One-Pot Green Synthesis and Bioapplication ofl-Arginine-Capped Superparamagnetic Fe3O4 Nanoparticles

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. (See supplementary material 1)


Introduction
In the last decade, inherently safer nanomaterials and nanostructured devices were widely fabricated with the ''green chemistry'' principles [1][2][3][4][5][6][7][8][9][10][11][12][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 [1]. 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 [14].
In the present work, we described a facile and green approach toward synthesis and stabilization of Fe 3 O 4 nanoparticles. Water and glycerol were used as environmentally benign solvents in the synthesis. Inartificial amino acid L-arginine was chosen as the nontoxic, renewable stabilizing agent.

Experimental Section
Materials 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.
Buffers and solutions used were listed below: Synthesis of L-Arginine-Capped Fe 3 O 4 Nanoparticles L-Arginine (3.0 g) and FeCl 3 (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 Teflonlined 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 unbound L-arginine, finally dried by vacuum freezedesiccation 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 lL Fe 3 O 4 nanoparticles suspension and 1 mL phosphate-buffered saline (PBS). Then, 10 lL 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.

Immunomagnetic Assay
The above store suspension (100 lL) was added to a tube and rinsed three times with washing buffer (PBST) in a magnetic field. Then, 100 lL 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 lL) 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.
Characterization XRD patterns were recorded on the X-ray diffractometer (Bruker D8) with a graphite monochromator and Cu Ka radiation (k = 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 carboncoated 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
Black products were prepared via a one-step solvothermal method. L-Arginine, an alkaline amino acid with guanidino group, was served as capped reagent in this reaction. The crystallinity and phase purity were determined by powder X-ray diffraction (XRD) as shown in supporting information. All diffraction peaks could be assigned to inverse spinel Fe 3 O 4 phase (JCPDS card . No other crystalline impurity was detected. The lattice constant calculated from this pattern was 8.389 Å , which is very close to Nanoscale Res Lett (2010) 5:302-307 303 the reported value. A representative TEM image of L-arginine-capped Fe 3 O 4 nanoparticles dispersed in acidic solution is shown in Fig. 1a, which indicates that Fe 3 O 4 nanoparticles have an average diameter of 13 nm. The highresolution transmission electron microscopy (HRTEM) image (Fig. 1b) suggests the crystalline nature of Fe 3 O 4 nanoparticles with a clearly resolved lattice spacing of around 0.483 nm, corresponding to that of (111) of inverse spinel Fe 3 O 4 crystal. All the spots of Fourier transformed pattern (Fig. 1c) obtained from the HRTEM image in Fig. 1b can be indexed as those peculiar to the 01 " 1 ½ zone axis of face centered cubic Fe 3 O 4 .
X-ray photoelectron spectroscopy (XPS) was used to further confirm the products. From spectra in Fig. 2a, [38,39]. This phenomenon confirms the product is Fe 3 O 4 rather than c-Fe 2 O 3 . As is shown in the magnetic hysteresis loop of L-arginine-capped Fe 3 O 4 nanoparticles (Fig. 3), the nanocrystals behave with superparamagnetism at room temperature with saturation magnetization of 49.9 emu g -1 and negligible remanence or coercivity.
The different dispersing state of Fe 3 O 4 nanoparticles in acidic and alkaline solutions can be clearly observed by naked eye, as shown by the supporting information Fig. S2. Fe 3 O 4 nanoparticles dispersed in an alkaline solution completely precipitated in a few minutes, while they are stable in an acidic solution for at least 1 month and could be moved by a magnet just like ferrofluid. When the suspensions were filtrated with 0.45 lm filtration membrane, we got colorless and transparent liquid as Fe 3 O 4 nanoparticles could not pass filtration membrane in alkaline solution. On the other hand, black and homogeneous solution was collected in acidic solution. L-Arginine is an inartificial amino acid. The amino group and the acid group could exist in the form of ammonium ions and carboxylate ions, respectively, under certain conditions [40]. It has been reported that both amine and acid groups are able to attach onto iron oxide surface [17,25]. When the guanidino group of L-arginine attaches onto the surface of iron oxide, the nanoparticles are expected to have distinct states in solutions with different pH value. Although the isoelectric point (pI) of pure L-arginine is 10.76 [40], the isoelectric point is expected to change for the attachment of the guanidino group in L-_arginine to Fe 3 O 4 nanoparticles. The new isoelectric point will be the average of the pKa of the carboxylic acid group and the pK b of the amine group [40] and therefore, ca. 5.61. As illustrated in Scheme 1, in an acidic solution, the L-arginine molecules exist in the cationic form due to the formation of ammonium ions. These ammonium ions may prevent formation of hydrogen bonds between Fe 3 O 4 nanoparticles. In an alkaline solution, surface-bound L-arginine molecules are negatively charged due to the formation of carboxylate ions which readily form hydrogen bonds with surface-bound amine groups of neighboring Fe 3 O 4 nanoparticles. This phenomenon is similar to the case of lysine-capped gold nanoparticles [41].
To demonstrate potential biomedical applications of L-arginine-capped Fe 3 O 4 nanoparticles, magnetite nanoparticles were bioconjugated with anti-CAP monoclonal antibody to form the immunomagnetic beads (IMB) via the classical EDC activation [42,43]. Then, they are used in the immunological test. The result showed that the mixture containing anti-CAP monoclonal antibody-labeled magnetic nanoparticles had a deep yellow color (Fig. 4 Fig. 4 left) at the same time and the absorbance was 0.065. It was suggested that L-arginine-capped Fe 3 O 4 nanoparticles were successfully attached to the anti-CAP monoclonal antibody.

Conclusions
We have synthesized L-arginine-capped superparamagnetic Fe 3 O 4 nanoparticles via a simple and green method in water and glycerol component solvent. The synthesized Fe 3 O 4 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.