Water-solublel-arginine-capped Fe3O4 nanoparticles were synthesized using a one-pot and green method. Nontoxic, renewable and inexpensive reagents including FeCl3,l-arginine, glycerol and water were chosen as raw materials. Fe3O4 nanoparticles show different dispersive states in acidic and alkaline solutions for the two distinct forms of surface bindingl-arginine. Powder X-ray diffraction and X-ray photoelectron spectroscopy were used to identify the structure of Fe3O4 nanocrystals. The products behave like superparamagnetism at room temperature with saturation magnetization of 49.9 emu g−1 and negligible remanence or coercivity. In the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, the anti-chloramphenicol monoclonal antibodies were connected to thel-arginine-capped magnetite nanoparticles. The as-prepared conjugates could be used in immunomagnetic assay.
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KeywordsMagnetite Superparamagnetic Solvothermal Amino acid Nanocrystals
In the last decade, inherently safer nanomaterials and nanostructured devices were widely fabricated with the “green chemistry” principles [1–13]. It is important to design synthetic methodologies that possess the minimization or even total elimination toxicity to the environment and human health in green chemistry [1, 14]. The nontoxic, renewable raw materials and environmentally benign solvents are generally considered in a green synthetic strategy . As society and environment can benefit from the products, green chemistry can convey a responsible attitude to public toward the development of nanoscience and nanotechnology .
Magnetite (Fe3O4) nanoparticles have attracted intensive interests for a wide range of fields, including magnetic fluids, immobilization of proteins, peptides and enzymes, immunoassays, drug or gene delivery magnetic resonance imaging, data storage, environmental remediation [15–25]. The Fe3O4 nanoparticles perform best in most of biomedicinal applications when the size of the nanoparticles is around 10–20 nm. In this range, an individual nanoparticle becomes a single magnetic domain and shows superparamagnetic behavior above blocking temperature [26, 27]. Large numbers of methods have been developed for the synthesis of high-quality Fe3O4 nanoparticles of various surface modifier based on the thermal decomposition of iron organometallic compounds in a high-boiling point organic solvent [28–37]. When those magnetite nanoparticles are applied in biomedical fields, surface post-treatments are usually needed.
In the present work, we described a facile and green approach toward synthesis and stabilization of Fe3O4 nanoparticles. Water and glycerol were used as environmentally benign solvents in the synthesis. Inartificial amino acidl-arginine was chosen as the nontoxic, renewable stabilizing agent.
Chloramphenicol (CAP) and 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma–Aldrich. o-Phenylenediamine (OPD) was purchased from Xinjingke Biotechnology. Hydrogen peroxide (30%) was supplied by Guangmang Chemical Co. The anti-CAP monoclonal antibody and HRP-CAP conjugates were produced by our lab. Other analytical grade chemicals were purchased from Shanghai Chemical Reagents Company. All of the chemicals were used as received without further purification.
Phosphate-buffered saline (PBS): 138 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4·H2O and 2.7 mM KCl, pH = 7.4.
Washing buffer (PBST): PBS containing 0.05 (v/v) Tween 20.
Citrate buffer: 19 mM citric acid, 33.5 mM Na2HPO4·H2O, pH = 5.0
Substrate solution: 5 mg OPD, 12.5 mL citrate buffer, 2.5 μL H2O2(30%).
Stopping solution: 2 N HCl.
Synthesis ofl-Arginine-Capped Fe3O4Nanoparticles
l-Arginine (3.0 g) and FeCl3(0.5 g) were added to a component solvent containing glycerol (10 mL) and water (10 mL). A transparent solution formed through sonication of this mixture. This solution was transferred into a Teflon-lined stainless steel autoclave with a capacity of 50 mL and maintained at 200°C for 6 h. Then, the autoclave was cooled to room temperature naturally. The product was washed with distilled water to remove residue of solvent and unboundl-arginine, finally dried by vacuum freeze-desiccation technology before characterization. During each step, the product was separated from the suspension by magnetic force.
Preparation of Magnetic Nanoparticles Conjugates
A solution was formed by mixing 250 μL Fe3O4 nanoparticles suspension and 1 mL phosphate-buffered saline (PBS). Then, 10 μL of anti-CAP monoclonal antibody and 1 mg of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added. Afterward, the mixture was incubated overnight with light shaking at room temperature. Excess EDC and the supernatant were removed by magnetic separation, and the precipitate was washed three times with PBS. Antibody-labeled magnetic nanoparticles were redispersed in PBS (1 mL) and stored at 4°C for use.
The above store suspension (100 μL) was added to a tube and rinsed three times with washing buffer (PBST) in a magnetic field. Then, 100 μL conjugates of chloramphenicol and horseradish peroxidase (CAP-HRP) were injected. The incubation was performed for 2 h at room temperature with constant shaking. The sample was washed three times with PBST as earlier. Substrate solution (100 μL) was added, and the reaction was kept for 15 min. Finally, stopping solution (2 N HCl) was used to stop the reaction, and the absorbance was determined at 492 nm. A comparative experiment was performed just replaced magnetic nanoparticles conjugates with unlabeled magnetic nanoparticles.
XRD patterns were recorded on the X-ray diffractometer (Bruker D8) with a graphite monochromator and Cu Kα radiation (λ = 1.5418 Å) in the range of 10–80° at room temperature. The morphology of the products was determined with transmission electron microscopy (JEM-100CXII) with an accelerating voltage of 80 kV. The nanocrystals dispersed in water were cast onto a carbon-coated copper grid. Magnetization measurements of the nanocomposites were performed with a Micromag 2900 at room temperature under ambient atmosphere. X-ray photoelectron spectra (XPS) were measured with X-Ray photoelectron spectroscopy XPS (ESCALAB 250). Enzyme immunoassay (ELISA) was performed with an automatic microplate reader KHB ST-360 from Shanghai Zhihua Medical Instrument Ltd.
Results and Discussion
We have synthesizedl-arginine-capped superparamagnetic Fe3O4 nanoparticles via a simple and green method in water and glycerol component solvent. The synthesized Fe3O4 nanoparticles have an average diameter of 13 nm and the saturation magnetization reaches to 49.9 emu g−1 with negligible remanence or coercivity. With superparamagnetic properties and the active groups on the surface of the nanoparticles, their application for magnetic separation and concentration in immunoassays were further demonstrated. These products are expected to have more extensive applications in biomedical fields.
Financial support from the Program for New Century Excellent Talents in University (NCET-06-0586), the Key Project of Chinese Ministry of Education (No. 109098), and the National Basic Research Program of China (973 Program 2005CB623601, 2007CB936602) is gratefully acknowledged. Prof. Xi acknowledges the financial support from the National Natural Science Foundation of China (No. 20675048), the National High-Tech Research and Developmental Program of China (863 Program, No. 07AA10Z435 and 2007AA06A407).