Fe₃O₄ nanoparticles were synthesized according to Ahmed et al. 11 with several modifications resulting in substantial quality improvements. All of the chemicals used in this research were of analytical grade and obtained from commercial sources. FeCl₂. 4H₂O, FeCl₃·6H₂O, sodium hydroxide, sulphuric acid, nitric acid, hydrochloric acid, N-tetra methyl ammonia hydroxide, formaldehyde (37%), ammonium hydroxide, sodium phosphate monobasic, sodium phosphate dibasic and hydrogen tetrachloroaurate(III) (HAuCl₄.4H2O, 99%) were obtained from Merck, Germany. Chitosan was prepared from Sigma-Aldrich, USA. Deionized water was obtained from Milli Q system and used throughout. The solutions of FeCl₃·6H₂O (4 ml, 2 M) and FeCl₂·4H₂O (2 ml, 2 M) were prepared in 250 ml flasks, added to a flat bottom beaker, and stirred at 30°C for 45 min. The Fe(III)/Fe(II) ratio was kept 2 throughout. Then, an aqueous ammonia solution (100 ml, 1 M) was added by droplet under the cover of N₂ gas and the pH of the solution was carefully adjusted up to 10. The solution was stirred for about 1 h until stable, black Fe₃O₄ particles appeared. Next, the particles were filtered and then rinsed with distilled water and then methanol until the pH reached 7. They were then dried in a vacuum oven at room temperature for 24 h.

The synthesis of Fe₃O₄-gold nanoparticles was carried out according to Cui et al. 12 with some modifications. First, Fe₃O₄ nanoparticles were dispersed in a 0.1 M HAuCl₄·4H₂O solution in a flat bottom beaker for 20 minutes using sonication, and then slowly mixed in a shaking incubator at 38°C to allow the adsorption of Au³⁺ into the Fe₃O₄ surface. Glucose was then added to the system as a reducing agent and the mixture was incubated at room temperature in a shaking incubator (200 rpm). The core-shell nanoparticles that formed were then washed with pure water until the pH reached 7.

Chitosan (200 mg) was added to 14 ml of acetic acid (1%, v/v) solution and stirred for 10 minutes at room temperature until it became a homogeneous viscous solution. Then, various concentrations of formaldehyde (2-10 ml, 5 M) were used to improve the gelation properties of the formed hydrogel. The prepared chitosan solution was simultanously added to the gold-coated magnetic nanoparticles being formed in the solution and incubated at room temperare with shaking in a shaking incubator (200 rpm) for 1.5 h leading to synthesis of the core-shell structure of Fe₃O₄-gold-Chitosan.

Fourier transformed infrared (FTIR) spectroscopy was carried out by a Bruker FTIR-6000 (Bruker, Germany) using KBr discs to investigate the interaction of functional groups in chitosan with the nanoparticles surface. The crystallographic characterization of nanoparticles was done by a powder X-ray diffraction (XRD) spectrometer (Bruker D8 Advance, Germany). Transmission electron microscopy (TEM) images to obtain the morphology and size of the nanoparticles were taken using a LEO920 TEM (Carl Zeiss, Germany). The topographic images of nanoparticles and their orientation in the chitosan texture were obtained by atomic force microscopy (AFM) (CSM-Bruker, Germany). The mean hydrodynamic diameter of nanoparticles was measured using Zetasizer (Malvern model, China),

Gold magnetite nanoparticles have an Fe₃O₄ core with an average size of 9.8 nm in diameter. The average size of Fe₃O₄ nanoparticles was measured using XRD with Cu Kα radiation at 1.540 Å (Figure 1). Magnetic seeds were synthesized using co-precipitation under controlled condition (pH = 10) and N₂ protection gas. The optimum mole Fe³⁺: Fe²⁺ ratio used was 2:1. The AFM image of Fe₃O₄ stabilized by chitosan is exhibited in Figure 2. AFM topographic images indicate physically dispersed Fe₃O₄ nanoparticles on chitosan gel (A), and immobilization of Fe₃O₄ nanoparticles in chitosan gel (B). The magnetite nanoparticles were oriented in one direction due to magnetic properties (Figure 2-B).

The FTIR spectra of chitosan, formaldehyde cross linked chitosan hydrogel and Fe₃O_4-chitosan hydrogel are shown in Figure 3. The broad band found at 3429 cm^-1 is due to overlapped -OH and -NH groups in chitosan. The band observed at 2902 cm^-1 is attributed to C-H bands. The band at approximately 1656 cm^-1 is due to amide band C-O stretching, along with N-H deformation, and at 1592 cm^-1, it is due to the characteristic peak of the NH₂ group. The absorption peaks at 1412 cm^-1 are characteristic of -CH₂- and, skeletal vibration involving C-O-C bridge stretching of the glucosamine residue is responsible for the band at 1107 cm^-1. The 1025 cm^-1 band is likely related to CH-OH bonds in cyclic compounds. The peaks that appeared at 587 and 477 cm^-1, are indicative of stretching, and the variation modes of Fe-O confirms the presence of crystalline Fe₃O₄ (Figure 3-c). For the Fe₃O₄ nanoparticles dispersed on the chitosan hydrogel, the FTIR spectrum confirmed considerable changes for the immobilized Fe₃O₄ nanoparticles based on the shape and frequencies of the bands, indicating the interaction of functional groups in chitosan with the Fe₃O₄ at the surface (Figure 3-d).

Gold provides stability for the magnetic nanoparticles in solution as well as providing a good inert surface for assisting the binding of various biomolecules 131415. The gold shell was synthesized by the reduction of Au³⁺ with glucose as a nontoxic, biocompatible reducing agent in the presence of Fe₃O₄ nanoparticles. When the Fe₃O₄ nanoparticles were gradually coated by gold, the color of the solution changed the black nano-magnetite particles (A) to reddish brown (B) (Figure 4). The magnetic properties of the Fe₃O₄-gold nanoparticles can be controlled by synthesis conditions. For example, saturation magnetization values for uncoated and coated Fe₃O₄ nanoparticles can be decreased with the formation of gold layer at different temperatures 9.

The XRD spectra of the Fe₃O₄-gold nanoparticles showed that they have an average diameter size of 15 nm. The diffraction peaks at 2θ° = 38.3°, 44.2°, 64.5°, 77.8°, and 81.7° are attributed to Fe-gold, which can be indexed to 111, 200, 220, 311, and 222 lattice planes of gold in a cubic phase, respectively. The absence of any diffraction peaks for Fe₃O₄ is most likely due to the heavy atom effect from gold as a result of the formation of gold-coated Fe₃O₄ nanoparticles. The diffraction peaks from Fe₃O₄ provide strong evidence for complete coverage of the magnetic core by gold (Figure 5).

Chitosan plays an important role in nanocomposite production via amino and hydroxyl groups, and stabilizes the produced nanoparticles. It seems that Au³⁺ ions were absorbed at first physically on the surface of Fe₃O₄, and then chemically by adding glucose and chitosan in order to retrieve its electron. Chitosan and glucose both act as reducing and stabilizing agents via the crowding method 1617 (Figure 6). The effect of various parameters including the amount of formaldehyde as cross linker, pH and temperature on the equilibrium water content (EWC %) of the formed chitosan hydrogel was evaluated. When the concentration of formaldehyde was increased, the equilibrium water content decreased (Figure 7a). This can be due to a decrease in the space between polymer chains. The maximum EWC% of the hydrogel was observed at pH 3 (Figure 7c), this is attributed to complete protonation of the amine groups of chitosan. The hydrogel exhibited an equilibrium water content (EWC %) in the range of 96-97.5% at pH 7 and temperature between 25-45°C (Figure 7b). The chitosan hydrogel showed maximum swelling at low pH and high temperature.

The TEM image of the core shell Fe₃O₄-gold nanoparticles stabilized by chitosan confirms the formation of core-shell Fe₃O₄-gold nanoparticles (Figure 8). The Fe₃O₄ core, after it was coated with the gold shell, was much darker than the pre-coated magnetite nanoparticles. TEM analysis revealed that the average particle size increased from 9.8 nm before gold coating to 15 nm after gold coating, respectively. The average diameter of nanoparticles was found to be about 25 ± 5 nm using dynamic light scattering (DLS) measurements (Figure 9). Of course, it seems that DLS is not accurate method for true size measurement of nanoparticles. The synthesized nanoparticles were uniformly dispersed in the sample and seemed to be spherical in structure. To obtain a monotonous, smooth gold-layer shell, glucose was used to reduce Au³⁺. Ultrasonic agitation was applied to give it uniform monodispersity and to prevent particle aggregation.

Up until now, considerable effort has gone into the formation of gold-coated magnetite nanoparticles, but the use of them is still restricted due to some problems in the way it is synthesized 2121819. In most cases, hydroxylamine, citrate, and borohydride have been used as reducing agents in combination with the reverse micelle technique for reducing gold salt nanoparticles 13182021. Tamer et al. 19 reported a two-step synthetic method in which the magnetite nanoparticles were coated with gold using the borohydride reduction of HAuCl₄ under sonication in order to achieve a better monodispersity and prevent aggregation problems. In this study, the use of the biopolymer chitosan as a template for the preparation of stable magnetite-gold core-shell monodisperse nanoparticles with a mean diameter of 15 nm was developed under mild temperature conditions.