Synthesis, photoluminescence and bioconjugation of rare-earth (Eu) complexes-embedded silica nanoparticles

Solid State Communications 142 (2007) 689–693 www.elsevier.com/locate/ssc

Synthesis, photoluminescence and bioconjugation of rare-earth (Eu) complexes-embedded silica nanoparticles Yan Wang b,c , Weiping Qin a,b,∗ , Jisen Zhang b , Chunyan Cao b,c , Jishuang Zhang b,c , Ye Jin b,c , Xinguang Ren b , Zhuhong Zheng b , Shaozhe L¨u b a State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China b Key Laboratory of Excited State Processes, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences,

Changchun 130033, China c Graduate School of Chinese Academy of Sciences, Beijing 100049, China

Received 15 December 2006; received in revised form 17 April 2007; accepted 26 April 2007 by B. Jusserand Available online 3 May 2007

Abstract Eu(DBM)3 Phen-embedded silica nanoparticles were synthesized in water-in-oil (W/O) microemulsion containing aqueous phase of Eu(DBM)3 Phen, surfactant Triton X-100, cosurfactant octanol and oil-phase cyclohexane. The size and morphology of the nanoparticles were characterized by transmission electron microscopy (TEM). The low-temperature time-resolved emission spectra indicate that the Eu complex in the silica nanoparticles have longer lifetime than that of the pure complex. Under 355 nm continuous excitation, the nanoparticles show high resistance to photobleaching. The free amino groups were attached to silica surfaces by copolymerization of 3-aminopropyl(triethoxy)silane. Preliminary results demonstrated that the silica-coated Eu complex nanoparticles can be a probe in the detection of biomolecular interactions. c 2007 Elsevier Ltd. All rights reserved.

PACS: 71.20.Eh; 71.20.Rv; 71.70.Ch; 81.40.Tv Keywords: A. Rare-earth complexes; A. Nanoparticles; A. Streptavidin; D. Luminescence

1. Introduction Fluorescence nanoparticles have been widely applied in biological and medical fields in order to elucidate the complex interplay of biomolecules, to monitor the interactions of multiple proteins or cells, to study the complexity and dynamics of biological interactions [1]. The representative fluorescence probes include conventional fluorescence dyes [2], quantum dots (QDs) [3–6], conjugated polymer nanoparticles [7,8] and dye-doped silica nanoparticles [9,10]. Another alternative class of colloidal nanoparticles as a potentially promising candidate for biolabeling is the class of rare-earth (RE) complexes-doped luminescent nanoparticles. It is well known ∗ Corresponding author at: State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China. Tel.: +86 431 85168240x8325; fax: +86 431 85168240x8325. E-mail address: [email protected] (W. Qin).

c 2007 Elsevier Ltd. All rights reserved. 0038-1098/$ - see front matter doi:10.1016/j.ssc.2007.04.038

that the luminescence of lanthanide ions comes from f–f transitions, which result in their unusual optical characteristics such as sharp emission bands, long radiative lifetime, but also low extinction coefficients (1–10 M−1 cm−1 ) [11]. The low extinction coefficients can be overcome by organic chromophores coordinated with lanthanide ions, which serve as antennas to absorb light, and then transfer energy to the metal ions. The RE chelates display large stokes shift and narrow emission bands that are crucial for low detection limits and high sensitivity in fluorescence detection. Particularly, their long fluorescence lifetimes (microto milliseconds range), which allow the removal of background fluorescence (autofluorescence) and increased assay sensitivity by time-resolved measurements, which give accurate and highly sensitive quantification of specific targets [12,13]. In addition, the lanthanide complexes are more photostable and suffer less from photobleaching as compared with organic fluorophores [14]. These unique properties make them ideal as specific reporters in fluorescence detection of biomolecules and

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high throughout assays [11,15–17]. Furthermore, lanthanide complexes can be used in multiplexing through synthesizing different RE ions with same organic chromophores [18]. As promising biological probes the RE complexes must contain a chelate serving as a scaffold for attachment of the antenna and a reactive group for coupling the chelate complex to biomolecules. These need a lot of work in synthesis, separation and purification. On the other hand, many RE complexes with high quantum efficiency are prevented from application in this field if their ligands do not have reactive group for bioconjugation. A simple and effective method is to grow a silica shell around the RE complexes, forming the so-called core–shell structure [14,19–24]. Based on the silica surface modification chemistry, the RE complexesembedded silica nanoparticles can be easily conjugated with biomolecules. In addition, optically encoded particles formed by incorporating multicolored dye molecules into small silica particles at precisely controlled ratios can be used in multiple bioanalysis. To date, QDs [25] and organic dye molecules [22] have formed optically encoded particles via doping different colored nanoparticles into one silica sphere. Generally, the biocompatible silica shell can be prepared by two synthetic routes: the St¨ober and microemulsion processes. Recently, our group has synthesized 300 nm Eu(DBM)3 Phen/silica using a modified St¨ober method [24]. Others, such as Yuan et al., have developed a luminescence silica nanosphere system based on lanthanide-luminescence probes using the water-in-oil (W/O) microemulsion method [14,19]. In this report, microemulsion method was used to prepare silica spheres doped with rare-earth complexes. The size of silica spheres is about 40 nm through TEM observations. Low-temperature time-resolved emission spectra showed that the complexes embedded in silica nanoparticles have longer luminescence lifetime of lanthanide than that of pure complex. Under 355 nm excitation, the silica nanoparticles have higher resistance to photobleaching than pure complex. The surface of silica shell was modified with amino groups by hydrolysis and copolymerization reactions of 3-aminopropyl(triethoxyl)silane (APS). The preliminary protein molecules recognized reveals that luminophore-embedded silica nanoparticles have potential applications in label biomolecules. 2. Experimental 2.1. Materials and measurement Europium oxide (Eu2 O3 , 99.999%) was purchased from Shanghai Yuelong New Materials Co., Ltd. Dibenzoylmethanate (DBM, 98%) came from Alfa-Asesar. 3-aminopropyl (triethoxyl)silane (APS, 97%) was obtained from Avocado Research chemical Ltd. Streptavidin was purchased from Promega Corporation. Phenanthroline (Phen), Tetraethyl orthosilicate (TEOS), Triton X-100, ammonium hydroxide (25%), sodium hydroxide, sulfuric acid, ethanol, octanol, cyclohexane, acetone, hexane, N , N -dimethylformamide (DMF) were all analytical reagent grade and were obtained from Beijing Chemical Corporation. Glutaraldehyde (AR), bovine serum albumin

(BSA), Tween 20, human IgG and bioinylated goat anti-human IgG were purchased from Beijing Dingguo Biotechnology Corporation. All chemical materials were purchased from commercial sources and used without further purification. The size and morphology of nanoparticles were characterized by TEM (JEM, 2000EX 200 KV). Room-temperature emission spectra were performed with a Hitachi F-4500 fluorescence spectrometer. The time-resolved emission spectra at 10 K were recorded with the samples mounted in the helium exchange gas chamber of a closed cycle refrigeration system. A 355 nm light generated from a third harmonic generator pumped by pulsed Nd:YAG laser was used as the excitation source. The CCD image of protein was taken by Olympus optical camera under 365 nm excitation from a high-pressure mercury lamp. 2.1.1. Preparation of luminophore-embedded silica nanoparticles Eu(DBM)3 Phen and silica-coated Eu(DBM)3 Phen were synthesized according to the processes reported in our previous publication [26]. 2.1.2. Preparation of capture antibody substrate Aldehyde glass slides were prepared according to the method reported in the literature [27,28]. Glass slides were first soaked in sulfuric acid and saturated sodium hydroxide solution for 24 h, respectively, rinsed with copious deionized water and dried in a stream of nitrogen. They were successively cleaned by ultrasonic waves for 15 min each in hexane, acetone and ethanol, dried at 80 ◦ C for 5 min. The surface of the slide was aminosilane-coated at 80 ◦ C overnight, using APS in dry xylene in the presence of a catalytic amount of sodium ethylate as aminosilanizing agent. After aminosilanizing, the slides were washed thoroughly with ethyl acetate and then the aminomodified chip immersed into 5% glutaraldehyde in phosphatebuffered saline (PBS, Tween 20 0.05%, pH = 7.4) for about 5 h. Then the slide was rinsed with deionized water and dried in a stream of nitrogen. 2.1.3. Bioconjugation of nanoparticles After drying in the air, the nanoparticles were conjugated with biomolecules. The experimental processes are as follows: 2.0 mg nanoparticles were suspended in 1.0 mL of 0.1 M PBS buffer (pH = 7.4), 300 µL of 0.8% glutaraldehyde and 10 µL BSA were added. The solution was stirred at 4 ◦ C over night and the nanoparticles were centrifuged and washed with PBS. The nanoparticles were suspended in 1.0 mL 0.1 M PBS again, and 0.5 mg of streptavidin and 200 µL 0.8% glutaraldehyde were added. The solution was stirred at 4 ◦ C overnight again. Then 1.0 mg NaBH4 was added, and the solution was stirred for another 2 h at room temperature. Finally, the streptavidincoated fluorescent nanoparticles were centrifuged, washed and used immediately. Human IgG with concentration of 1.0 mg/mL was spotted manually on an aldehyde glass slide. The spotted sample was allowed to incubate for 2 h at 37 ◦ C. After washing with PBST (PBS containing 0.05% Tween 20) buffer three times,

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Fig. 2. Emission spectra of (A) Eu(DBM)3 Phen complex and (B) Eu(DBM)3 Phen-embedded silica nanoparticles under 355 nm excitation.

Fig. 1. TEM image of amino groups-modified silica-coated Eu(DBM)3 Phen complex.

the glass slide was put into 0.1% BSA solution and reacted for 1 h in order to passivate the active sites on the slide. Biotinylated goat anti-human IgG was spotted in the same site with a concentration of 15 µg/mL and incubated for 0.5 h. After washing, the streptavidin-coated fluorescent nanoparticles were spotted and reacted for 1 h. At last, the glass slide was washed with PBST and deionized water. 3. Results and discussion The W/O microemulsion method is simple and efficient in preparation of embedded nanoparticles. In our experiment, the hydrolysis and copolymerization of TEOS and APS simultaneously occurred in a W/O microemulsion containing aqueous solution of Eu(DBM)3 Phen, surfactant Triton X-100, cosurfactant octanol and oil-phase cyclohexane. Fig. 1 shows the TEM image of the nanoparticles, from which it can be seen that the europium chelate molecules are embedded inside the silica network as dark dots. This is because the europium chelate molecules aggregated in the water pool of the microemulsion while the silica network was forming. The diameter of nanoparticles is nearly 40 nm and the sample presents regular spherical shape and good dispersibility. The nanoparticles’ size could vary with the well-controlled concentration of the reagents, reaction time and the ratio of water-to-oil [14,21,29,30]. It has to be noted that, in our samples, the tedious surface modification procedure of the silica shell was left out due to the direct formation of free amino groups by hydrolysis and the copolymerization reactions of APS. Fig. 2 presents emission spectra under 355 nm excitation. The emission peak at 580 nm is due to the 5 D0 → 7 F0 transition of Eu3+ ions. The 5 D0 → 7 F1 transition of

Eu3+ ions at about 590 nm is the magnetic dipole transition. The strongest emission at 611 nm is due to the force electric dipole transition (5 D0 → 7 F2 ), which is allowed only in the condition that the europium ions occupy a site without an inversion center and are hypersensitive to crystal environment, in terms of the Judd–Ofelt theory [31,32]. In the emission spectrum of the silica-coated nanoparticles, the peaks of 616 and 623 nm corresponding to the Stark splitting of 5 D0 → 7 F2 transition cannot be resolved due to the homogeneous broadening at room temperature. The intensity ratio of I5D0→7F2 /I5D0→7F1 is reduced in silica-coated nanoparticles. Generally, the I5D0→7F2 /I5D0→7F1 serves as an effective spectroscopic probe of the site symmetry in which europium is situated, that is, the higher the ratio, the lower the site symmetry [33–35]. In addition, after the growth of silica shell material, emission lines of 5 D1 → 7 F J (J = 0–3) (broad peak from 520 to 560 nm) and 5 D2 → 7 F J (J = 0–2) (peaks at 467 nm, 472 nm, and 491 nm, respectively) transitions can also be observed. These lines become intense after the silica-coated europium complex, indicating reduced quenching of the 5 D1 , 5 D level. 2 The low-temperature (10 K) time-resolved emission spectra of pure Eu(DBM)3 Phen and Eu(DBM)3 Phen-embedded silica with delay time from 10 to 500 µs are shown in Fig. 3. It can be seen that the intensity of the 5 D0 → 7 F2 peak dramatically decreased in the pure europium complex with the delay time increase. However, the intensity decreased slowly in nanoparticles, indicating that the lifetime of nanoparticles was longer than that of Eu(DBM)3 Phen, which can further be seen from logarithmic curves of the normalized intensity at 611 nm of Eu(DBM)3 Phen complex and Eu(DBM)3 Phenembedded silica nanoparticles as a function of time (Fig. 3(c)). In our experiment, the Eu complex was encapsulated into the silica spheres that isolated the complex from surrounding environment and caused a decrease of the nonradiative transition rate [36]. In addition, Li et al. described that the relatively rigid structure of the silica sphere may limit the vibrations of the ligands, which leads to a decrease of the nonradiative transition rate caused by ligand vibrations [37]. The decrease of nonradiative transition rate increases the luminescence quantum efficiency. Long luminescence lifetime

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Fig. 4. Photobleaching effect of (A) Eu(DBM)3 Phen complex and (B) Eu (DBM)3 Phen-embedded silica nanoparticles.

Fig. 5. CCD image of protein molecules recognized.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Time-resolved emission spectra of (A) Eu(DBM)3 Phen complex and (B) Eu(DBM)3 Phen-embedded silica nanoparticles. (C) Logarithmic curves of the normalized intensity of Eu(DBM)3 Phen complex () and Eu(DBM)3 Phenembedded silica nanoparticles ( ) at 611 nm as a function of time.

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and high luminescence quantum efficiency are necessary for an ideal biological probe. To evaluate the photostability of the silica-coated Eu(DBM)3 Phen fluorescent nanoparticles, the photobleaching experiments of Eu complex and silica-coated nanoparticles are performed as described in other reports [38]. The samples were soaked in PBS buffer and excited at room temperature by 355 nm light from a xenon lamp. The emission intensity was recorded and the results are shown in Fig. 4. When samples were irradiated for 1 h, the fluorescence intensity decreased by about 24% for Eu3+ complexes and 13% for silica-coated nanoparticles. The high photostability of silica-coated nanoparticles is due to the fact that the silica layer isolates the complex from the outside environment and effectively protects the complexes from photodecomposition. To demonstrate the utility of the Eu(DBM)3 Phen-embedded silica nanoparticles as labeling material in biological detection,

a preliminary experiment was carried out. The experiment employed streptavidin–biotin system (ABS), a well known and powerful tool for research and analysis, as the model system. The streptavidin was conjugated to the nanoparticles and biotinylated goat anti-human IgG was immobilized on a chemically modified glass slide. In order to decrease the effect of strong steric hindrance between the rigid nanoparticles and the solid-phase surface, the amino groups on the surface of the nanoparticles are conjugated to BSA to form a flexible BSA ‘bridge’ between the nanoparticles and streptavidin. The BSA-coated nanoparticles were conjugated to streptavidin by coupling the amino groups of the BSA-coated nanoparticles and of streptavidin with glutaraldehyde. The streptavidin-coated on the fluorescent nanoparticles was demonstrated by its specific binding with a biotinylated goat anti-human IgG spotted on a glass slide. The resulting CCD image is given in Fig. 5. The red spots come from the luminescence of europium complex. The image can confirm the specific interaction between streptavidin of nanoparticles and biotin on the glass slide. 4. Conclusion In conclusion, the Eu(DBM)3 Phen-embedded silica nanoparticles were successfully prepared by W/O microemulsion. The sphere size is 40 nm, as characterized by TEM. Under 355 nm

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excitation, the silica-coated nanoparticles present strong characteristic emission of europium ions. The nanoparticles have higher photostability and longer luminescent lifetime than those of pure complex. The preliminary protein molecules recognized reveal that nanoparticles can be used as probes to label biomolecules. Acknowledgement The authors thank the National Science Foundation of China for supporting this work. (Grant Nos. are 10474096 and 50672030). References [1] I. Johnson, Histochem. J. 30 (3) (1998) 123–140. [2] J.I. Seifert, R.E. Connor, S.A. Kushon, M. Wang, B.A. Armitage, J. Am. Chem. Soc. 121 (1999) 2987–2995. [3] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science 281 (1998) 2013–2016. [4] W.C.W. Chan, S. Nie, Science 281 (1998) 2016–2018. [5] J.R. Taylor, M.M. Fang, S. Nie, Anal. Chem. 72 (2000) 1979–1986. [6] G.P. Mitchell, C.A. Mirkin, R.L. Letsinger, J. Am. Chem. Soc. 121 (1999) 8122–8123. [7] C. Wu, H. Peng, Y. Jiang, J. McNeill, J. Phys. Chem. B 110 (2006) 14148–14154. [8] C. Wu, C. Szymanski, J. McNeil, Langmuir 22 (2006) 2956–2960. [9] S. Santra, P. Zhang, K. Wang, R. Tapec, W. Tan, Anal. Chem. 73 (2001) 4988–4993. [10] S. Stantra, K. Wang, R. Tapec, W. Tan, J. Biomed. Opt. 6 (2001) 160–166. [11] P.R. Selvin, Annu. Rev. Biophys. Biomol. 31 (2002) 275–302. [12] S. Petoud, S.M. Cohen, J.C.G. Bunzli, K.N. Raymond, J. Am. Chem. Soc. 125 (2003) 13324–13325. [13] A.K. Saha, K. Kross, E.D. Kloszewski, D.A. Upson, J.L. Toner, R.A. Snow, C.D.V. Black, V.C. Desai, J. Am. Chem. Soc. 115 (1993) 11032–11033.

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