Article pubs.acs.org/cm
Preparation, Characterization, and Surface Modification of Trifluoroethyl Ester-Terminated Silicon Nanoparticles Wouter Biesta,† Barend van Lagen,† Veronique S. Gevaert,‡ Antonius T. M. Marcelis,† Jos M. J. Paulusse,†,⊥ Michel W. F. Nielen,†,§ and Han Zuilhof*,†,∥ †
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands Macromolecular and Organic Chemistry, Department of Chemical Engineering and Chemistry, Eindhoven University, P.O. Box 513, 5600 MB Eindhoven, The Netherlands § RIKILT−Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands ∥ Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah, Saudi Arabia ‡
S Supporting Information *
ABSTRACT: Platinum-catalyzed hydrosilylation of hydrogen-terminated silicon nanoparticles (Si NPs) with 2,2,2-trifluoroethyl 4-pentenoate gave well-defined nanoparticles (NPs) with surface groups that are reactive toward amines. The particles showed a diameter of 1.4 ± 0.2 nm as revealed by transmission electron microscopy (TEM) measurements. Characterization with 1H, 13C and heteronuclear NMR techniques revealed that the trifluoroethyl pentenoate group is attached to the Si NP surface via the terminal carbon atom. The trifluoroethyl ester is reactive toward primary amines, allowing for additional surface functionalization. Modification of the Si NPs was performed with benzylamine, 1,2-diaminoethane, and propargylamine. The modification gave a complete substitution of the trifluoroethyl group to amide groups. The modified Si NPs were characterized in detail by a series of one-dimensional (1-D) and two-dimensional (2-D) NMR techniques and by FTIR. The propargylamide-terminated Si NPs were further functionalized with an azide-terminated fluorescent dye (Azide-Fluor 585 sulphorhodamine) using a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). Gel permeation chromatography and time-resolved fluorescence anisotropy spectroscopy of the dye reveal a significant increase in the hydrodynamic radius upon clicking of the dye. Additionally, NMR spectroscopy reveals the presence of a 1,2,3-triazole ring in the product, which confirms that the increase in the hydrodynamic radius is caused by the attachment of the dye to the Si NP surface via the CuAAC reaction. KEYWORDS: silicon, nanoparticles, bioconjugation, NMR, click chemistry
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inherent nontoxicity of the silicon core.18,19 To prevent degradation of the Si NPs, they are typically coated with a hydrocarbon shell20 or by a layer of silicon oxide by exposing the freshly prepared Si NPs to air.21 Up to now, a large number of methods has been developed for the preparation of well-defined Si NPs using top-down and bottom-up techniques.16 The bottom-up approaches are based on dissociation,21−24 reduction,25−27 and oxidation28 of molecular silicon precursors. This approach allows for the use of solution chemistry techniques with a wide variety of silicon sources and reducing or oxidizing agents.29 Silicon tetrachloride can be reduced with lithium aluminum hydride in a one-pot reaction in the presence of tetraoctylammonium bromide (TOAB) as a surfactant,30 yielding small and relatively monodisperse particles.20 A similar approach was used by Rosso-Vasic et al. in which the reduction takes place in a TOAB-containing solution under sonication.26 The method initially yields Si NPs with a hydrogen-terminated surface. To
INTRODUCTION Fluorescent semiconductor nanoparticles, also coined quantum dots, have received significant attention over the past decades from many fields, such as biology,1,2 analytical chemistry,3 solar cell research,4 and optoelectronics.5 They exhibit superior resistance against photobleaching and photochemical degradation, and their size-dependent fluorescence in combination with broad excitation bands make them extremely useful for multiplex fluorescence analysis and bioimaging techniques.6,7 However, quantum dots are often made of elements that are inherently toxic, even at low concentrations, such as cadmium, selenium, lead, and/or arsenic.8,9 For medical or biological applications it is therefore crucial to prevent leaching of these elements to the surrounding medium. This is typically achieved by attaching a biocompatible shell around the particles, which makes the quantum dots suitable for in vivo applications. Examples are shells of silica,10 polyethylene glycols,11 surfactants,12 and proteins.13 Ever since the discovery of photoluminescence in nanosized crystalline silicon,14,15 silicon nanoparticles (Si NPs) are considered a promising alternative to these toxic quantum dots,16,17 as rigorous toxicological tests have shown the © 2012 American Chemical Society
Received: July 2, 2012 Revised: October 23, 2012 Published: November 7, 2012 4311
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stabilize the surface and to prevent oxidation of the particle, a hydrocarbon shell may be attached by a hydrosilylation reaction with terminal alkenes or alkynes, in analogy with the passivation of planar silicon surfaces.31−33 For application in biological systems, the Si NPs need to be rendered water-soluble and suitable for the conjugation of biologically interesting compounds. Using techniques developed in the field of silicon surface chemistry,31,34 it is possible to modify the surface properties of the coated Si NPs by performing surface chemistry. Water-soluble Si NPs have been synthesized via the attachment of amine35,36 or carboxylic acid19,37,38 groups and by growing poly(acrylic acid) chains on the Si NP surface.39,40 Several experiments have shown the suitability of these Si NPs for bioimaging of multiple generations of cells by only staining the mother cells.36 Recently, some indirect approaches have been developed to introduce hydrophobic Si NPs in aqueous media. Horrocks and co-workers showed that a minimal amount of a polar organic cosolvent like tetrahydrofuran (THF) or dimethylsulfoxide (DMSO) can be used to obtain a nonaggregated dispersion of nonpolar Si NPs in aqueous medium.41 Another approach to disperse hydrophobic Si NPs in aqueous media is by using surfactants.42 Covalent attachment of functional (bio)molecules still remains a major challenge in the development of Si NPs for biological and analytical purposes. Initial attempts were carried out in the group of Reipa to attach DNA and streptavidin to alkyl-terminated Si NPs.43,44 First, the passivating alkyl layer was functionalized via reaction with highly reactive carbene species, which then allowed attachment of NHS esters. Subsequently, these reactive esters were functionalized with amine-terminated DNA strands and streptavidin to demonstrate the proof of principle. Our group has shown that alkylamine-terminated Si NPs are also reactive toward activated esters.45 In this way ruthenium dyes provided with an activated NHS-moiety were attached to Si NPs through different alkyl spacers. Energy transfer studies on these Si NPs revealed a correlation between the transfer efficiency and the alkyl chain length, indicating a covalent attachment of the dye to the Si NPs. Recently, terminal alkene groups have been introduced as an interesting functional group for conjugation reactions. Tilley and co-workers have shown that Si NPs with terminal alkene groups can be converted into epoxides and subsequently into diol groups.46 Lee and co-workers have activated poly(acrylic acid)-terminated Si NPs via EDC/NHS chemistry and conjugated goat-anti mouse antibodies to the activated surface of the Si NPs.47 Ruizendaal et al. have shown that alkeneterminated Si NPs can be functionalized with various thiolterminated oligoethylene glycols and biofunctional moieties including DNA via the versatile thiol−ene reaction.48 In this paper the synthesis and full NMR characterization of novel reactive trifluoroethyl ester-terminated Si NPs is presented. The trifluoroethyl ester moiety is able to react with primary amines, as is shown for a number of primary amines (see Figure 1).49 We provide the first highly detailed NMR characterization of any alkyl-coated Si NP with a series of 1 H and 13C 1D-NMR spectroscopies, as well as 2D-COSY, HSQC, and HMBC NMR experiments using 1H, 13C, and 29Si resonances, revealing the mode of attachment of the alkyl chains to the Si NP surface. An alkyne-terminated NP obtained with this reaction was further conjugated with an azidecontaining dye, and its attachment to the NP was demonstrated
Figure 1. Synthesis of trifluoroethyl ester-terminated Si NPs (TFE Si NPs) and their further conversion with amines.
by GPC, fluorescence anisotropy measurements, and NMR spectroscopy.
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EXPERIMENTAL SECTION
Toluene (anhydrous 99.8%), SiCl4 (99.998% pure based on trace metal analysis), tetraoctylammonium bromide (98%), 4-pentenoic acid (>98%), ethylenediamine (99.5%), propargylamine (98%), and benzylamine (99%) were purchased from Aldrich; lithium aluminum hydride (1 M in tetrahydrofuran), dicyclohexylcarbodiimide (99%) and 4-dimethylaminopyridine (99%) were obtained from Acros; 2,2,2trifluoroethanol (99%) was obtained from ABCR; hexane (HPLC grade) and ethyl acetate were obtained from Biosolve; Azide-Fluor 585 was purchased from Jena Bioscience (Jena, Germany). All chemicals were used as received. Chloroplatinic acid (99.995% trace metal basis) was purchased from Aldrich and diluted with methanol to a 0.05 M solution. Column chromatography was carried out on a Biotage Isolera flash purification system using SNAP KP-Sil cartridges. Preparative gel permeation chromatography was performed on a Varian PLGel 5 μm 100 Å column. Transmission electron microscopy (TEM) was performed on a Tecnai G2 Sphera TEM (FEI) operated at 200 kV. Samples for TEM were prepared by dropcasting a diluted solution of the nanoparticles on a carbon coated copper TEM grid (Agar Scientific) and drying. Images were analyzed with ImageJ version 1.42e software. FT-IR measurements were recorded with a Bruker Alpha-P FTIR diamond ATR spectrometer. High resolution positive ion mass spectra were recorded using a Thermo Fisher Scientific model Exactive orbitrap mass spectrometer equipped with an IonSense Direct Analysis in Real Time (DART) atmospheric pressure ionization probe and operated with helium as ionization gas. All NMR spectra were recorded on a Bruker Avance III NMR spectrometer equipped with a dual band probe. The samples were measured as solutions in normal quartz NMR tubes. UV−vis spectra were recorded on a Cary 50 UV− vis spectrophotometer. Fluorescence spectroscopy was measured on an Edinburgh instruments F900 fluorescence spectrometer. All measurements were carried out using Elma 1 cm quartz cuvettes with acetonitrile as solvent. For time-resolved measurements of the fluorescent dye-modified nanoparticles, samples were excited with a Picoquant pulsed laser diode at a wavelength of 501 nm. Synthesis. 2,2,2-Trifluoroethyl 4-Pentenoate. A solution of 4pentenoic acid (30.0 g, 299 mmol), 2,2,2-trifluoroethanol (75.0 g, 749 mmol), and 4-dimethylaminopyridine (7.92 g, 64.8 mmol) in dichloromethane (300 mL) was stirred at 0 °C. A solution of dicyclohexylcarbodiimide (75.0 g, 364 mmol) in dichloromethane (200 mL) was added. Immediately after addition, dicyclohexylurea formed as a white precipitate. The mixture was stirred overnight, after 4312
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Figure 2. (A) 80 kV TEM image of TFE Si NPs on a carbon grid. The black spots represent individual Si NPs indicated by the arrows. (B) TEM image-derived size distribution of the Si NPs . (−CH2-CH2−Si). IR (cm−1): 2929, 2869, 1756, 1453, 1411, 1281, 1240, 1161, 1123, 1066. Conjugation of Primary Amines to Si NPs. Benzylamine. A mixture of Si NPs (20 mg), 1 mL of benzylamine, and 1.5 mL of CDCl3 was stirred for 24 h at 50 °C. Afterward, the mixture was diluted with dichloromethane to 15 mL and washed 3 times with 5 mL of 1 M aqueous hydrochloric acid. The organic layer was separated, dried over MgSO4, and filtered. The filtrate was concentrated under reduced pressure yielding the functionalized Si NPs as a colorless waxy solid. Yield: 24 mg. 1H NMR (CDCl3, 400 MHz, ppm): δ 7.24 (Ar), 4.33 (OC-NH−CH 2 -Ar), 2.19 (−CH 2 CO−), 1.61 (SiCH2CH2CH2-), 1.33 (Si-CH2CH2-), 0.90 (unknown Si-attached compound), 0.51 (Si−CH2-). 13C NMR (CDCl3, 100 MHz, ppm): 174.06 (CO), 138.16 (C−Ar), 126.97−128.53 (CH−Ar), 43.59 (OC-NH-CH 2 −Ar), 35.74 (−CH 2 CO−), 29.09 (SiCH2CH2CH2-), 22.59 (Si-CH2CH2-), 15.95 (Si-CH2-). Ethylenediamine. The Si NPs (20 mg) were dissolved in CDCl3 (1 mL), after which ethylenediamine (1 mL) was added. The mixture was stirred at room temperature for 3 h, after which the reagents and solvents were evaporated under reduced pressure yielding a colorless waxy solid. 1H NMR (D2O), 400 MHz, ppm): δ 3.22 (-OCNHCH2CH2NH2), 2.73 (-OC-NHCH2CH2NH2), 2.20 (−CH2C O−), 1.55 (Si-CH2CH2CH2-), 1.30 (Si-CH2CH2-), 0.54 (Si-CH2-). 13 C NMR (CDCl3, 100 MHz, ppm): δ 40.68 (-OC-NHCH 2 CH 2 NH 2 ), 39.76 (-OC-NH−CH 2 CH 2 NH 2 ), 35.54 (−CH2CO−), 28.90 (Si-CH2CH2CH2-), 21.78 (Si-CH2CH2-), 14.54 (Si-CH2-). Propargylamine. A mixture of propargylamine (1.0 mL) and Si NPs (30 mg) in CDCl3 (1.5 mL) was stirred for 48 h at 50 °C, after which solvent and excess propargylamine were removed under reduced pressure. The residue was dissolved in dichloromethane and washed 3 times with 5 mL aqueous hydrochloric acid (1 M). The organic layer was separated, dried with MgSO4, filtered and concentrated under reduced pressure. Yield: 10 mg Si NPs as a yellowish waxy solid. 1H NMR (CDCl3, 400 MHz, ppm): δ 3.79 (-OC-NH−CH2-CC), 2.18 (−CH2CO- and CC-H), 1.59 (Si-CH2CH2CH2-), 1.31 (Si-CH2CH2-), 0.49 (Si−CH2-). 13C NMR (CDCl3, 100 MHz, ppm): δ 173.52(CO), 77.32 (CH2-CC), 71.24 (-CCH), 35.87 (CH2CO−), 29.10 (-OC-NH-CH2−C C), 28.90 (Si-CH2CH2CH2-), 22.70 (Si-CH2CH2-), 15.20 (Si-CH2-). CuAAC Click Reaction. The propargylamide-terminated Si NPs (10 mg) were dissolved together with 3 mg of CuSO4, 7 mg of sodium ascorbate, and 5 mg of Azide-Fluor 585 in a 24 mL 1:1 MeOH/water mixture for 24 h at room temperature. Afterward, the mixture was concentrated under reduced pressure, dispersed in acetonitrile, and filtered. The filtrate was collected, and the clicked product was purified
which the white solid was filtered off. Solvent was carefully removed under reduced pressure (500 mbar) and upon distillation under reduced pressure (∼70 °C, 100−110 mbar; boiling point at standard pressure: 140 °C) the product was isolated as a colorless liquid. Yield: 41.0 g (224 mmol, 75%). 1H NMR (CDCl3, 400 MHz, ppm): δ 5.81 (m, 1H, CH2CH-), 5.06 (m, 2H, CH2CH-), 4.65 (q, J = 8.4 Hz, 2H, O−CH2CF3), 2.52 (t, J = 7.2 Hz, 2H, CH2-CO−), 2.40 (q, J = 6.8 Hz, 2H, CH−CH2-CH2−); 13C NMR (CDCl3, 100 MHz, ppm): δ 171.33 (s, -CO−), 135.90 (s, CH2CH-), 122.93 (q, J = 277 Hz, −O−CH2-CF3), 115.96 (s, H2CCH−), 60.19 (q, J = 37.0 Hz, -OCH2CF3), 32.93 (s, CH−CH2-CH2-), 28.52 (s, CH-CH2−CH2-); IR (cm−1): 3038, 2979, 2925, 2854, 1758, 1643, 1413, 1429, 1282, 1160, 975. Mass spectrometry: (DART) [M+H]+ calculated: 183.0627; found: 183.0625. Trifluoroethyl Ester-Terminated Si NPs. Dry and degassed toluene (100 mL) was placed in a three-necked flask with tetraoctylammonium bromide (3.00 g, 5.74 mmol) under an argon atmosphere. The solution was sonicated for 30 min, and silicon tetrachloride (200 μL, 280 mg, 1.76 mmol) was added via a syringe, after which the solution was sonicated for another 30 min. Subsequently, lithium aluminum hydride (LAH) (4.6 mL, 1 M in THF, 4.6 mmol) was added to the mixture, which was sonicated again for 30 min. Caution! During this reaction some silane (SiH4) gas evolves, which may ignite spontaneously when it comes in contact with air. Excess LAH was carefully quenched by addition of dry methanol (60 mL). After the quenching, the mixture was sonicated for another 15 min. Finally, the hydride-terminated particles were passivated through addition of chloroplatinic acid (H2PtCl6, 80 μL, 0.05 M in dry methanol) and 2,2,2-trifluoroethyl 4-pentenoate (25.0 g, 137 mmol) and the mixture was sonicated for 60 min. Solvent was removed under reduced pressure. The residual solids were dispersed in 10% ethyl acetate in hexane, and the suspension was filtered. The filtrate was concentrated under reduced pressure, and the remaining solids were dispersed in hexane. The suspension was filtered again, and the filtrate was concentrated. After column chromatography of the residue (gradient, 15−100% ethyl acetate in hexane), the NPs were obtained as a colorless waxy solid (30−50 mg). 1 H NMR (CDCl3, 400 MHz, ppm): δ 4.46 (q, J = 8.4 Hz, 2H, CF3CH2O), 3.50 (s, H3C−O−CO−), 3.43 (s, Si−O−CH3), 2.42 (t, J = 7.6 Hz, 2H, CH2-CO−), 2.32 (t, J = 7.6 Hz, H3C−O−CO−CH2-), 1.69 (m, J = 7.4 Hz, 2H, Si-CH2CH2CH2-), 1.40 (m, 2H, SiCH2CH2CH2−), 0.61 (m, 2H, Si−CH2CH2CH2−). 13C NMR (CDCl3, 100 MHz, ppm): δ 172.26 (O-CO−C), 122.96 (q, J = 276 Hz, -OCH2CF3), 60.06 (q, J = 37 Hz, −OCH2CF3), 33.15 (−COCH2-), 28.98 (−CO−CH2-CH2-), 26.20 (−CH2−CH2−Si), 15.00 4313
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by preparative gel permeation chromatography using acetonitrile as eluent. Yield: 2 mg as a bright purple film.
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RESULTS AND DISCUSSION Hydrogen-terminated silicon nanoparticles (Si NPs) were synthesized in a one-pot reaction (Figure 1) according to the procedure of Rosso-Vasic et al.26 Under an inert atmosphere, silicon tetrachloride was reduced under sonication at room temperature with LiAlH4 in the presence of tetraoctylammonium bromide. After quenching the reaction with methanol, the hydrogen-terminated Si NPs were provided with a hydrocarbon shell by the in situ Pt-catalyzed hydrosilylation34,50 with the terminal alkene 2,2,2-trifluoroethyl 4-pentenoate (for spectra see Supporting Information, Figures S1−S3 and Table S1). The reactions were performed under an inert atmosphere and with a large excess of alkene to avoid undesired side reactions as much as possible. After this passivation step, the salts and surfactants were removed by precipitation and filtration after which the NPs were isolated with normal phase silica gel column chromatography, yielding 30−50 mg of Si NPs per batch. This purification process removed salts, surfactants, Pt catalyst, and small molecule side products. The average size and size distribution were determined using TEM images of the Si NPs. For the TEM images a solution of the Si NPs was dropcast on a carbon-coated copper grid and dried. The visualization of the Si NP cores and their size determination is a challenging task because of the small size of the NPs, the low contrast of silicon versus the carbon grid background, and the hydrocarbon coating on the silicon core.51 The image depicted in Figure 2A, recorded at 80 kV, displays Si NPs as small black spots. A measurement of the diameter of 260 Si NPs yields the distribution as displayed in Figure 2B. From this distribution an average size of the Si NPs is derived of 1.4 ± 0.2 nm, in line with the sizes of alkyl-terminated Si NPs obtained with the original procedure.26 Also the fluorescence of the trifluoroester-terminated Si NP does not differ markedly from what was reported before for Si NPs synthesized in this manner (λem = ∼300 nm at λexc = 276 nm).26 XPS spectra of the NP reveal C and Si signals with intensities and energies that are comparable with previously found data.26 Furthermore, elemental analyses confirm the presence of Si cores in the sample. Detailed characterization of the hydrocarbon shell of NPs with NMR is often hampered by interfering impurities, the presence of covalently attached side products, and a weak resolution or broad peaks in the NMR spectra.46,48,51,52 A detailed and thorough investigation of the spectra is often required to be able to interpret the unclear picture that is initially provided by the spectrum. The 1H NMR spectrum of the trifluoroethyl ester-terminated Si NPs (Figure 3) shows five distinct signals with similar integrations, labeled a−e. Together with the absence of alkene signals this indicates attachment of the terminal carbon atom of 2,2,2-trifluoroethyl-4-pentenoate to the silicon surface. Attachment of the terminal carbon was confirmed by 2D COSY NMR spectroscopy (see Supporting Information, Figure S5). The shift of peak e in the spectrum at 4.46 ppm, which is assigned to the trifluoroethyl protons, corresponds nicely to the shift of the same group of 2,2,2trifluoroethyl 4-pentenoate (4.65 ppm; see Supporting Information, Figure S1). Besides the signal from the trifluoroethyl group, a small signal (f) is observed at 3.67 ppm, which is assigned to a methyl ester. This signal is thought to result from transesterification of the trifluoroethyl ester into
Figure 3. 1H NMR spectrum of trifluoroethyl ester-terminated Si NPs. Peaks a−e are assigned to the linearly attached carbon chain; peaks f and g originate from a small amount of trifluoroethyl ester that was converted into methyl ester; peak h is assigned to −OCH3 groups bound directly to the Si NP surface.
the methyl ester during the quenching step with methanol, in which methoxide anions form. The methyl ester is accompanied by a signal at 2.36 ppm (g), corresponding to protons next to the carbonyl moiety. The fact that the signals are broad is additional proof that the groups form part of a nanoparticle. Different groups are present in slightly different environments. Together with the slower rotation of the nanoparticles as compared to small molecules this results in broadening of the signals. Furthermore, the broadening of the signals is largest for the CH2 group attached to the Si-core and least for the CH2 group of the trifluoroethyl ester group. DOSY spectroscopy reveals that all signals a−h in the 1H NMR spectrum of Figure 3 are connected to the same particle. The diffusion is slower than that of solvent molecules. 13 C NMR spectroscopy reveals seven signals in agreement with the seven carbons of the alkyl chain, labeled a−g in Supporting Information, Figure S4. Heteronuclear Single Quantum Coherence (HSQC) NMR spectroscopy and 1 H-13C Heteronuclear Multiple Bond Correlation (HMBC) spectra revealed correlations of these carbons with the expected signals in the 1H NMR spectrum (Supporting Information, Figures S6 and S7). A 13C-DEPT 135 experiment (see Supporting Information, Figure S4), which allows for distinguishing tertiary from secondary carbon atoms, indicated that peaks a−f originate from CH2 groups, supporting a linear attachment of the alkyl chain to the silicon surface via the terminal carbon atom. An 1H-29Si HMBC spectrum was recorded to further investigate the mode of attachment of the alkyl chain to the Si NP surface. In this experiment, the 29Si signal is measured through the 1H channel of the spectrometer and the coupling of the 1H to 29Si nuclei is measured, which means that only signals from 29Si nuclei are detected that couple with 1H.53 In the spectrum (Figure 4) the 1H NMR signal at 0.60 ppm, which corresponds to the protons at position a in the 1H NMR spectrum of Figure 3, and the signal at 1.43 ppm, corresponding to the protons at position b, show a strong interaction with the main 29Si-signal at −13.0 ppm (both encircled in Figure 4). These interactions show that the attachment of the alkyl chain occurs via the terminal carbon 4314
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Figure 4. 1H-29Si HMBC NMR spectrum of the TFE Si NPs. The encircled signals are characteristic for the attachment of the hydrocarbon shell to the Si NP surface.
Figure 5. Infrared spectra of the TFE Si NPs (TFE), benzylamide Si NPs (benzyl), propargylamide Si NPs (alkyne), and aminoethylamideterminated Si NPs (amine).
atom to a silicon atom that displays a signal at −13.0 ppm with respect to TMS. An interaction with the silicon signal is also observed for the 1 H signal at 3.48 ppm. This proton signal couples to a 13C signal at 49.9 ppm, as is seen in the HSQC spectrum (Supporting Information, Figure S6). This signal originates from a methoxide moiety attached directly to the silicon surface. The attachment of methoxide to the silicon surface is most likely caused by the platinum catalyst that is used. The platinum catalyst is able to covalently bind water to the hydrogen-terminated silicon surface.50 We propose that the methoxide groups are formed on the Si NP surface by a similar Pt-catalyzed reaction of methoxide anions, which are present after the quenching of the hydride reagent, with the Hterminated silicon surface. It cannot be excluded that during workup some or all of the remaining Si−H sites on the surface
of the nanoparticle are converted into Si−OH groups, because of their high reactivity. The 29Si signals at about +7 and +15 ppm that correlate with the α-CH2 group probably result from 29 Si nuclei that are further away and interact through 3 bonds. Their chemical shift suggest that they could be attached to an oxygen atom, for example, via an −OCH3 or −OH group. The intensity of the Si signal is however no indication for the amount of oxidized groups. These results also indicate that the alkyl groups are not bound to a single Si atom, but to a Si aggregate, that is, a Si nanoparticle. The FTIR spectrum of the TFE Si NPs (see Figure 5, black line) displays the features of the ester groups that are attached to the Si NPs. The CO stretch vibration of the trifluoroethyl ester is located at 1756 cm−1 and the C−O bending vibrations are located at 1281 and 1240 cm−1. The frequencies of the ester 4315
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attached amines and the absence of bridged structures. The remaining primary amine functionality is therefore available for further conjugation of the NP surface. In the IR spectrum, the CO stretch vibration of the ester disappeared and a new C O vibration appeared at 1634 cm−1, corresponding to the presence of an amide. The N−H stretch vibrations of the free amine are visible as a shoulder under the major N−H vibration of the amide bond at 3277 cm−1. The 13C NMR, 1H-13C HSQC, and 1H-13C HMBC NMR spectra also confirm the conversion to the aminoethylamide groups (spectra Supporting Information, Figures S13−S15). Finally, propargylamine was coupled to the Si NPs. In this way, Si NPs with terminal alkyne groups were prepared that are suitable for further modification with, for example, alkyne− azide or alkyne-thiol “click” reactions. Similar reaction conditions were employed as for the benzylamine coupling. Full conversion was confirmed with NMR spectroscopy, and the modified Si NPs were isolated by removal of the excess reagents under reduced pressure, followed by an extraction with acid to remove the last traces of free propargylamine. The 1H NMR spectrum shows complete disappearance of the trifluoroethyl ester signals and the appearance of an alkyneproton signal at δ = 2.15 ppm and the CH2−N group at 4.05 ppm, which are the expected shifts for such groups (see Supporting Information, Figure S16). The infrared spectrum also shows a shift of the carbonyl CO stretch to 1649 cm−1; moreover N−H stretch and bend vibrations are observed at 3288 and 1540 cm−1. The 13C NMR, 1H-13C HSQC and 1 H-13C HMBC NMR spectra also confirm the conversion to the propargylamide groups (spectra Supporting Information, Figures S17−S19). The fluorescence properties of these modified Si NP do not change significantly upon substitution of the ester functionality. Apparently the Si core centered fluorescence is dominated by wave functions that are not interacting strongly with the alkyl termini, for example, in contrast to what has been observed previously for alkylamine-terminated Si nanoparticles.36 The propargylamide-terminated Si NPs are suitable for the attachments of all kinds of azide-terminated (bio)molecules via CuAAC click chemistry.54,55 We have explored the feasibility of CuAAC chemistry by attaching the azide-containing fluorescent dye Azide-Fluor 585 (Figure 6) in a 1: 1 methanol/water
moieties correspond well with the characteristic vibrations of 2,2,2-trifluoroethyl 4-pentenoate. Surface Modifications of the Si NPs. Trifluoroethyl esters are slightly activated esters, which display smooth reactivity toward primary amines without the need for a coupling reagent.49 To investigate the reactivity of the esters on the Si NP surface, the Si NPs were reacted with several amines to obtain NPs with different terminal groups on their surfaces. An overview of the conjugation reactions that were performed is depicted in Figure 1. First, the conjugation reaction was tested with benzylamine, which gives clear signals in the aromatic region of the 1H NMR spectrum (Supporting Information, Figure S8). Experimentally, we found that for a fast and clean reaction the amine should be added in large excess, up to 100 equiv, and additionally the mixture is heated to 50 °C. Full conversion of the trifluoroethyl ester to the benzylamide was achieved in 24 h, as indicated by the 1H NMR spectrum. Furthermore, XPS and elemental analyses showed the disappearance of F and the appearance of N. The excess of benzylamine was easily removed by extraction with dilute acid, yielding the pure functionalized Si NPs. Compared to the 1H NMR of the TFE Si NPs in Figure 3, the quartet e of the trifluoroethyl group at δ = 4.46 ppm is no longer observed, and a new signal has appeared at δ = 4.36 ppm, which corresponds with the expected shift of a benzyl group attached to an amide. The signal d is slightly shifted upfield from δ = 2.35 ppm to 2.30 ppm. The aromatic signals of the phenyl moiety are observed at approximately δ = 7.26 ppm. The expected N−H signal is lacking in the 1H NMR spectrum, most likely because these signals are broadened too much or not seen because of exchange with solvent. However, the FTIR spectrum shows distinct features of the amide group (Figure 5; red line). The characteristic CO stretch signal corresponding to the ester at 1756 cm−1 has disappeared and a new CO stretch signal is found at 1643 cm−1, which is expected upon formation of an amide bond. The bands observed at 3288 cm−1 (N−H stretching) and 1548 cm−1 (N−H bending) are also characteristic for the amide bond. We finally note that after all reactions with amines the IR spectra of the resulting amidefunctionalized NPs show a weak band at 1090 cm−1, assignable to a Si−O stretch vibration,21 which indicates that formation of some oxide groups on the surface of the Si NPs took place during the synthesis, either as Si-OCH3 groups as also seen in the 1H NMR spectra or maybe some Si−OH groups. The absence of Si−H stretching vibrations at about 2200 cm−1 might also be an indication for this, although such signals are weak and are usually also not observed upon modification of hydrogen-terminated flat Si surfaces with alkenes.32 Overall, the level of oxidation seems low, since only weak bands attributable to Si−O stretching vibrations are seen. The 13C NMR, 1H-13C HSQC, and 1H-13C HMBC NMR spectra also confirm the conversion to the benzylamide groups (spectra Supporting Information, Figures S9−S11). Coupling with 1,2-diaminoethane was carried out analogously, yielding full conversion already after 3 h without heating. The decrease in reaction time is probably due to the higher nucleophilicity of the base. In this case purification consisted of removing the excess reagents under reduced pressure. The modified Si NPs are highly polar and consequently only soluble in polar solvents. The 1H NMR spectrum, recorded in D2O (Supporting Information, Figure S12), shows six signals (four signals from the acid part and two from the amine part). This indicates the presence of linearly
Figure 6. CuAAC-catalyzed reaction of propargyl-terminated Si NPs with the Azide-Fluor 585 dye.
mixture. After concentration and filtration of the reaction mixture in acetonitrile, the filtrate was further purified with preparative GPC using acetonitrile as eluent. The fraction of fluorescent material that had a shorter retention time (8.2 min) in the GPC fractionation than the original Azide-Fluor 585 dye material (11.2 min) was isolated and further investigated (for chromatograms see Supporting Information, Figure S22). The 4316
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shorter retention time suggests an increase in hydrodynamic radius, which would be expected upon modifying a relatively small NP with relatively large surface groups. The steady-state excitation and emission fluorescence spectra of this fraction show similar features as those of the original dye molecule (Supporting Information, Figure S20), confirming the presence of the dye on the NP. Also the time-resolved fluorescence lifetime is similar to the original dye (Supporting Information, Figure S21). The 1H NMR spectrum (Figure 7), measured in
Article
ASSOCIATED CONTENT
S Supporting Information *
1
H, 13C NMR and IR spectra of 2,2,2-trifluoroethyl 4pentenoate (Figures S1−S3; Table S1), 1H and 13C NMR spectra of the silicon nanoparticles (Figures S4−S19) and chromatograms and fluorescence spectra of the dye-modified nanoparticles (Figures S20−S22). 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]. Phone: 31-317-482961. Present Address ⊥
Department of Biomedical Chemistry, MIRA Institute of Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project was financially supported by the IPOP Bionanotechnology program of the VLAG graduate school of Wageningen University and Research Center. The authors wish to thank dr. Willem Haasnoot and dr. Loes Ruizendaal for many fruitful discussions.
Figure 7. 1H NMR spectrum (CD3OD) of the aromatic region of the dye-functionalized Si NPs (A) and the free Azide Fluor 585 dye (B). In spectrum A the two signals at 6.78 and 6.89 ppm56,57 are attributed to the triazole ring that is formed in the CuAAC click reaction.
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methanol-d4, shows the appearance of two additional signals at 6.78 ppm and 6.89 ppm, which indicate the presence of a triazole ring.56 The shorter retention time on GPC and the 1H NMR data unambiguously show that the attachment of the dye to the Si NP surface via the CuAAC click reaction was successful. Given the broad applicability of both amide-forming reactions and alkyne-based click reactions, we thus believe that this provides a highly versatile entry into functionalized Si NPs.
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REFERENCES
CONCLUSIONS
We present the synthesis and full characterization of welldefined, monodisperse (1.4 ± 0.2 nm) and reactive trifluoroethyl ester-terminated Si nanoparticles, and their subsequent conjugation with a range of functional primary amines. A set of 1D- and 2D-NMR techniques has been used to characterize the nature of the surface attachment, and clearly point to a Si−C bond forming reaction with the terminal carbon atom of the alkene. Also the products of the conjugation with various amines have been characterized extensively with different 1D- and 2D-NMR techniques. This reveals that the trifluoroethyl ester-terminated Si NPs reacts readily and with full conversion with a variety of primary amines, leading to welldefined Si NPs with different functional surface groups. Reaction with an amine containing an alkyne functionality resulted in the formation of alkyne-functionalized Si NP, which could be modified further with azides through a CuAAC “click” reaction. These results show that trifluoroethyl ester-terminated Si NPs are an excellent platform for the facile functionalization of Si NP surfaces. 4317
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