Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains
- First Online:
- 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
- 296 Views
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.
KeywordsUp-conversion phosphorAvidinOligosaccharide chainsFluorescence probeNanoparticlesBiomolecules
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.
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.
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
Functionalization of NaYF4:Yb3+, Er3+ nanoparticles with SiO2–NH2 groups and conjugation with avidin
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.
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).