Article pubs.acs.org/JPCC
Pluronic-Silica (PluS) Nanoparticles Doped with Multiple Dyes Featuring Complete Energy Transfer Enrico Rampazzo,† Sara Bonacchi,† Damiano Genovese,† Riccardo Juris,† Marco Montalti,† Veronica Paterlini,† Nelsi Zaccheroni,† Cécile Dumas-Verdes,‡ Gilles Clavier,‡ Rachel Méallet-Renault,‡ and Luca Prodi*,† †
Dipartimento di Chimica “G. Ciamician”, Università degli Studi di Bologna, Via Selmi 2, 40126 Bologna, Italy PPSM, ENS Cachan, CNRS UMR8531, 61 av Président Wilson, F-94230 Cachan, France
‡
S Supporting Information *
ABSTRACT: We report here the design of two sets of multifluorophoric silica nanoparticles, observing unprecedented efficiencies in the energy-transfer processes among the doping dyes. These nanomaterials show a very high overall sensitization, allowing under a single wavelength excitation to obtain many different colors (one per nanoparticle) in emission with negligible crosstalk. Moreover, each particle can present very large and tunable pseudo-Stokes shifts (up to 435 nm), a very high brightness even exciting the bluest donor, and a negligible residual emission intensity from all donor dyes. All these features, combined with colloidal stability and synthetic method reliability, make these multicomponent nanoparticles very promising for multiplex analysis and for all the diagnostic techniques requiring high sensitivity associated with a large Stokes shift.
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variability.5 Nevertheless, multiplex assays benefit most from the tandem dyes, especially in the fields of flow cytometry and histochemistry.6 The great advances in nanotechnology7 have provided the knowledge to find reasonable solutions to overcome these problems. Among the new nanostructures proposed, different kinds of nanoparticles have been exploited as bright luminescent signaling units such as quantum dots (QDs),7g dye-doped polymers,8 or silica nanoparticles (NPs). In our opinion, these last ones are particularly promising for their high versatility.2b With proper engineering, in fact, they can be prepared in different architectures to obtain suitable properties for specific applications. The luminophores, for example, can be attached on their surface to be able to interact with species in solution, or they can be confined inside the silica matrix to protect them by the environment, thus increasing their photostability.9 In the framework of multiple detection, for instance, sets of silica NPs each doped with a different commercial dye that can be excited by the same laser have been already proposed,10 but the restricted choice of suitable luminophores limits this approach and the possibility to finely tune the photophysical properties of the system.9 A more versatile multifluorophoric approach was brilliantly used by Wang and co-workers to prepare bar-coding tags for multiplex analysis.11 In these cases, the partial ET among the doping
INTRODUCTION Luminescence is a highly sensitive and noninvasive technique, ideal for labeling and sensing in many fields of strong social impact such as environmental and food analysis, security, and medical diagnostics.1 Fluorescence detection involving molecular dyes, however, can present also some drawbacks such as photodegradation and interferences due to background signals. These problems can become particularly severe when the analyte is diluted and the matrix is complex, as it is typically the case in biomedical applications. A possible solution is the simultaneous detection of many biological events or markers; in recent years, different ways have been explored to provide luminescent materials, generally on the basis of multifluorophoric approaches, able to answer to all these requirements.2 These materials typically take advantage of Förster resonance energy transfer (FRET) processes to obtain systems presenting a large separation between the excitation and the emission maxima (pseudo-Stokes-shift) and, therefore, a very high signal-to-noise ratio thanks to the reduced interferences from Rayleigh-Tyndall and Raman bands.1a,3 Commercial tandem dyes, for example, are a combination of two covalently conjugated fluorochromes (a donor and an acceptor) that present by means of FRET processes a large pseudo-Stokes shift. This strategy, taking profit from two different moieties for the absorption and emission, enlarges the possibilities to obtain systems endowed with high brightness. However, the balance of their communication, their resistance to photobleaching, and their stability in biological environment4 still remains very delicate and are sometimes altered by manufacturing © 2014 American Chemical Society
Received: February 7, 2014 Revised: April 3, 2014 Published: April 4, 2014 9261
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fluorophores has been brilliantly exploited to obtain a multiband emission for each NP under a single wavelength excitation to simultaneously reveal several events or analytes, such as different cells and bacteria.12 It is important to underline that a complete energy transfer has not yet been achieved in the cited examples or in systems based on organic nanoparticles13 and polymeric nanoparticles.14 This result would, however, represent a very valuable feature, as mentioned by Wang and Tan,11a allowing to increase even further the number of possible analytical applications. Starting from previous results on FRET based nanoparticles, and thanks to the precise structure of the Pluronic-silica (PluS) nanoparticles developed in our laboratory,15 we designed NPs doped with various dyes featuring optimized photophysics to reach, for the first time, an almost complete ET among them. Importantly, such materials would change the paradigm of multiplex analysis because their only analytical signal is the emission of the lowestenergy fluorophore, and multiple analysis can be performed in a single measurement with single excitation and without any separation step or bar-coding decodification. In addition, in flow cytometry, this feature allows the use of only one emission channel per kind of NP without the typical interferences observed in the case of multiband emission. Furthermore, a significant gain in the signal-to-noise ratio is obtained because of a high pseudo-Stokes shift.
nanoparticle solution diluted with water (1:50). The NP TEM images show that only the silica cores present sufficient contrast to appear in the images. The size distribution was obtained analyzing images with a block of several hundred nanoparticles, see Figure 1 (left). The obtained histogram was fitted according to a Gaussian distribution obtaining an average diameter of (10 ± 2) nm for the silica nanoparticle core.
Figure 1. TEM image of core−shell silica-PEG nanoparticles (scale bar 200 nm) and silica core size distribution, d = (10 ± 2) nm.
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MATERIALS AND METHODS Nanoparticles Synthesis. In a typical preparation of core− shell silica-PEG (poly(ethylene glycol)) nanoparticles,16 100 mg of Pluronic F127 and the desired amount of the silanized dyes (see Figure S12 and Table S1 of the Supporting Information) were carefully solubilized with 1.0−2.0 mL of dichloromethane in a 20 mL glass scintillation vial. The solvent was evaporated from the homogeneous solution by means of a gentle nitrogen flow and subsequently under vacuum at room temperature. NaCl (68.6 mg) was added to the solid residue, and the mixture was solubilized at 25 °C under magnetic stirring with 1565 μL of acetic acid 1 M. Tetraethyl orthosilicate (TEOS, 179 μL, 0.8 mmol) was then added to the resulting aqueous homogeneous solution followed by chlorotrimethylsilane (TMSCL, 10 μL, 0.08 mmol) after 180 min. The mixture was kept under stirring for 48 h at 25 °C before dialysis treatments. The dialysis purification steps were carried out versus water on a precise amount of nanoparticle solution (1500 μL) finally diluted to a total volume of 5 mL with water. Nanoparticle Characterization. The determination of the nanoparticle hydrodynamic diameter distributions was carried out through dynamic light scattering (DLS) measurements employing a Malvern Nano ZS instrument equipped with a 633 nm laser diode. Samples were housed in disposable polystyrene cuvettes of 1 cm optical path length using water as solvent. The width of DLS hydrodynamic diameter distribution is indicated by PdI (polydispersion index). In case of a monomodal distribution (Gaussian) calculated by means of cumulant analysis, PdI = (σ/Zavg)2, where σ is the width of the distribution and Zavg is the average diameter of the particles’ population, respectively (Figure S13 of the Supporting Information). For transmission electron microscopy (TEM) investigations, a Philips CM 100 transmission electron microscope operating at 80 kV was used. Standard 3.05 mm copper grids (400 mesh) covered by a Formvar support film were dried up under vacuum after deposition of a drop of
FRET Model Calculations. Intermolecular distances were calculated with a homemade Matlab routine. A simple geometric model is used in which the NPs are approximated to be perfect spheres of 11 nm in diameter and in which dyes are considered dimensionless points randomly dispersed in the inner volume. Furthermore, the model accounts for a Poisson distribution of dyes in a large number of NPs (calculations were run on a set of 2000 NPs, a number that ensures high reproducibility >97% of the resulting information). Poisson distribution can indeed affect significantly the calculations for low doping degrees, as it is the case for the PluS NPs presented here, and it must be thus taken into account. Orientation is not considered at this stage. We calculated FRET efficiency from the intermolecular distances using 50 angstroms as the Förster radius, a value close to typical Förster radii that we obtained for the dyes involved in this study. Fluorescence Imaging. Fluorescence imaging of NPs physically adsorbed on glass fibers was carried out with two different setups using different detectors and imaging modes but the same excitation wavelength. Confocal Setup. We used a Nikon Eclipse C1 system equipped with a Nikon Eclipse TE2000 inverted fluorescence microscope, a 405 nm laser source for excitation, and four different single-photon avalanche diode (SPAD) detectors to observe emission in four different spectral regions selected through bandpass filters at 425−475, 500−550, 570−620, and 660−735 nm (Figure S17 of the Supporting Information). Wide-Field Setup. We used an Olympus IX71 inverted microscope, a 405 nm excitation laser, an objective with 10× magnification from Leica, and a color charge-coupled device (CCD) to observe fluorescence (Basler scA640-70gc), Figure 4.
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RESULTS AND DISCUSSION We designed a set of Pluronic-silica (PluS) nanoparticles doped with four kinds of dyes. In each NP, the excitation energy is funneled to the lowest energy dye, yielding four different emission colors with negligible residual emission from the 9262
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Figure 2. Chemical structures of dyes used in the NP preparation.
higher energy dyes and, thus, a very low cross talking. The present system works in water, and all the particles of a set, although they present different emissions, can be excited at the same absorption maximum. These NPs have been obtained through a versatile surfactant aided strategy using Pluronic F127 micelles as templates. These nanomaterials are extremely stable even from a photochemical point of view,2,16,17 are reproducible in size and properties with a silica core of 10 nm diameter (see Figure 1) and an outer PEG shell (overall hydrodynamic diameter = 25 nm), and display the typical advantages of both silica and PEG, such as water solubility and nontoxicity.16,17 To obtain highly efficient ET among two or more doping dyes, we have selected fluorophores with suitable photophysical propertieshigh molar absorption coefficients ε, high fluorescence quantum yield Φ, and good overlap integral according to Förster theory18 between each two dyes acting as a couple of energy donor and acceptorand have functionalized them with a trialkoxysilane moiety for covalent linking to the silica matrix (see Figure 2 and Figures S12−14 and Table S1 of the Supporting Information). We first investigated the influence of the inclusion of the dyes in the NPs on their photophysical properties. The most significant values are listed in Table 1. In general, the shape and position of the absorption and of the fluorescence bands of the dyes in the NPs are very similar to the ones of the free luminophores in ethanol solution (as could be expected for a transition mostly π−π* in nature) presenting only very small red shifts (400 000 M−1 cm−1) at 405 nm, high brightness, and four distinguishable emission bands with negligible crosstalk (Figure 3). To prove the generality of this approach, we also prepared a second totally analogous set of nanoparticles. In this series, B2, C1, and C2 were introduced with a doping degree of 0.1% while the first donor D2 was introduced at 0.2%. Similar or even better results could be obtained with this new set of NPs; in particular, the D2+B2+C1+C2 doped system presents a totally negligible emission in all the visible range and a very high emission in the near-infrared (NIR) (see Figure S15 of the Supporting Information). In these NPs, the difference among the emission wavelength of the lower energy acceptor and the absorption excitation wavelength (pseudo-Stokes shift) is 11 300 cm−1 (435 nm, Table 2), to our knowledge, the largest value reported so far in literature for silica NPs. To prove with additional experimental evidence the almost complete absence of cross interferences in these materials, we imaged different glass fibers after physical adsorption of D1@ NP, (D1+B1)@NP, (D1+B1+R)@NP, and (D1+B1+R+C1) @NP (blue, green, orange, and red fluorescence, respectively) on their surfaces. The sets of fibers, all excited at the same wavelength (405 nm), show a fluorescence emission of four different colors, being perfectly distinguishable with a common microscopy setup either with a color CCD (wide-field fluorescence, Figure 5) or with a set of filters and detectors (confocal microscopy, Figures S17−S18 of the Supporting Information).
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ASSOCIATED CONTENT
* Supporting Information S
Chemicals, ultrafiltration experiments, chromophore synthesis, details on nanoparticle synthesis and characterization, photophysical experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +39 051 2099456. Notes
The authors declare no competing financial interest. 9265
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ACKNOWLEDGMENTS This work has been supported by the European Commission and Italian CNR through the Eranet Plus programm (“NanoSci-E+”, INOFEO project), by MIUR (PRIN2009Z9ASCA and PON 01_01078 projects), and by Consorzio SPINNER.
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ABBREVIATIONS ET, energy transfer; FRET, Förster resonance energy transfer; NP, nanoparticle; PluS NPs, pluronic-silica nanoparticles; PEG, poly(ethylene glycol); TEOS, tetraethyl orthosilicate; TMSCl, chlorotrimethylsilane; DLS, dynamic light scattering; TEM, transmission electron microscopy
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