilä, I. A.; Lövgren, T. N. Co-Fluorescence Effect in Time-Resolved Fluoroimmunoassays. A Review. Analyst 1992, 117, 1061−1069. (7) Hagan, A. K.; Zuchner, T. Lanthanide-Based Time-Resolved Luminescence Immunoassays. Anal. Bioanal. Chem. 2011, 400, 2847− 2864. (8) Reddy, M. L. P.; Divya, V.; Pavithran, R. Visible-Light Sensitized Luminescent europium(III)-β-Diketonate Complexes: Bioprobes for Cellular Imaging. Dalton Trans. 2013, 42, 15249−15262. (9) Tian, L.; Dai, Z.; Ye, Z.; Song, B.; Yuan, J. Preparation and Functionalization of a Visible-Light-Excited Europium ComplexModified Luminescent Protein for Cell Imaging Applications. Analyst 2014, 139, 1162−1167. (10) Feng, J.; Zhang, H. Hybrid Materials Based on Lanthanide Organic Complexes: A Review. Chem. Soc. Rev. 2013, 42, 387−410. (11) Binnemans, K. Lanthanide-Based Luminescent Hybrid Materials. Chem. Rev. 2009, 109, 4283−4374. (12) Tian, L.; Dai, Z.; Zhang, L.; Zhang, R.; Ye, Z.; Wu, J.; Jin, D.; Yuan, J. Preparation and Time-Gated Luminescence Bioimaging Applications of Long Wavelength-Excited Silica-Encapsulated Europium Nanoparticles. Nanoscale 2012, 4, 3551. (13) Duarte, A. P.; Mauline, L.; Gressier, M.; Dexpert-Ghys, J.; Roques, C.; Caiut, J. M. A.; Deffune, E.; Maia, D. C. G.; Carlos, I. Z.; Ferreira, A. A. P.; et al. Organosilylated Complex [Eu(TTA)3(BpySi)]: A Bifunctional Moiety for the Engeneering of Luminescent SilicaBased Nanoparticles for Bioimaging. Langmuir 2013, 29, 5878−5888. (14) Peng, H.; Stich, M. I. J.; Yu, J.; Sun, L. N.; Fischer, L. H.; Wolfbeis, O. S. Luminescent Europium(III) Nanoparticles for Sensing and Imaging of Temperature in the Physiological Range. Adv. Mater. 2010, 22, 716−719. (15) Sun, W.; Yu, J.; Deng, R.; Rong, Y.; Fujimoto, B.; Wu, C.; Zhang, H.; Chiu, D. T. Semiconducting Polymer Dots Doped with Europium Complexes Showing Ultranarrow Emission and Long Luminescence Lifetime for Time-Gated Cellular Imaging. Angew. Chem., Int. Ed. 2013, 52, 11294−11297. (16) Li, Q.; Zhang, J.; Sun, W.; Yu, J.; Wu, C.; Qin, W.; Chiu, D. T. Europium-Complex-Grafted Polymer Dots for Amplified Quenching and Cellular Imaging Applications. Langmuir 2014, 30, 8607−8614. (17) Peng, H.; Wu, C.; Jiang, Y.; Huang, S.; McNeill, J. Highly Luminescent Eu3+ Chelate Nanoparticles Prepared by a Reprecipitation−Encapsulation Method. Langmuir 2007, 23, 1591−1595. (18) Wen, X.; Li, M.; Wang, Y.; Zhang, J.; Fu, L.; Hao, R.; Ma, Y.; Ai, X. Colloidal Nanoparticles of a Europium Complex with Enhanced Luminescent Properties. Langmuir 2008, 24, 6932−6936. (19) Härmä, H.; Graf, C.; Hänninen, P. Synthesis and Characterization of Core-Shell Europium(III)-Silica Nanoparticles. J. Nanopart. Res. 2008, 10, 1221−1224. (20) Stein, G.; Wurzberg, E. Energy Gap Law in the Solvent Isotope Effect on Radiationless Transitions of Rare Earth Ions. J. Chem. Phys. 1975, 62, 208−213. (21) Ermolaev, V. L.; Sveshnikova, E. B. The Application of Luminescence-Kinetic Methods in the Study of the Formation of Lanthanide Ion Complexes in Solution. Russ. Chem. Rev. 1994, 63, 905−922. (22) Placide, V.; Bui, A. T.; Grichine, A.; Duperray, A.; Pitrat, D.; Andraud, C.; Maury, O. Two-Photon Multiplexing Bio-Imaging Using a Combination of Eu- and Tb-Bioprobes. Dalton Trans. 2015, 44, 4918−4924. (23) Selvin, P. R.; Rana, T. M.; Hearst, J. E. Luminescence Resonance Energy Transfer. J. Am. Chem. Soc. 1994, 116, 6029−6030. (24) Selvin, P. R.; Hearst, J. E. Luminescence Energy Transfer Using a Terbium Chelate: Improvements on Fluorescence Energy Transfer. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 10024−10028.

CONCLUSIONS Chelate NPs doped with NB or Ox-170 have been prepared by two different reprecipitation methods, one of which was utilized for the first time, yielding a different structure of the chelates. The reprecipitation of presynthesized chelates results in formation of chelate NPs with longer luminescence lifetime than that of spontaneously formed chelates. The analysis of undoped NP luminescence decay has shown that NPs from presynthesized chelates have monoexponential luminescence decay in contrast to NPs produced from spontaneously formed chelates. The monoexponential luminescence decay indicates that M2 NPs are composed from homogeneous chelates. The existence of FRET between Eu3+ chelates and dyes is confirmed by the quenching and lifetime shortening of Eu3+ luminescence. The analysis of chelate luminescence quenching shows high efficiency of the energy transfer as an individual dye molecule quench luminescence of more than 100 Eu3+ chelates. On the basis of the molar extinction coefficient and quantum yield, the fluorescence brightness of 10 nm dye-doped chelate NPs is calculated to be 50 times higher than that of a single Rhodamine 6G molecule. Time-resolved measurements of NPs fluorescence reveal the presence of delayed dye fluorescence with microsecond lifetimes that opens a way to use these NPs in time-gated bioassays. The longer lifetime of Eu3+ luminescence in M2 NPs provides the longer lifetimes of delayed dye fluorescence which is preferable for time-gated applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b03648. Fluorescence spectra, particle height histogram of M2 NPs, absorption spectra of undoped NPs, and dye-doped NPs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (L.Y.M.). ORCID

Leonid Yu. Mironov: 0000-0001-9291-7614 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Present work is performed with financial support of the Ministry of Education and Science of the Russian Federation (project RFMEFI58715X0012). The present work is a part of the 382-PiGnano project of the ERA.Net RUS Plus 2013-2018 initiative under Consortium Agreement with Swiss Federal Laboratories for Materials Science and Technology (Switzerland) and Hamburg University of Technology (Germany).

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REFERENCES

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