The synthesis and characterization of monodispersed chitosan-coated Fe3O4 nanoparticles via a facile one-step solvothermal process for adsorption of bovine serum albumin

Preparation of magnetic nanoparticles coated with chitosan (CS-coated Fe3O4 NPs) in one step by the solvothermal method in the presence of different amounts of added chitosan is reported here. The magnetic property of the obtained magnetic composite nanoparticles was confirmed by X-ray diffraction (XRD) and magnetic measurements (VSM). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) allowed the identification of spherical nanoparticles with about 150 nm in average diameter. Characterization of the products by Fourier transform infrared spectroscopy (FTIR) demonstrated that CS-coated Fe3O4 NPs were obtained. Chitosan content in the obtained nanocomposites was estimated by thermogravimetric analysis (TGA). The adsorption properties of the CS-coated Fe3O4 NPs for bovine serum albumin (BSA) were investigated under different concentrations of BSA. Compared with naked Fe3O4 nanoparticles, the CS-coated Fe3O4 NPs showed a higher BSA adsorption capacity (96.5 mg/g) and a fast adsorption rate (45 min) in aqueous solutions. This work demonstrates that the prepared magnetic nanoparticles have promising applications in enzyme and protein immobilization.


Background
In the past several decades, magnetic nanomaterials of iron oxides (Fe 3 O 4 NPs) have attracted much research interest due to their potential applications in magnetic storage, catalysis, electrochemistry, drug delivery, medical diagnostics, and therapeutics based on their unique magnetic, physiochemical, and optical properties [1][2][3][4][5]. Among the various methods for the preparation of Fe 3 O 4 NPs, the solvothermal approach is one of great significance [6][7][8][9]. Under the solvothermal conditions, Fe 3 O 4 NPs were usually composed of multiple singledomain magnetic nanocrystals. To date, the solvothermal method was developed for the preparation of magnetite spheres with strong magnetization through the hydrolysis and reduction of iron chloride in ethylene glycol at high temperatures. However, producing Fe 3 O 4 NPs with specific functional groups on the surface and acceptable size distribution without particle aggregation has consistently been a problem. Thus, a variety of modifiers were added to the reaction mixtures to control the size of Fe 3 O 4 NPs and improve the colloidal stability and biocompatibility, such as poly(acrylic acid) (PAA) [10], polyethyleneimine (PEI) [11,12], polyethylene glycol (PEG) [13], and other biocompatible polymers [14,15]. These modifiers are usually polymers bearing carboxylate or other charged groups. During the formation process of Fe 3 O 4 NPs, these charged groups can coordinate with iron cations in solution, and affect the nucleation and aggregation of the nanocrystals, resulting in the formation of Fe 3 O 4 NPs with controllable grain size and self-assembled structures. Compared with the types of polymers mentioned above, chitosan has been intensively studied as a base material for magnetic carriers because of its significant biological and chemical properties. The conventional method for preparing Fe 3 O 4 NPs coated with chitosan is the coprecipitation method that involves obtaining the magnetic nanoparticles, followed by chitosan coating. Several research teams have tried to simplify the procedure to obtain Fe 3 O 4 NPs coated with chitosan in one step [16][17][18][19][20]. However, there are very few reports on the synthesis of magnetic nanoparticles coated with chitosan (CScoated Fe 3 O 4 NPs) by a one-step solvothermal process.
In this paper, we report the preparation of monodispersed CS-coated Fe 3 O 4 NPs in the presence of different amounts of added chitosan via a facile one-step solvothermal process. A detailed characterization of the products was carried out to demonstrate the feasibility of this method for obtaining CS-coated Fe 3 O 4 NPs. Bovine serum albumin (BSA) isolation experiments were used to demonstrate the potential of the materials for adsorption.

Characterization
Transmission electron microscopy (TEM) images were obtained with a JEM-2100 transmission electron microscope (Jeol Ltd., Tokyo, Japan). X-ray diffraction (XRD) analysis was performed using a Dmax-2500 (Rigaku Corporation, Tokyo, Japan). Magnetic measurements (VSM) were studied using a vibrating sample magnetometer (Lake Shore Company, Westerville, OH, USA) at room temperature. Scanning electron microscopy (SEM) images were carried out on a Philips XL30 microscope (Amsterdam, The Netherlands). The zeta potential of these particles was measured by dynamic light scattering (DLS) with a Delsa™ NanoC Particle Size Analyzer (Beckman Coulter, Fullerton, CA, USA). Thermogravimetric analysis (TGA) of the nanocomposite and chitosan was       min to reach equilibrium. After reaching adsorption equilibrium, the supernatant and the solid were separated by using a permanent magnet. BSA concentrations were measured by a UV-2501PC spectrophotometer at 595 nm. The amounts of BSA adsorbed on the magnetic adsorbents were calculated from mass balance. The standard curve of BSA is Y = 0.867X + 0.033(R 2 = 0.9975).

Results and discussion
All reactions rendered a black powder at the end of the process. However, a difference between the composite nanoparticles loaded with different amounts of chitosan was visually detected. Figure 1 presents photos of Fe 3 O 4 coated with different amounts of chitosan. As shown in Figure 1a, the suspension color changed from black to tan and then turned to black with increasing amount of chitosan. Moreover, with increasing amount of chitosan of more than 1.25 g, there were lots of nonmagnetic black powder under the bottle (Figure 1e,f ), which may be caused by the oxidization and aggregation of excessive chitosan.
The functional groups of chitosan are very important for various applications, especially for biotechnological purposes. Therefore, the present functional groups should be kept even if the shape was changed into a new form; FTIR analyses were carried out. The FTIR spectra of MFCS-0, MFCS-1/3, MFCS-1/2, MFCS-2/3, and pure CS are given in Figure 2, which were exhaustively washed and magnetically recovered so that all the chitosan in the final products are chemically bound to the magnetic nanoparticles. In the spectrum of naked Fe 3 O 4 (Figure 2a), the absorption at 586 cm −1 is assigned to the characteristic band of the Fe-O group [21]. For pure CS (Figure 2e) (440)) were observed [23]. As shown in Figure 4b,c,d, these characteristic peaks can be seen in the composite magnetic nanoparticles, while the broad peak at 2θ = 17°to 27°was ascribed to chitosan, which indicated the existence of an amorphous structure [17].  As seen in Figure 5, the surfaces of the spheres appear rough and composed of many small nanoparticles. However, the spheres tend to be uniform, and the surface of the nanoparticles became smoother with increasing weight ratios of chitosan/Fe from 0 to 1/2 (Figure 5a,b,c). When the weight ratio of chitosan/Fe was from 2/3 to 1, the CS-coated Fe 3 O 4 NPs became morphologically rough and irregular and exhibited loss of structural cohesion (Figure 5d,e,f). In Figure 6, the spheres became smaller with increasing weight ratios of chitosan/Fe from 0 to 2/3.
The stability of the CS-coated Fe 3 O 4 NPs in this work was studied. We chose representative water, phosphatebuffered saline (PBS) plus 10% (v/v) fetal bovine serum, PBS, and NaCl (1.0 mol/L) as media in which CS-coated Fe 3 O 4 NPs were dispersed to systematically investigate their stability by UV-visible absorbance spectroscopy at a fixed wavelength (450 nm). If nanoparticles are not stable and sedimentate rapidly, they can be monitored by a decreased absorbance as a function of time. Figure 7 shows that the CS-coated Fe 3 O 4 NPs dispersed in water, PBS, and PBS plus 10% (v/v) fetal bovine serum present excellent stability, whereas those dispersed in high concentration of NaCl exhibit poor stability. These results suggest that the CS-coated Fe 3 O 4 NPs dispersed in high concentration of NaCl aggregate rapidly, which is confirmed by the DLS result, as seen in Table 1.
The electrostatic interaction of the magnetic nanoparticles can be controlled by variation in their surface charges, which can be determined by measuring the zeta potential of these particles. Compared with that of naked Fe 3 O 4 NPs (Figure 8a), the zeta potential of MFCS-1/2 possessed a higher positive charge (Figure 8b). This may be caused by the hydrogen of the amino group (-NH 2 ) in chitosan. Thus, this indicated that the modification with CS on Fe 3 O 4 NPs was successful.
The magnetic properties of the as-synthesized NPs after being coated with CS are a prerequisite for magnetic guiding application. To gain a better understanding of the magnetic properties of the as-synthesized NPs, the magnetization curves of different amounts of CS coated on the surface of the Fe 3 O 4 NPs were measured. As shown in Figure 9, the saturation magnetization values of the CS-coated Fe 3 O 4 NPs synthesized with chitosan: MFCS-0, MFCS-1/3, MFCS-1/2, and MFCS-2/3, were 64.2, 52.5, 30.8, and 20.5 emu g −1 , respectively. This trend can likely be attributed to the higher weight fraction of chitosan.
In the experiment, Fe(OH) 3 was formed through the hydrolysis of FeCl 3 · 6H 2 O, then Fe(OH) 2 was obtained through the reduction of Fe(OH) 3 with ethylene glycol at high temperature, and finally Fe(OH) 3 and the newly produced Fe(OH) 2 formed a more stable Fe 3 O 4 phase. As reported by Burke [24], Fe(III) ions are attached to the chitosan's surface by forming a complex compound in which Fe(III) ions act as the metal center while the ligands are the amine and -OH of chitosan. Therefore, the    possible reaction describing the formation mechanism of the CS-coated Fe 3 O 4 NPs can be expressed by Figure 10.
In order to investigate the adsorption capabilities and adsorption rate of the CS-coated Fe 3 O 4 NPs, 10 mg of dried CS-coated Fe 3 O 4 NPs were added into a 10.0-mL BSA aqueous solution. As illustrated in Figure 11a, the amount of adsorbed BSA increased with elapsed immersion time. Compared with naked Fe 3 O 4 nanoparticles (Figure 11a), the CS-coated Fe 3 O 4 NPs showed a higher BSA adsorption capacity (96.5 mg/g) and a fast adsorption rate (45 min) in aqueous solutions. This is due to the higher initial BSA concentration that provides a higher driving force for the molecules from the solution to the amide-functionalized CS-coated Fe 3 O 4 NPs [25], resulting in more collisions between BSA molecules and active sites on the CS-coated Fe 3 O 4 composites.

Conclusions
In summary, a facile one-step solvothermal method was developed to prepare CS-coated Fe 3 O 4 NPs with tunable magnetism, sizes, suspension stability, and surface charge. The size of the nanoparticles was about 150 nm, and chitosan made up 40% to 48.0% of the weight of the modified Fe 3 O 4 NPs. Compared with Fe 3 O 4 nanoparticles, the CScoated Fe 3 O 4 NPs showed a higher BSA adsorption capacity. This work revealed a promising method for the recovery of slaughtered animal blood by using magnetic separation technology.