Organic Functionalization of Luminescent Oxide Nanoparticles toward

Sep 5, 2008 - An example of application of the amino-functionalized particles is given for the coupling with α-bungarotoxins. The average number (up ...
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Organic Functionalization of Luminescent Oxide Nanoparticles toward Their Application As Biological Probes Domitille Giaume,† Me´lanie Poggi,† Didier Casanova,‡ Genevie`ve Mialon,† Khalid Lahlil,† Antigoni Alexandrou,‡ Thierry Gacoin,*,† and Jean-Pierre Boilot† Laboratoire de Physique de la Matie`re Condense´e, Ecole Polytechnique, CNRS, Route de Saclay, 91128 Palaiseau, France, and Laboratoire d’Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM, Route de Saclay, 91128 Palaiseau, France ReceiVed May 20, 2008. ReVised Manuscript ReceiVed June 30, 2008 Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with R-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.

Introduction The development of inorganic luminescent nanoparticles offers a unique opportunity for biologists to use their high photostability for the dynamic tracking of individual species during in Vitro or in ViVo experiments.1 In the past decade, a huge amount of work has been done in this field, mostly using quantum dots, and an increasing number of results has indeed shown the high potentiality of nanoparticles to help understand some fundamental processes in biology.2 For all these experiments, it is clear that the main difficulty concerns the chemical functionalization of the particle surface, whose aim is to specifically target the desired biological function, while maintaining the colloidal stability of the particles in an aqueous medium with a high ionic strength. In most cases, the first step of the functionalization strategy consists in covering the surface of the particles with chemical functions (mostly amino-, thio-, or carboxylate) that can be further used to graft the particles with biological species using common coupling reactions.3,4 Up to now, most studies have been performed on luminescent quantum dots, with a major difficulty arising from the presence, at their surface, of tightly bound amphiphilic molecules such as trioctylphosphine oxide, which seem to be required to have particles with optimized optical properties.1 Rare earth doped oxide nanoparticles represent another class of highly luminescent systems that are the subject * To whom correspondence should be addressed. Tel: +33 (0)1 69 33 46 56. Fax: +33(0)1 69 33 47 99. E-mail: [email protected]. † Laboratoire de Physique de la Matie`re Condense´e. ‡ Laboratoire d’Optique et Biosciences. (1) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science. 2005, 307, 538–544. (2) Courty, S.; Luccardini, C.; Bellaiche, Y.; Cappello, G.; Dahan, M. Nano Lett. 2006, 6, 1491–1495. (3) Kapanidis, A. N.; Weiss, S. J. Chem. Phys. 2002, 117, 10953. (4) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128–4158.

of investigations in our group5-7 and in others.8-10 These particles may be obtained directly in water using simple reactions of precipitation starting from precursor salts.6 The spectroscopic features of the particle emission are roughly similar to the related bulk materials, i.e., very narrow emission lines with a millisecond lifetime of the excited states. Bulk defects and surface states were shown to affect the emission yield, but quantum yields of more than 20% are commonly observed for bare particles in water without any specific passivation treatment.11 In addition to the absence of emission intermittency, the high emission yield of these particles without having to perform complex surface treatments, and the availability of aqueous suspensions directly after their synthesis are clear motivations for the study of their application as biological probes. Although the luminosity of these particles is mainly limited by their low absorption cross section under visible light excitation, results obtained in our groups have shown that single particles with size down to 14 nm could be easily detected using a conventional wide-field optical microscope with a reasonable excitation power at 466 nm.12 We could thus (5) Buissette, V.; Giaume, D.; Gacoin, T.; Boilot, J.-P. J. Mater. Chem. 2006, 16, 529–539. (6) Huignard, A.; Gacoin, T.; Boilot, J.-P. Chem. Mater. 2000, 12, 1090–1094. (7) Buissette, V.; Moreau, M.; Gacoin, T.; Boilot, J.-P.; Chane-Ching, J.-Y.; Le Mercier, T. Chem. Mater. 2004, 16, 3767–3773. (8) Haase, M.; Riwotzki, K.; Meyssamy, H.; Kornowski, A. J. Alloys Compd. 2000, 303-304191-197. (9) Louis, C.; Bazzi, R.; Marquette, C. A.; Bridot, J-L.; Roux, S.; Ledoux, G.; Mercier, B.; Blum, L.; Perriat, P.; Tillement, O. Chem. Mater. 2005, 17, 1673– 1682. (10) Sivakumar, S.; Diamente, P. R.; Van Veggel, F. C. J. M. Chem.;Eur. J. 2006, 12, 5878–5884. (11) Huignard, A.; Buissette, V.; Franville, A.-C.; Gacoin, T.; Boilot, J.-P. J. Phys. Chem. B 2003, 107, 6754–6759. (12) Casanova, D.; Giaume, D.; Beaurepaire, E.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. Appl. Phys. Lett. 2006, 89, 253103. (13) Beaurepaire, E.; Buissette, V.; Sauviat, M.-P.; Mercuri, A.; Martin, J.-L.; Lahlil, K.; Giaume, D.; Huignard, A.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. Nano Lett. 2004, 11, 2079–2083.

10.1021/la8015468 CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

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Figure 1. Schematic representation of reaction leading to the encapsulation of the particles with an amino- or epoxy-silane.

show that these particles are indeed promising single-biomolecule labels.13,15 As mentioned above, applications of inorganic particles require the preliminary grafting at their surface of organic or bio-organic functions. Different strategies may be used such as encapsulation with functional polymers16 or direct grafting of bifunctional ligands.17 The latter method has been studied in the case of luminescent lanthanum phosphate nanoparticles in the early report by Meiser et al.18 In our case, we used the approach that relies on the encapsulation of the particles with a thin layer of functional polysiloxane. So-called silane coupling agents present the advantage to provide a covalent grafting of reactive functions with a high surface coverage, and benefit from the versatility of silane chemistry. They have been largely developed for the surface modification of various substrates such as flat silica or silicon substrates for adhesion promotion19-21 or biochips,22,23 or silica gel for chromatography.24-27 Functionalization of colloidal nanoparticles has also been studied in many systems such as silica,28-32 boehmite,33 maghemite,34,35 and quantum dots.36 It has also been used in the case of rare earth doped compounds such as gadolinium oxide9 and lanthanum fluoride.10 (14) Casanova, D.; Giaume, D.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. J. Phys. Chem. B 2006, 110, 19264–19270. (15) Casanova, D.; Giaume, D.; Moreau, M.; Martin, J.-L.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. J. Am. Chem. Soc. 2007, 129, 12592–12593. (16) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. J. Mater. Chem. 2004, 14, 2161–2175. (17) Traina, C. A.; Schwartz, J. Langmuir 2007, 23, 9158–9161. (18) Meiser, F.; Cortez, C.; Caruso, F. Angew. Chem., Int. Ed. 2004, 43, 5954– 5957. (19) Howarter, J. A.; Youngblood, J. P. Langmuir 2006, 22, 11142–11147. (20) Luzinov, I.; Julthongpiput, D.; Liebman-Vinson, A.; Cregger, T.; Foster, M. D.; Tsukruk, V. V. Langmuir 2000, 16, 504–516. (21) Tsukruk, V. V.; Luzinov, I.; Julthongpiput, D. Langmuir 1999, 15, 3029– 3032. (22) Yakovlava, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emne´us, J. Anal. Chem. 2002, 74, 2994–3004. (23) Wong, K. Y.; Krull, U. J. Anal. Bioanal Chem. 2005, 383, 187–200. (24) Jal, P. K.; Patel, S.; Mishra, B. K. Talanta 2004, 62, 1005–1028. (25) Vrancken, K. C.; Van Der Voort, P.; Possemiers, K.; Vansant, E. F. J. Colloid Interface Sci. 1995, 174, 86–91. (26) Shimizu, I.; Yoshino, A.; Okabayashi, H.; Nishio, E.; O’Connor, C. J. J. Chem. Soc., Faraday Trans. 1997, 93, 1971–1979. (27) Ramos, M. A.; Gil, M. H.; Schacht, E.; Matthys, G.; Mondelaers, W.; Figueiredo, M. M. Powder Technol. 1998, 99, 79–85. (28) Douce, J.; Boilot, J.-P.; Biteau, J.; Scodellaro, L.; Jimenez, A. Thin Solid Films 2004, 466, 114–122. (29) Csogo¨r, Zs.; Nacken, M.; Sameti, M.; Lehr, C.-M.; Schmidt, H. Mater. Sci. Eng., C 2003, 23, 93–97. (30) Van Blaaderen, A.; Vrij, A. J. Colloid Interface Sci. 1993, 156, 1–18. (31) Daniels, M. W.; Francis, L. F. J. Colloid Interface Sci. 1998, 205, 191– 200. (32) Gellermann, C.; Storch, W.; Wolter, H. J. Sol-Gel Sci. Technol. 1997, 8, 173–176. (33) Philipse, A. P.; Nechifor, A.-M.; Patmamanoharan, C. Langmuir 1994, 10, 4451–4458. (34) Philipse, A. P.; Van Bruggen, M. P. B; Patmamanoharan, C. Langmuir 1994, 10, 92–99. (35) Flesch, C.; Joubert, M.; Bourgeay-Lami, E.; Mornet, S.; Duguet, E.; Delaite, C.; Dumas, P. Colloids Surf. A: Phys. Eng. Aspects 2005, 262, 150–157. (36) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861–8871.

The purpose of this work is to fully describe the application of this functionalization strategy in the case of luminescent YVO4 nanoparticles that have been shown to be attractive new biological labels.13-15 In many papers dealing with the surface derivatization of colloidal nanoparticles, the efficiency of the functionalization process is only shown indirectly through the modification of the particle interactions with their environment (colloidal stability, specific or nonspecific binding to substrates). No clear and quantitative characterizations of the surface chemistry of the particles are usually provided in terms of the number of surface reactive functions that are effectively present at the surface of the particles. The aim of this work is to provide this characterization in the specific case of lanthanide-ion doped YVO4 particles that are functionalized with amino- and epoxy-silanes. These functions were chosen because their chemistry is wellknown and they are already commonly used for the versatile surface functionalization of substrates such as biochips for further coupling reactions with organic or bio-organic species of interest.22,23 The grafting process relies on the sol-gel condensation of glycidoxypropyltrimethoxysilane (GPTMS) or aminopropyltriethoxysilane (APTES) at the surface of the particles, leading to an expected structure as decribed in the reaction scheme given in Figure 1. The main aim of this paper is to describe the reaction processes that have been used to achieve the surface modification of the particles and to provide an accurate determination of the amount of grafted functions available for the further coupling of the bio-organic species that are to be studied in luminescence labeling experiments.

Experimental Section Synthesis of the YVO4:Eu Colloidal Particles.6 An aqueous solution of 0.1 M sodium orthovanadate is freshly prepared, and its pH is adjusted between 12.6 and 13 with a 1 M NaOH solution. The same volume of an aqueous solution of yttrium and europium nitrate with the desired europium content ([Y3+] + [Eu3+] ) 0.1 M) is then added dropwise using a peristaltic pump under vigorous stirring. A milky precipitate appears immediately after the addition, corresponding to the formation of the solid phase. The solution is then left under stirring for 30 min until the pH is stabilized to about 8-9. The solution is then purified by dialysis against pure water until its conductivity is below 100 µS · cm-1. The final solution is then sonicated with a 450W Branson sonifier for 5 min, leading to a homogeneous slightly diffusing colloidal suspension. Deposition of the Primary Silicate Layer. To one volume of as-synthesized YVO4 nanoparticles with a concentration of 40 mM (expressed as the total vanadate amount) is added one volume of an aqueous solution of tetramethylammonium silicate (TMASi; pH: 12, TMASi/VO43- molar ratio equal to 9). The resulting solution, whose initial pH is about 11, is left under stirring overnight and finally dialyzed against water until the conductivity is about 100 µS · cm-1. Trialkoxysilane (GPTMS or APTES) Shell Deposition. In a 500 mL three-necked flask are introduced 225 mL of absolute ethanol and 5 equiv (as compared to the total vanadate amount) of trialkoxysilane (either GPTMS or APTES). The solution is heated

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Figure 2. (left) Transmission electron microscopy image of the YVO4:Eu nanoparticles; (right) size distribution and log-normal fits of the prolate ellipsoidal particles dimension (major (b) and minor (9) axis).

BS3 and the same concentration of triethylamine in a 1 mL DMSO solution under Ar atmosphere and stirred for 3 days at 50 °C. The nanoparticle-BS3 conjugates were then transferred into a pH 7.4 phosphate buffer solution (7.74 mM NaH2PO4 and 2.26 mM Na2HPO4) by centrifugation/redispersion (discussion on the stability of the NHS ester under these experimental conditions is given in the Supporting Information). For the protein-coupling step, we used the R-bungarotoxinAlexa488 conjugate (Molecular Probes) at various concentrations. After 2 h of reaction, the free R-bungarotoxins are separated from the nanoparticle-R-bungarotoxin conjugates by three centrifugations/ redispersions (16 100 g, 70 min each).

under reflux, and 75 mL of the colloidal solution of silicated nanoparticles ([VO43-] ) 3 mM) at pH 9 are added dropwise with a peristaltic pump. The total addition time is about 2 h. The solution is then left under reflux and gentle stirring during 24 h. The resulting solution is purified either by dialysis against water or by three successive centrifugation/dispersion steps in a water/ethanol mixture (1:3, v/v). Azo Dye Grafting on Accessible Surface Epoxy Functions of GPTMS Silanized Particles. Purified GPTMS silanized particles are dispersed in a water/ethanol mixture (1:3, v/v). Five equivalents of N-methylaniline (as compared to the total vanadate amount) are then added, and the resulting solution is left under stirring at 80 °C for 24 h. The resulting solution is then dialyzed against water to remove the excess N-methylaniline, and further concentrated under vacuum. Five equivalents of nitrobenzenediazonium tetrafluoroborate (as compared to the total vanadate amount) are added in the same volume of acetic acid. After a few hours under mild stirring, a deep red turbid solution is observed. The observed precipitate is washed several times by centrifugation, and the particles are finally dispersed in dimethyl sulfoxide (DMSO) leading to a clear red solution. Fluoresceine Dye Grafting on Accessible Surface Amino Functions of APTES Silanized Particles. The protocol used for the fluoresceine grafting reaction was adapted from the one given by Molecular Probes starting from fluoresceine isothiocyanate (FITC).37 A colloidal suspension of APTES silanized nanoparticles with a total VO43- concentration of 1.3 mM is prepared in a ethanol/ water (3:1 v/v) solution. To one volume of the obtained suspension is added one volume of ethanol containing 50 equiv of FITC with respect to the total vanadate concentration. The obtained solution is left under stirring at 40 °C for two days. The suspension is then purified four to five times by centrifugation/dispersion in ethanol to remove the excess FITC. Finally, 50 µL of phosphate buffer solution (pH ) 8) is added into 1 mL of the obtained solution, and colorimetric titration of the remaining FITC is performed.37 Protein Coupling with APTES Functionalized Particles. Coupling of proteins to the APTES functionalized nanoparticles was achieved using a homobifunctional amine-reactive cross-linker (bis(sulfosuccinimidyl)suberate, BS3, Pierce).15 The cross-linker is first reacted with amino-groups at the surface of the particles and, in a second step, with amino groups at the protein surface. The conjugation of the cross-linker to the nanoparticles takes place in 100% DMSO (Prolabo). Using three successive centrifugations at 16 100 g during 70 min and redispersions of the precipitate in DMSO, we achieved a complete transfer into DMSO of the APTES functionalized nanoparticles formerly in water. A large excess of BS3 was used for the nanoparticle-BS3 reaction to avoid nanoparticle cross-linking. Typically, a 0.15-µM concentration of nanoparticles ([VO43- ) 27 mM]) was reacted with a 27 mM concentration of

The YVO4:Eu particles studied in this work are prepared through a simple coprecipitation from precursor salts in aqueous solution. The process and the structural characterization of the particles are described extensively in our previous paper.6 As shown in Figure 2, the final colloidal suspension consists of dispersed particles exhibiting a prolate ellipsoid shape. Their size follows a log-normal distribution with average axial dimensions of 19 nm (σ ) 7 nm) and 32 nm (σ )12 nm), respectively. At the end of the synthesis, i.e., after purification by dialysis, the conductivity is about 100 µS · cm-2, the pH is 8.3, and the zeta-potential is around -10 mV. Primary Silicate Layer Deposition. The conditions used for the silane encapsulation were chosen to optimize the density of reactive surface functions for the lowest possible thickness of the added layer. Contrary to other authors,9,10 we consequently chose not to perform this encapsulation in the presence of tetraalkoxysilane that potentially allows improving the efficiency of the silane polymerization, but leads to shells with a much larger thickness. In some of the previous work devoted to silane functionalization of non siliceous compounds, deposition of the trialkoxysilane layer is achieved directly on the surface of the oxide by reaction with surface hydroxyl groups.38 Preliminary studies performed on our YVO4 system did not provide satisfactory results, probably because of insufficient interactions between the surface of our particles and the hydrolyzed alkoxides, leading mostly to the formation of separate siloxane particles or clusters. This problem was circumvented by following the route used by Philipse and co-workers in the case of boehmite needles.33 These authors performed a first step of deposition of a very thin shell of silica that acts as a primary layer, enhancing the

(37) Invitrogen Technical Resources-Manuals and Product Inserts, product #F143 http://probes.invitrogen.com/media/pis/mp00143.pdf.

(38) Abboud, M.; Turner, M.; Duguet, E.; Fontanille, M. J. Mater. Chem. 1997, 7, 1527–2532.

Results and Discussion

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Figure 3. Evolution of the IR spectra from a dried drop of solution.

interactions and thus the targeted deposition of the functional trialkoxysilane at the surface of the particles. The silica deposition is simply achieved by the addition of an excess of a basic silicate solution into the colloidal suspension of particles. Contrary to the work of Philipse,33 we used TMASi instead of sodium silicate since a higher stability was observed in this case for the silicated particles in the alcoholic medium used for the further silane deposition step. The initial large excess of TMASi used for the silicate deposition is removed by dialysis. Since the vanadate and the silicate vibration bands in the infrared (IR) region are both intense and well-separated, the elimination of the excess silicate and the presence of remaining adsorbed species can be conveniently followed by the IR spectra of a dried drop of solution taken after different times of dialysis (Figure 3). From calibration curves measured for the ratio between the 814 cm-1 absorption band from the vanadate and the 1120 cm-1 band from silica, a quantitative estimation of the amount of remaining silicate is obtained. At the end of the purification, i.e., for a conductivity of 100 µS · cm-1, the r ) NSilicate/NYVO4 molar ratio is found to be 1.3. This value is consistent with the amount found by chemical analysis of the sample (r )1.5). The amount found if the purification is achieved by successive centrifugation/ washing of the particles is only slightly lower (r )1.1). This shows that the remaining silicate is not present as separate particles but is indeed located at the surface of the particles. It was nevertheless noted that the extensive purification of the particles leads to an almost complete removal of the silicate species. This can be explained either as a reversible adsorption of the silicate species or a progressive dissolution of the silicate as the purification proceeds. In any case, this justifies why the purification was stopped at a relatively high value of conductivity (100 µS · cm-2). To detect the presence of adsorbed silica species on the nanoparticles, we performed zeta potential measurements at different pH values (see Figure 4). We showed that the point of zero charge (PZC) of the silicated particles lies at pH ) 4, slightly below the one found for the initial particles (pH ) 5.6). This value is shifted toward the PZC of silica beads (pH ) 2.5) without reaching it, confirming the idea that silica is present on the particles as small clusters that are adsorbed at their surface, without forming a complete shell. Determination of the Total Amount of Grafted Silane. Functionalization of the silicated particles by the trialkoxysilanes (GPTMS or APTES) was performed following a procedure adapted from the work of Philipse et al.33 The surface modification was clearly detected by zeta-potential measurements (Figure 4). Starting from silicated particles with a PZC of 4, the reaction with APTES provides particles with a PZC around 9, corresponding to a high surface coverage with amino-groups. In the case of GPTMS, no net surface charge could be measured at any pH, as expected in the presence of neutral glycidoxypropyl functions at the surface of the particles.

Figure 4. Dependence with pH of the zeta potential of bare particles (0), silicated particles ([), GPTMS silanized particles (×), and APTES silanized particles (b).

Figure 5. Relative weight loss (150-550 °C) for (a) silicated particles, (b) nonpurified GPTMS silanized particles, (c) GPTMS silanized particles purified by dialysis, (d) GPTMS silanized particles purified by successive centrifugation/dispersion, (e) nonpurified APTES silanized particles, and (f) APTES silanized particles purified by successive centrifugation/ dispersion steps.

While part of the introduced silane has been grafted on the nanoparticle surface, another part almost inevitably reacted outside of the particle surface, forming small silane aggregates in the surrounding solution. Careful characterization of the nanoparticle functionalization then requires the removal of excess partially condensed silane. This purification was achieved either by dialysis or by sedimentation using three successive steps of centrifugation/dispersion. The efficiencies of these two purification processes were compared using thermogravimetric experiments under oxygen. The thermogravimetric weight loss data are presented in Figure 5 in the case of GPTMS- and APTEStreated particles. Up to 150 °C, most of the weight loss corresponds to desorption of the solvent and to the release of water from the condensation of silanol groups into siloxane bonds.38 In the case of silicated nanoparticles, i.e., before grafting of the functional silane, the total weight loss between 150 and 550 °C is found to be around 7%. This corresponds to the removal of residual adsorbed water, or water resulting from condensation reaction of silanols. The larger weight losses observed on the GPTMS and APTES silanized samples between 150 and 550 °C are attributed to removal of the organic part of the silane. The amplitude of the weight loss is obviously an indication of the total amount of

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Figure 6. IR spectra of pure GPTMS (a), pure APTES (b), initial YVO4: Eu nanoparticles (c), silicated (d), GPTMS silanized (e), and APTES silanized nanoparticles (f).

silane (grafted or not) present in the sample, and thus of the efficiency of the purification process. The GPTMS and APTES silanized samples purified by dialysis present a weight loss of 25.4% and 32%, respectively, much larger than the weight loss for the samples purified by centrifugation/dispersion (15% and 11%, respectively). This clearly shows the better efficiency of the latter method for the purification process, since a nonnegligible part of the silane condensed separately from the surface of the particles. Further characterizations were then performed on samples purified by centrifugation/dispersion steps. Figure 6 displays the IR spectra of the different samples and the corresponding references. The presence of grafted silane is attested by the CH bands lying between 2900 and 3000 cm-1 and, in the case of the APTES silanized sample, the bands between 1400 and 1700 cm-1.39 We could also observe a clear change in the relative intensity of the δSi-O-Si band at 1120 cm-1 and the ν3/V-O-V band at 814 cm-1 as compared to the initial silicated particles. While a higher relative intensity is observed in the case of the APTES silanized sample, indicating a larger amount of deposited silane, the GPTMS silanized sample shows a clear decrease of this relative intensity, which could only be understood by a partial desorption of the silicate from the nanoparticle surface during the grafting process. This was confirmed by chemical analysis, which gives a r ) NSi/NYVO4 molar ratio of about 0.27, lower than the ratio measured on the initial silicated sample (r ) 1.5). We must then conclude that a significant part of the silicate has been leached out of the surface during the silanization treatment, probably as a consequence of its condensation with the silane in excess. The full characterization of the silanized particles then requires determining both the amount of silicate that remained adsorbed on the particle after the grafting process and the amount of grafted silane F-Si, either GP-Si or A-Si, for the partially condensed GPTMS and APTES, respectively. Knowing the total amount of silicon species on the particles, the information to be obtained is the ratio x ) NF-Si/Nsilicate between the functionalized silane F-Si and the silicate. We attempted to characterize our samples by 29Si NMR experiments, as described in other systems.28,31 Spectra are given as Supporting Information. On the spectrum of the unpurified GPTMS grafted sample, both silicon from the (39) Shimizu, I.; Okabayashi, H.; Taga, K.; Nishio, E.; O’Connor, C. J. Vib. Spectrosc. 1997, 14, 113–123.

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silicate and GP-Si are clearly observed and may be quantified. But the spectrum from the purified sample, for which the amount of material is limited, the signal-to-noise ratio is too poor to conveniently perform an accurate determination of x. We then focused on the analysis of the data from thermogravimetric analysis (TGA). This technique has been shown to conveniently provide almost quantitative determination of silane grafting efficiency on many other systems.28,32,35,38 The results are nevertheless usually overestimated because the condensation of residual silanols is neglected and the weight loss is supposed to show only the thermal degradation of the organic group borne by the silane.35 In our case, the analysis is even more complex since we know neither the relative amount of silane to silicate nor their condensation state. We nevertheless show hereafter that such analysis performed on silicated nanoparticles, unpurified silanized particles, and the purified ones may provide accurate information. From the weight loss measured between 150 and 550 °C and using the r ) NSi/NYVO4 molar ratio as determined by chemical analysis or IR, one can determine the total apparent weight loss per mole of silicon FT (details for the calculation are given in the Supporting Information). In the case of the silicated sample, FT is found to be 15 g · mol-1. Neglecting the removal of residual tetramethylammonium (as justified by the absence of significant signal on the IR spectrum), FT depends on the degree of condensation of the silicate prior to the thermal treatment. Its value should then lye between 36 g · mol-1 for an uncondensed silicate Si(OH)4, and 0 g · mol-1 for a fully condensed silicate SiO2. The measured value is thus coherent with a partially condensed silicate adsorbed on the surface. Concerning the unpurified GPTMS silanized sample, FT is found to be 107 g · mol-1. It may be expressed as FT ) (Fsilicate + x · FGP-Si)/(1 + x), where Fsilicate and FGP-Si are the average weight losses per mole of grafted silicate and GPTMS, respectively, and x ) NGP-Si/Nsilicate. As the grafted silicate may have condensed with the functionalized silane during the silanization step, Fsilicate should then lie between 0 and 15 g · mol-1. Moreover, x ) NGP-Si/Nsilicate is known to be 3.33 as it was fixed by the experimental conditions. We thus obtain FGP-Si ) 134-139 g · mol-1 depending on Fsilicate. This value should correspond to the weight loss occurring during the reaction [GP-Si(OX)2-O]f SiO2. A fully condensed silane, corresponding to X ) Si, leads to a weight loss of 107 g · mol-1, while the highest weight loss should be found for X ) C2H5 where the silane has not been fully hydrolyzed and the methoxy groups have been replaced by ethoxy groups from the solvent through a transesterification reaction. This latter case corresponds to a maximum expected weight loss of 181 g · mol-1. (X ) H leads to 125 g · mol-1, and X ) CH3 leads to 153 g · mol-1). Finally, the purified GPTMS silanized sample gives FT ) 86 g · mol-1. Considering the same state of hydrolysis/condensation for the silane and the silicate as in the unpurified sample, i.e., a value of FGP-Si ) 134-139 g · mol-1 and Fsilicate ) 0-15 g · mol-1, we obtain a value of x ) NF-Si/Nsilicate, lying between 1.3 and 1.8. Chemical analysis of this sample gives an r ) NSi/NYVO4 value of 0.27. The molar ratio of grafted GPTMS is then found to be NGP-Si/NYVO4 ) 0.15-0.17, while the amount of remaining silicate is Nsilicate/NYVO4 ) 0.1-0.12. The case of APTES silanized nanoparticles was treated the same way. Analysis of the unpurified APTES silanized sample leads to FT ) 44 g · mol-1. The experimental conditions used during the functionalization process give us the value of x ) 3.33, and we consider here also that the silicate can only be

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Table 1. Summary of the Results Obtained for the Characterization of the Particles Functionalized with Epoxy- or Aminosilanes sample

silicated

GPTMS unpurified

GPTMS purified

APTES unpurified

APTES purified

∆m r ) NSi/NYVO4b x ) NF-Si/NSilicatec FΤ ) ∆m/NSid Fsilicate (g · mol-1)e FF-Si (g · mol-1)f Nsilicate/NYVO4g NF-Si/NYVO4h σF-Si (molecules · nm-2)i e (nm)j δ (groups · nm-2)k

7% 1.5 0 15 15

54% 6.5 3.33 107 0-15 134-139

9.5% 0.27 1.3-1.8 86 0-15 134-139 0.10-0.12 0.15-0.17 12-14 2.7-3.1 0.4

32,3% 6.5 3.33 44 0-15 53-57

10.7% 1.2 0.69-0.83 28 0-15 53-57 0.57-0.86 0.34-0.63 28-51 4.5-8.0 1.9

a

1.5

a TGA relative weight loss between 150 and 550 °C. b Total silicon-to-vanadate molar ratio as determined by chemical analysis. c Molar ratio between the functionalized silane and the silicate. d TGA relative weight loss per mole of silicon atom. e TGA relative weight loss for a mole of deposited silicate and organosilane, respectively. f TGA relative weight loss for a mole of deposited silicate and organosilane, respectively. g Molar ratio of silicate and organosilane with respect to one YVO4 formula unit. h Molar ratio of silicate and organosilane with respect to one YVO4 formula unit. i Number of organosilane molecules per surface unit on the particle. j Equivalent thickness of the siloxane layer. k Number of functions with a chemical accessibility per surface unit on the particles.

Figure 7. General representation of the chemical structure of the siloxane shell indicating functional groups that are or not accessible for further chemical or biochemical coupling reactions.

further condensed during the grafting process (Fsilicate e 15 g · mol-1). This leads to a relative weight loss per aminosilane of FA-Si ) 53-57 g · mol-1. This corresponds to the weight loss during the calcination reaction of [A-Si(OX)2-O]- f SiO2. A fully condensed silane (X ) Si) would lead to a weight loss of 50 g · mol-1, while a completely hydrolyzed grafted silane (X ) H) loses 68 g · mol-1, and a nonhydrolyzed silane loses 124 g · mol-1. The experimental value obtained (FA-Si ) 53-57 g · mol-1) thus indicates that most of the silane molecules are fully condensed. After the purification process, the FT value is found to be 28 g · mol-1. We still assume that the hydrolysis-condensation state of the silicate and the silane remained unchanged, so that Fsilicate ) 0-15 g · mol-1, and FA-Si ) 53-57 g · mol-1. This leads to a value of x ) NA-Si/Nsilicate between 1.12 and 0.44. Chemical analysis of this sample gives an r ) NSi/NYVO4 value of 1.2. The amount of grafted aminosilane is then found to be NA-Si/NYVO4 ) 0.34-0.63, and the amount of silicate is Nsilicate/NYVO4 ) 0.57-0.86. From all the previous results, summarized in Table 1, one can reasonably assume that the particles may be seen as shown in Figure 7. The good dispersion state of the particles was checked using dynamic light scattering experiments performed on the colloidal suspensions. The measured average size (30 nm) is similar to the one of the initial particles, giving evidence that particles are well dispersed in solution and that silanization did not induce a significant particle aggregation. Taking into account the size distribution of the particles, the average surface and volume of the particles are found to be 2300 nm2 and 15000 nm3, respectively (details of the calculation are given in the Supporting Information). Considering four VO4 units for a cell volume of 0.319 nm3, the average number of

vanadates per particle can then be estimated to be around 188 000. From the values calculated for NF-Si/NYVO4, the average number of grafted organosilane F-Si per surface unit on the particle (σF-Si) can be calculated: σGP-Si ) 12 to 14 molecules · nm-2 in the case of GPTMS-treated nanoparticles and σA-Si ) 28 -51 molecules · nm-2 in the case of APTES-treated ones. Assuming a homogeneous reticulated shell with a functionalized silane matrix density of 1 g · cm-3,28 the estimated values of the slab thickness is 2.7-3.1 nm with GPTMS and 4.5-8.0 nm in the case of APTES. It can be noted that the significant difference between the shells thicknesses formed by the two precursors probably arises from their different chemical properties. The glycidoxypropyl function has a low polarity, so that most interactions between the silane and the surface of the particles occur through the silanols leading to siloxane bond formation, and leaving the organic part at the outer part of the surface. This leads to a progressive increase of glycidoxypropyl functions at the surface of the shell and thus to a self-limited growth of the shell. Similar behavior may occur in the case of the aminosilane, but a thicker shell is obtained probably as a result of the interaction between the amino groups and silanols from the growing shell. Chemical Accessibility of the Epoxy and Amino Functions. Considering that the functional silane is present at the surface of the particles as a layer with a thickness higher than the one expected for a monolayer, it is clear that not all the reactive epoxy or amino functions are oriented outside of the particle/ solvent interface, and part of them are sterically hindered inside the shell. Moreover, in the case of APTES, the amino groups may be directly bonded to the surface through complexation of cations or electrostatic interactions. Such interactions with the

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Giaume et al.

Scheme 1. Successive Reactions Employed for the Azo Dye Labeling of Chemically Accessible Epoxy Groups

GPTMS functionalization are less probable, but the experimental conditions used for the deposition of the silane may lead to an opening of the epoxy groups, thus annihilating their reactivity.40-42 The issue is then to determine the number of functions that are indeed potentially reactive toward a further functionalization of the particles. The opening of part of the epoxy rings during the GPTMS deposition step was indeed evidenced by 13C NMR spectroscopy performed on the unpurified sample. The spectrum showed that this opening occurs mainly through nucleophilic reactions with water and ethanol, producing diol and glycolether derivatives, respectively. This experiment could nevertheless not be achieved on the purified sample, for which the too small amount of silane leads to a poor signal-to-noise ratio. Moreover, proton decoupled 13C NMR spectroscopy does not allow a quantitative determination of the different contributions in the spectrum. In order to obtain a rough estimation of the amount of opened epoxy rings after the silanization step, the experiment was achieved under exactly the same conditions, but replacing the GPTMS by glycidylisopropylether. This molecule was chosen assuming a similar reactivity toward epoxy opening, and considering that this compound as well as the opened ring byproduct can be easily detected by vapor phase chromatography. It was found that, at the end of the experiment, 74% of the epoxy was still present, while 8% had been opened by ethanol and 18% by water. Following the strategy commonly used in biology, the determination of the number of reactive surface functions was determined using a colorimetric titration. In both cases (GP-Si and A-Si particles), the experiment consists of grafting a dye molecule bearing a function that is known to react efficiently with the epoxy or the amino functions, respectively. The dye molecules that have not reacted with the nanoparticles are removed by centrifugation. The number of grafted molecules is finally determined either by colorimetry or fluorescence, providing the amount of reactive surface functions at the surface of the silanized particles. In the case of the epoxy-functionalized particles, we used a deep red azoic dye obtained after a two-step process (Scheme 1).43 In a first step, N-methyl aniline reacts with the epoxy groups, then nitrobenzenediazonium tetrafluoroborate is added, leading to the formation of the deep red azo dye. This two-step process was preferred to the direct grafting of the azo dye, whose reactivity toward the amino group was found to be strongly decreased by the azo bond. A preliminary experiment was performed and fully characterized using glycidylisopropylether, showing that the two (40) Calleri, E.; Massolini, G.; Lubda, D.; Temporini, C.; Loiodice, F.; Caccialanza, G. J. Chromatogr., A 2004, 1031, 93–100. (41) Bro¨nsted, J. N.; Kilpatrick, M. J. Am. Chem. Soc. 1929, 51, 428–461. (42) Pritchard, J. G.; Long, F. A. J. Am. Chem. Soc. 1956, 78, 2667–2670. (43) Vollardt, P.; Schore, N. Traite´ de Chimie Organique, 3rd ed.;Translation by P. Depovere; De Broeck University: Brussels, Belgium, 1999; ISBN 2-80412153-X 1019.

steps of the reactions have a yield of 97% and 73%, respectively. Assuming a similar reactivity at the surface of the particles, the yield of formation of grafted azo dye as compared to the number of accessible epoxy functions should be 70%. The final concentration of surface azo dye was determined by colorimetry, assuming a molar extinction coefficient of the grafted dye (ε460 nm ) 32 500 L · mol-1 · cm-1) identical to the one of the free dye in solution. The concentration of vanadate groups in solution was determined from absorbance measurements. We thus deduced a dye/VO43- molar ratio of 5 × 10-3. Taking into account the yield of the grafted dye synthesis and based on the average number of vanadates per nanoparticle of 188 000, this corresponds to an average of about 940 reactive surface epoxy groups per nanoparticle and about 0.4 reactive surface epoxy groups per square nanometer. This corresponds to about 3% of the total grafted epoxy groups. Concerning the amino-functionalized particles, the number of accessible amino functions was determined using the reaction with FITC.9,44,45 The protocol used for the FITC coupling reaction was adapted from the one given by Molecular Probes.37 The reaction was achieved in a 3/1 ethanol/water solution in order to preserve the stability of the particles, and the pH was adjusted to about 8 to take into account the pH-dependent spectroscopic properties of this molecule. Colorimetric titration was performed under the same conditions, using calibration curves providing the extinction coefficient of FITC: ε280nm ) 14 680 L · mol-1 · cm-1 and ε500nm ) 75 650 L · mol-1 · cm-1. From our colorimetric titration, the grafted fluoresceine-tovanadate molar ratio was found to be about 23 × 10-3. This corresponds to about 1.9 reactive amino functions per square nanometer and to an average of about 4300 reactive amino groups per nanoparticle. This corresponds to a fraction of about 5% of the total grafted amine, consistent with a multilayer deposition of the silane. It can be noted that this density of surface reactive amino functions is of the same order of magnitude as what is found in the case of biochips, for which an optimized surface coverage of 2.1 amine · nm-2 was reported.46 Biological Accessibility of the Epoxy and Amino Functions. The main purpose of the surface functionalization of the nanoparticles by amino- and epoxy- functions is the further grafting of biological species for labeling applications and tracking using fluorescence microscopy. First, results on YVO4:Eu nanoparticles were obtained using epoxy-functionalized particles for the biological labeling of sodium channels in living cells.13 In this case, guanidinium groups were covalently reacted with the dangling epoxy functions at the surface of the particles. The guanidinium group is the active part of two naturally occurring (44) Schmitt, M.; Wagner, J.; Jung, G.; Hempelmann, R. J. Colloid Interface Sci. 2007, 311, 425–429. (45) Arroyo-Hernandez, M.; Pe´rez-Rigueiro, J.; Martı´nez-Duart, J. M. Mater. Sci. Eng. C 2006, 26, 938–941. (46) Jianying, Z.; Yuhan, L.; Haiquan, G.; Lianxun, G. Chin. J. Anal. Chem. 2006, 34, 1235–1238.

Organic Functionalization of Luminescent Oxide NPs

Langmuir, Vol. 24, No. 19, 2008 11025

Figure 8. Scheme showing the coupling of proteins to nanoparticles using a homobifunctional (X-X) or a heterobifunctional (X-Y) cross-linker (left) and the distribution of the number of proteins per nanoparticle determined for an ensemble of 379 single nanoparticles.

toxins, tetrodotoxin and saxitoxin, which selectively and potently block voltage-dependent sodium channels by plugging the channel mouth.47-49 The efficiency of the functionalization was mainly attested by the physiological properties of the particles that behaved like artificial toxins targeting and inhibiting the sodium channels in a similar way as the natural toxins.13 In this work, only the coupling of proteins onto aminofunctionalized particles was studied. The chemistry of this system appears to be much more versatile compared to epoxy functions, mainly because of the existence of a variety of coupling (crosslinking) agents that can react with different chemical functions present on the proteins (amino, thiols, carboxylate, etc.) under mild conditions. This approach was first tested on a relatively small protein (R-bungarotoxin) that is commercially available with an organic fluorescent tag (Alexa 488).15 This allowed testing the coupling of this protein with the amino-functionalized nanoparticles through the use of a homobifunctional cross-linker, namely BS3 (Pierce), which can react in a first step with amino groups on the particle surface and, in a second step, with amino groups on the protein surface through its two succinimidyl ester groups [see Figure 8 (left)]. The use of a protein tagged with an organic fluorescent marker allows the accurate quantitative determination of the number of grafted proteins per particle and the efficiency of the grafting process. Determination of the average number of grafted R-bungarotoxin was determined on a purified sample (i.e., after removal of excess protein), using the emission intensity of the Alexa tag and a calibration curve obtained in the same medium. Depending mostly on the initial protein/nanoparticle concentration, the average number of grafted R-bungarotoxins per nanoparticle could be adjusted from 0 up to about 8 proteins per nanoparticle. A control experiment performed on nanoparticles coupled with BS3 that has been hydrolyzed led to a coupling efficiency decreased by a factor of 30, indicating that the coupling proceeds mainly through covalent binding between the BS3 and the protein. We furthermore obtained similar results for coupling to sulfhydryl groups of R-bungarotoxin using a heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC, Pierce), instead of BS3. The emission spectra and decay times of the nanoparticles before and after coupling to the proteins are shown in the Supporting Information. The numbers of reactive epoxy and amino groups available at the surface of an average nanoparticle (940 and 4300 for epoxy and amino groups, respectively, as determined above) are largely sufficient to couple the desired number of proteins per nanoparticle. Indeed, the maximum number of proteins that can be (47) Hille, B. Biophys. J. 1975, 15, 615–619. (48) Kao, C. Y. Ann. N.Y. Acad. Sci. 1986, 479, 52–67. (49) Narahashi, T. J. Pharmacol. Exp. Ther. 2000, 294, 1–26.

coupled will rather be determined by the size of the protein and limited by the steric hindrance between different proteins. Considering that these particles are promising for applications as single-molecule labels, the desired protein-nanoparticle ratio is usually 1:1 in order to avoid effects such as receptor crosslinking. For such applications, the ultimate characterization is the determination of the number of proteins per particle at the single particle level and of the distribution of this number for the total particle population. This characterization was achieved on our system as described in more detail elsewhere15 through the observation of the emission from single particles deposited from the colloidal solution onto a silica slide. It can be noted that observation of single particles as checked by their emission intensity12 is a clear indication of the good dispersion state of the particles, which are thus not agglomerated after all the functionalization steps. The basic principle of the determination of the distribution of the number of proteins per particle is to measure the successive photobleaching of individual Alexa dye molecules that label each protein grafted on single nanoparticles observed by fluorescence microscopy. Figure 8 (right) shows the distribution of the protein-nanoparticle ratio obtained from this characterization at the single-particle level. Only 4% of the particles are not grafted to a protein, which confirms the good homogeneity of all the silane functionalization process, considering that part of this fraction may also result from the Poissonian distribution of the protein number. Although the average protein-nanoparticle ratio for this sample was 8, the maximum of the distribution lies at 3 proteins per nanoparticle. This difference is due to the presence of large nanoparticles bearing a large number of proteins (35% of the nanoparticles are coupled to more than 5 proteins-data not shown in Figure 8) and demonstrates the importance of a single-particle determination of the protein-particle coupling ratio.

Conclusion This work was devoted to the full characterization of the functionalization of luminescent YVO4:Eu nanoparticles developed for their further application in biological tagging and tracking experiments. The strategy that was used is the encapsulation of the particles with a thin shell of organosiloxane that was polymerized either from APTES or GPTES precursors. The deposition process was achieved after the preliminary adsorption of silicates that act as a primary layer for the targeted deposition of the organosilanes at the surface of the particles. This deposition leads to the formation of organosilane shells with thicknesses of a few nanometers. The careful characterization of the functionalization was achieved using conventional analytical techniques (chemical analysis, TGA, NMR, and IR spectroscopy) with the aim to determine as precisely as possible the amount of grafted silane and the number of amino or epoxy functions that are

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indeed accessible for the further coupling of the particles with chemical or biological species. The density of reactive epoxyor amino-functions was found to be respectively 0.4 and 1.9 functions/nm2, which is on the order of magnitude of the surface densities found in biochips after an optimized silanization process. Coupling with proteins was investigated in the case of R-bungarotoxin that was previously labeled with an Alexa fluorescent dye. Up to 8 proteins per particle were coupled on average, and the exact number of proteins per particle at the single particle level was determined using the stepwise photobleaching of the Alexa dye molecules.15 Current development of this work concerns applications of these particles to address biological issues such as real-time tracking of single biomolecules labeled by YVO4:Eu nanoparticles.

Giaume et al.

Acknowledgment. We thank S. Tu¨rkcan for the lifetime measurements, and the Fonds National pour la Science (ACI Dynamique et Re´activite´ des Assemblages Biologiques), the De´le´gation Ge´ne´rale pour l’Armement (D.C.), and the Ecole Polytechnique (Monge fellowship, D.G.) for financial support. Supporting Information Available: Stability of NHS ester from BS3 in pH 7.4 phosphate buffer; 29Si NMR spectra of purified and unpurified GPTMS treated samples; calculations of the total apparent weight loss per mole of silicon and the average particle surface and volume for a prolate ellipsoid; emission spectra and decay times of nanoparticles before and after coupling to proteins. This material is available free of charge via the Internet at http://pubs.acs.org. LA8015468