Bioconjugation of Ln3+-Doped LaF3 Nanoparticles to Avidin

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Bioconjugation of Ln3+-Doped LaF3 Nanoparticles to Avidin Peter R. Diamente,† Robert D. Burke,‡ and Frank C. J. M. van Veggel*,† UniVersity of Victoria, Department of Chemistry, P.O. Box 3065, Victoria, British Columbia, Canada V8W 3V6, and UniVersity of Victoria, Department of Biology and Biochemistry & Microbiology, P.O. Box 3020, Victoria, British Columbia, Canada V8W 3N5 ReceiVed September 22, 2005. In Final Form: NoVember 25, 2005 The binding of Eu3+-doped LaF3 nanoparticles with biotin moieties at the surface of the stabilizing ligand layer to avidin, immobilized on cross-linked aragose beads, is described. The biotin moieties were attached to the nanoparticles by reaction of an activated ester with the amino groups on the surface of the nanoparticles resulting from the 2-aminoethyl phosphate ligands that were coordinated to the surface through the phosphate end. This strategy of employing the reactions of amines with activated esters provides a general platform to modify the surface of the 2-aminophosphate stabilized Ln3+-doped LaF3 nanoparticles with biologically relevant groups. Significant suppression of nonspecific binding to the avidin modified aragose beads has been realized by the incorporation of poly(ethylene glycol) units via the same reaction of a primary amine with an activated ester. The particle size distribution of the functionalized nanoparticles was within 10-50 nm, with a quantum yield of 19% in H2O for the LaF3 nanoparticles codoped with Ce3+ and Tb3+. A discreet, 4 unit poly(ethylene glycol) spaced heterobifunctional cross-linker, functionalized with biotin and N-hydroxysuccinimide at opposite termini, was covalently linked to the 2-aminoethyl phosphate ligand via the N-hydroxysuccinimide activated ester, making an amide bond, imparting biological activity to the particle. Modification of the remaining unreacted amino groups of the stabilizing ligands was done with Me(OCH2CH2)3CH2CH2(CdO)s NHS (NHS ) N-hydroxysuccinimide).

Introduction Water-soluble, highly luminescent nanoparticles have come to the forefront as promising alternative materials for biotechnological applications, due to the increased ease of processability and functionalization with biologically active components. Materials such as quantum dots,1-4 and gold nanoparticles,5-8 are making their way into the realm of luminescent labeling and imaging applications9 due to their high photostability, biocompatible properties, size, and composition-tunable luminescence emission from visible to near-infrared wavelengths.10-15 * Corresponding author. E-mail: [email protected]. Telephone: (250) 7217184. † Department of Chemistry. ‡ Department of Biology and Biochemistry & Microbiology. (1) Meiser, F.; Cortez, C.; Caruso, F. Angew. Chem., Int. Ed. 2004, 43, 59545957. (2) Ow, B.; Larson, D. R.; Sriastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. Nano Lett. 2005, 1, 113-117. (3) Gaponik, N.; Radtchenko, I. L.; Gerstenberger, M. R.; Fedutik, Y. A.; Sukhorukov, G. B.; Rogach, A. L. Nano Lett. 2003, 3, 369-372. (4) Pellegrino, T.; Kudera, S.; Kiedl, T.; Javier, A. M.; Manno, L.; Parak, W. J. Small 2004, 1, 48-63. (5) Parak, W. J.; Gerion, D.; Pellegrino, T.; Znachet, D.; Micheel, C.; Williams, S. C.; Boudreau, R.; LeGros, M. A.; Larabell, C. A.; Alivisatos, A. P. Nanotechnology 2003, 14, 15-27. (6) Jianrong, C.; Yuquig, M.; Nongyue, H.; Xiaohua, W.; Sijiao, L. Biotechnol. AdV. 2004, 22, 505-518. (7) Nam, J.-M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932-5933. (8) Willner, I.; Willner, B. Pure Appl. Chem. 2002, 74, 1773-1783. (9) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 8, 969-976. Lim, Y. T.; Kim, S.; Nakayama, A.; Stott, N. E.; Bawendi, M. G.; Frangioni, J. V. Mol. Imag. 2003, 2, 50-64. Green, M. Angew. Chem., Int. Ed. 2004, 43, 4219-4313. (10) Wang, C.-W.; Moffitt, M. G. Langmuir 2005, 21, 2465-2473. (11) Ba¨umle, M.; Stamou, D.; Segura, J.-M.; Hovious, R.; Vogel, H. Langmuir 2004, 20, 3828-3831. (12) Meziani, J. M.; Pathak, P.; Harruff, B. A.; Hurezeanu, R.; Sun, Y.-P. Langmuir 2005, 21, 2008-2011. (13) Charvet, N.; Reiss, P.; Roget, A.; Dupuis, A.; Gru¨nwald, D.; Carayon, S.; Chandezon, F.; Livache, T. J. Mater. Chem. 2004, 14, 2638-2642. (14) Powe, A. M.; Fletcher, K. A.; St. Luce, N. N.; Lowry, M.; Neal, S.; McCarroll, M. E.; Oldham, P. B.; McGown, L. B.; Warner, I. M. Anal. Chem. 2004, 76, 4614-4634.

Use of water-soluble, lanthanide-based luminescent probes has also come to the forefront as an alternative to the metal- and semiconductor-based labels, and these probes are typically used in three different directions: (1) lanthanide chelates,16 (2) polystyrene nanoparticles impregnated with lanthanide chelates,17 and (3) lanthanide-based nanoparticles.18 Work done by Weibel et al.19 has developed lanthanide chelates based on glutamic acid skeleton systems, whereby activation of the appended carboxylate function of the glutamate moiety, in the form of an Nhydroxysuccinimidyl ester, allows for the covalent linking of the complexes to primary amino groups of biological compounds, such as bovine serum albumin. Others, such as Ha¨rma¨ et al.,20 have capitalized on using carboxyl-modified polystyrene nanoparticles (∼107 nm) that are impregnated with Eu3+- and Tb3+based chelates. The resulting nanoparticles contain about 30 000 chelates yielding very intense luminescence, with a luminescent lifetime of 720 µs (for Eu3+), rivaling the chelates used in traditional dissociation enhanced fluoroimmunoassy methods. The third form consists of various inorganic matrixes, such as silica-coated YVO4:Eu nanoparticles functionalized with guanidinium for sodium channel targeting by Beaurepaire et al.21 Meiser et al.22 have developed LaPO4:Ce/Tb nanoparticles functionalized with streptavidin for biotin-streptavidin binding studies. In a (15) Sun, C.; Yang, J.; Ki, L.; Wu, X.; Liu, Y.; Liu, S. J. Chromatogr., B 2004, 803, 173-190. (16) Selvin, P. R. Annu. ReV. Biophys. Biomol. Struct. 2002, 31, 275-302. (17) Huhtinen, P.; Vaarno, J.; Soukka, T.; Lo¨vgren, T.; Ha¨rma¨, H. Nanotechnology 2004, 15, 1708-1715. Ye, Z.; Tan, M.; Wang, G.; Yuan, J. Anal. Chem. 2004, 76, 513-518. Tan, M.; Ye, Z.; Wang, G.; Yuan, J. Chem. Mater. 2004, 16, 2494-2498. Matsuya, T.; Tashiro, S.; Hoshino, N.; Shibata, N.; Nagasaki, Y.; Kataoka, K. Anal. Chem. 2003, 75, 6124-6132. (18) 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. (19) Weibel, N.; Charbonnie`re, L. L.; Guardigli, M.; Roda, A.; Ziessel, R. J. Am. Chem. Soc. 2004, 126, 4888-4896. (20) Soukka, T.; Ha¨rma¨, H.; Paukkunen, J.; Lo¨vgren, T. Anal. Chem. 2001, 73, 2254-2260. (21) Beaurepaire, E.; Buisette, V.; Sauviat, M.-P.; Giaume, D.; Lahill, K.; Mercuri, A.; Casanova, D.; Huignard, A.; Martin, J.-L.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. Nano Lett. 2004, 11, 2079-2083.

10.1021/la052589r CCC: $33.50 © 2006 American Chemical Society Published on Web 12/30/2005

Bioconjugation of Ln3+-Doped LaF3 Nanoparticles to AVidin

very recent contribution, Li and co-workers23 demonstrated that an Er3+/Yb3+ upconverting nanoparticle label could be used in fluorescence resonance energy transfer (FRET) type analysis, whereby the emission of the upconverting nanoparticle is quenched by the gold nanoparticles, with both nanoparticles functionalized with biotin for biotin-avidin detection and quantification. Recently, we developed a series of water-soluble, highly luminescent Ln3+-doped LaF3 nanoparticles (Ln3+ ) Eu3+, Er3+ and Ce3+/Tb3+), which were prepared in a series of one-pot syntheses, with an average nanoparticle size less than 20 nm and whose luminescent lifetimes were among the longest reported for water-soluble, lanthanide-based matrix systems.24 This work shows that water solubility can readily be achieved by either 2-aminoethyl phosphate or oligoethylene glycol based ligands. It further shows through the use of a model N-hydroxysuccinimide activated ester that the primary amines on the surface of the Ln3+-doped LaF3 can be converted into amide bonds. Neither this work nor work done by others shows that Ln3+-doped LaF3 can indeed be turned into biolabels with specific binding to a protein; in the current work we use the well-known biotinavidin systems as a proof of principle. Of the three major directions of lanthanide probes currently being studied for biological applications, the nanoparticles are the least well developed. LaF3-based nanoparticles could have a number of advantages as probes used in bioconjugation applications over other biolabels. First, the optical properties seen for each lanthanide ion in the nanoparticle consist of nonoverlapping absorption and emission lines that do not change position with particle size and, thus, do not interfere with the optical properties of other Ln3+ ions, offering multiplexing capabilities. Second, the inherent long-lived luminescent lifetimes (µs to ms range) prevent interference from any spontaneous background emission sources (natural fluorescence of proteins is within 1-10 ns25). Third, the spectroscopic selectivity of the nanoparticles can be extended beyond the range of interferences from biological systems, by means of doping with, for example, Er3+, Nd3+, Pr3+, Yb3+, or Ho3+ for near-infrared emission lines.26-28 Finally, the optical robustness of the nanoparticle is due to the radiative transitions within the [Xe]4fn configuration of the Ln3+ ions (the partially filled 4f orbitals are shielded from the environment by the filled 5s and 5p orbitals, minimizing effects of the crystal field), resulting in long-term stability of the nanoparticle signal because there are no chemical bonds that can be broken in the photocycle, resulting in high quantum yields. To expand the versatility of the particles, here we report the binding of Eu3+-doped LaF3-based nanoparticles, whereby the 2-aminoethyl phosphate surface of the nanoparticle has been functionalized with one heterobifunctional cross-linking derivative, a “biotin-spacer-activated ester” (2), referred to as poly(ethylene glycol)-biotin, and compound 4, having an activated ester and a methyl ester-terminal triethylene glycol unit, referred to as PEG-ME. Modification of the nanoparticle surface with both of the above heterobifunctional cross-linking ligands resulted (22) Meiser, F.; Cortez, C.; Caruso, F. Angew Chem., Int. Ed. 2004, 43, 59545957. (23) Wang, L.; Yan, R.; Huo, Z.; Wang, L.; Zeng, J.; Bao, J.; Wang, X.; Peng, Q.; Li, Y. Angew. Chem., Int. Ed. 2005, 44, 6054-6057. (24) Diamente, P. R.; van Veggel, F. C. J. M. J. Fluor. 2005, 4, 543-551. Diamente, P. R. Development of Water-Soluble Ln3+-doped LaF3 Nanoparticles as Potential Biolabels. M.Sc. Thesis, University of Victoria, Victoria, British Columbia, Canada, 2005. (25) Tan, M.; Ye, Z.; Wang, G.; Yuan, J. Chem. Mater. 2004, 16, 2494-2498. (26) Hebbink, G. A.; Stouwdam, J. W.; Reinhoudt, D. N.; van Veggel, F. C. J. M. AdV. Mater. 2002, 16, 1147-1150. (27) Stouwdam, J. W.; van Veggel, F. C. J. M. Nano Lett. 2002, 7, 733-737. (28) Driesen, K.; van Deun, R.; Go¨rller-Walrand, C.; Binnemans, K. Chem. Mater. 2004, 16, 1531-1535.

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in specific binding with almost complete elimination of nonspecific adsorption. The reaction of surface amino groups with activated esters makes this into a platform technology to introduce biological functionality because of the abundance or ready availability of activated esters. Use of the poly(ethylene glycol) spacer in the heterobifunctional cross-linker offers three important benefits:29,30 (1) it increases water solubility in aqueous environments, (2) it minimizes particle aggregation and/or agglomeration in highly concentrated electrolyte solutions, and (3) it helps minimize nonspecific interaction between the nanoparticle and biological macromolecules (such as proteins and enzymes).31 These are particularly important points to consider since nonspecific adsorption onto proteins could affect the sensitivity of immunoassays and result in irreversible and uncontrolled aggregation of nanoparticles. Consequently, the need for the PEG-ME ligand on the nanoparticle surface, in addition to the poly(ethylene glycol)-based biotin ligand, is twofold: to minimize the effects of the nonspecific binding resulting from the nanoparticle’s 2-aminoethyl phosphatestabilizing ligand and, second, to prevent even further particle aggregation in the buffer solutions. Furthermore, though cross-linking reagents are not luminescent themselves, they offer three main distinct advantages over traditionally used fluorophores for biological detection: (1) they permit protein immobilization on surfaces for increased isolation efficiency without affecting the protein activity, (2) they allow for facile attachment of highly luminescent probes for increased signal intensity in relation to the background signal (ideal for immunoassays), and (3) they are more widely applicable due to their versatility in end-group functionalization.

Results and Discussion Synthesis of Biotin-Based Ligands. The specific binding of the nanoparticle to avidin was achieved by using a poly(ethylene glycol)-based heterobifunctional cross-linker (compound 2), functionalized with N-hydroxysuccinimide and biotin at opposite termini (refer to Figure 1 for reaction schemes). The activated ester of the N-hydroxysuccinimide unit allows for the covalent attachment of the heterobifunctional cross-linker to the primary amines present on the outer extremity of the nanoparticle’s 2-aminoethyl phosphate (1‚(2H+)) capping ligand. Briefly, the synthesis of the ligand mixture used (at a predefined final ligand ratio of 1‚(2H+) and 3 that is equivalent to a 9:1 molar ratio, respectively) is as follows: 1‚(2H+) was dissolved in 4 mL of water at 37 °C, neutralized with NaOH(aq), followed by the addition of 2 at a 1:0.1 molar ratio of ligands 1‚(2H+) and 2, respectively. The solution was stirred for 1 h, and product was isolated by precipitating with 25 mL of acetone, centrifuged, triturated in acetone, and dried overnight. The 1H NMR spectrum in Figure 2 shows the expected peaks of the ligand mixture 1‚(2H+):3‚(2H+), at 3.9 ppm (POCH2CH2NH2) and 3.8 ppm (POCH2CH2NHR), respectively, and corresponds to a ligand mixture with a molar ratio of ∼10:1, respectively, which was further supported by 13C NMR. The 31P (29) Woghiren, C.; Sharma, B.; Stein, S. Bioconjugate Chem. 1993, 4, 314318. Gaertner, H. F.; Offord, R. E. Bioconjugate Chem. 1996, 7, 38-44. Akiyama, Y.; Otsuka, H.; Nagasaki, Y.; Kato, M.; Kataoka, K. Bioconjugate Chem. 2000, 11, 947-950. Zhang, S.; Du, J.; Sun, R.; Li, X.; Yang, D.; Zhang, S.; Xiong, C.; Peng, Y. React. Funct. Polym. 2003, 56, 17-25. (30) Veronese, F. M. Biomaterials 2001, 22, 405-417. Roberts, M. J.; Bently, M. D. AdV. Drug. DeliVery ReV. 2002, 54, 459-476. Otsuka, H.; Nagasaki, Y.; Kataoka, K. AdV. Drug. DeliVery ReV. 2003, 5, 403-419. Caliceti, P.; Chinol, M.; Roldo, M.; Veronese, F. M.; Semenzato, S.; Paganelli, G. J. Controlled Release 2002, 83, 97-108. Greenwald, R. B.; Choe, Y. H.; McGuire, J.; Conover, C. D. AdV. Drug DeliVery 2003, 55, 217-250. (31) Zheng, M.; Li, Z.; Huang, X. Langmuir 2004, 20, 4226-4235.

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Figure 1. (a) Schematic diagram of the synthesis of ligand 3‚(2H+) from ligands 1‚(2H+) and 2. (b) Schematic diagram of the surface functionalization of 1:3‚LaF3:Eu with 4, resulting in 3:5‚LaF3:Eu.

Figure 2. 1H NMR of the ligand mixture 1‚(2H+):3‚(2H+) at a molar ratio of 1:0.1, respectively, in D2O.

NMR spectrum shows the two peaks of 1‚(2H+) (3.7 ppm) and 3‚(2H+) (2.8 ppm), with an integration of ∼10 to 1, corresponding to the presence of both ligands in their expected molar ratios. Mass spectrometry analysis of the ligand mixture 1‚(2H+):3‚

(2H+) shows the two expected peaks of 140.0 and 613.2 (M H)-, respectively. Synthesis of Biotinylated Nanoparticles. The above ligand mixture was redissolved in 25 mL of water, neutralized, heated

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Figure 3. (left) Emission spectrum at 1 nm resolution of 1:3‚LaF3:Eu in H2O. The inset, measured at 0.05 nm resolution, shows the 5D0 w 7F0 transition at 578 nm and its deconvolution. λex ) 397 nm. (right) Decay curve of 1:3‚LaF3:Eu in H2O. λex ) 464 nm, and λem ) 591 nm.

to 37 °C, followed by the addition of NaF and the Ln3+ salts, and allowed to stir for 16 h until a clear solution was obtained. The particles were isolated by removing the water under vacuum until the product was reduced to a pastelike consistency. The product was redissolved with 5 mL of water, precipitated with 25 mL of acetone, and isolated by centrifugation followed by washing with acetone and drying. Removal of 1 and 3 from the nanoparticle surface to estimate the molar ratio of the ligands on the surface of the nanoparticle was accomplished by adding 0.3 mL of citrate buffer solution (buffer pH ∼ 6) to the NMR tube and letting it sit for 2 days. The resulting 1H NMR ratio of the ligand mixture 1‚(2H+) and 3‚(2H+) was calculated to be approximately 10%, which is in accordance with what was expected due to the fact that both ligands are linear structures in solution, resulting in minimal steric hindrance of the two ligands after surface coordination. This shows that there is no selectivity for the binding of one ligand over the other on the nanoparticle surface, most likely resulting in a completely statistical mixture of the two ligands throughout the nanoparticles. Dynamic light scattering experiments on the nanoparticles gave an effective diameter of 14.6 nm ((3 nm) in H2O, which is in accordance with previously reported Ln3+-doped LaF3 nanoparticles with only ligand 1, which had a particle size of 12 nm and a size distribution between 10 and 40 nm.24 Spectroscopic Analysis of the Nanoparticles. Qualitative information about the nature and symmetry of the Eu3+ ion in the nanoparticle was determined by analyzing both the shape of the nondegenerate 5D0-7F0 transition at 578 nm and the I7F2/I7F1 intensity ratio, which in this case was calculated to be 1.6.32 In comparing the I7F2/I7F1 intensity ratio of approximately 1 for bulk LaF3:Eu nanoparticles, the value of 1.6 is due to the fact that, within the nanoparticle, a large portion of the Eu3+ ions located near the surface of the nanoparticle experience a more asymmetric crystal field, which increases the transition probability of the allowed electrical dipole (5D0 f 7F2 transition), resulting in an increase in its intensity.27,33,34 Furthermore, due to the fact that the 5D0 and 7F0 states are both nondegenerate, only a single Gaussian-shaped peak for the transition should appear if all the (32) Werts, M. H. V.; Jukes, R. T. F.; Verhoeven, J. W. Phys. Chem. Chem. Phys. 2002, 4, 1542-1548. (33) Klink, S. I.; Hebbink, G. A.; Grave, L.; Oude Alink, P. G. B.; van Veggel, F. C. J. M.; Werts, M. H. V. J. Phys. Chem. A 2002, 106, 3681-3689. (34) Sudarsan, V.; van Veggel, F. C. J. M.; Herring, R. A.; Raudsepp, M. J. Mater. Chem. 2005, 15, 1332-1342.

Eu3+ ions were in the same crystal field. The inset in Figure 3 (left side inset), which was measured separately at a resolution of 0.05 nm, shows a peak at 578 nm emission that could be deconvoluted into two Gaussians, indicating that the Eu3+ ions are located in more than one crystal field within the nanoparticle, which is consistent with the calculated I7F2/I7F1 intensity ratio. As shown in Figure 3 (right side), the luminescent lifetimes of the nanoparticles were 6.5 ms (51%), 2.9 ms (41%), and 0.9 ms (8%), which are in agreement with those for 1‚LaF3:Eu, at 5.9 ms (50%), 2.5 ms (39%), and 0.9 ms (11%).24 The reason for the multiexponential decay of these nanoparticles has been discussed by us in several recent papers.34-37 As for quantum yields, due to the low absorption coefficients of most Ln3+ ions, estimation of quantum yields is not easily feasible, and therefore, use of nanoparticles with a La0.4F3:Ce0.45,Tb0.15 (LaF3:Ce,Tb) matrix, stabilized with the respective ligand mixture, was carried out. Ce3+ ions have a relatively broad absorption band from 200 to 300 nm with an allowed 4f-5d transition, and it is known that Ce3+ undergoes energy transfer to other Ln3+ ions, in particular with Tb3+, which emits in the visible (green) region.32,36,38 A quantum yield of 19%, in H2O, was calculated with 1:3‚LaF3:Ce,Tb, which is in agreement with the value calculated for 1‚LaF3:Ce,Tb, with the particle size of 1:3‚LaF3:Ce,Tb being within experimental error ((5 nm) of that of 1:3‚LaF3:Eu. To test the near-infrared luminescent capabilities of the particles, the synthesis of 1‚LaF3:Nd was carried out, the emission spectrum of which (in H2O) in Figure 4, shows a broad peak for the 4F3/2-4I11/2 transition at 1064 nm, with a luminescent decay time (in D2O) of 18 µs and an estimated quantum yield of ∼4%. The broad peak is a result of the fact that this spectrum had to be recorded at a very low resolution due to the low intensity of the signal. This low intensity is also reflected in the low quantum yield. There must be extensive quenching of the excited Nd3+ ions by absorbed waters on the surface of the LaF3, when compared to the cases of similar Nd3+-doped LaF3 nanoparticles that are soluble in organic solvents.34-37 In general, the Ln3+ (35) Stouwdam, J. W.; Hebbink, G. A.; Huskens, J.; van Veggel, F. C. J. M. Chem. Mater. 2003, 15, 4604-4616. (36) Stouwdam, J. W.; van Veggel, F. C. J. M. Langmuir 2004, 20, 1176311771. (37) Stouwdam, J. W.; Raudsepp, M.; van Veggel, F. C. J. M. Langmuir 2005, 21, 7003-7008. (38) Ko¨mpe, K.; Borchert, H.; Storz, J.; Lobo, A.; Adam, S.; Mo¨mmer, T.; Haase, M. Angew. Chem., Int. Ed. 2003, 42, 5513-5516.

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Figure 4. Emission spectrum of 1‚LaF3:Nd in H2O. λex ) 810 nm.

ions that emit in the near-infrared are highly susceptible to quenching by overtones of the OH vibration (V ) 3400 cm-1 of the fundamental mode).39 However, the surface-related quenching effects can be reduced by the formation of core-shell nanoparticles, where the growth of a layer of LaF3 around the core minimizes contact between the dopant ions and sources of nonradiative quenching processes.36 Due to the robustness of the synthesis protocol, as already demonstrated with Eu3+-doped, Ce3+/Tb3+-doped and Er3+-doped (reported elsewhere24) nanoparticles, the size of the Nd3+-doped nanoparticles can be assumed to be within 10-15 nm, with an overall size distribution in accordance with the case of the above nanoparticles. Modification of the Surface-Bound Amino Groups. Preliminary biotin-avidin binding control experiments, exposing 1‚LaF3:Eu to the avidin-agarose beads, resulted in high levels of nonspecific binding based on the intensity of the europium signal, due to the terminating -NH3+ group of 1 under physiological conditions. Consequently, to suppress this effect, the residual unmodified ligands 1 on the nanoparticle surface were reacted with PEG-ME (4), which features a discreet 3 unit poly(ethylene glycol) spacer capped with an unreactive -OCH3 functional group. Briefly, the procedure for modifying the nanoparticle surface via a direct surface reaction to yield 3:5‚ LaF3:Eu is as follows (and schematically illustrated in Figure 1b): 1:3‚LaF3:Eu was dissolved in 4 mL of water at 37 °C, neutralized with NH4OH(aq), followed by the addition of 4, and reacted for 1 h. Isolation of the nanoparticle was done by removing the water by rotary evaporation until a slurry remained, then redissolving in 5 mL of water, precipitating with acetone, centrifuging, triturating in acetone, and drying overnight. 1H NMR analysis of the particles 3:5‚LaF :Eu gave the expected 3 peak of 3.8 ppm (POCH2CH2NHR) with a large poly(ethylene glycol) peak at ca. 3.5 ppm, arising from the presence of both poly(ethylene glycol)-based ligands on the nanoparticle surface. Only a small peak at 3.9 ppm (POCH2CH2NH2) was present, indicating that there still remained a small amount of unreacted 2-aminoethyl phosphate (around the 5% level) on the nanoparticle surface. Atomic force microscopy of the nanoparticles gave an average size of 10-15 nm, with a size distribution from 10 to 50 nm (Figure S3 in the Supporting Information). Features beyond 50 nm were not included in the histogram because of particle (39) Hofstraat, J. W.; Oude Wolbers, M. P.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Werts, M. H. V.; Verhoeven, J. W. J. Fluor. 1998, 8, 301. Werts, M. H. V.; Jukes, R. T. F.; Verhoeven, J. W. Phys. Chem. Chem. Phys. 2002, 4, 15421548.

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Figure 5. Emission spectrum at 1 nm resolution of 3:5‚LaF3:Eu in H2O. The inset, measuered at 0.05 nm resolution, shows the 5D0 w 7F transition at 578 nm and its deconvolution. λ ) 397 nm. 0 ex

aggregation, which is in accordance to the results obtained for both 1‚LaF3:Eu and 1:3‚LaF3:Eu. This is expected due to the fact that no alteration to the nanoparticle core occurs; thus, the observed increase in particle diameter is a result of the brushlike coating of ligand on the nanoparticle surface. Spectroscopic analysis of the particles, shown in Figure 5, gave the same emission spectrum as 1‚LaF3:Eu, with an I7F2/I7F1 intensity ratio of 1.6 and with luminescent decay lifetimes of 6.5 ms (54%), 2.7 ms (36%), and 0.9 ms (7%), which are all within experimental error ((5%) of the case of 1‚LaF3:Eu (vide supra). Thus, the surface reaction does not affect the luminescence properties of the Eu3+, indicating that the inorganic core of the nanoparticle is not altered. Biotin-Avidin Binding. To test the ability for the biotinfunctionalized nanoparticles to be bound to a biological system, avidin cross-linked agarose beads were used as a model for nanoparticle immobilization on a biologically active surface. In principle, due to the fact that the nanoparticles have biotinterminated ligands on their outer surface (vide supra), biotin binding to the avidin-agarose beads with a large excess of nanoparticles should be nearly quantitative and irreversible, given that the biotin-avidin has an affinity constant in the range of 1015 M-1, which is one of the highest known in nature between a protein and a ligand.40 The emission spectrum of the Eu3+ signal from the bound 3:5‚LaF3:Eu nanoparticles to the avidin-agarose beads is shown in Figure 6, line a, which indicates that specific binding has been achieved. Also shown in Figure 6, line b, is the emission spectrum of the control experiment, whereby nanoparticles 5‚LaF3:Eu that lack biotin moieties were exposed to the avidin-agarose beads in the same manner as the biotin-based nanoparticles 3:5‚LaF3: Eu, resulting in a small but measurable amount of the Eu3+ signal. This implies that that some nonspecific binding does still occur. The latter could be a result of the fact that there are still some remaining ammonium groups at the surface in combination with a relatively thin protective layer around the LaF3 nanoparticle. We expect that forcing the reaction to completion by longer reaction times and excess 4 will solve this problem. Another aspect that needs further investigation is if it is beneficial to use branched ligands to achieve a better coverage of the space surrounding the strongly curved surface of the nanoparticles. Attempts using Nd3+-doped (and Er3+-doped) LaF3 nanoparticles for near-infrared emission capabilities on the avidin(40) Wilchek, M.; Bayer, E. A. Biomol. Eng. 1999, 16, 1-4.

Bioconjugation of Ln3+-Doped LaF3 Nanoparticles to AVidin

Langmuir, Vol. 22, No. 4, 2006 1787 solution, with Φref ) 55%.41 The equation used to calculate the quantum yield is defined below, where n the is refractive index, I Φ)

Figure 6. Emission spectrum of (a) 3:5‚LaF3:Eu specifically bound and (b) 5‚LaF3:Eu nonspecifically bound to the avidin-coated agarose beads. The spectra are offset for clarity. λex ) 397 nm.

agarose beads proved unsuccessful, which is attributed to the low quantum yield of both of the nanoparticles, probably caused by quenching due to the ligands and more importantly water that are on or near the surface. Core-shell particles, with a layer of undoped LaF3 around the Ln3+-doped LaF3 core, thus having the ligands and water further away from the luminescent Ln3+ ions, will most likely be advantageous here.

Conclusions A two-step synthesis of highly luminescent, Eu3+-doped LaF3 nanoparticles was achieved, whereby the nanoparticle surface was successfully biotinylated with a poly(ethylene glycol)-based, heterobifunctional cross-linker. The spectroscopic properties of the functionalized nanoparticles showed little deviation from those of previously reported unfunctionalized Ln3+-doped LaF3 nanoparticles (with measured luminescent lifetimes up to 6.5 ms for Eu3+-doped nanoparticles and a quantum yield of 19% for Ce3+/Tb3+-doped nanoparticles, which were within experimental error of the case of unmodified nanoparticles). These results show the high level of structural and optical robustness of the nanoparticles to surface modifications. The use of methoxyterminated poly(ethylene glycol)-based ligands on the surface of the nanoparticle showed an almost complete suppression of the nonspecific binding to avidin cross-linked agarose beads. Further modification of the nanoparticles, via a core-shell system, should improve the quantum yield of the Nd3+- and Er3+-doped nanoparticles, allowing for improved near-infrared emission and detection in an aqueous environment. The use of terminal amino groups on the ligands that stabilize the Ln3+-doped LaF3 nanoparticles and activated esters provides a simple and general approach to introduce biologically active moieties. Methods and Materials Chemicals of the highest purity were obtained from Aldrich and used without further purification. The avidin cross-linked agarose beads, from Aldrich, consisted of avidin immobilized on a crosslinked 6% beaded agarose. All heterobifunctional cross-linkers were obtained from Quanta Biodesign and used without further purification. All water used was distilled water. All nanoparticles were made with LaF3 at 5% Eu3+ or Nd3+ atom doping on the total Ln3+ amount. Nanoparticles used for quantum yield calculations were made with an LaF3:Ce,Tb matrix, with La3+ at 40%, Ce3+ at 45%, and Tb3+ at 15% atom doping on the total Ln3+ amount. The reference material for quantum yield calculations was quinone sulfate in a 1 M H2SO4

[

]

n2sampleIsampleAref n2refIrefAsample

Φref

is the measured intensity, A is the absorbance of the solution, and Φref is the quantum yield of the reference material. The error was estimated at 2% for duplicate measurements. Steady-state fluorescence analyses were done using an Edinburgh Instruments FLS 920 fluorescence system, which was equipped a CW 450W xenon arc lamp via an M300 single grating monochromator for Eu3+-doped nanoparticles or a Coherent 810 nm diode laser at the 1.05 A power setting (via a Keithley 2400-C source meter) for Nd3+-doped nanoparticles. A red-sensitive Peltier-cooled Hamamatsu R955 photomultiplier tube (PMT), with a photon-counting interface, was used for analyses between 200 and 850 nm, and a N2-cooled (-80 °C) Hamamatsu R5509 PMT was used for analyses between 800 and 1700 nm. All emission analyses in the visible region were measured with a 1 nm resolution. All emission analyses in the nearinfrared region were measured with a 10 nm resolution. All spectra were corrected for detector sensitivity. Lifetime analyses for Eu3+doped nanoparticles were done by exciting the solution with a 10 Hz Q-Switched Quantel Brilliant, pumped by a Nd:YAG laser, with an optical range from 410 to 2400 nm, and collecting the emission using the respective detector mentioned above. Decay curves were measured with a 0.2 ms and a 0.01 ms lamp trigger delay for the R955 PMT. Lifetime analyses for Nd3+-doped nanoparticles were done by exciting the solution with a Coherent 810 nm diode laser at the 1.05 A power setting, with a 10-slot optical chopping disk at 22 000 Hz, and collecting the emission using the respective detector mentioned above. Steady-state spectra of Eu3+-doped materials were measured by excitation at 397 nm (5L6 level), and lifetimes were measured by excitation at 464 nm (5D2 level). Emission is predominantly from the 5D0 level and thus not influenced by the excitation wavelength. All lifetime analyses were calculated using the Edinburgh Instruments F900 tailfit software, and signal intensities greater than 1% of the maximum intensity were included and were fitted so as to obtain χ2 values from 1.0 to 1.3. If two exponentials did not give an acceptable fit, three exponentials were taken. Reported lifetime and percent contribution are only treated in a qualitative sense, and errors were estimated to be 5% based on duplicate measurements. Peak deconvolution of the high resolution 578 nm peak was done by fitting with two standard Gaussian distributions, from 757 to 579.5 nm, using software from Originlab (Origin 7.5). All 1H and 13C NMR analysis were done using a Bruker 300 MHz NMR instrument, and 31P NMR was done on a Bruker AMX 350 MHz instrument. Chemical shifts of 31P NMR were measured relative to an external standard of 85% H3PO4. No NMR analysis was carried out on Nd3+- and Ce3+,Tb3+-based nanoparticles due to severe line broadening. All mass spectrum analyses were carried out on a Kratos Concept Analytical instrument, in a glycine matrix by negative mode liquid secondary ion mass spectrometry (LSIMS). Atomic force microscopy was done using a Thermomicroscope Explorer in contact mode, with a silicon nitride cantilever (0.01-0.5 N/m). The set point was set to -7 nA at 1000 lines resolution, and the PID settings were 1.0, 0.5, and 0, respectively. Samples were deposited from an ethanol suspension on a freshly cleaved mica substrate, and the ethanol was allowed to fully dry. The particle size distribution was based on a minimum of 150 particles. Dynamic light scattering experiments were carried out on a Brookhaven Instruments photon correlation spectrometer equipped with a BI-200SM goniometer, a BI-9000AT digital autocorrelator, and a Melles Griot He-Ne laser (632.8 nm) with a maximum power output of 75 mW. All water and nanoparticle solutions were filtered through 0.45 µm Teflon syringe filters. Sample vials used for measurements were rinsed three times with the above filtered water. The final sample concentration used was 0.5 mg‚mL-1. DLS experiments were done at a 90° angle. (41) Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107-1114.

1788 Langmuir, Vol. 22, No. 4, 2006

Figure 7. Atom labeling of ligand 3‚(2H+). 1‚LaF3:Eu. A solution of 1‚(2H+) (0.14 g, 1.02 mmol) in 25 mL of water was neutralized with NH4OH(aq), followed by the addition of NaF (0.13 g, 3.00 mmol). The solution was heated to 37 °C. Then a solution of La(NO3)3‚6H2O (0.54 g, 1.26 mmol) and Eu(NO3)3‚ 5H2O (0.03 g, 0.04 mmol) dissolved together in 2 mL of water was added dropwise, and the resulting solution was stirred at 37 °C for 16 h, yielding a clear solution. Isolation of the particles was done by removing the water until the product was reduced to a pastelike consistency, redissolving with 5 mL of water, and precipitating with 25 mL of acetone. The particles were then isolated by centrifugation, the supernatant was poured off, and the remaining precipitate was then triturated with acetone, separated by centrifugation, and dried under reduced pressure. 1H NMR: δ (D2O) 4.0 (bs, 2H, POCH2CH2NH3+), 3.2 (bs, 2H, POCH2CH2NH3+). 13C NMR: δ (D2O) 61.0 (bs, POCH2CH2NH3+), 40.0 (bs, POCH2CH2NH3+). 31P NMR: δ (D2O) 1.3 (bs, O3PO-R), -1.5 (very bs, O3PO-R). For comparison, the NMR data for 1‚(2H+) are given here as well. 1H NMR: δ (D2O) 4.1 (dt, 3JHP ) 7.1 Hz, 3Jvic ) 5.7 Hz, 2H, POCH2CH2NH3+), 3.2 (t, 3Jvic ) 5.7 Hz, 2H, POCH2CH2NH3+). 13C NMR: δ (D2O) 61.1 (d, 2JCP ) 5.0 Hz, POCH2CH2NH3+), 40.2 (d, 3JCP ) 8.2 Hz, POCH2CH2NH3+). 31P NMR: δ (D2O) 0.4 (s, O3PO-R). 1‚LaF3:Ce,Tb. The same procedure as that for 1‚LaF3:Eu was used, but with Ce(NO3)3‚5H2O (0.58 g, 1.31 mmol), Tb(NO3)3‚ 6H2O (0.21 g, 0.55 mmol), and La(NO3)3‚6H2O (0.55 g, 1.26 mmol). 1‚LaF3:Nd. The same procedure as that for 1‚LaF3:Eu was used, but with Nd(NO3)3‚6H2O (0.03 g, 0.27 mmol). 3‚(2H+). Ligand 1‚(2H+) (0.01 g, 0.08 mmol, Aldrich) was dissolved in 4 mL of water and neutralized with NaOH(aq), followed by the addition of 2 (0.05 g, 0.08 mmol, Quanta Biodesign). The solution was stirred for 1 h at 37 °C, and product was isolated by precipitating with 25 mL of acetone, centrifuging the precipitate, and drying under reduced pressure. Please refer to Figure 7 for atom labeling. 1H NMR: δ (D2O) 4.6 (dd, 3Jcis ) 7.6 Hz, 2Jgem ) 5.4 Hz, 1H, Hb), 4.4 (dd, 3Jcis ) 8.0 Hz, 2Jgem ) 4.5 Hz, 1H, Hc), 3.9 (dt, 3J 3 3 HP ) 7.4 Hz, Jvic ) 6.7 Hz, 1H*, POCH2CH2NHR), 3.8 (t, Jvic ) 5.9 Hz, 2H, Hc10), 3.7 (range) (m, 12H, (C2H4O)3), 3.5 (t, 3Jvic ) 5.3 Hz, 2H, Hc11), 3.45 (t, 3Jvic ) 5.0 Hz, 1H*, POCH2CH2NHR), 3.38 (t, 3Jvic ) 5.0 Hz, 2H, Hc9), 3.38-3.32 (m, 1H, Ha), 3.0 (dd, 3J 2 3 cis ) 13.0 Hz, Jgem ) 4.5 Hz, 1H, Hd), 2.8 (dd, Jcis ) 13.0 Hz, 1H, He), 2.6 (t, 3Jvic ) 6.0 Hz, 2H, Hc12), 2.3 (t, 3Jcis ) 6.6 Hz, 2H, Hc1), 1.7 (m, 2H, Hc2), 1.5 (m, 2H, Hc3), 1.4 (m, 2H, Hc4). 31P NMR: δ (D2O) 2.8 (s, O3PO-R-biotin). MS (LSIMS) ) 613.2 (M - H+)-. 1‚(2H+):3‚(2H+) (with the Ratio of Ligands 1 and 3 Equal to a 9:1 Molar Ratio). The same procedure as that for 3‚(2H+) was

Diamente et al. used, but with 1‚(2H+) (0.14 g, 1.02 mmol, Aldrich) and 2 (0.05 g, 0.08 mmol, Quanta Biodesign). 1H NMR: δ (D2O) 4.6 (dd, 3Jcis ) 7.6 Hz, 2Jgem ) 5.4 Hz, 1H, Hb), 4.4 (dd, 3Jcis ) 8.0 Hz, 2Jgem ) 4.5 Hz, 1H, Hc), 4.0 (dt, 3JHP ) 7.4 Hz, 3Jvic ) 6.7 Hz, 10H*, POCH2CH2NH2), 3.9 (dt, 3JHP ) 7.4 Hz, 3Jvic ) 6.7 Hz, 1H*, POCH2CH2NHR), 3.8 (t, 3Jvic ) 5.9 Hz, 2H, Hc10), 3.7 (range) (m, 12H, (C2H4O)3), 3.5 (t, 3Jvic ) 5.3 Hz, 2H, Hc11), 3.45 (t, 3Jvic ) 5.0 Hz, 1H*, POCH2CH2NHR), 3.38 (t, 3Jvic ) 5.0 Hz, 2H, Hc9), 3.38-3.32 (m, 1H, Ha), 3.2 (t, 3Jvic ) 5.0 Hz, 10H*, POCH2CH2NH2), 3.0 (dd, 3J 2 3 cis ) 13.0 Hz, Jgem ) 4.5 Hz, 1H, Hd), 2.8 (dd, Jcis ) 13.0 Hz, 3 3 1H, He), 2.6 (t, Jvic ) 6.0 Hz, 2H, Hc12), 2.3 (t, Jcis ) 6.6 Hz, 2H, Hc1), 1.7 (m, 2H, Hc2), 1.5 (m, 2H, Hc3), 1.4 (m, 2H, Hc4). *The number of protons of 1 are in relation to 3 for clarity. 31P NMR: δ (D2O) 3.7 (s, O3PO-R), 2.8 (s, O3PO-R-biotin). 1:3‚LaF3:Eu (with the Ratio of Ligands 1 and 3 Equal to a 9:1 Molar Ratio). The same procedure as that used for 1‚LaF3:Eu, but with the ligand mixture 1‚(2H+):3‚(2H+). 1H NMR: δ (D2O) 4.7-4.5 (bs, Hb, Hc) 4.1-3.7 (bs, OCH2CH2N-), 3.6-3.4 (bs, Hc10, (PEG)3, Hc11), 3.3-2.2 (bs, OCH2CH2N-, Ha, Hd, Hc12, and Hc1), 2.0-1.0 (bs, Hc2-Hc4). 1:3‚LaF3:Ce,Tb (with the Ratio of Ligands 1 and 3 Equal to a 9:1 Molar Ratio). The same procedure as that for 1:3‚LaF3:Eu, but with Ce(NO3)3‚5H2O (0.58 g, 1.31 mmol), Tb(NO3)3‚6H2O (0.21 g, 0.55 mmol), and La(NO3)3‚6H2O (0.55 g, 1.26 mmol). 3:5‚LaF3:Eu (via Surface Functionalization with 4). 1:3‚LaF3: Eu (10 mg) was dissolved in 4 mL of H2O, neutralized with NH4OH(aq), and heated to 37 °C, followed by the direct addition of 4 (8.1 mg, 0.2 mmol). The solution was allowed to stir for 1 h. Isolation of the particles was done by removing the water until the product was reduced to a slurrylike consistency. Then the product was redissolved with 5 mL of water and precipitated with 25 mL of acetone. 1H NMR: δ (D2O) 4.7-4.5 (bs, Hb, Hc), 4.1-3.7 (bs, OCH2CH2N-), 3.6-3.4 (bs, Hc10, (PEG), Hc11), 3.4 (s, -OCH3), 3.3-2.2 (bs, OCH2CH2N-, Ha, Hd, Hc12, and Hc1), 2.0-1.0 (bs, Hc2-Hc4). Nanoparticle-Avidin Binding. 3:5‚LaF3:Eu (10 mg) was dissolved in 4 mL of 0.05 M borate buffer solution, followed by the addition of avidin cross-linked agarose beads (supplied from Aldrich) (100 µL). The solution was allowed to incubate for 0.5 h at 21 °C under constant shaking. The avidin-agarose beads were washed three times with the borate buffer solution, resuspended in 4 mL of borate buffer solution, and transferred to a cuvette for fluorescence measurements. The same procedure was used for the control experiments.

Supporting Information Available: Emission spectra of 3:5‚ LaF3:Ce,Tb and quinone sulfate and NMR spectra, AFM images, and particle size distribution of 3:5‚LaF3:Eu. This material is available free of charge via the Internet at http://pubs.acs.org. LA052589R