Journal of Nanoparticle Research

, Volume 11, Issue 4, pp 821–829

Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains

Authors

  • Deyan Kong
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
    • Graduate School of the Chinese Academy of Sciences
  • Zewei Quan
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
  • Jun Yang
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
  • Piaoping Yang
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
  • Chunxia Li
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
    • State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied ChemistryChinese Academy of Sciences
Research Paper

DOI: 10.1007/s11051-008-9437-5

Cite this article as:
Kong, D., Quan, Z., Yang, J. et al. J Nanopart Res (2009) 11: 821. doi:10.1007/s11051-008-9437-5

Abstract

NaYF4:Yb3+, Er3+ nanoparticles were successfully prepared by a polyol process using diethyleneglycol (DEG) as solvent. After being functionalized with SiO2–NH2 layer, these NaYF4:Yb3+, Er3+ nanoparticles can conjugate with activated avidin molecules (activated by the oxidation of the oligosaccharide chain). The as-formed NaYF4:Yb3+, Er3+ nanoparticles, NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups, avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), UV/Vis absorption spectra, and up-conversion luminescence spectra, respectively. The biofunctionalization of the NaYF4:Yb3+, Er3+ nanoparticles has less effect on their luminescence properties, i.e., they still show the up-conversion emission (from Er3+, with 4S3/2 → 4I15/2 at ~540 nm and 4F9/2 → 4I15/2 at ~653 nm), indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.

Keywords

Up-conversion phosphorAvidinOligosaccharide chainsFluorescence probeNanoparticlesBiomolecules

Introduction

Since the introduction of biospecific affinity chromatography, the immobilization of biomolecules onto insoluble matrices has been the subject of extensive research (Rao et al. 1998). Recently, biomolecules conjugation onto inorganic nanoparticles has been paid more and more attention (Storhoff and Mirkin 1999; Niemeyer 2001; Maxwell et al. 2002; Katz and Willner 2004). The major requirements for the immobilization are the production of a stable linkage between the matrix and the biomolecule and retention of specific characteristics of the immobilized species, i.e., biological activity (O’Shannessy and Hoffman 1987; O’Shannessy and Wilchek 1990). Glycoconjugates contain glycoproteins (enzymes, antibodies, hormones, receptors, proteoglycans, etc.), glycolipids, and nucleosides and nucleotides (O’Shannessy and Hoffman 1987). The common feature is the presence of one or more sugar units covalently linked to a nonsugar unit. The oligosaccharide is in many instances not involved in the specific biological activity and is often situated at a sites(s) far removed from the “active site” such as glycoenzymes and antibodies. It is necessary to directly covalently immobilize glycoconjugates on the matrix by oligosaccharide chains. The method relies on the specific oxidation of the oligosaccharide by chemical or enzymatic means resulting in the formation of aldehydes. The aldehydes thus produced may in turn be condensed with suitable nucleophiles such as primary amines or hydrazides of the matrices. The conjugation to inorganic nanoparticles with glycoconjugate molecules has generally been carried out by means of esterification process or glutaraldehyde modified nanoparticles. The method of reaction of oligosaccharide of glycoconjugates with nanoparticles has not been reported. The high affinity constant between the glycoprotein avidin and the vitamin biotin prompted attention to the scientists (Bayer and Wilchek 1980).

Up-conversion phosphors NaYF4:Yb3+, Er3+ were chosen as the inorganic nanoparticles. Up-conversion technique methodology has high potential value in diagnostic pathology and offers advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology (Zijlmans et al. 1999; Lu et al. 2004). Up-converting fluorescence labels possess several distinct advantages compared to down-converting phosphors: (1) up-converting occurs within the host crystal; therefore, the optical properties of the phosphors are anti-Stokes emission of discrete wavelengths, and not significantly influenced by their environment (e.g., buffer pH or assay temperature) (Yi et al. 2004). Consequently, the detection process is unaffected by the sampled fluid and is robust with respect to sampling conditions or samples such as whole blood, plasma, urine, sputum, or tissue homogenates (Niedbala et al. 2001); (2) excitation is performed using an infrared laser, which is compact, power-rich, and also inexpensive (Hirai et al. 2002); (3) the fluorescence from biological samples (background) upon excitation with IR radiation is extremely low as the interfering biomolecules absorb in the UV (not the IR) region; (4) due to the large wavelength separation between excitation and emission, the optical train is very simple, and so there is no need for time-resolved detection; (5) simultaneous detection of multiple analytes can be realized since different colors of visible light can be obtained from combination between different lanthanide dopants and the host matrix (Heer et al. 2004) under the excitation of the same IR laser (Wang et al. 2006a, b) in the analysis of biological and environmental samples, and especially for fluorescence imaging in vivo (Wang et al. 2005). In this article, avidin was used as model glycoprotein, which conjugates with the NaYF4:Yb3+, Er3+ nanoparticles functionalized with SiO2–NH2 layer. The biofunctionalization of the NaYF4:Yb3+, Er3+ nanoparticles has less effect on their up-conversion luminescence properties from Er3+, indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.

Experimental

Materials

Y2O3, Yb2O3, Er2O3 (99.99%, Shanghai Yuelong New Materials Co., Ltd.), NH4F (96.0%, Beijing Beihua Chemicals Co., Ltd.), CH3COONa (analytical reagent, A. R., Beijing Beihua Fine Chemicals Co., Ltd.) were used as starting materials and diethyleneglycol (DEG) (A. R., Beijing Yili Fine Chemicals Co., Ltd.) as the solvent for the preparation of NaYF4: 20% Yb3+, 2% Er3+ nanoparticles, respectively. Y(CH3COO)3, Yb(CH3COO)3, Er(CH3COO)3 were prepared by dissolving Y2O3, Yb2O3, Er2O3 in acetic acid, respectively.

Tetraethoxysilane (TEOS, A. R., Beijing Beihua Fine Chemicals Co., Ltd.), 3-aminopropyltriethoxysilane (APTES, 99%, Aldrich Chemical Inc.), ammonia solution (25%, A. R., NH4OH, Beijing Beihua Fine Chemicals Co., Ltd.) were used as the materials for the amino-functionalization of NaYF4:Yb3+, Er3+ nanoparticles with isopropyl alcohol (A. R., Beijing Beihua Fine Chemicals Co., Ltd.) as the solvent.

Avidin (Calbiochem–Novabiochem Co.), sodium m-periodate (A. R., Beijing Beihua Fine Chemicals Co., Ltd.), ethylene glycol (A. R., Beijing Beihua Fine Chemicals Co., Ltd.) were used as the materials for the oxidation of biomolecules with sodium acetate buffer as the solvent; then dialysis bag (Baiao Biotechnology Co., Ltd.) was utilized to filter small molecules.

Synthesis of NaYF4:Yb3+, Er3+ nanoparticles

The doping concentrations of Yb3+, Er 3+ were 20 and 2 mol% of Y3+ in NaYF4 host, respectively (Boyer et al. 2006; Mai et al. 2006). Typically, 16 mmol NH4F was dissolved in 30 mL DEG in an oil bath at 70 °C to form a clear solution (solution A). At the same time, 30 mL DEG containing 4 mmol of CH3COONa, 3.12 mmol of Y(CH3COO)3, 0.8 mmol of Yb(CH3COO)3, and 0.08 mmol of Er(CH3COO)3 in 250 mL round-bottomed flask was stirred and heated to 100 °C in the oil bath in an Ar atmosphere (solution B). When solution B turned clear, the temperature was increased to 180 °C. Then, solution A was injected into it, and the mixture was kept stirring for 1 h at 180 °C. The obtained suspension was cooled to room temperature and diluted with 120 mL ethanol. The NaYF4:Yb3+, Er3+ nanoparticles were obtained by centrifugation at a speed of 4500 rpm. Then, they were redispersed in ethanol and centrifuged several times to remove the loosely absorbed solvent molecules on their surfaces. Finally, the obtained nanoparticles were dried at 70 °C in air.

Functionalization of NaYF4:Yb3+, Er3+ nanoparticles with SiO2–NH2 layers

In a typical procedure (Stöber et al. 1968; Ohmori and Matijevié 1992, 1993), 0.2206 g NaYF4:Yb3+, Er3+ nanoparticles were added into 200 mL isopropyl alcohol solution containing 0.45 mol dm−3 NH4OH and 3.05 mol dm−3 H2O; then the suspension was stirred 40 °C for 30 min. Then 0.4 mmol TEOS and 0.4 mmol APTES were added into the suspension simultaneously, and the mixture was stirred for 2 h at 40 °C. The product was achieved by centrifugation and washing process as described in the above section. In this way, the amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were obtained.

Conjugation of avidin with the amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles

One milligram of avidin was dissolved in 2 mL of 0.1 M sodium acetate buffer (pH 5.0), and then 0.01 mL of sodium m-periodate (0.5 M) was added. The reaction was carried out for 2 h at 4 °C, after which ethylene glycol (0.2 mL) was added. The solution was dialyzed overnight. The contents of the dialysis bag were then allowed to interact with the NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups (0.0370 g) for 3 h at room temperature. Finally, 0.8 mg of NaBH4 was added to stabilize the bioconjugate. The product was achieved by centrifugation and washing process as described in the above section.

The whole reaction and formation processes for the functionalization of NaYF4:Yb3+, Er3+ nanoparticles with amino (–NH2) groups and the subsequent conjugation with avidin on NaYF4:Yb3+, Er3+ nanoparticles are shown in Scheme 1.
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Scheme 1

The diagram showing the whole reaction and formation processes for the functionalization of NaYF4:Yb3+, Er3+ nanoparticles with amino (–NH2) groups, the specific oxidation of the oligosaccharide of avidin, and then conjugation of oxidated avidin with the NaYF4:Yb3+, Er3+ nanoparticles

Characterization

X-ray diffraction (XRD) was carried out on a Rigaku-Dmax 2500 diffractometer with Cu Kα radiation (λ = 0.15405 nm). The accelerating voltage and emission current were 40 kV and 200 mA. Fourier transform infrared (FT-IR) spectra were measured with Perkin-Elmer 580B infrared spectrophotometer with the KBr pellet technique. The UV/Vis absorption spectra were measured on a TU-1901 spectrophotometer. The UC emission spectra were obtained using a 980 nm laser from an optical parametric oscillator (OPO, Continuum Surelite, USA) as the excitation source and detected by R955 (HAMAMATSU) from 400 to 900 nm. TEM images were obtained using a JEOL 2010 transmission electron microscope operating at 200 kV. Samples for TEM were prepared by depositing a drop of an ethanol suspension of the powders onto a carbon-coated copper grid and dried in air. The surface morphology of biofunctionalized nanoparticles was investigated by atomic force microscopy (AFM). The AFM experiments were performed with a SPA-300HV atomic force microscope (AFM) with an SPI 3800N controller (Seiko Instruments Industry Co., Ltd.). Pyramid-like Si3N4 tips, mounted on 100 μm triangle cantilevers with spring constants of 0.09 N/m, were applied for contact mode experiments of AFM. All the measurements were performed at room temperature.

Results and discussion

Structure and morphological properties

XRD and TEM were employed to characterize the structure and morphology of the as-prepared NaYF4:Yb3+, Er3+ sample and those functionalized with the SiO2–NH2. Figure 1 shows XRD patterns of the as-prepared NaYF4:Yb3+, Er3+ sample and the standard data for bulk α-NaYF4 as a reference. The results of XRD indicate that the NaYF4:Yb3+, Er3+ sample is crystallized well and all the peaks are in good agreement with cubic phase structure known from bulk α-NaYF4 phase (JCPDS card no. 06-342). The calculated crystal cell parameters of the α-NaYF4:Yb3+, Er3+ sample (a = 0.545 nm) according to their XRD data are in good agreement with the reported values for bulk NaYF4 crystal (a = 0.547 nm). In Fig. 1, the diffraction peaks for NaYF4:Yb3+, Er3+ sample are broadened due to the smaller crystallite size. The crystallite size can be estimated from the Scherrer equation, D = 0.90λ/βcosθ, where D is the average grain size, λ is the X-ray wavelength (0.15405 nm), and θ and β are the diffraction angle and full-width at half-maximum (FWHM) of an observed peak, respectively (Birks and Friedman 1946). The strongest peak (111) at 2θ = 28.08° was used to calculate the average crystallite size (D) of the NaYF4:Yb3+, Er3+ sample, and the estimated crystallite size is around 12.6 nm.
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Fig. 1

XRD patterns of the as-prepared NaYF4:Yb3+, Er3+ sample and the standard data for bulk α-NaYF4 as a reference (JCPDS card no. 06-342)

Figure 2 shows the TEM micrographs for the as-prepared NaYF4:Yb3+, Er3+ sample (a, b), NaYF4:Yb3+, Er3+ functionalized with SiO2–NH2 layers (c), and the AFM micrographs for the NaYF4:Yb3+, Er3+ bonded with avidin (d), respectively. From the low magnification TEM micrograph (Fig. 2a), it can be seen that the as-prepared NaYF4:Yb3+, Er3+ sample consists of nanosized particles with a spherical morphology, and the average diameter of the particles is 12 nm. The size of the NaYF4:Yb3+, Er3+ nanoparticles is basically consistent with that calculated from XRD peak via the Scherrer equation. The high resolution TEM micrograph (Fig. 2b) clearly displays the lattice resolved fringes with a constant spacing of 0.293 nm ascribed to the (220) plane of NaYF4, indicative of the high crystallinity of these NaYF4:Yb3+, Er3+ nanoparticles. The NaYF4:Yb3+, Er3+ nanoparticles functionalized with SiO2–NH2 layers (Fig. 2c) still keep the sphere morphology with the average diameter 13 nm, respectively, which are slightly larger than the bare NaYF4:Yb3+, Er3+ nanoparticles due to the additional silica layer (with NH2 groups) deposited on the NaYF4:Yb3+, Er3+ nanoparticles. Although the silica layer cannot be well resolved due to its thin characteristics, we can basically confirm that the silica treatment did not create large micron-size agglomerates of NaYF4 nanoparticles and silica particles, i.e., most of the silica materials were coated on the NaYF4 nanoparticles, as reported previously for other systems (Goldman et al. 2002a, b; Feng et al. 2003; Wang et al. 2006a, b; Meiser et al. 2004). The AFM micrograph (Fig. 2d) shows that the biofunctionalized NaYF4:Yb3+, Er3+ nanoparticles became much bigger. The size of the avidin conjugated NaYF4:Yb3+, Er3+ nanoparticles (Fig. 2d) is in the range of 35–100 nm, mostly between 45 nm and 75 nm. The organic avidin molecules with dimensions around 6.0 nm × 5.5 nm × 4.0 nm (Deschamps et al. 1992) cannot be distinguished clearly by the TEM image (but can be proved by the FT-IR spectra to some extent, see next part).
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Fig. 2

TEM micrographs for the as-prepared NaYF4:Yb3+, Er3+ sample with low (a) and high (b) resolution, NaYF4:Yb3+, Er3+ functionalized with SiO2–NH2 layers (c), and the AFM micrographs for the NaYF4:Yb3+, Er3+ bonded with avidin (d)

Functionalization of NaYF4:Yb3+, Er3+ nanoparticles with SiO2–NH2 groups and conjugation with avidin

The FT-IR spectra were used to characterize the functionalization of NaYF4:Yb3+, Er3+ nanoparticles with –NH2 groups and their further conjugation with avidin. Figure 3 shows the FT-IR spectra of the as-prepared NaYF4:Yb3+, Er3+ nanoparticles (a), NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups (b), avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles (c), respectively. In Fig. 3a for the as-prepared NaYF4:Yb3+, Er3+ nanoparticles, the broad absorption band peaking at 3472 cm−1 is due to the stretching vibration of hydroxyl group (–OH), and the absorption bands at 1452 and 745 cm−1 are assigned to the scissoring and rocking vibrations of CH2 groups, respectively (Rao 1963). The peak at 1082 cm−1 attributes to the stretching vibration of C–O band in DEG. The band around 1638 cm−1 is due to H2O absorbed in the sample (Toneguzzo et al. 1999). These results indicate that the as-formed NaYF4:Yb3+, Er3+ nanoparticles, which were prepared by using DEG as the solvent (Feldmann and Jungk 2001; Feldmann 2003, 2005; Eiden-Assmann and Maret 2004), contain a large amount of –OH groups on the surfaces. After the reaction of NaYF4:Yb3+, Er3+ nanoparticles with APTES and TEOS in NH4OH aqueous solution, the FT-IR spectrum (Fig. 3b) shows broad absorption peak at 3404 cm−1 due to the asymmetric stretching vibration of the primary amino group (–NH2, it becomes weaker than that of –OH absorption near the same region) and 1566 and 769 cm−1 due to the scissoring and wagging vibrations of the NH2 groups, respectively. The strong absorption bands in the region 1150–1000 cm−1 (two peaks at 1138, 1044 cm−1) arise from the Si–O–Si asymmetric vibration. The weak peak at 2937 cm−1 is due to the asymmetrical stretching vibration modes of CH2 group. From these results, it can be confirmed that after the functionalization reaction with APTES and TEOS, the NaYF4:Yb3+, Er3+ nanoparticles have been covered with a silica layers containing the functional –NH2 groups on their surface due to the polycondensation process between TEOS and APTES (Buining et al. 1997; Gerion et al. 2001). After the reaction of avidin with the amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles, the FT-IR spectrum (Fig. 3c) shows sharp peaks at 3434 (ν NH), 2926 (νas CH2), 1642 (ν C=O, the amide I absorption), 1564 (δ N–H, the amide II absorption), 1215 (COOH band, the amide III absorption), 1156 (ν CO), and 1049 cm−1(ν Si–O), respectively (Bellamy 1975). This demonstrates that the avidin has been successfully conjugated on the NaYF4:Yb3+, Er3+ nanoparticles. The covalent coupling between the avidin molecules and the NaYF4:Yb3+, Er3+ nanoparticles is stable and irreversible. The avidin conjugated NaYF4:Yb3+, Er3+ nanoparticles can be easily connected with some proteins or nucleic acid. Biotin can conjugate with avidin proteins with outstanding selectivity and specificity; in addition, biotin can easily conjugate on other biomolecules, such as nucleic acid, antibodies (Goldman et al. 2002a, b).
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Fig. 3

The FT-IR spectra of the as-prepared NaYF4:Yb3+, Er3+ nanoparticles (a), NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups (b), avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles (c)

Photoluminescent properties

The absorption spectra with a concentration of 1.25 g/L in the deionized water for the above bare and functionalized NaYF4:Yb3+, Er3+ nanoparticles are shown in Fig. 4a–c, respectively. The absorption spectrum of the as-prepared NaYF4:Yb3+, Er3+ nanoparticles in Fig. 4a shows no absorption in the UV–Vis range. However, the absorption peaks are present after the surface functionalization with NH2 groups (Fig. 4b) and the subsequent conjugation with avidin (Fig. 4c). The decrease of absorption intensity in Fig. 4c is ascribed to the fact that the NaYF4:Yb3+, Er3+ nanoparticles functionalized with NH2 groups have been covered by avidin.
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Fig. 4

The absorption spectra of the as-prepared NaYF4:Yb3+, Er3+ nanoparticles (a), those functionalization with NH2 groups (b), and the subsequent conjugation with avidin (c) (with the same concentration of 1.25 g/L in deionized water)

The visible up-conversion spectra of NaYF4:Yb3+, Er3+ nanoparticles and avidin conjugating on the NaYF4:Yb3+, Er3+ nanoparticles under 980 nm excitation are shown in Fig. 5a and b, respectively. The spectra correspond to what has been reported previously for Er3+ upconversion in NaYF4:Yb3+, Er3+ nanoparticles (Wang and Li 2007). Up-conversion luminescence were observed from the 2H11/2 → 4I15/2 (~522 nm), 4S3/2 → 4I15/2 (~540 nm), 4F9/2 → 4I15/2 (~653 nm). These transitions are generated through an efficient energy transfer process involving Yb3+ → Er3+ ions. A schematic energy level diagram, up-conversion excitation, and visible emission processes for the Yb3+–Er3+ systems are shown in Fig. 6. The 980 nm light is absorbed by the 2F7/2 → 2F5/2 of Yb3+, and the electrons of Er3+ are first excited from the 4I15/2 level to the 4I11/2 level via excitation energy transfer from the 2F5/2 level of Yb3+, then to the 4F7/2 level of Er3+ by absorbing the energy of another electron from Yb3+ (2F5/2). The excited electrons of the 4F7/2 (Er3+) level can relax nonradiatively to the emitting 2H11/2, 4S3/2, and 4F9/2 levels (Suyver et al. 2006). The 4F9/2 level may also be populated from the 4I13/2 level of the Er3+ ion by absorption of a 980-nm photon. After conjugation on avidin, the luminescent properties do not change greatly, indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.
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Fig. 5

The visible up-conversion spectra of NaYF4:Yb3+, Er3+ nanoparticles (a) and avidin conjugating on the NaYF4:Yb3+, Er3+ nanoparticles (b) under 980 nm laser excitation

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Fig. 6

Schematic energy level diagrams, up-conversion excitation, and visible emission processes for the Yb3+–Er3+ systems. Full, dashed, and curly arrows indicate radiative, nonradiative energy transfer, and multiphonon relaxation processes, respectively

Conclusions

In conclusion, the well-crystallized NaYF4:Yb3+, Er3+ nanoparticles have been successfully synthesized via a polyol process. These nanoparticles can be functionalized by amino groups by silica coating process and further conjugated with avidin (activated by oxidation of oligosaccharide unit). The biofunctionalized NaYF4:Yb3+, Er3+ nanoparticles could remain their up-conversion luminescence intensity, pointing out the great potential for these NaYF4:Yb3+, Er3+ nanoparticles as fluorescence probes for biological system.

Acknowledgments

This project is financially supported by the foundation of “Bairen Jihua” of Chinese Academy of Sciences, the MOST of China (2003CB314707, 2007CB935502), and the National Natural Science Foundation of China (50572103, 20431030, 00610227).

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